ANTI-DIABETIC EFFECTS OF THE LEAF EXTRACT OF Parquetina nigrescens (AFZEL.) BULLOCK, PHYTOL AND SQUALENE IN ALLOXAN-INDUCED DIABETES IN MALE WISTAR RATS

Abstract

Diabetes mellitus is a disorder with episodes of hyperglycaemia. Although, Parquetina nigrescens has been reported to reduce blood glucose, the mechanisms of its normoglycaemic effect have not been fully elucidated. This study was designed to investigate the mechanisms of the anti-diabetic activity of Methanol Extract of Parquetina nigrescens (MEPN) and the effects of two of its constituents (phytol and squalene) on renal damage in alloxan-induced diabetes using male Wistar rats. Parquetina nigrescens leaves were collected from Ibadan, Oyo State and authenticated at the Department of Botany, University of Ibadan (UIH No.: 22475). The leaves were air dried, pulverised, extracted by maceration in methanol and concentrated. An aliquot of MEPN was analysed using Gas Chromatography-Mass Spectrometry to identify the chemical constituents. Diabetes was induced by single intraperitoneal injection of alloxan monohydrate (120 mg/kg) after an overnight fasting. Fifty male Wistar rats (90-130 g) were used in two studies to test for the antidiabetic effects of MEPN, phytol and squalene. Twenty-five rats were used in study one and were grouped into five (n=5); Control, Diabetic untreated (DU), Diabetes+MEPN (100 mg/kg), Diabetes+MEPN (200 mg/kg) and Diabetes+metformin (100 mg/kg). In study two, analytical grade of phytol and squalene were used. Twenty-five rats grouped into five (n=5); Control, DU, Diabetes+phytol (250 mg/kg), Diabetes+squalene (3 mg/kg) and Diabetes+metformin (100 mg/kg) were used. Treatments were administered orally for 4 weeks in both studies. Fasting Blood Glucose (FBG), Liver Glycogen (LG) and Insulin Concentration (IC) were assessed using glucose oxidase, anthrone and ELISA methods, respectively. Renal diacylglycerol, Protein Kinase C-Alpha (PKC-α), and Transforming Growth Factor-Beta (TGF-β) were measured using ELISA while Bcl-2, p16, p53 and COX-2 in kidney tissues were assessed using immunohistochemistry. Data were analysed using descriptive statistics and ANOVA at p<0.05. The GC-MS revealed the presence of phytol (4.8%) and squalene (8.0%) in MEPN. In Diabetes+MEPN (200 mg/kg), FBG (95.80±19.57 mg/dL) decreased significantly compared to DU (396.00±17.55 mg/dL). The LG (3.10±0.18 mg/mL) and IC (4.87±0.18 μU/mL) in Diabetes+MEPN (200 mg/kg) increased significantly compared to DU (2.09±0.09 mg/mL and 1.57±0.15 μU/mL, respectively). In Diabetes+phytol, FBG (101.60±4.44 mg/dL) decreased significantly while LG (32.61±2.18 mg/mL) and IC (4.53±1.22 μU/mL) increased significantly compared to DU (358.82±16.80 mg/dL, 13.15±1.70 mg/mL and 1.52±0.35 μU/mL, respectively). In Diabetes+squalene, FBG (125.17±2.78 mg/dL) decreased significantly while LG (47.67±9.00 mg/mL) and IC (3.79±0.16 μU/mL) increased significantly compared to DU. Also, diacylglycerol (12.66±1.88 ng/mL), PKC-α (3.56±0.12 ng/mL) and TGF-β (78.33±4.03 pg/mL) decreased significantly only in Diabetes+phytol compared to DU (34.94±1.33 ng/mL, 8.79±0.29 ng/mL and 168.90±2.16 pg/mL, respectively). The presence of anti-apoptotic protein (Bcl 2) was increased in Diabetes+phytol compared to DU. Pro-apoptotic protein presence (p16, p53 and COX-2) were reduced in Diabetic+phytol compared to DU. vi The mechanism of antidiabetic activity of methanol extract of Parquetina nigrescens, phytol and squalene was through decreased blood glucose, increased liver glycogen and insulin concentration. Phytol exhibited reno-protective activity by decreasing the level of diacylglycerol, protein kinase C-alpha and transforming growth factor-beta in the diabetic male Wistar rats.