Chapter one

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 type 1 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 type 1 diabetes, which causes approximately 10% of diabetes mellitus cases in North America and Europe.(Rather KI April 2007) 1.1.3. Type 2 diabetes: Type 2 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. Type 2 diabetes is the most common type.

1.1.4. Gestational diabetes: Gestational diabetes mellitus (GDM) resembles type 2 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 type 2 diabetes later in life (Robbins Basic Pathology).

1.2. Signs and Symptoms Polyuria (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 renourish Weight 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

Symptoms

Figure 1: Hypoglycemic (www.ihc.com/diabetes)

Table 1: Type 2

Difference between Type 1 & Diabetes

1.3. Prevalence of Diabetes Comparison of type 1 and 2 diabetes Feature Type 1 diabetes Onset Sudden Age at onset Any age Body habitues Ketoacidosis Autoantibodies Endogenous insulin Concordance

(mostly young) Thin or normal Common Usually present Low or absent 50%

Type 2 diabetes Gradual Mostly in adults Often obese Rare Absent Normal, decreased, increased 90%

in identical twins 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/Territory 1. India 2. China 3. U.S.A 4. Russia 5.Brazil 6. Germany 7. Pakistan 8. Japan 9. Indonesia 10. Mexico

2010 (millions) 50.8 43.2 26.8 9.6 7.6 7.5 7.1 7.1 7.0 6.8

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- ƙB 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 platelet–von Willebrand (vWF) factor and platelet–fibrin 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)

Hyperglycemia Can Cause Serious Long-Term Problems

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

1.5. Management 1.5.1. Diabetes management Diabetes 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 wellmanaged 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 world’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.

(Scartezzini, P. et al. 2000) Medicinal plants are the most exclusive source of life saving drugs for the majority of the world’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.

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) Agent Sulfonylureas

Metformin

Mechanism

Site of action

Main advantages Stimulating Pancreatic beta effective insulin production cells Inexpensive by inhibiting the KATP channel Decreases Weight loss insulin resistance Does not cause Liver hypoglycemia

Main side effects Hypoglycemia Weight gain GI symptoms, including diarrhea, nausea,

abdominal pain Lactic acidosis Acarbose

Thiazolidinediones

Reduces GI tract intestinal glucose absorption

Low risk

Reduce insulin Fat, muscle resistance by activating PPARÎł

Metallic taste GI symptoms, including diarrhea, abdominal cramping, flatulence Hepatoxicity

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 WĂśhler and Liebig.

The name "Alloxan" emerged from an amalgamation of the words "Allantoin" and "Oxalsäure" (oxalic acid).

1.7.3. 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. (A. Mrozikiewicz at 1994).

1.7.4. 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)

1.8. Pain 1.8.1. 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).

1.9 TYPES OF PAIN NOCICEPTIVE

NEUROPATHIC

Visceral

Somatic • bones, joints • connective tissues • muscles

• Organs – heart, liver, pancreas, gut, etc.

Deafferentation

COMPONENT Steady, Dysesthetic

Sympathetic Maintained

DESCRIPTORS • Burning, Tingling • Constant, Aching • Squeezing, Itching

Peripheral

EXAMPLES • Diabetic neuropathy • Post-Herpetic Neuropathy

• Allodynia • Hypersthesia Paroxysmal, Neuralgic

• Stabbing

• trigeminal neuralgia

• Shock-like, electric

• may be a component of any neuropathic pain

• Shooting • Lancinating

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: Cancer Musculoskeletal pain  Osteoarthritis  Rheumatoid arthritis  Low back pain

 Failed back surgery  Fractures & osteoporosis Nerve pains due to    

Diabetic neuropathy Peripheral blood vessel disorders and stroke Herpetic infection Trauma

Somatic pain:  Originates from bones, muscles, tendons or blood vessels and is often known as musculoskeletal pain  Usually sharp, well-localized  Can be reproduced by touching or moving the involved area  Usually of longer duration Coetaneous pain  Is due to injury of the skin or the superficial tissues  Usually well-described, localized pain of short duration Visceral pain  Originates from the internal organs of the body’s 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 cramping 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.

1.10.2. 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:

Diclofenac

The exact mechanism of action is not entirely primary mechanism responsible for its anti-inflammatory,

known, but it is thought that the 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 Linnaeus 2.1.3. Common name or Local name: Common name: Bengali: োসোনোলী Sonali, Bandarlati, Amaltas, Golden shower tree, Indian Laburnum • Hindi: अमलतास Amaltas • Manipuri: চহুঈ Chahui • Tamil: ொகொன்றைற Konrai • Malayalam: Vishu konnai • Marathi: बहावा Bahava • Mizo: Ngaingaw •, Urdu:‫ املتاس‬Amaltas (MMPND2005).

2.2. Taxonomic Classification Kingdom : Division : Class : Subclass : (unranked) : Order : Family : Subfamily: Tribe : Subtribe : Genus : Species :

Plantae Magnoliophyta Magnoliopsida Rosidae Eurosids I Fabales Fabaceae Caesalpinioideae Cassieae Cassiinae Cassia C. fistula

2.3. Various Parts of Cassia fistula:

Leaves & Flower

Stem Bark

Fruit

Flowers

Figure 8: Images of various parts of Cassia fistula

Part of Plant

2.4. Biogeographically distribution of Cassia fistula(Tropical Plant Database /www.ildis.org)

  

  

  

  

  

  

  

     

  

 

= Less commonly found = Commonly found

Figure 9: Biogeographically distribution of Cassia fistula in Bangladesh 2.5. 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 Brazil

as a laxative, analgesic, anti-inflammatory

Dominican as a laxative, vermifuge Republic India

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

Java

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

Mexico

as a laxative

Panama

for diabetes

Peru

as an astringent, laxative, purgative

Venezuela as an astringent, laxative, purgative Elsewhere for 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 parts Heartwood

