J Complement Integr Med. 2015; 12(2): 117–125

Mahmood Ahmad Khan, Mythily Subramaneyaan, Vinod Kumar Arora, Basu Dev Banerjee and Rafat Sultana Ahmed*

Effect of Withania somnifera (Ashwagandha) root extract on amelioration of oxidative stress and autoantibodies production in collagen-induced arthritic rats Abstract Background: Rheumatoid arthritis is an inflammatory autoimmune disorder. Withania somnifera Dunal (Solanaceae) (WS), is a common medicinal plant used in traditional systems of medicine for the treatment of arthritis, and is an ingredient of anti-arthritic polyherbal formulations such as Habb-e-Asgand® and Arthritin™. In the present study, we evaluated the antioxidant and anti-arthritic activity of aqueous extract of WS root (WSAq) in collagen-induced arthritic (CIA) rats. Methods: CIA rats were treated by using three doses of WSAq (100, 200, 300 mg/kg b. wt., orally) and methotrexate (MTX, 0.25 mg/kg b. wt. i.p.) as a standard reference drug for 20 days. The anti-arthritic effect was assayed by measuring the arthritic index, autoantibodies such as rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (a-CCP), anti-nuclear antibody (ANA), anti-collagen type II antibody (a-CII) and inflammatory marker like C-reactive protein (CRP). The oxidative stress parameters were also measured. Results: Treatment with WSAq resulted in a dose-dependent reduction in arthritic index, autoantibodies and CRP (p < 0.05) with maximum effect at dose of 300 mg/kg b. wt. and the results were comparable to that of MTXtreated rats. Similarly, oxidative stress in CIA rats was ameliorated by treatment with different doses of WSAq, as evidenced by a decrease in lipid peroxidation and glutathione-S-transferase activity and an increase in the *Corresponding author: Rafat Sultana Ahmed, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi, India, E-mail: [email protected] Mahmood Ahmad Khan, Mythily Subramaneyaan, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi, India Vinod Kumar Arora, Department of Pathology, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi, India Basu Dev Banerjee, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi, India

glutathione content and ferric-reducing ability of plasma (p < 0.05). Conclusions: The results showed that WSAq exhibited antioxidant and anti-arthritic activity and reduced inflammation in CIA rats and suggests the potential use of this plant in the treatment of arthritis. Keywords: Ashwagandha, herbal medicine, reactive oxygen species, rheumatoid arthritis DOI 10.1515/jcim-2014-0075 Received December 6, 2014; accepted February 25, 2015; previously published online March 18, 2015

Introduction Rheumatoid arthritis (RA) is an inflammatory disease characterized by chronic inflammation of the synovial joints associated with proliferation of synovial cells and infiltration of activated immuno-inflammatory cells including memory T cells, macrophages and plasma cells leading to progressive destruction of cartilage and bones [1]. The allopathic drugs which are commonly used in the treatment of RA have several side effects and toxicity including some risk of infections in subset of patients being treated with biological response modifiers [2]. As a result of this, due to their less/nil side effects and toxicity, use of alternative treatments based on natural plant products and herbal combinations is on the rise. Withania somnifera Dunal (Solanaceae) (WS) commonly known as Ashwagandha, is an important medicinal plant that has been used in Ayurvedic and indigenous medicine for over 3,000 years [3]. WS roots are a rich source of Withanolides (steroids) and much of Withania’s pharmacological activity has been attributed to two main Withanolides, Withaferin A and Withanolide D. A number of studies have reported that WS possesses various biological activities such as antioxidant, anti-inflammatory, immunomodulatory and antitumor property [4, 5]. WS is

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traditionally used in the treatment of arthritis and is a constituent of various formulations in Ayurveda (Arthritin™) and Unani medicine (Habb-e-asgand®), used for the treatment of various physiological and musculoskeletal disorders. Recent studies from our laboratory showed the anti-arthritic activity of Habb-e-asgand, a commonly used antiarthritic Unani formulation containing WS (Asgand) as a major constituent [6]. Taking into consideration its immunomodulatory, anti-inflammatory activity, the present study was designed to assess the anti-arthritic activity of aqueous extract of WS (WSAq) root in collagen-induced arthritis (CIA) model.

Induction of arthritis Arthritis in rats was induced by immunization with type II collagen (CII) (Sigma-Aldrich, USA) as described by Campo et al. [7]. Briefly, collagen was dissolved in 0.1 M acetic acid by gently stirring overnight at 4 °C and emulsified with equal amounts of complete Freund’s adjuvant (CFA). Each rat was immunized with 0.2 mL (200 μg of CII) of the emulsion by subcutaneous (sc) injection at the base of the tail. To ensure a high incidence and severity of arthritis a booster injection with 0.2 mL (200 μg of CII) emulsion was given 14 days after immunization. After the second immunization, development of arthritic symptoms was observed, and recorded on the 25th day.

