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Dr. Jayeshree Daoo * Pranali Panchal, **
* Research Guide, Principal, ICLES Jhunjhunwala College, Vashi, Navi Mumbai- India **Research Student Department Of Zoology, G.N. Khalsa College, Mautnga, Mumbai 17,
Abstract The methanol extract of leaves of Moringa oleifera was evaluated for its antidiabetic efficacy in streptozotocin induced diabetic rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin ( 45 mg/kg body weight). Methanol extract administered orally at dose of 300 mg/kg body weight for 35 days in diabetic rats. Blood glucose level, liver glycogen, muscle glycogen, insulin as well as initial and final changes in body weight were evaluated. Experimental studies showed significant antidiabetic efficacy of extract in case of reduction of blood glucose level in diabetic rats. Significant improvement was observed in liver and muscle glycogen , insulin level and body weight profiles in methanol extract treated diabetic rats . The effect of the extract at 300 mg/kg body weight was comparable to that of standard drug glibenclamide ( 5 mg/kg body weight). Key words : Streptozotocin, antidiabetic, Moringa oleifera, glibenclamide
Introduction Diabetes is a complex multisystemic disorder characterized by a relative or absolute insufficiency of insulin secretion and disturbances in carbohydrate, protein, lipid metabolism. [1,2] It is an insidious disease. Although the prevalence of diabetes is increasing, diabetes is not homogenously distributed throughout the population.[3] Diabetes means siphon and mellitus stands for sweet. Chronic hyperglycemia during diabetes causes gyration of body proteins that in turn leads to secondary complications affecting eyes (retinopathy),kidneys (nephropathy), nerves (neuropathy) and arteries (atherosclerotic vascular disease).The International Diabetes Federation has predicted that the number of indivisuals with diabetes will increase from 240 million in 2007 to 380 million in 2025 with 80% of the disease burden in low and middle-income countries.[4] It also estimated that the number of diabetic patients in India more than doubled from 19 million in 1995 to 40.9 million in 2007 and is projected to increase to 69.9 million by 2025. So India is the diabetes capital of the world. According to recent estimation, the global population is approaching the midst of diabetes pandemic. Although various oral hypoglycemic drugs exist alongside insulin, still there is no promising therapy to cure diabetes. On the other hand from time immemorial, man depended on plants as medicine. From historical perspective, it is evident that the fascination for plants is as old as mankind itself. The plant kingdom represents a rich storehouse of organic compounds, many of which have been used for medicinal purposes and could serve as lead for the development of novel agents having good efficacy in various pathological disorders in the coming years.[5] Many of traditional medicinal plants have been used successfully since ancient times to treat diabetes and related complications because plants have been the major source of drugs for the treatment of diabetes mellitus in Indian system of medicine and other ancient systems in the world, though their biologically active compounds are unknown. [6] Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes due to their effectiveness, less side effects and relatively low cost. [7] Botanical products can improve glucose metabolism and the overall condition of individuals with diabetes not only by hypoglycemic effects but also by improving lipid metabolism, antioxidant status and capillary function. [8] Recently numerous traditional medicinal plants were tested for their antidiabetic potential in the experimental animals. In the present investigation is directed to the exploration of antidiabetic efficacy of the methanol extract of leaves of Moringa oleifera . Moringa oleifera is the most widely cultivated species of a monogeneric family, the moringaceae and the most nutrient rich plant on the earth. Three non-governmental organizations in particular- Trees for Life, Church World Service and Educational Concerns for hunger Orgnization- have advocated Moringa as natural nutrition for the tropics. [9] Moringa oleifera is mentioned in the Shushruta Sanhita which was written in the beginning of the first century A.D. there is evidence that the cultivation of this tree in India dates back many thousands of years and all over world due to high protein, vitamins, carbohydrate content of entire plant ,high value of nutrition for both humans and live stock. [10,11] It is also called Drumstick Tree or Horse Radish Tree.It is soft wood tree, short slender, deciduos, perennial tree with drooping branches, corky bark, leaves feathery pale green, compound, tripinnate. All parts are used in Ayurvedic system of medicine. [12] The plant was reported to contain various amino acids, fatty acids, vitamins and its constituents such as leaf, flower, fruit and bark have been anecdotally used as herbal medicine in the treatment for inflammation, paralysis and hypertension, psychosis, eye diseases, fever and as an aphrodisiac. It is highly potent anti-inflammatory, hepatoprotective, antihyperytensive and anti-tumor [13,14] which for centuries has been advocated for traditional and industrial uses. All parts of the Moringa tree are edible and have long been consumed by humans. It is mothers best friend because it increases womans milk production. It is reported to elicit good clinical response in children suffering from upper respiratory tract infection .[15] Leaves can be eaten fresh,cooked, refrigeration, and reportedly without loss of nutritional value. [9] Leaves have been reported to be a rich source of beta carotene, protein, amino acid, vitamin C, calcium and potassium. [12] This makes Moringa leaves an ideal, safe and natural treatment with wide medicinal uses such as cleaning the body from toxins, strengthening the immune system, support to Gout and Arthritis supporting treatment and much more. Leaves contain- 7 types of vitamins, 6 types of minerals, 18 types of Amino Acids, 46 types of anti-oxidants. Leaves can help maintain normal blood sugar level. The leaves and fruits are found to have hypocholesterolaemic activity in Wistar rats and rabbits. [16]
MATERIALS AND METHODS Plant Material Leaves of Moringa oleifera were collected in the month of February 2009 from Bhabha Atomic Research Centre campus, Mumbai, Maharashtra, India. The plant was authenticated by Dr. Harshad Pandit, Head Dept. Botany, G.N. Khalsa College, Matunga, Mumbai, India. A voucher specimen no. is PZ/1503b.