Chemical Constituents Fistucacidin (3,4,7,8,4’-

References Padmanabha Rao, 1965

Bark

Pentahydroxyflavan Oxyanthraquinone,

Rani et al., 1998

Leaves

Dihydroxyanthraquinone (-) epiafzelechin, (-) epiafzelechin-3-

Kashiwada et al., 1996; Kaji

Oglucoside,

et al.,

(-) epicatechin, procyanidin

1968; Kashiwada et al.,

B2, biflavonoids, triflavonoids, rhein,

1996;

rhein glucosidal, sennoside A,

Mahesh et al., 1984.

sennoside B, chrysophanol, physcion tetramer (with free glycol unit), rhein,

Narayanan and Seshadri,

fistulin, alkaloids, triterpenes

1972;

Flowers

Kumar et al., 1966; GuriFruit pulp

Rheine, volatile oil, waxy and resinous

Fakim et al., Liptak and Szentagali, 1937

Pods

Derivatives Fistulic acid,

Misra et al., 1997

3-formyl-1-hydroxy-8-

methoxy anthaquinone, 3B-hydroxy-17Seeds Reproductive

norpimar-8(9)-en-15-one Chrysophanol Proanthocyanidins, flavonoids

organs:

Khana and Chandra, 1984 Luximon-Ramma et al., 2002

flower bud, flower, pod Vegetative

Rhamnetin-3-O-gentiobioside

Vaishnav and Gupta, 1996

organs: young 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 parts Bark

Pharmacological activities Hepatoprotective

References G. Parthasarathy et al.

Activity,cardioprotective activity,Anti- 2009,Khatib N.A. et al . inflammatory

and

Anti-oxidant 2010 Raju Ilavarasan et al.

activity, Ant diabetic Activity,Anti- 2005, T. Ranjith Vimalraj et bacterial activity

al 2009,S.N. Malpani et al 2010

Fruits

Immunomodulatory,Toxicity

Nafisa Hasan Ali et al. 2008, M.

Fruit pulp Pod

potential, Antioxidant activity Analgesic

A. Akanamu2004.

Bhatnagar M et al.2010 N.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 noninsulin 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 β-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 world’s 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 world’s 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 No

Name of Plant

1.

Allium cepa (I) Juice (100g), orally

Clinical Trial

Results

20 diabetic patients Reduction and

20

Reference

of 144

normal blood sugar in

healthy controls

diabetics

No

alteration in blood

sugar in controls

(ii) Aqueous extract (25-50gm)

20

healthy No

volunteers (Fasting fasting and

2.

3.

Clerodendron

6.

blood but

induced

reduced the rise

hyperglycemia)

in blood sugar on

glucose loading phlomoides 33 diabetic patients Reduction in 146

(Alcoholic extract)

and

10normal fasting

Cinnamomum tamale

volunteers 5 diabetic patients

4 times for 15 days) Coccinia indica

sugar Reduction

41 diabetic patients

on 147

Reduction

in 148

blood sugar

Ficus bengallensis

12

(aqueous extract of bark)

volunteers,

Momordica charantia

blood

blood glucose

(powder 3gm twice daily) 5.

on 145

adrenaline sugar

(leaves powder 3 teaspoons 4.

effect

normal No

effect

6 normal

in 149

human,

diabetic

patients mild activity in

and

controls diabetic patients

6

patients Normal controls 25 No

significant 150

(fruits powder 100gm daily patients of diabetes effect in either for 2 weeks)

mellitus

cases

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 alloxaninduced 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 method 4.1. 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 째C. 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 40-45째C. 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 (22-25°C, 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 Âľl/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 Design In 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 mice

After 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 mice For 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 Analysis Data were expressed as mean Âą Standard error of mean (SEM). Statistical comparison was performed by one-way ANOVA, followed by Dunettâ&#x20AC;&#x2122;s Multiple Comparison. Results considered as significant when p values were less than 0.05 (p<0.05). Statistical calculations and the graph were prepared using Graph Pad Prism Software version 5 (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

PHYTOCHeMICal SCReeNING 5. Photochemical Screening 5.1. Introduction The subject of phytochemistry or plant chemistry has developed in recent years as a distinct discipline, somewhere in between natural product organic chemistry and plant biochemistry is closely related to both. It is concerned with the enormous variety of organic substances that are elaborated and accumulated by plant and deals with the chemical structures of these substances, their biosynthesis, turnover and metabolism, their natural distribution and their biological function.

In all these operations, methods are needed for separation, purification and identification of different constituents present in plants. Thus advances in our understanding of phytochemistry are directly related to the successful exploitation of known techniques, and the continuing development of new techniques to solve outstanding problems as they appear.

As a result of modern extraction, isolation techniques and pharmacological testing procedure, new plant drugs usually find their way into medicine as purified substances rather than in the form galenical preparations. The precise mode of extraction naturally depends on the texture and water content of the plant material being extracted. There are two types of procedure for obtaining organic constituentsa. Cold extraction &

b. Hot extraction.

The extract obtained is then concentrated and constituents are separated by different methods such as chromatography. As a standard precaution against loss of material, concentrated extracts should be stored in the refrigerator (Ghani A, 1998).

5.2. Reagents used for the different chemical group test The following reagents were used for the different chemical group test (Ghani, 1998; Harborne, 1984).

5.2.1. i) Mayer’s reagent 1.36 gm mercuric iodide in 60 ml of water was mixed with a solution containing 5 gm of potassium iodide in 20 ml of water.

5.2.2. ii) Dragendroff’s Reagent 1.7 gm basic bismuth nitrate and 20 gm tartaric acid were dissolved in 80 ml water. This solution was mixed with a solution containing 16 gm potassium iodide and 40 ml water.

5.2.3. iii) Fehling’s solution A 34.64 gm copper sulphate was dissolved in a mixture of 0.50 ml of sulphuric acid and sufficient water to produce 500 ml.

