Ethnomedicine: A Source of Complementary Therapeutics by Research Signpost

Ethnomedicine: A Source of Complementary Therapeutics 2010 Editor

Debprasad Chattopadhyay ICMR Virus Unit, ID & BG Hospital, GB-4 First Floor, 57 Dr Suresh C Banerjee Road Beliaghata, Kolkata

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Research Signpost 2010; Rights Reserved Research Signpost T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Debprasad Chattopadhyay Managing Editor S.G. Pandalai Publication Manager A. Gayathri Research Signpost and the Editor assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-308-0390-6

Autobiography of the Editor Dr. Debprasad Chattopadhyay did his Ph.D. in Pharmaceutical Microbiology from Jadavpur University, Kolkata in 1989 after his M.Sc. and then moved to London Hospital Medical College, London as a Visiting Fellow. After a brief training at London under Late Professor J.D. Williams and Dr Jette Elisabeth Kristiansen at Satens Serum Institute, Copenhagen he returned to India and joined the Indian Institute of Chemical Biology (CSIR), Kolkata and then in the Regional Medical Research Centre (Indian Council of Medical Research), Port Blair in 1993 as a Research Scientist. In 1997 he moved to ICMR Virus Unit, Kolkata, at which he is presently working as an Assistant Director (Scientist). He made significant contribution in public health research, particularly in antimicrobial drug development. His in depth studies on ethnomedicinal practices of the Onge, Nicobarese, and Shompen tribes of Andaman & Nicobar Island (by establishing personal relationship with them), help in recording the endemic, threatened and rare flowering plants of Bay Islands. Utilizing the tribal Knowledge-base, he has investigated the scientific basis of those medicaments and identified four herbal leads with antimicrobial, anti-inflammatory, antipyretic, antipsychotic and sperm motility-inhibiting activities. His group has purified and characterized a bioflavonoid that can inhibit the in vitro proliferation of Plasmodium falciparum, the agent of deadly malaria, and is highly effective against the chloroquine-resistant P. falciparum strain. His another contribution is the demonstration of antibacterial activity of methdilazine, a phenothiazine, that produce synergism with aminoglycoside antibiotics. This combination is useful in controlling bacterial resistance as methdilazine alters membrane permeability of bacteria thereby facilitate the entry of antibiotics within the bacterial cell. He has contributed more than 50 research papers, 5 chapters and 5 Review in highly prestigious International Journals like Biotechnology Annual Review, Mini Review in Medicinal Chemistry; New Biotechnology etc., and Books like Modern Phytomedicine: Turning Medicinal Plants into Drugs; New Strategies Combating Bacterial Infection (Wiley-VCH, Germany), Evaluation of Herbal Medicinal Products: Perspectives of Quality, Safety and Efficacy (Royal Pharmaceutical Press, Great Britain) etc. and is the editor of the reference book Ethnomedicine, A Source of Complementary Therapeutics (Research Signpost, India) and

Phytotherapy in Diabetes and Hypertension (Bentham Science, USA). He is associated with 18 International peer reviewed Journals as Reviewer and Editorial Board Member of three Journals, published by Bentham Science, Elsevier Science, Global Science Book Publications, Academic Press etc. As Guest Faculty, Examiner, paper setter, Ph.D. guide he is attached with Universities like Jadavpur University, Kolkata; Aligarh Muslim University, Aligarh, Dr MGR University, Chennai etc. He has received awards from the Indian Association of Medical Microbiologists (1988), CSIR Research Associateship (1989-1992), Dr R.V. Rajam Medal from the Indian Association for the Study of Sexually transmitted Diseases & AIDS (1998), International Society of Chemotherapy (1991), Overseas Award from the World Conference on Dosing of Antiinfectives, Germany (2004), Professor Amiya Bose Oration Award from Indian Dietetic Association (2008 and 2009), Indian Science Communicator Award in 2nd Rastriya Vigyan Sancharak Sammelan 2009 by the Department of Science & Technology, Government of India. Dr Chattopadhyay also chaired the scientific session in the International Conference on Drug Delivery and Drug Targeting Research, Kolkata in 2008, and International conference on Herbal Medicine Evaluation of Quality, Efficacy and Safety, Bangalore in 2009. He is the member of different Selection Committee for Government funded research projects, and for the selection of teaching faculty of University and Colleges. He has four patents in his credit and handled several projects of national importance, including ethnomedicinal projects with grass root innovators.

Foreword The use of medicinal herbs and herbal medicine is an age-old tradition and the recent progress in modern therapeutics has stimulated the use of natural product worldwide for diverse ailments and diseases. The educated public and health professionals have enormous interests in the medicinal uses of herbs but, unfortunately, there is a great deal of confusion about their identification, effectivity, therapeutic dosage, toxicity, standardization and regulation. According to WHO, ethnomedicine is popular in all regions of the developing world and its use is rapidly expanding in the developed countries, for example, in China traditional herbal preparation account for 30-50% of the total medicinal consumption. In Ghana, Mali, Nigeria and Zambia, the first line treatment for 60% of children with malaria is the use of herbal medicine. In San Francisco, London and South Africa, 70% of people living with HIV/AIDS use traditional medicine. Today the annual global market for herbal medicine stands at over US $60 billion. Western trained physicians should not ignore the impact of ethnomedicine on their patients. Human evolution across time represents a fascinating story in the parlance of biology. However, the journey was not trouble free. In our sustenance and perpetuation many odds like disease posed serious threat towards very survival of mankind. Many a time’s history has witnessed epidemics leading to the loss of huge lives across continents imposing high economic, social and psychological costs. Therefore, it was important to prevent diseases, maintain and restore the health of those who fall ill. Every human community responded to this challenge by developing a system for health care. Hence, the medicinal system reflects the interaction and synergy of social and cultural system. Since prehistoric times, human beings have found remedies within their habitat and have adopted different therapeutic strategies depending upon climatic, phytogeographic, floral and faunal characteristics as well as their cultural and socio-structural typologies. Traditional systems thus, contain beliefs and practices in order to avoid, prevent or avert ailments, which constitute traditional preventive medicine. The medicinal systems based on cultural framework thus created a new discipline of “ethnomedicine”. (eth·no·med·i·cine/eth″ no-med´ĭ-sin/ Ethnomedicine eth ·nō·me ·di·sin) or ethnic medicine or “folk medicine” is the medical systems based on the cultural beliefs and practices of specific ethnic groups or particular culture and concern about the care and treatment of illness. The study of etiology of disease, practitioners and their role in health care, and types of treatment administered are the purview of ethnomedicine, which helps in the

search for new cures and wellness among indigenous people. At large, ethnomedicine is a sub-field of ethnobotany or medical anthropology of all cultures either written (e.g. Traditional Chinese Medicine, Ayurveda), or orally transmitted over the centuries. In the scientific arena, it deals with the use of several health promoting practices and natural products for the maintenance of optimal physical and emotional health, as well as prevention and treatment of diseases. The term “traditional” imply repetition from generation to generation developed on careful observation by traditional healers in a given generation of indigenous people, compare their personal experiences with what they have been told by their teachers and neighbours, conduct experiments to test the reliability of their knowledge, aa well as to enhance and improvise their findings. The “tradition” about traditional knowledge is not its antiquity but the way of acquiring and use, which is unique to each indigenous culture. In almost all ethnomedicinal system the cause of diseases are commonly described as “natural” and “magical” or “supernatural”, and illness caused by angry deities, ghosts, ancestors and witches fall into the first category; while those due to an upset in body humors and consequently lose of bodily equilibrium fall into second. This stands in contrast to that of natural causes, where illness is explained in impersonal, systemic terms. The intrusion of heat or cold into or their loss from the body upsets the basic equilibrium; the balance of humors off the dosha of Ayurveda, and the Yin and Yang of Chinese medicine must be restored if the patient is to recover. The natural environment is a living incubator where the components like land, sea, atmosphere, the flora and the fauna are linked and interact with human being in an intrinsic manner. Therefore, plants play a participatory role in healing. A healer’s power is determined by the magnitude of his understanding of the natural laws for the benefit of patients and the whole community. Ethnomedicine in many cultures is used as healing traditions over centuries, embraces on the belief that the earth plays a vital role in one's spiritual, emotional, and physical well-being, and these methods are effective in many cases as biomedical treatment. The western medicine is much more technical and analytical, while traditional practices are much more holistic. Good health, disease, success or misfortune are not seen as random but arise as a result of the individuals’ actions and the balance or imbalance between the individual and the social environment. Use of herbal remedies in Africa, South America and Asia, some parts of Europe represents a long history of human interactions with the environment. Plants used in traditional medicine contain a wide range of metabolites that can be used to treat chronic as well as infectious diseases. A vast knowledge about the use of plants against different illnesses may be expected to have accumulated in areas where the use of plants is still of

great importance. The medicinal value of plants lies in either alone or in combination of some phytochemicals that produce a definite physiological action on the human body. The most important of these bioactive compounds are alkaloids, flavanoids, tannins and phenolics. Medicinal plants or its components used in traditional medicines are usually less or non-toxic due to time tested selection, and traditional dosage in liquid form to encourage the use of extremely low concentration of the active ingredients. However, the problem of toxicity arises due to human manipulation to increase the accumulation of the active compound for increased bioactivity. Therapeutically active molecules in plants exists in a mixture with other phytochemicals like tannins, carbohydrates, amino acids, proteins, vitamins, trace metals, etc. Moreover, the human body is well acquainted to these natural extracts, most of which, in other forms, are consumed as food. Thus, usually these herbs do not upset or inflict toxicity in the body, but help in maintaining the physiological homeostasis by and large. Single isolated compound as drugs possess all other substances with which they co-exist in the plant in natura. It has been suggested that the reactivity of a single pure compound as drug with the bodyâ&#x20AC;&#x2122;s physiological medium leads to the manifestation of drug toxicity. Phytomedicines with a little amount of processing can promote the healthy development of the body (health food) since it contains not only the active drug molecule, but also other substances required to maintain the overall physiological functions of the body synergistically. This is why â&#x20AC;&#x2DC;bitter leafâ&#x20AC;&#x2122; Vernonia amygdalina can be used as a food and as a drug for diabetes without apparent toxicity. However, the purified extracts or concentrated isolates are considered as medicines and must be subjected to rigorous standardization used to test medicinal agents. Phytochemical studies must be tailored to match the biological activity while the chemical studies should provide information that help in standardization and quality control of the finished product. Research interest and activities on ethnomedicine have increased tremendously in recent time and scientific research has made important contribution to the understanding of traditional subsistence, medical knowledge, wisdom and practice. The explosion of ethnomedicinal literature help in increased awareness among people, the recognition of indigenous health concepts as a means of ethnic identities, the search for new treatments and technologies. The ethnomedicine have long been ignored by biomedical practitioners as the chemical composition, dosages and toxicity of ethnomedical plants are not clearly defined. However, ethnomedicinal uses of plants are one of the most successful criteria in finding new therapeutic agents. Some outstanding drugs developed from the ethnomedicinal uses include: vinblastine and vincristine from Catharanthus roseus for treating lymphoma and leukaemias, reserpine from

Rauwolfia serpentina for hypertension, aspirin from Salix purpurea for inflammation, pain and thrombosis and quinine from Cinchona pubescens for treating malaria. This reference book is an attempt to summarize the current knowledge of promising ethnomedicines and their phytophores, to compounds tested against diverse diseases. The therapeutic properties and structure activity relationship (SAR) of some important and potentially useful ethnomedicines is addressed with a focus on how these ethnic knowledge can led to the development of useful therapeutic lead for preclinical or clinical evaluation. In general it is a snapshot of different areas of research on the role of phytochemicals in health, comprehensively presented and is useful of tidbits of knowledge or ideas for research, covering the ethnomedicines uses in the management of several diseases, particularly, infectious diseases (like viral, bacterial and fungal) and lifestyle related disorders mostly validated by modern scientific methods. In depth information prepared by experts all over the globe traces the evolution of herbal drugs with civilization and their use as antiviral, antibacterial, antifungal, antiparasitic, antioxidants, anticancerous, chemopreventors, memory enhancers, neuroprotective, immunomodulator, laxatives, analgesic and anti-inflammatory disorders, along with safety issues and toxic effects. The special emphasis covers twelve important topics like Diseases that need new drugs, role of ethnomedicine as complementary therapeutics, ethnomedicnal plants in parasitic infections, ethnomedicines for the development of anti-herpesvirus agents, ethnomedicinal plants derived antibacterials, recent advances in antimicrobial effects of essential oils, ethnomedicnal plants to fight neoplastic diseases, recent advances on ethnomedicinal immunomodulatory agents, plant based treatment of Alzheimerâ&#x20AC;&#x2122;s disease, ethnomedicinal plants as antiinflammatory and analgesic agents, ethnomedicnal plants as laxative drugs and the relationship of ethnomedicines with pharmacogenomics. This book offers researchers working on diverse aspects of medicinal plants with a complete coverage of botany, ethnology, pharmacology, toxicology and medicinal properties, and provides essential source material to all working in the fields of botany, pharmacy, traditional systems of medicine and drug industry. This book is the outcome of extensive consultations among biomedical scientists and clinicians and I am immensely grateful to those colleagues for their support in developing the concept. My thanks go to Dr. Shankar Pandalai, Managing Editor and A. Gayathri, Publication Manager of the Research Signpost for their suggestions and help in producing this book. With great pleasure and respect, I extend my sincere thanks and indebtedness to all the contributors, particularly Professor Joseph Molnar, Dr Iqbal Ahmad,

Professor George Varughese, Dr. Mahmud Tareq Hassan Khan, Dr. Biswajit Mukherjee, Dr. Pulok Mukherjee, Professor C.K.K. Nair, Professor Ilkay Orhan, Professor Wandee Gritsanapan, Dr. Mahiuddin Alamgir and Dr. M. Anilkumar, for their response, excellent updated contributions and consistent cooperation as well as patience. I express my deep gratitude to all those scientific colleagues and teachers, who not only contributed their work but also, help me in reviewing the manuscripts time to time. I am indebted to the Officer in-Charge of the ICMR Virus Unit, Kolkata and the Research Signpost, Thiruvanandapuram, India for their support and interest to made this volume possible.

Dr. D. Chattopadhyay

Contents

Chapter 1 Diseases that need new drugs: Need of the hour Debprasad Chattopadhyay, Paromita Bag and Sujit K Bhattacharya

Chapter 2 Ethno medicine in complementary therapeutics Pulok K. Mukherjee, S. Ponnusankar and M. Venkatesh

Chapter 3 Ethnomedicinal plants in parasitic infections Varughese George, Sabulal Baby and Anil John J

Chapter 4 Ethnomedicines for the development of anti-herpesvirus agents Debprasad Chattopadhyay, Sonali Das, Sekhar Chakraborty and Sujit K Bhattacharya

117

Chapter 5 Ethnomedicinal plants derived antibacterials and their prospects Maryam Zahin, F. Aqil, M. S. A. Khan and Iqbal Ahmad

149

Chapter 6 Recent advances in research of antimicrobial effects of essential oils and plant derived compounds on bacteria Zsuzsanna Schelz, Judit Hohmann and Joseph Molnar

179

Chapter 7 Ethnomedicinal plants to fight neoplastic diseases C. K. K. Nair, P. Divyasree and G. Gopakumar

203

Chapter 8 Recent advances on the ethnomedicinal plants as immunomodulatory agents Mahiuddin Alamgir and Shaikh Jamal Uddin

227

Chapter 9 An update on plant-originated treatment for Alzheimer’s disease Ilkay Orhan, Gürdal Orhan and Bilge Şener

245

Chapter 10 Ethnomedicinal plants as anti-inflammatory and analgesic agents M. Anilkumar

267

Chapter 11 Ethnomedicinal plants popularly used in Thailand as laxative drugs Wandee Gritsanapan

295

Chapter 12 Ethnomedicines and pharmacogenomics Biswajit Mukherjee, Biswadip Sinha and Soma Ghosh

317

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 1-28 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

1. Diseases that need new drugs: Need of the hour 1

Debprasad Chattopadhyay1, Paromita Bag1 and Sujit K Bhattacharya2

ICMR Virus Unit, ID & BG Hospital, GB-4, First Floor, 57 Dr Suresh C Banerjee Road, Beliaghata Kolkata; 2Additional Director General, Indian Council of Medical Research, Ansari Nagar New Delhi 110029, India

Abstract. Studies on new drugs for malaria, trypanosomiasis, filariasis, tuberculosis, schistosomiasis, leshmaniasis and amoebiasis came almost to a standstill; while there is no suitable drug to stop the emerging, re-emerging and the drug-resistant pathogens. On the other hand, the clinical efficacy of many ethnomedicines or compounds of natural origin was not yet evaluated and the composition of many traditional preparations was only crudely analysed. Pharmaceutical scientists are experiencing difficulty in identifying new lead structures in the finite world of chemical diversity as most synthetic drugs have unacceptable side effects. While many ethnomedicinal molecules like artemisinin, baccosides, curcumine, picrosides, piperidines, phyllanthins, psoralens, quinghaosu, rauwolfia alkaloids, steroidal lactones and glycosides, ursolic acid, withanolides showed impressive successes. The need of the hour is to develop new effective drugs against protozoal (malaria, trypanosomiasis, filariasis, schistosomiasis, leshmaniasis and amoebiasis), emerging and re-emerging viral and bacterial diseases, drug resistant microbes, chronic and difficult-to-treat diseases (like cancers, cardiovascular disease, diabetes, rheumatism etc), and prion diseases. This review will highlight the pros and corn of those diseases that need the new drugs and the recent research on the line. Correspondence/Reprint request: Dr. Debprasad Chattopadhyay, ICMR Virus Unit, ID & BG Hospital, GB-4 First Floor, 57 Dr Suresh C Banerjee Road, Beliaghata, Kolkata. E-mail: debprasadc@yahoo.co.in

Debprasad Chattopadhyay et al.

Introduction Disease and drug A disease is a pathological condition or abnormality of body (cell, organ, or system) and or mind of an organism resulting from infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms that causes discomfort, dysfunction, distress, or death to the person afflicted or those in contact. Literally, a disease refers to the invasion of the body by pathogens, hence, Pathology is the study of diseases, while the systematic classification of diseases is referred to as Nosology; but the knowledge about diseases and their treatments is Medicine. Medical science distinguishes a disease, having a known specific cause(s), called its etiology, and a syndrome is a collection of signs or symptoms that occur together. Many different intrinsic (genetic defects or nutritional deficiencies) or extrinsic (environmental exposure, such as second-hand smoke) factors alone or together can cause disease, while for many a cause cannot be identified. These factors are broadly categorized into: social, psychological, chemical and biological. Biological causes is considered as a spectrum where a disease is either caused by genetic factors (e.g. CAG repeats in Huntingtin gene causes Huntington's Disease) or environmental factors (like toxic acetaldehyde in cigarette smoke and dioxins from Orange) and infectious agents (e.g. smallpox virus, poliovirus, bacteria). In between, genes (e.g. NOD2/CARD15) and environmental factors (Gut microbiota) interact to cause disease (inflammatory bowel disease, Crohn's Disease). Koch's postulates is used to determine whether a disease is caused by an infectious agent, while family inheritance pattern determine the genetic factors (e.g., inheritance of hemophilia in the British Royal Family). Now Apoliprotein E (ApoE) gene is found to be a susceptibility gene for Alzheimer's disease (Bekris et al., 2008). Similarly segregation of genes or genetic markers like single nucleotide polymorphism (SNP) or expressed sequence tag throughout the genome contributes many diseases (McVean et al., 2005; Skelding et al., 2007). Hence, disease can be broadly classified on the basis of its cause as: genetic diseases, infectious diseases, and non-infectious (including lifestyle related) diseases. An infectious diseases is the result of attack by a acellular (prion, virus) or cellular organism (bacteria, fungus, parasites etc.), a genetic diseases is due to defect in genetic makeup or genetic change, while the lifestyle related disorders are due to change in several physiological and environmental factors. The word "drug" is etymologically derived from the Dutch/low German word "droog" (means dry), since historically most drugs were dried plant

Diseases that need new drugs: Need of the hour

parts. A drug is any substance that can modify a chemical process or processes in the body, like to treat an illness, relieve a symptom, enhance a performance or ability, or to alter states of mind. Drug can be a biological substance (natural), synthetic or semi-synthetic, that is taken for non-dietary needs, usually synthesized as secondary metabolites of living organisms (bacteria, fungi, higher plants and animals), or outside of an organism, but introduced into an organism to produce its action. That is, when taken into the body, it will produce some effects or alter some bodily functions (relieving symptoms, curing diseases or as preventive medicine etc). The endogenous biochemicals (such as hormones) can bind to the same receptor in the cell, producing the same effect as a drug. Thus, drug is merely an artificial definition that distinguishes whether that molecule is synthesized within or outside an organism. For instance, insulin is a hormone that is synthesized by the pancreas inside the body, but if it is introduced into the body from outside, it is considered as a drug.

Problems and prospect The Pharmaceutical research got momentum when natural product chemists and pharmacologists, began to unravel the chemistry of traditional medicines. Scientific advancement led to the identification of many molecules as novel compounds and many new drugs against infections, cancers, ulcers, heart diseases were emerged; while many developed through random screening of plants; and many others resulted from sharp-eyed observations of scientists (Patwardhan et al., 2004). Studies on new drugs for malaria, trypanosomiasis, filariasis, tuberculosis, schistosomiasis, leshmaniasis and amoebiasis came almost to a standstill; while there is no suitable drug to stop the emerging and re-emerging microbes, particularly the drug-resistant pathogens. On the other hand, the clinical efficacy of many ethnomedicines was not yet evaluated and the composition of many traditional preparations was only crudely analysed. Pharmaceutical scientists are experiencing difficulty in identifying new lead structures in the finite world of chemical diversity as most synthetic drugs have unacceptable side effects. On the other hand, ethnomedicinal molecules like quinghaosu, artemisinin, rauwolfia alkaloids, psoralens, holarrhena alkaloids, guggulsterons, mucuna pruriens, piperidines, baccosides, picrosides, phyllanthins, curcumine, withanolides, steroidal lactones and glycosides showed impressive successes. A whole range of chronic and difficult-to-treat diseases such as cancers, cardiovascular disease, diabetes, rheumatism, AIDS, neglected tropical diseases and diseases caused by drug resistant microbes require new effective drugs.

Debprasad Chattopadhyay et al.

A major problem with traditional, indigenous medicine is its reliability and standardization, whereas most of the modern medicine have unacceptable side effect, high cost, and unsuitable for all conditions. In the bioprospecting of new medicine, the use of indigenous medicine is broken down, as the indigenous population has been marginalized or limited to small groups or small geographical areas, as in Africa. The Ayurveda and Chinese traditional medicine (CTM) are â&#x20AC;&#x2DC;great traditionsâ&#x20AC;&#x2122;, while the traditions of Africa, Tibet, Chakma (Chakma Tilaka Chikitsa of Chakma tribes, Bangladesh) are excellent repository of knowledge. However, researchers usually exploited poisonous sources because it is relatively easy to demonstrate; spread easily by word of mouth; and can differentiate between ordinary and extraordinary materials. But a considerable time is required to demonstrate true medicinal activities of a plant with a proven safety profile. As the Ayurveda and CTM have relatively organized database and easy to test by modern methods, hence have an important role in the bioprospecting of new medicines. Table 1 shows the major communicable and non communicable diseases that need new drugs. Table 1. The diseases that can cause major devastation worldwide and need new drugs.

Infectious diseases and its global burden For centuries infectious diseases pose major challenges to human progress and survival, and remain the leading causes of death and disability worldwide. Periodic emergence of new and old infection epidemics greatly magnifies the

Diseases that need new drugs: Need of the hour

global burden of infections. Studies of these emerging infections reveal the evolutionary properties of pathogens and the dynamic relationships between microbes, their hosts and environment (Morens et al., 2004). The infections that have newly appeared in a population or existed previously and now rapidly increasing in incidence or geographic range is called emerging infection (EI). Acquired immune deficiency syndrome (AIDS) caused by human immune deficiency virus (HIV) was first recognized in 1981, and as a global killer HIV/AIDS now threatens to surpass the Black Death of the 14th century and the 1918-1920 influenza pandemic, each of which killed nearly 50 million people. Of the 'newly emerging' and 're-emerging/resurging' diseases after HIV/AIDS, the monkeypox, severe acute respiratory syndrome (SARS) in 2003, bird flu in 2006 and swine flu in 2009 had a worldwide impact; while the 2001 anthrax bioterrorist attack in US was a 'deliberately emerging' diseases. Actually emergence results from dynamic interactions between rapidly evolving infectious agents and changes in environment and host behaviour that provide such agents a favourable ecological niche (Morens et al., 2004). It was estimated that infectious disease alone contribute about 15 million (>25% of 57 million) annual deaths worldwide, and an additional millions of deaths from past infections (like streptococcal rheumatic heart disease) and complications with chronic infections, such as liver failure and hepatocellular carcinoma in people with hepatitis B or C (WHO Report 2004). The burden of morbidity (ill health) and mortality in infectious diseases is more in developing countries (Guerrant & Blackwood, 1999), particularly on infants and children (about 3 million children die each year from malaria and diarrhoeal diseases); while in developed nations mortality in infectious disease disproportionately affects indigenous and disadvantaged minorities (Butler et al., 2001).

Old microbes cause new diseases and the newly emerging infections Some infections once caused familiar diseases, but now causing new or uncommon diseases as found with Streptococcus pyogenes (caused a fatal pandemic of scarlet and puerperal fevers between 1830 and 1900) is now rare (Katz & Morens, 1992), and replaced by streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever (Musser & Krause, 1998). Sometimes the diseases causing ability of a new microbe are delayed, as found with Koch-Weeks bacillus discovered by Robert Koch in 1883. More than a century later, a virulent clonal variant of Koch-Weeks bacillus or Haemophilus influenzae biogroup aegyptius was found to be responsible for Brazilian purpuric fever, a fatal emerging infection (Musser & Selander, 1990). Although the basis of emergences and severity of S. pyogenes and H. influenzae biogroup aegyptius are not fully known, but complex microbial genetic events are suspected. PCR

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studies demonstrate that a unique plasmid, and chromosomal regions of H. influenzae biogroup aegyptius are responsible (Li et al., 2003). Similarly the virulence in S. pyogenes is due to factors like M1 surface protein, M proteins, bacteriophage-encoded superantigen toxins and a sic (streptococcal inhibitor of complement) protein selected by human mucosal factors (Reid et al., 2001; Beres et al., 2003). Evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection (Reid et al., 2001; Beres et al., 2003). Newly emerging infectious on the otherhand are those which are not previously recognized in man, but now cause due to genetic mutation (microbes), genetic recombination or reassortment (viruses), changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behaviour (human movement and urbanization), and environmental changes. The microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them (Chattopadhyay & Naik, 2007).

Some chronic diseases have microbial aetiology Infectious agents associated with chronic diseases are one of the most challenging categories, as evident from the associations of hepatitis B and C with chronic liver damage and hepatocellular carcinoma, certain genotypes of human papillomaviruses (HPV) with cervical cancer, Epstein-Barr virus with Burkitt's lymphoma (largely in Africa) and nasopharyngeal carcinoma (in China), human herpesvirus 8 with Kaposi sarcoma, and Helicobacter pylori with gastric ulcers and gastric cancer (Chang et al, 1994; Parsonnet, 1999; Sanders & Peura, 2002). Interestingly cardiovascular disease and diabetes mellitus, a major cause of global death and disability, is now suggesting infectious aetiologies (Fredricks & Relman, 1998). Re-emerging/resurging infections and global spread Re-emerging and resurging infections existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. Re-emergence is caused by microbial evolutionary vigour, zoonotic encounters and environmental encroachment, and re-emergences or cyclical resurgences of some diseases may also be climate-related, for example, the El Ni単o/Southern Oscillation phenomenon is associated with resurgences of cholera and malaria (Kovats et al., 2003). World is now a global village and

Diseases that need new drugs: Need of the hour

travel has an important role in bringing people into contact with infectious agents (Cliff et al., 2002). An increase in travel-associated diseases, anticipated in 1933 (Massey, 1933), is now demonstrated by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in 1981 (Morens, 1998), epidemics of meningococcal meningitis associated with the Hajj, exportation of epidemic SARS from Guangdong Province of China to Hong Kong to Beijing, Hanoi, Singapore, Toronto and elsewhere (Peiris et al, 2003), Bird flu and swine flu from Europe to Asia in 2006 and 2009. The persistent spread of HIV along air trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme (Quinn, 1994; Daley et al, 1999; Ronald, 1995).

Prion diseases A prion is an abnormal, transmissible agent that induces abnormal folding of normal cellular proteins in the brain, leading to rapidly progressive brain damage and death. Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive neurodegenerative disorders that affect man and animals, with a long incubation periods, spongiform changes with neuronal loss, that failed to induce inflammatory response (Aguzzi & Heikenwalder, 2006). Injections of ground-up brain tissue from an affected animal or man into another animal transmit the disease, suggesting that the disease is caused by a virus like agent. But no genome was identified from such brain tissues, and treatment with UV light (that destroy DNA) does not reduce its infectiousness, indicate that the causative agent is not a virus. To date, the evidence indicates that the infectious agent of TSEs is a protein (Aguzzi & Heikenwalder, 2006), called â&#x20AC;&#x153;prion proteinsâ&#x20AC;? (PrP) or simply prions by Stanley Prusiner that bring him the Nobel Prize in 1997 (Prusiner, 2001). The normal cellular protein (PrPC) are transmembrane glycoprotein on the surface of certain cells (neural and hematopoietic stem cells) is easily soluble, digested by proteases and is encoded by a gene PRNP located on chromosome no 20. While the abnormal, diseaseproducing protein PrPSc (for scrapie) has the same amino acid sequence in their primary structure but secondary structure contain beta conformation, insoluble in solvents (except the strongest solvents) and resistant to proteases. When PrPSc comes in contact with PrPC, it converts the three-dimensional configuration of PrPC into more of itself, and bind to each other forming aggregates (Mead, 2006). It is not clear whether these aggregates directly cause the cell damage or are cellular dame is a side effect of the disease process. Prion diseases are either inherited or infectious. Inherited Prion Diseases are spontaneous and inherited with infectious maladies. It includes (i) Creutzfeldt-Jakob disease (CJD): 10â&#x20AC;&#x201C;15% are inherited (the patient comes from a family in which the disease has appeared

Debprasad Chattopadhyay et al.

before) as an autosomal dominant mutated PRNP gene. (ii) GerstmannStr채ussler-Scheinker disease (GSS) caused by the inheritance of a PRNP gene with a mutations encoding leucine instead of proline at position 102 (P102L) or valine instead of alanine at position 117 (A117V). (iii) Fatal Familial Insomnia (FFI), a rare disorder inherited by a PRNP gene with asparagine instead of aspartate at position 178 (D178N), the susceptibility polymorphism of methionine at position 129 of the PRNP gene; and extracts from autopsied brains of FFI victims can transmit the disease to transgenic mice (Johnson, 2005). Infectious Prion Diseases includes (i) Kuru, first found among the Fore tribe in Papua New Guinea whose rituals included eating the brain tissue of their recently deceased members. The disease is now disappeared as this practice was halted. (ii) Scrapie of sheep (and goats) transmitted from animal to animal by contaminated feed with nerve tissue, or by injection of brain tissue. (iii) Bovine Spongiform Encephalopathy (BSE) or "Mad Cow Disease", found in Great Britain in 1985, among cattle feed that contained brain tissue from sheep infected with scrapie (Aguzzi, 2006), and was declined as the use of such food was banned after epidemic in 1992. Another similar disease Variant Creutzfeldt-Jakob disease (vCJD) was also noticed in Great Britain cattle herds. Even though the cow and human PRNP genes differ at 30 codons, their prion sequences (all patients are homozygous for the susceptibility polymorphism of methionine at position 129) suggests that these patients (155 by 2005) acquired the disease from eating contaminated beef (Glatzel et al., 2005). The changes in slaughter techniques that mix cattle nervous tissue in beef for human consumption in 1989 now restrict the disease. Prion diseases are fatal, and death occurs within a year after the first symptoms (Will et al., 1999; Carsten Korth et al., 2001). There is no suitable therapy exists till date and devising approaches to the therapy of prion diseases is extremely difficult because of the mysterious nature of the infectious agent, its composition, structure, mode of replication and the mechanism of pathogenesis. As the disease affects the brain parenchyma, so the therapeutic agents need to cross the brain-blood barrier or to be introduced directly into the cerebrospinal fluid or brain tissue, and the disease is noticed only after onset of severe clinical symptoms (Prusiner, 2001). However, many compounds can inhibit prion propagation in rodents (Kimberlin & Walker, 1983; Dickinson et al, 1975; Diringer & Ehlers, 1991; Ehlers & Diringer, 1984) but treatment with these compounds was ineffective, like congo red inhibit PrPSc formation (Caughey & Race, 1992; Caspi et al., 1998; Priola et al., 2000) in rodents but ineffective when neurologic signs appear (Ingrosso et al., 1995). Because the blood-brain barrier restricts the access of many molecules to the CNS, and thus agents or drugs that can cross this barrier have been screened for their ability to inhibit PrPSc formation. The acridine and

Diseases that need new drugs: Need of the hour

phenothiazine derivatives (a tricyclic scaffold and a side chain moiety) used against psychoses (Delay et al., 1952) showed that chlorpromazine inhibited PrPSc formation (at 3 ÂľM), whereas quinacrine (a structural antecedent of the phenothiazines) was 10 times more potent due to its aliphatic side chain (Korth et al., 2001). Earlier reports also indicated the potent inhibition of PrPSc formation by acridine and chlorpromazine (Roikhel, et al., 1984; Dees et al., 1985). Quinacrine and other lysomotrophic reagents added to ScN2a cells can clear PrPSc (Doh-urs et al., 2000), as quinacrine, can cross the blood brain barrier, and for the treatment of CJD and other prion diseases, with a daily oral tolerable dose of 1001,000 mg (Phuan et al., 2007). To date, several compounds have been tested that decrease PrPres concentration in scrapie-infected cell lines or prolong the incubation period in vivo. These include sulfated polyanions (Farquhar et al., 1999; Huang et al., 2002), amphotericin B derivatives (Adjou et al., 2000), Congo red (Demaimay et al., 1998), tetracyclic compounds (Forloni et al., 2002), tetrapyrroles (Priola et al., 2000), branched polyamines (Supattapone et al., 2001) and Î˛-sheet breakers derived from PrP peptides (Soto et al., 2000). Antimalarial quinacrine, antipsychotic chlorpromazine, some tricyclic derivatives with an aliphatic side chain were found as efficient inhibitors of PrPres formation in murine neuroblastoma cells chronically infected with the Chandler scrapie isolate (Barret et al., 2003; Korthy et al., 2001). Nevertheless, none of these agents are effective in clinical phase, thereby restricting an evidence-based rationale for their use in the treatment. Certain non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclo-oxygenase (cox) that metabolizes arachidonic acid to prostaglandins, is protective against the toxic effects of prion protein, or extracts containing infectious prions (PrP), inhibit neuronal PGE production and protect neuroblastoma cells and primary cortical neurones against prions (Bate et al., 2002; Ludewigs et al, 2007).

Virus infection control Viruses are acellular, ultramicroscopic, metabolically inert nucleoprotein particles containing gene strands of either RNA or DNA, with or without a lipid envelope (Chattopadhyay et al., 1999) Unlike free-living bacteria, viruses are obligate intracellular parasites, utilize the host cell machinery to propagate and can cause ailments as benign as a common wart, as irritating as a cold, or as deadly as the bloody African fever. The viruses that cause Lassa and Ebola fever and AIDS spread easily, kill swiftly and have no cure or vaccine. Viruses have numerous invasion strategies and each strain has its own unique configuration of surface molecules (Chattopadhyay et al., 1999), enabling them to enter into host cells by precisely fitting their surface molecules with the molecules of the target cells. The genetic variation,

Debprasad Chattopadhyay et al.

variety of transmission, efficient replication and the ability to persist within the host are the major evolutionary advantage of viruses. As a consequence, viruses have adapted to all forms of life and have occupied numerous ecological niches resulting in widespread diseases in humans, livestock and plants (Chattopadhyay et al., 1999; Wagner et al., 1999; Chattopadhyay & Naik, 2007). Despite the continuous advances made in antiviral therapy, viral diseases become the leading cause of death globally. To control viral infections, either prophylactic or therapeutic measures are used. As a metabolically inert particle virus requires a metabolic pathway of living cells to replicate, which makes it difficult to design a treatment that attacks the virions or its replication, without affecting the host (Chattopadhyay et al., 1999; Wagner et al., 1999). Although numerous compounds have been tested on different viruses, only 37 licensed antivirals are in the market (Table 2). But the development of antivirals from natural sources is less explored, probably because there are very few specific viral targets for small molecules to interact with. Fortunately, many viruses have unique features in their structure or replication cycles that can be the potential target, as evident with nucleoside analogue acycloguanosine (acyclovir) which specifically blocks certain viral Table 2. Current armamentarium for the chemotherapy of viral infections (37 licensed antivirals). Agents I. Anti-HIV Nucleoside reverse transcriptase inhibitors (NSRTI) Nucleotide reverse transcriptase inhibitor (NTRTI) Non-nucleoside reverse transcriptase inhibitors (NNSRTI) Protease inhibitors (PI) Entry inhibitor (EI) II. Anti-HBV (Chronic infections) III. Anti-herpesvirus

Number 19 07 01 03 07 01 02 12

IV. Anti-Influenza virus 03 V. Anti-RSV

VI. Anti-HCV

Name of the Antiviral Agent Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine Tenofovir disoproxil fumarate Nevirapine, Delavirdine, Efavirenz Saquinavir, Ritonavir, Indinavir, Nelfinavir, Aamprenavir, Lopinavir, Atazanavir Enfuvirtide Lamivudine, Adefovir dipivoxil Acyclovir, Valaciclovir, Penciclovir (topical), Famciclovir, Idoxuridine, Trifluridine (both topical), Brivudin (HSV and VZV); Ganciclovir, Valganciclovir, Foscarnet, Cidofovir, Fomivirsen (intravitreal injection for CMV in immunosuppressed AIDS patients with CMV retinitis) Amantadine, Rimantadine, Neuraminidase inhibitors: Zanamivir, Oseltamivir Ribavirin (topically as aerosol) Combination of Ribavirin with (pegylated) interferon-alpha

Diseases that need new drugs: Need of the hour

enzymes of herpes viruses (Chattopadhyay et al., 1999; Wagner et al., 1999; De Clercq, 2004; Chattopadhyay & Naik, 2007) that play the key role in triggering disease. In recent years, the demand for new antiviral strategies has increased markedly for several contributing factors, including the ever-increasing prevalence of chronic viral infections like HIV, Hepatitis B virus (HBV) and Hepatitis C virus (HCV), and the emergence of new viruses such as the SARS coronavirus. The potential danger of hemorrhagic fever viruses and eradicated viruses such as variola virus being used as bioterrorist weapons has also increased the profile of antiviral drug discovery. The cellular and viral targets of several virus families for drug development are depicted in Table 3. Several viral diseases like AIDS, Influenza, Cytomegalovirus, Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hepatitis B and C, Herpes viruses, Human papilloma viruses, Lassa fever, Marburg hemorrhagic fever, Rabies, Rubella, SARS, West Nile, Yellow fever, Viral encephalitis, gastroenteritis, meningitis and viral pneumonia need effective therapy. However, Table 3. Viral and Cellular targets for antiviral agents. VIRUS

VIRAL TARGET

Parvovirus Polyomavirus Papillomavirus Adenovirus α-Herpesvirus β-Herpesvirus γ-Herpesvirus

DNA polymerase DNA polymerase DNA polymerase DNA polymerase DNA polymerase, Thymidine kinase, Helicaseprimase DNA polymerase, Protein kinase, Terminase DNA polymerase DNA & RNA polymerase DNA polymerase (RT) Capsid RNA polymerase RNA polymerase RNA polymerase RNA polymerase RNA polymerase RNA polymerase RNA polymerase RNA polymerase, RNA helicase, viral protease Matrix protein, Neuraminidase Fusion polypeptide Spike (S) protein, RNA polymerase (replicase), helicase, protease gp41, RT, Protease, gp120, Integrase, Transcription transactivator (TAT)

Poxvirus Hepadnavirus Picornavirus Flavivirus Arenavirus Bunyavirus Togavirus Rhabdovirus Filovirus Hepacivirus Orthomyxovirus Paramyxovirus Coronavirus Reovirus Retrovirus

CELLULAR TARGET Cellular factors 4 enzymes* 4 enzymes* -do-do-do-do4 enzymes* 4 enzymes* IntegrationTranscription factors

*Inosine 5’ monophosphate (IMP) dehydrogenase, S-adenosylhomocysteine (SAH) hydrolase, Oritidine 5’phosphate (OMP) decarboxylase, Cytosine 5’-triphosphate (CTP) synthétase. [Reproduce from De Clercq, E. (2004) Nature Review 2: 710-720].

Debprasad Chattopadhyay et al.

currently the attention is directed towards the influenza viruses that cross the host barrier. The constant burden of influenza each year goes unnoticed, and every few years there is a blip on the curve of excess deaths associated with influenza caused by influenza virus A and B. Influenza A viruses are endemic gastrointestinal viruses of wild waterfowl, have evolved to jump species into domestic fowl, farm animals and humans. Periodic gene segment reassortments between human and animal viruses produce important antigenic changes (referred to as 'shifts'), that can lead to deadly pandemics, as occurred in 1888, 1918, 1957 and 1968 (Shortridge et al., 2003; Webster, 2001). In intervening years, shifted viruses undergo continual antigenic changes (called 'drifts'), and thereby partially escape human immunity raised by previously circulating viruses. Influenza A has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. It remains mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. Before 1997, mild cases of human disease associated with avian influenza viruses were occasionally reported (Subbarao & Katz, 2000), but now it is frequent and even severe. Avian influenza has made dead-end jumps to humans, as noticed in 1997 Hong Kong outbreak with H5N1, 2003 H7N7 epidemic in the Netherlands, the 2003-2004 H5N1 and H7N3 epizootics in Asia, and occasional cases of H9N2. Meanwhile, back-switches of human H3N2 viruses have emerged in pigs, from which both doubly mixed (pig-human) and triply mixed (pig-human-avian) viruses isolated (Shortridge et al., 2003; Webster, 2001). Such enzootic/zoonotic mixing occurs in influenza pandemic of 1918-1920 caused by H1N1 with an avianlike receptor-binding site (Stevens et al., 2004). The virulence genes of this virus were reported from 85-year-old pathology specimens and from frozen corpses (Reid & Taubenberger, 2003). It is clear that by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing, adaptation, and ecological factors bring humans into contact with animals and each other for the spread and emergence of influenza viruses. In April 29, 2009, another serious influenza Swine flu has pushed WHO to raise its pandemic alert to level 6. Swine influenza is a highly contagious respiratory disease in pigs caused by swine influenza A viruses, and human swine influenza A (H1N1) have been reported worldwide (Dunham et al., 2009). In 2009, influenza like illness were first reported in Mexico on March 18, and subsequently confirmed as swine influenza A from 19 states of Mexico. Although 18 Mexican cases have been confirmed as Swine Influenza A/H1N1 (12 are genetically identical to Swine Influenza A/H1N1 from California), approximately 1,600 cases and 103 deaths have been attributed to

Diseases that need new drugs: Need of the hour

swine influenza (Dunham et al., 2009), and several cases were subsequently confirmed from US, Canada, Scotland, France, Israel and Brazil (Baz et al., 2009). Outbreaks of swine influenza are common in pigs year-round and humans have become infected as a result of close contact with infected pigs. However, the current virus is a novel influenza A (H1N1) not previously identified in humans, and appears to be spread by human-to-human transmission (Baz et al., 2009).

Bacterial diseases Out of several bacterial diseases Anthrax, Bacterial meningitis, Brucellosis, Campylobacteriosis, Cholera, Diphtheria, Legionellosis, Leprosy (Hansen's Disease), Leptospirosis, Listeriosis, Lyme Disease, Methicillin resistant Staphylococcus aureus (MRSA) infection, Nocardiosis, Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Gonorrhea, Tuberculosis, Typhoid Fever are particularly important from the drug development point of view. Of these emerging superbugs like MRSA, tuberculosis, Enterococcus, Streptococcus and Gonococcus are notorious.

Drug resistant super-bug microbes Drug resistance, causing microbial and viral re-emergence, may result from mutation (in viruses and M. tuberculosis), or from acquisition of extraneous genes through transformation or infection with plasmids (in bacteria). Sequential emergences of Staphylococcus aureus (â&#x20AC;&#x153;flesh-eating bacteriaâ&#x20AC;? colonizes to 25-30% general population in nose or skin surface, causing minor skin infections, pimples or boils, to fatal pneumonia or sepsis; and for many years treated with penicillin and methicillin) are resistant to sulpha drugs (1940s), penicillin (1950s), methicillin (1980s) and to vancomycin (CDC, 2002), a last line of antibiotic defense for these drugresistant bugs (Klevens et al., 2007). Similarly nosocomial Enterococcus faecalis became fully resistant to vancomycin by 1988, and then transferred vanA resistance genes to co-infecting staphylococci (CDC, 2002). Methicillinresistant staphylococci are also isolated from livestock that fed with growthpromoting antibiotics (Lee, 2003), possibly contributing to resistance problems in man. Many other important microbes like Streptococcus pneumoniae and Neisseria gonorrhoeae have also become effective 'resistors (Neu, 1992). Drug-resistant Streptococcus pneumoniae, a common cause of ear infections in children, meningitis, systemic infection, and pneumonia

Debprasad Chattopadhyay et al.

showed resistant to penicillin and related antibiotics and are responsible for a large percentage of death and sickness globally. Antibiotic resistance is being detected in many different bacteria like Pseudomonas aeruginosa (opportunistic bacteria that infects immunocompromised host), Streptococcus pyogenes (flesh-eating bug infect throat, impetigo, and cause scarlet fever), and Proteus vulgaris (cause urinary tract infections) at alarming rates. Drugresistant Enterococcus faecalis and E. faecium, found in the bowel and female genital tract cause urinary tract infections, blood infections, and meningitis, and is fatal in immunocompromised individuals like infants and elderly. Several strains of drug-resistant enterococci have emerged in the last 30 years, including resistant to penicillin, vancomycin, and linezolid (Hyucke et al., 1998). Drug-resistant Neisseria gonorrhoea has outsmarted all but one class of antibiotics, and hence, the Infectious Diseases Society (IDSA) called for new antibiotics to treat this drug-resistant â&#x20AC;&#x153;superbugâ&#x20AC;? that develop widespread resistance to powerful fluoroquinolones and now only treatable by cephalosporins (MMWR 2006). Treatment options for gonorrhea and other superbugs have become dangerously few. As per CDCâ&#x20AC;&#x2122;s estimate gonorrhea is the second most common infection in the world. Any sexually active person can become infected, and because most people do not have obvious symptoms, they unknowingly spread it to others. The highest reported rates of infection are among sexually active teenagers and young adults. Because of its inflammatory nature gonorrhea puts people at greater risk of contracting HIV/AIDS, and HIV-infected people with gonorrhea are more likely to transmit HIV to others. In women, gonorrhea can cause pelvic inflammatory disease and ectopic pregnancy, while in men, it can lead to infertility. It can only be prevented by using condoms consistently and correctly, and by abstaining from sexual intercourse. Hence, the main strategy for controlling gonorrhea is to find infected patients and partners and treat them early to interrupt the chain of transmission, and we need to rely on inexpensive drugs which are easy to administer. Now situation is very perilous especially for the patients allergic to cephalosporins. In recent years, drug-resistant strains of gonorrhea have become more common like many other bacteria that quickly mutate and produce as many as 500,000 generations during one human generation. In order to survive, these bacteria develop resistance to antibiotics. Other infections that are becoming difficult to treat because of drug resistance are Acinetobacter baumannii, a cause of hospital-acquired pneumonia and among soldiers returning from Iraq and Afghanistan; and vancomycin-resistant Enterococcus faecium (VRE), a major cause of infections in bloodstream, heart, meningitis, and intra-abdomen. The development of resistant strains of bacteria also limits the long-term market potential for an

Diseases that need new drugs: Need of the hour

antibiotic. Unless we develop more incentives for drug development, we will soon return to the pre-antibiotic era, when acquiring a bacterial infection was often a death sentence. Drug-resistant tuberculosis (MDR- and XDR-TB), caused by Mycobacterium tuberculosis, a horrific wasting disease acquired by inhalation and cause pulmonary tuberculosis, and even spread to other organs with various presentations (meningitis, Pott's Disease etc.). Prior to the discovery of antibiotics, tuberculosis was untreatable. However, with the widespread use of antibiotics since 1940s, multidrug-resistant tuberculosis (MDR-TB) has emerged and is a leading cause of death, particularly among HIV-infected individuals. MDR-TB strains of M. tuberculosis are resistant to isoniazid and rifampicin, and sometimes to second-line (XRD-TB) medications and individuals with HIV are at greatest risk (Fact Sheet. MDR TB and XDR TB, CDC, 2008). Tuberculosis, one of the most deadly re-emerging diseases, accounts for 3 million global deaths annually and is the leading killer of adults today. The discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in many nations. By the 1980s, when tuberculosis had re-emerged in the era of HIV/AIDS, local and state health departments lacked field, laboratory and clinical staff to reinvent its control programmes (Committee on EMTH, 1992). The remarkable re-emergence of tuberculosis was fuelled by the immune deficiencient people with AIDS, which greatly increases the risk of latent M. tuberculosis infections progressing to active disease, and transmitted to others. Inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drugresistant and multidrug-resistant strains (Espinal, 2003), and a need for more expensive treatment like directly observed therapy (DOT). For over a century tuberculosis was a disease of poverty, associated with crowding and inadequate hygiene, and the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. Immune deficiency associated with AIDS, cancer chemotherapy, immune-mediated diseases and transplantation has contributed to an enormous increase in the numbers of immunosuppressed people globally (more than 1% of the world's population), setting the stage for the reemergence of many opportunistic infections. HIV is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens like Pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesviruses (Jones et al. 1999). Breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immunemediated diseases and for transplantation (Vento Cainelli, 2003; Singh, 2003) can also leave patients susceptible to opportunistic infections. Human organ

Debprasad Chattopadhyay et al.

transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes (Chapman, 2003). Moreover, fungal diseases like Aspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcosis, Tinea pedis and Histoplasmosis need new effective drugs also.

Re-emerging zoonotic and vector-borne diseases The emergence of zoonotic and vector-borne diseases is also associated with human behaviours and environmental perturbation. In 2003, monkeypox, an endemic infection of African rodents crossed the Atlantic with exported pets that shipped from Texas to infect people throughout the US Midwest (CDC, 2003). Lyme disease, caused by Borrelia burgdorferi, reemerged as a result of suburban expansion, which brought people into increasing contact with deer, deer mice and ticks. Similarly, tick-borne encephalitis re-emerged in Russia when weekend getaways (dachas) drew city dwellers into contact with forest ticks. In 1999 emergences of encephalitis due to West Nile virus in the US and in Russia (Nash et al. 2001; Platonov et al., 2001) reflect abundances of eclectic vector mosquitoes and avian hosts, probably carried by migratory birds and travellers. The remarkable geographical spread of West Nile virus from Western Hemisphere reflects an unfortunate confluence of viral promiscuity and ecological diversity (Komar, 2003). Although humans are dead-end hosts for West Nile virus, the risk of infection is greatly increased by marked zoonotic viral amplification and environmental persistence. Though West Nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally (Gubler, 1998). A 2001â&#x20AC;&#x201C;2002 epidemic in Hawaii is a reminder that dengue has also re-emerged in areas once considered to be dengue-free. Usually transmitted by Aedes aegypti mosquitoes, dengue has recently been transmitted by Aedes albopictus, a vector switch of potential significance with respect to dengue re-emergence (Gubler, 1998). In many parts of the world like US, India A. albopictus is spreading into areas where A. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting more people at risk of dengue infection. Dengue re-emergence is further complicated by increases in a serious form of the disease, dengue haemorrhagic fever and dengue shock syndrome, due to the evolution of dengue viruses to escape high population immunity, seen in increased viral virulence and human antibody-dependent enhancement of viral infection (Morens, 1994).

Diseases that need new drugs: Need of the hour

Cholera is an important cause of mortality, and several complex factors determine its re-emergence. Both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto- and zooplankton, algae and crustaceans. These environmental strains seem to act as reservoirs for human virulence genes (genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (TCP) factor for attachment), and to undergo gene transfer events that lead to new strains with more virulence gene combinations. These result in periodic cholera emergences causing epidemics and pandemics (Faruque & Nair, 2002). Thus, although the disease cholera appeared to be clinically and epidemiologically stable since the third pandemic in the 1840s, modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments.

Neglected diseases (DNDi): Matching needs and opportunities Neglected diseases such as leishmaniasis, trypanosomiasis, Chagas disease and malaria have a devastating impact on the world's poor. These treatable, tropical diseases have been progressively marginalized by research programme decision makers. Unfortunately, people suffering from these diseases do not constitute a market lucrative enough to attract investment in research and development for new drugs. For decades if not centuries, Malaria is a major killer, and is still killing between one and two million people a year. The victims are mostly developing-country babies, particularly African babies. Drugs for Neglected Diseases Initiative (DNDi) are addressing this lack of new, improved drugs for neglected patients with an alternative model of research and development. This model matches patients' needs with gaps identified in the drug development pipeline. Parasitic infections are a major global health problem, particularly for lessdeveloped nations, where it causes a substantial economic burden. The global prevalence of parasitic infections is more than 50% due to factors like population, poverty and pollution, that leads to poor sanitation and health education, inadequate control of vector, infection and agricultural waste, global travel, population migration, military operation and development of resistant parasitic and vectors to the existing drugs or chemicals. Most of these infections are neglected because their effects on human health are more subtle, except malaria that causes high morbidity and mortality without treatment. Chemotherapy remains the single most effective, efficient and inexpensive method to control most of the parasitic infections despite encouraging progress in identifying promising molecular targets for vaccines. The global effort of last 50 years is unable to provide safe and effective drugs for the treatment of serious parasitic

Debprasad Chattopadhyay et al.

infections like Trypanosomiasis, Leishmaniasis, and drug resistant Malaria. An ideal antiparasitic agent should be safe at high therapeutic dose, easily given by oral route in a single or divided doses in same day, chemically stable under climatic conditions of use, inexpensive and do not induce of drug resistance. Till now a very few agents meet all these criteria for mass chemotherapy. Most antiparasitic agents have been discovered by screening natural products and synthetic compounds in appropriate animal model. But this traditional approach needs to be complemented by research on host and parasite biology for selective exploitation of chemotherapeutic advantage. Tremendous advances in molecular biology, pharmacology and genomics in the last 20 years, can help to understand the interaction between host and parasite and the use of improved methods for in vitro screening with the demonstration of efficacy and safety in man. Though the most concerned parasitic infections are the African trypanosomiasis, Amebiasis, Ascariasis, Chagas Disease, Cryptosporidiosis, Cysticercosis, Dracunculiasis, Echinococcosis, Enterobiasis, Fascioliasis, Filariasis and freeliving amebic infection, Giardiasis, Gnathostomiasis, Hymenolepiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis, Onchocerciasis, Pediculosis, Pinworm, Scabies, Schistosomiasis, Taeniasis, Toxoplasmosis, Trichuriasis; but the major human infections that need new agents for effective treatment are listed in Table 4. Plasmodium falciparum malaria, neglected for several decades, is now among the most important re-emerging diseases. Years of effective use of DDT (dichlorodiphenyltrichloroethane) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. Consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine. Research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets. Gaps in the pipeline: At present DNDi has nine projects (long, medium, and short-term) to fill the gaps in the drug development pipeline at different stage (early discovery, the stage before drugs enter clinical development, and at the point where drugs should reach patients). Four long-term projects will identify new lead compounds that can kill trypanosoma and/or leishmania parasites, and one on combining existing anti-leishmanial drugs. The remaining four short-term projects with existing drugs: like, the registration of paromomycin (an antibiotic) for visceral leishmaniasis in Africa (in collaboration with Institute of One World Health and WHO/TDR); the evaluation of nifurtimox (a drug for Chagas disease), in combination with eflornithine to treat second stage sleeping sickness (in collaboration with

Diseases that need new drugs: Need of the hour

Table 4. Drugs used for Chemotherapy of Parasitic Infections. Infection & Parasite I. Protozoan Amebiasis Entamoeba histolytica Invasive Amebiasis Balantidiasis Balantidium coli Giardiasis Giardia lambia Leishmaniasis Leishmania brasiliensis L. maxicana (American mucocutaneous & cutaneous Leishmaniasis) L. donovani (Visceral Kalaazar) L. tropica (Cutaneous Oriental sore) Malaria Plasmodium falciparum, P. malariae, P. ovale, P. vivax (Chloroquine sensitive) P. vivax and P. ovale P. falciparum (CQ or multidrug resistant) Pneumocystosis Pneumocystis carinii Trichomoniasis Trichomonas vaginalis Trypanosomiasis Trypanosoma cruzi (Chagasâ&#x20AC;&#x2122; Disease) T. rhodesiense; T. gambiense (Sleeping sickness) II. Metazoan Helminth A. Nematode (Roundworm) Ascariasis Ascaris lumbricoides

Year

Drug of choice 1st Line

2nd Line

Diloxanide fluroate Metronidazole + Diloxanide

Tinidazole plus Diloxanide

Tetracycline Metronidazole or Quinacrine

Stibogluconate sodium

Amphotericin B

1959

1940

Stibogluconate Stibogluconate

Present situation Iodoquinol & Dehydroemetine have potentially toxicity

1959

Most develop resistance Less effective

1955

Less effective

1920

Less effective

Most develop resistance

Pentamidine isothionate, -

Chloroquine phosphate (CQ)

1934

Primaquine phosphate after CQ Quinine sulfate or dihydrochloride

1937

Trimethoprinsulfamethoxazole

Pentamidine isethionate

Metronidazole

Nifurtimox

Benznidazole

1921

Less effective

Suramin

Pentamidine isethionate

1920

Less effective

Mebendazole, pyrantelpamoate or ivermectin

Piperazine citrate

Most develop resistance Less effective

Quinine plus doxycycline 1920

Less effective Both sexual partners should be treated

Less effective

Debprasad Chattopadhyay et al.

Table 4. Continued Capillariasis Capillaria philippeiensis Dracunculiasis Dracunculus medinensis (Guinea worm) Enterobiasis Enterobias vermicularis (Pin worm) Filariasis Wuchereria brancrofti, Brugia malayi, Loa loa Dipetalonema perstans, Onchocerca volvulus Hookworm Necator americanus Ancylostoma duodenale Strongyloidiasis Strongyloides stercoralis Toxocariasis Toxocara Species Trichinosis Trichinella spiralis Trichuriasis Tricuris trichura (Whipworm) B. Cestode (Tapeworm) Taeniasis Taenia saginata (beef) Taenia solium (pork) Diphyllobothriasis Diphyllobothrium latum (fish) Hymenolepiasis Hymenolepis nana (Dwarf) Echinococcosis Echinococcus granulosus Echinococcus multilocularis C. Trematode (Fluke) Schistosomiasis (Blood) Schistosoma haematobium S. japonicum S. mansoni, S. mekongi S. intercalatum

Less effective Mebendazole

1971

Mebendazole

1971

Less effective

Pyrantel pamoate or mebendazole

Ivermectin

1983

Less effective

Diethylcarbamazine (DEC) Ivermectin

DEC

DEC does not kill 1947 microfilariae in nodule & hydrocele.

Mebendazole or Pyrantelpamoate

1972

Thiabendazole or Ivermectin

Immunosuppressive 1978 patients are at risk

Thiabendazole or Diethylcarbamazine

1978

Efficacy is quaestionable

Thiabendazole

1978

Less effective

Mebendazole or Ivermectin

Oxantel pamoate/ Thiabendazole

1972

Less effective

Niclosamide Praziquantel Niclosamide or Praziquantel

Niclosamide

1960

Less effective

1961

Less effective

Niclosamide or Praziquantel

1961

Less effective

Mebendazole/ Albendazole Mebendazole

1975

Less effective

Praziquantel Praziquantel Praziquantel Praziquantel

Metrifonate Oxamniquine -

1972 1962 1977

Less effective

Diseases that need new drugs: Need of the hour

Table 4. Continued Intestinal Fluke Fasciolopsis buski, Heterophyes heterophyes, Metagonimus yokogawai Liver Fluke Clonorchis sinensis, Opisthorchis felineus, O. viverrini, Fasciola hepatica Lung Fluke Paragonimus Species, P. westermani, P. kellicotti

Praziquantel

1979

Less effective

Praziquantel

1979

Less effective

Not effective

Praziquantel

1979

Less effective

WHO/TDR and Bayer); and two fixed-dose artesunate combination therapies of artesunate/amodiaquine and artesunate/ mefloquine against chloroquineresistant malaria in Africa and Asia (in collaboration with 7 medical research institutes across the world).

Enhancing drug discovery Discovering â&#x20AC;&#x2DC;leadâ&#x20AC;&#x2122; compounds having potential to be a drugs is a critical step for sustainable innovative products (Figure 1); and the establishment of public-private partnerships will helped to stimulate R&D for some neglected diseases, particularly the high-risk early discovery phase. TDR is helping to fill this gap through a coordinated initiative aimed at generating new lead compounds for tropical diseases. Hence, TDR effort is the establishment of multi-disciplinary networks and partnerships between researchers in industry and the public sector in both developed and developing countries.

A need for novel drugs The need for new, effective and affordable drugs to treat parasitic infection is one of the issues facing global health today. Available drugs to treat these diseases, such as malaria, African sleeping sickness, Chagas disease, leishmaniasis, filariasis, onchocerciasis and schistosomiasis, are limited by factors ranging from parasite resistance to safety, compliance and cost. Products representing entirely new innovations in medicinal chemistry are presently lacking. More effective diagnostics also are needed for these diseases, which often are misdiagnosed or diagnosed too late. New generations of public-private partnerships have provided one kind of response to these challenges by supporting accelerated development and clinical testing for

Debprasad Chattopadhyay et al.

Figure 1. The innovation cycle.

potential new products already in the pipeline. These partnerships include TDR-initiated ventures such as the Medicines for Malaria Venture, the Foundation for Innovative Diagnostics, as well as others such as the DNDi, the Global Alliance for TB Drug Development, and the Institute for One World Health. Still, there remains a need for new chemical entities, or ‘lead’ structures, having potential to become innovative treatments, and thus new approaches to ‘drug discovery’ for tropical diseases are required. To develop a drug, first discover it. The paucity of quality lead compounds feeding the development pipeline for malaria, we need to fill this gap not only for malaria but for other neglected diseases. Now the biology of many pathogens and their vectors, especially their genome sequences are known. That has to be translated into lead compounds for innovative treatment. Scientists seem that the fight against neglected disease is a moral obligation; and sustaining the availability of products for the poor. These networks are interactive and cover different aspects of the process. The networks includes: (i) Compound Evaluation Network: This is the engine of TDR’s drug discovery effforts, that strengthened and access to compound through industry collaborations with TDR, and screen over 20000 compounds per year. The network includes: Swiss Tropical Institute, Basel; London School of Hygiene and Tropical Medicine; Northwick Park Institute

Diseases that need new drugs: Need of the hour

for Medical Research, London; Theodor Bilharz Research Institute, Cairo; and the University of Antwerp. (ii) Medicinal Chemistry and Pharmacokinetics Networks: This network will further progress the ‘hits’ or ‘leads’ identified through the compound evaluation network. The medicinal chemistry work will be done by the pharmaceutical companies like Pfizer, Serono and Pharmacopeia. Academic institutions include University of Cape Town (South Africa), University of Dundee (UK), University of Nebraska, Ohio State University and St. Jude Children’s Research Hospital (USA). The pharmacokinetics/metabolism network will provides essential pharmacology data for medicinal chemists to guide further synthetic work, in Monash University (Australia), Pfizer, Serono and Pharmacopeia (USA). (iii) Drug Target Portfolio Network: This network creates a globally accessible database of prioritized list of drug targets across the range of parasites focused by TDR research. This will use parasite genome sequencing, and help in drug discovery based on molecular targets. The database is thus an important point in the search for new drugs and is freely accessible. It includes the University of Pennsylvania (USA), Sanger Centre (Cambridge, UK), the Walter Eliza and Hall Institute for Medical Research (Melbourne, Australia), and the Institute for Research in Biotechnology (Argentina). (iv) Helminth Initiative for Drug Discovery: This aims to support academic and industry partners to develop new technologies for the search of new drugs against Helminth such as schistosomiasis, onchocerciasis and lymphatic filariasis, and thus fills a gap for dedicated public-private partnerships for antihelmintic product.

The multiple tracks of drug discovery To discover new drugs for tropical diseases one of the fastest approaches is to examine how established drugs might be used or adapted as treatments. This need ‘whole organism’ screening of a known drug/compound against the parasites (e.g., anticancer compound eflornithine and miltefosine for African trypanosomiasis and visceral leishmaniasis). Another approach is the ‘piggy-back’ strategy, useful when a molecular target of a parasite is known, and thus provides a chemical ‘starting point’ for investigation (e.g., the histone deacetylase inhibitors developed for cancer treatment is now explored for anti-malarial drug research). A more long-term strategy involves ‘de novo’ discovery of new chemical entities based on protein or enzyme targets. ‘High throughput’ screening, virtual screening linked to cheminformatics, and x-ray crystallography is the part of this target-based, ‘rational’ approach. High-throughput screening exposes a parasite protein or enzyme to thousands of compounds in automated process. Industry will play a leading role in

Debprasad Chattopadhyay et al.

developing this technology, and can be applied in academic environments. Rational drug discovery is challenging but arguably has great potential for identifying drugs with novel modes of action.

Conclusion The ancient traditions like Indian traditional medicines Ayurveda, Siddha and Unani and the Chinese Traditional medicine (CTM) has a sound philosophical and experiential basis (Dahanukar & Thatte, 2000; Chopra & Doiphode, 2002). The Indian Atharvaveda (1200 BC), Charak Samhita and Sushrut Samhita (1000–500 BC) represent a medical pluralism with a holistic approach that contain detailed descriptions of over 700 herbs and their use (Dash & Sharama, 2001) and still remain dominant over modern medicine, especially for the treatment of chronic diseases (Waxler-Morrison, 1988). Out of estimated 50 thousand Indian plant species, nearly 700 are used in indigenous medicine, whereas Chinese medicine contains over 1500 plant species (Chin et al., 2006). Today several studies identified memory enhancer from Bacopa, antiinflammatory from Curcuma, cardiotonic from Asclepias, hypolipedemic agents from Commiphora, and hepatoprotective from Picrorhiza (Jain, 1994). It is now known that ayurvedic medicines can be customized to an individual constitution, and be used in the bioprospecting for new sources of medicine with its unique holistic approach (Amadea, 1990). Combining the strengths of ethnomedicinal knowledge with the power of combinatorial sciences and high throughput screening scientists can generate structure-activity libraries. While the experiential database can provide the new functional leads that reduce time, money and toxicity, the three main hurdles in drug development. The pharmacoepidemiological evidence on safety and practice of traditional medicine is underway in many countries, like Indian Golden Triangle Approach, proposed in the Chitrakoot Declaration for Ayurveda medicine, and the development of standardized herbal formulations by the Council for Scientific and Industrial Research, Government of India, under the New Millennium Indian Technology Leadership Initiative (Mashelkar, 2003). In randomized controlled trials clinical efficacy of the ayurvedic formulations for rheumatoid arthritis, osteoarthritis, hepatoprotective, hypolipedemic, asthma, and Parkinson’s diseases were established (Vaidya et al., 2001; Chopra et al., 2000). Thus the traditional knowledge database allows researchers to use a well-tested and safe botanical material, and the normal drug discovery course of ‘laboratories to clinics’ alters into ‘clinics to laboratories’ a reverse pharmacology approach (Vaidya, 2002), where safety is the most important starting point and efficacy is a matter of validation (Patwardhan et al., 2004). In drug

Diseases that need new drugs: Need of the hour

discovery and therapeutics there is a positive trend towards holistic health, integrative sciences, and systems biology approaches (Anand, 2003). Thus, a golden triangle consisting of ethnomedicine, modern medicine and modern science will converge to form a real discovery engine that can result in newer, safer, cheaper and effective therapies.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Adjou, K.T., Privat, N., Demart, S., Deslys, J.P., Seman, M., Hauw, J.J., and Dormont, D. 2000. J. Comp. Pathol., 122(1), 3-8. Aguzzi, A. 2006. J. Neurochem., 97(6), 1726-1739. Aguzzi, A., and Heikenwalder, M. 2006. Nat. Rev. Microbiol., 4(10), 765-775. Anand, N. 2003. CSIR Diamond Jubilee Symposium on Rasayana Drugs, CDRI, Lucknow, India. Amadea, M. 1990. The Ayurvedic Cookbook, Lotus Press, Santa Fe. Bate, C., Rutherford, S., Gravenor, M., Reid, S., and Williams, A. 2002. Neuroreport., 13(15), 1933-1938. Barret, A., Tagliavini, F., Forloni, G., Bate, C., Salmona, M., Colombo, L., De Luigi, A., Limido, L., Suardi, S., Rossi, G., Auvré, F., Adjou, K.T., Salès, N., Williams, A., Lasmézas, C., and Deslys, J.P. 2003. J.Virol., 77(15), 8462-8469. Baz, M., Abed, Y., Nehmé, B., and Boivin, G., 2009. Antimicrob. Agents Chemother., 53, 791-793. Beres, S.B. 2003. Proc. Natl Acad. Sci., USA 99, 10078-10083. Bekris, L.M., Millard, S.P., Galloway, N.M., Vuletic, S., Albers, J.J., Li, G., Galasko, D.R., DeCarli, C., Farlow, M.R., Clark, C.M., Quinn, J.F., Kaye, J.A., Schellenberg, G.D., Tsuang, D., Peskind, E.R., and Yu, C.E. 2008. Journal of Alzheimer's disease, 13(3), 255-266. Butler, J.C., 2001. Emerg. Infect. Dis., 7 (suppl.), 554-555. Korth, C., May, B.C.H., Cohen, F.E., and Prusiner, S.B. 2001. 98 (17), 98369841. Caughey, B., Race, R.E. 1992. J. Neurochem., 59, 768-771. Caspi, S., Sasson, S.B., Taraboulos, A., and Gabizon, R. 1998. J Biol Chem., 273, 3484-3489. Centers for Disease Control and Prevention, 2002. Staphylococcus aureus resistant to vancomycin- United States, 2002. MMWR 51, 565-567. Centers for Disease Control and Prevention, 2003. Multistate outbreak of monkeypox Illinois, Indiana, and Wisconsin, 2003. MMWR 52, 537-540. Chapman, L.E., 2003. Curr. Top. Microbiol. Immunol., 278, 23-45. Chattopadhyay, D., Chakraborty, M.S., and Saha, G.C. 1999, Indian J Sex Transmit Dis., 20, 54. Chattopadhyay, D., and Naik, T. 2007, Mini Rev. Med. Chem., 7(3), 275. Chang, Y. 1994. Science, 266, 1865-1869. Chin, Y.W., Balunas, M.J., and Chai, H.B., Kinghorn, A.D.2006, AAPS Journal., 8(2), 239.

Debprasad Chattopadhyay et al.

22. Chopra, A., Lavin, P., Patwardhan, B., and Chitre, D. 2000, J. Rheumatol., 27, 1365. 23. Chopra, A., and Doiphode, V. 2002, Med. Clin. North Am., 86, 75. 24. Cliff, A., Haggett, P. and Smallman-Raynor, M. 2002. In: Island Epidemics Ch. 6, 165-236, Oxford Univ. Press, Oxford. 25. Committee on Emerging Microbial Threats to Health, 1992. Emerging Infections. Microbial Threats to Health in the United States (Lederberg J, Shope RE, Oaks SC eds). National Academy Press, Washington DC. 26. Dahanukar, S., and Thatte, U. 2000, Ayurveda Revisited, Popular Prakashan, Mumbai, 3rd edn. 27. Daley, C.L. 1999. in The AIDS Knowledge Base 3rd edn (eds Cohen PT, Sande MA & Volberding PA) Ch. 3, 23-52, Lippincott, Williams and Wilkins, Philadelphia. 28. Dash, B., and Sharama, B.K., 2001, Charak Samhita, Chaukhamba Sanskrit Series, Varanasi, India, 7th edn. 29. Demaimay, R., J. Harper, H., and Gordon, D. 1998, J. Neurochem, 71, 2534-254. 30. De Clercq, E. 2004, Nat. Rev. Microbiol., 2(9), 704. 31. Dees, C., Wade, W. F., German, T. L., and Marsh, R. F. 1985, J. Gen. Virol., 66, 845-849. 32. Delay, J., Deniker, P., and Harl, J.M. 1952, Ann. Med. Psychol., 110, 267-273. 33. Dickinson, A.G., Fraser, H., and Outram, G.W. 1975, Nature, 256, 732-733. 34. Diringer, H., and Ehlers, B. 1991, J. Gen. Virol., 1991, 72,457-460. 35. Doh-urs, K., Iwaki, T., and Caughey, B. 2000, J. Virol., 74, 4894-4897. 36. Dunham, E.J., Dugan, V.G., Kaser, E.K., Perkins, S.E., Brown, I.H., Holmes, E.C., and Taubenberger, J.K. 2009, J. Virol., JVI.02565-08v1. 37. Ehlers, B., and Diringer, H. 1984, J. Gen. Virol., 65, 1325-1330. 38. Espinal, M.A. 2003, Tuberculosis 83, 44-51. 39. Fact Sheet. Multidrug-Resistant, (MDR TB) and Extensively Drug-Resistant Tuberculosis (XDR TB). Division of Tuberculosis Elimination. CDC. 11 August 2008. 40. Farquhar, C., Dickinson, A., and Bruce, M. 1999, Lancet, 353, 117. 41. Faruque, S.M., and Nair, G.B. 2002, Microbiol. Immunol. 46, 59-66. 42. Fredricks, D.N., and Relman, D.A. 1998. In: Current Clinical Topics in Infectious Diseases Vol. 18 (eds Remington JS & Swartz MN) 180â&#x2C6;&#x2019;200, Blackwell Science, Malden, Massachusetts. 43. Forloni, G., Iussich, S., Awan, T., Colombo, L., Angeretti, N., Girola, L., Bertani, I., Poli, G., Caramelli, M., Bruzzone, M.G., Farina, L., Limido, L., Rossi, G., Giaccone, G., Ironside, J.W., Bugiani, O., Salmona, M., and Tagliavini. F. 2002, Proc. Natl. Acad. Sci., 9, 10849-10854. 44. Glatzel, M., Stoeck. K., Seeger, H., Luhrs, T., and Aguzzi, A. 2005, Arch. Neurol., 62(4), 545-52. 45. Gubler, D.J. 1998, Dengue and dengue haemorrhagic fever. Clin. Microbiol. Rev. 11, 480-497. 46. Guerrant, R.L., and Blackwood, B.L. 1999, Clin. Infect. Dis. 28, 966-986. 47. Huang, F.P., Farquhar, C.F., Mabbott, N.A., Bruce, M.E., MacPherson, G.G. 2002, J Gen Virol., 83(Pt 1), 267-271.

Diseases that need new drugs: Need of the hour

48. Hyucke, Mark, M., Daniel, F., Sahm, and Michael, S. 1998. Emerging Infectious Diseases, 4, 239-249. 49. Ingrosso, L., Ladogana, A., and Pocchiari, M. 1995, J. Virol., 69, 506-508. 50. Jain, S.K., 1994, Ciba. Found. Symp., 185, 153-164. 51. Johnson, R.T. 2005, Lancet Neurol., 4(10), 635-642. 52. Jones, J.L. 1999, MMWR 48 (CDC Surveillance Summary no. SS-2), 1-22. 53. Katz, A.R., and Morens, D.M. 1992, Clin. Infect. Dis., 14, 298-307. 54. Kimberlin, R.H., and Walker, C.A. 1983, Arch. Virol., 78, 9-18. 55. Klevens, R., Monina. 2007, JAMA 298, 1763-1771. 56. Komar, N. 2003. Adv. Virus Res. 61, 185-234. 57. Korth, C., May, B.C.H., Cohen F.E and Prusiner S.B. 2001, Proc. Natl. Acad. Sci., 98(17), 9836-9841. 58. Kovats, R.S., Bouma, M.J., Hajat, S., Worrall, E., and Haines, A. 2003, Lancet 362, 1481-1489. 59. Lee, J.H. 2003, Appl. Environ. Microbiol., 69, 6489-6494. 60. Li, M.S., Farrant, J.L., Langford, P.R., and Kroll, J.S. 2003, Mol. Microbiol., 47, 1101-1111. 61. Ludewigs, H., Zuber, C., Vana, K., Nikles, D., Zerr, I., and Weiss S. 2007, Expert Review of Anti-infective Therapy 5(4), 613-630. DOI 10.1586/14787210.5.4.613. 62. Massey, A. 1933. Epidemiology in Relation to Air Travel, H. K. Lewis and Co., London. 63. Mashelkar, R.A. 2003, Chitrakoot Declaration, National Botanical Research Institute Convention. 64. McVean, G., Spencer, C.C., and Chaixn, R. 2005. Genet., 1 (4): 54e. 65. Mead, S. 2006. Eur. J. Hum. Genet, 14(3), 273-281. 66. Morbidity and Mortality Weekly Report (MMWR), April 13, 2006. 67. Morens, D.M. 1994, Clin. Infect. Dis., 19, 500-512. 68. Morens, D.M. 1998, Pac. Health Dialog, 5, 147-153. 69. Morens, D.M., Folkers, G.K., and Fauci, A.S. 2004, Nature 430, 242-249. 70. Musser, J.M., and Krause, R.M. 1998, In: Emerging Infections. Biomedical Research Reports (ed. Krause, R. M.) 185â&#x2C6;&#x2019;218, Academic Press, San Diego. 71. Musser, J.M., and Selander, R.K. 1990, J. Infect. Dis., 161, 130-133. 72. Nash, D. 2001, N. Engl. J. Med., 344, 1807-1814. 73. Neu, H.C. 1992, Science 257, 1064-1072. 74. Parsonnet, J. (Ed., 1999) In: Microbes and Malignancy: Infection as a Cause of Human Cancers, Oxford University Press, New York. 75. Patwardhan, B., Chopra, A., and Vaidya, A.D.B. 2003, Current Sci. 84, 1165. 76. Patwardhan, B., Vaidya, A.D.B., and Chorghade, M. 2004, Current Science 86(6), 789-799. 77. Peiris, J.S.M., Yuen, K.Y., Osterhaus, A.D.M.E., and StĂśhr, K. 2003, New England J. Med., 349, 2431-2441. 78. Phuan, P.W., Zorn, J.A., Safar, J., Giles, K., Prusiner, S.B. Cohen, F.E., May, B.C.H. 2007, J Gen Virol, 88(Pt4), 1392-1401. 79. Platonov, A.E. 2001. Emerg. Infect. Dis., 7, 128-132. 80. Priola, S.A., Raines, A., Caughey, W.S. 2000, Science, 287(5457), 1503-1506. 81. Prusiner, S.B. 2001, New England J Med, 344(20), 1516-1526.

Debprasad Chattopadhyay et al.

82. Murray J. 2002, BMJ, 325(7373): 1128. New Engl J Med. 344(20):1548-1551, 2001; New Engl J Med. 345(11):840-841, 2001. 83. Quinn, T.C. 1994, Proc. Natl Acad. Sci., USA 91, 2407-2014. 84. Reid, S.D., Hoe, N.P., Smoot, L.M., and Musser, J.M. 2001, J. Clin. Invest., 107, 393-399. 85. Reid, A.H., and Taubenberger, J.K. 2003, J. Gen. Virol., 84, 2285-2292. 86. Roikhel, V.M., Fokina, G.I., and Pogodina, V.V. 1984, Acta Virol., 28(4), 321-324. 87. Ronald, A.R. 1995, Infect. Dis. Clin. North Am., 9, 287-296. 88. Sanders, M.K., and Peura, D.A. 2002, Curr. Gastroenterol. Rep., 4, 448-454. 89. Skelding, K.A., Gerhard, G.S., Simari, R.D., and Holmes, D.R. 2007, Nat. Clin. Pract. Cardiovasc. Med., 4 (3), 136-142. 90. Shortridge, K.F., Peiris, J.S., and Guan, Y. 2003, J. Appl.Microbiol., 94 (suppl.), 70S-79S. 91. Singh, N. 2003, Lancet Infect. Dis., 3, 156-161. 92. Soto, C., Kascsak, R.J., SaborĂo, G.P., Aucouturier, P., Wisniewski, T., Prelli, F., Kascsak, R., Mendez, E., Harris, D.A., Ironside, J.,Tagliavini, F., Carp, R.I., and Frangione, B. 2000, Lancet. 355, 192-197. 93. Stevens, J. 2004, Science, 303, 1866-1870. 94. Subbarao, K., and Katz, J. 2000, Cell Mol. Life Sci., 571, 1770-1784. 95. Supattapone, S., Wille, H., Uyechi, L., Safar, J., Tremblay, P., Szoka, F.C., Cohen, F.E., Prusiner, S.B., and Scott, M.R. 2001, Journal of Virology, 75(7), 3453-3461. 96. Vaidya, A.D.B., Vaidya, R.A., and Nagaral, S.I., 2001, J. Assoc. Physicians India, 49, 534. 97. Vaidya, A. 2002, Reverse pharmacology approach, CSIR NMITLI Herbal Drug Development Programme. 98. Vento, S., and Cainelli, F. 2003, Lancet Oncol., 4, 595-604. 99. Wagner, E.K., and Hewlett, M.J., 1999, Basic Virology. Blackwell Science, Malden. 100. Waxler-Morrison, N.E. 1988, Soc. Sci. Med., 27(5), 531-544. 101. Webster, R.G. 2001, Science, 293, 1773-1775. 102. World Health Organization, GenĂ¨ve, 2004. 103. Will, R.G., Alpers, M.P., Dormont, D., Schonberger, L.B., and Tateishi, J., 1999, Prion Biology and Diseases. Prusiner S.B., editor, Plainview, N.Y., Cold Spring Harbour Lab. Press, 465-507.

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Ethnomedicine: A Source of Complementary Therapeutics, 2010: 29-52 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

2. Ethno medicine in complementary therapeutics Pulok K. Mukherjee, S. Ponnusankar and M. Venkatesh School of Natural Product Studies, Dept. of Pharmaceutical Technology, Jadavpur University Kolkata 700032, India

1. Introduction The relationship between man and plants has been very close throughout the development of human culture. The evidences of the use of various plants for various ailments by our ancestors, indicates that the plant derived medicines, are from rich traditions of ancient civilizations and scientific heritage. A great number of these natural products have come to us from the scientific study of remedies traditionally employed by various cultures. At no time in the development of man kind, there has been more rapid and more deeply meaningful progress was made in our understanding of plants and their chemical constituents. The gradual sophistication of phytochemistry, pharmacology etc and the hope for scientific remedies from plant sources setup a tendency concerning the potential value of natural products. The plants of various regions have been well documented for their medicinal values Correspondence/Reprint request: Dr. Pulok K. Mukherjee, School of Natural Product Studies, Dept. of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India. E-mail: pulokm@gmail.com

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but there is still need for proper documentation of these plants because such type of study basically lies in finding out the usage of plants or its parts for betterment understanding from ethno-medical uses. Herbs have provided the basis for the great medical systems in human history of Hippocrates and Galen and the great Ayurveda of the Indian subcontinent, Traditional Chinese Medicine of Chinese medical system, Islamic medical system over two millennia and many other cultural traditions that were often hybrids of the various systems of medicine. All these systems were formed in large part by the peculiar characteristics of the respective materia medica, plants have clearly demanded and been granted their own therapeutic approach (1). In India for drug development from ethno medicine there is a strong historical base, where from the ancient literature on Indian system of medicine several documents on therapeutics can be derived. The classical Indian text like Rig-Veda, Atherveda, Charak Samhita and Sushruta Samhita are the evidences of the use of plants by our ancestors (Table 1). Table 1. The Historicity and the available ancient literature on Indian ancient system of medicine – Ayurveda. Ayurveda is the most ancient system of medicine, its antiquity going back to the Vedas, surviving today through the classic texts Name of texts Author Historicity Subject Brihattrayi- The three major texts: 1. Charaka Samhita Charaka 1000 – 700 BC Philosophy & Medicine 2. Sushruta Samhita Sushruta 1000 – 600 BC Practice of Surgery 3. Vagbhatta Vagbhatta 300 – 600 AD Medicine and Samhitas therapeutics Laghuttrayi – The three major texts 1. Madhav Nidana Madhavkara 900 AD Diagnostics 2. Sarangadhar Sarangdhara 1300 AD Medicine Samhita 3. Bhava Prakasa Bhava Misra 1600 AD Drugs and Herbs

Herbal medicine is a triumph of popular therapeutic diversity and used as complementary therapies in many developing countries. Until the discovery of modern medicines, any system of medicine that relieved the patient and their ailment(s) was considered to be a therapeutic system without further investigation. Whether the system was properly analyzed, researched or organized did not matter. But with the rapid development of conventional medicines, this ethno medicinal plants and its knowledge required to be

Ethno medicine in complementary therapeutics

proved scientifically with proper evidence to prove the therapeutic efficacy of the ethnic system of medicine to be used as complementary therapies. Medicinal uses of plant and animal species have been practiced for centuries in many parts of the world. Even today, hundreds of millions of people, mostly in developing countries, derive a significant part of their subsistence needs and income from gathered plant and animal products. Gathering of high value products such as mushrooms (morels, matsutake, truffles), medicinal plants (ginseng, black cohosh, goldenseal) also continues in developed countries for cultural and economic reasons (2). Ethno medicine has been utilized for a long time for various disorders, now the time has come to use these resources as complementary therapeutics based on scientific validations. Several approaches for the development of drugs from ethnic medicine are given in Figure 1. The era of grand systems has probably passed but it may be time to develop a new coherent approach to herb use for a scientific age. Apart from a general view that herbs are safer, there has been only a fragmentary rationale for using them as medicines in modern times. Standardization of traditional formulations (used by various cultures, tribal and ethnic groups)

Evidence based approach To Science based mechanism understanding

Multiprone approach

Drug discovery engine Lead potentials identification Ethnopharmacology approach

Chemical biology approach System biology approach Combinatorial library Personalized medicine

Figure 1. Ethno medicine â&#x20AC;&#x201C; approaches in drug development.

