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| ORIGINAL ARTICLE |
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| Year : 2013 | Volume
: 32
| Issue : 4 | Page : 193-198 |
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Study of antihyperglycaemic activity of medicinal plant extracts in alloxan induced diabetic rats
Anoja P Attanayake1, Kamani A. P. W. Jayatilaka1, Chitra Pathirana1, Lakmini K. B. Mudduwa2
1 Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka
2 Department of Pathology, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka
| Date of Web Publication | 6-May-2014 |
Correspondence Address:
Anoja P Attanayake
Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Galle
Sri Lanka
 Source of Support: University Grants Commission,
Sri Lanka (UGC/ICD/CRF 2009/2/5)., Conflict of Interest: None  | Check |
DOI: 10.4103/0257-7941.131970
Background: Diabetes mellitus, for a long time, has been treated with plant derived medicines in Sri Lanka.
Aim: The aim of this study is to determine the efficacy and dose response of oral antihyperglycaemic activity of eight Sri Lankan medicinal plant extracts, which are used to treat diabetes in traditional medicine in diabetic rats.
Materials and Methods: Medicinal plants selected for the study on the basis of documented effectiveness and wide use among traditional Ayurveda physicians in the Southern region of Sri Lanka for the treatment of diabetes mellitus. The effect of different doses of aqueous stem bark extracts of Spondias pinnata (Anacardiaceae), Kokoona zeylanica (Celastraceae), Syzygium caryophyllatum (Myrtaceae), Gmelina arborea (Verbenaceae), aerial part extracts of Scoparia dulcis (Scrophulariaceae), Sida alnifolia (Malvaceae), leaf extract of Coccinia grandis (Cucurbitaceae) and root extract of Languas galanga (Zingiberaceae) on oral glucose tolerance test was evaluated. A single dose of 0.25, 0.50, 0.75, 1.00, 1.25, 2.00 g/kg of plant extract was administered orally to alloxan induced (150 mg/kg, ip) diabetic Wistar rats (n = 6). Glibenclamide (0.50 mg/kg) was used as the standard drug. The acute effect was evaluated over a 4 h period using area under the oral glucose tolerance curve.
Statistical Analysis: The results were evaluated by analysis of variance followed by Dunnett's test.
Results: The eight plant extracts showed statistically significant dose dependent improvement on glucose tolerance (P < 0.05). The optimum effective dose on glucose tolerance for six extracts was found to be 1.00 g/kg in diabetic rats with the exception of C. grandis: 0.75 g/kg and L. galanga: 1.25 g/kg.
C onclusion: The aqueous extract of G. arborea, S. pinnata, K. zeylanica, S. caryophyllatum, S. dulcis, S. alnifolia, L. galanga and C. grandis possess potent acute antihyperglycaemic activity in alloxan induced diabetic rats. Keywords: Antihyperglycaemic activity, blood glucose, diabetes mellitus, oral glucose tolerance test
How to cite this article:
Attanayake AP, Jayatilaka KA, Pathirana C, Mudduwa LK. Study of antihyperglycaemic activity of medicinal plant extracts in alloxan induced diabetic rats. Ancient Sci Life 2013;32:193-8 |
| Introduction | |  |
Diabetes mellitus is the most important non-infective epidemic to hit the globe in the present millennium and at present has a world-wide incidence of 5% in the general population. Despite the great strides made in understanding and management of diabetes, the disease and disease related complications are increasing unabated due to multiple defects in its pathophysiology. [1] Diabetes results from defects in insulin secretion, insulin sensitivity or both and includes a group of metabolic disorders characterized by hyperglycaemia and abnormalities in carbohydrate, fat and protein metabolism. [2]
Thus far, there is no promising therapy to cure diabetes mellitus. [3] In addition to this, current antidiabetic drugs usually have adverse side-effects and decreased efficacy over time. [4] Though different types of oral hypoglycaemic agents as insulin releasers, insulin sensitizers and glucosidase inhibitors are available for the treatment of diabetes mellitus, there is a increased demand by patients to use herbal medicines with fewer side effects. [5] Thus, renewed appreciation and scientific interest is now booming throughout the world in the study of medicinal plants, due to their perceived effectiveness, lesser side-effects in clinical practice and relatively low costs in their treatment. Although hundreds of plants are used in the world to prevent or cure diseases, scientific evidence is lacking in most of the cases. [6]
Herbal medicine is part of the Sri Lankan culture as in many developing countries of the world. The rich Sri Lankan flora of medicinal plants contributes to this practice. About 126 plants belonging to 51 families are used to treat diabetic patients in Sri Lanka and more than 600 species are documented currently as antihyperglycaemic in traditional medicine. [7] However, only a small fraction of the antidiabetic plants used in Sri Lankan traditional medicine are pharmacologically evaluated for their in vivo efficacy. In the present study, we selected eight medicinal plant extracts, which are widely used to treat diabetes mellitus in traditional medicine. There is no scientific data available on the complete range of dose response of selected plant extracts including the therapeutic dose of each extract on glucose tolerance in diabetic rats. Medicinal plants/parts selected are listed in [Table 1]. Therefore, the present study aims towards the comparative screening of eight Sri Lankan medicinal plant extracts for efficacy and dose response on glucose tolerance in alloxan induced diabetic rats.