5.2.4. iv) Fehling’s solution B 176 gm of sodium potassium tartarate and 77 gm of sodium hydroxide were dissolved in sufficient water to produce 500 ml. Equal volume of above solution were mixed at the time of use.

5.2.5. v) Benedicts Reagent 1.73 gm cupric sulphate, 1.73 gm sodium citrate and 10 gm anhydrous sodium carbonate were dissolved in water and the volume was made up to 100 ml with water.

5.2.6. Vi) Molisch Reagent 2.5 gm of pure α-naphthol was dissolved in 25 ml of ethanol.

5.2.7. Vii) Libermann-Burchard Reagent

5 ml acetic anhydride was carefully mixed under cooling with 5ml concentrated sulphuric acid. This mixture was added cautiously to 50 ml absolute ethanol with cooling.

5.3. Tests performed for identifying different chemical groups The following tests were performed for identifying different chemical groups.

5.3.1. Tests for tannins  Ferric Chloride Test 5 ml solution of the extract was taken in a test tube. Then 1 ml of 5% Ferric chloride solution was added.  Potassium dichromate test 5 ml solution of the extract was taken in a test tube. Then 1 ml of 10% Potassium dichromate solution was added.  Lead Acetate Test 1 ml of 10% Lead acetate solution was added to 5 ml of extract solution.

5.3.2. Test for Flavonoids A few drops of concentrated hydrochloric acid were added to a small amount of extract of the plant material.

5.3.3. Test for Saponins 1 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes.

5.3.4. Test for gums 5 ml solution of the extract was taken and then molisch reagent and sulphuric acid were added.

5.3.5. Tests for Steroids  Libermann-Burchard test 1 ml solution of chloroform extract was taken and then added 2 ml Libermann-Burchard reagent.  Sulphuric acid test 1 ml solution of chloroform extract was taken and then 1ml Sulphuric acid was added.

5.3.6. Tests for alkaloids

 Mayer’s test 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Mayer’s reagent was added.  Dragendroff’s test 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Dragendroff’s reagent was added.  Wagner’s test 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of iodine solution (Wagner’s reagent) was added.  Hager’s test 2 ml. solution of the extract and 0.2 ml. of dilute hydrochloric acid were taken in a test tube. Then 1 ml of picric acid solution (Hager’s reagent) was added.

5.3.7. Tests for Carbohydrates i) Benedict’s Test (Test for Reducing Sugar) 0.5 ml of aqueous extract of the plant material was taken in a test tube. 5 ml of Benedict’s solution was added to the test tube, boiled for 5 minutes and allowed to cool spontaneously. ii) Fehling’s Test (Standard Test for Reducing Sugar) 2 ml of aqueous extract of the plant material was added to 1ml of a mixture of equal volumes of Fehling’s solutions A and B, boiled for few minutes. iii) Combined Reducing Sugar Test 1 ml of aqueous extract of plant material was boiled with 2 ml of dilute HCl acid for 5 minutes. The mixture was cooled and neutralized with NaOH solution and performed the Fehling’s test as described above.

5.4. Investigations The crude extract was subjected for chemical group tests and identified for - steroids, alkaloids, tannins, gums, flavonoids, reducing sugar and saponins. Results of different group tests are given in table

5.4.1. Results of different chemical group test of the ethanol extract of Cassia fistula Linn.

Sample Tests for Alkaloids:

Test solution Observation Inference 1 ml of Mayer’s reagent. Yellowish buff colored Presence

 2 ml solution of the extract and

0.2

ml

of

precipitate was not alkaloid.

dilute

obtained.

hydrochloric acid  2 ml solution of the extract 1 ml of Dragendroff’s and

0.2

ml

hydrochloric acid.  2 ml solution

of

dilute reagent. of

observed.

Yellowish precipitate Presence

extract and 0.2 ml of dilute

was not obtained.

1 ml sulphuric acid

observed

extract.

Test for Saponins:

added

to

the was not formed.

of

of

Flavonoids.

extract Shaken in a graduated One-centimeter layer Presence

 1 ml solution of the extract cylinder for 15 minutes.

of

steroid.

Few drops of conc. HCl Immediate red color Presence

 1 ml solution of ethanol was

of

alkaloid.

Red color was not Absence

 1 ml solution of chloroform extract Test for Flavonoids:

Orange brown Presence precipitate was not alkaloid.

the 1 ml of Picric acid

hydrochloric acid.

Test for Steroids:

of

of foam was formed.

of

saponins.

was diluted with distilled water to 20 ml.

Tests for Tannins:

1 ml of 5% Ferric Greenish

 5 ml solution of extract.

chloride solution.

 5 ml solution of extract.

formed.

1 ml of 10% potassium Yellow precipitate was Presence

black Presence

precipitate

dichromate solution.

was tannins.

obtained.

1 ml of 10% lead acetate Yellow precipitate

Test for Reducing Sugars:

solution.

1 ml equal volume of Brick

 2 ml solution of aqueous Fehling’s extract.

A

and

Presence

of

tannins. colored Absence

of

B precipitate was not reducing

solution.

Boiled for 5 minutes on

found.

of

tannins.

 5 ml solution of extract.

red

of

sugars.

a boiling water bath.

Test for Gums :

Molisch

reagent

ď&#x192;&#x2DC; 5 ml solution of extract.

sulfuric

acid

and Red violet ring was Presence were produced

added.

at

of

the gums

junction of two liquids.

5.5. Results & Discussions Physiochemical studies showed that alkaloid, flavonoids, tannins, Saponin & Gum are present in the ethanol extract of the plant. The experimental findings from the study showed that the ethanol extract has some important groups and secondary metabolites that can show extensively pharmacological activity.

