7 introduction

ANTIDIABETIC AND ANALGESIC STUDIES ON

Polyalthia longifolia

INTRODUCTION 1.1. General Introduction Medicine is the wonder of the world and blessings for mankind. From the very ancient time men used various plant parts as their wound healing. Pharmacy, the science and practice of medicine and its primary source plays an important role in identifying the new molecule of drug through both synthetically and from that of natural origin. The history of drug from natural sources is very significant and well known. By trial and error, primitive man must have acquired knowledge that was useful in determining which plants and animals possessed food value and which were to be avoided because they were unpalatable, poisonous, or dangerous.

Medicinal plants continue to be an important therapeutic aid for alleviating ailments of humankind. Search for eternal health and longevity and to seek remedy to relieve pain and discomfort prompted the early man to explore his immediate natural surrounding and tried many plants, animal products and minerals and developed a variety of therapeutic agents.

Over millennia that followed the effective agents amongst them were selected by the process of trial, error, empirical reasoning and even by experimentation. These efforts have gone in history by the name discovery of 'medicine'. In many eastern cultures such as those of India, China and the Arab/Persian world this experience was systematically recorded and incorporated into regular system of medicine that refined and developed and became a part of the Material Medical of these countries. The ancient civilization of India, China, Greece, Arab and other countries of the world developed their systems of medicine independent of each other but all of them were predominantly plant based. But the theoretical foundation and the insights and in depth understanding on the practice of medicine that we find in Ayurveda is much superior among organized ancient systems of medicine. According to past records, Babylonians (about 3,000 B.C.) were aware of a large number of medicinal plants and their properties. Some of the plants are still used today in the same way and for the same purposes. The earliest mention of the medicinal use of plants in the Indian

subcontinent is found in the Rig Veda (4,500–1,600 B.C.), which noted that the Indo-Aryans used the Soma plant (Amanita muscaria), a narcotic and hallucinogenic mushroom, as a medicinal agent. The Vedas made many references to the healing powers of sharpagandha (Rauvolfia serpentine), while a comprehensive Indian herbal book, the Charaka Samhita, cites more than 500 medicinal plants (Gani, 1998; Shamshad, 2004).

1.1.1. Plants in traditional medicine It is estimated that 70-80% of people worldwide rely chiefly on traditional, largely herbal medicine to meet their primary healthcare needs (Farnsworth & Soejarto, 1991; Pei Shengji, 2001). The global demand for herbal medicine is not only large, but growing (Srivastava, 2000). The market for Ayurvedic medicines is estimated to be expanding at 20% annually in

India (Subrat, 2002), while the quantity of medicinal plants obtained from just one province of China (Yunnan) has grown by 10 times in the last 10 years (Pei Shengji, 2002b). An example of increased pressure on collecting grounds is provided by the Gori valley in the Indian Himalayas, where the annual period of MAP harvesting has increased from 2 to 5 months (Uniyal & et. al., 2002). Factors contributing to the growth in demand for traditional medicine

include the increasing human population and the frequently inadequate provision of Western (allopathic) medicine in developing countries.

1.1.2. Status of medicinal plants in Bangladesh About 500 medicinal plants have been reported to occur in Bangladesh. Almost 80% of rural population is dependent on medicinal plants for their primary health care. The local people conserve traditional knowledge through their experience and practices, which is handed down orally without any documentation. The over exploitation of wild medicinal plants has become a threat to its extinction. In Bangladesh there is no systematic cultivation process of conservation strategies about medicinal plants. There is no government policy or rules and regulations about the medicinal plants cultivation conservation and marketing. There are almost 422 herbal medicinal companies using medicinal plants as raw materials mostly by importing from abroad (S Khan & M Huq, 1975).

1.1.3. Medicinal Plants in World Market

The largest global markets for MAPs are China, France, Germany, Italy, Japan, Spain, the UK and the US. Japan has the highest per capita consumption of botanical medicines in the world (Laird, 1999). The International Council for Medicinal and Aromatic Plants expects world growth during 2001 and 2002 to be approximately 8-10 per cent a year (Srivastava, 2000). In 1999, the world market for herbal remedies was US$19.4 billion, with Europe in the

lead (US$6.7 billion), followed by Asia (US$5.1 billion), North America (US$4.0 billion), Japan (US$2.2 billion) and the rest of the world (US$1.4 billion) (Laird and Pierce, 2002). India is a major exporter of raw MAPs and processed plant-based drugs. Exports of crude drugs from India in 1994-95 were valued at US$53,219 million and of essential oils US$13,250 million (Lambert et al., 1997). Overall sales of botanical medicine products in China in 1995 were

estimated at US$5 billion (Laird, 1999). The botanical medicine market in Japan in 1996 was estimated at US$2.4 billion.

1.1.4. Herbal drug research: bioactivity guided approach Books on herbal medicinal practice report numerous medicinal plants, which are still not investigated. These plants can be subjected to pharmacologic screening as per their traditional use to evaluate their utility. In case of significant result, chromatographic and spectroscopic methods can be applied to isolate the responsible agent.

Bioactivity guided approach has three characteristic phases of investigation.

First: Biological activity is detected in crude material, and a bioassay system is set up to permit the identification of active fractions and discarding the inactive ones.

Second: The crude material is fractionated by the most appropriate chemical procedures, all fractions are tested, and active fractions are further fractionated, and so on, until pure compounds are obtained.

Third: The chemical structures of pure compounds are determined (Goldstein, 1974).

1.1.5. Approaches to new product discovery This subject is covered authoritatively in some recent publications (Laird and Pierce, 2002; Laird & ten Kate, 2002; Ten Kate & Laird, 1999) and partially elsewhere in this paper. Several

stages are involved in the process of prospecting the chemical properties of plants to discover drugs or other novel products. First, unless discoveries are fortuitous, decisions are made about which plants to sample and how to sample them. Sampling may be in the field or from ex situ collections, the latter perhaps represented by plants growing in botanical gardens or by dried specimens in herbaria. These decisions are based on published and unpublished information, including sometimes knowledge of local medical uses and about the relative difficulty of undertaking research in different contexts. The next step involves isolation of chemical fractions for automated screening, for example the in vitro testing of activity against cell lines. Promising results may lead to further tests, including perhaps clinical trials, and these may result in the development, including licensing, of marketable products. As an alternative to chemical screening, there is growing interest today in screening extract from plants for genetic information, a branch of science set to grow spectacularly (Hamilton, 2003).

Traditional practitioner dispensing his own medicines is being gradually shifted to herbal drug stores which are profit-oriented. As a result, there is no guarantee of the authenticity and quantity of plant material used in the preparations. The qualities of traditional medicines so produced vary widely and may not even be effective. Therefore, there is a need to select proper and appropriate technologies for the industrial production of traditional medicines such that the effectiveness of the preparation is maintained.

Traditional methods used have many disadvantages which could be corrected by selecting the suitable technologies. It has to be stated that the traditional methods were dependent on the status of technology that was available at that time. It therefore follows that these can be modified and improved using the technologies available today to make them more effective, stable, reproducible, controlled and in dosage forms that can easily be transported or taken to office.

Hence the introduction of appropriate, simple and low-cost technologies should be encouraged maintaining as much as possible the labor-intensive nature of such activities, conservation of biodiversity through small-scale production and preservation of cultural knowledge. Use of sophisticated modern technology will alienate the traditional practitioners as he has no control over such production methods. Even in the use of

appropriate technologies, the practitioner who produces these drugs has to be educated about the advantages of using such production and quality control methods. One major concern in introducing modern technology for the production of traditional medicines is whether the final preparation will be acceptable to the practitioner who has sole faith in extemporaneous preparations. This problem has to be overcome by a process of education, whereby the disadvantage of the old methods and the advantage of the new methods can be imparted. The value of medicinal plant as a source of foreign exchange for developing countries depends on the use of those plants as raw materials in the pharmaceutical industry.

These raw materials are used to: 

Isolate pure active compounds for formulation into drugs (guinini, reserpine, digoxin etc.)



Isolate intermediates for the production of semi-synthetic drugs.



Prepare standardized galenicals (abstracts, powders, tinctures etc.) If one is to produce known pure phytopharmaceutical used in modern medicine more processing stages and more sophisticated machinery are required.