2. Biodiversity of ethno medicinal plants Biodiversity has been touted as a mechanism for both discovering new pharmaceutical product and saving endangered ecosystem. Since time immemorial, people have gathered plant and animal resources for their needs. However, medicinal plants play a central role, not only as traditional medicines used in many cultures, but also trade commodities which meet the

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demand of often distant markets (1). There has been considerable recent interest in the search of medicines from ethnic resources. In nature, these medicines are prepared by plants as metabolites. These chemical compounds might be of considerable commercial value if adapted to industrial, agricultural and particularly preparation of phytomedicines and pharmaceuticals. Bio-diversity responds to a number of new, emerging concerns including, the result of new developments in technology, in particular, biotechnology and information technology, and the ongoing degradation of the environment, inevitably accompanied by an erosion of biological diversity (3). Biodiversity encompasses all biological entities occurring as an interacting system in a habitat or ecosystem and plants constitute a very important segment of such biological systems. Demand for a wide variety of wild species is increasing with growth in human needs, numbers and commercial trade. With the increased realization that some wild species are being over-exploited, a number of agencies are recommending that wild species be brought into cultivation systems (4). Despite the increasing use of medicinal plants, their future is being threatened by complacency concerning their conservation. Reserves of herbs and stocks of medicinal plants in developing countries are diminishing, several important species are in danger of extinction as a result of growing trade demands for safer and cheaper healthcare products and new plant-based therapeutic markets in preference to more expensive target-specific drugs and biopharmaceuticals. The number of plant species which have at one time or another been used in some countries for medicinal purposes can only be estimated. Enumerations of the WHO from the late 1970â&#x20AC;&#x2122;s listed 21,000 medicinal species are used in various parts of the globe (5). Developing countries like China and India have a vast biodiversity of medicinal and aromatic plants. In China alone 4,941 of 26, 092 native species are used as drugs in Chinese traditional medicine. If this proportion is calculated for other well-known medicinal floras and then applied to the global total of 4,22,000 flowering plant species are used for various purposes and it can be estimated that the number of plant species used for medicinal purposes is more than 50 000 (6, 7). In India, medicinal and aromatic plants have been in use in one form or another, under indigenous systems of medicine like Ayurvedha, Siddha and Unani etc. India is having a well-recorded and well practiced knowledge of traditional herbal medicine (8). India officially recognizes over 3000 plants for their medicinal value. It is generally estimated that over 6000 plants in India are in use in traditional, folk and herbal medicine, representing about 75% of the medicinal needs of the third world countries (9). India is one of the 12 mega biodiversity centers having 45, 000 plant species; its diversity is

Ethno medicine in complementary therapeutics

unmatched due to the 16 different agroclimatic zones, 10 vegetative zones, and 15 biotic provinces. The country has a rich floral diversity and reported to have15,000-18,000 flowering plants, 23,000 fungi, 25,000 algae, 1,600 lichens, 1,800 bryophtes and 30 million micro-organisms (1).

3. Plant derived pharmaceuticals and its delivery Many plant based drugs were used as complementary therapies in industrial countries that were originally discovered by folk healers. Around 60% medicinally useful formulations and other health products, which are either derived or developed from plant origin dominate the global market of health care products. Quinine from cinchona tree had its origin in the royal households of the South American Incas. In the early 1500s, Indian fever bark was one of the first medicinal plants to find appreciative consumers in Europe. Taken from the cinchona tree (Cinchona officinalis), the bark was used as an infusion by native people of the Andes and Amazon highlands to treat fever (10). In Andean cultures, the leaves of the coca tree have been primarily chewed to obtain perceived benefits. From ancient times, indigenous people have added alkaline materials such as crushed seashells or burnt plant ashes to the leaves in order to accentuate the pharmacologically active moiety of coca. Pot curare arrowhead poison used in the East Amazon is predominately from the species Strychnos guianensis. Tube curare in the West Amazon is from Chrondrodendron tomentosum; curare in modern medicine is made from this and named as tubocurarine. The jaborandi tree (Pilocarpus jaborandi) secretes alkaloid- rich oil. Several substances are extracted from this aromatic oil, including the alkaloid pilocarpine, a weapon against the blinding disease, glaucoma(11). American Indians on the island of Guadeloupe used pineapple (Ananas comosos) poultices to reduce inflammation in wounds and other skin injuries, to aid digestion and to cure stomachache (11). In Ayurveda and other Indian systems of medicines, use of different plants for treating ailments was based on the fact that the additive or synergistic effects of the secondary metabolites present in those plants enhance therapeutic viability of the phytoconstituents. This knowledge and experiential database can provide new functional leads to reduce time, money and toxicity â&#x20AC;&#x201C; the three main hurdles in drug development. These records are particularly valuable, since effectively these medicines have been tested for thousands of years on people. Efforts are underway to establish pharmacoepidemiological evidence base regarding safety and practice of Ayurvedic medicines. Randomized controlled clinical trials for rheumatoid and

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osteoarthritis, hepatoprotectives, hypolipedemic agents, asthma, Parkinsonâ&#x20AC;&#x2122;s disease and many other disorders have reasonably established clinical efficacy. Many conventional drugs originate from plant sources: a century ago, most of the few effective drugs were plant based. Examples include artemisinin, atropine, digoxin, ephedrine, gallanthamine, morphine, physostigmine, quinine, reserpine, salicylic acid, sennoside, Taxol, vincristine, vinblastine, glycyrrhizin, and psoralen (12). Similarly a second antimalarial in addition to quinine is now available for cerebral malaria resistant to chloroquine. Other analogues of artemisinin are now being evaluated and arteether and artemether were found to be more effective. Flavopiridol is totally synthetic, but the basis of it is rohitukine which is isolated from Dysoxylum binectariferum Hook. f. (Meliaceae), which is phylogenetically related to the Ayurvedic plant D. malabaricum Bedd. used for rheumatoid arthritis. The successful introduction of these plants into modern therapeutics indicates that other discoveries are waiting to be made. The plants used in Indian Systems of Medicine showed the presence of a variety of chemical entities, belonging to different classes (13). Combining the strengths of the knowledge base of complementary alternative medicines like Ayurveda with the dramatic power of combinatorial sciences and High Throughput Screening (HTS) will help in the generation of structureâ&#x20AC;&#x201C;activity libraries. The development of drugs from ethnic plants continues, with drug companies engaged in large scale pharmacologic screening of herbs (14). There is a revival of interest in Ayurvedic herbal products at a global level; herbs such as turmeric, neem, ginger, holi basil and ashwagandha are a few examples of what is gaining popularity among modern physicians. 3.1. Novel delivery of phytoconstituents Every nation is seeking health care beyond the traditional boundaries of modern medicine; turning to self-medication in the form of herbal remedies (15,16). Now-a-days extensive research in novel drug delivery systems is going on to improve the therapeutic efficacy of the existing natural molecules. Toxicity and limited absorption of different phytoconstituents obtained from herbs are crucial problems in exploring their real potentials against different diseases. Value added formulation, as its name indicates is a formulation with added value, which gives better therapeutic efficacy of its main chemical constituents inside our body. The development of value added herbal formulations having better absorption and utilization profiles in our body is of paramount importance (17). To minimize drug degradation and loss during herbal drug consumption and to

Ethno medicine in complementary therapeutics

increase herbal bioavailability, various drug delivery and drug targeting systems are currently under development. Among them, Phytosomes are advanced forms of herbal products that are better absorbed, utilized, and as a result produce better effects than conventional herbal extracts. Phytosomes are produced via a patented process whereby the individual components of an herbal extract are bound to phosphatidylcholine (18). The Phytosome process has been applied to many popular herbal extracts including Ginkgo biloba, grape seed, milk thistle, and green tea (19). Improved therapeutic efficacy of phytosomes can be best presented with the results obtained from the studies with Ginkgo biloba phytosomes. A representation of phytosomes as effective delivery systems for herbal constituents has been shown in Figure 2. It has been reported that Ginkgo phytosome produced better results compared to the conventional extracts (20, 21). There are also other phytosomal formulations like Grape seed phytosome, glycyrrhetinic acid phytosome, hawthorn phytosome, Panax ginseng phytosome etc. which have been proved to be a trust-worthy and useful pharmaceutical product keeping in view, their improved therapeutic activities in phytosome form. Phytosomes have also got tremendous impact in skin care products and cosmetology. Enhanced microcirculation provided by the phytosomes is very much useful for skin care and opened a new avenue in cosmetic science with significant upper hand over the crude extracts or uncomplexed phytoconstituents, used in this field (22-24). Our recent studies with phospholipids complex of different potent phytomolecules like Quercetin,

Figure 2. Drug activity enhancement through phytosomes drug development.

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naringenin and curcumin showed that the therapeutic efficacy, in terms of free radical scavenging activity of the molecules increased upon complexation with phospholipids and the phospholipid complexes at different dose levels showed better effects than respective free molecules at same doses (25-27).

4. Bioactive phytoconstituents â&#x20AC;&#x201C; lead for drug development Research into the isolated plant constituents is of great importance for the development of bioactive substances from ethnic medicine. With the emergence of latest technologies and enhanced knowledge about the isolated plant constituents, characterization and analytical tools, lot of compounds are efficiently isolated from potential plants and have been of great contribution to the drug discovery from ethnic plants. Separating a medicinal herb into its constituents cannot often explain exactly the way in which it works in the natural form. The whole herb is worth more than the sum of its components. A plant contains hundreds of chemical constituents that interact in a complex way to produce therapeutic effects of the remedy. We may not understand the detailed mechanism, in which a particular herb works â&#x20AC;&#x201C; even though its medicinal benefits are well established. With the development of standardization tools like HPTLC, HPLC, LC MS/MS etc., several plant extracts as well as their formulations, will be standardized in a better way to enhance the bioactivity of the ethnic medicines (9). In the development of drugs and therapeutics, the use of plants as medicines has involved the isolation of active compounds, beginning with the isolation of morphine from opium in the early 19th century (28, 29). Drug discovery from medicinal plants led to the isolation of early drugs such as cocaine, codeine, digitoxin, and quinine, in addition to morphine, of which some are still in use (30,31). Plant based drugs provide outstanding contribution to modern therapeutics; for example: serpentine isolated from the root of Indian plant Rauwolfia serpentina in 1953, was a revolutionary event in the treatment of hypertension and lowering of blood pressure. During 1950-1970 approximately 100 plants based new drugs were introduced in the USA drug market including deserpidine, reseinnamine, reserpine, vinblastine and vincristine etc which are derived from Ethnomedicinal plants. From 1971 to 1990 new drugs such as etoposide, E-guggulsterone, teniposide, nabilone, plaunotol, Z-guggulsterone, lectinan, artemisinin and ginkgolides appeared all over the world. Drugs introduced from 1991 to 1995 include paciltaxel, toptecan, gomishin, irinotecan etc. Drugs isolated from ethnic medicines can serve not only as new drugs themselves but also as drug leads. In the recent past, many bioactive phytoconstituents were isolated from natural products or derived from natural

Ethno medicine in complementary therapeutics

products. The following are some of the plant derived drugs available in market after the approval of USFDA and few in early stages of clinical trials. The bioactive phytoconstituents derived from ethnic medicine showed the presence of variety of chemical entities, belonging to different classes as represented in Figure 3 and were made available in the market. O O

HO N

Arteether (1)

Galanthamine (2) F

N+

S O

N+

Br-

Nitisinone (3)

Tiotropium (4) HO

F F

O O

N O

O O

NH O

Morphine-6-glucuronide [M6G] (5) NH2

Vinflunine (6)

O O

O OH

Exatecan (7)

Calanolide A (8) O O

OH O

O N

Betulinic acid (9)

O O

Pervilleine A (10)

Figure 3. Several potent bio-active phytoconstituents from ethnic medicine.

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Arteether (1), [trade name Artemotil] is a potent antimalarial drug and is derived from artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (Asteraceae), a plant used in traditional Chinese medicine (TCM) (32). Galanthamine (2), [trade name Reminyl] is a natural product discovered through an ethnobotanical lead and first isolated from Galanthus woronowii Losinsk. (Amaryllidaceae) in Russia in the early 1950s (33). It is approved for the treatment of Alzheimerâ&#x20AC;&#x2122;s disease, slowing the process of neurological degeneration by inhibiting Acetylcholinesterase (AChE) as well as binding to and modulating the nicotinic acetylcholine receptor (nAChR)(34). Nitisinone (3), [trade name Orfadin] is a newly released medicinal plantderived drug that works on the rare inherited disease, tyrosinaemia, demonstrating the usefulness of natural products as lead structures (35). Nitisinone is a modification of mesotrione, an herbicide based on the natural product leptospermone, a constituent of Callistemon citrinus Stapf. (Myrtaceae). All three of these triketones inhibit the same enzyme, 4-hydroxyphenylpyruvate dehydrogenase (HPPD), in both humans and maize. Tiotropium (4), [trade name Spiriva] has been introduced to the United States market for treatment of chronic obstructive pulmonary disease (COPD) (36). Tiotroprium is an inhaled anticholinergic bronchodilator, based on ipratropium, a derivative of atropine that has been isolated from Atropa belladonna L. (Solanaceae) and other members of the Solanaceae family. Tiotropium has shown increased efficacy and longer lasting effects when compared with other available COPD medications (37). Compounds M6G (5) is in later stages of Phase III clinical trials and its subtle modifications of drugs currently in clinical use (38). M6G or morphine-6glucuronide (5) is a metabolite of morphine from Papaver somniferum L. (Papaveraceae) and will be used as an alternate pain medication with fewer side effects than morphine (39). Vinflunine (6) is a modification of vinblastine from Catharanthus roseus (L.) G. Don (Apocynaceae) for use as an anticancer agent with improved efficacy (40). Exatecan (7) is an analog of camptothecin from Camptotheca acuminata Decne. (Nyssaceae) and is being developed as an anticancer agent (41). Modifications of existing natural products exemplify the importance of drug discovery from medicinal plants as NCEs and as possible new drug leads. Calanolide A (8) is a dipyranocoumarin natural product isolated from Calophyllum lanigerum var. austrocoriaceum (Whitmore) P.F. Stevens (Clusiaceae), a Malaysian rainforest tree (42). Calanolide A is an anti-HIV drug with a unique and specific mechanism of action as a non-nucleoside reverse transcriptase inhibitor (NNRTI) of type-1 HIV and is effective against AZTresistant strains of HIV (43). Calanolide A is currently undergoing Phase II clinical trials (44). Drug discovery from medicinal plants has played an important role in the treatment of various diseases including life threatening conditions such as

Ethno medicine in complementary therapeutics

cancer. Most new clinically useful bioactive phytoconstituents are from plant secondary metabolites and their derivatives (38). Anticancer agents from plants currently in clinical use can be categorized into four main classes of compounds: vinca (or Catharanthus) alkaloids, epipodophyllotoxins, taxanes, and camptothecins. Vinblastine and vincristine were isolated from Catharanthus roseus (L.) G. Don (Apocynaceae) (formerly Vinca rosea L.) and have been used clinically for over 40 years. Vinblastine isolated from the Catharanthus rosesus is used for the treatment of Hodgkins, choriocarcinoma, non-hodgkins lymphomas, leukemia in children, testicular and neck cancer. Vincristine is recommended for acute lymphocytic leukemia in childhood advanced stages of hodgkins, lymophosarcoma, small cell lung, cervical and breast cancer(9). Podophyllotoxin was isolated from the resin of Podophyllum peltatum L. (Berberidaceae) but was found to be too toxic in mice so derivatives were made with the first clinically approved drug being etoposide. The epipodophyllotoxins bind tubulin, causing DNA strand breaks during the G2 phase of the cell cycle by irreversibly inhibiting DNA topoisomerase II (45). Podophyllotoxin is also a constituent of Phodophyllum emodi currently used against testicular, small cell lung cancer and lymphomas. Indian indigenous tree of Nothapodytes nimmoniana (Mappia foetida) are mostly used in Japan for the treatment of cervical cancer. Teniposide and etoposide isolated from Podophyllum species are used for testicular and lung cancer. Paclitaxel (Taxol) isolated from Taxus brevifolia Nutt. (Taxaceae) is used for the treatment of metastatic ovarian cancer and lung cancer. The taxanes, including paclitaxel and derivatives, act by binding tubulin without allowing depolymerization or interfering with tubulin assembly (46). The above drugs came into use through the screening study of medicinal plants because they showed less side effects, were cost effective and possessed better compatibility. Camptothecin was isolated from Camptotheca acuminata Decne. (Nyssaceae) but originally showed unacceptable myelosuppression. Interest in camptothecin was revived when it was found to act by selective inhibition of topoisomerase I, involved in cleavage and reassembly of DNA (41). Together, the taxanes and the camptothecins accounted for approximately one-third of the global anticancer market. Several of these plant derived compounds are currently undergoing further investigation including betulinic acid (9), pervilleine A(10), and silvestrol. Betulinic acid, a pentacyclic triterpene, is a common secondary metabolite of plants, primarily from Betula species (Betulaceae). Betulinic acid was isolated from Ziziphus mauritiana Lam. (Rhamnaceae) collected in Zimbabwe (47). The ethyl acetate-soluble extract displayed selective cytotoxicity against human melanoma cells (MEL-2). Betulinic acid was isolated using

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bioassay-guided fractionation including silica gel chromatography and crystallization techniques. Pervilleine A, along with eight other tropane alkaloids, was isolated from the roots of Erythroxylum pervillei Baill. (Erythroxylaceae) collected in southern Madagascar(48). The chloroformsoluble extract was found to be selectively cytotoxic against a multi-drug resistant (MDR) oral epidermoid cancer cell line (KB-V1) in the presence of the anticancer agent vinblastine. The pervilleines were isolated using bioassay-guided fractionation including silica gel chromatography and aluminum oxide chromatography. Silvestrol was first isolated from the fruits of Aglaia sylvestris (M. Roemer) Merrill (Meliaceae) (later re-identified as Aglaia foveolata Pannell) collected in Indonesia(49). The chloroform-soluble extract was found to be cytotoxic to several human cancer cell lines and, more importantly, the extract was active in the P-388 in vivo test system. Bioassay-guided fractionation was performed using silica gel chromatography and reversed-phase high-pressure liquid chromatography (HPLC) leading to the isolation of silvestrol.

5. Drug development from ethnomedicine Drug development from ethnic medicinal plants has evolved to include various fields of inquiry and numerous methods of analysis. The process typically begins with a botanist, ethnobotanist, ethnopharmacologist, or plant ecologist who collects and identifies the plant(s) of interest. Collection may involve species with known biological activity for which active compound(s) have not been isolated (e.g., traditionally used herbal remedies) or may involve taxa collected randomly for a large screening program. Phytochemists (natural product chemists) prepare extracts from the plant materials, subject these extracts to biological screening in pharmacologically relevant assays, and commence the process of isolation and characterization of the active compound(s) through bioassay-guided fractionation. Molecular biology has become essential to medicinal plant drug discovery through the determination and implementation of appropriate screening assays directed towards physiologically relevant molecular targets (9). Despite the recent interest in molecular modeling, combinatorial chemistry, and other techniques by pharmaceutical companies and funding organizations, ethnic medicines remain an important source of new drugs, new drug leads, and new chemical entities (NCEs) (30, 31). Numerous methods have been utilized to acquire compounds for drug discovery including isolation from plants, synthetic chemistry, combinatorial chemistry, and molecular modeling (50-52). Natural products provided a starting point for new synthetic compounds, with diverse structures and often with multiple stereocenters that can be challenging

Ethno medicine in complementary therapeutics

synthetically(53-55). Many structural features common to natural products (e.g., chiral centers, aromatic rings, complex ring systems, degree of molecule saturation, and number and ratio of heteroatoms) have been shown to be highly relevant to drug discovery efforts (55-58). Furthermore, since the escalation of interest in combinatorial chemistry and the subsequent realization that these compound libraries may not always be very diverse, many synthetic and medicinal chemists are exploring the creation of natural product and natural-product like libraries that combine the structural features of natural products with the compound-generating potential of combinatorial chemistry (59-61). Several factors have contributed to the revival of interest in plant derived products which include, undisputed clinical efficacy of the product, compounds with less direct therapeutic potential may offer new molecular templates for the design of more effective drugs (1), as an alternative to established therapy and a valuable, inexpensive sources of â&#x20AC;&#x153;feed stockâ&#x20AC;? molecules that can be really transformed into drugs. Biodiversity is a major source for drug development which fuels this reviving interest. For the exploration of this resource for new leads for drug development, high throughput screening for bioactivity has great potential. However, for an efficient exploration of this resource new methods are required that enable the rapid identification or false-positives and knownactive compounds. This can help in developing several new chemical entities (NCE) from ethno medicine. 5.1. Combinatorial biosynthesis of medicinal plant secondary metabolites The approach to combine genes from different microorganisms for the production of new and interesting metabolites has become known as combinatorial biosynthesis. It is now possible to combine various genes and extend the realm of combinatorial biosynthesis far beyond the biosynthesis. The diversification of products will increase dramatically when genes of very different origins are used. However, there is no need to concentrate on new compounds only; there are many interesting natural products, of which the application (e.g. as a drug or fine chemical) is hampered by its availability. The biodiversity is endless and there are still possibilities to enlarge the diversity from a chemical point of view, by combining genes and products from different sources that in nature would never meet. This strategy will deliver compounds that are not influenced by selection pressures, by a habitat, or the biochemical limitations of an organism (such as compartmentalization or storage). These compounds can be selected for a specific pharmaceutical mode of action or an activity can be adjusted to a more specific pharmaceutical demand.

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There are several pharmaceuticals in the market that are highly expensive, due to the fact that these compounds are only found in rare plants and often in extreme low concentrations. Podophyllotoxin and paclitaxel are clear examples of pharmaceuticals that can only be produced through the isolation from plants. To achieve a sustainable source of such compounds scientists all over the world have been experimenting with biotechnological approaches aiming at the development of an alternative production system. With this aim in mind, combinatorial biosynthetic strategies are expected to yield interesting alternatives in the near future. With regard to the production of podophyllotoxin it has been shown that plant cell cultures of Linum flavum L. can be used to convert deoxypodophyllotoxin, a major lignan of Anthriscus sylvestris L. into 6-methoxypodophyllotoxin. The combination of the product of one species and the enzymes of another species to yield a desired product is a good example of combinatorial biosynthesis (62). 5.2. Ethno pharmacology approach Ethnopharmacologic approach is based on botany, chemistry and pharmacology (observation, identification, description and experimental investigation) but other disciplines have made vital contributions. Based on these considerations, ethnopharmacology is defined as â&#x20AC;&#x153;the interdisciplinary scientific exploration of biologically active agents traditionally employed or observed by manâ&#x20AC;?. This study of traditional drugs is not meant to advocate a return to the use of these remedies in their aboriginal form, or to exploit traditional medicine. The objectives of ethnopharmacology are to rescue and document an important cultural heritage before it is lost, and to investigate and evaluate the agents employed. Thus, it plays an immense role in evaluation of natural products and more particularly the herbal drugs from traditional and folklore resources. Field observations and descriptions of the use and effects of traditional remedies, botanical identification, phytochemical and pharmacological studies are all within the scope of ethnopharmacology. It is essential that anthropologists interested in ethnopharmacology seek contact and collaboration with experts in botany, chemistry and pharmacology. Such a multidisciplinary approach presents added advantages. Even in recent times an anthropologist can give a detailed composition of an African poison ordeal without bothering about the chemical composition of the poisonous drink used or even its plant origin. The identification of medicinal plants and other traditional drugs is of course a crucial point, and good ethnopharmacological research can only be based on properly prepared voucher specimens, carefully authenticated by experts. Wherever possible, phytochemical studies

Ethno medicine in complementary therapeutics

on medicinal plants should be followed by a careful search for the biological activities of the compounds isolated. When biologically active principles have been found, the findings must be interpreted in the light of the traditional use. It is impossible to establish a dose-effect relationship unless the original drug preparations are analyzed and evaluated chemically and pharmacologically. In ethnopharmacological research, it is essential to have a proper sampling and analysis methods, and this necessity requires close cooperation by pharmacologists, with anthropologists and ethnobotanists on the one hand and specialists in chemical analysis on the another (1). Most traditional drugs are administered as mixtures of many components, and with today knowledge of the many possible interactions between drugs, and between food and drugs, ethnopharmacological research must deal with this aspect too. Additive, synergistic, or antagonistic effects are all possible. Various admixtures have also been shown to affect the bioavailability of pharmacologically active principles. Pharmacological studies of traditional medicinal agents should be therefore initiated prior to, or in parallel with, chemical research and should guide the isolation of active principles. Field observations of traditional therapies and the pharmacological effects in humans should be carried out by trained pharmacologists, and when interesting activity is found, controlled experiments should be initiated. Ethnopharmacology is not just a science of the past using an outmoded approach. It still constitutes a scientific backbone in the development of active therapeutics based upon traditional medicine of various ethnic groups with the ultimate aim of validating these traditional preparations, either through the isolation of active substances or through pharmacological findings. 5.3. Chemical biology approach Unlike modern drugs in form of single chemical ingredient, ethnic medicines are usually derived from aqueous extracts of a few herbs and contain hundreds of chemical compounds. Modern clinical trial proved that a complex formulation composed of up to 20 herbs had greater efficacy than single herb used (63). Obviously, there exists certain relation between biological activity and chemical composition of herbal medicine and it is called as quantitative composition- activity relationship (QCAR) (64). Experimental studies, such as random controlled trials (RCT), often provide the most trust-worthy methods for establishing causal relationships from data, in which one or more variables is changed (typically random) to measure its effect on other variables. In recent years, the relation between active ingredients of herb medicine and biological activity is one type of

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causal relationship was attempted. When the amount of active components in certain formulation varied, its therapeutical effect was correspondingly changed. Thus, causal analysis method can be employed in studying relationship of chemical composition and bioactivity of herbs and help to discover active components. There are few methods available for discovering causal relationships, in which the actual process of controlled experiment could be stimulate through series of conditional dependence tests. A recent approach called â&#x20AC;&#x153;STEPCARDâ&#x20AC;? {stepwise causal adjacent relationship discovery} method has been developed to overcome the disadvantage of existing causal discovery algorithm, as well as the unreliability of traditional statistical methods, e.g. stepwise regression. The main idea of STEPCARD is using conditional dependency test to determine causal adjacent relationships between explanatory variables and predictor. For a given data set containing chemical composition matrix (parameter X) and bioactivity information matrix (parameter y), STEPCARD algorithm can be applied to choose the components or components combinations most correlative to the biological activity of original formulation (comparison drug). The computational results of STEPCARD algorithm dealing with chemical and biological data, it represents the minimal significant level used in conditional independent test to pick out at least one variable. But there is lack of scientific approach to study correlations of their chemical constitution and pharmacological mechanism. However, this work affords a new strategy to identify active component or component combinations of ethnic medicine and will be helpful to accelerate the speed of new drug discovery. 5.4. Genomics approach Completion of human genome project and role of genomic and proteomics have revolutionized natural products based drug discovery. Several business collaborations are taking place around the world for genetic targets of small-molecule drugs for new discovery leads. This strategy is primarily coming from the pressing need to increase productivity and success rate of new drug discovery. Molecular markers identify the plant at genomic level and establish new standards in standardization and quality control of botanicals. In order to get better quality of herbal drugs generating breeds for disease resistance plant is inevitable, for which marker assisted selection (MAS) is employed. To authenticate the plant species and their adulterant technique like Sequence Characterized Amplified Region (SCAR) analysis was employed (65). Molecular techniques are more superior in sensitivity as well as specificity than conventional techniques (66). Molecular markers such as Random Amplified Polymorphic DNAs (RAPDs) also plays an

Ethno medicine in complementary therapeutics

important role in assessment of genetic variability and in strain identification (67) and differentiation from its related species. Ultra-HTS (uHTS) assays require an accurate and reliable means of fluid handling in the submicroliter volume range. This relates to the design of instrumentation for dispensing fluids, as well as assay plates. In a chemical genomic research approach, ultra-high throughput screening of genomic targets takes place early in the drug discovery process, before target validation to generate drug leads (68). As more genomes have been sequenced and gene functions elucidated, time has come to bring the valuable ethnic medicinal knowledge to the rapidly expanding genomic landscapes. It is envisioned that systematic identification and characterization of gene targets could lead to deeper appreciation of the chemo-diversity in ethnic medicinal plants. 5.5. In-silico approach Bioinformatics has gained popularity during the last few years to describe tools and techniques for storing, handling, and communicating the massive and ever increasing amounts of scientific (primarily biological) data. It is made possible by dramatic improvements in computational power and computer accessibility. Bioinformatics has become a major scientific discipline and applications in other fields are currently underway. It is essential that informatics technology be devoted to increasing our understanding of earthâ&#x20AC;&#x2122;s biodiversity, and to develop new tools for archiving global diversity. The need for dynamically updated databases to support informed conservation decision-making is becoming increasingly recognized (69). Traditional approaches to gathering and disseminating ethnobotanical information are clearly inadequate to deal with the global increase of ethnobotanical research data. Consequently, numerous electronic databases have recently been developed to disseminate information on plant uses. However, because these databases were designed and developed independently, they are often oriented toward particular user groups (e.g. students, researchers, or the general public). As a consequence, the information they contain is very variable in its content and quality, often only having a regional or cultural focus. Although the current ethnobotanical databases are of great value in promoting the awareness of the need to record and conduct ethnobotanical research, they are insufficient, especially for collaborative and comparative analysis. A coordinated global approach is necessary to currently manage the increasing amount of bio-cultural knowledge being documented by ethnobiologists worldwide. The growing number of independent ethnobotanical databases is itself a compelling argument for a unified coordinated data management system.

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The system’s primary goals would be to function as both a comprehensive digital library with a search engine for retrieving information on the present, past and future uses of plants, providing a “one-stop educational resource” for users. As new technologies for data retrieval should be developed, and the system could evolve into a meta-database – that is, a system that allows researchers to analyze comparative data pooled from multiple databases. The lack of a unified approach and standard data model for recording research data has led to a paucity of comparative ethnobotanical studies that not only examine different uses of the same type of plants in different cultures, but also compare the ways plants figure in different world views (70). Few papers published by leading scientific journals address comparative analysis of possible patterns of medicinal plant selection and use by humans across cultures, regions or hemispheres. Indeed the absence of readily accessible comparative sources of ethnobotanical data has been recognized as a serious hindrance (71). In fact, a dynamic resource would encourage a unified approach by facilitating a greater opportunity for comparative analysis of research data through direct contributions by members of the ethnobiological research community. 5.6. Reverse pharmacology approach The ethno-medicine is based on its use for many years and its clinical existence is presumed. For bringing more objectivity and to confirm ethnic claims, systematic clinical trials are necessary. In normal drug discovery course “laboratories to clinic” approach is followed, in herbal medicine research “clinics to laboratories” approach – a true reverse pharmacology approach is followed. In latter, clinical experiences, observations or available data becomes a starting point, where as with conventional drug research it comes at the end. Reverse pharmacology is the science of integrating documented clinical/experiential hits, into leads by transdisciplinary exploratory studies and further developing these into drug candidates by experimental and clinical research. In reverse pharmacology approach process safety remains the most important starting point and efficacy becomes a matter of validation. The scope of reverse pharmacology is to understand the mechanisms of action at multiple levels of biological organization and to optimize safety, efficacy and acceptability of the leads in natural products, based on relevant science. Thus the drug discovery based on Ayurveda or ethnic medicine should follow a ‘reverse pharmacology’ path (72).

Ethno medicine in complementary therapeutics

5.7. Systems biology approach Systems biology aims at understanding biological complexity by unbiased measurements of as many as possible parameters, without having any hypothesis. Such measurement can be of very different kind, on the level of the genome, transcriptome, proteome, metabolome as well as physiological parameters, such as blood pressure, pulse, pain, fever, weight, length, gender and age. By the use of suitable biostatistical methods such as multivariate analysis and principle component analysis these data can be analyzed, and for example correlations between certain parameters can be made. One of the key technologies in the systems biology is metabolomics. Metabolomics aims at qualitatively and quantitatively determining as many compounds as possible in an organism. This can be in extracts of tissues, but also in body fluids such as serum or urine in case of humans. Chromatographic methods in combination with mass spectrometry (MS), mass spectrometry with electron spray ionization (MS-ESI), and nuclear magnetic resonance spectrometry (NMR) etc are used for such analyses. By combining the results of such analyses with other parameters novel correlations can be found, for example a relation between the occurrence of certain compounds in extracts and a biological activity. Analysis of metabolites in urine by means of 1H NMR is already extensively applied for studying toxicity of drugs. Also for the quality control of botanicals the metabolomics approach is very promising tool. The said technique was applied to metabolic profiling by means of 1H NMR in the quality control of Ginkgo biloba pharmaceutical preparations. Beside a recognizable pattern the quantitative analysis of ginkgolides and bilobalide could be done with a 5 min acquisition time of the spectrum, without the need of any elaborate sample preparation. Also for other preparations such as Strychnos, Ephedra and Cannabis this method found to be suitable. Such studies are the first step on a long way to a better understanding of the activity of medicinal plants. Because of the many years of documented use, one may start immediately from clinical trials, in that way also shortening the whole processes of developing a novel drug (73). 5.8. Personalized approach Despite all the improvement in technology the number of novel drugs coming to the market is decreasing every year, because almost for all known targets drugs are available, and to find a better one which is still affordable for the patients is getting increasingly difficult. The costs are now upto 1 billion Euros for developing a novel drugs and the total duration is 12 â&#x20AC;&#x201C; 15

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years. That means only for major diseases it is worth while to develop novel drugs. Diseases of the developing countries, such as malaria, TB are consequently not the targets for drug development by big pharmaceutical companies. In fact, the approach of â&#x20AC;&#x153;single target single compoundâ&#x20AC;? does not work anymore. Novel approaches should be thought of. Our ancestors did not discover active plants, but they also developed a holistic approach in their medical systems, for example, well-known Ayurvedic medicine, Traditional Chinese medicine, Siddha medicine etc each have principles in treating the disease. This is among others reflected in a tailor-made prescription that is made for each individual patient after an extensive diagnosis, a concept that now is also considered to be of the interest for the pharmaceutical industry. Pharmacogenomics and pharmacogenetics research, will in the coming years lead to a more individualized pharmacotherapy. The holistic traditional approach requires also a holistic way of studying it. The reductionist approach of the modern drug development will not be able to detect activity in case of the presence of several (may be weak) active compounds, synergy between compounds and pro-drugs. The reductionist approach is so far used to try to proof activity, i.e., studying the activity of known targets such as receptor binding assays. This approach failed for example in case of St Johnâ&#x20AC;&#x2122;s wort. Despite positive results in clinical trials, no single active compound has been found that can explain the proven clinical activity. That pro-drugs exist is best proven by one of the most successful drug salicylate. Based on the use of Salix bark as an analgesic preparation, this compound was developed at the end of the 19th century. However, in Salix bark there is no salicylate, instead there is a glucoside of salicylic alcohol that first needs to be hydrolyzed and then oxidized before the active compound is formed. This all happens in our bodies, a clear example of a pro-drug that never will be found be the reductionist high throughput screening methods. Only in vivo systems will be able to detect such activities. Until some 30 years ago in vivo pharmacology was the most important tool for drug development, and all compounds such as morphine, atropine, salicylate, and reserpine were found through such in vivo pharmacology. In fact our ancestors probably developed drugs only by testing plants on themselves; the reductionist trend in drug development is only of the past century. So, go back to a more holistic personalized approach in drug development, particularly to come to evidence-based medicinal plants (73).

6. Conclusion In many part of the developing countries, with ethnic groups and informal settlements, ethnic healers are the important source(s) for the treatment of

Ethno medicine in complementary therapeutics

various diseases, such as cut wounds, skin infection, swelling, aging, mental illness, asthma, diabetes, jaundice, scabies, eczema, snake bite, gastric ulcer etc,. They provide instructions to local people on how to prepare medicine from herbal or they prepare and give it to the patients. There are no records and the information is mainly passed on verbally from generation to generation. The local knowledge on various medicinal plants, ethnomedicinal preparations are useful resources, which may be scientifically evaluated and disseminated for efficacious drug development and improved health status. Thus traditional knowledge from various part of the world provides a good source of drug discovery for the future. Future directions for traditional herbal medicines may have several paths including: • • • • •

Development of human safety and activity data based on pharmacoepidemiology approach and its documentation. Reverse pharmacology path for rapid R & D for natural drugs Advanced multicentric and trans-disciplinary network of drug discovery from ethnic medicine Making standardized herbal extracts to therapeutic equivalence acceptance Develop active plant principles to form basis for combinatorial chemistry and high throughput screening

Evaluation of traditional medicines clinically is difficult, in addition if herbs are prepared traditionally, selecting placebo for comparison will be problematic, and herbal formulation usually take longer to work than conventional medicines or drugs. At the same time, a single medicinal plant can contain hundreds of natural constituents and they may have synergistic effect. Establishing which constituent is responsible for effect be time consuming and expensive. Although pre-clinical and clinical studies of botanicals are unique in many respects, they should be carried out and evaluated the same basic criteria applied to other types of investigations related to any drug development and should take into considerations of international initiatives and existing guidelines. Challenges in clinical research of botanicals are multiple and vary according to the type of the product (i.e single vs combination) and the history of use (i.e., new vs traditional). Yet given the worldwide popularity of herbal medicines, a widely applicable, appropriate and effective means of evaluating herbal medicines with limited resources is urgently needed. Considering the synergistic, antagonistic and interacting activities of natural health products particularly botanicals the need for adequate research and development of these products has a strong impact on the research community, the

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pharmaceutical industry and the consumers. Reliable standardization and manufacturing processes, analytical methods, pharmacological test systems and well-designed clinical studies are essential for cost-effective development of botanicals and should follow international initiatives and existing guidelines.

7. Acknowledgement Authors wish to thank All India Council for Technical Education (AICTE), New Delhi for providing financial assistance through RPS scheme to School of Natural Product Studies, Jadavpur University, Kolkata and providing QIP - Fellowship to Sri S Ponnusankar

8. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19.

Mukherjee, P. K. 2002, Quality control of herbal drugs â&#x20AC;&#x201C; an approach to evaluation of botanicals, Business Horizons, New Delhi. Jones, E. T., Mc Lain, R, J., and Weigand, J. 2002, Nontimber forest products in the United States, University Press of Kansas, USA. Romulo, R. N., and Ierece, M. L. 2007, J. Ethnobiol. Ethnomed., 3, 14. Lambert, J., Srivastava, J., and Vietmeyer, N. 1997, Medicinal plants â&#x20AC;&#x201C; rescuing a global heritage, World Bank technical paper 355, Washington DC, USA. Penso, G. 1980, WHO inventory of medicine plants used in different countries, WHO, Geneva, Switzerland. Sandhya Wakdikar, S. 2004, Electronic J. Biotechnology, 7, 217-223. Kamboj, V. P. 2000, Curr. Sci., 78, 35. Balunas, M.J., and Kinghom, A.D. 2005, Life. Sci., 78, 431. King, S. 1992, Pac. Discovery., 45, 23. Patwardhan, B., Ashok, D.B., and Chorghade, M. 2004, Curr. Sci., 86, 789. Mukherjee, P. K., Rai, S., Mukherjee, K., Hylands, P. J., and Hider, R. C. 2007, Expert. Opin. Drug. Discov., 2, 633. Mukherjee, P. K., 2003, Clin. Res. Regul. Aff., 20, 249. Mukherjee, P. K., Sahu, M., and Suresh, B., 1998, The Eastern Pharmacist, 42, 21. Gold, J. L., Laxer, D. A., and Rochon, P. A. 2000, Ann. R. Coll. Physicians. Surg. Can., 33, 497. Mukherjee, P. K., 2001, Drug. Inf. J., 35, 623. Mukherjee, P. K. 2005, Promotion and development of botanicals with international coordination: exploring quality, safety, efficacy and regulations, Allied Book Agency., Kolkata, India. Mukherjee, P. K., and Wahile, A. 2006, J. Ethnopharmacol., 103, 25. Bombardelli, E., Curri, S, B., Della Loggia, R., et al. 1989, Fitoterapia., 60, 1. Della Loggia, R., Sosa, S., Tubaro, A, Morazzoni, P., Bombardelli, E., and Griffini, A. 1996, Fitoterapia., 67, 257.

Ethno medicine in complementary therapeutics

20. Arpaia, G., Bombardelli, E, Curri, S. B., Della Loggia, R. 1989, Fitoterapia., 60 (Suppl 1), 11. 21. Bombardelli, E., and Spelta, M. 1991, Cosmet. Toiletries., 106, 69. 22. Bombardelli, E., and Spelta, M., Della Loggia, R., Sosa, S., Tubaro, A. 1991, Fitoterapia., 62, 115. 23. Bombardelli, E., Cristoni, A., Morazzoni, P. 1994, Fitoterapia, 65, 387. 24. Maiti, K., Mukherjee, K., Gantait, A., Saha, B. P., Mukherjee, P. K. 2007, Int. J. Pharm., 330, 155. 25. Maiti, K., Mukherjee, K., Gantait, A., Saha, B. P., Mukherjee, P. K. 2006, J. Pharm. Pharmacol., 58, 1227. 26. Maiti, K., Mukherjee, K., Gantait, A., Ahamed, K. H. N., Saha, B. P., Mukherjee, P. K. 2005, Iranian. J. Pharmacol. Ther., 4, 84. 27. Kinghorn, A. D. 2001, J. Pharm. Pharmacol., 53, 135. 28. Samuelsson, G. 2004, Drugs of Natural origin: a textbook of Pharmacognosy, Swedish Pharmaceutical Press, Stockholm. 29. Newman, D, J., Cragg, G. M., Snader, K. M. 2000, Nat. Prod. Rep., 17, 215. 30. Butler, M. S. 2004, J. Nat. Prod., 67, 2121. 31. Graul, A. I. 2001, Drug News Perspect., 14, 12. 32. Heinrich, M., Teoh, H. L. 2004, J. Ethnopharmacol., 92, 147. 33. Pirttila, T., Wilcock, G., Truyen, L., Damaraju, C. V. 2004, Eur. J. Neurol., 11, 734. 34. Frantz, S., and Smith, A. 2003, Nat. Rev. Drug. Discov., 2, 95. 35. Frantz, S. 2005, Nat. Rev. Drug. Discov., 4, 95. 36. Mundy, c., Kirkpatrick, P. 2004, Nat. Rev. Drug. Discov., 3, 643. 37. Butler, M. S. 2004, J. Nat. Prod., 67, 2141. 38. Lotsch, J., Geisslinger, G. 2001, Clin. Pharmacokinet., 40, 485. 39. Okouneva, T., Hill, B. T., Wilson, L., Jordan, M. A. 2003, Mol. Cancer. Ther., 2, 427. 40. Cragg, G. M., Newman, D. J. 2004, J. Nat. Prod., 67, 232. 41. Yang, S. S., Cragg, G. M., Newman, D. J., Bader, J. P. 2001, J. Nat. Prod., 64, 265. 42. Yu, D., Suzuki, M., Xie, L., Morris- Natschke, S. L., Lee, K. H. 2003, Med. Res. Rev., 23, 322. 43. Creagh, T., Ruckle, J. L., Tolbert, D. T., Giltner, J., Eiznhamer, D. A., Dutta, B., Flavin, M.T., Xu, Z. Q. 2001, Antimicrob. Agents. Chemother., 45, 1379. 44. Gordaliza, M., Garcia, P. A., del Corral, J. M., Castro, M. A., Goomez-Zurita, M. A. 2004, Toxicon., 44, 441. 45. Horwitz, S. B. 2004, J. Nat. Prod., 67, 136. 46. Pisha, E., Chai, H., Lee, I. S., Chagwedera, T. E., Farnsworth, N. R., Cordell, G. A., Beecher, C. W., Fong, H. S., Kinghorn, A. D., Brown, D. M., Wani, M. C., wall, M. E., Hieken, t. J., Das Gupta, T. K., Pezzuto, J. M. 1995, Nat. Med., 1, 1046. 47. Silva, G. L, Cui, B., Chavez, D., You, M., Chai, H. B., Rasoanaiyo, P., Lynn, S. M., Oâ&#x20AC;&#x2122; Neill, M. J., Lewis, J. A., Besterman, J. M., Monks, A., Farnsworth, N. R., Cordell, G. A., Pezzuto, J. M., Kinglhorn, A. D. 2001, J. Nat. Prod., 64, 1514.

Pulok K. Mukherjee et al.

48. Hwang, B. Y., Su, B. N., Chai, H., Mi, q., Kardono, L. B., Afriastini, J. J., Riswan, S., Santarsiero, B. D., Mesecar, A. D., Wild, R., Fairchild, C. R., Vite, G. D., Rose, W. C., Farnsworth, N. R., Cordell, G. A., Pezzuto, J. M., Swanson, S. M., Kinghorn, A. D. 2004, J. Org. Chem., 69, 3350. 49. Ley, S. V., and Baxendale, I. R. 2002, Nat. Rev. Drug. Discov., 1, 573. 50. Geysen, H. M., Schoenen, F., Wagner, R. 2003, Nat. Rev. Drug. Discov., 2, 222. 51. Lombardino, J. G., Lowe III, J. A. 2004, Nat. Rev. Drug. Discov, 3, 853. 52. Clardy, J., Walsh, C. 2004, Nature., 432, 829. 53. Peterson, E. A., and Overman., L. E. 2004, Proc. Natl. Acad. Sci. USA., 101, 11943. 54. Koehn, F. E., and Carter, G. T. 2005, Nat. Rev. Drug. Discov., 4, 206. 55. Lee, M. L., and Schneider, G. 2001, J. Comb. Chem., 3, 284. 56. Feher, M., and Schmidt, J. M. 2003, J. Chem. Inf. Comput. Sci., 43, 218. 57. Piggott, A. M., and Karuso, P. 2004, Comb. Chem. High. Throughput. Scr., 7, 607. 58. Burke, M. D., Berger, E. M., Schreiber, S. L. 2004, J. Am. Chem. Soc., 126, 14095. 59. Ganesan, A. 2004, Curr. Opin. Biotech., 15, 584. 60. Tan, D. S. 2004, Comb. Chem. High. Throughput. Scr., 7, 631. 61. Mattijis, K. J. et al. 2006, Biomol. Eng., 23, 265. 62. Oka, H., Yamamoto, S., Kuroki, T., et al. 1995, Cancer., 76, 743. 63. Fan, X. H., Cheng, Y. Y. 2004, Chem. J. Chinese. U., 25, 2004. 64. Wang, J., Ha, w. Y., Ngan, F. N., et al. 2001, Planta Med., 67, 781. 65. Techen, N., Khan, I. A., Pan, Z. 2006, Planta Med., 72, 241. 66. Kumar, S., Prasad, K. V., Choudhary, M. L. 2006, Curr. Sci., 90, 1108. 67. Croston. G. E. 2002, Trends. Biotechnol., 20, 110. 68. Smith, T., Biotani, L., Bibby, c., Brackett, D., Corsi, F., da Fonesca, G. A. B., Gascon, C., Dixon, M. G., Hilton-Taylor, c., Mace, G., Mittermier, R. A., Rabinovich, J., Richardson, B. J., Rylands, A., Stein, B., Stuart, S., Thomsen, J., Wilson, C. 2000, Science, 289, 2073. 69. Balick, M. J. and Cox, P. A. 1996, Plants, People, and Culture: The Science of Ethnobotany, Scientific American, New York. 70. Thomas, M. B., Lin, N., and Beck, H.W. 2001, Pharm. Biol., 39 Supplement, 41. 71. Vaidya, A. D. B., Vaidya, R. A., and Nagaral, S. I. 2001, J. Assoc. Physicians. India., 49, 534. 72. Verpoorte, R., Kim, H. K., Choi, Y. H. 2005, International conference on promotion and development of botanicals with International coordination: exploring quality, safety, efficacy and regulations, P. K. Mukherjee (Ed.), Allied Book Agency, Kolkata, 10.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 53-116 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

3. Ethnomedicinal plants in parasitic infections Varughese George, Sabulal Baby and Anil John J Phytochemistry and Phytopharmacology Division, Tropical Botanic Garden and Research Institute Pacha-Palode, Thiruvananthapuram - 695 562, Kerala, India

Abstract. Parasitic infections are common in the tropical regions of Africa, Asia and Latin America and relatively less common in other countries. Parasites that infect humans are various protozoa and helminths. Parasitic diseases disable their hosts and render them incapable of leading normal lives. In certain cases they cause mortality of the affected human hosts. The drugs currently available for parasitic infections are effective in many cases. But they also have limitations such as toxic side effects, high costs and the resistance developed by the parasitic organisms against these drugs. Bioactive plant metabolites may offer cheap, cost effective and easily affordable drugs against parasitic infections. Further, indigenous knowledge systems are valuable resources for the selection of plants for drug prospecting. In this article an attempt is made to review recent advances in the use of ethnomedicinal plants against parasitic infections such as malaria, leishmaniasis, giardiasis, amoebiasis, trypanosomiasis and helminthiasis. Correspondence/Reprint request: Dr. Varughese George, Amity Institute of Phytochemistry and Phytomedicine, 3-Ravi Nagar, Ambalamukku, Peroorkada P.O., Thiruvananthapuram-695005, Kerala, India E-mail: georgedrv@yahoo.co.in, sabulal@gmail.com

Varughese George et al.

Introduction There are a number of parasitic infections that affect lives of millions of people in the tropical countries of Asia, Africa and Latin America. These diseases disable the affected population rendering them incapable of attending to their normal livelihood activities causing tremendous economic hardship to the families, societies and governments. Parasitic infections cause major public health problems leading to morbidity and sometimes mortality of the victims. Traditional societies use a number of medicinal plants to treat parasitic infections. These plants being locally available, the therapeutic preparations from these plants are cheap, cost effective and easily affordable. Moreover, they conform to the social and ethnic ethos of the society. Traditional wisdom or what is usually called indigenous knowledge is thus an invaluable resource base on which one can confidently rely upon in the selection of plants for drug prospecting. This strategy has definitely helped researchers in discovering a number of useful therapeutic agents. It has been pointed out that 28% of all new chemical entities launched on to the market between 1981-2002 had their origin from natural products (1). Further, 24% of the new chemical entities introduced into the market during this period were synthetic or natural mimic compounds originating from pharmacophores related to natural products (2). Thus natural products are important sources for new drugs and are excellent lead candidates for synthetic modification during drug development. Since secondary metabolites from natural sources have been elaborated with in living systems they are often perceived as showing more druglikeness and biological friendliness than totally synthetic molecules (3), making them good candidates for further drug development. A good number of exhaustive reviews are available in literature on the antiprotozoal and antihelminthic activities of medicinal plants. Hence, herein we focus on some selected studies on the antiprotozoal activities of ethnomedicinal plants used by various cultures to combat certain protozoa caused diseases particularly malaria, leishmaniasis, giardiasis, amoebiasis and trypanosomiasis. The role of medicinal plants in combating human parasitic nematodes is also briefly reviewed.

Malaria Malaria is among the most widespread parasitic infections causing serious public health problems in developing countries situated in the tropical belts of Asia, Africa and South America. The disease is mainly caused by the parasite Plasmodium falciparum that is transmitted by the malaria vector mosquito Anophelus gambiae. Other malaria causing parasites are P. vivax,

Ethnomedicinal plants in parasitic infections

P. malariae and P. ovale. More than a third of the worldâ&#x20AC;&#x2122;s population live in malaria affected areas and one billion people are estimated to carry parasites at any one time. It is estimated that malaria is directly responsible for the deaths of 1-2 million people each year (4). Common characteristic symptoms of malaria are alternating episodes of fever and asymptomatic phases associated with symptoms like chills, headache, myalgia, joint pains, sweating and anaemia. Rigors are common and splenomegaly is a frequent consequence (5). The majority of malaria deaths are due to cerebral malaria and other complications following infection with P. falciparum that is transmitted by the female mosquitoes of the genus Anophelus. Most of the deaths occurring in Africa are in children under the age of 5 years (6). Quinine, the first antimalarial drug, was isolated from Cinchona bark in 1820. In 1940 chloroquine was synthesized and it is the most widely used antimalarial drug. It works by joining with the ferriprotoporphyrin IX in the parasite thereby antagonising the polymerization of this toxic metabolite into inert crystals of haemozoin. Another synthetic antimalarial drug is mefloquine, the mechanism of action of which is similar to that of chloroquine. Primaquine is given as a follow up drug after treatment with chloroquine to eradicate the liver hypnozoites of P. vivax and P. ovale. Halofantrine, which also has a mechanism of action similar to that of chloroquine, is administered in cases of uncomplicated multiresistant P. falciparum malaria. Atovaquone is administered in synergistic combination with proguanil to treat uncomplicated P. falciparum malaria. Artemisinin and its derivatives are used for both uncomplicated and severe P. falciparum malaria (Fig. 1). The most commonly used derivatives of artemisinin are artemether, arteether and artesunate (7). Artemisinin, an endoperoxide sesquiterpene lactone, was isolated from the Chinese herb Artemisia annua in 1972 (8). The structure of artemisinin was established in 1979 (9). A. annua has been used in traditional Chinese medicine as a remedy for cold and fevers. It is now grown in many countries including India. Artemisinin group of drugs are characterized by quick reduction of fever, fast clearing of parasites in blood and no significant side effects. The endoperoxide group of artemisinin is essential for its activity (Fig. 1). When the malarial parasite infects a red blood cell, it consumes the hemoglobin and liberates the iron-porphyrin complex, heme. This heme complex causes the reductive activation of the endoperoxide bridge of artemisinin and generates high-valent iron-oxo species. These iron-oxo species trigger a sequence of reactions producing reactive oxygen radicals. These reactive radicals cause the death of the malarial parasite. More specific details of the structure activity relationship of artemisinin is still an area of active research (10-20).

Varughese George et al.

Figure 1

N H 3C

CH3O N

N Chloroquine

Quinine (1)

(2)

CH3

H2 N

NH N

CF3

CH3O

CF3

Primaquine

Mefloquine (3)

(4)

CF3 O

Cl N OH

OH Halofantrine (5)

O Atovaquone (6)

Ethnomedicinal plants in parasitic infections

CH3 H 3C

H N

CH3

NH O

Proguanil

Artemisinin

(7)

(8)

CH3

CH3 H 3C

H3C

O O

CH3

CH3 O

CH3

CH3 Artemether

Arteether

(9)

(10)

CH3 H 3C

CH3 O

HO O Artesunate (11) Figure 1. Structures of antimalarial drugs.

Varughese George et al.

Artemisinin drugs have a short half-life of 1-4 h. But, due to their strong antiplasmodial activity, they reduce the biomass of the existing parasites by 95% at each dosage of administration. It also kills the sexual stages of the malarial parasite. Residual parasites are eliminated by the host immunity. But, fake drugs are prevalent and this could result in development of artemisinin-resistant strains (21). For malaria, WHO recommends combinations of drugs containing artemisinin derivatives to overcome the resistance to conventional monotherapies. Examples of these therapeutic combinations are: (i) artemether, (ii) artesunate-maodiaqine, (iii) artesunate-sulphdoxinepyremethamine, (iv) artesunate-mefloquine and (v) amodiquine-sulphdoxinepyremethamine (22). Since conventional antimalarial drugs are rapidly loosing their effectiveness due to the resistance developed by the malarial parasites there is an increasing demand for developing new antimalarials (23). From very early times, plants have been the only weapon to combat malarial parasites. Hence, there is a strong conviction among researchers that plants may provide alternate, safe and effective remedies for the treatment of malaria. Recent efforts have resulted in the isolation and characterization of a number of antimalarial metabolites with diverse structural features from plants. A compilation of plant extracts and isolates which showed antimalarial properties is given in Table 1.

Leishmaniasis Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania and is transmitted by certain species of sand fly. Twelve million people in at least 88 countries are infected by these parasites annually. Approx. 350 million people are living in leishmaniasis-affected areas (115). About 1.5 million new cases of cutaneous leishmaniasis are occurring annually throughout the world (115). The number of new cases of visceral leishmaniasis annually is approx. 5,00,000 (116). Leishmaniasis is found from the rain forests in Central and South America to the deserts in West Asia (117). Leishmania spp. have a single free flagellum, rod-shaped kinetoplasts, a single nucleus, mitochondrion and rough endoplasmatic reticulum. The size of the parasite varies not only between species but also between amastigote and promastigote forms. The disease is transmitted by female sand flies of Phlebotomus spp. or the Lutzomyia spp. The sand fly takes up the pathogenic Leishmania spp. from infected hosts such as horses, dogs, sloths, rats etc. (118). In the infected sand fly, the amastigotes migrate to the alimentary canal where they mature and differentiate into motile prom astigotes. Then, they transit away from the midgut region to the

Table 1. Antimalarial extracts and compounds from medicinal plants.

Ethnomedicinal plants in parasitic infections 59

Table 1. Continued

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Table 1. Continued

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pharynx or the proboscis of the sand fly. While feeding, the sand fly injects the promastigotes into the blood stream of the victim and there they will be phagocytized (119). The parasite is equipped to evade the digestive enzymes present in the vacuole. Leishmania spp. have a membrane bound molecule known as the lipophosphoglycan and it helps intracellular survival (120, 121). Once inside the macrophage, the parasite resides in the phagolysosomal or the parasitophorous vacuole. Here, it transforms back into the infectious amastigote. Amastigotes replicate and are released back into the blood stream where similar cycles commence. Parasites are known to infect macrophages within the skin, viscera and blood tissues. Dissemination of the diseases is local or systemic depending on the Leishmania spp. involved (122). Three common clinical manifestations (cutaneous, mucocutaneous and visceral leishmaniasis) and two rare forms (recidua and post-kala azar dermal leishmaniasis) of the disease exist. Cutaneous leishmaniasis (oriental sore) is the most common form of leishmaniasis. It is a skin infection caused by the parasite that is transmitted by sand fly bites. The manifestation results in external lesions in the outer epidermal layers. The infection cures itself spontaneously in 4-6 months, except in the case of the incurable diffuse cutaneous leishmaniasis. There are about 20 species of Leishmania that may cause cutaneous leishmaniasis. The most common are L. mexican complex, L. tropica and L. major (123). Mucocutaneous leishmaniasis (espundia, chiclerâ&#x20AC;&#x2122;s ulcer) results in ulcers within the pharyngeal and nasal mucosa and also self cures within months. The extensive scarring and degradation of tissues never completely heal. It is most commonly found in the forest areas of central and south America and the organisms responsible for the diseases belong to the L. braziliensis complex (124). Visceral leishmaniasis (Kala-azar, black fever, black sickness) is the most severe form of leishmaniasis. It is the second-largest parasitic killer in the world after malaria. It is responsible for an estimated 60,000 deaths each year around the world out of half-million infections. The parasite migrates to the visceral organs such as liver, spleen and bone marrow. If left untreated will almost always result in the death of the mammalian host. Signs and symptoms include fever, weight loss, anemia and substantial swelling of the liver and spleen. L. chagasi and L. donovani are responsible for visceral leishamaniasis (125). Recidua leishmaniasis (chronic relapsing) is a cutaneous manifestation that is responsible for the presence of chronic lesions in the epidermis. L. tropica is most often associated with this disease. Post-Kala-azar dermal leishmaniasis, another rare form of the disease, is often encountered after a successful recovery from visceral leishmaniasis (126).

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Figure 2. Structures of antileishmanic drugs.

The most common drugs recommended for the treatment of leishmaniasis are the pentavalent antimonials sodium stibogluconate and meglumine antimoniate. These antimonials are effective drugs, but the limitations are the requirement for prolonged parenteral administration up to 28 days, the variable efficacy against visceral and cutaneous leishmaniasis and the emergence of significant resistance. Other drugs for leishmaniasis are amphotericin B, pentamidine, miltefosine etc. (Fig. 2). Amphotericin B, a polyene antibiotic, is highly effective for the treatment of antimonial-resistant L. donovani visceral leishmaniasis. However, toxicity and the requirement of slow parenteral infusion extending over four hours are its limitations. The use of pentamidine is also limited by its toxicity. The most significant advance has been the effective oral treatment of visceral leishmaniasis with miltefosine, an alkylphosphocholine, originally developed as an anticancer drug (127). Miltefosine (Fig. 2) was originally developed for the treatment of cutaneous metastasis from mammary carcinomas

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(128, 129). But it has proved to be an effective treatment for human visceral leishmaniasis (130). It is potentially the first oral treatment of human leishmaniasis (131). The leishmaniacidal activities of miltefosine have been associated with perturbation of the alkyl-phospholipid metabolism and the biosynthesis of alkyl-anchored glycolipids and glycoproteins (132). Earlier studies have indicated that miltefosine is causing apoptosis-like death in all the forms of Leishmania parasite. Studies are now in progress to find the possible targets of miltefosine action. Verma and Dey, 2004 proved that L. donovani undergoes apoptosis-like cell death due to miltefosine treatment by a combination of several techniques, including propidium iodide and in situ TUNEL staining, DNA condensation and fragmentation assay (133). Further, Verma et al., 2007 studied the effect of miltefosine in arsenite-resistant L. donovani (Ld-As20) promastigotes (134). These studies showed that miltefosine induces programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Thus, this study also showed apoptosis-like death in arsenite-resistant L. donovani by miltefosine. Since miltefosine has been in clinical use in recent years as an antileishmanial therapy, a better understanding of the mechanism that regulate cell death may help us to design new therapeutic strategies against Leishmania parasites. Further, several structure-activity correlation studies on currently used antileishmanic drugs are in progress. These drugs currently available for treatment of leishmaniasis have limitations due to several factors such as toxicity, route of administration, expense and due to resistance developed by Leishmania spp. Hence there is a need to develop new effective drugs with reduced toxicity and affordable prices. Plants traditionally used by different cultures may provide cheap and effective alternatives to the currently used drugs. A selected compilation of the progress achieved in the search for antileishmanic extracts and compounds from medicinal plants is presented in Table 2. Rocha et al, 2005 recently reviewed studies on natural products with antileishmanial activity (173). This excellent article lists 101 plants, their geographical distribution, plant parts utilized, type of extracts and compounds and the organisms tested. Rocha et al. covers 288 compounds isolated from higher plants and microorganisms classified into appropriate chemical groups such as alkaloids, flavonoids, terpenes, steroids, lactones, quinines, iridoids, lignans etc. These recent studies underline the scope for further investigation on ethnomedically used antileishmanic plants for the discovery of safe and effective drugs.

Table 2. Antileishmanic extracts and compounds from medicinal plants.

74 Varughese George et al.

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Trypanosomiasis Trypanosomiasis is a group of diseases in vertebrates caused by parasitic protozoan trypanosomes of the genus Trypanosoma. Trypanosomes are a group of kinetoplastid protozoa, distinguished by having only a single flagellum. They are classified under the subkingdom of Protozoa, phylum Sarcomastigophora, order Kinetoplastida and family Trypanosomatidae. Common features of trypanosomatids are a flagellum and a kinetoplast, a small organelle consisting of a condensed network of circular DNA. All members of the Trypanosomatidae family are parasitic with a very diverse range of hosts including vertebrates, invertebrates, plants etc. (174). African trypanosomiasis is also known as sleeping sickness (175). The disease was largely controlled in the 1960s. But it reemerged in the 1980s and today about 60 million people are exposed to it (176). There are two forms of African trypanosomiasis caused by two morphologically identical parasites. Trypanosoma brucei gambiense causes primarily a human chronic disease and is endemic to west and central African countries. T. b. rhodesiense has a huge animal reservoir and is primarily zoonotic. It causes acute illness in people in eastern and southern African countries (177). T. b. gambiense and T. b. rhodesiense are morphologically similar (178). Sleeping sickness is transmitted by tsetse fly of the genus Glossina. The most common vectors are Glossina morsitans, G. pallidipes, G. fuscipes and G. palpalis. The infected tsetse fly carries the metacyclic trypomastigote of T. b. gambiense or T. b. rhodesiense in their salivary gland. Infection occurs when the tsetse fly bites an individual. The metacyclic trypomastigotes rapidly transform into bloodstream trypomastigotes within the extracellular spaces in the subcutaneous tissue. The trypomastigotes eventually find their way into the bloodstream and the lymphatics, where they continue the replication cycle. Wild animals and cattle are important reservoir hosts for T. b. rhodesiense. For T. b. gambiense the main reservoir is humans (179). Initially the trypanosomes are present extracellularly in the subcutaneous tissue at the site of the bite of the tsetse fly and give rise to papular and later ulcerating lesion, often called a chancre. In the first stage trypanosomes enter the bloodstream and multiply there. This stage is accompanied by fever and lymphoid hyperplasia leading to enlargement of the spleen and especially of the cervical lymph nodes. The second stage involves central nervous invasion associated with intermittent fever. Trypanosomes in the cerebrospinal fluid produce diffuse meningoencephalitis. The central nervous system lesions are accompanied by headache, apathy, wasting of musculature, tremors, inability to walk and eventually to somnolescence, paralysis, coma and death, usually after a course of 1-3 years (180).

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Figure 3. Structures of trypanosomiasis drugs.

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The drugs currently used for the treatment of sleeping sickness are suramin, pentamidine, melarsoprol, eflornithine, nifurtimox etc. (Fig. 3) (181). Suramin, a polysulfonated naphthylurea, was introduced in the early 1920s and it remains the drug of choice for treatment of the early phase of T. b. rhodesiense infections (182). It is a medicinal drug developed by Oskar Dressel and Richard Kothe of Bayer in 1916 (183). It is also under investigation as treatment for prostate cancer. Suramin is used to treat the first stage of infection of human African trypanosomiasis, when the parasites are largely found in the blood. But, it is considered ineffective once the parasite has invaded the central nervous system. This is due to the inability of suramin to cross the blood-brain barrier and the bloodcerebrospinal fluid barrier in sufficient quantities to reach active concentrations in the target tissues (184). But, there is evidence that suramin can reach specific parts of the CNS, as it successfully cleared Trypanosoma brucei rhodesiense from the cerebral cortex of infected mice (185). Suramin is thought to slowly enter the parasite by receptor-mediated endocytosis, which is possibly linked to host low-density lipoprotein endocytosis. Further, LDL receptors are expressed at the blood-brain barrier and are believed to be involved in the transcytosis of LDL from the blood to the brain (184). Coadministration of suramin with drugs that are active against the CNS stage of the parasite such as melarsoprol, nifurtimox, and eflornithine, has been shown to improve cure rates (184). Pentamidine was first introduced in 1949. The drug is only used as the second line drug when therapy with suramin is contraindicated (182). The modes of action of these drugs are not well understood (186). Melarsoprol was introduced in 1949 for the treatment of late-stage sleeping sickness. But, it causes serious side effects such as reactive encephalopathy in 5-10 % of the cases. It also causes vomiting, abdominal colic and peripheral neuropathy. The mechanism of action of melarsoprol could be the combination of trypanothione depletion and the inhibition of trypanothione reductase (187). Eflornithine is the drug of choice for treatment of late-stage sleeping sickness caused by T. b. gambiense. The drug is not recommended for T. b. rhodesiense infections. The inhibition of polyamine biosynthesis by eflornithine triggers a range of downstream biochemical effects causing the trypanocidal effect (188). Nifurtimox is another drug registered for the treatment of sleeping sickness. Side effects are common for nifurtimox and 50% of patients are unable to complete its full course of treatment. But, it has been used in the treatment of late-stage sleeping sickness where eflornithine or melarsoprol are ineffective (189). American trypanosomiasis (chagas disease) is caused by Trypanosoma cruzi and is a major public health problem in Latin America (190). The disease is

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a zoonosis, which afflicts a variety of small mammals. The parasite is transmitted among its hosts by hematophagus reduvild bugs. Human disease occurs, when the bugs establish a habitat in human dwellings (191). Currently, there are 18-20 million people infected and another 40 million people are at risk of acquiring the disease (192). T. cruzi differs from other trypanosomes in that it has an intracellular amastigote stage in cardiac muscle and other tissues as well as trypomastigote forms in the circulating blood (193). The trypomastigotes are approx. 20 Âľm in length. The nucleus is generally centrally positioned and the large oval kinetoplast is located posteriorly. In stained blood films they characteristically assume a C or U shape. The infective stage of T. cruzi is the metacyclic trypomastigote. It is 15 Âľm in length and possesses a single nucleus and flagellum. The disease is transmitted by reduvild bugs of Rhidnius spp. or Triatoma spp. etc. (194). Infection occurs shortly after an infected bug bites an individual. Its feces contain the infective trypomastigotes. The host experiences a mild itching sensation and rubs the trypomastigotes into the bite wound. Trypomastigotes enter a wide variety of cells and transform into amastigotes. The amastigote is 3-5 Âľm in diameter and does not possess an external flagellum. The host becomes hypersensitive to the parasite as the result of the cellular destruction at the site of initial infection. Some amastigotes transform into trypomastigotes and after being released into the peripheral blood, they infect other sites in the body. The bug becomes infected, when it takes a blood meal from an individual harbouring trypomastigotes. Trypomastigotes transform into epimastigotes within the midgut of the bug. Epimastigotes differentiate into metacyclic trypomastigotes within the hindgut. This is the infective stage of the parasite (195). The entry of T. cruzi into human body is accompanied by inflammation at the point of entry. If this occurs in the eye there may be conjunctivitis, unilateral palpebral oedema and satellite adenopathy. Manifestations of generalised infection occur with fever, tachycardia, lymphadenopathy and oedema. The acute congenital phase may be symptomless or may be associated with jaundice, skin haemorrhages and neurological signs. After 2-4 months the acute clinical manifestation disappears and the disease enters the chronic phase, generally starting with a long period of clinical latency, which lasts 10-30 years or throughout life. After this period many infected patients present manifestations related to the involvement of certain organs such as heart, oesophagus, colon and nervous system. Heart involvement is the major aspect of chagas disease because of its characteristics, frequency and consequences, and is also the source of most controversies. About 20-30 % of the total chagasic population in endemic areas has symptomless heart disease and these patients may live for many years. Heart disease worsens in some of them, with increasing arrythmias or heart failure (196). The drugs used for the treatment of American trypanosomiasis are nifurtimox and benznidazole (197).

Table 3. Antitrypanosomal compounds and extracts from medicinal plants.

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Since most of the drugs currently available for treatment of African trypanosomiasis and American trypanosomiasis are toxic, attempts are being made in laboratories around the world to discover new, safe and cost effective molecules from medicinal plants with an ethnomedical history. Results of a few selected studies are presented in Table 3. Studies, up to mid 1995, on plant metabolites active against T. cruzi has been reviewed by Sepulveda-Boza and Cassels (208). Fournet et al., 1994 reported trypanocidal activity of 43 plants collected from Bolivia against T. cruzi (211). In another review, Arias et al., 1995 covered studies on trypanocidal activity of plant secondary metabolites with moderate to high activity in in vivo and in vitro bioassays against Trypanosomia spp. (212). Giardiasis Giardiasis is considered the most common protozoal infection in humans. It occurs frequently in both developing and industrialized countries. Worldwide incidence is believed to range between 20-60 percent with 2-7 percent in developed nations. Giardia is a common human parasite which can cause significant morbidity. Giardiasis is caused by the protozoan parasite Giardia lamblia. G. lamblia was first described in 1681 by the Dutch microscopist Antonie van Leeuwenhoek who observed the protozoan in one of his own diarrheic stools (213). Giardia can exist in two distinct forms: the cyst and the trophozoite. Cysts are dormant forms responsible for the transmission of giardiasis (214). They are excreted from an infected host with the feces and are exceptionally hardy and capable of tolerating extremes of pH and temperature. Transmission to humans usually occurs through the ingestion of cysts in contaminated water or food or via direct fecal-oral contact. Ingestion of a sufficient number of cysts is required to cause infection. Signs and symptoms usually begin within 6-15 days of contact with the organism. Once ingested, cysts pass into the stomach, where they are exposed to gastric acid. The low pH in the stomach and pancreatic proteases found in the proximal small intestine promote rapid excystation within minutes of reaching the duodenum. Typically, each cyst gives rise to two trophozoites. Trophozoites are the vegetative form of giardia (214). They are able to colonize and rapidly replicate in the gastrointestinal tract as well as cause gastrointestinal symptoms. Symptoms of giardia infestation include abdominal pain, nausea, anorexia, diarrhoea, vomiting, flatulence, eructation and fatigue. Signs include weight loss, abdominal distension and tenderness, pale watery stools, malodorous flatulence and signs of malabsorption. Less common symptoms include low-grade fever, chills, headaches urticaria and polyarthritis. Mucous- and blood-tinged feces are rarely found. Symptoms usually range in

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severity from mild to extreme. A significant proportion of infected individuals are completely asymptomatic. In some individuals giardiasis is short-lasting and resolves spontaneously, whereas in others infection can be prolonged and debilitating (215). Giardiasis is managed by the antibiotics metronidazole, tinidazole, benzimidazole etc. (Fig. 4) (216). Of the nitroimidazoles, the mechanism of killing of Giardia by metronidazole has been the most thoroughly studied. Metronidazole utilizes the anaerobic metabolic pathways present in Giardia (216). The drug enters the trophozoite and once it is within the cell, electron transport protein ferredoxins from the parasite donate electrons to the nitro group of the drug (217). The drug becomes activated by reduction of this nitro group (218). A gradient favoring the intracellular transport of metronidazole is established by this reduction reaction. Reduced metronidazole serves as a terminal electron acceptor, which binds covalently to DNA macromolecules (219). This results in DNA damage in the form of loss of helical structure, impaired template function and strand breakage, with subsequent trophozoite death. In addition to this effect, metronidazole inhibits trophozoite respiration (220). The reductive activation of metronidazole may also lead to toxic radicals, which react with essential cellular components (217). Trophozoites within cysts may be less affected by nitroimidazoles, possibly because of poor penetration of drug through the cyst wall (221). Resistance to metronidazole has been induced in vitro. It correlates with decreased activity of parasite pyruvate:ferredoxin oxidoreductase, which is required for reductive activation of nitroimidazoles (222). Metronidazole is quickly and completely absorbed after oral administration and penetrates body tissues and secretions such as saliva, breast milk, semen and vaginal secretions. The drug is metabolized mainly in the liver and is excreted in the urine (223). In vitro assays for nitroimidazole drug susceptibility have been performed with G. lamblia since 1980. Using microscopic evaluation of parasite morphology and mobility, Jokipii and

Figure 4. Structures of giardiasis drugs.

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Jokipii, 1980 first demonstrated that metronidazole and tinidazole were effective (224). Subsequently, morphology, growth inhibition, [3H]thymidine incorporation, serum killing, vitaldye exclusion, inhibition of adherence, metabolic and colorimetric assays have been employed to measure the in vitro response of the drug to many therapeutic agents (225). However, as indicated by the variety of assays used, there is no standard for in vitro testing, making it difficult to compare results and apply in vitro findings to the clinical setting. Of the nitroimidazoles, tinidazole and metronidazole have consistently demonstrated the greatest in vitro activity (226-228). The disease is best managed by nutritional intervention and phytotherapy. Nutritional management is done by adding probiotics such as food items contaning Lactobacillus, fructooligosaccharides, dietary fiber, wheat germ etc. Reducing diary products contaning lactose may help to control giardiasis. Medicinal plants which can help to control giardiasis are garlic, Piper longum, berberin containing herbs etc. (229). Pippali rasayana, the Ayurvedic product, may also help in controlling giardiasis. Some flavonoid and tannin containing plants such as Origanum vulgate, Psidium guajava, Mangifera indica and Plantago major are also used for the control of diarrhoea. A review of plants used as antigiardial drugs are presented in Table 4. Amoebiasis Amoebiasis is the second leading cause of death from parasitic disease worldwide. WHO has defined amoebiasis as â&#x20AC;&#x2DC;a condition in which a patient is harbouring the organism Entamoeba histolytica in the bowelâ&#x20AC;&#x2122;. E. histolytica is ingested via water or food contaminated with human feces (233). The incubation period for amoebic dysentery is usually 1-4 weeks but may be shorter or substantially longer. About 90% of infected persons are asymptomatic. There are two basic types of amoebiasis: intestinal and extraintestinal disease, which may exist simultaneously. In symptomatic intestinal amoebiasis common signs and symptoms are fever, gradual onset of colicky abdominal pain, increased number of stools, jaundice, anorexia, weight loss, and tenesmus. Severe infections may have an acute onset and be characterized by severe abdominal pain, frequent and profuse bloody diarrhoea, more rapid weight loss and the potential for dehydration. Rarely, a form of chronic amoebic colonitis develops which mimics irritable bowel disease (234). The most common extraintestinal amoebiasis is hepatic amoebiasis (235). Symptoms of hepatic amoebiasis include a gradual or acute onset of fever, right upper quadrant pain, hepatomegaly and tenderness, nausea and vomiting, anorexia, weight loss and malaise. Intercostal tenderness is common. Prompt treatment is necessary to prevent the hepatic abscess from rupturing. Other extraintestinal infections include perianal skin

Table 4. Antigiardial extracts and compounds from medicinal plants.

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infections and rare metastatic infections to the brain, lungs and genitalia (236, 237). Untreated patients may develop megacolon, vomiting, high fever, dehydration and circulatory collapse. Perforation may occur leading to peritonitis, pericarditis and pleurisy (237). Microscopy is the most widespread method for diagnosis of amoebisis. In asymptomatic infections E. histolytica cyst is distinguished from the cysts of nonpathogenic intestinal protozoa (such as E. coli) in the stools. But, this is not a very accurate testing method. In symptomatic infections multiple stool examinations are done to detect the trophozoites. Detection of trophozoites containing digested red blood cells is diagnostic. In patients with dysentery the WBC count is increased but not in patients with mild colitis. Endoscopy is used when stools are negative. Another detection method, serology testing, is positive only in severe intestinal infections. Recently kits have been introduced to detect the presence of amoebic proteins and DNA in the feces (238). Development of new antiamoebic drugs is still in infancy and vaccine development appears to be distant dream. In future, the development of drug resistance may seriously affect the control of amoebiasis (239, 240). Antiamoebic drugs are classified into three groups: luminal, tissue, and mixed amoebicides. Metronidazole is the major drug of choice and other nitroimidazole derived compounds like tinidazole, secnidazole and ornidazole are equally effective. Diloxanide furoate, diiodohydroxyquin, paromomycin, emetine and chloroquine have also been used as alternate drugs. Metronidazole, tinidazole and other 5-nitroimidazole agents which kill the trophozoites by alterations in the protoplasmic organelles of the amoeba are ineffective in treating cyst passers. Chloroquine acts on the vegetative forms of the parasite and kills it by inhibiting DNA synthesis, emetine kills the trophozoites mainly by inhibiting protein synthesis. Dehydroemetine, emetine and metronidazole act on amoebas in the bowel wall but not in the lumen. Diloxanide furoate, idoquinol and paromomycin act on amoebas in the bowel lumen (Fig. 5). Tetracycline inhibits bacterial growth in both the bowel wall and lumen. Metronidazole effects both the bowel wall and lumen but if given alone has a 50% fail rate as it needs a luminal amoebicide to augment its action (237, 241). Prevention is essential and includes hand washing, proper food handling, and boiling questionable water to 55oC. Research on the antiamoebic effect of various plants in Africa has shown that swallowing fresh whole papaya seeds can prevent amoebiasis. Two tablespoons of fresh papaya seeds twice a week may reduce the incidence of amoebiasis (242, 243). A series of natural constituents with amoebicidal activity isolated from medicinal plants have been listed (244, 245). A review of plant species assessed in vitro for antiamoebic activity or both antiamoebic and antiplasmodial properties has been published (246). Table 5 lists amoebicidal properties of extracts and pure compounds from medicinal plants.

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Figure 5. Structures of antiamoebic drugs.

Helminthiasis The common nematodes infesting humans are hookworm, pinworm, threadworm, whipworm and tapeworm. Anthelmintics are drugs that expel parasitic worms (helminths) from the body by either killing or stunning them. A traditional remedy of this type is often called a vermifuge or vermicide. Chemical control of helminths coupled with improved management has been an important worm-control strategy throughout the world. Increasing problems due to the development of drug resistance have led to the screening of medicinal plants for anthelmintic activity (249). Several medicinal plants have been in use

Table 5. Amoebicidal extracts and compounds from medicinal plants.

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Table 5. Continued

100

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102

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103

104

Figure 6

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Figure 6

105

106

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Figure 6. Chemical structures of selected antiparasitic natural products.

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to control intestinal worms in traditional and alternative medicines. Some of the common plants used by different cultures for control and elimination of intestinal worms are Rhicinous communis (castor oil), black walnut, Artemisia absynthium (wormwood), Artemisia vulgaris (wormwood, santonin), Syzygium aromaticum (clove), Tanacetum vulgare (tansy tea), Hagenia abyssinica (Hagenia), Nigella sativa (kalonji seeds), Dryopteris filix-mas (the male fern), Plumeria acutifolia (Plumeria) and P. rubra (250). Although these plants are antihelmintic if not used in correct dose, they can be poisonous as well as dangerous to humans.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Newmann DJ, Cragg GM, Snader KM (2003). J. Nat. Prod. 66: 1022-1037. Newmann DJ, Cragg GM, Snader KM (2000). Nat. Prod. Rep. 17: 215-234. Koehn FE, Carter GT (2005). Nat. Rev. Drug. Discov. 4: 206-220. Bradley DJ (1995). The epidemiology of malaria in the tropics and in travelers, In: G Pasvol (ed), Malaria, pp. 211-226. London: Bailliere Tindall. Senn MW (2006). Structure and evaluation of biologically active constituents of Cussonia zimmerannii Harms. Ph. D. Thesis, University of Basel. Winstanley PA (2000). Parasitol. Today 16: 146-153. Rosenthal PJ (2003). J. Exp. Biol. 206: 3735-3744. Woerdenbag HJ, Lugt C B, Pras N (1990). Pharm. Weekbl. Sci. 12: 169-181. Anonymous (1979). Chin. Med. J. 92: 811-816. Avery MA, Gao F, Chong WK, Mehrotra S, Milhous WK (1993). J. Med. Chem. 36: 4264-4275. Posner GH, McGarvey DJ, Oh CH, Kumar N, Meshnick SR, Asawamahasadka W (1995). J. Med. Chem. 38: 607-612. Avery MA, Bonk JD, Chong WK, Mehrotra S, Miller R, Milhous W, Goins DK, Venkatesan S, Wyandt C, Khan I (1995). J. Med. Chem. 38: 5038-5044. Avery MA, Fan P, Karle JM, Bonk JD, Miller R, Goins DK (1996). J. Med. Chem. 39: 1885-1897. Avery, MA, Mehrotra, S, Bonk, JD, Vroman, JA, Goins, DK, Miller R (1996). J. Med. Chem. 39: 2900-2906. Avery, MA, Mehrotra, S, Johnson, TL, Bonk, JD, Vroman, JA, Miller, R (1996). J. Med. Chem. 39: 4149-4155. Paitayatat S, Tarnchompoo B, Thebtaranonth Y, Yuthavong Y (1997). J. Med. Chem. 40: 633-638. Woolfrey JR, Avery MA, Doweyko AM (1998). J. Comput. Aided Mol. Des. 12: 165-181. Avery, MA, Alvim-Gaston, M, Rodrigues, CR, Barreiro, EJ, Cohen, FE, Sabnis, YA, Woolfrey, JR (2002). J. Med. Chem. 45: 292-303. Avery MA, Alvim-Gaston M, Vroman JA, Wu B, Ager A, Peters W, Robinson BL, Charman W (2002). J. Med. Chem. 45: 4321-4335. Avery MA, Muraleedharan KM, Desai PV, Bandyopadhyaya AK, Furtado MM, Tekwani BL. (2003) J. Med. Chem. 46: 4244-4258.

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21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

Varughese George et al.