Preparation of Methanol Extract Methanol extract of Moringa leaves was prepared by Soxhlet method with 50 gm powder + 300 ml methanol. Leaves of Moringa oleifera were shade dried, the leaf powder was prepared using home grinder no. IS: 4250CM/L-7480681 Orange Company. The extract was stored in desicator for use in subsequent experiment. Animals Healthy Wistar strain male albino rats weighing 240-280 gms and 10-12 weeks old were used for the study. Animals were procured from Haffkines Biopharmaceuticals Private Limited (HBPL), Parel, Mumbai. The animals were acclimatized to laboratory conditions for 7 days prior to experiment. They were housed in polypropylene cages at an ambient temperature of 25-30 C with a 12 hour each of dark and light cycle in the animal room. The rats were fed with standard pellet diet ( Amrut Feed) and water ad libitum. The principles of Laboratory Animals Care were followed and instructions given by institutional animal ethical committee (525/02/a/CPCSEA) were followed throughout the experiment. Six rats were used for all studies in each group.
Chemicals Streptozotocin, a most widely used chemical diabetogen was purchased from SRL Chemicals, Mumbai, India. All reagents used in the experiment were of analytical grade. Glibenclamide a standard antidiabetic agent was procured from chemist, Mumbai.
Induction of diabetes with STZ Diabetes was induced in overnight fasted Wistar strain male albino rats weighing 240-280 gms by intraperitoneal injection of freshly prepared STZ in 0.1 M citrate buffer pH=4.5 [13] ( 45 mg/kg body weight). Fasting blood glucose level was estimated at the time of induction of diabetes and post prandinal glucose (PPG) was checked regularly each alternate day till 35 days and showed stable blood sugar level they were used for antidiabetic study.
Experimental Design Aniamls were divided into four groups of six rats each. They are i) Normal group ii) Test (Moringa lvs extract) group iii)Standard group (glibenclamide) iv) Diabetic control group. Normal group received only distilled water. Test group was administered methanol extract of Moringa oleifera leaves at dose of 300mg/kg body weight(once a day daily) by oral route. Standard group was treated with standard drug glibenclamide at an oral dose of 5 mg/kg body weight. Diabetic control group was treated with only vehicle 0.5% CMC solution at 1ml/kg once a day daily. [18] The blood sugar level(mg/dL) was estimated every alternate day. Liver and muscle glycogen determined by Amyloglucosibase. [ 19 ] and insulin was evaluated by Chemiluminescence method. Initial and final body weight was also recorded. [20]
RESULTS Acute Toxicity Studies The albino swiss mice were treated with Moringa oleifera for a period of 14 days for toxicity study. The toxicity studies revealed nontoxic nature of the Moringa oleifera at a concentration of 2 and 5gm/kg of body weight/day for a period of 14 days. There was no morphological changes like condition of fur, skin damage, subcutaneous swelling,eye dullness, pupil diameter, ptosis, condition of teeth, breathing distress, gait, weight loss, etc. There was no lethality or any toxic reactions found at either doses selected till the end of treatment period. Body Weight The changes in initial and final body weight are listed in Table 2. observed data indicates significant improvement of body weight profile ( P< 0.05 at 300 mg/kg body weight) in extract and glibenclamide treated diabetic rats with respect to diabetic control group. Fig 1: Comparison of blood glucose of normal, diabetic, std drug treated and Moringa oleifera treated rats
Table 1: Effect of methanol extract of Moringa oleifera leaves on liver glycogen, Muscle glycogen , Insulin of STZ- induced diabetic rats
Group Liver Glycogen Muscle Glycogen Insulin Normal Control 51.940.88 9.60.31 16.460.17 Diabetic Control 18.450.33 1.860.12 5.330.21 Diabetic+ MOME 300 mg/kg 45.180.79 8. 430.33 14.00.28 Diabetic + Std Drug 5mg/kg 46.190.14 9.50.33 12.460.15
Table 2: Effect of methanol extract of leaves of Moringa oleifera on body weight profile of STZ-induced diabetic rats Group Initial Body weight ( Gram) Final Body weight (Gram) Normal control 236.6625.23 2855.47 Diabetic control 241.6622.06 134.164.91 Daibetic+MOME 300 mg/kg 237.520.91 207.58.21 Daibetic +Std drug 5 mg/kg 235.8319.08 235.0018.7
