IN VIVO ASSESSMENT OF ANTI-ARTHRITIC ACTIVITY OF WITHANIA COAGULANS DUNAL FRUITS

* Archana K. Shendkar 1 , Sugandha G. Chaudhari 1 and Dr. Yogesh K. Shendkar 2 . 1. Department of Pharmacology, Dr.L.H.Hiranandani College of Pharmacy, Ulhasnagar, District-Thane, India. 2. Department of Medicine, Virar Homeopathic Medical College, Virar, District-Thane, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The aim of this study was to determine in-vivo anti-arthritic activity of methanolic and hydro alcoholic extract of Withania Coagulans Dunal fruits i.e. Withania Coagulans Methanolic Extract (WCME) and Withania Coagulans Hydro alcoholic Extract (WCHAE). Phytochemical analysis has indicated presence of alkaloids, flavonoids, glycosides, steroids, saponins, fixed oil, carbohydrates. Both the extracts have showed anti-arthritic activity by Complete Freund"s adjuvant induced arthritis (CFA) in rats method.Treatment with WCME-200mg/kg, WCME-500mg/kg, WCHAE-450 mg/kg caused significant (p<0.01) inhibition of the arthritis index score, paw edema volume, joint area and pain perception parameters like dorsal flexion and motility along with rise in stair climb score in comparison with disease control (CFA induced) groups on 28 th day. Both the extracts showed the good anti-inflammatory activity but WCHAE showed the most significant anti-arthritic activity than WCME. The findings of the present study suggested that Withania Coagulans dunal fruits could be a potential natural source of ant-arthritis activity which is very important against treatment of rheumatoid arthritis disease.

…………………………………………………………………………………………………….... Introduction:-
RA is chronic inflammatory and autoimmune diseases affects millions of people across the world leading to suffering, economic loss and premature death. Common inflammatory diseases include asthma, chronic obstructive pulmonary diseases (COPD), allergic rhinitis, osteoarthritis, inflammatory bowel diseases (IBD) and psoriasis. Inflammatory diseases including different types of rheumatic diseases are a major cause of mortality of the working force throughout the world. This has been called the "king of the human miseries." Inflammation is the reaction of living tissues to injury, infection or irritation of foreign substance. Lysosomal enzyme released during the inflammation leads to the tissue injury which is responsible for pathological condition of rheumatoid arthritis. Inflammation manifest in the form of painful swelling associated with some changes in skin covering at the site. It can be classified either acute or chronic. Acute inflammation is initial response of body to harmful stimuli and prolonged inflammation is known as chronic inflammation. [1] The immune system is a well-organized and well-regulated system. The deregulation of the immune system may lead to the development of RA with destructive inflammatory polyarticular joint potentially resulting in progressive

iii) Joint Thickness
Joint thickness (perimeter) was measured on 7 th , 14 th , 21 st and 28 th day by using micrometer screw gauge. The mean changes in joint thickness, were calculated on respective days.

B) Assessment of Pain Perception Parameter i) Arthritic Dorsal Flexion Pain Test
Ankle joint was gently flexed dorsally until the toes touched the front of the leg for 5 times with an inter-test interval of 5 seconds. Pain was scored zero when the animal showed neither squeaking nor quick leg-withdrawal. The scores was 1 when either reaction appeared, and scored 2 when both reactions appeared. All the groups were evaluated in this manner on 7 th , 14 th , 21 st and on 28 th day.

ii) Stair Climbing Activity
Overnight fasting animals were trained for one week to climb a staircase with steps at 5, 10 and 15 cm having water at second step and food at the third step. Climbing ability of the rats in the above groups scored 0, if the rats did not climb ; 1, if the rats climbed onto step-1; 2, if the rats climbed onto step -2 and 3, if the rats could climb all the three steps. All the groups were evaluated in this manner on 7 th , 14 th , 21 st , and on 28 th day.

