Chapter one

Pain

ANTIDIABETIC AND ANALGESIC STUDIES OF ETHANOLIC EXTRACT OF GOLDEN SHOWER PLANT Cassia fistula Linn.INTRODUCTION

Introduction

1.1. Diabetes

1.1.1. Definition: 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 long-term complications of DM affecting kidney, retina, heart and nervous system (David, M.N. et al. 1997).1.1.2. Type 1 Diabetes:Type 1 diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas leading to insulin deficiency. This type of diabetes can be further classified as immune-mediated or idiopathic. The majority of type1 diabetes is of the immune-mediated nature, where beta cell loss is a T-cell mediated autoimmune attack. There is no known preventive measure against type1 diabetes, which causes approximately 10% of diabetes mellitus cases in North America and Europe.(Rather KI April 2007)1.1.3. Type 2 diabetes:Type2 diabetes mellitus is characterized by insulin resistance which may be combined with relatively reduced insulin secretion. The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor. However, the specific defects are not known. Diabetes mellitus due to a known defect are classified separately. Type2 diabetes is the most common type.1.1.4. Gestational diabetes:

Gestational diabetes mellitus (GDM) resembles type2 diabetes in several respects, involving a combination of relatively inadequate insulin secretion and responsiveness. It occurs in about 2%5% of all pregnancies and may improve or disappear after delivery. Gestational diabetes is fully treatable but requires careful medical supervision throughout the pregnancy. About 20%50% of affected women develop type2 diabetes later in life (Robbins Basic Pathology). 1.2. Signs and SymptomsPolyuria (frequent urination)

Glucose concentration in blood is high

Reabsorbing of glucose in the proximal renal tubuli is incomplete, glucose remains in urine

Osmotic pressure of urine increases (J. Goldman-Levine et al 2005). Inhibits reabsorbing of water by kidney, resulting in increase urine production

Dehydration

Lost water volume in kidney replaced from water held in body, increased thirst and increased fluid intake polydipsia (A. Barnett 2003)Polyphagia

Increased appetite, no glucose delivered to muscles, tissues, body sends signal to brain to eat something to renourishWeight loss and weakness

Glucose cannot participate in crib cycle to be used as energy, use of fat as alternative energy source (T. Levien et al 2002).Vision changes

changes shapes of lens in eye

Figure 1: Hypoglycemic Symptoms (www.ihc.com/diabetes) Table 1: Difference between Type 1 & Type 2 Diabetes

Comparison of type 1 and 2 diabetes

FeatureType 1 diabetesType 2 diabetes

OnsetSuddenGradual

Age at onsetAny age

(mostly young)Mostly in adults

Body habituesThin or normalOften obese

KetoacidosisCommonRare

AutoantibodiesUsually presentAbsent

Endogenous insulinLow or absentNormal, decreased, increased

Concordancein identical twins50%90%

1.3. Prevalence of Diabetes

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 population all over the world [3]. At present, there are an estimated 246 million people with diabetes in the world, of whom about 80% reside in developing countries (Adapted from the Report of the American Diabetes Association, ADA 2002) 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 [4]. 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% (Rajshahi Diabetic Association, 2005). So, the diabetes is a global disease with a huge adverse impact on health and mortality.

Table 2: Number of People with diabetes between 20 to 79 years

(International Diabetes Federation)Country/Territory2010 (millions)

1. India50.8

2. China43.2

3. U.S.A26.8

4. Russia9.6

5.Brazil7.6

6. Germany7.5

7. Pakistan7.1

8. Japan7.1

9. Indonesia7.0

10. Mexico6.8

1.4. Pathphysiology 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 (ET-1), activation of transcription factors such as NF-B and AP-1, and increased production of prothrombotic factors such as tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1).(Collins T et al 2001).

Figure 2: The metabolic abnormalities that characterize diabetes. (King GL. 1996)

1.4.1. 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 plateletvon Willebrand (vWF) factor and plateletfibrin interaction. (Li Y et al 2001)The bioavailability of NO is decreased. 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 (Ceriello A et al. 1995).