Furthermore safety and pollution aspects have to be considered. Certain plants are rich sources of intermediates used in the production of drugs. The primary processing of parts of plants containing the intermediate could be carried out in the country of origin thus retaining some value of the resource material. Processed products (galenicals) from plants could be standardized fluid/ solid extract or powders or tinctures. Standardized extract of many plants are widely used in health care. Some of these have to be formulated for incorporation in modern dosage forms. New formulations require some development work, particularly on account of the nature of the processed products. Plant extract are difficult to granulate, sensitive to moisture and prone to microbial contamination. Hence the types of excipients to be used and the processing parameters have to be determined (Planning commission, 2000).

1.2. Diabetes 1.2.1. Definition

Diabetes mellitus (DM) is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion, or action, or both. The term diabetes, without qualification, usually refers to diabetes mellitus, which roughly translates to excessive sweet urine (known as "glycosuria") and excessive muscle loss in the ancient world. Elevated levels of blood glucose (hyperglycemia) lead to spillage of glucose into the urine, hence the term sweet urine. When our food is digested the glucose makes its way into our bloodstream. Our cells use the glucose for energy and growth. However, glucose cannot enter our cells without insulin being present – insulin makes it possible for our cells to take in the glucose. Insulin is a hormone that is produced by the pancreas. After eating, the pancreas automatically releases an adequate quantity of insulin to move the glucose present in our blood into the cells, and lowers the blood sugar level (Ruchi Mathur et al).

1.2.2. Classification Most cases of diabetes fall into three broad categories: Type 1 Diabetes: Type 1 diabetes was also called insulin dependent diabetes mellitus (IDDM), or juvenile onset diabetes mellitus. In type 1 diabetes, the pancreas undergoes an autoimmune attack by the body itself, and is rendered incapable of making insulin.

Abnormal antibodies have been found in the majority of patients with type 1 diabetes.

Antibodies are proteins in the blood that are part of the body's immune system. The patient with type 1 diabetes must rely on insulin medication for survival.

Type 2 Diabetes: Type 2 diabetes was also referred to as non-insulin dependent diabetes mellitus (NIDDM), or adult onset diabetes mellitus (AODM). In type 2 diabetes, patients can still produce insulin, but do so relatively inadequately for their body's needs, particularly in the face of insulin resistance. In many cases this actually means the pancreas produces larger than normal quantities of insulin. A major feature of type 2 diabetes is a lack of sensitivity to insulin by the cells of the body (particularly fat and muscle cells).

Table-1: Type 1 versus type 2 diabetes mellitus (American Diabetes Association, 2002) Parameter

Type 1

Type 2

Clinical

Onset <20 years

Onset>30 years

Normal weight

Obesity

Markedly

decreased

blood Increased blood insulin(early); normal to

insulin

moderate decreased insulin (late) No antibodies to islet cells

Antibodies to islet cells

Ketoacidosis rare; nonketotic hyperosmolar coma

Genetics

Ketoacidosis common Autoimmune destruction of B- Insulin

Pathogenesis cells mediated Absolute insulin deficiency 30% to 70% concordance in twins Linkage to MHC class ll HLA

Islet cells

resistance

in

skeletal

muscle,

adipose tissue and liver by T cells and humoral mediators β-cell dysfunction and relative insulin deficiency 50% to 90% concordance in twins

genes

No HLA linkage

Insulitis early

Linkage to candidate “diabetogenic” genes No insulitis

Marked atrophy and fibrosis

Focal atrophy and amyloid deposition

β-cell depletion

Mild β-cell depletion

Gestational diabetes: Gestational diabetes (GDM) is defined as a carbohydrate intolerance that normally develops during the 24th through the 32nd week of pregnancy (Thomas R Moore, 2005). This condition affects 2% to 5% of all pregnant women and is the most

common disease affecting pregnancy. Gestational diabetes often can be controlled by diet, but insulin is sometimes necessary to maintain glycemic control. An elevated blood glucose level during pregnancy is associated with an increase in complications for both mother and child. Following pregnancy, normal blood glucose tolerance usually returns. Women who

have had gestational diabetes have a 40% to 60% chance of developing diabetes in the next 5-10 years.

1.2.3. Impact of diabetes Over time, diabetes can lead to blindness, kidney failure, and nerve damage. These types of damage are the result of damage to small vessels, referred to as micro vascular disease.

Diabetes is also an important factor in accelerating the hardening and narrowing of the arteries (atherosclerosis), leading to strokes, coronary heart disease, and other large blood vessel diseases. This is referred to as macro vascular disease. Diabetes affects approximately 17 million people (about 8% of the population) in the United States. In addition, an estimated additional 12 million people in the United States have diabetes and don't even know it.

From an economic perspective, the total annual cost of diabetes in 1997 was estimated to be 98 billion dollars in the United States. The per capita cost resulting from diabetes in 1997 amounted to $10,071.00; while healthcare costs for people without diabetes incurred a per capita cost of $2,699.00. During this same year, 13.9 million days of hospital stay were attributed to diabetes, while 30.3 million physician office visits were diabetes related.

Remember, these numbers reflect only the population in the United States. Globally, the statistics are staggering. Diabetes is the third leading cause of death in the United States after heart disease and cancer (Clinical Trials).

1.2.4. Long-term Complications of Diabetes Mellitus Diabetics often develop kidney failure (nephropathy), lesions of the eye (retinopathy) and atrophy of the peripheral nerves (neuropathy). Generally, these processes occur because the walls of the capillaries that supply these tissues with blood and nutrients thicken. The molecular mechanisms leading to these late complications of diabetes have not been established conclusively (National Diabetes Advisory Board, 1983). Over the years there has been considerable debate on whether the lesions that develop within the diabeticâ&#x20AC;&#x2122;s retina, kidneys, nerves, and vascular system are due to a disorder in the structure and function of

blood vessels or whether they are a consequence of prolonged hyperglycemia caused by inadequate metabolic control. The long term complications of diabetes are shown below: Table-2: Complications of diabetes mellitus and their management (John R. White, 1992) Body Location Eyes

Description Treatment Retinopathy, cataract formation, Strict control of blood glucose to glaucoma, and periodic visual avoid need for treatment via disturbances; leading cause of laser

Mouth

photocoagulation,

new blindness vasectomy Gingivitis, increased incidence of Strict control and daily hygiene dental cavities and periodontal see dentist, floss, brush, and

Reproductive

disease water-pik often Increased incidence of large Strict control before and during

System

babies, stillbirths, miscarriages, pregnancy

(pregnancy)

neonatal

Nervous system

defects Motor, sensory and autonomic Strict control, daily foot care,

deaths,

neuropathy

congenital

leading

to surgery,

tricycle

anti-

impotency, neurogenic bladder, depressants and phenothiazines Vascular System

parathesias, gangrene Large vessel disease

Skin

microangiopathy artery bypass surgery Numerous infections and specific Strict control, daily hygiene

and Strict blood glucose control,

lesions due to small vessel disease, increased lipids in blood, Kidneys

and pruritus Diabetic

glomerulosclerosis Strict control, eventually diet

causing nephropathy

low in proteins, prednisone,

dialysis Reticuloendothelial Diabetics have a higher incidence Strict control and aggressive Systems (infections)

of

cystitis

tuberculosis,

skin

infections; more difficult time overcoming infections; moniliasis common in diabetic women

anti-infective therapy

1.2.5. Common signs of diabetes Some of the common 'early warning' signs of diabetes are (www.disabled-world.com) 1. Excessive thirst: The first symptom of diabetes is often excessive thirst that is unrelated to exercise, hot weather, or short-term illness.

2. Excessive hunger: You are still hungry all the time even though you've eaten.

3. Frequent urination: Frequent urination is often noticed because you must wake up repeatedly during the night.

4. Fatigue: Tiredness and fatigue, possibly severe enough to make you fall asleep unexpectedly after meals, is one of the most common symptoms of diabetes.

5. Sudden weight loss: Rapid and/or sudden weight loss (any dramatic change in weight is a sign to visit a doctor).

1.2.6. Mechanism of Vascular Complications induced by Diabetes Diabetes mellitus affects approximately 100 million personâ&#x20AC;&#x2122;s worldwide (Amos AF, 1997). Five to ten percent have type 1 (formerly known as insulin-dependent) and 90% to 95% have type 2 (nonâ&#x20AC;&#x201C;insulin-dependent) diabetes mellitus. It is likely that the incidence of type 2 diabetes will rise as a consequence of lifestyle patterns contributing to obesity (Mokdad AH, 2001). Cardiovascular physicians are encountering many of these patients because vascular

diseases are the principal causes of death and disability in people with diabetes. The macro vascular manifestations include atherosclerosis and medial calcification. The micro vascular consequences, retinopathy and nephropathy, are major causes of blindness and end-stage renal failure. Physicians must be cognizant of the salient features of diabetic vascular disease in order to treat these patients most effectively. The present review will focus on the relationship of

diabetes

mellitus

and

atherosclerotic

vascular

disease,

highlighting pathophysiology and molecular mechanisms (Part I) and clinical manifestations and management strategies (Part II).

