Diabetes mellitus (DM) represents a series of metabolic conditions associated with hyperglycemia and caused by defects in insulin secretion, and/ insulin action. Exposure to chronic hyperglycemia may result in microvascular complications in the retina, kidney or peripheral nerves. According to the World Health Organization (WHO) global burden of disease, more than 176 million people are diabetic with about two thirds of these living in developing countries. With a long course and serious complications that often result in high incidences of mobility and mortality rate, the treatment of diabetes is often costly. The management of this disease is not without side effects and this is a challenge to the medical system. This has led the researches to seek new antidiabetic agents from plants. Acetone extract of 8 plants namely Terminalia sericea Burch. Ex DC, Euclea natalensis A.DC, Warbugia salutaris Bertol.f.) Chiov., Artemisia afra Jacq.ex Willd., Aloe ferox Mill, Sclerocarya birrea (A.Richi.) Hochst. subsp. caffra , Spirostachys Africana Sond and Psidium guajava L were evaluated for antidiabetic and antioxidant properties. In addition extracts were tested for cytotoxicity. Different parts of all these plants are traditionally used in South Africa for diabetes treatment. Plants were selected based on ethnobotanical information and phytochemical constituents. For determining inhibitory activity against each enzyme (α-glucosidase and α- amylase), all extracts were tested at concentration that ranged from 2x10-5 to 0.2mg/ml for α-glucosidase and 0.025 to 1.25mg/ml for α-amylase and fifty percent inhibition or higher was taken as significant (p<0.05). The extracts of A. ferox and S. africana showed no inhibition against α-glucosidase at the highest concentration tested (0.2mg/ml) whereas A. afra showed weak inhibition (47.15%). T. sericea showed to be a potent inhibitor of α-glucosidase exhibiting 97.44 % inhibition of the enzyme (p<0.05). W. salutaris, S birrea and E. natalensis also showed good activity on α-glucosidase as they demonstrated 71.84; 97.44 and 92.60 % inhibition respectively (p<0.05). Other plant extracts such as A. ferox and S. africana did not exhibit any activity on α-glucosidase. T. sericea and S. birrea showed the best inhibitory activity on α-amylase enzyme, exhibiting 91.91 and 94.94 % inhibition respectively at 1.25mg/ml. A. afra, E. natalensis, P. guajava and W. salutaris also showed good inhibitory activity on -amylase enzyme at 1.25mg/ml which was the highest concentration tested (p<0.05). Low levels of plasma antioxidants is a risk factor associated with diabetes therefore, it has been suggested that plant-based medicines that contain antioxidant properties add an advantage in curbing complications that arise during DM aetiology. The antioxidant activity of plant extracts was carried out using 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) assay. Six plant extracts which showed good α-glucosidase and α-amylase inhibitory activity were evaluated for antioxidant activity. The radical scavenging activity was measured in terms of the amount of antioxidants necessary to decrease the initial DPPH absorbance (EC50). The EC50 is the amount of antioxidants necessary to decrease initial DPPH absorbance by 50%. All 6 tested plant extracts showed good activity. W. salutaris and T. sericea demonstrated the highest activity exhibiting EC50 values of 5.08 and 5.56βg/ml respectively as compared to ascorbic acid/Vitamin C (EC50=2.52μg/ml), a well- known potent antioxidant. This was followed by P. guajava (EC50=6.97μg/ml); E. natalensis (EC50=8.46μg/ml) and S. birrea (EC50=9.41μg/ml). A. ferox showed EC50 value of 48.53μg/ml. It has been suggested that plant extracts and compounds must undergo toxicity test for safety before drug discovery is taken into consideration. Due to the large number of plants screened in this study and limited resources in our laboratory, only the acetone extract of T. sericea (which demonstrated good α-glucosidase and -amylase inhibitory activities) was tested for cytotoxicity. Acetone extract of T. sericea demonstrated moderate toxicity against primary vervet monkey kidney cells (VK) cells exhibiting IC50 values of 20.94 μg/ml when tested at 400μg/ml. Consequently, the acetone extract of T. sericea was selected for the isolation and identification of bioactive compounds. A bio-assay guided fractionation of the acetone extract of T. sericea led to the isolation of 4 pure compounds namely β-sitosterol, β-sitosterol-3-acetate, lupeol and 3-onestigmasterol and two sets of mixtures of isomers (epicatechin-catechin; MI1 and epigallocatechingallocatechin; MI2). Antidiabetic, antioxidant and cytotoxicity activities of isolated compounds were evaluated. μ–Sitosterol and lupeol showed best inhibitory activity on α-glucosidase exhibiting 50% inhibitory concentration (IC50) value of 54.50 μM and 66.48 μM respectively (p<0.05). This was followed by the MI2; epigallocatechin-gallocatechin (IC50=119.34 μM); β-sitosterol-3-acetate (IC50=129.34 μM); 3-one-stigmasterol (IC50=164.87 μM) and the MI1; epicatechin-catechin (IC50=255.76 μM). During the evaluation of purified compound’s inhibitory activity on α-amylase, compounds of interest were lupeol and β-sitosterol which exhibited IC50 values of 140.72 μM and 216.02 μM respectively as compared to the positive drug-control acarbose (IC50=65.25 μM). Epicatechincatechin and epigallocatechin-gallocatechin also demonstrated α-amylase inhibitory properties and the IC50 values were found to be lower than 100μg/ml. Epigallocatechin-gallocatechin, epicatechin-catechin and lupeol showed good free radical scavenging activity as they inhibited DPPH by 98.19; 96.98 and 70.90 % at 100ìg/ml respectively (p<0.05). The DPPH scavenging activity was very low in case of 3-one-stigmasterol (21.5% inhibition), whilst β-sitosterol and its derivative β-sitosterol-3-acetate did not show any activity. During cytotoxicity evaluation of pure compounds against monkey kidney cells, all the compounds except β-sitosterol did not inhibit the growth of these cells lines at the highest concentration tested (200μg/ml). β-Sitosterol showed moderate toxicity exhibiting IC50 values of 197.72 μM. β- Sitosterol-3-acetate, epicatechin-catechin, lupeol and epigallocatechin-gallocatechin were found to be non-toxic to Vero cells as 100% cell viability was observed when Vero cells were exposed to these samples at 200μg/ml. The compounds isolated and the extract of T. sericea demonstrated significant antidiabetic and antioxidant properties as compared to well known drugs acarbose (a known -glucosidase and α- amylase inhibitor) and Vitamin C (a well known antioxidant). This study is the first to report α- glucosidase, α-amylase and antioxidant properties of epicatechin-catechin, epigallocatechingallocatechin, β-sitosterol-3-acetate and stigma-4-ene-3-one isolated from T. sericea. In addition, epicatechin-catechin, epigallocatechin-gallocatechin, β-sitosterol-3-acetate and stigma-4-ene-3-one are isolated from T. sericea for the first time. Overall all results scientifically validated the traditional use of the bark of T. sericea for diabetes in South Africa. / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/28896 |
| Date | 21 October 2009 |
| Creators | Nkobole, Nolitha Khanya |
| Contributors | Prof N Lall, s27466958@tuks.co.za |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2009, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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