Figure 3: Alteration of platelet function and coagulation factors in diabetes. (Vinik AI et al 2001)

Figure 4: Long-term Complications of Diabetes Mellitus (American Diabetes Associationwww.diabetes.org)

1.5. Management 1.5.1. Diabetes managementDiabetes mellitus is a chronic disease which is difficult to cure. Management concentrates on keeping blood sugar levels as close to normal ("euglycemia") as possible without presenting undue patient danger. This can usually be with close dietary management, exercise, and use of appropriate medications (insulin only in the case of type 1 diabetes mellitus. Oral medications may be used in the case of type 2 diabetes, as well as insulin).Patient education, understanding, and participation is vital since the complications of diabetes are far less common and less severe in people who have well-managed blood sugar levels. Wider health problems may accelerate the deleterious effects of diabetes. These include smoking, elevated cholesterol levels, obesity, high blood pressure, and lack of regular exercise. (Nathan DM et al 2005)1.5.2. Management of Diabetes with Alternative Medicines

Medicinal plants are the most exclusive source of life saving drugs for the majority of the worlds 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.(Scartezzini, P. et al. 2000)Medicinal plants are the most exclusive source of life saving drugs for the majority of the worlds 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. In the past decade, research has been focused on scientific evaluation of traditional drugs of plant origin and screening of more effective and safe hypoglycemic agents has continued to be an important area.

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.

The following table compares some common anti-diabetic agents, generalizing classes although there may be substantial variation in individual drugs of each class: Table 3: Anti Diabetic agents (Richard D. Howland et al 2006)AgentMechanismSite of actionMain advantagesMain side effects

SulfonylureasStimulating insulin production by inhibiting the KATP channelPancreatic beta cellseffective

InexpensiveHypoglycemiaWeight gain

MetforminDecreases insulin resistanceLiverWeight loss

Does not cause hypoglycemiaGI symptoms, including diarrhea, nausea, abdominal pain

Lactic acidosisMetallic taste

AcarboseReduces intestinal glucose absorptionGI tractLow riskGI symptoms, including diarrhea, abdominal cramping, flatulence

ThiazolidinedionesReduce insulin resistance by activating PPAR-Fat, muscleHepatoxicity

1.6. Diabetic nerve pain is a growing problem

Diabetes can destroy small blood vessels, which in turn can damage the nervous system, and these damaged nerves can cause pain. When a person has pain that is caused by nerve damage from diabetes, it is called Diabetic Nerve Pain. About 8% of Americans have diabetes. Unfortunately, this number is only growing. As would be expected, the number of people suffering from Diabetic Nerve Pain has also increased. The American Diabetes Association reports that about 50% of people with diabetes have some form of nerve damage known as diabetic neuropathy. Diabetic Nerve Pain is a common diabetes complication, as are kidney and eye (retinopathy) conditions.

The most common type of diabetic neuropathy is peripheral neuropathy (burning, throbbing, or painful tingling in your hands or feet). In the early stages of peripheral neuropathy, some people have no signs. Some may have numbness or tingling in the feet. Because nerve damage can occur over several years, these cases may go unnoticed. The patient may only become aware of neuropathy if the nerve damage gets worse and becomes painful.

Diabetic nerve damage to the feet, sometimes called diabetic neuropathy, or more correctly, diabetic distal symmetric sensory polyneuropathy, frequently causes people with diabetes to lose sensation in their feet, which is usually describe as "numbness." Diabetes leg pain, as well as the problem in the foot is not felt until some damage may have been done. Neuropathy is a complication of this disease after years of uncontrolled high blood sugar. This lowers the sensitivity to pain thus posing a danger that damage could happen without feeling a warning.

When the diabetic loses a lot of this throbbing sensation, he has not really lost the feeling completely but rather the sensation is at a different level so that by the time the real sensation becomes uncomfortable, the damage may have already occurred.

The poor circulation to the lower extremities caused by the complication of this disease leads to chronic skin ulcers, numbness and burning of the lower legs and feet. These along with the diabetes leg pain can be painful. When left untreated, this could result in gangrene that may require amputation.

The feeling of the diabetes leg pain may be in the form of cramps while walking, predominantly in the area of the calf muscle. This may be the sign of the circulatory problem. The other signals are redness of the feet and darkening of the skin when the legs are in a dependent situation.