1.2.6.1. Pathophysiology of Diabetic Vascular Disease The metabolic abnormalities that characterize diabetes particularly hyperglycemia, free fatty acids, and insulin resistance, provoke molecular mechanisms that alter the function

and structure of blood vessels. These include increased oxidative stress, disturbances of intracellular signal transduction (such as activation of PKC), and activation of RAGE. Consequently, there is decreased availability of NO, increased production of endothelin (ET1), activation of transcription factors such as NF-ƙB and AP-1, and increased production of prothrombotic factors such as tissue factor (TF) and plasminogen activator inhibitor-1 (PAI1) (Libby P.et al, 2000).

1.2.6.1.1. Hyperglycemia and NO Hyperglycemia and endothelium-derived vasoactive substances. Hyperglycemia decreased the bioavailability of nitric oxide (NO) and prostacyclin (PGI 2), and increased the synthesis of vasoconstrictor prostanoids and endothelin (ET-1) via multiple mechanisms, as discussed in the text. PLC indicates phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; eNOS, endothelial nitric oxide synthase; Thr, thrombin; NAD(P)H Ox, nicotinamide adenine dinucleotide phosphate oxidase; O2-, superoxide anion; ONOO-, peroxynitrite; MCP-1, monocyte chemoattractant protein-1; NF- B, nuclear factor kappa ß; TNF, tumor necrosis factor; ILs, interleukins; and COX-2, cyclooxygenase-2 (Nishikawa T, 2000).

1.2.6.1.2. Free Fatty Acid Liberation and Endothelial Function Circulating levels of free fatty acids are elevated in diabetes because of their excess liberation from adipose tissue and diminished uptake by skeletal muscle. Free fatty acids may

impair endothelial

function

through

several

mechanisms,

including

increased production of oxygen-derived free radicals, activation of PKC, and exacerbation of dyslipidemia (Dresner A, 1999). Infusion of free fatty acids reduces endothelium-dependent vasodilation in animal models and in humans in vivo. Co-infusion of the antioxidant ascorbic acid improves endothelium-dependent vasodilation in humans treated with free fatty acids, which indicates that oxidative stress mediates the abnormality. Elevation of free fatty acid concentrations

activate

PKC

and

decrease

insulin

receptor

substrate-1–

associated phosphatidylinosital-3 kinase activity. These effects on signal transduction may decrease NOS activity as discussed above. The liver responds to free fatty acid flux by increasing very-low-density lipoprotein production and cholesterol ester synthesis (Sniderman AD, 2001). This increased production of triglyceride-rich proteins and the

diminished clearance by lipoprotein lipase results in hypertriglyceridemia, which is typically observed in diabetes. Elevated triglyceride concentrations lower HDL by promoting cholesterol transport from HDL to very-low-density lipoprotein. These abnormalities change LDL morphology, increasing the amount of the more atherogenic, small, dense LDL. Both hypertriglyceridemia and low HDL have been associated with endothelial dysfunction (Snider man A et al, 1978).

1.2.6.1.3. Insulin Resistance and NO Type 2 diabetes mellitus is characterized by insulin resistance. Insulin stimulates NO production from endothelial cells by increasing the activity of NOS via activation of phosphatidylinositol-3 kinase and Akt kinase. Thus, in healthy subjects, insulin increases endothelium-dependent

(NO-mediated)

vasodilation. In

insulin-resistant

subjects,

endothelium-dependent vasodilatation is reduced. Furthermore, insulin-mediated glucose disposal correlates inversely with the severity of the impairment in endothelium-dependent vasodilatation. Drug therapies that increase insulin sensitivity, such as motormen and the thiazolidinediones, improve endothelium-dependent vasodilation. Abnormal endotheliumdependent vasodilation in insulin-resistant states may be explained by alterations in intracellular signaling that reduce the production of NO. Specifically, insulin signal transduction via the phosphatidylinositol-3 kinase pathway is impaired, and insulin is less able to activate NOS and produce NO. Insulin signaling via the mitogen-activated protein kinase pathway remains intact. Mitogen-activated protein kinase activation is associated with increased endothelia production and a greater level of inflammation and thrombosis (Zeng G et al, 1996).

Also, insulin resistance is associated with elevations in free fatty acid levels. Abdominal adipose tissue, the type found prominently in type 2 diabetes, is more insulin resistant and releases more free fatty acids compared with the type of adipose in other locations.

Activating lipoprotein lipase to metabolize these free fatty acids increases insulin sensitivity (Oliver FJ 1991).Thus, free fatty acidâ&#x20AC;&#x201C;induced alterations in intracellular signaling, as discussed

previously, may also contribute to decreased NOS activity and reduced production of NO in insulin-resistant states such as type 2 diabetes.

1.2.6.1.4. Diabetes and Vascular Smooth Muscle Function (Suzuki LA et al, 2001) The impact of diabetes mellitus on vascular function is not limited to the endothelium. In patients with type 2 diabetes mellitus, the vasodilator response to exogenous NO donors is diminished. Moreover, vasoconstrictor responsiveness to exogenous vasoconstrictors, such as endothelin-1, is reduced. Deregulation of vascular smooth muscle function is exacerbated by impairments in sympathetic nervous system function. Diabetes increases PKC activity, NFB production, and generation of oxygen-derived free radicals in vascular smooth muscle, akin to these effects in endothelial cells. Moreover, diabetes heightens migration of vascular smooth muscle cells into nascent atherosclerotic lesions, where they replicate and produce extracellular matrix—important steps in mature lesion formation. Vascular smooth muscle cell apoptosis in atherosclerotic lesions is also increased, such that patients with diabetes tend to have fewer smooth muscle cells in the lesions, which increases the propensity for plaque rupture. In persons with diabetes, elaboration of cytokines diminishes vascular smooth muscle synthesis of collagen and increases production of matrix metalloproteinase, yielding an increased tendency for plaque destabilization and rupture.

1.2.6.1.5. Diabetes, Thrombosis and Coagulation Platelet function and plasma coagulation factors are altered in diabetes, favoring platelet aggregation and a propensity for thrombosis. There is increased expression of glycoprotein Ib and IIb/IIIa, augmenting both platelet–von Willebrand (vWF) factor and platelet–fibrin interaction. The bioavailability of NO is decreased (Vinik AI et al, 2001). Coagulation factors, such as tissue factor, factor VII, and thrombin, are increased; plasminogen activator inhibitor (PAI-1) is increased; and endogenous anticoagulants such as thrombomodulin are decreased (Li Y, 2001).

Figure-1: Alteration of platelet function and coagulation factors in diabetes (Vinik AI, 2001) 1.2.7. Alloxan 1.2.7.1. Definition Alloxan (2, 4, 5, 6-tetraoxypyrimidine; 2, 4, 5, 6-pyrimidinetetrone) is an oxygenated pyramiding derivative. It is present as alloxan hydrate in aqueous solution (Merck Index, 11th Edition, 281).

1.2.7.2. History Alloxan was originally isolated in 1818 by Brugnatelli and was named in 1838 by Wohler and Liebig. The name "Alloxan" emerged from an amalgamation of the words "Allantoin" and "Oxals채ure" (oxalic acid).

1.2.7.3. Biological effects Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species.

This causes an insulin-dependent diabetes mellitus (called "Alloxan Diabetes") in these animals, with characteristics similar to type 1 diabetes in humans. Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter (Lenzen S, 2008). Alloxan, in the presence

of intracellular thiols, generates reactive oxygen species (ROS) in a cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is initiated by free radicals formed in this redox reaction. One study suggests that alloxan does not cause diabetes in humans. Others found a significant difference in alloxan plasma levels in children with and without diabetes Type 1 (Mrozikiewicz et al, 1994).

1.2.7.4. Impact upon beta cells Because it selectively kills the insulin-producing beta-cells found in the pancreas, alloxan is used to induce diabetes in laboratory animals. This occurs most likely because of selective uptake of the compound due to its structural similarity to glucose as well as the beta-cell's highly efficient uptake mechanism (GLUT2) (Tyrberg B et al, 2001).

However, alloxan is not toxic to the human beta-cell, even in very high doses, probably due to differing glucose uptake mechanisms in humans and rodents. Alloxan is, however, toxic to the liver and the kidneys in high doses (Eizirik D et al, 1994).

