EVALUATION OF FRACTIONATED METHANOL EXTRACTS OF VERNONIA AMYGDALINA AND OCIMUM GRATISSIMUM LEAVES IN TYPE 2 DIABETIC WISTAR ALBINO RATS
EVALUATION OF FRACTIONATED METHANOL EXTRACTS OF VERNONIA AMYGDALINA AND OCIMUM GRATISSIMUM LEAVES IN TYPE 2 DIABETIC WISTAR ALBINO RATS
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Date
2017-10
Authors
OKODUWA, STANLEY IROBEKHIAN REUBEN
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Abstract
Type 2 diabetes (T2D) accounts for 90-95 % of the present 422 million estimated cases of diabetes mellitus worldwide, yet greater part of anti-diabetic research is on Type 1 diabetes. This study evaluated the effects of fractionated methanol extracts of Vernonia amygdalina (VA) and Ocimum gratissimum (OG) leaves in Type 2 diabetic rats. In the 1st phase, the hypoglycemic efficacy of extracts obtained from three extraction methods [Soxhlet Extraction (SE), Cold Maceration (CM) and Microwave-Assisted-Extraction (MAE) was investigated. It was performed on each of 50 g of VA and OG leaves. The 2nd phase developed rat model with features of human T2D using sixty male albino rats divided into two groups and fed commercially available normal diet feed (NDF) and water or fortified diet feed (FDF) with 20 % fructose solution as drinking water. After 6 weeks of dietary regimen, both groups were each divided into 5 subgroups of 6 rats. Each subgroup received single injection (ip) of streptozotocin (STZ) (0, 25, 35, 45, 55 mg/kg b.w.) dissolved in citrate buffer (pH 4.5) after overnight fast. The minimum oral therapeutic dose of the crude extracts in graded dose (100, 150, 200 etc mg/kg b.w.) were evaluated in the diabetic rats over 4 h period. In the 3rd phase the activity guided fractionation of methanol extracts of VA and OG leaves using organic solvents of increasing polarity (n-hexane, chloroform, ethyl acetate, n-butanol and water) was conducted. Daily treatment of diabetic rats with the fractionated methanol extracts of VA and OG leaves (250 mg/kg b.w.) lasted for 28 days. The tolerance to oral glucose loading (2 g/kg bw) was examined after 28 days. The 4th phase isolated the antidiabetic bioactive components of the plants. The most active fractionated methanol extract of each plants were subjected to column chromatography. Column subfractions with similar Rf values were pooled together. The pooled column sub-fractions obtained were administered orally (10 mg/kg b.w) to T2D model rats. Metformin and normal saline were used as positive and negative controls respectively. The most active column sub-fractions with hypoglycemic activity were analyzed using Fourier transform infrared and Nuclear magnetic resonance spectroscopy for identification and structural elucidation of the bioactive component(s). The result showed that the CM extract gave significantly (p<0.05) higher hypoglycemic effect within 14 days, hence, was employed for further investigations. The FDF-fed rats exhibited significant (p<0.05) increase in body weight, total cholesterol, triglycerides and blood glucose compared to the NDF-fed rats. Ten days post STZ-induction, the groups treated with STZ (45 and 55 mg/kg) developed frank hyperglycemia with <46.8 % serum-insulin, a severe deficiency typical of Type 1 diabetes. The NDF groups induced with 25 and 35 mg/kg b.w STZ with 75.7 and 64.4 % serum insulin respectively had
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relatively normoglycemia, whereas the FDF group induced with 35 mg/kg b.w STZ (FDF35) had 85.8 % serum insulin and were notably hyperglycemia (>300 mg/dL). This FDF35 rats were stable over 6-weeks post diabetes confirmation and sensitive to T2D drugs (glibenclamide, metformin and pioglitazone) and were selected for further investigation. The VA and OG extracts dose of 250 mg/kg b.w significantly (p<0.05) reduced (>25%) the blood glucose of diabetic rats by 33.23 and 29.25 % respectively at 4 h post-administration, hence was used in the study. The chloroform fraction of fractionated methanol extract of VA (VACF) and n-butanol fraction of fractionated methanol extract of OG (OGBF) emerged as the most active in significantly (p<0.05) reducing blood glucose of diabetic rats by 66.70 ± 6.50 % and 63.52 ± 4.84 % respectively after oral treatment for 28 days as compared to the untreated diabetic control (-50.45+11.46 %). The Area under the glucose tolerance curve showed that treatment with VACF and OGBF significantly (p<0.05) reduced blood glucose of diabetic rats by 49.01 % and 48.05 % respectively, after oral glucose loading. Nine pooled column sub-fractions (C1-C9) were isolated from VACF and four (B1-B4) from OGBF. The column sub-fractions C5 and B3 were found to be the most potent hypoglycaemic in reducing blood glucose by 12.55 ± 3.55 % and 9.32 ± 0.51 % respectively at 4 h post-oral administration, when compared to the positive (18.07 ± 1.20 %) and negative (-1.99 ± 0.43 %) controls. The spectroscopic data analysis reveals that, the isolated bioactive compounds C5 and B3 were 11β,13-dihydrovernolide and 5-hydroxyhex-3-enoic acid respectively. The isolated compounds are part of the bioactive components present in VA and OG leaves which are responsible for the anti-diabetic activities. Further research is needed in the development of these compounds for pharmaceutical use.
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A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY,
FACULTY OF LIFE SCIENCES, AHMADU BELLO UNIVERSITY, ZARIA, NIGERIA
Keywords
EVALUATION,, FRACTIONATED METHANOL,, VERNONIA AMYGDALINA,, OCIMUM GRATISSIMUM LEAVES,, TYPE 2 DIABETIC WISTAR,, ALBINO RATS,