ANTI ARTHRITIC ACTIVITY OF ACETONE EXTRACT OF TERMINALIA CHEBULA
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1 Research paper Abstract ANTI ARTHRITIC ACTIVITY OF ACETONE EXTRACT OF TERMINALIA CHEBULA Y. Roja Ramani*, A Panda, B Rath, S Pradhan Dept. of Pharmacology M.K.C.G.Medical College, Berhampur, Orissa yrramani@yahoo.com OBJECTIVE: To study the anti-arthritic activity of Acetone Extract of fruits of Terminalia chebula (TCE) in wistar rats in an experimental model of rheumatoid arthritis. MATERIAL AND METHODS: After necessary approval from Institutional Animal Ethics Committee (IAEC), 28 wistar albino rats of either sex ( gm) were randomly divide into 7 groups. Arthritis was induced in all animals by intradermal injection of Complete Freund s Adjuvant (CFA) 0.1ml in plantar surface of left hind paw. The vehicle, TCE (graded doses) and indomethacin (reference standard) was administered p.o from day1 to day13. Changes in paw edema, joint thickness and body weight were measured every alternate day till 21 st day. Erythrocyte sedimentation rate (ESR) and rheumatoid factor (RF) were measured on day0 and day 21. RESULTS: In this current study the Acetone Extract of Terminalia chebula fruits had a better effect on controlling CFA induced arthritis. TCE showed good reduction in paw edema and joint thickness. It produced a reduction of ESR values and RF values comparable to the dexamethasone treated group. Therefore an Acetone Extract of Terminalia chebula fruit has got definite effect in reducing the inflammatory components as well as the above cited results also focus on its immunomodulatory role. CONCLUSION: The results of the present study empirically indicate that Terminalia chebula may be effective in treatment of rheumatoid arthritis and that it supports the common belief prevailing in traditional medicines world wide. Key words: Terminalia chebula, CFA induced arthritis, arthritic index, Rheumatoid factor. Introduction: Arthritic group of disorders has already started showing an upward curve with increasingly sedentary life style. It is estimated that over 13 million Indians are afflicted with some form of arthritis every year. Increase in the geriatric population, changed food habits, chemicals and insecticides, pollution and wide array of factors play an important role in the pathogenesis of this crippling and agonizing syndrome. However, judicious management of arthritis can effectively alleviate symptoms, delay the progression of the disease and minimize the suffering. Rheumatoid arthritis is a type of auto immune disease and is a major cause of disability. Pathogenesis of rheumatoid arthritis is focused on auto antibodies, immune complexes, T-cell-mediated antigen-specific responses and T-cell-independent cytokine networks. Based on the pathogenic mechanisms, specific therapeutic interventions can be designed to suppress synovial inflammation and joint destruction in rheumatoid arthritis 1. Modern medicine uses various drugs like Methotrexate, Cortisones like Dexamethasone etc and NSAIDS like Phenylbutazone and Indomethacin to relive the pain and inflammation in Rheumatoid arthritis at the cost of characteristic profile of side effects. In recent years, there is an
2 upsurge in the clinical usage of indigenous drugs in chronic arthritic disorders, because of their efficacy and being free from serious toxic effects. It is interesting to note that, according to a recent survey conducted by WHO, approximately 80% of world population relies mainly on the traditional medicines for the primary health care. 2 Herbal drugs are believed to enhance the natural resistance of the body against infection 3 and their immunomodulatory activities have been reported in numerous plants. 4 Moreover constant increase in the antibiotic resistant strains and various side effects caused by the synthetic drugs have prompted scientists to look for herbal Immunomodulators to treat various infections. 