1.2.8. Drug Management of Diabetes Mellitus Most commonly employed oral hypoglycemic agents are sulfonylureas and biguanides.

These drugs however have disadvantages such as primary and secondary failure of efficacy as the potential for induction of severe hypoglycemia (Ceriello A, 1990). The toxicity of oral ant diabetic agents differs widely in clinical manifestations, severity, and treatment. Despite the introduction of hypoglycemic agents from natural and synthetic sources, diabetes and its secondary complications continue to be a medical problem in the world population.

Table-3: Relative advantage and disadvantage of parenteral and oral hypoglycemic agents (Lippincottâ&#x20AC;&#x2122;s Pharmacology, 3rd edition)

Drugs

Advantage

Disadvantage

Parenteral hypoglycemic agent, insulin

1. Used in both juvenile and 1. Frequent injection. adult diabetes.

2. Insulin flow peripheral tissue 2. Effective even color β-cells before reaching the liver are completely destroyed.

3. Can be used in diabetes - Allergy with complication.

- Hypoglycemic Shock 4. Can be used in pregnancy Sulfonylurea’s derivative

1. Easy oral administration

1. Cannot be used for Juvenile

2. Endogenous insulin first diabetes reaches the liver, so more 2. Sulfonylurea’s are not used active. when 3. Side effects such as allergic 40 I.U of insulin is needed per day. reaction, resistance grow are 3. It does not give in diabetes rise. complication ketoacidosis, gangrene.

4. Sulfonylurea does not give in emergency like shock, major surgery, acute injection, and acute sleock.

Biguanides

1. As supplement Sulfonylureas.

to

2. In over weight diabetes of

a 1. Hepatic and renal disease 2. In pregnancy ketosis and lactic acidosis, patient may die.

3. To smooth out the effect of 3. GIT upset. insulin. α-Glucosidase inhibitors

1. Effective diabetes.

to

Type

2 1. Abdominal pain 2. Flatulence and diarrhoea

Acarbose Thiazolidinedi Ones Rosiglitazone

1. More effective in Type 2 1. Hepatotoxic diabetes than others.

2. Weight gain 3. Fluid retention

1.3. Pain Pain is “an unpleasant sensory and emotional experience associated with actual or potential damage, or described in terms of such damage. Pain is always subjective. Each individual

learns the application of the word through experiences relating to injury in early life.â&#x20AC;? (Lynn B, 1984)

In 1994, the International Association for the Study of Pain (IASP) defined pain as an unpleasant sensory or emotional experience arising from real or probable tissue damage. In other words, the perception of pain is, in part, a psychological response to noxious stimuli.

This definition addresses the complex nature of pain and moves away from the earlier dualistic idea that pain is either psychogenic (of mental origin) or somatogenic (of physical origin). The contemporary view characterizes pain as multidimensional; the central nervous system, emotions, cognitions (thoughts), and beliefs are simultaneously involved (Raj, PP, 2007).

1.3.1. Types of Pain The International Association for the Study of Pain (IASP) classification system describes pain according to five categories: duration and severity, anatomical location, body system involved, cause, and temporal characteristics (intermittent, constant, etc.) Pain can either be acute (immediate and short-term) or chronic (long-term, lasting more than three months). Various pains are treated differently, based on severity and type. Pain can also be divided into categories that help explain its origin in the body and its effects on the body (Woolf et al, 1998). The types of pain include nerve or neuropathic, nociceptive, or

psychogenic.

Acute pain: Acute pain is a normal sensation in the nervous system to alert the individual to possible injury. Acute pain is triggered by a stimulus, such as getting cut by a knife, getting burned, or falling on a rock. Acute pain is frequently associated with anxiety, tachycardia (fast heart rate), and increased respiratory rate, increased blood pressure, diaphoresis (sweating), and dilated pupils (Main, 2001). Acute pain, for the most part, results from injury to tissues and/or inflammation. Acute pain generally has a sudden onset. For example, after trauma or surgery, acute pain may be accompanied by anxiety or emotional distress.

Chronic pain: Chronic pain is resistant to most medical treatments. Chronic pain can, and often does, cause severe problems for the individual. Pain signals keep firing in the nervous system for weeks, months, even years. Initial injuries, such as an infection, sprained back, or

sprained muscle, may cause acute pain that may lead to chronic pain. There may be an ongoing cause of pain, such as in back pain, arthritis, diabetes (diabetic neuropathy), or cancer (Coda, 2001). Some individuals suffer chronic pain in the absence of any past injury or evidence of body damage. Many chronic pain conditions affect older adults.

Common chronic pain complaints include headache, lower back pain, cancer pain, arthritis pain, neuropathic pain (pain resulting from damage to the peripheral nerves or to the central nervous system itself), and psychogenic pain (pain not due to past disease or injury or any visible sign of damage inside or outside the nervous system) (Bogduk, 1994) Malignant Pain: Pain associated with a malignant disease such as carcinoma. Pain in cancer patients can be caused by the disease itself, its treatment, e.g. surgery and radiotherapy or can be completely unrelated e.g. osteoarthritis or migraine.

Pain can be nociceptive, non-nociveptive, somatic, visceral, neuropathic, or sympathetic.

Look at the table below: Nociceptive Pain: Specific pain receptors are stimulated. These receptors sense temperature (hot/cold), vibration, stretch, and chemicals released from damaged cells (Coda, 2001).

Somatic Pain: A type of nociceptive pain. Pain felt on the skin, muscle, joints, bones and ligaments is called somatic pain. The term musculo-skeletal pain means somatic pain. The pain receptors are sensitive to temperature (hot/cold), vibration, and stretch (in the muscles). They are also sensitive to inflammation, as would happen if you cut yourself, sprain something that causes tissue damage. Pain as a result of lack of oxygen, as in ischemic muscle cramps, are a type of nociceptive pain. Somatic pain is generally sharp and well localized - if you touch it or move the affected area the pain will worsen.

Visceral Pain: A type of nociceptive pain. It is felt in the internal organs and main body cavities. The cavities are divided into the thorax (lungs and heart), abdomen (bowels, spleen, liver and kidneys), and the pelvis (ovaries, bladder, and the womb). The pain receptors

-

nociceptors

-

sense

inflammation,

stretch

and

ischemia

(oxygen

starvation). Visceral pain is more difficult to localize than somatic pain. The sensation is

more likely to be a vague deep ache. Colicky and cramping sensations are generally types of visceral pain. Visceral pain commonly refers to some type of back pain - pelvic pain generally refers to the lower back, abdominal pain to the mid-back, and thoracic pain to the upper back.

Nerve Pain or Neuropathic Pain: Nerve pain is also known as neuropathic pain. It is a type of non-nociceptive pain. It comes from within the nervous system itself. People often refer to it as pinched nerve, or trapped nerve. The pain can originate from the nerves between the tissues and the spinal cord (peripheral nervous system) and the nerves between the spinal cord and the brain (central nervous system, or CNS) (Torrance N, 2006). Neuropathic pain can be caused by nerve degeneration, as might be the case in a stroke, multiplesclerosis, or oxygen starvation. It could be due to a trapped nerve, meaning there is pressure on the nerve. A torn or slipped disc will cause nerve inflammation, which will trigger neuropathic pain. Nerve infection, such as shingles, can also cause neuropathic pain. Pain that comes from the nervous system is called non-nociceptive because there are no specific pain receptors. Nociceptive in this text means responding to pain. When a nerve is injured it becomes unstable and its signaling system becomes muddled and haphazard.

The brain interprets these abnormal signals as pain. This randomness can also cause other sensations, such as numbness, pins and needles, tingling, and hypersensitivity to temperature, vibration and touch. The pain can sometimes be unpredictable because of this (Dworkin RH, 2005)

Sympathetic Pain: The sympathetic nervous system controls our blood flow to our skin and muscles, perspiration (sweating) by the skin, and how quickly the peripheral nervous system works. Sympathetic pain occurs generally after a fracture or a soft tissue injury of the limbs.

This pain is non-nociceptive - there are no specific pain receptors. As with neuropathic pain, the nerve is injured, becomes unstable and fires off random, chaotic, abnormal signals to the brain, which interprets them as pain. Generally with this kind of pain the skin and the area around the injury become extremely sensitive. The pain often becomes so intense that the sufferer daren't use the affected arm or leg. Lack of limb use after a time can cause other problems, such as muscle wasting, osteoporosis, and stiffness in the joints.

