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Antihyperglycemic activity studies on methanol extract of Petrea volubilis L. (Verbenaceae) leaves and Excoecaria agallocha L. (Euphorbiaceae) stems.

Introduction

Petrea volubilis L. (Verbenaceae, local name: nilmoni lota) is a vinous plant found in a scattered manner in various regions of Bangladesh. The plant is comparatively little known and there is a total absence of any significant scientific studies on its pharmacological properties or its phytochemical constituents. Excoecaria agallocha L. (Euphorbiaceae, local name: gewa) is a common mangrove species of the Sunderbans forest region, which covers portions of the three coastal south-western districts of Bangladesh, namely Bagerhat, Khulna and Satkhira. Both plants are used by the folk medicinal practitioners of Bangladesh for treatment of a variety of ailments including diabetes.

The constituents of E. agallocha and some of their pharmacological properties have been well described in the scientific literature. A novel phorbol ester, 12-deoxyphorbol 13-(3E,5E-decadienoate), with anti-human immunodeficiency virus properties, has been described from leaves and stems of the plant (Erickson et al., 1995). From the resinous wood of the plant, a number of diterpenes have been reported, of which several exhibited anti-tumor promoting activity in vivo in a two-stage carcinogenesis test of mouse tumor using 7,12-dimethylbenz[a]-anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter (Konishi et al., 1998; Konoshima et al., 2001). Antioxidant activity has been reported for methanolic extract of leaves (Masuda et al., 1999). Triterpenoids, including taraxerone, b-amyrin acetate, taraxerol, acetylaleuritolic acid, as well as steroids like b-sitostenone and b-sitosterol have been reported from stems and twigs of the plant (Tian et al., 2008). From the stems and twigs of the plant, diterpenoids have further been reported with properties of tumor necrosis factor-a inhibitory expression induced by lipopolysaccharide in mouse macrophages Raw 264.7 cells (Li et al., 2010). Extract of leaves of the plant reportedly demonstrated anti-plasmodial activity against Plasmodium falciparum (Ravikumar et al., 2010).

Considering the use of the two plants, P. volubilis and E. agallocha in the folk medicinal system of Bangladesh for treatment of diabetes, we decided to evaluate the antihyperglycemic potential of the plants in oral glucose tolerance tests in glucose-challenged mice. The leaves of the former and stems of the latter were selected for antihyperglycemic studies, because these are the plant parts used in folk medicine for their antidiabetic effects.

Materials and Methods

Plant material and extraction

The leaves of P. volubilis were collected from Dhaka district in August 2010. The plant was taxonomically identified by Bangladesh National Herbarium at Dhaka (Accession Number 35,048). The leaves of P. volubilis were air-dried in the shade for 120 hours, grounded into a fine powder, and were extracted with methanol at a ratio of 1:3 (w/v). After 24 hours, the mixture was filtered; filtrate was collected and the residue was again extracted with methanol at a ratio of 1:2 (w/v) for 24 hours. Filtrates were combined and evaporated to dryness. The initial weight of dried leaf powder used for extraction was 100g; the final weight of the extract was 3.94g.

The stems of E. agallocha were collected from the Sunderbans forest region in Satkhira district during January 2010. The plant was taxonomically identified by Bangladesh National Herbarium at Dhaka (Accession Number 34,971). Stems were sliced into small pieces, dried and pulverized to a fine powder. 100g of the stem powder was extracted for 24 hours with methanol at a ratio of 1:4 (w/v). After 24 hours, the mixture was filtered; filtrate was collected and the residue was again extracted with methanol at a ratio of 1:4 (w/v) for 24 hours. Filtrates were combined and evaporated to dryness. The final weight of the extract was 7.04g.

Chemicals and Drugs

Glacial acetic acid was obtained from Sigma Chemicals, USA; glibenclamide and glucose were obtained from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals were of analytical grade.

Animals

In the present study, Swiss albino mice (male), which weighed between 14-20g were used. The animals were obtained from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). All animals were kept under ambient temperature with 12h light followed by a 12h dark cycle. The animals were acclimatized for one week prior to actual experiments. The study was conducted following approval by the Institutional Animal Ethical Committee of University of Development Alternative, Dhaka, Bangladesh.

Antihyperglycemic activity

Glucose tolerance property of methanol extract of P. volubilis leaves and E. agallocha stems was determined as per the procedure previously described by Joy and Kuttan (1999) with minor modifications. In brief, fasted mice were grouped into eight groups. The various groups received different treatments like Group-I received vehicle (1% Tween 80 in water, 10 ml/kg body weight) and served as control, Group-II received standard drug (glibenclamide, 10 mg/kg body weight), Groups III-VI received the methanol extract of P. volubilis leaves at four different doses of 50, 100, 200 and 400 mg/kg body weight; Groups VII and VIII were administered methanol extract of E. agallocha stems at doses of 200 and 400 mg per kg body weight, respectively. Each mouse was weighed and doses adjusted accordingly prior to administration of vehicle, standard drug, and test samples. All substances were orally administered. Following a period of one hour, all mice were orally administered 2 g glucose/kg of body weight. Blood samples were collected two hours after the glucose administration through puncturing heart. Serum glucose levels were measured by glucose oxidase method (Venkatesh et al., 2004).