5.5.1. Results of chemical group tests

Tested

Alkaloids

Steroids

Saponin

Tannins

Flavonoids

groups

Reducing

Gums

Sugars

Ethanol Extract of

+

-

+

+

+

-

+

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

ReSUlTS aND DISCUSSIONS 6.1. RESULTS The effect of the ethanolic extract of Cassia fistula on the fasting blood glucose tolerance was investigated in normal and alloxan-induced diabetic mice using metformin HCl as standard ant diabetic agent.

6.1.1. Ant diabetic activity of CF extract on normal and alloxan induced diabetic mice

The blood samples were analyzed for glucose content at 0, 30 60, 90, 120 minutes, respectively. In case of alloxan induced diabetic mice metformin-HCl reduced blood glucose level was significantly higher in diabetic compared to normal mice. The blood sugar levels in mice of Group CF 250 and Group CF 500 were lowered significantly (p<0.05) were comparable to diabetic control and the effects were dose-dependent. Group CF 250 and Group CF 500 mice showed significant glucose lowering

Blood S ugar Level (mmol/l)

efficacy between 60-90 min and were comparable to diabetic standard.

Normal Control

25

Diabetic Control Metf ormin-HCl CF-250

20

CF-500

*

15

* * *

*

10

** *

5 0

0

30

60

90

120

Time in Minutes

Figure 10 : Effect of C. fistula extract on the alloxan induced diabetic mice. Data expressed in Mean Âą SEM. Each group comprised of 5 animals. Control group received 0.5% methyl cellulose and standard group received 100mg/kg Metformin. *p<0.05 compared with diabetic control.

The effect of C fistula extract in glucose induced hyperglycemia in normal mice shown in figure 2. The blood samples were analyzed for glucose content at 0, 30 60, 90, 120 minutes, respectively. The blood sugar were reduced in Group CF 250 and Group CF 500 significantly (p<0.05) and were comparable to Blood S ugar Level (mmol/l)

normal mice and the effects were dose-dependent.

20

Normal Control Metf ormin-HCl

*

15

* 10

CF-250 CF-500

* ** *

5 0

0

30

60 Tim e in Minute s

90

120

Figure 11: Effect of C. fistula extracts on the glucose-induced hyperglycemia in normal mice.

Data expressed in Mean ± SEM. Each group comprised of 5 animals. Control group received 0.5% methyl cellulose and standard group received 100 mg/kg Metformin. *p<0.05 compared with normal control.

6.1.2. Analgesic activity of CF extract on Swiss albino mice The effect of the ethanolic extract of Cassia fistula was investigated on acetic acid-induced writhing response in mice using Diclofenac-Na as reference standard drug. In writhing test the initial writhing of control mice, Group II, III and Group IV were recorded and they were found 48±2.3, 10.5±2.1, 28.34 ± 2.46, and 18.67 ± 3.53 per 20 min., respectively. The analgesic activity of extract at 200 mg/kg and 400 mg/kg dose level were reduced 42 and 63% writhing inhibitory response but diclofenac was observed 80% of that in Group II. So CF 200 and CF 400 mg/kg was found significantly (p<0.05) inhibitory response to pain induced by acetic acid when compared to control group I. Analgesic effect of the extract showed dose dependent antinociception against chemical induced pain in mice (Figure 3).

However the reference drug Diclofenac-Na was more potent than the plant extract at all dose level.

60 Control Diclofenac Na CF-200

40

CF-400

* *

20

% of writhing

* 0 Control

Diclofenac Na CF-200

CF-400

Figure 12: Effect of C. fistula extract on writhing response induced by acetic acid in mice. The results are expressed in Mean ± SEM. Each group comprised of 5 animals. * indicate significant when compared with control (p<0.05).

6.1.3. Physiochemical screening test results

Phytochemical studies showed the presence of alkaloid, flavonoids, tannins, Saponin & Gum in ethanol extract of the plant C. fistula (Table 8).

Table 8: Phytochemical test results of C. fistula stem bark extract Tested Alkaloid Group Ethanol

+

Steroid -

Saponin +

Flavonoid +

Tannin

Reducing

Gums

+

sugar -

+

Extract of C. fistula Note: + indicates the presence of the tested group, - indicates the absence of the tested group

6.2. DISCUSSION Diabetes mellitus is the worldâ&#x20AC;&#x2122;s largest growing metabolic disease characterized by high blood glucose levels due to absolute or relative deficiency of circulating insulin levels (Holmann R.R et al 1991). 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). Diabetes is a global disease with a huge adverse impact on the health and mortality. Traditional plant medicines are used throughout the world for the treatment of diabetic mellitus. The study of such medicine might offer a natural key to unlock for the future.

In the light of the literature on Cassia fistula made an attempt for the first time to study the antibiabetic and analgesic effect of ethanolic extract of bark in mice. The experiment showed that Glucose Tolerance Test (OGTT) measures the body ability to use glucose, the bodyâ&#x20AC;&#x2122;s main source of energy (Du Vigneaud 1925). This test can be used to diagnose prediabetes and diabetes. Glucose lowering effects were found after oral administration of ethanolic extracts in mice (Figure 1 and figure 2). This may be due to the presence of hypoglycemic flavonoids, triterpines or saponin glycosides that also requiring further investigation. The extracts have the properties to stimulate or regenerate the Ă&#x;-cell for the secretion of insulin and are most effective for controlling diabetes by various mechanisms which may finally lead to improvement of carbohydrate metabolizing enzymes towards the re-establishment of normal blood glucose level. Induction of diabetes with alloxan was associated with decrease in hepatic glycogen, which could be attributed to decrease in the availability of the active form of enzyme

glycogen synthesize probably because of low levels of insulin [Gold A.H 1970, Goel RK 2004]. In the present study, C. fistula restored the depressed hepatic glycogen levels possibly by increasing the level of insulin. Decreased activities of the enzymes involved in glucose homeostasis in liver and kidney such as hexokinase has been reported in diabetic animal resulting in depletion of liver and muscle glycogen content [Grover JK 2000]. Treatment with plant extracts might increase the level of enzyme to the control level indicating an over-all increase in glucose influx. The exact mechanism of action needs further investigation.

