International Journal of Research in Pharmacology & Pharmacotherapeutics

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1 International Journal of Research in Pharmacology & Pharmacotherapeutics ISSN Print: IJRPP Special Issue 1 Oct - Dec ISSN Online: Journal Home page: Research article Open Access Curative Effect of Kaempferol-5-O-β-D-glucopyranoside from Methanolic Extract of Indigofera aspalathoides Vahl ex DC against Adjuvant Induced Arthritis in Albino rats. S.Swarnalatha 1, S.Gayathri 2, S. Lavanya 2, G.Kavithalya 2, A. Puratchikody 2* 1 Drug Discovery and Development Research Group, Department of Pharmaceutical Technology, Anna University Chennai, Bharathidasan Institute of Technology, Tiruchirappalli Department of Pharmacology, AadhiBhagawan College of Pharmacy, Rantham, Cheyyar *Corresponding author: Ayarivan Puratchikody puratchikodypharma@gmail.com ABSTRACT To investigate the in vitro anti-arthritic potential of isolated compound Kaempferol-5-O-β-D-glucopyranoside (K-5- β-d-g) from methanolic extract of aerial parts of Indigofera aspalathoides Vahl ex DC. (Papilionaceae) using Wistarrats. Anti-arthritic activity of K-5-β-D-g was evaluated using Freund s complete adjuvant model. The parameters such as foot pad thickness, body weight measurement, rheumatoid factor, spleen index assessment, hematological estimation and histopathology of synovial joints were examined. Methotrexate was used as standard (5mg/kg b.w.p.o). Administration of K-5-β-D-g at the doses of 10, 20, 50, 100 mg/kg b. w. p. o exhibited statistically significant (p < 0.001) inhibition of the paw edema, increase in body weight, decrease in arthritic, rheumatoid and spleen index factors. The results of haematological parameters showed that the treatment of K-5-β- D-g at varied doses significantly increased (P < 0.001) the Heamoglobin (Hb) level, Red Blood Cells (RBCs) count, decreased White Blood Cells (WBCs) count and Erythrocyte Sedimentation Rate (ESR). All these treatment groups were compared with arthritis treated groups. Histopathological analysis demonstrated that K-5-β-D-g had comparable anti-arthritic activity with methotrexate. The results obtained suggestedthatk-5-β-d-g from the methanol extract of aerial parts of I. aspalathoidesvahl ex DC. Holds significant anti-arthritic activity by inhibiting various inflammatory mediators and there by act on multiple targets to achieve optimal effects. Keywords: Indigofera aspalathoidesvahl ex DC.Kaempferol-5-O-β-D-glucopyranoside, Rheumatoid arthritis, Freund s complete adjuvant-induced arthritis. INTRODUCTION Rheumatoid arthritis (RA) is a systemic autoimmune disease that causes chronic inflammation of connective tissue primarily in the joints that involves synovial proliferation and cartilage destruction [1]. In RA, bone deformations and disability of joint function occurred due to ~ 9~

2 progressive erosion of articular cartilage and infiltration of auto antibodies in the synovial joint. It leads to severe pain, swelling and redness within the joints [2]. Amongst the various experimental animal models of arthritis, induction of arthritis by Freund s complete adjuvant (FCA) is considered to be one of the standardized methods which mimic the human pathophysiological state including chronic swelling in multiple joints due to accumulation of inflammatory cells, bone and joint destruction. The drugs that are used commonly for the treatment of arthritis include glucocorticoids namely cortisone and prednisone, NSAIDS like ibuprofen and naproxen, disease modifying anti-inflammatory and anti-rheumatic drugs like methotrexate and leflunomide and newer therapies such as biological response modifiers like tumor necrosis factor, alpha blocking agents, which are often required to inhibit the underlying immune processes [3]. However, besides high costs, all of these drugs are associated with numerous side effects, severe adverse reactions and toxicity [4].In recent