1.3.2. Impact of Pain

Pain can be essentially divided into 2 broad categories: adaptive and maladaptive.

Adaptive pain contributes to survival by protecting the organism from injury or promoting healing when injury has occurred. Maladaptive pain, in contrast, is an expression of the pathologic operation of the nervous system; it is pain as disease (Plainer J, 2002).

Everything about pain is not bad or detrimental. There are deeper and essential rewards for biological systems through pain processes. Among the higher organisms of the evolution tree, the sensory system has the role of informing the brain about the state of the external environment and the internal milieu of the organism. As pain is a sensory perception, it constitutes an alarm that ultimately has the role of helping to protect the organism: it both triggers reactions and induces learned avoidance behaviors, which may decrease whatever it causing the pain and, as a result, may limit the potentially damaging consequences (Woolf C J, 2000).

1.3.3. Causes and Symptoms 1.3.3.1. Causes Acute pain can usually be linked directly to the noxious influence or injury that caused the pain, like the pain you feel after burning your skin or following a surgical intervention (Tagliazucchi E, 2010).

For chronic pain the connection is far more difficult to establish as the original cause of pain might not exist any longer and the nerves may have become oversensitive and react already to the slightest stimulus, which would not cause any pain in otherwise healthy subjects (Keay, 2000).

Sometimes intensive, multi-disciplinary examination may be needed to reveal the underlying cause.

Somatic pain: â&#x20AC;˘

Originates from bones, muscles, tendons or blood vessels and is often known as musculo-skeletal pain

•

Usually sharp, well-localized

•

Can be reproduced by touching or moving the involved area

•

Usually of longer duration

Cutaneous pain •

Is due to injury of the skin or the superficial tissues

•

Usually well-described, localized pain of short duration

Peripheral neuropathy •

Means that the peripheral nerves are not working properly

•

Is usually the result of an injury to or a disease process, such as diabetes associated with loss of function in the nerve

•

Often starts in the hand and feet and often affects the body symmetrically

Entrapment of a nerve •

A pinched or trapped nerve due to compression in the spine or elsewhere in the body, such as elbow, shoulder, wrist or foot

Phantom limb pain •

Sensation of pain from a limb that has been lost or from which no longer physical signals are being received

•

Reported after amputation or in quadriplegics

Chronic central neuropathic pain •

Can follow traumatic spinal cord injury or diseases of the brain itself, like stroke.

Other causes •

Other causes with ensuing damage of the nervous tissue include post-herpes infection.

1.3.3.2. Symptoms

Symptoms vary depending on the site of pain and are treated medically. However, there are common symptoms associated with pain disorder regardless of the site (Brand, P; Yancey, 1997)

•

inactivity, passivity, and/or disability

•

increased pain requiring clinical intervention

•

insomnia and fatigue

•

disrupted social relationships at home, work, or school

•

depression and/or anxiety

1.3.4. Analgesics An analgesic (colloquially known as painkillers) is any member of the diverse group of drugs used to relieve pain and to achieve analgesia. This derives from Greek an-, "without", and -algia, "pain".

Analgesic drugs act in various ways on the peripheral and central nervous system; they include paracetamol (acetaminophen), the nonsteroidal anti-inflammatory drugs (NSAIDs) such as the salicylates, narcotic drugs such as morphine, synthetic drugs with narcotic properties such as tramadol, and various others (Anonymous, 1990). The pain relief induced by analgesics occurs either by blocking pain signals going to the brain or by interfering with the brain's interpretation of the signals, without producing anesthesia or loss of consciousness (Dworkin RH, 2003).

There are basically two kinds of analgesics: non-narcotics and narcotics.

1.3.4.1. Non-Narcotic Analgesics Acetaminophen is the most commonly used over-the-counter, non-narcotic analgesic.

Acetaminophen is a popular pain-reliever because it is both effective for mild to moderate pain relief and relatively inexpensive. It must be emphasized though that the safety of acetaminophen is tied to proper use of the drug (use according to specific prescribing instructions). If acetaminophen is not used according to the directions on the label, serious side effects and possible fatal consequences can occur (Bertolini A, 2006). For example,

taking more than 4000 mg/day or using it long term can increase the risk of liver damage.

The risk of liver damage with acetaminophen use is also increased by ingesting alcohol.

Make sure you discuss with your doctor the maximum allowable dose of acetaminophen and any other guidelines for its use.

Many people do not realize that acetaminophen is found in more than 600 over-thecounter drugs. It can be found in combination with other active ingredients in many cold, sinus, and cough medications. The cumulative effect of acetaminophen must be considered if you are talking multiple drugs which contain acetaminophen (Hillier, Keith; 2001).

How can acetaminophen damage the liver? Acetaminophen changes into metabolites which are eliminated from the body. By taking more than the recommended maximum daily dose of acetaminophen, more toxic metabolites are produced than can be eliminated (David MA, 2001)

1.3.4.2. Narcotic Analgesics There are two types of narcotic analgesics: the opiates and the opioids (derivatives of opiates). Opiates are the alkaloids found in opium (a white liquid extract of unripe seeds of the poppy plant) (Driessen B, 1992) Opioids are any medication which binds to opioid receptors in the central nervous system or gastrointestinal tract. According to Wikipedia, there are four broad classes of opioids: Endogenous opioid peptides (produced in the body: endorphins, dynorphins, and enkephalins) â&#x20AC;˘

Opium alkaloids (morphine, codeine, thebaine)

â&#x20AC;˘

Semi-synthetic

opioids

(heroin,

oxycodone,

hydrocodone,

dihydrocodeine,

hydromorphone, oxymorphone, nicomorphine) â&#x20AC;˘

Fully synthetic opioids (pethidine or demerol, methadone, fentanyl, propoxyphene, pentazocine, buprenorphine, butorphanol, tramadol, and more)

Opioids are used in medicine as strong analgesics, for relief of severe or chronic pain.

Interestingly, there is no upper limit for the dosage of opioids used to achieve pain relief, but the dose must be increased gradually to allow for the development of tolerance to adverse effects (for example, respiratory depression) (Reimann W, 1998). According to eMedicine, "Some people with intense pain get such high doses that the same dose would be fatal if taken by someone who was not suffering from pain."

There have been debates over the addictive potential of opioids vs. the benefit of their analgesic properties for treating non-malignant chronic pain, such as chronic arthritis. Some experts believe opioids can be taken safely for years with minimal risk of addiction or toxic side effects. The enhanced quality of life which opioids may provide the patient must be considered.

Table-4: Some non-steroidal drugs used to remove pain Type

Drug

Salicylates

Aspirin, Choline magnesium trisalicylate, Diflunisal, Salsalate

Coxibs

Celecoxib

Others

Diclofenac, Etodolac,Fenoprofen,Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen,

Ketorolac, Meclofenamate,

acid, Meloxicam, Nabumetone,

Mefenamic Naproxen,

Oxaprozin, Piroxicam,Sulindac,Tolmetin

1.3.4.3. Principle of Diclofenac In this method, acetic acid is administered intra-peritoneal to the experimental animals to create pain sensation. As a result, the animals squirms their body at regular interval out of pain. This squirm to contraction of the body is termed as â&#x20AC;&#x153;Writhingâ&#x20AC;?. As long as the animals feel pain, they continue to give writhing. Each writhing is counted and taken as an indication of pain sensation. Any substance that has got analgesic activity is supposed to lessen the number of writhing inhibition of positive control was taken as standard and compared with

test samples and control. As positive control, any standard NSAID drug can be used. In the present study, diclofenac was used to serve the purpose (wiki/Diclofenac).

Figure-2: Structure of diclofenac (wiki/Diclofenac) 1.3.4.3.1. Pharmacological Properties of Diclofenac Diclofenac has analgesic, antipyretic, and anti-inflammatory activities. Its potency against COX-2 is substantially faster than that of indomethacin, naproxen, or several other NSAID. In addition, diclofenac appears to reduce intracellular concentrations of free leukocytes, perhaps by altering its uptake. The selectivity of diclofenac for COX-2 resembles that of celecoxib (Dutta NK, 2000). Indeed the incidence of serious gastrointestinal adverse effects did not differ between celecoxib and diclofenac in the clinical trial. Furthermore observational studies have the possibility of a cardiovascular hazard from chronic therapy with diclofenac. A large-scale randomized under way.

