Journal of Innovations in Pharmaceuticals and Biological Sciences JIPBS www.jipbs.com Original Article Combined effect of Coccinia indica leaf extract with acarbose in type II diabetes induced neuropathy in rats Sanket Kohli*, Prem N. Kumar Department of Pharmacology, Krupanidhi College of Pharmacy, Banglore-560035. Abstract Uncontrolled diabetic can leads to various acute and chronic complications such as neuropathy, nephropathy, gastropathy, retinopathy etc. Combination of Coccinia indica with low dose of acarbose can be administered which would be useful in diabetic neuropathic pain by restoring blood glucose level and antioxidant status. Low dose of acarbose is given to avoid the hypoglycemic effect. Adult male Spraque Dawley rats weighing 200-250gm were selected for the study. They are divided into 6 groups with 6 animals each. Type 2 diabetes was induced by two weeks of HFD treatment followed by low dose 35mg/kg of STZ dissolved in 0.1M/l of citrate buffer through i.p route. Blood glucose estimation is done on every 15 days once. Single and multiple dose studies of ethanolic extract of Coccinia indica and acarbose was performed to assess the antihyperglycemic activity. Diabetic rats were treated with acarbose, quercetin, coccinia indica and its combination with acarbose for period of 7 weeks. Eddy’s hot plate test and tail immersion test were performed to assess the antinociceptive activity of the drugs. At the end of the treatment antioxidant status in the sciatic nerve was performed to find out the antioxidant status. Diabetic rats treated with Coccinia indica alone and in combination with low dose of acarbose produced significant decrease in the blood glucose level after 7 weeks of treatment. Untreated diabetic rats showed significant hypersensitivity towards thermal stimuli when compared with normal control. Histopathological studies proved that there is no damage in the sciatic nerve of the groups treated with the ethanolic extract of coccinia indica and its combination with low dose of acarbose. Key words : Diabetic neuropathy, ethanolic extract of coccinia indica leaf, Hyperalgesia, Sciatic nerve, nociception *Corresponding Author: Sanket Kohli, Department of Pharmacology, Krupanidhi College of Pharmacy, Bangalore- 560035. Mobile No. : 9739844120. Email : [email protected] 1. Introduction DIABETES is a major health diabetes. It is a group of diseases problem today, as approximately 5% of characterized by high blood glucose level, the world’s population suffers from which results from defects in Insulin ©JIPBS, All rights reserved Innovational Publishers www.innovationalpublishers.com Sanket Kohli, JIPBS, Vol 1 (2), 77-87, 2014 Secretion or action or both.[1] Pre- mitochondrial membrane potential is diabetes’ or better called ‘potential associated with induction of reactive diabetes’ is a condition that occurs when a oxygen species (ROS). [6]. It has been person’s blood glucose levels are higher documented that the degree and duration than normal but not high enough for a of hyperglycaemia are primary risk factors diagnosis of type 2 diabetes. The for diabetic neuropathy but at present, the development of peripheral neuropathy is therapeutic strategy available is strict the most common complication of Type 2 glycemic control.[7] The strong glycemic DM and occurs in 50% of the subjects control reduces the incidence of micro based on epidemiological data. [2] Diabetic vascular complication.[8]. Hence in the neuropathy occurs through diverse present study Coccinia indica and its pathogenic mechanisms most of them combination with low dose Acarbose are initiated by hyperglycaemia and oxidative used to obtain adequate glucose control. stress.[3] Coccinia indica commonly known as Little gourd or Rantondli in Marathi, Bimba in Sanskrit and Kandutikibel in Hindi belonging to family Cucurbitaceae It is indigenous to Bengal and other parts of India.Also grows abundantly all over India, Tropical Africa, Australia, Fiji and throughout the oriental countries. The plant has also been used extensively in Ayurvedic and Unani practice in the Indian subcontinent (Wealth of India, 1992). Hypoglycaemic action of Coccinia indica could be due to Figure No.1 Coccinia Indica Plant potentiating the insulin effect of plasma by 2. Material and Methodology increasing the pancreatic secretion of insulin from the existing β-cells.[4] Experimental Animals: Coccinia indica is also believed to bring Male Sprague Dawley Rats weighing about its antidiabetic action by stimulating 200-250 g were housed at 25° ± 5°C in a glucose transport.