iii) Motility Test
The motility patterns of rats was observed for a period of 5 minutes on plane surface and score 0, if the rat walked easily, score 1, if rat walked with more difficulty and scored 2, if rat walked with more difficulty and avoided touching the toes of the inflamed paw to the floor. All the groups were evaluated in this manner on 7 th , 14 th , 21 st , and on day 28 th . [11,12]

D) Histopathological Analysis of Arthritic Rats
On 28 th day all animals were sacrificed and their hind limbs were removed surgically and fixed in 10 % buffered formalin. Then limbs were decalcified in 15 % formic acid for histopathological study. [15]

E) Radiological Analysis of Arthritic Rats
Before sacrificing the animals, X-rays were taken on 14 th and 28 th day at the joint of hind paw of the animals for evaluating the bone and cartilage damage. Radiographs were taken using X-ray apparatus (Siemen-60 MA, Germany) and industrial X-Ray film (Fuji photo film, Japan). The X-ray apparatus was operated at 220 V with a 40 V peak, 0.2 second exposure time, and a 60 cm tube to film distance for anterior-posterior projection. [15]

F) Analysis of Serum Transaminases
The blood was collected after sacrificing the animals of all groups on 28 th day. It is then centrifuged (REMI) and serum was then analyzed for lysosomal enzymes such as serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) by using SGPT, SGOT and ALP analysis kit. [16] i Sample (μl) Working reagent Sample 500 50 As mentioned in the above table blank, standard and sample was prepared by considering 500 μl of working reagent and 50 μl each of distilled water, standard and sample respectively, later all the samples were aspirated individually and absorbance was recorded at 340 nm.

ii) Estimation of Serum Glutamate Pyruvate Transaminase Activity (SGPT/ALT) [U.V. Kinetic Method] Principle
Serum Glutamate Pyruvate Transaminase (SGPT or ALT) catalyses the reversible transfer of amino group from Lalanine to 2-oglutarate forming pyruvate and L-glutamate. The pyruvate produced is reduced to L-Lactate by lactate dehydrogenase (LDH) and NADH. The oxidation of NADH in this reaction is measured as a decreased in the absorbance of NADH at 340 nm which is proportional to the SGPT activity. The rate of decrease in concentration of NADH measured by UV kinetic method is proportional to catalytic activity of ALT present in the serum sample.  As mentioned in the above SGOT, the same procedure had been followed.

iii) Estimation of Alkaline Phosphatase Activity (ALP) Principle
Phosphatase is catalyses the hydrolysis of phosphates. Alkaline and acid phosphatases are differentiated by pH range of their optimum activities. The serum alkaline phosphatase hydrolyses p-nitro phenyl phosphate in the presence of oxidizing agent Mg +2 In the phosphatase determination, p-nitrophenylphosphate is used as substrate, which is dissociated by the enzyme into p-nitrophenolate and phosphate. The phenolate liberation occurs as yellow anion, which is then determined by using UV method. The amount of p-nitrophenolate liberated in unit time is directly proportional to the phosphatase activity.
Reaction p-Nitrophenyl phosphate ALP, Mg +2 p-Nitrophenol + Phosphate Procedure Table 3:-Preparation of Blank, STD and Sample for ALP Pipette Sample (μl) Working reagent Sample 500 10 As mentioned in the above blank, standard sample was prepared by considering 500 μl of working reagent and 10 μl of each of distilled water, standard and sample respectively, later all the samples were aspirated individually and absorbance was recorded at 405 nm.

Statistical Data Analysis
Data obtained were subjected to one way analysis of variance (ANOVA) followed by Dunnet's test *p < 0. 05, **p < 0.01 was considered significant Results:-

Preliminary Phytochemical Evaluation
Phytochemical studies of methanolic extract and hydro alcoholic extract of fruits part of Withania Coagulans Dunal showed the presence of carbohydrates, saponin glycosides, flavonoids, alkaloids, glycosides, steroidal compounds, fixed oils.
TLC Profile R f value found to be 0.6 for WCME and 0.65 for WCHAE which is similar to that of R f value of Withaferin A (i.e.0.6-0.65) hence it shows the presences of Withaferin A in the extracts.