| Materials and Methods | | |
Chemicals
Alloxan monohydrate, D-glucose and glibenclamide were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Chemicals were of analytical grade and used without any purification. A Sanyo Gallenkamp (model SP65) spectrophotometer was used for spectrophotometric measurements.
Plant material
Stem bark of Gmelina arborea, Spondias pinnata, Kokoona zeylanica, Syzygium caryophyllatum, arial parts of Scoparia dulcis, Sida alnifolia, and root of Languas galanga and leaf of Coccinia grandis were collected during May-June 2011 from the southern region of Sri Lanka.
The identity of plants was identified by comparing authentic samples at the National Herbarium, Royal Botanical Gardens, Peradeniya, Sri Lanka. A voucher specimen has been deposited at the Department of Biochemistry, Faculty of Medicine, University of Ruhuna, Sri Lanka (FM/Attanayake/2011/1-8).
Preparation of the aqueous plant extract
Selected plant parts were cut into small pieces, dried at 40°C until a constant weight was reached and coarsely ground. Powdered plant material (50.00 g) was dissolved in 400.0 mL of distilled water and refluxed for 4 h. The mixture was strained through cheese-cloth and the final volume was adjusted to 50.0 mL. A single dose of 0.25, 0.50, 0.75, 1.00, 1.25, 2.00 g/kg was administered orally to diabetic test rats.
Animals
Healthy Wistar albino rats with 220 ± 25 g body weights were used to carry out experiments. They were housed in standard environmental conditions at the animal house of Faculty of Medicine, University of Ruhuna, Sri Lanka (temperature 25°C ± 2°C and relative humidity: 55-65% and 12 h light/dark cycle). Rats were fed with standard diet with free access to water before and during the experiment. The rats were randomized into various groups and allowed to acclimatize for a period of 7 days under standard environmental conditions before the commencement of the experiment. The animals described as fasting were deprived of food and water for 16 h ad libitum. All protocols used in this study were approved by the Ethics Committee of Faculty of Medicine; University of Ruhuna, Sri Lanka guided by the Council for International Organizations of Medical Sciences international guiding principles of biomedical research involving animals.
Induction of diabetes
Diabetes was induced in 16 h fasted rats by intraperitoneal administration of alloxan monohydrate dissolved in sterile saline at a dose of 150 mg/kg. The rats were maintained on 5% D-glucose solution for the next 24 h. Rats were allowed to stabilize for 3 days and blood samples were drawn from the tail vein on the 3 rd day to determine the blood glucose concentrations to confirm the development of diabetes mellitus. Rats with fasting blood glucose value of 9.70 mmol/L (equal to fasting serum glucose concentration of 11.0 mmol/L) or above were considered as hyperglycaemic and used for the experiments.
Experimental design
Rats were randomly divided into 11 groups containing six animals in each group. Each group (III-X) consisted of diabetic rats with six subgroups (a-f) receiving an oral single dose of 0.25, 0.50, 0.75, 1.00, 1.25, 2.00 g/kg of relevant plant extract through a stomach tube. All animals were fasted before the treatment.
Group I: Untreated healthy rats; received distilled water.
Group II: Untreated diabetic rats; received distilled water.
Group III (a-f): Diabetic rats; received the bark extract of G. arborea.
Group IV (a-f): Diabetic rats; received the bark extract of S. pinnata.
Group V (a-f): Diabetic rats; received the bark extract of K. zeylanica.
Group VI (a-f): Diabetic rats; received the bark extract of S. caryophyllatum.
Group VII (a-f): Diabetic rats; received aerial plant extract of S. dulcis.
