Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 4, 2011, pp. 393-397

Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 393-397

Research Article

Evaluation of the anti-inflammatory activity of Aegle marmelos (Bilwa) root

Jyoti M Benni, MK Jayanthi, RN Suresha

Department of Pharmacology, Jagadguru Sri Shivarathreeshwara Medical College (JSS University), Mysore, India

Correspondence Address: Jyoti M Benni Department of Pharmacology, Jagadguru Sri Shivarathreeshwara Medical College (JSS University), Mysore India benni_jyoti@yahoo.co.in

Date of Submission: 26-Jul-2010
Date of Decision: 12-Feb-2011
Date of Acceptance: 25-Apr-2011

Code Number: ph11107

DOI: 10.4103/0253-7613.83108

Abstract

Keywords: Aegle marmelos; anti-inflammatory; carrageenan; granuloma; edema

Introduction

Pain and inflammation is a common complaint in most patients suffering from disease conditions. Inflammation is a host defense mechanism to combat or overcome the invading pathogen or the foreign particles. Non-steroidal anti-inflammatory drugs (NSAIDs) make up one of the largest groups of drugs used for pain and inflammation. ^[1] Currently available anti-inflammatory agents are associated with unwanted side effects and have their own limitations. About 34-46% of the users of NSAIDs usually sustain some gastrointestinal damage due to the inhibition of the protective cyclooxygenase enzyme in gastric mucosa. ^[2] The added advantages of indigenous medicinal treatment would include its complementary nature to the conventional treatment making latter safer, well tolerated and economical remedy for acute and chronic inflammatory conditions.

Aegle marmelos (Bilwa) is an Indian plant, which has enormous therapeutic value in traditional systems of medicine. ^[3] Bilwa belongs to the family Rutaceae and grows wild, in dry forest, outer Himalayas and Shivaliks. Bilwa is a medium to large sized deciduous glabrous, armed tree with axillary and 2.5 cm long alternate trifoliate leaves, short flowers and globular fruits. ^[4] The crude extracts of Bilwa have shown various activities including antidiabetic, ^[5] antioxidant, ^[5] anti-inflammatory, ^[6] analgesic, ^[6] antiulcer, ^[7] antimicrobial, ^[8] antihyperlipidaemic, ^[9] anticancer, ^[10] antiviral, ^[11] radioprotective ^[12] and antispermatogenic properties. ^[13]

From the previous studies, methanol extract of Aegle marmelos leaves showed significant anti-inflammatory activity at a dose of 100 mg/kg. ^[6],[14] However, studies on aqueous extract of root and its evaluation in different anti-inflammatory models are sparse. Hence, here is an attempt to evaluate the anti-inflammatory activity of aqueous extract of Bilwa root bark in albino rats and to compare the activity between the acute and chronic inflammatory animal models.

Material and Methods

Materials

Preparation of Extract

The root bark coarse powder was authenticated and procured. Aqueous extract was prepared in distilled water in the ratio of 1:16, soaked overnight and boiled next day until the volume reduced to 1/4 ^th , cooled and drained. ^[15] The extract was prepared freshly on the day of experiment.

Phytochemical Test

The freshly prepared aqueous extract of A. marmelos was subjected to phytochemical screening tests for various constituents. A pilot study of extract with different doses (5mg/kg, 50 mg/kg, 100mg/kg and 200mg/kg) was done to estimate the dose for study. The ant-inflammatory activity was significant at dose of 100mg/kg and hence the same dose was used in this study.

Chemicals

Indomethacin (Sigma), Carrageenan (Sigma) and all other chemicals were of analytical grade.

Animals

Adult albino rats of either sex weighing between 150 to 250 grams were randomly selected from central animal facility. Animals were housed in groups of 3, at an ambient temperature of 25±1°C with ad libitum access to food and water. The study protocol was approved by Institutional Animal Ethics Committee.

Methods

Animals were randomly divided into 3 groups of 6 rats each; I group: Control (1ml of Vehicle, 2%

Gum acacia suspension); II group: Standard drug (Indomethacin 100mg/kg); III group: Test drug (Bilwa 100 mg/kg). Following models were used to screen the anti-inflammatory activity of Bilwa.

Carrageenan Induced Rat Paw Edema Animal Model ^[16],[17]

In this method, rats were divided in 3 groups of six animals each. The animals were pretreated with drugs orally 1 hr before the experiment. 0.05 ml of 1% carrageenan was injected aseptically into the subplantar surface of right hind paw of each rat. Paw edema was measured by Mercury Plethysmograph (UGO Basile, Italy) at ′0′hour and at the end of ′4′hours. The difference between the zero and 4 hours gives the actual edema. Percentage inhibition (protection) against edema formation was taken as an index of acute anti-inflammatory activity.