Assessment of severity of arthritis

Materials and methods Plant material Fresh roots of WS were obtained in the month of March from the local market of Delhi and authenticated by Dr. Sunita Garg [Chief Scientist & Head, Raw Materials and Herbarium, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi]. The voucher specimen (NISCAIR/RHMD/ 2013/2198/204) was deposited in the raw material and herbarium museum, NISCAIR, Delhi. Root of WS was air dried at room temperature. Dried roots were crushed in an electric grinder machine to a fine powder and stored in an air-tight container, until further use.

Rats were observed daily for the severity and progression of arthritis. The progression was measured by arthritic index (AI). The measurement was carried out prior to the onset of arthritis and then every week after induction of arthritis. The level of inflammation of each paw was graded as scores from 0 to 15 (one point for each swollen or red toe, one point for mid foot digit or knuckle, and five points for swollen ankle). The scores of the four paws were added. A total score of 60 for each rat was considered as RA induction [8] and referred to as AI. The severity of the clinical arthritis in each affected paw was graded at the end of the treatment protocol by the same macroscopic scoring system.

Grouping and treatment The induced animals were randomly divided into six groups (6 animals each).

Preparation of extract Soxhlet extraction: Extraction was performed using Soxhlet apparatus with accurately weighed 50 g root powder of WS in each lot for 14 h. Extraction was performed separately with 500 mL distilled water as the extracting solvent at room temperature. Total 400 g of root powder was used for extraction. After extraction the extracts were pooled, concentrated in a lyophilizer and stored in refrigerator at 4 °C. The percentage yield of dry root WSAq was 4.37 % of dry weight of starting material. The lyophilized root extract was used for further studies.

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Test animals Male Wistar albino rats of body weight 100–150 g were obtained from the Central Animal House facility of the institute. The animals were allowed to acclimatize for one week before the initiation of the experiment. They were randomly divided into five groups and maintained under standard conditions (light:dark 14:10 h, ambient temperature 22  2 °C, humidity 40–45 % and free access to food and water). All animals received humane care in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC Approval No. IAEC/2012-03 dated May 1, 2012).

Group I – Control (non-induced þ normal saline). Group II – Induced (treated with CII, for induction of RA) þ normal Saline. Group III – Induced þ Treated with Methotrexate (MTX, 0.25 mg/kg b. wt. once every week after induction of arthritis) [9] Group IV – Induced þ Treated with WS, aqueous extract (WSAq, 100 mg/kg b. wt.), orally daily for 20 days after induction of arthritis Group V – Induced þ Treated with WS aqueous extract (WSAq, 200 mg/kg b. wt.) orally daily for 20 days after induction of arthritis Group VI – Induced þ Treated with WS aqueous extract (WSAq, 300 mg/kg b. wt.) orally daily for 20 days after induction of arthritis

The induction and treatment protocol was followed as mentioned in Figure 1. The dose of WSAq was selected after literature survey and preliminary studies.

Assessment of body weight Body weight was measured periodically. The change in body weight of each animal was calculated after the onset of arthritis and after subsequent treatment.

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Figure 1: The induction and treatment protocol of CIA rats.

Collection of samples At the end of treatment, the rats were fasted overnight and blood was collected by retro-orbital vein puncture for biochemical studies. The whole blood was utilized to determine the glutathione (GSH) content. Serum was separated and used for estimation of glutathione-S-transferase (GST) activity, ferric-reducing ability of plasma (FRAP) and the extent of lipid peroxidation (LPO), autoantibodies and C-reactive protein (CRP).

Biochemical analysis

working FRAP reagent was prepared by adding 300 mM/l acetate buffer (pH 3.6), 10 mM/l 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 40 mM/l HCl and 20 mM/l FeCl3·6H2O in ratio of 10:1:1 respectively. Sample was added, thoroughly mixed and the absorbance was monitored at 593 nm for 4 min. The values are expressed as µM/mL. Assessment of anti-inflammatory activity: The level of serum CRP was measured by using standard ELISA kit according to the manufacturer’s instructions (Ray biotech, Norcross, Georgia, USA).

