Anti-Inflammatory Effect of the Embelia tsjeriam -Cottam Fruit Extracts.

Sambrekar Sudhir N.¹* Patil P.A.² and Suhas A. Patil¹

¹Dept of Pharmacology, Maratha Mandal’s College of Pharmacy, Belgaum, India.

²Dept of Pharmacology, K.L.E’s Institute of Dental Sciences Bangalore.India.

ABSTRACT:

Extracts of Embelia tsjeriam-cottam Fruits were investigated for anti-inflammatory activity with acute and chronic models. In the doses of 200 mg kg-¹exhibited significant (P < 0.001) anti-inflammatory activity in all the models tested. The alcohol extract of Embelia tsjeriam-cottam at 200 mg kg-1 showed maximum inhibition of paw volume 1.21 ± 0.1 in carrageenan-induced rat paw edema while the standard drug Diclofenac it was 1.18 ± 0.1 after 6 hrs of carrageenan injection. In the chronic inflammatory model alcoholic extract inhibited the granuloma weight by 35.50 %, whereas the Diclofenac inhibited 46.18%.

KEYWORDS: Anti-inflammatory activity, carrageenan induced rat paw, cotton pellet induced granuloma, Embelia tsjeriam-cottam

INTRODUCTION:

Inflammation is a local response of living mammalian tissues to the injury. It is a body defense reaction in order to eliminate or limit the spread of injurious agents. 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. Edema formation in the paw is the result of a synergism between various inflammatory mediators that increase vascular permeability and/or the mediators that increase blood flow. Several experimental models of paw edema have been described. Carrageenan-induced paw edema is widely used for determining the acute phase of inflammation. Histamine, 5-hydroxytryptamine and bradykinin are the first detectable mediators in the early phase of carrageenan-induced inflammation whereas prostaglandins are detectable in the late phase of inflammation. Drugs which are in use presently for the management of pain and inflammatory conditions are either narcotics e.g. opioids or non-narcotics e.g. salicylates and corticosteroids e.g. hydrocortisone. All of these drugs possess well known side and toxic effects. Moreover, synthetic drugs are very expensive to develop and whose cost of development ranges from 0.5 to 5 million dollars. On the contrary many medicines of plant origin had been used since long time without any adverse effects. Exploring the healing power of plants is an ancient concept. For centuries people have been trying to alleviate and treat disease with different plant extracts and formulations. It is therefore essential that efforts should be made to introduce new medicinal plants to develop cheaper drugs. Plants represent still a large untapped source of structurally novel compounds that might serve as lead for the development of novel drugs. Screening of the plants for their biological activity is done on the basis of either their chemotaxonomic investigation or ethnobotanical knowledge for a particular disease.

Identification of a particular compound against a specific disease is a challenging long process. Importance of the plant lies in their biologically active principles. There are two types of plant chemicals, primary metabolites such as sugars, proteins, amino acids, chlorophylls etc. The other category of chemicals is called secondary metabolites, which includes alkaloids, terpenoids, saponins and phenolic compounds. These chemicals exert a significant physiological effect on the mammalian system. A lot of references are available in the field of ethnomedicinal plants used as anti-inflammatory drugs.

Embelia tsjeriam-cottam is cultivated in India, Myanmar and Sri lanka. In India it is common in the most forests of Karnataka, Kerala and Tamilnadu. It is propagated by seeds. Fruit contains embelin, quercitol, fatty ingredients and vilangin. Fruits of Embelia tsjeriam used as appetizer; carminative, anthelmintic, taenifuge, antispasmodic alexiteric, laxative, alterative; cures tumours, ascites, bronchitis, mental diseases, dyspnoea, heart diseases, blood purifier, fever, tonsil, urinary discharges, useed in snake-bite, stomach disorders,Anti-inflammatory, Jaundice, hemicrania, wound, analgesic, purgative indolent ulcer of the mouth and the gums. Sushruta describes the use of fruits along with liquorice root for the purpose of strengthening the body and preventing the effect of age.^1,2,3