4 Emphasis is also laid on the integration of indigenous health care systems with modern health facilities. Millions of households in rural and urban areas of our country consume traditional diets, use home remedies and follow health customs based on the principles of traditional systems of medicines 5. To date, the goal of treatment in rheumatoid arthritis is to reduce joint inflammation and pain, maximize joint function, and prevent joint destruction and deformity. Optimal treatment for the disease involves a combination of medications, rest, joint strengthening exercises, joint protection, and patient (and family) education 6.Thus a drug for treatment of this disease is needed which will control joint inflammation, relieve joint pain and protect the arthritic joint from damage without causing serious adverse effects. Terminalia chebula is a plant species belonging to the genus Terminalia, family Combretaceae. It is also known as Haritaki (Sanskrit and Bengali), Harad (Hindi). The fruit of the tree has been used as traditional medicine for household remedy against various human ailments, since antiquity. 7 Terminalia chebula exhibits significant antibacterial 8,9 hepatoprotective 10, cardioprotective 11,12 antimutagenic/anticarcinogenic 13,14, cytoprotective 15, radioprotective 16, antioxidant 17, antianaphylactic 18 and immunomodulatory 19 effects indicating that it is a safe substance to be used as a drug ordinarily. Terminalia chebula, one of the components of Triphala, was shown to be a potent hyaluronidase and collagenase inhibitor that prevented degradation of cartilage. Studies also demonstrate its noncytotoxic and non genotoxic effect both invitro and in vivo. In this context the present study was undertaken to assess the antiarthritic activity of acetone extract of fruits of Terminalia chebula (TCE) in wistar albino rats by using adjuvant induced arthritis model a well known experimental model for rheumatoid arthritis. The anti arthritic effect was accessed by measuring changes in paw edema, joint thickness, body weight, secondary lesions, ESR, and rheumatoid factor. Materials and methods: After necessary approval from Institutional Animal Ethics Committee (IAEC), 28 wistar albino rats of either sex ( gm) were used for the study. Arthritis was induced in all animals by intradermal injection of Complete Freund s adjuvant 0.1ml in plantar surface of left hind paw. Then they were randomly divided into 7 groups (n=4). The vehicle (distilled water), TCE (test drug) and indomethacin / dexamethasone (reference standards) were administered p.o to different groups from day1 to day13. Changes in paw edema, joint thickness were measured by the plethysmometer and micrometer respectively every alternate day till 21 st day and tabulated. ESR (modified micro capillary tube method) and Rheumatoid Factor were measured on day 21. Change in body weight and appearance of any secondary lesions were also observed through out the study period every alternate day.
3 PREPARATION OF TERMINALIA CHEBULA EXTRACT Fruits of Terminalia chebula were collected, dried and powdered by grinding. Approximately 500 g of powder was taken in a beaker, treated with 4 litre of 70% acetone and agitated at a room temperature for about 2 hours. It was then filtrated and the filtrate was evaporated to get a solid substance, which was subsequently dissolved in 1 litre of water and 1 lit of ethyl acetate to obtain an extract. The extracted ethyl acetate layer was separated by using separating funnel. The separated layer was filtered and dried by evaporation finally to get a solid matter; obtained extract was stored under cool conditions. 20 Yield- 20gms, %yield- 4% Experimental protocol: Group Drug Dose(mg/kg) Nature I Distilled water 1ml Control II Dexamethasone 0.1 Standard 1 III Indomethacin 4 Standard 2 IV TCE Test 1 V TCE Test 2 VI TCE Test 3 VII TCE Test 4 Induction of arthritis: Arthritis was induced in all 28 rats by injecting 0.1ml of Complete Freund s Adjuvant (Sigma Aldrich USA) intradermal in the plantar surface of left hind paw. Acute inflammation in the form of paw edema was produced after injecting CFA suspension. Assessment of arthritis: Arthritis was assessed by measuring the following parameters: 1. Left hind paw edema by plethysmometer. The % inhibition of paw edema was calculated by the formula used by Pillai et al. in % inhibition = V C -V T / V C X 100, Where V C = paw edema of control group & V T = paw edema of drug treated group.