[5] Blood glucose well-ventilated animal house under 12:12 lowering activity was may be due to the h light dark cycle. Institutional Animal inhibition of intestinal glucose uptake, Ethics Committee approved the insulin secreting property, insulinotropic experimental protocol. The animals were activity of the component present in the maintained under standard conditions in extract. an animal house as per the guidelines of Committee for the Purpose of Control and The diabetic rats when treated with Supervision on Experiments on Animals combination of Coccinia indica leaf with (CPCSEA). The Institutional ethical acarbose exhibited significant decrease in committee approved the experimental blood glucose level when compared to protocol (2013/ PCOL / 004) diabetic control without causing hypoglycemia. Increasing evidence Extraction of Coccinia indica: The extract indicates that a change in the inner of Coccinia indica (Batch No. CIL/12005) 78 Innovational Publishers www.innovationalpublishers.com Sanket Kohli, JIPBS, Vol 1 (2), 77-87, 2014 was collected from the Green Chem each rat of different groups. Glucose industry, Bangalore solution (2 g/kg P.O) was given after 30 min after the administration of the drug. Induction of Diabetes: Single and Four more samples were taken at 30, 60, multiple dose study in normal and diabetic 90 and 120 min after glucose . rats [9] administration. All blood samples were Single dose study: Normal and diabetic checked with the help of accucheck rats will be administered with a single glucometer. [11] dose of plant extract and the selected oral hypoglycemic agents (OHA). The blood Treatment Groups: Effect of Coccinia glucose level will be estimated just prior to indica leaf extract alone and in administration of plant extract and OHA combination with Acarbose in and at 1, 2 and 4 h after administration. streptozotocin induced diabetic rats: n = 6 Glucose levels will be estimated as Group 1: Normal control, rats receive mentioned above. saline/vehicle. Repeated dose study: The same groups Group 2: Diabetic control. (Single dose study) of normal animals will be continued with the same dose levels of Group 3: Diabetic rats treated with plant extract and OHA once daily, for 11 Coccinia indica (200mg/kg)[12] leaf days. The glucose levels of all the animals extract. will be measured on 3, 5, 7, 9 and 11th day of the treatment period Group 4: Diabetic rats treated with Acarbose (Low dose) [13] Development of high fat diet (HFD) fed / Low dose streptozotocin Treated type Group 5: Diabetic rats with Coccinia indica 2 diabetic rats: [10] - The animals were (200mg/kg) leaf extract + Acarbose fed with HFD once a day for two weeks (5mg/kg) followed by I.P injection of streptozotocin Group 6: Diabetic rats treated with (35mg/kg) dissolved in 0.5M/l citrate Quercetin (50mg/kg p.o.). buffer ( pH: 4.4) after overnight fasting. STZ injected animals were then given 5% At the end of the treatment period, the w/v glucose solution for 5-6 hours rats will be sacrificed by decapitation and following the injection to prevent initial the sciatic nerve will be excised and drug induced hypoglycemic mortality. The histopathological studies will be carried rats with non fasted plasma level ≥ out. 300mg/dl were considered diabetic. The blood Sample was collected from tail vein Neuropathic pain models- and blood glucose was checked using Thermal stimuli- glucose diagnostic kit (Accucheck) Hot-plate test (Eddy’s): Standardization and selection of low dose The hyperalgesic response on the for standard drugs: hot-plate is considered to result from a Oral Glucose Tolerance Test: After combination of central and peripheral overnight fasting (18hrs), a 0-min blood mechanisms. In this test, animals will be sample was taken from the tip of the tail of individually placed on a hot-plate (Eddy’s 79 Innovational Publishers www.innovationalpublishers.com Sanket Kohli, JIPBS, Vol 1 (2), 77-87, 2014 hot-plate) with the temperature adjusted Samples of sciatic nerve were kept to 55 ± 1ºC. The latency to the first sign of in the fixative solution (10% formalin) and paw licking or jump response to avoid the cut into 4um thickness. Staining was done heat will be taken as an index of the pain by using hematoxylin and eosin. Nerve threshold; the cut-off time will be 15 sec in sections were analysed under light order to avoid damage to the paw. The microscope (400 x) for axonal degeneration responses will be recorded one week post and vascular defects [17]. STZ injection[14]. Care of diabetic animals: Tail-Flick Latency Test Since diabetic animals drink large The tail-flick measurement will be amount of fluid and produce large volume made using the Ugo Basile tail-flick unit of urine, the bedding was changed (Ugo Basile, Comerio, Italy) based on a frequently, usually every day and, in some modification of a method described circumstances, more than once per day. originally by D'Amour and Smith (1941) Diabetic rats should have sufficient food [15]. The tail-flick unit consisted of an and water; hence six diabetic rats had infrared heat source (50-W bulb) of been housed per cage. adjustable intensity that will be set at 40 units (determined to elicit tail-flick latency 3. Results of 2–4 s as baseline in naive animals). The Feed consumption and water infrared source will be focused to the base consumption of diabetic rats increased of the tail. The latency time (in seconds) significantly when compared to the normal required by the rat to reach the thermal control groups.