Cassia fistula has not been subjected to pharmacological investigations so far analgesic screening to provide scientific justification to its traditional claim in various pains. Therefore the present study has shown to establish remarkable analgesic potential of Cassia fistula (figure 3). 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 by the action of phospholipase A2 and other acyl hydrolaseâ&#x20AC;&#x2122;s [Ahmed F et al 2006]. The Prostaglandins mainly prostacyclin and prostaglandin-E have been reported to be responsible for pain sensation by exciting the A-fibres.

Activities in the A-fibres cause a sensation of sharp well localization pain. Any agent that lowers the number of writhing will demonstrate analgesia preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition [Brown JE 1998]. The response is thought to be mediated by peritoneal mast cells, acid sensing ion channels and the prostaglandin pathway [Voilley N 2004, Hossain MM et al 2006]. Flavonoids being powerful antioxidants [Brown JE 1998] are reported to play a role in analgesic activity by targeting prostaglandins [Rajanarayana KMS 2001]. Overall the analgesic action of Cassia fistula is assumed to be due to inhibition of prostaglandin synthesis.

The phytochemical screening of the plant C. fistula stem bark showed the presence of steroids, triterpenoids, flavonoids, glycosides, saponins and tannins. The ethanol extracts of the plant C. fistula bark exhibited significant antihyperglycemic and analgesic activity in alloxan-induced diabetic mice when compared with normal control. The activity was comparable to that of the effect produced by Metformin.

The present investigation established that the stem bark of the plant C. fistula have bioactive principles with anti-diabetic and analgesic potentials. However, further investigations were warranted to isolate bioactive compounds, to observe their effects on diabetic model and to find out the possible mechanism action for their beneficial effects both in normal and alloxan-induced diabetic mice.

CONCLUSION The present study was designed to evaluate ant diabetic and analgesic effects of ethanolic extract of Cassia fistula (CF) stem barks in mice. The analgesic effect of extract was evaluated by acetic acid induced writhing test method while ant diabetic effect was investigated by oral glucose tolerance test (OGTT) in normal and alloxan induced diabetes animal models. Diclofenac (80 mg/kg) and metformin (150 mg/kg) were used as reference drugs for comparison. The extract significantly (P<0.05) reduced blood sugar level in alloxan induced diabetic (hyperglycaemic) and glucose induced (normoglycaemic) mice at 250 mg/kg and 500 mg/kg body weight (b. wt.) of ethanolic extract respectively. The glucose tolerance results showed significant (p<0.05) improved 50.7% and 66% at the dose 250 mg/kg and 500 mg/kg body weight (b. wt.) of ethanolic extract respectively. On the Other hand, the analgesic activity of extract at 200 mg/kg and 400 mg/kg dose level were produced 42% and 63% writhing inhibitory response but diclofenac was observed 80% of that when compared to control group. The plant's extract produced dose-dependent, significant (P<0.05) analgesic effects against chemically induced nociceptive pain in mice. However a glucose tolerance hypoglycemic test is comparable to diabetic control group and effect is a dose dependent. The phytochemical screening of the plant C. fistula stem bark showed the presence of steroids, triterpenoids, flavonoids, glycosides, saponins and tannins.

The findings of this experimental animal study indicate that Cassia fistula stem-bark ethanolic extract possesses analgesic and hypoglycemic properties; and thus lend pharmacological credence to the anecdotal, folkloric, ethno medical uses of the plant in the treatment and/or management of painful, arthritic, inflammatory conditions, as well as in the management and/or control of type 2 diabetes mellitus.

We conclude that ethanolic extract of C. fistula possesses hypoglycemic and strong analgesic activity.

However, further studies are necessary to examine underlying mechanism of ant diabetic and analgesic effects and to isolate the active compound responsible for these pharmacological activities.

REFERENCES Adapted from the Report of the American Diabetes Association (ADA) Expert Committee on the diagnosis and Classification of Diabetes Mellitus. 2002, Diabetic Care 25 (Suppl. 1): S5-S20.

Agriculture Handbook 165. 1960. Index of plant diseases in the United States. USGPO. Washington.

Ahmed F, Hossain MH, Rahman AA and Sahid IZ. 2006, Antinociceptive and sedative effects of the bark of Cerbera odollam Gaetin. Ori. Pharm.

Allen, O.N. and Allen, E.K. 1981. The Leguminosae. The University of Wisconsin Press. 812 p.

Ajgaonkar SS. 48.Stamler, I. Vaccaro, O, Neaton, J.D., Wentworth, D, 1993. Diabetes, other risk factors and 12-yrs cardiovascular mortality for ment screened in the multiple risk factor intervention tria l.

Diabetes Care 15, 434-444.

Alarcon-Aguilera FG, Roman-Ramos R, Perez-Gutierrez S, Aguilar- Conteras A, Contreras-Weber cc, Flories-Saenz JL, 1998, study of the anti- hyperglycemic effect of plants used as antidiabetics. J Ethnopharmacol; 61: 101-110.

Abu Sayeed M, Abbas Ali M, Astaq Mohal Khan GRM, Rahman MS (1999). Studies on the characterization and glyceride composition of Cassia fistula seed oil. Bangladesh J. Sci. Indust. Res. 34:144-148.

Adegoke GO, Vijay Kumar M, Gopal Krishna AG, Varadaraj MC, Sambaiah K, Lokesh BR (1998).

Antioxidants and lipid oxidation infoods: A critical appraisal. J. Food Sci. & Technol. 35:283-298.

Agrawal GD, Rizvi SAI, Gupta PC, Tewari JD (1972). Structure of Fistulic acid a new colouring matter from the pods of Cassia fistula.Planta Med. 2:150-155.