years; extensive studies have been focused towards the traditional system of medicine for the discovery of drugs that are long acting with minimum side effects [5]. However, herbal medicines are being accepted and used increasingly by general populations because of the ethnic acceptability and compatibility having fewer side effects [6]. It is imperative to examine the therapeutic activity of herbal products against autoimmune disorders as well as other conditions involving inflammation 7. Plants and isolated compounds from plants have long been used in medicine [8]. In addition, active plant principles serve as important sources for new drugs, such as templates for synthetic drugs, and as intermediates used in the production of semisynthetic drugs [9]. The plant, Indigofera aspalathoides Vahl ex DC, belongs to family Papilionaceae, is a low under shrub distributed throughout South India and Sri Lanka [10]. It is found to be active against transplantable tumors and inflammations [11]. The whole plant is an ingredient of an oily preparation used for dandruff, syphilis and other skin infections [12]. In Siddha system of medicine, this plant is prescribed for eczema, psoriasis, boils, burns, wounds, ulcers and used as an antidote to snake venom [13]. In view of these reports and as a continuation of our earlier studies on immunomodulatory activity [14], we have herein reported the curative effects of isolated compound Kaempferol-5-O-β-D-glucopyranoside (K-5-β-D-g) from I. aspalathoides Vahl on in vitro arthritic models of rodents. MATERIALS AND METHOD Plant material Fresh aerial parts of I.aspalathoideswas collected from the herbal garden of Tirunelveli district of Tamilnadu during the month of January The plant material was identified and authenticated by Dr. J. Jayaraman, Director, Plant Anatomy Research Centre, West Tambaram, Chennai. Voucher specimen (No. PARC/2011/886) was preserved for future references. Animals Wistar albino rats ranging between g of either sex were used. Rats were maintained in stainless steel cages under constant conditions of temperature (23 ± 2 C), relative humidity (60 ± 2%) and lighting (12 h light / dark cycle). The animals had free access to water ad libitum and fed with pellet diet (Lipton India Ltd., Mumbai, India.) except 1 h before and during the experiments. All experimental procedures were carried out in strict accordance with the guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 685/PO/02/a/CPCSEA/ ) and were approved by the Institutional Animal Ethics Committee (IAEC) from the KMCH College of Pharmacy, Coimbatore (KMCH / PhD /17/ , dated ). Drugs and chemicals All the reagents used were of analytical grade. Methotrexate was obtained from Akums Drugs & Pharmaceuticals Ltd, Haridwar, India. Freund s complete adjuvant (FCA) was purchased from Sigma Aldrich, St.Louis, USA. Acute oral toxicity test (AOT) AOT for K-5-β-D-g was performed in Wistar albino rats as per OECD guideline 425 (OECD, 2008). Animals were divided into 4 groups of 3 animals in each. Female, nulliparous and nonpregnant rats, weeks and weighing between g, in addition, weeks, male wistar rats, and weighing g, were selected for this study. The animals were kept fasting overnight ~ 10~

3 providing only with water, after which the compound K-5-β-D-g were administered orally. The doses selected were 175 mg/kg, 550 mg/kg, and 2000 mg/kg. Another one group received distilled water as vehicle control. Their weight were taken and recorded before administration. The dose at which mortality was observed in two out of three rats was considered as toxic dose. All the animals were observed twice daily for health condition, morbidity and mortality for 14 days. Based on the result obtained from this study, the dose for this study was fixed [15]. Freund s Complete Adjuvant Induced (FCA) Arthritis Wistar albino rats of either sex weighing between 200 to 300 g were taken and divided into seven groups each