IJPRD, 2011; Vol 3(4): June 2011 (38 - 45)

International Standard Serial Number 0974 9446

-------------------------------------------------------------------------------------------------------------------------------------------------AEGLE MARMELOS SUPPRESSES INFLAMMATION AND CARTILAGE DESTRUCTION IN COLLAGEN-INDUCED ARTHRITIC RAT. Hardik P. Trivedi1*, Nimish L. Pathak1, Mahendra G. Gavaniya1, Anjana K. Patel1, Dr. Harshkant D. Trivedi1, Nitin. M Panchal1
1

C.U. Shah College of Pharmacy and Research Wadhwan, Gujarat-363030

Correspondence to Author ABSTRACT Aegle marmelos has multiple applications in Indian traditional medicine because of its anti-pyretic, anti-inflammatory, antidiabetic and antimicrobial activities and also have immunosuppressive activity. However, no study on the anti-arthritic activity of Aegle mamrmelos has been reported in vivo. Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic inflammation characterized by hyperplasia of synovial cells in affected joints, which ultimately leads to the destruction of cartilage and bone. We investigated therapeutic efficacy of Aegle marmelos in treating Rheumatoid Arthritis (RA) using collagen-induced arthritis (CIA) animal model. Arthritis was induced in wistar rats by immunization with bovine type II collagen. CIA rats were treated daily with oral administration of different doses of Methanolic extract of A.marmelos (MAM) beginning on the day after the onset of arthritis (day 21st, the therapeutic treatment) until day 45. The results showed that treatment with MAM markedly reduced paw swelling and arthritic index even in the established CIA. Radiologic and histopathologic changes in the arthritic joints were also significantly reduced in the MAM-treated versus vehicle-treated rats. Moreover, Rheumatoid factor was significantly reduced in MAM treated group in compared to disease control group. Hence, our studies demonstrate safety, and effectiveness of A.marmemlos as an anti-arthritic agent, which makes A.marmelos a strong candidate for further research on rheumatoid arthritis (RA).

Hardik P. Trivedi C.U. Shah College of Pharmacy and Research Wadhwan, Gujarat-363030

Email Hardik_trivedi23@yahoo.com Key Words Arthritis, Collagen Induced arthritis, Aegle Marmelos, Cartilage destruction

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International Journal of Pharmaceutical Research & Development

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INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory disease involving multiple joints. The main pathology of the affected synovial tissue consists of hyperplasia and subintimal infiltration of T and B lymphocytes. Synovial tissue hyperplasia forms the pannus tissue that irreversibly destroys the cartilage and bone in the affected joint. Collagen-induced arthritis (CIA) rat is a widely studied animal model of inflammatory polyarthritis with similarities to RA. CIA is induced after immunization of susceptible strains of rat with bovine type II collagen (CII) in Incomplete Freunds adjuvant (IFA), and the resulting disease is primarily mediated by an autoimmune response. [1,2] The significance of the model lies in the fact that CII is the major constituent protein of the cartilage in the diarthrodial jointsthe primary site affected in RA.[2] Drug therapy for RA is based on two principal approaches: symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs).[3] However, most of the currently available drugs are primarily directed towards the control of pain and/or the inflammation associated with joint synovitis, but do little to interfere with the underlying immuneinflammatory events, and consequently also do little to block the disease progression and reduce cartilage and bone destruction of joints. A systematic review of randomized placebo-controlled trials conducted recently demonstrates that the published evidence supports only the efficacies of nine agents in Western medicine, i.e., infliximab, cyclosporin, sulphasalzine, leflunomide, methotrexate, parenteral gold, corticosteroids, auranofin and IL-1 receptor antagonist, in decreasing radiological progression in RA.[4] Nevertheless, a variety of problems exist with these drugs. For example, the use of methotrexate and leflunomide is impeded by their long-term side-effects and toxicity,[5] while cytokines antagonists, despite substantial efficacy and clinical improvement, entail high cost and incur hypersensitivity to medications and infections.[6] Consequently, there is dramatically growing interest in herbal medicines among persons with RA and in the RA research community.[7] In fact, herbal medicine is being widely used virtually around the world for treatment of rheumatic and arthritic