Nerve pain is different from other types of pain, like pain from a muscle ache or sprained ankle. Common pain medicines like aspirin may not work for nerve pain. However, there is a prescription treatment option. This treatment is clinically proven to provide effective relief from the burning, throbbing, or painful tingling of Diabetic Nerve Pain. However, diabetic neuropathy occasionally creates a severe burning pain, or other very unpleasant sensations, that are extremely frustrating.

1.7. Alloxan

1.7.1. Definition

Alloxan (2,4,5,6-tetraoxypyrimidine; 2,4,5,6-pyrimidinetetrone) is an oxygenated pyrimidine derivative. It is present as alloxan hydrate in aqueous solution. ( Merck Index, 11th Edition, 281).

Figure 5: Alloxan

1.7.2. History

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

1.7.3. Biological effectsAlloxan 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. (A. Mrozikiewicz at 1994).1.7.4. Impact upon beta cellsBecause 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)1.8. Pain1.8.1. Definition: An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.Pain motivates us to withdraw from potentially damaging situations, protect a damaged body part while it heals, and avoid those situations in the future. Most pain resolves promptly once the painful stimulus is removed and the body has healed, but sometimes pain persists despite removal of the stimulus and apparent healing of the body and sometimes pain arises in the absence of any detectable stimulus, damage or disease. ("International Association for the Study of Pain. Pain Definitions". Retrieved 12 October 2010.) 1.8.2. Clinical Terms For The Sensory Disturbances Associated With Pain :( Mike Halos WRHA Palliative Care) Dysesthesia An unpleasant abnormal sensation, whether spontaneous or evoked.

Allodynia Pain due to a stimulus which does not normally provoke pain, such as pain caused by light touch to the skin

Hyperalgesia An increased response to a stimulus which is normally painful

Hyperesthesia - Increased sensitivity to stimulation, excluding the special senses. Hyperesthesia includes both allodynia and hyperalgesia, but the more specific terms should be used wherever they are applicable. (von Baeyer CL; Pain Research and Management 11(3) 2006; p.157-162).

Figure 6: Mechanism of pain (Hinz, B et al)1.10. Causes and Symptoms: 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. (Keay, KA et al 2000)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.

Sometimes intensive, multi-disciplinary examination may be needed to reveal the underlying cause.(Coda, BA et al 2001).1.10.1. Frequent causes of pain:

CancerMusculoskeletal pain Osteoarthritis Rheumatoid arthritis Low back pain Failed back surgery Fractures & osteoporosisNerve pains due to Diabetic neuropathy Peripheral blood vessel disorders and stroke Herpetic infection TraumaSomatic pain:

Originates from bones, muscles, tendons or blood vessels and is often known asmusculo-skeletal pain Usually sharp, well-localized Can be reproduced by touching or moving the involved area Usually of longer durationCoetaneous pain

Is due to injury of the skin or the superficial tissues

Usually well-described, localized pain of short durationVisceral pain

Originates from the internal organs of the bodys cavities such as thorax (heart and lungs), abdomen (liver, kidneys, spleen and bowels) and pelvis (ovaries, bladder and womb)

More aching, vague and often difficult to localize Of longer duration

Sometimes colicky or crampingPeripheral 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 symmetricallyEntrapment 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 footPhantom 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 quadriplegicsChronic 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.

1.10.2. 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 1997) negative or distorted cognition, such as feeling helpless or hopeless with respect to pain and its management

inactivity, passivity, and/or disability

increased pain requiring clinical intervention (Cox JJ et al 2006). insomnia and fatigue