It was calculated by:

The percent inhibition of edema = 100 x (1- Vt / Vc)

Where, Vc = mean paw edema volume in the control group.

Vt = mean paw edema volume in the drug treated group.

Cotton Pellet Induced Granuloma Animal Model ^[16],[18]

This is an established animal model to screen the chronic anti-inflammatory activity of the drugs. Four sterile cotton pellets weighing 10mg each were implanted subcutaneously in both the axilla and groin of each rat. Rats were fed with the respective drug daily for 7 days along with free access to water and food ad libitum. Later the animal were sacrificed on the 8 ^th day and the cotton pellets with granulation tissue were removed, cleaned of the extraneous tissue and dried in a hot air oven to a constant weight and the dry granuloma weight was determined. The dry weight of the granuloma (i.e. the amount of actual granulation tissue formed) was calculated by noting the difference in the dry weight of the cotton pellets recorded before and after implantation. The animal weight was also recorded on day 1 as initial weight and on day 8 as the final animal weight. The percentage change of granuloma weight relative to vehicle control group was taken as an index of chronic anti-inflammatory activity.

It was calculated by

Percent anti-granuloma activity = 100 x (1- Wt / Wc) .

Where, Wt= mean dry weight of granuloma in drug treated group

Wc= mean dry weight of granuloma in drug untreated control group.

Statistical Analysis

Results were expressed as mean ± Standard deviation (SD). Statistical analysis was performed using One-way analysis of variance (ANOVA) followed by Scheffe′s post hoc test. P <0.05 was considered statistically significant. All the statistical methods were carried out through the SPSS for Windows (version 16).

Results

Preliminary phytochemical screening of aqueous extract of Aegle marmelos root revealed the presence of alkaloids and glycosides.

Carrageenan-Induced Rat Paw Edema

The aqueous extract of Bilwa (100mg/kg) and indomethacin (100mg/kg) both significantly inhibited carrageenan induced rat paw edema (P < 0.001). The maximum inhibition of paw edema was observed in both indomethacin and Bilwa at the end of four hours when compared to the control group. Anti-inflammatory activity is expressed as Percent Inhibition (PI). The PI with Bilwa and indomethacin were 35.7% and 51.5% respectively [Table - 1] and [Figure - 1].

Cotton Pellet Induced Granuloma

The indomethacin (100mg/kg) showed significant (P < 0.001) anti-inflammatory activity in granuloma induced by cotton pellets. Bilwa (100g/kg) did not show significant anti-inflammatory activity in reducing granuloma formation, compared to control group but showed a trend of anti-inflammatory activity (P > 0.05). The PI with Bilwa and indomethacin were 9.2% and 24.7% respectively [Table - 2] and [Figure - 2]a.

Effect on Body Weight

The body weight of all animals was recorded on day of implantation and on the day of removing the cotton pellets. The weight loss was observed more in indomethacin treated animals than the control group. The body weight in Bilwa treated animals showed negligible loss of weight and the results obtained, however were statistically non-significant (P > 0.05) [Figure - 2]b.

Discussion

Inflammation is the integral part of the body′s defence mechanism. Acute inflammation is characterized by vasodilatation, exudation of plasma, release of various inflammatory mediators, cytokines, growth factors and emigration of leukocytes. While the features of chronic inflammation includes infiltration of mononuclear cells, proliferation of fibroblasts, blood vessels and increased connective tissue formation. Tissue infection is a prototype of inflammatory response. Anti-inflammatory drugs inhibit different stages of inflammation.