diseases.[7,8,9,10,11] Thus, herbal medicines constitute a potentially important avenue leading to novel therapeutic agents for RA that may not only prevent structural damage of arthritic joints caused by tissue and bone breakdown, but also be safe, relatively inexpensive, highly tolerated, and convenient for many patients.Aegle marmelos (AM) a highly reputed ayurvedic medicinal tree commonly known as bael fruit tree is found all over India . The tree is endowed with various medicinal properties. Several studies on different parts of AM showed the plant possess antidiarrhoel [12], antidiabetic [13], anticancer [14], radio protective [15], antifungal [16], antimicrobial [17], antimicrofilarial [18], and anti-inflammatory, anti-pyretic, and analgesic activities [19]. AM has been used in nervous disorder and as tonic for brain [20, 21]. Phytochemical analysis of AM leaves have shown to contain several bioactive compounds including essential oil (e.g marmenol, - caryophyllene, - humulene), triterpenoids (e.g. lupeol, & - sitostrelo), flavoinds (e.g rutin), alkaloids (rutacin, aegeline, aegelinine) coumarins (e.g- marmesinin, umbeliferone), condensed tannins, anthocyanins and flavoinds glycosides [19, 22-24] MATERIAL AND METHODS Plant Material The disease free fresh plant material (Leaves) was collected in the month of September 2009 from surrounding area of Wadhwan, Gujarat and authenticated at Botany Department of Gujarat University. After authentication, fresh leaves were collected in bulk from the tree, shade dried, pulverized and passed through sieve no.40 to obtain coarse powder. Preparation of the Extract The powdered leaves (400 gm.) were subjected to continuous hot extraction with methanol in Soxhlet extractor for 48 hrs, followed by concentrating extract under vacuum. The extracts were stored in airtight bottle until use. Animals The study was carried out with adult female Wistar rats weighing 180250 g. Animals were acclimatized to the experimental conditions in cages and kept under standard environmental conditions (22 3C; 12/12 h

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International Journal of Pharmaceutical Research & Development light/dark cycle). Rats were allowed to feed and water ad libitum. Induction of CIA and MAM treatment Arthritis was induced in female Wistar rats using the method described previously.[25] Collagen was dissolved overnight at 4C in 0.1M Acetic acid to prepare concentration 2mg/ml. This solution was added drop wise to an equal volume of chilled incomplete Freunds adjuvant (IFA). On day first, animals (five groups, each containing six animals) receive total 0.5 ml collagen in 0.5 ml, equally divided in 5 sites. All injections are intradermal, one at the base of each appendages and one in the nape of neck. Seven days post-immunization, the animals receive identical booster injections. Control animals receive only the incomplete Freunds adjuvant diluted with 0.1 M acetic acid. In the therapeutic treatment protocol for the established CIA, treatment with MAM, Dexamethazone, and vehicle were initiated on the day after the onset of arthritis (day 21) and continued once daily until day 45 of the experiment. Rats were treated orally with different dose of MAM (200, 400, 600 mg/kg of body weight), Dexamethasone (1mg/kg) and vehicle until day 45 of experiment. Evaluation of the development of arthritis Rats were inspected daily for the onset of arthritis characterized by edema and/or erythema in the paws. The incidence and severity of arthritis were evaluated using a system of arthritic scoring, and measurement of bi-hind paw volumes every 2 or 3 days beginning on the day when arthritic signs were first visible. Animals were observed for presence or absence of nodules in different organs like ear, fore paw, hind paw, nose and tail. Animal were score 0 for absence and 1 for presence of nodules. 5 was the potential maximum of combined arthritic score per animal. Hind paw volume was measured using plethysmometer. Paw volumes of both hind limbs were recorded from 21st day to 45th day at four day interval using mercury column [26] plethysmometer. Rheumatoid factor The latex turbidimetry method was used in the present study using RF turbilatex kit of SPINREACT Company. Calibration was carried out for linear range up to 100 IU/ml. The reading of RF factor of all the groups obtained was compared with the control animals and was expressed as IU/ml RF.
[27]