Acute Toxicity Studies
WCME and WCHAE did not show any toxic or deleterious effects up to 2000 mg/ kg oral dose. As the rats were administered up to maximal possible dose, the LD 50 value of WCME and WCHAE could not be determined.

a) Effect of WCE on Arthritis Index Score in FCA Induced Arthritis
Effect of WCE on arthritis index was summarized in Table 4. Significant increased in arthritis index was observed in FCA treated group of animal when compared with normal animals. FCA increased arthritis index were significantly (p<0.05, p<0.01, p<0.01 and p<0.01) reduced by treatment with WCME-200, WCME-500, WCHAE-450 and 2065 indomethacin (10 mg/kg) on 28 th day in comparison with disease control group in FCA induced arthritis model respectively.

) Effect of WCE on Paw Edema in FCA Induced Arthritis
Effect of WCE on paw edema was summarized in Table 4. In present study it was observed that treatment of WCME-500 and WCHAE-450 significantly (p<0.01) reduce the paw edema volume (ml) on 28 th day in comparison with disease control group in FCA induced arthritis model. Here, in agreement with other studies, WCME and WCHAE effectively inhibited the development of edema, a characteristic sign of inflammation, in arthritic hind of arthritic rats.  Values are expressed in mean ± SEM (n=6), *p<0.05, **p<0.01 vs Disease Control group, Data analyzed by One way ANOVA test followed by Dunnet"s multiple test for comparison. Treatment of various groups is as follows, N: Normal, DC: Disease Control, STD: Standard, TA1: WCME-200 mg/kg, TA2: WCME-500 mg/kg, TB1: WCHAE-450 mg/kg

c) Effect of WCE on Joint Diameter (thickness) in FCA Induced Arthritis
In present study it was observed that treatment of WCME-500 and WCHAE-450 significantly (p<0.05 and p<0.01) reduce the paw edema volume (ml) on 28 th day respectively in comparison with disease control group in FCA induced arthritis model.  Values are expressed in mean ± SEM (n=6), *p<0.05, **p<0.01 vs Disease Control group, Data analyzed by One way ANOVA test followed by Dunnet"s multiple test for comparison. Treatment of various groups is as follows, N: Normal, DC: Disease Control, STD: Standard, TA1: WCME-200 mg/kg, TA2: WCME-500 mg/kg, TB1: WCHAE-450 mg/kg

d) Effect of WCE on Dorsal Flexion Score in FCA Induced Arthritis
In present study it has been observed that FCA induced increased dorsal flexion score were significantly (p<0.05, p<0.01, p<0.01 and p<0.01) reduced by treatment with WCME-200, WCME-500, WCHAE-450 and indomethacin (10 mg/kg) on 28 th day in comparison with disease control group in FCA induced arthritis model respectively. As reduction in response given by arthritic animals on dorsal flexion was observed, hence it may be concluded that the level of inflammation is decreased with the progression of WCE treatment. Values are expressed in mean ± SEM (n=6), *p<0.05, **p<0.01 vs Disease Control group, Data analyzed by One way ANOVA test followed by Dunnet"s multiple test for comparison. Treatment of various groups is as follows, DC: Disease Control, STD: Standard, TA1: WCME-200 mg/kg, TA2: WCME-500 mg/kg, TB1: WCHAE-450 mg/kg