Saxena S, Pant N, Jain DC, Bhakuni RS (2003). Curr. Sci. 85: 1314-1329. Olliaro PL, Taylor WRJ (2003). J. Exp. Biol. 206: 3753-3759. White NJ (2004). J. Clin. Invest. 113: 1084-1092. Perez HA, De La Rosa M, Apitz R (1994). Antimicrob. Agents Chemother. 38: 337-339. Campell WE, Nayar JJ, Gammom DW, Bastida J, Codina C, Viladomat F, Smith PJ, Albrecht CF (1998). Planta Med. 64: 91-93. Campell WE, Nair JJ, Gammon DW, Codina C, Bastida J, Viladomat F, Smith PJ, Albrecht CF (2000). Phytochemistry 53: 587-591. Likhitwitayawuid K, Angerhofer CK, Chai H, Pezzuto JM, Cordell, GA (1993). J. Nat. Prod. 56: 1331-1338. Ahmed MS, Gulal AM, Ross SA, Ferreira D, Elsohly MA, Ibrahim ARS, Mossa JS, El-Feraly FS (2001). Phytochemistry 58: 599-602. Horgen FD, Madulid DA, Angerhofer CK, Pezzuto JM, Soejarto DD, Farnsworth NR (1997). Phytomedicine 4: 353-361. Francois G, Bringmann G, Doehez C, Schneider C, Timperman G, Ake Assi L (1995). J. Ethnopharmacol. 46: 115-120. Bringmann G, Saeb W, God R, Schaffer M, Francois G, Peters K, Peters E-M, Proksch P, Hostettmann K, Ake Assi L (1998). Phytochemistry 49: 1667-1673. Francois G, Bringmann G, Phillipson JD, Ake Assi L, Doehez M, Rubenacker M, Schneider C, Wery M, Warhurst DC, Kirby GC (1994). Phytochemistry 35: 1461-1464. Bringmann G, Gunther C, Saeb W, Mies J, Brun R, Ake Assi L (2000). Phytochemistry 54: 337-346. Hallock YF, Cardellina JH, Schaffer M, Bringmann G, Francois G, Boyd MR (1998). Bioorg. Med. Chem. Lett. 8: 1729-1734. Hallock YF, Manfredi KP, Blunt JW, Cardellina JH, Schaffer M, Gulden KP, Bringmann G, Lee AY, Clardy J, Francois G, Boyd MR (1994). J. Org. Chem. 59: 6349-6355. Hallock YF, Cardellina JH, Schaffer M, Stahl M, Bringmann G, Francois G, Boyd MR (1997). Tetrahedron 53: 8121-8128. Bringmann G, Saeb W, Ake Assi L, Francois G, Narayanan ASS, Peters K, Peters E-M (1997). Planta Med. 63: 255-257. Liang XT, Yu DQ, Wu WL, Deng HC (1979). Acta Chim. Sin. 37: 215-230. Muhammad I, Dunbar DC, Takmatsu S, Walker LA, Clarke AM (2001). J. Nat. Prod. 64: 559-562. Wright CW, Allen D, Cai Y, Phillipson JD, Said JM, Kirby GC, Warhurst DC (1992). Phytother. Res. 6: 121-124. Kaewpradub N, Kirby GC, Steele JCP, Houghton PJ (1999). Planta Med. 65: 690-694. Francois G, Ake Assi L, Holenz J, Bringmann G (1996). J. Ethnopharmacol. 54: 113-117. Kapadia GJ, Angerhofer CK, Ansa-Asamoah R (1993). Planta Med. 59: 565-566. Zhang HJ, Tamez PA, Hoang VD, Tan GT, Hung NV, Xuan LT, Huong LN, Cuong NM, Thao DT, Soejatto DD, Fong HHS, Pezzuto JM (2001). J. Nat. Prod. 64: 772-777.

Ethnomedicinal plants in parasitic infections

109

45. Oketch-Rabah HA, Dossaji SF, Christensen SB, Frydenvang K, Lemmich E (1997). J. Nat. Prod. 60: 1017-1022. 46. Dagne E, Steglich W (1984). Phytochemistry 23: 1729-1731. 47. Ye ZG, Van Dyke K, Yang BZ (1994). Drug Dev. Res. 31: 229. 48. Wright CW, Marshall SJ, Russell PF, Anderson MM, Phillipson JD, Kirby GC, Warhurst DC, Schiff PL (2000). J. Nat. Prod. 63: 1638-1640. 49. Weiss CR, Moideen SVK, Croft SL, Houghton PJ (2000). J. Nat. Prod. 63: 1306-1309. 50. Figueiredo JN, Raz B, Sequine U (1998). J. Nat. Prod. 61: 718-723. 51. Pavanand K, Webster HK, Yongvanitchit K, Kun-Anake A, Dechatiwongse T, Nutakul W, Vansiddhi J (1989). Phytother. Res. 3: 136-139. 52. Pollack Y, Segal R, Golenser J (1990). Parasitol. Res. 76: 570-572. 53. Valsaraj R, Pushpangadan P, Smitt UW, Adserson A, Christensen SB, Sittie A, Nyman U, Nielsen C, Olsen CE (1997). J. Nat. Prod. 60: 739-742. 54. van Agtmacl MA, Eggelte TA, van Boxtel CJ (1999). Trends Pharm. Sci. 20: 199. 55. Robert A, Dechy-Cabaret O, Cazelles J, Meunier B (2002). Acc. Chem. Res. 35: 167-174. 56. Wu Y (2002). Acc. Chem. Res. 35: 255-259. 57. Chanphen R, Thebtaranonth Y, Wanauppathamkal S, Yuthavong Y (1998). J. Nat. Prod. 61: 1146-1147. 58. Rucker G, Schenkel EP, Manns D, Mayer R, Heiden K, Heinzmann BM (1996). Planta Med. 62: 565-566. 59. Francois G, Passreiter CM, Woerdenbag HJ, van Loveren M (1996). Planta Med. 62: 126-129. 60. Oketch-Rabah HA, Lemmich E, Dossaji SF, Theander TG, Olsen CE, Cornett C, Kharazmi A, Christensen SB (1997). J. Nat. Prod. 60: 458-461. 61. Yu HW, Wright CW, Cai Y, Phillipson AD, Kirby GC, Warhurst DC (1994). Phytother. Res. 8: 436-438. 62. Weenen H, Nkunya MHH, Bray DH, Mwasumbi LB, Kinalb O, Kilimali VAEB, Wijnberg JBPA (1990). Planta Med. 56: 371-373. 63. Okunji CO, Iwu MM, Jackson JE, Tally JD (1996). Adv. Exp. Med. Biol. 404: 415-428. 64. Hazra B, Ghosh R, Banerjee A, Kirby GC, Warhurst DC, Phillipson JD (1995). Phytother. Res. 9: 72-74. 65. Kraft C, Janett-Siems K, Siems K, Solis PN, Gupta MP, Bienzle U, Eich E (2001). Phytochemistry 58: 769-774. 66. Kraft C, Janett-Siems K, Siems K, Gupta MP, Bienzle U, Eich E (2000). J. Ethnopharmacol. 73: 131-135. 67. Kittakoop P, Kirtikara K, Tanticharoen M, Thebtaranonth Y (2000). Phytochemistry 55: 349-352. 68. Rasoanaivo P, Ratsimamanga-Urveg S, Rafatro H, Ramanitrahasimbola D, Palazzino G, Galeffi C, Nicoletti M (1998). Planta Med. 64: 58-62. 69. Ratsimamanga-Urveg S, Rasoanaivo P, Rafatro H, Robijaona B, RakotoRatsimamanga A (1994). Ann. Trop. Med. Parasitol. 88: 271. 70. Achanbach H, Waibel R, Nkunya MNH, Weenen H. (1992). Phytochemistry 31: 3781-3784.

110

Varughese George et al.

71. Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD (1997). Planta Med. 63: 270-272. 72. Siad IM, Latiff A, Partridge SJ, Phillipson JD (1991). Planta Med. 57: 389. 73. Deck LM, Royer RE, Chamblee VB, Hernandez YM, Malone RR, Torres JE, Hunsaker LA, Piper RC, Makler MT, Vander Jagi DL (1998). J. Med. Chem. 41: 3879-3887. 74. MacKinnon S, Durst T, Arnason JT, Angerhofer C, Pezutto J, Sanchez-Vindas PE, Poveds LJ, Gbeassor M (1997). J. Nat. Prod. 60: 336-341. 75. Joshi P, Rojatkar SR, Nagasampagi BA (1998). J. Med. Arom. Plant Sci. 20: 1000. 76. Likhitwitayawuid K, Dej-adisai S, Jongbunprasert V, Krungkrai J (1999). Planta Med. 65: 754-756. 77. Jennet-Siems K, Siems K, Jakupovic J, Solis PN, Gupta MP, Mockenhaupt FP, Bienzleueich E (2000). Planta Med. 66: 384-385. 78. Likhitwitayawuid K, Kaewamatawong R, Ruangrungsi N, Krungkrai J (1998). Planta Med. 64: 237-241. 79. Cimanga K, De Bruyne T, Lasure A, Van Poel B, Pieters L, Clacys M, Vanden Berghe D, Kambu K, Tona L, Vlietinck AJ (1996). Planta Med. 62: 22-27. 80. Cimanga K, De Bruyne T, Pieters L, Vlietinck AJ (1997). J. Nat. Prod. 60: 688-691. 81. Grellier P, Ramiaramanana L, Millcrioux V, Deharo E, Schrevel J, Frappier F, Trigalo F, Bodo B, Pousset JL (1996). Phytother. Res. 10: 317-321. 82. Wright CW, Phillipson JD, Awe SO, Kirby GC, Warhurst DC, Quetin-Leclercq J, Angenot L (1996). Phytother. Res. 10: 361-363. 83. Kirby GC, Paine A, Warhurst DC, Noamese BK, Phillipson JD (1995). Phytother. Res. 9: 359-363. 84. Jennet-Siems K, Mockenhaupt FP, Bienzle U, Gupta MP, Eich E (1999). Trop. Med. Int. Health 4: 611-615. 85. Lin L-Z, Hu S-F, Zaw K, Angerhofer CK, Chai H, Pezzuto JM, Cordell GA (1994). J. Nat. Prod. 57: 1430-1436. 86. Kitagawa I, Wei H, Nagao S, Mahmud T, Hori K, Kobayashi M, Uji T, Shibuya H (1996). Chem. Pharm. Bull. 44: 1162-1167. 87. Sauvain M, Moretti C, Bravo J-A, Kallapa J, Munos MH, Ruiz E, Richard B, Le Men-Olivier L (1996). Phytother. Res. 10: 198-201. 88. Furukawa H, Ito C, Ono T, Wu T-S (1993). J. Chem. Soc. Perkin Trans. I, 471. 89. Kitagawa I, Mahmud T, Simanjuntak P, Hori K, Uji T, Shibuya H (1994). Chem. Pharm. Bull. 42: 1416-1421. 90. Pavanand K, Nutakul W, Dechatiwongse T, Yoshihira K, Yongvanitchit K, Scovill JP, Flippen-Anderson JL, Gilardi R, George C, Kanchanapec P, Webster HK (1986). Planta Med. 52: 108-111. 91. Oâ&#x20AC;&#x2122;Neill MJ, Bray DH, Boardman P, Chan KL, Phillipson JD (1987). J. Nat. Prod. 50: 41-48. 92. Cabral JA, McChesney JD, Milhous WK (1993). J. Nat. Prod. 56: 1954-1961. 93. Moretti C, Deharo E, Sauvain M, Jardel C, David PT, Gasquet M (1994). J. Ethnopharmacol. 43: 57-61. 94. Pouvelle B, Farley PJ, Long CA, Taraschi TF (1994). J. Clin. Invest. 94: 413-417.

Ethnomedicinal plants in parasitic infections

111

95. Takaya Y, Kurumada KI, Takeuji Y, Kim H-S, Shibata S, Ikemoto N, Wataya Y, Oshima Y (1998). Tetrahedron Lett. 39: 1361-1364. 96. Takaya Y, Takeuji Y, Akasaka M, Nakajawasai O, Tadano T, Kisara K, Kim HS, Wataya Y, Niwa M, Oshima Y (2000). Tetrahedron 56: 7673-7678. 97. Kamchonwonpaisan S, Nilannonta C, Tarnchompoo B, Thevtaranonth C, Thevtaranonth Y, Yuthavong Y, Kongsacree P, Clardy J (1995). Tetrahedron Lett. 36: 1821-1824. 98. Wright CW, Allen D, Cai Y, Phillipson JD, Said IM, Kirby GC, Warhurst DC (1992). Phytother. Res. 6: 121-124. 99. Wright CW, Oâ&#x20AC;&#x2122;Neill MJ, Phillipson JD, Warhurst DC (1988). Chemother. 32: 1725-1729. 100. Hooper M, Kirby GC, Kulkarni MM (1990). Eur. J. Med. Chem. 25: 717-723. 101. Wright CW, Anderson MM, Allen D, Phillipson JD, Kirby GC, Warhurst DC, Chang HR (1993). J. Eukaryotic Microbiol. 40: 244-246. 102. Wright CW, Allen D, Cai Y, Chen Z, Phillipson JD, Kirby G, Warhurst D, Tits M, Angenot L (1994). Phytother. Res. 8: 149-152. 103. Wright CW, Bray, DH, O'Neill MJ, Warhurst DC, Phillipson JD, QuetinLeclercq J, Angenot L (1991). Planta Med. 57: 337-340. 104. Simonsen, HT, Nordskjold JB, Smitt, UW, Nyman, U, Pushpangadan P, Joshi P, George V (2001). J. Ethnopharmacol. 74: 195-204. 105. Tasdemir D, Brun R, Perozzo R, Donmez AA (2005). Phytother. Res. 19: 162-166. 106. Sairafianpour M, Christensen J, Staerk D, Budnik BA, Kharazmi A, Bagherzadeh K, Jaroszewski JW (2001). J. Nat. Prod. 64: 1398-1403. 107. Nguyen-Pouplin J, Tran H, Tran H, Phan TA, Dolecek C, Farrar J, Tran TH, Caron P, Bodo B, Grellier P (2007). J. Ethnopharmacol. 109: 417-427. 108. Mbatchi SF, Mbatchi B, Banzouzi JT, Bansimba T, Nsonde-Ntandou GF, Quamba JM, Berry A, Benoit-Vical F (2006). J. Ethnopharmacol. 104: 168-174. 109. Wube AA, Bucar F, Asres K, Gibbons S, Rattray L, Croft SL (2005). Phytother. Res. 19: 472-476. 110. Boyom, FF, Ngouana V, Zollo PH, Menut C, Bessiere JM, Gut J, Rosenthal PJ (2003). Phytochemistry 64: 1269-1275. 111. Tran OL, Tezuka Y, Ueda JY, Nguyen NT, Maruyama Y, Begum K, Kim HS, Wataya Y, Tran OK, Kadota S (2003). J. Ethnopharmacol. 86: 249-252. 112. Muthaura CN, Rukunga GM, Chhabra SC, Omar SA, Guantai AN, Gathirwa JW, Tolo FM, Mwitari PG, Keter LK, Kirira PG, Kimani CW, Mungai GM, Njagi ENM (2007). J. Ethnopharmacol. 122: 545-551. 113. Krief S, Martin M-T, Grellier P, Kasenene J, Sevenet T (2004). Antimicrob. Agents Chemother. 48: 3196-3199. 114. Rukunga GM, Muregi FW, Tolo FM, Omar SA, Mvitari P, Muthaura CN, Omlin F, Lwande W, Hassanali A, Githure J, Iraqi FW, Mungai GM, Kraus W, KofiTsekpo WM (2007). Fitoterapia 78: 455-459. 115. Shaw J (2007). Mem. Inst. Oswaldo Cruz. 102: 541-547. 116. Guerin PJ, Olliaro P, Sundar S, Boelaert M, Croft SL, Desjeux P, Wasunna MK, Bryceson AD (2002). Lancet Infect. Dis. 2: 494-501. 117. Dedet JP (2001). Med. Mal. Infect. 31: 178-183. 118. Berman JD (1997). Clin. Infect. Dis. 24: 684-703.

112

Varughese George et al.

119. Mau J (1990). J. Leukocyte Biol. 47: 187-193. 120. Rittig MG, Bogdan C (2000). Parasitol. Today 16: 292-297. 121. Svobodovรก M, Bates PA, Volf P (1997). Acta Tropica 68: 23-35. 122. Murray HW, Berman JD, Davies CR, Saravia NG (2005). The Lancet 366: 1561-1577. 123. Hepburn NC (2001). Curr. Opin. Infect. Dis. 14:151-154. 124. Camuset G, Remy V, Hansmann Y, Christmann D, de Albuquerque CG, Casseb GAS (2007). Med. Mal. Infect. 37: 343-346. 125. Marty P, Rosenthal E (2002). Expert. Opin. Pharmacother. 3: 1101-1108. 126. Ramesh V (1993). Australas. J. Dermatol. 34: 35-35. 127. Ouellette M, Drummelsmith J, Papadopoulou B (2004). Drug Resist. Update 7: 257-266. 128. Hilgard P, Klenner T, Stekar J, Unger C (1993). Cancer Chemother. Pharmacol. 32: 90-95. 129. Leonard R, Hardy J, van Tienhoven G, Houston S, Simmonds P, David M, Mansi J (2001). J. Clin. Oncol. 19: 4150-4159. 130. Sundar S, Jha TK, Thakur CP, Engel J, Sindermann H, Fischer C, Junge K, Bryceson A, Bermam J (2002). N. Engl. J. Med. 347:1739-1746. 131. Soto J, Toledo J, Gutierez P, Nicholls RS, Padila J, Engel J, Fischer C, Voss A, Berman J (2001). Clin. Infect. Dis. 33: E57-E61. 132. Lux H, Heise N, Klenner T, Hart D, Opperdoes FR (2000). Mol. Biochem. Parasitol. 111: 1-14. 133. Verma NK, Dey CS (2004). Antimicrob. Agents Chemother. 48: 3010-3015. 134. Verma NK, Singh G, Dey CS (2007). Exp. Parasitol. 116: 1-13. 135. Georgopoulou K, Smirlis D, Bisti S, Xingi E, Skaltsounis L, Soteriadou K. (2007). Planta Med. 73: 1081-1088. 136. Toma CC, Ollivier E, Delmas F, DiGiorgio C, Balansard G (2007). Rev. Med. Chir. Soc. Med. Nat. Iasi 111: 285-289. 137. Peraza-Sanchez SR, Cen-Pacheco F, Noh-Chimal A, May-Pat F, Sima-Polanco P, Dumonteil E, Garcia-Miss MR, Mut-Martin M (2007). Fitoterapia 78: 315-318. 138. Castillo D, Arevalo J, Herrera F, Ruiz C, Rojas R, Rengifo E, Vaisberg A, Lock O, Lemesre JL, Gornitzka H, Sauvainm (2007). J. Ethnopharmacol. 112: 410-414. 139. Ndjako-Lenta B, Vonthron-Senecheau C, Fongang-Soh R, Tantangmo F, Ngouela S, Kaiser M, Tsamo E, Anton R, Weniger B (2007). J. Ethnopharmacol. 111: 8-12. 140. Mesquita ML, Desrivot J, Bories C, Fournet A, Paula JE, Grellier P, Espindola LS (2005). Mem. Inst. Oswaldo Cruz. 100: 783-787. 141. Tasdemir D, Brun R, Perozzo R, Donmez AA, (2005). Phytother. Res. 19: 162-166. 142. Singh N, Mishra PK, Kapil A, Arya KR, Maurya R, Dube A (2005). J. Ethnopharmacol. 98: 83-88. 143. Germonprez N, Maes L, Van Puyvelde L, Van Tri M, Tuan DA, De Kimpe N (2005). J. Med. Chem. 48: 32-37.

Ethnomedicinal plants in parasitic infections

113

144. Bringmann G, Dreyer M, Faber JH, Dalsgaard PW, Staerk D, Jaroszewski JW, Ndangalasi H, Mbago F, Brun R, Christensen SB (2004). J. Nat. Prod. 67: 743-748. 145. Bringmann G, Messer K, Brun R, Mudogo V (2002). J. Nat. Prod. 65: 1096-1101. 146. Sairafianpour M, Christensen J, Staerk D, Budnik BA, Kharazmi A, Bagherzadeh K, Jaroszewski JW (2001). J. Nat. Prod. 64: 1398-1403. 147. Kayser O, Kolodziej H, Kiderlen AF (2001). Phytother. Res. 15: 122-126. 148. Ghazanfari T, Hassan ZM, Ebtekar M, Ahmadiani A, Naderi G, Azar A (2000). Scand. J. Immunol. 52: 491-495. 149. Da-Silva SA, Costa SS, Rossi-Bergmann B (1999). Parasitology 118: 578-582. 150. Akendengue B, Ngou-Milama E, Laurens A, Hocquemiller R. (1999). Parasite 6: 3-8. 151. Waechter AI, Ferreira ME, Fournet A, Rojas de Arias A, Nakayama H, Torres S, Hocquemiller R, CavĂŠ A (1997). Planta Med. 63: 433-435. 152. Franca F, Lago EL, Marsden PD (1996). Rev. Soc. Bras. Med. Trop. 29: 229-232. 153. Fournet A, Barrios AA, Munoz V, Hocquemiller R, Roblot F, Cave A (1994). Planta Med. 60: 8-12. 154. Puri A, Saxena RP, Sumati Guru PY, Kulshreshtha DK, Saxena KC, Dhawan BN (1992). Planta Med. 58: 528-532. 155. Fournet A, Angelo A, Munoz V, Roblot F, Hocquemiller R, Cave A (1992). J. Ethnopharmacol. 37: 159-164. 156. Tandon JS, Srivastava V, Guru PY (1991). J. Nat. Prod. 54: 1102-1104. 157. Tahir AE, Ibrahim AM, Satti GMH, Theander TG, Kharasmi A, Khslid AS (1998). Phytother. Res. 12: 576-579. 158. Singha UK, Guru PY, Sen AB, Tandon JS (1992). Int. J. Pharmacog. 30: 289-295. 159. Iwu MM, Jackson JE, Tally JD, Klayman DL (1992). Planta Med. 58: 436-441. 160. Martin T, Villaescusa L, Gasquet M, Delma F, Bartolome C, Diaz-Lanza AM, Ollivier E, Balansard G (1998). Pharm. Biol. 36: 56-62. 161. Abreu PM, Martins ES, Kayser O, Bindseil KU, Siems K, Seemann A, Frevet J (1999). Phytomedicine 6: 187-195. 162. Jaramillo MC, Arango GJ, Gonzalez MC, Rovledo SM, Velez ID (2000). Fitoterapia 71: 183-186. 163. Waechter AI, Ferreira ME, Fournet A, Arias AR, Nakayama H, Torres S, Hocquemiller R, Cave A (1997). Planta Med. 63: 433-435. 164. Sulsen VP, Cazorla SI, Frank FM, Redko FC, Anesini CA, Coussio JD, Malchiodi EL, Martino VS, Muschietti LV (2007). Am J. Trop. Med. Hyg. 77: 654-659. 165. Fournet A, Ferreira ME, De Arias AR, De Ortiz ST, Fuentes S, Nakayama H, Schinini A, Hocquemiller R (1996). Antimicrob. Agents Chemother. 40: 2447-2451. 166. Truiti MCT, Ferreira ICP, Zamuner MUM, Nakamura CV, Sarragiotto MH, Souza MC (2005). Braz. J. Med. Biol. Res. 38: 1873-1878. 167. Maes L, Berghe DV, Germonprez N, Quirijnen L, Cos P, De Kimpe N, Van Puyvelde L (2001). Antimicrob. Agents Chemother. 48: 130-136.

114

Varughese George et al.

168. Habtemariam S (2003). BMC Pharmacol. 3: 6. 169. Khalid FA, Abdalla NM, Mohomed HEO, Toum AM, Magzoub MMA, Ali MS (2004). Turkiye Parazitol. Derg. 28: 129-132. 170. Ndjakou LB, Vonthron-Senecheau C, Fongang SR, Tantangmo F, Ngouela S, Kaiser M, Tsamo E, Anton R, Weniger B (2007). J. Ethnopharmacol. 111: 8-12. 171. Mesquita ML, Desrivot J, Bories C, Fournet A, Paula JE, Grellier P, Espindola LS (2005). Mem. Inst. Oswaldo Cruz. (Brazil) 100: 783-787. 172. Tasdemir D, Brun R, Perozzo R, Donmez AA (2005). Phytother. Res. 19: 162-166. 173. Rocha LG, Almeida JRGS, Macedo RO, Barbosa-Filho JM (2005). Phytomedicine 12: 514-535. 174. Barrett MP, Burchmore RJS, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ, Krishna S (2003). The Lancet 362: 1469-1480. 175. Stich A, Abel PM, Krishna S (2002). BMJ 325: 203-206. 176. Hide G (1999). Clin. Microbiol. Rev. 12: 112-125. 177. Hill GC, Shimer SP, Caughey B, Sauer LS (1978). Science 202: 763-765. 178. Briggs LJ, McKean PG, Baines A, Moreira-Leite F, Davidge J, Vaughan S, Gull K (2004). J. Cell. Sci. 117: 1641-1651. 179. Hutchinson OC, Fevre EM, Carrington M, Welburn SC (2003). Lancet Infect. Dis. 3: 42-45. 180. Kennedy PGE, Rodgers J, Bradley B, Hunt SP, Gettinby G, Leeman SE, Felipe CD, Murray M (2003). Brain 126: 1683-1690. 181. Nok AJ (2003). Parasitol. Res. 90: 71-79. 182. Denise H, Barrett MP (2001). Biochem Pharmacol. 61: 1-5. 183. Stein CA, LaRocca, RV, Thomas, R, McAtee, N, Myers CE (1989). J. Clin. Oncol. 7: 499-508. 184. Sanderson L, Khan A, Thomas S (2007). Antimicrob. Agents Chemother. 51: 3136-3146. 185. Raseroka BH, Ormerod WE (1986). Trans. R. Soc. Trop. Med. Hyg. 80: 634-641. 186. Bray PG, Barrett MP, Ward SA, de Koning HP (2003). Trends Parasitol. 19: 232-239. 187. Burri C, Nkunku S, Merolle A, Smith T, Blum J, Brun R (2000). The Lancet 355: 1419-1425. 188. Milord F, Pepin J, Loko L, Ethier L, Mpia B (1992). Lancet 340: 652-655. 189. Pepin J, Milord F, Mpia B, Meurice F, Ethier L, Degroof D, Bruneel H. (1989). Trans. R. Soc. Trop. Med. Hyg. 83: 514-517. 190. Kirchhoff, LV (1993). N. Engl. J. Med. 329: 639-644. 191. Tyler KM, Engman DM (2001). Int. J. Parasitol. 31: 472-480. 192. Woody NC, Woody HB (1955). J. Am. Med. Assoc. 159: 676-677. 193. Tibayrenc M, Ward P, Moya A, Ayala FJ (1986). Proc. Natl. Acad. Sci. U S A. 83: 115-119. 194. Tyler KM, Engman DM (2001). Int. J. Parasitol. 31: 472-480. 195. Serrano AA, Schenkman S, Yoshida N, Mehlert A, Richardson JM, Ferguson MAJ (1995). J. Biol. Chem. 270: 27244-27253. 196. Bellotti G, Bocchi EA, de Moraes AV, Higuchi M.de-L, Barbero-Marcial M, Sosa E, Esteves-Filho A, Kalil R, Weiss R, Jatene A, Pileggi F (1996). Am. Heart J. 131: 301-307.

Ethnomedicinal plants in parasitic infections

115

197. Urbina JA (2001). Curr. Opin. Infect. Dis. 14: 733-741. 198. Mbava AW, Nwosu CO, Onyeyili PA (2007). J. Ethnopharmacol. 111: 526-530. 199. Kamanzi AK, Schmid C, Brun R, Kone MW, Traore D (2004). J. Ethnopharmacol. 90: 221-227. 200. Okpekon T, Yolou S, Gleye C, Roblot F, Loiseau P, Bories C, Grellier P, Frappier F, Laurens A, Hocquemiller R (2004). J. Ethnopharmacol. 90: 91-97. 201. Scio E, Ribeiro A, Alves TM, Romanha AJ, Shin YG, Cordell GA, Zani CL (2003) J. Nat. Prod. 66: 634-637. 202. Uchiyama N, kiuchi F, Ito M, Honda G, Takeda Y, Khodzhimatov OK, Ashurmetov OA (2003). J. Nat. Prod. 66: 128-131. 203. Grael CF, Vichnewski W, Souza GE, Lopes JL, Albuquerque S, Cunha WR (2000). Phytother. Res. 14: 203-206. 204. Bastos JK, Albuquerque S, Siva ML (1999). Planta Med. 65: 541-544. 205. Berger I, Barrientos AC, Caceres A, Hernandez M, Rastrelli L, Passreiter CM, Kubelka W (1998). J. Ethnopharmacol 62: 107-115. 206. Freiburghaus F, Kaminsky R, Nkunya MH, Brun R (1996). J. Ethnopharmacol. 55: 1-11. 207. Freiburghaus F, Ogwal EN, Nkunya MH, Kaminsky R, Brun R (1996). Trop. Med. Int. Health 1: 765-771. 208. Sepulveda-Boza S, Cassels BK (1996). Planta Med. 62: 98-105. 209. Nok AJ, Williams S, Onyenekwe PC (1996). Parasitol. Res. 82: 634-637. 210. Talakal TS, Dwivedi SK, Sharma SR (1995). J. Ethnopharmacol. 49: 141-145. 211. Fournet A, Barrios AA, Munoz V (1994). J. Ethnopharmacol. 41: 19-37. 212. Rojas de Arias A, Ferro E, Inchausti A, Ascurra M, Acosta N, Rodriguez E, Fournet A (1995). J. Ethnopharmacol. 45: 35-41. 213. Elmendorf HG, Dawson SC, McCaffery JM (2003). Int. J. Parasitol. 33: 3-28. 214. Gillin FD, Reiner DS, McCaffery JM (1996). Annu. Rev. Microbiol. 50: 679-670. 215. Hawrelak J (2003). Altern. Med. Rev. 8: 129-142. 216. Gardner TB, Hill DR (2001). Clin. Microbiol. Rev. 14: 114-128. 217. Upcroft J, Upcroft P (1998). Bioessays 20: 256-263. 218. Samuelson, J (1999). Antimicrob. Agents Chemother. 43: 1533-1541. 219. Muller, M (1983). Surgery 93: 165-171. 220. Paget TA, Jarroll EL, Manning P, Lindmark DG, Lloyd D (1989). J. Gen. Microbiol. 135: 145-154. 221. Thompson RCA, Reynoldson JA, Mendis AH (1993). Adv. Parasitol. 32: 71-160. 222. Upcroft, JA., Upcroft P. (1993). Parasitol. Today 9: 187-190. 223. Lau AH, Lam NP, Piscitelli SC, Wilkes L, Danzinger LH (1992). Clin. Pharmacokinet. 23: 328-364. 224. Jokipii L, Jokipii AMM (1980). J. Infect. Dis. 141: 317-325. 225. Gardner TB, Hill DR (2001). Clin. Microbiol. Rev. 14: 114-128. 226. Gordts B, Hemelhof, W, Asselman, C., Butzler JP (1985). Antimicrob. Agents Chemother. 28: 378-380. 227. Crouch AA, Seow WK, Thong YH (1986). Trans. R. Soc. Trop. Med. Hyg. 80: 893-896. 228. Majewska AC, Kasprzak W, De Jonckheere, JF, Kaczmarek, E. (1991). Trans. R. Soc. Trop. Med. Hyg. 85: 67-69.

116

Varughese George et al.

229. Amaral FMM, Ribeiro MN S, Barbosa-Filho JM, Reis AS, Nascimento FRF, Macedo RO (2006). Brazilian Journal of Pharmacognosy 16: 696-720. 230. Tripathi DM, Gupta N, Lakshmi V, Saxena KC, Agrawal AK (1999). Phytother. Res. 13: 561-565. 231. Soffar SA, Mokhtar GM (1991). J. Egypt Soc. Parasitol. 21: 497-502. 232. Barbosa E, Calzada F, Campos R (2007). J. Ethnopharmacol. 109: 552-554. 233. Stanley SL.Jr. (2003). The Lancet 361: 1025-1034. 234. Martﾄｱﾌ］ez-Palomo A, Espinosa-Cantellano M (1998). Parasitol. Today 14: 1-3. 235. Salles JM, Moraes LA, Salles MC (2003). Braz. J. Infect. Dis. 7: 96-110. 236. Centre for Disease Control and Prevention (1999). http://www.cdc.gov/ncidod/ dpd/amebias.htm. 237. Goldsmith RS (1999). In: Tierney LM, McPhee SJ, Papadakis MA (Eds.). Current Medical Diagnosis and Treatment 38th Edition, pp. 1356-1361. Stamford Connecticut; Appleton and Lange. 238. Bruckner DA (1992). Clin. Microbiol. Rev. 5: 356-369. 239. Bansal D, Malla N, Mahajan RC (2006). Indian J. Med. Res. 123: 115-118. 240. Mahajan RC, Sehgal R, Ganguly NK (1991). Indian Rev. Life Sci. 11: 139-67. 241. Wiessman S, Salata R (2000). In: Behrman R, Kliegman R, Jenson H (Eds.). Nelson textbook of pediatrics, 16th Edition, pp.1035-1036. Philadelphia: W.B. Saunders Company. 242. Rosenblatt, JE (1999). Mayo Clin. Proc. 74: 1161-1175. 243. Sohni Y, Kaimal P, Bhatt R (1995). J. Ethnopharmacol. 19, 29-32. 244. Tona L, Kambu K, Ngimbi N, Cimanga K, Vlietinck, A (1998). J. Ethnopharmacol. 61: 57-65. 245. Keene AT, Harris A, Phillipson JD, Warhurst DC, (1986). Planta Med. 52: 278-285. 246. Di Stasi LC (1995). Parassitologia 37: 29-39. 247. Sharma P, Sharma JD (2001). Phytother. Res. 15: 1-17. 248. Streliaeva AV, Chebyshev NV, Sadykov VM (2000). Med. Parazitol. (Mosk) 4: 33-35. 249. Das P, Sinhababu SP, Dam T (2006). J. Altern. Complement. Med. 12: 299-301. 250. Bohand X, Aupee O. (2006). Med. Trop. (Mars) 66: 329-330.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 117-147 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

4. Ethnomedicines for the development of anti-herpesvirus agents Debprasad Chattopadhyay, Sonali Das, Sekhar Chakraborty and Sujit K Bhattacharya ICMR Virus Unit, ID & BG Hospital, GB-4, 57 Dr Suresh C Banerjee Road, Beliaghata, Kolkata 700010; Division of Virology, National Institute of Cholera & Enteric Diseases, Kolkata, and Indian Council of Medical Research, Ansari Nagar, New Delhi 110029, India

Abstract. The Herpesviruses are important human pathogens that can cause mild to severe lifelong infections with high morbidity. Moreover, Herpes simplex virus type 2 (HSV-2) has been reported to be responsible for increased transmission of human immunodeficiency virus (HIV) and progression of AIDS. Therefore, the discovery of novel anti-herpesvirus drugs deserves great efforts. Ethnomedicines have been used as source of candidate drugs in many diseases, but the development of antivirals from natural sources is less explored probably because there are a very few specific viral targets for the natural molecules to interact with. The nucleoside analog acyclovir, a widely used antiherpes drug, unable to cure the life long or recurrent infections and often leads to the development of viral resistance coupled with the side effects, recurrence and viral latency. On the other hand, a wide array of herbal medicaments of many medicinal system have shown high levels of antiherpes virus activities, and many of them Correspondence/Reprint request: Dr. Debprasad Chattopadhyay, Fellow, British Society for Antimicrobial Chemotherapy (England), Assistant Director, ICMR Virus Unit, I.D. & B.G. Hospital Campus, General Block: 4, First floor, 57, Dr. Suresh C. Banerjee Road, Beliaghata, Kolkata 700 010, India. E-mail: debprasadc@yahoo.co.in

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have complementary or overlapping mechanisms of action, either inhibiting viral entry, replication, viral genome synthesis, maturation or assembly. This review will summarize some of those promising plant extracts and isolated compounds, with proven in vitro and documented in vivo anti-herpesvirus activities.

Introduction Over the centuries herbal medicinal products formed the basis of medicaments in many civilizations [Chattopadhyay, 2006; Chattopadhyay & Bhattacharya, 2008]. The traditional healers have long used plant products to prevent or cure infectious conditions and now clinical microbiologists are interested in the herbal products as (i) the effective life span of antimicrobials is limited, (ii) many microbial diseases are intractable to most of the antimicrobials and (iii) the problems of drug resistance, latency and recurrence. Moreover, the rapid spread of emerging and reemerging infectious diseases demand intensive investigation into plant products. Additionally the rapid rate of species extinction leads to irretrievable loss of structurally diverse and potentially useful phytochemicals. The secondary metabolites of plants are species/strain specific with diverse structures and bioactivities, synthesized mainly for defense against predators, as the natural version of chemical warfare [Chattopadhyay & Naik, 2007]. This review will describe some of the promising extracts and compounds plant and, having anti-herpes virus activity, with proven in vitro and some documented in vivo activities.

The herpesvirus The Herpesviruses belongs to Herpesviridae, a family of DNA viruses that cause diseases in humans and animals. In Greek word herpein means "to creep", referring to the latent, re-occurring and lytic infections typical of these viruses. There are eight distinct viruses, presented in Table 1, in this family known to cause disease in humans. Viruses of the herpes group are morphologically indistinguishable, share many common features of intracellular development, but differ widely in biologic properties. All human herpesviruses (HHV) contain a large doublestranded, linear DNA with 100-200 genes encased within an icosahedral protein capsid wrapped in a lipid bilayer envelope, called a virion. Following the binding of viral envelope glycoproteins to host cell membrane receptors, the virion is internalized and dismantled, allowing viral DNA to migrate to the host cell nucleus, where viral DNA replication and transcription occurs. One replication cycle of herpesvirus depends upon a number of steps, like: (i) virion entry, (ii) expression of immediate-early (Îą) genes such as infected

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Table 1. Members of Human Herpesviridae.

cell protein (ICP) 0 and 4, (iii) early (β1, β2) genes including DNA polymerase and thymidine kinase, (iv) late genes (γ1, γ2) containing glycoprotein B (gB), C (gC), ICP5, and (v) unpaired DNA replication [Sandri-Goldin, 2006]. During symptomatic infection, infected cells transcribe lytic viral genes, but sometimes a small number of latency associated transcript (LAT) genes accumulate, which help the virus to persist in the host cell indefinitely. The primary infection is a self-limited period of illness, but long-term latency is symptom-free. Following reactivation, transcription of viral genes switches from LAT to multiple lytic genes that lead to enhanced replication and virion production. Herpesviruses cause localized skin infections of the mucosal epithelia of the oral cavity, pharynx, oesophagus and the eye, or genitals, depending upon the type involved [Habif, 2004]. Moreover, the herpesvirus establish latent infections that can be periodically reactivated, and sometimes produce serious infections of the central nervous system like acute encephalitis and meningitis, and can be fatal in immune deficient patients [Sandri-Goldin, 2006]. The immediate-early genes of HSV can also activate the genes of HIV [Ostrove et al., 1987], varicella-zoster virus [Felser et al., 1988] or human papillomavirus type 18 [Gius & Laimins, 1989], causing a significant risk factor for transmission of HIV/AIDS [Corey et al., 2004]. The herpesvirus can also lead to scarification, a major cause of blindness in developing nations [Habif, 2004; Sandri-Goldin, 2006]. Moreover, the HSV-2 is also known as an oncogenic virus as it can convert infected cells into tumor cells [Habif, 2004]. The search for selective antiherpesvirus agents is an urgent need as the problems like viral resistance, conflicting efficacy in recurrent infection and immunocompromised patients with available antiherpes drugs remain unresolved. Moreover, herpesviruses (HSV-1 and HSV-2) spread silently (asymptomatic), cause opportunistic infections in immunocompromised (especially cancer and HIV infected) patients, and develop resistance to acyclovir.

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The herpesvirus causes a lifelong infection with high morbidity, and is under-diagnosed due to its mild and asymptomatic nature. HSV alone affects more than one third of the world's population1 and is responsible for a wide array of human disease, with effects ranging from discomfort to death. Before the 1970s, when acyclovir (ACV) was introduced as an antiviral drug, cutaneous HSV infection was managed with drying agents and other local care. Newer antiviral drugs with once-daily dosage benefits have emerged during the past several years. Famciclovir and valacyclovir offer effective and convenient therapeutic choices but are often more expensive than acyclovir. Although no cure is available till date, nucleoside analogues acyclovir [9-(2-hydroxyethoxymethyl) guanosine], is widely used, as it selectively phosphorylated by thymidine kinase (TK) enzyme in infected cells. However, acyclovir-resistant herpesvirus was isolated in immunocompromised patients. Moreover, acyclovir is not suitable in neonatal infections and the infections caused by other members of Herpesviridae [Habif, 2004]. Therefore, new antiherpesvirus agents that can substitute or complement acyclovir group of drugs are highly desirable. Several herbal medicinal products are potential sources of functional foods and have various bioactivities like immunomodulatory and antitumor functions. Although the development of antiherpetic agents from herbal source is less explored probably because there are a very few specific viral targets for small natural molecules to interact with. However, several studies showed that phloroglucinol [Arisawa et al., 1990], anthraquinones [Sydiskis et al., 1991], polysaccharides [Marchetti et al., 1996], triterpenes and saponins [Simõnes et al., 1999], and polyphenols [Kuo et al., 2002; Chattopadhyay & Naik, 2007] isolated from several plants inhibit the replication of herpesviruses. A large number of plant-derived and synthetic antiherpes virus agents have also been described [Ferrea et al., 1993; Bourne et al., 1999; Ikeda et al., 2000; Jassim & Naji, 2003; Chattopadhyay & Khan, 2008] and several works is in progress to identify plants and their active components having anti-herpesvirus activity with an aim to prevent the transmission of sexually transmitted infections (STI’s), as well as to develop complementary antiherpes virus agent. A topical preparation from Glycyrrhiza glabra (liquorice root) containing triterpene glycyrrhetinic acid (Glycyrrhizin) used for the prevention and treatment of herpes outbreaks was found to inhibit acyclovir resistant HSV-1, by induction of CD4+ T cells [Utsunomiya et al., 1995]. Again oryzacystatin from rice plant (Oryzae sativa) showed in vitro and in vivo anti HSV-1 activity by inhibiting proteinase enzyme of herpesviruses [Aoki et al., 1995]. When 19 plant-derived compounds were tested by plaque reduction assay against HSV-2, it was found that eugenol, cineole, curcumin and carrageenan lambda type IV (ED50 ≤ 7.0 mg/ml), provided significant protection (P < 0.05) in intravaginal HSV-2 infected mouse and guinea pigs [Bourne et al., 1999]. Interestingly a thiazolylsulfonamide

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BAY57-1293 inhibit helicase-primase of herpesvirus with potent in vitro and in vivo antiherpetic activity [Betz et al., 2002]; while n-docosanol is approved as a topical agent for herpes labialis by FDA [Sacks et al., 2001]. All these findings indicated that the herbal products are still potential sources for new antiherpetic agents. Due to the amazing structural diversity and broad range of bioactivities herbal medicinal products can be explored as a source of complementary antiherpetic agents, as many of them inhibit several steps of replication cycle and certain cellular factors of herpesviruses [Chattopadhyay & Khan, 2008]. The objective of this review is to summarize the potential uses of natural products, especially derived from herbal sources, for the prevention and treatment of infections caused by herpesviruses, especially the HSV-1 and HSV-2.

Screening models for herbal anti-HSV agents and their value in drug discovery In vitro primary screening assays Several in vitro and in vivo methods are available to study the antiherpetic activities of plant-derived products, but the most commonly used in vitro method for preliminary screening of extracts or compounds is the study of cytopathic effect (CPE) by plaque reduction assay on HSV-infected cells. Here the Hep2 or Vero Cells were grown in suitable cell culture media with incubation at 37 °C for 24 h. Confluent cell monolayers are then infected with 100-200 plaque-forming units (PFU) of the virus. After 1h incubation (to allow viral adsorption), the cells are washed with phosphate buffer saline (PBS) and overlaid with agar in cell culture medium containing twofold dilutions of the extracts or test compounds, and recultured at 370C, until plaques appeared. Finally the monolayer cells are fixed with formalin, stained, dried and the number of plaques are microscopically counted. The percentage inhibition of plaque formation [(mean number of plaques in control − mean number of plaques in test)/(mean number of plaques in control) × 100], or the effective concentration for 50% plaque reduction [EC50; the lowest extract concentration that reduced plaque number by 50% in the treated cultures compared to untreated ones], or the 50% inhibitory concentration [IC50; the extract concentration required to reduce the virus plaque number by 50%] is calculated. When different herbal preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) were analyzed by plaque reduction assay, it was observed that the ethanolic extract of Rheum officinale and methanol extract of Paeonia suffruticosa inhibit attachment and penetration of HSV-1; while the aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibit attachment and replication of HSV-1 and HSV-2 [Hsiang et al.,

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2001], indicating that these herbs can be the potential source for new antiHSV lead. The pioneering work of Vanden Berghe and his group showed that the inhibition of cytopathic effect on Vero cell monolayer infected with HSV can be measured by end-point titration method [Vanden Berghe et al., 1993], which is also helpful to determine virucidal activity after preincubations of test compound and virus [Vlietinck et al., 1997; Apers et al., 2002]. The 50% end point titration (Fig. 1) was performed on confluent monolayers of Vero cells (104 cells per well) infected with serial ten-fold dilutions (107 TCD50/ml) of virus suspension and the first monolayer of cells was infected with multiplicity of infection (MOI) of 10 to 10-4 by serial ten-fold dilution. The virus was allowed to absorb for 1 hr at 370C, and then serial two-fold dilutions of extract or test compound (in maintenance medium, supplemented with 2% foetal bovine serum and antibiotics) were added. The plates were incubated (370C) and viral CPE was recorded by light microscopy for 7 days (Fig. 1). It is important to run cytotoxicity control (uninfected but treated cells), and cell control (uninfected untreated cell) at each treatment concentration, while the virus control (infected but untreated) at each viral dilution. Toxic doses (CT) of the test extract or compound are considered to be dilutions that cause destruction of monolayer of cells, so that no virus titer can be determined. The antiviral activity is expressed as the virus titer reduction at the maximum non-toxic dose (MNTD) of the test extract or substance, i.e., the highest concentration that does not affect the monolayers under test conditions. In this method virus titer reduction factors (RF, the ratio of the virus titer reduction in the absence and presence of the MNTD of the test sample) of 103 to 104 indicate a pronounced antiviral activity and are suitable as selection criteria for further investigation of the said extract or compound. It was observed that the antiviral activity should be present in at least two subsequent dilutions of the test substance, otherwise the activity is likely to be due to its toxicity, or the activity is only virucidal. The extracellular virucidal activity can also be determined by titration method of the residual infectious virus at room temperature after incubation of the test compound with virus suspension (106 TCD50/ml) during 1 h at 370C [Vanden Berghe et al., 1993; Apers et al., 2001]. Another rapid and sensitive in vitro procedure of evaluating anti-HSV agents is based on spectrophotometrical assessment for viability of virus- and mock-infected cells via in situ reduction of a tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and is proved to have similar sensitivity like plaque reduction assay [Sudo et al., 1994]. It not only significantly simplifies the assay procedures, but also allowing the evaluation of larger numbers of compounds at a time. Kira et al. [1995] reported the development of another highly sensitive assay system using a suspension cell line derived from human myeloma cells, that are sensitive for several HSV strains such as HSV-1: standard

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Figure 1. The in vitro protocol for testing anti-viral extracts.

strain KOS, ACV-resistant A4D, clinical isolate Hangai and HSV-2 standard strain G; and many known antiherpesvirus compounds [acyclovir, sorivudine, arabinoside, 9-(1,3-dihydroxy-2-propoxy)methyl guanine, phosphonoformate and dextran sulfate] and produce good results when tested against KOS and G strains of HSV. Kurokawa et al. [1999] reported the in vitro anti-HSV activity of Rhus javanica extracts by plaque reduction assay against wild-type, acyclovirphosphonoacetic acid-resistant, thymidine kinase-deficient HSV-1 and wild-type HSV-2 (EC50 2.6-3.9Âľg/ml) and the in vivo efficacy of the cutaneously infected mice with HSV-1.

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High throughput screening assays High capacity anti-HSV drug screening assay is available for the primary analysis of compounds in a 96 well microtiter assay. The basic assay methods involve infection of Vero cells with HSV-1 or HSV-2 in the presence of test compounds. The ability of the compounds to inhibit HSV-induced cell killing is measured five days post-infection using the tetrazolium dye MTS. Mitochondrial enzymes of viable cells convert MTS to a soluble, colored formazan. The quantitation of the amount of the formazan product present in each well of the microtiter plate is then determined spectrophotometrically at 490/650 nm. While the toxicity of the test compounds to host cells is measured concurrently in the same microtiter plate. Data can be analyzed by a statistical software program along with determinations of the efficacy (IC50), toxicity (TC50) and selectivity (therapeutic index, TI) of the compounds. Several primary screens are also available for the evaluation of compounds against human CMV. A high capacity drug screening assay similar to the anti-HSV assay is used for CMV. The MRC-5 cells are infected with HCMV in the presence of drugs for a period of seven days. Compound efficacy and toxicity is then measured using MTS. Standard Plaque Reduction assay as well as an ELISA is also available. For VZV the primary screen currently available is a standard Plaque Reduction assay. While for EBV a moderate throughput PCR-based assay is developed to identify inhibitors of EBV using P3HR1 cells, a cell line that is latently infected with EBV. Lytic virus replication spontaneously occurs in approximately 5% of the cell population resulting in the release of virus particles from the cells. The P3HR1 cells are incubated with compounds for a period of six days; supernatant virus is collected and quantitated using TaqMan PCR methodology. Compound toxicity is evaluated in parallel using MTS. A virus induced cytopathic effects (CPE)inhibition assay is employed to evaluate compounds against HHV-6. Uninfected HSB-2 cells are co-cultured with HHV-6 infected HSB-2 cells in the presence of test compounds. After six days incubation, CPE inhibition is determined by microscopic inspection of the cultures. Upon infection and replication of HHV-6 in HSB-2 cells, the cells increase in size and become light refractory. These changes are readily apparent by microscopic examination of the wells which allows for the quantitation of the numbers of infected cells in each of the cultures. Compound toxicity is evaluated in parallel using MTS. An HHV-6 ELISA is also developed. A moderate throughput PCR-based assay to identify inhibitors of HHV-8, using BCBL-1 cells, a cell line that is latently infected with HHV-8, is developed. Lytic virus replication is induced using the phorbol ester TPA, resulting in the release of virus particles from the cells. BCBL-1 cells are incubated with compounds for a period of four days. Supernatant virus is collected and quantitated using TaqMan PCR methodology. Compound toxicity is evaluated in parallel using MTS.

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Secondary testing assays Compounds or extracts that are evaluated for their ability to inhibit herpesvirus infection needs a variety of phenotypically distinct susceptible cell lines including Vero, MRC-5, HFF, BHK, HEp-2 as well as other human and mammalian cell lines. To evaluate compounds that inhibit replication of viruses need a panel of virus isolates, including clinical and/or drug resistant herpesvirus isolates. Currently there are ten HSV-1, five HSV-2, sixteen HCMV, five VZV, two EBV, two HHV-6 and two HHV-8 isolates are used throughout the globe. To study the titer reduction of HSV-1, HSV-2, HCMV and VZV supernatant virus from drug treated cultures is collected and titrated to determine the level of reduction in virus produced as a result of drug treatment. This is an in vitro model for estimating the fold log reduction in virus that could be obtained by antiviral treatment in vivo. The extracts or compounds can also be evaluated for their activity in combination with other drugs including other known herpesvirus inhibitors, or HIV-1 inhibitors or any other compound of interest. Interactions (synergy, additivity, antagonism) of the compounds can be evaluated in terms of antiviral efficacy and toxicity.

Studies on the mechanism of action Compounds can also be evaluated for activity when challenged with different amounts of virus like HSV-1, HSV-2, HCMV and VZV ranging from very low to very high multiplicity of infection. Time of addition and time of removal: Compounds are added or removed from cultures at various times pre- and postinfection. By comparison with other known herpesvirus inhibitors, this allows to determine the relative point in the virus life cycle that is being inhibited (immediate early, early, late functions, DNA polymerization, etc.). This standard technique typically used early during the process of determining mechanism of action as it allows one to narrow in on a smaller target window of activity for further experimentation. It also allows for an easy way to determine if a compound is acting by a unique or novel mechanism compared to other known inhibitors. Furthermore, time of removal studies allow one to determine the reversibility of a compounds activity. Analysis of viral DNA: The effect of compounds on the production of viral DNA can be evaluated using various hybridization techniques, PCR or TaqMan PCR. Analysis of viral proteins: The effect of compounds on the production of immediate early, early and late viral proteins can be evaluated using Western blots and/or Flow cytometry. Selection and characterization of drug-resistant virus isolates: Resistant virus isolates are selected in tissue culture by serial passage of the virus in the presence of gradually increasing concentrations of the compound. Resistance evaluations can be performed in any of the available cell lines with a variety of virus isolates. In addition, resistance selection can be evaluated using combinations of anti-HSV

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agents to evaluate the relative ability of the virus to become resistant to multiple agents that might be used in the clinic. Furthermore, these studies can be performed as part of a directed research project to determine the location of the drug resistance mutations that develop in the viral genome and transfer of the mutations into susceptible virus strains to demonstrate they are responsible for drug resistance (Marker Transfer Experiments).

In vivo model As the HSV infections of mice provide a good model for human disease, we will describe here the different mouse models as well as with other animals that provide receptors for HSV entry and expression of viral glycoproteins that influence disease and pathogenicity in man. To test the in vivo toxicity and efficacy of the herbal products in animal several models have been developed. The most commonly used method of in vivo toxicity determination is dermal toxicity testing of the extract or substance, usually done by skin irritation test; while the efficacy of any extract or compound is measured by cutaneous lesion development in guinea pigs (Fig. 2). For dermal toxicity testing of any extract or formulations guinea pigs of either sex (200-250 g) is used. After removing the body hair from the dorsal side of the animal, the naked skin (6 cm Ă&#x2014; 7 cm) is washed with warm water, dried and abraded with dermal (Seven-Star) needles. The extract or formulations of different potency is then applied to the abraded area of cohorts of animals (n = 5) usually at the rate of 2 g per animal. After 24 h, the extract is removed with warm water and the animals are examined for erythema and or edema 1 h later, upto the next 72 h. To study the extract potency on HSV-1 induced cutaneous lesion, the abraded dorsal area of the animals is first divided into four quadrants and each of the quadrants will then infected with 30 Âľl of 10-fold diluted HSV-1, and the animals are observed upto 10 days for typical herpes lesion development. Once the lesion developed, the Cohorts of fresh animals (n = 15) will be infected as above and treated with the extract or formulations, control drug (acyclovir) or vehicle control to the infected area with sterile cotton swabs twice daily for 6-days (Fig. 2). The extent of lesion should be scored daily as: 1.0-1.6 lesions on 1/4 of infected area; 1.7-2.4 lesions on 1/2 of infected area; 2.5-3.2 lesions on 3/4 of infected area; and 3.3-4.0 lesions on the entire infected area [Zhang et al., 2007]. A fast, simple reactivation model to study the ocular herpesvirus infection and latency was successfully established by Gordon et al., [1990] in New Zealand female rabbits. Each unscarified rabbit eye was inoculated with a suspension of thymidine-kinase-positive HSV-1W strain (5 X 104 pfu/eye) into the lower fornix, following a topical anesthesia with eye drops. The HSV-1W establishes latency and reactivates in a manner similar to mouse pathogenic strain HSV-1 McKrae [Gordon et al., 1986]. Successful inoculation (100%) of eyes was found on day 7 with typical herpetic dendritic ulcers and significant HSV-1 titer (104pfu/ml) and viral shedding can be

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Figure 2. In vivo testing protocols for anti-HSV extract.

determined by neutralization test. After satisfactory anesthesia the globe was proptosed with a wooden cotton applicator and an operating microscope is used to facilitate all surgical manipulations. The intrastromal injection (by a no. 30 short bevel needle attached to a 0.25-ml tuberculin syringe) into the central corneal stroma. One group will receive deionized sterile endotoxin free water, another group will get 100 Âľl air while the third group receive no injection, and for all three groups, the needle should be carefully withdrawn, and the proptosed globe gently returned to the orbit by gentle digital pressure. The anterior chamber injection with deionized sterile water is made at the limbus, inserted into the anterior chamber parallel to the iris plane. Needle is carefully withdrawn, and the insertion site will be pressed with a cotton swab for 30 sec, to avoid aqueous loss. This pressure also returned the proptosed globe to its proper place in the orbit.

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For topical administration of 100 ml deionized sterile water will be made onto the cornea of the proptosed globe by a pipette and the globe will return to the orbit by gentle digital pressure. Viral Shedding (detection of latent HSV-1 after reactivation and induced shedding into the tear film) is determined by swabbing the eyes 2 days prior to treatment and for 7 consecutive days after treatment. Each eye swab is mixed with 0.3 ml MEM (modified Eagle's medium with Earle's salt, 10% newborn calf serum, 1% penicillinstreptomycin, 1% Fungizone), vortexed, and the eluant is plated onto a Vero cell monolayer. After a 1-hr adsorption period, an additional 1.5 ml media is added to the well, and the plate can be examined daily for 7 days for the progressive CPE characteristic of HSV-1. Random HSV-1 isolates can be confirmed by neutralization [Gordon et al., 1983; Gordon et al., 1990]. To test the in vivo efficacy of an extract or formulation against HSV-2, the Genital herpes model was developed in random breed BALB/c female mice or in female Sigmodon hispidus (cotton) rats [Yim et al., 2005] by intravaginal inoculation of HSV-2 in anesthetized inbred 6-week-old mice or rats. After oneweek acclimatization at room temperature (23±30C) the animals (10 animals for each dilution) are inoculated with HSV-2 (30 µl of 10−3 virus stock) to the vagina by a size 12 needle, and the animals and observe for 12 days to develop vaginitis or lethality to determine the median lethal dose (LD50). To test the efficacy of the extract or formulation, fresh batches of animals are infected with 10 LD50 dose of the virus (105 PFU) as described above. Following inoculation, a vaginal cotton swab sample is collected from each animal, transferred to 0.5 ml of PBS and stored at -20°C. The animals are divided into test groups (different potency), positive control group (acyclovir), negative control group (solvent or base cream), and one no treatment (virus control) group as well as an additional group of uninfected control. Symptoms of viral vaginitis (topical edema of the vaginal tract with turbid secretions) will be observed on the third day of infection. Treatment began on day 3 post-infection, by applying the extract or formulations to the vaginal tract with cotton swabs, at a dose of 2 mg per mouse twice daily for a 6-day period [Zhang et al., 2007]. Mortality and the number of days for mortality to occur are recorded. From day one following the completion of the treatment, as well as from the deceased animals immediately following their death vaginal swab samples should be collected (Fig. 2). The vaginal samples are then diluted five times in MEM and used to infect Vero cells. Samples that gave positive CPE is considered positive for HSV-2 [Zhang et al., 2007]. A polysaccharide lignin-carbohydrate complex from Prunella vulgaris (PPS-2b), when tested by the plaque reduction assay showed strong activities against HSV1 and HSV-2, as the complex block HSV binding and penetration; while a cream with semi-purified fraction of P. vulgaris showed a significant reduction (P<0.01) in skin lesions and animal (P<0.01) mortality in a HSV-1 skin lesion guinea pigs model and HSV-2 genital infection model in BALB/c mice, indicating that this complex had potent anti-HSV activity [Zhang et al., 2007].

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In vitro and in vivo antiherpetic activity of crude herbal extracts During the last 40 years many broad based screening programs was undertaken throughout the world to evaluate the in vitro and in vivo antiviral activity of hundreds of herbal products and many of them revealed strong antiherpesvirus activity, while some can be used as a lead for the development of anti-herpes virus agents [Yarnell & Abascal, 2005; Khan et al., 2005; Chattopadhyay et al., 2006; Chattopadhyay & Khan, 2008]. These recent reviews reported the in vitro and sometimes in vivo antiherpes virus activities of many plant extracts like Pongamia pinnata inn, Beta vulgaris, Callisia grasilis, Annona sp (CC50 49.6x103 mg/ml), Polygonium punctatum, Lithraea molleiodes, Sebastiania braseiliensis, S. klotzschiana (ED50 39-169 µg/ml), Eupatorium articulum (125-250 µg/ml), Melaleuca leucadendron fruits, Nephelium lappaceum pericarp (199µg/ml), Barleria lupulina Lind, Clinacanthus nutans (Burm.f) Lindua, Nepeta nepetella, N. coerulea, N. tuberosa (150-500 µg/mL), Byrsonima verbascifolia (2.5 µg/ml), Holoptelia integrifolia (750 mg/kg per day), Myrica rubra, Thea (Camellia) sinensis, Pterocarya stenoptera, Boswellia ameero, Boswellia elongata, Buxus hildebrandtii, Cissus hamaderohensis, Cleome socotrana, Dracaena cinnabari, Exacum affine, Jatropha unicostata and Kalanchoe farinacea (IC50 0.7-12.5 µg/mL) mainly against HSV-1 and HSV-2. The garlic extracts showed strong inhibitory activity against human cytomegalovirus (HCMV) [Khan et al., 2005; Chattopadhyay & Bhattacharya, 2008; Chattopadhyay & Khan, 2008]. Interestingly the i.p. administration of black seed (Nigella sativa) oil to BALB/c mice strikingly inhibit HCMV in vitro murine cytomegalovirus (MCMV) titers in spleen and liver [Salem & Hossain, 2000]; while the extract of Terminalia chebula not only significantly inhibits HSV in vivo, but also the replication of HCMV in vitro and murine CMV in immunosuppressed mice [Chattopadhyay & Khan, 2008]. The search for natural antivirals was actually initiated by the Boots drug company, England in 1952. However, Canadian researchers first reported the antiviral activities of grape, apple and strawberry juices against HSV and other viruses; while leaf extract of Azadirachta indica inhibit DNA viruses like smallpox, chicken pox, poxvirus and HSV [Khan et al., 2005]. The British Columbian ethnomedicines Cardamine angulata, Conocephalum conicum, Polypodium glycyrrhiza showed anti-HSV-1 activity [McCutcheon et al., 1995]; while strong anti-HSV activity was found with Byrsonima verbascifolia extract, a folk remedy for skin infections [Rao et al., 1969]. The aqueous extracts of Nepeta nepetella, Dittrichia viscosa and Sanguisorba minor magnolii of Iberian Peninsula inhibit vesicular stomatitis virus (VSV) and HSV-1 at 50-125 µg/ml [Glatthaar-Saalmuller et al., 2001]; while the Nepalese ethnomedicine Nerium indicum inhibit HSV and influenza A virus [Alche et al., 2000]; but the Chinese antipyretic and antiinflammatory folk medicine Rheum officinale and Paeonia

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suffruticosa prevent HSV attachment and penetration [Hsiang et al., 2001]. On the otherhand, Senecio ambavilla, a folk remedy of La Reunion Island had antiHSV-1 and anti-poliovirus activities [Rajbhandari et al., 2001]. The extracts of Aglaia odorata, Moringa oleifera and Ventilago denticulate, folk medicine of Thailand, inhibit thymidine kinase-deficient and phosphonoacetate-resistant HSV-1 and delayed the development of skin lesions at 750 mg/kg/per dose, increase the mean survival times and reduced the mortality of infected mice similar to acyclovir [Fortin et al., 2002]. The Taiwan folk remedy Boussingaultia gracilis and Serissa japonica extract can inhibit HSV and adenoviruses (ADV) 3, 8 and 11. [Chiang et al., 2003]. Interestingly the viral adsorption, replication and transcription of HSV-1and HSV-2 were inhibited by Ceratostigma willmattianum extract, an ethnomedicine of China, with IC50 of 29.46 and 9.2 mg/L respectively [Chen et al., 2004]. The extracts of Senna petersiana, a folk remedy for sexually transmitted diseases, have strong anti-HSV activity [Dong et al., 2004]. The aqueous extract of Carissa edulis (Forssk.) Vahl (Apocynaceae) root from Kenya, significantly (100%) inhibited plaque formation in Vero E6 cells infected with 100PFU of wild type HSV (7401H of HSV-1 and Ito-1262 strains of HSV-2) or resistant HSV strains (TK(-) 7401H and AP(r) 7401H of HSV-1) at 50 Âľg/ml in vitro with minimal cell cytotoxicity (CC50 480 Âľg/ml). The extract at an oral dose of 250 mg/kg significantly (50%) delayed the onset of HSV infections in a murine model using Balb/C mice, cutaneously infected with wild type or resistant strains of HSV. It also increased the mean survival time (28-35%) and reduced the mortality rate (70-90%) of infected mice [Tshikalange et al., 2005]. This potent anti-HSV activity can be exploited for the development of an anti-HSV drug. Methanol and hot-aqueous extracts of 25 different plant species, used in Yemeni traditional medicine, and growing in the island Soqotra when tested in two in vitro viral systems using HSV-1 in Vero cells and influenza virus A in MDCK cells, the extract showed higher HSV-1 sensitivity at non-cytotoxic concentrations, against the extracts.

In vitro and in vivo antiherpetic activity for herbal extracts and some potential phytochemicals Due to the amazing structural diversity and broad range of bioactivities compounds from herbal medicinal products alone or in combinations can be explored as a source of complementary antivirals, as many of them are reported to inhibit certain steps of replication cycle and cellular factors of many DNA viruses, particularly the members of herpesviridae. A list of some potential herbal extracts along with the isolated compounds having antiherpesvirus activities with probable mode of action are presented in Table 2.

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Table 2. Some important Ethnomedicinal extracts and compounds having antiherpes virus activity.

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Table 2. Continued

Phenolics and polyphenols The simplest bioactive polyphenols with a single substituted phenolic ring belongs to phenylpropane that are in the highest oxidation state and have wide range of antiviral activities. The aqueous extract of Geranium sanguineum L. aerial roots containing polyphenolic complex catechins and condensed tannins was reported to inhibit both HSV-1 and HSV-2 (EC50 3.6-6.2 µg/ml) and at MIC90 (120µg/ml) it strongly inactivate HSV-1Kupka strain in plaque reduction assay, and delayed the development of herpetic vesicles in guinea pig [Serkedjieva & Ivancheva, 1999]. Propolis, a crude extract of the balsam of various trees inhibits acyclovir-resistant HSV-1 and VZV due to the synergistic action of a mixture of terpenoids, flavonoids, benzoic acids esters, and phenolic acid esters [Huleihel & Isanu, 2002]. Interestingly the anticancer, antiinflammatory and immunomodulating agent caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives inhibit 70% of plaque formation in HSV at 10M dose, while at higher concentration it inhibits RNA, protein synthesis and proliferation of HSV [Huleihel & Isanu, 2002]. The aqueous extract of Plantago major L., a popular medicine of Ayurvedic, traditional Chinese medicine (TCM) and Chakma Talika Chikitsa of Bangladesh, used for treating ailments from cold to viral hepatitis, showed anti-HSV activity. Interestingly the isolated caffeic acid, chlorogenic acid and rosmarinic acid of P. major exhibited strongest activity against both HSV-1 (EC50 15.3µg/ml) and HSV-2 (EC50 87.3µg/m) [Chiang et al., 2002]. The SAR studies revealed that the site(s) and number of -OH groups on phenols are responsible for their antiviral activity. The Myrica rubra barks containing prodelphinidin-di-O-gallate inhibits HSV-2 attachment and penetration, and reduces viral infectivity by affecting the late stage of infection [Cheng et al., 2003]. Similarly Homalium cochinchinensis root bark containing cochinolide B, tremulacin and tremuloidin, inhibit HSV-1

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and HSV-2 [Ishikawa et al., 2004]. The in vitro anti-HSV activity of Vaccinium vitis-idaea Linn (Ericaceae) by XTT assay (IC50 73.3±14.5 µM) and plaque reduction assay (IC50 41.9±2.0 µM for HSV-1 and 62.8±6.3 µM for HSV-2) is found to be due to proanthocyanidin A-1 blocks HSV-2 infection at non-toxic dose (CC50 282.1±27.5 µM) by inhibiting viral attachment and penetration and affects the late stage(s) of infection [Cheng and Lin-Chun, 2005]. Similarly proanthocyanidins isolated from Hamamelis virginiana bark and oligomeric procyanidin C1 of Crataegus sinaica showed remarkable anti-HSV-1 activity by plaque reduction assay [Shahat et al., 2002]. It was observed that proanthocyanidins C1 nonspecifically bind proteins, but selectively inhibit nuclear factor kappa B (NFkB)-dependent gene expression, that modulate apoptosis [Cos et al., 2004]. A Xanthohumol-enriched Humulus lupulus (hop) extract showed moderate antiviral activity against HSV-1, HSV-2 and CMV in plaque reduction assay [Buckwold et al., 2004]. An interesting SAR is noted with dimeric procyanidins and related polyphenols, where epicatechin- containing dimers showed pronounced anti-HSV activities, as the ortho-trihydroxyl groups in the B-ring with the double interflavan linkages lead to a significant increase of anti-HSV activity. The flavonoids and dimeric stilbenes from Artocarpus gomezianus Wall, phloroglucinols from Mallotus pallidus Airy Shaw, and coumarins from Triphasia trifolia (Burm.f.) inhibit both HSV-1 and HSV-2 due to bis-hydroxyphenyl moiety [Likhitwitayawuid et al., 2005], which can be the potential target for anti-HSV drugs development. Sakagami et al. [2005] opines that the virucidal activity of polyphenols is due to their association with proteins and/or host cell surfaces, resulting in reduction or prevention of viral adsorption. The health promoting, disease-preventing dietary compounds flavonoids are hydroxylated phenolics contain one carbonyl group, are therapeutically useful or can be used as prototypes for drug development. The flavonoids occur as a C6-C3 unit linked to an aromatic ring and are synthesized in response to microbial infections, and hence showed broad spectrum of antimicrobial activity. Some recent reviews reported that flavonoids can inhibit diverse viruses, including herpesviruses, both in vitro and in vivo by direct inactivation or blocking replication [Khan et al., 2005; Chattopadhyay & Khan, 2008]. Flavonoids like quercetin, procyanidin, and pelargonidin are virucidal to HSV while catechin and hesperidins can inactivate HSV [Kaul et al., 1985]. Similarly quercetin, galangin, naringenin, kaempferol and 3-methyl Kaempferol are potent antiherpetic compounds, as these agents inhibit viral attachment and penetration and augmentation of their activity depends on the degree of sulfation [Sarisky et al., 2002]. The extract of Sapium sebiferum containing methyl gallate and methyl3,4,5-trihydroxybenzoate are the potent and specific inhibitor of Herpes viruses; while phloroglucinol of Mallotus japonicus can only reduce HSV plaque formation [Arisawa et al., 1990]. The hesperidin of orange (Citrus aurantium L.) and grape (Vitis vinifera L.) can inhibit HSV replication; while catechin inhibits infectivity of HSV-1, but quercetin inhibit both, as the small structural differences

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of these compounds are critical to their activity. The Centella asiatica L., Maclura cochinchinensis Cornor, and Mangifera indica L. used as herpesvirus remedies in Thailand can inhibit HSV-1 and HSV-2 virion production in plaque inhibition assay. Combinations of each of these extracts with acyclovir resulted in synergistic or additive interaction in a dose dependent manner, probably due to the active constituent asiaticoside of C. asiatica and mangiferin of M. indica with good therapeutic potential [Yoosook et al., 2000]. Bunyapraphatsara et al., 2000 reported that the morin, a flavonoid group, isolated from ethyl acetate extract of Maclura cochinchinensis have powerful anti-HSV-2 activity (EC50 38.553.5µg/ml), due to their free hydroxyl groups. The extract of Rhus succedanea and Garcinia multiflora containing amentoflavone and robustaflavone, inhibits in vitro growth of HSV, while VZV were inhibited by rhusflavanone and succedaneflavanone [Chattopadhyay, 2006]. It was reported that the quercetin from Caesalpinia pulcherrima Swartz had a broad anti- herpesvirus activity (EC50 24.3-50mg/l), by inhibiting early stage of multiplication [Chiang et al., 2003], suggesting its potential use for anti-HSV drug development. Similarly the isoquercitrin of Waldsteinia fragarioides had significant anti-HSV activity [Khan et al., 2005]. When 18 flavonoids of five classes were tested against HSV-1 and HSV-2, epicatechin (EC), epicatechin gallate (ECG) and quercetin (flavanols); genistein (isoflavone) and naringenin (flavanone) showed a high level of CPE inhibitory activity. EC, ECG, galangin and kaempferol showed strong anti-HSV activity, while catechin, epigallocatechin, epigallocatechin gallate, naringenin, apigenin, chrysin, baicalin, fisetin, myricetin, quercetin, and genistein had moderate effect on HSV-1 in plaque reduction assay. Hence, flavanols and flavonols appeared to be more active than flavones, probably due to their structural differences. Furthermore, Vero cells treated with ECG and galangin before virus adsorption led to a enhancement of inhibition by yield reduction assay, indicating that an intracellular effect may also be involved [Lyu et al., 2005]. Hence, the potent antiviral flavonoids appear to block viral DNA/RNA polymerase, where the degree of inhibition depends on the structure and side chain. A recent study with ent-epiafzelechin-(4α->8)-epiafzelechin (EEE) from fresh leaves of Cassia javanica L. agnes de Wit revealed the inhibition of HSV-2 replication in a dose-dependent manner (IC50 83·8±10·9µM) by inhibiting penetration and replication at the late stage of viral life cycle [Cheng et al., 2006]. The evidence of oxidative stress in virus-infected individuals, particularly in chronic lifelong HSV infections, indicates that antioxidants like flavonoids and proanthocyanidins with low oral bioavailability may have some role in controlling disease progression [Chattopadhyay & Khan, 2008]. A recent report revealed that sodium rutin sulfate (SRS), a sulfated derivative of the flavonol glycoside rutin inhibit HSV-1 (IC50 88.3 ± 0.1 µM) at CC50 >3.0 mM in human genital ME180, HeLa and primary human foreskin fibroblast cell lines, indicating its novel candidature for the development of anti-HSV microbicide [Tao et al., 2007].

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Coumarins The double-ring phenolic compounds made up of fused benzene and Îą-pyrone rings are called coumarins. Coumarins are reported to have antithrombotic, anti-inflammatory, antioxidant, anticarcinogenic, antiallergic, vasodilatory and hepatoprotective activities [Chattopadhyay & Bhattacharya, 2008]; even some are enzyme inhibitors or precursors of toxic substances, and in defense against infection. As coumarins can stimulate macrophages so can exert an indirect effect on viral infections like inhibition of recurrences of cold sores caused by HSV-1 [Khan et al., 2005; Chattopadhyay, 2006]. The topical tree Clausena heptaphylla leaves, widely used in China, India and Vietnam to treat fever, showed antiviral activity against HSV-1 and HSV-2 at 0.5 mg/ml; while its ethyl acetate extract inhibit replication of both HSV types [Huy et al., 2004]. Hence, this plant can be a potential candidate for anti-HSV agent. The coumarin derivatives 1,4-dihydropyridine-5-carboxylic acid and pyridine-5-carboxylic acid from some herbal products showed antiviral activities against CMV (AD-169 and Davis strain), HSV-1 (KOS, F, Mclntyre, TK-B2006, TK-VMW1837, TK-Cheng C158/77, TK-Field C137/101), HSV-2 (G, 196, Lyons), and VZV (TK+ OKA, TK+ YS, TK- 07/1, TK- YS/R strains) [Patent WO 01/14370, Rephartox B.V]. Hydroxylated coumarins, a type of phytoalecins are produced by many plants as defense in response to microbial infection. A polyphenolic phytoalexin resveratrol (3,5,4'-trihydroxystilbene) is produced by the enzyme stilbene synthase, in cis- (Z) and trans- (E) form. The transform can undergo isomerisation to the cis- form in presence of heat or ultraviolet irradiation in grapes, berries, plums, peanuts, Vaccinium species, pines and knotweed. Resveratrol having anti-cancer, anti-inflammatory, anti-aging and neuroprotective activities is reported to inhibits HSV-1 and HSV-2 replication by suppressing NF-kappaB activation, an essential step of its life cycle during infection [Gregory et al., 2004]. Further study by Electromobility shift assays demonstrated that resveratrol suppressed NFkB activation of both HSV types as well as acyclovir resistant HSV-1 in a dose dependent and reversible manner, impairing expression of essential immediate-early, early and late genes and synthesis of DNA [Faith et al., 2006]. Similarly oxyresveratrol of Millettia erythrocalyx and Artocarpus lakoocha inhibited herpesvirus replication [Likhitwitayawuid et al., 2005a].

Essential oils and terpenoids Many essential oils containing isoprene derivatives are called terpenes, and when they have additional elements like oxygen they are termed as terpenoids. Both essential oils and terpenoids have antiviral activities against many viruses [Khan et al., 2005]. The monoterpenes essential oil Isoborneol, isolated from Australian tree tea Melaleuca alternifolia and many other plants, can block

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HSV-1 replication within 30 min of exposure. Isoborneol can inhibit glycosylation of viral polypeptides and gB without affecting the glycosylation of host cell polypeptides; while pulegone of the same species inhibit HSV-1 replication [Primo et al., 2001], indicating that isoborneol is an interesting compound for anti-HSV drug development. On the otherhand Santalum album oil had a dose dependent anti-HSV-1 activity, but essential oil of Italian food plant Santolina insularis inhibits cell-to-cell transmission of herpes viruses [Khan et al., 2005]. Again, the terpinen-4-ol of M. alternifolia oil used as antimicrobial preservative in cosmetics, exhibited strong virucidal activity against HSV-1 and HSV-2, and at noncytotoxic dose plaque formation was reduced by 98.2% (HSV1) and 93.0% (HSV-2); the viruses before or during adsorption by viral suspension tests. The IC50 concentration for plaque formation in RC 37 cell was 0.0008 (HSV-1) and 0.009% (HSV-2). Although the active antiherpes components of tree tea oil is not yet fully characterized but its application in recurrent herpes infection is promising. The essential oil of Artemisia douglasiana and Eupatorium patens inhibit HSV-1 (65-125 ppm) while Melissa officinalis oil inhibits HSV-2 replication [Khan et al., 2005; Chattopadhyay & Khan, 2008]; while the volatile oil 1,8-cineole and terpinen-4-ol of Melaleuca armillaris are the effective virucidal agents [Farag et al., 2004]. The essential oil obtained from Artemisia arborescence is reported to inhibit HSV-1 (IC50 24µg/ml) and HSV-2 (4.1 µg/ml) at CC50 132 µg/ml in MTT reduction assay and CC50/IC50 ratio of 55 for HSV-1, and 32.2 for HSV-2 with a direct virucidal effect, inhibiting the virus and cell to cell virus diffusion [Saddi et al., 2007]. A diterpenoid scopadulcic acid B, isolated from Scoparia dulcis L. is found to inhibit HSV-1 replication in vitro by interfering with early events of viral growth, while in hamster it delayed the appearance of herpetic lesions and the survival time at 100-200 mg/kg/day dose [Chattopadhyay & Khan, 2008]. Another diterpene putranjivain A of Euphorbia jolkini significantly reduced infectivity (IC50 6.3µM), attachment and penetration and the late stage of HSV-2 replication [Cheng et al., 2004]. The stem bark of Glyptopetalum sclerocarpum containing a sesquiterpene sclerocarpic acid, showed antiviral activity against HSV-1 and HSV-2 [Chattopadhyay, 2006]; while the quassinoids inhibit Epstein-Barr virus [Chattopadhyay & Khan, 2008]. It was observed that the triterpenes betulinic acid and moronic acid of Rhus javanica inhibit acyclovir-resistant, thymidine kinase-deficient and wild-type HSV-1 (EC50 of 2.6-3.9µg/ml). Further study revealed that the oral administered of moronic acid to cutaneously infected mice with HSV-1 significantly retarded skin lesions and/or prolonged the mean survival times at non-cytotoxic dose by suppressing virus yields in the brain [Kurokawa et al., 1999]. Hence, moronic acid can be a good anti-HSV lead with a different mechanism of action than acyclovir. Interestingly both aqueous and ethanolic extract of Ocimum basilicum (basil), an Indian medicine, along with purified apigenin, linalool and ursolic acid showed strong anti-HSV-1 activity.

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The ursolic acid showed the strongest activity against HSV-1 (EC50 6.6mg/L), while apigenin revealed the highest activity against HSV-2, by blocking the infection process and the replication phase [Chiang et al., 2005], indicating its potential candidature as antiherpes agent. Again the tetracyclic triterpenes lupenone of Euphorbia segetalis strongly inhibit plaque formation of HSV-1 and HSV-2 [Chattopadhyay, 2006]. It was found that a tetranortriterpenoid (limonoid) meliacine (7α-acetoxymeliaca-14,20,22-trien-3-one), isolated from Melia azedarach L. leaves inhibit HSV-1 replication in vitro. Further study revealed that meliacine exerts potent anti-HSV-1 activity by inhibiting ICPs, DNA synthesis, nucleocapsids assembly and late stages of HSV life cycle. Ultrastructural analysis of infected cells showed that meliacine treatment results accumulation of unenveloped nucleocapsids in the cytoplasmic vesicles of infected cells, suggesting that it also block the syntheses of viral DNA and its maturation [Alche et al., 2000]. The corneal herpetic stromal keratitis, a leading cause of human blindness, when developed in Balb/c mice by HSV-1 (KOS strain) and treated with meliacine topically, it was found that meliacine significantly reduced the development of keratitis and the histological damage in corneas. The results revealed that the viral titers in eyes of infected and treated mice were 2-fold lower than the corresponding controls [Alche et al., 2000]. Another limonoid terpene 28-deacetylsendanin of Melia azedarach fruit showed anti-HSV-1 activity (IC50 1.46µg/ml) at 400µg/ml.

Saponin The triterpenoid saponin of oleanane group inhibits DNA synthesis, while the ursane group inhibits capsid protein synthesis of HSV-1. Apers et al [2001] observed that alcoholic extract of Maesa lanceolata leaves contain a maesasaponin VI2 that showed virucidal effect against HSV (reduction factor ≥103 at 50 µg/ml), due to diacylation of replicating enzymes. On the otherhand the saponin glycosides (spirostane, tomatidane, solasodane, nuatigenin, ergostane and furostane dimers) of some Solanum species inhibit HSV-1, probably for their oligosacchride moiety [Khan et al., 2005].

Tannins Tannins are polymeric phenolics (MW 500-3000), when combine with the collagen of animal skins it can form leather, or precipitate gelatin from solution. Tannins are grouped into hydrolyzable and condensed tannins. Hydrolyzable tannins are based on gallic acid, while the condensed tannins are derived from flavonoid monomers. It is believed that the consumption of tannin-containing beverages, like green teas and red wines, can cure or prevent a variety of illnesss. Some recent reviews reported that tannins can stimulate phagocytic cells, inhibit

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tumor and wide range of microbes by forming complex with microbial proteins. It can inactivate virus adsorption, transport proteins, polysaccharides and viral enzymes [Chattopadhyay, 2006; Chattopadhyay & Naik, 2007]. It was found that Eucalyptus grandis extract contains euglobal-G1 and G3 that can inhibit EpsteinBarr virus, like quassinoids (ailantinol B, ailantinol C, and ailanthone). While the eugenol and eugeniin from Geum japonicum and Syzygium aromaticum block viral DNA polymerase and thereby inhibit acyclovir-resistant TK-deficient HSV-1, wild HSV-2 and Epstein-Barr virus multiplication [Chattopadhyay & Khan, 2008]. A detailed study on ellagitannins from Phyllanthus myrtifolius and P. urinaria (Euphorbiaceae) demonstrated the inhibition of Epstein-Barr virus DNA polymerase, probably due to corilagin moiety of these tannins. Terminalia arjuna Linn bark contains the virucidal hydrolysable tannin casuarinin. At 25µM dose casuarinin reduce viral titers upto 105 fold (IC50 3.6±0.9 and 1.5±0.2) in XTT and Plaque reduction assay, and at C50 89±1.0µM it inhibit HSV-2 attachment and penetration [Cheng et al., 2002]. In a bioassay-guided fractionation study the alcoholic extract of Limonium sinensi significantly suppressed HSV-1 multiplication due to a tannin samarangenin B. While extracts of Bupleurum rigidum and Scrophularia scorodonia inhibit the cellular viability of HSV-1at the non-toxic limit concentrations due to some saikosaponins, iridoids and phenylpropanoid glycoside like verbascoside (53.6%), 8acetylharpagide (32.1%), harpagoside (43.3%), scorodioside (47.8%) and buddlejasaponin IV (56.9%) at 25-500µg/ml [Kuo et al., 2002; Chattopadhyay & Khan, 2008].

Lignans Lignans are dimmers of phenylpropanoids with two C-6, C-3 units linked to β, and β‫׳‬, and are reported to have antiviral activities. The Larrea tridentates, Rhinacanthus nasutus and Kadsura matsudai extracts showed anti-herpes activities due to the lignan nordehydroguanoferate; while Rhus javanica lignans exhibit anti-HSV-2 activity similar to acyclovir. The cones of various pine trees (Pinus parviflora Sieb. et Zucc, P. densiflora Sieb. et Zucc., P. thunbergii Parl., P. elliottii var. Elliottii, P. taeda L., P. caribaea var. Hondurenses, P. sylvestris L.) or seed shell of P. parviflora and P. armadii Franch inhibited the proliferation of HSV, due to lignin-carbohydrate complexes. It was observed that the antiHSV activity was maximum when lignin was added at the time of virus adsorption [Sakagami et al., 2005]. In a bioassay-guided study with the Chinese herb Chamaecyparis obtusa, yielded yatein (C22H23O7; MW 399), a lignan that significantly inhibit HSV-1 multiplication in HeLa cells. It was found that yatein can impended the levels of gB and gC mRNA expression in HeLa cells, can arrest DNA replication, inhibit the expression of alpha gene, ICP0 and ICP4 genes, arresting DNA synthesis and expression of structural protein of HSV-1 [Kuo et al., 2006].

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Alkaloids Alkaloids are the heterocyclic nitrogen compounds and have antiviral activities against many viruses. The MeOH extract of a Chinese and Mongolian folklore Stephania cepharantha HAYATA root tubers, contain bisbenzylisoquinoline, protoberberine, morphine and proaporphine alkaloids with in vivo anti-tumor, antiinflammatory, antiallergic and immunomodulating activity showed potent anti-HSV-1 activity (IC5018 µg/ml). Further study revealed that the biscoclaurine alkaloid cepharanthine of S. cepharantha inhibited HSV-1 [Nawawi et al., 1999; Chattopadhyay, 2006]. Again N-methylcrotsparine showed antiviral active against HSV-1, TK-deficient (acyclovir resistant) HSV-1, and HSV-2 with IC50 7.8, 9.9 and 8.7 µg/ml respectively by plaque reduction assay; while the alkaloid FK-3000 was more promising anti-HSV candidate [Nawawi et al., 1999]. As cepharanthine had strong antiviral activity against both RNA and DNA viruses and hence, it may be a potential lead for new antiviral development. The ethanol extracts of Nelumbo nucifera (Lotus) Gaertn seed, used throughout China, Egypt, Middle East and India for over 1000 years in gastrointestinal and bleeding disorders, can significantly inhibit HSV-1 and HSV-2 replication at 100µg/ml concentration with IC50 50µg/ml and IC50 62.0±8.9µg/ml respectively; while its subfractions NN-B-5 from bioactive butanol part showed the highest activity at 50 µg/ml by inhibiting TK deficient HSV-1 replication in HeLa cells [Kuo et al., 2005]. The Ophirrhoza nicobarica, folklore of Shompen and Nicobarese tribes of Grate Nicobar Islands, India contain a β-carboline alkoloid Harmine, which is found to inhibit plaque formation of HSV-1 and HSV-2, and delayed the eclipse phase of HSV replication at 300µg/ml [Chattopadhyay et al., 2006].