disrupted social relationships at home, work, or school

depression and/or anxiety

1.11. Pain management

Pain management (also called pain medicine; algiatry) is a branch of medicine employing an interdisciplinary approach for easing the suffering and improving the quality of life of those living with pain. The typical pain management team includes medical practitioners, clinical psychologists, physiotherapists, (Hardy, Paul A. J. 1997) occupational therapists, and nurse practitioners.Pain sometimes resolves promptly once the underlying trauma or pathology has healed, and is treated by one practitioner, with drugs such as analgesics and (occasionally) anxiolytics. Effective management of long term pain, however, frequently requires the coordinated efforts of the management team. (Main, Chris J. et al 2000)Medicine treats injury and pathology to support and speed healing; and treats distressing symptoms such as pain to relieve suffering during treatment and healing. When a painful injury or pathology is resistant to treatment and persists, when pain persists after the injury or pathology has healed, and when medical science cannot identify the cause of pain, the task of medicine is to relieve suffering. Treatment approaches to long term pain include pharmacologic measures, such as analgesics, tricyclic antidepressants and anticonvulsants, interventional procedures, physical therapy, physical exercise, application of ice and/or heat, and psychological measures, such as biofeedback and cognitive behavioral therapy. (Hienhaus, Ole; Cole, B. Eliot 2002).An analgesic (also known as a painkiller) is any member of the group of drugs used to relieve pain (achieve analgesia). Analgesic drugs act in various ways on the peripheral and central nervous systems; they include paracetamol (para-acetylaminophenol, also known in the US as acetaminophen), the non-steroidal anti-inflammatory drugs (NSAIDs) such as the salicylates, and opioid drugs such as morphine and opium. They are distinct from anesthetics, which reversibly eliminate sensation.( Dworkin RH et al 2003). Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) taken to reduce inflammation and as an analgesic in certain conditions.

Figure 7: DiclofenacThe exact mechanism of action is not entirely known, but it is thought that the primary mechanism responsible for its anti-inflammatory, antipyretic, and analgesic action is inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (COX). It also appears to exhibit bacteriostatic activity by inhibiting bacterial DNA synthesis.(Dutta NK et al 2000).Inhibition of COX also decreases prostaglandins in the epithelium of the stomach, making it more sensitive to corrosion by gastric acid.This is also the main side effect of diclofenac. Diclofenac has a low to moderate preference to block the COX2-isoenzyme (approximately 10-fold) and is said to have therefore a somewhat lower incidence of gastrointestinal complaints than noted with indomethacin and aspirin. (Fowler PD et al 1983).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 delage for those medications is to 100-200mg, given in several divided doses. (Mazumdar K et al 2006). Diclofenac also is useful for short term treatment of acute musculoskeletal pain, postoperative pain, and dysmenorrheal. 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 misprostol. (Dutta NK et al 2006).But it is not effective for the treatment of diabetic neuropathic pain. For this reason, it is necessary to findings of natural and safe plant medicine for the treatment of diabetic neuropathic complication.Plant preview

2. Plant preview

2.1. Cassia fistula

2.1.1. Botanical features

Cassia fistula also called Golden Shower Tree is a flowering plant in the family Fabaceae, native to southern Asia, from southern Pakistan east through India to Myanmar and south to Sri Lanka. It is the national tree of Thailand and its flower is Thailand's national flower. (Karanth KS. et al September 2006).

2.1.2. Botanical name/ Scientific name: Cassia fistula Linnaeus2.1.3. Common name or Local name:

Common name: Bengali: Sonali, Bandarlati, Amaltas, Golden shower tree, Indian Laburnum Hindi: Amaltas Manipuri: Chahui Tamil: Konrai Malayalam: Vishu konnai Marathi: Bahava Mizo: Ngaingaw , Urdu: Amaltas (MMPND2005).

2.2. Taxonomic Classification

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Subclass : Rosidae

(unranked) : Eurosids I

Order : Fabales

Family : Fabaceae

Subfamily: Caesalpinioideae

Tribe : Cassieae

Subtribe : Cassiinae

Genus : Cassia

Species : C. fistula

2.3. Various Parts of Cassia fistula:

Leaves & Flower Stem Bark

Fruit

Flowers Part of Plant

Figure 8: Images of various parts of Cassia fistula2.4. Biogeographically distribution of Cassia fistula(

other Web sites." \t "_blank"

HYPERLINK "http://www.rain-tree.com/canafistula.htm" \o "PLANTS is not

responsible for the content or availability of other Web sites." \t "_blank" Tropical Plant Database /

is not responsible for the content or availability of other Web sites." \t "_blank" www.ildis.org)

Figure 9: Biogeographically distribution of Cassia fistula in Bangladesh2.5. Description (Allen O.N. et al 1981)Deciduous tree 10 m tall, the bole to 5 m, to 1 m DBH. Leaves alternate, pinnate, 30-40 cm long, with 4-8 pairs of ovate leaflets, 7.5-15 cm long, 2-5 cm broad, entire, the petiolules 2-6 mm long. Flowers yellow, in long drooping terminal clusters (racemes); petals 5, yellow; sepals 5, green, the individual flower stalks 3-6 cm long. Stamens 10, three with longer stalks. Fruits pendulous, cylindrical, brown, septate, 25-50 cm long, 1.5-3 cm in diameter, with 25-100 seeds. Seeds lenticular, light brown, lustrous. Leaves: Alternate, petiolate, Leaflets ovate. The leaves are deciduous, or semi-evergreen. 15-60 cm long, pinnate with 3-8 pairs of leaflets. Each leaflet 7-21 cm long and 4-9 cm broad.

