International journal of scientific and innovative research 2017; 4(2)p issn 2347 2189, e issn 2347

VOLUME – 4, ISSUE – 2 (JULY-DECEMBER) 2016

Editorial

I am happy to convey that second issue of third volume of "International Journal of Scientific and Innovative Research (IJSIR)", a bi-annual journal has been published by Sky Institute, Lucknow in an effort to promote multidisciplinary scientific and innovative research of societal benefit. This journal covers all branches of science, technology, engineering, health, agriculture and management. Research articles in the field of education are also encouraged in order to promote educational technology aiming at improvement in present educational system. As research and development (R & D) has been playing a significant role in overall development of society, continuous multidisciplinary innovative research in science and technology is needed to address the challenges in context to changing environmental conditions in the present era of gradual increase in industrial and technological advancement at global level. Efforts should be made to develop eco-friendly technologies in order to provide solutions for developing socially, economically and culturally sustainable society.

The present issue of International Journal of Scientific and Innovative Research (IJSIR) contains 12 research papers I articles covering different areas of science and technology. All these papers are well written and informative in content. I express my sincere thanks and gratefulness to Mr. Mohit Bajpai, Chairman, Sky Institute, Lucknow (U.P.), India for his support in publishing it. I express my thanks to members of Committee for Editorial Assistance Dr. B.C.Tripathi, Dr. Pankaj Verma, Shri Sanjay Pandey, Shri Sanjay Dixit and Mr. Shamshul Hasan Khan for their hard work and devotion in giving the final shape to the journal. I am thankful to all faculty members, scientists and research scholars of different universities, research organizations and technical institutions for contributing their research articles for publication in the present issue of the journal. The help provided by faculty members and supporting staff of Sky Institute in publishing the present volume of the journal is also acknowledged. I hope scientists, academicians and young researchers will be greatly benefited by this publication for their research work.

I request humbly to the readers and contributors of our journal to continue encouraging us for regular publication of the journal. Any suggestion and comment for the improvement in the quality of the journal are always welcome.

Dr. B. R. Pandey Editor-in-Chief

International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

EDITOR-IN-CHIEF Dr. B.R. Pandey Director (Research) Sky Institute, Kursi Road, Lucknow, U.P, India

Dean, Faculty of Science & Technology, Sai Nath University, Jharkhand, India Former Joint Director, Council of Science & Technology, UP, Lucknow (Department of Science and Technology, UP Government), India Former Professor, International Institute of Herbal Medicine (IIHM), Lucknow, U.P., India E-mail Id: editorijsir02@gmail.com, Mobile-: 9794849800

COMMITTEE FOR EDITORIAL ASSISTANCE Dr. B.C.Tripathi Assistant Prof., Deptt. of Education, Rama P.G. College, Chinhat, Lucknow, Uttar Pradesh

Dr. Pankaj Verma Senior Research Fellow, Deptt. of Oral & Maxillofacial Surgery, Faculty of Dental Sciences, K.G. Medical University, Lucknow, Uttar Pradesh

Shri Sanjay Pandey Assistant Prof., National Institute of Fashion Technology, Raebareli, Uttar Pradesh

Shri Ashish Tiwari Research Scholar, Sai Nath University, Ranchi, Jharkhand

Shri Sanjay Dixit Scientist, Sky Institute, Lucknow, Uttar Pradesh

Shamshul Hasan Khan Scientist, Sky Institute, Lucknow, Uttar Pradesh

ADVISORY BOARD Prof.(Dr.)S. P. Ojha

Prof. (Dr.) R. L. Singh

Former Vice Chancellor, CCS Meerut University, Meerut, Uttar Pradesh

Prof & Head, Department of Biochemistry & Coordinator Biotechnology Program , Dr. R. M. L. University Faizabad, Uttar Pradesh

Prof.(Dr.)V.K. Srivastava Former Prof & Head, Deptt. of Community Medicine King George Medical

Dr. Sarita Verma

University, Lucknow.

Former Director, Integral Institute of Medical Sciences & Research, Integral University, Lucknow Former Vice -Chancellor, Texila American University, Georgetown, Guyana, South America

Head, Deptt. of Home Sci., Mahila P.G. College, Kanpur, Uttar Pradesh

Prof.(Dr.) M.I. Khan Prof & Head, Deptt. of Mechanical Engg., Integral University, Lucknow, Uttar Pradesh

Prof. (Dr.) S.K. Avasthi Former Director, H.B.T.I., Kanpur, Uttar Pradesh

Prof.(Dr.) Amrika Singh Prof & Head (Chemistry), Deptt. of Applied Sciences, Institute of Engg. & Technology, Sitapur Road, Lucknow, Uttar Pradesh

Prof. (Dr.) U.N. Dwivedi Prof & Ex- Head, Deptt of Biochemistry, Former Pro- Vice Chancellor, Former Dean, Faculty of Science, University of Lucknow, Lucknow, U.P.

Prof. (Dr.) U.K. Misra Head, Deptt. of Neurology, Ex Dean, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, U.P.

Dr. A.K. Gupta Former Deputy Director General, Indian Council of Medical Research (ICMR), Ansari Nagar, New Delhi

Prof.(Dr.) V.K.Tondon Former Prof & Head, Deptt. of Chemistry, Ex- Dean Faculty of Science, University of Lucknow, Lucknow, Uttar Pradesh

Prof. (Dr.) Amod Kumar Tiwari, Prof.- Director, Bhabha Institute of Engg.& Technology, Kanpur, U.P.

Prof.(Dr.) Chandra Dhar Dwivedi Former Prof. & Chairman, Deptt. of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Borokings, South Dakota, USA

Prof. (Dr.) Vimal Kishore Prof. & Chairman, Deptt. of Basic Pharmaceutical Sciences, Xevier College of Pharmacy, University of Louisiana, 7325, Palmetto Street New Orlens, Louisiana USA

Prof. (Dr.) S.P. Singh Former Prof & Head, Deptt. of Pharmacology, G. S. V. M. Medical College, Kanpur, Uttar Pradesh

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Prof. (Dr.) S.K.Agarwal Pro. & Ex-Head, Deptt. of Biochemistry, Lucknow University, Lucknow, U.P.

Dr. Bharat Sah Director, National Institute of Fashion Technology, Raebareli, Uttar Pradesh

Prof. (Dr.)N.S. Verma Prof., Deptt. of Physiology, K. G. Medical University, Lucknow, Uttar Pradesh

Prof. (Dr.)A.K. Tripathi Prof. & Head, Deptt. of Clinical Hematology & Medical Oncology, K. G. Medical University, Lucknow, Uttar Pradesh

Prof.(Dr.)C.M. Pandey Prof. & Head, Deptt. of Biostatistics & Health Informatics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

Dr. Rupesh Chaturvedi Associate Prof., School of Biotechnology, Jawaharlal Nehru University, New Delhi, Former Asstt. Prof., Deptt. of Pharmaceutical Sciences , College of Pharmacy, Vanderbilt University, Tennessee, USA

Dr. S.Sinha Asstt. Prof. Deptt. of Internal Medicine, CD University, C. David Giffen School of Medi., University of California, Los Angeles, USA

Dr. K.Raman Principal Scientist, Martek Biosciences Corporation, 6480 Dobbin Road, Columbia, MD 21045, USA Dr. P.K.Agarwal Editor –in – Chief, Natural Product Communication, Natural Product Inc 7963, Anderson Park Lane West Terville, OH, USA

Dr. R.K.Singh, Chief Scientist, Division of Toxicology, CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow, Uttar Pradesh

Dr. Mohd. Tarique Prof., Deptt of Physical Edu., Lucknow University, Lucknow, Uttar Pradesh

International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

EDITORIAL BOARD Prof.(Dr.) Y.B. Tripathi

Dr. Vinod Singh

Prof. & Head, Deptt. of Medicinal Chemistry,Institute of Medical Sciences, Banaras Hindu University Varanasi, Uttar Pradesh

Assoc. Prof. & Head, Deptt. of Microbiology, Baruktulla University, Bhopal, Madhya Pradesh

Prof.(Dr.) R.K. Singh

Dr. K.K.Verma

Prof. & Head , Deptt. of Biochemistry, Shri Guru Ram RaiInstitute of Medical & Health Sciences, Dehradun, Uttarakhand & Former Prof. & Head, Department of Biochemistry, K. G. Medical University, Lucknow, U.P.

Assoc. Prof., Deptt. of Physics & Electronics.Dr. R. M. L. Awadh University , Faizabad,Uttar Pradesh

Prof. (Dr.) R.S.Diwedi Former Director, National Research Centre for Groundnut (NRCG) , ICAR, Junagarh, Gujarat & Former Principal Scientist – Head, Deptt. of Plant Physiology, Indian Institute of Sugarcane Research, Lucknow, Uttar Pradesh

Dr. Atul Gupta Senior Scientist, CSIR- Central Institute of Medicinal & Aromatic Plants, Lucknow, Uttar Pradesh

Dr. Saudan Singh,

Prof. (Dr.) Nuzhat Husain

Senior Principal Scientist, CSIR- Central Institute of Medicinal & Aromatic Plants , Lucknow, Uttar Pradesh

Prof. & Head , Deptt of Pathology & Acting Director, R. M. L. Institute of Medical Sciences, Lucknow,Uttar Pradesh

Dr. S.K.Tiwari

Prof. (Dr.) Amita Jain

Senior Principal Scientist ,CSIR- National Botanical Research Institute, Lucknow, Uttar Pradesh

Prof. Deptt. of Microbiology, K.G. Medical University, Lucknow, U.P.

Dr. Shivani Pandey,

Dr. Sudhir Mahrotra

Asstt. Prof., Deptt. of Biochemistry,K.G.Medical University, Lucknow, U.P.

Associate Prof., Deptt. of Biochemistry, Lucknow University, Lucknow, U.P.

Dr. B.C. Yadav,

Prof. (Dr.) Vibha Singh

Lucknow Associate Prof. & Coordinator, Deptt. of Applied Physics, School for Physical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, U.P.

Prof., Deptt. of Oral & Maxillofacial Surgery, Faculty of Dental Sciences, K. G. Medical University, Lucknow, Uttar Pradesh

Dr. Anchal Srivastava,

Prof. (Dr.) U.S. Pal

Prof., Deptt of Physics, Lucknow University,Lucknow, Uttar Pradesh

Prof. & Head, Deptt. of Oral & Maxillofacial Surgery, Faculty of Dental Sciences, K. G. Medical University, Lucknow, Uttar Pradesh

Dr. Shalini Bariar

Prof. (Dr. ) K.K. Pant

Associate Professor, Thakur Institute of Management Studies and Research,, Mumbai, India Dr.A.K.Pandey

Prof. & Head, Deptt. of Pharmacology & Therapeutics, K. G. Medical University, Lucknow, Uttar Pradesh

Principal Scientist, National Bureau of Fish Genetic Resources,Lucknow, U.P.

Dr. C.M.K.Tripathi

Dr.S.K.Pandey

Former Deputy Director & Head, Division of Fermentation Technology, CSIR- Central Drug Research Institute , Lucknow, Uttar Pradesh

Dr. Suneet Kumar Awasthi,

G.M. LML Factory, Kanpur Uttar Pradesh

Dr. R.D. Tripathi

Asst. Prof, Deptt.of Physics J.P. University, Noida, Uttar Pradesh

Chief Scientist & ProfessorPlant Ecology & Environmental Science Division, Uttar Pradesh CSIR-National Botanical Research Institute, Lucknow, U.P.

Dr.G. N. Pandey

Prof.(Dr.) Ashwani K. Srivastav Prof. & Head, Deptt. of Biosciences, Integral University,Lucknow, Former Senior Scientist, Birbal Sbahani Institute Paleobotany, Lucknow, U.P.

Prof.(Dr.) L. Pandey Prof. & Head , Postgraduate Deptt . of Physics,Former Dean, Faculty of Science, Rani Durgawati University, Jabalpur, Madhya Pradesh, India

Prof .(Dr.) Bali Ram Prof., Deptt. of Chemistry, Banaras Hindu University, Varanasi, Uttar Pradesh

Asst. Prof, Deptt. of Physics Amity University, Noida ,Uttar Pradesh

Dr. Mukesh Verma Asst. Prof., Deptt. of Physical Education, Dr. R.M.L. Avadh University, Faizabad, Uttar Pradesh

Dr. Abhay Singh, Head, Physical Education, Delhi Public School, Lucknow Uttar Pradesh

Dr. Santosh Gaur Asst. Prof. Deptt. of Physical Education, Jawahar Lal Nehru P.G. College, Barabanki, Uttar Pradesh

Prof.(Dr.) J.P.N.Rai

Dr.Sanjeev Kumar Jha

Prof.& Head, Deptt. of Environmental Sciences, G.B. Pant University of Agr. & Technology, Pant Nagar, Uttarakhand

Senior Scientist, DEOACC Patna

Prof.(Dr. )R. S. Dubey

Dr. Shivlok Singh Scientist, DEOACC, Lucknow, Uttar Pradesh

Prof. & Head, Deptt. of Biochemistry, Banaras Hindu University, Varanasi, U.P.

Dr. Anurag Tripathi,

Prof. (Dr.) Omkar

Asstt . Prof. , Deptt. of Electrical Engg., Institute of Engg. & Technology, Sitapur Road, Lucknow, Uttar Pradesh

Deptt. of Zoology, Lucknow University, Lucknow, Uttar Pradesh

Prof.(Dr.) Sudhir Kumar

Prof. V.P.Sharma

Prof., Deptt. of Zoology, Lucknow University, Lucknow, Uttar Pradesh

Senior Principal Scientist, CSIR-Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh

Prof.(Dr.) Naveen Khare Prof., Deptt. of Chemistry, Lucknow University, Lucknow, Uttar Pradesh

Dr. Krishna Gopal

Prof.(Dr.) S. M. Natu

Former Deputy Director & Head , Aquatic Toxicology Division, CSIR- Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh

Prof., Deptt. of Pathalogy,K.G. Medical University, Lucknow, Uttar Pradesh

Dr. Kusum Lata Mishra,

Dr. S.P. Shukla

In-charge, Coagulation Laboratory, Deptt. of Pathology, K.G. Medical University, Lucknow, Uttar Pradesh

Prof. , Deptt. of Civil Engg., Institute of Engg. & Technology, Sitapur Road , Lucknow, Uttar Pradesh

Prof.(Dr.)V.K. Sharma, Prof., Deptt. of Chemistry, Lucknow University, Uttar Pradesh

Dr. Ajay Mishra Associate Prof. , Deptt. of Geology, Lucknow University, Lucknow , U. P.

Prof.(Dr.) R.K. Shukla

Dr. Ashutosh Singh

Prof., Deptt. of Physics, Lucknow University, Lucknow Uttar Pradesh

Prof., Deptt. of Chemistry,Saket P.G. College, Ayodhya, Faizabad, U. P.

Prof.(Dr.)Anil Gaur

Dr. S.K. Singh

Prof., Deptt. of Biotechnology & Genetic Engg., G.B. Pant University of Agr. & Technology, Pant Nagar, Uttarakhand

Shri Sudesh Bhat

Principal, Gita College of Education , Nimbari, Panipat, Haryana

Dr. Mahesh Pal

Advisor (Education), Sky Institute, Lucknow, Uttar Pradesh

Principal Scientist ,Phytochemistry Division, CSIR- National Botanical Research Institute, Lucknow, Uttar Pradesh

Dr. Krishna Gopal Asst. Prof., Deptt. of English,Rama University, Kanpur, Uttar Pradesh

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International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

ABOUT EDITOR-IN- CHIEF : DR. B. R. PANDEY Dr. B. R. Pandey is a well-known academician and scientist with brilliant academic career and research accomplishments. He has done M.Sc. (organic chemistry) from Banaras Hindu University, Varanasi, India in the year 1972. He has done PhD in Medicinal Chemistry under the guidance of world renowned Biochemist & Medicinal Chemist, Professor S.S. Parmar, Professor of Medicinal Chemistry & Chemical Pharmacology, Department of Pharmacology & Therapeutics, K. G. Medical College, Lucknow (Presently K. G. Medical University), Faculty of Medicine, University of Lucknow, Lucknow, India in the year 1976. Dr. Pandey has all throughout first class educational qualifications and his research interest covers medicinal chemistry, biochemical pharmacology, neurochemistry, neuro-toxicology, environmental chemistry, herbal medicine & natural products. He is having extensive research experience of more than 40 years and published several research papers in peer reviewed journals of international repute. His research particularly on the studies of central nervous system acting drugs and anti-inflammatory drugs and their biochemical mode of action using animal models and enzymes such as monoamine oxidase, acetylcholine esterase, purine catabolizing enzymes , proteolytic enzymes, membrane stabilizing enzymes, respiratory enzymes, microsomal enzymes etc. has been well recognized as evidenced by his research publications. Further, his research on developing herbal medicines has been found very useful in prevention and treatment of chronic diseases and other refractory diseases for which modern system of medicine have no permanent cure. He has worked on the position of Joint Director, Council of Science & Technology, U.P., Lucknow, Department of Science & Technology, Uttar Pradesh Government, India from the year 1979 to 2011, where he successfully executed several R & D projects in various disciplines of Science & Technology including chemical & pharmaceutical sciences, medical sciences, biological sciences, environmental sciences etc. During his tenure as Joint Director, he has been instrumental in launching and implementing important schemes: Young Scientists Scheme, Young Scientist Visiting Fellowship Scheme, Establishment of Centre of Excellence- Encephalitis Research Centre of Excellence in Sanjay Gandhi Post Graduate Institute of Medical Sciences ( SGPGIMS), Lucknow , U. P. India ; Centre of Excellence in Materials Science ( nano materials) in Z. H. College of Engg. & Technology, Aligarh Muslim University, Aligarh, U.P. India, Establishment of Patent Information Centre in the premises of Council of Science & Technology , U.P. He has also worked on the post of Secretary ( as additional charge ) , Council of Science & Technology, U.P. several times and functioned as Administrative Head of the Organization. Prior to taking over the position of Joint Director, Council of Science & Technology, U.P. in the year 1979, he has worked as Junior Research Fellow/ Senior Research Fellow (Council of Scientific & Industrial Research, New Delhi ), Assistant Research Officer ( Jawaharlal Nehru Laboratory of Molecular Biology) at Department of Pharmacology & Therapeutics, K.G. Medical College (presently K. G. Medical University), Faculty of Medicine, University of Lucknow, Lucknow, India from the year 1972 to 1979 and involved in multidisciplinary biomedical research leading to drug development .

He has worked as Visiting Scientist / Faculty in the Department of Physiology, School of Medicine, University of North Dakota, Grand Forks, North Dakota, USA and also visited scientific institutions in Sweden, U.K. and U.S.A. under Training Program on Capacity Building in Environmental Research Management (World Bank Funding Project). After his superannuation in the year 2011, he has been associated with International Institute of Herbal Medicine (IIHM), Lucknow, India as Professor and is presently associated with Sky Institute, Lucknow, India as Director (Research) and Dean, Faculty of Science & Technology, Sai Nath University, Jharkhand, India and involved in programs related to higher education and research of scientific & technological fields. He has organized several national and international www.ijsir.co.in

International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

conferences. He has actively participated in national and international conferences, symposia and workshops and presented research papers and chaired scientific / technical sessions. He is life member and fellow of many scientific societies such as National Academy of Sciences India, Society of Toxicology of India, Indian Academy of Neurosciences, Bioved Research Society India, International Society for Herbal Medicine (ISHM), Society of Biological Sciences and Rural Development, India. He has been member of several scientific expert committees/ advisory committees to evaluate scientific research proposals. Dr. Pandey has been actively associated with various universities and institutions in India as examiner for conducting graduate, post graduate and doctoral level examinations in disciplines like chemical sciences, pharmaceutical sciences, biochemical sciences, biotechnology and allied areas and member of Board of Studies for the academic development in the department. He has been approved research supervisor for guiding research in chemistry, biotechnology and related areas from various universities of India leading to PhD Degree. In view of his vast research and administrative experience and broad R & D vision, Dr. Pandey has been associated with International Journal of Scientific & Innovative Research (IJSIR) as Editor-inChief.

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International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

FROM THE DESK OF CHAIRMAN, SKY INSTITUTE

It is my privilege to state that I have great desire to contribute to the development of our country and to bring about social transformation through education, higher learning and research. This inner feeling prompted me to establish Sky Institute in Lucknow (Uttar Pradesh), the city known for its rich cultural heritage and vibrant academic institutions of higher learning. Sky Institute, since its inception in the year 2006, has been functioning to impart various educational and training courses with a vision to improving lives through education, research and innovation. The institute provides a professional learning environment that acts as a catalyst, for the exponential growth of student as well as extracurricular abilities. It conducts regular courses at the level of graduate and post graduate followed by research courses leading to M Phil and PhD in all subjects in association with universities .

I feel great pleasure to highlight that Sky Institute has started to publish a bi-annual journal “International Journal of Scientific and Innovative Research ( IJSIR ) which encourages to publish research articles in all branches of science, technology, engineering, health, agriculture and management. Research articles in the field of education are also considered in order to improve educational standard in educational institutions with innovative technologies. First volume of the journal has been successfully published. The present issue of second volume of the journal contains useful and informative research articles which may be interesting to readers and educational and research organizations. The association of eminent faculty and scientists of reputed organizations with our journal is highly appreciable.

I call upon all the students who are willing to join various programs/courses being run at Sky Institute in association with selected universities, to strive hard to gain knowledge, transform it into skills with right attitude and inculcate the habit of learning, which will drive them to self directed learning.

My best wishes to all the aspiring students.

Mohit Bajpai Chairman

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International Journal of Scientific and Innovative Research 2016; 4(2) P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

CONTENTS GEOMETRICAL AND CONFORMATIONAL STERIC AFFINITY INTERACTION STUDIES OF ANTICANCER DRUGS WITH HEXANUCLEOTIDE SEQUENCES OF DNA BY FLUORESCENCE AND ABSORPTION SPECTROPHOTOMETRY. Sulabh Kharbanda

1

PREVALENCE AND STUDY OF DIFFERENT STAGES OF CHRONIC KIDNEY DISEASES (CKD) IN PATIENTS. Shailza Verma, Dheeraj Upadhyay, Rahul Singh, Neha Pandey, Saurabh Singh,Vishwajeet Pandey, Sulabh Kharbanda

9

HONEY- GOLDEN LIQUID FOR HEALTH Vibha Singh

16

PHENYLHYDRAZINE INDUCED HAEMATOTOXICITY AND IT'S RECOVERY BY FUMARIA INDICA PLANT EXTRACT IN CHARLES FOSTER RATS Raj Kumar, Vivek Kumar Mishra, Anil K.Meena, Pallavi Singh, R.L.Singh and R .K. Singh

22

IN VIVO ASSESSMENT OF ANTI-GENOTOXIC POTENTIAL OF ASPARAGUS ADSCENDENS Sakshi Jaiswal, Anil K. Meena, Pallavi Singh, R.L. Singh and & R.K. Singh

32

BENZENE INDUCED HAEMATOTOXICITY AND IT'S AMELIORATION BY QUERCETIN IN SPRAGUE DAWLEY RATS Vivek Kumar Mishra, Raj Kumar, Anil K. Meena, Pallavi Singh, R.L. Singh & R.K. Singh

43

ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OFEUGENIA JAMBOLANUM, BUTEA MONOSPERMA AND CASSIA AURICULATA LEAVES EXTRACT Kiran Abha Singh, Anup Kumar

52

REHABILITATION OF SICK INDUSTRIAL UNITS Niaz Ahmed Siddiqui

63

EMERGING MARKETING STRATEGIES IN TEXTILE INDUSTRIES IN INDIA WITH SPECIAL REFERENCE TO BRAND BUILDING Monika Yagnik Merh

73

DEVELOPMENT AND SCOPE OF TOURISM'S SECTOR IN UTTAR PRADESH Rashmi Mishra, Kamlesh Kumar Shukla, Ishvinder Singh Ahluwalia

78

VARIOUS ASPECTS OF MARKETING OF FINANCIAL SERVICES Kumar Kautilya

83

RE-ORIENTING THE ROLE AND REVAMPING OF FOOD CORPORATION OF INDIA Shantanu Kumar Srivastava

87

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International Journal of Scientific and Innovative Research 2016; 4(2) : 1‐8 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

GEOMETRICAL AND CONFORMATIONAL STERIC AFFINITY INTERACTION STUDIES OF ANTICANCER DRUGS WITH HEXANUCLEOTIDE SEQUENCES OF DNA BY FLUORESCENCE AND ABSORPTION SPECTROPHOTOMETRY. *Sulabh Kharbanda Department of Biotechnology, Saroj Institute of Technology and Management, Lucknow, India *Address for correspondence: Dr. Sulabh Kharbanda, Assistant Professor, Saroj Institute of Technology and Management, Lucknow, India. Email ID: sulabhchandra2000@gmail.com

ABSTRACT The anticancer drugs like adriamycin,epiadriamycin and daunomycin intercalate with different sequences of DNA and provide hindrance to the replication of DNA in cancerous cells by interacting with DNA gyrase, DNA helicase and DNA topoisomerase which help in the winding and unwinding of DNA respectively. The drugs bind in the major groove and minor groove of DNA according to the base specificity of nucleotides in DNA. Various forces that help in the stabilization of drug with DNA are electrostatic forces like salt bridges, dipole forces like hydrogen bonds, entropic forces like hydrophobic effect, base stacking forces like dispersion forces. Adriamycin has a higher affinity, cooperativity and conformational stability during binding to poly(dA.dT) than epiadriamycin and daunomycin Further studies were done to study the binding of daunomycin, adriamycin, and epiadriamycin to hexanucleotide sequence d-CGATCG by absorption and fluorescence measurements and energy minimization studies were done with AMBER force field to study the hydrogen binding interactions between drug and DNA sequence. In addition to these studies,the affinity of binding of hexanucleotide sequences like d-CGATCA, and dCGTACA to drugs adriamycin, epiadriamycin and daunomycin was studied and compared with dCGATCG sequence. Sugar moiety in drug molecules interacts more strongly with d-CGATCA than with dCGTACA due to the formation of direct hydrogen bonds between d-CGATCA and not in altered nucleotide conformation sequence d-CGTACA. In sequence d-CGATCG interaction with drug epiadriamycin, the 4'hydroxyl group is inverted in the sugar molecule as compared to the adriamycin drug where the 4hydroxyl group position helps in the formation of hydrogen bonds with adenine N atom in the DNA hexanucleotide sequence d-CGATCG. Keywords- Hexanucleotide drug; DNA; Absorption spectroscopy; Fluorescence spectroscopy; Adriamycin; Epiadriamycin; Daunomycin; Anticancer drugs; Intercalation; Drug-DNA binding.

INTRODUCTION Aromatic ring derivative molecules like daunomycin, epiadriamycin and adriamycin are anticancerous anthracycline drugs that are obtained from Streptomyces peucetius strains of bacteria through the process of fermentation.These drugs are effective in the treatment of acute lymphoblastic leukemia, and myelogenous leukemia. These drugs intercalate in the major groove and minor groove of various DNA sequences and inhibit the replication of DNA during the S-phase of cell cycle. The structure of these drugs consists of a planar, hydrophobic tetracycline ring structure linked to a sugar moiety www.ijsir.co.in

with a glycosidic bond linkage. The anthracycline derivative has four fused rings (A-D).

Epiadriamycin molecule has 4' hydroxyl (OH) group inversion on the sugar moiety as compared to Adriamycin and Daunomycin molecules. There are various forces between drug-DNA complexes such as electrostatic forces, hydrogen bonds, hydrophobic effects, salt bridges, and dispersion forces. Electrostatic forces depend upon the concentration of salt solutions as the concentration increases, the electrostatic force decreases. The electrostatic forces vary where there is a presence of water and or its absence and the forces are much 1

International Journal of Scientific and Innovative Research 2016; 4(2) : 1‐8 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

stronger in the absence of water molecules due to the dielectric constant and polarity of water which forms hydrogen bonds and weakens the electrostatic forces. The C-11 H–atom of hydroxyl groups in ring C forms a hydrogen bond with ring B oxygen atom in drug molecules and the C-9 carbonyl oxygen atoms in ring D of the drug molecule also forms hydrogen bonds with guanine NH2–group (amine) nucleotide group in the DNA molecule. The other effects include the hydrophobic effects caused due to the hydrophobic aromatic anthracycline rings of drug molecules when water molecules surround them. These water molecules are released during the formation of clusters and stacking of aromatic drug molecules leading to the formation of aggregates. Further these interactions stabilize the entropy of the system and decrease the free energy. Further interaction studies were studied in the intercalation of salmon sperm DNA with daunomycin[1] .The effect of increase in the concentration of drug daunomycin was studied by spectrophotometric absorption spectroscopy to analyze the affinity of intercalation of this drug with salmon sperm DNA sequence and the concentration of bound, unbound drug and extinction coefficients of the free and DNA bound drugs like daunomycin was analyzed [2]. experimentally Absorption spectrophotometry The results of the intercalation of the drug daunomycin with salmon sperm DNA was [2] analyzed experimentally by absorption spectrophotometry at wavelength 480nm in 10mM MES buffer, pH=6.2 at 250C. Further linear plots and scatchard plots were drawn for the spectrophotometric data according to the equation given below, n=n m a x -(1/K a )(n/D f ), where n=number of moles of drug bound per mole of DNA sequence, nmax= represents the maximum binding capacity of the drug with DNA, Ka= association constant for the drug-DNA complex, and Df= is the concentration of unbound drug.

Further hydrogen bonding of the amino group present at the 3' position of the sugar moiety in the drug molecule with the nucleotide of the salmon sperm DNA was studied and the substitution of the amino group at the 3' position of the sugar of drug molecule with peptides lead to an alternation in the binding affinity of the drug with DNA, and 2

weakened the interaction and intercalation of the drug with DNA. Further absorption experiments were done to study the intercalation of daunomycin drug with DNA sequence [3]. The total concentration of the drug was calculated from the Lambert-Beer's Law at 540nm wavelength, Ct= A540/ 540 where A540=absorbance at 540nm, ϵ540= molar extinction [3] coefficient at 540nm . Further formula was used to calculate the amount of bound drug= C b = A/∆ =(Ao-Aobservance)( f- d), the amount of free drug was calculated by taking the difference of total drug concentration (C t ) – the bound drug concentration (Cb),Ao is the absorbance of drug at a known concentration and Aobserved is the absorbance at a particular ratio of DNA to drug. Further for absorption studies the concentration of free drug was calculated according to formula Cf=Ct[(A/AoP)/(1-P,) where Cf is the concentration of free drug ,Ct is the concentration of bound drug, A is the absorbance at a given ratio of nucleic acid binding to drug, A o is the absorbance of drug at concentration 10µM , P=Aα /Ao where Aα is the absorbance when all the drug is bound to DNA sequence. In addition to these experiments fluorescence studies were done at wavelength λ exitation =480nm and λ emmission =555nm. Ratio of Fluorescent intensity of drug daunomycin before and after binding to DNA sequence was used to calculate the amount the concentration of bound drug to DNA, Cf=Ct(I/I0-P)/(1-P), where Ct is the concentration of bound drug and P=is the ratio of quantum yield of fluorescence of total bound drug to DNA to the fluorescent intensity of bound drug at concentration 10 µM, where P=Iα /Io..Analysis of data was done ahead by drawing the graphs of r/Cf where r=number of moles of bound daunomycin per mole of DNA base pair and C f is the concentration of free drug(unbound drug), The calculation of this ratio was explained by the algorithm loop of Crothers (1968) and closed form of Mc Ghee and Von Hippel (1974), r/Cf=Ki(1nr)[(1-nr)/{1-(n-1)}]n-1 , where Ki is the intrinsic binding constant and n is exclusion parameter in base pairs. The experiments were continued ahead by analyzing the equilibrium binding of daunomycin and adriamycin with calf thymus [4] DNA . The absorbance spectra of daunomycin drug in free state and bound state from wavelength 480nm to 505 nm shows a red shift and fluorescence spectra was calculated at 555nm and www.ijsir.co.in

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592nm for excitation and emission of the drug daunomycin . The concentration ratio of drug to DNA varies from 1:6 to 1:10 along with the variation in temperature and ionic strength of salts.