Bilwa is one of the most frequently used drug in the traditional and folklore systems of medicine. The decoction of the Bilwa roots, root bark and stem bark are used to cure palpitations, abdominal pain, fever, urinary troubles, hypochondriasis and melancholia. ^[19],[20] Preliminary phytochemical screening of aqueous extract of Aegle marmelos root revealed the presence of alkaloids and glycosides. Studies have shown the evidenceof the presence of various chemical constituents in the Bilwa root. Coumarins ^[21] like marmin, marmesinin, umbelliferone, skimmianine alkaloid ^[22] and triterpenes ^[21] like b-sitosterol and lupeol were identified. Marmin, a coumarin isolated from the roots of Bilwa (1g/kg p.o.) showed anti-inflammatory effect against carrageenan induced inflammation in rats. ^[23] Marmin, marmesin, umbelliferine and skimmianine are identified from the bark and roots which contribute to the anti-inflammatory property of Bilwa. ^[24],[25] Lupeol, a pentacyclic triterpenes showed reduction in paw swelling by 39% compared to 35% by indomethacin. ^[25],[26] Oxidative stress and neutrophil infiltration are common for inflammatory diseases and it is now accepted that gastric ulcer is mainly caused by oxidative stress. Intra gastric administration of marmin at a dose of 25mg/kg body weight exhibited antiulcer activity in experimental ulcer models. ^[7] In another study the in vitro antibacterial potential of chloroform extract of the root of A. marmelos (Correa) Linn was determined by agar dilution and disc diffusion techniques. The in vitro activity was found to be comparable to that of ciprofloxacin. Further root extract treated animals showed significant inhibitory activity against castor oil induced diarrhea. ^[8] Therefore the additional property of antimicrobial along with anti-inflammatory activity of Bilwa root may have synergistic effects on the inflammation induced by the invading pathogens.

Carrageenan is regarded as an established phlogistic agent/oedemogen and edema induced by the subplantar injection of carrageenan in the rat hind paw is reported to have been inhibited by a number of steroidal and non-steroidal anti-inflammatory drugs. ^[16] It has a biphasic effect. The first phase is due to release of histamine and serotonin (5-HT) (0-2 hr), plateau phase is maintained by a kinin like substance (3hr) and second accelerating phase of swelling is attributed to PG release (>4hr). ^[27] In our study the aqueous extract of A. marmelos 100 mg/kg, p.o. significantly reduced edema induced by the carrageenan. The percent inhibition of paw edema by indomethacin was 51.5% while that of Bilwa is 35.7%. Hence Bilwa showed 69.32% anti-inflammatory activity as that of standard Indomethacin under the present experimental conditions. The results obtained in this study are in concurrence with the study done by Udupa et al, where aqueous extract of root was used in a dose of 5mg/100g body weight i.p once daily. ^[28] The probable mechanism of the acute anti-inflammatory activity might be due to the inhibition of release of mediators like histamine, serotonin and prostaglandins. This activity probably will be due to the chemical constituents like marmin, lupeol etc.

Cotton wool granuloma is a method for testing the proliferative phase i.e granuloma formation, provoked by the subcutaneous implantation of compressed cotton pellets. After few days histologically gaint cells and undifferentiated connective tissue can be observed along with fluid infiltration. The amount of newly formed connective tissue can be measured after removal and weighing the dried pellets. ^[16],[18] The percent inhibition of granuloma formation by indomethacin was 24.7% and that of Bilwa was 9%. Hence Bilwa showed 37.24% (P >0.05) anti-granuloma activity as that of standard indomethacin (P < 0.05) under the present experimental conditions. The additional finding, body weight loss observed in the indomethacin group than the Bilwa treated animals, suggest that indomethacin induced gastritis might be responsible for the reduced food intake and leading to loss of weight. Though the Bilwa showed less chronic anti-inflammatory activity compared to indomethacin further assessment of Bilwa with change in the dosage, solvent extracts and other chronic inflammatory models, will throw a more light on its chronic anti-inflammatory activity.

Studies on Bilwa root are fewer and studies on aqueous extract of root bark on different anti-inflammatory models are sparse. Hence the present study has attempted to fill these lacunae of this invaluable drug. The standardization of the extracts, identification and isolation of active principles and pharmacological studies of these needs to studied further. To conclude, a large number of studies on Bilwa have showed antidiabetic, antiulcer, anti-inflammatory, analgesic, antipyretic, antidiarrhoeal, antihyperlipidaemic, antioxidant, anticancer, antimicrobial and radioprotective properties. In the present study aqueous extract of Aegle marmelos root has showed promising results in acute model however these were less effective in the chronic model of experimental inflammation when compared to the standard indomethacin . These studies are valuable for identifying lead compounds for anti-inflammatory drugs, keeping in mind the side effects of NSAIDs and corticosteroids. Further human studies are needed to prove the safety and efficacy of long term administration of aqueous extract of Aegle marmelos as potential anti-inflammatory agent in routine clinical practice.

Acknowledgement

We are thankful to ′The Sadvaidyasala Private Limited′, Nanjangud, Mysore district, Karnataka for providing the Bilwa coarse root powder for the study.

References

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