Group VIII (a-f): Diabetic rats; received the aerial plant extract of S. alnifolia.
Group IX (a-f): Diabetic rats; received the root extract of L. galanga.
Group X (a-f): Diabetic rats; received the leaf extract of C. grandis.
Group XI: Diabetic rats; received the standard drug glibenclamide (0.50 mg/kg).
Acute toxicity study
Acute toxicity testing was performed for plant extracts following the Organization for Economic Cooperation and Development guideline 423, fixed dose procedure. [8] The healthy rats were observed after administration of a single oral dose of 0.25, 0.50, 0.75, 1.00, 1.25, 2.00 g/kg extract for any adverse signs and symptoms at hourly intervals for the next 24 h and thereafter for 2 days.
Effect of aqueous extract of different plants on oral glucose tolerance
The rats of all test groups were given an oral dose of glucose (3.00 g/kg) 30 min after drug administration. Blood samples were collected from the tail vein just prior to administration of extract/drug (0) and 1 st , 2 nd , 3 rd and 4 th h subsequently. Blood glucose concentration was measured immediately by the glucose - oxidase method using glucose assay kit. [9] The acute effect was evaluated over a 4 h period using area under the oral glucose tolerance curve. Curves of blood glucose concentration (mmol/L) vs. time intervals (h) were constructed. Area under the curve value was calculated with the formulae in the trapezoidal method. [10]
Statistical analysis
All results were expressed as mean ± standard error of mean. The data were analysed using analysis of variance and the mean values for each group were compared by Dunnett's multiple comparison test.
| Results | | |
Acute toxicity study
Acute toxicity studies revealed that the administration of stem bark extract of G. arborea, S. pinnata, K. zeylanica, S. caryophyllatum, aerial part extract of S. dulcis, S. alnifolia, root extract of L. galanga and leaf extract of C. grandis did not produce significant changes in the behaviour of animals. All the animals were physically active and no death was observed up to the dose of 2.00 g/kg which may be considered as a therapeutic advantage.
Effect of aqueous extract of different plants on oral glucose tolerance
The improvement on glucose tolerance with plant extracts was dose dependent. The mean total area under the curve values of untreated healthy, untreated diabetic and diabetic test rats for the range of doses 0.25-2.00 g/kg is shown in [Table 2]. | Table 2: Total area under the oral glucose tolerance curve values of diabetic test rats
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A significant improvement in glucose tolerance with extracts of G. arborea, S. pinnata, K. zeylanica, S. caryophyllatum, S. dulcis and S. alnifolia at doses of 1.00, 1.25 and 2.00 g/kg was found in diabetic rats. The improvement on glucose tolerance at doses of 0.25, 0. 50, 0.75 g/kg was statistically non-significant for the above six plant extracts (P > 0.05). The root extract of L. galanga and the leaf extract of C. grandis showed optimum effectiveness at the dose of 1.25 g/kg and 0.75 g/kg respectively (P < 0.05). In addition, when compared to untreated diabetic rats the area under the curve during 4 h period was significantly decreased in both plant extract treated (at and above the therapeutic dose) and glibenclamide treated diabetic rats.
The oral glucose tolerance curves for normal untreated and diabetic treated rats at the optimum effective dose are shown in [Figure 1]a and b. The improvement on glucose tolerance at the optimum effective dose was in the decreasing order of G. arborea, S. pinnata, S. dulcis, alnifolia, S. caryophyllatum, L. galanga and C. grandis in alloxan induced diabetic rats [Table 3]. | Table 3: Percentage of improvement on glucose tolerance of diabetic test rats treated with different plant extracts at the optimum effective dose and the diabetic rats treated with glibenclamide (0.5 mg/kg)
Click here to view |
| Discussion | | |
Medicinal plants selected for the screening was based on popularity, documented effectiveness and applicability among medical practitioners in Sri Lanka. The traditional usages of medicinal plants are commonly in the form of aqueous extracts prepared singly or as combinations. In this study, plant extracts were prepared in the traditional way and assumed to be relevant medicinally and nutritionally. The human therapeutic dose of each plant extract was considered in selecting the range of doses used in this study. The human therapeutic dose was extrapolated to rats according to the standard guidelines. [11] The overall improvement on glucose tolerance with each of the plant extract over a period of 4h was evaluated using the total area under oral glucose tolerance curve, which has been followed by many authors. [12],[13]
Experimental diabetes in animals has provided considerable insight into the physiological and biochemical derangements of the diabetic state. Alloxan is widely used as a diabetogenic agent in experimental animals. In addition alloxan induced rat model has been used to study the antidiabetic effects of several plant products. [14] Alloxan induces diabetes mellitus by destroying the insulin producing beta cells of the islets of Langerhans in the pancreas. Moreover, diabetogenic effect of alloxan is due to the excess production of reactive oxygen species leading to cytotoxicity in pancreatic beta cells, which reduces the synthesis and release of insulin. [15] This effect was seen in the current study through the elevation of blood glucose concentration in alloxan induced diabetic rats compared with healthy rats.