Biochemical Parameters 1)Blood Glucose : Blood glucose levels in rats of different groups are shown in Fig 1. The effect of methanol extract of Moringa oleifera leaves on blood glucose level of STZ induced diabetic rats was observed. The glucose level was significantly high in diabetic control group. On the other hand , the level of blood glucose was significantly decreased in treated groups ( Moringa extract and Glibenclamide) compared with normal and control groups.
2)Glycogen: The liver glycogen, muscle glycogen of control and experimental groups of rats were evaluate after the termination of experiment i.e 35 th day and represented in Table 1 .The liver ,muscle glycogen significantly decreased in diabetic group , when compared to normal control group, and significantly increased after the treatment of Moringa oleifera and std. drug glibenclamide, compared to diabetic group. 3)Insulin: Insulin level of control and experimental groups of rats was estimated and presented in Table 1. The insulin level was significantly decreased in diabetic group when compared with normal group. Treatment with methanol extract of Moringa oleifera and standard drug glibenclamide significantly increased the level of insulin when compared with diabetic group.
DISCUSSION The STZ is a broad-spectrum antibiotic, glucosamine-nitrosourea compound extracted from Streptomyces acromogenes. [21] It is toxic to the insulin-producing beta cells of the pancreas in mammals. STZ induces sever and irreversible hyperglycemia in experimental animal. [22] So it is suggested the drug as an animal model of diabetes. Rats with STZ-induced diabetes have reduced body weight, hyperglycemia, and hypoinslinemia because of damaged insulin-secreting cells in pancreatic islets. [23] The body weight of STZ-induced diabetic male wistar rats were decreased and recovered after the treatment. In our study the body weight was increased in Moringa oleifera and glibenclamide treated diabetic rats. The toxicity studies revealed nontoxic nature of the Moringa oleifera. The treatment of anti-diabetic medicinal plant extract to the STZ-induced male albino wistar rats, that activated the cells return to normal. The glibenclamide is a standard antidiabetic drug, used to compare the antihyperglycemic property in experimental rats. Glibenclamide stimulate insulin secretion from pancreatic cells by inhibiting ATP-sensitive potassium channels in pancreatic cell membrane. [21] The blood glucose level increases in diabetic rat. [25] An extract from the Moringa leaf has been shown to effective in lowering blood sugar levels within 3 hrs. ingestion, though less effectively than the standard hypoglycemic drug, glibenclamide. [26] In our study glibenclamide and Moringa oleifera treated diabetic rats by STZ induction showed decrease in blood glucose level. Simultaneously it was observed increased plasma insulin level . Insulin was increased in Moringa oleifera treated and glibenclamide treated diabetic rats. The glycogen content is decreases in liver and muscle of diabetic rats [25] and recovered ( increased) significantly in Moringa oleifera treated diabetic rats and glibenclamide treated diabetic rats.
STATASTICAL ANALYSIS Data were stastically calculated by utilizing one-way ANOVA and expressed as S.E.M..The significance of the difference between the means of test and control studies was established by Dunnetts t-test. P values less than 0.05 were considered significant. CONCLUSION The administration of Moringa oleifera is a significant restoration of the blood glucose, liver and muscle glycogen and insulin. The result of this investigation revealed that methanol extract of leaves of Moringa oleifera possesses significant antidiabetic activity in STZ-induced diabetic rats in dose dependent manner. The Moringa oleifera enhance the glycolytic enzymes and controls the glucose metabolism in the liver tissues of STZ- induced diabetic rats that leads to normoglycemic . The extract restore body weight also.
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