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Radiography Female wistar rats were sacrificed on 45 day of collagen administration and legs were removed and placed on formalin containing plastic bag. This plastic bag was kept at a distance of 90 cm from the X-ray source was and Radiographic analysis of arthritic and treated animal hind paw were performed by X-ray machine with a 300-mA exposition for 0.01 s. An investigator blinded for the treatment regimen performed radiograph score. The following radiograph criteria were considered: These scores (destroyed or intact joint) were used as a quantal test for bone necrosis. Radiographs were carefully examined using a stereo microscope and abnormalities were graded as follows: (i) Periosteaic reaction, 0 - 3 (None, Slight, Moderate, Marked); (ii) Erosions, 0 - 3 (None, Few, Many Small, Many Large); (iii) Joint space narrowing, 0 - 3 (None, Minimal, Moderate, Marked); (iv) Joint space destruction, 0 - 3 (None, Minimal, Extensive, Ankylosis). Bone destruction was scored on the patella as described previously. Histological processing and assessment of arthritis damage Rats were killed by ether anesthesia. Knee joints were removed and fixed for 4 days in 4% formaldehyde. After decalcification in 5 % formic acid, the specimens were processed for paraffin embedding tissue sections (7 m thick) and were stained with haematoxylin and eosin, or safranin. An experienced pathologist, unaware of the different drug treatments scored the condition of tibiotarsal joints. Histopathological changes were scored using the following parameters. Infiltration of cells was scored on a scale from 0 to 3, depending on the amount of inflammatory cells in the synovial tissues. Inflammatory cells in the joint cavity were graded on a scale from 0 to 3 and expressed as exudate. A characteristic parameter in arthritis is the progressive loss of articular cartilage. This destruction was separately graded on a scale from 0 to 3, ranging from the appearance of dead chondrocytes (empty lacunae) to complete loss of the articular cartilage. Bone erosion was scored on a scale ranging from 0 to 3, ranging from no abnormalities to complete loss of cortical and
[26]

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International Journal of Pharmaceutical Research & Development trabecular bone of the femoral head. Cartilage and bone destruction by pannus formation was scored ranging from 0, no change; 1, mild change (pannus invasion within cartilage); 2, moderate change (pannus invasion into cartilage/subchondral bone); 3, severe change (pannus invasion into the subchondral bone); and vascularity (0, almost no blood vessels; 1, a few blood vessels; 2, some blood vessels; 3, many blood vessels). Histopathological changes in the knee joints were scored in the femur region on 5 semiserial sections of the joint, spaced 70 m apart. Scoring was performed on decoded slides by two observers, as described earlier. RESULTS Following the primary and booster injections of CII/IFA, rats developed arthritis from day 21st onwards, and treatment with MAM, Dexamethazone(1 mg/kg), and vehicle were initiated on day 21st after the primary immunization and continued until day 45th of the experiment. As shown in Fig. 1, even after the onset of arthritis, MAM at high doses (200, 400 and 600 mg/kg) did markedly reduce hind paw volume of the arthritic rats in a dose-dependent manner as compared to the vehicle-treated arthritic rats. Arthritic index and rheumatoid factor were significantly decreased in treatment with MAM (200 mg/kg, 400 mg/kg and 600mg/kg) and dexamethasone (1 mg/kg) treated animal as compare to disease control treatment. Bone destruction, which is a common feature of arthritis, was examined by radiological analysis. Collagen administered rats had developed definite joint space narrowing of the intertarsal joints, diffuse soft tissue swelling that included the digits, diffuse demineralization of bone, marked periosteal thickening, and cystic enlargement of bone and extensive erosions produced narrowing or pseudo widening of all joint spaces. In contrast, in rats treated with MAM attenuate abnormalities consisted of asymmetric soft tissue swelling and small erosions, periosteal thickening, and minimal joint space narrowing, predominantly localized to the proximal areas of the paws. As shown in Fig 5 a [NC]: Histology of synovial joint of normal control rat with intact morphology of synovium and synovial lining Fig 5 b [DC]: CA induced disease control rat showed plenty of lymphocytic infiltration
[28-31]