containing six animals. On day zero, all rats were administered with 0.2 ml of Freund s complete adjuvant (FCA) into the sub plantar region of the left hind paw. The adjuvant contained heat killed Mycobacterium tuberculosis suspended in sterile paraffin oil thoroughly grinding with mortar and pestle to give a concentration of 1mg/mL.Dosing with the test and standard compounds was started on the first day and continued for 12 days according to the following schedule: Group I: Normal control (0.5 ml normal saline), Group II: Disease control (arthritic treated), Group III VI: K-5-β-D-g (10, 20, 50, 100 mg/kg b.w.p.o.), Group VII: Methotrexate (5mg / kg b.w.p.o.). From day 13 th to 21 st, the animals were not dosed with the test compounds or the standard 16. The following parameters were measured. Evaluation of foot pad thickness and body weight changes Wistar albino rats were weighed using digital weighing balance andfoot pad thickness of left hind limbs were recorded on the day of FCA injection, again measured on 3 rd, 7 th, 14 th and 21 st day using mercury column Plethysmometer (UgoBasile, Italy) and changes in body weight also measured [17]. Arthritic Index Inflammation in each paw was graded according to the extent of erythematic and edema of the periarticular tissues using a scale of 0 4. Animals were scored 0 for no inflammation, 1 for unequivocal inflammation of one joint of the paw, 2 for unequivocal inflammation of at least two joints of the paw or moderate inflammation of one joint, 3 for severe inflammation of one or more joints, 4 for maximum inflammation of one or more joints in the paw. The arthritis score for each rat on day 0 was determined to be 0. The scores for each paw were then added to get the total arthritis score on day 22, designated as the arthritic index [18]. Rheumatoid Factor On the day 22 nd blood was withdrawn from the each animal through retro-orbital plexus under light ether anesthesia. The blood was collected into vials containing EDTA and the RF of synovial joints was analyzed. The latex turbidimetry method was used in the present study using RF turbilatex kit of SPINREACT. Latex particles coated with gamma globulin are agglutinated when mixed with samples containing RF. The agglutination causes an absorbance change, dependent upon the RF content of sample that can be quantified by comparison from a calibrator of known RF concentration. Calibration was carried out for linear range up to 100 IU/ML. The results of RF factor of all the groups obtained was compared with the control animals and was expressed as IU/MI [19]. Spleen-Index At the end of the experiment, after sampled for serum separation, all rats were sacrificed by ether anesthesia [20]. The spleen of all the rats were removed and weighed immediately. The spleen indexes were calculated using the following formula: Spleen Index =Spleen weight of CFA rat/ Body weight of CFA rat Spleen weight of normal rat/body weight of normal rat Hematological estimation The collected blood samples after serum separation were subjected to hematological parameter. In which, hemoglobin content was estimated by the method of Austin and Drabkin. Red Blood Cell (RBC) and White Blood Cell (WBC) counts were estimated according to the method of Chesbrough and MC Arthur in an improved Neubauer chamber. Estimation of Erythrocyte Sedimentation Rate (ESR) was carried out by the method of Westergren [21]. Histopathology Wistar rats were sacrificed by cervical decapitation on 22 nd day, the proximal ~ 11~