Flowers: Bracteates, Ebeacteate, zygomorphic, Bisexual & Hypogenous. The flowers are produced in pendulous racemes. 20-40 cm long, each flower 4-7 cm diameter with five yellow petals of equal size and shape.

Contin.. Calyx - Sepals 5, Polysepalous, yellowish green in colour. Corolla - Petals 5, Polypetalous, petal smallest. Androecium - Stamens 10, arranged in two whorls of 5 each, Anthers dorsifixed. Gynoecium - Ovary superior, monocarpellary, Unilocular.

Fruit & seed: A legume, cylindrical in shape and divided into segments. Seed - Large, circular in shape. The seeds are poisonous.

2.6. Traditional Medicinal Uses In Ayurvedic medicine, Golden Shower Tree is known as "disease killer". Its fruitpulp is used as mild laxative. as well as cardiac conditions and stomach problems such as acid reflux. flowers used for fever, root as a diuretic. The bark and leaves are used for skin diseases.

In Ayurvedic medicine systems, the seeds are recognized as antibilious, aperitif, carminative, and laxative while the root is used for curing adenopathy, burning sensations, leprosy, skin diseases, syphilis, and andtubercularglands.The bark has been employed in tanning, often in conjunction with avaram. The drug "cassia fistula", a mild laxative, is obtained from the sweetish pulp around the seed.

The leaves of the tree is used for erysipelas, malaria, rheumatism, and ulcers, the buds are used for biliousness, constipation, fever, leprosy, and skin disease and the fruit for abdominal pain, constipation, fever, heart disease, and leprosy. Thus every part of this plant is recognized for its medicinal properties (Sharma, 1979).

There are many Cassia species worldwide which are used in herbal medicine systems. This particular family of plants is used widely for their laxative actions. Canafistula is no exception... it is often used as a highly effective moderate laxative that is safe even for children. However, in large doses, the leaves and bark can cause vomiting, nausea, abdominal pain and cramps. Canafistula is also employed as a remedy for tumors of the abdomen, glands, liver, stomach, and throat, for burns, cancer, constipation, convulsions, delirium, diarrhea, dysuria, epilepsy, gravel, hematuria, pimples, and glandular tumors. In Ayurvedic medicine systems, the seeds are attributed with antibilious, aperitif, carminative, and laxative properties while the the root is used for adenopathy, burning sensations, leprosy, skin diseases, syphilis, and tubercular glands. The leaves are employed there for erysipelas, malaria, rheumatism, and ulcers. In Brazilian herbal medicine, the seeds are used as a laxative and the leaves and/or bark is used for pain and inflammation. Table 4: WORLDWIDE ETHNOMEDICAL USES (Tropical Plant database).WORLDWIDE ETHNOMEDICAL USES

Brazilas a laxative, analgesic, anti-inflammatory

DominicanRepublicas a laxative, vermifuge

Indiafor burns, cancer, convulsion, delirium, diarrhea, dysuria, epilepsy, gravel, hematuria, pimples, syphilis

Javafor carbuncles, dermatosis, herpes, wounds; as a purgative, laxative

Mexicoas a laxative

Panamafor diabetes

Peruas an astringent, laxative, purgative

Venezuelaas an astringent, laxative, purgative

Elsewherefor constipation, flu, fumitory, tumors; as an aperients, laxative, purgative

2.7. Chemical Literature Review of Plant

Table 5 : Chemical compounds isolated from Cassia fistula Linn.Plant partsChemical ConstituentsReferences

HeartwoodFistucacidin (3,4,7,8,4-

PentahydroxyflavanPadmanabha Rao, 1965

BarkOxyanthraquinone,

DihydroxyanthraquinoneRani et al., 1998

Leaves(-) epiafzelechin, (-) epiafzelechin-3-Oglucoside,

(-) epicatechin, procyanidin

B2, biflavonoids, triflavonoids, rhein,

rhein glucosidal, sennoside A,

sennoside B, chrysophanol, physcionKashiwada et al., 1996; Kaji et al.,

1968; Kashiwada et al., 1996;

Mahesh et al., 1984.

