Combined Effect of Trigonella Foenum Graceum Seed Extract with Oral Hypoglycemics in Diabetic

“COMBINED EFFECT OF TRIGONELLA FOENUM GRACEUM SEED EXTRACT WITH ORAL HYPOGLYCEMICS IN DIABETIC NEUROPATHIC PAIN”. a) BRIEF RESUME OF THE INTENDED WORK: Need of study: Hyperglycemia is the pathophysiologic mechanism responsible for type1 and type2 diabetes and persistent hyperglycemia in diabetics despite appropriate therapeutic measures lead to the development of secondary complication like diabetic neuropathy1. Diabetic neuropathy is the most common complication reported in more than 50% of diabetic type2 patients. Improving glycemic control reduces incidence of type2 diabetes2. Pain associated with diabetic neuropathy can occur spontaneously or as a result of exposure to mild painful stimuli (hyperalgesia) or to stimuli not normally perceived as painful (allodynia). Brown and co-workers identified patients with diabetic sensory neuropathy had distal burning pain in extremities3. Several pathways contribute to the development of diabetic neuropathy. Oxidative stress has been suggested as a major contributor to the diabetic neuropathy4. Inflammatory process with severe nerve ischemic lesion seems responsible for severe form of peripheral diabetic neuropathy. Thickening or hyalinisation of walls of small blood vessels and duplication of basal lamina around endothelial suggests a role for nerve ischemia5. Several mechanisms including are auto oxidative glycosylation, formation of advanced glycation end products and increased oxidative stress6. Quercetin, a natural antioxidant improves the neuronal blood flow through reactive oxygen species scavenging or by its direct vasorelaxant properties. Any agent which can scavenge reactive oxygen species can be used as powerful analgesic drug for neuropathic pain7. Neuropathic pain is refractory to treatment with conventional analgesics such as opiates and non-steroidal anti-inflammatory drugs8-9 as well as antidepressant agents10. Hypersensitivity to noxious stimulus is due to impaired blood flow and thus vasodilator treatment has been demonstrated to reduce allodynia in diabetic rats11. It has been reported that the analgesic effect of Trigonella foenum graecum (Fenugreek) on tail-flick test may be due to the presence of flavonoids. Trigonella foenum graecum seed powder has also been reported to possess antinociceptive activity in diabetic neuropathy12. Recent studies have reported the antinociceptive property of Emblica officinalis Gaertan (Amla) in high fat diet-fed/low dose streptozotocin induced diabetic neuropathy in rats. It was reported that the curative and preventive property of amla extract in diabetic neuropathy may be due to its improvement in glucose intolerance and antioxidant property13. Oral hypoglycemics is been used frequently in the treatment of type2 diabetic patients. To attain adequate glucose control combined pharmacological therapy is used and so drug-drug interactions must be carefully considered when herb is added as an adjuvant to oral hypoglycemic agents. Glucose-lowering effects of various combinations of oral hypoglycemic agents have been reported14. Apart from combining two or more oral hypoglycemic agents, physicians also recommend use of anti-diabetic herbal drugs along with oral hypoglycemic which may lead to hypoglycemia and has to be monitored. Hence the present study is necessary to evaluate the role of Trigonella foenum graecum seed extract in combination with oral hypoglycemic agents (low dose) in the treatment of peripheral diabetic neuropathic pain in rats as well as to avoid hypoglycemia .

