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A pharmacological study on antinociceptive and anti-hyperglycemic effects of methanol extract of leaves of Phyllanthus reticulatus poir. In Swiss albino mice.

Introduction

Phyllanthus reticulatus Poir. (Family: Euphorbiaceae, local name: chitki, pan chitki) is a large climbing shrub growing from 8-10 feet in height. The plant is found in the wild as well as fallow lands of Bangladesh. It is also present in India, where it is used in the traditional medicinal system of the country for treatment of a variety of ailments including small pox, syphilis, asthma, diarrhea, and bleeding from gums (Kirtikar, K.R., and Basu, B.D., 2003; The Wealth of India, 2005). In Bangladesh, the traditional medicinal practitioners (Kavirajes) use various parts of the plant like leaf, bark, root, stem, and fruit for treatment of edema, constipation, helminthiasis, dysentery, diarrhea, pain, and kidney, gall bladder, liver and gastrointestinal disorders of diabetic patients.

The phytochemicals, lupeol, lupeol acetate, and stigmasterol have been reported to be present in the plant Jamal et al., (2008) Dichloromethane extract of leaves of the plant revealed presence of three compounds, (5R*,6R*)-4,6-dimethoxycarbonyl-5-[2 ,3 ,4 -trihydroxy-6 -(methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran-2-one along with 3,4,3 -tri-O-methylellagic acid, and methyl gallate. The first compound reportedly demonstrated weak insecticidal activity against Spodoptera frugiperda Pojchaijongdee et al., (2010).

Ethanol extract of the plant has been observed to demonstrate hepatoprotective activity against carbon tetrachloride-induced liver damages in rats Dae et al., (2008). Petroleum ether and ethanol extracts of leaves of the plant reportedly showed hypoglycemic effect in alloxan-induced diabetic mice, albeit at a high dose of 1000 mg/kg body weight. Phytochemical contents of the leaves included presence of terpenoid glycosides, protein, carbohydrates but absence of alkaloids and steroids Kumar et al., (2008). Anti-plasmodial activity has been reported for extracts of the plant against both chloroquine-sensitive (K67) and chloroquine-resistant (ENT 36) strains of Plasmodium falciparum Omulokoli et al., (1997).

Considering the folk medicinal uses of Phyllanthus reticulatus in Bangladesh, the objective of the present study was to evaluate the antinociceptive and anti-hyperglycemic effects of methanol extract of the leaves, respectively, in acetic acid-induced gastric pain writhing model, and oral glucose tolerance tests in glucose-loaded mice.

Materials and Methods

Plant material and extraction

The leaves of Phyllanthus reticulatus Poir. were collected from Gazipur, Bangladesh in December, 2009. The plant was taxonomically identified by Mr. Manzur-ul-Kadir Mia, ex-Principal Scientific Officer and Curator of Bangladesh National Herbarium at Dhaka. The leaves of Phyllanthus reticulatus 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 hrs, 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 hrs. Filtrates were combined and evaporated to dryness.

Chemicals and Drugs

Glacial acetic acid was obtained from Sigma Chemicals, USA; aspirin, 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 20-25g were used. The animals were obtained from International Centre for Diarrheal 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.

Acetic acid-induced writhing method

Antinociceptive activity of methanol extract of Phyllanthus reticulatus leaves was examined using previously described procedures of Deb et al., (2010) with minor modifications. Pain was induced in mice in the writhing test through intraperitoneal administration of 1% acetic acid at a dose of 10 ml/kg body weight. Mice were separated into five groups of six mice each. Group-I served as control and was administered vehicle (1% Tween 80 in water, 10 ml/kg body weight). The standard drug, aspirin was administered to Group-II mice at a dose of 200 mg/kg body weight. Groups-III to V received extract, respectively at 50, 100 and 200 mg extract/kg body weight orally 30 min before acetic acid injection. A period of 5 minutes was given to each animal to ensure bio-availability of acetic acid, following which period, the number of writhings was counted for 10 min.

Anti-hyperglycemic activity

Glucose tolerance property of Phyllanthus reticulatus leaves was determined as per the procedure previously described by Joy and Kuttan, (1999) with minor modifications. In brief, fasted mice were grouped into five groups of eight mice each. 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) and the other three groups (III-V) received the methanol extract of Phyllanthus reticulatus leaves at three different doses of 100, 200 and 400 mg/kg body weight. 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).