Flowerstetramer (with free glycol unit), rhein,

fistulin, alkaloids, triterpenesNarayanan and Seshadri, 1972;

Kumar et al., 1966; Guri-Fakim et al.,

Fruit pulpRheine, volatile oil, waxy and resinous

DerivativesLiptak and Szentagali, 1937

PodsFistulic acid, 3-formyl-1-hydroxy-8-methoxy anthaquinone, 3B-hydroxy-17-norpimar-8(9)-en-15-oneMisra et al., 1997

SeedsChrysophanolKhana and Chandra, 1984

Reproductive organs: flower bud,

flower, podProanthocyanidins, flavonoidsLuximon-Ramma et al., 2002

Vegetative organs: young Rhamnetin-3-O-gentiobiosideVaishnav and Gupta, 1996

2.8. Biological Literature Review of Cassia fistula Linn.Experimental studies demonstrated that the cassia fistula have the following pharmacological properties (Table 6). Table 6: Reported biological activities of Cassia fistula Linn.

Plant partsPharmacological activitiesReferences

BarkHepatoprotective Activity,cardioprotective activity,Anti-inflammatory and Anti-oxidant activity, Ant diabetic Activity,Anti-bacterial activityG. Parthasarathy et al. 2009,Khatib N.A. et al .2010 Raju Ilavarasan et al. 2005, T. Ranjith Vimalraj et al 2009,S.N. Malpani et al 2010

FruitsImmunomodulatory,Toxicity potential,Nafisa Hasan Ali et al. 2008, M. A. Akanamu2004.

Fruit pulpAntioxidant activityBhatnagar M et al.2010

PodAnalgesicN.W. Sheikh et al. 2010

PURPOSE OF RESEARCH

3. Purpose of Research

3.1. Background of the study

Diabetes mellitus is a chronic disease characterized by high blood glucose levels due to absolute or relative deficiency of circulating insulin levels (Holmann R.R et al). Diabetes can be divided into two main groups based on their requirements of insulin: insulin dependent diabetes mellitus (IDDM or Type 1), and non-insulin dependent diabetes mellitus (NIDDM or Type 2). However, other types of diabetes have also been identified. Maturity Onset Diabetes of the Young (MODY) is now classified as Type 3 and gestational diabetes classified as Type 4. NIDDM accounts for about 90 percent of diabetic cases (World Health Organization 2002), manifested by insulin resistance and -Cell dysfunction are the metabolic abnormalities in the type 2 diabetes. Glycemic control is one of the targets for managing diabetes mellitus. Several studies have confirmed that effective control of blood glucose levels in type 2 diabetes substantially decrease the risk of developing diabetic complications. (Tanko Y., et al., 2008)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. According to the WHO, more than 70% of the worlds population must use traditional medicine to satisfy their principal health needs. A great number of medicinal plants used in the control of diabetes mellitus have been reported (Bailey C. J.1989). There are various medicinal plants in the world, which are the potentials sources of the drugs. The discovery of the widely used hypoglycemic drug, metformin came from the traditional approach through the use of Cassia fistula. (J. K. Grover et al. 2002)

Medicinal plants are the most exclusive source of life saving drugs for the majority of the worlds population. In developing countries 80% population are using traditional medicine in primary medical problems (Grover J.K. 2002), 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. In the past decade, research has been focused on scientific evaluation of traditional drugs of plant origin and screening of more effective and safe hypoglycemic agents has continued to be an important area.