Various plots were studied and analyzed for the binding of drug to DNA by calculating the ratios of r/Cf with r at various concentrations of drug from 30 0 7 µm at different temperatures of 15 C to 25 C. The sigmoid curves were plotted with following characteristics of r and free drug concentration (Cf) with drug concentration in the range from 4.4 µm to 5.5 µm for adriamycin. The result suggested that the sigmoid curve decreased as the concentration of [4] the adriamycin decreased and next graphical analysis of fractional drug saturation and DNA concentration revealed that as the concentration of daunomycin was increased, the sigmoid curve of fractional saturation decreased.[4]

Cooperativity behavior of the binding of anticancer drugs with different sequences of DNA Experiments suggested that the binding of adriamycin to DNA takes place by a positive cooperative mechanism and a positive slope is formed in the curve of saturation kinetics of drug[5] DNA interaction and is dependent upon the ionic concentration of salts in the solution. Various sequences of DNA nucleotides were compared and it was shown that the positive cooperativity decreases in the order as follows poly(dGdT)—poly(dA-dC) > poly(dAdT)---poly(dA[5] dT),>poly(dG-dC)---poly(dG-dC) . The binding of the drug decreases with the increase in the salt concentration from 0.1M to 0.2M. Studies revealed that daunomycin molecules interact immediately upstream of the DNA. Further the interaction of drug daunomycin with sequence dCGATCG has more pronounced stacking pattern than interaction of sequence dTGATCA with daunomycin drug.

Studies revealed that daunomycin binds to DNA specific nucleotides for every two base pairs, and the binding affinity varies for the drug as it was observed for guanine nucleotide along with 5'methyl group cytosine. The X-ray crystal structure was developed after the intercalation of drug daunomycin-(CGTACG) 2 with 2-NH 2 amine group with variation in ionic strength and polyelectrolyte concentration with different ionic [8] strength . Crystallization studies of 4'www.ijsir.co.in

epiadriamycin with hexamer dCGATCG revealed that one drug molecule binds at each CpG site and the sugar molecule attached to the anthracycline ring of drug binds to the minor groove of DNA whereas spermine molecules bind to the major groove of DNA.[9]There is variation in the binding affinity of drug with hexamer sequence dCGATCG than with sequence dCGTACG. Direct hydrogen bonds are formed in the drug-dCGATCG complex and not in drug-CGTACG complex in the minor groove of DNA. In the major groove of DNA, the sequence dCGATCG forms a complex with [6] spermine protein but not in dCGTACG sequence .

The methylene and amino groups of spermine lead to the formation of Van-der-Waals forces in the drug-dCGATCG complex but not in drugdCGTACG complex[6].Studies ahead proved that adriamycin drug has much more binding affinity for hexanucleotide d(TATATA)2 than for hexanucleotide d(CGCGCG)2 although in hexanucleotide d(CGTACG)2 strongest affinity for this sequence is observed than for other nucleotides along with affinity of interaction at the 9'OH position in drug and the DNA sequence[10]. Base pair triplet in the hexanucleotide sequence defines the affinity of interaction of the drug-DNA complex than the presence of different types of two base pairs during drug-DNA intercalation studies.

MATERIALS AND METHODS Calf Thymus D N A and hexanucleotides d(CGATCG) were intercalated with adriamycin, epiadriamycin and daunomycin anticancer drugs and the complex was mixed and the results were analyzed by spectrophotometer and spectro fluorometer. The concentrations of drugs were taken in the range of 10µM. The concentration was100µM for spectrophotometry and 50µM for spectrofluorometry studies ( D N A ).0.1M phosphate buffer was used at pH=7.1 with 5mM EDTA which was diluted to 20mM phosphate b u ff e r a n d 1 m M E D TA . A b s o r p t i o n spectrophotometry was performed on Carry 100 Bio(Varian) UV-visible spectrophotometer with peltier thermostatted cell holder and quartz cuvette (path length=1cm).Further titration was done by mixing 20µl of DNA to a drug solution 9-11µM and recording the spectrum in the wavelength range of 200nm-800nm after each addition.

Spectrofluorometry was performed by adding 3ml 3

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RESULTS AND DISCUSSION 1. Absorption spectrophotometry of DNA sequence dCGATCG with drug Adriamycin

Fig- no.1 Graph plot of 1/DNA (dCGATCG) Vs 1/A0-A of drug Adriamycin.

The interaction of drug adriamycin with calf thymus D N A was studied by absorption spectrophotometry. The kinetics of intercalation of the drug with DNA was studied and the wavelength scanning showed a change in the absorption peak from 480nm to 503 nm, called as the bathochromic (red shift). Further isosbestic point was observed at 540nm which is the point on the absorption curve at which the concentration of bound drug and unbound drug is the same. Scatter plots were plotted for r and r/Cf values as already discussed.

Absorption spectrophotometry graphs were plotted for intercalation of adriamycin drug with sequence dCGATCG(Fig no.1, Fig no.2), graphs for epiadriamycin drug with sequence dCGATCG(Fig no. 3,4), graphs for daunomycin with dCGATCG (Fig no. 5,6) for different concentration ranges and fluorescent studies were done for the intercalation of drug adriamycin with sequence dCGATCG, epiadriamycin with sequence dCGATCG, drug daunomycin with sequence dCGATCG, and all these fluorometric intensity graphs showed the same results as the spectrophotometric graphs already discussed.

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It was analyzed from the graphs that the binding of the drugs adriamycin, epiadriamycin, and daunomycin with calf thymus DNA observes noncooperative kinetics and the binding of the drugs with sequence dC G AT C G observes cooperative kinetics The binding sites for the intercalation of epiadriamycin with DNA was found out to be maximum, then for daunomycin and least for adriamycin whereas the association constant i.e the strength of the binding of drug is maximum for daunomycin ,then for epiadriamycin and least for adriamycin as compared to the other research works with different concentration limits of drug and sequences of nucleotides of DNA[10].The work can be compared to previous works where the affinity and the strength of binding of the anticancer drug adriamycin was analyzed with different sequences of DNA[10]. In the graph of intercalation of drug adriamycin with DNA sequence dCGATCG, the number of binding sites of drug to DNA is equal to 0.52 and the Y-axis depiction of n/Cf value which is equal to n/ Kassociation 6 is 2.14 x 10 .

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Fig no.2 Scatter plot of r (x-axis) Vs r/Cf (y-axis) for dCGATCG with drug Adriamycin.

2.

Absorption spectrophotometry of DNA sequence dCGATCG with drug epiadriamycin .

Fig no. 3 Graph plot of 1/DNA(x-axis) (dCGATCG) Vs 1/Ao-A(y-axis) with epiadriamycin drug.

In the same way graph of intercalation of epiadriamycin with DNA sequence was studied by absorption spectrophotometry (Fig no. 3, 4).The

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number of binding sites of drug on DNA is 0.63, and the Y-intercept (Fig no. 4) gives the value of r/Cf which is equal to n/Kassociation equal to 2.73 x 106.

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Fig no. 4 Scatter plot of r Vs r/Cf for dCGATCG with drug epiadriamycin drug.

Further absorption spectrophotometry was performed by mixing daunomycin drug with DNA sequence dCGATCG was calculated to be 0.52. The Y-axis value from fig no. 6 which gives the value of r/Cf (or n/Kassociation) is equal to 4.72 x 106.

Fig no.5 Graph plot of 1/DNA (dCGATCG) Vs 1/A0-A of drug daunomycin.

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3. Absorption spectrophotometry of DNA sequence dCGATCG with daunomycin

Fig no. 6 Scatter plot of r Vs r/Cf for dCGATCG with drug daunomycin drug.

Further spectrofluorometric quenching studies were done for the anticancer drugs adriamycin,

epiadriamycin and daunomycin as depicted in the graphical figure no. 7,8.

Fig no 7. The graph shows the ratio of DNA/Drug concentration to the percentage (%) fluorescence intensity.

[7]

Studies were compared with intercalation of daunomycin with poly(dG-dC) sequence .

Fig no.8 Graph of 1/DNA (x-axis) concentration of dCGATCG and % fluorescence quenching of drugs. www.ijsir.co.in

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CONCLUSION The paper discusses about the strength, affinity, steric and conformational interaction of anticancer drugs like adriamycin, epiadriamycin and daunomycin with different hexanucleotide sequences of DNA by absorption spectrophotometry and fluorescent spectroscopy techniques.

The drugs bind to the major groove and minor groove of the DNA as it is elucidated in this scientific article and prevent the replication of DNA by intercalating with it and further inhibiting the activity of enzymes like gyrase, topoisomerase in the cells.

ACKNOWLEDGEMENTS Author is highly grateful to the faculty members, Head of department and research scholars of Indian Institute of Technology, Roorkee for providing the necessary inputs for experiments.

REFERENCES 1. Gabbay E.J., Grier D, Fingerle R.E., Reimer R., levy R, Pearce S.W., Wilson W.D., (1976) Interaction specificity of the anthracyclines with deoxyribonucleic acid, Biochemistry, May 18, 15(10), 2062-2070.

2. Hyman W. R and Davidson N., (1967), The binding of actinomycin to crab dAT ; the nature of the DNA binding site, Biochemical and Biophysical Research communications, 26(2), 116-120.

3. Chaires B.J., Dattagupta N., Crothers D.M., (1985) Self-association of daunomycin August, 21(17), Biochemistry, 3927-3932.

4. Barcelo F., Martorell Jordi., Gavilanes F.,

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B,Gonzalez-Ros J.,(1988) Equilibrium binding studies of Daunomycin and Adriamycin in calf thymus DNA, Biochemical Pharmacology, 37,11, 2133-2138.

5. Graves D.E., Krugh T.R., (1983) Adriamycin and daunorubicin bind in a cooperative manner to deoxyribonucleic acid, Biochemistry 2, 22(16),3941-3947.

6. Frederick C.A., Williams L.D., Ughetto G., Van der Marel G.A., van Boom J.H., Rich A., Wang A., (1990) Structural comparison of anticancer drug-D N A complexes: adriamycin and daunomycin ,Biochemistry, 29, 2538-2549.

7. Chaires J.B.,(1986), Allosteric conversion of Z D N A to an intercalated right-handed conformation by daunomycin, Journal of Biological Chemistry, July 5, 261(19), 88998907.

8. Xodo L.E., Manzini.G., Ruggiero.J., and Quadrifoglio.F.,(1988), On the interaction of daunomycin with synthetic alternating DNAs: Sequence specificity and polyelectrolyte on the intercalation equilibrium, Biopolymers, November, 27(11),1839-1857.

9. Rich A., Ughetto G., Frederick C.A., Williams L.D.,(1990), Ternary interactions of spermine with DNA:4' epiadriamycin and other DNA: anthracycline complexes, Nucleic Acid Research, 18(18),5533-5541.

10. Pullman B., Gresh N., Chen K.S., (1986) A theoretical investigation on the sequence specific binding of adriamycin to double stranded polynucleotides, Nucleic Acid Research, February, 14(5),2251-2267.

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PREVALENCE AND STUDY OF DIFFERENT STAGES OF CHRONIC KIDNEY DISEASES (CKD) IN PATIENTS. 1

1

1

1

Shailza Verma , Dheeraj Upadhyay ,Rahul Singh ,Neha Pandey , 1 1 2 Saurabh Singh ,Vishwajeet Pandey ,*Sulabh Kharbanda 1

Graduate Student,Department of Biotechnology, Saroj Institute of Technology and Management, 2 Lucknow, Uttar Pradesh, India, Assistant Professor, Department of Biotechnology, Saroj Institute of Technology and Management, Lucknow,Uttar Pradesh, India. *Address for correspondence: Dr. Sulabh Kharbanda,Assistant Professor, Department of Biotechnology, Saroj Institute of Technology and Management, Lucknow, India.

Email ID: sulabhchandra2000@gmail.com ABSTRACT Chronic kidney diseases are quite prevalent in USA and western countries due to hypertension and diabetes. Majority of the population in USA and western countries is affected by chronic kidney diseases leading to increased levels of creatinine and albumin in blood due to increase in filtration pressure inside the juxtaglomerular apparatus of kidney. In India also the rate of disease affliction has increased tremendously to 7%, with a range varying from 5%-13% in various regions like Jharkhand and Chandigarh.The symptoms of kidney disease include changes in the color of urine to darker in appearance with froth due to the presence of albumins as a result of increase in porosity of basement cell membrane cells and podocytes with changes in pressure affecting the levels of antidiuretic hormone, angiotensinogen and renin in kidney along with cirrhosis and the appearance of blood in urine. In addition to this there is retention of fluid in legs, feet, face, hand and ankles of patients suffering from chronic kidney diseases.

Keywords:CKD; Nephritis; Nephropathy; Anemia; GFR; Biomarker; Cell Signaling; Transplant.

INTRODUCTION Chronic kidney disease (CKD) is prevalent in many countries of the world like USA, India etc.The major causes of chronic kidney diseases [22] include lack of iron in diet which causes anemia , [1] hypertension, diabetes and stress .Patients were divided into groups in which one group was given oral dose of iron(ferritin) and second group was given intravenous dose of iron. The levels of albumin, hemoglobin, proteinuria, transferrin,and glomerular filtration rate(GFR) were unchanged in both the groups of patient samples analysed by plasma iothalamatemeglumine method but the levels of serum ferritin and logarithmic-ratio of concentration of protein in urine andcreatinine were changed and increased and decreased for oral group and intravenous administered iron group respectively. Various infected samples from skin, bone, lung, sepsis, urinary tract infections (UTI), cardiovascular diseases, angina, stroke, and cancer related patient samples. The rate values for the ratio of intravenous iron to oral dose of iron and p-values www.ijsir.co.in

were reported to be as follows for each sample (depending on the number of samples in each group which were adjusted accordingly like skin-3.79, and p value-0.013, lung value-4.53,and p-value0.022, bone value-0.59, and p-value 0.2 (values adjusted further), urinary tract infections value(less number of samples)-2.37, and p-value-0.2 (samples-less and less harmful effects), sepsis (sample-more and more harmful effects) value2.59, p value-0.056, cardiovascular-value-2.51,pvalue-is equal to-0.001,arrhythmias value-1.29, pvalue-0.2,angina-value-1.03,p(statistical)-value0.2, hyperkalemia- value-0.69, p-value-0.2, cancer related samples value-2.07, p-value-0.2 [1] . A comparative study was done for the analysis of CKD in patient samples from 48 hospitals and consisting of 4712 testing specimens. The samples were analysed for different types of diseases associated with kidney damage as mentioned in [2] Table 1 below .

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SNo.

Disease name

Percentage (%)

1.

Diabetes

41%

2

Hypertension

22%

3

Chronic glomerular nephritis

16%

4

Chronic interstitial disease

5.4%

5

Ischaemic nephropathy

5.4%

6

Obstructive uropathy

2.7%

Table 1: The percentage of occurrence of different diseases associated along with kidney damage in patient samples in A.I.I.M.S, New Delhi, India [2].

REVIEW OF LITERATURE Diagnosis of Chronic Kidney Diseases The diagnosis of chronic kidney diseases includesimbalances in the concentrations of ions, metabolites and hormones in the blood, and the kidney and changes in the glomerular filtration pressure (GFR). A decrease in the concentration of iron causes anemia and damage to the kidney along with decrease in the secretion of erythropoietin.

There is increase in the levels of phosphate in kidney leading to a condition of hyperphosphatemia and less secretion of phosphate in blood, changes in the concentration of calcium, parathyroid hormone due to alteration in the levels of parathyroid hormone leading to bone mineral metabolism imbalance. Further hypertension is observed in patients with CKD which further leads to cardiovascular disease with altered blood pressure. Diabetes also leads to the development of this disease[3].The high levels of glucose metabolite in blood leads to changes in the glomerular filtration pressure in juxtaglomerular apparatus of kidney, loop of henle, distal convoluted tubule and proximal convoluted tubule. The alterations in the levels of ions like sodium, potassium in the blood and kidney due to the impaired transport in the aquaporin membrane channel in kidney cells also leads to the development of kidney diseases due to the impaired osmolality concentration gradient.

The levels of urea,creatinine and albumin are also

altered in kidney diseases.The renin-angiotensinaldosterone system regulates the blood pressure in the arteries and filtration pressure in the kidney [4] which gets altered in kidney diseases .

Angiotensin converting enzyme (ACE-1) converts the angiotensin-I to angiotensin-II which constricts the blood vessels in the kidney leading to changes in the filtration rate of different metabolites like glucose, maltose, sucrose, albumin, urea, uric acid, hippuric acid and creatinine. ACE-1 inhibitor drugs and angiotensin receptor blockers have been used to treat hypertension due to altered blood pressure [4].The flow chart depicted in figure no.1 gives the detailed analysis of the changes in filtration pressure in kidney cells(nephrons).The secretion of angiotensin and aldosterone in the renal cells leads to the constriction of blood cells which alters the filtration pressure in kidney juxtaglomerular apparatus cells. This mechanism leads to an alteration in the balance of sodium, potassium ions across the membrane channels of nephrons and increase in the absorption of sodium ions and excretion of potassium ionsbetween blood, interstitial fluid and tubules of kidney cells[5].

There is absorption, assimilation and excretion of different ions and metabolites in different parts of kidney namely, loop of henle, proximal convoluted tubule, distal convoluted tubule and the collecting duct before they are excreted in the form of urine from the urinary bladder.

Secretion of angiotensin by kidney cells and endothelin-1 by blood vessels.

Vasoconstriction of blood vessels in kidney due to renin-angiotensin and lack of proper functioning of endothelial cells lining blood vessels due to generation of free radicals.

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Changes in glomerular filtration pressure in juxtaglomerular apparatus in kidney due to diabetes, hypertension, anemia in patients, metabolic-acidosis, bone and mineral changes in calcium and phosphate leading to metabolism disorder, and accumulation of uric acid, albumin in urine.

Damage to kidney cells (nephrons) leading to cell death and changes in sodium,potassium transport of ions from membranes of kidney cells to interstitial fluid and blood.

Figure 1:The flow-chart depicting the changes in the filtration pressure in kidney cells (nephrons).

Different stages in the development of chronic kidney diseases.

The different stages of progression of kidney disease are mentioned in table no.2 below along with decrease in the glomerular filtration rate[3].

SNo. 1.

2.

3.

4.

5.

Stage of kidney disease Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Glomerular filtration rate volume 2

90ml/min per 1.73m and albuminuria 2 60ml/min-80ml/min per 1.73m 30ml/min-59ml/min per 1.73m2 15ml/min-29ml/min per 1.73m2 Less than 15ml/min per 1.73m2 with end stage renal disease

Table 2: The stages of progression of kidney disease along with glomerular filtration rate. Major types of Kidney diseases leading to chronic destruction of nephrons.

Granulointerstitialnephritis (GIN)

IgA nephropathy

In this condition, there is inflammation at the site of infection inside the nephrons which leads to the aggregation of giant cells with multinucleated condition. There is formation of granuloma inside the nephrons with accumulation of eosinophils at that site. Prednisolone drug is used for the treatment of this nephritis condition[6].

In this condition, there is deposition of IgA antibody and C3 protein in the mesangial cells of the kidney and along the walls of glomerulus cells.

In this there is a defect in the O-glycosylation of IgA antibody during post-translational modification of IgA chain and IgA-IgG aggregates are formed by the recognition of these epitopic regions on IgA by the immune system of the body leading to the generation of IgG antibodies complexed with IgA in the kidney cells leading to destruction and apoptosis of kidney cells. Galactosyltransfease enzyme is responsible for the modification and addition of O-linked carbohydrate groups to the IgA antibody. The complement system C3-C4-C5C9 gets activated along with the release of tumor necrosis factor (TNF-alpha),TGF-beta, monocyte chemokine factor-1, macrophage migration inhibitory factor, interleukin(I L-6) which promotes inflammation at the site of injury. There are clusters of CD71-IgA complex, CD89-IgA complexes formed in the mesangial cells in the kidney[9]. Drugs used for treatment of this disease include cyclosporins[10,20].

Acuteinterstitialnephritis (AIN)

In this disease condition, the interstitial cells of kidney develop edema, which is the accumulation of fluid along with inflammation of these cells. The skin develops rashes, fever and destruction of nephrons by bacteria. Mycophenolate mofetil drug [7] is used for the treatment of this disease .

Chronic tubulointerstitial nephritis.

In this condition, there is destruction of glomerulus cells of the kidney leading to apoptosis of these cells. They are caused by bacteria,fungi,virus or protozoan infection in kidney cells.It is identified by observing inflammation, interstitial fibrosis, and apoptosis, atrophy and degradation of proximal and distal tubular cells. Skin rashes, fever develops in patients with increase in eosinophils count in blood.

It is also caused by heavy metal toxicity[8,21]. www.ijsir.co.in

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Lupus nephritis

It is caused by an auto-immune disease called as systemic lupus erythematosus which causes damage to the kidney cells. The symptoms include blood in the urine, frothy appearance and bubbling in urine and pain and swelling in the hands and legs of the body.

Focal segmental glomerulonephritis.

In this disease, lesions are formed which leads to the destruction of podocytes in kidney cells.

Further crescent shaped pauci-immune glomerular immune complexes are formed after infection in kidney cells. The levesl of urokinase plasminogen activator protein(uPAR) increases after inflammation in podocytes. Rituximab, a monoclonal antibody binds to the CD20 receptor of B-cells and is used for the treatment of focal segmental [17] glomerulonephritis . Further HIV virus infection also causes glomerulonephritis.

Diabetic Nephropathy

In this condition, there is increase in the amount of

urinary albumin concentration from a stage of less albumin concentration called as microalbuminuria (20µg/min) to a stage of increase in albumin concentration and the condition is called as macroalbuminuria- (200µg/min).There is also an increase in the amount of blood glucose due to diabetes and the condition is called as hyperglycemia. Drugs used for the treatment of diabetic nephropathy include repaglinide and nateglinide.

There is a chance of developing anemia and levels of low density lipoprotein go down (less than 70mg/dl). Sulodexide and pimagedine drugs reduce the excretion of proteins and albumin during [18] diabetic nephropathy .

Drug-induced nephrotoxicity and chronic kidney diseases There are various side-effects and harmful effects of drugs or medicines taken during infection caused to the body. The side-effects or harmful effects caused by drugs in kidney are listed in table no.3 [11] given below .

SNo. Type of Drug

Disease caused in kidney

1

Aminoglycosides

Chronic tubule- interstitial nephritis

2

vancomycin

Tubular destruction

3

acyclovir

Crystal formation

4

AmphotericinB

Renal tubular acidosis, necrosis

5

ciprofloxacin

Needle crystals

6

Pencillins and cephalosporins

Toxic tubular injury

7

Antiretroviral drugs

Crystalline deposition

8

cetuximab

Renal magnesium wasting and hypomagnesia.

9

pamidronate

Focal segmental glomerulosclerosis

Table 3: Drug induced nephrotoxicity and chronic kidney disease

Cell signaling in chronic kidney diseases (CKD) There are various cell signaling pathways which are activated during chronic kidney disease diagnosis in nephrons. The cell signaling molecule

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cluster is mentioned in table no.4 as mentioned [12] below .It depicts the name of cell signaling molecule and the receptor signaling pathway activated during the infection in kidney.

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SNo.

Name of cell signaling molecule

Signaling pathway

1

clusterin

Tumour growth factor-beta

2 3

Epidermal growth factor pathway Lipocalin

Hypoxia inducing factor(Hif - 1) Hif-1, Lcn2, EGF

4

Hepatocyte growth factor pathway

Nuclear factor -kappa B, tumour necrosis factor alpha (less CKD progression), and apoptotic proteins like Bcl-2(more CKD progression)

Table 4: Cell signaling molecules in chronic kidney diseases Biomarkers of chronic kidney disease (CKD) There are various markers used for the analysis of chronic kidney diseases as mentioned in table no.5 below[13,14,19].

S.No. Name of biomarker molecule in kidney disease 1

Podocin

2

nephrin

3

podocalyxin

4

Cystatin

5

lipocalin

6

Kidney injury molecule-1

7

N-acetyl glucosaminidase

8

Beta-microglobulin

9

Albumin, transferrin, Liver FABP(liver type fatty acid binding protein)

10

Tenascin,TIMP-1

11

Asymmetric dimethyl arginine

Table 5: Biomarkers involved in chronic kidney disease Treatment of kidney diseases The treatment for end-stage kidney disease involves continuous renal replacement therapy (CRRT), peritoneal dialysis, and intermittent hemodialysis. Peritoneal dialysis method involves the use of catheter to remove solutes in circulation in blood from kidney. Intermittent hemodialysis involves the movement of solutes and ions across the membrane by means of concentration gradient in blood and kidney cells. This method involves www.ijsir.co.in

hemodialysis, hemofiltration, and combination of two methods. Various salts like saline, anticoagulants like heparin, buffer like citrate, and anticoagulant drugs like danaparoid, argatobran and [15] nafomostatmesilate is used for renal dialysis .

Various immunosuppressive drugs like tacrolimus is used during kidney transplantation to prevent organ rejection and auto-immune response in patients[16].

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CONCLUSIONS [1,20]

1. CKD leads to the development of anemia

2.

3.

4.

5.

6.

in patients which was further studied by administering iron-sucrose in patients and measuring the glomerular filtration rates(GFR)[1,20].

A population survey was conducted in patients from A.I.I.M.S, New delhi which indicated the percentage of factors responsible for prevalence of chronic kidney diseases in the statistical study and predominant factor for [2] CKD was found out to be diabetes .

Various others diseases associated with CKD include parathyroidism, bone-mineral associated disorder, decreased erythropoetin synthesis, anemia due to iron deficiency, calcification of skull, and cardiovascular diseases after statistical study in patients[3].

Various biomarkers indicate the diagnosis of chronic kidney disease[13,14,19] in patient samples.

The chronic kidney diseases are characterized as of various types as discussed [6,7,8,9,17,18].

Many different types of immunosuppressive drugs are used for kidney dialysis, replacement and transplantation[10,11,15,16].

REFERENCES 1. Rajiv Agarwal, MD, John W. Kusek, PhD, and Maria K. Pappas, BA,A randomized trial of intravenous and oral iron in chronic kidney disease, Kidney Int. 2015 October ; 88(4): 905–914. doi:10.1038/ki.2015.163 2. Dash and Sanjay Aggarwal, Incidence of chronic kidney disease in India, Advance Access publication 11, October-2005.

3. Chronic Kidney Disease and Its Complications Robert Thomas, Abbas Kanso, and John R. Sedor, –Prim Care. 2008 Jun; 35(2): 329vii., doi: 10.1016/j.pop.2008.01.008.

4. S a m i h H N a s r , J a i r a d h a k r i s h n a n , VivetteDD'Agati,Bacterial infection related glomerulonephritis in adults, Kidney International Journal ,may 2013, Volume 83, Issue 5, Pages 792-803.

5. Atlas SA, The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition-,J Manag Care Pharm. 2007 Oct;13 (8 Suppl B):9-20.

6. Nicola Joss, Scott Morris, Barbara Young, and Colin Geddes Granulomatous Interstitial 14

Nephritis, Clin J Am SocNephrol2:222–230, 2007, December 6, 2006.

7. Insara Jaffer Sathick, LadanZand, Afrin N. Kamal, Suzanne M. Norby, Vesna D. Garovic, Acute interstitial nephritis: etiology, pathogenesis, diagnosis, treatment and prognosis, Vol. 5 Issue 1 |, Jan-Dec 2013 | pp. 1 – 20, Nephrology research and reviews.

8. Chronic and tubulointerstitial nephritis, Sergey V. B r o d s k y, T i b o r N a d a s k y, C h a p t e r 25,Hepistall's Pathology of the Kidney.

9. Samuels JA, StrippoliGF,Craig JC, Schena FP, Molony DA IgA NephropathyJonathan Barratt a n d J o h n F e e h a l l y, I m m u n i t y a n d Immunosuppressive agents for treating IgA nephropathy., Cochrane Database Syst Rev. 2003;(4):CD003965.

10. ChabovaV, TesarV, Zabka J, Rychlik I, Merta M, Jirsa M Jr,Stejskalova A, Long term treatment of IgA nephropathy by cyclosporine A,Renal failure, Jan 22(1):55-62.

11. George Sunny Pazhayattil, AnushreeC Shirali, Drug-induced impairment of renal function, International Journal of Nephrology and Renovascular Disease 2014:7.

12. Zeba Khan,Manoj Pandey Role of kidney biomarkers of chronic kidney disease: An update, Saudi Journal of Biological Sciences (2014) 21, 294–299.

13. Robert G. Fassett, Sree K. Venuthurupalli, Glenda C. Gobe, Jeff S. Coombes, Matthew A. Cooper and Wendy E. Hoy ,Biomarkers in chronic kidney disease: a review, Kidney International (2011) 80, 806–821;published online 22 June 2011.

14. Sun Young Kim and AreeMoon, Drug-Induced Nephrotoxicity and Its Biomarkers,BiomolTher 20(3), 268-272 (2012).

15. NeeshPannu, RT Noel Gibney, Renal replacement therapy in the intensive care unit,Therapeutics and Clinical risk management, 2005:1(2),141-150.

16. Robles-Piedras AL, Gonzalez-Lopez EH, Tacrolimus levels in adult patients with Renal transplant, Proc West PharmacolSoc, 2009, 52:33-34.

17. Agnes Fogo: Causes and pathogenesis of focal segmental glomerulonephritis,Nat Rev Nephrol. 2015 February; 11(2):76–87.

18. Jorge L Gross, MirelaAzevedo, Sandra P Silveiro, Luis Henrique Canani, Maria www.ijsir.co.in

International Journal of Scientific and Innovative Research 2016; 4(2) : 9‐15 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

LuziaCaramori, and Themis Zeimanovitz Diabetic nephropathy: Diagnosis, Prevention, and treatment, Diabetes Care, 2005, Jan, 28(1): 164-176.

19. Rajiv Agarwal, Kevin L. Duffin, Dennis A. Laska, James R. Voelker, Matthew D. Breyer, and Peter G. Mitchell A prospective study of multiple protein biomarkers to predict progression in diabetic chronic kidney disease, Nephrol Dial Transplant (2014) 0: 1–10,doi: 10.1093/ndt/gfu255.

20. Vecchio M, Bonerba B, Palmer SC, Craig JC,

www.ijsir.co.in

Ruospo M, Samuels JA, Molony DA, Schena F, Strippoli GFM, Kidney and transplant group, Cochrane group, 3,Aug 2015.

21. Chronic Tubulointerstitial Nephritis, Edgar V. Lerma, Chapter 37 Diagnosis and Treatment, Nephrology and Hypertension,Access Medical Online.

22. S h e t h N i d h i V, S h a i l a N , C l i n i c o Hemotological studies of Chronic Kidney disease, International Journal of Science and Research, 2014.

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HONEY- GOLDEN LIQUID FOR HEALTH *Vibha Singh Department of Oral and Maxillofacial Surgery, K.G. Medical University, Lucknow, U.P. India. *Address for correspondence : Dr Vibha Singh, Professor, Department of Oral and Maxillofacial Surgery, K.G. Medical University, Lucknow, U.P. India.

Email ID : vibhasinghraghuvanshi@gmail.com ABSTRACT Honey is one of the oldest traditional medicines considers to be important in the treatment of several human ailments. Treatment of infection has been practiced since the origin of mankind. In most of the ancient cultures honey has been used for both nutritional and medical purpose. Honey was used to treat infected wounds as long ago as 2000 years before Bactria was discovered to be the cause of infection. In 50 AD Dioscorides described honey as being good for all rotten and hollow ulcers. Honey was often used for wound dressing in the early decade of 20th century but after second world war it as replaced by more modern and sophistatcated products despite plethora of literature discribing the healing properties of honey.

Keywords : Antioxidant; Apitherapy;Wound Healing INTRODUCTION Honey therapy is known as Apitherapy.As per Rigveda this herb born of honey dripped in honey, sweetened by honey is the remedy for all injuries.

Let's every wing that blow drops honey , let the rivers and streams recreate honey let all our medicine turn in to honey , let dawn and the evening be full of honey our nourishes this sky above be full of honey let our trees be honey let the sun be honey let our cows secrete honey.[1] Honey is one of the oldest traditional medicines considers to be important in the treatment of several human ailments. Treatment of infection has been practiced since the origin of mankind. In most of the ancient cultures honey has been used for both nutritional and medical purpose. Honey was used to treat infected wounds as long ago as 2000 years before Bactria was discovered to be the cause of infection. In 50 AD Dioscorides described honey as [2] being good for all rotten and hollow ulcers.

In Hinduism the Madhu (Honey) is one of the five ingredients of Panchamrit the five nectars the other four are ghee , sugar and butter milk . In the temples honey is poured over the deities in ritual called Madhuabhiseka. Jatakarma is performed to welcome the child in the Hindu family by putting some drops of honey in the childs mouth and [1] whispering name of god in the ear of child.

Honey is sweet sticky material produced by bees following the collection of nectar and honey dew.

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Its healing properties have been known for hundreds of years. It was often used in early decades of the 20th century but after the Second World War it was gradually supplanted by more modern and sophisticated products .Although it was widely used in treating wounds by ancient civilizations and it is still utilized in remote communities for the same purpose. The alternative medicine branch known as Apitherapy offers treatment based on honey and other bees product against disease including bacterial infections.