Results and Discussion

The methanol extract of leaves of P. volubilis, when administered to glucose-challenged mice demonstrated dose-dependent and significant antihyperglycemic activity. The results are shown in Table 1. The concentration of serum glucose in control mice was 97.01 [+ or -] 5.13 mg/dl (mean [+ or -] SEM). The standard antihyperglycemic drug, glibenclamide, administered at a dose of 10 mg per kg body weight, reduced serum glucose levels to 39.80 [+ or -] 1.84 mg/dl. The highest reduction in serum glucose levels with methanol extract of P. volubilis leaves was observed at a dose of 400 mg per kg body weight. At this dose, serum glucose level in mice was observed to be 41.98 [+ or -] 1.52, which was nearly equivalent to that observed with glibenclamide.

Dose for dose, the methanol extract of stems of E. agallocha exhibited greater potency in the reduction of serum glucose levels. At a dose of 200 mg extract per kg body weight, serum glucose levels in mice was reduced to 26.25 [+ or -] 3.16 mg/dl, which was significantly lower than the level observed with glibenclamide. At the highest dose evaluated of 400 mg per kg body weight, serum glucose level in mice was further reduced to 23.29 [+ or -] 2.22 mg/dl (Table 1). It appears from the results obtained in the present study that the mangrove species plant E. agallocha possess considerable antidiabetic potential and can form the basis for further scientific studies for isolation of antihyperglycemic constituent(s).

Reduction of serum glucose levels by a plant extract can occur by several mechanisms. The extract may increase the pancreatic secretion of insulin, or the extract may increase the uptake of glucose (Nyunai et al., 2009; Farjou et al., 1987). It is also possible that the extract may inhibit glucose absorption in gut, which in turn will reduce the presence of glucose in serum (Bhowmik et al., 2009). The actual mechanism through which the crude extracts of leaves of P. volubilis and stems of E. agallocha lowered serum glucose levels in glucose-loaded mice needs further investigations. It is to be noted in this regard that E. agallocha has been reported to contain b-amyrin acetate (Tian et al., 2008). The compound a-amyrin acetate has been shown to possess antihyperglycemic properties in several studies (Singh et al., 2009; Narender et al., 2009). Whether b-amyrin acetate has similar properties like a-amyrin acetate is not clear but needs to be elucidated.

The results obtained in the present study also validate the folk medicinal uses of both plants for treatment of diabetes in Bangladesh. Indigenous use of plants can form and have often in the past formed the basis of discovery of modern allopathic drugs. Our study highlights the importance of scientifically evaluating indigenous uses of antidiabetic plants of Bangladesh. Diabetes is a debilitating disease for which no modern cure is known, and which affects millions of people in all countries of the world. As such, it is important that further research is done on medicinal plants that have been used for centuries by indigenous peoples or folk medicinal practitioners for treatment of diabetes.

References

Bhowmik, A., L.A. Khan, M. Akhter and B. Rokeya, 2009. Studies on the antidiabetic effects of Mangifera indica stem-barks and leaves on nondiabetic, type 1 and type 2 diabetic model rats. Bangladesh Journal of Pharmacology, 4: 110-114.

Erickson, K.L., J.A. Beutler, J.H. Cardellina, 2nd., J.B. McMahon, D.J. Newman and M.R Boyd, 1995. A novel phorbol ester from Excoecaria agallocha. Journal of Natural Products, 58: 769-772.

Farjou, I.B., M. Al-Ani and S.Y. Guirgea, 1987. Lowering of blood glucose of diabetic rats by Artemisia extract. Journal of the Faculty of Medicine, 92: 137-147.

Joy, K.L. and R.J. Kuttan, 1999. Anti-diabetic activity of Picrorrhiza kurroa extract. Journal of Ethnopharmacology, 67: 143-148.

Konishi, T., M. Takasaki, H. Tokuda, S. Kiyosawa and T. Konoshima, 1998. Anti-tumor promoting activity of diterpenes from Excoecaria agallocha. Biological & Pharmaceutical Bulletin, 21: 993-996.

Konoshima, T., T. Konishi, M. Takasaki, K. Yamazoe and H. Tokuda, 2001. Anti-tumor-promoting activity of the diterpene from Excoecaria agallocha. II. Biological & Pharmaceutical Bulletin, 24: 1440-1442.

