Evaluation of antiinflammatory activity of methanol extract of Phyllanthus amarus in experimental animal models

*Corresponding Author:

B. M. Patil
Dept of Pharmacology, K. L. E. S’s College of Pharmacy, Belgaum-590 010, India.
E-mail: bmpatil59@hotmail.com

Abstract

The effects of methanol extract of Phyllanthus amarus on different phases of inflammation were examined. Investigations were performed using different phlogistic agents-induced paw edema, carrageenan-induced air-pouch inflammation and cotton pellet granuloma in rats. Methanol extract of Phyllanthus amarus significantly inhibited carrageenan, bradykinin, serotonin and prostaglandin E1-induced paw edema, but failed to inhibit the histamine-induced paw edema. Maximum inhibition was observed in prostaglandin E1-induced paw edema. In carrageenan air-pouch model, methanol extract of Phyllanthus amarus significantly reduced the volume of exudate and migration of neutrophils and monocytes. The extract significantly decreased formation of granuloma tissue in chronic inflammation model. The present study revealed that methanol extract of Phyllanthus amarus inhibits all the phases of inflammation.

Introduction

Inflammation is a local response of living mammalian tissues to injury. It is a body defense reaction in order to eliminate or limit the spread of injurious agent. There are various components to an inflammatory reaction that can contribute to the associated symptoms and tissue injury. Edema formation, leukocyte infiltration and granuloma formation represent such components of inflammation [1].

Phyllanthus amarus (P. amarus, family Euphorbiaceae) is traditionally used for the treatment of hepatitis, diabetes and dropsy [2,3]. P. amarus was extensively investigated against viral hepatitis [4-7]. Recently Kiemer et al. have Ereported antiinflammatory potential of this traditionally employed herbal medicine8. They have reported that standardized extract of P. amarus inhibited iNOS, COX-2 and TNF-α, in RAW 264.7 cell lines and human whole blood [8]. Further, in carrageenan-induced inflammation, methanol extract of Phyllanthus amarus (MEPA) significantly inhibited the paw edema [9]. However effect of P. amarus on different phases of inflammation has not been studied. In the present study, we have evaluated the effect of MEPA on acute inflammation using different inflammatory mediators-induced paw edema, subacute inflammation (leukocyte infiltration and exudation) using carrageenan air-pouch model and chronic inflammation by cotton pellet-induced granuloma formation in rats.

Materials and Methods

The plant Phyllanthus amarus was collected in the month of July from Western Ghat forests near Jamboti, Belgaum Dist., Karnataka, India. The plant material was taxonomically identified by the Department of Botany, Karnataka University, Dharwad, India. Bradykinin, histamine, and serotonin were obtained from Sigma Chemical Co. (St. Louis, MO, USA): Prostaglandin E₁ was obtained from Cayman Chemical Co. (Michigan, USA),Carrageenan was obtained from Himedia Chemical Co. (Mumbai, India). Indomethacin was obtained as a gift sample from Micro Labs (Mumbai, India).

Male Wistar rats weighing 150-200 g were procured from National Institute of Nutrition, Hyderabad, India. They were acclimatized to laboratory conditions for a week prior to the initiation of the experiment. They were fed on standard rat feed and given free access to water. Twelve hours before the start of the experiment, rats were deprived of food, but given free access to water. The experiment was carried out according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and Institutional Animal Ethical Committee approved all the procedures.

Preparation of methanol extract

The whole plant was shade dried and subjected to size reduction to get a coarse powder. The powdered material was subjected to successive extraction in a Soxhlet apparatus, using methanol (75%) as solvent at 50°. The extract was then evaporated on a rotary evaporator.

Phlogistic agents-induced paw edema

The rats were divided into different groups of six each.Acute inflammation was induced by intraplantar administration of 0.1 ml of carrageenan (1%) or chemical mediators viz; histamine (0.1%), serotonin (0.1%), bradykinin (0.002%) and prostaglandin E₁ (0.0001%). Rats were treated with either vehicle or MEPA (250 mg/kg, p.o.), 1 h before administration of phlogistic agents. The paw volume was measured prior to injection of phlogistic agent (0 h) and then at predetermined interval for each agent. For carrageenan, bradykinin and prostaglandin E₁, the interval was 3 h, whereas, for serotonin and histamine they were 1 h and 2 h, respectively. Paw volume was measured using digital Plethysmometer (UGO BASIL, Italy). Change in the paw volume was measured and antiinflammatory activity was calculated by using the formula: % Inhibition of inflammation = 1-(Vt/Vc)×100, where, Vt represents the of inflammation = 1-(Vt/Vc)×100, where, Vt represents the change in the paw volume in MEPA treated group and Vc represents the change in the paw volume in the corresponding vehicle treated control group.

Carrageenan air-pouch model

The rats were divided into three groups (n=6). Air-pouch was produced according to the method described by Salvemini et al.[10]. Briefly, rats were anesthetized and air cavities were produced by subcutaneous injection of 20 ml of sterile air into the intrascapular area of the back (0 d). An additional 10 ml of air was injected into the cavity every 3 d (3^rd and 6^th d) to keep the space open. On the 7^th d, 2ml of 1% solution of carrageenan dissolved in saline was injected directly into the pouch to induce an inflammatory response. The rats were orally pre-treated with either vehicle or MEPA or indomethacin 2 h prior to the injection of carrageenan into the air-pouch. The second dose of treatment was repeated after 24 h of the first treatment. Forty-eight hours after carrageenan injection, the rats were anesthetized with ether and the pouch was carefully opened by a small incision. The volume of exudate was collected and measured. An aliquot of the exudate was used for quantification of leukocyte concentration using a hemocytometer and differential cell count was performed using a manual cell counter after staining with Wright’s stain. The results were expressed as the total number of neutrophils and monocytes.

Cotton pellet-induced granuloma

The rats were divided into three groups (n=6). After shaving the fur, the rats were anesthetized with ether and 20 mg of sterile cotton pellets were surgically inserted in the groin region. The MEPA (250 mg/kg, p.o.) or indomethacin (10 mg/kg, p.o.) or vehicle was administered for 7 consecutive days from the day of the cotton pellet implantation. The animals were anesthetized on the 8th d and the cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37° for 24 h and dried at 60° for constant weight. Increment in the dry weight of the pellets was taken as measure of granuloma formation [11].

statistical analyses

All data were expressed as mean±SEM. The statistical analyses were performed using Student’s ‘t’ test. p<0.05 was considered as significant.

Results

MEPA significantly (p<0.05) inhibited carrageenan,bradykinin,serotonin and prostaglandin E₁-induced paw edema, but failed to inhibit histamine-induced paw e edema when compared to vehicle treated control group (Table 1). Maximum inhibition (47%) was found in prostaglandin E₁-induced paw edema, whereas, it was 37% in carrageenan induced paw edema. In case of bradykinin and serotonin-induced paw edema, the MEPA showed 20.74% and 31.32% inhibition, respectively relative to vehicle treated control group.