In the past 25 years or so more and more studies have been carried out all over the world to gain a better insight into the efficacious ingredients of honey, in the light of current state of knowledge honey deserves more than our passing curiosity as clinician we should honey full attention especially in the hospital environment where the bacteria's become resistant to antibiotics.

The honey have both anti-inflammatory and antibiotic properties, antioxidant and have greater influences on healing wound. Honey has been proven to create favourable condition in the wound bed autolytic debridement and presence of substance that promote and accelerate the healing process.[3] Honey has been a well-known medicament since ancient times but recently there has been a resurgence of interest in using honey in the www.ijsir.co.in

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management of wounds. The excellent antimicrobial properties of honey have been reported in various studies.[4] ANTIOXIDANT ACTIVITY The antioxidant activity of natural honey is due to the presence of a variety of compounds such as flavonoids namely, apigenin, kaempferol, pinocembrin quercetin, chrysin, galangin, hesperetin etc. , phenolic acids such as ellagic, pcoumaric, caffeic, and ferulic acids), ascorbic acid, catalase, tocopherols, superoxide dismutase, [5] reduced glutathione, amino acids and selenium The anti-oxidant activity has been shown to strongly correlate with the phenolic content of the partucilar honey. Gheldofet. Al. reported a linear correlation between phenolic content and oxygen radical absorbance capacity (ORAC).[6] In addition to neutralization of free radicals, honey also exerts its anti-oxidant action by inhibiting the formation of free radicals, catalysed by metal ions such as iron and copper. Flavonoids and other polyphenols, common constituents of honey have the potential to trap these metal ions in complexes, preventing the generation of free radicals .Honey contains both hydrophilic and lipophilic antioxidants which may act at different cellular/ physiologic levels making it the ideal source of anti-oxidants. [7] Given its complex composition and diverse medicinal properties coupled with its impressive safety profile, it is not surprising that honey has found a very significant role in modern wound care.

Several randomized controlled trials, investigating the effect of honey on minor burns demonstrated accelerated healing time compared with conventional dressings, such as silver sulfadiazine It was even found to be superior to nonconventional dressings such as amniotic membrane [8] and potato peel. Furthermore, Analysis of current evince also indicates the superiority of honey in superficial and partial thickness burns therapy Further a recent Cochrane review reported high quality evidence that honey dressings heal partial thickness burns more quickly than conventional dressings Honey compared with 1% silver sulfadiazine cream in the treatment of superficial and partial thickness burns Till date, majority of the studies conducted speak in favour of using honey for management of radiation-induced oral mucositis in head and neck cancer patients receiving radiotherapy, especially in light of the www.ijsir.co.in

fact that the conventional therapies have failed to offer a comprehensive and effective management for oral complications.[9,10,11,12] In a meta-analysis, Cho et. Al. reported oral administration of honey after radiotherapy could prevent moderate to severe mucositis and associated weight loss.

In their meta-analysis, Song et. Al reported an overall, pooled relative risk of developing severe Mucositis to be almost 80% lower in patients treated with honey compared to patients in the control groups based on the studies included in the [13] review.

ANTIMICROBIAL AND WOUND HEALING PROPERTIES Honey selected for medicinal use should be produced under hygienic condition from traceable source with minimal contamination by pesticide antibiotics or pollutants .

The antimicrobial nature of all honey is clearly demonstrated by the ability to remain unspoiled by micro organism. The inhibitory effect of pure honey on various Gram-positive and Gramnegative bacteria and found that most pathogenic bacteria failed to grow in honey at a concentration of 40% and above, In particular, Salmonella Shigella, Enteropathogenic Escherichia coli and Vibrio cholera.[4] Honey can be divided in to those whose activity was confined to their high sugar content low moisture content and acidity or those that exclusively generated low levels of hydrogen peroxide on dilution or those that retained activity that was independent of synthesis of hydrogen peroxide on dilution.

The types of honey reported in the literature peroxidase and non-peroxidase honey the ability to generate hydrogen peroxide has been shown due to the oxidation of glucose by glucose oxidase which is an enzyme secreted by the bees at its deposits nectar and honey dew in to the hive. Peroxidase honey is not uncommon. Antibacterial activity of 42 Canadian honey against two bacterial species shows that all the activity was associated with the production of hydrogen peroxide. Non peroxide honey however are less common Manuka honey from New Zealand and Jelly bush honey from Australia are two common examples of nonperoxide honey which are postulated to possess unidentified active components in addition to the production of hydrogen peroxide.[14] 17

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The wound healing properties of honey are because of three physical properties a very high concentration of glucose and fructose (osmotic action) a low Ph value (3.2 -5.4) and low water content and four important ingredients honey has three pharmacological characteristics antimicrobial, wound healing and antiinflammatory. Honey promotes healing by maintaining an environment which is moist (18%) water and acidic. Its osmotic properties resulting from high level of simple sugar (80%) honey helps clear away the necrotic tissue in the wound bed .

This prevents the dressing sticking to the wound and protects the granulation edges and this turn stimulates cell division mitosis, because of this high glucose fructose concentration honey deprives the inflammatory bacteria of their nourishment . Instead of metabolizing the amino acids the amine and sulphur containing decomposition products of amino acid causes putrid smell the bacteria concentrate on the producing lactic acid and acidifying environment through this mechanism honey is able to inhibit development of pathogenic bacteria that causes infection.

The presence of glucose oxidase enzymes leads to the formation of hydrogen peroxide in the honey in accordance with following chemical reaction Glucose + water = gluconic acid + hydrogen peroxide.

This release very gradually after 12 hours the concentration is 4 to 5 microgram after 24 hour and this is sufficient to disinfect wound to set autolytic debridement in motion and promote granulation without any risk of toxic effect that would be caused by too high concentration of hydro radicals.

A second ingredient with antibacterial properties this is one non peroxide was demonstrated by Professor Thomas Henle at Germany in 2008. This is substance which have been known for many years because it is present in all food stuffs with higher sugar content Methylglyoxal. This is one of the dicarbonyl components that are formatted through Maillard reaction which occurs in all products that have very high sugar content. It varies according to the geographic origin and the type of honey .Depending on the MGO content which can range from 3-4 microgram to 750-800 microgram per gram honey the honey will have weaker or strong effect on a narrower or wider spectrum of 18

bacteria particularly on the methicillin resistant Staphylococcus aureus strain, the vancomycin resistant enterococci and pseudomonas aeruginnisa which are unaffected by present day antibiotics.

The third effective ingredient was demonstrated by Dr Zaat in 2009 A minute quantity of the substance ranging from 2-3 nanogram per gram honey is present in all types of honey . This substance is similar to human beta defensin 1 (HBD-1) protein a peptide molecule with cationic properties which play an antimicrobial role by aggregation and destruction of the host cell behaving like a true peptide antibiotic.

A fourth group of substance which al so play an important role in wound healing are the flavonoid a group of molecules belonging to the polyphenols which are known to be effective against type 1 radicals .

At high concentration these substances reduces any inflammation present and moderate the pain the important of these effects during the wound healing process should not be under rated as they make the [15] episode more bearable for the patient.

COMPOSTION Honey comprise of 40% glucose, 40% fructose, 20% water with organic acid , vitamins enzymes and minerals. It has specific weight 1.4 and ph 3.4.

Treatment with honey is simple and inexpensive and it's not needs to be sterilize as it already possesses a bactericidal property. Because of its high viscosity it forms a physical barrier creating moist environment which appears to be helpful and accelerate wound healing.[16] The exact molecular mechanism of wound healing using honey is yet to be elucidated .various studies shows that it acts by reducing ROS levels beside this it exert antibacterial activity and low pH and high free acid content may assist wound healing with honey .

The types of wound and degree of severity al so affect efficacy selected honey should be used in sufficient quantity so that it remains there if diluted with wound exudates .It should cover and extend beyond the wound margin, the effect of wound healing is result of combined effect of chemical debridement of dead and devitalized tissue from ulcer by catalase, absorption of edema by hygroscopic properties of honey and promotion of granulation and epithelisation from wound edges. www.ijsir.co.in

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Even raw honey can be used directly for wound care when all of the above facilities are not available for sterilization of the honey. Wound care by honey should be supervised by physician or experienced wound care nurse in patients with significant co morbidities. Any co morbidity problem which contribute to the problem in wound healing should be diagnosed and treated. It should be contact with the wound for at least 12 hours, preferably for 24 hours. If dressing is not appropriate the honey may be wash out of the wound by exudates.

In the case of infected wound systemic antibiotic should be administered after local swab has been taken or if fever or local sign revealed soft tissue infection. The use of honey never has been observed to foster bacterial résistance.

There are some adverse effect related to use of honey. Stinging pain after administration of honey is reported in 5% of cases, this can be managed by applying Local anaesthetic cream but it causes vasoconstriction which can result in reduction in local perfusion.

It can also be used preoperatively to prepare wounds for reconstruction or to make it possible to use smaller or less complex graft. This will help in reducing post-operative morbidity of the donor site. The effect of treatment should be evaluated [17] continually for a period of up to two weeks.

Types of wound, care protocols and pharmaceutical requirements for the medicinal use of honey Honey therapy canal so play a palliative role in improving quality of life for terminal patients with decubitus ulcers. It helps in changing dressing without any pain and al so eliminates unpleasant foul odour .But if debridement is inadequate honey dressing will not help because it does not replace good quality basic wound care.[18] There is increase in use of honey as a dressing on infected wounds burns and ulcers but there is some common concern that risk of botulism form clostridial spores sometimes present in honey. It is established fact that antibacterial activity is heat labile so would be destroyed if honey will be sterilized buy autoclaving. So effect of gama radiation in sterilization of honey and its antibacterial properties was studied by Molan PC et al showed that 25 kGy of gama radiation was sufficient to achieve sterility without affecting its antibacterial properties. [19] Use of honey in www.ijsir.co.in

management of damaged intestinal mucosa, It promote the repair of damaged intestinal mucosa stimulate the growth of new tissue and work as an anti-inflammatory agent. It also reduces symptoms of inflammation when applied to the wounds. Raw honey contains copious amount of compound like flavonoids and other poly phenols which may [20] function as antioxidants.

The mucositis a side effect of chemo and radiotherapy that affects the entire gastrointestinal tract from the mouth to the anus .the cancer treatment breaks down the epithelial cell lining the tract leaving the patient prone to ulceration and infection. The use of honey in oral mucositis followed by radiotherapy is very effective in terms of ulcer healing and quality of life improvement.[21] The use of honey in the cases of alveolar osteitis dry socket followed by dental extraction and use of honey in oral mucosistis.. The symptomatic relief was significant, and results were quite promising both of the studies were done in the department of [21] oral and maxillofacial surgery K.G.M.U. .Honey has a complex chemical composition and neither the identities of all of its inhibitory components nor its mechanisms of action are yet completely understood. Laboratory tests have demonstrated the effective inhibition of a wide range of microbial species, with both antibioticsensitive and antibiotic-resistant bacteria showing susceptibility. The publication of case reports of the eradication of MRSA from patients give validity to in vitro observations, but large scale clinical trials are needed to establish its clinical efficacy. With the increased availability of licensed wound care products containing honey, clinical use is expected to increase and further evidence will become available. Honey seems to have the potential to clear infection as well as being an effective prophylactic agent that may contribute to reducing the risks of cross-infection. Time will demonstrate whether the present optimism about honey is [22] justified.

The theapeutic effectiveness can be guaranteed by measuring the peroxidase activity and antibacterial properties against a selection of bacteria . active ingradient contents of honey depends on the production area and the way in which the honey is collected and processed the bee keeping production and collection of honey needs to be carried out under strictly controlled conditions.

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It is essential that honey that is produced in protected natural areas such as Regional Park is selected and tested for pesticides and heavy metals.

Absence of bactrial contimainartion yeast and botulism spore. Honey is quickly contaminated when it is exposed to the air especially in the polluted atmosphere .Sterlization with gama rays is neccessary to produce honey with bacterial burden of no more than 30CFU/gram.

Presence of pollen may cause alllergic reaction in some patients .In moderate climate regions honey can be stored at room temperature but temperature 25 degree should be avoided optimum storage temperature cis between 12-25 degree temperature . it should al so be protected against UV rays as there are chances of changed in honey by UV rays.

In more than 500 publications th clinical use of honey in open wound in the literature no adverse reaction have been noted . some times a localised stinging sensation described by some patients . trhis may be due to acidity of the honey as it has not [23] been reported when acidity is nutrilsed.

CONCLUSIONS Honey was often used for wound dressing in the early decade of 20th century but after second world war it as replaced by more modern and sophistatcated products despite plethora of literature discribing the healing properties of honey. There are many possible reasons why the pharmaceutical companies has ingnpred this natural product including lack of knowledge lack of research in to medicinal use of honey .Honey seems to be have the potential to clear infection as well as being in effective prophylactic agent that may contribute to reducing the risk of cross infection.

There are some practical consideration which shoould be tken care of before using honey for clinical cases. The amount of honey required for the wound dressing should depends on the amount of exudate released from wound will result in dilution of the dressing. If there is no exudate dressing needs to change twice /week. It should be applied to absorbent dressing because if applied directly on the wound it tends to rub off bfore secondary dressing is applied. Soaking of honey in to direct dressing facilitiated by warming honey to body tempertaure or adding one part with 20 part of honey. For moderately to heavy exudated wound 20

a secondary dressing may be needed .Honey can be used to treat cavity wound by using adhesive film dressing .A low adhrent dressing helps prevent he honey sticking to the wound but it must be porous to allow antibacterial component of the honey to diffuse freely in to the wound bed.

Honey can be safely filled in to cavities and sinuses . it is water soluble and easily rinse out any any residue are biodegradable. For sinuses catheter or syring can be used for applying honey . honey dressing needs to extend beyond the inflammed area surrounding a wound.

REFERENCES 1. Nayik et al. UJP 2014, 03 (01): Page 5-8 www.ujponline.com .Universal Journal of Pharmacy, 03(01), Jan-Feb 2014 .

2. Gunther RT. The Greek Herbal of Dioscorides The Greek Herbal of Dioscorides. New York: Hafner, 1934 (reprinted 1959).

3. GMS Krankenhhyg Interdiszip. 2007; 2(2): Doc51. Published online 2007 Dec 28.

4. Mohammad Javed Ansari, Ahmad Al-Ghamdi, Salma Usmani, Noori S. Al-Waili, Deepak Sharma, AdgabaNuru, and Yehya Al-Attal , Effect of Jujube Honey on Candida albicans Growth and Biofilm Formation. Archives of Medical Research 44 (2013) 352e360.

5. Ahmad Oryan, EsmatAlemzadeh , Ali Moshiri, Biological properties and therapeutic activities of honey in wound healing: A narrative review and meta-analysis. Journal of Tissue Viability (2016) 25, 98-118.

6. Gheldof N Wang XH , Engeseth NJ identification and quantification of antioxidant component of honey from various floral sources. J.Agric Food Chem 2002 Oct 9 50 (21) 5870-7.

7. Subrahmanyam M. A prospective randomised clinical and histological study of superficial burn wound healing with honey and silver sulfadiazine. Burns 1998;24:157-61.

8. Subrahmanyam M. Honey dressing versus boiled potato peel in the treatment of burns: a pro Dec;51(2-3):121-34.

9. doi: 10.1080/10376178.2016.1171727. Epub 2016 Apr Mashhood AA, Khan TA, Sami AN. s p e c t i v e r a n d o m i z e d s t u d y. B u r n s 1996;22:491-3. . J Pak AssocDermatol 2006;16:14-9. www.ijsir.co.in

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10. Bangroo AK, Katri R, Chauhan S. Honey

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dressing in pediatric burns. J Indian AssocPediatrSurg 2005;10:172-5.

Subrahmanyam M, Sahapure AG, Nagane NS, Bhagwat VR, Ganu JV. Effects of topical application of honey on burn wound healing.

Ann Burns Fire Disasters 2001;14(3).

Subrahmanyam M. Honey-impregnated gauze versus amniotic membrane in the treatmentof burns. Burns 1994;20:331-3.

Song JJ, Twumasi-Ankrah P, Salcido R. Systematic review and meta-analysis on the use of honey to protect from the effects of radiationinduced oral mucositis. Adv Skin Wound Care 2012;25(1):23-8.

GMS Krankenhhyg Interdiszip. 2007; 2(2): Doc51.Published online 2007 Dec 28.

Dr David LECHAUX, gastrointestinal surgeon - Hôpital Yves Le Foll 22000 STBRIEUC (France). Previously specialist in internal medicine at various hospitals in Rennes (Fr). Chairman of the Food and Nutrition Liaison Committee Topical Application of Honey in The Treatment of Wound Healing: A Metaanalysis B Medhi, A Puri*, S Upadhyay,** L Kaman***PMCID: PMC2686636 Arne Simon Kirsten Traynor Kai Santos Gisela Blaser UdoBode and Peter Molan Medical Honey for Wound care Still a latest Resort .A Evid Based Complement Alternat Med 2009

www.ijsir.co.in

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Jun 6(2) 165-173.

Dr David Lechaux, gastrointestinal surgeon Hôpital Yves Le Foll 22000 ST-BRIEUC (France). Previously specialist in internal medicine at various hospitals in Rennes (Fr).

Chairman of the Food and Nutrition Liaison Committee.

Molan PC Allen K The effect of gama irradiation on antibacterial activity of honey .

J Pharma Pharmacol 1996 Nov 48 (11)1206-9.

Manisha Deb Mandal and Shyam APADA Mandal -Honey ita medicinal property and antibacterial activity Asian Pac J Trop Biomed 2011 Biomed 2011Apr 1(2) 154-160 Vibha Singh ,US PAL , Nikita V Soni . Honey a sweet approach to avlveolar osteitis National journal of maxillofacial surgery 20144 Jan 5(1) 31 -4.

Rose Cooper Centre foe Biomedical Sciences Cardiff Scool of health Sciences University of Wales Institute Cardiff . United Kingdom GMS Krankenhaushyg Interdiszip 2007 2(2) D o c 5 1 . http://www.egms.de/de/journals/dgkh/20072/dgkh000084.shtml Honey and wound care dr david lechaux D.Lechaux Yves Le Foll Hopital St Briuc. D. Lechaux, Yves Le Foll Hôpital, St--Brieuc

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PHENYLHYDRAZINE INDUCED HAEMATOTOXICITY AND IT'S RECOVERY BY FUMARIA INDICA PLANT EXTRACT IN CHARLES FOSTER RATS Raj Kumar1, Vivek Kumar Mishra1, Anil K.Meena1, Pallavi Singh2, R.L.Singh3and *R .K. Singh1 1

Division of Toxicology ,CSIR-Central Drug Research Institute,Lucknow-226031, Uttar Pradesh, India, 2 3 Department of Biotechnology, CEAT, IILM Academy, Greater Noida , Uttar Pradesh, India, Dr. Ram Manohar Lohia Avadh University, Faizabad, Uttar Pradesh, India * Address for Correspondence: Dr. R.K. Singh , Chief Scientist & Professor , Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow-226031, Uttar Pradesh, India, Email ID : rktox@yahoo.com

ABSTRACT Phenyl hydrazine (PHZ) is a potent chemical causing haemolytic anaemia in various tissues at various levels. Phenyl hydrazine is used as a model for the induction of haemolytic anaemia in Charles foster rats.

It causes the destruction of red blood cells by oxidative stress within erythrocytes and changes at cellular level resulting haemolytic anaemia. Flowers of Fumaria indica showed the remarkable haemo protective activity against the haemolytic anaemia. Methanolic extract of Fumaria indica showed the haemoprotective activity against phenyl hydrazine induced haemolytic anaemia in Charles foster rats at different doses. The plant extract contains this haemoprotective activity due to the presence of high phenolic, flavonoid and alkaloid contents.

Keywords: Phenyl Hydrazine (PHZ); Haemolytic Anaemia; Red Blood Cells; Oxidative Stress; Cytochrome P450, Fumaria Indica ; Medicinal and Pharmacological Activities INTRODUCTION Blood is a body fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. In vertebrates, it is composed of blood cells suspended in blood plasma. Plasma, which constitutes 55% of blood fluid, is mostly water and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide and blood cells themselves. Albumin is the main protein in plasma, and it functions to regulate the colloidal osmotic pressure of blood. The blood cells are mainly red blood cells (also called RBCs or erythrocytes), white blood cells (also called WBCs or leukocytes) and platelets. The most abundant cells in vertebrate blood are red blood cells. These contain haemoglobin, an ironcontaining protein, which facilitates oxygen transport by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. In contrast, carbon dioxide is mostly transported extracellular as bicarbonate ion transported in plasma. The word toxicity is used to describe the potential for a material to produce injury in 22

biological system. Phenyl hydrazine is inducing to formation of reactive oxygen species, and it changes the haemoglobin in oxihaemoglobin.

Phenyl hydrazine causes lipid peroxidation and degration of membrane skeleton and cause haemolytic anaemia. It also decreases haemoglobin level, red blood cell concentration and packed cell volume, and impairs erythrocyte deformability.

Due to lipid peroxidation, RBCs enter in the spleen and causes splenomegaly. Methanolic extract of the plant Fumaria indica contains monomethy fumarate, which showed significant protection against hepatotoxicity induced by carbon tetrachloride, paracetamol and rifampicin in vivo. The plant extract is used as antibacterial, antiimplantation anti estrogenic activity and anticancer. The Fumaria indica treats fever and influenza. Fumaria indica stem is used as a tonic. It is also used in scrofula, constipation, and jaundice.

It is also used as a component of various herbal product such as esano capsule and Ayurveda capsule. www.ijsir.co.in

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1. Contents of Blood The blood of all vertebrates contains the following contents.  Erythrocytes (RBC)  Leukocytes (WBC)  Platelets (Thrombocyte)  Erythrocyte (RBC) Red blood cells also called erythrocytes are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen (O2) to the body tissues—via blood flow through the circulatory system. RBCs take up oxygen in the lungs or gills and release it into tissues while squeezing through the body's capillaries.  Leukocytes (WBC) White blood cells (WBCs), also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from a multi-potent cell in the bone marrow known as a hematopoietic stem cell. Leukocytes are found throughout the body, including the blood and lymphatic system.  Platelet Platelet also called thrombocytes are a component

of blood whose function is to stop bleeding by clumping and clotting blood vessel injuries.

Platelets have no cell nucleus: they are fragments of cytoplasm that are derived from the megakaryocytes, of the bone marrow, and then enter the circulation.

These inactivated platelets are biconvex discoid (lens-shaped) structures, 2–3 µm in greatest diameter.

2. Phenyl hydrazine Phenyl hydrazine (PHZ) was the first hydrazine derivative characterized by Hermann Emil Fischer in 1875. This compound used worldwide mainly as a chemical intermediate in pharmaceutical, agrochemical, and chemical industries. PHZ, C6H8N2 has a molecular weight 108; it exists as yellow to pale brown crystal or as a yellowish oily liquid, with a freezing point of 19.60C and a boiling 0 point of 243 C. PHZ metabolism seems to occur via ring hydroxylation and conjugation, excretion.

PHZ derivatives were used firstly as antipyretics but the toxin action on red blood cells made their used dangerous. For many years, phenyl hydrazine is used for experimental induction of anaemia in animals until Morawitz and Pratt suggested it as a drug for polycythaemia Vera (Falconer 1933), a clonal disorder (Spivak 2002) which is known by a net increase in a total number of erythrocyte in the body.

Chemical formula‐ C6H8N2 Molar mass‐ 108.14g/mol Boiling point ‐ 243.5 °C Mel ng point ‐ 19.5C Colour‐ Yellow oily Liquid

Fig.1: Structure of Phenyl hydrazeen 1. Mechanism of Phenyl hydrazine (PHZ) induced haematotoxicity PHZ reacts with carbonyl group (C=O) which is common among biological molecule so it directly attaches with biological molecule. PHZ is taken up by the inhalation, orally, and dermal roots in

animals and humans. After absorption, PHZ is rapidly taken up by the red blood cells. In RBCs, PHZ interacts with hemoglobin and cytochrome P450 and causes oxidation reaction and generates destructive free radicals that are mainly responsible for hemolysis.

Phenyl hydrazine Oxidation of oxy hemoglobin www.ijsir.co.in

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p – HYDROXYPHENYLHYDRAZINE (It causes hemolysis and lipid peroxidation of RBCs) FORMATION OF PHENYL FREE RADICAL (REACTIVE OXYGEN SPECIES) (Released in to haemoglobin) CONVERSION OF OXYHAEMOGLOBIN IN TO METHEMOGLOBIN, HEMICHROMES AND OTHER HEMOGLOBIN BREAKDOWN PRODUCTS (Oxidative alteration of RBC protein)

HEINZ BODIES FORMATION (Formation of phenyl radical and the replacement of hemi with phenyl- substituted protoporphyrin causes destabilization of hemoglobin to induce Heinz Bodies)

UPTAKE OF RBCs BY MACROPHAGES IN SPLEEN

TRANSLOCATION OF PHOSPHATIDYLSERINE FROM THE INNER TO OUTER OF THE PLASMA MEMBRANE

PHAGOCYTOSIS OF CELL UNDER PROGRAMMED DEATH BY MACROPHAGES Fig.2: Process of Phenyl hydrazine induced haematotoxicity

1. Haemolytic anaemia Hemolysis is the type of destructive process in which destruction or removal of red blood Cells occurs from the circulation before their normal life period of 120 days. When the haemolysis exceeds to be lifelong asymptomatic condition, it often causes anaemia. And this particular condition is known as haemolytic anaemia. Hemolytic anaemia can be extrinsic or intrinsic.  Extrinsic haemolytic anaemia Extrinsic hemolytic anaemia is also known as autoimmune hemolytic anaemia. This type of anaemia develops when the spleen traps and destroys healthy red blood cells. It can also come from red blood cell destruction due to infection, tumours, autoimmune disorders, medication side effects, leukemia, lymphoma.  Intrinsic hemolytic anaemia Intrinsic hemolytic anaemia develops when 24

the red blood cells produced by the body are defective. This condition is often inherited, such as in people with sickle cell anaemia or thalassemia. Anyone of any age can develop hemolytic anaemia. However, according to the Na onal Heart, Lung, and Blood Ins tute (NHLBI), hemolytic anaemia seems to affect more African Americans than Caucasians.  Causes of Hemolytic Anaemia Some underlying causes of extrinsic hemolytic anaemia are enlarged spleen, hepatitis, E p s t e i n - B a r r v i r u s , t y p h o i d , f e v e r, Escherichia coli, streptococcus, leukaemia, lymphoma.

 Symptoms of Hemolytic Anaemia These are some common symptoms haemolytic anaemia. These are paleness of the skin, fatigue, fever, light-headedness, dizziness, weakness or inability to do physical activity. There are some less common signs www.ijsir.co.in

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and symptoms that are seen in patients with hemolytic anaemia include: dark urine, yellowing of the skin and the whites of the eyes (jaundice), heart murmur, increased heart rate, enlarged spleen and enlarged liver.

2. Fumaria indica Fumaria indica is found throughout India and is one of the more commonly used herbs in Indian medicine. The plant is sold under the name pitpapra in Ayurvedic bazaars and used in the preparation of various traditional formulae such as parpatadya kawatha and parpatadya arista. It is also used in the Unani system of medicine and incorporated into trifalashahtara.

 Habitat The plant is distributed throughout India, particularly on the banks of the Ganges and in the Himalayas up to an altitude of 2700 m. It is also distributed in the higher elevations of the Mysore plateau and Nilgiris. It is also found in Europe, Africa and many other Asian countries.  Common name Indian Fumitory Garo: Pid-papra, Shahtra Hindi: Papara, Pit papra Malayalam: Parpatakam Nepali: Dhukure Sanskrit: Parpata, Parpatakah Telugu: Chatarashi.  Classification

Kingdom Class Order Family Genus Species

: Plantae : Angiosperms : Ranunculales : Papaveraceae : Fumaria : F. indica

Fig.3: Plant (Flower) of Fumaria indica and Classification  Botanical description It is an annual, diffuse herb, up to 30 cm high, with grooved branchlets. The leaves are pale green, 2-3 pinnatisect, 5-7 cm long. Flowers are asymmetrical, pale pink or white with purple tips, in terminal or leaf-opposed racemes, with a filiform style and a two-lobed stigma. The calyx consists of two Ian ceo late sepals which are much smaller than the corolla tube. The fruit is a small, indehiscent nutlet, rugose when dry, rounded at the top with two pits and containing one seed.

1. Medicinal and pharmacological activities  Hepatoprotective activity: A methanolic extract of the plant yielded monomethyl-fumarate, which showed significant protection against hepatotoxicity induced by carbon tetrachloride, paracetamol and rifampicin in vivo. In an in vitro screening using thioaceta-mide-induced hepatotoxicity, the drug exhibited similar activity. www.ijsir.co.in

 Anticonvulsant activity: Fumariline, a spirobenzylisoquinoline alkaloid from the herb, showed a significant, dosedependent anticonvulsant activity when tested using maximal electroshock-induced seizures.  Analgesic and anti-inflammatory activity : Fumariline showed anti-nociceptive activity in experimental animals, producing a dosedependent activity measured as an increase in the latent period of the tail flick response (analgesic index). Alkaloids including narceimine, narlumidine and adlumidine exhibited anti-inflammatory activity. Protopine nitrate, an alkaloidal salt present in the plant, is a natural central nervous system stimulant.  Antipsychotic activity: 1- Tetrahydrocoptisine present in Fumaria indica exhibited neuroleptic activity. The antipsychotic activity of the constituent is like that of chlorpromazine in laboratory animals.

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 Antifungal activity: Protopine, 1-tetrahydrocoptisine, narlumidine and narlumicine from Fumaria indica were assessed against a number of spore-germinating plant pathogenic fungi. Narlumidine and protopine showed the most potent antifungal activity.  Hypotensive activity: An extract of the plant was found to have a relaxant effect and produced a moderate fall in blood pressure in experimental animals. The major alkaloid protopine had a similar potency to that of papaverine. In another study, protopine exhibited marked relaxation on ileum and intestine of the experimental animals.  Hypoglycaemic activity: Fumaria indica, administered orally to alloxaninduced diabetic animals, resulted in a significant reduction of the blood sugar levels.

MATTERIALS AND METHODS 1. Experimental animal  Selection and Grouping of Animals A total number of 20 young Charles foster rats (140-160 kg ) were taken from the Division Laboratory Animals, CDRI, Lucknow .The Healthy animals (rats) were selected on the basis of their regular health check up and record of their body weight and randomly divided into different experimental groups. Each group has four healthy male Charles foster rats and out of these groups one group of animal served as control.  Housing and Feeding Conditions Each group of animals were housed together in polypropylene, auto clavable cage of steel wire and mesh lid which having facility for attaching water bottle and also for keeping food pellets.

Bedding of husk was present in the autoclaved cages and this husk was changed daily. The experimental animal room, in which cages were kept, maintained with temperature and relative humidity at the target level of 24±2c and between 30-70 percent, respectively. During the whole study, 12 hours cycle of lighting and darkness was maintained artificially in the room. For animal feeding, laboratory pellet diet 26

was manufactured at CDRI and provided ad lib through an appropriate container size made in the wire mesh lid of the cages. Water was also provided ad libtium with the help of water feeding bottles which fitted with a nozzle.

2. Plant Material  Collection and Authentication of the Plant: Plants were collected from local commercial source of Lucknow (U.P.), India and the authentication of plant materials have been done in Botany Division of CSIR-CDRI, Lucknow.  Preparation of plant extract: Extract of the authenticated plants material have been prepared in our laboratory at Division of toxicology, CSIR – CDRI, Lucknow. The plant material that was collected from local commercial source of Lucknow was dried under shade and coarsely powdered with the help of grinder. The 1.5 Kg powdered plant material was shocked in 5 litter ethanol initial for 48 hr. and regularly mixed time to time.

After 48hrs the extract was filtered by filter paper and the extract was dried under in oven at 370C temperature. The dried plant extract was stored in refrigerator for further use.

3. Assessment of Haemoprotective activity  Experimental design A total number of 20 rats which were divided into groups and each group contained 5 rats kept in different cages were administered phenyl hydrazine at a dose of 10 mg/kg of body weight for seven consecutive days, resulted haemolytic anaemia. These rats were again received methanolic plant extract at different dose for again 7 consecutive days for detecting the ameliorative effect of Fume against the haemolytic anaemia.

For this, Group I served as control received only distilled water. Group II, III, and IV were administered phenyl hydrazine at a dose of 10 mg/kg of body weight for seven consecutive days for the induction of Anaemia. After seven Days, the rat of Group II was received 200 mg/kg Fumaria indicaextract. Group III rats were received 400 mg/kg Fumaria indica extract. Group IV rats received 600 mg/kg Fumaria indica . www.ijsir.co.in

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RESULTS 1. Body Weight The uniform gain was seen in the body weight of control group animals, but remaining three group of Phenyl hydrazine treated animal

showing the significant decrease in body wt after 7 day treatment Phenyl hydrazine at dose 10mg/kg body weight .The increase in the body weight was observed after the treatment of 200, 400,600 mg/kg plant extract of Fumaria indica.