Abuja A, Parshad R, Kaushik JP (1988). Anthraquinones from calluscultures of Cassia fistula. Fitoter.

LIX (49): 496-500.

Alam MM, Siddiqui MB, Hussian W (1990). Treatment of diabetes through herbal drugs in rural India.

Fitoter. 61: 240â&#x2C6;&#x2019;242.

Aruoma OI (2003). Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mut. Res. 544: 203-215.11th Edition, 281.

AmericanDiabetesAssociationATTN: NationalCallCenter1701NorthBeauregardStreet

AmericanDieteticAssociation120SouthRiversidePlazaSuite2000Chicago, Illinois60606-6995 Alloxan monohydrate Submitted by A. V. Holmgren and Wilhelm Wenner1. Checked by T. L. Cairns and R. W. Upson. (1963); Organic Syntheses, Coll. Vol. 4, p.23 Vol. 32, p.6 (1952).Link Alloxantin dehydrate R. Stuart Tipson Organic Syntheses, Coll. Vol. 4, p.25; Vol. 33, p.3 online article Anonymous (1990). Cancer pain relief and palliative care; report of a WHO expert committee. World Health Organization Technical Report Series, 804. Geneva, Switzerland: World Health Organization. pp. 1–75. ISBN 924120804X. Barnett, 2003, Diabetic Medicine, 20, 873-885 Browne, F.G. 1968. Pests and diseases of forest plantations trees.

Clarendon Press, Oxford. B. Cheatham, C. Kahn, 1995, Endocrine Reviews, 16, 117-142 Bamigbade TA, Davidson C, Langford RM, Stamford JA (September 1997). "Actions of tramadol, its enantiomers and principal metabolite, O-desmethyltramadol, on serotonin (5-HT) efflux and uptake in the rat dorsal raphe nucleus". British Journal of Anaesthesia 79 (3): 352–6. PMID 9389855.

Bailey C. J and C. Day, (1989), Diabetes Care 12, 553. Doi: 10.2337/ diacare.12.8.553.

142. J. K. Grover, S. Yadav, and V. Vats, J. Ethnopharmacol. 81, 81(2002). Doi: 10.1016/S03788741(02)00059-4.

CollinsT, Cybulsky MI.2001; NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest. 107: 255–264.

Coda, BA; Bonica, JJ (2001). "General considerations of acute pain". In Loeser, D; Bonica, Dworkin RH, Backonja` M, Rowbotham MC, et al. (2003). "Advances in neuropathic pain: diagnosis, mechanisms, and treatment recommendations". Arch. Neurol. 60 (11): 1524–34..

Driessen B, Reimann W (January 1992). "Interaction of the central analgesic, tramadol, with the uptake and release of 5-hydroxytryptamine in the rat brain in vitro". British Journal of Pharmacology 105 (1): 147–51. PMC 1908625. PMID 1596676.

David, M.N., M James. E.S. Daniel., 1997. The epidemiology of cardiovascular disease in type 2 diabetes mellitus, how sweet it is... or is it, Lancet, 350 (Suppl. 1):, S14-S19.

Duke, J.A. and Wain, K.K. 1981. Medicinal plants of the world. Computer index with more than 85,000 entries. 3 vols.

Kerala (2008): Kerala Symbols. Database on state of environment.

Du XL, Edelstein D, Dimmeler S, et al. 2001, Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. ; 108: 1341–1348.

Desco MC, Asensi M, Marquez R, et al. 2002; Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes. 51: 1118–11124.

De Man FH, Weverling-Rijnsburger AW, van der Laarse A, et al. 2000; Not acute but chronic hypertriglyceridemia is associated with impaired endothelium-dependent vasodilatation: reversal after lipid-lowering therapy by atorvastatin. Arterioscler Thrombi Vasc Biol. 20: 744–750.

Dutta NK, Annadurai S, Mazumdar K, Dastidar SG, Kristiansen JE, Molnar J, Martins M, Amaral L. (2007). "Potential management of resistant microbial infections with a novel non-antibiotic: the antiinflammatory drug diclofenac sodium". Int. J. Antimicrob. Agents 30 (3): 242–249.

Eizirik D, Pipeleers D, Ling Z, Welsh N, Hellerström C, Anderson A (1994). "Major species differences between humans and rodents in the susceptibility to pancreatic beta-cell injury". Proc Natl Acad Sci U S A 91 (20): 9253–6. doi:10.1073/pnas.91.20.9253. PMID 7937750.

Elizabeth D Agabegi; Agabegi, Steven S. (2008). Step-Up to Medicine (Step-Up Series). Hagerstwon, MD: Lippincott Williams & Wilkins. ISBN 0-7817-7153-6.

Extract on Blood Glucose Levels of Streptozocin-Induced Diabetic Wistar Rats. International Journal of Applied Research in Natural Products, Vol. 1(2), pp. 32-36.

Fowler PD, Shad forth MF, Crook PR, John VA (1983). "Plasma and synovial fluid concentrations of diclofenac sodium and its major hydroxylated metabolites during long-term treatment of rheumatoid arthritis". Eur. J. Clin. Pharmacol. 25 (3): 389–94.

Ferri C, Pittoni V, Piccoli A, et al. Insulin stimulates endothelin-1 secretion from human endothelial cells and modulates its circulating levels in vivo. J Clin Endocrinol Metab. 1995; 80: 829–835.

Gani, M. 1998. Medicinal Plants of Bangladesh: Chemical Constituents and Uses of 460 Species.

Asiatic Society of Bangladesh, Dhaka, Bangladesh.

Gobbi M, Moia M, Pirona L, et al. (September 2002). "P-Methylthioamphetamine and 1-(mchlorophenyl) piperazine, two non-neurotoxin 5-HT releasers in vivo, differ from neurotoxin amphetamine derivatives in their mode of action at 5-HT nerve endings in vitro". Journal of Neurochemistry 82 (6): 1435–43. 2004, Oxford Textbook of Palliative Medicine, 3rd ed. (Doyle D, Hanks G, Cherney I and Calman K, eds. Oxford University Press,).