Preparation of extract:

Embelia tsjeriam-cottam fruits collected from open field around Belgaum city in the month of May - June were identified and authenticated by the taxonomist Dr. Harsha Hegde and the herbarium (voucher no. RMRC-487) has been preserved at Regional Medical Research Centre, Belgaum a unit of Indian council for medical research New Delhi. Dried fruits were powdered to moderately coarse grade. Petroleum ether, chloroform, alcohol and aqueous extracts of fruits were obtained by using soxhlet extractor. The extraction was continued for 12 cycles or until the solvent in the thimble was clears. After evaporating the solvent, the dark brown semisolid extract was obtained .The phytochemical analysis was carried out and further extract was kept in an air tight container at 4°C for future use.

Phytochemical analysis:

The extracts were subjected to phytochemical analysis for constituent identification using standard protocol.⁴

Animals:

Female Wister rats 180-200 g of either sex were used for Anti-inflammatory study and Swiss albino mice of either sex weighing between 20-30 gms for acute toxicity study to determine LD₅₀ of various extracts. Animals were procured from Venkateshwara Enterprises, Bangalore. The animals were kept in the standard polypropylene cages and provided with food and water ad libitum. The animals were acclimatized for a period of 14 days prior to perform the experiments. The experimental protocols were approved by Institutional Animal Ethical Committee (Resolution No 1/2/2007, dated 23-11-2007).

Acute Oral Toxicity study:⁵

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co–operation and Development (OECD), received draft guidelines 425, received from Committee for the Purpose of Control and Supervision of Experiments on Animals ( CPCSEA) , Ministry of Social Justice and Empowerment, Government of India. Swiss albino mice weighing between 20-30 gms were fasted over night prior to the acute experimental procedure. The principle, which is based on a stepwise procedure with the use of a minimum number of animals per step. The LD₅₀ of different extracts were determined. The therapeutic dose was calculated as1/10^th of the lethal dose for further investigation

Carrageenan induced rat paw edema:⁶

The rats were divided into four groups, each group consisting of six animals. Edema was induced by subplantar injection of 0.1% freshly prepared carrageenan suspension into the right hind paw of each rat. The paw volume was measured at 1 h, 3 h and at 6^thhour after the injection of carrageenan, using a plethysmometer.⁷The alcoholic and aqueous extract of Embelia tsjeriam-cottam at 200 mg kg-1 doses were administered orally to first two groups of rats. The third and fourth groups of rats received 5 ml kg-1 of 2% w/v Tween 80 orally as vehicle control and 50 mg kg-1 diclofenac as standard respectively, for assessing comparative pharmacological significance. Drug pretreatment was given 1 h before the injection of carrageenan.

Cotton pellet induced granuloma:⁸

The four groups of rats, six in each group was included in this study. After shaving off the fur the animals were anaesthetized. Sterile preweighed cotton pellets (50 ± 1 mg) were implanted in the axilla region of each rat through a single needle incision.⁷ Alcoholic and aqueous extract of Embelia tsjeriam-cottam at a dose of 200 mg kg-1, diclofenac at 50 mg kg-1 (standard) and 5 ml kg-1 of 2%w/v Tween 80 (control) were administered orally to the respective group of animals for seven consecutive days, from the day of cotton-pellet implantation. On the eighth day, the animals were anaesthetized again; the cotton pellets were removed surgically and made free from extraneous tissues. The pellets were incubated at 37 °C for 24 h and dried at 60°C to constant weight. The increase in the dry weight of the pellets was taken as a measure of granuloma formation.

Statistical analysis:

Results are expressed as Mean ± S.E.M. The difference between experimental groups was compared by One-way Analysis of Variance (ANOVA) followed by Dunnett’s test. The results were considered statistically significant when P < 0.05.

Table-1: Phytochemical Screening of Embelia tsjeriam-cottam.

Extracts	Steroid	Carbo hydrate	Alkaloides	Saponin	Tannin	Flavonoid
Alcohol	+++	-	++	+	+++	++
Aqueous	-	-	+	-	++	+

+++ = High concentration, ++ = medium concentration, + = low concentration, - = absent.