PHZ Induced Haematotoxicity and It's Recovery by FI Plant Extract in CF Rat Average of all groups body weight 10Mg/kg b.wt PHZ & Control - 1% Gum Acecea , 200(II)+400(III)+600(IV) Mg/kg b.wt FI, Average of b.wt. Initial, Middle, Final Group I

Group II

Group III

Group IV

Initial

201.8

199.6

217.4

224.4

Middle

206.8

193

212

218

Final

211.4

201.6

217.2

225.6

Table 1: Group-I, II, III, IV Body weight (Average)

Fig.4: Group-I, II, III, IV Body weight (Average) 3. Hematology After the treatment of 7 day phenyl hydrazine at the dose of 10mg/kg body weight showed adverse effect on blood profile of animas. The 50 reduction was seen in the values of Hemoglobin and total www.ijsir.co.in

RBC count in the phenyl hydrazine treated animals. The WBC count was slightly increased in phenyl hydrazine treated animals as compare to control animals.

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Heamoglobin PHZ Induced haematotoxicity and its amelioration by Fumaria indica plant extract

Group-I

Group-II

Group-III

Group-IV

Initial Middle Final Initial Middle Final Initial Middle Final Initial Middle Final Mean 16.2

16.5

14

14.4

7.72

14.5

14.4

7.46

14.4

15.4

6.9

14.3

0.91

1.48

1.62

0.65

0.54

0.35

1.39

0.5

0.82

0.65

1.24

0.57

±SD

Table 2: Heamoglobin percentage (Initial, Middle, Final) of Group- I,II,III,IV

Fig.5: Heamoglobin percentage (Initial, Middle,Final) of Group- I,II,III,I RBC PHZ Induced haematotoxicity and its amelioration by Fumaria indica plant extract Group-I Group-II Group-III Group-IV

Initial Middle Final Initial Middle Final Initial Middle Final Initial Middle Final

Mean

6.84

7.24

6.87

7.02

2.26

6.11

6.72

1.89

5.84

6.38

2.4

5.59

±SD

0.76

1.25

0.86

0.42

0.68

0.31

0.77

0.81

0.48

0.49

0.41

0.46

Table 3: Total-RBC Count. (Initial, Middle, Final) of Group- I,II,III,IV 28

www.ijsir.co.in

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Fig.6: Total-RBC Count. (Initial, Middle, Final) of Group- I,II,III,IV WBC PHZ Induced haematotoxicity and its amelioration by Fumaria indica plant extract

Group-I

Group-II

Group-III

Group-IV

Initial Middle Final Initial Middle Final Initial Middle Final Initial Middle Final

Mean

18.2

17.48

17.9 6

17.28

26.16

19.1 6

17.4

26.24

19.0 4

17.3

25.54

18.6 8

±SD

0.7

1.03

1.04

0.73

1.19

0.86

0.9

0.95

0.85

1.26

1.65

0.61

Table-4: Total-WBC Count. (Initial, Middle, Final) of Group- I, II, III, IV

Fig.7: Total-WBC Count. (Initial, Middle, Final) of Group- I, II, III, IV www.ijsir.co.in

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DISCUSSION In this experimental study, we have shown that after phenyl hydrazine treatment at the dose 10mg/kg body weight in Group-II, III, IV group of rats after 7 days the induction of Haemolytic anaemia has been observed After treatment with phenyl hydrazine we have ameliorate the haemolytic anaemia by giving the different dose of methanolic plant extract of fumaria indica to the charles foster rats. The different doses of fumaria indica are 200mg, 400mg,and 600mg per kg body weight. In comparison to control ,the other groups showed the significant decrease of Hemoglobin and RBC and increase in the WBC. After dosing of Fumaria indica plant extract, we see increase in level of Heamoglobin and RBC and the number of WBC is decreased. We can identify that in anaemic condition the Hemoglobin and RBC is decreased and the number of WBC is increased.

The decreasing level of Hemoglobin and RBC has shown that phenyl hydrazine causes haematotoxicity and disrupted Hemoglobin and RBCs. The increasing number of WBC shows that the in body of rats any disease is caused because in case of any disease the WBC is increased. WBC also gives protection against any foreign particle and disease. In the study of body weight, we see the middle body weight is decreased in treated group in comparison to initial body weight and control group. The effect of Phenyl-hydrazine on spleen, in comparison to control, the size of other group's spleen is significantly increased, bed PHZ caused damage in the RBC and dead RBCs are stored in the spleen. So the size of spleen is increased in comparison to control and treated group organs. In our study, PHZ was administered by oral route at dose 10 mg/kg body weight for 7 days. After 7 days in PHZ treated animals, a significant decrease were observed in the values of T-RBC and Hgb level decreased and the level of WBC increased. The initial body weight of control group rats is slightly increased but the weight of treated groups rats is decreased. From the decrease in the body weight it is clear the animal is in anemic condition and results on blood parameter also shows that in haematology parameters of PHZ treated animals 50% reduction were seen in the values of T-RBC and Hgb, so we can say that the effect of PHZ is fully on HgB and RBC but 50% increase were seen in the values of WBC. The decrease in RBC and 30

Hemoglobin showed anaemic condition in rats.

These anaemic animals were used as a model in Haemoprotective activity of Fumaria indica.

The Present study was conducted to observe the effect of PHZ and Fumaria indica on drug metabolising enzymes in rats which showed that values of Hb, total RBC increased. Fumaria indica plant extract increased the level of HgB and RBC and it decreased the level of WBC. The various blood cells are produced at a rate of approximately one million to 3 million per second in a healthy adult, this characteristic makes hematopoietic tissue particularly sensitive target for cytotoxic and antibiotic agents such as those used to treat cancer, infection and immune mediated disorders. Animals of control and treated groups remained generally active and healthy throughout the experiment.

There was no redness or discharge from the mucosal membranes and body orifices. No mortality was seen in any of the groups either control or treated. There was no significant gain or loss in the body weight of the rats as observed throughout the experiment. There were no regular variations in the average 24-hour water and food intake of the rats of both control and treated group monitored. In comparison to initial food and water intake the middle food and water intake is increased in middle and final in control group. But in treated group we see the middle food and water intake is deceased in all treated group. And after giving of plant extract the final food and water intake is increased. This increase can be attributed to the ameliorative effect of Fumria indica. Haemat-otoxic effect of PHZ was studied in rats at the dose level 10 mg/kg b. wt. PHZ effect was well defined in treated group characterized by decrease in RBC count, Hb% and WBC count which were indicators of anaemia.

Previous studies suggested that the number of WBC increased in the condition of anaemia. These effects were dose related and reversible.

REFERENCES 1. Falconer E: Treatment of anaemia: the reticulocyte response to venesection .phenyl hydrazine and radiation. Ann. Intern. Med. 7: 172-189,1933.

2. Houstan AH, Murad A: Erythropoiesis in fishrecovery of the gold fish Carassius auratus from acute anaemia. Can.J. Zool. 73:411418,1995. www.ijsir.co.in

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3. Hassan R. Medicinal Plants (Importance and Uses). Pharmaceutica Analytica Acta, 3:10, 2012.

4. Chopra A., Doiphode V. Ayurvedic medicine: Core concept, therapeutic principles and current relevance. Med. Chin. North Am. 86: 75-89, 2002.

5. Tyler V. E., Brady L.R., Robbers J. E., Pharmacognosy. 9th ed. Philadelphia: Lea & Febiger, 1988.

6. Oubre A.Y., Carlson t. J., King S. R., Reaven G. M. From plant to patient: an Ethnomedical approach to the identification of new drugs for the treatment of NIDDM. Diabetologia, 40:614–617, 1997.

7. Dhaliwal G., Cornett A. P., Tierney M. L. Hemolytic Anemia. American Family Physician, 69:11; 2599-2606, 2004.

8. Shukla P., Yadav K. N.,, Singh P., Bansode W. F., Singh K. R. Phenylhydrazine induced toxicity: a review on its Haematotoxicity.

International Journal of Basic and Applied Medical Sciences, 2:2:86-91, 2012.

9. Polsthettiwar V., Varma R. S. Polystyrene sulfonic acid catalyzed greener synthesis of hydrozones in aqeous medium using microwaves. Available (http: //dx.doi.org/ 10.1016/j.tetlet.2007.06.038.), 2012.

10. Cary R., Dobson S., Brooke I. Phenyl hydrazine. Concise International Chemical Assessment Document 19, 1-26, 2000.

11. Pandey K., Meena K. A., Jain A., Singh K. R. Molecular Mechanism of Phenyl hydrazine

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Induced Haematotoxicity: A Review.

American journal of Phytomedicine and Clinical Therapeutics, 2(3):390-394, 2014.

12. Maruthappan V, Shree KS. Antinuclear activity of aqueous suspension of fumaria indica flower aginestgastric ulcers in albino rats. Journal of Pharmacy Research. 2010; 3(1):17-20.

13. Telford RD, Sly GJ, Hahn AG, Cunningham RB, Bryant C, Smith JA (January 2003).

Footstrike is the major cause of hemolysis during running. J. Appl. Physiol. 94 (1): 38–42. doi:10.1152/japplphysiol. 00631.2001. PMID 12391035.

14. Kolb S, Vranckx R, Huisse MG, Michel JB, Meilhac O (July 2007). The phosphatidylserine receptor mediates phagocytosis by vascular smooth muscle cells. The Journal of Pathology212 (3): 249–59. doi:10.1002/ path.2190. PMID 17534843.

15. Bosman GJ, Willekens FL, Werre JM (2005).

Erythrocyte aging: a more than superficial re s e m b l a n c e t o a p o p t o s i s ? . C e l l u l a r Physiology and Biochemistry 16 (1–3): 1–8. doi:10.1159/000087725. PMID 16121027.

16. Josef Berger, Alternative haematotoxicological testing. J Appl Biomed Volume 8 (2010), No 1, p 19-22.DOI 10.2478/v10136009-0003-y 17. Jerry L. Spivak , Polycythemia vera: myths, mechanisms and management. Blood, 15 December 2002 Volume 100, Number 13, 4272- 4290

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IN VIVO ASSESSMENT OF ANTI-GENOTOXIC POTENTIAL OF ASPARAGUS ADSCENDENS 1

1

2

3

Sakshi Jaiswal , Anil K. Meena , Pallavi Singh , R.L. Singh and & *R.K. Singh

1

1

2

Division of Toxicology,CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India, Biotechnology, CEAT, IILM Academy, Greater Noida, Uttar Pradesh, India, 3Dr. Ram Manohar Lohia Avadh University, Faizabad, Uttar Pradesh, India *Address for correspondences : Dr. R. K. Singh, Chief Scientist & Professor, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India, Email ID : rktox@yahoo.com ABSTRACT The protective effect of Asparagus adscendens was evaluated on genotoxicity induced by an alkylating agent Cyclophosphamide. The present study deals with the anti-genotoxic effect on DNA and chromosome damage. This study was carried out by using chromosome aberration assay and micronucleus test. Different group of mice were treated with Asparagus adscendens for 14 days and 24 hour after the last injection with Cyclophosphamide at dose of 70mg/kg body weight and Colchicines at dose of 4mg/kg before two hours of the autopsy. In Chromosomal Aberration assay, a significant difference was seen in the % aberration in chromosome treated with Asparagus adscendens as compared to control group . The same results were also observed in the frequency of micronucleus data of all animals .The results of this study strongly recommended that the plant Asparagus adscendens may be used as an anti-genotoxic agent, but further study is required to establish its biochemical mechanism of action.

Keywords : Cyclophosphamide; Genotoxicity; Asparagus Adscendens; Colchicines; Antioxidant; Antimutagens; Chromosome Aberration Assay; Micronucleus Assay INTRODUCTION Genotoxicity is the property of chemical agents that damages the genetic information within a cell causing muta ons, which may lead to cancer. All mutagens are genotoxic; but not all genotoxic substances are mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct D N A damage leading to mutations. The permanent, heritable changes can affect either soma c cells of the organism or germ cells to be passed on to future generations.[1] Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis. However, the damage may not always be fixed leading to mutagenesis. This DNA damage can be in the form of single and doublestrand breaks, loss of excision repair, cross-linking, alkali-labile sites, point mutations, and structural and numerical chromosomal aberrations.[2] The compromised integrity of the genetic material causes cancer. As a result, many modern techniques such as Ames Assay, in vitro and in vivo Toxicology Test, and Comet Assay have been developed to assess the chemicals' potential to cause DNA 32

damage that may lead to cancer.The antimutagenic effect of certain naturally occurring compounds extracted from plants has been well established in bacteria and mammalian cells . [3,4] Bacterial short-term tests, used to detect environmental mutagens, are recommended for identifying antimutagens. [3,5] Combined with mammalian enzymes, they provide information about the kind of metabolic activation or detoxification that the agent may undergo in vivo.

Cyclophosphamide (C 7 H 1 5 Cl 2 N 2 0 2 P) having molecular mass 261.086 g/mol is used as a chemotherapeutic and immunosuppressive agent for the treatment of a neoplastic as well as autoimmune diseases. There are several reports indicating the carcinogenic effects of Cycloph[6] osphamide (CP) in humans and animals . It causes genotoxicity in mice bone marrow cells.

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents. Structural chromosome www.ijsir.co.in

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aberrations may be of two types, chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also occur. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour suppressor genes are involved in cancer in humans and experimental systems. Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this guideline.

This chromosome aberration assay is also a powerful classical cytogenetic tool for genotoxicity testing and especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues. An in vivo test is also useful for further investigation of a mutagenic effect detected by an in vitro test. [7] Asparagus adscendens is mainly known for its phytoestrogenic properties. With an increasing realization that hormone replacement therapy with synthetic oestrogens is neither as safe nor as effective as previously envisaged, the interest in plant-derived oestrogens has increased tremendously making Asparagus adscendens particularly important. The plant has been shown to be useful in the treatment of neurodegenerative disorders and in alcohol abstinence-induced withdrawal symptoms.

MATERIALS AND METHOD Experimental Design The mice were divided into four groups and were subjected to the following chemical treatment.

Group I : Control Group II : Cyclophasphamide + Colchicine (70 mg/kg +4mg/kg) Group III : Asparagus adscendens + Colchicines (400 mg/Kg+ 4mg/kg) Group 1V : A s p a r a g u s a d s c e n d e n s + C P + Colchicine (400 mg/Kg+70mg/ kg+4mg/kg) www.ijsir.co.in

Chromosome Aberration Assay Procedure for Bone Marrow Chromosome Aberration Assay : (1) Sample Collection The animals were sacrificed by cervical dislocation 24 h after the last dose of Cyclophosphamide with single IP injection of colchicine before two hours of the autopsy and both the femur bones were removed clean by cotton and the bone marrow was extracted in 1% sodium citrate using 25 g needle flushed thoroughly. It was then further processed (Chromosomal assay).

(2) Slide Preparation The mice are treated with the Asparagus Adscendens and then exposed to Cyclophosphamide with colchicine. The mice are then sacrificed by cervical dislocation. The animal is dissected open and both femur bones are removed and cleaned by cotton gauge from the adhering muscle and tissue. The cap of the bone is broken open and the bone marrow tissue is retrieved. This is then transferred carefully into a centrifuge tube containing 1% sodium citrate using 25 g needle and centrifuged at 2000 rpm for 10 min . the supernatant being subsequently removed and the pelleted cells xed by addition of 5ml hypotonic

solution of (0.075M) KCl. To the residual cell pellet and mix the material thoroughly with the help of pasture pipette and incubate the cells in water bath for 20 minutes at 37° C. Centrifuged the tubes after 20 minute at 1000rpm for 10 minutes, discarded the supernatant and add 5ml/tube freshly prepared chilled carnooy's fixative (Methanol:Acetic Acid in 3:1 Ratio).

Re-suspend the cells gently and refrigerate for 12hrs at 4° C. Put the clean glass slide in refrigerator. Centrifuge the mixed cells for 10 minutes at 1000rpm and discard the supernatant, add 0 .3 to 0 .4 µl fresh chilled carnoy's fixative to each tube and re-suspend the cells, take out the slides from the freeze. Dropped the above prepared cell suspension with help of siliconiged Pasteur pipette for the height of near bout 6 feet on clean chilled microscope slide drop by drop at different places on the slide and left to air dried on hot plate at 40° C and then stained with Giemsa in a phosphate buffer.

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(3) Staining Protocol 1. The slide was air-dried and stained with 10% Giemsa stains for 10 minute.

2. Washed in plain water to remove excess stain.

3. Dehydrate with acetone and treated acetone: xylene (1:1) and then with pure xylene.

4. Mounted in DPX mountant for making of permanent slides.

(4) Slide Scoring Two slides were prepared from each animal. Slides were coded and observed under 100x magnification with oil. At least 100 metaphase were observed from each group for the assessment of chrosomal abnormality both in number and structure. The ring chromosome and chromosomal break was taken as chrosomal aberration, count both two type of aberration in cell out of 100 metaphases.

Fig. 1: Photomicrograph showing ring and break chromosome in metaphage cells of Balb/c mice Micronucleus Assay Procedure for Bone Marrow Micronucleus Assay : (1) Sample Collection The animals were sacrificed by cervical dislocation 24 h after the last dose of Cyclophosphamide and both the femur bones were removed clean by cotton and the bone marrow was extracted in FCS (fetal calf serum) and flushed thoroughly. It was then further processed (Micronucleus Test).

(2) Slide Preparation The mice are treated with the Asparagus Adscendens and then exposed to Cyclophosphamide. The mice are then sacrificed twentyfour hours later by cervical dislocation. The animal is dissected open and both femur bones are removed and cleaned by cotton gauge from the adhering muscle and tissue. The cap of the bone is broken open and the bone marrow tissue is retrieved. This is then transferred carefully into a centrifuge tube containing fetal calf serum (FCS) using 25 g needle and 34

centrifuged at 1000 rpm for 5 min. The supernatant is then removed with the help of a Pasteur pipette. The residue left behind is mixed thoroughly with the help of Pasteur pipette and smeared onto a clean slide. The slide is air-dried. These air-dried smear slides are stained in May-Grunwald and Giemsa stains following a specific procedure.

(3) Staining Protocol 1. The slides were immersed in 100% MayGrunwald stain for 5 min.

2. It was then immersed in 50% May-Grunwald stain for 3 min.

3. Washed in plain water to remove excess stain -3 times.

4. Immersed in Giemsa stain (1:6) for 10 min.

5. Washed in plain water to remove excess stain -3 times.

6. Immersed in Xylene for 5 min. (dehydrating agent).

7. Mounted in DPX mountant for making of permanent slides. www.ijsir.co.in

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(4) Slide Scoring Two slides were prepared from each animal.

Slides were coded and observed under 100 x magnification with oil. At least 1000 polychromatic erythrocytes (PCEs) with or without micronuclei and the corresponding number of normochromatic erythrocytes (NCEs) were scored from each slide. PCEs,

micronucleated polychromatic erythrocytes (mn-PCEs), NCEs, micronucleated normochromatic erythrocytes (mn-NCEs) were recorded from each slide. PCEs are immature erythrocytes which are bluish in colour while the N C Es are mature erythrocytes which are golden in colour.

Figure 2: Photomicrograph showing Polychromatic and normochromatic micronucleted cells in bone marrow of Balb/c mice.

RESULTS Body Weight rd After protective treatment with different dose of Asparagus adscendens in group 3 for 14 consecutive nd th days showed increase in body weight as compared to the Group-2 (C.P) and 4 (As . A + CP).

CP Induced Genotoxicity and It's Recovery by AA Plant Extract in Balb/c Mice Table 2: Group-1 Control

Table3 :Group-2 + Ve Ctrl (CP 70 mg/kg)

Initial

Final

1M

22.0

22.0

4M

28.00

27.60

2M

26.8

26.8

5M

28.80

27.18

3M

24.8

24.6

6M

28.91

27.71

Mean

24.53

24.47

Mean

28.57

27.50

±SD

2.41

2.40

±SD

0.50

0.28

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Initial

Final

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Table 4: Group-3 AA 400mg/kg

Table 5: Group-4 (AA 400+CP 70 mg/kg)

Initial

Final

Initial

Final

7M

31.40

33.39

10M

38.80

36.00

8M

31.20

32.8.1

11M

39.60

38.20

9M

32.40

32.85

12M

38.40

37.20

Mean

31.67

33.12

Mean

38.93

37.13

±SD

0.64

0.38

±SD

0.61

1.10

Table 6: Average body wt. of all different groups of mice

Group-1

Group-2 +Ve

Group-3 AA

Control

Ctrl(CP 70 mg/kg)

400mg/kg

Group-4(AA 400+CP 70 mg/kg)

Initial

Final

Initial

Final

Initial

Final

Initial

Final

Mean

24.53

24.47

28.57

27.50

31.67

33.12

38.93

37.13

±SD

2.41

2.40

0.50

0.28

0.64

0.38

0.61

1.10

Figure 3 : Average Body wt. of all different groups of mice 36

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Food Intake Record st On the 1 day (before giving any dose of As. A and C.P) of study, there is no significant difference in water uptake in all group of mice. After 14 days

nd

treatment with the dose of CP (G-2 ) and C.P+AA th rd (G-4 ) mice as compared to the G-3 consumed significantly high amount of food except .

CP Induced Genotoxicity and It's Recovery by AA Plant Extract in Balb/c Mice Food intake of all four group of mice Table 7 : Initial Food Intake Total Pellet

Pellet

Total Consumed/

Average Pellet/

Given gm.

Remained(gm)

Group/Day/(gm)

Mice/Day

GI

150

138

12.0

4.00

Cage-2

G II

150

139

11.0

3.66

Cages-3

G III

150

137

13.0

4.33

Cages-4

G IV

150

137.5

12.5

4.16

Cages

Group

Cages 1

Table 8 : Final Food Intake

Cages

Group

Total Pellet

Pellet

Given gm.

Remained(gm)

Total Consumed/Group/Day /(gm)

Average Pellet/Mice/Day

Cages 1

GI

150

137.5

12.5

4.16

Cages-2

G II

150

137

13.0

4.33

Cages-3

G III

150

136.4

13.6

4.53

Cages-4

G IV

150

136

14.0

4.66

Average Food intake of all four group of mice Table9 : Average Food Intake/animal/Day Groups

Initial

Final

GROUP- I Control

4.00

4.16

GROUP- II +Ve Control

3.66

4.33

GROUP- III 400 mg/Kg AA

4.33

4.53

GROUP- IV 400mg/kg AA+70mg/Kg CP

4.16

4.66

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Figure 4 : Average Food intake of all four group of mice Water Intake Record st On the 1 day before giving any dose of A.A and CP of study there is no significant difference in water intake in all group of mice but after 14 days

treatment with the dose of C.P Group.II and rd th nd C.P+A.A Group 3 and 4 mice of group 2 C.P control significantly consumed high amount of water as compared to all other groups of mice.

Water intake of all four group of mice Table 10 : Initial Water Intake Total water Water Total Consumed/

Cage No.

Group

Average

Given (gm)

Remained(gm)

Group/Day/(gm)

Water/Mice/Day

Cage- 1

GI

200

186.0

14.0

4.66

Cage-2

G II

200

186.5

13.5

4.50

Cage-3

G III

200

185.8

14.2

4.73

Cage-4

G IV

200

186.2

13.8

4.60

Table 11 : Final Water Intake Cage

Total Group

No.

38

Water Given gm.

Water

Total Consumed/

Average

Remained(gm)

Group/Day/(gm)

Water/Mice/Day

Cage-1

GI

200

185.0

15.0

5.00

Cage-2

G II

200

184.8

15.2

5.06

Cage-3

G III

200

184.5

15.5

5.16

Cage-4

G IV

200

184.4

15.9

5.30 www.ijsir.co.in

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Average Water intake of all Four group of mice

Table 12 :Average Water and Intake/animal/Day Groups

Initial

Final

GROUP- I Control

4.66

5.00

GROUP- II +Ve Control

4.50

5.06

GROUP- III 400 mg/Kg AA

4.73

5.16

GROUP- IV 400mg/kg AA+70mg/Kg CP

4.60

5.30

Figure 5 : Average Water intake of all four groups of mice Chromosomal Aberration Assay In chromosomal aberration assay, analyzed 100 metaphase/group, the ring chromosome and chromosomal breaks was taken as a parameter for chromosomal aberration assay. In every group count the number of ring chromosome and chromosomal breaks out of 100 metaphase and calculate the % chromosomal aberration in every group. In the positive control 63% aberration was seen that is significantly different from the % aberration(11%) of control. The animals treated

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with AA at dose 400mg/kg body weight showing the 15% aberration that is not significantly different form the control. The animals of group-IV was initially treated with AA at doses of 400mg/kg for the period of 7 days and before 24hrs of the last dose the animals also treated with a single intra peritoneal injection of 70mg/kg Cyclophaspamide , in this group the % of aberration was 19 % that is significantly different from the Positive control but control.

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Table 13 : Showing the Number of Different Chromosomal aberrations and the % of Aberration in all groups

Groups

Total Number Ring % Chromosomal of Metaphase Chromosome Chromosomal Break Aberration Analysed

GROUP- I Control

100

3

8

11

GROUP- II +Ve Control

100

15

48

63

GROUP- III 400 mg/Kg AA

100

5

10

15

GROUP- IV 400mg/Kg AA+70mg/Kg CP

100

7

12

19

* Data are significantly different than control (p<0.001). # Data are significantly different than CP control (p<0.001).

Fig 6 : Showing the frequency of Different Chromosomal aberrations and the % of Aberration in all groups Micronucleus Assay Asparagus adscendens is medicinal plant that shows many medicinal activities. In this study the frequency of mn-PCE in control group was 0.245±0.082. The frequency of mn-PCEs in asparagus treated groups 400 mg/kg was 0.584±0.076. As expected in cyclophosphamide group the frequency of mn-PCEs was significantly 40

increased up to 1.989±0.076. A group of mice treated with orally administered of asparagus 400 mg/kg for the period of one week and then treated with a single injection of cyclophosphamide before the 24 hrs of the last dose significant reductions in the frequency of mn-PCEs as compared to cyclophosphamide exposed group alone. The frequency was 0.740±0.048(Table-14) www.ijsir.co.in

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Table 14 : Effect of Asparagus adscendens of average micro nucleated polychromatic erythrocytes (mn-PCE) induced by cyclophaspamide in Balb/c mice Group

Polychromatic erythrocytes (PCEs)

Mg/kg

PCEs

mn‐PCEs

% mn‐PCEs

Control(1)

2010

2

0.100

Control(2)

2005

1

0.050

Control(3)

2012

2

0.099

Pooled

6027

5

0.245±0.082

CP‐50 (1)

2014

13

0.645

CP‐50 (2)

2011

12

0.597

CP‐50 (3)

2010

15

0.746

Pooled

6071

40*

1.989±0.076

AA 400(1)

2014

3

0.149

AA 400(2)

2075

5

0.241

AA 400 (3)

2060

4

0.194

Pooled

6149

12

0.584±0.064

AA 400+CP 70(1)

2034

5

0.246

AA 400+CP 70(2)

2012

4

0.199

AA 400+CP 70 (3)

2033

6

0.295

Pooled

6079

15

0.740±0.048

* Data are significantly different than control (p<0.001). # Data are significantly different than CP control (p<0.001).

Figure 7 : Effect of Asparagus adscendens on frequency of bone marrow micronucleated polychromatic erythrocytes (mn-PCE) induced by cyclophosphamide in Balb/c mice www.ijsir.co.in

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DISSCUSSION Genotoxicity studies have been frequently conducted on bone marrow in mice for the evaluatation of mutagenic potential associated with acute or chronic exposure to aqueous plant extract and also for the anti-gentoxic potential of drugs, food products and plant products. Recently, particular attention has been devoted to the M.N assay in order to identify substances with genotoxic activity. This test allows the detection of DNA damage such as single and double-strand breaks after acute and/or chronic exposure to a genotoxic agent . Chromosome aberration assay has been used for the validation of M.N Assay results.

Genotoxicity treatments include: alkylating agent that modifies DNA bases and thus interferes with DNA replication and transcription; intercalating agents that wedge into the spaces between the nucleotides in the DNA double helix and interfere with transcription and replication; and enzyme inhibitors which induce D N A damage by inhibiting key enzymes such as the topoisomerases which are involved in D N A replication.

Cyclophosphamide( C P ) is believed to be involved in induction of DNA damage through the inhibition of topoisomerase II and free radical generation by redox reactions. We found that C P induced a significant increase in DNA damage score and the frequency of chromosome abnormalities by breaking the metaphase chromosomes, In addition, we also found that C P shows similar effect in micronucleus assay, the treatment of CP at dose of 70 mg/kg body weight shows a significant increase in the number of micro nucleated polychromatic erythrocyte. Natural antioxidants have been used to prevent chromosome damage induced as a strategy to attenuate the toxicity in bone marrow with agents such as CP. In many studies, Asparagus adscendens (AA) has shown good antioxidant activities . Due to antioxidant potential of Asparagus adscendens (AA), we use this plant for the antigentoxic potential against CP induced genotoxicity, In our study, the results showed that ethanolic extract of AA at dose 400mg/kg did not show any genotoxic effect on mice bone marrow that suggests that the substances present in the extract are not clastogenic nor do they promote DNA damage. The one week treatment of AA at dose of 400mg/kg day induced only 15% 42

aberration that is not significantly different from the control data (11%) . The same effect was also seen in the results of micronucleus assay. The animals were initially treated with AA plant extract for one week and also given a single intra peritoneal injection of CP at dose 70 mg/kg before 24hrs of last dose. The frequency of micro nucleated polychromatic erythrocytes and the % aberration was not significantly different from the control data, these results showed the protective effect of AA aqueous plant extract on genotoxicity induced

by CP. Our results demonstrate that Asparagus adscendens aqueous plant extracts have no genotoxic effect on bone marrow and can provide effective protection to mice bone marrow against CP induced DNA damage. Our findings indicate that further investigations are necessary to evaluate the in vivo benefits of Asparagus adscendens aqueous plant extract.

REFERENCES 1. Kolle, Susanne (2012-06-01). "Genotoxicity and Carcinogenicity". BASF The Chemical Company. Retrieved 2013-03-16.

2. "Genotoxicity: Validated Non-animal Alternatives". AltTox.org. 2011-06-20.

Retrieved 2013-03-16.

3. Kuroda, Y., 1990. Antimutagenic activity of vitamins in cultured mammalian cells. Basic Life Sci., 52: 233-256.

4. Bootman, J. S. & Robertson, J. S. (1988).

Sequence analysis of the hemagglutinin of B/Ann Arbo r/I/86, an epidemiologically significant variant of influenza B virus.

Virology 166, 271-274.

5. Kada T, Kaneko K, Matsuzaki S, Matsuzaki T and Hara Y (1985) Detection and chemical identification of natural bioantimutagens. A case of green tea factor. Mutation Research 150:127-132.

6. Ember I, Raposa T, Varga C, Kiss I. Effect of different cytostatic protocols on oncogene expression in CBA/Ca mice. Anticancer Res. 1995; 15:1285–1288. [PubMed] 7. www.ijpab.com.

8. http://www.merriam-webster.com/dictionary/ toxicity.

9. Toxicity Endpoints & Tests. AltTox.org.

Retrieved 25 February 2012. www.ijsir.co.in

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BENZENE INDUCED HAEMATOTOXICITY AND IT'S AMELIORATION BY QUERCETIN IN SPRAGUE DAWLEY RATS 1

1

1

2

3

Vivek Kumar Mishra , Raj Kumar , Anil K. Meena , Pallavi Singh , R.L. Singh & *R.K. Singh

1

1

2

Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India, 3 Department of Biotechnology , CEAT, IILM Academy , Greater Noida , Uttar Pradesh, India, Dr.Ram Manohar Lohia Avadh University Faizabad , Uttar Pradesh, India *Address for correspondences : Dr. R. K. Singh, Chief Scientist & Professor, Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India, Email ID : rktox@yahoo.com ABSTRACT Benzene toxicity involves both bone marrow depression and leukemogenesis caused by benzene damage to multiple classes of hematopoietic cells and a variety of hematopoietic cell functions. Study of the relationship between the metabolism and toxicity of benzene indicates that several metabolites of benzene play significant roles in generating benzene toxicity. Benzene is metabolized, primarily in the liver, to a variety of hydroxylated and ring-opened products that are transported to the bone marrow where subsequent secondary metabolism occurs. Two potential mechanisms by which benzene metabolites may damage cellular macromolecules to induce toxicity include the covalent binding of reactive metabolites of benzene and the capacity of benzene metabolites to induce oxidative damage. The Quercetin showed the remarkable hemoprotective activity against the anaemia. Methanolic extract of showed the hemoprotective activity against benzene induced anaemia in Sprague dawley rats at different doses. The plant extract contains this hem protective activity due to the presence of high phenolic, flavonoid and alkaloid contents.