Gohl, B. 1981. Tropical feeds. Feed information summaries and nutritive values. FAO Animal Production and Health Series 12. FAO, Rome.

Grover J.K., Yadav S., and Vats V. 2002, Medicinal plants of India with ant diabetic potential. J, Ethnopharmacol, 81, p, 81 Goel RK, Mahajan M.P. and Kulkarni SK. J. Pharm. 2004, Sci 7: 80.

Grover JK, Vats V. and Rathi SS. 2000, Ethnopharmacol 73(3): 461.

Hardy, Paul A. J. (1997). Chronic pain management: the essentials. U.K.: Greenwich Medical Media.

Hinz,B.; Cheremina, O.; Brune, K. (2008). "Acetaminophen (paracetamol) is a selective cyclooxygenase-2 inhibitor in man.". The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 22(2): 383–390

Hinz,B.;Cheremina,O.;Brune, K. (2008). "Acetaminophen (paracetamol) is a selective cyclooxygenase2 inhibitor in man.". The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 22(2): 383–390.

Hartwell, J.L. 1967-1971. Plants used against cancer. A survey. Lloydia 30-34.

Holmann R.R., Turner R.C. 1991 ‘Textbook of diabetes’ Oxford: Blackwell, 142. http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/index.html Hortus Third. 1976. A concise dictionary of plants cultivated in the United States and Canada.

MacMillan Publishing Co., Inc., New York.

HI-Plants of Hawaii (HEAR) IntermountainHealthCareIHCDiabetesManagementProgram Phone:1-800-442-5305 www.ihc.com/diabetes International Diabetes Federation International Legume Database & Information Service (ILDIS) (2005): Genus Cassia. Version 10.01, November 2005. Retrieved 2007-DEC-20.

JJ. Bonica's management of pain (3 ed.). Philadelphia: Lippincott Williams & Wilkins Jackson TS, Xu A, Vita JA, et al. Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations. Circ Res. 1998; 83: 916–922.

J. Brange, 1997, Diabetologia, 40, S48-S53 J. Goldman-Levine, K. Lee, 2005, the Annals of Pharmacotherapy, 39, 502-507 Keay, KA; Clement, CI; Bandler, R (2000). "The neuroanatomy of cardiac nociceptive pathways" In Horst, GJT. The nervous system and the heart. Totowa, New Jersey: Humana Press. p. 304.

Kuhn FE, Mohler ER, Satler LF, et al. 1991; Effects of high-density lipoprotein on acetylcholine-induced coronary vasoreactivity. Am J Cardiol. 68: 1425–1430.

Kirtikar, K.R. and Basu, B.D. 1975. Indian medicinal plants. 4 vols. 2nd ed. Jayyed Press, New Delhi.

King GL1996; The role of hyperglycaemia and hyperinsulinaemia in causing vascular dysfunction in diabetes. Ann Med. 28: 427–432.

King H. Abubert RE, Herman WH., 1998. Global burden of diabetes 1995-2025, prevalence, numerical estimates and projection. Diabetes care 21:1414-31.

Karanth KS, Duttaroy D, Yelur SV, Jain AP (September 2006). "Colorectal foreign body—a case report". J Indian Med Assoc 104 (9): 528, 530. PMID 17388014.

Lambert, J., Srivastava, J. & Vietmeyer, N. (1997). Medicinal plants: rescuing a global heritage. The World Bank, Washington, D.C., USA.

Lenzen, S: 2008, the mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 51, 216- 226, (Review) Lin KY, Ito A, Asagami T, et al. 2002; impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation. 106: 987– 992.

Lambert, P (2002). "What is Type1 Diabetes?". Medicine 30: 1–5. Doi: 10.1383/medc.30.1.1.28264.

Luzi, L., 1998. Pancreas transplantation and diabetic complications. New England Journal of Medicine, 339: 115-117.

MMPND (2005) Multilingual Multiscript Plant Name Database (MMPND) (2005): Cassia fistula L.. Version of 12/01/2005. Retrieved 2007-DEC-20.

Mrozikiewicz, D. Kielstrokczewska-Mrozikiewicz, Z. Lstrokowicki, E. Chmara, K. Korzeniowska and p.

M. Mrozikiewicz: 1994 Blood levels of alloxan in children with insulin-dependent diabetes mellitus. Acta Diabetologica 31, 236-237, (Rapid report)

M. Dunn, BioMetals 2005, 18, 295-303 Mohamed AK, Bierhaus A, Schiekofer S, et al. 1999; The role of oxidative stress and NF-kappa activation in late diabetic complications. Biofactors. 10: 157–167.

Mike Harlos MD, CCFP, FCFP Professor and Section Head, Palliative Medicine, University of Manitoba Medical Director, WRHA Palliative Care Medical Director, Pediatric Symptom Management Service Mazumdar K, Dutta NK, Dastidar SG, Motohashi N, Shirataki Y (2006). "Diclofenac in the management of E. coli urinary tract infections". In Vivo 20 (5): 613–619.

Main, Chris J.; Spanswick, Chris C. (2000). Pain management: an interdisciplinary approach. Churchill Livingstone. ISBN 0 443 05683 8.

Nalawadi, U.G., Bhandary, K.R., and Chandrashekar, T. 1977. Germination of Cassia fistula (Linn.) seeds could be improved by treatment with sulphuric acid for 20 Minutes. Current Research. Hort.

Abstr. 46. 3645:1975. 4: 3: 42-43.

Nomura S, Shouzu A, Omoto S, et al. 2000; Significance of chemokines and activated platelets in patients with diabetes. Clin Exp Immunol. 121: 437–443.