Statistical analysis

Experimental values are expressed as mean [+ or -] SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.

Results and Discussion

Antinociceptive activity

The methanol extract of leaves of Phyllanthus reticulatus showed significant antinociceptive activity when administered to mice in acetic acid-induced gastric pain writhing tests. Maximum inhibition of writhing (39.1%) was observed at an extract dose of 200 mg/kg body weight. The standard drug, aspirin, when administered at a dose of 200 mg/kg body weight inhibited writhings by 50.4%. The results, shown in Table 1 demonstrate that the methanolic extract of leaves of Phyllanthus reticulatus could be used as a treatment for pain.

Acetic acid-induced writhing test is a suitable detector for both central and peripheral analgesia Shanmugasundaram and Venkataraman, (2005). Intraperitoneal administration of acetic acid (1%) leads to pain and inflammation involving a mechanism in which prostaglandins, mainly prostacyclines (PGI2) and prostaglandin-E (PG-E) are produced, which have been reported to be responsible for excitation of the Ad-nerve fibers, leading to sensation of pain (Reynolds, 1982; Rang and Dale, 1993). Analgesia will be demonstrated then by any agent that lowers the number of writhing by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition. Leaf extract of Phyllanthus reticulatus significantly caused reduction in the number of abdominal constrictions as well as stretching of hind limbs induced by the intraperitoneal injection of acetic acid. The results suggest that the methanolic extract of leaves may contain components, which when administered leads to inhibition of prostaglandin synthesis. The responsible component has not been identified in the present study and further laboratory investigations are on-going to identify the responsible components.

Anti-hyperglycemic effect

The results from the present study showed that the methanol extract of Phyllanthus reticulatus leaves exhibited dose-dependent and significant anti-hyperglycemic activity in glucose-induced hyperglycemic mice. Even at the lowest dose of the extract tested (100 mg/kg body weight) serum glucose levels were lowered by 18.4%. The maximum serum glucose lowering effect was found with the dose of 400 mg extract/kg body weight (35.0%). The standard drug, glibenclamide at a dose of 10 mg/kg body weight lowered serum glucose level by 57.8% (Table 2). The anti-hyperglycemic effect observed following administration of methanolic leaf extract suggests that the extract may potentiate pancreatic secretion of insulin or increase the glucose uptake (Nyunai et al., 2009; Farjou et al., 1987), or inhibit glucose absorption in gut Bhowmik et al., (2009). The exact mechanism of action needs to be elucidated and can form the basis for further experiments.

The results of the present study demonstrate that the leaves of Phyllanthus reticulatus contains phytocomponents, which when administered, can lead to antinociceptive and anti-hyperglycemic effects in mice models. Overall, the results validate the folk medicinal use of the plant in Bangladesh as treatment of pain, or various organ complications in diabetic patients.

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.

Das, B.K., S. Bepary, B.K. Datta, A.A. Chowdhury, M.S. Ali and A.S. Rouf, 2008. Hepatoprotective activity of Phyllanthus reticulatus. Pakistan Journal of Pharmaceutical Sciences, 21: 333-337.

Deb, D., S. Dev, A.K. Das, H. Khanam, M. Banu, M. Shahriar, A. Ashraf and M.S.K. Choudhuri, 2010. Anti-nociceptive, Anti-inflammatory and anti-diarrheal activity of crude root extract of Lasia spinosa Linn. (Family- Araceae). Latin American Journal of Pharmacy, (in press).

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.

Jamal, A.K., W.A. Yaacob and L.B. Din, 2008. A chemical study on Phyllanthus reticulatus. Journal of Physical Science, 19: 45-50.

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

Kirtikar, K.R. and B.D. Basu, 2003. Indian Medicinal Plants. Dehradun: International Book Distributors, pp: 3060.

Kumar, S., D. Kumar, R.R. Deshmukh, P.D. Lokhande, S.N. More and V.D. Rangari, 2008. Antidiabetic potential of Phyllanthus reticulatus in alloxan-induced diabetic mice. Fitoterapia, 79: 21-23.