Keywords: Benzene; Bone Marrow Depression; Leukemogenesis; Hematopoietic Cells; Cellular Macromolecules; Oxidative Damage; Plastic Anaemia ; Quercetin ; Hemoprotective Activity; AntiOxidant; Anti-Artherogenic ; Anti-Carcinogenic Properties INTRODUCTION Blood is a body fluid of animals that delivers necessary substances (nutrients and oxygen) to the cells and maintains homeostasis and transports metabolic products away from the cells. The average adult's body contains about 5 liters of blood. Blood is separated in to 2 distinct layers.

 Plasma  Formed Elements Plasma: Plasma is the yellow straw coloured liquid component of blood in which the blood cells are suspended normally.

Formed Elements: Formed elements are the cells and cell-like structures in the blood that constitute about 45% of our blood volume. There are three types of formed elements:  Red blood cells (RBC)  Leukocytes (WBC) www.ijsir.co.in

 Platelets RBCs are the most abundant cells in the blood-there are about 5 million of them per cubic of blood.

They are formed in the bone marrow and have a lifetime of about 3 months . Red bloodcells are important mainly because theycontain Hemoglobin, a protein substance that attaches to oxygen and transports this element to body cells and tissues .Haematopoiesis is the process of forming blood cellular components. All cellular blood components are derived from haemat-opoietic stem cells. In a healthy adult person, approximately 1011 –1012 new blood cells are produced. Primarily the Bone marrow, spleen, tonsils, and lymph nodes, involved in the formation of blood. Hemotoxins (hematotoxins) are the toxins that destroy red blood cells, disrupt blood clotting, and cause organ degeneration and generalized tissue damage.

Hemotoxins not only damage the blood but also damage other tissues. Injury from a hematotoxins 43

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causes very painful injury. Sometimes these toxins can cause permanent damage and create death.

Benzene a white to oily liquid, with a boiling point of 80.10°C. It is soluble in water (25°C) and miscible with alcohol, ether, chloroform, benzene, and acetone [3]. Excessive exposure to benzene has been known for more than a century to damage the bone marrow resulting in decrease in the numbers of circulating blood cells, and ultimately, a plastic anaemia. Of more recent vintage has been the appreciation that an alternative outcome of benzene exposure has been the development of one or more types of leukemia. While many investigators agree that the array of toxic metabolites, generated in the liver or in the bone marrow, can lead to traumatic bone marrow injury, the more subtle mechanisms leading to leukaemia have yet to be critically dissected. This problem appears to have more general interest because of the recognition that so-called “second cancer” that results from prior treatment with alkyl ting agents to yield tumour remissions, often results in a type of leukaemia reminiscent of benzene-induced leukaemia .Benzene induced target organs are bone marrow , central nervous system and immune system which were determined by using the haematological parameters like total leukocyte count and differential leukocyte (WBC) and also different bone-marrow progenitor cells.Many of the modern medicines are produced indirectly from medicinal plants. Plants are directly used for medicinal purposes because medicinal plants are the resources of new drugs. Medicinal plants help us to understand plant toxicity and protect humans and animals from natural poisons. The medicinal effects of plants are due to metabolites especially secondary compounds (Terpenoids, non-protein amino acids, amines, gynogenic glycosides, glucosinolates, and alkaloids and phenolics) produced by plant species. Quercetin is a bioflavonoid found in fruits and vegetables, but highest levels are found in apples and onions. Like many other bioflavonoid, Quercetin has antioxidant, anti-artherogenic, and anti-carcinogenic properties. Quercetin is also retroactive, with some of the same abilities as caffeine but less potent.

There is a divide between the effects seen in quercetin in in vitro (cell cultured) studies and in vivo (in living) studies, with cell studies showing great results that are not that amazing in humans or 44

animals. This is mostly due to quercetin having low oral bioavailability (low percentage of the compound is absorbed and put to use), but could also be due to in vitro studies using a form of quercetin called 'quercetin aglycone' whereas this particular form is never found in the blood, even after ingested, as it gets changed in the liver. Many studies also note a high range of differences between people who ingest the same amount of quercetin, suggesting a large degree of variability is possible with supplementation. Quercetin has GRAS (Generally Recognized As Safe) status, and no side-effects have yet been noted in doses of a few grams a day in either humans or animals. Quercetin is a flavonol found in many fruits, vegetables, leaves and grains. It can be used as an ingredient in supplements, beverages, or foods.

Blood is a fluid connective tissue constituting about 7% of our total body weight. Main Components of blood are plasma and formed elements. Formed elements are cells and their derivatives that contain about 45% of the total blood volume .The elements are categories in to 3 types:  Erythrocytes - red blood cells (5 X 3 106/mm )  Leukocytes - white blood cells (5-10 X 103/mm3)  Thrombocytes – platelets (150-200 X 3 103/mm ) Haematopoiesis Haematopoiesis (Hemopoiesis) is the process in which blood cells are generated in a stepwise manner from the immature cells that subsequently entered into the blood which is circulating and peripheral organs again for maturation.

Blood Disorder A blood cell disorder is a condition in which there is a problem with your red blood cells, white blood cells, or the smaller, circulating cells called platelets, which are critical for clot formation. All three cell types form in the bone marrow, which is the soft tissue inside your bones (I) Anaemia Anaemia is a condition in which there is a deficiency of red blood cells or of haemoglobin in the blood resulting in pallor and weariness.

Anemia, also spelled Anemic, is usually defined as www.ijsir.co.in

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a decrease in the amount of red blood cells (RBCs) or hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen. There are three main types of anemia: that due to blood loss , that due to decreased red blood cell production, and that due to increased red blood cell breakdown. Causes of blood loss include trauma and gastrointestinal bleeding, among others.

(II) Bone marrow aspiration Bone marrow aspiration is a procedure that involves taking a sample from the soft tissue inside the bones. Bone marrow is the spongy tissue found inside bones. It contains cells that produce white blood cells, red blood cells, and platelets inside larger bones such as the spine, breastbone, hips, ribs, skull . White blood cells help fight infection.

Red blood cells carry oxygen and nutrients.

Platelets enable your blood to clot. If a complete blood count shows that the number or function of red blood cells, white blood cells, or platelets is abnormally high or low, your doctor may want to examine your bone marrow to help figure out the cause. Bone marrow aspiration is often performed with a bone marrow biopsy, which uses a different type of needle to remove tissue from your bone marrow.

(III) Leukemia A malignant progressive disease in which the bone marrow and other blood-forming organs produce increased numbers of immature or abnormal leucocytes. These suppress the production of normal blood cells, leading to anaemia and other symptoms.

Types of leukemia There are four main types of leukaemia (I) Acute myeloid leukaemia (AML) Acute myeloid leukaemia (AML), also known as acute myelogenous leukaemia, acute myeloblastic

leukaemia, acute granulocytic leukaemia or acute lymphocytic leukaemia, is a fast-growing form of cancer of the blood and bone marrow. ( II ) Chronic myeloid leukaemia (CML) Also known as chronic myelogenous leukemia, chronic myeloid leukemia (CML) is a form of cancer that affects the bone marrow and blood. It begins in the blood-forming cells of the bone marrow and then, over time, spreads to the blood .

(III) Acute lymphocytic leukaemia (ALL) Acute lymphocytic leukemia (ALL), also called acute lymphoblastic leukemia and acute lymphoid leukemia, is a blood cancer that results when abnormal white blood cells (leukemia cells) accumulate in the bone marrow.

(IV) Chronic lymphocytic leukaemia (CLL) Chronic lymphocytic leukemia (CLL) is a typically slow-growing cancer which begins in lymphocytes in the bone marrow and extends into the blood. It can also spread to lymph nodes and organs such as the liver and spleen.

Benzene Benzene is an important organic chemical compound with the chemical formula C6H6. The benzene molecule is composed of six carbon atoms joined in a ring with one hydrogen atom attached to each.

Because it contains only carbon and hydrogen atoms, benzene is classed as a hydrocarbon. The suggestion that benzene exposure could result in leukaemia was more difficult to establish than the demonstration that benzene could induce a plastic anaemia. Benzene-induced decreases in blood cells could be observed within a few months after exposure was initiated . However, there is a lag time of perhaps years between initial benzene exposure and the development of leukaemia

Fig. 1 : Structure of Benzene www.ijsir.co.in

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Xenobiotic metabolism usually modifies chemicals to make them more water-soluble by introducing oxygen or other polar groups into the potentially dangerous chemical. The process was termed “detoxication” by R.T. Williams but unfortunately metabolic transformation by the xenobiotic metabolism system often leads to more toxic metabolites .

The Target Organ The Hematopoietic System of Bone Marrow Haematopoiesis is a complex process that occurs in the bone marrow and insures that the proper number and type of circulating blood cells can be produced during one's lifetime. Normally the levels of effective blood cells in the circulation are maintained by a system in the bone marrow that is comprised of hematopoietic stem cells (HSCs) and their progeny. HSCs have been termed pluripotent because they can give rise to erythrocytes, each of the leukocyte lineages, and platelets via directed differentiation. These processes are controlled by a variety of factors including cytokines, and growth factors of various types that are released from cells within the marrow or from other sites within the body, e.g., erythropoietin, which stimulates red cell production is a product of the kidney.

Antioxidants Antioxidants are the species of molecule which inhibits the oxidative stress of other molecules.

Oxidation reaction is the type of chemical reaction in which species of hydrogen or electrons are transferred from a substance to an oxidizing agent. Free radicals are generated by the oxidation reactions. These free radicals will initiate a chain of reactions. This chain of reaction causes damage or death to the cell.

Quercetin Quercetin is a plant pigment (flavonoid). It is found in many plants and foods, such as red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's worth, American elder, and others. Buckwheat tea has a large amount of Quercetin. People use Quercetin as a medicine. Quercetin is used for treating conditions of the heart and blood vessels including “hardening of the arteries” (atherosclerosis), high cholesterol, heart disease, and circulation problems. It is also used for diabetes, cataracts, hay 46

fever, peptic ulcer, schizophrenia, inflammation, asthma , gout , viral infections , chronic fatigue syndrome ( CFS ), preventing cancer , and for treating chronic infections of the prostate. Quercetin

is also used to increase endurance and improve athletic performance Chemical and Physical Properties : Chemical formula : C15H10O7 Molar mass : 302.236 g/mol Appearance: yellow crystalline powder 3 Density: 1.799 g/cm Melting point : 316 °C (601 °F; 589 K) Solubility : Practically insoluble in water; soluble in aqueous alkaline solution Antioxidant activity Quercetin, a plant-derived glycone form of falconoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of Quercetin. The reduction potential of Quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of Quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monotypic leukemia cells was determined by flow cytometry using CMH2DCFDA dye. The results were compared with curcumas, a natural product exhibiting a similar range of reported health benefits.

Antigenotoxic activity Genotoxicity is the toxicity as genetic level. In this type of toxicity mutation occurs in the main genetic material that is DNA which is recognized by different types of tests like micronucleus tests and comet assay. Quercetin showed the potential against genotoxicity caused by different chemicals due to the presence of secondary metabolites Hemoprotective activity Quercetin showed the hemoprotective activity against benzene induced haemolytic anaemia which was determined by using the haematological parameter like Erythrocyte (RBC), total and differential leukocyte ( W B C ), Hematocrit (Hct), Haemoglobin (Hb). Various studies have investigated the hemoprotective www.ijsir.co.in

International Journal of Scientific and Innovative Research 2016; 4(2) : 43‐51 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

activities of Quercetin. In this experiment , metabolic Quercetin has shown the haemoprotective activity in rats. This haemoprotective effect of Quercetin was due the presence of phenolic, flavonoids, alkaloids, saponinsis and steroids, samples. It is previously mentioned that all the above secondary metabolites have good antioxidant activity.

different cages were administered .

Group I: Control Group II: 400 mg/Kg Benzene + 10 mg/Kg Quercetin Group III: 400 mg/Kg Benzene + 20 mg/Kg Quercetin Group IV: 400 mg/Kg Benzene + 40 mg/Kg Quercetin

MATERIALS AND METHODS Selection and Grouping of Animals A total number of 20 young SD rats (150-175 gm) were taken from the Division of Laboratory Animals, CDRI, Lucknow .The healthy animals (rats) were selected on the basis of their regular health check up and record of their body weight and randomly divided into different experimental groups. Each group has four healthy male Charles foster rats and out of these groups one group of animal served as control.

Haematology The sample of blood was collected at 0, 7th and 14th day through tail of the rat and haematology was done. In this process, different parameters of blood have been checked at regular time intervals with the help of fully automatic haematology analyzer MS-9 (Melet Schlosing). The haematological parameters which were analyzed by haematology analyzer are as: Haemoglobin (Hb), Total Red Blood Cell Count (T-RBC). Total Leukocyte Count (TLC)

Marking for Identification of Animals For the identification of rats colour coding of fur was done at different sites for giving a unique identity to each rat. Initial temporary marking of animals was done by 1% aq.Eosin, and the final I'd.

Marking was done by saturated aqueous solution of picric acid.

RESULTS Body Weight The comparable gain was seen in the body wt. of th control group at day 0 today 14 , but a significant decrease was seen in the body wt. of animals treated with 400 mg/kg body wt. benzene for initial 7 Days. After the dosing of Quercetin at 10, 20 and 40 mg/kg body wt. a comparable gain was seen in different groups, but the gain showing irregular manner.

Experimental Design A total number of 20 rats which were divided into 4 groups and each group contained 5 rats kept in

Table 1 : Body Weight

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Group I

Group II

Group III

Group IV

Initial

205

260

271

310

Middle

279

250

265

290

Final

300

310

320

345

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International Journal of Scientific and Innovative Research 2016; 4(1) : 43‐51 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

Fig. 2 (Body Weight) and this particular condition showed the haemolytic anaemia in rats. Benzene-induced haemolytic anaemia in SD rats was ameliorated by giving the different doses of the extract of Quercetin for consecutive 7 days and showed a significant increase in RBC, Hgb and decrease in MCV level as compared to benzene treated groups.

Haematology Haematological parameter was observed on 0, 7th, and 14th day of the study period. At day 0, there was no significant decrease in RBC, Hgb and in MCV level. After 7 days, benzene dosing, all groups except control showed a significant (p<0.05) decrease in RBC, Hgb and increase in MCV level

Benzene induced Haemolytic Anaemia and its Amelioration by Quercetin in Sprague Dawley Rats ( Haematology(Hgb) (g/dl) Table 2: Hgb Group‐I

Group‐II

Ini al Middle Final Ini al Middle

Group‐III Final

Group‐IV

Ini al Middle Final Ini al Middle Final

Mean

16

16.18

15.12

16.24

7.52

14.64

16.33

7.85

14.72

16.18

6.49

14.72

±SD

0.94

1.17

0.65

0.94

0.22

0.49

0.99

0.48

0.77

1.17

0.17

0.56

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Fig. 3: Hgb

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Table -3 Rbc (x106/mm ) Group‐II

Group‐III

Group‐IV

Initial Middle Final Initial Middle Final Initial Middle Final Initial Middle Final Mean

6.65

6.64

7.34

6.66

3.13

5.35

6.64

2.66

4.78

6.64

2.11

5.14

±SD

1.01

1.01

0.86

1.01

0.36

0.33

1.01

0.36

0.52

1.01

0.73

0.84

Fig.4: Rbc 3

3

Table 4 : Haematology WBC(x10 /mm ) Group‐I

Group‐II

Ini al Middle Final Ini al Middle Mean ±SD

16.65 0.62

17.9 0.71

15.65 1.15

17.13 0.71

24.17 1.45

Group‐III Final 11.58 1.29

Group‐IV

Ini al Middle Final Ini al Middle Final 17.09 0.71

24.68 0.93

11.35 0.75

17.09 0.71

24.81 0.65

11.43 1.16

Fig 5 : WBC www.ijsir.co.in

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DISCUSSION Benzene derivatives were used firstly as antipyretics but the toxic action on the hemopoietic system. The exposure of benzene induced the bone marrow depression in all living beings. In delayed condition leukemic condition was observed in the benzene exposed volunteers. Benzene decreases haemoglobin level, red blood cell concentration, and packed cell volume, and also increases the number of immature white blood cells in the blood stream. Benzene, the commercial use of which dates to the late nineteenth century, was one of the earliest industrial chemicals demonstrated to affect the health of large numbers of workers . Exposure of benzene varied from low concentrations, thought to be without effect in humans, to high concentrations, ranging up to many hundreds of parts per million (ppm), which resulted, in workers, displaying decrease in the numbers of erythrocytes, leucocytes and/or thrombocytes in circulating.

Blood Decreased numbers of all three cell types were termed pancytopenia and was usually found to be the result of aplastic anemia. Thus, it was agreed that the bone marrow was the target organ for benzene toxicity. We now recognize that benzene exposure can also lead to the development of myelodysplasia , a pre-leukemic state, and to one of the many types of blood cancers termed the leukemias.

For more than a century our main concern regarding benzene and human health has been the danger. Furthermore, we have little information on exposure to benzene in gasoline, cigarette smoke or other potential sources . In our study, benzene was administered to animals by oral route at dose 400 mg/kg body weight for 7 days. After 7 Days in benzene treated animals a significant decrease was observed in the values of T-RBC and Hgb, and increase was observed in the value of WBC. The initial body weight of rats was slightly increased in both control and treated animals but no significant difference were seen in the treated groups. In haematology parameters of benzene treated animals , 52% reduction were seen in the values of T-RBC, 35% reduction were seen in the values of Hgb, but 50% increase were seen in the values of WBC. The increase in immature WBC showed leukemic condition in rats. These leukemic animals were used as a model in activity of Quercetin.

Present study was conducted to observe the effect 50

of benzene and Quercetin drug metabolising enzymes in rats which showed that values of Hgb and total RBC increased. The various blood cells (erythrocytes, granulocytes, platelets) are produced at a rate of approximately one million to 3 million per second in a healthy adult, this characteristic makes hematopoietic tissue particularly sensitive target for cytotoxic and antibiotic agents such as those used to treat cancer, infection and immune mediated disorders. Animals of control and treated groups remained generally active and healthy throughout the experiment.

There was no redness or discharge from the mucosal membranes and body orifices. No mortality was seen in any of the groups either control or treated. There was no significant gain or loss in the body weight of the rats as observed throughout the experiment. There were no regular variations in the average 24-hour water and food intake of the rats of both control and treated group monitored. However group IV animals where benzene was administered along with Quercetin showed a slight increase in erythrocyte (RBC), Haemoglobin (Hb), Hct, MCV and lymphocyte count value. This increase can be attributed to the ameliorative effect of Quercetin. Haematotoxic effect of benzene was studied in rats at the dose level 400 mg/kg b. wt. Benzene effect was well defined in treated group characterized by decrease in RBC count, Hb% and WBC count which were indicators of anaemia. Previous studies suggested that bone marrow showed erythroid maturation arrest, vacuolation, hypocellular marrow and erythroid depletion in benzene treated rats at different dose levels. These effects were dose related and reversible. In human, effect of benzene was studied by the rapid inhibition of hemat-opoiesis with reduced haemoglobin and infrequent decrease in WBC and platelet count. Hence, the findings of the present study showed that the doses given of concentration 400 mg/kg can bring about no histopathological effect in any organ of rats.In this experiment after benzene treatment at the dose 400mg/kg body weight in 3 group (benzene treated) of rats , induction of leukemia and anaemia has been observed. After treatments with benzene we have ameliorate the leukemia and anaemia by giving the different dose of Quercetin to the Spragu dawley rats, the different doses of Quercetin are 10mg, 20mg and 40mg per kg body weight. www.ijsir.co.in

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REFERENCES 1. Hunter, D. The Diseases of Occupations, 3rd ed.; Little Brown and Co.: Boston, MA, USA, 1962.

2. Newell, L.C. Faraday's discovery of benzene. J. Chem. Educ. 1926, 3, 1248–1253.

3. World Bank Group. Coke Manufacturing: Pollution Prevention and Abatement Handbook; World 4. Batchelder, H.R. Chemicals from coal. Ind. Eng. Chem. Prod. Res. Dev. 1970, 9, 341–343.

5. Production of major commodity chemicals.

Chem. Eng. News 1991, 77, 35.

6. United States Environmental Protection

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Agency (USEPA). Carcinogenic Effects of Benzene: An 7. Mitchell AE, Hong YJ, Koh E, Barrett DM, Bryant DE, Denison RF, Kaffka S (Jul 2007).

"Ten-year comparison of the influence of organic and conventional crop management practices on the content of flavonoids in tomatoes". Journal of Agricultural and Food Chemistry 55 (15): 6154–9. doi : 10.1021/ jf070344. PMID 17590007.

8. Jeong EJ, et al Inhibitory cons tuents of Euonymus alatus leaves and twigs on nitric oxide produc on in BV2 microglia cells . Food

ChemToxicol. (2011)

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International Journal of Scientific and Innovative Research 2016; 4(2) : 52‐61 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

ANTIBACTERIAL AND ANTIOXIDANT ACTIVITIES OFEUGENIA JAMBOLANUM, BUTEA MONOSPERMA AND CASSIA AURICULATA LEAVES EXTRACT Kiran Abha Singh1, *Anup Kumar2 1

Kiran Abha Singh, Department of Life Sciences, Singhania University, Jhunjhunu, Rajasthan, India,2Anup Kumar, Central Institute of Medicine and Aromatic Plants (CIMAP), Lucknow, India *Address for correspondence : Anup Kumar, Central Institute of Medicine and Aromatic Plants (CIMAP), Lucknow, India, E-mail ID: akumar2554@gmail.com

ABSTRACT Present work investigated antibacterial and antioxidant activities of Eugenia jambolanum, Butea Monosperma and Cassia auriculata leaves extract. In antibacterial activity,ethyl acetate and methanol extracts obtained from leaves of Eugenia jambolanum, Butea Monosperm and Cassia auriculata and tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. The antibacterial activity was evaluated based on the diameter of inhibition zone (in millimeters) using disk diffusion and well diffusion method. The minimal inhibitory concentration of the plant extracts against these bacteria was assessed using broth dilution method. Result shows that leaves extracts of Eugenia jambolanum, Butea Monosperma and Cassia auriculatashowed varying degrees of antibacterial activity on the microorganisms tested as above. Therefore, they can be used as an external antiseptic in the prevention and treatment of bacterial infections caused by these pathogenic bacteria. The antioxidant activity was determined by in vitro methods such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, Reducing power and Nitric oxide radical scavenging of the Ethyl acetate extract from leaves of Eugenia jambolanum, Butea Monosperma and Cassia auriculata. It was observed that the Ethyl acetate extract has possessed prominent significant scavenging activity as DPPH, Reducing power and Nitric oxide scavenging when compared to the reference standard ascorbic acid. Ascorbic acid also has shown significantly increase in the scavenging activity. Qualitative results reveal that ethyl acetate extract has the maximum with strong antioxidant activity indicates its scope for utilization in food and biological systems.

Keywords: Antibacterial; Antioxidant;Eugenia Jambolanum;Butea Monosperma; Cassia Auriculata INTRODUCTION Nature has provided abundant plant wealth which possess medicinal virtues for all living creatures.

The use of plants with pharmaceutical properties has received increased interest nowadays from both homeopathic and allopathic branches (Joy et al.; 2012). The medicinal plants play an important role in public health especially in developing countries where it is believed that the intense utilization of plants with therapeutic action does not lead to intoxication (Mossi et al.; 2009).

Microorganisms are closely associated with the health and welfare of human beings. Some are beneficial and some are detrimental. Infectious diseases are the leading cause of death worldwide.

The herbal remedies of traditional healing systems 52

around the world can be utilized as an important source for the discovery of new antibiotics (Okpekon et al.; 2004).Some traditional remedies have already produced compounds that are effective against clinically important strains of bacteria. Plants possessing antibacterial activity for various diseases are being studied by various methods to evaluate their antibacterial property. An attempt has been made to assess the antibacterial properties of Eugenia jambolanum; Butea Monosperma and Cassia auriculata for potential antibacterial activity against Bacillus subtilis; Staphylococcus aureus; Escherichia coli; Pseudomonas aeruginosa and Salmonella typhi. www.ijsir.co.in

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Reactive species play a dual role in human as both toxic and beneficial compounds. At low or moderate levels, reactive species exert beneficial effects on cellular redox signaling and immune function but at high concentrations they produce oxidative stress, a harmful process that can damage cell function and structures. Plants are the potential source of natural antioxidants. Natural antioxidants are the secondary metabolites of plants and are found in all parts of plants. The Ethyl acetate extract of Eugenia jambolanum, Butea Monosperma and Cassia auriculata leaves were evaluated for their DPPH radical scavenging, Nitric oxide radical scavenging and reducing power activities.

MATERIALS AND METHODS Collection of Plant Materials The fully mature Eugenia jambolanum, Butea Monosperma and Cassia auriculata were collected in September and October 2015 from Sonbhadra District of Uttar Pradesh,India from a single tree. The leaves were identified and authenticated wide voucher specimen by Department of Botany,Singhania University, Jhunjhunu,Rajasthan, India. The plant samples were thoroughly washed in the running tap water to remove adhering dust particles and dried under the shades for about two weeks. It was grounded into a coarse powder and stored in an airtight container which can be used for the further investigations.

Test microorganisms The microorganisms used for antibacterial activity evaluation were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh,India), which were maintained on Nutrient broth media. They were Gram-positive bacteria such as Bacillus subtilis (MTCC 8561), Staphylococcus aureus (MTCC-96) and Gramnegative bacteria such Escherichia coli (MTCC729),Pseudomonas aeruginosa (MTCC-647) and Salmonella typhi (MTCC-733).

Culture medium and inoculums The stock cultures of microorganisms used in this study were maintained on Plate Count Agar slants at 4°C. Inoculum was prepared by suspending a loop full of bacterial cultures into 10 ml of nutrient www.ijsir.co.in

broth and was incubated at 37°C for 24 hours. On the next day Muller- Hinton agar (MHA) was sterilized in a flask and cooled to 45-50°C and was distributed by pipette (20 ml) into each sterile petri dish and swirled to distribute the medium homogeneously. About 0.1 ml of bacterial suspension was taken and poured into petri plates containing 20 ml nutrient agar medium. Using the L-shaped sterile glass spreader bacterial suspensions were spread to get a uniform lawn culture.

In vitro determination of antibacterial activity Stock cultures were maintained at 4°C on slants of nutrient agar. Active cultures for experiment was prepared by transferring a loopful of colonies from the stock culture to peptone water and incubated for 4h at 37°C. Antibacterial activity was determined by agar disc diffusion method (Atata et al; 2003).

Standard suspension of bacteria was inoculated on the surface of Muller-Hinton (Hymenia) agar plates. Dimethyl Sulphoxide and Methanol (1:1) was used to dissolve the plant extract. Sterilized filter paper discs (5mm) containing 20μL of each extract (100mg/ml) was arranged on the surface of the inoculated plates and incubated at 37°C for 1824h. Along with this 50μg streptomycin disc was studied for antimicrobial activity as a positive control whereas the solvent used for preparing extract was used as a negative control. At the end of incubation,inhibition zones formed around the disc were measured with Himedia zone scale.The study was performed in triplicate and the mean values were presented.

PLANT EXTRACTS ACTIVITY ASSAY

Paper disc method Diameter of zone of inhibition was determined using the paper disc diffusion method as described

by Lai et al. (2009) and Adedapo et al. (2008). A swab of the bacteria suspension was spread on to petri plates containing nutrient agar media. Sterile filter paper discs (6 mm in diameter) was impregnated with 50 μL/ml extracts and for each organism placed on seeded agar.The plates were incubated at 37°C for 24 h. The ethanol served as negative control while the standard streptomycin 50μL/ml disc was used as positive controls.

Antimicrobial activity was indicated by the presence of clear inhibition zone around the discs. The assay 53

International Journal of Scientific and Innovative Research 2016; 4(2) : 52‐61 P‐ISSN 2347‐2189, E‐ ISSN 2347‐4971

was repeated thrice and mean of three experiments was recorded.MIC was defined as the lowest concentration of extract that inhibit visible growth.

Well diffusion method The agar well diffusion method was used for the antimicrobial evaluations. According to Obeidat et al., an inoculum suspension was swabbed uniformly to solidified 20 ml Mueller-Hinton Agar for bacteria and the inoculum was allowed to dry for 5 min. Holes of 6 mm in diameter were made in the seeded agar using glass pasteur pipettes.

Aliquot of 20 μL from each plant crude extract was added into each well on the seeded medium and allowed to stand on the bench for 1 h for proper diffusion.The plates were incubated at 37°C over night and examined for the zone of inhibition. The diameter of the inhibition zone was measured in millimeters (mm).

Determinationof MICof plant extract by broth dilution assay Ethyl acetate and methanol extracts of plants were determined for their MIC values using a standard protocol of Andrews, 2001. Nutrient broth was used as the medium to culture bacteria. One ml of this broth was added to the numbered tubes 1–9.

Reducing power assay The reducing power of a complex acts as a marker of its potential antioxidant activity. The reducing properties are generally connected with the presence of reductones which show the evidence of antioxidant activity by breaking the chain reactions by donating the hydrogen atoms (Venkatachalam et al., 2012).The reducing power was determined according to the method of Oyaizu (1986). Various concentrations of mushroommethanolic extracts (2.5 ml) were mixed with 2.5 ml of 200 mmol/l sodium phosphate buffer (pH 6.6) and2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50º C for 20 min. After 2.5 ml of 10% trichloroaceticacid (w/v) were added; the mixture was centrifuged at 650 rpm for 10 min. The upper layer (5 ml) was mixed with5 ml deionised water and 1 ml of 0.1% of ferric chloride and the absorbance was measured at 700 nm: higher absorbance indicates higher reducing power. The 54

One ml of the stock culture was added to tube 1 and serially diluted until tube number 7. The last 1 ml of tube 7 was discarded. Tube number 8 was used as a negative control and the tube 9 as a positive control.

The bacterial inoculum was cultured in nutrient broth and incubated overnight. All the tubes were inoculated with 1 ml of the test bacteria media except tube number 8 and incubated for 24 hrs at 37°C DPPH radical scavenging activity DPPH free radical scavenging is a conventional mechanism used for screening the antioxidant activity of the plant extracts.The DPPH radical scavenging activity of different extracts were measured in terms of hydrogen donating or radical scavenging ability using a stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl). Different concentrations of each extracts and standard were taken in different vials. 3 ml of DPPH solution (2mg/ml) were rapidly mixed with plant extracts.

The mixture was then vortex mixed vigorously and left for 30 min at room temperature in the dark. The absorbance was read at 517 nm. Ascorbic acid was used as reference standard. The radical scavenging activity was expressed as percent inhibition and was calculated using the following formula.

assays were carried out in triplicate and the results are expressed as mean values ± standard deviations.Higher absorbance indicates higher reducing power. Ascorbic acid was used as reference compound. All tests were carried out in triplicate.

Nitric oxide radical scavenging (NO) assay Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions which were measured using the Griess reaction reagent(Green et al.,1982).Nitric oxide is a very unstable species under aerobic condition. It reacts with O2 to produce stable products, nitrate and nitrite through intermediates. It also plays an important role in the pathogenesis of pain, inflammation, neural signal transmission,immune response , control of vasodialation and blood pressuren (Divya and Mini, 2011).3.0 ml of 10mM sodiumnitroprusside www.ijsir.co.in

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in phosphate buffer was added to 2.0ml of extract and reference compound in different concentrations (25 - 400 μg/ml). The resulting solutions were then incubated at 25°C for 60 min.

A similar procedure was repeated with methanol as blank which served as control. To 5.0 ml of the incubated sample,5.0ml of Griessreagent (1% sulphanilamide, 0.1%naphthyethylenediamine dihydrochloride in 2%H 3 PO 3 ) is added and absorbance of the chromophore formed was measured at 546 nm.Percent inhibition of the nitrite oxide generated was measured by comparing the absorbance values of control and test preparations.

Ascorbicacid can be used as a positive control. All tests were carried out in triplicate.

RESULTS AND DISCUSSION Antibacterial activity from leaves of Eugenia jambolanum The results of the effects of ethyl acetate and methanol extracts of the Eugenia jambolanum using 50 μL of 10 mg/mL of plant extracts against the Staphylococcus aureus ,Salmonella typhi Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis using Disc diffusion and Well diffusion method are presented in Table 1. It is shown that ethyl acetate and methanol extracts have the highest zone of inhibition on S. aureus in both method.