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[]in synovial lining with severe inflammation and marked angiogenesis [] studied with proliferation of synovial cells []Fig 5 c [DEXA]: Dexamethasone [5 mg/kg] treated rats showed significant protection with mild lymphocytic infiltration [] with no evidence of thickening of synovial lining and angiogenesis Fig 5 d MAM200: MAM 200 treated rats showed milder angiogenesis [], lymphocytic infiltration [] and synovial lining thickening[]. Fig 5 e MAM400: MAM400 treated rats showed milder angiogenesis [], synovial lining thickening [] with no evidence of lymphocytic infiltration. Fig 5 f MAM600: MAM600 treated rats showed milder lymphocytic infiltration [] and synovial-lining thickening [] with no evidence of angiogenesis. DISCUSSION RA is a complicated refractory autoimmune disease characterized by a number of the inflammatory and destructive events such as joint pain and swelling, synovial hyperplasia, pannus formation, cartilage and bone erosions, joint malformation.[32] Although various therapeutic approaches, such as cytokine-, receptor-, signal-transduction- or cell-type directed intervention, have been done, the inflammatory and destructive processes of RA are impeded partly, but rarely blocked fully, which reflects both the complexity and multiplicity of the pathogenesis. These findings indicate that treatment targeting individual molecules might not suffice to stop the ongoing events, while combination therapy, which interferes with multiple targets, might be more effective for the treatment of RA. [33] In this regard, herbal medicines, comprising multiple active components with broad ranging pharmacological activities, represent an ancient model of combination therapy that could serve us well today. Evaluation of the inflammatory stratus in RA is reflected by inflammation in the hind paw. Progression of disease in MAM treated group shows reduction in edema in dose dependent manner as compare to tissues control animals. Symmetric involvement of small hand joints (especially proximal interphalangeal and metacarpophalangeal), foot joints (metatarsophalangeal), wrists, elbows, and ankles is typical, but initial manifestations may occur in any joint. Inflammation and / or nodules are observed on ears, nose, and tail, fore paws and hind paws. Arthritic index

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International Journal of Pharmaceutical Research & Development is the average of the score given to severity of the lesions in these places. This gives full picture of the disease. [34] MAM treated animal showed significant lesser arthritic index as compared with disease control animals. Prominent immunological abnormalities that may be important in pathogenesis of RA include immune complexes are found in joint fluid cells and in vasculitis. Plasma cells produce antibodies e.g., rheumatoid factor (RF) that contribute to these complexes. Serum rheumatoid factor (RF) is the immunological expression of an individual's immune system reaction to the presence of an immunoglobulin molecule that is recognized as "non-self." This response to the "non-self" immunoglobulin results in the presence of immune complexes. These, in turn, bind complement and may eventually lead to synovium, cartilage, and bone destruction. Higher the levels of serum rheumatoid factor, higher are the development of inflammation.[35] Serum rheumatoid factor (RF) measures the amount of antibody IgM titer present in the serum.[36] MAM treated animal showed significantly lesser serum RF when compared to disease control animals. Bone destruction, which is a common feature of arthritis, was examined by radiological analysis. CA treated rats had developed definite joint space narrowing of the intertarsal joints, diffuse soft tissue swelling that included the digits, diffuse demineralization of bone, marked periosteal thickening, and cystic enlargement of bone and extensive erosions produced narrowing or pseudowidening of all joint spaces. Despite a similar clinical course of arthritis, disease control rats suffered from more pronounced bone destruction than MAM treated group. RA is characterized by synovial tissue leukocyte ingress and angiogenesis. [37] The disease is thought to occur as an immunological response to as yet unidentified antigen. Even in early RA, some of the earliest histological observations are blood vessels. [38] A mononuclear infiltrate characterizes the synovial tissue along with a luxuriant vasculature. Angiogenesis is integral to formation of the inflammatory pannus and without angiogenesis, leukocyte ingress could not occur. Changes in the density of blood vessels in the synovium and alterations in endothelial proliferative responses in RA have been shown in a range of studies. The number

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of synovial blood vessels has been found to correlate with hyperplasia of synovial cells, infiltration of mononuclear cells, and indices of joint tenderness.[39] Histopathology study of synovial joint showed that treatment with Aegle marmelos Group decreased vascularity, lymphocytic infiltration with less rheumatoid inflammation and angiogenesis, with no thickening of synovial membrane and absence of lymphoid follicles. As compared to disease control and orally treated animals. Our data suggested that Aegle marmelos possesses significant antiarthritic activity. The possible mode of anti- arthritic activity of Methanolic extract of Aegle marmelos appears to be, Possessing antiinflammatory activity showed in arthritic parameters like Paw edema, Arthritic index, Rheumatoid factor, improving bone erosion. Acknowledgements The authors are thankful to the Director, Principal and management of C U Shah College of Pharmacy and Research, Wadhwan for providing necessary facilities to carry out this work. Our Acknowledgement to Dr. H A Solanki for their technical assistance in the plant collection and Identification. References: 1. Haqqi TM, David CS. T-cell receptor V beta genes repertoire in mice: Possible role in resistance and susceptibility to type II collagen-induced arthritis. J. Autoimmun. 1990, 3, 113121. 2. Myers LK, Rosloniec EF, Cremer MA, Kang AH. Collagen induced arthritis, an animal model of autoimmunity. Life Sci. 1997, 61, 18611878. 3. Smolen JS, Steiner G. Therapeutic strategies for rheumatoid arthritis. Nature Reviews. Drug Discovery 2003, 2, 473488. 4. Jones G, Halbert J, Crotty M, Shanahan EM, Batterham M, Ahern M. The effect of treatment on radiological progression in rheumatoid arthritis: a systematic review of randomized placebo-controlled trials. Rheumatology 2003, 42, 613. 5. Kremer JM, Methotrexate and leflunomide: biochemical basis for combination

