Anti-hyperglycemic activity studies on leaves and stems of Areca catechu L. (Arecaceae).
Areca catechu L. (Arecaceae) (local name: supari) is more commonly known as the areca palm or betel nut palm because its fruit is chewed often with betel leaf. The palm tree can grow up to 20 meters tall, and is cultivated throughout the tropical parts of the Indian sub-continent, including Bangladesh (Satyavatia et al, 1976). The fruits of the palm (nuts) are known to be in use for over two thousand years (Bradley, 1979) and are chewed either by themselves for breath freshening effect, digestive aid, expelling intestinal worms (Cawte, 1985), or as in Bangladesh, with betel leaf, lime and other ingredients as a digestive aid and stimulant effect. Betel nut with betel leaf chewing is possibly the biggest addiction in Bangladesh, more so than tobacco. Chewing of the nut reportedly caused a depressive effect on the heart and hypotension (Kapoor, 1990; Lin et al., 2002).
A number of alkaloids have been reported to be present in the nut. These include arecoline, arecaidine, guvacoline, guvacine, arecolidine, and choline (Chu, 2001; Farnsworth, 1976). Presence of phenolic compounds like hydroxychavicol and safrole, and phytochemicals like tannins, gallic acid, catechin, -sitosterol, gum and amino acids have been reported in betel nut (Wang et al., 1997; Duke, 1992).
The anti-depressant effects of ethanol extract of betel nuts have been reported (Dar and Khatoon, 1997). Cholinomimetic and acetylcholinesterase inhibitory constituents have also been reported in betel nut (Gilani et al., 2004). Arecoline, an alkaloid present in betel nut reportedly showed hypoglycemic activities (Chempakam, 1993). The compound also reportedly enhanced uptake of 2-deoxyglucose dose-dependently in L6 myotubes and caused increased expression of glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3K) genes suggesting that the compound can replace commercially available anti-diabetic drugs (Prabhakar and Doble, 2010). On the other hand, it has been reported that arecoline significantly attenuated in 3T3-L1 adipocytes insulin-induced uptake of glucose (Hsu et al., 2010). Clearly, more studies are necessary for accurate evaluation of betel nut extract or isolated constituents like arecoline from nut for any possible anti-diabetic effects.
Contrary to betel nut and some of its constituents, there is an absolute dearth of pharmacological activity studies on effect of betel leaf and stem extracts. The leaves and stems are used in folk medicine of Bangladesh for the treatment of intestinal pain, ulcer, dental disease, diarrhea, gout, and constipation, but so far no scientific study has been done with regard to their anti-hyperglycemic effects. It was the objective of the present study to evaluate the anti-hyperglycemic activity of leaves and stems of the plant in a rodent model.
MATERIALS AND METHODS
Collection of plant material:
The leaves and stems of Areca catechu were collected in December 2009 from Gazipur district, Bangladesh. Identification of the leaves and stems was done at the Bangladesh National Herbarium, Mirpur, Dhaka (Accession No. 35,030) and sample specimens have been kept over there.
Preparation of the test samples:
The dried leaves and stems of Acacia catechu were separately air-dried in the shade and pulverized into a fine powder. The powder was then mixed with methanol at a ratio of 1:3 (w/v). After 24 hours, the mixtures were filtered; filtrate was collected and the residue was again mixed with fresh methanol at a ratio of 1:2 (w/v) for 24 hours. After filtration, filtrates were combined and evaporated to dryness (approximate yields were 11.2% for leaves and 4.1% for stems) using rotary evaporator. 1% Tween 80 in water was used to suspend the extracts prior to administration.
Preliminary phytochemical screening:
Preliminary phytochemical screening (Kokate, 1994; Harborne, 1998) revealed the presence of presence of phenolic compounds, alkaloids, glycosides, and tannins.
Swiss albino mice (male), weighing 25-30g bred in the animal house of ICDDR,B (International Centre for Diarrheal Disease and Research, Bangladesh) were used for the present experiments. All animals were acclimatized one week prior to the experiments. The animals were housed under standard laboratory conditions (relative humidity 55-65%, room temperature 25.0 [+ or -] 2[degrees]C, and 12 hrs light-dark cycles). The animals were fed with standard diet from ICDDR,B and had free access to water. The study was approved by the Institutional Animal Ethical Committee of the University of Development Alternative, Dhaka, Bangladesh.
Anti-hyperglycemic activity test:
Anti-hyperglycemic activities of the extracts were studied through the glucose tolerance test method. Glucose tolerance test was performed following the procedure described by Joy and Kuttan (1999) with minor modifications. Briefly, fasted mice were divided into nine groups of six mice each. Each group received a particular treatment like group-I served as control and received vehicle (1% Tween 80 in water, 10 ml x [kg.sup.-1] body weight), while group-II received standard drug (glibenclamide, 10 mg x [kg.sup.-1] body weight). Groups III-VI received stem extract at four different doses of 50, 100, 200 and 400 mg extract x [kg.sup.-1] body weight, respectively. Groups VII-IX received leaf extract at doses of 100, 200 and 400 mg extract x [kg.sup.-1] body weight, respectively. Each mouse was weighed properly and the doses of the test samples, standard drug, and control materials were adjusted accordingly. Test samples, control, and glibenclamide were given orally. After one hour, all mice were orally treated with 2 g x [kg.sup.-1] of glucose. Blood samples were collected two hours after glucose administration. Serum was separated and blood glucose levels were measured immediately by glucose oxidase method (Venkatesh et al, 2004).