Microorganisms

Diameter of inhibition zones (mm/50 μL) Disc diffusion method Well diffusion methods Ethyl Acetate Methanol Ethyl Acetate Methanol Staphylococcus aureus 14 16 15 17 Salmonella typhi 11 12 13 14 Pseudomonas aeruginosa 11 12 10 13 Escherichia coli 14 15 12 14 Bacillus subtilis 12 13 13 14

Used concentrations: 50 μL of 10 mg/mL of plant extracts Table 1: Antimicrobial Activity of Eugenia jambolanum Escherichia coli and Bacillus subtilis. During Antibacterial activity from leaves of Butea antimicrobial study;methanolic extracts showed Monosperma high zone of inhibition compare toethyl acetate Ethyl acetate and methanol extracts of the Butea extractagainst all organisms in cup plate method Monosperma were showed significant zone of was shown in table 2. inhibition against the Staphylococcus aureus; Salmonella typhiPseudomonas aeruginosa; Diameter of inhibition zones (mm/50 μL) Disc diffusion method Well diffusion methods Ethyl Acetate Methanol Ethyl Acetate Methanol Staphylococcus aureus 10 13 11 15 Salmonella typhi 11 14 11 15 Pseudomonas aeruginosa 12 12 12 14 Escherichia coli 15 15 14 16 Bacillus subtilis 13 14 14 12 Microorganisms

Used concentrations: 50 μL of 10 mg/mL of plant extracts Table 2: Antimicrobial Activity of Butea Monosperma Antibacterial activity from leaves of Cassia auriculata The ethyl acetate and methanol extracts from leaves of Cassia auriculata showed a good inhibition against all the bacterial strains tested. www.ijsir.co.in

The gram-positive bacteria were sensitive with gram-negative bacteria.The leaf extracts of Cassia auriculata showing the zone of inhibition in millimeters; for gram positive and gram-negative bacteria are summarized in Table 3.

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Diameter of inhibition zones (mm/50 μL) Disc diffusion method Well diffusion methods Ethyl Acetate Methanol Ethyl Acetate Methanol Staphylococcus aureus 14 15 15 16 Salmonella typhi 13 13 12 15 Pseudomonas aeruginosa 11 14 10 15 Escherichia coli 13 15 14 14 Bacillus subtilis 14 16 13 17 Used concentrations: 50 μL of 10 mg/mL of plant extracts Microorganisms

Table 3: Antimicrobial Activity of Cassia auriculata Many naturally occurring compounds found in plants have been shown to possess antimicrobial functions and serve as a source of antimicrobial agents against pathogens (Kumar et al.; 2006).

Bacterial infectious diseases represent an important cause of morbidity and mortality worldwide. Therefore; the development of new antimicrobial agents for the treatment of bacterial infections is of increasing interest.

Microorganisms

S. aureus S.typhi P.aeruginosa E.coli B. subtilis

Eugenia jambolanum 33 49 57 72 74

Minimum inhibitory concentration (MIC) The results of MIC values for ethyl acetate and methanol extracts of the Eugenia jambolanum,Butea Monosperma,Cassia auriculata, Aegle marmelos and Cassia fistula against the Staphylococcus aureus, Salmonella typhi Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis were shown in Table 4 and 5.

MIC (mg/mL) for ethyl acetateextract Butea Monosperma Cassia auriculata 38 66 57 46 70

46 50 17 11 10

Table 4: MIC value of ethyl acetate extract from leaves of Plant Microorganisms

S. aureus S.typhi P.aeruginosa E.coli B. subtilis

Eugenia jambolanum 37 49 60 70 76

MIC (mg/mL) for methanolic extract Butea Cassia auriculata Monosperma 40 50 66 50 57 15 45 12 70 12

Table 5:MIC value of Methanol extract from leaves of Plant used to evaluate the free radical scavenging ability All plants showed a good inhibition against all the of various natural products and has been accepted bacterial strains tested.The present study indicates as a model compound for free radical originating in that the phytochemicals of these plants have lipids.The scavenging effects of Ethyl acetate significant inhibition for all the bacterial strains extract from leaves of Eugenia jambolanum, Butea tested and justified the medicinal use of these plant Monosperma and Cassia auriculata on DPPH leaves and further study is required to find out the were examined at the different concentrations (20, active component which is of utmost medicinal 40, 60, 80,100μg/ml) and were shown in graph 1 to value against this range of microorganisms.

3.

DPPH radical scavenging activity The DPPH radical scavenging has been widely 56 www.ijsir.co.in

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Ascorbic acid was used as a reference standard. It was observed that the DPPH Radical scavenging activity of these plants were as comparable to that of standard ascorbic acid (graph 1 to 3).The Ethyl acetate extract of the plant showed promising free radical scavenging effect of D P P H in a concentration dependant manner up to a concentration of 100μg / ml. The % of inhibition was increased with increasing concentration of the extract.Concentration of the sample necessary to decrease the initial concentration of DPPH by 50% (IC50) under the experimental condition can be

calculated from the graph 1 to 3. A lower IC50 value denoted a higher antioxidant activity. Ethyl acetate extract from leaves of Eugenia jambolanum (IC50value is 14.2μg/ml); Butea Monosperma(IC50 value is 70.0μg/ml) and Cassiaauriculata (IC50value is 36.2μg/ml) showed promising free radical scavenging activity by DPPH method as compared the positive standard (ascorbic acid) for Eugenia jambolanum (IC50 value is 12.0 μg/ml), Butea Monosperma (IC50 value is 36.1 μg/ml) and Cassia auriculata(IC50 value is 38.3 μg/ml).

Graph 1: Graph displaying DPPH radical scavenging activity of ethyl acetate extract of Eugenia jambolanum.

Graph 2: Graph displaying DPPH radical scavenging activity of ethyl acetate extract of Butea Monosperma. www.ijsir.co.in

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Graph 3: Graph displaying DPPH radical scavenging activity of Ethyl acetate extract of Cassia auriculata.

Reducing power assay Ethyl acetate extract from leaves of Eugenia jambolanum, Butea Monosperma, Cassia auriculata, Aegle marmelos and Cassia fistulaon Reducing power were examined at the different

concentrations (20, 40, 60, 80, 100μg/ml) and were shown in graph 4 to 6.Ascorbic acid was used as a reference standard.Reducing capacity of extract were compared with ascorbic acid for the reduction of Fe3+ - Fe2+.

Graph 4:Graph displaying Reducing power assay of Ethyl acetate extract of Eugenia jambolanum

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Graph 5: Graph displaying Reducing power assay of Ethyl acetate extract of Butea Monosperma

Graph 6: Graph displaying Reducing power assay of Ethyl acetate extract of Cassia auriculata Nitric oxide radical scavengingactivity Percent inhibition of the nitrite oxide generated was measured by comparing the absorbance values of control and test preparations and ascorbic acid was used as a positive control.The scavenging effects of Ethyl acetate extract from leaves of Eugenia

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jambolanum, Butea Monosperma, Cassia auriculata, Aegle marmelos and Cassia fistulaon Nitric oxide radical scavenging activity were examined at the different concentrations (20,40,60, 80,100μg/ml) and were shown in graph 7 to 9.

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Graph 7: Graph displaying Nitric oxide scavenging activity of Ethyl acetate extract of Eugenia jambolanum

Graph 8: Graph displaying Nitric oxide scavenging activity of Ethyl acetate extract of Butea Monosperma.

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Graph 9: Graph displaying Nitric oxide scavenging activity of Ethyl acetate extract of Cassia auriculata CONCLUSION The leaf extracts of Eugenia jambolanum,Butea Monosperma andCassia auriculata showed varying degrees of antibacterial activity on the microorganisms tested as Staphylococcus aureus, Salmonella typhi Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis. The activity of leaf methanol extract was found to be quite good compare to ethyl acetate extract therefore they can be used as an external antiseptic in the prevention and treatment of bacterial infections caused by these pathogenic bacteria,which have developed resistance to antibiotics. This study demonstrated that the methanolic leaf extracts of these plants are as effective as modern medicine to combat pathogenic microorganisms.It was also observed that ethyl acetate has possessed prominent significant scavenging activity as DPPH ,Nitric oxide scavenging and Reducing power when compared to the reference standard ascorbic acid.

Ascorbic acid also has shown significant increase in the scavenging activity.

REFERENCES 1. Okpekon; T.;Yolou; S.;Gleye; C.;Roblot; F.;Loiseau; P.;Bories; C.;Grellier; F.;Frappier; F.; Laurens; A.;Hocquemiller; R. (2004): Antiparasitic activities of medicinal plants used in Ivory Coast. J. Ethnopharmacol. 90: 91-97.

2. Atata R F ; Sani A. and Ajewole S M (2003);Biokemistri;15(2) ;84-92.

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Antioxidative; tyrosinase inhibiting and antibacterial activities of leaf extracts from medicinal ferns. Biosci Biotechnol Biochem; 2009; 73; P. 1362-1366.

Adedapo; A.A.;Jimoh; F.O.;Koduru; S.; Afolayan; A.J. and Masika; P.J.; Antibacterial and antioxidant properties of the methanol extracts of the leaves and stems of Calpurnia aurea. BMC Complementary and Alternative Medicine; 2008; 8; P. 53 Obeidat. M;Shatnawi. M; Al-alawi. M; AlZu`bi. E; Al-Dmoor. H; Al-Qudah. M; ElQudah. J and Otri. I (2012). Antimicrobial Activity of Crude Extracts of Some Plant Leaves. Research Journal of Microbiology; Vol.7: 59-67.

Andrews; J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy; 48 (suppl 1); 5-16.

Venkatachalam U;Muthukrishnan S. Free radical scavenging activity of ethanolic extract of Desmodiumgangeticum; Journal of Acute Medicine; 2; 2012; 36-42.

Oyaizu M. Studies on product of browning reaction prepared from glucose amine; Japan Journal of Nutrition; 7; 1986; 307-315.

Green LC; Wagner DA; Glogowski J; Skipper PL; Wishnok JS; Tannenbaum SR. Analysis of nitrate; nitrite and nitrate in biological fluids; 61

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Analytical Biochemistry; 126; 1982; 131–138.

10. Divya BT and Mini S. In vitro radical scavenging activity of different extracts of Butea Monospermabark. Int J Curr Pharm Res; 3; 2011; 114-116.

11. Kumaran; A.; Joel Karunakaran; R. 2007.

Antioxidant activity of Cassia auriculata flowers;Fitoterapia 78: 46-47.

12. Joy V; Paul M; Peter J;Yesu JR; Ramesh

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(2012). Medicinal values of avaram (Cassia Auriculata Linn.): A Review. Int J Curr Pharm Res; Vol 4; Vol 4; Issue 2; 1-3.

13. Mossi AJ. Mazutti; Paroul M.;Corazza N; Dariva ML;Cansian C & Oliveira RL (2009).

Chemical variation of tannins and triterpenes in Brazilian populations of Maytenusilicifolia Mart. Ex Reiss Brazilian Journal of Biology 69 (2).

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REHABILITATION OF SICK INDUSTRIAL UNITS *Niaz Ahmed Siddiqui Department of Mechanical Engineering, Integral University, Lucknow, Uttar Pradesh, India *Address for correspondence : Dr. Niaz Ahmed Siddiqui, Department of Mechanical Engineering, Integral University, Lucknow, Uttar Pradesh, India, Email ID: niazsiddiqui@yahoo.com ABSTRACT Industrial units , going sick, have very adverse effect on growth and economy of the country. It results into (i) under utilization of capital assets, (ii) poor business scenario, (iii) investors holding back their investments (iv) large scale unemployment and (v) decrease in profitability of banks/financial institutions.

Hence, prevention of sickness and rehabilitation of sickunits assume greater importance. Government of India enacted a law 'Sick IndustrialCompanies (Special Provisions) Act, 1985' (SICA). The main objective of SICA is to determine sickness and expedite the revival of viable units or closure of unviable units. The Board of Industrial and Financial Reconstruction (BIFR) was set up in Jan.1987. The role of BIFR as envisaged under SICA is (i) timely detection of sick units, (ii) determination of various measures to be taken in respect of sick unit and (iii) enforecement of such measures.

There may be many causes of sickness – (i) external factors, where management has little control and (ii) internal factors, where management has more or full control. It is seen that liquidity and constraints is the root cause of sickness. Any inustrial unit going sick gives many disturbing signals in different areas of business. If these signals are noticed and controlled, the unit may be prevented from going sick. Few of these signals are in different areas, like (i) stock inspection, (ii) bankledger, (iii) market report, (iv) financial statement etc. A unit once declared sick would have already consumed huge scarce resources.

In order to utilise the assets and infrastructure already created for the unit, the unit is to be revived from sickness. Before attempting to rehabilitise a sick unit, a detailed viability study is to be made. It is advisable not to go for any revival programme if there are doubts. The viability study can be made on four important aspect – (i) technical, (ii) commercial, (iii) managerial and (iv) financial factors. In the process of rehabilitation of a sick unit, certain important information and its detailed analysis is very much required. These informations may be listed as (i) objectives and features of rehabilitation scheme, (ii) decision on rehabilitation and (iii) preparation of rehabilitation scheme which needs a detailed study/analysis like – (i) assesment of additional funds, (ii) preparation/arrangement of cash flow with creditors, (iii) marketing arrangement, (iv) recovery of debts, (v) disposal of unwanted assets etc.

Keywords : Act ; Account; Asset; Assesment; Bank; Board; Borrower; Company; Capital; Cash; Creditors; Commercial; Development; Division; Debt; Disposal; Economy; Entrepreneurship; Enforcement; Equity; Finance; Funds; Features; Feasibilty; Holders; Industrial; Investor; Loss; Land; Labour; Liquidity; Ledger; Management; Market; Production; Promoter; Potential; Performance; Rehabilitation; Reconstruction; Remedial; Regulations; Registrar; Sick; State; Schedule; Signal; Stock; Technical; Unit; Unemployment,; Unrest; Viable.

INTRODUCTION Industrial Units that have gone sick have very adverse effect on the economy of the country. The following are the bad effects of sick industrial unitsa. It results into under utilization of capital assets which is a drain/loss of capital for any country.

It adversely affects the process of capital generation for poor and developing countries. www.ijsir.co.in

b. The entrepreneurship level goes down. In a production system; land, labour and capital are considered as the factors of production. It is only the skill of project promoters that brings together the factors of production for accomplishing the task of nation building.

Increase in industrial sickness slows down the pace of country's development.

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c. The investor's confidence shakes up badly.

Hence, capital is not invested for productive use. d. Industrial unit sickness results into large scale unemployment and industrial unrest. e. Profitability of banks and financial organizations gets affected adversely since they do not get back their funds invested in projects that have gone sick. They also do not earn interest on their invested funds. Since their funds get blocked in sick projects, banks/ financial organizations could not recycle their funds which results that even a good project is not funded by them.

Therefore, prevention of sickness and rehabilitation of sick projects assume greater importance.

DEFINITION The Sick Industrial Companies (Special Provisions) Act 1985, defines sick industrial company as an industrial company which has at the end of any financial year accumulated losses equal to more than its entire net worth.

Government companies having State or Central Government share holding of 51% or more are kept outside the purview of the Act. Also small scale industrial units and ancillary units are kept outside the purview of the Act. BOARD FOR INDUSTRIAL AND FINANCIAL RECONSTRUCTION (BIFR) In the wake of sickness in the country's industrial climate prevailing in the eighties, the Government of India set up in 1981, a committee of experts to examine the matter and recommend suitable remedies. Based on the recommendations of the committee, the Government of India enacted a special legislation namely, the Sick Industrial Companies (Special Provisions) Act 1985, commonly known as SICA. The main objective of SICA is to determine sickness and expedite the revival of potentially viable units or closure of unviable units. It was expected that by revival, idle investments in sick units will become productive and by closure, the locked up investments in unviable units would get released for productive use elsewhere.

The Sick Industrial Companies (Special Provisions) Act 1985 was enacted with a view to securing the timely detection of sick and potential 64

sick companies,, the speedy determination by a body of experts of the preventive, remedial and other measures which need to be taken with respect to such companies and the expeditious enforcement of the measures so determined and for matters connected therewith or incidental thereto.

The Board for Industrial and Financial Reconstruction (BIFR) was set up in January 1987 th and functional with effect from 15 May 1987. The Appellate Authority for Industrial and Financial Reconstruction (AAIFR) was constituted in April 1987. Government companies were brought under the purview of SICA in 1991 when extensive changes were made in the Act including the changes in the criteria for determining industrial sickness.

SICA applies to companies both in public and private sectors owning industrial undertakings – a. Pertaining to industries specified in the First Schedule to the Industries Development and Regulation Act 1951 (IDR Act) except the industries relating to ships and other vessels drawn by power. b. Not being small scale industrial undertakings or ancillary industrial undertakings, as defined in section 3(j) of the IDR Act. c. The criteria to determine sickness in an industrial company are – (i) The accumulated losses of the company to be equal to or more than its net worth i.e. its paid up capital plus its free reserves. (ii) The company should have completed five years after incorporation under the Companies Act 1956. (iii) It should have 50 or more workers on any day of the 12 months preceding the end of the financial year with reference to which sickness is claimed. (iv) It should have a factory license.

The role of BIFR as envisaged in Sick Industrial Companies Act (Special Provision) is: a. Securing the timely detection of sick and potentially sick companies. b. Speedy determination by a group of experts of the various measures to be taken in respect of the sick company. c. Expeditious enforcement of such measures.

BIFR has a Chairman and may have a maximum of 14 members, drawn from www.ijsir.co.in

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various fields, including banking, labour, accountancy, economics etc. It functions like a court and has constituted four benches.

1. Reporting to BIFR The Board of Directors of a sick unit is required to report the sickness to the BIFR within 60 days of finalization of audited account. BIFR has a prescribed format for this report.

Any other interested person/party can also report the facts of sickness of the unit to BIFR. Such interested parties may be bank/financial institution that has lent loan to the company.

The BIFR has prescribed a different format for the report to be submitted by such interested parties.

When a company has been financed by a group of banks, it is the lead bank that should report to BIFR about the sickness under information to other contributing banks.

2. Enquiry by the BIFR When a case is reported to BIFR, it is verified by the Registrar of BIFR as to whether the case falls under the provisions of Sick Industrial Companies (Special Provisions) Act 1985. If so, the BIFR accepts the case and notifies a date for hearing. BIFR, then invites the representatives of the sick unit, the representatives of concerned financial institutions/banks, central/state governments, trade unions etc. for the hearing. Enquiry is made u/s 16 of the Act. After the hearing, the BIFR itself may conduct a study or assigned the work to an authorized agency to determine the facts. The enquiry should be completed within 60 days. Based on the enquiry report, BIFR declares whether the unit is sick or not.

3. Viability of Sick Units A sick unit may be considered viable, if it would be in a position, after implementing a relief package spread over a period not exceeding five years from the commencement of the package from banks, financial institutions, Government (central/state) and other concerned agencies, as may be necessary, to continue to service its repayment obligations as agreed upon including those forming part of the package, without the help of the concessions after the www.ijsir.co.in

aforesaid period.

The repayment period of restructured debts should not exceed seven years from the date of implementation of the package. In case of small decentralized sector units, the period of relief and repayment period of restructured debts will be two years and three years respectively.

Based on the norms specified above, it is for the banks/financial institutions to decide whether a sick unit is potentially viable or not. The viability study of the unit should be carried out and decision on rehabilitation or otherwise should be taken expeditiously on receipt of complete information on all relevant aspect from the management of the unit. It is of utmost importance to take measures to ensure that sickness is arrested at the initial stage itself.

The management of the units should be advised about their primary responsibility to inform the banks/financial institutions if they face problems which could lead to sickness and also to restore the units to normal health. The branch officials who are familiar with the day-to-day operations in the borrowal accounts should also identify the early warning signals by making visits to the units and initiate corrective steps promptly.

CAUSES OF SICKNESS Causes of industrial sickness have to be viewed from the general background of an industrial economy which experience prosperity and depression over a period of time. At any point of time, the problems of industries are not u n i f o r m . H o w e v e r, f a c t o r s g e n e r a l l y responsible for the problems can be divided into – a. External Factors These factors are those over which the management of the organization has little control. Government's plans and actions, failure of monsoon which affects agriculture and allied industries, emergence of strong competitors etc. are some of the external factors. b. Internal Factors These are those factors which are within the control of the management of the organization.

These include faulty planning, implementation of the project, lack of inventory and cost 65

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control, wrong selection of the site, faulty management accounting, managerial deficiency etc.

Though sickness may be caused either by internal or external factors, sometimes, the management may be able to revamp its organization, plan suitable strategies and take on the external factors to reduce their impact.

Causes of sickness can vary from area to area, size to size of units and product to product.

Sickness can originate right from the stage of conception or implementation of the project. In general, a unit becomes sick when it cannot generate adequate cash surplus from internal sources. Internal cash generation is the most important indicator of the unit.

Decline in the internal cash generation happens because of the following reasons : 1.

Drop in production on account of –

problems of production lack of orders lack of raw material 2. Poor cash management 3. Increased cost of production 4. Delayed payments by customers 5. Diversion of funds for non-productive purposes Thus, the causes of industrial sickness are numerous and varied. While studying causes, it could be seen that liquidity and constraint is the root cause of sickness. With continuous cash losses, the unit fails to meet its obligations and in this process, the unit faces a major threat from the suppliers of material. Loss in liquidity arises due to diversion of short–term funds for acquiring longterm assets. Due to worsening liquidity position the unit will have to operate at a lower capacity and lose its major customers. Thus, unit is said to have lost viability and considered sick.   

CAUSES OF SICK UNITS LACK OF WORKING FUNDS

DROP IN INTERNAL CASH GENERATION

DROP IN PRODUCTION

CASH MANAGEMENT

INCREASED COST OF PRODUCTION

a. Deliberate diversion of funds. b. Well intended but unwise diversion c. Poor collections d. Unproductive expenditure e. Unplanned payments to creditors f. High inventory PROBLEMS IN PRODUCTION

a. Machine breakdowna. b. Poor quality raw material c. Poor labour productivityc. d. Power shortaged. e. Lack of Production, Planninge

LACK OF ORDERS

a. Competition b. recession c. Low quality technically d. Irregular deliveries e. Poor marketing efforts

LACK OF RAW MATERIAL

a. National or regional shortage b. high cost c. Overdue payments is suppliers d. Poor quality e. Uncertain suppliers

And Controlf. Obsolescencef.

Lack of planning f. Delayed supplies from Sub-contractors g. Poor industrial relations.

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INCREASED COST OF RAW MATERIAL

INCREASED OVERHEAD COSTS

a. Increased costs not covered in sellinga. prices due to faulty costingb. Unutilized capacity b. Large order booked at fixed pricesc. c. High material waste

a. Inefficient production. b. Heavy borrowings, high interest charges d. Increased administration or selling costs. e. Unplanned capital f. New product development or diversification.

SIGNALS OF SICKNESS There are several signals of sickness of an industrial unit. These signals are obtained from few sources. These include – i. Stock Inspection ii. Bank Ledger iii. Discussion with Borrower iv. Market Report v. Financial Statement It is attempted to show how Banks generally obtain signals from these sources.

1. Stock Inspection By comparing the stock statement of a borrower with his stock register, it is possible to ensure whether the stock position as declared by him is in accordance with the books of accounts. If there is a major variation, a signal is thrown. In case of large borrowers, the verification of stock includes: i. Stock kept at the factory premise ii. Finished stock at the sales depots of the company iii. Raw materials sent to outsiders for conversion or processing iv. Stock in transit Besides physical verification of stock, it is necessary to ensure i. Purchase price of raw material is same as indicated in the invoices ii. Position of goods in process and finished stock as, on the given date, is duly certified by the production manager iii. Pricing policy of the company and methods of valuation of stock is properly adopted.

Any major variation in stock value as per the stock statement on one hand and books of accounts on the other, gives an indication of misappropriation of stock.

1. The physical verification of stock ensures the www.ijsir.co.in

2.

3.

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5.

6.

end use of loans. If the stock is inadequate, it indicates that the funds are not fully utilized for the agreed purpose.

If any stock not related to business is stored in the factory store, it indicates about trading activity.

If the stock statements are deliberately not submitted for a long time, it shows that stock may not be adequately held.

If the company has maintained excessive stock than required, it also indicates that the company is indulging in trading practices.

If the company has taken dual finance from two different bankers on the same stock, it indicates the dishonesty of the borrowers.

It is difficult to ensure the quality of the stock.

But, if there is no movement of a major part of finished goods, it shows that the stock is outdated and not saleable.

2. Study of Bank Ledger 1. Poor turnover in an account indicates that either the sales are being routed through other banks or have dropped. In either case, the officer should make an enquiry. Otherwise, it may lead to misuse of funds by the borrower.

2. Cheques for large and round amount, postdated cheques and cheques frequently issued in favour of those parties not related to business, may need to be examined carefully. A dialogue with the borrower could be initiated to find out the root cause and to take remedial steps.

3. The bill account reveals the major transactions of the business. Frequent return of bills may mean that the borrower's goods are being rejected by purchasers or the purchasers are sending payments directly to the borrower.

When the bills are received back, the borrower is promptly advised that the bills have been returned by the collecting branch for the reasons, 'payment not forthcoming' and that he 67

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5.

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should replenish his account forthwith. Often, banks are more worried about the account and not the reasons for non-payment.

If there has been irregularity in the account for a longer period, it indicates that outflows are in excess of inflows of cash.

If there are no operations in the account during a part of the year, it indicates that the unit has stopped working during that period. This is a signal for analysing the reasons for the inactive operations in the account.

The behaviour of the account during the year would help in getting the signal. The actual drawings should be related to the business requirements. It is expected that the unit normally requires more funds during the busy season. It is also expected that the unit has to maintain a minimum level of stock and, therefore, it requires minimum amount of funds all through the period. Any variation in these expectations would throw a signal.

If there is a heavy withdrawal of cash, this is a signal of sickness.

Discussion with Borrowers Major breakdown in plant and machinery Labor strike Change in management Sudden death/illness of Partner/Director Disputes among Partners/Directors Frequent reconstitution of the Firm/Board Frequent requests for enhancement of limits

4.

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2.

3.

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Market Reports Recession in industry Unfavourable position of the inputs Unsatisfactory reports about the party Sharp fall in prices Unfavourable changes in government policy as regards imports, exports, price fixation, etc.

6. Routing transactions to other banks.

5.

1.

2.

3.

4.

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Financial Statements and other Data Unsatisfactory trend in profits Rise in book debts Shortage of working funds Unsatisfactory position of equity Diversion of short term

NEED FOR REHABILITATION PROGRAMME A unit once declared sick would have already 68

consumed huge scarce resources. In order to utilize the assets and infrastructure already created for the unit, the unit is to be revived from sickness.

The unit must have had some weak areas which could have been the cause for sickness. Inspite of this, rehabilitating a sick unit is worth considering since the cost of setting up a new unit might be substantially higher as compared to the cost of rehabilitating a viable sick unit. It is important to know the factors that were responsible for leading the unit to sickness. They can be properly addressed in the revival package.

Revival of a sick unit may be justified in view of the socio-economic objectives such as the followinga. The unit may be in a sector that is vital to the economy. Abandoning the unit may lead to other socio-economic adverse effects. b. Many ancillary units may be dependent on the units that have gone sick. Unless the sick unit is revived, it will have a chain effect of all such dependent ancillary units becoming sick. c. Banks and financial institutions would have heavily invested their money in the sick units.

In order to get back the investments of banks and financial institutions, the unit is to be revived and made to work again and generate profit. Though banks and financial institutions that support a revival programme for the sick unit may be required to fund the unit again, they will be prepared to implement revival packages if they are convinced that they will, apart from getting back their present investment with interest, also get back their past investments too.

Viability study for rehabilitation proposal Before attempting to rehabilitate a sick unit, a detailed and through viability study is to be made to ensure that the revival programme will be a success. It is not advisable to go far any revival programme if there are doubts that need further study.

The viability study shall enquire into the Technical, Commercial, Managerial and Financial factors.

1. Technical Factor a. Study the manufacturing process used by the unit. Ascertain if any new process has since been developed. Explore the necessity of switching over to the latest manufacturing process and study the cost, benefit aspects of such switchover. www.ijsir.co.in

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b. Study the production capacity of different production sections and ensure if the production capacity of different sections are perfectly balanced. The imbalances, if any, are to be addressed first. The capacity of the project can be substantially enhanced without much investments by adding machines/equipments required for balancing.

2. Commercial Factor a. Commercial failure of a project is mainly due to problems relating to the product itself, viz. defects in product design which may lead to consumer's dislike. Such situations indicate that the products offered by competitors have better features that attract consumers. Hence, the scope for product improvement for consumer's acceptability and the cost involved are to be studied. b. Inspite of consumer's liking of the product, if the project has gone sick, it is certain that the profit margins would also be low. Minor modifications in designing and packaging of the product with upward revision in price may be accepted by the consumer which may bring better returns to the company. This aspect may be studied by the carrying out test marketing for the improved product. c. Every product follows a life cycle which passes through four stages, viz.  Introduction  Rapid Expansion  Maturity  Decline Profit margins are low and sign of sickness appear when the product is in its 'decline' stage. Product improvements can only sustain the products at this stage. The decline once started cannot be contained for long inspite of product improvements. Product diversification may prove to be a feasible solution.

Hence for rehabilitating a unit whose product has already reached its 'decline' stage, the feasibility of switching over to diversified products making use of the existing production facilities is to be studied.

The cost-benefit analysis of additional investments needed for product diversification and additional expected benefits are to be analyzed.

3. Managerial Factor A good project in the hands of a poor management team turns the project bad. Similarly, a good management is capable of making a average www.ijsir.co.in

project, a success. Hence, the first thing is to study whether the sickness is due to reasons beyond the control of the present management or due to the poor management.

If the sickness is due to reasons beyond the control of management but the management is still committed to the project and is serious about reviving the unit. The management's commitment and seriousness may be indicated, if  It has agreed to inject additional funds to revive the unit.  It has agreed to strengthen the existing management by inducting professionals as Directors at various functional areas like technical/finance/marketing/ research and development etc.

The managerial appraisal will suggest the required changes in the existing organizational set up with an aim to reduce the man power without affecting the organizational efficiency.

4. Financial Factor Since appraisal of other areas have financial commitment in one form or the other, financial appraisal assumes greater importance. All aspects of financial investments need to be considered and analyzed.

When a project that has long term debt becomes sick, it becomes necessary to ease the burden of debt. This necessitates restructuring of the debts. In general, banks and financial institutions offer the following concessions in their package of rehabilitation assistance. a. Reduction in interest rate of existing loans. b. Conversion of short term loans into long term loans. c. Conversion of part of long term loans into equity. d. Funding of overdue, unpaid interest and making it repayable in easy instalments . The funded interest component may carry concessional rate of interest or no interest at all. e. Offering a revised schedule of repayment for the principal components of term loan. f. Sanction of additional loan to meet the additional capital expenditure. g. Enhancement of working capital limits and regularizing the irregular portion of working capital.

If any asset is found not useful, it should be disposed off. The amount thus realized should be used in the rehabilitation programme.

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PROCESS OF REHABILITATION In general, a sick unit is defined in terms of its capacity to generate internal funds. A sick unit fails to generate internal funds on a regular basis and depends on its survival on frequent infusion of external funds. Hence, a need for detailed discussions on rehabilitation is very much felt.

1. Objectives of Rehabilitation Scheme (a) To enable the unit to operate at a profitable level. (b) To adjust the irregularity in the account, if any, according to a phased programme.

Thus, the rehabilitation scheme involves two aspects –  Preparation of feasibility report before implementing the scheme.  Careful monitoring of the performance of the unit during the nursing period.

2. Features of Rehabilitation Scheme (a) The scheme should be carefully drawn and all doubts must be clarified before chalking out the same. It must be supported by the bank and the borrowers. (b) As the unit is already heavily debt burdened, the scheme should be based on well calculated breakeven point. Under rehabilitation, the unit should operate at a much higher level than before and in this regard, the following questions must be answeredQ. Is it possible to operate at a higher level under present market conditions?

Q . Will present plant and machinery allow manufacturing output at higher level of activity?

Q. Can present organization cope with increased level of activity? (c) To be able to operate at higher level, additional finance will be necessary and especially in cases where the net worth of the unit has become almost negative. In such cases, the end use of funds must be carefully monitored. (d) The repayment programme must be carefully worked out, enough funds must be available to the unit to operate at the desired level with a view to ensuring continued internal generation of surplus. (e) The rehabilitation should be decided by the concerned authorities within a reasonable time.

The longer the decision making period, the greater would be magnitude of the problem.