NationalDiabetesInformationClearinghouse1InformationWayBethesda,MD20892-3560 Phone:1-800-860-8747 www.diabetes.niddk.nih.gov Nathan DM, Cleary PA, Backlund JY, et al. (December 2005). "Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes". The New England Journal of Medicine 353 (25):

2643–53.

doi:10.1056/NEJMoa052187.

PMC 2637991.

PMID 16371630.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2637991.

Oliver FJ, de la Rubia G, Feener EP, et al. 1991;Stimulation of endothelin-1 gene expression by insulin in endothelial cells. J Biol Chem. 266: 23251–23256.

Perry, L.M. 1980. Medicinal plants of east and Southeast Asia. MIT Press, Cambridge Rajshahi Diabetic Association, 19 th Annual General Meeting; 2005.

Reimann W, Schneider F (May 1998). "Induction of 5-hydroxytryptamine release by tramadol, fenfluramine and reserpine". European Journal of Pharmacology 349 (2-3): 199–203. doi:10.1016/S0014-2999(98)00195-2. PMID 9671098.

Roskoski, J.P., Gonzalez, G.C., Dias, M.I.F., Tejeda, E.P., and Vargas-Mena y Amezcua. 1980.

Shamshad, R. 2004. Role of Youth in the Conservation of Traditional Medicinal Plants. Knowledge Marketplace reports of the 3rd IUCN World Conservation Congress, Bangkok, Thailand 17-25 November, 2004, pp.

Scartezzini, P. and Sproni, E., 2000. Review on some plants of Indian traditional medicine with antioxidant activity, J Ethnopharmacol, 71, 23-43.

S. Taylor, A. Cama, D. Accili, F. Barbetti, M. Quon, M. De La Luz Sierra, Y. Suzuki, E. Koller, R. LevyToledano, E. Wertheimer, V. Moncada, H. Kadowaki, T. Kadowaki, 1992, Endocrine Reviews, 13, 566595

Tanko Y., et al., 2008. Hypoglycemic Activity of Methanolic Stem Bark of Adansonnia digitations World Health Organization 2002. Diabetes Mellitus fact sheet 138. Geneva.

Timimi FK, Ting HH, Haley EA, et al. 1998; Vitamin C improves endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 31: 552–557.

"The effect of intensive diabetes therapy on the development and progression of neuropathy. The Diabetes Control and Complications Trial Research Group". Annals of Internal Medicine 122 (8): 561– 8.April1995.doi:10.1059/0003-4819-122-8-199504150-00001(inactive2009-10-31).PMID 7887548. http://www.annals.org/cgi/pmidlookup?view=long&pmid=7887548.

Tropical plant Database

Tagliazucchi E, Balenzuela P, Fraiman D, Chialvo DR (2010). "Brain resting state is disrupted in chronic back pain patients.". Neurosci Lett 485 (1): 26–31.

Tyrberg B, Anderson A, Borg L (2001). "Species differences in susceptibility of transplanted and cultured pancreatic islets to the beta-cell toxin alloxan". Gen Comp Endocrinol 122 (3): 238–51. doi:10.1006/gcen.2001.7638. PMID 11356036.

Tropical Plant Database Tesfamariam B, Brown ML, Cohen RA. 1991; Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. J Clin Invest. 87: 1643–1648.

Thienhaus, Ole; Cole, B. Eliot (2002). "The classification of pain". In Weiner, Richard S,. Pain management: A practical guide for clinicians. CRC Press. p. 29. ISBN 0849309263.

T. Levien, D. Baker, J. White Jr., R. Campbell, 2002, The Annals of Pharmacotherapy, 36, 10191027http://en.wikipedia.org/wiki/Diabetes

Uniyal, S. K., Awasthi, A. & Rawat, G. S. (2002). Current status and distribution of commercially exploited medicinal and aromatic plants in upper Gori valley, Kumaon Himalaya, Uttaranchal. Current Science 82, 1246-1252.

U. S. Department of Agriculture, William Saunders; June 5, 1891, Catalogue of Economic Plants in the Collection of the U. S. Department of Agriculture; Washington D. C.; ILDIS (2005) U. Derewenda, Z. Derewenda, E. Dodson, G. Dodson, X. Bing, J. Markussen, 1991, Journal of Molecular Biology, 220, 425-433 Utta NK, Annadurai S, Mazumdar K, Dastidar SG, Kristiansen JE, Molnar J, Martins M, Amaral L (2000). "The anti-bacterial action of diclofenac shown by inhibition of DNA synthesis". Int. J. Antimicrob.

Agents 14 (3): 249–51.

Vinik AI, Erbas T, Park TS, et al. Diabetes Care. 2001; Platelet dysfunction in type 2 diabetes. 24: 1476–1485. von Baeyer CL; Pain Research and Management 11(3) 2006; p.157-162 Vet drug 'killing Asian vultures'", BBC News, 28 January 2004, webpage: BBC583. www.ildis.org Voilley N. 2004, cids-Sensing Ion channels (ASICs): New target for the analgesic effects of Non Steroidal Anti-Inflammatory Drugs (NSAIDs). Curr. Drug Targets Inflam. Allerg. 3:71-79. wikimedia.org/wiki/Cassia fistula. Author index .org/cassia. wareseeker.com/free-cassia-fistula/.

Woody tropical legumes: potential sources of forage, firewood, and soil enrichment. p. 135-155. In: SERI: Tree crops for energy co-production on farms. SERI/CP-622-1086. USGPO. Washington.

W. Duckworth, R. Bennet, F. Hamel, 1998, Endocrine Reviews, 19, 608-624 Z. Vajo, J. Fawcett, W. Duckworth, 2001, Endocrine Reviews, , 22, 706-717 Zeiher AM, Fisslthaler B, Schray-Utz B, 1995, Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ Res.; 76: 980–986.

Libby P. 2000 Changing concepts of atherogenesis. J Intern Med.; 247: 349–358.