Figure-3: Synthesis of prostaglandins and leucotrienes (diclofenac blocks prostaglandin synthesis by inhibition of cyclooxygenase) 1.3.4.3.2. Therapeutic uses of Diclofenac

Diclofenac is approved in the states for the long term symptomatic treatment of rheumatoid arthritis osteoarthritis and alkylosing spondylitis. Three formations are available, an intermediate release potassium salt deluge for those medications is to 100-200mg, given in several divided doses (Mazumdar K, 2006). Diclofenac also is useful for short term treatment of acute musculoskeletal pain, postoperative pain, and dysmennorrhea. Diclofenac is also available in combination with misprotostol, a PGE analog. This retains the efficacy of Diclofenac while reducing the frequency of gastrointestinal ulcers and added misoprostol (Dutta NK, 2007)

1.4. Purpose of study 1.4.1. Background Diabetes mellitus (DM) is a metabolic syndrome with multiple etiology, is characterized by chronic hyperglycemia together with disturbances in carbohydrate, protein and fat metabolism results from a decrease in circulating concentration of insulin (insulin deficiency), a decrease in the response of peripheral tissues to insulin (insulin resistance) or both. Hyperglycemia is an important factor in the development and progression of longterm complications of DM affecting kidney, retina, heart and nervous system (David, 1997) DM is a multi-factorial disease that has a significant impact on the health, quality of life and life expectancy of patients as well as on the health care system. DM is the commonest clinical disorder affecting nearly 10% of the populations all over the world (Kuboki k, 2000).

In the present world, the number of people with diabetes is expected to be 221 million over the 13 year period from 1997 to 2010. According to World Health Organization (WHO), the diabetic population is likely to increase by 35% by the year 2025. DM occurs at any stage of life from infancy to old age and the occurrence of type-I diabetes is about 10% whereas type-II diabetes accounts for around 90% of diabetes cases.

The prevalence of DM is

increasing rapidly in developing countries than in the developed nations. India and China will be the leading countries in their annual incidence rates for diabetes mellitus by the year 2025 due to their high population (King H, 1998). In Bangladesh, the situation is the most vulnerable and it has been estimated that the number of diabetes will rise from 3.2 million in 2000 to 11.7 million by the year 2030. Diabetes is the fourth leading cause of death in

developed countries. In 2005, WHO reported that around 1.1 million people were died of diabetic complicacy, among which 80% from developing countries and it has also been suggested that the death rate will increase up to 50%. So, diabetes is a global disease with a huge adverse impact on health and mortality.

Most commonly employed oral hypoglycemic agents are sulfonylureas and biguanides.

These drugs however have disadvantages such as primary and secondary failure of efficacy as well as the potential for induction of severe hypoglycemia. The toxicity of oral ant diabetic agents differs widely in clinical manifestations, severity, and treatment. Despite the introduction of hypoglycemic agents from natural and synthetic sources, diabetes and its secondary complications continue to be a major medical problem in the world population.

There is a need, therefore for new compounds that may effectively reduce insulin resistance or potentiate insulin action in genetically diabetic or obese individuals. The search for such drugs with a potential to reduce long-term complications of diabetes is, therefore of current interest.

Medicinal plants are the most exclusive source of life saving drugs for the majority of the worldâ&#x20AC;&#x2122;s population. Virtually, the use of traditional medicine is the mainstay of primary healthcare in all developing countries. A number of indigenous medicinal plants have been found to be useful to manage diabetes. In the last few years there has been an exponential growth in the field of herbal medicine, and these drugs are gaining popularity both in developing and developed countries because of their natural origin and less side effects.

WHO also recommended and encouraged this practice, especially in countries where access to the conventional treatment of diabetes is not adequate.

With growing interest

worldwide in medicinal plant as a source of medicine, there is need to introduce new important plants of established therapeutic values used either in modern or traditional system of medicine. In recent years, there has been a renewed interest to screen such plant materials, especially to examine the long-term beneficial effects of plant materials, to identify the active principle and to understand the mechanism of action, which is at present unclear.

According to the ethnobotanical surveys more than 800 plants are used worldwide in traditional medicine to treat diabetes. The hypoglycemic activity of many these plants has

been confirmed in hundreds of studies in experimental animals and several studies in diabetic patients. Bangladesh is a country with rich plant resources and an ancient history of traditional medicines. Although a large number of medicinal plants have been already tested for their anti-diabetic and anti-hyperlipidemic activity, many remain to be scientifically established. In the past decade, research has been focused on scientific evaluation of traditional drugs of plant origin and screening of more effective and safe antidiabetic drugs has continued to be an important area.

Medicinal plants are the most exclusive source of life saving drugs for the majority of the worldâ&#x20AC;&#x2122;s population. In developing countries 80% population are using traditional medicine in primary medical problems.

However, lots of herbs are now being used in the

management of DM. Bangladesh is endowed with the wealth of medicinally important plants and has ancient herbal treatment methods where traditional alternative medicines are popularly practiced among the large segment of its population. With growing interest worldwide in medicinal plant as a source of medicine, there is need to introduce new important plants of established therapeutic values used either in modern or traditional system of medicine.

Most of the people of our country have no or little access to allopathic medication due to their low-income in respect to the high cost of allopathic medicine. They can hardly afford to spend much money for the prevention and cure of their diseases. As a result, about 7080% of the population of our country still has to depend on the indigenous systems for the maintenance of their health. (Yusuf et al., 1994). This survey indicates an extensive use of medicinal plants, most of which are served in a crude and substandard form by people. The use of such drug is dangerous and threatens public health. Thus the analysis of medicinal plants for exploring of chemical entities and their biological screening is the current need for standardization of herbal medication.

1.4.2. Aims and Objectives The research work was undertaken to evaluate the hypoglycemic effect of Polyalthia longifolia in normal and alloxan-induced diabetic mice and analgesic effect. The most widely used experimental procedures were followed:

 To examine the effects of plant extract on blood glucose level both in normal and alloxan-induced diabetic mice.  To determine the effects of plant extract the analgesic activity for its central and peripheral pharmacological actions using acetic acid induced writhing test in mice.  Seeking for a new analgesic and hypoglycemic drug in Polyalthia long folia.  Exploration of possible newer medicinal activities of Polyalthia long folia.  Finally, find out the possible mechanism action of the plant extract for their beneficial effect both in normal and alloxan-induced diabetic mice.

And the present study was designed to evaluate analgesic and hypoglycemic activity to the plant extract of the Polyalthia long folia leaf.

PLANT PREVIEW 2.1. Plants Botanical Name: Polyalthia long folia Family: Annonaceae Common Names: (Polyalthia long folia - Ashok.htm) Ashok, False Ashok, Mast Tree • Bangla: Debdaru • Hindi: Ashok • Marathi: Devdar • Malayalam: Hemapushpam • Telugu: Devdaru • Tamil: Vansulam • Assamese: Umboi • Kannada: Ubbina • Sanskrit: Putrajiva • Konkani: Asok 2.1.1. Taxonomic Classification of Polyalthia long folia (Internet-2) Kingdom: Plantae Subkingdom: Viridaeplantae

Phylum: Tracheophyta Subphylum: Euphyllophytina Class: Magnoliopsida Subclass: Magnoliidae Order: Annonales Family: Annonaceae Genus: Polyalthia Species: longifolia Botanical Name: Polyalthia long folia

2.1.2. Botanical Features of Polyalthia long folia Polyalthia long folia a tall, attractive, bushy evergreen tree, of the family Annonaceae with smooth, dark bark and undulate leaves. The wood is white or whitish-yellow, light, very flexible, fairly close and even-grained. The wood is used for various purposes including making drums, pencils, small boxes and matches. Bark of this tree contains medicinal properties, which is used as a febrifuge (Banglapedia) Polyalthia long folia is a lofty evergreen tree, commonly planted due to its effectiveness in alleviating noise pollution. It exhibits symmetrical pyramidal growth with willowy weeping pendulous branches and long narrow lanceolate leaves with undulate margins. The tree is known to grow over 30 ft in height (Wikipedia).

Leaves: Fresh leaves are a coppery brown color and are soft and delicate to touch, as the leaves grow older the color becomes a light green and finally a dark green. The leaves are shaped like a lance and have wavy edges. The leaves are good and for ornamental decoration and used in festivals (Wikipedia).

Flowers: In spring the tree is covered with delicate star-like pale green flowers. The flowers last for a short period, usually two to three weeks, are not conspicuous due to their color.

Flowers are yellowish-green and normally arranged in clusters (Wikipedia).

Fruit: Fruit are borne in clusters of 10-20. Initially green but turning purple or black when ripe. These are loved by birds, such as the Asian Koel Eudynamys scolopaceus and bats including the flying foxes (Wikipedia).