Epidemiological studies and clinical trials strongly support that hyperglycaemia is the principal cause of diabetic complications. [16] The sustained reduction in hyperglycaemia or effective glucose control is the key in preventing/reversing the disease. Therefore, glucose loaded hyperglycaemic model was selected to screen the antihyperglycaemic effects of plant extracts. [17] The routine biochemical marker used in the diagnosis and progress monitoring during the management of diabetes mellitus in clinical and experimental settings is blood or serum/plasma glucose concentration. The results obtained for blood glucose concentration of diabetic untreated rats showed high values in oral glucose tolerance test and high total area under the curve values as compared with healthy untreated rats. These results are in accordance with the findings of several authors using alloxan induced diabetic rats. [18],[19]
The optimum effective dose for aqueous extracts of G. arborea, S. pinnata, K. zeylanica, S. caryophyllatum, S. dulcis and S. alnifolia was found to be 1.00 g/kg and was approximately equivalent to the therapeutic dose of relevant extract. The root extract of L. galanga and leaf extract of C. grandis showed optimum effectiveness at the dose of 1.25 g/kg and 0.75 g/kg respectively. The leaf extract of C. grandis appeared to be a potent antihyperglycaemic agent in diabetic rats. The optimum effective dose was subsequently used to determine the long-term efficacy and the mechanism of action of these plant extracts. The glucose lowering effect produced by plant extracts increases significantly with time. This is well manipulated through the total area under the curve over a period of time. Low total area under the curve reflects high efficacy or improvement on glucose tolerance of the extract. The leaf extract of C. grandis showed an improvement of 31.84% at the dose of 0.75 g/kg over a glucose load, which is comparable with the improvement produced by glibenclamide. In response to a glucose load, the excessive amount of glucose in the blood induces insulin secretion. This secreted insulin stimulates the peripheral glucose utilization. In all diabetic treated groups the blood glucose concentration was always lower than the blood glucose concentration of diabetic untreated rats at each time interval. This may be due to the supportive action of glucose utilization by these plant extracts.
Diabetic rats treated with glibenclamide showed a significant reduction in the total area under the curve and blood glucose concentration in oral glucose tolerance test. The improvement in glucose tolerance was 39.28% compared with diabetic untreated rats and the values are comparable with previous studies using animal models. [20] Glibenclamide inhibits adenosine triphosphate (ATP)-dependent potassium channels of pancreatic beta cells, which augment insulin secretion and increase the sensitivity of the insulin dependent tissues, i.e. the liver, muscles and adipose tissues resulting in glucose utilization. [21]
Further studies are in progress to elucidate mechanisms of antihyperglycaemic activity of the plant extracts.
| Conclusion | | |
This study revealed that the optimum effective dose to produce the antihyperglycaemic effect shown by the improvement of glucose tolerance in alloxan induced diabetic rats to be 1.00 g/kg for stem bark extract of G. arborea, S. pinnata, K. zeylanica, S. caryophyllatum, arial part extract of S. dulcis, S. alnifolia, 0.75 g/kg for the leaf extract of C. grandis and 1.25 g/kg for the root extract of L. galanga. The results of the acute toxicity study suggest that the eight plant extracts are safe in rats up to a dose of 2.00 g/kg. The improvement on glucose tolerance of the leaf extract of C. grandis at the dose of 0.75 g/kg was comparable with the effect of glibenclamide (0.5 mg/kg).
| Acknowledgements | | |
The authors wish to thank Dr. D.A.B.N. Gunarathne, Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Sri Lanka for his guidance in statistical data analysis, Mrs. B.M.S. Malkanthie and Mr. G.H.J.M. Priyashantha, Faculty of Medicine, University of Ruhuna, Sri Lanka for technical assistance.
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[Figure 1]
[Table 1], [Table 2], [Table 3]
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