The Biochemical Effects of Cussonia Arborea Root Bark Extract in Alloxan-Induced Diabetic Rats

Abstract

Diabetes mellitus is a group of metabolic disorders characterized by chronic hyperglycemic condition resulting from defects in insulin secretion, insulin action or both. It is a growing public health concern worldwide affecting humans and animals. Synthetic drugs available for the treatment of the ailment have serious side effects, complicated mode of intake and are costly. Literature search revealed that Cussonia arborea is used folklorically in the management of Diabetes mellitus. The aim of this study is to isolate, characterize and elucidate the active principle responsible for its hypoglycaemic activity using alloxan-induced diabetic rats. The root bark of C. arborea (2 kg) was extracted with 80% methanol by cold maceration method. Acute toxicity study was done in 35 rats assigned into 7 groups of 5 rats per group. Groups 1, 2,3,4,5 and 6 rats were orally administered with graded doses (500, 1000, 2000, 3000, 4000, 5000 mg/kg bw) of the extract respectively. The rats in group 7 received 10 ml/kg distilled water (DW) to serve as negative control group. They were observed closely for 48 hours for signs of toxicity. Assessment of hypoglycemic activities of the extract was done using oral glucose tolerance test (OGTT), acute and chronic antidiabetic studies. In OGTT, 30 rats were randomly assigned into 5 groups of six rats per group. Groups 1, 2 and 3 rats received 250, 500 and 1000 mg/kg bw of the extract respectively while groups 4 and 5 rats received 2 mg/kg bw glibenclamide and 10 ml/kg DW respectively after 18 h fasting and prior to 2000 mg/kg of glucose load. The fasting blood glucose (FBG) levels of the rats were determined after 30, 60, 120 and 180 min post glucose challenge. Diabetes was induced by single intraperitoneal administration of alloxan monohydrate at the dose of 160 mg/kg bw. Rats with FBG levels above 126 mg/dl (7 mMol/L) were considered diabetic. Thirty male albino Wistar rats assigned into 6 groups of 5 rats per group were used for acute antidiabetic studies. Groups 1, 2, 3, 4 and 5 were diabetic rats treated with 250, 500 1000 mg/kg bw of the extract, 2 mg/kg bw glibenclamide and 10 ml/kg DW respectively while group 6 rats were non diabetic but administered with 10 ml/kg DW. The FBG levels of the rats were determined 1 h, 3 h, 6 h and 24 h post treatment. Seventy two (72) male albino wistar rats weighing between 100 and 105 g were assigned into six groups of 12 rats per group for chronic antidiabetic studies. Groups 1, 2, 3, 4 and 5 rats were made diabetic as described earlier and treated with 62.5, 125, 250 mg/kg bw of the extracts, 2 mg/kg bw glibenclamide and 10 ml/kg DW respectively while the non diabetic group 6 rats received 10 ml/kg DW and served as normal control rats. The treatment was daily through the oral route for 84 days. The biochemical (aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglyceride, high density lipoprotein (HDL), very low density lipoprotein (VLDL), low density lipoprotein (LDL), total protein, albumin, globulin, blood urea nitrogen, creatinine, total bilirubin, conjugated bilirubin, unconjugated bilirubin, malondiadehyde (MDA), superoxide dismutase (SOD), catalase, and reduced glutathione) and haematological (red blood cell (RBC), haemoglobin (Hb), packed cell volume (PCV), white blood cell (WBC), differential leucocytes (lymphocytes, neutrophils, basophils, eosinophils and monocytes), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)) parameters were assayed on days 28, 56 and 84 post treatment while the FBG values and weight changes were determined every two weeks. The glycosylated haemoglobin values were measured on days 42, 56 and 84 post treatment. Three rats per group were humanly sacrificed on days 28, 56 and 84 for the assessment of the histomorphology of various organs (pancreas, kidney, liver and heart). In vitro antioxidant assay was carried out using ferric reducing antioxidant power (FRAP) and diphenyl picryl hydrazyl (DPPH) photometric assay models. Chromatographic separation of the plant extract was done using column and thin layer chromatographic techniques. The 1H proton and 13carbon nuclear magnetic resonance (NMR) were used for structural elucidation of the active hypoglycaemic principle. No sign of toxicity was observed after acute toxicity test. The phytochemical analysis revealed the presence of alkaloids, flavonoids, glycosides, saponins, tannins and terpenes. The mean FBG level of the rats treated with 250 mg/kg bw of the extract in acute anti-diabetic study, at 3 h post extract administration was significantly (p<0.05) lower than the FBG before administration of the extract. In the chronic anti-diabetic study, the mean FBG level of rats treated with 125 mg/kg bw of the extract reduced significantly (p<0.05), the post induction FBG from 315.33± 10.08 mg/dl to 93.00 ±8.50 mg/dl, 14 days post administration and ameliorated the glycosylation of haemoglobin compared to the negative control group. Administration of 250 mg/kg bw of the extract in glucose challenged rats reduced the post challenge glucose level from 147.66 ±1.85 mg/dl to 83.00 ±3.5 mg/dl, 180 min post treatment. Assessment of the biochemical and haematological parameters in chronically-treated diabetic rats showed that the administration of 125 mg/kg bw of the extract, significantly (p<0.05) reduced the activities of AST, ALT and the levels of total cholesterol, triglyceride, VLDL, LDL BUN, creatinine, total bilirubin, malondialdehyde but significantly (p<0.05) increased the activities of SOD, catalase and the levels of HDL, total protein, conjugated bilirubin, reduced glutathione , RBC, Hb and PCV when compared to the diabetic untreated group (negative control). The extract yielded seven fractions. The FBG of the rats treated with 12.5 mg/kg bw of fraction 2 was significantly (p<0.05) lower compared to the negative control group. Subfraction 1 (2 mg/kg) of fraction 2 significantly (p<0.05) reduced the FBG levels from 310.00±5.77 mg/dl to 74.00±0.57 mg/dl. Rats treated with 125 mg/kg bw of the extract showed milder histopathologic lesions in various tissues compared with negative control. The DPPH in vitro antioxidant assay showed a concentration dependent activities with 400 µg/ml of the extract and ascorbic acid showing 74% and 92% activities respectively. The FRAP values of the extract and ascorbic acid at 400µg/ml were 1.60 and 2µm respectively. The NMR spectroscopy revealed a pentacyclic triterpenoid, {(3β)-3-hydroxyolean-12-en-28-oic acid} as the active compound. The study showed that the root bark extract of C. arborea did not only possess antihyperglycaemic, hypolipidemic, antioxidant and antianemic properties but also reduced haemoglobin glycosylation and ameliorated the severe degenerative lesions in the pancreas, kidney, liver and cardiac myocytes occasioned by Diabetes mellitus.