Lectins, polypeptides and polysaccharides Lectins are natural proteins that target the sugar moieties of various glycoproteins and are widespread in higher plants. The most prominent class of monocots mannose-specific lectins of Cymbidium hybrid, Epipactis helleborine and Listera ovata showed a marked anti-HCMV activity, while the (GlcNAc) n-specific lectin from Urtica dioica was inhibitory to CMV-induced cytopathicity at an EC50 of 0.3-9 µg/ml [Balzarini et al., 1992]. The antimicrobial peptides are often positively charged and contain disulfide bonds. Recently a 2kD peptide from seeds of Sorghum bicolor L strongly inhibited the replication of HSV-1, dose-dependently, at 10-50µM, after incubation, with EC50 and EC90 values of 6.25 and 15.25µM, respectively. The IC50 value of the 2kD peptide against Vero cells was 250µM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent CPE reduction at 6.25 to 50µM. The presence of this peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC50 values of

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12.5, 6.25, and 6.25µM, respectively. Similar results were observed when this 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1, while this peptide had weak activity against non-enveloped poliovirus type 1. Thus these results indicate that 2kD peptide of Sorghum bicolor was able to inhibit the initiation and the spread of infection, and also had an in vitro prophylactic effect against HSV-1 infection [Filho et al., 2008]. The sulphated galactans from a marine alga Bostrychia montagnei are found to inhibit herpes virus multiplication in cell culture and replication in vitro [Duarte et al., 2001]. While the polysaccharide of Cedrela tubiflora leaves inhibit HSV-2 replication at non-cytotoxic dose, indicating that the anti-HSV activity of polysaccharides correlates with molecular weight and sulfate content. Recently Thompson and Dragar [2004] reported that a seaweed Undaria pinnatida containing a sulfated polysaccharide galactofucan had anti-HSV-1activity. Further study revealed that it inhibit viral binding and entry into the host cell (IC50 32µg/ml), and highly active against HSV-2 (IC50 0.5µg/ml; P<0.001). On the other hand a water soluble anionic polysaccharide from Prunella vulgaris L. (Labiatae) inhibit HSV-1 at 100 µg/ml and HSV-2 at 10 µg/ml by plaque inhibition assay [Xu et al., 1999] while the anionic polysaccharide of the same herb collected from Japan showed specific anti-HSV activity (IC50 10µg/ml) by competing for cell surface receptor [Chiu et al., 2004]. Interestingly the polysaccharide fraction prepared from P. vulgaris revealed that the viral antigen increased time-dependently in the infected HSV-1 and HSV-2 cells, and polysaccharide fraction (25-100µg/ml) reduced such antigen expression (EC50 20.6 and 20.1µg/ml), along with the antigen expression of acyclovir-resistant strain of HSV-1 (24.8-92.6%). It shows that polysaccharide fraction has a different mode of anti-HSV action from acyclovir [Chiu et al., 2004].

Conclusions The diseases caused by human herpesviruses (HSV, VZV, CMV, EBV and Kaposi’s Sarcoma- associated herpes virus) are the global concern for their contiguous nature, recurrence ability, mutability and silent epidemic potential. These diseases are sometimes fatal, especially in neonates and immunocompromised patients. The ‘cure’ or development of effective vaccines are not yet possible, hence, cost effective natural or complementary antivirals to prevent resistance development with reduced toxicity are of immediate need. Moreover, the antivirals used against herpesviruses are expensive with high toxicity. Therefore, development of safe, effective and inexpensive antivirals is among the top global priorities of drug development. Hence, scientists from divergent fields are investigating herbal medicinal products, with an eye to their antiherpesvirus usefulness. The extensive research for last 4 decades leads to the discovery of a few agents with antiherpes virus activity. The polyphenolic Caffeic acid, CAPE, Catechin, Quercetin, Epicatechin dimers, Procyanidins, Apigenin,

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Robustaflavone, Glycyrrhizin, Phloroglucinols, Epiafzelechin; triterpenoids Ursolic acid, Betulinic acid, Glycyrrhetinic acid, Resveratrol; essential oils Eugenol, Isoborneol; tannins like Ellagitannin and Samarangenin B; lignan Yatein; alkaloids Cepharanthine and Harmine; Sulfated galactans and a few Lectins can be used as lead compounds because of their specific in vitro and in vivo activity with low toxicity and significant structure activity relationship. Some natural compounds are reported to have potential to interfere with particular herpesvirus enzymes (DNA polymerase inhibition by eugenol, eugeniin and ellagitannins while helicase-primase inhibition by thiazolylsulphonamide), RNA and protein synthesis (CAPE, procyanidin C1), DNA replication (prodelphinidine-di-O-gallate, quercetin, epiafzelechin, isoborneol, samarangenin B, resveratrol, scopadulcic acid, apigenin, ursolic acid, putranjivain A), cellular fusion (essential oils, epicatechin gallate, galangin), attachment and penetration (prodelphinidine di-o-gallate, epicaafzetechin, casuarinin, putranjivain A, procyanidin A1, polyssacharride-lignan complex), virus entry and target cell binding (flavones, flavonols, terpenoid). Some may interfere in more than one step of herpesvirus life cycle, resulting complementary mechanisms of action to the existing antiviral drugs. Although no plant-derived drug is currently in clinical use to treat herpesvirus diseases, thiazolylsulfonamide, n-docosanol, phloroglucinol, prodelphinidin-di-O-gallate are promising herbal product/natural product-derived candidates in preclinical and clinical trials. Interestingly most of these compounds can block virus entry into host cells and or specific cellular enzymes, alone or sometimes in combination with acyclovir, which is a very important aspect in the context of viral drug resistance and limited life span of antiviral drugs. The compounds having alternative mechanism of action, unlike synthetic antivirals, can be the potential candidates to tackle the threats posed by drug resistant herpesviruses, as it is quite difficult to eliminate herpesvirus diseases by the available antivirals till date.

Acknowledgement The authors wish to acknowledge Dr. Sujit K. Bhattacharya, the Additional Director General, Indian Council of Medical Research, New Delhi and the Officer-in Charge, ICMR Virus Unit, Kolkata, for their kind help and encouragement during the preparation of this manuscript.

References 1. 2.

Alche L E, Berra A, Veloso M J, Coto C E. (2000). Treatment with meliacine, a plant derived antiviral, prevents the development of herpetic stromal keratitis in mice. J Med Virol 61(4):474-480. Apers S, Baronikova S, Sindambiwe J B, Witvrouw M, De Clercq E, Vanden Berghe D, Van Marck E, Vlietinck A, Pieters L. (2001). Antiviral, haemolytic and

142

3. 4.

5. 6.

7. 8. 9.

10. 11.

12. 13. 14.

15.

Debprasad Chattopadhyay et al.

molluscicidal activities of triterpenoid saponins from Maesa lanceolata: establishment of structure -activity relationship. Planta Medica 67:528-532. Apers S, Cimanga K, Vanden Berghe D, Meenen E V, Longanga A O, Foriers A, Vlietinck A, Pieters L. (2002). Antiviral activity of Simalikalactone D, a quassinoid from Quassia africana. Planta Medica 68:20-24. Aoki, H., Akaike, T., Abe, K., Kuroda, M., Arai, S., Okamura, R. I., Negi, A., and Maeda, H. (1995). Antiviral effect of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo. Antimicrob. Agents Chemother., 39, 846-849. Arisawa M, Fujita A, Hayashi T, Hayashi K, Ochiai H, Morita N. (1990). Cytotoxic and antiherpetic activity of phloroglucinol derivatives from Mallotus japonicus (Euphorbiaceae). Chem Pharm Bull (Tokyo) 38(6):1624-1626. PMID: 2170038. Balzarini J, Neyts J, Schols D, Hosoya M, Van Damme E, Peumans W, De Clercq E. (1992). The mannose specific plant lectins from Cymbidium hybrid and Epipactis helleborine and the (N-acetylglucosamine) n-specific plant lectins from Urtica dioca are potent and selective inhibitors of HIV and cytomegalovirus replication in vitro. Antiviral Res 18(2): 191-207. Betz U A K, Fischer R, Kleymann G, Hendrix M, Rübsamen-Waigmann H. (2002). Potent in vivo antiviral activity of the herpes simplex virus primase-helicase inhibitor BAY 57-1293. Antimicrob Agents Chemother 46: 1766-1772. Bourne K Z, Bourne N, Reising S F, Stanberry L R. (1999). Plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2. Antiviral Res. 42, 219-226. Buckwold V E, Wilson R J, Nalca A, Beer B B, Voss T G, Turpin J A, Buckheit R W, Wei J, Wenzel-Mathers M, Walton E M, Smith R J, Pallansch M, Ward P, Wells J, Chuvala L, Sloane S, Paulman R, Russell J, Hartman T, Ptak R (2004). Antiviral activity of Hop constituents against a series of DNA and RNA viruses. Antiviral Res 61(1): 57-62. Bunyapraphatsara N, Dechsree S, Yoosook C, Herunsalee A¸ Panpisutchai Y. (2000). Anti-herpes simplex virus activity of Maclura cochinchinensis. Phytomedicine 6: 421-424. Chattopadhyay D. (2006). Ethnomedicinal Antivirals: Scope and Opportunity. Chapter 15. In: Modern Phytomedicine: Turning Medicinal Plants into Drugs. pp. 313-338. Ahmad I., Aquil F., Owais M. (Eds.), ISBN: 978-3-527-31530-7. WileyVCH Verlag GmbH & Co. KGaA, Boschstraße 12, Weinheim, Germany. Chattopadhyay D, Arunachalam G, Mandal A B, Bhattacharya S K. (2006). Dose dependent therapeutic antiinfectives from ethnomedicines of Bay Islands. Chemother 52: 151-157. Chattopadhyay D, Naik T N. (2007). Antivirals of Ethnomedicinal Origin: Structureactivity Relationship and Scope. Mini Rev Med Chem 7(3): 275-301 (Review). Chattopadhyay D, Bhattacharya S K. (2008). Ethnopharmacology: A new engine for the development of antivirals from naturaceuticals. In: Handbook of Ethnopharmacology, Eddouks M. Ed., Research Signpost, Trivandrum, India, ISBN: 978-81-308-0213-8, pp. 129-197. Chattopadhyay D, Khan M T H. (2008). Ethnomedicines and ethnomedicinal phytophores against Herpesviruses. Biotechnology Annual Review, Volume 14,

Ethnomedicinal products in the development of anti-herpesvirus agents

16.

17. 18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29.

143

Chapter 12: 297-349 (in Press). ISSN 1387-2656 DOI: 10.1016/S13872656(08)00012-4. Chen T, Jia W, Yang F, Xie Y, Yang W, Zeng W, Zhang Z, Li H, Jiang S, Yang Z, Chen J. (2004). Experimental study on the antiviral mechanism of Ceratostigma willmattianum against herpes simplex virus type 1 in vitro. Zhongguo Zhong Yao Za Zhi. (China J. Chinese Materia Medica). 29:882-886. 15575210. Cheng H Y, Lin C C, Lin T C. (2002). Antiherpes simplex virus type 2 activity of Casuarinin from the bark of Terminalia arjuna Linn. Antiviral Res 55(3):447-455. PMID: 12206882. Cheng H Y, Lin T C, Ishimaru K, Yang C M, Wang K C, Lin C C. (2003). In vitro antiviral activity of prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra. Planta Med 69(10): 953-956. Cheng H Y, Lin T C, Yang C M, Wang K C, Lin L T, Lin C C. (2004). Putranjivain A from Euphorbia jolkini inhibits both virus entry and late stage replication of herpes simplex virus type 2 in vitro. J Antimicrob Chemother 53: 577-583. Cheng H Y, and LinChun C. (2005). The antiherpes simplex viruses activity of extracts and compounds of natural products. Journal of Traditional Medicines 22 (Suppl.1):129-132. Cheng H Y, Yang C M, Lin T C, Shieh D E, Lin C C. (2006). ent-Epiafzelechin(4alpha-->8)-epiafzelechin extracted from Cassia javanica inhibits HSV-2 replication. J Med Microbio. 55: 201-206. Chiang L C, Chiang W, Chang M Y, Ng L T, Lin C C. (2002). Antiviral activity of Plantago major extracts and related compounds in vitro. Antiviral Res. 55:53-62. PMID: 12076751. Chiang L C, Cheng H Y, Liu M C, Chiang W, Lin C C. (2003). In vitro antiherpes simplex viruses and anti-adenoviruses activity of twelve traditionally used medicinal plants in Taiwan. Biol Pharm Bull 26(11):1600-1604. PMID: 12729671. Chiang L C, Ng L T, Cheng P W, Chiang W, Lin C C. (2005). Antiviral activities of extracts and selected pure constituents of Ocimum basilicum. Clin Exp Pharmacol Physiol 32: 811-816. Chiu Lawrence C-M, Zhu W, Ooi Vincent E-C. (2004). A polysaccharide fraction from medicinal herb Prunella vulgaris downregulates the expression of herpes simplex virus antigen in Vero cells. J Ethnopharmacol 93(1): 63-68. Corey L, Wald A, Celum C L, Quinn T C. (2004). The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J. Acquir. Immune Defic. Syndr 35: 435-445. Cos P, de Bruyne T, Hermans N, Apers S, Vanden Berghe D, Vlietinck A J. (2004). Proanthocyanidins in health care: current and new trends. In: Current medicinal chemistry 11: 1345-1359. Dong, Y., Li, H., Yao, Z., Tian, W., Han, Z., Qiu, H., and Piao, Y. (2004). The respiratory syncytial virus (RSV) effect of Radix Glycyrrhizae in vitro. Zhong Yao Cai., 27(6): 425-417. Duarte M E, Noseda D G, Noseda M D, Tulio S, Pujil C A, Damonte E B. (2001). Inhibitory effect of sulfated galactans from the marine alga Bostrychia montagnei on herpes simplex virus replication in vitro. Phytomedicine 8(1): 53-58.

144

Debprasad Chattopadhyay et al.

30. Faith S A, Sweet T J, Bailey E, Booth T, Docherty J J. (2006). Resveratrol suppresses nuclear factor-kappaB in herpes simplex virus infected cells. Antiviral Res 72(3): 24251. Epub 2006 Jul 14. 31. Farag R S, Shalaby A S, El-Baroty G A, Ibrahim N A, Ali, M A, Hassan E M. (2004). Chemical and biological evaluation of the essential oils of different Melaleuca species. Phytother Res 18: 30-35. 32. Felser J, Kichington P R, Inchauspe G, Straus S E, Ostrove J M. (1988). Cell line containing varicella-zoster virus open reading frame 62 and expressing the 'IE' 175 protein complement ICP4 mutants of herpes simplex virus type 1. J Virol 62: 20762082. 33. Ferrea G, Canessa A, Sampietro F, Cruciani M, Romussi G, Bassetti D. (1993). In vitro activity of a Combretum micranthum extract against herpes simplex virus types 1 and 2. Antivir Res 21: 317-325. 34. Fortin H, Vigor C, Lohezic L D F, Robinm V, Le Bossem B, Boustiem J, Amoros M. (2002). In vitro antiviral activity of thirty-six plants from La Reunion Island. Fitoterapia, 73(4): 346-350. 35. Filho I C, Cortez D A G, Ueda-Nakamura T, Nakamura C V, Filho B P D. (2008). Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor. Phytomedicine 15(3): 202-208. 36. Glatthaar-Saalmuller B, Sacher F, Esperester A. (2001). Antiviral activity of an extract derived from roots of Eleutherococcus senticosus. Antivir. Res., 50: 223-228. 37. Gius D, Laimins L A. (1989). Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and phorbol ester. J. Virol. 63: 555-563. 38. Gordon Y J, Armstrong J A, Brown S I, Becker Y (1983). The role of herpesvirus type 1 thymidine kinase in experimental ocular infections. Am J Ophthalmol 95:175181. 39. Gordon Y J, Araullo-Cruz T P, Romanowski E, Ruziczka L, Balouris C, Oren J, Cheng K P, Kim S. (1986). The development of an improved reactivation model for the study of HSV-1 latency. Invest Ophthalmol Vis Sci 27:1230-1234. 40. Gordon Y J, Romanowski E, Araullo-Cruz T. (1990). A Fast, Simple Reactivation Method for the Study of HSV-1 Latency in the Rabbit Ocular Model. Investigative Ophthalmology & Visual Science, Vol. 31(5): 921-924. 41. Gregory D, Hargett D, Holmes D, Money E, Bachenheimer S L. (2004). Efficient replication by HSV-1 involves activation of the IkappaB kinase-IkappaB-RelA/p65 pathway. J. Virol. 78: 13582-13590. 42. Habif T. (2004). Warts, Herpes Simplex, and Other Viral Infections. Clinical Dermatology, 4th Edition. Ed. Thomas Habif. New York, Mosby, pp. 381-388. 43. Hsiang C Y, Hsieh C L, Wu S L, Lai I L, Ho T Y. (2001). Inhibitory effect of antipyretic and anti-inflammatory herbs on herpes simplex virus replication. Am J Chin Med. 29(3-4):459-467. 44. Huleihel M, Isanu V. (2002). Anti-Herpes Simplex Virus effect of an aqueous extract of Propolis. IMAJ 4:923-927. 45. Huy L D, Caple R, Kamperdick C, Diep N T, Karim R. (2004). Isomeranzin against Herpes simplex virus in vitro from Clausena heptaphylla (Roxb.) W. & Arn: isolation, structure and biological assay. Journal of Chemistry 42 (1): 115-120.

Ethnomedicinal products in the development of anti-herpesvirus agents

145

46. Ikeda T, Ando J, Miyazono A, Zhu XH, Tsumagari H. Nohara T, Yokomizo K, Uyeda M. (2000). Anti-herpes virus activity of Solanum steroidal glycosides. Biol Pharm Bull. 23(3): 363-364. 47. Ishikawa T, Nishigaya K, Takami K, Uchikoshi H, Chen I S, Tsai I L. (2004). Isolation of Salicin derivatives from Homalium cochinchinensis and their antiviral activities. J. Nat. Prod., 67(4): 659-663. 48. Jassim S A A, Naji M A. (2003). Novel antiviral agents: a medicinal plant perspectiveJ. Appl. Microbiol., 95(3), 412-427. 49. Kaul T N Jr, Middletown E, Ogra P L (1985). Antiviral effect of flavonoids on human viruses. J. Med. Virol. 15:71-79. 50. Khan M T, Ather A, Thompson K D, Gambari R. (2005). Extracts and molecules from medicinal plants against herpes simplex viruses. Antivir Res 67: 107-119. 51. Kira T, Kakefuda A, Awano H, Shuto S, Matsuda A, Baba M, Saneyoshi M, Shigeta S. (1995). Development of anti-HSV screening system using suspension cell line and screening several nucleoside analogues in this method. Antiviral Res. 26(3): 309. 52. Kuo Y C, Lin L C, Tsai W J, Chou C J, Kung S H, Ho Y H. (2002). Samaragenin B identified from Limonium sinensis suppressed heroes simplex virus type 1 replication in Vero cells by regulation of viral macromolecular synthesis. Antimicrob. Agents Chemother 46: 2854-2864. 53. Kuo Y C, Lin Y L, Liu C P, Tsai W J. (2005). Herpes simplex virus type 1 propagation in HeLa cells interrupted by Nelumbo nucifera. J Biomed Sci 12(6): 1021-1034. 54. Kuo Y C, Kuo Y H, Lin Y L, Tsai W J. (2006). Yatein from Chamaecyparis obtusa suppresses herpes simplex virus type 1 replication in HeLa cells by interruption the immediate-early gene expression. Antiviral Res. 70(3): 112-120. 55. Kuo et al., 2006. 56. Kurokawa M, Basnet P, Ohsugi M, Hozumi T, Kadota S, Namba T, Kawana T, Shiraki K. (1999). Anti-herpes simplex virus activity of moronic acid purified from Rhus javanica in vitro and in vivo. J Pharmacol Exp Ther 289: 72-78. 57. Likhitwitayawuid K, Supudompol B, Sritularak B, Lipipun V, Rapp K, Schinazi RF. (2005). Phenolics with Anti-HSV and Anti-HIV Activities from Artocarpus gomezianus, Mallotus pallidus, and Triphasia trifolia. Pharmaceutical Biology, 43(8): 651-657. 58. Likhitwitayawuid K, Sritularak B, Benchanak K, Lipipun V, Mathew J, Schinazi R F(2005a). Phenolics with antiviral activity of Millettia erythrocalyx and Artocarpus lakoocha. Nat Prod Res 19: 177-182. 59. Lyu S-Y, Rhim J-Y, Park W-B. (2005). Antiherpetic Activities of Flavonoids against Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) In Vitro. Arch Pharm Res 28(11): 1293-1301. 60. Marchetti M, Pisani S, Pietropaola V, Seganti L, Nicoletti R, Degener A, Orsi N. (1996). Antiviral effect of a polysaccharide from Sclerotium glucanicum towards herpes simplex virus type 1 infection. Planta Med 62: 303-307. 61. McCutcheon A R, Roberts T E, Gibbons E, Ellis S M, Babiuk L A, Hancock R E. and Towers G H. (1995) Antiviral screening of British Columbian medicinal plants. J Ethnopharmacol 49, 101-110.

146

Debprasad Chattopadhyay et al.

62. Nawawi A, Ma C, Nakamura N, Hattori M, Kurokawa M, Shirak K, Kashiwada N, Ono M. (1999). Anti-herpes simplex virus activity of alkaloids isolated from Stephania cepharantha. Biol Phar Bull. 22(3): 268-274. 63. Ostrove J M, Leonard J, Weck K E, Radson A B, Gendelman H E. (1987). Activation of the human immunodeficiency virus by herpes simplex virus type 1. J Virol 61: 3726-3732. 64. Primo V, Rovera M, Zanon S, Oliva M, Demo M, Daghero J, Sabini L (2001). Determination of the antibacterial and antiviral activity of the essential oil from Minthostachys verticillata (Griseb.) Epling. Revista Argentina de Microbiol. 33(2): 113-117. 65. Rajbhandari M, Wegner U, Julich M, Schopke T, Mentel R. (2001). Screening of Nepalese medicinal plants for antiviral activity. J. Ethnopharmacol., 74(3), 251-255. 66. Rao A R, Kumar S S V, Paramasivam T B, Kamalakshi S, Parashuraman A R, Shanta B. (1969). Study of antiviral activity of tender leaves of margosa tree (Melia azadirachta) on vaccinia and variola virus- a preliminary report, Indian J. Med. Res., 57(3): 495-502. 67. Sacks S L, Thisted R A, Jones T M, Barbarash R A, Mikolich D J, Ruoff G E, Jorizzo J L, Gunnill L B, Katz D H, Khalil M H, Morrow P R, Yakatan G J, Pope L E Berg J E. (2001). Clinical efficacy of topical docosanol 10% cream for herpes simplex labialis: a multicenter, randomized, placebo-controlled trial. J. Am. Acad. Dermatol. 45: 222-230. 68. Saddi M, Sanna A, Cottiglia F, Chisu L, Casu L, Bonsignore L, De Logu A. (2007). Antiherpes activity of Artemisia arborescence essential oil and inhibition of lateral diffusion in Vero cells. Ann Clin Microbiol Antimicrob. 6(1): 10. 69. Shahat A A, Cos P, De Bruyne T, Apers S, Hammouda F M, Ismail S I, Azzam S, Claeys M, Goovaerts E, Pieters L, Vanden Berghe D, Vlietinck A J. (2002). Antiviral and antioxidant activity of flavonoids and proanthocyanidins from Crataegus sinaica. Planta Med 68: 539-541. 70. Sakagami H, Hashimoto K, Suzuki F, Ogiwara T, Satoh K, Ito H, Hatano T, Takashi Y, Fujisawa S. (2005). Molecular requirements of lignin窶田arbohydrate complexes for expression of unique biological activities. Phytochemistry 66(17): 2108-2120. Review. 71. Salem M L, Hossain M S. (2000). Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. Inter J Immunopharmacol 22(9): 729-740. 72. Sandri-Goldin R M. (editor). (2006). Alpha Herpesviruses: Molecular and Cellular Biology. Caister Academic Press. ISBN 978-1-904455-09-7. 73. Sarisky R T, Crosson P, Cano R, Quail M R, Nguyen T T, Wittrock R J, Bacon T H, Sacks S L, Caspers-Velu L, Hodinka R L, Leary J J. (2002). Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility. J Clin Virol 23: 191-200. 74. Serkedjieva J, Ivancheva S. (1999). Antiherpes virus activity of extracts from the medicinal plant Geranium sanguineum L. J Ethnopharmacol 64(1): 59-68. 75. Simﾃｵnes C M O, Amoros M, Girre L. (1999). Mechanism of antiviral activity of triterpenoid saponins. Phytother. Res 21: 317-325.

Ethnomedicinal products in the development of anti-herpesvirus agents

147

76. Sudo K, Konno K, Yokota T, Shigeta S. (1994). A sensitive assay system screening antiviral compounds against herpes simplex virus type 1 and type 2, J. Virol. Methods 49(2): 169-178. 77. Sydiskis R J, Owen D G, Lohr J L,. Rosler K H, Blomster R N. (1991). Inactivation of enveloped viruses by anthraquinones extracted from plants. Antimicrob Agents Chemother 35: 2463-2466. 78. Tao J, Hu Q, Yang J, Li R, Li X, Lu C, Chen C, Wang L, Shattock R. and Ben K. (2007). In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate Antiviral Research, 75(3): 227-233. 79. Thompson K D, Dragar C. (2004). Antiviral activity of Undaria pinnatifida against herpes simplex virus. Phytother Res. 18: 551-555. 80. Tshikalange T E, Meyer J J, Hussein A A. (2005). Antimicrobial activity, toxicity and the isolation of a bioactive compound from plants used to treat sexually transmitted diseases. J. Ethnopharmacol., 96(3): 515-519. 81. Utsunomiya T, Kobayashi M, Herndon D N, Pollard R B, Suzuki F. (1995). Glycyrrhizin (20 beta-carboxy-11-oxo-30-norolean-12-en-3 beta-yl-2-O-ﾎｲ-dglucopyranuronosyl-alpha-d-glucopyranosiduronic acid) improves the resistance of thermally injured mice to opportunistic infection of HSV-1. Immunol. Lett. 44 (1), 59-66. 82. Vanden Berghe D A R, Haemers A, Vlietinck A J. (1993). Antiviral agents from higher plants and an example of structure- activity relationship of 3-methoxyflavones. In: Colegate S M, Milyneux R J., editors. Bioactive Natural Products: Detection, Isolation, and Structural Determination. Boca Raton: CRC Press, 405-440. 83. Vlietinck A J, de Bruyne T, vanden Berghe DA. (1997). Plant substances as antiviral agents. Current Organic Chemistry, 1(1997), 307-344. 84. Yarnell E, Abascal K. (2005). Herbs for Treating Herpes Zoster Infections. Alternative & Complementary Therapies. 11(3): 131-134. 85. Yim K C, Carroll C J, Tuyama A, Cheshenko N, Carlucci M J, Porter D D, Prince G A., Herold B C. (2005). The Cotton Rat Provides a Novel Model To Study Genital Herpes Infection and To Evaluate Preventive Strategies. Journal of Virology 79(23):14632-14639. 86. Yoosook C, Bunyapraphatsara N, Boonyakiat Y, Kantasuk C. (2000). Anti-herpes simplex virus activities of crude water extracts of Thai medicinal plants. Phytomedicine 6(6): 411-419. PMID: 10715843. 87. Xu H X, Lee S H, Lee S F, White R L, and Blay J. (1999). Isolation and characterization of an anti HSV polysaccharide from Prunella vulgaris. Antiviral Res 44(1): 43-54. 88. Zhang Y, But P-H, Ooi E-C.V, Xu H-Xi, Delaney G D, Lee Spencer H S. and Lee S F. (2007). Chemical properties, mode of action, and in vivo anti-herpes activities of a lignin窶田arbohydrate complex from Prunella vulgaris. Antiviral Research 75(3): 242-249.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 149-178 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

5. Ethnomedicinal plants derived antibacterials and their prospects Maryam Zahin, F. Aqil*, M. S. A. Khan and Iqbal Ahmad Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University Aligarh-202002, India

Abstract. The use of plants, plant extracts or plant derived pure chemicals to treat disease is a therapeutic modality which has stood the test of time, indeed today many pharmacological classes of drugs include a natural product prototype. Ethnomedicinal plants contribution in primary health care are well established and there is a revival of interest in such plants as source of novel therapeutic compounds, evidence based herbal drug / phytomedicine. The uses of various medicinal plants in management of infectious diseases including bacterial are well known and validated in many cases. The antimicrobial activity of medicinal plants extracts or metabolites are comparable with antibiotic in several cases in vitro. The plant derived antibacterials are yet to be evaluated for their therapeutic efficacy. Development of multidrug resistance (MDR) in pathogenic bacteria has created immense clinical problem in the treatment of bacterial diseases which resulted in increase interest in plant antibacterial and antipathogenic compounds to combat resistance problem. Therefore, medicinal plants after primary antibacterial screening have now been evaluated for their activity against various multidrug resistant bacteria such as methicillin resistant Present address: Dr. F. Aqil, 580 S. Preston St., Brown Cancer Center, Baxter Research Building II, University of Louisville, Louisville, KY 40202, USA Correspondence/Reprint request: Dr. Iqbal Ahmad, Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002, India. E-mail: Iqbalahmad8@yahoo.co.in

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Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRSE), MDR Mycobacterium tuberculosis and enteric bacteria producing extended spectrum Î˛ lactamases and also resistant to non Î˛-lactam antibiotics, Pseudomonas aeruginosa, Helicobacter pylori and others. The present review has mainly focussed on the ethnomedicinal plants as a source of bioactive compounds especially antibacterial active against drug resistant bacteria reported from different parts of the world. Potential Indian medicinal plants with such activity have also been identified. It is interesting to observe that plants exhibiting broad spectrum antibacterial activity are effective almost equally both against drug resistant and drug sensitive bacteria. Antibacterial compounds identified have shown promising activity in vitro. However, efficacy of antibacterial compounds in vivo against bacterial infection, pharmacology, drug interactions and toxicity has to be evaluated. Similarly, plant derived products which can enhance the antibiotic activity, by their synergistic interaction or reversing drug resistance or decreasing virulence and pathogenicity are other alternative strategies to combat drug resistance problem. Further, clinical trials of standardized plant extracts and phytocompounds with their anti-infective potential have to be conducted to exploit them effectively.

1. Introduction Plants have formed the basis of traditional systems of medicine that have been in existence for thousands of years and continue to provide mankind with new remedies [1] The use of natural products with therapeutic properties is as ancient as human civilization and for a long time, mineral, plant and animal products were the main source of drugs [2]. For centuries, people have used plants for healing. Plant products as part of food or botanical portions and powder have been used with varying success to cure and prevent diseases throughout history [3]. Ethnomedicinal plants are used both for primary health care and for treating chronic diseases such as AIDS, cancer, hepatitis disorders, heart and old age related diseases like memory loss, osteoporosis and diabetic wound. In the Indian coded system (Ayurveda, Unani, Siddha, Amchi), Ayurveda currently utilizes as many as 1000 single drugs and over 8000 compound formulations of recognized merit .Similarly, 600-700 plants are utilized by other systems like Unani, Siddha and Amchi. [4]. Ayurveda is perhaps the most ancient of all traditional systems of medicine. Probably it is older than the Chinese medicine. It is considered to be the origin of systemized medicine. The first record written on clay tablets in cuneiform are from Mesopotamia and date from about 2600B.C.The history of medicinal plants date back to Rigveda, perhaps the oldest repository of human knowledge which was written around 4500-1600B.C. The Ayurveda, developed around 2500B.C. described the detail account of many drugs, which are even in use today. Ancient Ayurveda, includes the comprehensive work of Chraka (1000 B.C.) and Sushruta (800 B.C.) and

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provide the base for the materia medica. Hippocrates (in the late fifth century B.C.) mentioned 300-400 medicinal plants [5]. In the first century A.D., Dioscorides wrote Demateria medica, a medicinal plant catalogue which became the prototype for modern pharmacopias. The Bible offers description of approx. 30 healing plants. The Mohammedan culture enriched the vegetable Materia medica, which was further improved by those in Greece, Arabia and Persia [6]. Hakim Ibn Sina (981-1037C.E.) known as Avicena in the West laid down the foundation of the Greco-Arab system of herbal medicine (Unani Tibb), based on the philosophy of individualized treatment considering the variation amongst the individuals similar to concept of pharmacogenetics in conventional medicine. This system of medicine has found its root in India and become well established system of Indian medicine [7]. In the beginning of nineteenth century, John Flemming contributed a paper on the medicinal plants of India, a monograph of great value. The scattered information on the subject was collected and placed before the medicinal professional [8]. Little attention was paid to the medicinal plants of India before the establishment of Asiatic Society of Bengal by Sir William Jones [9]. Later on series of publications were made by various workers notably by Sir George Watt â&#x20AC;&#x153;A dictionary of economic products of Indiaâ&#x20AC;? published in 1889-1904, which provides the information on the medicinal plants as well as on its different parts. In the year 1962, Glossary of Indian Medicinal Plants was published by CSIR, India, giving traditional uses against various diseases and ailments and various active ingredients are also characterized [10]. The flora of India comprises about 45,000 of plants species, from unicellular blue green algae to flowering plants. These comprises approx. 11% of total species of flora of the world and out of which, about 4% are the flowering plants. The biogeographic position of India is unique as we have diverse ecosystem, ranging from the humid tropics of Western Ghats to the Alpine Zone of Himalaya and from the dry deserts of Rajasthan to the tidal mangroves of the Sunderbans and hence India is endowed with a rich flora [11]. Natural products and their derivatives represent more than 50% of all the drugs for the clinical use in the world. Higher plants contribute no less than 25% of the total. During the last 40 years, at least a dozen potential drugs have been derived from flowering plants. Plants have interdependent pathways that lead to the synthesis of numerous metabolites. Some of these metabolites are physiologically active and are being exploited for human and animal use, as they are being succeeded for various therapeutic properties and as source of new drugs. Recent findings showed their useful properties like anticancer, antitumour,

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antimutagenic, antioxidant, hepato-protective antiviral, antimalarial, antidysenteric, antiseptic, antistress and immunotherapeutic, antibacterial, antifungal and several more pharmacological actions and novel bioactivities such as anti quorum sensing activity effects of medicinal plants [12-19]. Excellent review articles on individual property of medicinal plants are available and is not the subject for this article. Therefore, we have tried to provide up to date information on medicinal plants especially on Indian medicinal plants used in traditional systems of medicine (Ayurveda and Unani Tibb) for their antibacterial activity with special reference to bioactive compounds characterized and their potency against multidrug resistant bacteria in vitro and possible exploitation in the management of bacterial infections caused by multidrug resistant bacteria.

2. Medicinal plants as source of bioactive and therapeutic compounds With the realization that ethnomedicinal plants are a repository of numerous potential medicines, concerted efforts from India, Pakistan, China and other countries around the globe have been made to evaluate scientifically these plants for various biological and therapeutic properties and alterative source of novel drugs. The traditional systems of medicine have now been recognized and accepted as alternative/complementary system of medicine for primary health care and for some chronic diseases. Although the first chemical substance to be isolated from plants was benzoic acid in 1560, the search for useful drugs of known structure did not begin until 1804 when morphine was separated from Papaver somniferum L. (Opium). Since then several drugs from higher plants have been discovered but less than 100 of defined structure are of common use today. About 55 drugs are widely employed in western medicine. Some of the drugs such as aspirin, atropine, artimesinin, colchicine, digoxin, ephedrine morphine, physostigmine, pilocarpine, quinine, quinidine, resperine, taxol, tubocurarine, and vincristine, and vinblastine are few examples of what medicinal plants given us in the past [7, 20].

3. Antibacterial activity of medicinal plants against multidrug resistant bacteria An increase in multi drug resistant bacteria has triggered immense interest in the search of new drugs or preparations from the natural sources including plants. Problematic groups of MDR bacteria include methicillin resistance S. aureus (MRSA), vancomycin resistant enterococci (VRE),

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extended spectrum Î˛-lactamases producing enteric bacteria (E. coli, Salmonella, Shigella and Klebsiella etc) and other multidrug resistant Pseudomonas, Campylobacter and Mycobacterium tuberculosis [21-25]. Antimicrobial activity of medicinal plants is widely spread and a large number of its secondary metabolites showed antimicrobial activity. There is a great structural diversity exist among antimicrobial phytocompounds [26]. Major groups of phytocompounds include essential oils and isolated compounds such as alkaloids, flavonoides, sesquiterpenes, lactones, diterpenes ,triterpenes or napthoquinones etc [27]. Considerable amount of work have been published on antimicrobial activity of medicinal plants from different parts of the world [14, 28-32]. Examples of such articles are from Brazil, Thailand, Turkey, India and so on [33-36]. Most of the studies are directed to see the activity against a variety of test bacteria including both pathogenic and non pathogenic strains. However, active compounds for antibacterial activity are yet to be determined in most of the cases. Encouraged with this trend of reports, many workers have made specific studies against pathogenic bacteria and also evaluated their therapeutic potential [37-39]. Similarly, several workers have made targeted screening against multidrug resistant bacteria such as methicillin resistant Staphylococcus aureus MRSA, VRE, Mycobacterium tuberculosis, Enteric bacteria and others[16, 27, 36, 40-43]. Some of the relevant studies are briefly summarized here. Hasegawa and workers demonstrated that various genoside and their aglycones isolated from Panx ginseng adversely affected efflux mediated tetracycline encoded resistance encoded by tetk genes in S. aureus clinical isolates. It was found that the tetracycline resistance was reversed by the non-toxic concentrations of these compounds [44]. Antibacterial activity of garlic and allicin was demonstrated against 40 strains of multiple drug resistance Shigella dysenteriae type 1, Shigella flexneri, enterotoxigenic E. coli and Vibrio cholerae [45]. In the same year, ajoene, a garlic derived sulphur containing compound had been isolated that prevent platelet aggregation, exhibited broad spectrum antimicrobial activity. Growth of S aureus and Lactobacillus platarum were inbibited by <20 Âľg/ml [126]. A compound z-4, 5, 7, 9 trithiadeo 1, 6 diane 9-oxide was isolated from oil macerated garlic extract. It showed broad antimicrobial activity against bacteria and yeast [46]. Furofuran lignans were isolated from the Propolis sp. The isolated lignan showed antibacterial activity against S. aureus and E. coli [47]. At our laboratory antimicrobial and phytochemical studies on 45 Indian medicinal plants against multidrug resistant human pathogens had been investigated. Among these, 40 plant extracts showed varied level of antimicrobial activity against one or more test bacteria. Overall broad spectrum antimicrobial

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activity was observed in 12 plants namely Lawsonia inermis, Eucalyptus sp. Holarrhena antidysenterica, Hemidesmus indicus, C. equistifolia, T. bellerica, T. chebula, Emblica officinalis, Camellia sinensis, S. aromaticum, Plumbago zeylanica and Punica granatum [40]. Antimicrobial activity of Allium sativum on three human pathogenic bacteria had been demonstrated . The extract showed activity against all three bacteria viz. S. aureus, S. typhimurium and Yersinia enterocolitica by disc diffusion method [48]. Petrol, acetone, chloroform and methanol extracts of the leaves and stems of two different varieties of Hypericum were tested by disc diffusion method. Both species Hypericum mysorence and Hypericum patulum showed broad spectrum activity against all six test bacteria [49]. The activity of aqueous and ethyl acetate extracts of the leaves of Piper regnellii was evaluated against Gram positive and Gram negative bacteria. Four compounds viz eupomatenoid–6, eupomatenoid-5, eupomatenoid-3 and conocarpan were isolated. The first two compounds showed very good activity with MIC of <3.12 µg/ml, while last compound showed MIC of 6.25 µg/ml against S. aureus and B. subtilis [50]. Antibacterial activity of 50 plant species of 33 families had been demonstrated which had documented in Iranian traditional medicine. Among the active plants, 32.6% were active against Gram -ve, 62% against Gram +ve and 47.3% against both groups [32]. The bactericidal activity of the whole extract of Eremophila duttonii was checked out by Time kill experiment and demonstrated that it could reduced the bacterial cells to the undetectable level [51]. Plumbago zeylanica extracts (ethyl acetate fraction) also showed the bactericidal activity against Helicobacter pylori. Four fold MIC concentrations of the extracts killed all the population with in the 4 hrs of incubation while the two fold concentration showed the similar effect in 8 hrs [52]. In-vitro inhibitory effect of roselle calyx and protocatechuic acid was found on the growth of methicillin resistant S. aureus. K. pneumoniae, P. aeruginosa and Acinetobacter brumannii. It was observed that both roselle calyx and protocatechuic acid inhibited effectively the growth of all test bacteria [53].Ten traditionally used Nigerian medicinal plants were tested against multidrug resistant S. typhi strains, of which six plants showed MIC and MBC in the range of 9-6 to 14 µg/ml and 40-33 µg/ml respectively against multi resistant S. typhi strains [54]. Recently, the antibacterial activity of medicinal plants had been reviewed against drug resistant bacteria as well as against Candida albicans. Many plants have been identified for characterizing the active principles [16].

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In vitro efficacy of 15 medicinal plant extracts against multi drug resistant enteric bacteria had been reported at our laboratory. The extracts of Acorus calamus, Hemidesmus indicus, Holarrhena antidysenterica, and Plumbago zeylanica demonstrated promising activity as compared to other tested plant extracts. Acetone fractions in most of the cases exhibited high potency as compared to ethyl acetate and ethanol fractions. Some of the extracts showed synergistic interaction with antibacterial drugs, tetracycline and ciprofloxacin. TLC bioautography showed the presence of alkaloids, phenols, and flavonoids as active constituents [55].

4. Anti-MRSA activity of medicinal plants The Anti-MRSA activity has been documented by few workers in certain phytocompounds and plant extracts like Camellia sinensis, Caesalpinia spinosa, Rosa eanina, Scutellara amoena, Arctostaphylos isvaris, Delonix regia, Erytherina variegata, Hypericum spp. etc. [53, 56-61]. Some of the relevant works has been summarized below. The antibacterial activity of phytoalexins, which are biosynthesized by plants and function as self defensive phyto-chemical against microbial infections has been screened based on the phytotherapeutic concept. Potent anti-MRSA activity was found in phytoalexins isolated from Sofora exigna, and these were chemically characterized as hydroxy-flavanones. Lower plants belonging to the leguminosae are abundant in flavonoid phytoalexins, which include flavanone derivatives, structurally analogues to the anti-MRSA flavonones [43, 62, 63]. The substituted flavanones were isolated from leguminous plants and compared their antibacterial activity against methicillin resistant S. aureus (MRSA). Among the thirteen flavanones tested, tetrahydroxy flavanones with these structural characteristics, isolated from Sofora exigna and Echinosphora koreensis showed activity and inhibited the growth of all MRSA strains at 3.13 to 6.25 Âľg/ml [64]. Anti-MRSA activity of Sofora flavonone G(SFG) and synergy between SFG and antibacterial agents against MRSA by means of minimum inhibitory concentration were demonstrated. The minimum inhibitory concentrations (MICs) of SFG against 27 strains of MRSA was found in the range of 3.13 to 6.25 Âľg/ml [65]. Several authors have demonstrated time kill kinetics of various plant extracts and essential oils against drug resistant bacteria. Tea tree oil showed a rapid decrease in the survival of different pathogenic drug resistant bacteria including Enterococcus faecalis, methicillin resistant S. aureus, and Pseudomonas aeruginosa in the first two hr of incubation. The oil killed the MSSA strains within the 30 min while two MRSA strains were killed in 1.5 hr

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and 3.5 hr. However, some of the Pseudomonas isolates showed 99% decrease in the viable count after 1 hr of incubation [66]. The effect of Helichrysum italicum extract was evaluated on growth and enzymatic activity of S. aureus. In the time kill curves of the isolates, dose of 10 times MIC, reduce the number of viable cells by almost 1 log10 unit within 1-2 h of exposure and between 2-4 h with four times MIC. No difference was shown between curves of MRSA and MSSA [41]. In a similar type of study, Azadirachta indica extracts killed all the bacterial cells of S. aureus at a concentration of 1mg/ ml. A higher concentration of 2mg/ ml had the same effect after 6hr [67]. Calozeyloxanthone, a compound from an endemic species of Sri Lanka, Calophyllum monii was obtained and found to be active against vancomycin resistant enterococci (VRE) and vancomycin sensitive enterococci (VSE) with MIC values of 6.25 µg/ml and 12.5 µg/ml [68]. The flavone and its derivatives had known to show weak antibacterial activity but dramatically intensified the susceptibility of β lactams in MRSA[69]. The susceptibility of methicillin susceptible and methicillin resistant staphylococci to Oregano essential oil, carvacrol and thymol was evaluated and their efficacy was described against 26 MSSA and 21 MRSA using an agar dilution method. It was further demonstrated that the comparison between the susceptibility of MSSA and MRSA to oregano oil. Carvacrol and thymol showed no significant difference [42]. An isoflavone from the roots of Erythrina variegata (Leguminosae) characterized as 2, 4-dihydroxy-8-γ-γ-dimethyl allyl 2” 2”-dimethyl pyrano [5”, 6”: 6, 7] isoflavone (bidwillon B) inhibited the growth of 12 MRSA strains with minimum inhibitory concentration of 3.13 to 6.25 mg/L, while MICs of mupirocin were 0.20-3.13 mg/L. Mupirocin is a naturally occurring agent produced by Pseudomonas fluorescens and has been successfully used to reduce substantially the nasal and hand carriage of MRSA[61, 70-72]. Mupirocin consists of a short fatty acid, (α-β-unsaturated carboxylic acid), the tail end of which appears to mimic isoleucin. It reversibly binds to isoleucyl tRNA synthetase and prevents the incorporation of isoleucine into growing polypeptide chain [73]. The purified ethyl galate isolated from a dried pod of tara (Caesalpinia spinosa) intensified β-lactam susceptibility in methicillin resistant and methicillin sensitive strains of S. aureus. The maximum activity of alkyl galate against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl galate with an MIC90 of 15.6µg/ml. At concentration lower than the MIC, alkyl galate synergistically elevate the susceptibility of MRSA and MSSA strains to β-lactams [74]. The six medicinal plants used in South-West Nigerian unorthodox medicine were tested for Anti-MRSA activity. Both water and ethanol extracts of

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T. avicennioides, P. discoideus, O. gratissium, and A. wilkesiana were effective on MRSA. The MIC and MBC of these plants ranged from 18.2 to 24 µg/ml and 30.4 to 43.0 µg/ml and 55.4 to 71.0 µg/ml were recorded for ethanol and water extract of B. fessuginea and A. conyzoides respectively [54]. The antibacterial activity of nine 2-arylbenzofurans isolated from Morus species against MRSA, MSSA and several other microorganisms were reported. Among these compounds, chalcomoraein (a leaf phytoalexin of mulberry tree) exhibited considerable antibacterial activity against MRSAs with MIC of 78µg/ml. In a similar type of study, compounds including benzopyran, xanthones were isolated first time from Garcinia nigroniala and showed anti-MRSA activity and MIC , 2 µg/ml [75]. Anti-MRSA activity of Indian medicinal plants have been conducted in our laboratory. The potential plant extracts include Plumbago zeylanica, Hemidesmus indicus, Acorus calamus, Camellia sinensis, Terminalia chebula, T. bellerica, and some other plants. [25,36]. Some of the Indian medicinal plants and their potential activity against drug resistant bacteria along with their active constituents are listed in Table1. For more details the readers are advised to see specific articles published elsewhere [16, 25, 26].

5. Synergy of medicinal plants with antibiotics In recent years, increasing attention has been given on investigating phytochemicals as possible medicinal agent against MDR bacteria. Plant extracts/ phytocompounds exhibiting strong antibacterial activity may interact with antibiotics. The interactions may be synergistic, neutral or antagonistic. Synergy with antibiotic is expected to be useful in antibiotic therapy. Although investigations in this direction are in their infancy, a number of phytocompounds exhibiting synergistic interactions with antibiotics have been isolated and characterized. Combinational effect of protoanemonum isolated from Ranunculus bulbosus with 22 antibiotics was evaluated. In one combination, protoanemonum-cefamendole showed strong synergism against S. aureus [76]. The synergistic activity was determined for two xanthones, α-mangostin and rubraxanthon isolated from Garcinia mangostana with antibiotics against MRSA strains [77]. Similarly, retin isolated from Sophora japonica could be hydrolyzed to quercetin which showed synergistic and additive effects with various antibiotics [78]. A synergistic interaction was also demonstrated against 11 MRSA strains with fraction inhibitory concentration indices of 0.5-0.75. The minimum bactericidal concentration (MBC) of mupirocin in the presence of bidwillon B (3.13 mg/L) was reduced to 0.05 to 1.56 mg/L. They suggested that bidwillon B

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Table 1. Antibacterial active compounds/class of compounds from common Indian medicinal plants.

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Table 1. Continued

may prove to be potent phytotherapeutic and/or combinational agent with mupirocin in the elimination of nasal and skin carriage of MRSA [61]. The antibacterial activity of 11 medicinal plants was investigated against several Gram negative and few Gram positive bacteria. Interestingly, the highest activity was observed in the extract of Caryophyllus aromaticus and Syzygium aromaticum. Interactions of active plant extracts with ampicillin, chloramphenicol and/ or tetracycline were determined in the synergism assay. Synergistic interactions were observed between antibiotics and extracts from clove, jambolan, pomegranate and thyme against Pseudomonas aeruginosa and Klebsiella pneumoniae [79]. Alpha-Mangostin isolated from the stem bark of Garcinia mangostana was found active against vancomycin resistance enterococci and MRSA with the MIC values of 6.25 µg/ml to 12.5 µg/ml. The compound showed synergistic activity with gentamycin against MRSA. However, partial synergism was found with ampicillin and minocycline [80]. Synergistic interaction of ethanolic extracts of Indian medicinal plants with β-lactam antibiotics, aminoglycosides and synthetic fluroquinilone indicated the synergistic interaction with one or more antibiotics (tetracycline, ciprofloxacin and chloramphenicol) but least with β-lactam antibiotics against S. aureus and E.coli [25, 55].

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6. Major groups of antibacterial phytocompounds Plants are solar powered biochemical factories which produce a large array of biological active metabolites that accumulate and are extractable. These plants organic compounds are classified as primary or secondary metabolites [81]. Secondary metabolites are biologically active compounds frequently present in small quantities compare to primary metabolites [82]. Although large quantities of secondary metabolites are not usually required due to their very strong biological activity and their selection by the external pressure during evolution, they are accumulated in plants due to continuous stimulation [83]. Secondary metabolites perform non essential functions in the plant and the information containing primary metabolites viz protein, DNA and RNA. Most secondary compounds function in defense against predators and pathogens. Some terpenoids give plants their odour, other (quinones and tannins) are responsible for plant pigments. Major group of phytocompounds and bioactive constituents of several plants have been described and documented by various authors [10,84,85]. Some of the representative groups of antimicrobially active phytocompounds are described below and also listed in table -1. Phenols and polyphenols Plants have an almost limitless ability to synthesize aromatic substances which are phenols or their oxygen substituted derivatives [86]. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total [5]. Phenols are among the largest group of secondary metabolites. They range from simple structures with one aromatic ring to complex polymers such as tannins and lignins. Simple phenols and phenolic acids: These compounds have a monocyclic ring with an alcoholic, aldehydic or carboxylic group. Cinnamic and caffeic acid are common representative of a wide group of phenylpropane derived compounds which are in highest oxidation state. Eugenol, the major constituent of Syzygium aromaticum is widely used in dentistry due to its antibacterial, anti-inflammatory and local anaesthetic activities [1]. The common herbs tarragon and thyme both contain caffeic acid, which is effective against bacteria [87, 88]. Gallic acid, the parent compound of the gallotannins, spread widely and can be isolated from various plants including Syzygium cordatum, Vitis vinifera and Rhus typhina etc. This compound demonstrated a variety of biological activities like antibacterial, antiviral, antifungal, antimutagenic and antitumor etc [85]. Catechol and pyrogallol both are hydroxylated phenols

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and found to be toxic to microorganisms. The site(s) and number of hydroxy groups on the phenol group are thought to be related to their relative toxicity to microorganisms. This evident that macerates hydroxylation results in increased toxicity [86].

Quinones Quinones are oxygen containing compounds that are oxidized homologue of aromatic derivatives. The quinines are yellow or orange pigments. Although they are widely distributed in higher plants, they contributed little to plant colour [85]. Quinones can be conveniently divided into three groups of increasing molecular size; the benzoquinones, the naphthoquinones and the anthraquinones. Naphthoquinones and anthraquinones have some medical importance [1]. Naphthoquinones are yellow and orange pigments from plants and found in the families like bignoniaceae, droseraceae, juglundaceae, plumbaginaceae and lythraceae etc. Plumbagin, a well known naphthoquinone, isolated from Plumbago zeylanica exhibited anti-bacterial and other biological activity [1,89]. Similarly Lawsone, a compound from Lawsonia inermis is a powerful fungicide and a good antibacterial agent [90]. Jaglone, a brown colour compound has shown its bioactive nature against groups of bacteria, fungi and viruses [85].

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Kazmi et al [91] described an anthraquinone from Cassia italica, a Pakistan tree, which was bacteriostatic to Bacillus anthracis, Corynebacterium pseudodiphthericum and Pseudomonas aeruginosa and bactericidal for Pseudomonas pseudomalliae. Hypericin, an anthraquinone from St. Johnâ&#x20AC;&#x2122;s wort (Hypericum perforatum) has received much attention as an antidepressent and duke reported in 1995 that it had general antimicrobial properties [92].

Tannins Tannins are a group of polymeric phenolics with a molecular weight of 500-3000 that occur widely in vascular plants. They have the ability to react with proteins. Chemically, there are two main types of tannins (1) condensed tannins and (2) hydrolysable tannins. These two classes are unevenly distributed in plant kingdom. Most tannins that have been purified and studied are biologically active [85]. The antimicrobial significance of this particular activity has not been explored. There is also evidence for direct inactivation of microorganisms, low tannin concentrations modify the morphology of germ tube of Crinipellis perniciosa [93]. Scalbert [94] extensively reviewed the antimicrobial properties of tannins. Thirty three studies listed by him showed the inhibitory activity of tannins to filamentous

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fungi, bacteria and yeast. In a study, tannins are considered partially responsible for the antibiotic activity of methanolic extracts of the bark of Terminalia alata [95]. Interestingly this activity was enhanced by UV light activation (320 to 900 nm at 5W/m2 for 2 hr.). Various authors have also shown biological activities of tannins especially in diarrhea [96].

Coumarins Coumarins are phenolic substances made of fused benzene and Îą-pyrone ring and have characteristic odour of hay and occur worldwide. Until year 2000, at least 1500 coumarins had been identified [1,26]. There are three major classes (1) the simple hydroxycoumarins such an umbelliferone (2) the furanocoumarins like angelicin and (3) the pyronocoumarins, such as decursinol [85]. Warferin is a particularly well known coumarin which is used both as an oral anticoagulant and also as a rodenticide. However, umbelliferone demonstrated strong antibacterial and antifungal properties [85]. Some coumarins are phytoalexins which are manufactured by plant in the event of an infection by bacteria and fungi [1,26]. In other study, hydroxycinnamic acid, related to coumarins, seems to be inhibitory to Gram positive bacteria [97]. General antimicrobial activity was documented in woodraff (Gallium odoratum) extracts [88]. Flavonols, flavones and flavonoids These compounds are very widely distributed in plants both as copigments to anthocyanins in petals and also in leaves of higher plants. Since these compounds are known to be synthesized by plants in response to microbial infection [98], it should not be surprising that they have been found

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in vitro to be effective antimicrobial substances against a variety of microorganisms. Catechins, a constituent of green tea deserve special mention as these flavonoides have been excessively researched. Many workers have demonstrated antibacterial activity of tea throughout the world [99] and reported that they contain a mixture of catechin compounds. These compounds inhibited Vibrio cholerae 01 in vitro, Streptococcus mutans, Shigella, methicillin resistant S. aureus and other microorganisms [100-102]. Nakahara et al demonstrated that catechins inactivated or inhibited the isolated bacterial glucotransferases in S. mutans [103]. An isoflavone found in a West African legume, Alpinumi soflavone prevents schitosomal infections when applied topically [104]. Hunter and Hull isolated phloretin from the apples and demonstrated its activity against a variety of microorganisms [105]. Similarly, galangin (3,5,7- trihydroxyflavone) derived from the perennial herbs Helichrysum aureunitens, seems to be a particularly useful compound since it has shown activity against a wide range of Gram positive bacteria as well as fungi [85] and viruses. Structures of some of the active constituents are given below.

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Terpenoids and essential oils The terpenoids comprise the largest group of natural plant products and more than twenty thousand such structures have been described from plant sources. Camphor, limonene, abscissic acid, aucubin, gossypol, gibberellic acid and Î˛-carotene are some typical plant terpenoids. There are a number of reports available on the biological activities especially antibacterial and antifungal activities of essential oils [95,106]. It was reported that 60% of essential oil derivatives examined to date were inhibitory to fungi, while 30% inhibited bacteria [107]. Menthol and euginol were found inhibitory to a variety of bacteria. The ethanol soluble fraction of purple prairie clover yields a terpenoid called petalostemumol, which showed excellent activity against Bacillus subtilis and S. aureus and lesser activity against Gram negative bacteria as well as Candida albicans. Some examples of this class structures are listed below.

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Alkaloids Alkaloids are principally of interest to human because of their medicinal properties and many are used as drugs e.g. atropine, codeine, morphine, vincristine etc. Diterpenoid alkaloids, mostly, isolated from plant of the Ranunculaceae are commonly found to have antimicrobial properties [108]. Berberine is a common representative of the alkaloid group. It is found to be active against several bacteria and potentially effective in inhibiting quorum sensing signals in Chromobacter violaceum and Pseudomonas aeruginosa [109]). Some classical examples are described below. Glycosides Glycosides are naturally occurring compounds which upon hydrolysis yield a sugar portion (glycon) and a non sugar portion (aglycon). On the basis of basic

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structure, there are various group of glycosides are known. These groups include steroidal glycosides, saponin glycosides, anthracene glycoside, cyanogen glycosides, flavoniod glycosides and resinous glycosides [110]. Seven saponin glycosides isolated from aerial part of Astragalus suberi have exhibited antibacterial activity against Gram positive and Gram negative bacteria with MIC values >100 µg/ml, while the minimum fungicidal concentration was found to be between 25 µg/ml to 50 µg/ml [111]. Luteolin, Luteolin 3’-O-β D-Glucopiranoside, and Luteolin 4’-0- β-DGlucopiranoside and three flavones from traditionally used plant Daucus carota showed antibacterial activity against Bacillus cereus and Lactobacillus plantarum [112]. In other study, strong antibacterial activity had been shown by anthrocyanin and proanthrocyanin fractions isolated from canberry juice against Enterococcus, Escherichia and Streptococcus mutans etc [113]. Various compounds have been known for their mode of action but many more are yet to be studied. Some examples of phytocompounds with their mode of action are documented [25, 26].

7. Need for control clinical trials Several hundreds of plants are used worldwide in traditional systems of medicine for treatments of bacterial infections. Many of these have subjected to

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in vitro screening but their efficacy has seldom been rigorously tested in controlled clinical trials. Only limited numbers of reports are available on clinical trials with varying level of efficacy. For example, Clinical trials of Allium sativum against Helicobacter pylori have shown variable results [114, 115]. One clinical trial is found against Staphylococcus aureus and Streptococcus pyogenes infections using 4% tea tree oil [116]. Tea extracts were tested against Streptococcus pyogenes infection [117]. Similarly, oils from tea tree oil and Ocimum gratissimum were tested against acne in clinical trials. The results from above clinical trials are in some cases encouraging but require more systemically designed controlled clinical trials to properly address the efficacy and safety issues [118]. The clinical trials should be preferably encouraged against infections caused by drug resistant bacteria both in animal system and human clinical trials.

8. Future prospects and new directions Extensive antimicrobial screening of ethnomedicinal plants of India and many other countries has led to identification of active compounds in some cases. However, in most of the screened plant extracts, the most active fraction is known but active compounds are yet to be characterized. In vitro and in vivo efficacy of the characterized extracts or compounds is to be worked out. MIC data of many plant extracts and phytocompounds are very high indicating the less activity than conventional antibiotics. However, many ingredients of essential oils and phytocompounds have showed comparable MIC values with antibiotics. An interesting observation is that majority of the active crude extracts and their fractions are almost equally active both against drug resistant and sensitive bacterial strains. On the other hand, there are interesting reports where the compound is not active (non antibacterial) or weak active but could enhance the activity of conventional antibiotics against drug resistant bacteria. Other interesting activity of plant extracts and/or phytocompounds which has eliminated the drug resistance plasmid from E.coli and antipathogenic properties (reduction in virulence and pathogenicity of bacterium) are the novel targets for screening of medicinal plants to obtain antibacterial / antipathogenic therapeutic compounds. Therefore, multi target based approaches of screening of medicinal plant extracts and herbal drugs are expected to yield novel activities. For this type of approach, an intelligent design of test system with simple requirement will be most effective. An example of novel activity includes the antiquorum sensing activity of medicinal plants recently reported by some workers. We have made preliminary investigations to screen 32 medicinal plants for their anti quorum sensing activity. Of these, ten plant extracts inhibited pigment production in Chromobacterium violaceum reporter strain. Further investigations are needed with more number of reporter strains and characterization of active compounds [119,120].

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Antibacterial and anti-pathogenic compounds as well as standardized plant extracts are to be tested for clinical trials both in animals and humans to evaluate their efficacy and safety and their therapeutic potential in controlling the infectious diseases caused by bacteria especially by multidrug resistant bacteria. Some of the future guidelines are suggested in this area of research are: a) There are greater need to develop simple, economical multitargeted approaches and methods to get novel and most effective combinations of biological activities. Activity against MDR bacteria and antibiotic reversal, plasmid elimination, virulence and pathogenicity reduction, inhibition of bacterial cell to cell communications (quorum sensing) are the potential targets. b) Synergy with antibiotic and herb-drug interactions should be studied in order to enhance the activity of older antibiotics and safe integration of herbal drug with modern medicine. c) Herbal preparation with known efficacy in traditional system of medicine must be standardized and can be directly tested for clinical trials both in animals and humans. However, isolated compounds with therapeutic efficacy should be regulated and tested for its toxicity and efficacy similar to that of modern medicine.

Acknowledgement We are thankful to Prof. Shamim Ahmad (JN Medical College, AMU), Prof. M. Saleemuddin (Interdisciplinary Biotechnology Unit, AMU) and Prof. Javed Musarrat, Faculty of Agricultural Sciences, AMU, Aligarh for their cooperation and encouragement.

References 1. 2. 3. 4. 5. 6. 7. 8.

Gurib-Fakim, A. 2006, Mol Aspects Med, 27, 1-93. De Pasquale, A. 1984, J Ethnopharmacol, 11, 1-16. Raskin, I., Ribnicky, D. M., Komarnystky, S., Ilic, N., Poulev, Borisjuk, Brinker, Moreno, D. A., Ripoll, C., Yakoby, N., O'Niel, J. M., Cornwell, T., Paetor, I. and riedlender, B. 2002, Trends Biotech. 20 (12), 1-10. Krishna, A. 2003, Proceedings of first National interactive meet on medicinal and aromatic plants (Eds) Mathur, A.K., Dwivedi, S., Patra, D.D. et al (CIMAP, Lucknow, India), 209-214. Schultes, R. E. 1978, Medicines from the Earth. W. A. R. Thomson (ed.), McGrawHill Book Co., New York, pp. 208. Arora, R. B. 1965, Indian Council of Medical Research, Delhi, Special report series No. 51, 1-117. Gilani A.H. and Atta-ur-Rahman. 2005, J.Ethnopharmacology, 100, 43-49. Flemming, J. 1910, Asiatic Researches, Vol. XI.

Antibacterials from medicinal plants

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

173

Mehmood, Z. 1998, Ph. D. Thesis, Studies on antimicrobial properties of some Indian medicinal plants submitted to the Aligarh Muslim University, Aligarh, India. Chopra, R. N., Nayer, S. L. and Chopra, I. C. 1992, Glossary of Indian Medicinal Plants, 3rd, Edt. Council of Scientific and Industrial Research, New Delhi, 7-246. Dahanukar, A. and Hazra, A. 1995, Heals with herbs, Ist Ed. Publication and information directorate (CSIR), New Delhi (India) 1-79. Farooq, S., Ahmad, I. and Pathak G. K., 1997, J Ethnopharmacol, 58, 109-116. Ahmad, I., Mehmood, Z. and Mohammad, F. 1998, J Ethnopharmacol, 62, 183-193. Mehmood, Z., Ahmad, I., Mohammad, F. and Ahmad, S. 1999, Pharmaceutical Biol, 37 (3), 237-242. Tiwari, A.K. 2004. Current Science, 86(25), 1092-1101. Nostro, A. 2006 In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley VCH, Germany, pp-199-226. Musarrat, J., Aqil, F. and Ahmad, I. 2006, In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley VCH, Germany, pp271-291. Chattopadhyay, D. 2006, In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley- VCH, Germany, pp, 313- 340. Aridogan, B.C. 2006, In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley -VCH, Germany, pp-341-356. Ahmad, I., Aqil, F., Farah, A. 2006, In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley- VCH, Germany, pp 59-76. Ahmad, I., Yadava, J.N.S. and Ahmad, S. 1994, Ind J Animal Sci, 64(5), 439-445. Davies, J. 1994, Science, 264, 375-382. Livemore, D. M. 1995, Clin Microbiol Rev, 8 (4), 557-584. Kariuki, S. and Hart, A. 2001, Curr Opin Infect Dis., 14, 579-586. Aqil, F. Ahmad, I. and Owais M. 2006, In: Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley-VCH, Germany, pp 59-79. Cowan M.M. 1999, Clin Microbiol Rev, 12, 564-582. Rios, J. L. and Recio, M. C. 2005, J Ethnopharmacol, 100, 80-84. Dhar, M. L., Dhar, M. M., Dhawan, B. N., Mehrotra, B. N. and Roy, C. 1968, Ind J Exp Biol, 5 (4), 232. Ikram, M. and Haq, I. 1980, Fitoterapia, 51 (5), 231. Dorman, H.J.D. and Deans, S.G. 2000, J Appl microbial, 88, 308-316. Aqil, F. and Ahmad, I. 2003. World Journal Microbiol Biotechnol, 19, 653-657. Bonjar, S. 2004, J Ethnopharmacol, 94, 301-305. Durate, M.C., Figueria, G.M., Sartoratto, A., Rehder, V.L., Delamelina C., 2005, J Ethnopharmacology, 97, 305-311. Wannissorn B., Jarikasem, S., Siriwangchal T., Thubthimthed, S. 2005, Fitoterapia, 76, 233-236. Uzun, E., Sariyar, G., Anderson, A., Karakoc, B., Otuk, G. Oktayolu, E., Prildar, S. 2004, Journal of Ethnopharmacology, 95, 287-296. Aqil, F., Ahmad, I. 2007, Methods Find Exp Clin Pharmacol, 29(2), 79-92. Oâ&#x20AC;&#x2122;Gara, E.A., Hill. D.J., Maslin, D.J. 2000, Applied and Environmental Micrbiology, 66, 2269-2273. Voravuthikunchai, S., Lortheeranuwat, A., Jeeju, W. 2004, J Ethnopharmacol, 2004, 94(1), 49-54.

174

Maryam Zahin et al.

39. Eloff, J.N., McGaw, J.N. 2006, Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley -VCH, Germany, pp 97-121. 40. Ahmad, I. and Beg, A. Z. 2001, J Ethnopharmacol, 74, 113-123. 41. Nostro, A., Bisignano, G., Cannatelli, M.A., Crisafi, G., Germano, M.P. and Alonzo, V. (2001). Int J Antimicrob Agents, 17, 517-520. 42. Nostro, A., Blanco, A.R., Cannatelli, M.A., Enea, V., Flamini, G., Morelli, I., Sudano Roccaro, A. and Alonzo, V. 2004, FEMS Microbiol Lett, 230, 191-195. 43. Sakagami, Y. 2006, In:Modern Phytomedicine: Turning Medicinal plants into drugs. Wiley 窶天CH, Germany, pp, 137-156. 44. Hasegawa, H., Matsumya,S. and Yamasak, K. 1995, Phytotherapy Research, 9, 260-263. 45. Ahsan, M., Chaudhary, A. K., Islam, S. N. and Ahmad, Z. U. 1996, Phytotherapy Res, 4 (10), 329-31. 46. Yoshida, H. Iwata, N. 1998, Biotech and Biochem, 62, 1014-1017. 47. Christov, R., Bankora, V. and Tejera, A. 1999, Fitoterapia, 40(1), 3-54. 48. Elizabeth, K. M. 2001, Ind J Microbiol, 41, 321-323. 49. Mukherjee, P. K., Saritha, G. S. and Suresh, B. 2002, Phytother Res, 16, 692-695. 50. Pessini, G.L., Filho, B.P.D., Nakamura, C.V. and Cortez, D.A.C. 2003, Mem Inst Oswaldo Cruz, Rio de Janeiro, 98(8), 1115-1120. 51. Shah, A. A., Hasan, F., Ahmed, S. and Hameed, A. 2004, Crit Rev Microbiol, 30 (1), 25-32. 52. Wang, Y. C. and Huang, T. L. 2005, FEMS Immunol Med Microbiol, 43, 407-412. 53. Liu, I. X., Durham, D. G. and Richards, R. M. E. 2000, J Pharmaceutical Pharmacolol, 52, 361-366. 54. Akinyemi, K. O., Oladapo, O., Okwara, C. E., Ibe, C. C., and Fasure, K. A. 2005, BMC Complementary Alter Med, 5, 1-7. 55. Ahmad, I., Aqil, F. 2007, Microbiological Research 162, 264-275. 56. Yam, T.S., Hamilton-Miller, J.M.T. and Shah, S.1998, J Antimicrob Chemother, 42, 211-216. 57. Shiota, S., Shimizu, M., Mizushima, T., Ito, H., Hatano, T., Yoshida, T., Tsuchia, 2000, FEMS Microbiol Letters, 185, 135-138. 58. Shimizu, M., Shiota, S., Mizushima, T., Ito, H, Hatano, T., Yoshida, T. and Tsuchiya, T. 2001, Antimicrob Agents and Chemother, 45, 11, 3198-3201. 59. Gibbons, S., Ohlendorf, S. B.and Johnsen, I. 2002, Fitoterapia, 73, 300-304. 60. Palombo, E. A. and Semple, S. J. 2002, J Basic Microbiol, 42, 444-448. 61. Sato, M., Tanaka, H., Yamaguchi, R., Kato, K. and Etoh, H. 2004, International J Antimicrob Agents 24, 43-48. 62. Iinuma, S., Tsuchiyam H., Sato, M., Yokoyama, J., Ohyama, M., Ohkawa, Y., Tanaka, T., Fujiwara, S. and Fujii, T. 1994, Journal of Pharmacy and Pharmacogology, 46, 892-895. 63. Sato, M., Tsuchiya, H., Miyazaki, T., Ohyama, M., Tanaka, T. and Linuma, M. 1995, Letters in App Microbiol, 21, 219-222. 64. Tsuichiya, H. Sato,Miyazaki, T.and Inuma, M. 1996, Journal of Ethnopharmacology, 50 (1); 27-34. 65. Sakagami, Y., Mimura, M., Kajimura, K., Yokoyama, H., Iinuma, M., Tanaka, T., and Ohyama, M. 1998, Letters Appl Microbiol, 27, 98-100.

Antibacterials from medicinal plants

175

66. May, J. Chen, C.H., King, A., Williams, L. and French, G.L. 2000, J Antimicrob Chemother, 45, 639-643. 67. Okemo, P. O., Mwatha W. E., Chhabra S. C. and Fabry, W. 2001, Afr J Sci Tech, 2, 113-118. 68. Sakagami, Y, Kajimura, K., Wijeesinghe, W. M. N. and Dharmarathe, H. R. W. 2002, Planta Medica, 68, 541-543. 69. Shibata, H., Shirakata, C., Kawasaki,H., Sato,Y., Kuwahara,T., Ohnishi, Y., Arakaki,N.and Higuti,T. 2003, Biological and Pharmaceutical Bulletin, 26, 14781483. 70. Hill, R.L.R., Duckworth, G.J. and Casewell, M.W. 1988, J Antimicrob Chemother, 22, 377-84. 71. Cederna, J.E., Terpenning, M.S., Ensberg, M., Bradley, S.F., and Kauffman, C.A., 1990, Infect Control Hosp Epidemiol, 11, 13-6. 72. Reagan, D.R., Doebbeling, B.N., Pfaller, M.A., Sheetz, C.T., Houston, A.K., Hollis, R.J. and Wenzel, R.P. 1991, Ann Intern, Med, 114, 101-6. 73. Casewell, M.W. and Hill, R.L.R., J. 1987, Antimicrob Chemother, 19, 1-5. 74. Shibata, H., Kondo, K., Katsuyama, R., Kawazoe, K., Sato, Y., Murakami, K., Takaishi, Y., Arakaki, N. and Higuti, T. 2005, Antimicrob Agents Chemother, 49, 549-555. 75. Fukai, T., Oku, Y., Hou, A.J., Yonekawa, M. and Terada, S. 2005, Phytomedicine, 12, 510-513. 76. Didry, N.; Dubreuil, L. and Pinkas, M. 1993, Phytotherapy Res, 7(1), 21-24. 77. Iinuma, M., Tosa, H., Tanake, T. and Miyauchi, K.I. 1996, J Pharmacy Pharmacol, 48 (8), 861-865. 78. Eo, S.K., Kum, Y.S., Lee, C.K., Lee, D.I., Hun, S.S. and Hoeji, Y. 1996, Neem News letter, 40, 653-559. 79. Nascimento, G. G. F., Lacatelli, J., Freitas, P.C. and Silva, G. 2000, Braz J Microbiol, 31, 247-256. 80. Sakagami, Y., Iinuma, Piyasena, K. G. N. P. and Dharmaratne. 2005, Phytomed, 12, 203-208. 81. Applezweig, N. 1980, Renewable resources: a systemic approach. Academic press, London, New York .pp, 369-378. 82. Farnsworth, N. R. and Bingel, A. S. 1977, New natural products and plant drugs with pharmacological, biological or therapeutical activity, Springer Berlin, Heidelberg, New York. 1-22. 83. Farnsworth, N. R. and Morris, R. W. 1976, American J Pharm, 148, 46-52. 84. Dixon, R. A. 2001, Nature, 411, 843-847. 85. Harborne, J. B. and Baxter, H. 1995, Phytochemical dictionary: a handbook of bioactive compounds from plants. Taylor & Francis Ltd, 4 John St., London. 86. Geissman, T. A. 1963, Pyrrole pigments, isoprenoid compounds and phenolic plant constituents, vol. 9. Elsevier, New York, N.Y., pp, 265. 87. Brantner, A., Males, Z. Pepeljnjak, S. and Antolic, A. 1996, J Ethnopharmacol, 52, 119-122. 88. Thomson, W. A. R. 1978, Medicines from the Earth. McGraw-Hill Book Co., Maidenhead, United Kingdom. 89. Beg, A. Z. and Ahmad, I. 2000, World J Microbiol Biotechnol, 16, 841-844. 90. Ali, M. 1996, Hamdard Medicus, 39, 43-48.

176

Maryam Zahin et al.

91. Kazmi, M. H., Malik, A., Hameed, S., Akhtar, N. and Noor, A.S., 1994, Phytochem, 36, 761-763. 92. Duke, J. A. 1985, Handbook of medicinal herbs. CRC Press, Inc., Boca Raton, Fla. 93. Brownlee, H. E., McEuen, A. R., Hedger, J. and Scott, I. M. 1990, Physiol Mol Plant Pathol, 36, 39-48. 94. Scalbert, A. 1991, Phytochem, 30, 3875-3883. 95. Taylor, R. S. L., Edel, F., Manandhar, N. P. and Towers, G. H. N. 1996, J Ethnopharmacol, 50, 97-102. 96. Bruneton, J. 1995, Pharmacognosy, Phytochemistry, Medicinal plants. Lavoisler Publishing Co. France, 265-380. 97. Fernandez, M. A., Garcia, M.D. and Saenz, M.T. 1996, J Ethnopharmacol, 53, 11-14. 98. Dixon, R. A., Dey, P. M. and Lamb. C. J. 1983, Adv Enzymol, 55, 1-69. 99. Toda, M., Okubo, S., Ohnishi, R. and Shimamura. T. 1989, Jpn J Bacteriol, 45, 561-566. 100. Borris, R. P. 1996, J Ethnopharmacol, 51, 29-38. 101. Batista, O., Duarte, A. Nascimento, J. and Simones, M. F. 1994, J Nat Prod, 57, 858-861. 102. Vijaya, K., Ananthan, S. and Nalini, R. 1995, J Ethnopharmacol, 49, 115-118. 103. Nakahara, K., Kawabata, S., Ono, H., Ogura, K., Tanaka, T., Ooshima, T. and Hamada, S. 1993, Appl Environ Microbiol, 59, 968-973. 104. Perrett, S., Whitfield, P. J., Sanderson, L. and Bartlett, A. 1995, J Ethnopharmacol, 47, 49-54. 105. Hunter, M. D. and Hull, L. A. 1993, Phytochem, 34, 1251-1254. 106. Tassou, C.C., Drosinos, E. H. and Nychas. G. J. E. 1995, J Appl Bacteriol, 78, 593-600. 107. Brantner, A. and Grein, E. 1994, J Ethnopharmacol, 44, 35-40. 108. Omulokoli, E., Khan, B. and Chhabra. S. C., 1997, J Ethnopharmacol. 56, 133-137. 109. Stermitz, F.R., Tawara-Matsuda, J., Lorenz, P., Mueller, P., Zenewicz, L., Lewis, K. 2000, J Nat Prod, 63, 1146-1149. 110. Iyenger, M.A. 1985, Study of Crude Drugs (2nd Ed.) College of Pharmaceutical Sciences. Kasturba Medical College, Manipal, India, 13-78. 111. Abbas, F. and Zayed, R. 2005, Z Naturforsch [C], 60 (11-12), 813-20. 112. Kumarasamy, Y., Nahar, L., Byres, M., Delazar, A. and Sarker, S.D. 2005, J Herb Pharmacother, 5(1), 61-72. 113. Leitao, D.P., Polizello, A.C., Ito, I.Y. and Spadaro, A.C. 2005, J Med Food, 8(1), 36-40. 114. Aydin, AQ., Ersoz, G., Tekesin, O., Akcicek, E., Tuncyurek, M. and Batur, Y. 1997, Turkish Journal of Gastroenterology, 8, 181-184. 115. Graham, D.Y., Anderson, S.Y. and Lang, T. 1994, American Journal of Gastroenterology, 94, 1200-1202. 116. Caelli, M., Porteous, J., Carson, C.F., Heller, R. and Riley, T.V. 2000, Journal of Hospital Infection, 46, 236-237. 117. Sharquie, K.E., Al-Turfi, I.and Al-Salloum, S.M. 2000, Journal of Dermatology, 27, 706-710. 118. Martin, K.W. and Ernst, E. 2003, Journal of Antimicrobial Chemotherapy, 51, 241-246.

Antibacterials from medicinal plants

177

119. Sameena, H. 2006. Qourum sensing inhibition and antimicrobial properties of certain medicinal plants and natural products. M.Sc Dissertation (Ag. Microbiology), submitted to Aligarh Muslim University, Aligarh, India. 120. Ahmad, I., Sameena, H. and Zahin, m. (2007). Bacterial quorum sensing inhibition by medicinal plant extracts: A novel target for anti infective compounds. Paper presented in 9th Indian Congress (Bioved Research and communication center), Allahabad, India. 121. Ficker, C, Smith, M.L, Akapagana, K, Gbeassor, M, Zhang, J, Durst, T, Assabgni, R. and Arnason, J.T. 2003, Phytother Res, 17, 897-902. 122. Eldeen, I.M.S., Elgorashi, E.E. and van Staden, J. 2005, Journal of Ethnopharmacology, 102 (3), 457-464. 123. Gupta, D., Khare, S.K. and Laha, A. 2004, Coloration Technology, 120 (4), 167-171. 124. McGaw, L.J., Jager, A.K. and van Staden, J. 2002, South African Journal of Botany, 68 (1), 31-35. 125. Grange, J.M., and Snell, N.J. 1996, J Ethnopharmacol, 50(1), 49-53. 126. Naganawa, R., Iwata, N., Ishikawa, K., Fucada, H., Fujino, T. and Suzukii, A. 1996, Appl Environ Microbiol, 59, 4238-4242. 127. San-blas, G., marino, I., San-blas, F. and apitz-Castro, R. 1993, J Med Vet Mycol, 31,133-141. 128. Martinez, M.J., Betancourt, J., Alonso-Gonzalez and Jauregi, A. 1996, J Ethnopharmacol, 52, 171-174. 129. Lee, K.K. and Choi, J.D 1999, International Journal of Cosmetic Science, 21(4), 275-284. 130. Govindachari, T.R., Suresh, G., Gopalakrishnan, G., Banumathy, G.B. and Masilamn, S. 1998, Phytoparasitica, 26(2), 1-8. 131. Surange, S.R. and Pendse, G.S. 1972, Journal of Research in Indian Medicine, 7, 1. 132. Mishra, A.N. and Tiwari, H.P. 1971, Phytochemistry, 10, 3318. 133. Okwu, D.E. and Josiah, C. 2006, African Journal of Biotechnology, 5(4), 357-361. 134. Mckenzie, R.A., Franke, F.O. and Duster, P.J. 1985, Antivet J, 64, 10-15. 135. Dickson, R.A., Houghton, P.J. and Hylands, P.J. 2007, Phytochemistry, 68(10), 1436-1441. 136. Yam, T.S., Shah, S. and Hamilton-Miller, J.M. 1997, FEMS Microbiol Lett, 152(1), 169-74. 137. Mohagheghzadeh, A., Faridi, P. and Ghasemi, Y. 2007, Food Chemistry, 100(3), 1217-1219. 138. Padmanabha Rao, T.V. and Venkateswarlu, V. 1965, Bull Nat Ins Sci, 31, 28-33. 139. Misra, T.R., Singh, R.S., Pandey, H.S. and Singh, B.K. 1997, Fitoterapia, LXVIII (58), 375. 140. Nishimura, H. and Satoh, A. 2006, Allelochemicals: Biological Control of Plant Pathogens and Diseases, Springer Netherlands 10.1007/1-4020-4447-X, 2. 141. Stange, R., Midland, S.L., Eckert, J.W. and Sims, J.J. 1993, J Nat Prod, 56, 1627-1629. 142. Apisariakul, A., Venittanakom, N. and Buddhasukh, D. 1995, J Ethnopharmaco,l 49, 163-169. 143. Aqil, F., Khan, M.S.A., Owais, M. and Ahmad, I. 2005, Journal of Basic Microbiology, 45, 114-123.

178

Maryam Zahin et al.