4 interphalangeal joints were removed, washed with saline and stored in 10% formalin. The interphalangeal joint sections were obtained, stained with eosin-haematoxylin and viewed under 100 X magnifications [22]. Statistical analysis The experimental results are expressed as mean ± SE. Statistical analysis of the data obtained from the experiment was carried out using the one way analysis of variance (ANOVA) followed by Dunnett s multiple range test. P-values < 0.05 was considered as statistically significant using SAS software -Version 6 (SAS Institute, Cary, North Carolina, USA). Table 1 - Effect of K-5-β-D-gon paw edema Mean paw thickness (mm) Treatment 0 day 3 th day 7 th day 14 th day 21 st day Group I 4.91± ± ± ± ±0.002 Group II 4.73±0.003*** 11.01±0.003*** 11.13±0.005*** 11.15±0.002*** 11.8±0.002*** Group III 4.9±0.04*** 8.21±0.04*** 7.73±0.03*** 6.03±0.02*** 5.26±0.02*** Group IV 4.66±0.034*** 8.72±0.054*** 7.28± 0.02*** 6.43±0.04*** 5.06±0.09*** Group V 4.65±0.03*** 8.86±0.02*** 8.13±0.02*** 7.3±0.02*** 6.12±0.03*** Group VI 4.68±0.02*** 8.66±0.02*** 7.75± 0.03*** 6.31±0.03*** 5.71±0.04*** Group VII 4.21±0.010*** 8.23±0.010*** 7.5±0.02*** 6.23±0.02*** 5.46±0.02*** N = 6, values were expressed as Mean ± SEM, ***p value <0.001 statistically significant, Group- I; Normal control. Group- II; Disease control.group III-VI; treatment group.group- VII; standard group. Table 2 - Effect of K-5-β-D-g on average body weight Average Body weight Treatment 0 th day 3 rd day 7 th day 14 th day 21 st day Group I ± ± ± ± ±0.72 Group II ±0.34** ±0.56** ±0.34** ±0.72** ±0.45** Group III ±0.84** ±0.96** ±0.24** ±0.92** ±0.32** Group IV ±0.74** ±0.46** ±0.54** ±0.82** ±0.52** Group V ±0.34** ±0.87** ±0.97** ±0.32** ±0.92** Group VI ±0.24** ±0.57** ±0.77** ±0.82** ±0.32** Group VII ±0.37** ±0.38** ±0.57** ±0.29** ±0.32** N = 6, values were expressed as Mean ± SEM, **p value <0.001 statistically significant, Group - I; Normal control. Group - II; Disease control. Group III -VI; treatment group. Group VII; standard group. ~ 12~

5 Table 3 -Effect of K-5-β-D-g on Hematological Parameter of CFA induced arthritis in albino rats Treatment Hb (g/dl) RBC ( 10 6/ mm 3 ) WBC ( 10 3/ mm 3 ) ESR(mm) Group I ± ± ± ± Group II 8.05 ± 0.034*** 3.70± 0.025*** ±0.030*** ± 0.025*** Group III 10.5 ± 0.025*** 4.20 ± 0.025*** 8.80 ± 0.025*** 8.51±0.030*** Group IV 11.8 ± 0.025*** 5.50 ±0.025 *** 7.50 ± 0.03*** 7.46±0.021*** Group V ± 0.004*** 5.76 ± 0.021*** 7.20 ± 0.02*** 6.11 ±0.03*** Group VI ± 0.004*** 6.10± 0.057*** 6.91 ± 0.030*** 6.60 ± 0.025*** Group VII ± 0.004*** 6.90 ± 0.057*** 6.78 ± 0.030*** 5.20 ±0.025*** N = 6, values were expressed as Mean ± SEM, ***p value <0.001 statistically significant, Group - I; Normal control. Group - II; Disease control. Group III VI; treatment group. Group VII; standard group. Fig. 1 - Effect ofk-5-β-d-g on arthritic index. N = 6, values were expressed as Mean ± SEM, ***p value <0.001 statistically significant, Group I; Normal control. Group II; Disease control. Group III VI; treatment group. Group VII; standard group ~ 13~

6 Fig. 2 -Effect of K-5-β-D-gon Rheumatoid factor. N = 6, values were expressed as Mean ± SEM, ***p value <0.001 statistically significant, Group I; Normal control. Group II; Disease control. Group III VI; treatment group. Group VII; standard group Fig. 3 -Effect of K-5-β-D-gon spleen index. N = 6, values were expressed as Mean ± SEM, ***p value <0.001 statistically significant, Group - I; Normal control. Group - II; Disease control. Group III VI; treatment group. Group -VII; standard group ~ 14~

7 Fig. 4-A: Normal control, B: Arthritic control,c : Std (Methotrexate (50 mg/kg), D, E, F, and G:Test compound (K- 5-β-D-g 5, 10, 50, 100 mg/kg). 1: Cartilage damage; 2: Vascular proliferation; 3: Minimal inflammation; 4: Severe inflammation. RESULTS Acute oral toxicity test (AOT) As per observations and calculations from Acute Oral Toxicity (OECD Guidelines 425), the LD 50 value of K-5-β-D-g was found at 1000 mg/kgb.w., and therefore, 1/10th of this dose (100 mg/kg b.w.) was taken as the test dose for further study. Freund s complete adjuvant induced rat paw edema The hind paw injected with Freund s complete adjuvant became gradually swollen and reached its peak on 21 st day (Table 1) whereas, in test compound K-5-β-D-g treated groups (10, 20, 50, 100 mg/kg) ~ 15~