e) Effect of WCE on Motility Score in FCA Induced Arthritis
In present study it has been observed that FCA induced increased motility score were significantly (p<0.05, p<0.01, p<0.01 and p<0.01) reduced by treatment with WCME-200, WCME-500, WCHAE-450 and indomethacin (10 mg/kg) on 28 th day in comparison with disease control group in FCA induced arthritis model respectively. It has been observed in radiological study that ankle joint space of arthritic animals is retained effectively by continuous WCE treatment which may be responsible for enhanced motility behavior of arthritic animals which may lead to less 2068 stiffness in joints. Hence it can be proposed that level of motility increases with progression of WCE treatment in FCA induced arthritis.  Values are expressed in mean ± SEM (n=6), *p<0.05, **p<0.01 vs Disease Control group, Data analyzed by One way ANOVA test followed by Dunnet"s multiple test for comparison. Treatment of various groups is as follows, DC: Disease Control, STD: Standard, TA1: WCME-200 mg/kg, TA2: WCME-500 mg/kg, TB1: WCHAE-450 mg/kg

f) Effect of WCE on Stair Climb Score in FCA Induced Arthritis
In present study it has been observed that FCA induced decreased motility score were significantly (p<0.05, p<0.01 and p<0.01) reduced by treatment with WCME-500, WCHAE-450 and indomethacin (10 mg/kg) on 28 th day in comparison with disease control group in FCA induced arthritis model respectively. As it has been observed in radiological study that ankle joint space of arthritic animals is retained effectively by continuous WCE treatment which may be responsible for enhanced motility behavior of arthritic animals which may lead to less stiffness in joints and more climbing capacity. 2069

g) Effect of WCE on Serum Transaminases in FCA Induced Arthritis
In present study it has been observed that treatment of WCE significantly decreased the raised level of serum transaminases like SGOT, SGPT and ALP. On 28 th day WCME-200, WCME-500, WCHAE-450 and standard significantly (p<0.01, p<0.01, p<0.01 and p<0.01) decrease the increased level of SGOT respectively. WCME-500, WCHAE-450 and standard significantly (p<0.05, p<0.01 and p<0.01) decrease the elevated and standard significantly (p<0.05, p<0.01 and p<0.01) decrease the elevated level of SGPT respectively. WCME-200, WCME-500, WCHAE-450 and standard significantly (p<0.01, p<0.01, p<0.01 and p<0.01) decrease the increased level of ALP in comparison to disease control group in FCA induced arthritis respectively.

i) Effect of WCE on Different Organ Weight in FCA Induced Arthritis
In case of liver arthritic rat shows increase in weight of liver whereas the Indomethacin and WCHAE-450 mg/kg shows significant (p<0.01 and p<0.05) decreased the weight. In case of increased weight of kidney, Indomethacin and WCHAE-450 mg/kg shows significant (p<0.01 and p<0.01) decrease in weight. In case of spleen Indomethacin, WCME-500 and WCHAE-450 mg/kg shows significant (p<0.01, p<0.01 and p<0.01) decrease in weight. Indomethacin, WCME-500 and WCHAE-450 mg/kg showed significant (p<0.01, p<0.05 and p<0.01) decrease in elevated weight of adrenal gland respectively. There were not significant changes observed in liver, kidney, spleen and adrenal gland weight in case of WCME-200 mg/kg.

j) Effect of WCE on Body Weight in FCA Induced Arthritis
In present study, it is clear from the data obtained that there is close relationship between the extent of joint inflammation and the degree of weight loss. In the first week after adjuvant injection, the arthritic rats showed marked weight loss, followed by normal weight gain in subsequent weeks whereas the WCME, WCHAE and standard drug treated groups did not show any weight loss. The significant (p<0.01) weight gain in the Indomethacin treated rats on 14 th and 28 th day. WCME-200 mg/kg treated group rats shows significant (p<0.05) weight gain on 28 th day whereas the WCME-500 mg/kg and WCHAE-450 mg/kg treated group rats shows significant (p<0.05 and p<0.01) weight gain on 14 th and 28 th day respectively.