144. Parekh, J. and Chanda, S.V. 2007, Turk J Biol, 31, 53-58. 145. Ağaoğlu, S., Dostbil, N. and Alemdar, S. 2005, YYÜ Vet Fak, 16 (2), 99-101. 146. Rani, P. and Khullar, N. 2004, Phytother Res, 18(8), 670-3. 147. Takahashi, T., Kokubo, R. and Sakaimo, M. 2004, Let Appl Microbiol, 39(1), 60-64. 148. Joy, B., Rajan, A. and Abraham, E. 2007, Phytotherapy Research, 21(5), 439-443 149. Qureshi, S, Rai, M.K. and Agrawal, S.C. 1997, Hindustan Antibiot Bull, 39(1-4), 56-60. 150. Dweck, A.C. 2001, Article for cosmetics & toiletries magazine ethnobotanical plants from Africa. Black Medicare Ltd, Iltshire, UK. 151. Bbosa, G.S., Kyegombe, D.B., Ogwal-Okeng, J., Bukenya-Ziraba, R., Odyek, O. and Waako, P. 2007, African Journal of Ecology, 45 (Suppl. 1), 13-16. 152. Schelz, Z., Molnas, J. and Hohmann, J. 2006, Fitoterapia, 77 (4), 279-285. 153. Park, K.M., You, J.S., Lee, H.Y, Baek, N.I. and Hwang, J.K. 2003, J Ethnopharmacol, 84(2-3), 181-5. 154. Parmar, C. and Kaushal, M.K. 1982, Wild Fruits. Kalyani Publishers, New Delhi, India. p. 45-48. 155. Narasimhan, B. and Dhake, A.S. 2006, Journal of Medicinal Food, 9(3), 395-399. 156. Khatune, N. A., Mossadik, M. A., Rahman M. M., Khondkar, P., Haque, M.E. and Gray, A.I. 2005, Dhaka University Journal of Pharmaceutical Sciences, 4(1). 157. Simoons, F. J. 1998 “Plants of Life, Plants of Death” The University of Wisconsin Press, Madison, Wisconsin. 158. Silva, M., Coimbra, H.S., Pereira, A.C., Almeida, V.A., Costa, E.S., Silva, R., Filho, A.A.M., Martins, C.H., Bastos, J.K. 2007, Phytother Res, 21(5), 420-422. 159. Pradhan, K.J., Variyar, P.S. and Bandekar, J.R. 1999, Lebensm-Wiss-Technol, 32 (2), 121-123. 160. Ansari, S. H., Ali, M., Velasco-Neuerela A. and Perez-Alonso, M. J. 1998, J Essent Oil Res, 10, 313. 161. Mossa, J.S., El-Feraly, F.S. and Muhammad, I. 2004, Phytother Res, 18(11), 934-7. 162. Moraes, M.R., Lata, H., Bedir, E., Maqbool, M. and Cushman, K. 2002, The American mayapple and its potential for podophyllotoxin production. Trends in new crops and new uses. ASHS Press, Alexandria, VA. p. 527-532. 163. Machadoa, T.B., Ivana C. R. Leala, Ana Claudia F. Amaral b, Kátia R. N. dos Santosc, Marlei G. da Silvac and Ricardo M. Kuster. 2002, J Braz Chem Soc 13 (5), 606-610. 164. Cai, L. and Wu, C.D. 1996, J Nat Prod, 59(10), 987-990. 165. Teixeira, C.C. and Fuchs, F.D. 2006, Journal of Ethnopharmacology, 108 (1), 16-19. 166. Singh, D.V., Verma, R.K., Gupta, M.M. and Kumar, S. 2002, Phytochemical Analysis, 13(4), 207-210. 167. Biradar, Y.S., Jagatap, S., Khandelwal, K.R. and Singhania, S.S. 2007, Evidencebased Complementary and Alternative Medicine. (eCAM Advance Access published online on August 23, 2007). 168. Chattopadhyay, R.R. and Bhattacharyya, S.K. 2007, Pharmacognosy Reviews, 1(1), 151-156. 169. Norajit, K., Laohakunjit, N. and Kerdchoechuen, O. 2007, Molecules, 12, 2047-2060. 170. Li, L.M., liao, X., peng, S.L. and Ding, L.S. 2005, Journal of Integrative Plant Biology, 47 (4), 494-498.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 179-201 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

6. Recent advances in research of antimicrobial effects of essential oils and plant derived compounds on bacteria 1

Zsuzsanna Schelz1, Judit Hohmann2 and Joseph Molnar1

Department of Medical Microbiology and Immunobiology, Faculty of Medicine University of Szeged, H-6720 Szeged, Dóm tér 10., Hungary; 2Department of Pharmacognosy Faculty of Pharmacy, University of Szeged, H-6720 Szeged, Eötvös u. 6., Hungary

Abstract. Antibiotic resistance is a great burden from medicinal and economic point of view, which stems from the overprescription and misuse of anti-infective drugs. It is further aggravated by the horizontal spread of resistance genes between bacterial species and genera. The increasing rate of antibiotic resistance of bacteria urges new attempts to overcome the problem. Antimicrobial agents with new mechanisms resistance modifiers of synthetic or natural origin would serve an alternative way of antimicrobial chemotherapy targeting the inhibition of bacterial growth and the spread of antibiotic resistance. This review reports the biological properties of essential oils and other plant derived compounds with special regard to their antiinfective features and resistance modifier activity, including antibacterial (gram-positive, gram-negative bacteria and Mycobacteria), antiplasmid activities both in vitro and in vivo.

Introduction Medicinal plants have been used for thousands of years; in the ancient times, plant remedies provided the medicinal armamentarium in the treatment Correspondence/Reprint request: Dr. Joseph Molnar, Department of Medical Microbiology and Immunobiology Faculty of Medicine, University of Szeged, H-6720 Szeged, Dóm tér 10., Hungary E-mail: molnarj@comser.szote.u-szeged.hu

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of diverse ailments. There is ample archaeological evidence that people of prehistoric time regularly employed medicinal plants, therefore the application of herbal drugs can be considered universal from the very early ages. Some of the earliest known written records deal with the subject of healing with medicinal substances. The ancient Egyptians of 3000 to 6000 years ago are credited with developing an elaborate and effective pharmacological collection of numerous curing materials obtained from natural resources. In the ancient Greece, Dioscorides is noted for assembling 24 detailed books on over 600 curative plants and their proper uses under the title De Materia Medica, the earliest known designation of that terminology [1]. The system of herbal medicine is built on the rich experience in use of herbs, and this knowledge has been accumulated since the birth of Chinese culture more than 2000 years ago. Thus, plants have an advantage that their application is based on long-term use by humans, often hundreds or thousands of years. The number of studies and surveys of which results can give sufficient information about the adverse effects and toxicity of legendary herbal remedies is increasing, due to the extended awareness and interest in complementary and alternative medicine. This trend can be attributed to several factors: dissatisfaction of patients with the high costs and potentially hazardous side effects of factory-made pharmaceuticals, wide availability of complementary medicinal products, which are often used in self-reported health problems without consultation with a health professional, the misconception about herbal remedies in terms of side effects and safely use. Nearly 1 in 5 people in the US population report using a herb for treatment of health conditions and/or health promotion. More than half did not disclose this information to their physician according to the 2002 National Health Interview Survey in the USA [2]. Based on the presence or absence of scientific evidence, there are two groups of how herbal products may be used: traditional use with limited documentation and established use, which is supported by scientific evidences [3]. Since most of the herbal drugs are Over The Counter (OTC) drugs and are obtainable at herbal drug suppliers, the established use would be the proper approach, therefore the elucidation of safety, toxicity, proper dosage, contamination, potential interactions with synthetic and other natural drugs and possible hazards would be essential [4,5]. Medicinal plants have always provided a stable source for medicines. Not only the herbs themselves but certain plant-derived compounds have served as lead molecules for further chemical modulation and natural products still continue to play a highly significant role in drug discovery and development process [6]. Herbal drugs have the advantage that they were used on a regular basis in the past and those products are still available in the same drug formulation (teas, lotions, powders, ointments, emollients, oils,

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dressings, cleansers) and plants, especially those with ethnopharmacological uses, have been the primary sources of medicines for early drug discovery [7,4]. It was proved by a study that 122 compounds of defined structure, obtained from only 94 species of plants, that are used globally as drugs and demonstrated that 80% of these had an ethnomedical use identical or related to the current use of the active element of the plant. Because these compounds are derived from only 94 species of plants, and a conservative estimate of the number of flowering plants occurring on the planet is 250,000, there should be an abundance of drugs, remaining to be discovered in these plants, which are hidden in undiscovered rain forests and in the oceans [8]. Several plants are part of our diet, which may exert medicinal properties as well. In addition, there is a sharp increase in the use of dietary supplements and great deals of them have a plant origin, however this field is understudied and there are not efficient food regulations. Therefore, the growing concern of food safety also necessitates the broader establishment of the biological activities and toxicity of herbs and plant derived purified compounds. Furthermore, there are clear trends to show that the mainstream in pharmaceutical research is moving away from single molecule or single target approach to combination and multiple target approaches, and for these attempt substances of natural origin are proved appropriate resource [9]. Essential oils are broadly studied of their anti-infective properties, of which importance is supported by their availability. Since essential oils are used in external and internal medicinal products, and are widely used in aromatherapies, in cosmetics as fragrances, there is a demand on a profound knowledge of their activities. In vitro experiments showed that certain essential oils and purified components are able to act against resistance mechanisms in bacteria and tumor cells via plasmid curing and inhibition of efflux processes. The available data in this respect are limited; therefore it would be worthwhile to establish a broader research in this field. The increasingly growing rate of antibiotic resistance of microorganisms necessitates the development and research of new antimicrobial agents or resistance modifiers. The kingdom of plants still provides a wide source for new drugs, therefore substances of herbal origin with antimicrobial properties may be potential candidates for the development of new anti-infective agents. Reversal of multidrug resistance may be another attempt to mitigate the spread of resistance. One approach is to interfere with the genetic material of bacteria, i.e. bacterial plasmids, which may encode the antibiotic resistance mechanisms. Terpenes of essential oils extracted from different herbs are proved to have antimicrobial activity, and some of them may act as resistance modifiers. The purpose of the present article is to review previously published and original data on the antimicrobial activities of the most common essential oils

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and purified components of these plant secondary metabolites, in order to sum up the versatile profile of their effectivity.

Antimicrobial effects of plant extracts and plant derived compounds Several plants exert antimicrobial activity, however this effect is much more pronounced when its crude extracts or purified components are studied. Most available data about the antimicrobial effect of a herb or plant extract are based on in vitro studies, but several herbs are used due to its antimicrobial effects in the complementary- or ethnomedicine based on empirical application in vivo [10]. Several Ayurvedic drugs have been investigated by Gautam et al and it was revealed that the ethnomedical use of herbal remedies for Mycobacterium related infection strongly correlated with their in vitro antibacterial activity against M. tuberculosis [11]. The main disadvantage of the results of in vitro studies that it is difficult to compare to each other because of the different test methods, different methods of extraction, test assays, and variation in chemical phytoconstituents in plants due to different agroclimatic conditions and plant phenotype. Shan et al investigated a series of dietary spices and herbal medicines in order to establish their antibacterial effects against bacteria, of which infections are related to food poisoning. Results emphasized that phenolic compounds significantly contributed to the antibacterial activity of the studied herbs [12]. This activity may be attributed to the enzyme inhibition by the more oxydized phenolic compounds possibly through reaction with sulfhydryl compounds or through more non-specific interactions with the protein. Cinnamic acid, caffeic acid, catechol and pyrogallol were shown to be toxic to microorganisms [13]. A great deal of flavonoids are synthesised by plants to fight against bacterial infections, therefore it is no surprise that they exert in vitro antimicrobial activity. Their effectivity is possibly due to their ability to form complex with extracellular soluble proteins and with bacterial cell walls [14,15]. Catechins deserve special mention, since they considerably contribute to the beneficial effects of oolong green teas. Beside their versatile activities, catechins exert antibacterial effects as well via DNA gyrase inhibition. Specific binding of selected catechins were proved to bind to the N-terminal fragment of gyrase B [16]. Furthermore, catechins are able to restore the susceptibility of resistance bacteria to different antibiotics e.g. tetracycline and beta-lactams, beta-lactamase inhibitors [17,18,19]. Tannins are polymeric water-soluble phenols that are common in higher herbaceous

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and woody plants. Tannins are known of their free radical scavenging activity and have antibacterial effects [20]. Hydrolysable tannins have antibacterial potential against Helicobacter pylori and it seems promising in the eradication of the bacterium without affecting intestinal bacterial flora [21]. Oligomeric proanthocyanidines are mainly used in vascular diseases and it is based on their ability to trap lipid peroxides and free radicals and that they non-competitively inhibit xanthine oxidase, which is a major generator of free radicals [22,23]. Among plant peptides, thionines have toxic effects on different grampositive and gram-negative pathogenic bacteria. Fabatins, which are recently identified peptide molecules from fava beans appeared to inhibit the growth of E. coli and P. aeruginosa [24]. The tea and petroleum extract of St Johnâ&#x20AC;&#x2122;s wort (Hypericum perforatum) were proved to have antibacterial effect against methicillin resistant Staphylococcus aureus strains, which are among the most common and most problematic resistant strains to eradicate. Most probably, hyperforin may be responsible for the beneficial effects, which is one of the active agents responsible for the antidepressant activity [25]. The fruit of Malpighia emarginata DC (Malpighiaceae) is called barbados cherry or acerola fruit. This healthy foodstuff is nutritionally attractive since it contains high amounts of vitamin C, beta-carotene, minerals, plant fibre and small amount of vitamin B. The extracts of the fruit has been used as folk medicines and shows biological effects including antioxidant activity for the prevention of age-related diseases. Motohashi et al examined the antibacterial and antifungal activity of the previously successively extracted fruit and found that the components extracted with acetone showed higher activity against S. aureus, while buthanol extracts had more pronounced antimicrobial effect against Candida albicans. The hexane extracts were effective against Helicobacter pylori with minimal inhibitory concentrations (MICs) between 17-27 Âľg/mL [26]. Similarly, to acerola fruit, the hypophasic and epiphasic extracts of the ripe fruit of spice paprika, Capsicum annuum and Valencia orange and Golden delicious apples were investigated. These extracts displayed potent anti-H. pylori activity with MICs comparable with metronidazole. In an in vitro study, the extracts of a special variant of Capsicum annuum was examined: the fruit of Russian Black sweet pepper was successively extracted and fractionated, and beside the anti-tumor activities, its effects were established against H. pylori. Those extracts and fractions eluted by less polar eluents, and which contain apolar components i.e. carotenoids, showed comparable antibacterial activities with metronidazole [27]. These fruits have a high carotenoid content, which compounds are well known of their antioxidant properties [28]. A high intake of carotenoids has been shown to prevent the development of H. pylori-

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associated diseases [29]. As free radicals play an important role in the pathogenesis of gastroduodenal mucosal inflammation, peptic ulcer disease and probably even gastric cancer, various micronutrients are considered to protect the gastric mucosa by scavenging the free radicals. It was found that the crude extract of Salvia officinalis reduced the minimum inhibitory concentration of aminoglycosides in vancomycin resistant enterococci, then the effective compound was isolated. Carnosol, the active compound showed weak antimicrobial activity and greatly reduced the MICs of various aminoglycosides [30]. Abietane diterpenoids extracted from Salvia sclarea were shown to be bacteriostatic and bactericid for the cultures of S. aureus and S. epidermidis strains, regardless to their antibiotic susceptibility profile [31]. Plants may have direct antimicrobial effects and may act indirectly by stimulating the defence mechanisms, stimulating the immune response. Echinacea is a well-known immunostimulant and anti-inflammatory drug. The immunostimulant effect is complex. It has been reported that E. purpurea has an interferon (IFN)-like effect, activating macrophages, T-lymphocytes and inducing the production of cytokines. Low concentration of Echinacea extracts induce higher levels of cytokines (TNF-Îą, IL-1, IL-6, IL-10) in macrophages, which is consistent with an immune activated antiviral effect [32,33].

Reversal of bacterial resistance by plant derived compounds Antibiotic resistance provides a great therapeutical and economic burden in the treatment of infectious diseases and it may threaten the success of antimicrobial chemotherapy. It is estimated that antibiotic resistance double hospital stay and morbidity. Single antibiotic resistance itself is a great problem, however the appearance of multiple antibiotic resistant (MDR) strains cause a more pronounced obstacle for patients and healthcare professionals [34,35]. Resistant strains appeared soon after the introduction of an antibiotic and initially showed up in hospitals, where most antibiotics were used, causing clinical difficulties for nosocomial treatment on a global scale. Antibiotic resistance has been unavoidable from an evolutionary perspective, since the antibiotic pressure provides the potential for resistant bacteria to acquire an important advantage. Discretionary use of antibiotics lead to the spread of resistant strains, which causes the most expressed problem at nosocomial circumstances, mainly in immunocompromized patients. Important contributing factors to community acquired infection with resistant bacteria may be the poor hand-hygiene among healthcare workers and device-associated infections.

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Antibiotics can be disseminated into the environment from agricultural sources as well, of which importance has recently been recognized and studied extensively. Antibiotics are widely used as growth promoters in veterinary practice in sub-clinical concentrations and this poses a potential risk for the selection of resistant bacterial strains, which may be transmitted to humans. Since the growing rate of bacterial resistance is of great medical and public concern, there is an urgent need to develop appropriate methods and protocols to overcome or at least reduce the incidence and spread of resistant strains [36,37,38]. The mechanisms of resistance show a versatile picture, however certain bacterial strains have intrinsic resistance against antimicrobial agents, acquired resistance against antimicrobial chemotherapy possess a larger risk of therapeutical failure. Antibiotic resistance in bacteria can be divided into the following major groups based on the mechanism involved: Resistance may develop due to the presence of an enzyme that inactivates (betalactamases) or modifies the antibiotic (aminoglycoside, due to the presence of an alternative enzyme for that inhibited by the antibiotic), modification of the antibiotic-target, reduced permeability and active efflux may also lead to resistance [39]. The genetic determinants encoding antimicrobial resistance can be located on the bacterial chromosome or on plasmids, which may replicate independently from the chromosome. Normally susceptible populations of bacteria may become resistant to antimicrobial agents through mutation and selection, or by acquiring from other bacteria the genetic information, that encodes resistance. The acquisition of such genetic elements may occur through transfer of genetic mechanisms i.e. conjugation, transduction and transformation [40]. These mechanisms are in the background of infectious resistance of bacteria. Development of novel antimicrobial agents or substances that target the resistance of bacteria on genetic or protein level would be a possible attempt to overcome the problem. Several in vitro studies prove that herbal medicines or plant-derived compounds may serve sources for this purpose. Many attempts has been made to investigate the potential role of plant extracts and some active compounds for their efficacy to combat the problems of drug resistance in bacteria by reversal of resistance, efflux inhibition, inhibition of biofilm formations, interference in bacterial quorum sensing etc. Some of these fin dings are discussed below. One attempt is the selective inhibition of efflux mechanisms related to antibiotic resistance in order to potentiate the chemotherapeutic agent. Martins et al found that the extract of Carpobrotus edulis is effective in this respect. The genus Carpobrotus is a perennial succulent horizontally low-

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growing sub-shrub. It is traditionally utilized for its medicinal properties: the leaf juice is used for a wide range of bacterial and fungal infections and in the treatment of sinusitis, diarrhoea, infantile eczema, tuberculosis and other internal chest conditions. The crude extract of the leaves was proved to have antibacterial effects against several human pathogenic bacteria. The methanol extract of that plant is able to enhance the killing activity of human peripheral blood monocyte-derived macrophages against intracellular S. aureus. It is hypothesised that it may be attributed to the ability of macrophages to concentrate the extract in vitro, which might be based on the inhibition of efflux functions [41,42,43,44]. A single pump can provide bacteria with resistance to a wide array of chemically and structurally diverse compounds. Microbial efflux was first reported for the efflux of tetracycline by E. coli. Five major families of efflux transporters can be distinguished: the major facilitator superfamily, the resistance-nodulation-division superfamily, the small MDR family, the ATP binding cassette family and the multiple antibiotic and toxin extrusion family. However there does not exist a combination of antibiotic and efflux inhibitor in the treatment of infections, plant derived compounds seem to be potential drug candidates in this respect. The antihypertensive plant alkaloid reserpine was first isolated from the roots of Rauwolfia vomitoria and its efflux inhibitory effect was demonstrated on various bacterial strains. However, this compound seemed ideal as an MDR modifier according to in vitro studies, the further investigation of this substance is highly limited by its neurotoxic effects in the concentration of efflux inhibition [45]. There have also been a number of methoxylated flavones and isoflavones described as putative inhibitors of MDR pumps. These effects are mainly exhibited by potentiation and not by direct inhibition [46]. In several microbial infections, biofilm formation plays a major role in the ineffectivity of antibiotic therapy, therefore the inhibition of biofilm formation would be a possible approach to find resistance modifiers. Biofilm formation is generally regulated in a population-density dependent manner via quorum sensing. An important consequence of biofilm growth is the markedly enhanced resistance to antimicrobial agents where biofilm-associated microorganisms are estimated to be 50 to 500 times more resistant than their planktonic counterparts. (Figure 1.) Quorum-sensing activities have been described for the sesquiterpenoid farnesol. In this role, farnesol produced extracellularly prevented the transition from yeast to hyphal growth in Candida albicans and greatly compromised biofilm formation by this fungus. Some studies indicated a possible interaction of farnesol with cell membranes of certain bacterial species including Streptococcus mutans. This natural compound has the potential to inhibit the development of

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Figure 1. Stages of biofilm formation. In certain microbial infections, biofilm formation plays an important role in the loss of activity of antimicrobial therapy, therefore the inhibition of quorum sensing mechanisms related to biofilm formation would serve a possible approach in the design of antibiotics with new targets. (http://biology. binghamton.edu/davies/images/biofilm.jpg).

dental caries via the inhibition of biofilm formation. The use of the compound would be a novel approach in oral hygiene, since this compound does not necessarily have a major effect on the viability of the oral flora population, but rather it may disrupt the accumulation and the polysaccharide content of dental plaque related to this common oral disease [47]. By monitoring changes in the amount of K+ ions in the presence of terpene alcohols, the rate of leakage of the ion from the bacterial cells was found to increase with increasing concentrations of the compounds added. Farnesol is naturally found in the essential oil of citrus fruits and was shown to devoid of toxic effects and non-mutagenic in vitro and in vivo. Inhibition of formation and accumulation of biofilm communities by affecting the synthesis of polysaccharides is an attractive route for preventing biofilm-related infections. Relatively low concentration of farnesol was sufficient to inhibit biofilm formation, as was shown by viability assays and fluorescence microscopy for both of MRSA and MSSA strains. The hydrophobic nature of farnesol favors its accumulation in the mambrane, possibly causing membrane leakage. It was also successful at enhancing the antibacterial efficacy of antibiotics to which S. aureus strains were somewhat susceptible. This ability of farnesol to sensitize S. aureus to such a heterogeneous group of antibiotics underlines the non-specific nature of this enhancing activity [48]. Farnesol may be a component of adjuvant therapy of skin infections [49,50,51]. The Australian red macroalga Delisea pulchra produces a range of halogenated furanone compounds that display antimicrobial properties. It is

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hypothesized that furanones of D. pulchra constitute a specific means of eukaryotic interference with bacterial signalling process via competing with the quorum-sensing signals. Some derivatives of these compounds were shown to repress quorum sensing in P. aeruginosa and reduce virulence factor expression [52,53]. Resistance of enterococci is a great concern in public health, several strains exhibit multiple antibiotic resistance including that to both ampicillin and vancomycin. Flavonoids i.e. galangin and 3,7-dihydroxyflavone were shown to restore the vancomycin sensitivity of Enterococcus faecalis and E. faecium, by lowering the MICs to the level of vancomycin sensitive strains. It is suggested that the alternatively synthesized disaccharide peptide (ala-lac) production might be initially inhibited by the presence of flavonoid. It seems likely that in the presence of both sub-MIC levels of flavonoids and vancomycin that the production of disaccharide peptides ala-ala and ala-lac were inhibited and cell wall biosynthesis was interrupted as a result of cessation of peptidoglycan synthesis [54].

Antibacterial effects of essential oils The antiseptic properties of aromatic plants and their extracts have been recognised since antiquity and are still used in the medicine, food and cosmetic industry. There appears to be a revival in the use of traditional approach to protecting livestock and food from disease and spoilage in industrial countries. Bearberry (Arctostaphylos uva-ursi) and cranberry juice (Vaccinium macrocarpon) to treat urinary tract infections is reported in different manuals of phytotherapy, while species such as lemon balm (Melissa officinalis), garlic (Allium sativum) and tea tree (Melaleuca alternifolia) are described as broad-spectrum antimicrobial agents. These therapeutic effects can be generally attributed to the essential oils of these plants rather than their extracts [55]. Essential oils are generally isolated from non-woody plants by distillation methods, mainly by steam or hydrodistillation. Essential oils are usually made up of terpenoids, specifically monoterpenes and sesquiterpenes, but diterpenes may also be present and a variety of low molecular weight aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones, coumarines and homologues of phenylpropanoids. Most terpenes are derived from the condensation of branched five-carbon isoprene units and are categorized according to the number of these units present in the carbon skeleton [56]. The mechanism of action of terpenes is not fully understood, it is assumed that membrane disruption by the lipophilic components is involved in the antibacterial action. In vitro studies proved that increasing the hydrophilicity of kaurene diterpenoids by the addition of a

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methyl group drastically reduced their antibacterial activities [57]. Terpenoids may serve as an example of lipid soluble agents which affect the activities of membrane-catalysed enzymes, for example their action on respiratory pathways. Certain components of essential oils can act as uncouplers, which interfere with proton translocation over a membrane vesicle and subsequently interrupt ADP phosphorylation. Specific terpenoids with functional groups, e.g. phenolic alcohols or aldehydes, also interfere with membrane-integrated or associated enzyme-proteins, stopping their production or activity [58]. The mode of action also depends on the microorganism and is mainly related to its cell wall structure. Gram-negative bacteria have intrinsic resistance against toxic components, since they have a permeability barrier against toxic agents. Hydrophobic macromolecules, such as essential oil constituents, are unable to penetrate the barrier. On the other hand, essential oils usually express low aqueous solubility, which prevents them from reaching a toxic level in cellular membranes [59]. In case of tea tree oil, the antimicrobial effect is based on the denaturation of membrane proteins, resulting in the outer membrane disruption, subsequent K+ leakage, respiration inhibition and cell lysis [60,61]. Essential oils are also able to inhibit the synthesis of DNA, RNA, proteins and polysaccharides in fungal and bacterial cells [62]. Since in vitro tests also proved the efficacy of certain essential oils as resistance modulators, there is every likelihood that these plant secondary metabolites might be used in the clinical practice as complementary agents in the treatment of resistance bacterial infections. Nevertheless their antimicrobial action is based on non-specific mechanisms (i.e. membrane disruption), thus the clinical use might be limited to superficial infections.

Antibacterial assays for essential oils For the establishment of antimicrobial properties of essential oils, conventional methods of testing are usually applied. The agar diffusion and broth dilution methods are the most widely used. The assessments of antimicrobial activities by these methods are limited due to the volatility, water insolubility and complexity of essential oils. The agar diffusion method is not considered a perfect method for essential oils, as their volatile components are likely to evaporate with the dispersing solvent during the incubation time, while their poorly soluble components do not diffuse well in the agar broth, but it still remains the most common technique. The inhibitory effect of essential oils in the test tubes and microtitre plates is measured turbidimetrically or with the count plate method. In experiments testing the essential oils activity towards microorganisms, the result depends mainly on

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the method used, however a number of other factors should also be considered. Culture conditions are predominantly influencing the study; therefore these should be precisely being stated in reports. [58].

Antimicrobial properties of selected essential oil producing herbs Peppermint (Mentha piperita) is a perennial flowering member of the mint family. The medicinal use of peppermint and other mint plants date back to the ancient Greece, where peppermint leaf was used internally as a digestive aid and for the management of gallbladder disease and it was also used in the treatment of upper respiratory diseases and for cough. Extracts of peppermint are used as flavouring in many alimentary and cosmetic products and in over the counter medicines. Peppermint oil has become a popular treatment for a variety of conditions. In vitro research shows peppermint oil to be effective in relaxing gastrointestinal smooth muscle. This finding has led to the popularity of enteric-coated peppermint formulations [63]. Since essential oils were found to exert antimicrobial effect on various bacterial strains in vitro, the beneficial effects may be partly attributed to this activity [64,65]. Studies examining the effect of peppermint oil on bowel motility have shown that mechanisms may include calcium channel blocking on a local level, causing smooth muscle relaxation [66,67]. Peppermint oil may reduce spasm during endoscopies and barium enema [68,69,70]. The antibacterial effect of peppermint oil can be used in those conditions which are in relation with the bacterial overgrowth of the small intestine. A number of functional somatic disorders are included like irritable bowel syndrome, fibromyalgia and chronic fatigue syndrome. In these conditions, peppermint oil preparations seem efficient to alleviate the symptoms [71,72]. The antimicrobial effects of peppermint oil and its components have been proved in several studies. The MIC values of the oil is relatively high (may reach 5.0 mg/mL depending on the source), therefore the medicinal use of the oil is limited [73]. Due to its toxicity, the external use of the oil would be preferable, but successful internal application was reported in enteric-coated form. Peppermint oil and its main constituent menthol was described as possible agent in reversal of multidrug resistance in bacteria via their plasmid curing effects. Bacterial plasmids may carry resistance genes for different antibiotics and structurally unrelated chemicals. These plasmids can be transferred by conjugation between different bacterial strains or genera, which are a major cause of the spread of multiple resistance to antibiotics of bacteria. The inhibition of this process by the elimination of resistance-gene

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carrying plasmids seems a promising perspective to overcome the problem. Molnar et al investigated a great number of chemical agents, predominantly tricyclic antipsychotic drugs and carried out a systematic assay in order to find structure-activity relationship in the antiplasmid activities of the investigated substances and it has been proposed that the HOMO orbital energy, the conjugated π-electron system of the tricyclic skeleton and the symmetric π-electron distribution to the L-molecular region and the superdelocalizability of the π-electron system on 10, 12, 13 atoms have special importance in the antiplasmid activity [74,75,76]. Promethazine was one of the most effective plasmid eliminators and was tested in vivo studies as well. Promethazine in combination with gentamycin reduced the number of recurrences in urinary tract infections as compared with the control (gentamycin only) group in children with frequently recurring pyelonephritis and had beneficial effects in urinary tract infections in adult patients [77,78,79,80]. Peppermint oil and menthol, the major component was tested for plasmid curing activity in in vitro experiments on the metabolic plasmid of E. coli F’lac K12 LE140. The plasmid curing activities were relatively high (Table 1.) [65]. Table 1. Antiplasmid activities of peppermint oil and menthol. The plasmid curing activities are designated in percentage.

Sample MTY Control Promethazine

Peppermint oil

Menthol

Concentration (mg/ml)

Bacterial growth after 24 h x108 CFU/5 ml

0 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.18 0.27 0.36 0.45 0.54 0.63 0.250 0.275 0.300 0.325 0.350 0.375 0.400

0 36.0 24.5 3.3 2.3 2.0 0.4 21.6 5.3 2.5 1.3 0.4 3.5 5.3 1.5 3.1 1.3 3.9 -

Plasmid elimination (%) 0.0 0.1 15.0 82.0 55.9 2.5 0 0.4 2.9 9.0 37.5 7.1 82.0 74.0 96.0 64.0 8.7 -

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Figure 2. Plasmid elimination of the Fâ&#x20AC;&#x2122;lac metabolic plasmid of E. coli K12 LE 140. Bacteria with functioning plasmid form dark colonies on the surface of eosine methylene blue agar, while those cells, of which Fâ&#x20AC;&#x2122;lac plasmid has been eliminated develop transparent colonies due to the loss lactose fermentation.

Garlic (Allium sativum L.) has had an important dietary and medicinal role for centuries. Most of its prophylactic and therapeutic effects are ascribed to specific oil- and water-soluble organosulfur compounds, which are responsible for the typical odour and flavour of garlic. During crushing or cutting the clove, the odourless amino acid alliin is cleaved by the enzyme allinase to yield allicin and other thiosulfinates that are the source of the characteristic odour of garlic. The crude extracts of garlic exhibit a broad antimicrobial spectrum against grampositive and gram-negative bacteria.[81,82] Gastric cancer is a major neoplastic diseases on a global scale. Helicobacter pylori is implicated in the aetiology of the development of the disease. It has been revealed that the incidence of stomach cancer is lower in individuals with a high intake of garlic vegetable. In vitro studies proved the anti-Helicobacter properties of garlic, therefore we may conclude that the lower incidence can be attributed to the antibacterial effect of the higher garlic intake, however there have not been made a sufficient number of randomized clinical trials [82]. Thiosulfonates play an important role in the antibacterial activity of garlic. It was shown that the antimicrobial activity of garlic is completely abolished when the thiosulfinates are removed from the extract. The major

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volatile component responsible for the antimicrobial effect is allicin. The antibacterial effect of allicin is of a broad spectrum, even resistant bacterial strains e.g. MRSA, MDR enterotoxigenic strains (ETEC, Enterococci, Shigellae) were found to be sensitive to allicin. Allicin also had an in vivo antibacterial activity against S. flexneri when tested in the rabbit model of experimental shigellosis [83]. The mechanism by which allicin exert its action is presumably based on the rapid reaction with thiol groups of thiolcontaining enzymes. It was also found in in vitro assays that allicin exhibits its activity via immediate and total inhibition of RNA synthesis, although DNA and protein syntheses are also partially inhibited, suggesting that RNA is the primary target [84]. Australian tea tree (Melaleuca alternifolia) lives on low-lying, swampy coastal ground in New South Wales in Australia and unlike several other Melaleuca species it does not occur naturally outside Australia. The essential oil of that plant is employed largely for its antimicrobial properties. Tea tree oil (TTO) is incorporated as active ingredient in many topical formulations used to treat cutaneous infections. The earliest reported use of M. alternifolia plant that presumably exhibited antibacterial activity was the traditional use by Australian aboriginals in New South Wales for coughs and colds. Several in vitro studies proved the antimicobial properties of TTO against grampositive and gram-negative bacterial strains [85,86,87,88]. The activity of TTO against antibiotic-resistant bacteria has attracted considerable interest, with methicillin-resistant Staphylococcus aureus receiving the most attention so far. Subsequent reports on the susceptibility of MRSA to TTO have not shown great differences compared to antibiotic-sensitive organisms [89]. Despite major advances in wound management, infection still remains an important factor in wound healing. In burns, majority of burns are due to complications with sepsis resulting from wound infection. A considerable proportion of wound become colonised by resistant strains of S. aureus. An in vitro study highlights the potential use of TTO impregnaterd dressings for treating wounds infected with MRSA [90]. The mechanism of TTO against microorganisms is partly elucidated. Via disruption of membrane integrity, it is able to permeabilize model liposomal membranes, and in bacterial cells inhibits respiration. Treatment of S. aureus with TTO resulted in leakage of K+ ions, sensitized bacteria to sodium chloride and produced morphological changes apparent electron microscopy, but not whole-cell lysis [91]. In contrast with the absence of whole-cell lysis in S. aureus, lysis occurs in E. coli after the treatment with TTO and this effect is further exacerbated by co-treatment with EDTA. All of these effects confirm that TTO compromises the structural and functional integrity of bacterial membranes. It has also been reported that the susceptibility of bacteria may differ in different growth

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phases, which suggests that in the effects of TTO targets other than the cell membrane may be involved [92]. In parallel with the characterization of in vitro antimicrobial activity of TTO, the clinical efficacy of the oil has also been the subject of investigation. In a single-blind randomized trial, the 5% lotion of tea tree oil (TTO) and benzoyl peroxide (control) were investigated in the treatment of acne. However, the benzoyl peroxide group showed a more rapid remission, the skin discomfort reported during the trial was less frequent in the TTO group [93]. A combination of a 4% tea tree oil nasal ointment and 5% TTO body wash was compared with a standard 2% mupirocin nasal ointment and triclosan body wash for the eradication of ethicillin resistant S. aureus carriage. TTO combination appeared to perform better than the standard combination [94]. In a randomized controlled trial of tea tree topical preparations versus standard preparations (mupirocin nasal ointment, chlorhexidine gluconate soap and silver sulfadiazine cream) were studied. Mupirocin was significantly more effective at clearing nasal carriage of MRSA than tea tree cream, but Tea tree treatment was more effective than chlorhexidine and silver sulfadiazine at clearing superficial skin sites and lesions. Since tea tree preparation was well tolerated by the treated patients, it may considered in regimens for eradication of MRSA carriage [95]. A patient with MRSA osteomyelitis was reported to have alternative anti-inflammatory therapy of a TTO preparation. During treatment, percutaneous TTO containing calcium sulphate pellets were given to the bone. Over a three-month period, the symptoms resolved with a healing response on X-ray [96]. Thyme is stated to possess carminative, antispasmodic, antitussive, expectorant, secretomotor, bactericidal, anthelmintic and astringent properties. Due to its antimicrobial properties, thyme oil is considered a potent food preservative. It has antilisterial activity in relatively low concentration. The effects on the bacterial cells were examined by electron microscopy and the following findings were taken: the bacterial cells shrank, cell wall exhibited budding scars and degenerative changes showing splitting of the wall layers. Lack of cytoplasm and cell membrane disruption was evident at an early stage. These results on the mechanism of action of thyme oil on the inactivation of L. monocytogenes will help in the development or modification of the processing conditions, or the implementation of a new preservation factor to complement those already employed in food preservation and safety [97,98]. Contamination of food comodities with aflatoxin resulting from fungal attack can occur before, after and during the harvest and storage operations. One of the characteristics of aflatoxin deactivation is that it should destroy the mycelia and spores of the toxic fungi, which may proliferate under favorable conditions. The essential oils of thymus species seem good candidates

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for that purpose. In an in vitro study, the essential oil-related inhbition in mycelial growth was observed to be associated with significantly decreased levels of aflatoxin production. 8- and 4-fold dilutions of the essential oils of T. eriocalyx and T. x-porlock are effective in complete retardation of fungal growth respectively and a somewhat lower concentrations are suficient to significantly inhibit aflatoxin production. Contribution of thymol to the positive results seems to be significant [99]. Origanum vulgare is a well known spice mainly in the Mediterraneum. Recently, origanum essential oil has been studied as a potential natural preservative in food manufacturing. Origanum essential oil exerts antimicrobial effect via cell membrane damage, with envelope disruption, formation of blebs and lack of cytoplasmic material. These alterations correlate with the ability of hydrocarbons to interact with hydrophobic structures, like bacterial membranes. These findings are based on investigations of E. coli and S. aureus by transmission electron microscopy, which allows to study the possible bacterial ultrastructural alterations [100].

Antimicrobial activities of individual oil components Essential oils are mainly composed of terpenoids: monoterpenes and sesquiterpenes. Terpenes have been reported to be very active against bacteria. Many essential oil components are chiral compounds. The enantiomers very often show different biological activity. (+)-pinene and (-)-pinene serve a good example, since these enantiomers act differently: (+)-pinen was found to exert more pronounced antimicrobial effect on different bacterial and fungal strains [58]. Carvacrol is one of the most common essential oil components which exert antibacterial effects, it is the major component of oregano and thyme. Carvacrol was investigated in E. coli O157:H7 ATCC 43895 for its effects on the protein synthesis. The presence of 1mM carvacrol during overnight incubation caused the bacterial strain to produce significant amounts of heat shock protein 60 (HSP60) and inhibited the flagellin synthesis highly significantly causing cells to be aflagellate and therefore nonmotile [101]. Addition of carvacrol of the culture of B. cereus results in an increase of membrane fluidity by changing their fatty acid and head-group composition. Carvacrol also caused depletion of the intracellular ATP pool and an increased permeability of the cell membrane for K+ upon exposure to carvacrol. K+ plays a role in the activation of cytoplasmic enzymes, the maintenance of turgor pressure, and possibly the regulation of the cytoplasmic pH. Different studies showed that an efflux of K+ is a first indication of membrane damage in bacteria. [102,103].

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The antifungal activities of carvacrol and eugenol were investigated in immunosuppressed rats for the treatment of oral candidiasis induced by Candida albicans. The anticandidal activity was established by microbiological and hystopathological methods. Nystatin was used as positive control. Microbiologically carvacrol and eugenol significantly reduced the number of colony forming units sampled from the oral cavity of rats treated. Histologically, the untreated control animals showed numerous hyphae on the epithelium of the dorsal surface of the tongue. In contrast, no hyphal colonisation was seen in carvacrol-treated animals, while in rats treated with eugenol, only a few focalized zones of the dorsal surface of the tongue was occupied by hyphae. Carvacrol and eugenol could be proposed as therapeutic agents in the treatment of oral candidiasis. [104] Elastase, a serine proteinase released by human neutrophils, can degrade a wide variety of biomacromolecules including elastin and is considered a biomarker of inflammatory diseases. Thymol, which is a potent antibacterial components of thyme essential oil can approach the ion channel proteins through the lipid phase of the membrane due to its hydrophobic nature and alters the local environment of calcium channels inhibiting capacitative calcium entry. This leads to a corresponding reduction in elastase. The antimicrobial, antioxidant and anti-elastase properties of thymol may have helpful effects in controlling the inflammatory processes present in many infections. [105] In a non-randomized clinical trial the extract of Thea assamica was tested on patients with impetigo contagiosa. The extract was as effective as the antibiotic control [106]. The essential oil of Chenopodium botrys has antibacterial activity on selected gram-positve and Gram-negative bacterial strains, furthermore the extract was proved to have antifungal activity against the ATCC strains of Aspergillus niger and Candida albicans [107].

Summary There is resurgence in the use of herbal medicines worldwide. An estimated one third of adults in the Western world use alternative therapies, including herbs. These herbs may be used either in their primary forms or combined in mixtures. In contrast to chemical drugs, herbs have sometimes been claimed to be non-toxic, because of their natural origin and long-term use as folk medicines. However, problems may arise due to intrinsic toxicity, adulteration, substitution, contamination, misidentification, drug-herb interactions and lack of standardization [108]. This unfavourable fact urges the study of medicinal plants and plant derived compounds used in medicine and food industry.

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A great deal of essential oils has beneficial properties, such as antioxidant, anti-inflammatory and antimicrobial properties. The main constituents of esential oils- mono- and sesquiterpenes including carbohydrates, alcohols, ethers, aldehydes and ketones â&#x20AC;&#x201C; are responsible for the fragrant and biological properties of aromatic and medicinal plants. Despite the development of antibiotics, bacterial and fungal infections are still a major issue in medicine, and the presence of multidrug resistant strains poses a great challenge. Recently, there has been a growing interest in natural products due to their availability and better biodegradebility. In this regard, essential oils may offer a great potential and these plant secondary metabolites may be used as alternative anti-infective and food preservatives [58]. The use of essential oils in the treatment of infectious diseases is limited due to their toxicity but in the treatment of superficial infections or respiratory diseases via aromatherapy may be possible applications. In food industry, the shelf-life of foods is often extended by the addition of antibacterial chemicals, but there is a need for preservatives, which do not alter the organoleptic properties of foods. Some of the essential oils seem good candidate for that purpose to prevent food spoilage as well.

References 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Cast, F. 2003, A Brief History of Drugs: From Plant Extracts to DNA Technology. The Practice of Medicinal Chemistry (Second Edition). Gardiner, P., Graham, R., Legedza, A.T., Ahn, A.C., Eisenberg, D.M., and Phillips, R.S. 2007, Altern. Ther. Health. Med., 13:22-9. Schilter, B., Andersson, C., Anton, R., Constable, A., Kleiner, J., O'Brien, J., Renwick, A.G., Korver, O., Smit, F., and Walker, R. 2003, Food. Chem. Toxicol., 41:1625-49. Halberstein, R.A., 2005, Ann. Epidemiol., 15:686-99. Ernst, E. 1998, Am. J. Med., 104:170-8. Newman, D.J., and Gragg, D.M. 2007, J Nat Prod., 70: 461-77. Chin, Y.W., Balunas, M.J., Chai, H.B., and Kinghorn, A.D. 2006, The AAPS J, 8: E239-53. Fabricant, D.S., and Farnsworth, N.R. 2001, Environ. Health. Perspect., 109S: 69-75. Patwardhan, B. 2005, J. Ethnopharmacol., 100: 50-52. Kumar, V.P., Chauhan, N.S., Padh, H., and Rajani, M. 2006, J. Ethnopharmacol. 107:182-188. Gautam, R., Saklani, A., and Jachak, S.M. 2007, J. Ethnopharmacol., 110: 200-34. Shan, B., Cai, Y.Z., Brooks, J.D., and Corke, H. 2007, Int. J. Food. Microbiol. In Press. Cowan, M.M. 1999, Clin. Microbiol. Rev., 12: 564-582. Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S., Tanigaki, S., Ohyama, M., Tanaka, T., and Iinuma, M. 1996, J. Ethnopharmacol., 50: 27-34. Cushnie, T.P., and Lamb, A.J. 2005, Int. J. Antimicrob. Agents., 26: 343-56.

198

Zsuzsanna Schelz et al.

16. Gradisar, H., Privstovsek, P., Plaper, A., and Jerala, R. 2007, J. Med. Chem., 50: 264-71. 17. Roccaro, A.S., Blanci, A.R., Giuliano, F., Rusciano, R., and Enea, V. 2004, Antimicrob. Agents. Chemother., 48:1968-73. 18. Stapleton, P.D., Shah, S., Anderson, J.C., Hara, Y., Hamilton-Muller, J.M., and Taylor, P.V. 2004, Int. J. Antimicrob. Agents., 23(5): 462-7. 19. Zhao, W.H., Asano, N., Hu, Z.Q., and Shimamura, T. 2003, J. Pharm. Pharmacol. 55: 735-40. 20. Akiyama, H., Fijii, K., Yamasaki, O., Oono, T., and Iwatsuki, K. 2001, J. Antimicrob. Chemother. 48: 487-491. 21. Funatogawa, K., Hayashi, S., Shimomura, H., Yoshida, T., Hatano, T., Ito, H., and Hirai, Y. 2004, Microbiol. Immunol. 48: 251-261. 22. Fine, A.M. 2000, Altern. Med. Rev. 5:144-51. 23. Okuda, T. 2005, Phytochemistry, 66:2012-2031. 24. Zhang, Y., and Lewis, 1997, K. FEMS Microbiol. Lett, 149:59-64. 25. Reichling, J., Weseler, A., and Saller, R. 2001, Pharmacopsychiatry, 34: S116-8. 26. Motohashi, N., Wakabayashi, H., Kurihara, T., Fukushima, H., Yamada, T., Kawase, M., Sohara, Y., Tani, S., Shirataki, Y., Sakagami, H., Satoh, K., Nakashima, H., Molnár, A., Spengler, G., Gyémánt, N., Ugocsai, K., and Molnár J. 2004, Phytother. Res., 18:212-223. 27. Shirataki, Y., Kawase, M., Sakagami, H., Nakashima, H., Tani, S., Tanaka, T., Sohara, Y., Schelz, Z., Molnar, J., and Motohashi, N. 2005, Anticancer. Res., 25: 1991-2000. 28. Molnár, P., Kawase, M., Satoh, K., Sohara, Y., Tanaka, T., Tani, S., Sakagami, H., Nakashima, H., Motohashi, N., Gyémánt, N., and Molnár J. 2005, Phytother. Res. 19: 700-707. 29. Zhang, Z.W., Patchett, S.E., Perrett, D., Domizio, P., and Farthing, M.J. 2000, Eur. J. Gastroenterol. Hepatol., 12: 497-503. 30. Horiuchi, K., Shiota, S., Kuroda, T., Hatano, T., Yoshida, T., and Tsuchiya, T. 2007, Biol. Pharm. Bull., 30: 287-90. 31. Kuzma, L., Rozalski, M., Walencka, E., Rosalska, B., and Wysokinska, H. 2007, Phytomedicine, 14: 31-5. 32. Mishima, S., Saito, K., Maruyama, H., Inoue, M., Yamashita, T., Ishida, T., and Gu, Y. 2004, Biol. Pharm. Bull. 27:1004-1009. 33. Burger, R.A., Torres, A.R., Warren, R.P., Caldwell, V.D., and Hughes, B.G. 1997, Int. J. Immunopharmac. 19:371-379. 34. Levy, S.B., and Marshall, B. 2004, Nat. Med., 10: S122-129. 35. Smith, M.A. 2005, Nurs. Clin. North. Am., 40: 63-75. 36. Barber, M. 1948, Lancet., 2: 641-644. 37. Crofton, J., and Mitchinson, D.A. 1948, Br. Med. J., 2:1009-1015. 38. Schneierson, SS. 1948, J. Bacteriol., 55:393-399. 39. Schmitz, F.J., and Fluit, A.C. Mechanisms of Antibacterial Resistance (Chapter 189) in Cohen & Powderly. Infectious Diseases, 2nd ed. 2004 Mosby, An Imprint of Elsevier. 40. Tenover, F.C. 2006, Am. J. Infect. Control., 34: S3-S10. 41. Martins, M., Ordway, D., Kristiansen, J., Viveiros, M., Leandro, C., Molnar, J., and Amaral, L. 2005, Fitoterapia, 76: 96-99.

Antimicrobial effects of essential oils

199

42. Ordway, D., Hohmann, J., Viveiros, M., Viveiros, A., Molnar, J., Leandro, C., Arroz, M.J., Gracio, M.A., and Amaral, L. 2003, Phytother. Res., 17: 512-9. 43. Watt, E., Pretorius, J.C. 2001, J. Ethnopharmacol., 76: 87-91. 44. Springfield, E.P., Amabeoku, G., Weitz, F., Mabusela, W., and Johnson, Q. 2003, Phytomedicine, 10: 434-9. 45. Amaral, L., Martins, M., and Viveiros, M. 2007, J. Antimicrob. Chemother. 59: 1237-46. 46. Stavri, M., Piddock, L.J., and Gibbons, S. 2007, J. Antimicrob. Chemother., 59: 1247-60. 47. Koo, H., Hayacibara, M.F., Schobel, B.D., Cury, J.A., Rosalen, P.L., Park, Y.K., Vacca-Smith, A.M., and Bowen, W.H. 2003, J. Antimicrob. Chemother. 52: 782-9. 48. Jabra-Rizk, M.A., Meiller, T.F., James, C.E., and Shirtliff, M.E. 2006, Antimicrob. Agents. Chemother., 50: 1463-9. 49. Akiyama, H., Oono, T., Huh, W.K., Yamasaki, O., Ogawa, S., Katsuyama, M., Ichikawa, H., and Iwatsuki, K. 2002, Chemotherapy, 48: 122-8. 50. Brehm-Stecher, B.F., and Johnson, E.A. 2003, Antimicrob. Agents. Chemother. 47: 3357-60. 51. Inoue, Y., Shiraishi, A., Hada, T., Hirose, K., Hamashima, H., and Shimada, J. 2004, FEMS Microbiol. Lett., 237: 325-31. 52. Hentzer, M., and Givskov, M. 2003, J. Clin. Invest. 112: 1300-7. 53. Hentzer, M., Riedel, K., Rasmussen, T.B. Heydorn, A., Andersen, J.B., Parsek, M.R., Rice, S.A., Eberl, L., Molin, S., Hoiby, N., Kjelleberg, S., and Givskov, M. 2002, Microbiology, 148: 87-102. 54. Liu, I.X., Durham, D.G., and Richards, R.M.E. 2001, J. Pharm. Pharmacol., 53: 129-132. 55. RĂos, J.L., and Recio, M.C. 2005, J. Ethnopharmacol., 100: 80-84. 56. Dorman, H.J., and Deans, S.G. 2000, J. Appl. Microbiol. 88(2): 308-16. 57. Mendoza, L., Wilkens, M., and Urzua, A. 1997, J. Ethnopharmacol., 58: 85-8. 58. Kalemba, D., and Kunicka, A. 2003, Curr. Med. Chem., 10: 213-829. 59. Mann, C.M., Cox, S.D., and Markham, J.L. 2000, Lett. Appl. Microbiol., 30: 294-7. 60. Cox, S.D., Gustafson, J.E., Mann, C.M., Markham, J.L., Liew, Y.C., Hartlan, R.P., Bell, H.C., Warmington, J.R., and Wyllie, S.G. 1998, Lett. Appl. Microbiol., 26: 355-8. 61. Hada, T., Inoue, Y., Shiraishi, A., and Hamashima, H. 2003, J. Microbiol. Methods. 53: 309-12. 62. Zani, F., Massimo, G., Benvenuti, S., Bianchi, A., Albasini, A., Melegari, M., Vampa, G., Bellotti, A., and Mazza, P. 1991, Planta Med., 57: 237-41. 63. Kligler, B., and Chaudhary, S. 2007, Am. Fam. Physician., 75: 1027-30. 64. Hammer, K.A., Carson, C.F., and Riley, T.V. 1999, J. Appl. Microbiol., 86: 985-990. 65. Schelz, Z., Hohmann, J., and Molnar, J. 2006, Fitoterapia, 77: 279-85. 66. Hills, J.M., and Aaronson, P.I. 1991, Gastroenterology, 101: 55-65. 67. Micklefield, G.H., Greving, I., and May, B. 2000, Phytother. Res. 14: 20-3. 68. Asao, T., Mochiki, E., Suzuki, H., Nakamura, J., Hirayama, I., Morinaga, N., Shoji, H., Shitara, Y., and Kuwano, H. 2001, Gastrointest. Endosc., 53: 172-7.

200

Zsuzsanna Schelz et al.

69. Yamamoto, N., Nakai, Y., Sasahira, N., Hirano, K., Tsujino, T., Isayama, H., Komatsu, Y., Tada, M., Yoshida, H., Kawabe, T., Hiki, N., Kamanishi, M., Kurosaka, H., and Omata, M. 2006, J. Gastroenterol. Hepatol., 21: 1394-8. 70. Mizuno, S., Kato, K., Ono, Y., Yano, K., Kurosaka, H., Takahashi, A., Abeta, H., Kushiro, T., Miyamoto, S., Kurihara, R., Hiki, N., Kaminishi, M., Iwasaki, A., and Arakawa, Y. 2006, J. Gastroenterol. Hepatol., 21: 1297-301. 71. Logan, A.C., and Beaulne, T.M. 2002, Altern. Med. Rev., 7: 410-7. 72. Grigoleit, H.G., and Grigoleit, P. 2006, Phyromedicine, 12: 601-6. 73. Iscan, G., Kirimer, N., Kürkcüoglu, M., Baser, K.H.C., and Demirci, F. 2002, J Agric. Food. Chem., 50: 3943-46. 74. Mándi, Y., Molnar, J., Holland, B., and Béládi, I. 1976, Genet. Res., 26: 109-111. 75. Molnár, J., Király, J., and Mándi, Y. 1975, Experientia, 31: 444-445. 76. Molnar, J., Domonkos, K., Mándi, Y., Földeák, S., and Holland, I.B. 1980, Phenothiazines and structurally related drugs: Basic and Clinical Studies, Usdin, Eckert and Forrest (Ed.). Elsevier-North Holland, Amsterdam, 115-118. 77. Molnar, J., Haszon, I., Bodrogi, T., Martonyi, E., and Turi, S. 1990, Int. Urol. Nephrol., 22: 405-11. 78. Kásler, M., Poczik, M., Molnar, J., and Ágoston, É. 1982, Urol. Nephrol. Szemle, 9: 130-133. 79. Molnár, J., Mucsi, I., and Kása, P. 1983, Zbl. Bakt. Hyg., I. Abt. Orig, 254: 388-396. 80. Molnar, J., Foldeak, S., Nakamura, M.J., Rausch, H., Domonkos, K., and Szabo, M. 1992, APMIS Suppl., 30: 24-31. 81. Adetumbi, M.A., and Lau, B.H. 1983, Med. Hypotheses., 12: 227-237. 82. Sivam, G.P. 2001, J. Nutr., 131: 1106S-8S. 83. Chowdhury, A.K., Ahsan, M., Islam, S.N., and Ahmed, Z.U. 1991, Indian. J. Med. Res., 93: 33-6. 84. Feldberg, R.S., Chang, S.C., Kotik, A.N., Nadler, M., Neuwirth, Z., Sundstrom, D.C., and Thompson, N,H. 1988, Antimicrob. Agents. Chemother., 32: 1763-8. 85. May, J., Chan, C.H., King, A., Williams, L., and French, G.L. 2000, J. Antimicrob. Chemother., 45: 639-643. 86. Brady, A., Loughlin, R., Gilpin, D., Kearney, P., and Tunney, M. 2006, J. Med. Microbiol., 55: 1375-80. 87. Papadopoulos, C.J., Carson, C.F., Hammer, K.A., and Riley, T.V. 2006, J. Antimicrob. Chemother., 58: 449-51. 88. Messager, S., Hammer, K.A., Carson, C.F., and Riley, T.V. 2005, J. Hosp. Infect., 59: 113-25. 89. Carson, C.F., Cookson, B.D., Farrelly, H.D., and Riley, T.V. 1995, J. Antimicrob. Chemother., 35: 421-4. 90. Edwards-Jones, V., Buck, R., Shawcross, S.G., Dawson, M.M., and Dunn, K. 2004, Burns, 30: 772-777. 91. Carson, C.F., Mee, B.J., and Riley, T.V. 2002, Antimicrob. Agents. Chemother. 46: 1914-20. 92. Gustafson, J.E., Liew ,Y.C., Chew, S., Markham, J., Bell, H.C., Wyllie, S.G., and Warmington, J.R. 1998, Lett. Appl. Microbiol., 26: 194-8. 93. Bassett, I.B., Pannowitz, D.L., and Barnetson, R.S. 1990, Med. J. Aust., 153: 455-8.

Antimicrobial effects of essential oils

201

94. Caelli, M., Porteous, J., Carson, C.F., Heller, R., and Riley, T.V. 2000, J. Hosp. Infect., 46(3): 236-7. 95. Dryden, M.S., Dailly, S., and Crouch, M. 2004, J. Hosp. Infect., 56: 283-6. 96. Sherry, E., Boeck, H., and Warnke, P.H. 2001, BMC Surg., 1:1. 97. Rasooli, I., Rezaei, M.B., and Allameh, A. 2006, Int. J. Infect. Dis., 10: 236-41. 98. Youdim, K.A., and Deans, S.G. 1999, Mechanisms of Ageing and Development, 109: 163-175. 99. Rasooli, I., and Abyaneh M.R. 2004, Food Control, 15: 479-483. 100. Becerril, R., Gomez-Lus, R., Goni, P., Lopez, P., and Nerin, C. 2007, Anal. Bioanal. Chem., 388: 1003-11. 101. Burt, S.A., van der Zee, R., Koets, A.P., de Graaff, A.M., van Knapen, F., Gaastra, W., Haagsman, H.P., and Veldhuizen, E.J. 2007, Appl. Environ. Microbiol. 73: 4484-90. 102. Ultee, A., Kets, E.P.W., Alberda, M., Hoekstra, F.A., and Smid, E.J. 2000, Arch. Microbiol., 174: 233-238. 103. Ultee, A., Kets, E..P, and Smid, E.J. 1999, Appl. Environ. Microbiol., 65: 4606-10. 104. Chami, N., Chami, F., Bennis, S., Trouillas, J., and Remmal, A. 2004, Braz. J. Infect. Dis., 8: 217-26. 105. Braga, P.C., Dal Sasso, M., Culici, M., Bianchi, T., Bordoni, L., and Marabini, L. 2006, Pharmacology, 77: 130-6. 106. Martin, K.W., and Ernst, E. 2003, J. Antimicrob. Chemother. 51: 241-246. 107. Maksimovicz, Z.A., Dordevic, S., and Mraovic, M. 2005, Fitoterapia, 76:112-4. 108. Zhou, S., Koh, H.L., Gao, Y., Gong, Z.Y., and Lee, E.J.D. 2004, Life Sciences, 74: 935-968.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 203-226 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

7. Ethnomedicinal plants to fight neoplastic diseases C. K. K. Nair, P. Divyasree and G. Gopakumar Department of Radiation Biology, Amala Cancer Research Centre Thrissur 680555, Kerala, India

Abstract. According to one of the ancient proverbs in India,â&#x20AC;? there is no plant on earth which has no medicinal property.â&#x20AC;? More than 90% of the compounds used in modern medicine have their origin from plant sources. A large number of plants have been used by man from ancient times as medicine for curing various ailments. In recent times there is an upsurge of interest and focus on the importance of medicinal plants and traditional health systems in solving the health care problems of the world. This article gives an over view and a brief account of the important ethno-medicinal plants used in treating neoplastic diseases.

Introduction Cancer is one of the most dreaded diseases of mankind and it is considered as an adversary of modernization and the pattern of socioeconomical life dominated by western medicine. Multidisciplinary scientific investigations are making best efforts to combat this disease, but a perfect cure is yet not realized in modern medicine. Recently, a greater emphasis has been given towards the researches on complementary and alternative medicine Correspondence/Reprint request: Dr. C. K. K. Nair, Department of Radiation Biology, Amala Cancer Research Centre, Thrissur 680555, Kerala, India. E-mail: ckknair@yahoo.com

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that deals with cancer management. Several studies employing methodologies of modern medicine have been conducted on a multitude of herbs of ethno-botanical importance (Dahanukar et al., 2000; Duke and Ayensu, 1985). Ayurveda, the traditional Indian system of medicine, has been successful from ancient times in using natural drugs, mostly herbal preparations, in preventing or suppressing various tumors using several lines of treatment. Thousands of herbal and traditional compounds are being screened worldwide to validate their use as anticancerous drugs (Diwanay et al., 2004; Liu et al., 1998; Premalatha and Govindarajan., 2005). The science of Ayurveda describes modalities on the curative aspects of cancers that have resemblance with clinical entities of arbuda and granthi mentioned in Sushrutha Samhita. An integrated approach is need of the day to manage cancer using the growing body of knowledge gained through scientific developments. The emerging integrative model of cancer treatment recognizes the importance of botanical medicine. The principles underlying herbal medicine are relatively simple, although they are not quite well understood and distinct from modern medicine.

Cancer therapy-a practical dilemma Any practical solution in combating cancer is of paramount importance. Many herbs have been evaluated in clinical studies and are currently being investigated phytochemically to understand their tumouricidal actions against various cancers. The traditional Indian system of medicine with its evolution through centuries has always fascinated practitioners and researchers on a scientifically proven research background. Herbal medicines have a vital role in the prevention and treatment of cancer. Some herbs protect the body from cancer by enhancing detoxification functions of the body (Bradstreet, 1997). Certain biological response modifiers derived from herbs are known to inhibit growth of cancer by modulating the activity of specific hormones and enzymes. Some herbs reduce toxic side effects of chemotherapy and radiotherapy and are often employed for cancer treatment (Kapoor 1990). Research work at the Sino-Vedic Research Centre, [Sino-Vedic Cancer Clinic, FB-12, Shivaji Enclave, New Delhi-110 027 (India)] aims to develop herbal formulations to boost immune system of the body against cancer, improve quality of life and prolong comfortable lifespan in the patients suffering from advanced stages of cancer. The recent upsurge of global interest in herbal medicines can be attributed to the spread of the traditional knowledge of the orient along with the realization and deeper understanding of the side effects and the waning effectiveness of some the conventional modern medicines such as antibiotics, which once had near-universal effectiveness against serious infections.

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The traditional Indian system of medicines, Ayurveda, uses about 2,000 plant species, while the Chinese Pharmacopoeia lists over 5,700 traditional medicines, most of which are of plant origin. Some of the ethnomedicinally important plants used to fight against cancer in traditional health care are listed below.

1. Andrographis paniculata Family: Acanthaceae Since ancient times, Andrographis paniculata (Kalmegh) is used as a wonder drug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and some other countries for multiple clinical applications. The plant extract exhibits anti-typhoid and anti-fungal activities. This plant is also reported to possess anti-hepatotoxic, anti-malarial and antiinflamatory properties, besides its general use as an immuno stimulant agent (Trivedi and Rawal, 2001). Andrographolide is the major constituent from the leaves which is a bicyclic diterpenoid lactone. Many in vitro studies have been conducted and andrographolide looks promising to be developed as a normal pharmacophore in cancer treatment (Kumar et al., 2004; Kumaran et al., 2003). Andrographolide treatment inhibited the proliferation of different tumor cell lines, representing various types of cancers. The compound exerts direct anticancer activity on cancer cells by cell cycle arrest at G0/G1 phase through induction of cell cycle inhibitory protein p27 and decreased expression of Cyclin dependent Kinase 4 (CDK4) (Rajagopal et al., 2003). Immunostimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes and production of interleukin 2 (Kumar et al., 2004). Andrographolide also enhanced the Tumor necrosis factor-Îą production and CD marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may contribute for its indirect anticancer activity (Kumar et al., 2004).

2. Aegle marmelos Family: Rutaceae Aegle marmelos is commonly known as Bael tree in India and considered sacred by Hindus. The extract of the plant is found to contain lupeol, a known tri-terpenoid, as a major bioactive component (Lambertini et al., 2005). It was found to stimulate and increase the expression of Era gene in MDA-MB231 Era â&#x20AC;&#x201C;negative breast cancer cells and also inhibited cell proliferation. Phytoconstituents derived from the fruit of Aegle marmelos were found to

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have strong anti-cancer activity against thyroid cancer (Lampronti et al., 2003). It is also used in the treatment of primitive neuro ectodermal tumors and malignant ascites, in addition it also possesses anti-viral and anti-inflamatory properties (Jagetia et al., 2005). The medicinal value of the bael fruit is enhanced due to the presence of tannin in its rind. A pyranocoumarin isolated from its seed gave significance protection against pylorus ligation and aspirin induced gastric ulcers in rats. (Dahanukar et al., 2000). Anticancer active principles derived from the herb Aegle marmelos, are used in the treatment of primitive neuro-ectodermal tumours (PNET) and also used in the treatment of various malignant tumours of brain and spinal cord (http://www.cancercliniconline.com/sinovedicanticancerherbs/htm).

3. Centella asiatica Family: Apiaceae Centella asiatica is a profusely branched prostrate herb consisting of active principles such as vallarine, asiaticoside, sitosterol, tannin, oxyasiaticoside (Brinkhaus et al., 2000). Asiaticoside stimulates the healing of chronic lesions such as ulcers, surgical wounds, fistula and gynecological and bladder lesions (Maquart and Francois, 1990). It is also used for treatment of psoriasis and found to be effective in destroying cultured cancer cells (Maquart and Francois, 1990). Centella asiatica protects from cancer by enhancing immune functions of the body (Punturee et al., 2007). The extract of the whole plant has shown strong anticancer activity (Yu et al., 2006). In Brazil, Centella asiatica is used to treat the uterine cancer (Yoshida et al., 2005).

4. Curcuma longa Family: Zingiberaceae Curcuma longa extract exhibits antioxidant properties and contains a yelloworange polyphenol (Srivastava et al., 1995). The strong anti-oxidant and antiinflammatory characteristics are its most obvious medicinal properties (Mukophadhyay et al., 1982). The major constituents of the extracts are curcumin sulphate and glucuronide. It causes apoptosis in various cancer cell types including skin, colon, fore-stomach, duodenum and ovary (Lee et al., 2002). The plant extract also possess anti-viral, anti-bacterial and anti-fungal activities (Babu et al., 2006). Curcumin, one of the most studied chemopreventive agent, is a natural compound extracted from Curcuma longa that allow suppression, retardation and invasion of carcinogenesis. Curcumin is also described as an antitumoral, anti-oxidant and anti-inflammatory agent capable of inducing apoptosis

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in numerous cellular systems (Duvoix et al., 2005). Curcumin can interfere with the cell growth cycle of A549 cell and suppress cell growth (Zhang et al., 2004). It has been shown to inhibit the growth of cancer by preventing production of harmful eicosanoid such as PGE-2 (Srivastava et al., 1995). The anticancer effect of curcumin has been demonstrated in all the steps of cancer development, ie,. initiation, promotion and progression of cancer (Mahady et al., 2002). In addition to inhibition of the genesis of cancer curcumin promotes the regression of cancer (Duvoix et al., 2005). Curcumin suppresses mutagenic effect of various mutagens including cigarette smoke condensates, 7, 12-dimethylbenz (a) anthracene (DMBA) and benzopyrene. Curcumin is found to decrease levels of urinary mutagens (Polasa et al., 1992). It also possesses anti-inflammatory and antioxidant properties (Mukophadhyay et al., 1982). The protective effects of Curcuma longa and its derivatives are partially due to direct antioxidant effect (Selvan et al., 1995). Studies have revealed that Curcuma longa inhibits production of nitrosamine that enhances natural antioxidant functions of the body (Churchill et al., 2000). Curcuma longa increases levels of glutathione and other non-protein sulfhydryls (Biswas et al., 2005). It acts directly on several enzymes. Curcumin is used to treat squamous cell carcinoma of the skin and the ulcerating oral cancer. Curcuma longa also prevents malignant transformation of leukoplakia (Cheng et al., 2001).

5. Heliotropium indicum Family: Boraginaceae Heliotropium indicum, a widely used indigenous plant in Ayurvedic medicine. The whole plant is used as a medicine. The leaves are useful in fever, urticaria, ulcers, wounds, localized inflammation, gonorrhoea, ringworm, rheumatism and erysipelas (Srinivas et al., 2000 and Reddy et al., 2002). The major constituents of the extract of Heliotropium indicum are tannins and alkaloids (Singh et al., 2003). Indicine-N-oxide derived from Heliotropium indicum has been found to have an antitumor activity and has been used in clinical trials as a chemotherapeutic agent for leukemia and solid tumors (Rao and Mcbride, 1968). Extracts of Heliotropium indicum showed significant activity in several experimental tumor systems (Kugelman et al., 1976).

6. Aloe vera Family: Aloaceae The plant Aloe vera has been used in several ayurvedic medicine. Aloe vera contains aloe-emodin, which activates the macrophages to fight cancer.

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Aloe vera also contains acemannan, which enhances activity of the immune cells against cancer. Aloe vera is found to inhibit metastasis (Lissoni et al., 1998). Lectin from Aloe, when injected directly into tumors activated the immune system to attack the cancer (Akev et al., 2007). Killer T cells, white blood cells that bind to invading cells and destroy them, began to attack tumor cells injected with lectin. Extract of Aloe has been prove to activate macrophages (white blood cells which "swallow" antigens), causing the release of immune-activating (and anticancer) substances such as interferons, interleukins and Tumor necrosis factor. In addition, it was found to promote the growth of normal (non- cancerous) cells and halt the growth of tumors (Choi and Chung, 2003). In addition the extract lowered the level of serum cholesterol, inflamation and arthritis and protected the body from oxidative stress (Saada et al., 2003).

7. Rubia cordifolia Family: Rubiaceae Rubia cordifolia (Linn) is a common plant found in southern parts of India and is used in indigenous systems of medicine. The extract of this plant has been reported to possess a significant antioxidant activity as well as immunomodulating properties (Joharapurkar et al., 2003). The major chemical constituents of the extract are rubiadin, rubierythrinic acid, purpurin, alizarin, pseudopurpurin and munjistin etc. (Tripathi et al., 1997). The extract of this plant shows anti-cancer ativity against a spectrum of tumor models such as leukemia, ascitic carcinoma, large intestinal and lung tumors, melanoma etc. (Adwankar and Chitinis, 1982). The extract of this plant has been reported to contain a number of cyclic hexapeptides with potent anti tumor activity (Wakita et al., 2004 and Hideji et al., 1984). The methanol extract of the herb has 80% inhibitory rate against ascitic S180 murine tumor (Kinghorn et al., 1999). In vitro test showed that water extract of the herb has 100% inhibitory rate against human cervical carcinoma JTC-26 cell line (Premalatha and Govindarajan, 2005).

8. Withania somnifera Family: Solanaceae The plant Withania somnifera is known in India as Ashwagandha, also called Indian ginseng. Extract of this herb is nontoxic and have been considered as an adaptogen having nonspecific activity to normalize physiological function, working on the Hypothalamic Pituitary Adrenal

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(HPA) axis and the neuroendocrine system (Thatte et al., 1999). The roots and berries of the plant are used in herbal medicine. In Ayurveda, the fresh roots are sometimes boiled in milk, prior to drying, in order to leach out undesirable constituents (Elsakka et al., 1990). The major chemical constituents are Anaferine alkaloid, Anahygrine, Beta-Sisterol, Chlorogenic acid (in leaf only), Cysteine (in fruit), Iron, Scopoletin, Somniferinine, Somniferiene, Tropanol, Withanine, Withananine and Withanolides A-Y (Steroidal lactones) (Elsakka et al., 1990). Among withanolides, which possess immunomodulatory activity, Withaferin A and Withanolide D found in Withania somnifera was reported to inhibit growth of cancer (Mathur et al., 2006). Studies have revealed that Withania somnifera enhances the therapeutic effect of radiotherapy. The principle constituents of its roots, withanolides have been believed to account for the multiple medicinal applications of ashwagandha (Dhar et al., 2006). These molecules are steroidal lactones with ergostane, which include withanone, withaferin, withanolides, withasomidienone, withanolide C and alkaloids about 0.2 %. The extract of this plant inhibited benzo(a)pyrene-induced forestomach papillomagenesis, showing up to 60 and 92% inhibition in tumor incidence and multiplicity, respectively (Wattenberg et al., 1980). Similarly, Withania inhibited 7,12-dimethylbenzanthracene-induced skin papillomagenesis, showing up to 45 and 71% inhibition in tumor incidence and multiplicity (Padmavathi et al., 2005). Thus the root extract has a chemopreventive efficacy against forestomach and skin carcinogenesis.

9. Ocimum sanctum Family: Lamiaceae The plant Ocimum sanctum, is called Tulsi in India is considered sacred by the Hindus. It is a tropical, much branched, annual herb. In several ancient systems of medicine including Ayurveda, Greek, Roman, Siddha and Unani, Ocimum has vast number of therapeutic applications such as in cardiopathy, haemopathy, leucoderma, asthma, bronchitis, catarrhal fever, otalgia, hepatopathy, lumbago, hiccups, ophthalmia, gastropathy, genitourinary disorders, ringworm, verminosis, skin diseases, etc. A variety of biologically active compounds have been isolated from the leaves of this plant and these include ursolic acid, apigenin, luteolin, ocimumosides A and B and ocimarin, apigenin, apigenin-7-O-β-D-glucopyranoside, apigenin-7-O-β-D-glucuronic acid, apigenin-7-O-β-D-glucuronic acid 6′′-methyl ester, luteolin-7-O-β-D-glucuronic acid 6′′-methyl ester, luteolin-7-O-β-D-glucopyranoside, luteolin-5-O-β-Dglucopyranoside, and 4-allyl-1-O-β-D-glucopyronosyl-2-hydroxybenzene, and two known cerebrosides (Gupta et al., 2007). Ocimum sanctum extract was found to be active against multidrug-resistant strains of Staphylococcus

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aureus that were also resistant to common beta lactam antibiotics (Barghava and Singh, 1981). Moreover Tulsi juice of fresh leaves has antibacterial, anti-oxidant, anti diabetic, antifungal, antitubercular, antistress, hypotensive, anti asthmatic and anti-inflammatory properties (Barghava and Singh, 1981) Anticancer and chemopreventive properties of Ocimum have been reported (Karthikeyan et al., 1999). Topical application of Ocimum extract significantly reduced the cumulative number of papillomas in 7,12-dimethylbenz(a) anthracene-induced skin papillomagenesis in rats (Prashar et al., 1994). A significant 2-fold elevation of reduced glutathione content and increased glutathione S-transferase activity was also observed in the skin of extract treated animals. Rat hepatocytes pretreated with the extract and then with DMBA showed significant reduction in DMBA-DNA adducts (Prashar et al., 1998). Pretreatment with 500 micrograms of the extract caused a 93% reduction in the mean values of DMBA-DNA adducts (Prashar et al., 1998). Similar effects were also noted with DMBAinduced hamster buccal pouch carcinogenesis (Karthikeyan, et al., 1999). The extract have been reported to shown increased activities of cytochrome p450, cytochrome b5, aryl hydrocarbon hydroxylase and glutathione S-transferase, all of which are important in the detoxification of carcinogens and mutagens (Prashar et al., 1994). Beneficial effects of the extract of this plant have also been reported in radiotherapy of human cancer (Ganasoundari et al., 1998).

10. Plumbago zeylanica Family: Plumbaginaceae The plant species Plumbago zeylanica (known in India as Chitraka), of the Plumbaginaceae family, is distributed as a weed in throughout the tropical and subtropical countries of the world. The family Plumbaginaceae consists of 10 genera and 280 species. The genus Plumbago includes 3 species, namely Plumbago indica L. (P. rosea L.) P. capensis L., and P. zeylanica L., which are distributed in several parts of India. The root of Plumbago zeylanica (Chitraka or Chitramulamu) has numerous therapeutic uses. In Indian system of medicine, Ayurveda the root of the plant is known to be abortifacient and having vesicant property. It is used as an appetizer and is diuretic. It is used for treating diarrhea, dysentery, piles and peptic ulcers. The bioassay-guided fractionation of the dichloromethane extract of aerial parts of Plumbago zeylanica led to the isolation of β-sitosterol, β-sitosteryl-3β-glucopyranoside, β-sitosteryl-3βglucopyranoside-6′-O-palmitate, lupenone, lupeol acetate, plumbagin and trilinolein (Nguyena et al., 2004). Plumbagin modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-κB, suggesting plumbagin might affect the NF-κB activation pathway (Santosh et al., 2006). Thus plumbagin inhibited NF-κB activation induced by TNF, and other carcinogens and

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inflammatory stimuli (eg. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1β, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-κB activation in certain tumor cells (Santosh et al., 2006). The suppression of NF-κB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IκB α kinase, IκB α phosphorylation, IκB α degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-κB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-β, and the p65 subunit of NF-κB (Chen et al., 2007). Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol (Santosh et al., 2006). Plumbagin down-regulated the expression and activity of NF-κB-regulated expressions of anti-apoptotic genes (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products (Hsu et al., 2006). This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion (Hsu et al., 2006).

11. Semecarpus anacardium Family: Anacardiaceae Semecarpus anacardium is a common tree of dry deciduous forests, easily recognized by large leaves and the red blaze exuding resin which blackens on exposure (Kirthikar and Basu, 1975). It is known as Bhallatak in India. The nuts of S. anacardium showed the presence of biflavonoids, phenolic compounds, bhilawanols, minerals, vitamins and amino acids. In addition to these, anacardic acid, cardol, catechol, anacardol and fixed oil were also found to be present in this plant (Premalatha and Sachdanandam, 1999). The fruits of the plant were reported to possess good anti-inflammatory agent and effective in various types of cancers (Chitinis et al., 1980). The nut extract of S. anacardium was found to reduce the extracellular matrix (ECM) which normally present at an elevated level in the early stage of invasion and was also responsible for the development of vascular bed, endothelial cell proliferation and invasion of tumour cells (Mathivadhani et al., 2007). The nut extract also caused the turnover restoration of the factors associated with matrix and expression of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 near to normal values (Mathivadhani et al., 2007). The stabilization of the ECM with the decreased activity of proteases might inhibit the epithelial–endothelial interaction and tumour cell migration thus, preventing the adjacent invasion and tumour growth resulting in antineoplastic activity (Mathivadhani et al., 2007). In addition to the anti cancer activity, plant extract of S. anacardium also possess immunomodulatory and anti-inflammatory properties (Ramprasath et al., 2006).

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12. Glycyrrhiza glabra Family: Fabaceae Glycyrrhiza glabra, otherwise known as Licorice been used in food and medicinal remedies for thousands of years (Naik et al., 2003). The major chemical constituents of the extract of this plant are glycyrrhizin, glabridin, glycyrrhetic acid, gycyrrhetinic acid and glycyrrhizic acid (Yamamura et al., 1992 and Obolentseva et al., 1999). Glycyrrhizin, one of the major component found in licorice has been reported to prove to reduce inflammation (Akamatsu et al., 1991). Glycyrrhizin and other licorice components appear to possess anticarcinogenic properties as well (Jung et al., 2001). The extract of the plant and glycyrrhizic acid possessed radioprotecting property (Shetty et al., 2002 and Gandhi et al., 2004). Although the exact mechanisms are still under investigation, research has demonstrated that they inhibit abnormal cell proliferation, as well as tumor formation and growth in breast (Shiota et al., 1999), liver and skin cancer (Nishino et al., 1984, Liu W et al., 1998). In traditional American herbalism it is used in the anti cancer formula. Licochalcone (LA) is a novel estrogenic flavanoid isolated from PC-SPES composition herb licorice root that was reported to show significant antitumor activity in various malignant human cell lines. LA induced modest level of apoptosis but had more pronounced effect on cell cycle progression, arresting cells in G2/M, accompanied by suppression of Cyclin B1 and cdc2. It also inhibited phosphorylation of Rb, specifically phosphorylation of S780 with no change of phosphorylation status of T821, decreased expression of transcription factor E2F concurrent with reduction of Cyclin D1, down-regulation of CDKs 4 and 6, but increased Cyclin E expression (Fu et al., 2004).

13. Piper longum Family: Piperaceae Piper longum sometimes called Indian Long Pepper, is a flowering vine cultivated for its fruit, which is usually dried and used as a spice and seasoning. The major chemical constituents of the dried fruits are piperine, piperlonguminine, sylvatine, guineensine, piperlongumine, filfiline, sitosterol, methyl piperate and a series of piperine-analog retrofractamides (Nakatani et al., 1986). The extract of Piper longum was found to inhibit significantly (50.6%) the number of tumor-directed capillaries induced by injecting B16F-10 melanoma cells. Administration of the methanolic extract of the plant was found to differentially regulate the level of proinflammatory cytokines like IL-1Î˛, IL-6, TNF-Îą, GM-and CSF which were found to be at

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an elevated levels during the development of cancer (Sunila and Kuttan, 2006). The level of IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) was increased significantly when the angiogenesis-induced animals were treated with the extract of P.longum. Moreover, P.longum was also able to inhibit the VEGF-induced proliferation, cell migration and capillary-like tube formation of primary cultured human endothelial cells (Sunila and Kuttan, 2006).

14. Tinospora cordifolia Family: Menispermaceae Tinospora cordifolia (Guduci) is one of the most versatile rejuvenative herbs and is called in Sanskrit by the name Guduci meaning the one which protects the body (Singh et al., 2003). It is also called as amrta or nectar, as it is extremely useful in strengthening the immune system of the body and keeping the functions of its various organs in harmony (Desai et al., 2002). The extract of the plant contain several bitter principles, glucoside and alkaloids, a glycoside â&#x20AC;&#x201C; giloin and a non-glucoside â&#x20AC;&#x201C; gilenin, gilosterol, alkakoid tinosporin, tinosporic acid, tinosporol and berberine, tinosporidine and sitosterol isolated from stem, cordifol, heptacosanol and octacosonal and a new furanoid diterpene â&#x20AC;&#x201C; tinosporide (Singh et al., 2003). Administration of the polysaccharide fraction from Tinospora cordifolia was found to be very effective in reducing the metastatic potential of B16F-10 melanoma cells (Leyon and Kuttan, 2004). Antiangiogenic activity was studied using B16F10 melanoma cell-induced capillary formation in animals (Leyon and Kuttan, 2004). Tinospora cordifolia is reported to have immunostimulatory properties (Mathew and Kuttan, 1999). It is considered as general tonic in Ayurveda. The positive effect of Tinospora cordifolia on leucocytes suggests its use as an adjuvant in cancer therapy (Leyon and Kuttan, 2004). Activation of macrophage by Tinospora cordifolia leads to increased in colony forming units, leading to leucocytosis and improvement in neutrophil function. It was found that the herbal mixture containing this plant was effective in treatment of advanced malignancies. In addition it helped in diseases like raktapitta, anaemia, cardiac debility, diabetes, sexual debility and spleenic disorders, due to vitiation of pitta (Singh et al., 2003).

15. Podophyllum hexandrum Family: Berberidaceae The perennial herb Podophyllum hexandrum (syn. Podophyllum emodi), bearing the common names Himalayan may apple or Indian may apple, is

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native to the lower elevations in and surrounding the Himalaya (Polunin and Stainton, 1984). The whole plant, especially the root, is cholagogue, cytostatic and purgative (Uphof, 1959). The plant contains podophyllin, which has an antimiotic effect, it interferes with cell division and can thus prevent the growth of cells (Chattopadhyay et al., 2004). It is, therefore, a possible drug for the treatment of cancer, especially in the treatment of ovarian cancer (Kumar et al., 2003). The roots of this plant also contain several important anti-cancer lignans, including podophyllin and berberine (Duke and Ayensu, 1985). The rhizome of the plant contains a resin, known generally and commercially as Indian Podophyllum Resin, which also can be processed to extract podophyllotoxin, or podophyllin. Podphyllotoxin is the most active cytotoxic natural product from this plant. It has been used as starting material for the synthesis of the anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly (Giri and Narasu, 2000). These drugs have been used to treat lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. (Giri and Narasu, 2000).

16. Phyllanthus niruri Family: Euphorbiaceae Phyllanthus niruri is a herb commonly known as stonebreaker. (Nishiura et al., 2004). Phyllanthus niruri has a long history in herbal medicine systems worldwide. The active compounds isolated from this plant are phyllanthin, hypophyllanthin, lignansniranthin, nirtetralin, quercetin and phyltetralin (Amir et al., 2003). The whole plant and its aerial parts have been used for many traditional remedies, mostly biliary and urinary (Naik and Juvekar, 2003). Some examples are kidney and gallbladder stones, hepatitis, colds, flu, tuberculosis, and other viral infections (Chopra et al., 1986). It has also been proven effective in liver diseases like jaundice and liver cancer (Chatterjee and Sil, 2006). The extract of the plant has been used for bacterial infections such as cystitis, prostatitis, venereal diseases, and urinary tract infections (Nishiura et al., 2006). It also assisted in reducing anemia symptoms, diabetes and hypertension (Nwanjo, 2007), and showed diuretic, analgesic, stomachic, antispasmodic, febrifugal, and cell protective properties (Unander et al., 1995). The plant extract was found to decrease the amount of hepatitis B virus found in the blood stream (Venkateswaran et al., 1987). The plant extract have been reported to block DNA polymerase, the enzyme needed for the hepatitis B virus to reproduce (Rajeshkumar et al., 2002).

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17. Terminalia arjuna Family: Combretaceae The genus Terminalia consists of large wooded trees and occurs in almost every part of India. Every part of the plant has useful medicinal properties (Bone K, 1996). Terminalia arjuna holds a reputed position in both Ayurvedic and Yunani Systems of Medicine. According to Ayurveda it is alexiteric, styptic, tonic, anthelmintic, and useful in fractures, uclers, heart diseases, biliousness, urinary discharges, asthma, tumours, leucoderma, anaemia, excessive prespiration etc. The extract of the plant has been used as expectorant, aphrodisiac, tonic and diuretic (Kapoor, 1990). The major chemical constituents are glucoside â&#x20AC;&#x201C; arjunetin, flavones â&#x20AC;&#x201C; arjunone, cerasidin, sitosterol, friedlin, methyl oleanolate, gallic, ellagic and arjunic acidsarjunetosides I, arjunetosides II, arjunetosides III and arjunetosides IV. Apart from this, Tannins and triterpenes have been found in the extract and were reported to show antigenotoxic or antimutagenic effects (Scassellati-Sforzolini et al., 1999). It was also found out that the flavone Luteolin isolated from T. arjuna has a well established record of inhibiting various cancer cell lines. (Pettit et al., 1996). The chemical Casuarinin isolated from the Bark of Terminalia arjuna inhibited breast cancer cell growth (Kuo et al., 2005). Moreover this could induce apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 Cells (Kuo et al., 2005).

18. Alstonia scholaris Family: Apocynaceae The plant Alstonia scholaris has been used in different system of traditional medicine for the treatment of diseases. The extract of the plant showed pharmacological activities ranging from antimalarial to anticancer properties (Gandhi and Vinayak, 1990). Almost all parts of the plant contained active principles and has been reported to contain various alkaloids, flavanoids and phenolic acid (Jagetia and Baliga, 2006). The major chemical components are Alstonine, Echitamine chloride, Villastonine and three alkaloids Ditamine, Echitamine or Ditaine, and Echitenines. In addition to these compound, several fatty and resinous substances were also present in the extract (Dung et al., 2001). Echitamine chloride showed anticancer activity in S-180, regression of tumor growth and fibro sarcoma (Kamarajan et al., 1991). Villastonine has antiamoebic activity and in vitro anticancer activity against human lung cancer cell lines MOR-P (adeno carcinoma) and COR-L23 (larger carcinoma) (Keawpradub et al., 1997). The extract of this

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plant also possessed antimutagenic and immuno modulating properties (Lim-Sylianco et al., 1990).