8 and std methotrexate group, the development of paw edema was inhibited significantly. Body weight changes Body weight in FCA treated rats was significantly reduced as compared to control animal (P < 0.001). The increase in body weight was observed in K-5-β- D-g at the dose of 10, 20, 50, 100 mg/kg, and Methotrexate 5 mg/kg when compared to arthritic treated rats (Table 2). Arthritic Index Administration of FCA in sub plantar region induced chronic disease of arthritic in arthritis treated rats which was significantly different from normal control group. Test drug treated group K-5-β-D-g (10, 20, 50, 100 mg/kg) produced statistically significant reduction (P < 0.001) in arthritic score compared to arthritis treated rats (Fig. 1). Rheumatoid factor and Spleen Index As shown in Figure 2, 3 rheumatoid factor and spleen index were significantly increased (P < 0.001) in all arthritic treated rats as compared to control rats. The rheumatoid factor and spleen index of K-5-β-D-g treated group (10, 20, 50, 100 mg/kg) and methotrexate treated group (5mg/kg) were significantly (P < 0.001) lesser than that of arthritic treated rats. Hematological parameters The changes in hematological parameters in adjuvant induced arthritic rats are shown in Table 3. There was a significant (p < 0.001) decrease in RBC count and hemoglobin, and increase in WBC count and ESR of arthritic rats as compared to control rats. The test compound has significantly brought back the altered hematological changes in both developing and developed phases of adjuvant induced arthritis. Histopathological Evaluation Histopathological of synovial joint of normal rats showed intact articular cartilage and normal synovial lining (Fig. 4A). The arthritic control rat joint showed cartilage damage, cellular infiltration and massive severe inflammation (Fig. 4B). However, the standard drug Methotrexate (5 mg/kg) has maintained intact articular cartilage and has reduced the number of inflamed cells like lymphocytes and esinophiles as compared to disease control (Fig. 4C). Test compound at the dose level of K-5-β-D-g (10 mg/kg) showed moderate edematous synovium, slight destructive lesions in articular cartilage of rat joint (Fig. 4D). Whereas, at 20 mg/kg, the rat joint showed increased vascular proliferation and synovial space thinning (Fig. 4E) and 50 mg/kg treated rat joint showed minimal inflammation, with no evidence of lymphocytic infiltration cells in synovium (Fig. 4F). The test drug 100 mg/kg at dose level of K-5-β-D-g showed significant protection in inflammatory condition at synovial lining with normal joint space and normal cellular infiltration in rat joint (Fig. 4G). DISCUSSION In the present study, the CFA model was chosen to induce arthritis in rats as it develops chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage and paw edema. Mediators responsible for the chronic inflammation involve release of cytokines (IL-1B and TNF-α), interferon s and platelet derived growth factor that can lead to severe disability 23. The determination of paw thickness is apparently simple, sensitive and quick procedure for evaluating the degree of inflammation and the therapeutic effects of drugs. On the 21 st day, a significant decrease in paw thickness was observed in K-5-β-D-g (10, 20, 50 and 100 mg/kg b.w.p.o.) and Methotrexate (5 mg/kg b.w.p.o.) Treated group as compared to the arthritic treated rats. During the course of study, change in body weight of rats was measured as one of the parameters to assess the severity of the disease and the response to therapy of test compoundk-5-β-d-g. It was observed from the Table 2, all the CFA treated rats body weight were decreased. The decrease in body weight of the rats may be due to alterations in the metabolic activities of diseased rats during the experimental period. Earlier findings suggested that absorption of 14C- glucose and 14C-leucine in rat s intestine was reduced in the case of inflamed rats but on the treatment with anti-inflammatory drugs, the decrease in absorption was nullified [24]. It indicated that the anti-inflammatory drugs correct the decreased absorption capacity of intestine during inflammation. Arthritic index includes the combined index of inflammation, formation of nodules and extent of spread of the disease to other organs [25]. In our ~ 16~