Bark: The bark is smooth and dark greyish-brown (Polyalthia longifolia - Ashok.htm).

Distribution: Found natively in Bangladesh, India and Sri Lanka. It is introduced in gardens in many tropical countries around the world (Banglapedia, Wikipedia).

Whole plant

Flowers

Leaves

Seeds and fruit

Figure-4: Images of Debdaru (Polyalthia long folia) (Banglapedia, Wikipedia).

2.1.3. Chemical Literature Reviews of Polyalthia longifolia The phytochemical studies have revealed the presence of various types of Chemical compounds in different parts of Polyalthia longifolia plants are shown in below Table-5: Chemical compounds isolated from Polyalthia longifolia (Internet-1) Chemical compositions Amino acids proline, L-glutamic acid, methionine Alkaloids pendulamine A, pendulamine B, penduline Antifungal Diterpenoids

Table-6: Reported biological activities of Polyalthia longifolia (Wikipedia, Internet-1) Plant Parts Pharmacological Activity Bark, seeds and Anti-diabetic, analgesic, leaves

hypotensive, hepatoprotective,

cytotoxic,

antioxidant, antibacterial,

antifungal, anticancer, antiulcer.

2.1.4. Traditional uses of Polyalthia longifolia (Internet-1) ď ś

Amino Acids: Study showed the seeds of Polyalthia longifolia to be a rich source of various amino acids - proline, L-glutamic acid, and methionine among others.

ď ś

Phytochemical: Study isolated a new clerodane diterpene from the bark of P. longilofia.

ď ś

Antimicrobial / Alkaloids: Study showed the root extract of P. longifolia to possess significant antibacterial activity. Study also led to the isolation of three new alkaloids: pendulamine A, pendulamine B and penduline along with other known compounds.



Hepatoprotective / Antiinflammatory: Of various solvent extract, study showed the methanol extract as the most potent, showing significant antiinflammatory (comparable to Diclofenac sodium) and hepatoprotective activity.



Antifungal: Diterpenoids isolated from the hexane extract of the seeds of P. longilfolia demonstrated significant antifungal activity.



Cytotoxic: Study isolated a new halimane diterpene and a new oxoprotoberberine alkaloid along with 20 known compounds, several of which were evaluated for cytotoxicity toward a small panel of human cell lines.



Anti-ulcer: Study showed the ethanol extract of Polyalthia longifolia to have significant antiulcer activity through reduction of gastric volume, free acidity and ulcer index. It showed 89.71% ulcer inhibition in HCl-ethanol induced ulcer and 95.3% ulcer protection index in stress-induced ulcer.



Antibacterial: Study of ethanol extract showed promising antibacterial activity against thirteen Gram-positive and nine Gram-negative organisms.



Antibacterial / Phytochemicals: Study revealed the presence of steroids, alkaloids, biterpenoids, carbohydrates, amino acids, essential oil, pheolics and flavonoids.

Highest antibacterial activity was seen with the hot aqueous (HAE) and methanol extract (ME) against K pneumonia, followed by E coli (HAE) and B subtilis (ME).



Hypotensive / Phytochemicals: Phytochemical studies yielded kolavenic acid, clerodane, liriodenine, lysicamine and bisclerodane and its isomer. Study showed the defatted 50% methanol extract of P longifolia root bark with significant ability to reduce blood pressure.

Uses: Polyalthia longifolia is used for fever, pain, skin diseases, hypertension, diabetes and helminthiasis. Bark used as febrifuge (Banglapedia).

MATERIALS AND METHODS 3.1. Plant Materials

Polyalthia longifolia leaves were collected from infront of the “National Parliament House”, Bangladesh during the month of January, 2011 and the plant authenticity was confirmed from the Bangladesh National Herbarium, Mirpur, Dhaka (Accession Number: DACB – 35392).

3.2. Preparation of Plant Extract The leaves of Polyalthia longifolia were shade dried for fifteen days at room temperature to ensure the active constituents free from decomposition . The dried leaves were powdered in an electrical grinder after overnight drying in an oven below 50°C. The powder was extracted with 96% ethanol at room temperature. The bottle were kept at room temperature and allowed to stand for 10 days with occasional shaking and stirring. When the solvent become concentrated, the liquid alcohol contents were filtered through cotton and then through filter paper (Whatman Fitter Paper No. 1). Finally, a highly concentrated ethanolic crude extract were obtained.

3.3. Drugs and Chemicals The standard drug, Metformin hydrochloride was the generous gift samples from Beximco Pharmaceuticals Ltd of Bangladesh. Alloxan monohydrate was purchased from Merck Schuchardt OHG, Germany. Blood samples analyzed for blood glucose content by using EZ Smart-168 glucose test meter (Tyson, Taiwan). Acetic acid was collected from laboratory of Bangladesh University. The standard drug Diclofenac-Na was purchased from Square Pharmaceuticals Limited of Bangladesh.

3.4. Experimental Animals Eight week-old Swice albino mice (27-30g) purchased from ICDDRB, Dhaka, Bangladesh and were housed in animals cages under standard environmental conditions (22-25°C, humidity 60-70%, 12 hr light: 12 hr dark cycle). The mice were feed with standard pellet diet taken from ICDDRB, Dhaka. The animals used in this study were cared in accordance with the guidelines on animal experimentation of our institute.

3.5. Induction of Diabetes

After fasting 16hr, diabetes was induced into mice by in intra-peritoneal injection (i. p.) of alloxan monohydrade (100 mg/kg), dissolved in saline (100 Âľl/mice, ip.). After 48hr, plasma glucose levels were measured by glucometer (Tyson, Taiwan) using a blood sample from tail-vein of mice. Mice with blood sugar level higher than 08.5-11.5 mmol/l are considered as diabetic. Age-matched healthy mice were used to examine the effects of extract on normal mice.

3.6. Experimental Protocols After induction of diabetes 25 mice were divided into five groups for the oral administration eitherI. Normal Control ( Normal Group, Vehicle 0.5% methyl cellulose, n = 4) II. Diabetic Control (Control Group, Vehicle 0.5% MC, n=4) III. Diabetic Standard (Standard Group, Metformin HCl, 100mg/kg, op. n=4) IV. Diabetic + Extract (250mg Group, 250mg/kg , n = 4) V. Diabetic + Extract (500mg Group, 500mg/kg , n = 4) For analgesic test all mice were divided into four groups. Each group comprises 4 mice.

Control group (received 0.5% methyl cellulose), Standard Group (received Diclofenac 75mg, 1ml), 250mg Group (received 250mg/kg extract) and 500mg Group (received 500mg/kg extract).

3.7. Oral Glucose Tolerance Test (OGTT) in diabetic mice The mice were fasted over-night and next day blood samples were taken from all groups of animals to estimate fasting blood glucose level (0 min). All mice received 1gm /kg glucose, after 1 hour of feeding of extract and/ drug and three more blood samples were collected at 30, 90 and 120 minutes intervals and blood glucose level was estimated in all the experiments by using glucometer.

3.8. Statistical Analysis

All values were expressed as mean ± Standard error of mean (SEM). Statistical comparison were performed by One-way analysis of variance (ANOVA), followed by using Tukeys test.

Results were considered as significant when p values less than 0.05 (p<0.05).

3.9. Acetic acid-induced writhing test for Analgesic activity The analgesic activity of the samples was also studied using acetic acid-induced writhing model in mice. Test samples and vehicle were administered orally 30 mins before intraperitoneal administration of 1% acetic acid but Diclofenac-Na was administered intraperitonially 15 mins, the mice were observed for specific contraction of body referred to as “writhing” for the next 10 mins (Ahmed F 6:344-348) 3.10. Phytochemical screening methods (Abdul Ghani: Medicinal plant and Bangladesh) 3.10.1. Test for Saponins Small quantities of an ethanolic extract of the plant material dissolved in minimum amount of distilled water and shaken in a graduated cylinder for 15 minutes. Formation of stable foam suggested the presence of saponins.

3.10.2. Test for alkaloids Hager’s Reagent (1 percent solution of picric acid): delayed production of crystalline yellow precipitate indicates the presence of alkaloid.

3.10.3. Test for Flavonoids A few drops of conc. HCl acid added to the extract. Immediate development of a red colour indicates the presence of flavonoids.

3.10.4. Test for glycosides Dissolve a small amount of the fresh or dried plant material in 1 ml of water and add a few drops of aqueous sodium hydroxide solution. A yellow color is formed in the presence of glycosides.