19. Asparagus racemosus Family: Liliaceae Asparagus racemosus has been used in traditional ayurvedic medicine, which is rich in phytoestrogens, a group of naturally occurring compounds that have a chemical structure very similar to estrogen. These phytoestrogens are estimated 100-500 times less potent in their estrogen effect than human estrogen. These were found to maintain a hormonal balance by acting as antiestrogen when the body's natural levels of the hormone was high and they acted as human estrogen when levels are low (Bopana and Saxena, 2007). Cancer cells also use estrogen to promote their growth (Herman et al., 1995). Asparagus therapy has been used to treat various forms of cancer and reversal of the disease has been reported in a number of cases (Diwanay et al., 2004). Asparagus contained more glutathione than any other food ever tested by the National Cancer Institute (Demirkol et al., 2004). It also contained high levels of Vitamins A and C, folic acid, iron, potassium and calcium. In addition, extract of Asparagus contained a protein called histone which can be believed to be active in controlling cell growth (Davies et al., 1996).

20. Catharanthus roseus Family: Apocynaceae Catharanthus roseus (Periwinkle) has been in use from ancient times for the treatment of blood pressure, diabetes mellitus, etc. in Indian system of medicine as well as in folk-lore medicinal practice (Gilman et al., 1985; Bhattacharya, 1988). The extract of this plant has been found to contain alkaloids having anti mitotic and anti microtubule activities. The vinca alkaloids include vinblastine, vincristine, vindesine and vinorelbine. They are dimeric compounds in which indole and dihydro indole nuclei are joined with other complex ring systems (Pearce 1990). They are used in acute leukemia, Hodgkinâ&#x20AC;&#x2122;s disease, non Hodgkinâ&#x20AC;&#x2122;s lymphoma, rhabdomyosarcoma, neuroblastoma, Swingâ&#x20AC;&#x2122;s sarcoma and Wilms tumour (Johnson et al., 1963; Johnson, 1968). Vincristine has been used to treat many solid tumours like breast, colon, cervical and neck and head cancers in combination with other drugs. A new vinca alkaloid derivative, S12363 (vinfosiltine), is 36 and 72 times more cytotoxic in vitro than vincristine

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and vinblastine, respectively (Adenis et al., 1995). Vinca alkaloids are involved in disruption of microtubules, inhibition of protein and nucleic acid synthesis, elevation of oxidized glutathione, alteration of lipid metabolism and membrane lipid content, elevation of cAMP and inhibition of calcium- calmodulin regulated cAMP phosphodiesterase (Wilson, 1975; Tucker et al., 1977). Vinblastine and vincristine, interfere with the dynamics of microtubules and have shown significant cell killing activity in a variety of tumor cells through induction of apoptosis (Huang et al., 2004). Vinca alkaloid induced apoptosis via a pathway independent of cell cycle arrest. Vincristine and analogues are still crucial in the treatment of hematological malignancies and few solid tumour like lung and colon tumours.

21. Taxus brevifolia Family: Taxaceae Taxus brevifolia (Pacific yew) is a medium sized evergreen conifer. Infusions, decoctions, and poultices of leaves and bark of the tree are used for treating lung problem, stomach ache, wounds and pain (Moerman, 1986). The extract of the plant contains paclitaxel, commonly known by the name taxol, a potent anticancer drug used to treat ovarian, breast, lung cancers and Kaposiâ&#x20AC;&#x2122;s sarcoma (Luck and Roche 2002; Ghamande et al., 2003; Sunwoo et al., 2001). Its structure was elucidated by Wani and Wall in 1971. The initial biological activity of this compound was related to the microtubule destabilizing properties of vinca-alkaloids. Studies have revealed a unique complimentary effect of its binding to polymerized tubulin, stabilizing it against disassembly and consequently inhibiting mitosis (Schiff et al., 1979, 1980). The remarkable stability of microtubules induced by paclitaxel is damaging to cells because of the perturbation in the dynamics of various microtubule dependent cytoplasmic structures that are required for cellular functions such as mitosis, maintenance of cellular morphology, shape changes, neurite formation, locomotion and secretion (Masurovsky et al., 1983; Thuret-Carahan et al., 1985). Paclitaxel also inhibits cells in G2 and M phase at lower concentrations and inhibits cells in interphase at higher concentrations (Fan, 1999).

Epilogue Man has been dependent on the flora and fauna around him for his food and medicine from time immemorial. Modern medicine and sophisticated therapeutic

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strategies, though effective in controlling and management of most of the diseases that plague humanity, do not reach all sections of human populations in all corners of the world due to the problems of affordability and as a consequence, large number of human populations still depend up on plant based traditional alternative systems of medicine to combat debilitating diseases. Several of the modern drugs have their origin from plants. Many compounds isolated from herbal extracts as such or with chemical modifications are used to treat various diseases in modern medicine because of their potent biological activities. Due to the awareness about the side effects and toxicity of the synthetic drugs used in modern medicine, there is an increasing interest in herbal drugs or traditional medicine in recent times, apart from the economic aspects. However rigorous safety and quality evaluation and comparative clinical studies using modern techniques are essential to validate the efficacy of several of the herbal traditional drugs. In case of herbal drugs proper standardization of the methods of production of raw materials, good agricultural practice, collection of plant material, good post harvest handling, good manufacturing practices, etc. have to be followed. Isolation and purification of biologically active components from the bulk extract and their use as in the case of modern medicine may not be practicable in traditional system of medicine as the yield of the ingredients in the extract could be very low or negligible. Also some compounds in the whole extract or preparation may have synergistic activity. Thus the wholesome use of herbal or traditional medicine may be more effective and advisable than the use of individual isolated purified components. To understand the basic mechanisms of the drug action, purified components may be essential. Plant kingdom with its enormous diversity is a store house of large number of compounds with potent therapeutic activities. In treatment of neoplastic diseases, use of herbal extracts are of particular relevance because of their cytotoxic, cytoprotective and chemopreventive activities. Herbal drugs with their long history are the only answer to provide an integrated health care for the suffering populations of humanity, particularly in the developing world, at affordable cost.

References 1. 2. 3.

Adenis A, Pion JM, Fumoleau P, Pouillart P, Marty M, Giroux B and Bonneterre J . Phase II study of a new vinca alkaloid derivative, S12363, in advanced breast cancer. Cancer Chemother and Pharmacol. 1995; 35(6): 527-528. Adwankar MK and Chitinis MP. In vivo anticancer activity of RC-18: a plant isolate from Rubia cordifolia Linn. against a spectrum of experimental tumor model. Chemother. 1982; 28(4):291-293. Akamatsu H, Komura J, Asada Y and Niwa Y. Mechanism of anti-inflammatory action of glycyrrhizin: effect on neutrophil functions including reactive oxygen species generation. Planta Med. 1991; 57:119-121.

Plants to fight neoplastic diseases

4. 5. 6.

7. 8. 9.

10. 11. 12. 13. 14. 15. 16.

17.

18. 19.

219

Akev N, Turkay G, Can A, Gurel A, Yildiz F, Yardibi H, Ekiz EE and Uzun H. Tumour preventive effect of Aloe vera leaf pulp lectin (Aloctin I) on Ehrlich ascites tumours in mice. Phytother Res. 2007; 21(11):1070-5. Amir M, Kumar S, Singh SK. Chemical and biological review of Phyllanthus niruri. Ind J Nat Prod. 2003; 19(4): 3-13. Babu KGD, Shanmugam V, Ravindranath SD and Joshi VP. Comparison of chemical composition and antifungal activity of Curcuma longa L. leaf oils produced by different water distillation techniques. Flav Frag J. 2006; 22(3): 191-196. Bhargava KP and Singh N. Antistress activity of Ocimum sanctum Linn. Ind J Med Res. 1981; 73: 443- 451. Bhattacharya S. Nayantara (Sada Bahar), in: Chiranjeeva Vanaushadi, Vol 9, Ist Ed, Ananda Publishers, Calcutta, 1988; pp3-7. Biswas SK, McClure D, Jimenez LA, Megson IL and Rahman I. Curcumin induces glutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity. Antioxid Redox Signal. 2005; 7(1-2):32-41. Bone K, Morgan M. Clinical applications of ayurvedic and chinese herbs: In monographs for the western herbal practitioner. Warwick, Australia. 1996; pp75-79. Bopana N and Saxena S. Asparagus racemosus: ethnopharmacological evaluation and conservation needs. J Ethno pharmacol. 2007; 110(1): 1-15. Bradstreet K. In Herbs for detoxification,Woodland Publications.1997. Brinkhaus B, Lindner M and Schuppan D. Chemical, pharmacological and clinical profile of the East Asian medical plant Centella asiatica. Phytomed. 2000; 7: 427-48. Chatterjee M and Sil PC. Hepatoprotective effect of aqueous extract of Phyllanthus niruri on nimesulide-induced oxidative stress in vivo. Ind J Biochem Biophys. 2006; 43(5): 299-305. Chattopadhyay S, Bisaria VS, Panda AK and Srivastava AK. Cytotoxicity of in vitro produced podophyllotoxin from Podophyllum hexandrum on human cancer cell line. Nat Prod Res. 2004; 18(1): 51-57. Chen YC, Tsai WJ, Wu MH, Lin LC and Kuo YC. Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-KB. Brit J Pharmacol. 2007; 150: 298-312. Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, Ming-Shiang W, Yu HS, Jee SH, Chen GS, Chen TM, Chen CA, Lai MK, Pu YS, Pan MH, Wang YJ, Tsai CC and Hsieh CY. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res. 2001; 21 (4B):2895-2900. Chitnis MP, Bhatia KG, Phatak MK and Kesava Rao KV. Anti-tumour activity of the extract of Semecarpus anacardium L. nuts in experimental tumor models. Ind J Exp Biol.1980 ;18(1):6-8. Chopra RN, Nayar SL and Chopra IC. In Ram PR, Malhotra BN (Ed) Glossary of Indian medicinal plants, CSIR, New Delhi Catholic press, Randi, India. 1986; pp 345-349.

220

C. K. K. Nair et al.

20. Churchill M, Chadburn A, Bilinski RT and Bertagnolli MM. Inhibition of intestinal tumors by curcumin is associated with changes in the intestinal immune cell profile. J Surg Res. 2000; 89(2):169-75. 21. Dahanukar SA, Kulkarni RA, Rege NN. Pharmacology of medicinal plants and natural products. Ind. J. Pharm. 2000; 32: S81-S118. 22. Davies KM, Seelye JF, Irving DE, Borst WM, Hurst PL and King GA. Sugar regulation of harvest-related genes in asparagus. Plant Physiol. 1996; 111(3):877-83. 23. Demirkol O, Adams C and Ercal N. Biologically important thiols in various vegetables and fruits. J Agric Food Chem. 2004; 52(26): 8151-8154. 24. Desai VR, Kamat JP and Sainis KB. An immunomodulator from Tinospora cordifolia with antioxidant activity in cell-free systems. Proc Indian Acad Sci (Chem Sci). 2002; 114(6): 713-719. 25. Dhar RS, Verma V, Suri KA, Sangwan RS, Satti NK, Kumar A, Tuli R and Qazi GN. Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochem. 2006; 67(20): 2269-2276. 26. Diwanay S, Chitre D and Patwardhan B. Immunoprotection by botanical drugs in cancer chemotherapy. J Eth pharmacol.2004; 90(1): 49-55. 27. Duke JA and Ayensu ES. Medicinal Plants of China.Reference Publications, Inc. 1985. 28. Dung NX, Ngoc PH, Rang DD, Nhan NT, Klinkb N and Leclercq P. Chemical composition of the volatile concentrate from the flowers of Vietnamese Alstonia scholaris (L.) R.Br. Apocynaceae. J Essen Oil Res.2001; 13(6): 424-426. 29. Duvoix A, Blasius R, Delhalle S, Schnekenburger M, Morceau F, Henry E, Dicato M and Diederich M. Chemopreventive and therapeutic effects of curcumin. Laboratoire de Biologie Moleculaire et Cellulaire du Cancer.Cancer Lett. 2005; 223(2):181-190. 30. Elsakka M, Grigorescu E, Stanescu U, Stanescu U and Dorneanu V. New data referring to chemistry of Withania somnifera species. Rev Med Chir Soc Med Nat Iasi.1990; 94(2): 385-387. 31. Fan W. Possible mechanisms of paclitaxel-induced apoptosis. Biochem Pharmacol. 1999; 57: 1215-1221. 32. Fu Y, Hsieh TC, Guo JQ, Kunicki J, Lee My WT, Darzynkiewicz Z, Wu JM. Licochalcone A. A novel flavonoid isolated from licorice root (Glycyrrhiza glabra), causes G2 and late-G1 arrests in androgen-independent PC-3 prostate cancer cells. Biochem Biophys Res Comm. 2004; 322(1): 263-270. 33. Ganasoundari A, Uma Devi P and Rao BSS. Enhancement of bone marrow radioprotection and reduction of WR-2721 toxicity by Ocimum sanctum. Mut Res. 1998; 397(2): 303-312. 34. Gandhi M and Vinayak VK. Preliminary evaluation of extracts of Alstonia scholaris bark for in vivo antimalarial activity in mice. J Eth pharmacol. 1990; 29(1): 51-57. 35. Gandhi NM, Maurya DK, Salvi V, Kapoor S, Mukherjee T and Nair CKK. Radioprotection of DNA by glycyrrhizic acid through scavenging free radicals. J Rad Res. 2004; 45, 461-468. 36. Ghamande S, Lele S, Marchetti D, Baker T and Odunsi K. Weekly paclitaxel: in patients with recurrent or persistent advanced ovarian cancer. Int J Gynecol Cancer. 2003; 13:142-147. 37. Gilman AG, Goodman LS, Rall TW et al: Vinca alkaloids, in: The Pharmacological Basis of Therapeutics, 7th Ed, Macmillan, New York, 1985; pp1277.

Plants to fight neoplastic diseases

221

38. Giri A and Narasu ML. Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs. Cytotechnology. 2000; 34(1-2): 17-26(10). 39. Gupta P, Yadav DK, Siripurapu KB, Palit G and Maurya R. Constituents of Ocimum sanctum with antistress activity. J Nat Prod . 2007; 70 (9):1410-1416. 40. Herman C, Adlercreutz T and Goldin BR. Soybean phytoestrogen intake and cancer risk. J Nutr. 1995; 125:757S-770S. 41. Hideji I, Koichi T, Noboru M, Shinpei K, Hiroo Y. isolation and antitumour activity of cyclic hexapeptides isolated from Rubiae radix. Chem Pharm Bull. 1984;32(1):284-290. 42. Hideji I, Koichi T, Noboru M, Shinpei K, Hiroo Y. Studies on antitumour cyclic hexapeptides RA obtained from Rubiae radix, Rubiaceae (IV): quantitative determination of RA-VII and RA-V in commercial Rubiae radix and collected plants. Planta Med .1984; 50: 313-316. 43. Hsu YL, Cho CY, Kuo PL, Huang YT and Lin CC. Plumbagin (5-Hydroxy-2methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. JPET. 2006; 318: 484-494. 44. Huang Y, Fang Y, Wu J, Dziadyk JM, Zhu X, Sui M and Fan W. Regulation of Vinca alkaloid-induced apoptosis by NF-kB/IkB pathway in human tumor cells. Mol Cancer Ther. 2004; 3: 271-277. 45. Jagetia GC and Baliga MS. Evaluation of anticancer activity of the alkaloid fraction of Alstonia scholaris (Sapthaparna) in vitro and in vivo. Phytother Res. 2006;20(2):103-109. 46. Jagetia GC, Venkatesh P and Baliga MS. Aegle marmelos (L.) correa Inhibits the proliferation of transplanted Ehrlich Ascites Carcinoma in mice. Biol Pharm Bull. 2005; 28 (1): 58. 47. Joharapurkar AA, Zambad SP, Wanjari MM, Umathe SN. In vivo evaluation of antioxidant activity of alcoholic extract of Rubia cordifolia Linn. and its influence on ethanol induced immunosuppression. Ind J Pharmacol. 2003; 35: 232-236. 48. Johnson IS, Amstrong JG, Gorman M and Burnett JP. The vinca alkaloids-a new class of oncolytic agents, Cancer Res. 1963; 23: pp1390-1427. 49. Johnson IS. Historical background of Vinca alkaloid research and areas of future interest. Cancer Chemother Rep. 1968; 52(4): 455-61. 50. Jung GD, Yang JY, Song ES and Park JW. Stimulation of melanogenesis by glycyrrhizin in B16 melanoma cells. Exp Mol Med, 2001; 33(3): 131-135. 51. Kamarajan P, Sekar N, Mathuram V and Govindasamy S. Antitumor effect of echitamine chloride on methylcholonthrene induced fibrosarcoma in rats. Biochem Int. 1991; 25(3): 491-498. 52. Kapoor LD. Handbook of Ayurvedic Medicinal Plants. Boca Raton, FL. CRC Press; 1990. 53. Karthikeyan K, Gunasekaran P, Ramamurthy N and Govindasamy S. Anticancer activity of Ocimum sanctum. Pharm-biol. 1999; 37 (4): 285-290. 54. Karthikeyan K, Ravichandran P and Govindasamy S. Chemopreventive effect of Ocimum sanctum on DMBA-induced hamster buccal pouch carcinogenesis. Oral Oncol. 1999; 35(1): 112-119.

222

C. K. K. Nair et al.

55. Keawpradub N, Houghton PJ, Eno-Amooquaye E and Burke PJ. Activity of extracts and alkaloids of Thai Alstonia species against human lung cancer cell lines. Planta Med. 1997; 63(2): 97-101. 56. Kinghorn AD, Farnsworth NR, Soejarto DD, Cordell GA, Pezzuto JM, Udeani GO, Wani MC, Wall ME, Navarro HA, Kramer RA, Menendez AT, Fairchild CR, Lane KE, Forenza S, Vyas DM, Lam KS and Shu YZ. Novel strategies for the discovery of plantderived anticancer agents. Pure Appl Chem. 1999; 71( 9):1611-1618. 57. Kirthikar KR and Basu BD. In Basu SN (Ed) Indian Medicinal Plants.Lalit Mohan Babu Publications, Allahabad. 1975; pp74-79. 58. Kugelman MM, Liu WC, Axelord M, Mc Bride TJ and Rao KV. Indicine NOxide, the antitumor principle of Heleotropium indicum. Economic Botany. 1976; 39(29):125-128. 59. Kumar PI, Rana SV, Samanta N and Goel HC. Enhancement of radiation-induced apoptosis by Podophyllum hexandrum. J Pharm Pharmacol. 2003; 55(9): 1267-73. 60. Kumar RA, Sridevi K, Kumar NV, Nanduri S and Rajagopal S. Anticancer and immunostimulatory compounds from Andrographis paniculata. J Eth pharmacol. 2004; 92(2-3): 291-295. 61. Kumaran KS, Thirugnanasambantham P, Viswanathan S and Ramamurthy MS. An HPLC method for the estimation of andrographolide in rabbit serum. Ind J Pharmacol. 2003; 35(2): 109-112. 62. Kuo PL, Hsu YL, Lin TC, Lin LT, Chang JK and Lin CC. Casuarinin from the bark of Terminalia arjuna induces apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 cells. Planta Med. 2005; 71(3):237-43. 63. Lambertini E, Lampronti I, Penolazzi, Letizia, Khan MTH, Ather, Arjumand, Giorgi, Gianluca, Roberto G and Roberta P. Expression of estrogen receptor Îą gene in breast cancer cells treated with transcription factor decoy is modulated by Bangladeshi natural plant extracts. Oncology Research Incorporating AntiCancer Drug Design. 2005; 15 (2): 69-79(11). 64. Lampronti I, Martello D, Bianchi N, Borgatti M, Lambertini E, Piva R, Jabbar S, Choudhuri MSK, Khan MTH and Gambari R. In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa. Phytomedicine. 2003; 10(4): 300-308(9). 65. Leyon PV and Kuttan G. Effect of Tinospora cordifolia on the cytokine profile of angiogenesis-induced animals. Int Immunopharmacol. . 2004; 4(13):1569-75. 66. Leyon PV and Kuttan G. Inhibitory effect of a polysaccharide from Tinospora cordifolia on experimental metastasis. J Eth pharmacol. 2004; 90(2-3): 233-237. 67. Lim-Sylianco CY, Jocano AP and Linn CM. Antimutagenicity of twenty Philippine plants using the micronucleus test in mice. Philippine J Sci. 1990; 117(3): 231-235. 68. Lissoni P, Giani L, Zerbini S, Trabattoni P and Rovelli F. Biotherapy with the pineal immunomodulating hormone melatonin versus melatonin plus Aloe vera in untreatable advanced solid neoplasm. Nat Immun.1998; 16:27-33. 69. Liu W, Kato M and Akhand A. The herbal medicine Sho-saiko-to inhibits the growth of malignant melanoma cells by up-regulating Fasmediated apoptosis and arresting cell cycle through down regulation of cyclin dependent kinases. Int J Oncol 1998; 12:1321-1326.

Plants to fight neoplastic diseases

223

70. Luck HJ, Roche H. Weekly paclitaxel: an effective and well-tolerated treatment in patients with advanced breast cancer. Crit Rev Oncol Hematol. 2002; 44: S15â&#x20AC;&#x201C;30. 71. Maquart and Francois-Xavier. Stimulation of Collagen Synthesis in Fibroblast Cultures by a Triterpene Extracted from Centella Asiatica. Connect Tissue Res. 1990; 24: 107-120. 72. Masurovsky EB, Peterson ER, Grain SM and Horwitz SB. Morphological alterations in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord ganglion structuresexposed to taxol. Neuroscience. 1983; 10: 491. 73. Mathew S and Kuttan G. Immunomodulatory and antitumour activities of Tinospora cordifolia. Fitoterapia. 1999; 70(1): 35-43. 74. Mathivadhani P, Shanthi P and Sachdanandam P. Effect of Semecarpus anacardium nut extract on ECM and proteases in mammary carcinoma rats. Vascul Pharmacol. 2007; 46(6): 419-426. 75. Mathur R, Gupta SK, Singh N, Mathur S, Kochupillai V and Velpandian T. Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis. J Eth pharmacol. 2006;105(3): 336-341. 76. Moerman DE. Medicinal plants of NativeAmerica. Technical Reports 19. University of Michigan Museum of Anthropology, Ann Arbor, MI. 1986. pp534 . 77. Mukophadhyay A, Basu N, Ghatak N and Gujral PK. Anti-inflammatory and irritant activities of curcumin analogues in rats. Agents and Actions. 1982; 12: 508-515. 78. Naik AD and Juvekar AR. Effects of alkaloidal extract of Phyllanthus niruri on HIV replication. Indian J Med Sci. 2003; 57(9): 387-93. 79. Naik GH, Priyadarsini I, Satav JG, Banavalikar MM, Sohoni DP, Biyani M K and Mohan H. Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine. Phytochemistry. 2003; 63(1): 97-104. 80. Nakatani N, Inatani R, Ohta H, Nishioka A. Chemical constituents of peppers (Piper spp.) and application to food preservation: naturally occurring antioxidative compounds. Environmental Health Perspectives.1986; 67: 135-142. 81. Nguyena AT, Malonne BH, Dueza R, Vanhaelen-Fastrea, Vanhaelena R and Fontaineb J. Cytotoxic constituents from Plumbago zeylanica. Fitoterapia. 2004; 75: 500-504. 82. Nishino H, Kitagawa K and Iwashima A. Antitumor-promoting activity of glycyrrhetic acid in mouse skin tumor formation induced by 7,12 dimethylbenz[a]anthracene plus teleocidin. Carcinogenesis. 1984; 5:1529-1530. 83. Nishiura JL, Campos AH, Boim MA, Heilberg IP and Schor N. Phyllanthus niruri normalizes elevated urinary calcium levels in calcium stone forming (CSF) patients. Urol Res. 2004; 32(5):362-366. 84. Nwanjo HU. Studies On The Effect Of Aqueous Extract Of Phyllanthus Niruri Leaf On Plasma Glucose Level And Some Hepatospecific Markers In Diabetic Wistar Rats. The Internet Journal of Laboratory Medicine. 2007; 2(2). 85. Obolentseva GV, Litvinenko VI and Ammosov AS. Pharmacological and therapeutic properties of licorice preparations (a review). Pharm Chem J. 1999; 33: 24-31. 86. Padmavathi B, Rath PC, Rao AR and Singh RP. Roots of Withania somnifera Inhibit Forestomach and Skin Carcinogenesis in Mice. Evid Based Complement Alternat Med. 2005; 2(1): 99-105.

224

C. K. K. Nair et al.

87. Pearce HL. Medicine chemistry of bisindole alkaloids from Catharanthus. In the alkaloids. Vol 37. Ed. By A. Brossi Orlando: Academic 1990; pp 145. 88. Pettit GR, Hoard MS, Doubek DL, Schmidt JM, Pettit RK, Tackett LP and Chapuis JC. Antineoplastic agents 338. The cancer cell growth inhibitory. Constituents of Terminalia arjuna (Combretaceae). J Eth pharmacol. 1996; 53(2):57-63. 89. Polasa K, Raghuram TC, Krishna TP and Krishnaswamy K. Effect of turmeric on urinary mutagens in smokers. Mutagenesis. 1992; 7(2): 107-109. 90. Polunin O and Stainton A. Flowers of the Himalayas. Oxford Universtiy Press. 1984. 91. Prashar R, Kumar A, Banerjee S and Rao AR. Chemopreventive action by an extract from Ocimum sanctum on mouse skin papillomagenesis and its enhancement of skin glutathione S-transferase activity and acid soluble sulfydryl level. Anticancer Drugs. 1994; 5(5):567-72. 92. Prashar R, Kumar A, Hewer A, Cole KJ, Davis W and Phillips DH. Inhibition by an extract of Ocimum sanctum of DNA-binding activity of 7,12-dimethylbenz [a]anthracene in rat hepatocytes in vitro. Cancer Lett. 1998; 128(2): 155-60. 93. Premalatha B and Govindarajan R. Cancer: an ayurvedic perspective. Pharmaco Res. 2005; 51: 19-30. 94. Premalatha B and Sachdanandam P. Semecarpus anacardium L. nut extract administration induces the in vivo antioxidant defence system in aflatoxin B1 mediated hepatocellular carcinoma. J Eth pharmacol. 1999; 66:131-139. 95. Punturee K, Wild CP, Kasinrerk W and Vinitketkumnuen U. Immunomodulatory activities of Centella asiatica and Rhinacanthus nasutus extracts. J Allergy Clin Immunol. 2007; 120(2): 293-299. 96. Qiao YF, Wang SX, Wu LJ, Li X and Zhu TR. Studies on antibacterial constituents from the roots of Rubia cordifolia L. Yao Xue Xue Bao. 1990; 25(11):834-9. 97. Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C and Rajagopalan R. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculatae. J Exp Therapeut Oncol. 2003; 3(3): 47-58. 98. Rajeshkumar NV, Joy KL, Kuttan G, Ramsewak RS, Nair MG and Kuttan R. Antitumour and anticarcinogenic activity of Phyllanthus amarus extract. J Eth pharmacol. 2002; 81(1): 17-22. 99. Ramprasath VR, Shanthi P, Sachdanandam P. Immunomodulatory and antiinflammatory effects of Semecarpus anacardium Linn. nut milk extract in experimental inflammatory conditions. Biol Pharm Bull. 2006; 29(4): 693-700. 100. Rao KV and Mcbride TJ. Indicine-N-Oxide as an antitumor and antileukemic agent for mice and rats. J Pharm Soc. 1968; 57: 1112-1117. 101. Reddy, Rao JS, Reddy PR and Mada S. Wound healing effects of Heliotropium indicum, Plumbago zeylanicum and Acalypha indica in rats. J Ethnopharmacol. 2002; 79(2): 249-51. 102. Saada HN, Ussama ZS and Mahdy AM. Effectiveness of Aloe vera on the antioxidant status of different tissues in irradiated rats. Pharmazie. 2003; 58(12):929-31. 103. Santosh KS, Haruyo I, Gautam S, Ahn-Kwang-Seok and Bharat BA. Plumbagin (5Hydroxy-2-methyl-1,4-naphthoquinone) Suppresses NF-[kappa]B activation and NF-[kappa]B-regulated gene products through modulation of p65 and

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225

I[kappa]B[alpha] kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem. 2006; 281(25): 1702317033. 104. Scassellati-Sforzolini G, Villarini LM, Moretti LM, Marcarelli LM, Pasquini R, Fatigoni C, Kaur LS, Kumar S and Grover IS. Antigenotoxic properties of Terminalia arjuna bark extracts. J Environ Pathol Toxicol Oncol. 1999; 18(2):119-25. 105. Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by Taxol. Nature (Lond). 1979; 277: 665-667. 106. Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci, USA.1980; 77: 1561-1565. 107. Selvan R, Subramanian L, Gayatri R and Angayarkanni N. Anti-oxidant activity of turmeric (Curcuma longa). J Eth pharmacol. 1995; 47(2): 59-67. 108. Seongwon Choi and Myung-Hee Chung MD. A review on the relationship between aloe vera components and their biologic effects. Seminars in Integrative Medicine. 2003;1(1) :53-62. 109. Shetty TK, Satav JG, and Nair CKK. Protection of DNA and microsomal membranes in vitro by Glycyrrhizia glabra L., against gamma irradiation. Phytother Res. 2002;16:576. 110. Shiota G, Harada K and Ishida M. Inhibition of hepatocellular carcinoma by glycyrrhizin in diethylnitrosamine-treated mice. Carcinogenesis.1999;20:5963. 111. Singh JP, Pandey DP, Pandey MB, Anita Singh and Singh R. Constituents Of Heliotropium Indicum. Orient J Chem. 2003; 19 ( 3 ). 112. Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro and Ghosh AC. Chemistry and medicinal properties Of Tinospora cordifolia (Guduchi). Ind J Pharmacol. 2003; 35: 83-91. 113. Srinivas K, Rao MEB and Rao SS. Anti-inflammatory activity Of Heliotropium Indicum Linn. and Leucas Aspera spreng. In albino rats. Ind J Pharmacol. 2000; 32: 37-38. 114. Srivastava KC, Bordia A and Verma SK: Curcumin, a major component of food spice tumeric (Curcuma longa) inhibits aggregation and alters eicosanoid metabolism in human blood platelets. Prostgland Leukotr Ess Fatty Acids. 1995; 52:223-227. 115. Sunila ES and Kuttan G. Piper longum inhibits VEGF and proinflammatory cytokines and tumor-induced angiogenesis in C57BL/6 mice. Int Immunopharm. 2006;6(5):733-741. 116. Sunwoo JB, Herscher LL, Kroog GS, et al. Concurrent paclitaxel and radiation in the treatment of locally advanced head and neck cancer. J Clin Oncol. 2001;19: 800-11. 117. Thatte UM and Dahanukar SA. Adaptogenic properties of six rasayana herbs used in ayurvedic medicine. Phyto Rese. 1999; 13: 275-291. 118. Thuret-Carahan J, Bossu JL, Feitz A, Langley K and Annis D. Effect of taxol on secretory cells: functional, morphological and electrophysiological correlates. J Cell Biol. 1985; 100: 1863. 119. Tripathi YB, Sharma M and Manickam M. Rubiadin, a new antioxidant from Rubia cordifolia. Ind J Biochem Biophys. 1997; 34(3):302-306.

226

C. K. K. Nair et al.

120. Trivedi NP and Rawal UM. Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice. Ind J Exp Biol. 2001; 39(1):41-46. 121. Tucker Rev, Owellen RJ, Harris SB. Correlation of cytotoxicity and mitotic spindle dissolution by vinblastin in mammalian cells. Cancer Res. 1977; 37: 43-46. 122. Unander DW, Webster GL and Blumberg BS. Usage and bioassays in Phyllanthus (Euphorbiaceae). IV. Clustering of antiviral uses and other effects. J Eth pharmacol. 1995; 45(1): 1-18. 123. Uphof JC. The Dictionary of Economic Plants. Weinheim. 1959. 124. Venkateswaran PS, Millman I and Blumberg BS. Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies. Proc Natl Acad Sci U S A. 1987; 84(1): 274-278. 125. Wakita K, Minami M, Venkateswarlu A, Sharma VM, Ramesh M, Akahane K. Antitumor bicyclic hexapeptide RA-VII modulates cyclin D1 protein level. AntiCancer Drugs. 2001; 12(5):433-439. 126. Wattenberg LW, Coccia JB and Lam LKT. Inhibitory effects of phenolic compounds on benzo(a)pyrene induced neoplasia. Cancer Res.1980; 40: 2820-3. 127. Wilson L. Microtubules as drug receptors. Pharmacological properties of Microtubule protein. Ann Acad Sci. 1975; 253-13. 128. Yamamura Y, Kawakami J, Santa T, et al. Pharmacokinetic profile of glycerrhizin in healthy volunteers by a new high-performance liquid chromatographic method. J Pharm Sci 1992; 81: 1042-1046. 129. Yoshida M, Fuchigami M, Nagao T, Okabe H, Matsunaga K, Takata J, Karube Y, Tsuchihashi R, Kinjo J, Mihashi K, and FujiokaT. Antiproliferative constituents from Umbelliferae plants VII.(1)active triterpenes and rosmarinic acid from Centella asiatica. Biol Pharm Bull. 2005; 28(1): 173-175. 130. Yu QL, Duan HQ, Takaishi Y and Gao WY. A Novel Triterpene from Centella asiatica. Molecules. 2006; 11: 661-665. 131. Zhang J, Qi H and Wu C. Research of anti-proliferation of curcumin on A549 human lung cancer cells and its mechanism. Zhong Yao Cai. 2004; 27(12): 923-927.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 227-244 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

8. Recent advances on the ethnomedicinal plants as immunomodulatory agents 1

Mahiuddin Alamgir1 and Shaikh Jamal Uddin2

School of Chemistry, The University of New South Wales, Sydney, NSW-2052, Australia School of Pharmacy, Griffith University, Gold Coast Campus, Queensland, QLD-4222, Australia

Abstract. Modulation of immune response to alleviate diseases has long since been of interest. Recent progress on the ethnomedicinal plants as immunomodulatory agent reported from 2000 to early 2008 is reviewed. Plant extracts have been widely investigated in this time in different parts of the world for their possible immunomodulatory properties. Some of the studies demonstrated to the isolation of potential bioactive molecule. Few have been tested as herbal formulations. Several plant extracts, compounds and formulations has been patented.

Abbreviations AIDS CB CD4 CNS CR3 DNA FITC

- Acquired immunodeficiency syndrome - Cannabinoid - Cluster of differentiation 4 - Central nervous system - Complement receptor 3 - Deoxyribonucleic acid - Fluorescein isothiocyanate

Correspondence/Reprint request: Dr. Mahiuddin Alamgir, School of Chemistry, The University of New South Wales, Sydney, NSW-2052, Australia. E-mail: m.alamgir@unsw.edu.au

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GSH H2O2 HIV IFN-γ IL INOS IκBα LPS MRNA MTT NFAT NF-κB NK NO PBL PBMCs PHA ROS TH THP-1 TLR4 TNF-α WBC

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- Glutathione - Hydrogen peroxide - Human immunodeficiency virus - Ιnterferon-gamma - Interleukin - Inducible nitric oxide synthase - Inhibitor of kappa B alpha - Lipopolysaccharide - Messenger ribonucleic acid - 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - Nuclear factor of activated T-cells - Nuclear factor-kappa B - Natural killer - Nitric oxide - Peripheral blood lymphocyte - Peripheral blood mononuclear cells - Phytohaemagglutinin - Reactive oxygen species - T helper cell - Human acute monocytic leukemia cell line - Toll-like receptor 4 - Tumor necrosis factor-alpha - White blood cells

1. Introduction Immune system is a remarkably sophisticated defence system within vertebrates, to protect them from invading agents. It is able to generate varieties of cells and molecules capable of recognizing and eliminating limitless varieties of foreign and undesirable agents. Modulation of the immune system denotes to any change in the immune response that can involve induction, expression, amplification or inhibition of any part or phase of the immune response. Thus, immunomodulator is a substance used for its effect on the immune system. There are generally of two types immunomodulators based on their effects: immunosuppressants and immunostimulators. They have the ability to mount an immune response or defend against pathogens or tumors. Immunopharmacology is a comparatively new and developing branch of pharmacology aims at searching for immunomodulators. The potential uses of immunodulators in clinical medicine include the reconstitution of immune deficiency (e.g. the treatment of AIDS) and the suppression of normal or excessive immune function (e.g. the treatment of graft rejection or autoimmune disease).

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Specific immunomodulators administered together with antigens known as immunological adjuvants to boost the immune response to the vaccine constituents. For instance, a plant origin saponin used in veterinary medicine. Whereas, the non-specific immunostimulators offer a generalized state of resistance to pathogens or tumors. Fungal product cyclosporin A selectively block the function of T lymphocyte and used to prevent graft rejection [1]. Medicinal plants and their active components have been shown to be an important source of immunomodulators. Thus the development of drugs for immunomodulation and antitumour activity from natural compounds has become an attractive project. Through these studies, we may not only find some promising immunomodulators, but also clarify the mechanism of clinical actions of some traditional medicines. A range of immunomodulatory agents from plants and fungi earlier has been reviewed [2, 3]. The present review will concentrate on recent developments on immunomodulatory activities of plants reported from 2000 to early 2008.

2.1. Immunomodulatory activity of crude plant extracts 2.1.1. Indian plants Fruits of Emblica officinalis (family: Euphorbiaceae) and whole plant of Evolvulus alsinoides (family: Convolvulaceae) has been extensively used in Indian Ayurvedic medicine for varieties of medical disorders. The immunomodulatory properties of Emblica offĂ˝cinalis and Evolvulus alsinoides were evaluated in adjuvant induced arthritic rat model. The crude aqueous extracts of both the herbs were administered intraperitonially following a repeated treatment profile. There was a significant reduction in swelling and redness of inflamed areas in treated animals than in untreated controls. The anti-inflammatory response of both extracts was determined by lymphocyte proliferation activity and histopathological severity of synovial hyperplasia. Both extracts showed a marked reduction in inflammation and edema. At cellular level immunosuppression occurred during the early phase of the disease. There was mild synovial hyperplasia and infiltration of few mononuclear cells in treated animals. The induction of nitric oxide synthase was significantly decreased in treated animals as compared to controls. These observations suggest that both the herbal extracts caused immunosuppression. Both are as potent as dexamethasone, a traditionally used immunosuppressant for arthritis [4]. Mehrota described in vitro immunosuppressive potential of ethanolic extract of Acorus calamus rhizome. Ethanolic extract of A. calamus inhibited proliferation of mitogen (phytohaemagglutinin) and antigen (purified protein

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derivative) stimulated human peripheral blood mononuclear cells (PBMCs). In addition, A. calamus extract inhibited growth of several cell lines of mouse and human origin. It also inhibited production of nitric oxide (NO), interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). Intracytoplasmic interferon-γ (IFN-γ) and expression of cell surface markers, CD16 and HLA-DR, on human PBMC, were not affected on treatment with A. calamus extract but CD25 expression was down regulated [5]. Crude extract of Tinospora cordifolia contained a polyclonal B cell mitogen which enhanced immune response in mice. An arabinogalactan polysaccharide, G1-4A from the stem of Tinospora cordifolia examined to modulate induced immunosuppression. Mice pre-treated with G1-4A exhibited protection against lipopolysaccharide (LPS) induced mortality [6]. Partially purified immunomodulator, G1-4A prevented lipid peroxidation and restored the activities of superoxide dismutase and catalase enzymes. Likewise, oxidative damage, induced by peroxynitrite, was also inhibited by partially purified immunomodulator similar to selective inhibitors of reactive oxygen species (ROS) like mannitol, superoxide dismutase, sodium azide and antioxidants, GSH and vitamin C [7]. In further studies, intraperitoneal administration of alcoholic extract of Tinospora cordifolia in Dalton's lymphoma bearing mice not only augmented the basic function of macrophages such as phagocytosis, but also their antigen presenting ability and secretion of IL-1, TNF and RNI. It was also indicated that the extract slow down the tumor growth and increases the life span of tumor bearing host, thus showing its anti tumor effect through destabilizing the membrane integrity of Dalton's lymphoma cells directly or indirectly. Thus, the study demonstrated alcoholic extract of Tinospora cordifolia activated tumor associated macrophages and showed antitumor effect on the spontaneous T-cell lymphoma and may have some clinical implications [8]. Ethanolic extract of Boerhaavia diffusa, a plant used in Indian traditional system of medicine, significantly inhibited the cell proliferation [9]. Extracts of B. diffusa roots inhibited human NK cell cytotoxicity in vitro, production of nitric oxide in mouse macrophage cells, interleukin-2 and tumor necrosis factor-α (TNF-α), in human PBMCs. Whereas, intracytoplasmic interferon-γ (IFN-γ) and cell surface markers such as CD16, CD25, and HLA-DR did not get affected on treatment with B. diffusa extract and demonstrates immunosuppressive potential of B. diffusa [10]. Aqueous leaves extract of biopesticidal plant Nyctanthes arbor-tristis has been found as a potent immunomodulator [11]. The extract has been evaluated as immunorestorative or anti-immunosuppressive agent in the malathion exposed immunosuppressed mice by studying various immunological parameters (humoral, cell mediated immune, numerical values of immunocytes

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and functions of phagocytes) in treated or untreated malathion-exposed mice. The results revealed that the immunological parameters which were suppressed with malathion either reverted back to normal or showed a trend towards normalcy, when treated with aqueous leaves extract of Nyctanthes arbor-tristis [12]. Methanol extract of Eclipta alba and Centella asiatica whole plant showed phagocytic index and antibody titer has been increased significantly. The F ratios of the phagocytic index and WBC count were also significant with a linearity in the dose-response relationship [13]. The ethanol extract of the root of the plant Cryptolepis buchanani caused significant stimulation of the delayed type hypersensitivity reaction and humoral antibody production in mice [14]. An aqueous extract of Rhodiola imbricata rhizome stimulated production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in human PBMCs as well as RAW 264.7 cell line. It also increased production of nitric oxide synergistically in combination with lipopolysaccharide in RAW 264.7. Furthermore, it increased the phosphorylated-IκB expression and activated the nuclear translocation of NF-κB in human PBMCs. Thus, Rhodiola most likely activated proinflammatory mediators via phosphorylated inhibitory κB and transcription factor NF-κB [15]. 2.1.2. Chinese plants Traditional herbal formulas, used to treat inflammatory arthritis in China include Boswellia carterii or Boswellia serrata. They both contain boswellic acids which have been shown to exhibit antiinflammatory and antiarthritic properties. B. carterii plant resin extract containing boswellic acids, where ethanol as a solvent resulted in significant cellular toxicity of TH1 cytokines (IL-2 and IFN-γ) and TH2 cytokines (IL-4 and IL-10) by murine splenocytes [16]. 2.1.3. Asian plants An aqueous extract (decoction) of Vietnamese plant Crinum latifolium L. retarded growth of 20-methylcholanthrene tumors (sarcomas) in rats. The inhibition of carcinogenesis had occurred probably due to the influence of immunomodulating and anti-tumor plant alkaloids and other biologically active components in the plant decoctions [17]. 2.1.4. Persian plants A group of Iranian medicinal plants Silybum marianum, Matricaria chamomilla, Calendula officinalis, Cichorium intybus and Dracocephalum

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kotschyi ethanolic extracts has been investigated on human peripheral blood lymphocytes and thymocytes; and on proliferative responsiveness of human lymphocytes to phytohemagglutinin. However, none of the extracts exhibited a direct mitogenic effect on human lymphocytes or thymocytes. Among the plants studied, C. intybus, C. officinalis and D. kotschyi showed an inhibitory effect on the proliferation of lymphocytes in the presence of PHA. Extract of M. chamomilla showed almost no stimulatory effect, whereas S. marianum displayed significant decrease in proliferation assay. In later, all the extracts except D. kotschyi enhanced the proliferation of lymphocytes after stimulation with the allogenic cells [18]. In a separate experiment, an aqueous extract of Calendula Officinalis showed in vitro human peripheral blood lymphocyte (PBL) proliferation and cytotoxic tumor cell activity [19]. Treatment of mice with the extract of Haussknechtia elymatica (family: Apioideae), decreased the footpad thickness indicating a dose-related inhibitory effect of H. elymatica on delayed hypersensitivity. The extract also significantly reduced the antibody titer after immunization with Sheep-RBC, human peripheral blood lymphocytes in the presence of mitogen and the production of IL-2 [20]. Amirghofran recently reported, methanolic extract of Stachys obtusicrena possess inhibitory effects on both cellular and humoral immune responses. S. obtusicrena showed a dose-related decrease in delayed type hypersensitivity and antibody responses in mice. In the in vitro study performed on the mitogen treated lymphocytes, the extract caused a dosedependent decline in [3H]-thymidine uptake and IL-2 levels in the culture supernatants of the activated lymphocytes [21]. 2.1.5. American plants Native American plant Echinacea purpurea (family: Asteraceae), known as the purple coneflower, is a medicinal plant widely used to treat a variety of illnesses, especially common cold, respiratory infections. Several studies indicated the ability of various Echinacea species to activate non-specific defence mechanisms and function as an immune stimulant [22]. However, the mechanism underlying Echinacea-induced immunomodulation remains largely unknown. Recent findings demonstrated that Echinacea extracts are potent activators of natural killer (NK) cells cytotoxicity. Echinacea augmented the frequency of NK target conjugates and activated the programming for lysis of NK cells [23]. Hall has reported that the complex aqueous Echinacea extract and the isolated high molecular weight constituents (polysaccharides) from this extract have effect on cytokine expression by macrophages [24]. It was reported alkylamides from Echinacea modulate TNF-Îą mRNA expression in human monocytes/macrophages via the cannabinoid

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type 2 (CB2) receptor [25]. However, the alkylamides dodeca-2E,4E,8Z,10Ztetraenoic acid isobutylamide and dodeca-2E,4E-dienoic acid isobutylamide bind to the CB2 receptor more strongly than the endogenous cannabinoids. Alkylamides from Echinacea are a new class of CB2-specific cannabinomimetics, which share the anti-inflammatory properties of anandamide and the cannabinoids from Cannabis sativa. Alkylamides and anandamide (Fig. 1) potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects were not exclusively related to CB2 binding [26]. Mollugo verticillata L. (family: Molluginaceae), a weed plant common in warm and/or wet regions of the American continent. The ethanolic extract of this plant showed immunostimulatory activity when peritoneal cells were stimulated in vitro with BCG antigen only. Ethanolic extracts directly increased NO release by peritoneal cells, but suppressed the immune response of these cells when treated with BCG antigen and Mycobacterium tuberculosis whole antigen [27]. Larrea divaricata Cav. (family: Zygophyllaceae) is a plant widely used as popular medicine to treat tumors, infections, and inflammatory diseases in America. The study showed, macrophages harvested from extract treated mice showed no signs of apoptosis. These cells showed a significant increase in NO and TNF-Îą release and exhibited the strongest expression of iNOS. Decoction also increased the phagocytosis of zymosan and the binding of LPS-FITC. The expression of CD14, TLR4 and CR3 was lower in macrophages of mice treated than in controls [28]. O O

N H O

N H

N H Alkylamides

Anandamide

Figure 1. Chemical structure of Alkylamides and anandamide.

2.1.6. European plants Methanol extract of a Portuguese plant Carpobrotus edulis (family: Aizoaceae) showed that the extract inhibit a verapamil sensitive efflux pump of mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. It also prime THP-1 human

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monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract induced the proliferation of THP-1 cells within one day of exposure to quantities normally associated with phytohaemagglutinin [29].

2.2. Immunomodulatory activity of plant derived compounds 2.2.1. Sterols and sterolins The phytosterols, β-sitosterol, and its glucoside (Fig. 2) enhanced the in vitro proliferative response of T-cells stimulated by sub-optimal concentrations of phytohaemagglutinin several fold at extremely low concentrations (femtogram level). A 100:1 (mass:mass) ratio of β-sitosterol: β-sitosterol glucoside showed higher stimulation than the individual sterols at the same concentration [30]. The mixture of the sterols (β-sitosterol) and sterolins (β-sitosterol glucoside) has the ability to enhance the cellular response of T lymphocytes both in vitro and in vivo. The mixture enhances the cytotoxic ability of natural killer (NK) cells against the target cancer cell line NK562. It has also been postulated that the sterol-sterolin mixture in a specific ratio could reinstate a balance between the TH1–TH2 cells, a delicate balance that determines the final outcome of an immune response. The same mixture inhibited the release of pro-inflammatory cytokines from endotoxin activated monocytes: interleukin-6 and tumour necrosis factor-α secretion [31]. In HIV-infected patients analysis of the CD4 cell-type (TH1 vs TH2-type) showed that those receiving the sterol-sterolin mixture maintained a favorable TH1 response, which implies that their cell-mediated response was possibly responsible for the viral control and inhibition of CD4 cell loss [32]. However, the cellular target of these molecules was not clearly described cytoplasmic receptors, gene regulation. In a different study, the total cells and eosinophils in the bronchoalveolar lavage fluid markedly decreased after β-sitosterol and lactose-β-sitosterol administration. They also mitigated the inflammation by eosinophil infiltration and mucus hypersecretion by goblet hyperplasia, inhibited the increased mRNA and protein expression of IL-4 and Et

Et Me R

Me Me R S

S H

R Pr-i

HO S S

H HO

β-sitosterol

S S

R R

S H

S S

O H

β-sitosterol glucoside

Figure 2. β-sitosterol and β-sitosterol glucoside.

R Pr-i

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IL-5 in the lung tissue and bronchoalveolar lavage fluid [33]. Immunomodulatory effect of a Î˛-sitosterol glycoside, daucosterol was also observed against disseminated candidiasis caused by Candida albicans caused by the CD4+ TH1 immune response [34]. 2.2.2. Cannabinoids Advances in understanding the physiology and pharmacology of the endogenous cannabinoid system have potentiated the interest of cannabinoid receptors as potential therapeutic targets. Cannabinoids have been shown to modulate a variety of immune cell functions and have therapeutic implications on central nervous system (CNS) inflammation, chronic inflammatory conditions such as arthritis, and may be therapeutically useful in treating autoimmune conditions such as multiple sclerosis. Many of these drug effects occur through cannabinoid receptor signalling mechanisms and the modulation of cytokines and other gene products [35]. Cannabidiol and cannabis-based medicines are potential therapeutic agents. Because the immune system has been widely demonstrated to be affected by psychoactive cannabinoids, such as â&#x2C6;&#x2020;9-tetrahydrocannabinol (Fig. 3). Cannabidiol significantly attenuated the elevation of IL-2, IL-4, IL-5, and IL-13 steady-state mRNA expression elicited by Ova challenge in the lungs. Plant derived immunomodulatory cannabinoids exhibited potential therapeutic utility in the treatment of allergic airway disease by inhibiting the expression of critical T cell cytokines and the associated inflammatory response [36]. Echinacea species with the cannabinoid (CB) receptor-binding lipophilic alkamides are the other best known herbal cannabinomimetics, which has been discussed earlier in this chapter. Me

HO OH

(CH 2 ) 4 Me

H Me

R R

Me (CH 2 ) 4

Me H2 C

Cannabidiol

9-Tetrahydrocannabinol

Figure 3. Cannabidiol and â&#x2C6;&#x2020;9-tetrahydrocannabinol.

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2.2.3. Polysaccharides Polysaccharides from plants have been the subject of study for a very long time mainly for their physical properties and industrial use based on these properties. Over the last 20 years there has been an ever increasing interest in the biological activity of biomolecules has led to new sources for interesting bioactive plant polysaccharides [37]. Botanical polysaccharides exhibit a number of beneficial therapeutic properties, and it is thought that the mechanisms involved in these effects are due to the modulation of innate immunity and, more specifically, macrophage function. Furthermore, botanical and microbial polysaccharides bind to common surface receptors and induce similar immunomodulatory responses in macrophages, suggesting that evolutionarily conserved polysaccharide structural features are shared between these organisms. Thus, the evaluation of botanical polysaccharides provides a unique opportunity for the discovery of novel therapeutic agents and adjuvants that exhibit beneficial immunomodulatory properties [38]. The immunomodulatory effects of the polysaccharide of Cistanche Deserticola have been evaluated by in vitro proliferation of murine thymus lymphocytes by MTT method. The enhancing effect of polysaccharide on murine thymus lymphocyte proliferation was related to its promotion on thymus intracellular Ca+2 delivering [39]. High molecular weight substances were isolated from Salicornia herbacea, which has been used to treat a variety of diseases including cancers in traditional oriental remedy. The active components of the extract have been described as polysaccharides, which not only activate monocytic cells strongly, but also induce differentiation of monocytic cells into macrophages [40]. 2.2.4. Alkaloids Plant bis-benzylisoquinoline alkaloid tetrandrine is active purified compound from dried tuberous root of the creeper Stephania tetrandra, is a potent immunomodulator used to treat rheumatic disorders, silicosis and hypertension in mainland China [41]. Tetrandrine effectively suppressed cytokine production and proliferation of CD28-costimulated T cells [42]. Recently, tetrandrine downregulated IκBα kinases- IκBα -NF-κB signalling pathway in human peripheral blood T cell. Compared to four tetrandrine analogs (tetrandrine, dauricine, berbamine and hemandezine; Fig. 4.), dauricine appeared as the most potent inhibition on CD28 but not on H2O2induced NF-κB DNA-binding activities [41, 43]. Recent studies showed tetrandrine might modulate lipopolysaccharide induced microglial activation by inhibiting the NF-κB-mediated release of inflammatory factors [44].

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Figure 4. Alkaloid immunomodulators.

Alcoholic extract of the fruits of well known spice (black pepper) Piper longum and its component alkaloid piperine exhibited immunomodulatory and antitumor activity to Dalton's lymphoma ascites cells and Ehrlich ascites carcinoma (EAC) cells. Piper longum extract and piperine increased the total WBC count, number of plaque forming cells, and bone marrow cellularity and Îą-esterase cells [45]. Chronic rejection after solid organ transplantation is the major cause of graft failure in the first postoperative year. The pathogenesis of this process remains poorly understood. To a rat model of cardiac transplantation the effect of retransplantation has been compared with the immunomodulatory effect of cyclosporin A and sinomenine, an alkaloid extract from the Chinese medical plant Sinomenium acutum. Treatment with either cyclosporin A or sinomenine prevented progression of vascular changes including myointimal proliferation, whereas combination therapy resulted in long-term graft survival and absence of the lesions mentioned above [46].

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2.2.5. Flavonoids Eupalitin-3-O-β-D-galactopyranoside (Fig. 5) purified from the ethanolic extract of Boerhaavia diffusa root inhibited phytohemagglutinin stimulated proliferation of peripheral blood mononuclear cells, two-way MLR and natural killer cell, as well as lipopolysaccharide induced NO production by RAW 264.7 It further inhibited production of phytohemagglutinin stimulated IL-2 at the protein and mRNA transcript levels; lipopolysaccharide stimulated TNF-α production in human peripheral blood mononuclear cells. It blocked the activation of DNA binding of nuclear factor-κB and AP-1, two major transcription factors centrally involved in expression of the IL-2 and IL-2R gene, which are necessary for T cell activation and proliferation. On the other hand, eupalitin showed little activity on the above experiments [47]. The extract of Apium graveolens var. dulce contained apiin as the major constituent (1.12%, wt./wt., of the ext.). The extract and apiin showed significant inhibitory activity on nitrite (NO) production in vitro and iNOS expression in LPS-activated J774.A1 cells. The findings suggested the properties of the extract in vivo were due to reduction of iNOS enzyme expression [48]. Bidens pilosa is an ethnical medicine for bacterial infection or immune modulation in Asia, America and Africa. Flavonoid, centaurein and its aglycone, centaureidin has been isolated from the butanol subfraction of Bidens pilosa. The study suggested that centaurein regulated IFN-γ transcription as an immunomodulator, probably via NFAT and NFκB in T cells [49]. OH

OH MeO

R R O

MeO

H OH

OH O

S S

R = H ; Eupalitin R = β-D-galactopyranoside; Eupalitin-3-O-β-D-galactopyranoside

R R

S O OH

Apiin OH OMe

OH O

R S

S R

MeO MeO

OMe

OH OH

OMe

Centaurein

Centaureidin

Figure 5. Flavonoid immunomodulators.

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2.2.6. Lectin A plant lectin from Viscum album has been previously shown to increase the number and cytotoxic activity of natural killer cells and to induce antitumor activity in animal models. Lectin-sugar interactions on the cell surface of immunocompetent cells can induce cytokine gene expression and protein synthesis [50]. Recently, plant lectin Viscum album agglutinin-I also demonstrated interesting potential therapeutic properties and immunomodulatory activities. Lectin alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species [51]. 2.2.7. Glycoprotein Immunomodulatory effect of a C3 binding glycoprotein has been isolated from the parasitic plant Cuscuta europea. The glycoprotein showed a dosedependent in vivo immunostimulation against mice immunized with sheep red blood cells. The in vitro stimulation was assessed by an increase in the number of hemolytic plaque forming cells and hemagglutination titers [52]. Later studies demonstrated that C3 binding glycoprotein induced proinflammatory and immunoregulatory cytokine production, in the highest degree IL-12, followed by IL-6 and in lower degree TNF-Îą. IL-12 quantity was significantly increased in glycoprotein stimulated cultures in comparison with LPS, PHA and PWM stimulated PBMC. The authors suggested that a part of the mechanism of action of C3 binding glycoprotein is mediated through NF-kB signal transduction pathway [53].

2.3. Herbal formulations as immunomodulators The increased neopterin production and tryptophan degradation in stimulated peripheral blood mononuclear cells was found to be significantly suppressed by several plant extracts, e.g., Uncaria tomentosa, Hypericum perforatum, green and black tea from Camellia sinensis and by the Tibetan herbal remedy PADMA 28. The plant compounds down-regulated TH1-type immune response by reducing the expression of the cytokine IFN-Îł [3]. Peanut allergy is potentially life threatening and there is no curative therapy for this disorder. FAHF-2 is a Chinese herbal formula completely eliminated anaphylaxis in mice allergic to peanut challenged as long as 5 weeks post therapy. This result was associated with down regulation of TH2 responses [54]. The immunomodulatory activities of Triphala, an Indian Ayurvedic formulation of three plants (Terminalia chebula, Terminalia belerica and

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Emblica officinalis) were assessed by testing the various neutrophil functions like adherence, phagocytosis (phagocytic index and avidity index and nitro blue tetrazolium) reduction in albino rats. Oral administration of Triphala stimulated the neutrophil functions in the immunized rats. Stress induced suppression in the neutrophil functions were also significantly prevented by Triphala [55].

2.4. Patented immunomodulators Polysaccharides Extracellular polysaccharides of Aphanothece halophytica has been patented for regulating immunity and treating and/or preventing pulmonitis [56]. A polysaccharide fraction (0.1-20%), extracted from callus plant tissue of Ungernia species was evaluated for its antimutagenic, immunomodulating, and antitumor activities. Per oral administration of the polysaccharide fraction at a daily dose during 20-30 days to mice enhanced effectiveness and safety of treatment [57]. Gulvel is extensively used in Ayurveda as a single or polyherbal formulation. Several plants of genus Tinospora such as T. cordifolia, T. malabarica, etc., are well known by this name. A process for the preparation of an immunomodulator from the above plants has been described. A branched polysaccharide, arabinogalactan was selectively precipitated from the polar extracts in aqueous medium by methanol. The active polysaccharide was further purified by high-performance gel permeation chromatography. It is polyclonally mitogenic to Î˛-cells, and augments antibody response as well as enhances T-cell responses to model antigens [58]. Coumarinolignoids A novel pharmaceutical composition consisting of a combination of three coumarinolignoids isolated from the seeds of the plant Cleome viscosa has been described to modulate humoral and cell mediated immune response [59]. Polyphenols Polyphenols are useful for prophylactic and therapeutic treatment of allergy. Apple polyphenol containing ~50% proanthocyanidin significantly promoted interferon-Îł formation later and inhibited IL-5 and IL-10 in ovalbumin-immunized murine spleen cells [60].

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Stilbenoids Extraction of pharmaceutically active stilbene derivatives (e.g., resveratrols, ε-viniferin, viniferin derivatives, hopeaphenol, and Ampelopepsin A) with immunomodulating activity from spermatophyte plants has been reported [61]. Herbal formulation An immunomodulator for the prevention and the treatment of AIDS comprises Cordyceps plants (such as Cordyceps militaris L., Brazil Cordyceps, and FENG Cordyceps), Radix Glycyrrhizae, Rhizoma Atractylodis Macrocephalae, Radix Angelicae Sinensis, Rhizoma Coptidis, Radix Aconiti Lateralis Preparata, Minor Decoction of Bupleurum, and AZT. This composition can maintain or improve the immunologic function of patients [62]. Fve proteins and peptides The Fve protein (i.e., a protein from the golden needle mushroom Flammulina velutipes) upregulated expression of TH1/TC1 cytokines such as interferon-γ and tumor necrosis factor-α; whereas down regulated expression of TH2/TC2 cytokines such as IL-4 and IL-13. In addition, it upregulated expression of T regulatory cell cytokines IL-10 and transforming growth factor-β. Furthermore, Fve proteins exhibited the following properties hemagglutination, lymphocyte aggregation, and lymphoproliferation. This peptide may be used as an immunomodulator, as an adjuvant, either alone or as a fusion protein with an antigen or allergen [63].

3. Conclusions Immunomodulation using medicinal plants can provide an alternative to conventional chemotherapy for a variety of diseases, especially when host defence mechanism has to be activated under the conditions of impaired immune response or when a selective immunosuppression is desired in situations like autoimmune disorders. There is great potential for the discovery of more specific immunomodulators which mimic or antagonize the biological effects of cytokines and interleukins, and the refinement of assays for these mediators will create specific and sensitive screens. Natural remedies should be revisited as important sources of novel ligands capable of targeting specific cellular receptors.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Bomford, R. 1988, Phytother. Res., 2, 159. Wagner, H. and Proksch, A., eds. 1985, Immunostimulatory drugs of fungi and higher plants. Economic and Medicinal Plant Research, ed. H. Wagner, H. Hikino, and N.R. Farnsworth. Vol. 1. Academic Press: London, 113. Winkler, C., Neurauter, G., Schroecksnadel, K., Wirieitner, B., and Fuchs, D. 2006, Recent Prog. Med. Plant., 11, 139. Ganju, L., Karan, D., Chanda, S., Srivastava, K.K., Sawhney, R.C., and Selvamurthy, W. 2003, Biomed. Pharmacother., 57, 296. Mehrotra, S., Mishra, K.P., Maurya, R., Srimal, R.C., Yadav, V.S., Pandey, R., and Singh, V.K. 2003, Int. Immunopharmacol., 3, 53. Desai, V.R., Ramkrishnan, R., Chintalwar, G.J., and Sainis, K.B. 2007, Int. Immunopharmacol., 7, 1375. Desai, V.R., Kamat, J.P., and Sainis, K.B. 2002, Proceedings - Indian Academy of Sciences, Chemical Sciences, 114, 713. Singh, N., Singh, S.M., and Shrivastava, P. 2004, Immunopharmacol. Immunotoxicol., 26, 145. Mungantiwar, A.A., Nair, A.M., Shinde, U.A., Dikshit, V.J., Saraf, M.N., Thakur, V.S., and Sainis, K.B. 1999, J. Ethnopharmacol., 65, 125. Mehrotra, S., Mishra, K.P., Maurya, R., Srimal, R.C., and Singh, V.K. 2002, Int. Immunopharmacol., 2, 987. Puri, A., Saxena, R., Saxena, R., Saxena, K., Srivastava, V., and Tandon, J. 1994, J. Ethnopharmacol., 42, 31. Bhatia, A. and Kaur, J. 2001, Int. J. Environ. Stud., 58, 197. Jayathirtha, M.G. and Mishra, S.H. 2004, Phytomedicine, 11, 361. Kaul, A., Bani, S., Zutshi, U., Suri, K.A., Satti, N.K., and Suri, O.P. 2003, Phytother. Res., 17, 1140. Mishra, Recent Prog. Med. Plant. K.P., Padwad, Y.S., Jain, M., Karan, D., Ganju, L., and Sawhney, R.C. 2006, Immunopharmacol. Immunotoxicol., 28, 201. Chevrier, M.R., Ryan, A.E., Lee, D.Y.W., Ma, Z., Zhang, W.-Y., and Via, C.S. 2005, Clin. Diagn. Lab. Immunol., 12, 575. Tram, N.T.N., Yanchev, I., Zvetkova, E., Dineva, J., Katzarova, E., Kostov, G., Svilenov, D., Ilieva, I., and Shalamanov, P. 2001, Exp. Pathol. Parasitol., 4, 9. Amirghofran, Z., Azadbakht, M., and Karimi, M.H. 2000, J. Ethnopharmacol., 72, 167. Jimenez-Medina, E., Garcia-Lora, A., Paco, L., Algarra, I., Collado, A., and Garrido, F. 2006, BMC Cancer, 6, 119. Amirghofran, Z., Azadmehr, A., and Javidnia, K. 2007, Iranian J. Immunol., 4, 26. Amirghofran, Z., Bahmani, M., Azadmehr, A., and Javidnia, K. 2007, Int. Med. J. Expt. Clin. Res., 13, 145. Hwang, S.A., Dasgupta, A., and Actor, J.K. 2004, Clin. Chim. Acta, 343, 161. Gan, X.-H., Zhang, L., Heber, D., and Bonavida, B. 2003, Int. Immunopharmacol., 3, 811.

Recent advances on the ethnomedicinal plants as immunomodulatory agents

243

24. Hall, A., Riek, N., Christensen, E., and Cech, N. 2003, Abstracts, 55th Southeast Regional Meeting of the American Chemical Society, Atlanta, GA, United States, November 16-19, 2003, 556. 25. Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. 2004, FEBS Lett., 577, 563. 26. Raduner, S., Majewska, A., Chen, J.-Z., Xie, X.-Q., Hamon, J., Faller, B., Altmann, K.-H., and Gertsch, J. 2006, J. Biol. Chem., 281, 14192. 27. Ferreira, A.P., Soares, G.L.G., Salgado, C.A., Goncalves, L.S., Teixeira, F.M., Teixeira, H.C., and Kaplan, M.A.C. 2003, Phytomedicine, 10, 154. 28. Davicino, R., Mattar, A., Casali, Y., Porporatto, C., Correa Silvia, G., and Micalizzi, B. 2007, Immunopharmacol. Immunotoxicol., 29, 351. 29. Ordway, D., Hohmann, J., Viveiros, M., Viveiros, A., Molnar, J., Leandro, C., Arroz Maria, J., Gracio Maria, A., and Amaral, L. 2003, Phytother. Res., 17, 512. 30. Bouic, P.J.D., Etsebeth, S., Liebenberg, R.W., Albrecht, C.F., Pegel, K., and Jaarsveld, P.P.V. 1996, Int. J. Immunopharmacol., 18, 693. 31. Bouic, P.J.D. 2002, Drug Discovery Today, 7, 775. 32. Bouic, P., Clark, A., Brittle, W., Lamprecht, J., Freestone, M., and Liebenberg, R. 2001, S. Afr. Med. J., 91, 848. 33. Yuk, J.E., Woo, J.S., Yun, C.-Y., Lee, J.-S., Kim, J.-H., Song, G.-Y., Yang, E.J., Hur, I.K., and Kim, I.S. 2007, Int. Immunopharmacol., 7, 1517. 34. Leea, J.-H., Leea, J.Y., Parka, J.H., Jungb, H.S., Kimb, J.S., Kangb, S.S., Kimb, Y.S., and Hana, Y. 2007, Vaccine, 25, 3834. 35. Woelkart, K., Salo-Ahen, O.M.H., and Bauer, R. 2008, Curr. Top. Med. Chem., 8, 173. 36. Jan, T.-R., Farraj, A.K., Harkema, J.R., and Kaminski, N.E. 2003, Toxicol. Appl. Pharmacol., 188, 24. 37. Paulsen, B.S. 2001, Curr. Org. Chem., 5, 939. 38. Schepetkin, I.A. and Quinn, M.T. 2006, Int. Immunopharmacol., 6, 317. 39. Zeng, Q., Zheng, Y., and Lu, Z. 2002, Zhejiang Daxue Xuebao, 31, 284. 40. Im, S.-A., Kim, K., and Lee, C.-K. 2006, Int. Immunopharmacol., 6, 1451. 41. Ho, L.-J., Juan, T.-Y., Chao, P., Wu, W.-L., Chang, D.-M., Chang, S.-Y., and Lai, J.-H. 2004, Br. J. Pharmacol., 143, 919. 42. Lai, J.H., Ho, L.J., Kwan, C.Y., Chang, D.M., and Lee, T.C. 1999, Transplantation, 68, 1383. 43. Lai, J.-H. 2002, Acta Pharmacol. Sin., 23, 1093. 44. Xue, Y., Wang, Y., Feng, D.-c., Xiao, B.-g., and Xu, L.-y. 2008, Acta Pharmacologica Sinica, 29, 245. 45. Sunila, E.S. and Kuttan, G. 2004, J. Ethnopharmacol., 90, 339. 46. Schneeberger, S., Mark, W., Seiler, R., Offner, F., Amberger, A., and Margreiter, R. 2000, Chir. Forum Exp. Klin. Forsch., 183. 47. Pandey, R., Maurya, R., Singh, G., Sathiamoorthy, B., and Naik, S. 2005, Int. Immunopharmacol., 5, 541. 48. Mencherini, T., Cau, A., Bianco, G., Della Loggia, R., Aquino, R.P., and Autore, G. 2007, J. Pharm. Pharmacol., 59, 891. 49. Chang, S.-L., Chiang, Y.-M., Chang, C.L.-T., Yeh, H.-H., Shyur, L.-F., Kuo, Y.H., Wu, T.-K., and Yang, W.-C. 2007, J. Ethnopharmacol., 112, 232.

244

Mahiuddin Alamgir & Shaikh Jamal Uddin

50. Hostanska, K., Hajto, T., Spagnoli, G., Fischer, J., Lentzen, H., and Herrmann, R. 1995, Nat. Immun., 14, 295. 51. Lavastre, V., Pelletier, M., Saller, R., Hostanska, K., and Girard, D. 2002, J. Immunol., 168, 1419. 52. Stanilova, S.A., Zhelev, Z.D., and Dobreva, Z.G. 2000, Int. J. Immunopharmacol., 22, 15. 53. Stanilova, S.A., Dobreva, Z.G., Slavov, E.S., and Miteva, L.D. 2005, Int. Immunopharmacol., 5, 723. 54. Srivastava, K., Kattan, J., Zou, Z., Li, J., Zhang, L., Wallenstein, S., Goldfarb, J., Sampson, H., and Li, X. 2005, J. Allergy Clin. Immunol., 115 171. 55. Srikumar, R., Jeya Parthasarathy, N., and Sheela Devi, R. 2005, Biol. Pharm. Bull., 28, 1398. 56. Zheng, W., Chu, C., and Cheng, Q. 2006, CN: 1771984, CAN: 145:89725. 57. Muzyka, V.A., Kuznetsov, V.A., Kolonina, I.V., and Tsekhanovskii, S.N. 2000, RU: 2147439, CAN: 135:147406. 58. Chintalwar, G.J., Jain, A., Sumariwalla, P.F., Ramakrishnan, R., Sipahimalani, A.T., Sainis, K.B., and Banerji, A. 2000, IN: 183805, CAN: 140:276140. 59. Khanuja, S.P.S., Pal, A., Chattopadhyay, S.K., Darokar, M.P., Patel, R.P., Gupta, A.K., Negi, A.S., Kaur, T., Tandon, S., Kahol, A.P., and Garg, A. 2007, WO: 2007066197, CAN: 147:58364. 60. Shoji, T., Kanda, T., Akiyama, H., Yonetani, T., and Aida, Y. 2005, JP: 2005082497, CAN: 142:309895. 61. Ravagnan, G., Falchetti, R., Lanzilli, G., Fuggetta, M.P., Tricarico, M., and Mattivi, F. 2001, WO: 2001091764, CAN: 136:11085. 62. Wang, J. 2001, CN: 1305823. 63. Chua, K.Y., Seow, S.V., and Kolatkar, P.R. 2004, WO: 2004092210, CAN: 141:378841.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 245-265 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

9. An update on plant-originated treatment for Alzheimer’s disease 1

Ilkay Orhan1, Gürdal Orhan2 and Bilge Şener1

Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey 2 Neurology Clinic of Numune Training and Research Hospital, Ministry of Health 06100 Ankara, Turkey

Abstract. Alzheimer’s disease (AD) is a progressive and fatal brain disorder named after German physician Alois Alzheimer, who first described it in 1906. AD is one of the most common forms of dementia which demolishes vital brain cells, causing troubles with memory, thinking, and behavior brutal enough to affect work, lifelong hobbies or social life. The disease has several stages from mild, moderate to severe-types. Unfortunately, there is no cure for AD at present and no treatment that may impede its progression. In fact, some medications are existing that can help with some symptoms, which can slow down the decline of memory, language, and thinking abilities. In the treatment of AD, the standardized extracts of Ginkgo biloba L. (Ginkgoceae) has been the most prescribed herbal medicine. In addition to that, galanthamine, an alkaloid from Galanthus nivalis L. (Amaryllidaceae), has been the most recent drug available in the market. On the other hand, huperzin A isolated from Huperzia serrata Thunb. (Lycopodiaceae) is also a quite promising alkaloid which will become available in the market in a very near future. This chapter intends to cover the latest scientific literature about AD treatment for new drug development from herbal sources. Correspondence/Reprint request: Dr. Ilkay Orhan, Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey. E-mail: iorhan@gazi.edu.tr

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Introduction Alzheimer’s disease (AD) is a progressive and neurodegenerative disease that primarily affects the elderly population over 65 years of age, and is estimated to account for 50–60% of dementia cases [1]. The prevalence has been found to raise exponentially with age, ranging from 3.0% in patients aged 65 to 74 years to as much as 47.2% for those aged 85 years [2,3]. The etiology of AD remains still unclear but some factors have been suggested that appear to reduce the incidence of the disease, or for which a hypothesis has been put forward based on scientific research [4]. Degeneration of cholinergic cortical neurons is one of the main reasons for the cognitive deficit in Alzheimer-type of dementia. The presence of extracellular plaques, which contain deposits of proteins and beta-amyloid, as well as intracellular neurofibrillatory tangles are also another hallmarks of the AD pathology [5]. In a considerable proportion of AD patients, there is also a slowing of motor activity and extrapyramidal dysfunction resembling that is seen in Parkinson’s disease [6]. Unfortunately, there has been no cure so far to stop AD, while only symptomatic treatment is available. The pathology of AD consists of a combination of genetic and non-genetic multifactors. Among them, one theory named as “the cholinergic hypothesis” has been suggested leading to discovery of “acetylcholinesterase (AChE) inhibitors”. The most effective treatments are therefore this type of the medications that attempt to increase the brain’s levels of acetylcholine, an important neurotransmitter whose levels decrease with onset of the disease. AChE inhibitors work by interfering with AChE, an enzyme that breaks down acetylcholine. Both the American Academy of Neurology and the British National Institute for Clinical Excellence have approved the use of AChE inhibitors in AD patients [7]. In addition to AChE inhibition, changes through CNS in the AD pathology have been identified as senile plaques and neurofibrillary tangles, oxidative and inflammatory processes and neurotransmitter interruptions. On the other hand, cell damage due to free radicals is partly responsible for acute tissue damage and chronic diseases, e.g. arthritis and inflammation, atherosclerosis, renal damage, cell aging, diabetes, and also associated with central nervous system disturbances. Consequently, strong antioxidants could contribute to recovery or protection of neurodegeneration, although the ability of neurons to regenerate is very limited in the central nervous system (CNS). However, neuronal systems appear to be especially sensitive to oxidation. Oxidative damage to biochemical structures of nerve cells has been found in a large number of neurodegenerative disorders [7]. At this point, the special extract of Ginkgo biloba has been used in AD treatment

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since a long time with a remarkable success for its antioxidant and vasodilator effects. In this review, we aim to put focus particularly on the most prescribed herbal medicines and compounds from plants used in the treatment of AD. Besides, the future-promising herbs or their pure components usable against AD will be mentioned.

I. Clinically used herbal-originated drugs in AD treatment I.1. Ginkgo biloba L. Numbers of scientific research have been carried out on G. biloba L. (Gingkoceae), one of the oldest plants living on earth with geological records indicating this plant has been growing on earth for 150–200 million years. The plant, the unique species of the genus Ginkgo, is 30 to 40 m high deciduous tree whose leaves are fan-shaped with bifurcated ribs. It is also one of the best-known medicinal plants with traditional use being recorded as early as 2800 BC in the traditional Chinese medicine (TCM). G. biloba was first introduced to Europe in 1690 by an ethnobotanist Engelbert Kaempfer [8]. The drugs that have been prescribed until now most often in Germany against dementia are the extracts of G. biloba and it was the second bestselling dietary supplement in the USA [9]. It was reported that about 7 billion dollars are spent annually on botanical medicines and G. biloba ranks first among herbal medications [10]. In China, France, and the USA, fifty million Ginkgo trees are cultivated, which yield more than 8000 tons of dried leaves every year to meet the commercial demand for G. biloba preparations [11]. The incidence of adverse drug reactions is a few percent and, thus, at least 10 times lower than the average with the AChE inhibitors [12]. Special extract of the plant, which is recognized medicinally, is widely referred to “EGb 761” and has been used in various cardiovascular diseases and central nervous system diseases including ischemia and dementia. G. biloba has been used in circulatory disorders since the 1960s, where it has also been used in TCM for respiratory disorders [13]. G. biloba has also been used traditionally in Iran to improve memory loss associated with blood circulation abnormalities [14]. Considering the involvement of oxidative stress and spreading free radical reactions in its pathology, AD is one of the best characterized neurological diseases [11,15–17]. Because in dementia depending on neuronal loss and impaired neurotransmission, a reduction in oxygen and glucose accompanied with production of free radicals and lipid peroxidation has been observed [18]. Moreover, a relationship between formation of hydrogen peroxide, accumulation of reactive oxygen species and oxidative stress and AD has been again mentioned [19].

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Despite of the use of G. biloba for its vasodilator and antioxidant effects on CNS, the mechanism of action with this plant has not been elucidated properly, yet. A major mechanism by which G. biloba is proposed to exert its effect is by action of multiple antioxidants present in the extract [20]. The acetone-water extract (EGb 761) prepared from the leaves is standardized according to its flavone glycosides (24%) and terpene lactones (6%), which provide it with unique polyvalent pharmacological action, and EGb 761 is commercially available in China and around the world. However, the plant generally contains flavonoid derivatives such as mono-, di-, and tri-glycosides of quercetin, isorhamnetin, 3-Omethylmyristicin, and kaempferol; biflavonoids including amentoflavon, bilobetin, ginkgetin, and isoginkgetin; proanthocyanidins, trilactonic diterpenes such as gingkolides A, B, C, J, and M; as well as trilactonic sesquiterpene bilabolide, ginkgolic acids, phenolic acids such as p-coumaric acid esters as well as coumarylflavones (Figure 1) [21-24]. Flavonoids are not only specific to G. biloba, but trilactone terpenes called bilobalides and ginkgolides A, B, C, J, and M are only found in this plant and used as the analytical markers for fingerprint analysis of its leaf extract. Interestingly, ginkgolide J was found only in the leaves, whereas ginkgolide M was found only in the root bark. A QSAR analysis on gingkolides was also able to explain why gingkolide B had the most potent inhibitory effect against platelet activating factor (PAF), which is said to play a role in aging neuropathogenesis [25]. Numerous investigations have been conducted regarding the potential of G. biloba in cognitive disorders. More recently, an in vitro study indicated that the extract had also an anti-amyloid aggregation effect suggesting another mechanism whereby G. biloba may be effective in preventing or delaying the development of AD [26]. In one study, it was stated that anti-amyloid effect of G. biloba is not probably due to interactions between trilactonic diterpenes and amyloid peptide (AÎ˛25-35) [27]. On the other hand, a new extract prepared from G. biloba coded as P8A was 70% enriched with trilactonic diterpenes were shown to prevent anti-amyloid effect via AÎ˛1-42 inhibition of long term potentiation in mouse hippocampal slices which was mostly attributed to ginkgolide J [28]. Most recent evidence also suggests a modulating role of the extract on alpha secretase, the enzyme that cuts the amyloid precursor protein and prevents amyloidogenic fragments from being produced [29]. Gingkolides were suggested to exert their neuroprotective effects through hypoxia-induced injury which is regulated by hypoxia-inducable factor (HIF-1) in cellular and systemic homeostasis [30].

Herbal treatment of Alzheimer's disease H O H3C

249 H

O O

O OH O

H3 C

O O

OH O

C(CH3)3

O O

Gingkolide A

H3C

O O

O OH O

H3C

O O

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C(CH3)3

Gingkolide B

O O

H3C

C(CH3)3 OH

Gingkolide J H O

O O

O OH

HO C(CH3)3

O H

Bilobalide Figure 1. Chemical structures of ginkgolides A, B, C, J, and bilobalide.

The typical dose of Ginkgo is 40 to 80 mg three times daily of the extract standardized to contain 24% Ginkgo flavone glycosides. It may take up to 6 weeks before yielding any results. Besides, Ginkgo should not be used with anticoagulants, or by patients with clotting problems, without medical supervision. G. biloba has not been reported to alter the pharmacokinetics of co-administered drugs but can lead to pharmacodynamic drug interactions [31,32].

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A quite number of clinical trial studies have been carried out on AD patients with G. biloba presenting the positive effect of this herbal medicine in cognitive deficits [33-39]. These studies underlined that the efficiency of G. biloba in Alzheimer-type of dementia is likely to result from synergistic interaction between flavonoids and terpene lactones. Therefore, many analytical studies were carried out on G. biloba samples or its preparations by HPLC, LC-MS or GC-MS [40-50]. The fingerprint of G. biloba is very important from the view point of its quality control. Because quality of the extracts is unacceptable if the fingerprint analysis by any method shows the lower quantity of the marker compounds as ginkgolides and bilobalide [47]. The presence of ginkgolide peaks in these profiles may reveal to what extend the ginkgolides contribute to the quality of the Ginkgo biloba extracts. Considering bioavailability of the extract, a study on G. biloba extract after oral administration of 80 mg of EGb 761 showed that the absolute bioavailabilities of ginkgolides A and B were over 80%, where that of ginkgolide C was quite low [51]. Bioavailability of bilobalide was 70% after administration of 120 mg EGb 761 extract [52]. These data were confirmed in another pharmacokinetic study where the mean bioavailabilities of ginkgolide A, ginkgolide B and bilobalide were determined as 80%, 88% and 79%, respectively [53]. The half-lives of ginkgolides A, B and bilobalide were found to be 4.5, 10.6, and 3.2 h, respectively. In brief, both pre-clinical and clinical studies have pointed out that cognitive-enhancing effect of the standardized leaf extract of G. biloba has been approved. Although the pharmacology of the plant is highly complex owing to its multiple active constituents, its cognitive-enhancing effects have been attributed to its platelet-activating factor antagonistic and its free-radical scavenger activities. Recent evidence also suggests that it may have direct effects on the cholinergic system which might help to clarify both its acute and chronic cognitive-enhancing effects. I.2. Galanthamine Galanthamine is an isoquinoline alkaloid isolated firstly from Galanthus woronowii (Amaryllidaceae), known as â&#x20AC;&#x153;snowdropâ&#x20AC;? (Figure 2) [54]. It was later on also isolated from some other members of the Amaryllidaceae family including Narcissus sp. and Leucojum aestivum, in which we showed the presence of galanthamine and other similar-structured isoquinoline alkaloids and determined their AChE inhibitory effects [55]. Apart from our study, G. elwesii and G. nivalis were also found to contain galanthamine in a notable amount [56,57]. Positive AChE inhibitory effects of several Amaryllidaceae alkaloids having galanthamine and lycorine skeletons were also reported elsewhere [58],

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CH3

H3CO

Figure 2. Structure of galanthamine.

which were in compliant with our study. For medical application, galanthamine has been isolated from plant material especially from L. aestivum until that the total chemical synthesis of galanthamine on an industrial scale has been economically achieved [59] and its biosynthetic pathway was fully explained by which 4'-O-methyl-N-norbelladine was the precursor [60]. Crystallographic structure and other conformational properties of the molecule were also reported [61]. Acetylcholinesterase (AChE) inhibitors, which are also called “anticholinesterase drugs”, have been recently approved as a promising treatment approach against AD. Galanthamine has been found to be the longacting and specific inhibitor of AChE enzyme and to potentiate cholinergic nicotinic neurotransmission by allosterically modulating nicotinic acetylcholine receptors which may be of additional value in the treatment of AD [55,6264]. Being one of the newest anticholinesterase drugs emerged in the market, it has been approved as its HBr salt for the first time in Austria (Nivalin®), later licensed as Reminyl® in the USA, some European countries as well as Turkey, and more recently under the name Razadyne®. Undoubtedly, any other drug class, AChE inhibitors may also differ from one another with respect to enormity and duration of effect. According to Giacobini [65], for some AChE inhibitors such as physostigmine, galanthamine, eptastigmine, and metrifonate, the relation between AChE inhibition and cognitive effect seem to be different than other AChE inhibitors including tacrine and donepezil in terms of efficacy and adverse effects. Although the most common side effect observed after galanthamine administration is nausea, it is possible to eliminate nausea by increasing the galanthamine dose slowly [66]. In addition, galanthamine was shown to be safe from the view point of hepatotoxicity [67]. Another advantage of galanthamine

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is its reversible and competitive inhibition on AChE. It has recently been introduced on the European market and approved by the Food and Drug Administration (FDA) for the treatment of AD in 2001. On the other hand, there have been more researches on derivatives of galanthamine. For instance; homodimeric and heterodimeric alkylene linked bis-galanthamine was synthesized and these derivatives were found to be more potent inhibitors of AChE than galanthamine and tacrine [68-70]. Moreover, another synthetic galanthamine derivative called (-)-9dehydrogalanthaminium bromide was shown to be the strong AChE inhibitor in vitro by Ellman method (Figure 3) [71] and in young and old rats, its cognitive-enhancing activity was also observed [72].