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3. Decision on Rehabilitation Keeping in view all the possible outcomes, risk involved and the irregularity in the account, bank decision should aim at promoting the business activity and not the borrower as an individual. The decision should be based on likelihood of possible recovery. Finally, bank can support the borrower provided he has equal interest in coming out of the sickness. In other words, bank decision to rehabilitation a unit could be justified if it can –  Generate adequate activity and employment.  Control the business activity effectively.  Commit additional funds, if necessary.  Elicit co-operation from workers, suppliers of materials etc.

4. Preparation of Rehabilitation Scheme (a) Assessment of Feasibility of the Project The scheme should be finalized after a detailed study of the sick unit and understanding the problems of the unit. In this context, the following points should be kept in mind – i. Is the project feasible i.e. to operate above the break even point? ii. Are the prevailing market prices competitive?

Is it necessary to change the pricing policy? iii. Are raw materials available to produce goods at the desired level of activity? iv. Are there any constraints such as power, transport, space, skilled workers etc? v. Are the existing machineries able to cope with the increased level of activity? vi. Is the borrower prepared to accept the financial discipline being imposed by the bank.

In all the above and any other related questions are positively answered, the project is feasible and should be considered for rehabilitation. (b) Assessment of Additional Funds Additional funds are required for the following i. Pending liabilities and payments to creditors. ii. Minimum funds required to purchase machineries to raise the productive capacity to the desired level. iii. Minimum workings capital requirements till cash cycle gets into motion.

Operating deficits in the short run arising as per cash flow estimates and any other shortage therein to be provided for. iv Operating deficits in the short run arising as per cash flow estimates and any other shortage therein to be provided for. www.ijsir.co.in

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Sources of Funds include i. Additional capital contribution from the borrower and deposits from the friends and relatives. ii. Disposal of excess stock. iii. Speedy recovery of outstanding bills. iv. Internal generation of funds. v. Additional working capital inputs. vi. Additional term loan for acquisition of fixed assets. (a) Preparation of Cash Flow Estimates Banks have to prepare a cash flow statement showing cash flow estimates during the rehabilitation period. The estimates should be prepared on the basis of the realistic considerations. The scheme must also have a provision to sanction ad hoc limit in case the process of credit sanction takes a long time and in the meanwhile, the unit may need a small amount for certain immediate payments. (b) Arrangements made with other Creditors Before finalizing the scheme, banks must seek co-operation from the creditors for supply of raw materials. It must be ensured that supply of raw materials will continue on regular basis and at economic price. It must be ensured that the creditors will continue to grant normal credit to the borrowers. (c) Marketing Arrangement While finalizing the scheme, banks have to study the marketing problems and offer suggestions. Such suggestions would include – I A product that has lost its name in the market with the trader but not with the ultimate users is easy to re-establish by restoring confidence of the former. ii. If existing sales agents are not interested in continuing or not needed because of high cost, there could be two options; either to appoint other suitable selling agents or to create a wide network of selling agents who could be used to realize the overdue debts under gradual collection arrangements through steady supplies of the product to such parties. iii. Freezing of existing bank borrowings: This system is suggested for smooth banking operations. Cash credit balance against noncurrent inventory and old debts should be separated and frozen. Interest thereon should also be kept separate and frozen. For the purpose of deciding on the new financial www.ijsir.co.in arrangements, the current assets which are in

use should only be considered. The distinction between frozen and operating accounts gives operational control through security of transactions at the lender's end. (d) Arrangement for Recovery of overdue Debts and Disposal of unwanted Assets In some cases, it is possible that certain assets which are unwanted, should be disposed off.

Banks have to identify such assets in consultation with the borrower. Banks must ensure that the borrower will make all efforts to recover over dues from customers. Before deciding on whether to rehabilitate the unit or not, banks should know what could be the realizable amount from the disposal of unwanted assets. (e) Arrangement made with Financial Institutions and Equity Holders for additional funds In case of the borrowers having facilities with more than one credit institution, the rehabilitation programme could be finalized with the consent of the consortium bankers.

The possibility of issuing additional equity shares should also be examined. Under the rehabilitation programme, the stake of other lenders should be finalized. (f) Arrangements for Management Performance It is often thought of replacing the old management with new management to overcome the failure of the former. For ensuring the performance, the action can be as drastic as removing the entire top management.

This is suggested in case of mismanagement, misuse of funds, wrong inheritance and other similar situation. Lenders have a right to participate in management and decision making; the critical positions are finance, purchase, sales and production. Success of these executives should be measured on the basis of growth of the borrower's business and safeguarding of lender's interest.

CONCLUSION There are numerous issues relating to rehabilitation. Firstly, there is wide spread sickness in industrial sector. Nearly, 99% of the total sick units in the industrial sector in the country are from Small Scale Industries (SSI) sector. Further, 22% of the total bank credit is blocked in these SSI units.

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is disheartening to note that the percentage of bank credit for the sick units in the last decade has declined drastically. This indicates that banks have given less preference to rehabilitation of sick units in SSI sector though sickness is on the rise.

Further, the number of sick units being found viable is also negligible. Hence there is a major challenge before banks as to how to strengthen rehabilitation. Towards this end, need is felt to carry out a detailed study of sick units in the sector. In particular, it is appropriate to draw lessons from both successful as well as failed cases in rehabilitation of sick units.

REFERENCES 1. Project management by Nagarajan, K., New

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2.

3.

4.

5.

6.

7.

Age International (P) Limited, Publishers, New Delhi.

How to Diagnose, Prevent and Cure Industrial Sickness by Kaveri V.S., Sultan Chand & Sons, New Delhi.

Annual Reports of SIDBI, IDBI Ltd, ICICI Bank and IFCI for different years.

Report of the Working Group to frame guidelines on Rehabilitation of Sick SSI units, appointed by RBI, 1986.

Report of the Working Group on Rehabilitation of Sick SSI units, appointed by RBI, 2000.

Report of the Internal Group to review guidelines on Credit Flow to Sick SSI sector, appointed by RBI, 2005.

Report of High Level Committee on Credit Flow to SSI sector, appointed by RBI, 2004

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EMERGING MARKETING STRATEGIES IN TEXTILE INDUSTRIES IN INDIA WITH SPECIAL REFERENCE TO BRAND BUILDING *Monika Yagnik Merh Department of Commrece, Kalicharan P.G. College, Lucknow, U.P., India *Address for correspondence: Dr. Monika Yagnik Merh, Assistant Professor, Department of Commrece, Kalicharan P.G. College, Lucknow, U.P., India, Email ID: docchani@yahoo.co.in

ABSTRACT The Indian textile industry is one of the largest in the world with a massive raw material and textiles manufacturing base. Our economy is largely dependent on the textile manufacturing and trade in addition to other major industries. A textile is the largest single industry in India (and amongst the biggest in the world), accounting for about 20% of the total industrial production. It provides direct employment to around 20 million people. Textile and clothing exports account for one-third of the total value of exports from the country. There are 1,227 textile mills with a spinning capacity of about 29 million spindles. The process of brand building in India has led to the emergence of the following main trends: Disappearance of the distinction between domestic and international, lmarkets.Expansion of organized retail, networks.

Localisation of global brands, Globalization of local brands. This paper discusses the present scenario of textile marketing especially Brand building in India, and its importance, current trends, and highlights certain problems related to Brand bulding.Further it highlights the improvements that make the textile marketing system most effective.

INTRODUCTION The Indian textile industry is one of the largest in the world with a massive raw material and textiles manufacturing base. Our economy is largely dependent on the textile manufacturing and trade in addition to other major industries. About 27% of the foreign exchange earnings are on account of export of textiles and clothing alone. The textiles and clothing sector contributes about 14% to the industrial production and 3% to the gross domestic product of the country. Around 8% of the total excise revenue collection is contributed by the textile industry. So much so, the textile industry accounts for as large as 21% of the total employment generated in the economy. Around 35 million people are directly employed in the textile manufacturing activities. Indirect employment including the manpower engaged in agricultural based raw-material production like cotton and related trade and handling could be stated to be around another 60 million.

A textile is the largest single industry in India (and amongst the biggest in the world), accounting for about 20% of the total industrial production. It provides direct employment to around 20 million www.ijsir.co.in

people. Textile and clothing exports account for one-third of the total value of exports from the country. There are 1,227 textile mills with a spinning capacity of about 29 million spindles.

While yarn is mostly produced in the mills, fabrics are produced in the powerloom and handloom sectors as well. The Indian textile industry continues to be predominantly based on cotton, with about 65% of raw materials consumed being cotton. The yearly output of cotton cloth was about 12.8 billion m (about 42 billion ft). The manufacture of jute products (1.1 million metric tons) ranks next in importance to cotton weaving.

Textile is one of India's oldest industries and has a formidable presence in the national economy inasmuch as it contributes to about 14 per cent of manufacturing value-addition, accounts for around one-third of our gross export earnings and provides gainful employment to millions of people. They include cotton and jute growers, artisans and weavers who are engaged in the organised as well as decentralised and household sectors spread across the entire country.

International trade in textiles and clothing has 73

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played an important role in the development process of many countries and has also facilitated their integration in to the world economy. In the Developed Countries, the process of industrialization and Subsequent prosperity in a way commenced with the mechanization of textile production in the early 19th Century. In the Developing Countries, on the other hand, the sector has come to occupy an important place in terms of its contribution to national output, employment and exports. Developing countries as a group account for more than one half of world exports of textiles and clothing.

As the latest WTO report (2006) states "In no other category of manufactured goods do developing countries enjoy such a large net exporting position" as they do in the textile sector.

IMPORTANCE OF TEXTILE INDUSTRY IN INDIA Like the other developing countries, the Textile industry in India also occupies an important place in the economy as shown below: KEY INDICATORS  Contributes 4% to the Gross Domestic Product (GDP)  Accounts for 17% of total Exports  Is the largest employment provider after Agriculture ( 82 million people direct/ indirect)  Market size of the Textile industry (exports & domestic) is US$ 52 billion, at present  Expected to reach US$ 110 billion by 2012 COVERS THE ENTIRE VALUE CHAIN RAW MATERIAL: Cotton Production estimated at 4.32 million Tons SPINNING: 37.5 million spindles WEAVING: 1.93 Million looms (excluding hand looms) APPAREL: Current level of exports - US$ 10 billion EMERGING TRENDS IN WORLD TRADE With the removal of the Quota system, in the year 2005, the textile and clothing industry is undergoing structural changes worldwide with production lines further shifting distinctly towards low cost producing countries with flexible production systems, to match the growing retail power.

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Perceived as a "third migration" this shift is seen more towards Asia- away from Europe, US and a large number of small suppliers who were "Quota rich" prior to 2005 and whose rising cost structures are increasingly precluding them from being able to compete.

A noteworthy feature of these emerging trends in international trade is that the developed countries even though exiting from direct manufacturing, continue to dominate it by controlling the retail end of the supply chain. The cost and price structure globally is being characterized by higher potential for profit from innovation, marketing, and retailing rather than production, assembly, finishing and packaging. Multiple store retailers are already selling 70% of the clothing in Western Europe and 85% in the US. The developing countries on the other hand, are becoming manufacturing hubs for textile products, and are increasingly getting themselves integrated with the global market place and offering capabilities not only in production capacities, but also in product development and efficient Supply Chain management.

Application of Technology In this emerging scenario, wide spread application of technology is required not only to upgrade the quality of products, determine consumer choices, but also to overcome locational disadvantages and reduce overhead costs on unsold inventories.

The developed countries are already focusing on niche products like protective clothing, clothing for medical use by developing competitiveness in novel "nanotechnology" coatings, greater adoption of Product Life-cycle Management (PLM) Systems, in order to deliver new "fast fashion" paradigms, while at the same time remaining steadfastly committed to lower production costs.

The textile industry in the developed countries is also restructuring itself in a manner so as to take advantage of product innovation. Some of the products, now being developed are jackets that cool the wearers down, warm them up, and send out soothing vibrations, textiles with healing and caring properties and protection from harmful radiation. Intelligent Textiles, Smart Clothing are receiving unprecedented attention and are in the realm of possibilities. www.ijsir.co.in

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Immense opportunities are also being seen in the entire gamut of Technical Textiles given the range and diversity of row material, processes, products and applications that they encompass. "Technical textiles" have been breaking new ground due to their cost effectiveness, durability, versatility, user friendliness, eco-properties. In fact, it is estimated that around 40% of all textiles made in Germany are now covered under the field of "Technical Textiles".

While the developed countries are seeking to upgrade their presence in the textiles and clothing sector by moving in to the field of technical textiles, the developing countries are equally concerned about the need to adapt themselves to the changing requirements of the consumers and move up the value-added supply chain by adopting innovative technologies and redefining the product mix.

INDIAN TEXTILE INDUSTRY: CHANGING PROFILE The Indian textile industry has embarked on an ambitious programme of modernization and technological upgradation in recent years to transform the textile sector from a state of low technology level to a producer of high technology products. Technological upgradation in India has resulted in:  A shift from commodity based trading to high value added fashion garments.  Vertical integration and horizontal consolidation of production process leading to lowering of manufacturing costs.  Improved productivity gains  Efficient supply chain management  Development of Economies of scale.

DEVELOPING BRANDS: THE INDIAN EXPERIENCE Greater reliance on technology has led to good quality products, innovative designs, refining of consumer choices and needs. These factors have also led to the emergence of Brand Consciousness.

As is well known, "Brands" occupy a significant place in international marketing as they give a product better identification, differentiate it from competition, create long-term loyalties and make possible premium pricing.

Unbranded products, on the other hand, do save on costs of packaging, selling &manufacturing. www.ijsir.co.in

However, a quantitative analysis of branded & unbranded products shows that costs so saved are far less than the margins that a branded good fetches. For instance, unbranded ladies knitted tops are being sold at US$ 2.50, whereas the same product with a few modifications & improvements fetches nearly US$ 20 when sold as a brand. The difference is thus nearly 9 times.

BRAND-BUILDING IN INDIA The process of brand building in India has led to the emergence of the following main trends:  Disappearance of the distinction between domestic and international markets.  Expansion of organized retail networks.  Localisation of global brands.  Globalization of local brands.

Disappearance of the distinction between Domestic & International Markets Purchasing power of the Indian consumer continues to increase due to India's increasing GDP Growth and "demographic dividend" in terms of youthful population leading to an expansion of the domestic market.

The size of the domestic market is expected to reach US $ 60 billion by 2012 from the present level of US 34.6 billion, growing at a CAG R of 10% MARKET ESTIMATES Market Size of Indian Textile Industry (2006) US$ 52 bn; CAGR - 13% Market Size of Indian Textile Industry (2012) US$110 bn; CAGR -13% Market Size of Domestic Market (2006) - US$ 34.6 bn; CAGR -10% Market Size of Domestic Market (2012) - US$ 60.0 bn; CAGR - 10% The cloth production is expected to grow at an annual rate of 13% and garments at the rate of 19%.

Further, with the reduction in tariffs and the proliferation of Regional Trade Agreements and Free Trade Arrangements, the distinction between the domestic markets and international markets will be narrowed considerably.

Manufacturing firms have recognized this trend, which has resulted in vast improvements in the quality of goods produced for domestic markets, which is on par with international standards.

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With consumers becoming aware of fashion trends worldwide, demand for branded products has been increasing. In fact available data in India shows a vast & significant shift to branded products in preference to tailor made products.

EXPANSION OF RETAIL NETWORKS The increase in GDP per capita, availability of higher disposable income, greater use of credit cards, more working female population have allied to a greater penetration of organized retail network in India. The organized retail industry in India is amongst the fastest growing retail sectors in the world growing at a rate of 22% per annum adding approximately 25 million consumers each year.

The sector is expected to increase its penetration from the present 3% to 12%-15% by the year 2012.

Consequently, awareness about brands will also increase to smaller cities and semi-urban areas (Tier II and Tier III cities) with organized retailers now targeting the rural markets. The expansion of retail networks with their growing emphasis on "lean retailing" i.e. maintenance of low inventories and "rapid replenishment" of merchandise and "full packaging" i.e. one-stop sourcing from "fabrics to fashion" has further encouraged consolidation and integration in the Indian textile industry.

LOCALISATION OF GLOBAL BRANDS India is expected to grow at about 8% until 2020 according to a new report by Goldman Sachs and overtake the economies of Italy, France and the UK by 2017 and emerge as the largest economy in the world after China by 2042.

Considering these prospects and the ongoing rapid globalization of the Indian economy, a large number of foreign brands are jostling for shelf space trying to catch the fancy of Indian consumers.

Further, the present policy permits 51% FDI in single brand retailing which may, in future, be extended to multiple brands. With the entry of major corporates in India into the retailing sector, the demand for international brands is expected to expand exponentially thereby not only popularizing the global brands, but also localizing them.

EMERGENCE OF LOCAL BRANDS Another interesting feature of the Indian Market is the 76 emergence of local brands. All the leading

textile and apparel firms have introduced domestic brands and are increasingly positioning themselves within the various segments in the domestic market. Prior to 2000, there were around 5-6 brands in India, prominent amongst them being Zodiac, Monte-Carlo, Raymond, Bombay Dyeing.

The market size of branded wear has since expanded on account of the continued increase in purchasing power, rapid increase in the consuming class, coupled with reduction in import tariffs. The competition has thus intensified in the Indian market with all the major producers of textiles and clothing products in India now working towards building local brands.

Some of the brands built in recent years are "Pantaloon", "Killer" Jeans, "Easios", "Tibre", "Colour Plus", ''Trigger" etc. Many of these brands have now reached a stage where they can look towards gaining a regional, if not a global presence.

Written by: New Cloth Market In order to gain global acceptance several Indian companies are investing overseas and acquiring International brands. For example, in the Home Textile market, Welspun industry has purchased "Christys", a UK Towel Brand; GHCL has acquired "Dan River" and "Rosebys", Creative Garments has purchased "Portico" brand to facilitate entry into the US and EU markets; Alok Industries has purchased "Hamsard", a UK based retail chain.

ISSUES IN BRAND BUILDING AND BRAND PROMOTION While efforts are being made to popularize global brands in the Indian domestic market and globalize local Indian brands in world markets, there are a number of issues, which need to be addressed not only in the creation of brands but also in sustaining them over a period of time.

These issues are:  Need to understand consumer needs and carefully differentiate the product offering.  Reconcile differences in brand/product development across countries leading thereby to different product cycles for too same product.  Choose between "standardization" or "customization" so as to take into account local sentiments and beliefs.

The popular adage Think Global, Act Local explains how to overcome the above issues. Coca www.ijsir.co.in

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Cola, which is truly a global brand has now come out with Think local, Act local slogan focusing on the need to customize the offerings as per the local requirements.

Another example is the way Mc.Donalds has changed its product mix in India to suit local flavors, tastes and purchasing power.

Joint ventures and collaborations could be a way of overcoming cultural constraints and managing the highly decentralized operations required to make a brand successful in the market. Indian companies are already exploring these possibilities by tying up with overseas companies to try leverage their reach in the global markets.

CONCLUSION The Indian textile industry is rapidly repositioning itself as a global player. Towards this end, Indian manufacturers are increasingly integrating their operations, both vertically and horizontally. Yarn makers, weavers are moving forward into producing finished goods like Home Textiles and Garments.

Simultaneously, small and medium knitwear exporters are integrating backwards into yarn processing and even spinning. Firms are adopting Information Technology to not only manage supplies, but also control production and enhance productivity.

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With the "explosion of expectations", driving consumer demand and investments in retailing, the Indian firms are now catering to requirements of the entire value chain from spinning to branded garments and home textiles. Coupled with increase in the purchasing power of the consumers, strong economic growth and demographic advantages, the stage is well set in India today, for production, sale and consumption of Branded goods.

Textiles are being increasingly viewed as Life Style products with consumers becoming more value conscious than price sensitive. With unprecedented levels of growth and investments taking place, India offers immense opportunities for brand building and brand promo REFERENCES 1. www.tradeget.com 2. www.ibef.org 3. www.bharattextile.com 4. www.texprocil.com 5. www.economywatch.com 6. www.marketresearch.com 7. www.pd.cpim.org 8. www.meaindia.nic.in 9. www.ezinearticles.com 10. www.indialine.com

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DEVELOPMENT AND SCOPE OF TOURISM SECTOR IN UTTAR PRADESH 1

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* Rashmi Mishra , Kamlesh Kumar Shukla , Ishvinder Singh Ahluwalia 1

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Department of Commerce, Kalicharan P.G. College, Lucknow, U.P. India , Research scholar, Sai Nath University , Ranchi, Jharkhand, India

*Address for correspondence : Dr. Rashmi Mishra , Assistant Professor, Department of Commerce, Kalicharan P.G. College, Lucknow, Uttar Pradesh, India ABSTRACT Tourism is the activities of societies traveling to and residing in places outside their usual atmosphere for not more than one successive year for relaxation, business and other commitments not related to the application of a movement waged from within the place stayed. Tourism is travel for entertaining or vacation purposes. Surrounded in the soul of India is Uttar Pradesh, a land where values have developed and faiths converge. The importance of Uttar Pradesh lies not only in this convergence, but also in the advent of cultural and religious customs along some of the chief rivers in the Indian sub-continent – the Ganga and the Yamuna. Throughout history, great cities have arisen and established along great rivers.

Within India, the Ganga and the Yamuna have nurtured a culture because of which religious faith, rituals, culture and intellectual enlightenment have evolved in places along the two rivers.

Keywords: Tourism, vacation, diverse range, Ecotourism, Rural Tourism, Tourism circuits INTRODUCTION Tourism is the activities of societies traveling to and residing in places outside their usual atmosphere for not more than one successive year for relaxation, business and other commitments not related to the application of a movement waged from within the place stayed. Tourism is travel for entertaining or vacation purposes. The World Tourism Organization defines travelers as people who "travel to and stay in places separate from their usual atmosphere for not more than one repeated year for vacation, commercial and other commitments not related to the exercise of an activity compensated from within the place stayed". Tourism has become a popular worldwide relaxation movement. In 2008, there were more than 903 million global traveler arrivals, with a progress of 6.6% as equated to 2007. Transnational tourist earnings were USD 856 billion in 2008.

Notwithstanding the doubts in the worldwide economy, arrivals raised at around 5% during the first four months of 2009, almost a similar growth than the same period in 2008. Uttar Pradesh is the fourth leading state in India with an estimated area of 2, 40,928 sq. km. It is also the densely inhabited state in the nation with a population of 199.5 million (2011). Uttar Pradesh is one of the most 78

favoured state for visitors in India with a steady position amongst the topmost states in relations of tourist arrivals. In 2014 it was ranked 2nd in terms of total tourist arrivals, 2nd in terms of domestic tourist arrivals and 3rd in terms of external tourist arrivals amid Indian states. The Tourism industry in Uttar Pradesh has a noteworthy role to the state's financial growth.

UNIQUENESS OF UTTAR PRADESH Uttar Pradesh is endowed with a diverse range of tourism offerings. These range from wonder of the world (Taj Mahal – Agra) to exclusive cultural and holy hotspots like Varanasi, Braj (Mathura, Vrindavan, Goverdhan), Awadh (Lucknow, Ayodhya). U.P. is the single state which is habitat of one of the wonders of the world “Taj Mahal” which is also a UNESCO world heritage site. U.P. is home to some very key Hindu pilgrim centres of India viz. Krishna Janmabhoomi (Mathura), Ram Janmabhoomi (Ayodhya), Sangam (Allahabad), Baba Vishwanath (Varanasi), Maa Vindhyavasani (Vindhyachal). Some important destinations connected to the life of Lord Buddha viz.

Kapilvastu, Sarnath, Shravasti, Kaushambi, Sankisa and Kushinagar are located in U.P. Uttar www.ijsir.co.in

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Pradesh has a excess of unique environment especially in the Terai area of Dudhwa National Park and Pilibhit Tiger Reserve, which is endowed with exclusive floral and faunal biodiversity.

Dudhwa National Park of U.P. is the only secure area (other than Kaziranga National Park, Assam) where one can get one horned rhinoceros beside with national animal tiger. It is also the only place in India where one can see 5 species of deer viz. sambhar deer, swamp deer, spotted deer, barking deer and hog deer. Branch of Tourism has just introduced the Uttar Pradesh Culture Arc which is gaining all round appreciation. It attaches three most important tourism hubs of U.P. i.e. Agra, Lucknow and Varanasi. It is bound to increase tourism as it covers the true soul of Uttar Pradesh, by offering a gamut of cultural, religious and heritage tourism assets. Apart from Heritage Arc Circuit, UP has some very interesting and potential tourism circuits and trails, such as Buddhist Circuit, Pilgrimage Circuit – Kashi - Sangam, Mathura – Vrindavan, Allahabad – Vindhyachal, Ayodhya Trail, Sufi Trail – Lucknow - Deva Sharif; Ittra Trail – Kannauj, The Great Awadh Circuit – Lucknow, Heritage Trail – Lucknow, Cycling Trail – Lucknow, Taj Nature Tour, Agra Heritage Tour, Agra – Braj Tour; Rhino – Tiger Circuit – Dudhwa National Park, Awadh Bird Trail, Mango Trail, etc.

The cuisine of Uttar Pradesh is just as diverse as its geography. The main genre of U.P is Awadhi, famed for its Dumpukht (food cooked on slow fire).Uttar Pradesh is also home to rich textiles, crafts, dance/drama, and legacy of musicians adding to the exquisiteness of tourism offerings in the state.

CHALLENGES FACED BY UTTAR PRADESH TOURISM i. Inadequate support infrastructure at tourist destinations ii. Inadequate road, rail and air connectivity to various tourist destinations iii. Inadequate availability of hotel rooms iv. Inadequate availability of skilled labor v. Limited availability of hygienic and quality food outlets, restaurants at destinations vi. Inadequate cleanliness at tourist destinations & surroundings including lack of clean public toilets vii. Poor visitor management at site especially religious destinations viii. Lack of conservation of Heritage Sites www.ijsir.co.in

ix. Limited availability of certified tourist guides x. Harassment of tourists from miscreants &

notorious elements xi. Safety of tourists ACTIVITIES COVERED UNDER THE TOURISM INDUSTRY  Hotels, Motels and Restaurants  Heritage Hotels  Wayside facilities on National Highways or  State Highways wherein restaurants and parking are available  Tourist resort/tourist village  Theme Park, Amusement park & Water parks  Nature Walk, City Walk, Heritage Walk, Cycle Tours etc  Production and marketing of traditional crafts and other artwork  Work related to maintenance of cultural and historical heritage  Promotion to establishment and running of museums  Tourism/ Hotel Management Institutes  Tourism activities related to environment conservation/jungle safari/jungle lodge etc  Homestay scheme (Bread and Breakfast)  Organizing and development of Adventure activities such as – trekking, rock climbing, water sports, boat race, skating, fishing, aero sports etc  Package tours, conducted tours  Caravan, cruise boats, Yatch, house boats and establishment of boats clubs  Establishment and operation of ropeway  Yoga, Ayurveda and Naturopathy institutes  Travel agent/Tour Operation Company.  Operation of helium and hot air balloon  Activities in Rural tourism  Spa & health resorts.  MICE convention center (minimum area of 10,000sq. ft) STRENGTHENING OF TOURISM TRANSPORT SYSTEM AIR TRANSPORT i. Air transport will be developed further to improve last mile connectivity to the tourist places in the State. All the major tourist places will be connected through the aerial route. At present air connectivity is available only for Agra, Lucknow, Varanasi and other places. ii. International Airports will be constructed in Agra and Kushinagar 79

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iii. Civil Airports will be established at Allahabad, Gorakhpur, Jhansi, Muradabad iv. ir taxi and helicopter services will be encouraged to facilitate travel of tourists v. Private entrepreneurs will be encouraged for operating the air transport services through the State's Intra-State Regional Connectivity policy ROAD TRANSPORT i. All major tourist destinations which lack good connectivity will be connected through good quality roads. All major destinations will be connected through 4 lane highways and where needed the existing 4 lanes will be upgraded to 6 lane highways. The Uttar Pradesh Tourism Department will actively pursue this with the National Highway Authority of India (NHAI), Uttar Pradesh State Highways Authority of India (UPSHAI) etc ii. The quality of all the main roads connecting the tourist places in the State will be significantly improved with the help of the Public Works Department. It is proposed that a separate head be created in the PWD budget for creating roads connecting major tourist destinations iii. Traffic Signages of International and National standards will be placed along major roads leading to tourist destinations iv. Public amenities like eateries, toilets, washrooms, ATMs, repair shops, petrol pumps etc. will be created at strategic locations v. Police and Highway Patrol to be ensured on all major state and national highways connecting major tourism destinations in co-ordination with Home department and Highway Authorities BUS SERVICE i. Department of Tourism will increase its offerings in terms of Tourists Coaches/Luxury Buses by creating tourism packages to major tourism destinations with the help of UPSTDC & private sector ii. Hop-on and Hop-off bus services will be introduced at tourist places RAIL TRANSPORT i. At present only a few major tourist destinations are connected to the National capital by superfast express trains. Efforts will be made in close co-ordination with Indian Railways to 80

connect all major tourist destinations with the National Capital Region with high-speed rail services ii. Efforts will be made to extend Shatabdi connectivity to Varanasi on the Heritage Arc (Agra – Lucknow– Varanasi) iii. Arrangements for special tourist trains will be made for important tourist destinations/ festivals in close co-ordination with Indian Railways.

DEVELOPMENT OF NEW TOURIST DESTINATIONS i. New tourist destinations and new circuits shall be identified and presented to tourists ii. In the Tourism Policy of 1998; seven circuits had been identified for tourism development.

These will be reorganized and new attractions will be added to them. The circuits identified in 1998 were:  Braj Circuit (Mathura, Vrindavan, Agra and other places connected to Lord Shri Krishna's life)  Bundelkhand Circuit (Jhansi, Lalitpur, Deogarh, Kalinjar, Chitrakoot and nearby areas)  Buddha Circuit (pilgrimage sites connected with Lord Buddha)  Vindhya Circuit (areas connected to Vindhyachal and Sonbhadra)  Awadh Circuit (Lucknow and Allahabad and areas in between)  Forest, Eco Tourism and Adventure Tourism Circuit (Wildlife sanctuaries, forests and ecotourism spots in the State)  Water Sports Circuit (places with possibilities of water sports)  The State Department will undertake InfraGap Analysis of the existing tourism circuits to assess the civic and tourist infrastructure, and shall improve and strengthen the same for better tourist satisfaction.  Seven new circuits will be added to this series –  Heritage Arc (Agra-Lucknow-Varanasi) region  Mahabharata Circuit (areas connected to the Mahabharata era)  Ramayana Circuit (areas connected to the Ramayana era)  Jain Circuit (areas connected to the Jain faith)  Sufi Circuit (prominent areas connected to Sufism) www.ijsir.co.in

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 Freedom Struggle Circuit (areas connected to

the independence movement)  Craft, Cuisine and Culture trail (cultural centers connected to handicrafts, cuisine and special cultural activities) SHORTAGE OF HOTEL ROOMS AT TOURIST DESTINATIONS i. Development of wayside amenities with the help of private sector participation along major tourist routes / circuits ii. To address shortage of accommodation facilities the department of tourism shall identify unutilized government properties/resthouses viz. Department of Irrigation, PWD, Forest amongst others will be utilized for tourism purposes iii. Efforts shall be made with local bodies and development authorities to make regulations/ bye-laws flexible towards encouraging private sector participation in establishing tourism units iv. Provisioning of land parcels for tourism activities will be ensured under the development plans of local development authorities including provision for hotels in the City Master plans v. Department of Tourism will be consulted in selection of land parcels for development of hotels.

DEVELOPMENT OF PILGRIMAGE TOURISM i. Strengthening and Upgradation of civic amenities and facilities at religious tourist destinations across the state ii. Strengthening the facilities at religious destinations will be done developing management trusts on the lines of Mata Vaishno Devi Shrine Board iii. The private sector shall be encouraged to participate towards creation of accommodation facilities atreligious destinations iv. With the help of local bodies regular cleanliness drives shall be ensured at religious destinations v. Creation of Tourist Facilitation Centres will be done to enhance the visitor experience at religious destinations. These centres shall ensure centralized booking facilities, tourist information, food etc. Such centres shall be set www.ijsir.co.in

up in the upcoming 10 years at Vrindavan, Vindhyachal, Ayodhaya, Naimisharanya, Varanasi etc.