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.

Omulokoli, E., B. Khan, and S.C. Chhabra, 1997. Antiplasmodial activity of four Kenyan medicinal plants. Journal of Ethnopharmacology, 56: 133-137.

Pojchaijongdee, N., U. Sotanaphun, S. Limsirichaikul and O. Poobrasert, 2010. Geraniinic acid derivative from the leaves of Phyllanthus reticulatus. Pharmaceutical Biology, 48: 740-744.

Reynolds, J.E.F., 1982. "Martindale: The Extra Pharmacopoeia", The Pharmaceutical Press, 28th edition; pp: 245. Rang, H.P. and M.M. Dale, 1993. "Pharmacology", 2nd edition. Churchill Livingstone Publishers, UK.

Shanmugasundaram, P. and S. Venkataraman, 2005. Anti-nociceptive activity of Hygrophilous auriculata (Schum) Heine. African Journal of Traditional Complementary and Alternative Medicines, 2: 62-69.

The Wealth of India, 2005. National Institute of Science Communication and Information Resources. Council of Scientific and Industrial Research, New Delhi, pp: 34.

Corresponding Author: 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

Mohammed Rahmatullah, Khokon Chandra Ghosh, Abdullah Al Mamun, Md. Tozammal Hossain, Salman Ahmed, Md. Ashikur Rahman, Bipasha Eva, Shahnaz Rahman, Majeedul H. Chowdhury; A Pharmacological Study on Antinociceptive and Anti-hyperglycemic Effects of Methanol Extract of Leaves of Phyllanthus Reticulatus Poir. In Swiss Albino Mice

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.

(1) Mohammed Rahmatullah, (1) Khokon Chandra Ghosh, (1) Abdullah Al Mamun, (1) Md. Tozammal Hossain, (1) Salman Ahmed, (1) Md. Ashikur Rahman, (1) Bipasha Eva, (1) Shahnaz Rahman, (2) Majeedul H. Chowdhury

(1) Faculty of Life Sciences, 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.
Table 1: Antinociceptive effect of crude methanol extract of
Phyllanthus reticulatus leaves in the acetic acid-induced gastric
pain model mice.

                            Dose (mg/kg      Mean no.
Groups                      body weight)     of writhing

Control (vehicle)           10 ml            11.5 [+ or -] 1.3
Aspirin                     200 mg           5.7 [+ or -] 1.1
Phyllanthus reticulatus     50 mg            7.7 [+ or -] 1.0
Phyllanthus reticulatus     100 mg           7.8 [+ or -] 1.0
Phyllanthus reticulatus     200 mg           7.0 [+ or -] 0.7

Groups                      Inhibition (%)

Control (vehicle)           --
Aspirin                     50.4 *
Phyllanthus reticulatus     33.0 *
Phyllanthus reticulatus     32.2 *
Phyllanthus reticulatus     39.1 *

All administrations were made orally. Values represented as
mean [+ or -] SEM, (n=6); * P < 0.05; * significant compared
to control.

Table 2: Effect of methanol extract of Phyllanthus reticulatus leaves
on serum glucose level in hyperglycemic mice.

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

Control                   10 ml          100.3 [+ or -] 4.9
Glibenclamide             10 mg          42.3 [+ or -] 5.2
Phyllanthus reticulatus   100 mg         81.8 [+ or -] 6.4
Phyllanthus reticulatus   200 mg         78.6 [+ or -] 7.8
Phyllanthus reticulatus   400 mg         65.2 [+ or -] 2.8

                          % lowering of serum
Treatment                 glucose level

Control                   --
Glibenclamide             57.8 *
Phyllanthus reticulatus   18.4 *
Phyllanthus reticulatus   21.6 *
Phyllanthus reticulatus   35.0 *

All administrations were made orally. Values represented as
mean [+ or -] SEM, (n=8); * P < 0.05; significant compared
to hyperglycemic control animals.
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Title Annotation:Original Article
Author:Rahmatullah, Mohammed; Ghosh, Khokon Chandra; Al Mamun, Abdullah; Hossain, Tozammal; Ahmed, Salman;
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9BANG
Date:Sep 1, 2010
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