CH3 N

OCH3 Figure 3. Structure of bromide salt of (-)-9-dehydrogalanthamine.

Several molecular docking studies have been carried out on galanthamine-AChE enzyme complex of different origins. The compound was found to exert 53-fold selectivity against human erythrocyte AChE than butyrylcholinesterase (BChE), whereas it displayed 10-fold lower potency for human brain AChE than erythrocyte AChE [73,74]. Besides, Greenblatt et al. [75] performed another docking study at molecular level with AChE of Torpedo californica showing the binding properties of the moleculeâ&#x20AC;&#x2122;s different moieties with aminoacid residues of the enzyme at 2.3 Ă&#x2026; resolution. All these studies have shown that galanthamine, a potent inhibitor of AChE, is an effective commercial drug used in the treatment of mild to moderate AD. This natural compound can be taken as a model to develop new AChE inhibitors for future studies.

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II. Future-promising herbal-originated molecules in AD treatment II.1. Huperzine A Huperzia serrata (Thunb. ex Murray) is one of the genera in Huperziaceae family (syn. Lycopodiaceae family). This genus, used for its memory-enhancing effect since ages in TCM, is well-known to contain a large group of alkaloids called “Lycopodium alkaloids” [76,77]. The plant, known as “Qian Ceng Ta” in Chinese, is mostly grown in subtropical regions of the world as well as in China naturally along the Yangtze River as well as the southern parts of the country. Unfortunately, the plant may go to extinction in a near future depending on extensive collection from the wild, because of it medical importance [78]. Huperzine A is an quinolizidine alkaloid isolated from H. serrata (syn. Lycopodium serratum Thunb. ex Murray) for the first time in 1986 by the Chinese researchers in Institute of Materia Medica in Shanghai (Figure 4) [79,80]. We have also analyzed this alkaloid in the Lycopodium species growing naturally in Turkey (L. clavatum L., L. selago L., L. annotinum L., L.alpinum L., L. complanatum ssp. chamaecyparissus A. (Br.) Döll.) by LCMS and GC-MS, however, it was not found in the Turkish counterparts of this genus, whereas we just detected lycopodine in L. clavatum L. as a major component [81,82]. Up to date a series of alkaloids named as huperzine A to P were isolated by different research groups, however, only huperzine A sparkled for its strong AChE inhibitory among them [80]. Apart from its in vitro activity, this compound was shown to display in vivo potent, selective, and reversible

CH3

H2N

CH3

O Figure 4. Structure of huperzine A.

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inhibitory effect on AChE using various experimental models in rats [83-85]. The activity of Huperzin A has been found to be as high as physostigmine, galanthamine, donepezil, and tacrine, the commercial drugs already used against AD, or even greater [86-88]. In another similar study by Cheng and Tang [89], huperzine A was compared to donepezil and tacrine on the radial maze performance in ethylcholine mustard aziridinium ion-treated rats and the results showed that huperzine A had a higher bioavailability and more selective inhibition on AChE activity in rat cortex and hippocampus. In 1996, a tablet from of huperzine A named as “shuangyiping” was developed for AD treatment in China [90]. More recently, huperzine A-loaded microspheres were also developed [91]. The treatment of AD patients with this compound caused a noticeable improvement of the symptoms in a number of clinical trials [92-100]. In another study on hypoperfused rats, huperzine A was found to possess the effect not only in cholinergic system being as highly stereoselective inhibitor of cortical AChE, but also on the oxygen free radical and energy metabolism [101,102]. All these data prompted scientists to develop synthetic derivatives of huperzine A to see if those derivatives had higher efficacy towards AChE. A semi-synthetic derivative of huperzine A coded as “ZT-1” was prepared by Chinese chemists in Institute of Materia Medica in Shanghai (Figure 5) and reported to have similar anti-AChE effect as huperzine A, whereas it exhibited lower anti-BChE effect along with less toxicity in mice as compared to huperzine A [103]. ZT-1 exerts similar properties to those of huperzine A in regard with the blood–brain barrier crossing ability, oral bioavailability, as well as durability of action. Huperzine A and other AChE inhibitors can produce marked effects via inhibition of AChE, delaying hydrolysis of acetylcholine, and enhancement the level of the neurotransmitter called acetylcholine in the synaptic cleft [88]. Besides, three-carbon bridge in the molecule is essential for the anti-AChE activity of the compound [104]. In addition to ZT-1, several other derivatives of CH3 H3CO

CH3

Figure 5. Structure of ZT-1, a semi-synthetic derivative of huperzine A.

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huperzine A were also synthesized. In particular, fluoro-analogs of the compound were quite effective in AChE inhibition in vitro [105,106]. The above-mentioned literature data cover that huperzine A is the mostpromising drug of herbal origin which will become available in the market in a near future, expectedly. II.2. Terpenes from Salvia species Evaluation of ethnopharmacological-based evidence is quite important in discovering new drug candidates. Salvia species (Lamiaceae), referred to “sage” in English, is well-known to enhance memory in European folk medicine [107]. Therefore, a number of studies have been so far performed on various species of this genus considering their anti-AChE potentials. For instance; Perry et al. [108] investigated AChE inhibitory effect of the essential oil obtained from S. lavandulaefolia using human erythrocyte AChE by spectrophotometric Ellman method and analyzed the chemical composition of the essential oil by gas chromatography-mass spectrometry (GC-MS). The oil was found to contain a number of monoterpenes such as (+)-α-pinene, α- and β-terpineol, citronellal, γterpinene, R-(+)-limonene, 1,8-cineole, 1R-(+)-camphor, linalool, 1S-(-)-βpinene, and geraniol. S. officinalis was also analyzed in that study applying the same method. As a result, pure forms of (+)-α-pinene (IC50= 0.63 mM) and 1,8cineole (IC50= 0.67 mM) were independently tested (Figure 6) and concluded that synergistic interactions of these two simple molecules have caused the high antiAChE activity of the two Salvia essential oils mentioned [109]. In addition to the essential oils of the Salvia species, the extracts prepared with different polarity solvents from this plant have been also screened by a number of research groups. In one screening study on traditionally-used Lebanese plants, the ethyl acetate extract of S. triloba had a weak anti-AChE activity whereas its ethanol extract displayed a notable affinity on γ-aminobutiric acid

Figure 6. Structures of α-pinene (left) and 1,8-cineole (right).

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(GABA)-benzodiazepine receptor site [110]. The ethanolic decoction of S. officinalis did not show any remarkable inhibition against AChE at 0.5 and 1.0 mg/ml, while it showed 57.2% inhibition at 5.0 mg/ml [111]. Rauter et al. reported a notable AChE inhibitory effect of S. sclareoides, an endemic plant to Portugal, at 10 Âľg/ml, while it showed potent anti-BChE activity at 1000 Âľg/ml [112]. This active acetone extract, which was shown to be rich in triterpenes, afforded a new lupine-type of triterpene. In our screening study on fourteen Salvia species growing in Turkey (S. albimaculata Hedge and Hub, S. aucheri Bentham var. canescens Boiss and Heldr, S. candidissima Vahl. ssp. occidentalis, S. ceratophylla L., S. cryptantha Montbret and Bentham, S. cyanescens Boiss and Bal., S. frigida Boiss, S. forskahlei L., S. halophila Hedge, S. migrostegia Boiss and Bal., S. multicaulis Vahl., S. sclarea L., S. syriaca L., S. verticillata L. ssp. amasiaca), we tested anticholinesterase activity of the petroleum ether, chloroform, ethyl acetate and methanol extracts of these species using Ellman method on a ELISA microplate reader [113]. Most of the species exerted remarkable inhibitory activity against AChE at 1.0 mg/ml, while they were more effective on BChE. On the other hand, the roots of S. miltiorrhiza, a famous Chinese plant, has been also found to afford some diterpene compounds with AChE inhibitory activities including tanshinone I and IIA, cryptotanshinone, and 15,16dihydrotanshinone (Figure 7) [114]. Tanshinone IIA was shown to exhibit antiamnesic effect in the Morris water maze [115]. In a recent study [116], tanshinone I, tanshinone IIA, cryptotanshinone, and 15, 16-dihydrotanshinone I were tested in mice on learning and memory impairments induced by scopolamine (1 mg/kg, i.p.) using passive avoidance tasks, where tacrine was the reference drug. All of the tested diterpenoids were effective in reversing scopolamine-induced cognitive impairments. Tanshinone I (2 mg/kg, p.o.) and tanshinone IIA (10 or 20 mg/kg, p.o.) were also reversed diazepam-induced cognitive dysfunctions. II.3. Alkaloids from various plant species Alkaloids, the nitrogen-containing compounds, have been generally observed to have a strong inhibition of AChE and BChE. Among the different classes of alkaloids, isoquinoline alkaloids have been reported to possess anti-AChE activity. The most famous anti-AChE example of this class is galanthamine as mentioned in above parts. In our preliminary screening study [117], we had screened a number of Turkish plants for their anticholinesterase activity. Among them, ten Fumaria species of Fumarioideae subfamily of Papaveraceae, which are F. asepala Boiss., F. capreolata L., F. cilicica Hausskn., F. densiflora DC., F. judaica Boiss.,

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CH3

H3C

CH3

Tanshinone I O

Tanshinone IIA O

CH3

Cryptotanshinone

CH3

H3C

CH3

15,16-Dihydrotanshinone

Figure 7. Structures of diterpenes from S. miltiorrhiza.

F. kralikii Jordan, F. macrocarpa Parlatore, F. parviflora Lam., F. petteri subsp. thuretii (Boiss.) Pugsley and F. vaillantii Lois., by Ellman method. Furthermore, F. vaillantii Lois. extract having 94.23% inhibition was applied to bioactivitydirected fractionation in order to isolate the constituents responsible for the activity [118]. Through the active fractions of the extract, 10 isoquinoline alkaloids were isolated by preparative thin layer chromatography (TLC). Among the alkaloids, ophiocarpine (IC50= 1.1 µM) had the most potent inhibitory activity followed by β-allocryptopine (IC50= 1.3 µM), berberine (IC50= 1.6 µM), ophiocarpine-N-oxide (IC50= 1.79 µM) and protopine (IC50= 1.8 µM) (Figure 8). The methanolic extract of the tubers of Corydalis ternata from Papaveraceae family, displayed a potent AChE inhibitory activity by Ellman method, which led to isolation of protopine as the active ingredient, as well [119]. Moreover, the

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O N

O OCH3

CH3

OCH3 OCH3

OCH3 Î˛-Allocryptopine

Ophiocarpine

O O

O OCH3

CH3

O O O

OCH3 Berberine

Protopine

Figure 8. Structures of the isoquinoline alkaloids from F. vaillantii.

same plant showed a remarkable scopolamine-induced anti-amnesic effect in passive avoidance test. In a very recent report, the methanolic extract prepared from the tubers of C. cava also tested for its anti-AChE activity and activity-guided fractionation of the extract led to isolation of three isoquinoline alkaloids; bulbocapnine, corydaline, and corydine as the active constituents [120]. Besides, several synthetic derivatives of bisbenzylisoquinoline alkaloid derivatives showed the inhibitory activity towards AChE in micromolar range [121]. On the other hand, 8-hydroxydihydrochelerythrine and 8hydroxydihydrosanguinarine, the isoquinoline derivatives isolated from the ethanolic extract of the aerial parts of Chelidonium majus L. of Papaveraceae, were reported to possess a potent AChE inhibitory activity [122]. In a screening study of a number of Korean plants, the methanolic extract of Evodia rutaecarpa (Rutaceae) were found to have AChE inhibitory activity by Ellman method, while it also exerted anti-amnesic effect in vivo. Bioactivity-guided fractionation of this extract afforded HCl salt of dehydroevodiamine, an indole-derivative alkaloid (Figure 9) [123,124].

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O N N H

N H3C Cl

Figure 9. Structure of dehydroevodiamine HCl. HO

H3C

N H H

HO N H3C

N H

Figure 10. Structure of juliflorine.

Another alkaloid, juliflorine isolated from Prosopis juliflora (Fabaceae), was found to inhibit both AChE and BChE in concentration-dependent manner (Figure 10) [125]. Detailed molecular docking studies were also performed on this alkaloid and this paper concluded that it is a quite promising drug candidate for AD depending on its anticholinesterase activity as well as calcium-channel blocking activity. Another important plant genus for its alkaloid content is Buxus sp. of Buxaceae. One study on B. hyrcana underlined the existence of two triterpenetype alkaloids, (+)-homomoenjodaramine (IC50=19.2 µM) along with (+)moenjodaramine (IC50=50.8 µM), which was shown to inhibit AChE potently (Figure 11) [126]. In addition, B. papillosa was also investigated in the same manner and buxakashmiramine (IC50= 25.4 µM), cycloprotobuxine C (IC50= 38.8 µM), and cyclovirobuxein A (IC50= 105.7 µM) were revealed to be the most potent inhibitors of AChE [127]. On the other hand, buxamine B and C were also tested in vitro for their AChE inhibitory activities and molecular docking studies were carried out. [128]. The results pointed out that buxamine C was 20-fold more potent than buxamine B.

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H H3C

H O

(A) R1= CH3, R2= H3C-CH-Nb(CH3)2 (B) R1= H, R2= H3C-CH-Nb(CH3)2 Figure 11. Structures of (+)-homomoenjodaramine (A) and (+)-moenjodaramine (B). H N 21

1 9

N H

H 30

Figure 12. Structure of (+)-buxabenzamidienine.

In our recent study, we have also examined the anticholinesterase activity of the aerial parts and roots of B. sempervirens growing in Turkey (unpublished data). Activity-guided fractionation of the alkaloid extracts of the both aerial parts and roots showed the highest inhibition and alkaloidal components appeared to be the compounds responsible for the activity. Finally, (+)-buxabenzamidienine was elucidated as the main alkaloid contributing to the high anti-AChE activity of the plant (Figure 12).

Conclusion Recognition of the medical and economic benefits of herbal medicinal products with health claims is growing worldwide. AD is one of the most common forms of dementia affecting elder population in particular.

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Several hypotheses about the pathogenesis of AD have been suggested up-todate. One is called “cholinergic hypothesis”, which has led to launch of AChE inhibitors. This class of drugs attempts to increase the brain’s levels of acetylcholine, an important neurotransmitter whose levels decrease with onset of disease. On the other hand, EGb 761, the standardized extract of G. biloba, was found to be less effective in the treatment of AD than cholinesterase inhibitors [12,129]. However, the side effects of cholinesterase inhibitors such as nausea and vomiting are not produced by G. biloba, which also represents a relatively cheaper cost of AD treatment. Currently, there are a number of AChE inhibitors available in the market. However, they can treat only mild to moderate levels of disease. Recently, more compounds of synthetic origins have launched in the market as well for AD treatment. One of them is memantine, sold as under the name of Ebixa®, is an effective drug, which is a N-methyl-di-aspartate (NMDA) receptor antagonist. Novel β- and γ-secretase inhibitors are also on the horizon against AD. Although AChE inhibitors and G. biloba seem to be the main choices for AD so far, new mechanisms are emerging day by day since the pathogenesis of the disease has not been fully understood, yet. There is still a need to develop more efficient drugs for AD. We hope that all these research in finding new drug molecules for AD may still continue to focus on plants as it has been and the compounds of herbal origin such as galanthamine and huperzine A as the model compounds may lead to discovery of new derivatives.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Francis, P.T., Palmer, A.M., Snape, M., and Wilcock, G.K. 1999, J. Neurol. Neurosurg. Psychiatry, 66, 137. Wernicke, T.F., and Reischies, F.M. 1994, Neurology, 44, 250. Williams, B.R., Nazarians, A., and Gill, M.A. 2003, Clin. Therap., 25, 1634. Howes, M.J.R., and Houghton, P. 2003, Pharmacol. Biochem. Behav., 75, 513. Weinstock, M., Gorodetsky, E., Poltyrev, T., Gross, A., Sagi, Y., and Youdim, M. 2003, Prog. Neuro-Psychopharmacol. Biol. Psych., 27, 555. Honig, L.S., and Mayeux, R. 2001, Aging (Milano), 13, 171. Behl, C., and Mosmann, B. 2002, Free Rad. Biol. Med., 33, 182. Bilia, A.R. 2002, Fitoterapia, 73, 276. De Jager, L.S., Perfetti, G.A., and Diachenko, G.W. 2006, J. Pharm. Biomed. Anal., 41, 1522. Nakanishi, K. 2005, Bioorg. Med. Chem., 13, 4987. Schmidt, W. 1997, Nature, 386, 755. Schulz, V. 2003, Phytomed., 10, 74. Kenner, D., and Requena, Y. 1996, Botanical medicine: a European professional perspective. Paradigm Publications, Brookline. Ross, I.A. 2001, Medicinal plants of the world. Chemical constituents, traditional and modern medicinal uses, Vol. 2, Humana Press, Clifton (NJ).

262

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

Ilkay Orhan et al.

Markesbery, W.R. 1999, Arch. Neurol., 56, 1449. Beal, M.F. 1995, Ann. Neurol., 38, 357. Christen, Y. 2000, Am. J. Clin. Nutr., 71(Suppl.), 621S. Kleijnen, J., and Knipschild, P. 1992, Lancet, 340, 1136. Behl, C., Davis, J.B., Lesley, R., and Schubert, D. 1994, Cell, 77, 817. Dorman, D., Cote, L., and Buck, W. 1992, Am. J. Vet. Res., 53, 138. Braquet, P. 1987, Drug Future, 12, 643. Lai, S.M., Chen, I.W., and Tsai, M.J. 2005, J. Chrom. A, 1092, 125. Tang, Y., Lou, F., Wang, J., Li, Y. and Zhuang, S. 2001, Phytochemistry, 58, 1251. Fuzzati, N., Pace, R., and Villa, F. 2003, Fitoterapia, 74, 247. Zhu, W., Chen, G., Hu, L., Luo, X., Gui, C., Luo, C., Puah, C.M., Chen, K., and Jiang, H. 2005, Bioorg. Med. Chem., 13, 313. Luo, Y., Smith, J.V., and Paramasivam, V. 2002, Proc. Natl. Acad. Sci. USA, 99, 12197. He, J., Petrovic, A.G., Dzyuba, S.V., Berova, N., Nakanishi, K., and Polavarapu, P.L. 2007, Spectrochim. Acta Part A (in press). Colciaghia, F., Borronib, B., and Zimmermanna, M. 2004, Neurobiol. Dis., 16, 454. Vitolo, O., Gong, B., Cao, Z., Ishii, H., Jaracz, S., Nakanishi, K., Arancio, O., Dzyuba, S.V., Lefort, R., and Shelanski, M. 2009, Neurobiol. Aging, 30, 257. Zhu, L., Wu, X.M., Yang, L., Du, F., and Qian, Z.M. 2007, Brain Res., 1166, 1. Duche, J.C., Barre, J., Guinot, P., Duchier, J., Cournot, A., and Tillement, J.P. 1989, Int. J. Clin. Pharmacol. Res., 9, 165. Izzo, A.A., Di Carlo, G., Borrelli, F., and Ernst, E. 2005, Int. J. Cardiol., 98, 1. Oken, B.S., Storzbach, D.M., and Kaye, J.A. 1998, Arch. Neurol., 55, 1409. Le Bars, P.L., Katz, M.M., Berman, N., Itil, T.M., Freedman, A.M., and Schatzberg, A.F. 1997, JAMA, 278, 1327. Kanowski, S., Herrmann, W.M., Stephan, K., Wierich, W., and Horr, R. 1996, Pharmacopsychiatry, 29, 47. Kleijnen, J., and Knipschild, P. 1992, Br. J. Clin. Pharmacol., 34, 352. Rai, G.S., Shovlin, C., and Wesnes, K.A. 1991, Curr. Med. Res. Opin., 12, 350. Hofferberth, B. 1989, Arzneimittelforsch., 39, 918. Fitzpatrick, A.L., Fried, L.P., Williamson, J., Crowley, P., Posey, D., Kwong, L., Bonk, J., Moyer, R., Chabot, J., Kidoguchi, L., Furberg, C.D., and De Kosky, S.T. 2006, Cont. Clin. Trials, 27, 541. Lang, Q., Yak, H.K., and Wai, C.M. 2001, Talanta, 54, 673. van Beek, T.A. 2002, J. Chrom. A, 967, 21. Li, W., and Fitzloff, J.F. 2002, J. Pharm. Biomed. Anal., 30, 67. Li, W., and Fitzloff, J.F., Farnsworth, N.R., and Fong, H.H.S. 2002, Phytomed., 9, 442. He, J., and Xie, B. 2002, J. Chrom. A, 943, 303. Tang, C., Wei, X., and Yin, C. 2003, J. Pharm. Biomed. Anal., 33, 811. Deng, F., and Zito, S.W. 2003, J. Chrom. A, 986, 121. van Nederkassel, A.M., Vijverman, V., Massart, D.L., and van der Heyden, Y. 2005, J. Chrom. A, 1085, 230. Mesbah, M.K., Khalifa, S.I., El-Gindy, A., and Tawfik, K.A. 2005, Il Farmaco, 60, 583.

Herbal treatment of Alzheimer's disease

263

49. Ji, Y.B., Xu, Q.S., Hu, Y.Z., and van der Heyden, Y. 2005, J. Chrom. A, 1066, 97. 50. Dubber, M.J., and Kanfer, I. 2006, J. Pharm. Biomed. Anal., 41, 135. 51. Bhattaram, V.A., Graefe, U., Kohlert, C., Veit, M., and Derendorf, H. 2002, Phytomed., 9 (Suppl. III), 1. 52. Kimbel, K.H. 1992, Lancet, 340, 1474. 53. Fourtillan, J.B., Brisson, A.M., Girault, J., Ingrand, I., Decourt, J.P., Drieu, K., Jouenne, P., and Biber, A. 1995, Therapie, 50, 137. 54. Heinrich, M., and Teoh, H.L. 2004, J. Ethnopharmacol., 92, 147. 55. Orhan, I., and Şener, B. 2003, Chem. Nat. Compds., 39, 383. 56. Berkov, S., Sidjimova, B., Evstatieva, L., and Popov, S. 2004, Phytochemistry, 65, 579. 57. Kaya, G.I., and Gözler, B. 2005, Fitoterapia, 76, 340. 58. Lopez, S., Bastida, J., Viladomat, F., and Codina, C. 2002, Life Sci., 71, 2521. 59. Czollner, L., Frantsits, W., Küenburg, B., Hedenig, U., Fröhlich, J., and Jordis, U. 1998, Tetrahedron Lett., 39, 2087. 60. Eichhorn, J., Takada, T., Kita, Y., and Zenk, M.H. 1998, Phytochemistry, 49, 1037. 61. Kone, S., Galland, N., Graton, J., Illien, B., Laurence, C., Guillou, C., and Le Questel, J.Y. 2006, Chem. Phys., 328, 307. 62. Bores, G.M., F.P. Huger, and Petko, W. 1996, J. Pharmacol. Exp. Ther., 277, 728. 63. Levin, E.D., and Simon, B.B. 1998, Psychopharmacol. (Berlin), 138, 217. 64. Tariot, P.N., P.R. Solomon, J.C. Morris, P. Kershaw, S. Lilienfeld, and Ding, C. 2000, Neurology, 54, 2269. 65. Giacobini, E. 1998, Neurochem. Int., 32, 413. 66. Raskind MA, Peskind ER, Wessel T, Yuan W, and the Galantamine USA-1 Study Group. 2000, Neurology, 54, 2261. 67. Guillou C, Mary A, Renko DZ, Gras E, and Thal C. 2000, Bioorg. Med. Chem. Lett., 10, 637. 68. Pang, Y.P., Quiram, P., Jelacic, T., Hong, F., and Brimijoin, S. 1996, J. Biol. Chem., 271, 23646. 69. Carlier, P.R., Du, D.M., Han, Y., Liu, J., and Pang, Y.P. 1999, Bioorg. Med. Chem. Lett., 9, 2335. 70. Guillou, C., Mary, A., Renko, D.Z., Gras, E., and Thal, C. 2000, Bioorg. Med. Chem. Lett., 10, 637. 71. Mary, A., Renko, D.Z., Guillou, C., and Thal, C. 1998, Bioorg. Med. Chem., 6, 1835. 72. Lamirault, L., Guillou, C., Thal, C., and Simon, H. 2003, Neurobiol. Learn. Mem., 80, 113. 73. Thomsen, T., and Kewitz, H. 1990, Life Sci., 46, 1553. 74. Thomsen, T., Kaden, B., Fischer, J.P., Bickel, U., Barz, H., Gusztoni, G., CervosNavarro, J., and Kewitz, H. 1991, Eur. J. Clin. Chem. Clin. Biochem., 29, 487. 75. Greenblatt, H.M., Kryger, G., Lewis, T., Silman, I., and Sussman, J.L. 1999, FEBS Lett., 463, 321. 76. MacLean, D.B. 1985. Lycopodium Alkaloids in “The Alkaloids” (Brossi, A., Ed.), Vol. 26, pp. 241-296, Academic Press, New York.

264

Ilkay Orhan et al.

77. Ayer, W.A. 1991, Nat. Prod. Rep., 8, 455. 78. Ma, X., Tan, C., Zhu, D., and Gang, D.R. 2006, J. Ethnopharmacol., 104, 54. 79. Liu, J.S., Yu, C.M., Zhou, Y.Z., Han, Y.Y., Wu, F.W., Qi, B.F., and Zhu, Y.L. 1986, Acta Chim. Sin., 44, 1035. 80. Liu, J.S., Zhu, Y.L., Yu, C.M., Zhou, Y.M., Han, Y.Y., Wu, F.W., and Qi, B.F. 1986, Can. J. Chem., 64, 837. 81. Orhan, I., Eroglu, Y., Terzioglu, S., and Şener, B. 2002, Proceedings of XIVth Meeting on Vegetable Crude Drugs, May 29-31, Eskişehir, Turkey. 82. Orhan, I., Küpeli, E., Şener, B., and Yeşilada, E. 2007, J. Ethnopharmacol., 109, 146. 83. Tang, X.C., Han, Y.F., Chen, X.P., and Zhu, X.D. 1986, Zhong. Yao Li Xue Bao, 7, 507. 84. Tang, X.C., De Sarno, P., Sugayo, K., and Giacobini, E. 1989, J. Neurosci. Res., 24, 276. 85. Cheng, D.H., Ren, H., and Tang, X.C. 1996, Neurorep., 8, 97. 86. Wang, Y.E., Yue, D.X., and Tang, X.C. 1986, Zhong. Yao Li Xue Bao, 7, 110. 87. Wang, H., and Tang, X.C. 1998, Zhong. Yao Li Xue Bao, 19, 27. 88. Ma, X., Tan, C., Zhu, D., Gang, D.R., and Xiao, P. 2007, J. Ethnopharmacol., 113, 15. 89. Cheng, D.H., and Tang, X.C. 1998, Pharmacol. Biochem. Behav., 60, 377. 90. Liu, W.H., Song, J.L., Liu, K., Chu, D.F., and Li, Y.X. 2005, J. Control. Rel., 107, 417. 91. Tang, X.C. 1996, Zhong. Yao Li Xue Bao, 17, 481. 92. Xu, S.S., Z.X. Gao, and Z. Weng. 1995, Acta Pharmacol. Sin., 16, 391. 93. Mazurek, A. 1999, Alternative Ther., 5, 97. 94. Xu, S.S., Cai, Z.Y., Qu, Z.W., Yang, R.M., Cai, Y.L., Wang, G.Q., Su, X.Q., Zhong, X.S., Cheng, R.Y., Xu,W.A., Li, J.X., and Feng, B. 1999, Zhong. Yao Li Xue Bao, 20, 486. 95. Chiu, H.F., and Zhang, M. 2000, Int. J. Ger. Psychiatry, 15, 947. 96. Zhang, Z., Wang, X., Chen, Q., Shu, L., Wang, J., and Shan, G. 2002, Zhong. Yi Xue Za Zhi, 82, 941. 97. Jiang, H., Luo, X., and Bai, D. 2003, Curr. Med. Chem., 10, 2231. 98. Kuang, M.Z., Xiao, W.M., Wang, S.F., and Li, R.X. 2004, Chin. J. Clin. Rehabilit., 8, 1216. 99. Wang, R.P., Zhao, Z.K., and Hu, L.L. 2004, Chin. J. Clin. Rehabilit., 8, 3892. 100. Zhu, X.Z., Li, X.Y., and Liu, J. 2004, Eur. J. Pharmacol., 500, 221. 101. Wang, L.M., Y.F. Han, and Tang, X.C. 2000, Eur. J. Pharmacol., 398, 65. 102. Zhang, Y.H., Chen, X.Q., Yang, H.H., Jin, G.Y., Bai, D.L., and Hu, G.Y. 2000, Neurosci. Lett., 295, 116. 103. Ma, X., Gang, D.R. 2004, Nat. Prod. Rep., 21, 752. 104. Kozikowski, A.P., Miller, C.P., Yamada, F., Pang, Y.P., Miller, J.H., McKinney, M., and Ball, R.G. 1991, J. Med. Chem., 34, 3399. 105. Kaneko, S., Nakajima, N., Shikano, M., Katoh, T., and Terashima, S. 1998, Tetrahedron, 54, 5485. 106. Zeng, F., Jiang, H., Tang, C., Chen, K., and Ji, R. 1998, Bioorg. Med. Chem. Lett., 8, 1661. 107. Perry, N.S.L., Court, G., Bidet, N., Court, J., and Perry, E.K. 1996, J. Ger. Psychiatry, 11, 1063.

Herbal treatment of Alzheimer's disease

265

108. Perry, N.S.L., Houghton, P.J., Theobald, A., Jenner, P., and Perry, E.K. 2000, J. Pharm. Pharmacol., 52, 895. 109. Savelev, S., Okello, O., Perry, N.S.L., Wilkins, R.M., and Perry, E.K. 2003, Pharmacol. Biochem. Behav., 75, 661. 110. Salah, S.M., and Jäger, A.K. 2005, J. Ethnopharmacol., 97, 145. 111. Ferreira, A., Proença, C., Serralheiro, M.L.M., and Araujo, M.E.M. 2006, J. Ethnopharmacol., 108, 31. 112. Rauter, A.P., Branco, I., Lopes, R.G., Justino, J., Silva, F.V.M., Noronha, J.P., Cabrita, E.J., Brouard, I., and Bermejo, J. 2007, Fitoterapia, 78, 474. 113. Orhan, I., Kartal, M., Naz, Q., Ejaz, A., Yilmaz, G., Kan, Y., Konuklugil, B., Şener, B., and Choudhary, M.I. 2007, Food Chem., 103, 1247. 114. Ren, Y., Houghton, P.J., Hider, R.C., and Howes, M.J. 2004, Planta Med., 63, 44. 115. Liu, X.J., and Wu, W.T. 1999, Acta Pharmacol. Sin., 20, 987. 116. Kim, D.H., Jeon, S.J., Jung, J.W., Lee, S., Yoon, B.H., Shin, B.Y., Son, K.H., Cheong, J.H., Kim, Y.S., Kang, S.S., Ko, K.H., and Ryu, J.H. 2007, Eur. J. Pharmacol., 574, 140. 117. Orhan, I., Şener, B., Choudhary, M.I., and Khalid, A. 2004, J. Ethnopharmacol., 91, 57. 118. Şener, B., and Orhan, I. 2004, J. Chem. Soc. Pakistan, 26, 313. 119. Kim, S.R., Hwang, S.Y., Jang, Y.P., Park, M.J., Markelonis, G.J., Oh, T.H., and Kim, Y.C. 1999, Planta Med., 65, 218. 120. Adsersen, A., Kjolbye, A., Dall, O., and Jäger, A.K. 2007, J. Ethnopharmacol., 113, 179. 121. Markmee, S., Ruchirawat, S., Prachyawarakorn, V., Ingkaninan, K., and Khorana, N. 2006, Bioorg. Med. Chem. Lett., 16, 2170. 122. Cho, K.M., Yoo, I.D., and Kim, W.G. 2006, Biol. Pharm. Bull., 29, 2317. 123. Park, C.H., Lee, Y.J., Lee, S.H., Choi, S.H., Kim, H.S., Jeong, S.J., Kim, S.S., and Suh, Y.H. 2000, J. Neurochem., 74, 244. 124. Wang, H.H., Chou, C.J., Liao, J.F., and Chen, C.F. 2001, Eur. J. Pharmacol., 413, 221. 125. Choudhary, M.I., Nawaz, S.A., Zaheer-ul-Haq, Azim, M.K., Ghayur, M.N., Lodhi, M.A., Jalil, S., Khalid, A., Ahmed, A., Rode, B.M., Atta-ur-Rahman, Gilani, A.H., and Ahmad, V.U. 2005, Biochem. Biophys. Res. Commun., 332, 1171. 126. Atta-ur-Rahman, Parveen, S., Khalid, A., Farooq, A., Ayattollahi, S.A.M., and Choudhary, M.I. 1998, Heterocycles, 49, 481. 127. Atta-ur-Rahman, Parveen, S., Khalid, A., Farooq, A., and Choudhary, M.I. 2001, Phytochemistry, 58, 963. 128. Khalid, A., Azim, M.K., Parveen, A., Atta-ur-Rahman, and Choudhary, M.I. 2005, Biochem. Biophys. Res. Commun., 331, 1528. 129. Hoerr, R. 2005, Phytomed., 12, 598.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 267-293 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

10. Ethnomedicinal plants as antiinflammatory and analgesic agents M. Anilkumar Department of Botany, Union Christian College, Aluva, Ernakulam- 683 102, Kerala, India

Abstract. The use of traditional medicine is expanding to newer horizons and plants still remain as the novel source of structurally important compounds that lead to the development of innovative drugs. India has about 45,000 plant species among which medicinal property has been attributed to several thousands. The traditional Indian system of medicine, the Ayurveda, mentions the use of plants in the treatment of various diseased conditions. Ethnobotanical research done in last few decades have revealed the anti-inflammatory and analgesic properties of plants cited in the traditional literature. Many herbal preparations are being prescribed as anti-inflammatory and analgesic in the traditional literature. The search for new anti-inflammatory and analgesic agents from the huge array of medicinal plant resources is intensifying. This is because such taxa may hold assurance for the discovery of novel therapeutic agents capable of suppressing, reducing or relieving pain as well as inflammation. This chapter reviews such plant species and their products that have shown experimental or clinical anti-inflammatory or analgesic activities, Correspondence/Reprint request: Dr. M. Anilkumar, Department of Botany, Union Christian College, Aluva Ernakulam- 683 102, Kerala, India. E- mail: anilphd@hotmail.com

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the possible mechanism of action and their therapeutic value. Some of the important taxa which are found effective as anti-inflammatory and analgesic agents are Ananas comosus (L.) Merr. Bosewellia serrata Roxb., Callophyllum inophyllum L., Calotropis gigantea (L.) R.Br., Calotropis procera (Ak.) R.Br., Camellia sinensis (L.) Kuntz., Cannabis sativa L., Centella asiatica (L.) Urban, Curcuma longa L., Euphorbia heterophylla L., Gastrodia elata Blume, Harpagophytum procumbens (Burch.) DC, Kalanchoe crenata Andr., Mangifera indica L., Mesua ferrea L., Plumeria accuminata W.T. Aiton., Ricinus communis Linn., Salix alba L., Sida cordifolia L., Sylibium marianum L., Spillanthes acmella Murr, Tripterygium wilfordii Hook f., Uncaria tomentosa (Willd.) DC, U. guianensis, J.F.Gmel and Zingiber officinale Roscoe. These plants have shown varying degrees of anti-inflammatory and analgesic activities.

Introduction India harbours about 15% out of the 20,000 medicinal plants of the world, of which 90% of them are found growing wild in different climatic conditions [1]. The tribal and rural populations of India largely depend on medicinal plants for their health care as well as for their livestock. This attracted the attention of several botanists that lead to an array of reports on ethnomedicine [2]. Medicinal plants are the main sources of chemical substances with potential therapeutic effects. The use of medicinal plants for the treatment of many diseases is associated with folk medicine from different parts of the world. Naturally occurring compounds from plants, fungi and microbes are still used in pharmaceutical preparations in pure or extracted forms. A lot of compounds were characterized from plants. The research into plants with alleged folkloric use as pain relievers and antiinflammatory agents is definitely a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs. The term inflammation is derived from the Latin word â&#x20AC;&#x201C; Inflammare, means burn. Any form of injury to the human body can elicit a series of chemical changes in the injured area. Earlier it was believed that inflammation was contemplated as a single disease caused by disturbances of body fluids. According to the modern concept, inflammation is a healthy process resulting from some disturbance or disease. The cardinal signs of inflammation are heat, redness, swelling, pain and loss of function. Inflammation usually involves a sequence of events which can be categorized under three phases viz. acute transient phase, delayed sub acute phase and chronic proliferate phase. In the first phase, inflammatory exudates develop due to enhanced vascular permeability and leads to local edema. It is followed by the migration of leukocytes and phagocytes from blood to vascular tissues which is the second phase, In the third phase, tissue degradation is followed by fibrosis.

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Inflammation results in the liberation of endogenous mediators like histamine, serotonin, bradykinin, prostaglandins etc. Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. These mediators even in small quantities can elicit pain response. Pain results in dropped muscular activities. In order to comprehend the inflammatory process, antagonists of mediators are generally employed in both Ayurveda and Allopathy treatment. Most of the antiinflammatory drugs now available are potential inhibitors of cyclooxygenase (COX) pathway of arachidonic acid metabolism which produces prostaglandins. Prostaglandins are hyperalgesic, potent vasodialators and also contribute to erythema, edema and pain. Hence for treating inflammatory diseases analgesic and anti-inflammatory agents are required. These points to the utilization of plants possessing anti-inflammatory and analgesic properties. Now a days herbal drugs are routinely used for curing diseases rather than chemically derived drugs having side effects. The drugs used in inflammatory disorders may be either with analgesic and insignificant antiinflammatory effects or with analgesic and mild to moderate antiinflammatory activity. These drugs can cause gastric or intestinal ulceration that can sometimes cause secondary anaemia. Inflammatory diseases include different types of rheumatic disorders such as rheumatic fever, rheumatoid arthritis, ankylosing spondylitis, polyarthritis nodosa, systemic lupus erythematosus and osteoarthritis. An array of drugs are available in the market to treat these disorders but only very few are free from toxicity. Gastrointestinal problems associated with the use of anti-inflammatory drugs are still an enduring dilemma of medical world. It is very important that profound research with ethnobotanical plants possessing anti-inflammatory and analgesic properties can definitely open up new vistas in inflammatory disorders. Purified natural compounds from plants can serve as template for the synthesis of new generation antiinflammatory drugs with low toxicity and higher therapeutic value. This chapter reviews such medicinal plants with anti-inflammatory and analgesic properties which have been used by our ancestors to cure many of their ailments.

Plants with anti-inflammatory and analgesic activity 1. Ananas comosus ( L.) Merr. (Bromeliaceae) Ananas comosus (L.) Merril (Pineapple) has been used as a medicinal plant in several native cultures and its major active principle, Bromelain, has been known chemically since 1876. Bromelain is a general name for a family of sulphydryl proteolytic compounds obtained from Ananas comosus L. It is

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usually distinguished as either fruit bromelain or stem bromelain depending on its source. The primary component of bromelain is a sulphydryl proteolytic fraction. It also contains peroxidase, acid phosphatase, several protease inhibitors and originally bound calcium. Eight basic proteolytically active components have been detected in the stem [3]. The two main components have been designated as F4 and F5. The proteinase, considered to be the most active fraction, has been designated as F9. It comprises about 2% of the total proteins. It is estimated that 50% of the proteins in F4 and F5 are glycosylated, whereas F9 was found to be unglycosylated. Since bromelain is derived from a natural source, different sources can exhibit variability in their physiological activity, even when their proteolytic activity is the same. Bromelain seems to have both direct as well as indirect actions involving other enzyme systems exerting its anti-inflammatory effect. It inhibits the inflammatory pain in rats in a dose dependent manner [4]. It reduces pain and inflammation associated with surgery, arthritis, trauma or sports injury [5]. Bromelain was the most potent of nine anti-inflammatory substances tested on experimental rats [6]. Treatment with bromelain have been shown to decrease significantly the heat evoked immunoreactive substance released and subsequent edema in a rat model [7]. Bromelain is used as a digestive aid as it helps to heal and regenerate mucus lining of the stomach [8]. Bromelain interferes with the arachidonic acid cascade there by preventing the formation of pro-inflammatory eicosanoids [9]. Non-steroidal anti-inflammatory drugs inhibit COX, which is required for the synthesis of two prostaglandins, resulting in a decrease in both pro and anti-inflammatory prostaglandins. Rather than blocking the arachidonic acid cascade at the enzyme COX, bromelain may selectively decrease thromboxane generation and change the ratio of thromboxane/prostacyclin in favour of prostacyclin [3]. Bromelain has been shown to inhibit prostaglandins even though its action is significantly weaker [10]. The anti edema, anti-inflammatory and coagulation inhibiting effects are due to an enhancement of the serum fibrinolytic and fibrinogenolytic activity. It blocks synthesis and lowers serum and tissue levels of kinin compounds [11]. Bromelain has been shown to reduce edema, accelerate healing and lowers pain and inflammation after surgery in clinical trials [12]. A randomized, double blind, parallel group trial compared the efficiency and safety of an oral combination of bromelain with diclofenac in patients with osteoarthritis of the knee. From this study, Akhtar concluded that bromelain can be considered as an effective and safer alternative to nonsteroidal anti-inflammatory drugs in the treatment of painful episodes of osteoarthritis [13].

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2. Boswellia serrata Roxb. (Burseraceae) Boswellia serrata Roxb has been used traditionally in Indian Ayurvedic medicine and is well known for its anti-inflammatory activity. The resinous gum of the bark is known as guggulu in Ayurveda and is also used in modern phytomedicine. It has been reported to be a powerful anti-inflammatory agent without the ulceration or irritation as observed in non-steroidal anti-inflammatory drugs [14]. Bosewellia has been shown to possess sedative, analgesic [15,16], anti-inflammatory [17,18] and anticancer [19,20,21] effects. The resin obtained from the plant is recommended for rheumatoid arthritis, osteoarthritis, fibromyositis and spondylitis. Patients treated with Bosewellia reported decreases in knee pain, joint swelling and increases in knee flexion and walking distance [22,23]. Four pentacyclic triterpene acids including the bioactive compound β-boswellic acid which interferes with leukotriene biosynthesis have been isolated from B. serrata. It is a specific and dose dependent inhibitor of 5lipoxygenase, 5-eicosatetraenoic acid and leukotriene B4 [24]. These chemical mediators of inflammation are implicated in the pathogenesis of many diseases including asthma [25], arthritis [26], colitis [27] and cancer [28]. Bosewellia inhibits human leukocyte elastase (HLE) under in vitro conditions [29]. HLE inhibitor medications have been developed for the treatment of asthma, emphysema and cystic fibrosis [30]. Bosewellic acids were found to be more potent inhibitors of human topoisomerases-I and II-α than chemotherapeutic agents that work largely by inhibition of these enzymes[31]. The mechanism of action of beta boswellic acid has been recently reported [32]. B. serrata extract can decrease the glycosaminoglycan degradation which keeps the cartilages in better condition thus preventing the progression of osteoarthritis [33]. The safety and efficacy of B. serrata extract in relieving osteoarthritis has been reported recently [16,34]. 3. Callophyllum inophyllum L. and Mesua ferrea L. (Clusiaceae) Callophyllum inophyllum L. and Mesua ferrea L. has been commonly used for the treatment of rheumatism, skin diseases, dysentery and bleeding piles [35]. The whole plant is medicinal and contains compounds such as xanthones, triterpenes, coumarins and glucosides. The anti-inflammatory effect of C. inophyllum was reported earlier [36]. Gopalakrishnan et al. reported the anti-inflammatory and Central Nervous System (CNS) depressant activities of xanthones from C. inophyllum and M. ferrea [37]. They have isolated different xanthones such as dehydrocycloguanandine, callophyllin–B, 6-deoxyjacareubin etc. All xanthones isolated produced signs of CNS depression characterized by ptosis, sedation, decreased spontaneous

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motor activity and loss of muscle tone. The CNS depressant effect was predominant at a dose level of 200 mg/kg. Similar findings were reported on the pharmacological activity of the xanthones from Garcinia mangostana [38,39]. The xanthones of Callophyllum and Mesua have been found to produce significant anti-inflammatory activity in normal as well as adrenalectomised rats by both intra-peritoneal and oral routes. Usually the anti-inflammatory agents in clinical use exhibit analgesic and antipyretic properties along with ulcerogenicity and impairment of blood clotting as side effects. But the xanthones of C. inophyllum and M. ferrea did not possess any such properties and thus points to the possibility of developing anti-inflammatory drugs of future use. 4. Calotropis gigantea (L.) R. Br. (Asclepiadaceae) Calotropis gigantea (L.) R. Br. is an important medicinal plant where all parts of the plant including the milky secretion have been claimed to possess varied medicinal uses [35,40,41,42]. It has been claimed to be useful in treating skin diseases and healing of wounds and ulcers [43,44]. Very recently, the plant is reported to possess analgesic and antipyretic activities [45]. The methanolic extract of Calotropis gigantea leaves revealed the anti-inflammatory activity in experimental rats using paw edema test [46]. Anti-inflammatory effects of aqueous extract of leaves and latex of C. procera were reported earlier [47,48]. 5. Calotropis procera (Ak.) R.Br. (Asclepiadaceae) Calotropis procera (Ait) R Br. is a well known medicinal plant in the traditional medicine system of India. It is used in the treatment of skin diseases, rheumatism and aches [49]. It has been reported to possess antiinflammatory, analgesic and weak antipyretic activities [50,51,52]. The latex was reported to be as potent as standard anti-inflammatory drug phenylbutazone in inhibiting inflammatory response induced by different inflammatory agents in acute and chronic models [50]. The anti-inflammatory activity of the latex of C. procera and its methanolic extract against various inflammatory mediators as well as on leucocyte flux induced by carrageenan in rat paw edema model have been reported recently [53]. 6. Camellia sinensis (L.) Kuntze (Theaceae) Camellia sinensis (L.) Kuntze is one of the most commonly consumed beverages in the world. The established pharmacological activity of the green tea extracts are attributed to its high content of polyphenols/catechins, mainly epigallocatechin-3-gallate (EGCG) [54]. The potential effect of green tea in

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arthritis on collagen type-II-induced arthritis in mice has been reported [55]. The anti-inflammatory effect of green tea polyphenols was reflected in a marked inhibition of the inflammatory mediators such as COX 2, interferon–γ and TNF-α in arthritic joints. Histopathological studies revealed a reduction in biochemical markers correlated with the marked reduction in inflammation in synovium. Studies have shown that most of the effects of green tea extracts are mimicked by its constituent polyphenol, EGCG [54,56]. Further studies have shown that EGCG inhibited the transcription factor, nuclear factor-kappa-B (NF-κ B) in conjunction with pro-inflammatory cytokines IL-1β-inducible nitric oxide synthase (Inos) and COX 2, resulting in reduction of nitric oxide and prostaglandin E2 (PGE2) in vitro [57,58]. It has also been identified that EGCG selectively inhibited the IL-1β-induced phosphorylation of c-Jun-N-terminal kinase (JNK) p46 isoform resulting in lower levels of phosphor-c-Jun and DNAbinding activity of activation protein-1 (AP-1), a transcription factor implicated in the inflammatory response in human OA chondrocytes [59]. The metalloproteinases (MMPs) produced by the activated chondrocytes in arthritic joints can result in cartilage degradation even though they are involved in remodeling [60]. In OA and RA joints, the levels of MMP1 and MMP13 were found to be significantly elevated [61,62]. Pretreatment of human OA chondrocytes with EGCG significantly inhibited the expression and activities of MMP-1 and MMP-13 in vitro [63]. EGCG was equally effective in inhibiting IL-1β-induced MMP-1, MMP-3 and MMP-13 in human tendon fibroplasts [64]. Catechins from green tea inhibited the degradation of human cartilage proteoglycan and type-II collagen and selectively inhibited the aggrecanases called a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -1, - 4 and -5 [65,66]. Further studies are required in this direction to investigate the anti-inflammatory effects of Camellia sinensis at the molecular level. Green tea catechins or related compounds could one day be useful as an ethnobotanical cure for the treatment of RA and OA [67]. 7. Cannabis sativa L. (Cannabinaceae) Cannabis sativa L. has been used in various preparations for their medicinal effects including anti-pyretic, anti-rheumatic, anti-allergic and analgesic purposes [68]. The traditional use of Cannabis as an analgesic, anti-asthmatic and anti-rheumatic drug is well established. Extracts of Cannabis have been shown to possess analgesic activity [69], and delta-1tetrahydrocannabinol (delta-1-THC), the psychoactive component of Cannabis and cannabinol (CBN), the bioactive compound, were shown to exhibit analgesic activity in vivo [70].

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It is possible that the anti-inflammatory and anti-asthmatic properties of this herb are mediated through effects on arachidonate metabolism [71]. The constituents of Cannabis are known to stimulate [72] and inhibit [73] prostaglandin releases by influencing enzymes of the arachidonate pathway [74]. The anti-inflammatory potential of two extracts of Cannabis, pure cannabinoids and olivetol (a cannabinoid biosynthetic precursor) in two models of inflammation, in an attempt to separate on a structural basis, the peripheral from the central action of these phenolic drugs, have been studied [71]. It is possible that the cannabinoids and their extracts are inhibiting both PBQ induced writhing and TPA induced erythema by effects on arachidonate release and metabolism. Cannabinoids and olivetol have been shown to inhibit PG mobilization and synthesis [72,73]. Cannabinoids stimulate and inhibit phospholipase A2 (PLA2) activity [74] as well as inducing an inhibition of cyclooxygenase and lipoxygenase [75]. The activity of Cannabis herb or resin is complex, in which activities can be demonstrated on at least three major enzymes of the arachidonate cascade. The results of the work reported by Formukong et al. [71] suggested that the response of the ethanolic extract cannot be solely due to cannflavon. Other structurally related phenolic substances may account for the higher activity seen either due to cumulative or synergistic effects upon cyclooxygenase. The activity of the petroleum ether extract is likely to be largely due to the presence of CBD and CBN. The cultivation of Cannabis plants rich in CBD and other phenolic substances would be useful not only as fiber producing plants but also for medicinal purposes in the treatment of certain inflammatory disorders. 8. Centella asiatica (L.) Urban It belongs to the family Apiaceae and is commonly found in parts of Asia and the Middle East. Centella has been used in traditional medicine in Asia for 100 years [75]. The major bioactive constituents are triterpene saponins mainly asiaticoside, sapogenin, asiatic acid, madecassoside and madecassic acid [76]. It is believed to have beneficial effects in improving memory and treating mental fatigue, anxiety and eczema [77]. In Ayurveda, Centella is effectively used in the treatment of inflammation, anaemia, asthma, blood disorders, bronchitis, fever, urinary discharge and splenomegaly [78]. The aqueous extract of Centella possesses antioxidant, cognitive enhancing and antiepileptic properties [79]. The water extract of the plant was used to study the anti-inflammatory and analgesic activity in adult male rats. The extract elicited dose dependent anti-inflammatory activity at 2 mg/kg concentration. This study revealed that the extract is similar to

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mefenamic acid and interestingly 10 mg/kg extract showed a significantly higher effect when compared to mefenamic acid. In the case of analgesic activity both hot plate and abdominal writhing test revealed positive effect and the mechanism might involve opioid receptors. The potency of antinoceception was less than morphine and aspirin at similar doses. Bioactive terpene acids such as asiatic acid and madecassic acid from the water-methanol extraction of Centella has been studied [80]. These compounds may be present in the extract which contributes to the anti-inflammatory and analgesic property in the study. These findings justify the traditional use of Centella in the treatment of inflammatory conditions. 9. Curcuma longa L. (Zingiberaceae) Curcuma longa L. is a perennial herb distributed throughout tropical and subtropical regions of the world. It is widely cultivated in Asiatic countries, mainly in India and China. As turmeric powder it has been in continuous use for its flavouring, as a spice in both vegetarian and non-vegetarian food preparations and has digestive properties [80]. Traditional Indian medicine claims the use of turmeric powder against biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorder, rheumatism and sinusitis [81]. The active principle in turmeric powder is curcumin (diferuloyl methane) and was isolated in the 19th century from the rhizomes. It is with yellow colour and is responsible for the anti-inflammatory effects. In Hindu medicine turmeric powder has been extensively used for the treatment of sprains and swellings caused by injury [82]. The traditional medicine in China uses C. longa L. in abdominal pains. Religious ceremonies still use turmeric in many forms. The major components of turmeric are curcuminoids which include mainly curcumin (diferuloyl methane), dimethoxy curcumin and bisdimethoxycurcumin. These substances can be classified as Curcuminoids, the analogues of diarylheptanoids. The major constituent, Curcumin, is the most important fraction of C. longa L. and its chemical structure was determined by Roughley and Whiting [83]. Curcumin is insoluble in ethanol, alkalies, ketone, acetic acid and chloroform and is insoluble in water. In the molecule of curcumin, the main aliphatic, unsaturated and the aryl group can be substituted or not. There is a great number of papers in the literature relating the activity of compounds extracted from C. longa L. being potent inhibitors of inflammation. The activity of curcumin and other semi synthetic analogues in experimental rats were demonstrated [84]. In Ayurveda, turmeric has been used for various medicinal conditions including rhinitis, wound healing, common cold, skin infections, liver and urinary tract diseases and as blood purifier [85,86]. It was found to be effective even when given by different routes of

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administration, including topically, orally and by inhalation. Studies on anti-inflammatory activities included in vitro, animal and human models. The laboratory studies have identified a number of different molecules involved in inflammation that are inhibited by curcumin including phospholipase, lipoxygenase, cyclooxygenase-2, leukotriens, thromboxane, prostaglandins, nitric oxide, collagenase, elastase, hyaluronidase, monocyte chemoattractant protein-1 (MCP-1), interferon–inducible protein, tumour necrosis factor (TNF) and interleukin-12 (IL-12). The anti-inflammatory activity demonstrated in the experiment may be due to inhibition of a number of different molecules that play a role in inflammation.86 In animal studies, oral administration of curcumin to rats decreased the levels of inflammatory glycoprotein with a reduction in paw inflammation [86,87]. Curcumin was also found to be inhibiting the carrageenan induced paw edema in mice and rats with an ED 50 dose 48 and 100.2 mg/kg respectively [84]. Curcumin increases glutathione transferase activity which is involved in detoxification of carcinogens and increases mRNA transcription of transforming growth factor beta-1. Curcumin is a potent inhibitor of the common transcription factor NF-κ B in several cell types [88,89,90]. Others reported that curcumin inhibits or modulates upstream pathways of the arachidonic acid cascade by inhibiting the catalytic activities of phospholipases [91,92,93]. Curcumin also inhibited the incorporation of arachidonic acid into membrane lipids, PGF2 production, leukotriene B4 and leukotriene C4 synthesis as well as the secretion of collagenase, elastase and hyaluronidase by macrophages [92]. IL-1β- induced up regulation of MMP-3 was inhibited by curcumin in a time dependent manner. In addition IL-1 β- induced decrease in type II collagen synthesis was also blocked by curcumin treatment. Based on this study it was concluded that curcumin antagonises crucial catabolic effects of IL-1 β- signaling that are known to contribute to the pathogenesis of osteoarthritis. 10. Euphorbia heterophylla L. (Euphorbiaceae) Euphorbia heterophylla is a local medicinal plant commonly known as ‘spurge weed’. It is used in ethnomedicine for the treatment of constipation, bronchitis and asthma [94]. It grows in semi humid places especially in cassava, cow pea and soyabean plantations. Phytochemical studies have revealed the presence of saponins, diterpenes and phorbolesters in the extracts. The antiinflammatory activity of the aqueous and methanolic extract of Euphorbia heterophylla were evaluated by carrageenin induced rat paw edema test [95,96]. The aqueous extract of Euphorbia showed significant anti-inflammatory activity (P<0.001) comparable to the reference drug [94]. But the methanolic extract did not show any appreciable anti-inflammatory activity. These studies were in

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agreement with the earlier investigations [97] suggesting the presence of a flavanoid, quercetin, which is a known anti-inflammatory agent. The significant level of anti-inflammatory activity of the aqueous extract could be attributed to high amount of flavanoids present in the extract. This study justifies the traditional use of Euphorbia in the treatment of inflammatory disease conditions such as asthma. 11. Gastrodia elata Blume (Orchidaceae) Gastrodia elata Blume is a very important traditional herbal medicine used to treat head ache, migraine, dizziness, epilepsy, rheumatism, neuralgia, paralysis and other disorders [98]. Phytochemical studies of this plant have revealed the presence of several phenolic compounds [99]. Lee et al.[100] has identified eight compounds by structure activity guided separation. The compounds are 4-hydrxy benzaldehyde (1), 4-hydroxy bezyl alcohol (2), benzyl alcohol (3), bis(4-hydroxy phenyl) methane (4), 4(4'â&#x20AC;&#x201C;hydroxybenzyloxy) benzyl methyl ether (5), 4-hydroxy-3-methoxy benzyl alcohol (6), 4 hydroxy-3-methoxy benzaldehyde (7) and 4-hydroxy-3-methoxy benzoic acid (8). The anti-inflammatory and analgesic activities of these phenolic extracts were studied using animal models [100]. They suggested that these phenolic compounds inhibited COX activity and silica induced reactive oxygen species (ROS) generation in a dose-dependent manner. Among these phenolic compounds the compound (7) was the most antiinflammatory and analgesic. Compound (7) significantly inhibited silica- induced ROS generation and compound (6) significantly increased 1,1-diphenyl -2-picryl hydroazyl (DPPH) radical scavenging activity. Compounds (1), (2) and (3) significantly inhibited the activity of COX. They concluded that phenolic compounds of Gastrodia elata are anti-inflammatory, which could be related to inhibition of COX activity and to anti-oxidant activity. Consideration of the structure-activity relationship of these compounds of G. elata on the antiinflammatory action revealed that both C-4 hydroxy and C-3 methoxy radicals of benzyl aldehyde play an important role in anti-inflammatory activities [99]. 12. Harpagophytum procumbens (Burch.) DC (Pedaliaceae) Harpagophytum procumbens (Burch.) DC is commonly known as Devilâ&#x20AC;&#x2122;s claw and is a native of South Africa. The root tubers of the plant are used in herbal preparations [101]. Leung and Foster [102] reported three iridoid glycosides viz. harpagoside, harpagide and procumbide and are responsible for the anti-inflammatory and analgesic actions. These glycosides effectively reduced OA pain and was comparable with that of the analgesic/ cartilage protective drug, Diacerhein [103]. H. procumbens at the rate of 600-120 mg/day was helpful in reducing low back pain [104]. The antiinflammatory and analgesic effect of the aqueous extract of H. procumbens

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was recently reported [105]. Research is going on in this line and new results are yet to come. 13. Kalanchoe crenata Andr. (Crassulaceae) Klanchoe crenata Andr. is commonly known as “never die” or “dog’s liver”. It has been traditionally used for the treatment of ear ache, small pox, head ache, inflammation, pain, asthma, palpitations, convulsion and general debility. Phytochemical studies using the aqueous and alcoholic extract of K. crenata revealed the presence of alkaloids and saponins [106]. Nguelefack et al. [107] demonstrated the antinoceceptive activity of the ethanolic extract of K. crenata against acetic acid, formalin and hot plate as well as pain models induced by pressure. The anti-inflammatory property of the leaf extract of K. crenata was scientifically validated [108]. They reported the presence of sterols, flavanoids and saponins in the different extracts which were responsible for the acute and chronic anti-inflammatory activity against various phlogistic agents. The nbutanol fraction of the extract was capable of inhibiting edema induced by histamine, serotonin and formalin. Since n-butanol fraction of the extract significantly inhibited inflammation, it can be thought to possess antiproliferative and antiarthritic activities similar to that of diclofenac, a COX inhibitor. The nonsteroidal anti-inflammatory activity of the extract prompted them to test for the ulcerogenic effect. Non-steroidal anti-inflammatory drugs are thought to impair the mucosal defense of the stomach and the intestine. They act by inhibition of COX and therefore inhibit the production of gastric prostaglandins which in turn leads to a reduction in the gastric mucus and an increase in mucosal permeability [109]. This can be attributed to the inhibition of COX. Hence, it was concluded from this study that the flavanoids in the n-butanol fraction was responsible for its pharmacological activities. Flavanoids have been reported earlier also for such similar activities [110,111]. 14. Mangifera indica L. (Anacardiaceae) Mangifera indica L. aqueous extract, known as Vimang in Cuba, is used to improve the quality of life in patients suffering from elevated stress. Garrido et al. [112] evaluated the analgesic and anti-inflammatory effects of Mangifera indica bark aqueous extract. Analgesia was determined using acetic acid induced abdominal constriction and formalin induced licking. Antiinflammatory effects were studied using carrageenin and formalin induced edema. They reported polyphenols in the extract which might be responsible for the effect. Vimang at a concentration of 50-1000 mg/kg p.o exhibited a potent and dose dependent antinoceceptive effect against acetic acid test in mice. It also dose dependently inhibited the second phase of formalin induced pain, but not in

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the first phase. Edema formation was significantly inhibited both in carrageenin and formalin models. These inhibitions were similar to those produced by indomethacin and sodium naproxen p.o. The anti-inflammatory effects of mangiferrin in M. indica L. has also been reported earlier [113]. 15. Plumeria accuminata W.T. Aiton (Apocyanaceae) Plumeria acuminata W.T. Aiton belongs to the family Apocynaceae and is widely distributed in Southern parts of India. In traditional medicine system different parts of the plant have been used in a variety of diseases. The milky juice is employed for the treatment of inflammation and rheumatism. The leaves are reported to have anti-inflammatory and rubefacient in rheumatism and have strong purgative effect. The methanol extract of Plumeria acuminata exhibited significant anti-inflammatory activity on the tested experimental models in both acute and chronic inflammation models [114]. Preliminary phytochemical screening of the methanol extract revealed the presence of steroids, flavanoids, tannins, alkaloids and glycosides. The Methanolic extract produced significant (P<0.001) anti-inflammatory activity and the results were comparable to that of indomethacin as a standard anti-inflammatory drug. Their studies indicated that the extract acted in later phases probably involving arachidonic acid metabolites which produce an edema dependent on neutrophil mobilization [115]. 16. Ricinus communis L. (Euphorbiaceae) Ricinus communis Linn. is a small tree distributed through out the tropics and warm temperate regions of the world [116]. In Indian traditional system of medicine different parts of this plant has been used to cure inflammation and liver disorders [40]. Various bioactivities of the plant such as hepatoprotective [117,118], hypoglycaemic [119], laxative [120], diuretic [121] and antibacterial [122,123] have been reported earlier. The plant was reported to contain flavanoids [124] and tannins [125]. The anti-inflammatory activity of the methanolic extract of Ricinus communis Linn. root was reported recently [126]. The methanolic extract at a dose of 250 mg/kg p.o exhibited significant (p<0.001) anti-inflammatory activity in carrageenin induced rat paw edema model and a higher dose of 500 mg/kg p.o also exhibited significant (p<0.001) activity in cotton pellet granuloma model in Wistar albino rats. Flavanoids have been reported to have anti-inflammatory and antiarthritic activity [127,128]. The anti-inflammatory activity of Ricinus can be attributed to the presence of phytochemicals such as flavanoids, alkaloids and tannins in the plant extract.

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17. Salix alba L. (Salicaceae) Salix alba L. is commonly known as the Willow tree and its bark contains heavy concentrations of salicin, a glycoside, which is the precursor of aspirin. In India willow has been used for centuries as a remedy for fever [129]. Willow is listed in the herbal remedies of ancient Egypt, in the Ebrus papyrus (1534 BC). It was in the mid 18th century the first written document on the analgesic property of willow was made [130]. Salicin is responsible for the anti-inflammatory and analgesic actions [131]. The consumption of herbal combination containing 100 mg willow bark for two months improved functioning via pain relief in OA. A trial study revealed that 1360 mg of willow bark extract per day (delivering 240 mg of salicin) for two weeks to be effective in treating pain associated with knee and hip [132]. A four week trial found that willow extract containing 240 mg of salicin was effective in reducing exacerbations of low back pain [133]. Some may develop stomach problems after the intake of willow extract, but the symptoms are less when compared to aspirin [134]. Those with ulcers and gastritis were advised to avoid willow extract [135]. 18. Sida cordifolia L. (Malvaceae) Sida cordifolia Linn is an extensively used herbal ingredient in the Ayurvedic system of medicine in the Indian subcontinent [40]. The antiinflammatory and analgesic activities of the water extract of the plant in animal models were reported [136]. The whole plant extract in water has been used in the treatment of rheumatism [137]. Phytochemical analyses from time to time have revealed the presence of ephedrine, vasicinol, vascicinone and N-methyl tryptophan [138,139,140]. Sutradhar et al. [141] reported the anti-inflammatory and analgesic properties of the different extracts of S. cordifolia Linn. They used chloroform, methanol, ethanol, hexane, dichloromethane, butanol and diethyl acetate extracts. Chloroform, methanol, ethyl acetate and butanol extracts showed significant activity in experimental models. In addition another chemical constituent (5' – Hydroxymethyl - 1'-(1,2,3,9-tetrahydro-pyrrolo[2,1-b] quinazolin -1- yl)-heptan-1-one) was reported141 from the aerial parts of Sida cordifolia. They also investigated the anti-inflammatory and analgesic activity of the compound in mice and rat respectively. The bioactivity thus reported was due to the inhibitory effect of the compound by the inhibition of COX enzyme leading to the inhibition of prostaglandin synthesis. 19. Silybum marianum (L.) Gaertn. (Asteraceae) Silybum marianum (L.) Gaertn. is an important medicinal plant commonly known as ‘Milk thistle’ or ‘St. Mary’s Thistle’. The anti-inflammatory

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activity of this plant has been reported earlier [142]. The plant extract was reported to contain an important bioactive principle, sylimarin, which belongs to the flavanolignan group and possess anticancer, antiinflammatory, antioxidant and immunomodulatory effects [143,144]. Even though the whole plant has been reported to possess anti-inflammatory activity, the activity of the dried leaf callus was also been reported recently [145]. They examined the anti-inflammatory activity of the methanolic extract of dried leaf callus using carrageenin and formalin induced rat paw edema models. The leaf and leaf callus of Sylibum marianum (L.) Gaertn. inhibited the formation of paw edema to significant levels (74% and 93.9%) at a dose of 100 mg/kg when administered orally. 20. Spilanthes acmella Murr. (Asteraceae) Spilanthes acmella Murr. is an indigenous herb growing as an annual throughout the tropics. The whole plant is claimed to possess medicinal properties. The flowers are chewed to relieve tooth ache and the crushed plant is used in rheumatism [146,40]. The plant is generally known as tooth ache plant [147]. Chakraborty et al. [148] evaluated the anti-inflammatory and analgesic activity of the aqueous extract of S. amcella. They reported the presence of flavanoids in the aqueous extract which was responsible for the significant anti-inflammatory and analgesic property of the plant. The extract produced dose dependent and significant inhibition of prostaglandins which are involved in the late phase of acute inflammation and pain perception [149]. Detailed studies using the extract may reveal the exact mechanism of action of the flavanoids responsible for the anti-inflammatory and analgesic activity. 21. Tripterygium wilfordii Hook F (Celastraceae) Tripterygium wilfordii Hook F is a perennial vine like plant that grows in China and Thaiwan. The root of the plant is medicinal and is used for the treatment of inflammatory diseases like rheumatoid arthritis, asthma, nephritis etc for centuries ago [150]. The ethanolic and ethyl acetate extract of the plant is used in the treatment of rheumatoid arthritis [151]. They also reported a prospective, double blind placebo-controlled study of the ethanolic and ethyl acetate extract in patients with rheumatoid arthritis. Another report also substantiated the efficacy of the extract in both clinical manifestations and laboratory findings [152]. They concluded that the extract at dosages up to 570 mg/day was safe and doses >360 mg/day were associated with clinical benefit in patients with RA. The only toxic effect reported in this study was diarrhoea.

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The anti-inflammatory effects of T. wilfordii was believed to be due to the presence of triptolide, the active ingredient [153]. Animal studies showed that triptolide inhibited the CIA in mice and rat [154,155]. A recent study has revealed that triptolide inhibited iNOS gene expression by down regulating NF-κ B-DNA binding activity and JNK pathway [156]. Triptolide was shown to inhibit LPS and cytokine induced expression of COX-2, MMP-3 and MMP-13 in articular chondrocytes [157] and IL-1, IL-17 and TNF-α induced expression of aggrecanase gene in human chondrocytes [158]. Another mechanism of anti-inflammatory effect may be by the suppression of adhesion molecules E- selectin, ICAM-1 and VCAM-1 [159]. These studies revealed a strong scientific explanation for the known beneficial use of T. wilfordii in rheumatoid arthritis. 22. Uncaria tomentosa (Willd.) DC. and U. guianensis J.F. Gmel (Rubiaceae) Uncaria tomentosa (Willd.) DC. and U. guianensis DC commonly known as Cat’s claw is a Peruvian vine with medicinal properties that are well documented in alternative medicine literature. The anti-inflammatory activity of Cat’s claw extract was reported earlier [160,161]. In Peruvian medicinal system, the extract of both species has been used interchangeably to treat inflammatory and non-inflammatory conditions. The chemical composition of U. tomentosa and U. guianensis vary and accordingly the anti-inflammatory effects are independent of one another [160]. The antiinflammatory activity of U. tomentosa is mainly due to the active constituent, pentacyclic oxindole alkaloid [161]. U. guianensis was found to be more potent in inhibiting TNF-α production by macrophages.162 The safety and pharmacological profile of Cat’s claw in animal models using in vitro bioassays were reported by many workers [163,164]. Animal studies have suggested that cat’s claw extract is protective to the gastrointestinal tract and even protect the gut from the damaging effects of NSAIDs [162]. These studies revealed that cat’s claw extract was effective in inhibiting lipopolysaccharide induced free radical production followed by lipid peroxidation. They also showed that TNF-α production and iNOS expression via NF-κ B expression were also inhibited by the extract. A further study for the long term efficacy and safety of this extract is needed to develop an effective anti-inflammatory drug from this plant. 23. Zingiber officinale Roscoe (Zingiberaceae) Zingiber officinale Rosc. is one of the most common constituents of diets world wide and is reported to possess antioxidant, anti-inflammatory, antiseptic

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and carminative properties [165]. In folk medicine it has been used against pain, inflammation, arthritis, urinary infections and gastrointestinal disorders [166]. Ayurveda supports the use of ginger to treat inflammatory and rheumatic disorders. The major constituents of ginger include volatile oils, oleoresin (gingerol), linoleic acids and trace elements such as magnesium, phosphorus and potassium. Most of the pharmacological activities of ginger can be attributed to the presence of gingerol and its analogues found in the rhizome extracts [166]. Ginger oil contains a mixture of constituents like monoterpenes and sesquiterpenes which were reported to have antiinflammatory and analgesic activities [166,167]. Ginger has been known to prevent relief in arthritis [168] and the intake of less than a tablespoon ginger every day for three months was enough to relieve pain in arthritis. The antiinflammatory and analgesic properties of ginger essential oils have been reported recently [169]. The anti-inflammatory activity of the ginger essential oil was determined by pleuricy test using carrageenan (200 Âľg /cavity) in experimental mice. Ginger essential oil and indomethacin in 200 and 500 mg/kg was significant in proving anti-inflammatory activity. The experimental data suggested that ginger essential oil does not have influence on cellsâ&#x20AC;&#x2122; recruitment different to that observed for other essential oils [170]. Gingerol has been reported to have anti-inflammatory actions, which include suppression of both COX metabolites of arachidonic acid [171]. Antiinflammatory activity of silica gel chromatography fractions of ginger have been reported [172]. Ginger extract administered daily for four weeks, either orally or intraperitoneally, caused significant reduction in prostaglandin E2 levels in experimental rats [171]. The efficiency of ginger in alleviating pain and associated symptoms in patients suffering from osteoarthritis has been reported recently [173]. According to them a highly purified and standardised extract of ginger had a statistically significant effect on reducing the symptoms associated with OA of the knee. There was a good safety profile also. The beneficial effects of ginger could be attributed to its ability to inhibit COX and LOX pathways resulting in the blockage of PGE2 and LTB4 production in affected joints [91,92,124]. The anti-inflammatory activity shown by ginger essential oil could be owing to the inhibition of prostaglandin release and hence ginger may act in a way similar to other nonsteroidal anti-inflammatory drugs which interfere with prostaglandin biosynthesis. Ginger has been a common ingredient in arthritic formulas to encounter gastrointestinal adverse drug reactions due to aspirin and other non-steroidal anti-inflammatory drugs. A detailed study is needed to reveal the mechanisms of action of these compounds.

Table 1. List of some of the important anti-inflammatory and analgesic plants with their family and isolated compound.

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Table 1. Continued

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Summary and conclusion In modern times the trend towards the use of alternative and complementary medicine is increasing and it offers unprecedented opportunities for the development of herbal medicine. Many of the Asian countries are taking full advantage of the links to the ancient cumulative wisdom of the traditional practitioners. Ethnobotanical knowledge of the past as well as present folk is of immense value to the development of newer drugs with virtually no or less adverse effects. Previous studies have contributed much in the understanding of the compound(s) responsible for the known anti-inflammatory and analgesic action, their mechanism of action and therapeutic values. Compounds such as Bromelain act as anti-inflammatory agent due to its fibrinolytic and fibrinogenolytic effects. Bosewellic acids play a key role in inhibiting 5-HETE and leukotriene B4 which are involved in the pathogenesis of asthma and arthritis. Xanthones are also implicated in the anti-inflammatory and analgesic effects. Unlike other anti-inflammatory/ analgesic agents, xanthones were reported to have very less or no side effects such as ulcerogenicity and blood clotting. Green tea catechins are another useful candidate for the treatment of inflammatory diseases like RA and OA. Cannabinol and related compounds from Cannabis sativa are potent analgesic, antiasthmatic and anti-heumatic agents. Terpene acids such as madecassic acid and asiatic acid from Centella asiatica has been reported to be an effective analgesic and anti-inflammatory compound. Curcumin is the most important ethnobotanical drug isolated from Curcuma longa and is reported to have a variety of medical applications including antiinflammatory activity. The pharmacological action of curcumin can be attributed to the inhibition of a number of inflammatory molecules including lipoxygenase, cycloxygenase, TNF, IL-1, IL-2, leukotrienes and prostaglandins. Pathways of anti-inflammatory activity of curcumin have been studied by many workers and they come out with different views. However, curcumin is involved in one or the other pathway of inflammatory cascade and execute its effect. Gastrodia elata, an orchidacean member is used in ethnomedicine to treat a variety of disorders and eight structurally different phenolic compounds were identified. These compounds were involved in the inhibition of COX activity, which was attributed to the presence of C-3 and C-4 methoxy and hydroxyl radicals respectively in them. Yet another compound, Salicin, from Salix alba was also found to be very effective anti-inflammatory and analgesic agent and was proved better than aspirin. Gingerol and its analogues in Zingiber officinale are potent antioxidant, antinoceceptive and anti-inflammatory agents. Gingerol inhibits COX and LOX pathways, thus blocking the PGE-2 and LTB-4 production in affected areas. Much of the current research trend is towards the isolation, purification, identification and characterization of active principle(s) from crude extracts

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of ethnomedicinal plants. However, there is a hidden fact that the different components present in the crude plant drugs may be more efficient and potent than any of the single purified compound which may help to nullify the toxic effects of individual constituents. Most of the commonly used modern medicines have originated from the plant sources. The incidence of arthritis and related diseases is increasing now due to the drastic changes that happened in the present life style. The quest for new botanicals as relief for these life style disorders would be a welcome step for the local and urban health care. Majority of the anti-inflammatory and analgesic compounds isolated from the above discussed medicinal plants are prone to some side effects for which addition of modern medicines or antidotes from plant sources are recommended. At the same time plants like Bosewellia, Callophyllum and Mesua yield such compounds free from side effects. The development of neutraceuticals from them could substitute the present generic market to a great extent.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Singh, H.B. 1997, Ethnobotany and Medicinal Plants of Indian Subcontinent, J. K. Maheswari (Ed.), Scientific Publishers, Jodhpur, India, 109. Ganesan, S., Venkateshan, G., and Banumathy, N. 2006, 5, 245. Kelly, G.S. 1996, Alt. Med. Rev., 1, 243. Innoue, K., Montonaga, A., and Dainaka, J. 1994, Essent. Fatty Acids, 51, 457. Klein, G., and Kullich, W. 1999, Wein. Med. Wochenscher, 149, 577. Uhlig, G., and Seifert, J. 1981, Fortschr. Med., 99, 554. Yonehara, N., Shibutani, T., and Inoki, R. 1987, J. Pharmacol. Exp. Ther., 242, 1071. Felton, G.E. 1980, Med. Hypotheses, 6, 1123. Vellini, M., Desideri, D., and Milanese, A., 1986, Arzneimittelforschung, 36, 110. Taussig, S.J., and Batkin, S. 1988, J. Ethnopharmacol., 22, 191. Lotz-Winter, H. 1990, Planta Med., 56, 249. Kamenicek, V., Holan P., and Franek, P. 2001, Acta. Chir. Orthop. Traumatol Cech., 68, 45. Akhtar, N.M. 2004, Clin. Rheumatol., 23, 410. Singh, G. 1986, Agents Actions, 18, 407. Menon, M.K., and Kar, A. 1971, Planta Med., 19, 333. Kimmathkar, N., Thawani, V., Hingrani, L, and Khiyani, R. 2003, Phytomed., 10, 3. Ammon, H.P. 2002, Wien. Med. Wochenscher, 152, 373. Dahmen, U. et al. 2001, Transplant Proc., 33, 539. Park, Y. S. et al. 2002, Adv. Exp. Med. Biol., 507, 387. Hostanska, K., Daum, G., and Saller, R., 2000, Anticancer Res., 22, 2853. Park, Y. S. et al. 2002, Planta Med. 68, 397. Etzel, R. 1996, Phytomed. 3, 91. Kulkarni, R.R., Patki, P.S. Jog, V.P., Gandage, S.G.and Patwardhan, B. 1991, J. Ethnopharmacol., 33, 91.

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24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

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Ammon, H.P, Mack, T., Singh G.B.and Safayhi, H. 1991, Planta Med., 57, 203. Kemp, J.P. 2003, Am. J. Respir. Med., 2, 139. Laufer, S. 2003, Curr. Opin. Rheumatol., 15, 623. Rask-Madesen, J. et al. 1992, Agents Actions, 37, 46. Stele V.E., Homes C.A., hawk E.T., et al., 1999. Cancer Epidemiol. Biomarkers Prev. 8, 467. Safayhi, H., Rall, B., Sailer, E.R., and Ammon, H.P. 1997, J. Pharmacol. Exp. Ther., 281, 460. Varga, M., Kapui, Z., Batori, S., Nagy, L.T., Vasvari-Debreczy, L., Mikus, E., Urban-Sabo, K., and Aranyi, P., 2003, Eur. J. Med. Chem., 38, 421. Syrovets, T. et al. 2000, Mol.Pharmacol., 58, 71. Sharma, I.S. et al. 2004, Phytomed., 11, 255. Reddy, G.K., Chandrkasan, G., and Dhar, S.C., 1989. Biochem, Pharmacol., 38, 3527. Sontakke, S., Thawani,V., Pimpalkhute, S., Babhulkar, S., and Hingorani, L. 2007, Indian J. Pharmacol., 39, 27. Nadkarni, K.M. 1954, The Indian Materia Medica, Vol. I & II, Popular Prakashan Pvt. Ltd., Mumbai. Saxena, R.C., Nath, R., Palit, G., Nigam, S.K., and Bhargava, K.P. 1979, Indian J. Pharmacol., 11, 39. Gopalkrishnan, C., Sankaranarayanan, D., Nizamudeen, S.K., Viswanathan, S, and Kameswaran, L. 1980, Indian J. Pharmacol., 12, 181. Sankaranarayanan, D., Gopalakrishnan, C., and Kameswaran, L., 1979, Arch. Int. Pharmacodyn., 239: 257. Sankaranarayanan, D., Gopalkrishnan, C., Kameswaran, L., and Arumugam S. 1979, Mediscope, 22, 65. Kirtikar, K. R., and Basu, B.D. 1935, Indian Medicinal Plants, International Book Distributors, 9/3, Dehradun, India. Jain, S.K. 1991, Dictionary of Indian Folk Medicine and Ethnobotany, Deep Publications, New Delhi. Oudhia, P. 2001, Calotropis gigantea: Useful weed, PankaLoudhia@usa.net. www.celestine India.com/Pankoudhia. Rasik, A.M., Gupta, A., Shukla, A., Dubey., M.P., Srivastava, S., Jain, H.K., Kulshrestha, D.K., and Rahubir, R. 1999, J. Ethnopharmacol., 68, 261. Begum, D., and Nath, S.C. 2000, J. Herbs Spices & Med.Plants, 7, 55. Chitme, H.R.,Chandra, R., and Kaushik, S., 2005, Pharmaceut. Biol. 39, 1. Poddar, A., Vadlamudi, V.P., Koley, K.M., and Dewangan, G. 2007, Aryavaidyan, 20, 178. Jangade, C.R., Raut, C.G., and Bisan, V.V. 1994, Livestock Advisor, 19, 29. Majumdar, P.K., and Kumar, V.L., 1997, Phytother. Res. 11, 166. Anonymous. 1992, Wealth of India: Raw materials. III. CSIR Publication and Information Directorate, New Delhi, 8. Sangraula, H., Dewan, S., and Kumar, V.L. 2002, Inflammopharmacol., 9, 257. Dewan, S., Kumar, S.,and Kumar, V.L. 2000, J. Ethnopharmacol., 73, 307. Dewan, S., Kumar, S., and Kumar, V.L. 2000, Indian J. Pharmacol., 32:252. Arya, S., and Kumar, V.L. 2005, Mediators Inflamm., 4, 228. Siddiqui, I.A., Afaq, F., Adhami, V, M., Ahmad, N., and Mukhtar, H. 2004, Antioxid. Redox. Signal, 6, 165.

Anti-inflammatory and analgesic plants

289

55. Haqqi, T.M. et al. 1999, Proc. Natl. Acad. Sci. USA, 96: 4524-4529. 56. Curtis, C.L., Harwood, J.L., Dent, C.M., and Caterson, B. 2004, Drug Discov. Today, 9, 165. 57. Singh, R., Ahmad, S., Islam, N., Goldberg, V.M., Haqqi, T.M. 2002, Arthritis Rhem., 46, 2079. 58. Ahmad, S., Rahman, A., Hasnain, A., Lalonde, M., Goldberg, V.M., Haqqi, T.M. 2002, Free Radc. Biol. Med., 33, 1097. 59. Singh, R., Ahmad, S., Malemud, C.J., Goldberg, V.M., and Haqqi, T.M. 2003, J. Orthop. Res., 21, 102. 60. Malemud, C.J., Islam, N., and Haqqi, T.M. 2003, Cells Tissues Organs, 174, 34. 61. Ishiguro, N., Kojima, T., and Poole, A.R. 2002, Nagoya J. Med. Sci., 65, 73. 62. Iannone, F., and Lapadula, G. 2003, Ageing Clin. Exp. Res., 15, 364. 63. Ahmad, S., Lalonde, M., Wang, N., Goldberg, V.M., and Haqqi, T.M. 2004, J. Pharmacol. Exp. Ther., 308, 763. 64. Corps, A.N., Curry, V.A., Buttle, D.J., Hazleman, B.L. and Riley, G.P. 2004, Matrix Biol., 23, 163. 65. Adcocks, C., Collin, P., Buttle, D.J. 2002, J. Nutr., 132, 341. 66. Vankemmelbeke, M.N. et al. 2003, Eur. J. Biochem., 270, 2394. 67. Ahmed, S., Anuntiyo., J., Malemud, C.J., and Haqqi, T.M., 2005, Alternat. Med., 2, 301. 68. Pars, G., Razdan, R.J., and Howes, J.F. 1977, Adv. Drug. Res.11. 69. Gill, E.W., Paton, W.D.M., and Pertwee, R.G. 1970, Nature, 228, 225. 70. Sanders, J.M., Jackson, D.M., and Starmer, G.A.1979, Psycopharmacol., 61, 281. 71. Formukong, E.A., Evans, A.T., and Evans, F. J. 1988, Inflammation, 12, 361-371. 72. White, H.L., and Tansik, R.L. 1980, Prostaglandins Med., 4, 409. 73. Barret, M.L., Gordon, D., and Evans, F.J. 1985, Biochem. Pharmacol., 34, 2019. 74. Evans, E.T., Formukong, E.A. and Evans F.J. 1987, FEBS Lett., 211, 119. 75. Evans, E.T., Formukong, E.A. and F.J. Evans, 1987, Biochem. Pharmacol., 36, 2035. 76. Cheng, C.L., Koo, and M.W.L. 2000, Life Sci., 67, 2647. 77. Inamdar, P.K., Yeole, R. D., Ghogare, A.B., and de Souza, N.J.1996, J.Chromatography, 742: 127-130. 78. Hamid, A.A., Shah, Z.M.D., Musa, R., and Muhammed, S. 2002, Food Chem., 77, 465. 79. Duke, J.A. 2001, Handbook of Medicinal Herbs, CRC Press, New York. 80. Somchit, M.N., Sulaiman, M.R., Zurani., A., Samsuddin, L., Somchit, N., Israf, D.A., and Moin, S. 2004, I. J.Pharmacol. 36, 377. 81. Govindarajan, V.S. 1980, CRC Cr. Rev. In. Fd. Sci. Nut., 199. 82. Ammon, H.P.T., Anazodo, M.I., Safayhi, H.I., Dhawan, B.N., and Srimal, R.C. 1992, Planta Med., 58, 26. 83. Ammon, H.P.T., and Wahl, M.A., 1991, Planta Med., 57, 1. 84. Roughley, P.J., Whiting, D.A. 1973, J. Chem. Soc. 20, 2379. 85. Mukhopadyay, A., Basu, N., Ghatak, N., and Gujral., P.K. 1982, Agents and Actions, 12, 508. 86. Aggrawal, B.B., and Shishodia, S., 2004, Ann. NY Acad. Sci. 1030, 434.