DEVELOPMENT ECOTOURISM & WILDLIFE TOURISM i. The Department of Tourism will provide civic and tourist amenities in national parks and wildlife sanctuaries jointly along with Forest Department to ensure visitor satisfaction ii. Nature Interpretation Centers, Nature Camps, Boating, Nature Tour Programs etc shall be organized iii. While encouraging wildlife tourism it shall be ensured that it does not disturb, deteriorate or have any negative impact on the environment iv. Planning for such destinations will be done by engaging the local communities to ensure equitable distribution of benefits and socioeconomic upliftment of the local population. v. Trained eco-tourism guides and naturalists shall be trained and certified in co-ordination with the Forest Department DEVELOPMENT ADVENTURE TOURISM i. Private sector participation will be encouraged to develop adventure tourism activities likerock climbing, bungee jumping, aero sports activities like hot air ballooning, paragliding, parasailing and water sports centers etc will be developed ii. There will be an arrangement of special incentives to start adventure tourism activities in Bundelkhand and Vindhya regions DEVELOPMENT CULTURAL TOURISM i. The Department will keep on organizing several Mahotsavas and events to promote cultural tourism ii. Financial assistance will be provided for organizing traditional fairs iii. The dates of fairs/festivals will be publicized for the next ten years in the publicity materials and events of the state DEVELOPMENT CRAFT / HANDLOOM AND TEXTILE TOURISM i. Moradabad's craft industry, Bhadohi's carpet industry, Agra's inlay work and Zardoji, Varanasi's silk industry, Gorakhpur's terracota, Nijamabad and Khurja's pottery industry and 81

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Lucknow's chikan industry are world famous. ii. Shilpgrams and Shilp Bazaars will be

established across the state to promote UP's rich Handicraft legacy iii. The state shall develop textile tourism circuits to promote the indigenous art by linking it to major tourist destinations RURAL TOURISM Under Rural Tourism Policy, villages will be identified which are associated with special art forms or handicraft, music genre with a view to introduce foreign tourists and encourage their stay in the State. This shall enable tourists to experience special village cuisine, culture and arts.

Comprehensive village development plans to be prepared to boost tourism in rural areas PROMOTION OF SELF EMPLOYMENT IN TOURISM SECTOR To enhance the employment opportunities in tourism industry a grant up to INR 10 Lakhs can be availed by fast food centers, souvenir shops, operation of buses, taxies, adventure sports, camp sites, garages, etc.

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REFERENCES 1. UTTAR PRADESH Tourism Policy Perspective & Tourism Policy-2016 2. Cultural Heritage of Jammu and Kashmir / K. Warikoo - 2009 3. Heritagescapes and Cultural Landscapes / Rana P B Singh (Ed.) & William Logan – 2011 4. Unknown Himalayas / Himanshu Joshi – 2008 5. Branding India : An Incredible Story / Amitabh Kant – 2009 6. Sacred Kerala : A Spiritual Pilgrimage / Dominique-Sila Khan - 2009 7. www.egyankosh.ac.in 8. tourism.indiabizclub.com 9. www.indiastat.com 10. www.mystikalindia.com 11. www.theindiatravelguide.com 12. www.tradechakra.com 13. www.mustseeindia.com

www.ijsir.co.in

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VARIOUS ASPECTS OF MARKETING OF FINANCIAL SERVICES *Kumar Kautilya Department of Commerce, Vidyant Hindu, P.G. College, Lucknow, U.P., India *Address for Corresponding: Dr. Kumar Kautilya, Assistant Professor, Department of Commerce, Vidyant Hindu, P.G. College, Lucknow, U.P. India ABSTRACT A unique aspect of financial services marketing which differentiates it from other marketing practices is the illusive notion of quality. In the traditional context of marketing manufactured goods, quality is typically objectively measured utilizing standard quality assessment methods and by assessing product defect rates on the production line. However, in the context of financial services, the notion of quality is a highly subjective phenomenon. The marketing of financial services is a unique and highly specialized branch of marketing. The practice of advertising, promoting, and selling financial products and services is in many ways far more complex than the selling of consumer packaged goods, automobiles, electronics, or other forms of goods or services. The environment in which financial services are marketed is becoming more competitive, making the task of marketing financial services increasingly challenging and specialized. Financial services marketers are challenged every day by the unique characteristics of the products they market. Advertising is a fundamental part of most successful marketing strategies in both financial and non-financial services. It is the primary mechanism by which marketers create awareness among consumers about their products and services. However, it has a special role in the marketing of financial services since financial services are generally intangible. The intangible nature of financial services stems from the fact that they cannot be touched, tasted, felt, or visualized. As a result, consumers' perceptions of quality are often based on the image associated with the company. the quality of manufactured goods might be easily visible to the consumer through the observation of the product's physical features, the quality of financial services is a largely unobservable construct. The training and knowledge of a financial advisor, the transaction accuracy of a credit card company, or the financial strength of an insurance company are largely unknown measures to the masses.

As a result, the financial services advertisers have to educate consumers on the unique and beneficial features of their services. Financial services advertising facilitates the differentiation of a company from its leading competitors. This is an especially important task when consumers may not possess the required background knowledge and product information to appreciate the merits and weaknesses of competing financial services. Advertising is one of the few ways to achieve differentiation in financial services.

Keywords: Marketing; Financial Services; Advertising; Consumer; Insurance; Pricing.

INTRODUCTION The marketing of financial services is a unique and highly specialized branch of marketing. The practice of advertising, promoting, and selling financial products and services is in many ways far more complex than the selling of consumer packaged goods, automobiles, electronics, or other forms of goods or services. The environment in which financial services are marketed is becoming more competitive, making the task of marketing financial services increasingly challenging and specialized. Financial services marketers are www.ijsir.co.in

challenged every day by the unique characteristics of the products they market. For example, often financial services cannot be visually communicated in advertisements as easily as consumer goods can. Furthermore, the relatively unexciting nature of financial services makes the task of attracting consumer attention and inspiring consumer desire a difficult one. However, the study of financial services marketing is in many ways far more fascinating than other areas of marketing.

There are many predictable behaviors that 83

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consumers often exhibit in their dealings with financial services providers. The predictability of these behaviors and the abundance of data on existing and potential customers enable a uniquely scientific approach to developing and executing successful strategies for the marketing of financial services, much more so than in other markets.

There is mounting evidence that suggests the environment in which financial services are marketed is becoming more complex and challenging. Below, we will discuss some of the evidence to illustrate the notable changes that characterize the financial services markets of today and tomorrow.

In 1999, the Indian financial services industry was deregulated, thus allowing financial institutions from a variety of backgrounds to participate in markets they had not traditionally been active in.

Industry deregulation was partially motivated by the argument that allowing financial services organizations to operate on a larger scale would result in numerous cost efficiencies that could then be passed on to the consumer in the form of lower prices.

In other words, the ability to serve a larger customer base with a wider array of products would lead to lower cost structures due to more efficient operating infrastructures. What is interesting, however, is that evidence for the beneficial effects of operating on a large scale in the form of a better value for the consumer or the shareholder has yet to be documented. In fact, a study suggests that smaller commercial banks, in comparison to their larger counterparts, are better able to serve their customer base and shareholders. Other studies also question the economic advantages of large-scale consolidations in the banking sector.

While the deregulation of financial services created an environment that fosters competition, several additional regulations which limit or control the marketing activities of financial services organizations have been implemented since then.

In addition, other regulations have been implemented in the past decade that address how information about consumers can be shared across financial institutions.

The result has been a monumental reduction in the volume of paper checks that banks need to handle, thereby decreasing processing time, and increasing the overall cost efficiency of the banking sector.

One of the factors that make the marketing of financial services unique is the fact that most 84

financial services have to be judged by consumers within the context of the current economic environment in which they are offered. The attractiveness of a savings product, for example, might be a function of the interest rates and expected rates of inflation. Similarly, investment options may largely relate to one's expectations of how the stock market might behave in the near and distant future. Other factors, such as the cost of energy, expectations of unemployment, exchange rate fluctuations, and general trends in the economy might encourage or discourage consumers from purchasing particular financial products and services. The overwhelming influence that economic forces have on the attractiveness of financial products and services greatly impacts their marketing.

The current economic environment in India is in a unique historical phase. For example, interest rates in recent years have been at their lowest levels in decades, a main contributor to the high levels of consumer borrowing. Furthermore, leading economic indicators, such as the price of crude oil, suggest that we may be heading towards an economic cycle where increased production costs due to high energy prices may limit economic activity. It is noteworthy to point out that history has shown that when similar spikes in energy prices have occurred in the past, as for example seen in the mid and late 1970s, the economy was subjected to strong recessionary forces. The increased cost of energy will in one way or another have an impact on consumers' budgets and reduce overall consumer spending. Therefore, subsequent effects on financial services which support such spending, for example by providing consumers with credit, will likely follow.

A unique aspect of financial services marketing which differentiates it from other marketing practices is the illusive notion of quality. In the traditional context of marketing manufactured goods, quality is typically objectively measured utilizing standard quality assessment methods and by assessing product defect rates on the production line. However, in the context of financial services, the notion of quality is a highly subjective phenomenon. For example, while the objective quality of an insurance company might be reflected by the willingness of the company to pay out customer claims, this measure is rarely known by the average customer. In the insurance business, the majority of policyholders do not utilize their policy www.ijsir.co.in

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benefits since the events being insured typically have low probabilities of occurrence. As a result, most policyholders never experience the process of filing a claim, and for those that do, the outcome of their experience may not be captured or recorded anywhere for others to examine and learn from. The net effect is that the most objective aspect of the quality of an insurance company, which is the protection it offers its policyholders in case of losses, may never be determined by the majority of consumers. Quality assessments in such a context are therefore not objective and largely based on subjective factors such as the customer's recognition of the name of the company or suggestions and advice provided by friends or insurance brokers.

Similarly, in the context of securities brokerage services, customers may not necessarily be able to determine whether the broker is providing them with the most objective and informed advice. The objective quality of a broker-recommended investment portfolio may not be evident for many years until the securities within that portfolio have exhibited their long-term characteristics. A similar issue can be identified in the context of tax returns.

While a tax accountant's ability to secure the highest tax refund is probably the most objective aspect of quality, a client may never be certain of having received the highest possible refund. Such an inquiry would require one to file taxes with multiple accountants as a means of "testing," which is a highly impractical exercise. In all these contexts, despite the important role that the financial services provider plays in securing the financial well-being of the customer, quality assessments by the customer may be driven by highly subjective aspects of the service experience such as the friendliness of then service providers or perceptions of the level of expertise portrayed in the service process.

Pricing is one of the most important decisions in the marketing of financial services. Price serves multiple roles for the financial services organization as well as for the individuals who use those services. To the financial services organization, price represents the sole source of revenues. Most activities that an organization undertakes represent costs and an outflow of funds.

When advertising, for example, one has to spend money purchasing advertising space in a www.ijsir.co.in

newspaper or media time on radio or TV. When employing staff in a sales department salaries and benefits need to be paid. All of these activities represent an outflow of funds, and the only way to recover these expenditures is through revenues obtained by charging prices for the financial services provided. It is critical not only to appreciate the importance of price, but also to be certain that one's prices are at optimal levels.

Pricing too low. or too high can have detrimental effects on profitability of financial services organizations.

In addition, price is the most visible component of the marketing strategy of a financial services organization. Unlike advertising style, product strategy, or sales force incentives, which might be difficult to quantify precisely, price is always presented numerically, and can be observed and compared by consumers, regulators, and competitors. Therefore, a second function of price is to communicate to the marketplace the identity, market positioning, and intentions of a financial services organization. Lowering of prices or an upward movement of premiums might signal a shift in marketing strategy to competitors and may provoke reactions from them. This fact raises the strategic importance of price and highlights the great impact that price has been found to have in shifting the balance of power among competing financial services providers.

A third function of price is to serve as a signal of quality to customers. As mentioned in earlier chapters, the quality of a financial service may be highly elusive and vague. Determining whether one insurance policy is better than another or if an investment advisor will provide recommendations that generate high returns on one's investment portfolio, is difficult if not impossible for many. It has been well established in consumer research that in such situations where quality is not clearly evident, consumers tend to rely on price as a proxy for quality. They might therefore assume that higher-priced financial services are of better quality, and the lowering of prices may not necessarily be associated with more positive consumer impressions of the financial service. The potential for this unexpected relationship between price and consumer demand in specific markets further highlights the critical importance of setting prices correctly in financial services.

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The complexity of financial services prices and the cost structure of financial services organizations have a great impact on how financial services pricing is practiced.

Advertising is a fundamental part of most successful marketing strategies in both financial and non-financial services. It is the primary mechanism by which marketers create aware-ness among consumers about their products and services. However, it has a special role in the marketing of financial services since financial services are generally intangible. The intangible nature of financial services stems from the fact that they cannot be touched, tasted, felt, or visualized.

As a result, consumers' perceptions of quality are often based on the image associated with the company. This place the burden of informing consumers about the beneficial aspects of a financial service on the shoulders of the advertiser.

While the quality of manufactured goods might be easily visible to the consumer through the observation of the product's physical features, the quality of financial services is a largely unobservable construct. The training and knowledge of a financial advisor, the transaction accuracy of a credit card company, or the financial strength of an insurance company are largely unknown measures to the masses. As a result, the financial services advertisers have to educate consumers on the unique and beneficial features of their services.

Financial services advertising facilitates the differentiation of a company from its leading competitors. This is an especially important task when consumers may not possess the required background knowledge and product information to appreciate the merits and weaknesses of competing financial services. Advertising is one of the few ways to achieve differentiation in financial services.

Advertising in financial services can be formally defined as marketing communications carried out through the mass media or through direct marketing means, with the intention of motivating the purchase of specific financial products or encouraging particular forms of financial behavior.

Various forms of media can be used to execute advertising campaigns. Broadcast media such as television and radio, as well as print media such as newspapers and magazines, are often used to execute advertising campaigns for financial 86

services. In addition, a growing trend in financial services marketing involves using direct advertising methods such as direct mail and direct e mail to elicit consumer responses.

These methods create a sense of personalization and help generate leads for subsequent sales. In addition, advertising may not only have the objective of selling specific products, but it may also be used simply to encourage specific forms of financial behavior in consumers. For example, advertising may be used to increase public awareness of the needs for retirement planning and savings, or to encourage the purchase of insurance products to protect oneself against catastrophic financial losses.

As insurers move to direct distribution and database marketing, new approaches to the business, integrating the marketing, under-writing and pricing activity will be increasingly important.

Many insurers today are adopting increasingly sophisticated approaches to direct marketing, especially for automobile insurance. Large, sophisticated customer databases and sophisticated analytic approaches are used to direct marketing efforts. The underwriting, pricing and marketing roles at these companies are evolving but are still largely segregated. Each area functions in a highlyspecialized area, linked to the others but still compartmentalized. Marketing analyzes customers and customer lists to predict response rates and thus profitability of the marketing activity. Actuarial analyzes experience data to estimate loss costs by type of insured. This is often done with special actuarial databases, making little use of more extensive customer databases.

To achieve optimal results, more integration of these three roles is needed. The actuary will need to have active hands on involvement in the marketing process, helping the marketers move beyond response prediction, to analysis of loss costs as well.

REFERENCES 1. Arya P.P. Tandon, B.B. "Human Resource Development", Deep and Deep Punlications, New Delhi, 1991.

2. www. wikipedia.org.

3. Goyal, "Developing of HRD", Indian Journal of Commerce, Vol. XL, No. 3, Jul.-Dec. 1987 4. Media Reports, Press Releases, Press Information Bureau. www.ijsir.co.in

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RE-ORIENTING THE ROLE AND REVAMPING OF FOOD CORPORATION OF INDIA *Shantanu Kumar Srivastava Department of Commerce,Vidyant Hindu P.G. College, Lucknow, U.P. India *Address for correspondence: Dr. Shantanu Kumar Srivastava, Department of Commerce,Vidyant Hindu P.G. College, Lucknow, U.P., India. E-mail ID: - shantanu0729@yahoo.co.in ABSTRACT The Government of India set up a High-Level committee (HLC) to restructuring the Food Corporation of India(FCI) with a view to improve its operational efficiency and financial management. The FCI in its present condition has become obsolete to achieve food security and maintaining the buffer stock level. The Food Ministry itself is not keen on breaking up the FCI into three separate entities handling procurement, distribution and storage of food grains. Among the options being considered by food department is breaking up the FCI into zone-wise. The other options being mulled is to unburden the FCI of the tasks of procurement of food grains by letting state governments handle the same. The HLC had wide consultations with various stakeholders in its several meeting in different parts of the country. It also invited comments through advertisements in newspapers and electronic media.

The major recommendations of HLC are on procurement related issues, on PDS and NFSA related issues, on stocking and movement related issues, on buffer stocking operations and liquidation policy, on labor related issues, on direct subsidy to farmers and on end to end computerization. PM Narendra Modi promised during the election campaign in 2014 to break up the FCI into three divisions for handling procurement, management and distribution. It is commonly perceived that FCI is plagued badly with several functional and cost inefficiencies, which need to be removed for efficient management of food grains and saving cost. The Re-orienting the role and Revamping the Food Corporation of India would comes as Asia's largest economy to tame near double-digit food inflation amongst the emerging economies. This Article highlights the major recommendations of High Level Committee on re-orienting the role and revamping the Food Corporation of India and the future prospects of FCI. KEYWORDS: Procurement; Storage; Distribution; Buffer Stock; High Level Committee (HLC); Below Poverty Line (BPL); National Food Security Act (NFSA); Minimum Support Prices (MSP); Public Distribution System (PDS); Private Entrepreneurs Guarantee (PEG); Covered and Plinth (CAP); Direct Payment System (DPS) INTRODUCTION FCI was set up in 1965 under the Food Corporation Act, 1964 against the backdrop of major shortage of foodgrains, especially wheat, in the country to fulfill the objectives of the food policy. These included effective price support operations for safeguarding interests of farmers, distribution of foodgrains throughout the country for Public Distribution System, and maintaining a satisfactory level of operational and buffer stocks of foodgrains to ensure national food security. Selfsufficiency in grains was the most pressing objective and keeping that in mind high yielding seeds of wheat were imported from Mexico.

Agricultural Price Commission was created in www.ijsir.co.in

1965 to recommend remunerative prices to farmers. The Government of India (GoI) set up a High Level Committee (HLC) in August, 2014 with Shri Shanta Kumar as the Chairman , six members and a special invitee to suggest restructuring or unbundling of FCI with a view to improve its operational efficiency and financial management. The Government's move to revamp the Food Corporation of India makes sense. The problem is not just that FCI has been synonymous with inefficiency and mismanagement, converting itself into the world's largest hoarder of grains and a primary driver of food inflation in the country. The problem is that the agency, in its present shape, has 87

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become an obsolete to achieve food security, jacking up the cost of stored foodgrains and therefore, of food subsidy, to unsustainable levels.

As the government looks to revamp the functioning of FCI, the food ministry is considering various options to restructure the Corporation and not just limit itself to unbundling. The Food Ministry itself is not keen on breaking up the Food Corporation into three separate entities handling procurement, distribution and storage of foodgrains. The reason behind it is that the FCI's share in overall procurement is already low and dividing it into three separate units won't do a world of food.

Among the options being considered by food department is breaking up the FCI zone-wise. But in this option there would be some problem of coordination as in India, foodgrains production and consumption centres are miles apart and arranging logistics for them such as railway rakes could become difficult in the absence of a centralized agency. The other options being mulled is to unburden the FCI of the tasks of procurement of foodgrains by letting state governments handle the same. This would mean limiting the FCI's task to storage and distribution of foodgrains. This option looks slightly feasible because already, the Corporation's share in total procurement is miniscule and handling it over to the states will not make much of a difference. The FCI's overall share in procurement of wheat is merely 12% - 14%and 1% - 3% in case of paddy. The rest is purchased by state agencies on behalf of the Corporation.

The HLC had wide consultations with various stakeholders in its several meeting in different parts of the country. It also invited comments through advertisements in newspapers and electronic media. HLC would like to gratefully acknowledge that it has benefitted immensely from this consultative process and many of its recommendations are based on very intensive discussions with stakeholders. In order to conceive re-orienting the role of FCI and its consequent restructuring, one has to revisit the basic objective with which FCI was created and what was the background of food situation at that time. It is against that backdrop, one has to see how FCI has achieved its objectives, what the current situation on foodgrain front is, what are the new challenges with regard to food security, and how best these challenges can be met with a reoriented or restructured institution like 88

FCI. The past history of FCI indicates that India has moved far away from the shortage of 1960s into surpluses of cereals n past 2010 period, but somehow the food management system, of which FCI is an integral part, has not been able to deliver on its objectives very efficiently. The benefits of procurement have not gone to larger number of farmers beyond a few states, and leakages in TPDS remain unacceptable high. Needless to say, this necessitates a re-look at the very role and functions of FCI within the ambit of overall foodmanagement systems, and concerns of food security.

The report of HLC states that none of three main basic objectives of FCI are being met. MSP benefits only 6% of farmer, PDS suffers from huge leakages and the buffer stocks are often far above the requirements leading to huge costs being incurred in storing them and foodgrains often rotting. The report admits that FCI has been in the phase of diseconomies of scale and has been due for restructuring and unbundling long back. It does not talk about FCI becoming an agency for innovation in Foodgrain Management System. But it has not recommended and unbundling of FCI either on functional lines on geographical lines. The report of the HLC on Re-orienting the Role and Restructuring of Food Corporation of India submitted to PM makes a slew of suggestions seemingly unrelated topics like the National Food Security Act (NFSA), cash transfers to farmers as well as Below Poverty Line (BPL) consumers, Minimum Support Prices (MSP), deregulating urea prices and much more.

MAJOR RECOMMENDATIONS OF HIGH LEVEL COMMITTEE (HLC) The major recommendations of HLC keeping in mind how procurement benefits can reach larger number of farmers, how PDS system can be reoriented to give better deal to economically vulnerable consumers at a lower cost and in a financially sustainable manner and finally how stocking and movement operations can be made more efficient and cost effective in not only feeding PDS but also in stabilizing foodgrains market are as follows: (A) On Procurement Related Issues India has set up a panel to suggest an overhaul of a state-run foodgrain procurement agency as part of its www.ijsir.co.in

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plan to reduce wastage and help control runaway food prices. HLC recommends that FCI hand over all procurement operations of wheat, paddy and rice to states that have gained sufficient experiences in this regard and have created reasonable infrastructure for procurement. These states are Andhra Pradesh, Chhattisgarh, Haryana, Madhya Pradesh, Odisha and Punjab. FCI will accept only the surplus from these state governments to be moved to deficit states. FCI should move on to help those states whose farmers suffer from distress sales at prices much below MSP and which are dominated by small holdings, like Eastern Uttar Pradesh, Bihar, West Bengal, Assam, etc. This is the belt from where second green revolution is expected and where FCI needs to be pro-active, mobilizing state and other agencies to provide benefits of MSPs procurement to larger number of farmers, especially small and marginal ones.

The government should procure only to maintain the minimum buffer norm and allow private trade to procure, store and distribute foodgrain. This means keeping the MSP below the procurement price. Consumption subsidy should be transferred to consumers as which they can use to buy foodgrains from any number of public and private outlets that compete for custom and so keep costs down.

This will excise inefficiencies such as FCI's loader salaries of Rs. 1 lakh a month and insufficient and inferior storage of foodgrains.

FCI must stop paying for extortionate state levies on grain purchase. FCI at the Centre should enter into an agreement with states before every procurement season regarding costing norms and basic rules for procurement.

Negotiable Warehouse Receipt System (NWRs) should be taken up on priority and sealed up quickly. Under this system, farmers can deposit their produce to the registered warehouse, and get say 80% advance from banks against their produce value of MSP. They can sell later when they feel prices are good for them. This will bring back the private sector, reduce massively the costs of storage to the government and be more compatible with a market economy. GoI can explore whether this system can be used to compensate the farmers www.ijsir.co.in

in case of market prices falling below MSP without physically handling large quantities of grain.

The GoI also needs to revisit its MSP Policy.

Currently MSPs are announced for 23 commodities but effectively price support operates primarily in wheat and rice and that too in selected states. This creates highly skewed incentive structure in favour of wheat and rice. While country is short of pulses and oilseeds, their prices often go below MSP without any effective price support. Further, trade policy works independently of MSP policy, and many a times, import of pulses come at prices much below their MSP. This hampers diversification. HLC recommends that pulses and oilseeds deserve priority and GoI must provide better price support operations for them.

The report is also silent on the role of artiya (the middlemens) in the whole procurement process. The commission to these artiyas runs into a few hundred crores and this is an unnecessary cost. By not dealing with these issues in detail, the report could attract the criticism that it was using FCI as a cover to push unpopular food economy reforms.

(B) On PDS and NFSA related issues HLC

recommends that GoI must take a second look at the NFSA, its commitments and implementation. The government should defer its implementation in states that have not computerized their PDS, put out the list of beneficiaries in their public domain for verification and set-up vigilance committees.

The Act must be amended to bring down its coverage to 40% of the population from the current 67%. These genuinely poor target household should get 7 kg. of subsidized food grains instead of 5 kg. and the central issue prices being frozen for three years at Rs. 3/2/1 kg. for rice/wheat/coarse cereals respectively HLC recommend while Antyodya households can be given grain at Rs. 3/2/1/ kg. for the time being, but pricing for priority household must be linked to MSP, say 50% of MSP. Else, HLC feels that the NFSA will put undue financial burden on the exchequer, and investments in agriculture and food space may suffer. HLC 89

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would recommend greater investments in agriculture in stabilizing production and building efficient value chains to help the poor as well as farmers.

HLC recommends that targeted beneficiaries under NFSA or TPDS are given 6 months ration immediately after the procurement season ends. This will save the consumers from various hassels of monthly arrival at FPS and also save on the storage costs of agencies.

Consumers can be given well designed bins at highly subsidized rates to keep the ration safely in their homes. HLC also recommend that there should be a gradual introduction of cash transfer in PDS, starting with large cities with more than one million populations. It should then be extended to grain surplus states and finally grain deficient should be allowed to opt for cash or physical grain distribution. This will be much more cost effective way to help the poor, without much distortion in the production basket, and in line with but international practices. There should be transparent liquidation policies to offload food stocks far in excess of buffer requirements in the open market. This should kick in automatically when FCI is faced with excess stocks and the Corporation should get greater flexibility in this. Government must revisit its MSP policy, which has a highly skewed incentive structure in favour of wheat and rice and that too in selected states.

(C) On Stocking and Movement Related Issues

HLC recommends that FCI should outsource its stocking operations to various agencies such as Central Warehousing Corporation, State Warehousing Corporation, Private Sector under Private Entrepreneurs Guarantee (PEG) schemes and even state government that are building silos through private sector on state lands. It should be done on competitive bidding basis, inviting various stakeholders and creating competition to bring down costs of storage. India needs more bulk handling facilities than it currently has. Many of FCI's old conventional storage that have existed for ling number of years can be converted to silos with the help of private sector and other stocking agencies. Better mechanization is 90

needed in all silos as well as conventional storages. Covered and plinth (CAP) storage should be gradually phased out with no grain stocks remaining in CAP for more than three months. Silo bag technology and conventional storages where ever possible should replace CAP. Movement of grains needs to be gradually containerized which will help reduce transit losses, and have faster turn-around-time by having more mechanized facilities at railway sidings.

(D) On Buffer Stocking Operations and

Liquidation Policy One of the key challenges for FCI has been to carry buffer stocks with FCI have been more than double the buffer stocking norms costing the nation thousands of crores of rupees losses without any worthwhile purpose being served.

The underlying reasons for this situation are many, starting with the exports bans to open ended procurement with distortions, but the key factor is that there is no pro-active liquidation policy. Excess stocks add to carrying costs of FCI and grain stored in the open runs the risk of getting spoilt. The government should see excess stocks in small lots to a large number of traders to bring down retail prices. It must maintain a security buffer but the market for grain must be put to work to achieve efficient, competitive procurement, storage, milling in the case of rice and distribution. The current system is extremely ad-hoc, slow and costs the nation heavily. A transparent liquidation policy is the need of hour, which should automatically kick-in when FCI is faced with surplus stocks than buffer norms.

(E) On Labour Related Issues Meanwhile the

employee union of FCI has threatened to launch a nationwide agitation if the government implement the committee's suggestion to lowering of staff strength and privatization of godowns and warehouses. FCI engages large number of workers (loaders) to get the job of loading/ unloading done smoothly and in time. Currently there are roughly 16,000 departmental workers, about 26,000 workers that operate under Direct www.ijsir.co.in

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Payment System (DPS), some under no no pay, and about one lakh contract workers. A departmental worker costs FCI about Rs. 79,500/ per month a DPS worker at Rs. 26,000/ per month and contract labour costs about Rs. 10,000/ per month. Some of the departmental labours (more than 300) have received wages (including arrears) even more than Rs. 4 lakhs/ per month. This happens because of the incentive system in notified depots, and widely used proxy labour. This is a major aberration and must be fixed, either by de-notifying these depots, or handling them over to states or private sector on service contracts, and by fixing a minimum limit on the incentives per person that will not allow him to work for more than say 1.25 times the work agreed with him.

These depots should be put on priority for mechanization so that reliance on departmental labour reduces. There should be a cap on the incentive system which allows loader to earn up to Rs. 4 lakh a month in some cases. HLC recommends that the condition of contract labour, which works the hardest and are the largest in number, should be improved by giving them better facilities.

(F) On Direct Subsidy to Farmers Since the

whole system of food management operates within the ambit of providing food security at a national as well as at household level, it must be realized that farmers need due incentives to raise productivity and overall food production in the country. Most of the OECD countries as well as large emerging economies do support their farmers. India also gives large subsidy on fertilizers. HLC recommends that farmers be given direct cash subsidy and fertilizers sector can then be deregulated. This would help plug diversion of urea to non-agricultural uses as well as to neighbouring countries, and help raise the efficiency of fertilizer use. It may be noted that this type of direct cash subsidy to farmers will go a long way to help those who take leave from money lenders at exorbitant interest rates to buy fertilizers or other inputs thus relieving some distress in the agrarian sector.

(G) On End to End Computerization :- HLC www.ijsir.co.in

recommends total end to end computerization of the entire food management system, starting from procurement from farmers, to stocking, movement and finally distribution through TPDS. It can be done on real time basis, and some states have done a commendable job on computerizing the procurement operations.

But its devoteailing with movement and distribution in TPDS has been a weak link, and that is where much of the diversions take place.

CONCLUSION PM Narendra Modi, who took charge in May 2014, after a landslide victory, promised during the election campaign to break up the Food Corporation of India into three divisions for handling procurement, management and distribution. It is commonly perceived that FCI is plagued today with several functional and cost inefficiencies, which need to be removed for efficient management of food grains and saving cost. The organization, which is central to running the world's largest public food distribution programme, has long been criticized for poor management of country's growing food stockpile.

Indeed many of the recommendations of the HLC are going to be unpopular, notwithstanding their soundness. But despite of its shortcomings, the report sets out a comprehensive overhaul of the food economy. This overhaul is long overdue. The government may have to bite many bullets in implementing these recommendations, but these need to be bitter.

The revamp comes as Asia's third largest economy is struggling for years now to tame near doubledigit food inflation the highest amongst the emerging economies. The new face of FCI will be akin to an agency for innovation in Food Management System with a primary focus to create competition in every segment of foodgrain supply chain, from procurement to stocking to movement and finally distribution in TPDS, so that overall costs of the system are substantially reduced, leakages plugged, and it serves larger number of farmers and consumers. In this endeavour it will make itself much leaner and nimble, focus on eastern states for procurement, upgrade the entire grain supply chain towards bulk handling and end to end computerization by bringing in investments and technical and managerial expertise from the 91

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private sector. It will be more business oriented with a pro-active liquidation policy to liquidate stocks in export markets, but HLC hopes that FCI can rise to this challenge and once again play its commendable role as it did during late 1960s and early 1970s.

REFERENCES Books:1. Dutt, Ruddar & Sundharam, K.P.M. – Indian Economy.

2. Gaur, K. D. – Development Planning.

3. Gerald M. Meir & James E. Rauch. – Leading Issues in Economic Development.

4. J h i n g a n , M . L . – T h e E c o n o m i c s o f Development and Planning.

5. Kalam, A.P.J. – India Vision 2020.

6. Madan, K.D.P. – Management of Corporations.

7. Misra, S.K. & Puri, V.K. – India Economy.

8. Radhakrishna, R. – India’s Public Distribution System, A National & International Perspective.

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9. Rudra , Ashok. – India Agricultural Economics.

10. Seth, M. L. – Theory & Practice of Economic Planning.

11. Streeten, Paul & Lipton, Michael. – The Crisis of Indian Planning.

Reports, Journals and Publications:1. Annual Budget Review Reports, 2015-16, 2017-18.

2. Annual Reports of FCI, 2013-14, 2014-15.

3. Food Corporation Acts, 1964.

4. The Food Corporation Rules & Regulations, 1965.

5. The Indian Journal of Economics.

6. The Indian Journal of Agricultural Economics.

7. Journal of Applied Economics & Management.

8. Economic and Political Weekly, various issues.

9. Handbook of Statistics on the Indian Economy 10. Agricultural Statistics at a Glance, Ministry of Agriculture.

11. Economic Survey, 2013-14, 2015-16.

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