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International Journal of Pharmaceutical Research & Development therapy in the treatment of rheumatoid arthritis. Seminars in Arthritis and Rheumatism 1999, 29, 1426. 6. Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annual Review of Immunology 2001, 19, 163 196. 7. Rao JK, Mihaliak K, Kroenke K, Bradley J, Tierney WM, Weinberger M, Use of complementary therapies for arthritis among patients of rheumatologists. Annals of Intern Medicine 1999, 131, 409416. 8. Shin SS, Jin M, Jung HJ, Kim B, Jeon H, Choi JJ, Kim JM, etal. Suppressive effects of PG201, an ethanol extract from herbs, on collagen-induced arthritis in mice. Rheumatology 2003, 42, 665 672. 9. Soeken KL, Miller SA, Ernst E. Herbal medicines for the treatment of rheumatoid arthritis: a systematic review. Rheumatology 2003, 42, 652 659. 10. Chang SH, Sung HC, Choi Y, Ko SY, Lee BE, Baek DH, etal. Suppressive effect of AIF, a water extract from three herbs, on collagen-induced arthritis in mice. International Immunopharmacology 2005, 5, 13651372. 11. Park KC, Park EJ, Kim ER, Kim Y, Chung SH, Cho BW, etal. Therapeutic effects of PG201, an ethanol extract from herbs, through cartilage protection on collagenase-induced arthritis in rabbits. Biochemical and Biophysical Research Communications 2005, 331, 14691477. 12. Shobha FG, Thomas M. Study of antidiarrhoeal activity of four medicinal plants in castor-oi induced diarrhea. J. Ethnopharmacol 2001, 76, 73-76. 13. Kamalakkannan N. Prince PS. Hypoglycaemic effect of water extracts of Aegle marmelos fruits in streptozotocin diabetic rats. J.Ethnopharmacol 2003, 87, 207-210. 14. Lotufu LV, Khan MT, Ather A, et al. Studies of the anticancer potential of plants used in Bangladeshi folk medicines. J. Ethnopharmacol 2005, 99, 21-23. 15. jagetia GC, Venkatesh P, Balinga MS. Evaluation of radio protective effect of bael leaf (Aegle

ISSN: 0974 9446 marmelos) extract in mice. Int J Radiat Biol 2004, 80, 231-290. 16. Rana BK, Jain AK. Evaluation of anti fungal activity of essential oil isolated from leaves of the A.marmelos. J. Ethnopharmacol 1997, 57, 2937. 17. Mazmudar S, Bhattacharya A, Majmudar AK, Pattnaik P, Tiwari M, Chaudhary S. Anti bacterial evolution of Aegle marmelos (Correa) Linn. Root extract. Phytother Res 2006, 20, 82-84. 18. Sahare KN, Anandhraman V, Meshram VG, et al. Antimicrofilarial activity of methanol extract of vitex negudndo and Aegle marmelos and their phytochemical analysis. Indian J Exp. Biol 1981, 3, 245-249. 19. Veerappan A, Shigeru M, Rengnathan D. Studies on the anti inflammatory, antipyretic and analgesic properties of the leaves of Aegle marmelos Corr. J. Ethnopharmacol 2005, 96, 159-163. 20. Raj KPS, Patel MR, Some medicinal plants of Camby and its immediate vicinity and their uses in Indian Indigenous system of medicine Indian Drugs 1978, 15, 145-152. 21. Maheshwari JK, Singh JP. Plants used in the ethno-medicine by the Koles of Allahabad district Uttar Pradesh. Bull Med Ethnobot Res 1984, 5,105-121. 22. Garg SN, Siddiqui MS, Agarwal SK. P- Meth-1-on 3, 5 diol, a new constituent of Aegle marmelos. Indian J Chem 1980, 19, 162. 23. Chakravorty RN, Dasgupta B. -Sitosterol from the leaves of Aegle marmelos Correa. J Indian Chem Soc 1958, 35, 194-196. 24. Ali MS, Pervez M. Marmenol: a 7geranylozycoumarin from the leaves of Aegle marmelos Nat Prod Res 2004, 14, 141-146. 25. Wang C, Dai Y, Yang J, Chou G, Wang C, Wang Z. Treatment with total alkaloids from Radix linderae reduces inflammation and joint destruction in type II collagen-induced model for rheumatoid arthritis. J Ethnopharmacol 2007, 111,322-8. 26. Pearson CM, Wood FD. Studies on polyarthritis and other lesions induced in rats by injection of mycobacterium adjuvant. I. General clinic and