Table 7: Some Bangladeshi medicinal plants subjected to clinical trials

NoName of PlantClinical TrialResultsReference

1.Allium cepa

(I) Juice (100g), orally20 diabetic patients and 20 normal healthy controlsReduction of blood sugar in diabetics No alteration in blood sugar in controls144

(ii) Aqueous extract

(25-50gm)20 healthy volunteers (Fasting and adrenaline induced hyperglycemia)No effect on fasting blood sugar but reduced the rise in blood sugar on glucose loading 145

2.Clerodendron phlomoides (Alcoholic extract)33 diabetic patients and 10normal volunteers Reduction in fasting blood sugar146

3.Cinnamomum tamale

(leaves powder 3 teaspoons 4 times for 15 days)5 diabetic patientsReduction on blood glucose147

4.Coccinia indica

(powder 3gm twice daily) 41 diabetic patientsReduction in blood sugar148

5.Ficus bengallensis

(aqueous extract of bark)12 normal volunteers, 6 diabetic patients and 6 controls patientsNo effect in normal human, mild activity in diabetic patients149

6.Momordica charantia

(fruits powder 100gm daily for 2 weeks)Normal controls 25 patients of diabetes mellitusNo significant effect in either cases150

According to the ethnobotanical surveys more than 800 plants are used worldwide in traditional medicine to treat diabetes (Ajgaonkar SS. Et al.1993). 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. Cassia fistula also known as sonali in Bengali, Golden shower tree in English. Simple phenolic compounds, tannins, quinones and derivatives occur in the overlapping cortical root cells. It is assumed that these cell layers present a physicochemical barrier because of their role in thwarting nematode gall formation (Allen and Allen, 1981). Agriculture Handbook #165 reports the tosspot, Phyllachora canafistulae, in Maryland, near its northern limit. The plant possesses antidiabetic, antioxidant, analgesic, flavonoid, modulation of humoral immunity, toxicity potentials, cardio protective, Anti-fungal, laxative, Purgative, demulcent, anti-bacterial, HPMC, phytochemicalconstituents(fistucacidin(3,4,7,8,4-flavanheartwoodadmanabha ao, 1965oxyanthraquinone,dihydroxyanthraquinonebark (rani et al., 1998), (-) epiafzelechin, (-) epiafzelechin-3-oglucoside,(-) epicatechin, procyanidin b2, biflavonoids, triflavonoids, rhein,rhein glucoside, sennoside a,sennoside b, chrysophanol, physcion,leaves (kashiwada et al., 1996); (kaji et al.,1968); (ashiwada et al., 1996;mahesh et al., 1984).kaempferol, leucopelargonidin tetramer (with free glycol unit), rhein,fistulin, alkaloids, triterpenesflowers narayanan and seshadri, , volatile oil, waxy and resinous derivatives fruit pulp (liptak and szentagali, 1937) fistulic acid, 3-formyl-1-hydroxy-8-methoxy anthaquinone, 3b-hydroxy-17-norpimar-8(9)-en-15-onepods (misra et al., 1997)chrysophanol seeds khana and chandra, 1984rhamnetin-3-o-gentiobioside roots (gupta, 1996)proanthocyanidins, flavonoids). (Abu Sayeed et al 1999)3.2. Aims and Objectives

This research work was undertaken to evaluate the ant diabetic and analgesic effects of ethanolic extract of the stem barks of plant Cassia fistula in normal and alloxan-induced diabetic mice. The most widely used experimental procedures were followed: To examine the effect of plant extract on blood glucose level both in normal and alloxan-induced diabetic mice. To evaluate the hypoglycemic effect of the plant extract on glucose induced hyperglycemic mice. To determine the analgesic activity of plant extract for its central and peripheral pharmacological action using acetic acid induced writhing test in mice. Finally, find out the possible mechanism action of the plant extract for their beneficial effect both in normal and alloxan-induced diabetic mice.Materials and method

4. Materials and method4.1. Plant Materials

Fresh stem barks of the plant Cassia fistula Linn. Was collected from Sirajgonj district during the month of March-April in 2011 and the plant authenticity were confirmed from the Bangladesh National Herbarium, Dhaka. 4.2. Preparation of Plant Extracts

The stem barks collected were washed and sun dried under shadow for several days. The dried stem barks were powdered in an electrical grinder after overnight drying in an oven below 50(C. The powdered plant barks were extracted with 96% ethanol at room temperature. The bottle were kept at room temperature and allowed to stand for several 7-10 days with occasional shaking and stirring. The extracts thus obtained were filtered through cotton and then through filter paper (Whatman Fitter Paper No. 1). The filtrate was defatted with petroleum ether for several times. Then, the defatted liquor was allowed to evaporate using rotary evaporator at temperature 40-45C. Finally, a highly concentrated ethanol extract were obtained and kept in desiccators to dry to give a solid mass (Yield 15g of extract from 800 g of plant powder material).