Table-2: Effect of fruit extract of Embelia tsjeriam-cottam on Carrageenan induced Rat Paw Edema.

		Paw volume in ml (MEAN ± SEM)
Group	Dose/kg b.w.	0 hr	1 hr	3 hr	6 hr
Control	1%Tween 80 1mlv/v	1.28 ± 0.01	1.82 ± 0.01	1.91 ± 0.02	2.05 ± 0.01
Standard	50	1.21 ± 0.01	1.68 ± 0.01***	1.28 ± 0.01***	1.18 ± 0.01***
Alcoholic Extract	200	1.21 ± 0.01	1.59 ± 0.01***	1.33 ± 0.01***	1.21 ± 0.01***
Aqueous .Extract	200	1.27 ± 0.01	1.70 ± 0.01**	1.77 ± 0.01**	1.52 ± 0.01**

Standard=Diclofenac.

Each value is the Mean ± S.E.M. for 6 rats . **=P < 0.01; ***P <0.001 compared with control.

Table-3: Effect of fruit extract of Embelia tsjeriam-cottam on Cotton Pellet-Induced Granuloma in Rats.

GROUPS	DOSE mg/kg	WEIGHT OF COTTON PELLET-DRY(mg)	% INHHIBITION
Control	Tween 80 1mlv/v	55.00 ± 0.20	-
Standard	50	29.60 ± 0.35***	46.18
Alcoholic Extract	200	35.47 ± 0.38***	35.50
Aqueous .Extract	200	46.15 ± 0.25**	16.09

Standard=diclofenac.

Each value is the Mean ± S.E.M. for 6 rats **=P < 0.01; ***P < 0.001 compared with control

RESULTS:

The phytochemical investigation results are given in Table-1. Phytochemical studies of various extract showed presence of sterols, flavonoids, tannins and alkaloids.

The activities of alcoholic and aqueous extracts of Embelia tsjeriam - cottam against Carrageenan induced rat Paw Edema are given in Table-2. Both the alcoholic and aqueous extracts administered orally (200mg/kg) showed significant inhibitory effect against edema formation, 1, 3 and 6 hours after carrageenan injection. The peak inhibitory effect of alcoholic extract of Embelia tsjeriam - cottam was comparable with Diclofenac treated group (50mg/kg).

The results of Cotton pellet induced granuloma test are given in Table-3 The Embelia tsjeriam - cottam showed similar significant (p<0.001) inhibition of granuloma formation, comparable to that of Diclofenac.

In all the above parameters used for the study aqueous extract also showed the anti-inflammatory effect but it was less significant (p < 0.01) as compared to alcoholic extract treated groups ( p <0.001).

DISCUSSION:

In spite of tremendous development in the field of synthetic drugs during recent era, they are found to have some or other side effects, whereas plants still hold their own unique place, by the way of having no side effects. Therefore, a systematic approach should be made to find out the efficacy of plants against inflammation so as to explore them as herbal anti-inflammatory agents. The enzyme, phospholipase A2, is known to be responsible for the formation of mediators of inflammation such as prostaglandins and leukotrienes which by attracting polymerphonuclear leucocytes to the site of inflammation would lead to tissue damage probably by the release of free radicals. Phospholipase A2 converts phospholipids in the cell membrane into arachidonic acid, which is highly reactive and is rapidly metabolized by cyclooxygenase (prostaglandin synthesis) to prostaglandins, which are major components that induce pain and inflammation.^9,10

It is well known that carrageenan induced paw edema is characterized by biphasic event with involvement of different inflammatory mediators. In the first phase (during the first 2 h after carrageenan injection), chemical mediators such as histamine and serotonin play role, while in second phase (3 – 4 h after carrageenan injection). kinin and prostaglandins are involved.¹¹As our results revealed that administration of ethanolic extract during all phases of inflammation, which is probably inhibition of different aspects and chemical mediators of inflammation. The cotton-pellet granuloma is widely used to evaluate the transudative and proliferative components of the chronic inflammation.