k) Radiographic Evaluation in FCA Induced Arthritis
In present study on 14 th and 28 th day, radiological assessment of rat paw through X-rays showed significant reduction in various imaging parameters like spur formation, bone erosion, soft connective tissue swelling around ankle joint, calcium deposition and increase in interspacing between the bones in case of ankle joint, in Standard Indomethacin, WCME-200, WCME-500 and WCHAE-450 groups respectively in comparison with disease control group.  450 mg/kg Prevented bone destruction and soft tissue swelling Therefore from above study it was observed that both WCME and WCHA extract treatment shows significant antiarthritic activity.

l) Histological Evaluation in FCA Induced Arthritis
Histopathological studies of left hind paw joints in normal control rats shown intact articular cartilage and normal synovial lining. Distorted articular cartilage, bone erosion, synovial membrane lining cells hyperplasia, and infiltration of the inflammatory cells like lymphocytes and eosinophils were abundant in synovium and joint space in disease control rats. It was found that standard, WCME-500 mg/kg and WCHAE-450 mg/kg showed better retention of joint parameters like very less disruption of synovial cartilage, reduction in synovial hyperplasia, bone erosion, tissue infiltration with lymphocytes and eosinophils. While in case of treatment of WCME-200 mg/kg group, above parameters were found to have slightly more effect in comparison with disease control group.

Discussion:-
In spite of discovery of several newer agents, the search for better anti-inflammatory drugs continues because of their existing side effects and none of them is suitable for prolonged used. These side effects of the antiinflammatory drugs are one of the major problems in developing medicine today. Therefore there need of development of new and more powerful drugs with fewer side effects. Therefore there is various alternatives like drug from herbal and animal origin were discovered with few side effects, herbal drugs like Vertex Negunda, Terminalia Arjuna [17] , Cassia Auriculata [18] had shown effective anti-inflammatory activity in experimental animal models of inflammation and arthritic condition. In this research work we have primarily focused on the investigation of Withania Coagulans dunal fruits extracts against inflammatory and immune system conditions associated with RA.
Rheumatoid arthritis is chronic, relapsing inflammatory and autoimmune multisystem illness that affects the joints. It is characterized by inflammation of the synovial membrane, pain and restricted joint movement, stiffness and swelling of joints. [19] The process produce an inflammatory response of synovium (synovitis) secondary to hyperplasia of synovial cells, excess synovial fluid and the development of pannus in the synovium. The pathology of disease process often leads to the destruction of articular cartilage and ankylosis of the joints. In RA immune system activates the lymphocytes and chemical messenger like cytokine, TNF, IL-1 and IL-6 are expressed in inflamed area. This deregulation of the immune system may lead to the development of rheumatoid arthritis. Autoimmunity plays a pivotal role in both its chronicity and progression.