4.3. Drugs and Chemicals

The standard drug, Metformin hydrochloride was the generous gift samples from Pacific Pharmaceuticals Ltd. Alloxan monohydrate was purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Blood samples analyzed for blood glucose content by using BioLand G-423 glucose test meter (BioLand, Germany). All chemicals and solvents were of reagent grade.

4.4. Experimental Animals

Six weeks Swiss albino mice (20-30g) of either sex were purchased from ICDDRB, Dhaka, Bangladesh and were housed in animals cages under standard environmental conditions (22-25C, humidity 60-70%, 12 h light: 12 h dark cycle). The mice were fed with standard pellet diet obtained from ICDDRB, Dhaka and water ad libitum. The animals used in this study were cared in accordance with the guidelines on animal experimentation of our institute. 4.5. Induction of Diabetes

After fasting 16h, diabetes was induced into mice by in intra-peritoneal injection (i. p.) of alloxan monohydrate (100 mg/kg), dissolved in saline (100 (l/mice, ip.). After 48h, plasma glucose levels were measured by glucometer (Bioland, Germany) using a blood sample from tail-vein of mice. Mice with blood sugar level higher than 8.5.5-11.5 mmol/l are considered as moderate diabetic.

4.6. Experimental DesignIn the experiment, a total of 45 mice were used. The diabetic animals were divided into five groups and each group comprises of five mice. Group I received vehicle 0.5% methyl cellulose stands for normal control. Group II received vehicle 0.5% methyl cellulose serves as diabetic control. Group III selected for diabetic standard drug group which received metformin orally at a dose of 100 mg/kg. Group IV and Group V were received 250 and 500 mg/kg body weight mice CF extract orally after chemical diabetes. 4.7. Antidiabetic activity tests

The animals of Group IV and Group V received oral administrations of bark extract of C. fistula at a dose 250 and 500 mg/kg/ml body weight using intrgastric tube. Group III received metformin (100 mg/kg body weight), while Group II serves as diabetic control (vehicle 0.5% MC). The blood samples were analyzed for blood glucose content by Glucometer.

4.7.1. Oral glucose tolerance test (OGTT) in diabetic miceAfter fasting for overnight, a baseline blood glucose level was estimated (0 minutes). Without delay, a glucose solution (2 gm/kg body weight) was administered by gavage. At the same time standard drug and plant extracts were administered orally to the respective animal groups. Four more blood samples were taken at 30, 60, 90, 120 minutes after glucose administration and blood glucose level was estimated in all the experiments by using glucometer (Bioland-423, Germany).4.7.2. Oral glucose tolerance test (OGTT) in glucose induced hyperglycemic miceFor oral glucose tolerance test (OGTT) animals were divided into four groups (each group comprises five mice). Group VI to Group IX was prepared for testing of hypoglycemic effect after glucose-induced hyperglycemia in mice Group VI received vehicle 0.5% methyl cellulose stands for normal control. Group VII received metformin orally at a dose of 100 mg/kg and Group VIII and Group IX were received 250 and 500 mg/kg body weight mice CF extract orally. Four more blood samples were taken at 30, 60, 90, 120 minutes after glucose administration and blood glucose level was estimated in all the experiments by using glucometer.4.8. Analgesic activity test

Mice were divided into four groups (each group comprises five mice). Group I served as vehicle control mice received vehicles (1% Tween 80 in water), Group II served as standard group received Diclofenac sodium (80 mg/kg i.p) as standard drug, Group III and Group IV received 250 and 500 mg/kg orally of CF extract respectively. The analgesic activity of the samples was studied using acetic acid-induced writhing model in mice. Writhing was induced in mice by intraperitoneal administration of 0.1 ml of 1% Acetic Acid. Extract and vehicle were administered orally 30 mins before intraperitoneal administration of 1% acetic acid but Diclofenac-Na was administered intraperitoneally 15 mins before injection of acetic acid. After an interval of 5 mins, the mice observed for specific contraction of body referred to as writhing for the next 10 minutes (Ahmed F. et al. 2004).

4.9. Statistical AnalysisData were expressed as mean Standard error of mean (SEM). Statistical comparison was performed by one-way ANOVA, followed by Dunetts Multiple Comparison. Results considered as significant when p values were less than 0.05 (p