9 study, the arthritic treated rats showed significantly elevated levels of arthritic index and serum rheumatoid factor compared to normal control animal. The treatment withk-5-β-d-g (10, 20, 50 and 100 mg/kg b.w.p.o.) and Methotrexate (5 mg/kg b.w.p.o.) significantly reduced the levels of inflammation and autoimmune stimulation. It is learnt from the literature that the rheumatoid factor generation in arthritis involves B-cells activation and several genetic predispositions to arthritic diseases. Higher the levels of serum rheumatoid factor, higher the development of inflammation. The results of the present study indicate that the anti-inflammatory effects of K-5-β-D-g may be due to the inhibition of activation of B cells [26]. The prompt decrease in arthritic index and rheumatoid factor level after treatment with test drug indicates the anti- arthritic potential. Spleen is a vital organ involved in immune responses. In adjuvant arthritis, spleen serves as the reservoir for the cells and antibody formation which involved in the immune response. Increase in the weight of spleen is associated with the splenomegaly, generalized lymphadenopathy and altered hepatic function [27]. In the present study, treatment withk- 5-β-D-g (10, 20, 50 and 100 mg/kg) significantly decreased the spleen weight which might be attributed to anti-inflammatory and immuno stimulant effect. Our previous report on immunomodulatory activity 13 corroborated that the compound K-5-β-D-g may possibly act by decreasing the synthesis of T cells mediators such as IL and TNF-α to reduce spleen weight. In the present investigation, it was found that decrease in RBC count and haemoglobinlevel which represents the anemic condition in arthritic rats. It was postulated that the abnormal storage of iron in the reticuloendothelial system and synovial tissue causes the failure of bone marrow which leads to anemia. The data obtained from this study indicate that the K-5-β-D-g (10, 20, 50 and 100 mg/kg) treated groups significantly restores the decreased level of RBC along with Hb showed its immunomodulation effect. White blood cells (WBCs) are a major component of the body s immune system. Increased white blood cell counts are a common feature of inflammatory reactions, especially those induced by microbial infection [28]. IL-IB increases the production of both granulocyte and macrophages colony stimulating factor. Our study showed that the significant increase in WBC count in adjuvant-induced arthritic rats which may be due to the stimulation of immune system against the invading antigens. Administration of test compound K-5-β-D-g leads to inhibition of leucocyte migration which may have beneficial effect on joint preservation. Erythrocyte sedimentation rate (ESR) is an estimate of the suspension stability of RBCs in plasma. ESR in the arthritis treated group is found to be several folds high when compared to drug treated groups. ESR is strongly related with the ability of red cells to aggregate into orderly stacks or rouleaux [29]. Increase in sedimentation rate during inflammation may be due to the presence of proteins in RBC which may affect repellant surface charges on red cells and cause them to aggregate into rouleaux. In our study, it was found that the test compound K-5-β-D-g can reduce the elevated level of ESR attributing to its anti-inflammatory potential. In addition to macroscopic examination and hematological estimation in the experimental animals, the histopathological examination of the arthritic interphalangeal joints also was performed. In this study, we observed extensive