Statistical analysis for anti-hyperglycemic activity:
Experimental results are expressed as mean [+ or -] SEM. For statistical comparison, Independent Sample t-test was carried out. A p value < 0.05 in all cases was considered to be statistically significant.
Acute toxicity study:
The study was carried out as has been previously described (Ganapaty et al., 2002) with minor modifications. Selected animals were divided into nine groups of six animals each. The control group received 1% Tween 80 in normal saline (2 ml x [kg.sup.-1] body weight). The other groups received respectively, 100, 200, 300, 600, 800, 1000, and 2000 mg leaf or stem methanolic extract x [kg.sup.-1] body weight. Animals were monitored closely after dosing for the next 8 hours for any behavioral changes and were kept under observation up to 14 days to find out if there is any mortality.
RESULTS AND DISCUSSION
Acute toxicity study:
Mice did not show any mortality with any of the extracts at tested doses till the end of 14 days of observation.
The results obtained from the present study indicate that the methanol extract of the leaves and stems of Areca catechu lowered serum glucose levels dose-dependently and significantly when compared to control (group-I) at all the doses examined. Dose for dose, the anti-hyperglycemic activity was more pronounced with methanolic leaf extract of the plant than the stem extract. Maximum hypoglycemic activity of methanol extract of Areca catechu leaves in glucose-induced hyperglycemic mice was observed with a 400 mg x [kg.sup.-1] dose (65.7% inhibition), while the standard drug, glibenclamide produced 71.4 % inhibitory activity at 10 mg x [kg.sup.-1] dose (Table 1) under the experimental conditions of the present study. The present preliminary experimental results indicated that leaves and stems of Areca catechu exhibited significant blood glucose lowering property in glucose-induced hyperglycemic mice. A known hypoglycemic agent--arecoline is reportedly present in nuts of the plant (Chempakam, 1993). Whether arecoline is present in leaves and stems of the plant is currently under investigation. This, however, dose not preclude the presence of other phytochemical components in leaves and stems of Areca catechu, which was responsible for the observed anti-hyperglycemic effect. The mechanism underlying the glucose lowering efficacy of Areca catechu is yet to be investigated and is currently going on in our laboratory. However, it is speculated that the glucose lowering activity of the extract may either be through potentiating insulin secretion by the pancreas or through increasing the uptake of glucose.
It is to be noted that antihyperglycemic activity of leaf extract of A. catechu has been reported in streptozotocin-induced diabetic rats (Mondal et al., 2012). The beneficial role of nut extract in alloxan diabetic rats has also been reported (Kavitha et al., 2013). Ethanolic extract of leaf has been shown to cause significant lowering of blood glucose in hyperglycemic Sprague Dawley rats (Wankhade and Nandi, 2013). Thus the results obtained in the present study are consistent with previous studies and suggest that constituents from A. catechu can prove to be valuable glucose lowering agents in diabetic patients.
Received 25 January 2014
Received in revised form 12 March 2014
Accepted 14 April 2014
Available online 5 May 2014
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Shinn Akhter, Maruf Hassan, Shahnaz Rahman, Afifa Sultana, Shejuty Shahreen, Joyonta Banik, Rownak Jahan, Mohammed Rahmatullah
Department of Biotechnology and Genetic Engineering, University of Development Alternative, Dhanmondi, Dhaka-1205, Bangladesh
Corresponding Author: Professor Dr. Mohammed Rahmatullah, Pro-Vice Chancellor and Dean, Faculty of Life Sciences, University of Development Alternative, House No. 78, Road No. 11A (new), Dhanmondi, Dhaka1205, Bangladesh
Tele: +88-01715032621 Fax: +88-02-815739 E-mail: firstname.lastname@example.org
Table 1: Effect of methanol extract of Areca catechu (leaf and stem) on serum glucose level in hyperglycemic mice. Treatment Dose Group I (Control, vehicle) 1% Tween 80 in water (10 ml x [kg.sup.-1] body weight) Group II (glibenclamide) 10 mg x [kg.sup.-1] body weight Group III (stem extract) 50 mg x [kg.sup.-1] body weight Group IV (stem extract) 100 mg x [kg.sup.-1] body weight Group V (stem extract) 200 mg x [kg.sup.-1] body weight Group VI (stem extract) 400 mg x [kg.sup.-1] body weight Group VII (leaf extract) 100 mg x [kg.sup.-1] body weight Group VIII (leaf extract) 200 mg x [kg.sup.-1] body weight Group IX (leaf extract) 400 mg x [kg.sup.-1] body weight Serum glucose level Treatment (mg x [dl.sup.-1]) % inhibition Group I (Control, vehicle) 84.2 [+ or -] 9.1 -- Group II (glibenclamide) 24.1 [+ or -] 2.5 * 71.4 Group III (stem extract) 48.7 [+ or -] 6.6 * 42.2 Group IV (stem extract) 36.4 [+ or -] 2.8 * 56.8 Group V (stem extract) 34.2 [+ or -] 3.3 * 59.4 Group VI (stem extract) 31.1 [+ or -] 3.7 * 63.1 Group VII (leaf extract) 34.7 [+ or -] 2.8 * 58.8 Group VIII (leaf extract) 32.9 [+ or -] 3.5 * 60.9 Group IX (leaf extract) 28.9 [+ or -] 2.5 * 65.7 Extracts and drug were given orally one hour before glucose administration and serum glucose level was measured two hours after glucose administration. Values are given as Mean [+ or -] S.E.M. from six mice in each group. * P < 0.05 is significant compared to hyperglycemic control animals.