3.10.5. Test for carbohydrate (Molischâ&#x20AC;&#x2122;s test) To 2 ml of an extract of the plant material in a test tube add 2 drops of freshly prepared 10% alcoholic solution of alpha-naphthol and mix thoroughly. Allow 2 ml of concentration sulphuric acid to flow down the side of the inclined test tube so that the acid forms a layer beneath the solution. A red or reddish violet ring is formed at the junction of the two layers indicates the presence of carbohydrate.

3.10.6. Test for Tannins 0.5 g of extract was dissolved in 5ml distilled water. Then few drops of 5% ferric chloride were added and blue-black colour indicates the presence of tannins.

3.10.7. Test for Gums 5 ml solution of the extract was taken and then molisch reagent and sulphuric acid were added. Red violet ring was produced at the junction of two liquids, which indicates the presence of gums.

Table-7: Results of chemical group tests Tested groups Alkaloids Steroids Saponins Tannins Flavonoids Reducing Sugars Gums

Ethanol Extract of Polyalthia longifolia + + + + +

Note: + = Indicates the presence of the tested group, - = Indicates the absence of the tested group.

RESULTS AND DISCUSSION 4.1. Results

4.1.1. Oral Glucose Tolerance Test (OGTT) of Polyalthia longifolia extract in alloxaninduced diabetic mice Table-8: Effect of the ethanolic extract of Polyalthia longifolia leaf on oral glucose tolerance test in diabetic mice Time

Normal

Control

Standard

250

mg 500

0 min 30 min 90 min 120 min

Group 5.65±0.38 5.65±0.38 5.86±0.52 5.82±0.22

Group 14.82±1.79 15±1.47 15.12±1.43 14.97±1.74

Group 15.62±1.31 6.07±0.56 4.97±0.17 4.87±0.15

Group 16.45±0.75 14.82±0.62 14.35±0.50 12.55±0.42

mg

Group 16.25±0.88 13.5±0.80 7.02±0.85 6.22±0.41

Values were expressed in Mean ± SEM value. Each group comprised 4 animals. Control Group received 0.5% Methyl cellulose and Standard Group received 100mg/kg Metformin.

0Min 30Mins 90Mins 120Mins

al G C ro on up tro S l G ta ro nd up ar d G 25 ro 0m up g G 50 r 0m ou p g G ro up

18 16 14 12 10 8 6 4 2 0

N or m

Blood Glucose Level (mM)

Oral Glucose Tolerance Test (OGTT)

Figure-5: Effect of the ethanolic extract of Polyalthia longifolia leaf on oral glucose tolerance test in diabetic mice.

After oral administration of glucose the blood glucose levels were significantly higher in diabetic and experimental groups of mice as shown in Table-8 and Figure-5. In diabetic control the peak increase in blood glucose concentration was observed after 30 min and remained high over the next hour. Mice treated with extract in Group (250mg/kg), Group

(500mg/kg) showed a significant decrease in blood glucose concentration at 90min and 120min compared with diabetic control mice. The pronounced effects were observed with Group (500mg/kg) and this effect like that of Standard Group.

4.1.2. Analgesic effect of Polyalthia longifolia extract on acetic acid-induced writhing in mice Table-9: Effects of the ethanolic extract of leaf of Polyalthia longifolia on acetic acidinduced writhing in mice Animal Group

Writhing

Control Group Standard Group 250mg Group 500mg Group

±SEM) 41.75±1.70 9.5±1.29 17.5±2.08 12.75±1.70

Counting

(Mean

Values are mean ± SEM, (n=4); p<0.05 Dunet test as compared to Control Group. Control Group animal received vehicle (1% Tween 80 in water), Standard Group received Diclofenac 75 mg/ kg body weight, 250mg Group and 500mg Group were treated with 250 and 500mg/kg body weight (p.o) of the crude extract of Polyalthia longifolia.

Analgesic Treatment 45 Number of Writhing

40 35 30

Control Group

25

Standard Group

20

250mg Group

15

500mg Group

10 5 0 1

Figure-6: Effects of the ethanolic extract of leaf of Polyalthia longifolia on acetic acidinduced writhing in mice.

Analgesic Activity

% of Writhing Inhibition

90 80 70 60

Standard Group

50

250mg Group

40

500mg Group

30 20 10 0 1

Figure-7: Percent of inhibition effects of the ethanolic extract of leaf of Polyalthia longifolia on acetic acid-induced writhing in mice.

Table-9 shows the effects of the extract of an acetic acid-induced writhing in mice. The oral administration of both doses of Polyalthia longifolia extract significantly (p<0.05) inhibited writhing response induced by acetic acid in a dose dependent manner compared. The effect

was dose dependent and the most significant effect observed with 500mg Group (500 mg/kg) which is very close to the standard group compared to control group.

4.2. Discussion Diabetes mellitus is one of the most common chronic disease and is associated with hyperglycemia, polyurea, polydipsia, polyphagia, weight loss, muscle weakness, hyperlipidemia and co-morbidities such as obesity, hypertension. Hyperglycemia and Hyperlipidemia are the two metabolic complications of both clinical and experimental diabetes (Bierman EL, 1975).

Alloxan, a β-cytotoxin, induces "chemical diabetes" (alloxan diabetes) in a wide variety of animal species by damaging the insulin secreting pancreatic β-cell, resulting in a decrease in endogenous insulin release, which paves the ways for the decreased utilization of glucose by the tissues (Omamoto H, 1981).

In the light of the literature on Polyalthia longifolia, we made an attempt for the first time to study the effect of Polyalthia longifolia extract in hyperglycemic mice. The experiment showed that, the extract have the properties to stimulate or regenerate the ß-cell for the secretion of insulin and are most effective for controlling diabetes by various mechanisms due to presence of hypoglycemic alkaloids, saponins and flavonoids.

Oral Glucose Tolerance Test (OGTT) measures the body ability to use glucose, the body’s main source of energy (Du Vigneaud, 1925). It can be used to diagnose prediabetes and diabetes. In our study, it is found that various fractions have also hypoglycemic effect in glucose induced hyperglycemic mice. The effects of extract on blood sugar levels are dose dependent.

Induction of diabetes with alloxan was associated with decrease in hepatic glycogen, which could be attributed to the decrease in the availability of the active form of enzyme glycogen synthetase probably because of low levels of insulin (A.H.Gold, 1970; R.K.Goel, 2004). In the present study, Polyalthia longifolia restored the depressed hepatic glycogen levels possibly by increasing the level of insulin. Our result showed that supplementation of diabetic mice with plant extract resulted in significant elevation in hepatic glycogen content.

Acetic acid-induced writhing model represent pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from phospholipids. The acetic acid-induced writhing model response is a sensitive procedure to evaluate peripherally acting analgesic. The response is thought to be mediated by peritoneal mast cells, acid sensing ion channels and the prostaglandin pathway (Voilley, 2004; Hossain, 2006). Preliminary photochemical screening reveals the presence of flavonoid,

alkaloids, tannins and saponins in the plant extract. So the observed analgesic activity may be attributed to these compounds.

Further studies is required for the detailed studies pharmacological investigations of the leaf constituents, which have many pharmacological activity reported traditionally and its exact mechanism of action.

CONCLUSION Natural products are a huge resource for medicine as shown with the use of plants in different pharmaceutical products. Therefore the investigation medicinal value of plants has become a matter of great significance. Particularly in preventing or treating serious health conditions such as Diabetes, cancer, acquired immune deficiency syndrome (AIDS), and hypercholesterolemia and against pain.

There is a lot of evidence to support the hypoglycaemic and analgesic assertions made of Polyalthia longifolia. A number of valuable studies have been conducted on the consequence of Polyalthia longifolia administration, and its acceptance into clinical medicine.

In the present study hypoglycemic effect was significant (from 16.25mM to 6.22 mM Âą SEM) reducing blood glucose level in 500mg extract (p<0.05) but no significant change observed in 250mg. Satisfactory analgesic activity was observed (p<0.05) in extract 500mg by 69.46% inhibition of writhing reflex and in 250mg was 58.08% compared to standard drug diclofenac 78.16% writhing inhibition. The present study indicates significant hypoglycemic and analgesic effects of Polyalthia longifolia.

In the light of our pharmacological studies of Polyalthia longifolia extract can be useful as an adjunct in the diabetes and analgesic treatment. Further investigations, look to be promising, while isolated purified Polyalthia longifolia constituents have received appropriate scrutiny investigations to examine underlying mechanism of antidiabetic and analgesic effects of Polyalthia longifolia.

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