proliferation of synovial cells, cartilage destruction and infiltration of leukocytes in synovial region as prominent histological features in the arthritic control samples. Similar observations were reported earlier that indicate the changes associated with arthritis, that is, proliferation of inflammatory cells with consequent cartilage degradation and erosion. The cartilage destruction is mainly triggered by proinflammatory molecules including leukotrienes, prostaglandins, proteinases, and oxidative species secreted by activated macrophages and fibroblasts in inflamed tissues [30]. The treatments which are capable of inhibiting the joint inflammation are considered to be therapeutically efficient for RA. The histological examination of the arthritic joints treated with methotrexate exhibited mild inflammatory cell migration. Since methotrexate is a potent disease modifying agent, act by inhibiting the binding of Interleukin 1 beta to its cell surface receptor. These mechanisms effectively suppress the inflammatory symptoms in arthritic joints. Qualitatively, similar changes were observed with the test compound K-5-β-D-g treated animals which suppress the chronic inflammatory cell infiltration, synovial hyperplasia, and cartilage destruction. ~ 17~

10 It is probable that the test compound may act by inhibiting the production of matrix degrading enzymes, that is, collagenases and matrix metalloproteinases from chondrocytes and synoviocytes. It is notable that gait deficits was observed in arthritic rats during macroscopic examination of disease progression and histological findings, and hence we suggest that the inflammatory processes and disease progression which are the major complications of arthritis. Therefore, it is hypothesized that to alleviate the symptoms of arthritic conditions, the compound must be able to reverse the gait deficits as seen in the arthritic rats. In this study, both the weight reduction and joint inflammation are seen as the commonly manifested observations in arthritic animals. Therefore, we also measured these two parameters to monitor the progression of disease over a time period taken by the control arthritic rats to demonstrate full blown arthritis symptoms. In Conclusion, anti-arthritic activity of K-5-β-D-g from methanolic extract of I. aspalathoidesappears to be possessing anti-inflammatory activity showed in arthritic parameters like paw edema, arthritic index, rheumatoid factor, improving bone erosion. All these results thus predict that the test compound provide pharmacological rationale for the traditional use of the drug against inflammatory disorders such as rheumatoid arthritis. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We wish to thank AadhiBhagawan College of Pharmacy, Cheyyar and Anna University, Chennai, Bharathidasan Institute of Technology, Tirchirappalli for their support of this research. REFERENCES [1]. Chang J, Medicinal herbs: drugs or dietary supplements, BiochemPharmacol, 59, 2000, 211. [2]. Newman DJ, Cragg GM &Snader KM, The influence of natural products upon drug discovery, Nat Prod Rep, 17, 2000, 234. [3]. Ohshima H &Bartsch H, Chronic infections and inflammatory processes as cancer risk factors, possible role of nitric oxide in carcinogenesis, Mutat Res, 305, 1994, 264. [4]. Rehman Q & Lane NE, Bone loss therapeutic approaches for preventing bone loss in inflammatory arthritis, Arth Res, 3, 2001, 227. [5]. Allen MJ, Biochemical markers of bone metabolism in animals: uses and limitations, ClinPathol, 32, 2003, 113. [6]. Yamamura K, Yonekawa T, Nakamura T, Yano S & Ueno K, The histamine H2-receptor antagonist, cimetedine, inhibits the articular osteopenia in rats with adjuvant - induced arthritis by suppressing the osteoclast differentiation induced by histamine, J PharmacolSci,92, 2003, 49. [7]. Simon LS &Yocum D, New and future drug therapies for rheumatoid arthritis, Rheumatology, 39, 2000, 42. [8]. Noreen Y, Serrano