Review of Literature: The flavonoids may bind directly to the opiate receptors or like opiates, they may inhibit neuronal transmitter receptors from activating pain neurons. Several mechanisms have been postulated for the protective effect of antioxidants in diabetic neuropathy. Antinociceptive activity of quercetin is probably through modulation of opioidergic mechanism15. Trigonella foenum graecum seeds possess significant hypoglycemic16 and antiatherosclerotic17 activity. Different flavonoids, namely, vitexin, tricin, naringenin, quercetin, and tricin-7-O-β-D-glucopyranoside, are reported to be present in fenugreek seeds18. Anti-oxidant activity of fenugreek is also been reported19. The analgesic effect of Trigonella foenum graecum on tail-flick test that is usually not produced by NSAIDs, suggest another mechanism for the extract. The presence of effective compounds such as flavonoids may be responsible for the mechanism involved in its analgesic activity on tail flick test20. 5- HT mediated spinal antinociception has also been shown to involve μ-opioid receptors21. Spinal 5-HT system is partially involved in the analgesia induced by Trigonella foenum-graecum extract (Leaf) in the second phase of formalin test and also indicates for co-existence of other analgesic mechanism22. Fenugreek seed shows the galoctogogue action23. 4-hydroxy isoleucine is a novel amino acid present which increase glucose stimulated insulin release by islet cells in both rats and humans24. It also contains saponins, alkaloids like trigonelline gentianine etc. It acts as an antidiabetic by delaying the gastric emptying, slow carbohydrate metabolism, improving peripheral glucose utilization25. Objective of study: The objective of the present study is to evaluate the antinociceptive action of Fenugreek seed extract and to delineate its possible adjuvant in diabetic neuropathic pain therapy. SPECIFIC OBJECTIVES: Primary outcome  To study the role of fenugreek seed extract in diabetic neuropathic pain.  To study whether oral hypoglycemics can play a vital role in diabetic neuropathic pain.  To study the effect of the fenugreek seed extract in combination with standard hypoglycemic agents in diabetic neuropathic pain.  To study the effect of fenugreek seed extract alone and in combination with oral hypoglycemic agents on antioxidant status. Secondary outcome To study the effect of fenugreek seed extract alone and in combination with oral hypoglycemic agents on structural integrity. b) MATERIALS AND METHODS: Source of Data: Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Krupanidhi College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and Journals. Method of Collection of Data: Fenugreek seed extraction26: Soxhlet apparatus is used for the extraction of fenugreek seeds to obtain polyphenolic rich extract. EXPERIMENTAL MODELS: 1. Oral glucose tolerance test27 : The oral glucose tolerance test will be performed on overnight fasted Sprague Dawley rats.Rats will be divided into different groups as follows: Group 1: Normal control, rats receive saline/vehicle. Group 2: Diabetic control. Group 3: Trigonella foenum graecum (200mg/kg). Group 4: Pioglitazone (10 mg/kg). Group 5: Glipizide (5mg/kg). Group 6: Acarbose (5mg/kg). Glucose will be fed 30 min and 1hr after the administration of extracts. Blood will be withdrawn from the retro orbital sinus under ether inhalation at 0, 30, 60, 90 and 120 min of glucose administration. Glucose levels will be estimated. 2. Single and multiple dose study in normal and diabetic rats.28 3. Antioxidant activity-Invitro and invivo29. 4. Development of high fat diet-fed/low dose streptozotocin treated type2 diabetic rats13. The animals were fed high fat diet(HFD),once a day for 2 weeks followed by i.p injection of streptozotocin(35mg/kg) dissolved in 1M/l citrate buffer (pH4.4) after overnight fasting. The rats with non fasting plasma glucose level of >300 mg/dl were considered diabetic. Blood sample was collected from tail vein and glucose was measured using glucose diagnostic kit (Accucheck, India).

5. Effect of fenugreek seeds extract alone and in combination with oral hypoglycemic agents in streptozotocin induced diabetic rats : Group 1: Normal control, rats receive saline/vehicle. Group 2: Diabetic control. Group 3: Diabetic rats treated with Trigonella foenum graecum seed extract30. Group 4: Diabetic rats treated with Pioglitazone31. Group 5: Diabetic rats treated with glipizide 32. Group 6: Diabetic rats treated with acarbose 33. Group 7: Diabetic rats treated with pioglitazone plus glipizide. Group 8: Diabetic rats treated with glipizide plus acarbose. Group 9: Diabetic rats treated with Trigonella foenum graecum seed extract plus pioglitazone (low dose). Group10: Diabetic rats treated with Trigonella foenum graecum seed extract plus glipizide(low dose) Group11: Diabetic rats with Trigonella foenum graecum seed extract plus acarbose (low dose) At the end of the treatment period the rats will be sacrificed by decapitation and the sciatic nerve will be excised and histopathological studies will be carried out. Lipid peroxidation, TBARS, superoxide dismutase (SOD), catalase and the reduced form of glutathione (GSH) will also be estimated from the sciatic nerve homogenate.

6. TREATMENT PROTOCOL A. Induction and assessment of diabetic induced neuropathy in rats. B. The following diabetic neuropathic animal models34 will be used to evaluate the antinociceptive effect of fenugreek seed extract. 1. Tail immersion (warm water) test. 2. Hot plate test. 3. Cold allodynia testing . 4. Formaldehyde solution testing.

Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly: YES Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animal model. Has ethical clearance been obtained from your institute Ethical Committee approval letter is enclosed

c) List of References:

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