The moist weight of the pellets correlates with transude, the dry weight of the pellet correlates with the amount of granulumatous tissues.^12,13 Chronic inflammation occurs by means of the development of proliferate cells These cells can be either spread or in granuloma form. Non-steroidal anti-inflammatory drugs decrease the size of granuloma which results from cellular reaction by inhibiting granulocyte infiltration, preventing generation of collagen fibers and suppressing mucopolysaccharides.^14,15The alcoholic extract of Embelia tsjeriam - cottam showed significant anti-inflammatory activity in cotton pellet induced granuloma and thus found to be effective in chronic inflammatory conditions, which reflected its efficacy in inhibiting the increase in the number of fibroblasts and synthesis of collagen and mucopolysaccharides during granuloma tissue formation.

CONCLUSION:

The recent studies of anti-inflammatory activity claims that the flavonoid promote significant anti-inflammatory property.¹⁶ From the above studies it is quite apparent that the alcoholic extract possesses significant anti-inflammatory activity because of presence of flavonoid. The study justifies the use of Embelia tsjeriam - cottam in inflammation as suggested in the folklore medicines

REFERENCES:

1. Kirtikar, Basu B.D.(1984) Indian Medicinal Plants. Vol-II 2^nd ed. Singh B and singh MP, Dehraun, 1475-81.

2. Khare, C P. Indian Medicinal Plants. Springer US. 2008: 168

3. Sharma PC, Yelne MB, DennisTJ (2002). Database on medicinal plants used in Ayurveda, vol.5.central council for research in Ayurveda and Siddha, govt. of India. New Delhi,478-99.

4. Trease GE, Evans MC(1983) Text Book of Pharmacognosy,12^th edition Balliere,Tindall:London,343-353.

5. OECD guidelines for Testing Chemicals, Guidelines 425, acute oral toxicity: Acute toxic toxic class method. Paris: (1996).

6. Winter CA, Risley EA, Nuss GW(1962). Carrageenin induced oedemain the hind paw of the rat as an assay for anti-inflammatory drug. Proc. Soc. Exptl. Biol. Med. 111:544-547

7. Mohammed M.et al. Studies on the anti-inflammatory,antipyretic and analgesic activities of Santonin.Japan J. Pharmacology,64:135-139

8. Winter, C.A. and Porter, C.C., Journal of American Pharmaceutical Association. 1957,46:515-519.

9. Higgs GA, Moncada S, Vane JR (1984). Eicosanoids in inflammation. Ann. Clin. Res. 16: 287-299.

10. Vane JR (1971). Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature New Biol.231-235.

11. Hernandez PM, Rabanal Gallego R (2002). Evaluation of the anti-inflammatory and analgesic activity of Sideritis anariensis var. pannosa in mice J. Ethnopharmacol. 81: 43-47.

12. Lowry OH, Rosebrough NJ, Far AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. J. Biol . Chem. 193: 265-275.

13. Castro J, Saseme H, Sussman H, Bullette P (1968). Diverse effect of SKF 52 and antioxidants on CCL4 induced changes in liver microsomal P-450 content and ethylmorphine metabolism. Life Sci.7: 129-136.

14. Della Loggia A, Tubaro A, Dri P, Zilli C, Del Negro P (1968). The role of flavonoids in the anti-inflammatory activity of Chamomilla recutita. Clin. Biol. Res. 213: 481-486.

15. Alcaraz MJ, Jimenez MJ (1988). Flavonoide, an antiinflammatory agents. Fitoterapia 59: 25-38.

16. Pandurangan A. Khosala RL, Hemalatha S.,Anti-inflammatory activity of an alkaloid from Solanum trilobatum on acute and chronic inflammation models. Nat Prod Res. 2011 Mar 11:1-10.(PubMed).

Received on 12.08.2011

Accepted on 01.08.2011

Research J. Pharmacology and Pharmacodynamics. 3(5): Sept –Oct. 2011, 246-249