Li, Y., J Liu, S. Yu, P. Proksch, J. Gu and W. Lin, 2010. TNF-a inhibitory diterpenoids from the Chinese mangrove plant Excoecaria agallocha L. Phytochemistry, 71: 2124-2131.

Masuda, T., S. Yonemori, Y. Oyama, Y. Takeda, T. Tanaka, T. Andoh, Shinohara, A., and Nakata, M., 1999. Evaluation of the antioxidant activity of environmental plants: activity of the leaf extracts from seashore plants. Journal of Agricultural and Food Chemistry, 47: 1749-1754.

Narender, T., T. Khaliq, A.B. Singh, Joshi, M.D., Mishra, P., Chaturvedi, J.P., Srivastava, A.K., Maurya, R., and Agarwal, S.C., 2009. Synthesis of a-amyrin derivatives and their in vivo antihyperglycemic activity. European Journal of Medicinal Chemistry, 44: 1215-1222.

Nyunai, N., N. Njikam, E.H. Addennebi, J.T. Mbaford and D. Lamnaouer, 2009. Hypoglycaemic and antihyperglycaemic activity of Ageratum conyzoides L. in rats. African Journal of Traditional Complementary and Alternative Medicines, 6: 123-130.

Ravikumar, S., S. Jacob Inbaneson, P. Suganthi, M. Venkatesan and A. Ramu, 2010. Mangrove plants as a source of lead compounds for the development of new antiplasmodial drugs from South East coast of India. Parasitology Research, (in press).

Singh, A.B., D.K. Yadav, R. Maurya and A.K. Srivastava, 2009. Antihyperglycemic activity of a-amyrin acetate in rats and db/db mice. Natural Product Research, 23: 876-882.

Tian, M.Q., G.M. Bao, N.Y. Ji, X.M. Li and B.G. Wang, 2008. Triterpenoids and steroids from Excoecaria agallocha. Zhongguo Zhong Yao Za Zhi, 33: 405-408.

Venkatesh, S., G.D. Reddy, Y.S.R. Reddy, D. Sathyavathy and B.M. Reddy, 2004. Effect of Helicteres isora root extracts on glucose tolerance in glucose-induced hyperglycemic rats. Fitoterapia, 75: 364-367.

Corresponding Author: Dr. Mohammed Rahmatullah, Pro-Vice Chancellor and Dean, Faculty of Life Sciences University of Development Alternative House No. 78, Road No. 11A (new) Dhanmondi, Dhaka-1205 Bangladesh Telephone: +88-01715032621; Fax: +88-02-815739 Email: rahamatm@hotmail.com

(1) Mahabuba Rahman, (1) Aziza Siddika, (1) Bithika Bhadra, (1) Shahnaz Rahman, (1) Bipasha Agarwala, (2) Majeedul H. Chowdhury, (1) Mohammed Rahmatullah

(1) Department of Biotechnology & Genetic Engineering, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh

(2) Present address: New York City College of Technology The City University of New York 300 Jay Street, Brooklyn, NY 11201, USA.

Mahabuba Rahman, Aziza Siddika, Bithika Bhadra, Shahnaz Rahman, Bipasha Agarwala, Majeedul H. Chowdhury, Mohammed Rahmatullah: Antihyperglycemic Activity Studies on Methanol Extract of Petrea Volubilis L. (Verbenaceae) Leaves and Excoecaria Agallocha L. (Euphorbiaceae) Stems
Table 1: Effect of methanol extract of P. volubilis leaves and
E. agallocha stems on serum glucose level in hyperglycemic mice.

Treatment       Dose (mg/kg body weight)   Serum glucose level (mg/dl)

Control         10 ml                       97.01 [+ or -] 5.13
Glibenclamide   10 mg                       39.80 [+ or -] 1.84 *
P. volubilis    50 mg                       72.87 [+ or -] 1.56 *
P. volubilis    100 mg                      59.60 [+ or -] 4.12 *
P. volubilis    200 mg                      58.73 [+ or -] 2.63 *
P. volubilis    400 mg                      41.98 [+ or -] 1.52 *
E. agallocha    200 mg                      26.25 [+ or -] 3.16 *
E. agallocha    400 mg                      23.29 [+ or -] 2.22 *

All administrations were made orally. Values represented as mean
[+ or -] SEM, (n=6 for P. volubilis, n = 7 for E. agallocha);
* P < 0.05; significant compared to hyperglycemic control animals.
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Title Annotation:Original Article
Author:Rahman, Mahabuba; Siddika, Aziza; Bhadra, Bithika; Rahman, Shahnaz; Agarwala, Bipasha; Chowdhury, Ma
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9BANG
Date:Sep 1, 2010
Words:2118
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