4 2. Tibio-tarsal joint thickness by micrometer(screwguage) % reduction in joint thickness = T C -T t / T C X 100, Where T C = joint thickness of control group and T t = joint thickness of drug treated group 3. Body weight: measured on each alternate day from day 0(arthritis induction) to day Secondary lesions: Any lesions like swelling, redness or nodules on the right hind paw, the fore paws, the ears, the nose and the tail, was observed within days in the non injected site was scored and tabulated. Arthritic index was calculated as the sum of the scores as indicated above for each animal. The average of the treated animals is compared with the control group. The total percentage change was calculated as: % inhibitions of the injected paw (day 5) + % inhibitions of the non-injected paw (day 21) + % change of the arthritic index. 5. ESR of each rat by modified micro capillary tube method on 21 st day. 6. Rheumatoid Factor on 21 st day. Statistical analysis: Data was analyzed by one way ANOVA followed by LSD post test and results discussed. p<0.05 was considered as significant. Results: EFFECT OF TERMINALIA CHEBULA EXTRACT ON PAW VOLUME IN CFA INDUCED ARTHRITIS Group Mean Paw Volume on different Days ± ± ± ± ± ± ±0.06 Control 0.95± ±0.05* 0.55±0.05* 0.45±0.10* 0.35±0.05* 0.15±0.05* 0.1±0.06* Dexamethasone 0.85± ±0.05* 0.45±0.13* 0.3±0.06* 0.2±0.0* 0.1±0.06* 0.05±0.05* Indomethacin TCE ± ± ± ± ±0.06* 0.35±0.05* 0.3±0.06* TCE ± ± ± ±0.05* 0.35±0.05* 0.2±0.05* 0.15±0.05* Values are expressed in mean ± SEM. * indicates p < 0.05; df =3
5 EFFECT OF TERMINALIA CHEBULA EXTRACT ON JOINT THICKNESS IN CFA INDUCED ARTHRITIS Group Mean joint thickness on different Days Control 9.89± ± ± ± ± ± ±0.84 Dexamethasone 8.69± ±0.91* 5.70±0.93* 4.98±0.64* 3.04±0.68* 1.06±0.26* 0.48±0.11* Indomethacin 9.38± ± ±0.86* 5.89±0.61* 3.69±0.95* 0.92±0.15* 0.63±0.11* TCE ± ± ± ±0.71* 6.43±0.91* 4.55±0.99* 3.80±0.88* TCE ± ± ± ±0.05* 4.75±0.92* 3.04±0.75* 2.24±0.64* Values are expressed in mean ± SEM. * indicates p < 0.05; df =3 EFFECT OF TERMINALIA CHEBULA EXTRACT ON ARTHRITIC INDEX Sl.no Groups Control Dexamethasone Indomethacin TCE 160 TCE Mean * * SEM * indicates p < 0.05; df =3
6 EFFECT OF TERMINALIA CHEBULA EXTRACT ON ESR Groups Sl.no Control Dexamethasone Indomethacin TCE 160 TCE Mean * 10.45* 11.8* 11* SEM * indicates p < 0.05; df =3 EFFECT OF TERMINALIA CHEBULA EXTRACT ON RHEUMATOID FACTOR Groups Sl.no no Control Dexamethasone Indomethacin TCE 160 TCE Mean * * 18.3* SE Discussion: * indicates p < 0.05; df =3 The present study was designed to evaluate the anti-arthritic activity of Acetone extract of Fruits of Terminalia chebula (TCE), which is very commonly used in Indian system of medicine. Freund s adjuvant is used for induction protocols of many experimental animal models of autoimmune disease like Rheumatoid Arthritis. Adjuvant arthritis in rat has been described by
7 Pearson and Wood (1959) exhibiting many similarities to human rheumatoid arthritis. This model was selected for its simplicity and facts can be easily presented. Terminalia chebula is reported to contain hydrolysable tannins such as chebuline, chebulagic acid, gallic acid that have produced anti arthritic effect 21. From the previous studies chebulagic acid from immature seeds of Terminalia chebula suppressed onset and progression of collagen-induced arthritis in mice and produced immune suppression via induction of transforming growth factor beta(tgfβ) and CD4+,CD25+ T cells indicating a new strategy to treat rheumatoid arthritis and other related diseases. In this study acetone extract of Fruits of Terminalia chebula in doses of 40 and 80 mg/kg did not have any effect on adjuvant induced arthritis where as 160 and 320 mg/kg showed antiinflammatory effect as it decreased the paw edema significantly (p<0.05). It also produced a dose dependent anti-inflammatory, anti-arthritic activity which was comparable to Dexamethasone treated group. Results of this study are in agreement to the available literatures. To enumerate, all the above parameters i.e. paw volume, joint thickness, ESR, Arthritic index, Rheumatoid factor, it was seen that Terminalia chebula produced significant anti-inflammatory, antiarthritic