G, Perera P &Bohlin L, Flavan-3-ols isolated from somemedicinal plants inhibiting COX-1 and COX-2 catalyzed prostaglandin biosynthesis, Planta Med, 64, 1998, 524. [9]. Verdine GL, The combinatorial chemistry of nature, Nature, 384, 1996, 13. [10]. Bojaxa RA, Henry J & Rosalie, Phytochemical, Pharmacognostical Antimicrobial Activity of Indigoferaaspalathoides, Int J Biological Techn, 1, 2010, 15. [11]. Rajkapoor B, Murugesh N, Chodon D &Sakthisekaran D, Chemoprevention of Nitosodiethylamine induced phenobarbitol promoted liver tumors in the rat by extract of Indigoferaaspalathoides, BiolPharma Bull, 28, 2005, 366. [12]. Kirtikar KR &Basu BD, Indian Medicinal plants, International Book Distributors, Dehra Dun, India 1975, 714. [13]. Rajkapoor B, Murugesh N, Kavimani S, Krishna DR, Ravichandran V Gobinath M, Harikrishnan N, Vidyasagar J, Declercq E, Scott G &Franzblau, Antimycobacerial antiviral and cytoxic studies of Indigoferaaspalathoides, PharmacognosyMagazine, 3, 2007, 166. [14]. Swarnalatha S, Umamaheswari A &Puratchikody A, Immunomodulatory activity of Kaempferol-5-O-β-Dglucopyranoside from IndigoferaaspalathoidesVahl ex DC, (Papilionaceae), Med Chem Res, 9, 2015, ~ 18~

11 [15]. OECD: Guidance document on acute oral toxicity testing, environmental health and safety monograph series on testing and assessment number 24, Paris [16]. Bhakuni DS, Sharma VN &Kaul KN, Bitter constituents of Luffaspecies, J Sci and Indus Research, 20, 1961, 361. [17]. Pearson Carl M & Wood Fae D, Studies of poly arthritis and other lesions induced in rats by injection of mycobacterial adjuvant. I General clinical and pathological characteristics and some modifying factors, ArthritisRheumatol, 2, 1959, 459. [18]. Singh S &Majumdar DK, Effect of fixed oils of Ocimum sanctum against experimentally induced arthritis and joint edema in laboratory animals, J Pharmacogn, 34, 1996, 222. [19]. Mehta A, Sethiya NK, Mehta C & Shah GB, Anti-arthritis activity of roots of HemidesmusindicusR.Br. (Anantmul) in rats, Asian Pac J Trop Med, 2012, 135. [20]. Chunxia C, Peng Z, Huifang P, Hanli R, Zehua H &Jizhou W, Extracts of Arisaemarhizomatum Fischer attenuate inflammatory response on collagen-induced arthritis in BALB mice, J Ethnopharmacol, 133, 2011, 582. [21]. Beaufay H, Amar Costecec A, ThinesSempoux D, Wibo M, Robbi M &Berthet J, Analytical study of microsomes and isolated subcellular membranes from rat liver subfraction of the microsomal fraction by isopyonic and differential centrifugation in density gradients, J Cell Biol, 61, 1974, 231. [22]. Taneja V, Taneja N, Paisansinsup T, Behrens M, Griffiths M &Luthra H, CD4 and CD8 T cells in susceptibility and protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis, J Immunol, 168, 2002, 75. [23]. Hazeena BV &Sadique J, Long term effect of herbal drug Withaniasomniferaon adjuvant induced arthritis in rats, Indian J ExpBiol, 26, 1988, 882. [24]. Pearson CM, Experimental joint disease observations on adjuvant induced arthritis, J Chronic Dis 16, 1963, 864. [25]. Winder CV, Lembke LA & Stephens MD, Comparative bioassay of drugs in adjuvant induced arthritis in rats: flufenamic acid, mefenamic acid and phenylbutazone, Arthritis Rheum, 12, 2005, 482. [26]. Gujral ML, Sareen KN, Tangri KK, Roy AK, Gupta GP &Amma MK, Antiarthritic activity of Glycyrrhizaglabra Linn, Indian J PhysiolPharmacol, 3, 1959, 47. [27]. Walz DT, Di Martino MJ &Misher A, Adjuvant-induced arthritis in rats. II. Drug effects on physiologic, biochemical and immunologic parameters, J PharmacolExpTher, 178, 1971, 231. [28]. Panayi GS, Lanchbury JS & Kingsley GH, The importance of the T cell in initiating and maintaining the chronic synovitis of rheumatoid arthritis, Arthritis Rhem, 35, 1992, 735. [29]. David G & Sykes AJ, Westergren and Wintrobe methods of estimating ESR compared, Br Med J, 2, 1951, [30]. Mowat AG, Haematological abnormalities in rheumatoid arthritis, Semin Arthritis Rheum, 1, 1971, 9. ~ 19~