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International Journal of Pharmaceutical Research & Development pathological characteristics and some modifying factors. Arth rheum. 1959, 2, 440-459. 27. Van de Berg WB, Joosten LAB, Helsen MMA, van de Loo FAJ. Amelioration of established murine collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol. 1994, 95, 237243. 28. Joosten LAB, Helsen MMA, Van de Loo FAJ, van den Berg WB. Anticytokine treatment of established collagen type II arthritis in DBA/1 mice: a comparative study using anti-TNF alpha, anti-IL-1 alpha, beta and IL-1Ra. Arthritis Rheum. 1996, 39, 797809. 29. Joosten LAB, Lubberts E, Helsen MMA, van den Berg WB. Dual role of IL-12 in early and late stages of murine collagen type II arthritis. J Immunol. 1997, 159, 40944102. 30. Taniguchi K, Kohsaka H, Inoue N, Terada Y, Ito H, Hirokawa K, Miyasaka N: Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.Nat Med. 1999, 5, 760-767. 31. Robert H, Shmerling, et al. The Americn Journal of Medicine. 1991, 91, 528-534. 32. Weyand CM. New insights into the pathogenesis of rheumatoid arthritis. Rheumatology 2000, 39 (1), 38. 33. Smolen JS, Steiner G. Therapeutic strategies for rheumatoid arthritis. Nature Reviews. Drug Discovery 2, 2003, 473 488. 34. Colpert KM. Evidence that adjuvant arthritis in the rat is associated with chronic pain: Pain. 1987, 28, 201-222 35. Koopman W, et al. Rheumatoid factor and human disease. Clin Immunol News.1990, 10,137-141. 36. Szekanecz Z, Szegedi G, Koch A. Angiogenesis in rheumatoid arthritis: pathogenic and clinical significance. J Invest Med. 1998, 46, 27-41. D. Angiogenesis and arthritis. 37. Walsh Rheumatology. 1999, 38, 103-112. 38. Rooney M, Condell D, Quinlan W, Daly L, Whelan A, Feighery C, Bresnihan B. Analysis of the histologic variation of synovitis in rheumatoid arthritis. Arthritis Rheum. 1988, 31,956-963.

ISSN: 0974 9446 39. Simon G. Alterations in joint space (arthritis) and associated bone change, in: Principles of Bone Xray Diagnosis, 2nd ed., Butlerworth & Co. Ltd., Great Britain, 1965, 157- 163.

Fig. 1: Effect of Methanolic extract of Aegle marmelos on paw edema in collagen induced arthritis in rat. Data are presented as Mean SEM (n=6), * P < 0.05, when compared with Disease control. (One Way ANOVA).

Fig. 2: A & B Effect of Methanolic extract of Aegle marmelos on arthritic index and rheumatoid factor in collagen induced arthritis in rat. Data are presented as Mean SEM (n=6), @ P < 0.0001, when compared with normal control, # P < 0.001, when compared with disease control. (One Way ANOVA)

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Fig. 4: Effect of Methanolic extract of Aegle marmelos on Radiographs of tibiotarsal joints Fig. 3: Effect of Methanolic extract of Aegle marmelos on radiographic score in collagen induced arthritis in rat. Data are presented as Mean SEM (n=6), @ P < 0.001, # P < 0.05 when compared with disease control, (One Way ANOVA)

Fig. 5: Describe histopathology of joints indicate treatment with methanolic extract of Aegle marmelos prevent bone erosion.

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