and immuno modulatory activities at a higher dose of 320mg/kg. Also, in contrast to the traditional NSAIDs which are reported to produce gastric ulceration and G.I. bleeding, TCE is reported to have gastro protective effect 22. Conclusion: To conclude acetone Extract of Terminalia chebula fruits had a better effect on controlling CFA induced arthritis. It produced a reduction of ESR values and Rheumatoid factor values comparable to the Dexamethasone treated group. Therefore this fruit has got definite effect in reducing the inflammatory components as well as the above cited results also focus on its immunomodulatory role. Further, extraction of the active ingredient responsible for the above cited results along with biochemical analyses, evaluating its role against various inflammatory mediators like TNF-α, and subsequent human trials may further elucidate the potential role of Terminalia chebula in treating rheumatoid arthritis patients. Reference: 1. Gary, Firestein S., Evolving concepts of rheumatoid arthritis, Bhutani K.K., Herbal medicines An enigma and challenge to science and directors for new initiatives, Ind. J. Nat. Prod., 2001, 19(1), pages Hassan J. O., Curtiss R., Infect. Immun., 62, (1994). 4. Atal C. K., Sharma M. L., Kaul A., Khajuria A., J. Ethanopharmacol., 41, (1986). 5. Satyavathi.G.V,Gupta.A.K,Tandon, Medicinal plants of India. Indian council of Medicinal Research, New Delhi, 1987, pages Goodman and Gilman s, Drugs for Rheumatoid arthritis:in The pharmacological basis of therapeutics 11 th edition, pages
8 7. Chattopadhyay R.R., Bhattacharya S.K., PHCOG REV: Plant Review Terminalia chebula, Pharmacognosy Reviews, 2007, Vol -1, Issue 1, Jan-May, Page Ahmad, Mehmood Z., Mohammad F., Screening of some Indian medicinal plants for their antimicrobial properties, J. Ethnopharmacol, 1998, 62(2), pages Malckzadeh F., Ehsanifar H., Shahamat N., Levin M., Colwell R.R., Antibacterial activity of black myrobalan (Terminalia chebula Retz.) against Helicobactor pyroli, Int. J. Antimicrob. Agent. 2001, 18(1), pages Tasduq S.S., Singh A.K., Salti N.K., Gupta D.K,Suri K., Terminalia chebula fruits prevent liver toxicity caused by sub-chronic adminstration of refampicin, isoniazid and pyrazinamide (PZA) in combination, Human and Exp. Toxicol., 2006, 25(3), pages Suchalatha S, Shyamadevi C.S., Protective effect of Terminalia chebula against experimental myocardial injury induced by isoproterenol, Ind. J Exp. Biol, 2004, 42(2), Hamada S., KataokaT., Woo J.T,Yamada A. et al., Immunosuppressive effects of Gallic acid and chebulic acid on CTL-mediated cytotoxicity. Biol. Pharm Bull. 1997, 20(9), pages Kaur S., Grover I.S., Singh M., Kaur S., Antimutagenesity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimerium. Mutagen Res. 1998, 419(1-3), pages Saleem, Hushum M., Harkonen P., Pihlaja K., Inhibition of cancer cell growth by crude extract and phenolics of Terminalia chebula fruit, J. Ethnopharmacol., 2002, 81, pages Na M, Bae M., Keng S.S., Min.B.S., et al., Cytoprotective effect on oxidative stress and inhibitory effect on cellular aging of Terminalia chebula fruit. Phytother. 2004, Res. 18(9), pages Gandhi N.M., Nayar C.K.K., Radiation protection by Terminalia chebula some mechanistic aspects. Molecular and Cellular Biochemistry, 2005, 277(1-2) pages Lee H.S., Won N.H., Kim K.H., H. Lee, W. Jun, K.W. Lee, Antioxidant effects of aqueous extract of Terminalia chebula in vivo and in vitro, Biol. Pharm, 2005, Bull.28 (9), pages Shin T.Y, Jeong H.G, kim D.K. etal., Inhibitory action of water soluble fraction of Terminalia chebula on systematic and local anaphylaxis.j. Ethnopharmacol., 2001, 74, pages Fulzele SV, Satturwar PM, Joshi SB, Dorle AK., Indian Journal of Pharmacology, 2003, 35, Compounds with Diphenoyl -Structure for The Treatment of Immune Diseases, (Wo/2006/022502). 21. Sang I.K.L., Pung M.H., Seung H.K., Suppression of the onset and progression of collagen-induced arthritis by chebulagic acid screened from a natural product library, Arthritis Rheum, 2005 Jan, 52, Ray P.G., Majumdar S.K., Antimicrobial activity of some Indian plants, Econ. Bot, 1976, pages
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