In Vivo Anti-Arthritic Potential of WCE :-Complete Freund's Adjuvant Induced Arthritis in Rats:-
The evaluation of WCE for anti-arthritic potential was done with CFA model which has been used to induce an arthritic immunopathological condition that displayed many pathological features of human RA. [19] Freund"s adjuvant arthritis can be produced in rats by administration of Mycobacterium Tuberculosis suspended in oil in subplantar region of rat paw. Bacterial peptidoglycan and muramyl dipeptides are responsible for arthritic condition. It occurs through cell mediated autoimmunity by structural mimicry between mycobacteria and cartilage proteoglycan in rats. [20] Liberation and overproduction of bradykinin, prostaglandin, and kinin in paw tissue. This model is also involves pannus formation, neutrophil infiltration and bone erosion. Arthritis typically persists during the first two week after its appearance. [21] Severely affected animals eventually develop chronic deforming joint lesion.
In FCA induced arthritis it was observed that WCHAE than WCME significantly inhibited the arthritis index score, paw edema volume, joint area and pain perception parameters like dorsal flexion and motility along with rise in stair climb score in comparison with disease control for 28 days. After 14 th day in FCA prominent inhibition was observed in all parameters. The swelling has been found to be increasing in the initial phase of inflammation and then becomes constant in two weeks. The change in paw volume has been found to be associated with an increase in granulocyte and monocytes. Because, the activation of macrophages results in the production of several cytokines including IL-1, IL-6, interferon-γ and TNF-α which have been implicated in immune arthritis.
Tissue damage was assessed by measuring the activity of enzyme in the serum. Liver impairment is also one of the features of adjuvant arthritis. The increase in the aminotransferase is due to their release from the cells of the damaged organ. Alkaline phosphtase activity has been reported to increase during the morphological and functional development of tissue. Thus ALP, SGOT, SGPT are good indices of liver and kidney impairment which is also considered as feature of adjuvant arthritis. To evaluate liver function, SGOT and SGPT levels were estimated. [22] SGOT and SGPT has been reported to play a vital role in the formation of biologically active chemical mediators such as bradykinin in inflammatory process confirmed a positive correlation between the increased activity of serum alkaline phosphatase and disease activity of RA. [23] Amino transferase and alkaline phosphtase were significantly reduced by WCHAE than WCME in arthritic rats. This reducing effect may be related to their anti-inflammatory and anti-arthritic effect. The decreased enzyme levels on WCME and WCHAE treatment emphasized decreased bone loss and organo protective role of WCME and WCHAE in CFA induced rats.
As the severity of arthritic condition increases level of inflammation rises in patient of RA. Along with rise in inflammation, level of WBC"s also increases by virtue of autoimmunity. Hence the measurement of WBC"s is an effective tool to assess the severity of disease state in RA patients. As a part of WBC"s, B cells in patients with rheumatoid arthritis are usually secrets large amount of auto-antibodies which can enhance tissue destruction and release of auto-antigens, indicating that B-cells are also critically important in the severity and length of disease. [24] The rise in WBC count is due to release of IL-1B increase the production of monocyte and colony stimulating factor. [25] WBC count increase in arthritic rat to kill invading pathogenic microorganism. A large number of leucocytes that generate free radicals are present in the region of inflammation. Hence, free radical measurement reflects the number of leucocytes present in the inflammatory condition. [26] It was observed that WCHAE than WCME significantly restored the increased WBC count to the normal in comparison to disease control group which may be possibly due to inhibition of matured bone marrow dendritic cell growth which might be responsible for innate immunity. This is probably due to inhibition of matured bone marrow dendritic cells, responsible for development of innate immunity and T-cell cytotoxicity by PLA 2. The increase in WBC count is an indication of increased 2077 autoimmunity and level of inflammation along with anemic condition of animal due to decrease in RBC,HB and HCT as iron is deposited in connective tissue.
Anemia arises due to abnormal storage of iron in reticuloendothelial system and synovial tissue due to which RBC count, HB count and HCT value decreases below normal level. [27] It was observed that RBC, Hb and Hematocrit values significantly increased in WCHAE than WCME, which might be due to the matured bone marrow cells respond to anemic condition by preventing abnormal deposition of iron in reticuloendothelial system and synovial tissue.