290

M. Anilkumar

87. Chainami-Wu, N. 2003, J. Altern. Complement. Med. 9, 161. 88. Joe, B., Rao, U.J., and Lokesh, B.R., 1997. Mol. Cell. Biochem. 169, 125. 89. Ranjan, D., Chen, C., Johnston, T.D., Jeon, H., and Nagabhushan, M. J. 2004. Surg. Res. 121: 171-177. 90. Liacini, A., Sylvester, J., Li, W.Q., and Safarullah, M. 2002, Matrix Biol., 21, 251. 91. Schulze-Tanzil, G., Mobasheri, A., SAendzik, J., John, T., and Shakibaei, M., 2004, Ann. NY Acad. Sci. 1030, 578. 92. Hong, J. et al. 2004, Carcinogenesis, 25, 1671. 93. Wallace, J.M. 2002, Integr. Cancer Ther. 1, 7. 94. Frondoza, C.G., Sohrabi, A., Polotsky, A., Phan, P.V., Hungerford, D.S. and Lindmark, L. In vitro Cell Dev. Biol. Anim. 40, 95. 95. Faloudun, A., Okunrobo., L.O., and Uzoamaka, N. 2006, Afr. J. Biotechnol., 5, 529. 96. Winter, C.A., Risley, E.A., and Nuss, G.W., 1962, Proc. Soc. Exp. Biol. Ther. 111, 544. 97. Adeyemi, O.O., Okop, S.O., and Ogunti, O.O. 2002, Phytotherapia, 73, 375. 98. Faloudun, A., Agbakwuru, E.O.P., and Ukoh, G.C., 2003, Pak. J. Sci. Res., 46, 471. 99. Tang, W., and Eisenbrand, G. 1992, Chinese drug of plant origin, Gastrodia elata Blume, Springer-Verlag, Berlin. 100. Hayashi, J., Sekine, T., Deguchi, S., Lin, Q., Horie, S., Tsuchiya, S., Yano., S., Wantanabe, K., and Ikegami., F. 2002, Phytochemistry. 59, 513. 101. Lee. J.Y., Jang, Y.W., Kang, S., Moon, H., Sim, S.S., and Kim, C.J. 2006, Arch. Pharm. Res. 29, 849. 102. Tyler, V.E. 1993. The Honest Herbal, Pharmaceutical Products Press, Binghamton, New York. 103. Leung, A.Y., and Foster, S., 1996, Encyclopedia of common natural ingredients used in food, drugs and cosmetics, John Wiley and Sons, New York. 104. Chantre, P., Cappelaere, A. and Leblan, D., 2000, Phytomed., 7, 177. 105. Chrubassik, S., Zimpfer, C., Schutt, U., and Ziegler, R. 1996, Phytomed., 3, 1. 106. Baghdikian, B., Lanhers, M.C., Fleurentin, J., Ollivier, E., Maillard, C., Balasard, G., and Mortier, F. 2001, Phytother. Res.15, 18. 107. Sofowora, A. 1993, Medicinal plants and traditional medicine in Africa, Polygraphic Ventures Ltd. Ibdan, 207. 108. Nguelefack, T.B., Fotio, L.A., Watch, P., Wansi., S., Dimo, T., and Kamayi, A., 2004, Phytother. Res., 18, 385. 109. Theophile, D., Agathe, L.F., Nguelefack, T.B., Asongalem, E.A., and Kamtchouing, P. 2006, Indian. J. Pharmacol. 38, 115. 110. Jain, N.K., Kulkarni, S.K., and Singh, A. 2002, Life Sci., 70, 2857. 111. Martini, N.D., Katerere, D.R.P., and Eloff, J.N. 2004, J. Ethnopharmacol., 93, 207. 112. Toker, G., Kupeli, E., Memizolu, M., and Yesilada, E. 2004, J. Ethnopharmacol., 95, 393. 113. Garrido, G., Gonzalez, D., Delporte, C., Backhouse, N., Quintero, G., NunezSells, A.J., and Morales, M.A. 2001, J. Ethnopharmacol., 76, 45. 114. Sankaranarayanan, D., Gopalkrishnan, C., and Kameswaran, L. 1979, Indian J. Pharmaceut. Sci., 42, 78. 115. Gupta, M., Mazumder, U.K., Gomathi, P., and Thamilselven, V. 2006, Complement. Altern. Med. 6, 36.

Anti-inflammatory and analgesic plants

291

116. Just, M.J., Reico, M.C., Giner, R.M., Cullar, M.J., Manez, S., and Bilia, A.R. 1998, Plant Med., 64, 404. 117. Ivan, A. 1998, Medicinal plans of the world, Ross Humana Press Inc., Totowa, NJ, 375. 118. Yanfg, L.L., Yen, K.Y., Kiso, Y., and Kikino, H. 1987, J. Ethnopharmacol., 19, 103. 119. Visen, P., Shukla, B., Patnaik, G., Tripathi, S., Kulshreshtha, D., Srimal., R., and Dhawan, B. 1992, Internatl. J. Pharmacognosy 30, 241. 120. Dhar, M.L., Dhar, M.M., Dhawan, B.N., Mehrota, B.N., and Ray, C. 1968, Indian J. Exp. Biol., 6, 232. 121. Cappaso, F., Mascolo, N., Izzo, A.A., and Gaginella, T.S. 1994, Brittish J. Pharmacol., 113, 1127. 122. Abraham, Z., Bhakuni, S.D., Gsarg, H.S., Goel, A.K., Mehrota, B.N., and Patnaik, G.K., 1986, Indian J. Exp. Biol., 24, 48. 123. Khan, M.R., Ndaalio, G., Nkunya, M.H.H., and Wevers, H. 1978, Pakisthan J. Sci. Res., 27, 189. 124. Verpoorte, R., and Dihal, P.P. 1987, J. Ethnopharmacol., 21, 315. 125. Kang, S.S., Cordell, A., Soejarto, D.D., and Fong, H.H.S. 1985, J. Natural Products, 48, 155. 126. Khogali, A., Barakat, S., and Abou-Zeid, H. 1992, Delta J. Sci., 16, 198. 127. Illavarasan, R., Mallika, M., and Venkataraman, S. 2006, J. Ethnopharmacol., 103, 478. 128. Rajendran, N.N., Thirugnanasambandam, P., Viswanathan, S., Parvathi, V., and Ramasamy, S. 2000, Indian J. Exp. Biol., 38, 182. 129. Li, D.W., Lee, E.B., Kang, S.S., Hyun, J.E., and Whang, W.K. 2002, Chemical Pharmaceutical Bulletin, 50, 900. 130. Chevallier, A. 1996, The encyclopedia of Medicinal plants, Dorling Kindersley, London, 96. 131. Sctcliffe, J.and Duin, N. 1992, A History of Medicine, Barnes and Noble Books, New York, 49. 132. Bradley, P.R.1992, British Herbal Compendium Vol. I, British Herbal Medicine Association, 222. 133. Schmid, B., Tschirdewahn, B., and Katter, I. 1998. FACT, 3:186. 134. Chrubassik, S., Eisenberg, E., and Balan, E. 2000, Am. J. Med., 109, 9. 135. Sontakke, S., Thawani, V., Gharpure, K.J., and Patel, S.B. 2005, Herbals in osteoarthritis, Milestone, 25. 136. Blumenthal., M., Busse, W.R., and Goldberg, A. 1998, Integrative Medicine Communications, 120. 137. Franzotti, E.M., Santos, C.V.F., Rodrigues, H.M.S.L., Maurao, R.H.V., Andrade, M.R., and Antoniolli, A.R. 2000, J. Ethnopharmacol., 72, 273. 138. Yusuf, M., and Kabir, M.1999, Medicinal plants of Bangladesh, Bangladesh Council of Scientific and Industrial Research, Dhaka, Bangladesh, 226. 139. Asha, B., and Banerjee, N.R. 1985, Curr. Sci., 54, 690. 140. Ghosh, S., and Dutt, A. 1930, J. Indian Chem. Soc., 7, 825. 141. Gunatilaka, A.A.L., Sotheeswaran, S., Balasubramaniam, S., Chandrasekhara, AI., and Badrasriyani, H.T.1980, Planta Medica, 22, 66.

292

M. Anilkumar

142. Suthradhar, R.K., Rahman, A.M., Ahmad, M., Bachar, S.C., Saha, A., and Guha, S.K. 2006, Iranian J. Pharmacol. Therap., 5, 175. 143. De LA Puerta, R. 1996, J. Pharm. Pharmacol., 48, 968. 144. Maghrani, M., Zeggwagh, N.A., Lemhadr, A., El Amraoui, M., Michael, J.B., and Eddouks, M. 2004, J. Ethnopharmacol., 91, 309. 145. Katiyar, S.K. 2005, Int. J. Oncol., 26,169. 146. Balian, S., Ahmad, S., and Zafar, R. 2006, J. Pharmacol., 38, 213. 147. Sinha, S.C.1996. Spilanthes acmella, Medicinal plants of Manipur, Manipur Association for Science and Society (MASS), Imphal. 148. Usher, G. 1984, Spilanthes acmella, A dictionary of plants used by man, CBS Publishers and Distributors, New Delhi, 551. 149. Chakraborty, A., Devi, R. K.B., Rita, S., Sharatchandra, and Singh, I. 2004, Indian J. Pharmacol., 36, 148. 150. Rajanarayana, K., Reddy, M.S., Chaluvadi, M.R., and Krishna, D.R. 2001, Indian J. Pharmacol., 33, 2. 151. Tao., X., and Lipsky, P.E. 2000, Rheum. Dis. Clin. North Am., 57, 1221. 152. Tao, X., Younger, J., Fan, F.Z., Wang, B., and Lipsky, P.E. 2002, Arthritis Rheum., 46,1735. 153. Cibere, J., Deng, Z., Lin Y., Ou, R., He, Y., Wang, Z., et al. 2003, J. Rheumatol., 30, 465. 154. Qui, D., and Kao, P.N. 2003, Drugs R D., 4, 1. 155. Gu, W.Z., and Brandwein, S.R.1998, Int. J. Immunopharmacol., 20, 389. 156. Gu, W.Z., Brandwein, S.R., and Banerjee, S.1992, J. Rheumatol., 19, 682. 157. Wang, B., Ma, L., Tao, X., and Lipsky, P.E. 2004, Arthritis Rheum., 50, 2995. 158. Sylvester, J., Liacini, A., Li, W.Q., Denhyanade, F., and Zafarullah, M. 2001, Mol. Pharmacol., 59,1196. 159. Liacini, A., Sylvester, J., and Zafarullah, M., 2005, Biochem Biophys. Res. Commun., 327, 320. 160. Chang, D.M., Kuo, S.A.Y, Lai, J.H., and Chang, M.L. 1999, Ann. Rheum. Dis., 58, 366. 161. Sandoval-Chacon, M., Thompson, J.H., Zhang, X.J., Manick, E.E., SadowskaKrowicka, H., Charbonnet, R.M., et al., 1998, Pharmacol. Ther., 12, 1279. 162. Williams, J.E., 2001, Altern. Med. Rev., 6, 567. 163. Picosaya, J., Rodriguez, Z., Bustamante, S.A., Okuhama, N.N., Miller, M.J. and Sandoval, M. 2001, Inflamm. Res., 50, 442. 164. Mur, E., Hartig F., Eibl, G., and Schirmer, M. 2002, J. Rheumatol., 29, 656. 165. Afzal, M.M., Al-Hadidi, D., Menon, M., Pesek, J., and Dhami, M.S. 2001, Drug Metabol. Drug Interact., 18,159. 166. Santa Maria, A., Lopez, A., Diaz, M.M., Alban, J., Galan, de Mera, A., Vincente Orellana, J.A., et al. 1997, J. Ethnopharmacol., 57, 183. 167. Tang, W., and Eisenbrand, G. 1992, Chinese drugs of plant origin. Chemistry, Pharmacology, and use in traditional and modern medicine, Springer-Verlag, Berlin. 168. Suekawa, M., Ishige, A., Yuasa, K., Sudo, K., Aburada, M., and Hosoya, E. 1984, J. Pharmacobiodyn. 7, 836. 169. Srivastava, K.C., and Mustafa, T.1992, Med. Hypotheses, 39, 342.

Anti-inflammatory and analgesic plants

293

170. Vendruscolo, A., Takaki, I., Bersani-Amado, L.E., Dantas, J.A., Bersani-Amado, C.A., and Cuman, R.K.N. 2006, Indian J. Pharmacol., 38, 58. 171. Abe, S., Maruyama, N., Hayama, K., Inouye, S., Oshima, H., and Yamaguchi, H., 2004, Mediat. Inflam., 13, 21. 172. Thompson, M., Al-Qattan, K.K., Al-Sawan, S.M., Alnaqeeb, M.A., Khan, I., and Ali, M. 2002, Essent. Fat Acid, 67, 475. 173. Jolad, S.D., Lantz, R.C., Solyom, A.M., Chen, G.J., Bates, R.B., and Timmermann, B.N. 2004, Phytochem. 65, 1937. 174. Altman, R.D., and Marcussen, K.C. 2001, Arthritis Rheum. 44, 2531. 175. Manga, H.M., Brkic, D., Marie, D.E.P., and Quetin-Leclercq, J. 2004, J. Ethnopharmacol., 92, 209. 176. Smith, C., Lombard, K.A., Peffley, E.B., and Liu, W. 2003, The Texas J. Agri. Nat. Resources, 16, 24. 177. Chatterjee, G.K. and Pal, S.P. 1984, Indian Drugs, 21, 413. 178. Tassman, G.C., Zafran, J.N., and Zayon, G.M. 1965. J. Dent. Med., 20, 51. 179. Zafra-Polo, M.C. and Blasquez, M.A. 2006, Phytotherapy Res., 5(2), 91. 180. Shen, T.Y. 1981, J. Med. Chem., 24, 1. 181. Huang, K.C., 1999, CRC Press, 199. 182. Bachkouse, N., Rosales, L., Apablaza, C., Goity, L., Erazo, S., Negrete, R., Theodoluz., Rodriguez, J., and C. Delporte. 2008. J. Ethnopharmacol., 263. 183. Koudou, J., Abena, A.A., Ngaissona, P. and Bessiere, J.M. 2005, Fitotherapia, 76, 700. 184. Jayaprakasam, B., Seeram, N.P., and Nair, M.G. 2003, Cancer Letters, 10, 11. 185. Kholi, K., Ali, J., Ansari, M.J., and Rehman, Z. 2005, Indian J. Pharmacol., 37, 141. 186. Srimal, R.C., and Dhawan, B.N. J. Pharm. Pharmacol., 25, 447. 187. Clavin, M., Gorzalczany, S., Mach, A., Munoz, E., Ferraro, G., Acevedo, C., and Martino, V. 2007, J. Ethnopharmacol., 112, 585. 188. Bhattacharya, S.K., Ghosal, S., Chaudhuri, R.K., and Sanyal., A.K. 1972, J. Pharm. Sci., 61, 1838. 189. Mitchell., A.E., Hong, Y.J., Koh, E., Barrett, D.M., Bryant,.E., Denison, R.F., and Kaffka, S. 2007, J. Agric. Food Chem., 55, 6154. 190. Bonjean, K., De Pauw-Guillet, M.C., Defresne, M.P., Colson, P., Houssier, C., Dassonneville, L., Baily, C., Greimers, R., Wright, C., Quetin-Leclerck, J., Tits, M., Angenot, L. 1998, Biochemistry, 37, 5136. 191. Emmanuel, S., Ignacimuthu, S., Perumalsamy, R., Amalraj, T. 2006, Fitotherapia, 77, 611.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 295-315 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

11. Ethnomedicinal plants popularly used in Thailand as laxative drugs Wandee Gritsanapan Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University Bangkok, Thailand

Abstract. Laxative drugs are usually organized according to their mode of action, i.e. bulk forming agents, stool softeners, lubricants, saline laxatives, stimulants including some combined formulations. Among them, bulk forming laxatives promote the mildest laxation. The popular bulk forming herbal laxatives in Thailand and Southeast Asian countries are dry fruits of hairy basil (Ocimum americanum) and Scaphium scaphigerum both of which contain high amounts of mucilage substances. Ripe fruits of banana and papaya which contain pectin, and the rhizome of Amorphophallus spp. containing high amounts of glucomannan polysaccharides, are also popularly used as bulk laxatives. Several plants containing anthraquinone glycosides, i.e. leaves and pods of Senna (Cassia angustifolia), pods of C. fistula, young leaves and flowers of Senna siamea, leaves and seeds of S. tora have been used for stimulant laxatives. The leaves of Senna alata, listed in the List of Herbal Medicinal Product of Thailand A.D. 2006, are recommended as a herbal laxative drug showing astringent properties, attributed to the presence of both anthraquinones and tannins as active components. Correspondence/Reprint request: Dr. Wandee Gritsanapan, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. E-mail: pywgs@mahidol.ac.th

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Castor oil, containing high content of ricinoleic acid, is a safe and effective herbal drug for treating constipation. Moreover, pulp from the ripe fruits of tamarind, which contains several organic acids, also promotes mild laxative action. Although these plants have been used as laxatives since ancient days, some of them have not been evaluated in terms of modern pharmacological techniques and also needed to be validated with appropriate clinical studies. Besides being consumed as drug preparations, the described laxative plants can also be consumed in forms of foods and drinks.

Introduction Laxatives are classified as agents which cause a more or less normal evacuation of the bowel without irritation or griping effects. They are most often taken for treating constipation [1]. Drugs in this category were formerly classified on their relative potency based on the amount of cramping they produced and the relative consistency of the stool. The order of potency is from drastic purgative > cathartic > laxative [2]. Well known laxatives are Senna, Psyllium seed and mineral oil. At present, the laxative products are usually organized according to their mode of action which are bulk forming agents, stool softeners, lubricants or emollients, hydrating agents (saline laxatives and hyperosmotic agents), stimulants, and combinations [3,4]. Laxatives may be used as oral or suppository forms. Bulk forming or bulking laxatives contain dietary fibers. They promote the mildest effect among laxatives. These agents cause the stool to be bulkier and to retain more water, as well as forming an emollient gel, making it easier for peristaltic action to move it along [3]. Bulking laxatives should be taken with plenty of water to prevent choking. This kind of laxative adds bulk and water to the stool. The larger stools help trigger the bewel to contract and move the stool out. Ethnomedicinal plants which have been popularly used in Thailand and Southeast Asian countries as bulk forming laxative drugs are dry fruits of hairy basil and of Scaphium scaphigerum (G. Don) Guib.& Planch. which contain high content of mucilage substances that can swell in water to a large volume in the same manner with Psyllium seed [5]. Other bulking laxatives are ripe banana, ripe papaya, rice bran, rhizomes of Amorphophallus spp. Bulking laxatives can be used every day to help ease symptoms of irritable bowel syndrome, hemorrhoids and other bowel problems [4]. Stool softener laxatives help to mix fluid into stools to soften them. This makes stools easier to pass out of the body. The example of this kind of laxative is docusate sodium. Lubricant laxatives work by coating the surface of the stools, so they can move out of the body more easily. Drugs in this category are glycerin and mineral oil.

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Saline laxatives draw fluid from nearby tissue into the bowel. This can soften stools and helps the bowel move them out. Magnesium hydroxide (milk of magnesia) is an example of saline laxatives. Stimulant laxatives are the harshest laxative. They cause the bowel to squeeze or contract to move the stools out [4]. Several plants and plant products produce stimulant laxative effect due to their anthraquinone glycosides. The most interesting derivatives of anthraquinone compounds for laxative action are the O-glycosides of dianthrones and anthraquinones, as well as the C-glycosides of anthrones. The free anthraquinone aglycones present in the drug or formed by initial gastric hydrolysis, upon reaching the intestine, are absorbed in the small intestine, glucoconjugated in the liver, and almost totally excreted in urine. The glycosides of anthraquinones and dianthrones which are polar compounds, water-soluble, and have a high molecular weight, are not resorbed nor hydrolyzed in the small intestine. They are hydrolyzed in the colon by the Î˛-glucosidase enzyme of the intestinal flora to give free anthraquinones which are further reduced to anthrones. The anthrones formed in situ are the active laxative form of anthraquinone compounds which affect intestinal motility and increase peristalsis of the colon and the sigmoid. They also affect the absorption of water and electrolytes [6]. The popular stimulant laxative herbal drugs are the leaves of Senna alata, leaves and pods of Senna (Cassia angustifolia), pods of C. fistula, young leaves and young flowers of Senna siamea, leaves and seeds of Senna tora, castor oil and Aloe. Some laxatives combine more than one type of active ingredients to produce a combination of the laxative effect. The pulp of ripe pods of tamarind which contain organic acids such as citric acid and tartaric acid, including mucilages is also used as a laxative drug. Laxatives should not be used for longer than l week except for bulk-forming laxatives. Long-term use or overuse of laxatives can cause health problems. Most of the time, constipation does not require treatment with laxatives. It will go away on its own or by making changes in diet and other habits such as eating enough fiber, drinking enough fluids and getting enough exercise [4].

Ethnomedicinal laxative plants popularly used in Thailand and some other Asian countries 1. Senna alata (L.) Roxb., Leguminosae-Caesalpinioideae Synonym: Cassia alata L. Common names: Candle bush, Ringworm bush, Calalabra bush, Chumhet thet, Khikhak, Lap muen luang, Mak kaling thet, Chumhet yai, Ta-see pho Part used: Mature leaves

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Senna alata leaves and flowers

Active constituents: Anthraquinones such as aloe-emodin, rhein, chrysophanol, emodin, isochrysophanol, 4,5-dihydroxy-1-hydroxymethylanthrone [5, 7]. Geographical distribution: Senna alata is a typical tropical plant, widespreads in Southeast Asian countries. It is a heliophilous and hygrophilous plant and often grows along rivers and streams. It prefers moisture and sunlight, no specific soil required, but it is not suitable to the mountainous regions with too cold climate. In Thailnd, it is often growing along ditches between ricefields and is distributed in all parts of the country. It grows fast during the rainy season [5, 8]. Senna alata is a shrub, about 1-2 m high. Leaves are paripinnate, 30-60 cm. long, consisting of 8-20 pairs of leaflets which are oblong or elliptic oblong 1-15 by 3-7 cm [5]. The flowering and fruiting period of this plant is during October- December. S. alata has been listed in The List of Herbal Medicinal Products of Thailand A.D, 2006 for laxative drug and has been used in primary healthcare for a herbal laxative drug and for treatment of fungal skin diseases. Thai herbal Phormacopoeia (THP)[9] and The Standard of ASEAN herbal medicines [10] recommended that the dried S. alata leaves should contain not less than 0.5% w/w of anthraquinone glycosides. Time of collection: The leaves and leaf-bearing branches, collected before flowering, are used fresh or dried as a laxative drug [8].

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Traditional recipes [5] 1. Eight to twelve leaves are dried in the sun, powdered, and put the powdered leaves in a bag. Macerate 1-2 teabags of 3 g of dried powder in a cup of boiling water for 2-5 minutes. Take the infusion at bedtime. 2. Twelve fresh or dried leaflets are coarsely cut and boiled with 2 glasses of water until l glass of decoction is obtained, and strain into a glass. Take the whole decoction as a single dose before bedtime. 3. A few fresh inflorescences are cooked in boiling water and taken with a special sauce. 4. Three to five branches with leaves are boiled with water (1.5 liter). The decoction is boiled until about one third of the water used is obtained. Salt is added to the infusion to make a slightly salty taste. One glass of the decoction is taken before bedtime for laxative action [5]. 5. In Vietnam, the decoction of S. alata dried leaves (20g), Rumex wallichii (20g) and Rhubarb (4-6g) is given orally for the treatment of constipation [8]. In our works [11], we analyzed the content of total anthraquinone glycosides in the leaves of S. alata collected in Thailand in all seasons. The results show that the leaves collected in Winter (November–February) and Summer (March–May) contain the highest amount of total antraquinone glycosides (1.24% dry weight) while the samples collected in Rainy season (June–October) contain only 0.16% dry weight. 2. Senna alexandrina Mill., Leguminosae-Caesalpinioideae Synonym: Cassia angustifolia Vahl Common names: Senna, Tinnevelly senna, Indian senna, Ma kham khaek. Part used: Dried leaflets and pods Active constituents: Antraquinone compounds such as aloe-emodin and its glycosides, physcion, rhein, rhein-8-glucoside, sennosides A, B [12]. The active principle causing peristaltic movement of the large intestine was thought to be rhein-anthrone [13]. World Health Organization recommended that dried Senna leaves and pods should contain not less than 2.5 and 2.2 %, respectively of hydroxyanthracene glycosides, calculated as sennoside B. Geographical distribution: Senna alexandrina is indigenous to Somaliland and Arabia. Now, it is commonly cultivated in tropical regions [5]. Senna is normally recognized as two species, Cassia acutifolia Delile and C. angustifolia Vahl. Most of Senna cultivated in Thailand is C. angustifolia which is a small shrub 0.6-1.5 m. high. Leaves are paripinnate,

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having 3-7 pairs of elliptic or lanceolate leaflets of 1.5-5 cm. long and 0.5-1.5 cm. wide. Flowers are yellow, in terminal racemes. Pods are flattened, about 4-7 cm long, 2 cm wide [5, 12]. Time of collection: The leaves are harvested before flowering. The pods are picked up just before the seeds are developed [5]. Senna is most frequently used as crude drug in several dosage forms such as powdered tablet, capsule and infusion tea. Senna pods are more stable and produce less spasmodic pain side effect than the leaves due to containing lesser content of free anthraquinones [14]. The effects of Senna are due to the hydroxyantracene glycosides, especially sennosides A and B. They are not absorbed in the upper intestinal tract. They are hydrolyzed by Î˛-glucosidase enzyme of the bacteria in the large intestine into the active derivative, rheinanthrone. The rhein-anthrone formed in situ affects on the motility of the large intestine by stimulation of peristaltic contraction and inhibition of local contraction, resulting in an accelerated colonic transit, thereby reducing fluid absorption. Also, rhein-anthrone has an influence on fluid and electrolyte absorption and secretion by the colon. It stimulates mucus and active chloride secretion, thus, fluid secretion is increased [15,16]. The action time of Senna is about 8-10 hours, and thus the drug shoud be taken before bedtime [15]. Toxicity: Senna may cause mild abdominal discomfort such as colic or cramps. Prolonged use or overdosage can result in diarrhoea. The major symptoms of overdose of Senna are griping and severe diarrhoea with consequent losses of fluid and electrolytes especially potassium. Potassium deficiency may lead to disorders of the heart and muscular weakness. Treatment should be supportive with generous amounts of fluid. Electrolytes, particularly potassium, should be monitored, especially in children and the elderly. Excessive use and abuse of Senna has been associated with finger clubbing and with the development of cachexia, and reduced serum globulin concentration [12, 13]. Doses Dried leaflets: 0.5-2.0 g (equivalent to 1-30 mg of hydroxyanthracene glycosides calculated as sennoside B). Dried pods: 4-12 pods (0.6-2.0 g, equivalent to 1-30 mg of hydroxyanthracene glycosides calculated as sennoside B) steeped in 150 ml of warm water for 6-12 hours. Leaf, liquid extract: 0.5-2.0 ml (1:1 in 25% alcohol) The powder prepared as oral infusion is taken daily at bedtime. Daily dose equivalent to 10-30 mg sennosides (calculated as sennoside B) taken at

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night, is recommended for adults and children over 10 years. Senna should be used for short-term (less than 2 weeks) treatment of occasional constipation. Use for more than 2 weeks requires medical attention [12, 13]. 3. Cassia fistula L., Leguminosae-Caesalpinioideae Common names: Golden shower, Indian laburnum, Pudding pine tree, Khun, Lom laeng, Ku-phe-ya, Chaiya phruek, Ratcha phruek Part used: Ripe pods Active constituents: Anthraquinone compounds such as rhein, aloe-emodin, aloein, sennosides [7]. Geographical distribution: Cassia fistula is a native plant of India, naturalized in Africa, West Indies and South America. In Thailand, it is found in mixed deciduous forest and often cultivated as ornamental plant throughout the country [1]. C. fistula is a small to medium size tree, about 10-15 m tall. Leaves are compound pinnate, about 15-25 cm long, bears 3-8 pairs of leaflets which are 7-12 cm long and 4-8 cm wide. Flowers are racemes about 20-40 cm long. Fruits are straight cylindrical pods, 20-60 cm long and 1.5-2 cm in diameter. The pod is dark green when young, turning dark brown to black when mature. The ripe pod contains dark color sweetish pulp and numerous yellowish-brown seeds [5].

Cassia fistula leaves and flowers

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Time of collection: The pods should be collected when ripe and carefully dried. The best pods are those which do not rattle when shaken. These possess the most pulp [1]. Traditional recipes 1. 2.

Pulp of the ripe pod (4 g) is boiled with water, and a little bit of salt is added. The extract is taken before breakfast or before bedtime [17]. The Pulp is dissolved in distilled water, filtered through a sieve. The extract is evaporated on a boiling water bath until a dark semisolid is obtained. Take 4-8 g of the drug before bedtime [18].

Quantitative analysis of total anthraquinone glycosides in the pulp of ripe pods of C. fistula was investigated in comparison with the content in the mature leaves. The results show that the content of total anthraquinone glycosides in the pod is 0.4% dry weight while the contents in the leaves collected in Winter, Summer and Rainy season are 0.5, 0.15 and 0.16% dry weight, respectively [11]. Thus, it is possible to use C. fistula leaves, which can be collected any time of the year especially in Winter, as a laxative drug instead of the ripe pods that have only once a year. 4. Senna siamea (Lam.) Irwin & Barneby, Leguminosae-Caesalpinioideae Synonym: Cassia siamea Lamk Common names: Cassod tree, Thai copper pod, Khilek, Khilek kaen, Khilek baan, Khilek luang, Khilek yai, Phak chee-lee

Senna siamea leaves and flowers

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Part used: Young leaves and young flowers Active constituents: Young leaves and young flowers of S. siamea contain rhein, chrysophanic acid, chrysophanol, physcion. From our work, we analyzed total anthaquinone glycosides in the leaves of S. siamea collected in Winter, Summer and Rainy season and found that the contents are 0.12, 0.08 and 0.06% dry weight, respectively [11]. Geographical distribution: Cassia siamea is native of Southeast Asia and widely cultivated in the tropics. The plants are found in various types of forests at lower altitudes [5]. Senna siamea is a medium size tree. Leaves are paripinnate, composed of 7-10 pairs of leaflets. Flowers are in large terminal panicles, yellow color. Young leaves and young flowers of S. siamea are popularly used as a vegetable in Khilek curry which promotes sleeping- aid and mild laxative effect. The tranquilizing effect of S. siamea is the effect of barakol which is developed from 5-acetonyl-7-hydroxy-2-methylchronone, a major constituent of the leaves and flowers of S. siamea, while the laxative action comes from anthraquinone derivatives [5, 7]. Time of collection: The young leaves and young flowers are collected for use as a laxative drug. Traditional recipes 1.

The fresh young leaves and young flowers are prepared as food by boiling with water at a ratio of 1:3 for 1 hour 2-3 times to reduce the bitterness. The water is then discarded and the boiled leaves are mixed with coconut milk and curry paste and cooked as a curry which is consumed with cooked rice as a food producing a mild laxative effect and sleeping-aid [19]. Fresh or dried leaves of S. siamea are boiled with water, and the extract is taken before breakfast.

5. Senna tora (L.) Roxb., Leguminosae-Caesalpinioideae Synonym: Cassia tora L. Common names: Foetid cassia, Chumhet thai, Chumhet khwai, Chumhet na, Chumhet lek, Phrom dan, Lap mue noi, Ya luek luen Part used: Leaves and dried ripe seeds Active constituents: Leaves of S. tora contain chrysophanic acid, emodin, rhein [7]. Seeds contain aloe-emodin, chrysophanol, emodin, physion, rhein [7, 20].

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Senna tora leaves and flowers

Geographical distribution: Senna tora is widely distributed up to the altitude of 1,000 m. The plant is hygrophilous and heliophilous, and often forms large populations in humid soils along riversides or on abandoned grounds. In Thailand, the plants are often found as a common weed [5, 8]. Senna tora is a herb or undershrub up to 1 m high. Leaves are paripinnate with 3 pairs of leaflets. The leaflets are 2-5 by 1.5-2 cm. Flowers are in axillary, short, 1-3 flowered racemes, yellow color. Pods are 10-15 by 0.5 cm. Each pod has 20-30 rhomboidal seeds, 5 mm in diameter [5]. Time of collection: The leaves are collected before flowering. The fruits are gathered when ripe in September-November, dried, rubbed for obtaining seeds which are collected and dried again to complete dryness. The seeds should be stir-fried or charred before use [8]. Traditional recipes [21, 22] 1. Leaves, 15-30 g, is boiled with a glass of water, add 1-2 dried fruits of cardamom and a little bit of salt to make better smell and taste of the extract, taken the extract before breakfast. 2. Boil 8-12 g of the dried seeds with a glass of water, add 1-2 dried cardamom fruits and a little bit of salt, take the extract before breakfast.

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Heat the dried seeds in a hot pan to produce nice smell. Make infusion by putting a glass of hot water and drink as tea.

6. Ricinus communis L., Euphorbiaceae Common names: Castor, Castor oil plant, Lahung, Mahong, Lahung daeng Part used: Castor oil from mature seeds Active constituents: Mature seeds contain 45-55% of castor oil of which 90% of fatty acids are ricinoleic acid, a monounsaturated, 18-carbon fatty acid with a hydroxyl functional group at the twelfth carbon. This functional group causes ricinoleic acid and castor oil to be unusually polar, and also allows chemical derivatization that is not practical with other biological oils. Castor oil also contains 3-4% of oleic acid and linoleic acid. The Castor seed contains ricin, which is a toxic protein. Ricin can be removed by cold pressing and filtering [3]. Geographical distribution: Ricinus communis is native to India. The principal producing countries are Brazil, India, China, the Soviet Union and Thailand [23]. Castor oil is a fixed oil obtained from the seeds of R. communis which is a shrub or small tree, up to 6 m high. The fruit is a three-celled thorny capsule containing one seed in each cell. The seeds show considerable differences in size and colour. They are oval, 8-18 mm long and 4-12 mm broad. The testa is smooth, thin and brittle. The colour may be grey, brown or black, or may be variously mottled with brown or black [23]. Preparation of castor oil: The seeds are deprived of their testas and the kernels are cold-expressed in suitable hydraulic presses. The oil is refined by steaming, filtration and bleaching. Cold expression yields about 33% of medicinal oil. Medicinal castor oil is a colourless or pale yellow liquid, with a slight odour, faintly acrid taste, and high viscosity [23]. Pure cold pressed castor oil is really tasteless and odorless. When additives are added to the pure cold pressed oil, it becomes adulterated and the taste and smell can be changed. At present, FDA recognizes castor oil as generally safe and effective for over-the-counter use as a laxative drug. When the castor oil is taken, it is converted into ricinoleic acid which is the active laxative agent, in the gut. It directly acts on intestinal mucosa or nerve plexus and alters water and electrolyte secretion. Castor oil is preferred when more complete evacuation is required [3]. Dose: 15 ml of castor oil as a purgative and lubricant [1].

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7. Aloe barbadensis Mill., Liliaceae Synonym: A. vera L. Part used: Dried latex from leaves Active constituents: Dried latex of the leaves of Aloe spp. has been known as Aloe which is a reddish-black glistening mass containing anthraquinone glycosides, aloin A and B. The bitter yellow latex is obtained from a pericyclic tubules beneath the epidermis of the leaves. The latex or juice is often drained from the transversely cut leaves, concentrated by boiling and solidified on cooling [23]. After being dried, it yields Aloe. Aloe is official in the USP XXII for a potent laxative drug. The official varieties of Aloes are the Cape from South Africa and Kenya, and the Curacao from the West Indian Islands of Curacao, Aruba and Bonaire [23]. Cape Aloe contains not less than 18%, while Curacao Aloe contains not less than 28% of hydroxyanthracene derivatives, expressed as barbaloin. Aloe is used in forms of powder and preparations for oral use for shortterm treatment of occasional constipation. The mechanism of action is stimulating colonic motility, augmenting propulsion and accelerating colonic transit, which reduces fluid absorption from the faecal mass [12]. The solid residue, Aloes, is mainly obtained from various species of Aloe i.e. Cape Aloe from Aloe ferox; Curacao Aloe from A. barbadensis, Socotrine and Zanzibar Aloes from A. perryi. The genus Aloe includes herbs, shrubs and trees, bearing spikes of white, yellow or red flowers. Aloe leaves are fleshy, strongly cuticularized, and are usually prickly at the margins [23]. Geographical distribution: Aloe spp. are native to Africa. They need an abundance of sunshine and a well drained, porous soil [23]. Dose: The dose recommended for adults and children aged over 10 years is 10-30 mg of hydroxyanthracene derivatives (calculated as aloin) once daily at night [24]. 8. Rheum officinale Baill. and R. palmatum L., Polygonaceae Common names: Rhubarb, Chinese Rhubarb Part used: Dried rhizomes and roots, deprived of periderm tissues. Active constituents: The dried rhizomes and roots contain anthraquinone glycosides and their aglycones such as aloe-emodin, emodin, chrysophanol, physcion, rhein and sennosides A and B. They also contain tannins including gallic acid, epicatechin gallate and catechin [24, 25]. The B.P. drug is required to contain not less than 3.0% of hydroxyanthraquinone derivatives calculated as rhein [23].

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Geographical distribution: Rheum officinale and R. palmatum or other species (excepting R. rhaponticum L.) are native to China and Tibet. The production of Rhubarb extends over a large area of China. R. officinale is collected chiefly in the mountainous area, Tibet, Szechuen and Hupeh of China. R. palmatum is abundant in the province of Kansu and Tibet [1]. R. officinale and R. palmatum are perennial herbs. Their underground portions consist of strong vertical rhizomes with fleshy, spreading roots. The above-ground portions consist of a number of long petioled leaves which arise from the rhizomes. The lamina is cordate to orbicular, entire or coarsely dentate (R. officinale) or palmately lobed (R. palmatum). The fruit is an achene with 3 broad thin wings and surrounded at its base by the remain of the perianth [1]. Time of collection: The rhizomes and roots are dug up late in September from plants 8-10 years old. The Shensi Rhubarb, yielded by R. palmatum, is esteemed as the finest variety [1]. Traditional uses Rhubarb has been used both as a laxative and an anti-diarrhoeal agents. The laxative action is dued to anthraquinone derivatives, while astringent action comes from tannin components. Its use always causes intestinal griping and is seldom employed as a laxative now a day [2]. Dose: Small doses (0.05-0.5g) have an anti-diarrhoeal action while larger doses, i.e., 1-3 g have laxative effect after 6-10 h used in the forms of powder, extract and tincture, frequently mixed with other laxatives [25]. 9. Ocimum americanum L., Labiatae Synonym: O. basilicum L. var. citratum, O. canum Sims Common names: Hairy basil, Mang lak, Kom ko khao, I tu Past used: Dried ripe seeds Active constituents: The seeds contain mucilages, pentosans, polysaccharides and sugars. Geographical distribution: Ocimum americanum are widely spread in tropical Africa, India, Ceylon to South China and Malaysia. They are distributed in open waste places and also commonly planted in gardens [5]. Ocimum americanum is an erect herb, 30-50 cm high. Leaves are simple, opposite, 2.5-5 by 1-2.5 cm. White or purple inflorescences are in terminal raceme-like, simple or branched 7-15 cm long. Fruits are composed of dry 1-seeded ellipsoid nutlets, 1-2 mm long, black, dotted [5].

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Time of collection: The black ripe seed is collected. Traditional uses The seeds are soaked in water until they are fully swollen. The swollen seeds are taken as laxative, or mixed with syrup and coconut milk, taken as a dessert. The outer seed coat can swell about 45 times and is not digested in the digestive tract, it acts as a bulk laxative. The dried mucilage powder from the seeds has been made in various dosage forms such as powder, capsule and tablet for a laxative drug [5]. The seeds may be used as a bulk laxative in post-operative patients [26]. When 2 teaspoonfuls of the seed is soaked with a glass (240 ml) of water and the mixture is taken, the laxative effect is found to be the same as taken 2 spoonfuls of Psyllium seeds. The seeds of O. americanum are used as a bulk laxative by increasing the quantity of faeces and lubricating the intestine. The seeds should be prepared with sufficient water for complete swelling before taking in order to prevent dehydration and intestinal obstruction [5]. 10. Scaphium scaphigerum (G.Don) Guib. & Planch., Sterculiaceae Synonyms: S. macropodum Beaum. Sterculia scaphigera Wall. Common names: Phung thalai, Samrong, Buk jong, Jong Part used: Dried ripe fruits Active constituents: The fruits of S. scaphigerum contain 56-69% of dietary fiber which can swell in water around 10 times. The dietary fiber is composed of approximately 8% of soluble fiber, 90% of insoluble fiber, and 1% of polysaccharides [27]. Geographical distribution: Scaphium scaphigerum is always found in the florest and mountain area. In Thailand, the plants are distributed in the West of the country, especially in Chantaburi province [27]. S. scaphigerum is a medium to large sized tree, up to 45 m high. Mature fruits are brown color, thin pericarp. A part of fruit expands as a boat shape wing, called â&#x20AC;&#x153;Sum paoâ&#x20AC;?, which can flow for a long distance. The mesocarp of the fruit contains mucilages which can be swollen very well in water yielding brown soft sponge material. This sponge is used to prepare a drink for health and acts as a bulk laxative [27]. Time of collection: The ripe fruits are gathered for using as a laxative drug. Traditional uses Macerate the dried mature fruits in water for 1-3 hours or until complete swelling, then separate the swollen sponge material from the seeds. The sponge is

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Fruit of S. scaphigerum showing sum pao

then mixed with syrup, honey or fruit juice and crushed ice to make as a cold drink or dessert and to promote bulk forming laxative effect [27]. 11. Plantago afra L., Plantaginaceae Synonym: Plantago psyllium L. Part used: Dried ripe seeds Active constituents: Mucilages Psyllium seed or Plantago seed is the dried ripe seed of Plantago afra L. or of P. indica (known in commerce as Spanish or French Psyllium seed), or of P. ovata Forskal (known in commerce as Blond Psyllium or Indian Plantago seed). Plantago seeds contain 10-30% mucilaginous hydrocolloid which is locallized in the outer seed-coat (husk) and is the major active constituent. The mucilage is composed of a soluble polysaccharide fraction containing mainly arabinoxylans (85%). It is neither absorbed nor digested in the intestinal tract [12]. Geographical distribution: Plantago afra and P. indica are native to West Mediterranean countries, while P. ovata is native to Asia and the Mediterranean countries. The plant is cultivated extensively in India and Pakistan, and adapts to western Europe and subtropical regions [12]. P. afra is an annual, erect, glandular-hairy caulescent herb, with an erect branching stem. It possesses whorls of flattened linear to linear-lanceolate leaves from the upper axils of which flowering stalks as long as the leaves arise. The flower is tetramerous with a calyx of 4 similar persistent, lanceolate sepals. The fruit is membranous, 2-celled and 2-seeded [12].

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Psyllium seed is the most popular herbal bulk laxative. It is official in USP XXII. It can swell in water to a large volume promoting bulk laxation and lubrication. It is necessary to drink large amount of water when taking Psyllium seeds [2]. Dose: Usually 2 teaspoonfuls (about 7.5 g) with a glass of water, juice, or milk, stir throughly and drink it quickly before the mixture thicken [2]. 12. Amorphophallus spp., Araceae Common name: Bok Part used: Rhizomes Active constituents: Mucilages, glucomannan Amorphophallus spp. is a herb with brown rhizome which is rich in glucomannan and mucilages. The species which have high content of glucomannan are A. konjac C. Koch., A corrugatus N.E.Br., A. kerrii N.E.Br., etc. Geographical distribution: In Thailand, Amorphophallus which contains glucomannan can be found only in the western parts of the country [22]. Glucomannan is a water-soluble polysaccharide which can absorb water about 60 times of its dry weight yielding a viscous gel. The gel can coat the wall of the stomach and intestine. It reduces glucose and lipid absorption from the digestive tract and promotes bulk forming laxative effect. The polysaccharides from Amorphophallus rhizome is popularly prepared as food, such as noodles, instant drinks, or snacks [28]. 13. Bran Bran is the course outer coat or hull of the grain of rice (Oryza sativa L.), wheat (Triticum aestivum L.) or oats (Avena sativa L.). Bran is a cheap and abundant source of water-insoluble dietary fiber. Active constituents: Bran contains about 27% dietary fiber. When bran is taken, the fiber passes through the gut somewhat like a large wet sponge, absorbing and holding water and other compounds. The great bulk sponge increases the size of stool and decreases the emptying time of the colon [29]. In the market, bran is available in various forms such as crude material, compressed tablet, cereal and bread. Dose: Dried bran 1-2 tablespoons is put in a cup of hot water or hot milk, stir throughly, add some salt, sugar or spices, and taken. Bran should be prepared with sufficient water to prevent intestinal obstruction [28, 29].

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14. Musa sapientum L., Musaceae Common names: Banana, Kluai Part used: Ripe fruits Active constituents: Pectin Geographical distribution: Musa sapientum is common in the tropics and is native to Southeast Asia. The plant requires well-drained and moist soil [5]. Musa sapientum is a large herb with underground stem. The aerial part consists of wide overlapping leaf sheaths. The leaves are large, spirally arranged, oblong. Fruits are berry, more than 10 cm long, cylindrical grouped in hands that are born in bunches, sweet when ripe [5]. The ripe fruits contain high content of pectin, a water-soluble dietary fiber. Pectin promotes bulk laxative effect by increasing the quantity of stool and lubricating the intestine [22]. Time of collection: The fully grown but unripe fruits are collected and left until fully ripen. Dose: Take 2-4 ripe fruits daily. 15. Carica papaya L., Caricaceae Common names: Papaya, Papaw, Melon tree, Malakoo Part used: Ripe fruit Active constituents: Pectin Geographical distribution: Carica papaya is native to tropical America, naturalized in Southern Florida, and found in tropical areas throughout the world. Carica papaya is a small tree, The trunk is non-woody and hollow. The large simple leaves, which are deeply lobed, are in a terminal cluster, alternate. The inflorescences consist of racemose cymes of yellow, staminate and pistillate flowers. The fruit is a large melon-like, edible berry. When ripe, the fruit is a very desirable food. Seeds are albuminous. Lacticiferous tubes occur in roots, stem, leaves and fruits. Shalow cuts made on the surface of fully grown but unripe fruits give a milky sap or latex which after collection and drying is known as crude papain [1, 29]. The ripe fruit contains pectin and is used as a mild bulk forming laxative. Normally, it is a popular sweet fruit. 16. Tamarindus indica L., Leguminosae-Caesalpinioideae Common names: Tamarind, Indian date, Ma kham, Ta lup, Mak-kaeng Part used: Ripe pods

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Fruits of Carica papaya

Pods of Tamarindus indica

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Active constituents: The ripe pods contain pectin, mucilages, organic acids (12-15%) including tartaric acid, citric acid, malic acid and lactic acid [22]. Geographical distribution: Tamarindus indica is native to Africa. It is cultivated in India, the East Indies, Egypt and all over the tropics for their edible fruits and as the ornamental trees [1,5]. Tamarindus indica is a large tree up to 30 m high, having spreading branches with alternate paripinnate leaves and lateral racemes of yellow flowers. Each leaf comprises 8-10 pairs of leaflets. Pods are oblong, slightly curved, 5-15 by 1-2.5 cm, reddish-brown. Seeds are dark brown, embedded in a thick, sticky brown pulp. A pulpy mass has a light reddish-brown color, changing with age to a dark brown or black. The pulp contains some branching fibers and numerous seeds. The pulp is used as a mild laxative for treatment of constipation [1, 5]. Time of collection: The ripe pods are gathered, deprived of the brittle outer portion of the pericarp. Traditional recipes [22] 1. 2. 3.

Fruit pulp 70 -120 g is taken with salt, then drink large amount of water. Fruit pulp is boiled with water, add sugar and a little bit of salt to prepare as a drink. Fruit pulp is dissolved in water and mixed with salt, then it is used as rectal enema.

Dose: 15 g

Conclusion Traditional medicine is one of the oldest form of healthcare which is mainly used in a holistic manner. For the people who live in the remote villages where it is difficult to obtain modern drugs, traditional medicines are still much more necessary. In some countries, traditional medicines are still a central part of the medical system, such as Ayurvedic medicines in India, traditional Chinese medicines and Thai traditional drugs. Although the described plants have been traditionally used as laxatives for a long time, some of them have not been reported on pharmacological and clinical studies. It is interesting to find out the active compounds and confirm the laxative action of these plants. Besides being consumed as laxative preparations, they are always consumed as foods and drinks. The high content of anthraquinone glycosides are found in several Cassia and Senna leaves which are collected in Winter and Summer. These results

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support traditional way of plant collection of Thai people that the leaf drugs should be collected before the flowering period. The effectiveness of the herbal medicines mainly depend upon the proper use of authentic raw materials. A confusion of plant identification may cause by one name refers to different plants or one plant may have different dialect names. Therefore, using of corrected plant, part, dose and method of preparation is very important for traditional herbal medicines. Moreover, traditional medicines necessitated the availability of standards to ensure their quality, efficacy and safety. Thus, standardized raw materials are necessary and have to be concerned for the good quality of traditional medicines. The most popular traditional stimulant laxatives in Thailand are the leaves of Senna alata and Senna alexandrina, prepared informs of tea or decoction, while the most popular bulk laxatives are the dried ripe fruits of Ocimum americanum and of Scaphium scaphigerum prepared as drinks or desserts.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

Youngken, H.W. 1950, Textbook of Pharmacognosy, The Blakiston Company, Philadelphia. Tyler, V.E. 1994, Herbs of Choice: The Therapeutic Use of Phytomedicinal, Pharmaceutical Products Press, Inc., New York. htpp://en.wikipedia.org/ , retrived on April 10, 2007. file://F:laxative paper from net/laxative from American Academy of family Physiâ&#x20AC;Ś., retrived on 10/4/2007. Fransworh, N.R. and Bunyapraphatsara, N., Eds., 1992, Thai Medicinal Plants Recommended for Primary Health Care System, Prachachon Co. Ltd., Bangkok. Bruneton, J. 1993, Pharmacognosy, Phytochemistry, Medicinal Plants, Intercept Limited, Paris. Gritsanapan, W. 1983, Mahidol Univ. J. Pharm. Sci., 10 (3), 90. National Institute of Materia Medica. 1999, Selected Medicinal Plants in Vietnam, Science and Technology Publishing House, Hanoi. Thai Herbal Pharmacopoeia, Vol. 1, 1995, Prachachon Co. Ltd., Bangkok. Standard of ASEAN Herbal Medicine, Vol.1, 1993, ASEAN Countries, Jakarta. Gritsanapan, W., Phadungrakwitya, R. and Nualkaew, S. 2005, Investigation of Alternative Anthraquinone Sources from Cassia spp. Growing in Thailand, P. K. Mukherjee (Ed.), International Conference Proceeding on Promotion and Development of Botanicals with International Coordination, Kolkata, 120. World Health Organization, 1999, WHO Monographs on Selected Medicinal Plants, Vol. 1, Geneva. Barnes, J., Anderson, L.A. and Phillipson, J. D. 2002, Herbal Medicines 2nd ed., The Pharmaceutical Press, London. Gilman, A.G.; Goodman, L.S. and Gilman, A. 1990, The Pharmacological Basis of Therapeutics, 6th edition, Macmillan Publishing Co., Inc, New York.

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15. Godding, E.W. 1988, Pharmacology, 36 (Suppl.1), 230. 16. Bradley, P.R. 1992, British Herbal Compendium, Vol.1, British Herbal Medicine Association, Bournemouth. 17. Wamanonth, M. and Supchareon, P., Eds., 1994, Medicinal Plants for Primary Healthcare, Ong Karn Taharn Pan Suk Printing, Bangkok. 18. Pongboonrod, S. 1979, Mai Thet Muang Thai, Krungthon Printing, Bangkok. 19. Padumanonda, T. and Gritsanapan, W. 2006, Southeast Asian J. Trop. Med. Public Health, 37 (2), 388. 20. Manojlovic, I.; Bogdanovic-Dusanovic, G.; Gritsanapan, W. and Manojlovic, N. 2006, Chem. Pap., 60(6), 466. 21. Muanwongyaat, P. 1991, Handbook for Using of Medicinal Plants, Medical Media Press, Bangkok. 22. Gritsanapan, W. 1998, Samunprai Naroo, Chulalongkorn University Press, Bangkok. 23. Evans, W.C. 1992, Trease and Evansâ&#x20AC;&#x2122; Pharmacognosy 13rd ed., Bailliere Tindall, London. 24. Barnes, J.; Anderson, L.A. and Phillipson J.D. 2002, Herbal Medicines, The Pharmaceutical; Press, London. 25. Stahl, E., Ed., 1969, Drug Analysis by Chromatography and Microscopy, Ann Arbor Science Publishers Inc., Michigan. 26. Muangman, V., et al., 1985, Ramathibodi Med. J., 8(4), 154. 27. Gritsanapan, W. 2007, Journal of the Pharmaceutical Association of Thailand Under the Royal Patronage, 55(1), 11. 28. Kijchareon, N. 2006, Thai Pharmaceutical and Health Science Journal, 1(2), 153. 29. Tyler, V.E. 1993, The Honest Herbal, Pharmaceutical Products Press, New York.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Ethnomedicine: A Source of Complementary Therapeutics, 2010: 317-332 ISBN: 978-81-308-0390-6 Editor: Debprasad Chattopadhyay

12. Ethnomedicines and pharmacogenomics Biswajit Mukherjee, Biswadip Sinha and Soma Ghosh

Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, India

Introduction In the later half of the past century, after the discovery of the structure of DNA by Watson and Crick, and the beginning of this century with the decoding of the human genome, a massive advance has been made in technological development in genomics, proteomics, metabolomics and the bioinformatics processing of the massive data sets generated. Functional analysis at the level of the genome (large-scale DNA sequencing that provides insight to the heterogeneity in coding regions of genes causing polymorphisms) of gene expression (transcriptomics), of protein (proteomics), and of metabolite network and fluxes (metabolomics) is now fully developed. Pharmacogenomics, originated from the terms pharmacology and genomics, deals with heredity and the effect of drug. Medicine provided therapeutic activities are the subjects of pharmacology and when its mode of action is described through its role in genes and proteins, it becomes the subject of genomics and proteomics, respectively. Precisely, an individualâ&#x20AC;&#x2122;s response to drugs due to the individualâ&#x20AC;&#x2122;s genetic Correspondence/Reprint request: Dr. Biswajit Mukherjee, Dept. of Pharmaceutical Technology, Jadavpur University, Kolkata-700 032, India. E-mail: biswajit55@yahoo.com

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inheritance is the pharmacogenomics. Although ethnomedicines have been in use through years, but in many cases the isolated and purified forms from them and even the extracts have been included in the modern medicinal practices. Thus, to establish a correlation between ethnomedicines and pharmacogenomics is interesting and exciting. Here an effort has been made to describe a relationship between ethnomedicines and pharmacogenomics. One correlation between them is that the knowledge of ethnomedicines is far from being complete and is not properly elucidated and pharmacogenomics is still in its infancy and is used in a limited degree today. Along with the several prospects of pharmacogenomic studies, e.g. drug discovery to very specific targets (exact gene(s) involved) in diseases, personalized medicines, advance screening of diseases, production of better vaccines etc., pharmacogenomics also can describe the genes involved and complexity of genetic variations, that affect drug response. Discovery of genetics makse it possible to understand the effect of ethnomedicines on various types of gene expressions in the body. The knowledge of relationship between ethnomedicines and gene expressions may provide insight into prevention of diseases and improvement of quality of life through ethnomedicines. We have summarized, analyzed and correlated below some of the studies available in the literature related to some ethnomedicinal substances and expressions of various genes in the body. Such studies can elevate ethnomedicines to an entirely new horizon.

Cell signaling and pharmacogenomics Today there is a remarkable progress in understanding the basic principle of gene control in higher organisms. Most genetic differences between people are complex and many genes contribute. Regulations for most genes consist of the control of the initiation of gene transcription and this essentially depends on gene regulatory proteins that specifically bind to specific DNA sequences. Effect of the individual chemical moiety or the combinatorial effect of the different chemical moieties as found in the plant extracts, on a large network of a number of positive and negative regulatory proteins controlled by various genes determines the drug response. This complex network of transcriptional signals either depends on or activates a complex protein kinase signal transduction cascade (Maroni et al, 2004). Some signaling molecules used by living system carry signal over long distances, whereas others can act locally to convey information in and between neighbouring cells. The drug molecules enter into cells by receptor protein. They then tranduce signals through intracellular signaling proteins which ultimately lead to synthesize target proteins such as metabolic enzymes, gene regulatory proteins and cytoskeletal proteins which provide altered

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metabolism, altered gene gene expression and altered cell shape or movement, respectively (Fig.1). Among the cell membrane receptor proteins, G-protein linked receptor and receptor tyrosine kinase play enormous roles in signal transduction of signal molecules such as drugs. G-proteins usually act through adenyl cyclase and cAMP via PKA and regulate gene regulatory proteins. Alternatively, G-protein communicates through inositol pyrophosphate or diacyl glycerol and protein kinase C or CaM-Kinase to regulate gene regulatory proteins and many other target proteins. Drug binding with receptor tyrosine kinase can also trigger phospholipase C-diacyl glycerol-PKC pathway to provide the same action. However in most cases drug molecules acting via the PDK receptor follow map kinase or phosphoinositol kinase pathway to provide drug action (Fig 2). One of the most important and extensively investigated signaling cascades responsible for the growth, differentiation and survival of the most cell types is MAP kinase pathway. Schematic presentation of this pathway is depicted in Fig 3 for general interest of readers. This pathway is known to participate intensively in drug molecule signaling (Boldt et al, 2002).

Figure 1. Signal transduction through intracellular signaling proteins (Alberts et al eds, 2002, Fig. 15-1).

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Figure 2. Cascading of signals through G protein linked receptor and receptor tyrosine kinase (Alberts et al eds, 2002, Fig. 15-61).

Figure 3. Schematic representation of the mammalian map kinase pathway (Johnson & Lapadat, 2002).

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Genetic changes of the enzymes responsible for drug-metabolism lead to metabolic alteration of a drug which lead to unprecedented or unpredicted outcome. As an example, Kalow and Bertilsson (1994) have compared the metabolism capacity of codeine in Swedish and Chinese population. The three primary metabolism pathways: glucuronidation, n-demethylation to form nor-codeine, and o-demethylation to form morphine, were found to differ between these two populations. The known ethnic variation of CYP2D6 could explain the slow morphine formation in Chinese population, but no single enzyme change is known to account for the metabolic differences in glucuronidation and in nor-codeine formation in them. The CYP2D6 is the most variable human cytochome. In a list of eleven different drug metabolizing CYPs, many carry several mutations and cause 97 protein changes (Cascorbi, 2003). This substantiates the reason of genetic variation and variable drug action within and between the species.

Ethnomedicines: Present perspectives Classical pharmacopoeial ethnomedicinal preparations like tinctures, fluid extracts etc. are of special importance for empirically prepared products. But their use in industrially prepared herbal medicines is very limited. The herbal medicines must be based on a uniform definition of the characteristics of the individual extract. Complex herbal products represent specific problems in standardization, harmonization and manufacturing. In numbers of cases they are manufactured in a non-industrial scale by many small manufacturing units. Such preparations maintain their manufacturing secrecy. The problem related to the confidentiality of data concerning the manufacture of an active ingredient is of paramount importance for the manufacture. Nevertheless, it is important for the licensing authority to achieve sufficient information to perform proper assessment of the quality of active ingredients. Repeatability is important in the validation of a method in industry whereas reproducibility of the method is more important for incorporating them in pharmacopoeia. Thus the development of suitable methodologies is significant. The development and assay procedures for herbal products are an important area to be taken care. The approach to general analytical methods has to be magnified compared to the currently available methods. The fast evolution in this field makes the description of such methods rapidly obsolete. Both the in vitro and bioassay studies need to judge every substance as a single entity. Standard biological references are also needed to establish the pharmacological activities of herbal components. Suitable methodology should be developed for each active constituent to emphasize the use of active stereoisomer and to restrict the inactive, toxic or less active chiral components. Toxicological

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testings by licensing authority may not be unnecessarily excessive, but should be stringent enough to assure the consumers regarding the safety profiles of such formulations. Again use of validated, product-specific methods rather than one of the official methods should be considered by the licensing authority to analyze each product.

Genetic modulation by ethnomedicines Bioactivities of the isolated and pure herbal compounds and the herbal extracts used in popular folk medicine have been studied by many scientists to understand the pharmacodynamics of them. A mixture of multiple phytocompounds or plant extract can antagonize a single compound isolated from the extract. Again, the cumulative effect of ingredients present in an extract may be different or more potential than the individual isolated active compound itself. Lack of enough scientific evidences and acceptance could not yet substantiate the importance of medicinal herb extracts or phytocompound mixture as defined therapeutic agents. Due to the advent of DNA microarray analysis of global gene expression profile, characterization of the bioactivities of the herbal extracts at the genetic levels is possible and this opens up new avenue to study the pharmacogenomic activities of various ethnomedicines. Yang et al (2004) have compared a plant extract fraction and a single phytocompound for the treatment of human breast carcinoma. They have demonstrated level of complexity in global gene expression pattern for both the extract and the isolated compound. Analysis of microarray data comprising sets of previously known genes and unknown genes showed that 39 known genes were upregulated and 20 genes were downregulated in the extract treated cells whereas 50 known genes were upregulated and 30 known genes were downregulated in the single phytocompound treated cells resulting in strong and specific inhibition of proliferation of human breast carcinoma. Had there been no differences between the extract and pure compound, both the extract and pure compound could have similar effects. A patientâ&#x20AC;&#x2122;s genetic constitution is one of the factors responsible for adverse effects of herbal preparations or ethnomedicine (De Smet, 1995; Huxtable, 1990). Thus knowledge of genetic variation in different ethnic groups could provide insight in pharmacological adverse effects of different herbal medicines. Watanabe et al (2001) have used microarray technique to study the effect of Ginkgo biloba on some gene expressions in cortex and hippocampus of mice and they have demonstrated about three fold changes in the expression of mRNAs. Some researchers have examined the effect of Chinese folk medicine Shosaiko-to (SST) on lung cytokines level and on

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lipopolysachcharide induced lung injury in mice, which are known to show different immune responses due to differences in genetic background (Ohtake et al, 2002). Their reports suggest that SST may have different, possibly even opposite effect on lung immunity in hosts with different genetic background. Oxidative stress is one of the reasons for damage of DNA structure. Melo et al (2001) have demonstrated that different plant crude extracts have preventive effect against the oxidative stress induced damage of E.coli AB 1157 (wild type) strain, resulting the increased survival of the E.coli strain. They suggested that compounds in the crude extract could form chelate with stannus ions, protecting against the oxidation and avoiding the generation of ROS (reactive oxygen species) and could scavenge the ROS generated by SnCl2 and/or the extract have oxidant compounds that could oxidize the stannus ions abolishing or reducing the SnCl2 effects. Researchers at premiere research institutes are now engaged in cellular and pharmacogenomic profiling of natural products. Konkimalla and Efferth (2008) have created anticancer natural product library from traditional Chinese medicine and they have performed cellular and pharmacogenomic profiling for the ten most cytotoxic natural products. They have studied one compound helebrin in details by identifying candidate genes which are predicted for sensitivity or resistance of cell lines to hellebrin. Efferth et al (2008) have identified miltirone from Saliva miltiorrhiza and by hierarchical cluster analysis, they have identified candidate genes upregulated or downregulated by miltirone. The study have also predicted sensitivity or resistance of cell lines to miltirone.

Some ethnomedicinal compounds, their pharmacology and pharmacogenomics Ethnomedicines are mainly plant chemicals. Health professionals are gradually recognizing the role of phytochemicals (chemicals found in plants) in health management. Some important compounds from these ethnomedicines have been discussed below. Although dearth of data makes it difficult to correlate the chemicals and the genes in and/or between species, we have tried to establish the role of the phytochemicals in modulation of various genes interacting with those chemicals in individual case specific studies.

Quinine and its derivatives The bark of Cinchona tree, growing in Peru, was introduced in Europe in the early 17th century as a cure for fever. Later it was realized to be a specific remedy for malaria. Quinine, isolated from Cinchona bark in 1820, replaced the crude preparation and continued to be the major antimalarial drug till

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1942. Later Chloroquine was prepared as a quinine derivative. The mechanism of action of Chloroquine at the genetic level is not clearly known. It is actively concentrated by sensitive intraerythrocytic plasmodia. Higher concentration is found in infected RBCs. Probably by accumulating in the acidic vesicles of the parasite and because of its weakly basic nature, it raises the vesicular pH and thereby interferes with degradation of haemoglobin by parasitic lysosomes (Yayon et al, 1985). Quinine has been found to inhibit conversion of toxic heme to nontoxic parasite pigment hemeazoin. Heme then damages the plasmodial membranes. Clumping of pigment and changes in parasite membranes follow. The earlier hypothesis that it acts by interfering with DNA function is now considered untenable. Quinine was also found to downregulate other genes like mdr1 gene expression in Chinese hamster ovary cell lines LR73 (Belhoussine et al, 1997). Though this has no connection with malaria treatment, however this claims that the drug has role to modulate genes.

Reserpine It is an alkaloid from the roots of Rauwolfia serpentine (sarpagandha) indigenous to India which has been used in â&#x20AC;&#x2DC;Ayurvedicâ&#x20AC;&#x2122; medicine for centuries. The pure alkaloid was isolated in 1955 and later found to cause catecholamine and 5-HT depletion. It was a popular antihypertensive of the late 1950s and early 1960s, but is now used more as a pharmacological tool than a therapeutic agent. Reserpine acts at the membrane of intraneuronal granules which store monoamines (noradrenaline, 5-HT, and dopamine) and irreversibly inhibits active amine transport resulting the gradual depletion and degradation of the monoamines by monoamine oxidase (MAO). The hypotensive action of Reserpine is primarily due to its effect on depletion of noradrenalin from peripheral adrenergic nerve endings described above. It produces slowly developing fall in blood pressure and bradycardia. Higher doses deplete catecholamines and 5-HT in the brain as well; causes sedation and mental depression. Antipsychotic effect (mild) and extrapyramidal symptoms are produced due to dopamine depletion. Reserpine can regulate the transcription of various genes such as genes for phenylethanolamine-nmethyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal glands (Schalling et al, 1988).

Cocaine It is a natural alkaloid from leaves of Erythroxylon coca, a South American plant growing on the foot hill of Andes. The natives of Peru and Bolivia habitually chew these leaves. Cocaine is a good surface anaesthetic and is rapidly absorbed from buccal mucous membrane. It was first used for ocular

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anaesthesia in 1984. Cocaine should never be injected. It is a protoplasmic poison and causes tissue necrosis. Cocaine produces prominent CNS stimulation with marked effect on mood and behaviour. It induces a sense of well being, delays fatigue and increases power of endurance. In the periphery, it blocks uptake of noradrenalin and adrenalin into adrenergic nerve endings, resulting in higher concentration of the transmitter around the receptors- sympathomimetic effect, potentiation of directly acting sympathomimetics and suppression of indirectly acting sympathomimetics. Local vasoconstriction, tachycardia, rise in blood pressure, and mydriasis are a reflection of its sympathomimetic action. Using DNA microarray technique, Scott et al (2004) have identified more than 400 human genes that are affected by long-term cocaine abuse. Shapshak et al (2006) have identified few sets of genes which were not stochastically arranged on the chromosomes, were in proximity to each other, and to other genes involved in neuropsychiatric diseases. They have hypothesized that these genes fall in transcriptionally isolated groups and that abused drugs like cocaine can trigger transcription of these genes substantially, coerce expression that may result in damage to the chromosome's control and organization of chromatin transcription machinery. Bannon et al (2005) have demonstrated that long-term cocaine use can alter gene expression within the nucleus accumbens and other brain regions playing a critical role in addiction and this alteration leads to long-term neurochemical, structural and behavioural changes. Wang and Uhl (1998) have investigated drug-induced neuroplastic behavioral responses. An approach termed subtracted differential display (SDD) was used to identify genes, expression of which is regulated by psychostimulants. They have found that rGbeta1 is one of the SDD products that encodes a rat G-protein beta subunit. The expression of this G-protein is upregulated by cocaine or amphetamine treatments in neurons of the nucleus accumbens shell region, a major center for psychostimulant effects in locomotor control and behavioral changes.

Allicin The health benefits of garlic are numerous, including cancer chemopreventive, antibiotic, anti-hypertensive, and cholesterol lowering properties. The intact garlic bulb contains an odorless amino acid, alliin, which is converted enzymatically by allinase into allicin when the garlic cloves are crushed. Allicin (responsible for the characteristic odor of fresh garlic) spontaneously decomposes to form numerous sulfur containing compounds which are likely responsible for the various medicinal effects specially chemopreventive activity. In Mycobacterium tuberculosis (MTB) infected human, allicin has been found to regulate MTB 85B gene. Therefore, it is

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claimed to be useful as an adjunct in treatment of tuberculosis (Hasan et al, 2007). Allicin induces cell arrest of gastric cancer in M phase, which may be related to the up-regulated expression of p21WAF1 and p16INK4 genes (Ha and Yuan, 2004).

Ginsenosides They are obtained from a perennial plant having effect on central nervous system (CNS), cardiovascular system, endocrine secretion, immune function, metabolism, bio-modulating action, and also acts as anti-stress and anti-aging. Most of the pharmacological effects of ginseng are attributed to ginsenosides (saponin glycoside). Ginsenosides have a steroid-like skeleton consisting of four trans-rings (Fig 4), with modifications from each other depending upon type of sugar unit (e.g. glucose, maltose and fructose), number of sugar moieties and sites of attachment of the hydroxyl group (e.g. C-3, C-6, or C-20). Ginsenosides are amphipathic in nature. The hydroxyl (-OH) group of ginsenosides allows interactions between the polar head of the membrane phospholipids as the β-OH group of cholesterol, while the hydrophobic steroid backbone can interact with the hydrophobic side chains of fatty acids. Indeed, these physicochemical interactions are greatly determined by the numbers and sites of polar hydroxyl groups on each ginsenoside. Moreover, ginsenosides have been shown to interact with numerous membrane proteins such as ion channels, transporters and receptors, which lead to a broad range of physiological activities. Ginsenoside Rg1 is one of the important compounds obtained from Panax ginseng. (Lu et al, 2004) have demonstrated that ginsenoside Rg1 affected the expressions of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-α stimulated human umbilical vein endothelial cells (HUVECs). They concluded that Ginsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-α stimulated HUVECs. Ginsenoside Rg1 regulates sets of genes (Table 1) in endothelial cells and protects endothelial cells from TNF-α activation. By microarray analysis they have provided valuable insights into the atheroprotective mechanism by gingsenoside Rg1. Morphine The dark brown, resinous material obtained from poppy (Papaver somniferum) capsule is called opium which has been known from ancient times. Opium eating became a social custom in China in the 18th century. Serturner, a pharmacist, isolated the active principle of opium in 1806 and

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Compound Protopanaxadiol type Rb1 Rb2 Rc Rd Rg3 Rh2 Compound K (C-K) 20 (S)-protopanaxidol (ppd)

-glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc -H -H

-H -H -H -H -H -H -H -H

-glc(6-1)glc -glc(6-1)ara(p) -glc(6-1)ara(f) -glc -H -H -glc -H

Protopanaxitriol type Re Rg1 Rg2 Rh1 F1 20(S)-protopanaxitriol (Ppt)

H H H H H H

-O-glc(2-1)rha -O-glc -O-glc(2-1)rha -O-glc -OH -OH

glc glc H H glc H

Figure 4. The chemical structure of ginsenosides (Yue et al, 2007). glc = glucosyl (C6H11O6-); rha = rhamnosyl (C6H11O5-); ara = arabinosyl (C5H9O5-); p = pyran; f = furan.

named it â&#x20AC;&#x2DC;morphineâ&#x20AC;&#x2122; after the Greek God of dreams Morpheus. Morphine has site specific depressant and stimulant actions in the CNS. It is a strong analgesic. Suppression of pain perception is selective, without affecting other sensations or producing proportionate generalized CNS depression. The analgesic action of morphine has spinal and supraspinal components. It acts

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Table 1. Ginsenoside Rg 1-induced alterations in gene expression in TNF-α stimulated endothelial cells (Lu et al, 2004). Name of the Rg1 regulated gene Cell Adherence ICAM-1 VCAM-1 E-selectin Vascular Constriction Endothelin receptor type B NOS3 Cell Growth Basic fibroblast growth factor Insulin like growth factor –I TGF-beta 1 Epidermal growth factor (beta urogastrone) VEGF-B VEGF-C Cyclin-dependant kinase inhibitor 1A (p21, Cip1) (CDKN1A) Insulin like growth factor binding protein 1(IGFBP1 Latent transforming growth factor beta binding protein 4) Coagulation PAT-1 Fibrinogen alpha chain Tissue factor pathway inhibitor Coagulation factor XIII, A1 Coagulation factor II (Thrombin) receptor (F2R) Signal Transduction PKC type beta 1 Early growth response 1 Phospholipase A2, group X (PLA2G10) Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3 C1) Fms related tyrosine kinase 1 (Vascular endothelial growth factor / vascular permeability factor receptor) (FLT 1) Guanine nucleotide binding protein (G protein), beta polypeptide 3 (GNB 3) Hepatocyte nuclear factor 4, alpha (HNF4A) A kinase (PRKA) anchor protein (gravin) 12 (AKAP12) Others lipase A, lysosomal acid, cholesterol esterase (LIPA) Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) Carboxyl ester lipase (bile salt-stimulated lipase) (CEL)

Gene bank access number

Ratio (drug/modle)*

XM_008897 X 53051 NM_000450

↓0.45 ↓0.63 ↓0.52

M74921 AF400594

↑1.85 ↑1.79

NM_002006 NM_00068 XM_008912 NM_001963 NM_003377 NM_005429 NM_000389

↓0.60 ↓0.65 ↓0.61 ↓0.54 ↓0.42 ↓0.62 ↑1.91

NM_000596

↓0.61

NM_003573

↓0.52

NM_000602 M 64982 XM_002672 NM_000129

↓0.65 ↓0.63 ↓0.56 ↓0.48

NM_001992

↓0.46

X 06318 NM_001964 NM_003561 NM_000176

↓0.62 ↓0.66 ↑1.70 ↓0.51

NM_002019

↓0.59

NM_002075

↓0.65

NM_000457

↓0.61

NM_005100

↑1.56

NM_000235

↑1.75

NM_001776

↓0.66

NM_001807

↑1.61

*mean of ratios from three independent experiments

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in the substantia gelatinosa of dorsal horn to inhibit release of excitatory transmitters from primary afferents carrying pain impulses. The action appears to be exerted through interneurones which are involved in the ‘gating’ of pain impulses. Release of substance P from the primary pain afferents in the spinal cord and its postsynaptic action on dorsal horn neurons is inhibited by morphine. Action at supraspinal sites in medulla, mid-brain, limbic and cortical areas may alter processing and interpretation of pain impulses as well as inhibitory impulses through descending pathways to the spinal cord. Miyagi et al (2000) have demonstrated that morphine induces the expression of CCR5 receptor which suggests a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations. Spijker et al (2004) have demonstrated that profound gene expression changes (krox-20, Arc, c-fos) occur not only during morphine exposure but also long after cessation of morphine administration (fra-2, Arc, c-fos). The phasespecific expression profiles highlight distinct activities of it on functional groups of genes. They have concluded that gene expression changes not only involve elements of synaptic transmission (receptors and interaction partners), but also include proteins implicated in neuronal plasticity.

Vinca alkaloids Vincristine and Vinblastine are two major alkaloids obtained from the plant Catharanthus roseus and other Catharanthus species. These are used as antimitotic and anti-microtubule agents. Synthetic Vincristine and Vinblastine are used as drugs in cancer therapy and as immunosuppressive agents. Vindesine and Vinorelbine are two other alkaloids obtained from Catharanthus sp. Periwinkle extracts and derivatives, such as vinpocetine, are also used as nootropic drugs also referred as cognitive enhancers or brain enhancers. Vinca alkaloids bind to the β-tubulin subunit of the α/β-tubulin heterodimer and inhibit polymerization of microtubules. Liu et al (2007) have demonstrated that Vincristine could induce a significant expression of HIF-1alpha gene in gastric cancer cell induced by an upregulated protein MGr1-Ag/37LRP.

Taxol Taxol was isolated from the bark of the Pacific yew tree, Taxus brevifolia. When it was developed commercially by Bristol-Myers Squibb (BMS) the generic name was changed to 'Paclitaxel' and the BMS compound is sold under the trademark 'Taxol'. Paclitaxel works by interfering with normal microtubule

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growth during cell division. Paclitaxel altered the expression pattern of genes involved in various biological functions including cell cycle regulation and cell proliferation, apoptosis, signal transduction and transcriptional regulation, fatty acid biosynthesis and sterol metabolism, and IFN-mediated signaling (Bani, 2004). Moos et al (1999) hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. The Inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to beneficial and adverse effects encountered during taxol administration.

Conclusion The genetic modulations by herbal compounds in isolated forms or in galenicals have been demonstrated by many above mentioned studies. Regulatory alteration of the genes varies degree of gene expressions in individuals. Some individuals become addicted quickly to morphine and others take more time to be addicted. This fact claims the differential behavioral problems of gene-regulatory mechanisms among humans. Therefore, it is important to correlate the modern science of pharmacogenomics with the ethnomedicines which remain the support of survival of humans through thousands of years. The area should be explored in a systematic, distinctively reliable and elaborative way to substantiate and authenticate the status of ethnomedicines in the present world, effects of them in individual patients when used for a long-term basis, their safety, effectiveness and credibility. Discoveries in genetics make it possible to understand the effects of ethnomedicines on various gene expressions in the body. This insight helps to prevent disease and improve quality of life through ethnomedicines. From the above discussion it is clear that there are numbers of ethnomedicines that can cure numerous life threatening diseases by interactions with genes. Thus a clear knowledge of pharmacogenomics and pharmacogenomics -ethnomedicine interaction may open a new era of medical science.

References 1. 2. 3. 4.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, Molecular Biology of the Cell, 4th Edition, Fig. 15-1, 2002, available online at www.garlandscience.com. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, Molecular Biology of the Cell, 4th Edition, Fig. 15-61, 2002, available online at www.garlandscience.com. Bani MR, Nicoletti MI, Alkharouf NW, Ghilardi C, Petersen D, Erba E, Sausville EA, Liu ET and Giavazzi R, Gene expression correlating with response to paclitaxel in ovarian carcinoma xenografts, Mol Cancer Ther, 2004, 3(2):111-121. Bannon M, Kapatos G and Albertson D, Gene Expression Profiling in the Brains of Human Cocaine Abusers, Addict Biol, 2005, 10(1): 119-126.

Ethnomedicines and pharmacogenomics

6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21.

331

Belhoussine R, Morjani H, Lahlil R, Manfait M, Evidence for reversal of multidrug resistance by quinine in LR73 cells without alteration of nuclear pirarubicin uptake and down-regulation of mdr1 gene expression, Int J Cancer, 1997, 73(4): 600-606. Boldt S, Weidle UH, Kolch W, The role of MAPK pathways in the action of chemotherapeutic drugs, Carcinogenesis, 2002, 23(11) :1831-1838. Cascorbi I, Pharmacogenetics of cytochrome p4502D6: genetic background and clinical implication, Eur J Clin Invest, 2003, 33(s2):17-22. De Smet PAGM, Health risks of herbal remedies, Drug Saf, 1995, 13: 81-93. Efferth T, Kahl S, Paulus K, Adams M, Rauh R, Boechzelt R, Hao X, Kaina B and Bauer R, Phytochemistry and pharmacogenomics of natural products derived from traditional chinese medicine and chinese materia medica with activity against tumor cells, Mol Cancer Thera, 2008, 7: 152-161. Ha MW and Yuan Y, Allicin induced cell cycle arrest in human gastric cancer cell lines, Zhonghua Liu Za Zhi, 2004, 26(10): 585-589. Hasan N, Siddiqui MU, Toossi Z, Khan S, Iqbal J and Islam N, Allicin induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes, Biochem and Biophy Res Comm, 2007, 355(2): 471-476. Huxtable RJ, The harmful potential of herbal and other plant products, Drug Saf, 1990, 5 (suppl 1): S126-S136. Johnson GL, Lapadat R, Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases, Science, 2002, 298 (5600): 1911-1912. Lu JP, Ma ZC, Yang J, Huang J, Wang SR and Wang SQ, Ginsenoside Rg1induced alterations in gene expression in TNF-Îą stimulated endothelial cells, Chin Med J (Engl). 2004, 117(6): 871-876. Kalow W and Bertilsson L, Interethnic factors affecting drug response, Adv Drug Res, 1994, 25:1-59. Konkimalla VB, Efferth T, Anti-cancer natural product library from traditional Chinese medicine, Comb Chem High Throughput Screen, 2008, 11(1):7-15. Liu L, Ning X, Sun L, Shi Y, Han S, Guo C, Chen Y, Sun S, Yin F, Wu K, Fan D, Involvement of MGr1-Ag/37LRP in the vincristine-induced HIF-1 expression in gastric cancer cells, Mol and Cellular Biochem, 2007, 303(1-2):151-160. Maroni PD, Koul S, Meacham RB, and Koul HK, Mitogen Activated Protein kinase signal transduction pathways in the prostate, Cell Commun Signal, 2004, 2(1): 5. Melo SF, Soares SF, Costa RF, Silva CR, Oliveira MBN, Bezerra RJAC, Caldeira-de-AraĂşjo A and Bernardo-Filho M, Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli, Mutation Res, 2001, 496(1-2): 33-38. Miyagi T, Chuang LF, Doi RH, Carlos MP, Torres JV, and Chuang RY, Morphine Induces Gene Expression of CCR5 in Human CEM x174 Lymphocytes, J Biol Chem, 2000, 275(40): 31305-31310. Moos PJ, Muskardin DT, Fitzpatrick FA, Effect of taxol and taxotere on gene expression in macrophages: induction of the prostaglandin H synthase-2 isoenzyme, J Immunol, 1999, 162(1):467-473.

332

Biswajit Mukherjee et al.

22. Ohtake N, Nakai Y, Yamamoto M, Ishige A, Sasaki H, Fukuda K, Hayashi S and Hayakawa S, The herbal medicine Shosaiko-to exerts different modulating effects on lung local immune responses among mouse strains, Int Immunopharmacol, 2002, 2(2-3): 357-66. 23. Schalling M, Dagerlind A, BrenĂŠ S, Hallman H, Djurfeldt M, Persson H, Terenius L, Goldstein M, Schlesinger D and HĂśkfelt T, Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine, Proc Natl Acad Sci USA, 1988 ,85(21): 8306-8310. 24. Scott et al, Society for Neuroscience's annual meeting, New Orleans, November, 2004. 25. Shapshak P, Duncan R, Nath A, Turchan J, Pandjassarame K, Rodriguez H, Duran EM, Ziegler F, Amaro E, Lewis A, Rodriguez A, Minagar A, Davis W, Seth R, Elkomy FF, Chiappelli F and Kazic T, Gene chromosomal organization and expression in cultured human neurons exposed to cocaine and HIV-1 proteins gp120 and tat: drug abuse and Neuro AIDS, Front Biosci, 2006, 11: 1774-1793. 26. Spijker S, Houtzager SWJ, De Gunst MCM, De Boer WPH, Schoffelmeer ANM and Smit AB, Morphine exposure and abstinence define specific stages of gene expression in the rat nucleus accumbens, FASEB, 2004,18: 848-850. 27. Wang XB and Uhl GR, Subtracted differential display: Genes with amphetaminealtered expression patterns include calcineurin, Mol Brain Res, 1998, 53(1-2): 344-347. 28. Watanabe CMH, Wolffram S, Ader P, Rimbach G, Packer L, Maguire JJ, Schultz PG and Gohil K, The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba, Proc Natl Acad Sci U S A, 2001, 98(12): 6577-6580. 29. Yang NS, Shyur LF, Chen CH, Wang SY and Tzeng CM, Medicinal herb extract and a single-compound drug confer similar complex pharmacogenomic activities in MCF-7 cells, J Biomed Sci, 2004, 11(3): 418-422. 30. Yayon A, Cabantchik ZI and Ginsburg H, Susceptibility of human malaria parasites to chloroquine is pH dependent, Proc Nati Acad Sci USA, 1985, 82(9): 2784-2788. 31. Yue PYK, Mak NK, Cheng YK, Leung KW, Ng TB, Fan DTP, Yeung HW and Wong RNS, Pharmacogenomics and the Yin/Yang actions of ginseng: antitumor, angiomodulating and steroid-like activities of ginsenosides, Chin Med, 2007, 2: 6 (doi:10.1186/1749-8546-2-6).