The increase in platelet count might be due to the stimulation of immune system against the invading pathogenic microorganism. This is evident by the infiltration of inflammatory mononuclear cells in the joint of arthritis rat. [28] T-lymphocytes have been reported to play a central role in the pathogenesis of rheumatoid arthritis. These cells comprise the majority of the lymphoid cells found in the rheumatoid synovium. In arthritic condition there is a moderate elevation in lymphocyte count. In all treated groups the lymphocyte count was suppressed significantly as compared to control. ESR is an estimate of the suspension stability of RBC's in plasma, related to the number of size of red cells and to the relative concentration of plasma proteins especially fibrinogen and the α and β globulins. The acute phase proteins in ESR share the property of showing elevations in the concentration in response to stress or inflammation that occurs like infection, injury, and surgery and tissue necrosis. So in arthritic condition, ESR is elevated. [13] All the treated group significantly reduced the elevated level of ESR in comparison to disease control group.
In a chronic inflammatory process, such as RA, large numbers of neutrophils are attracted across the synovial membrane, and become activated. Neutrophils possess a range of potent proteinases and hydrolases, and have the ability to generate a series of reactive oxygen intermediates (ROI) via the combined activities of NADPH (reduced form) oxidase and myeloperoxidase (MPO). [29,30] If the neutrophils are not efficiently depleted, their production of inflammatory mediators, such as ROI, could prolong the inflammatory reaction. In fact, inappropriate release of ROI from activated neutrophils is responsible for joint damage observed in RA. [31] Beside ROI and inflammatory mediators, neutrophils are also an important source of proteolytic enzymes which play a role in degradation of articular structures. In present study all the treated group significantly reduced the elevated level of neutrophils in comparison to disease control group.
The changes in the body weight were monitored as apparent indicator of arthritic symptoms and the loss of body weight usually began to appear at the onset stage of arthritis. In the present study, the body weight decreased in arthritis induced animals might be linked to the systemic or local action of cytokines such as TNF-α, since TNF-α has been closely related to the loss in body weight occurring in animals suffering from chronic inflammation. The loss of body weight observed in CFA induced arthritic rats may be due to the reduced absorption of 14 C-glucose and 14 C-leucine in the rat intestine. WCHAE than WCHAE showed significant increase in body weight reveals the restoration of absorption capacity of the intestine in the arthritic animals. [32] In adjuvant treated rats an increase in lipid peroxide level was seen in liver and kidney. Liver and kidney weight were increased in AIA rats. WCHAE than WCME alter these values near to normal. Spleen is a vital organ serves as the reservoir for the cells and antibody formation which involved in the immune response. In adjuvant arthritis, spleen Increased in the weight of spleen is associated with the splenomegaly, generalized lymphadenopathy and altered hepatic function. In disease control group weight of spleen was significantly increased. [15] In case of spleen and adrenal gland weight both of this result were significant in WCHAE than WCME treated rats. Radiological changes in RA condition are useful diagnostic measure which indicates the severity of disease. Soft tissue swelling is the earlier radiographic sign, whereas the prominent radiographic changes like bone erosion narrowing of joints spaces can be observed only in the developed stages of arthritis. WCHAE than WCME groups X-rays showed significant reduction in various imaging parameters like hyperostosis, bone erosion, soft connective tissue swelling around ankle joint and increase in interspacing between the bones in case of ankle joint in comparison with disease control group.
In present study histological assessment of joint showed very less disruption of synovial cartilage, reduction in synovial hyperplasia, bone erosion, tissue infiltration with lymphocytes and eosinophils in case of WCME, WCHAE and standard treatment groups in comparison with disease control group in FCA induced arthritis.
2078 Thus, to summarize, WCME and WCHAE of fruits showed significant anti-arthritic and immunosuppressive activities probably by virtue of an underlying antioxidant activity.

Conclusion:-
 WCME oral treatment at both the doses-250 mg/kg and 500 mg/kg demonstrated significant anti-arthritic activity.  WCME-500 mg/kg provides more significant anti-arthritic activity and is comparable to the standard drug indomethacin.  Whereas the WCHAE-450 mg/kg shows highly significant anti-arthritic activity and is comparable to the standard drug indomethacin.
Phytochemical constituents like flavonoides, alkaloids, saponin, glycosides, steroids and withanolides were already reported to anti-inflammatory, immunosuppressant and anti-arthritic activity and these constituents were present in both WCME and WCHAE. Hence these chemical constituents can be accounted for the observed anti-arthritis activity.
The present study aims to bring about value addition to the health benefits of Withania Coagulans dunal fruits establish it as a potent "functional food" and promote its use in people"s diets. Besides, it is cheap, readily available to all strata of society, with medicinal properties attributed to it. Future research may be carried out to isolate the active constituents and bioactivity evaluation may be carried out.