Statistical analysis

Assessment of anti-arthritic activity: The biomarkers of RA such as rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (a-CCP) and antinuclear antibody (ANA), anti-collagen type II antibody (a-CII) were measured in rat serum by standard ELISA kits (Omega Diagnostics Ltd, Alva, Scotland, UK, and Ray Biotech, Norcross, Georgia, USA and Chondrex Inc Redmond, USA) as per manufacturer’s instructions, using a micro plate reader (ThermoScientific, USA). Assessment of oxidative stress parameters: The extent of LPO was determined by measuring the malondialdehyde (MDA) levels in serum as described by Satoh [10]. Briefly, 0.5 mL serum was precipitated with 20 % trichloroacetic acid (TCA). The precipitate was suspended in 0.05 N sulfuric acid and thiobarbituric acid (TBA) (0.07 % in 1 M sodium sulfate) and incubated in boiling water bath for 30 min. The MDA–TBA adduct thus formed was extracted with butanol and measured spectrophotometrically at 532 nm. The results are expressed as nM/mL. Total GSH content in whole blood was estimated as described by Tietze [11]. Briefly, the reaction mixture (1 mL) contained 25 µl of hemolysate, NADPH (0.2 µM/mL in 0.01 M/0.005 M phosphate EDTA buffer [pH 7.5]) and glutathione reductase (1 unit). On addition of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), the resulting chromophoric product, i.e., 2-nitro,5-thiobenzoic acid absorbance was measured at 412 nm. The result is expressed as nM/mL blood. Serum GST (EC 2.51.18) activity was measured spectrophotometrically by the method of Habig et al. [12]. Briefly, the assay mixture contained in a total volume of 3 mL, 1 mM CDNB (1-chloro-2, 4-dinitrobenzene) in ethanol (final concentration of ethanol not less than 4 %), 0.001 M GSH, 0.1 M potassium phosphate buffer (pH 6.5) and serum sample. The formation of the resulting adduct (GS-2, 4 dinitrophenyl glutathione) was monitored by noting the net increase in the absorbance at 340 nm against reagent blank for 0–5 min. The enzyme activity was determined by using the extinction coefficient 9.6/mM/cm and the results are expressed as Unit/mg protein. FRAP was determined by measuring the ability of plasma to reduce Fe3 þ to Fe2 þ as described by Benzie and Strain [13]. Briefly, the

All values are expressed as mean  SD. Data were analyzed by one-way ANOVA using SPSS version 16 (SPSS, Chicago, IL, USA) statistical program and the individual comparisons of the treatment were obtained by Tukey’s test for multiple comparisons. All p-values were two-sided. A difference of p < 0.05 was considered to be statistically significant.

Results Effect of WSAq on body weight In our study we found that there was a marked decrease in the body weight in arthritic rats. Oral administration of WSAq prevented the loss of body weight in CIA rats (Figure 2).

Effect of WSAq on severity of arthritis AI is a type of scoring system which represents the severity and advancement of arthritis that is used to assess the efficacy of treatments. In the CIA group, rats showed an increase in AI. WSAq dose dependently remits the progression of arthritic score in CIA rats in a dose-dependent manner, as evidenced by a decrease in AI (Figure 3).

Anti-arthritic activity of WSAq The autoantibodies such as RF, a-CCP, ANA and a-CII found in autoimmune-related disorders such as arthritis

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Figure 2: Effect of different doses of WSAq and methotrexate on body weight in CIA (n ¼ 6 in each group) rats.

Figure 3: Ameliorative effect of different doses of WSAq and methotrexate on arthritic index in CIA (n ¼ 6 per group) rats. *Significantly different from induced group (p < 0.05). WSAq100, WSAq200, WSAq300; Aqueous root extract of Withania Somnifera at a dose of 100, 200, 300 mg/kg b.wt respectively and Methotrexate (MTX) (0.25 mg/kg b. wt).

were significantly elevated (p < 0.05) in CIA rats as compared to control rats. Treatment with WSAq reduced the elevated level of autoantibodies (p < 0.05) in a dosedependent manner, which was comparable to that of MTX-treated rats. The maximum decrease was found in WSAq300, i.e., 45.53 %, 40.51 %, 50.56 % and 50.21 %, respectively, for RF, a-CCP, ANA and a-CII (Figure 4).

Anti-inflammatory activity of WSAq Serum CRP is one of the most important acute-phase protein and is an exquisitely sensitive systemic marker of inflammation and tissue damage. In this study, CRP levels were found to be significantly increased (p < 0.05) in CIA rats. Administration of WSAq showed a decrease (p < 0.05) in

CRP levels dose dependently, and the maximum decrease was found in WSAq300, i.e., nearly twofold. The results were comparable to that of MTX-treated rats (Figure 5).

Antioxidant activity of WSAq MDA levels were measured to know the oxidative damage of membrane lipids caused by reactive oxygen species (ROS). The level of MDA was elevated (p < 0.05) in CIA rats as compared to control group. At all the three doses of WSAq the level of MDA was decreased significantly (37.23 %, 49.21 %, 54.69 % for doses 100, 200 and 300 mg/kg b. wt., respectively) and the results were comparable to that of MTX-treated rats (Figure 6(A)). FRAP was measured to find the total antioxidant capacity. In CIA rats there was a significant decrease in the level

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Figure 4: Effect of different doses of WSAq and methotrexate on rheumatoid factor (RF, A), anti-cyclic citrullinated peptide antibody (a-CCP, B), anti-nuclear antibody (ANA, C), anti-collagen type II (a-CII, D) in CIA (n ¼ 6 per group) rats. *Significantly different from control; asignificantly different from induced group (p < 0.05). WSAq100, WSAq200, WSAq300; Aqueous root extract of Withania Somnifera at a dose of 100, 200, 300 mg/kg b.wt respectively and Methotrexate (MTX) (0.25 mg/kg b. wt).

Figure 5: Ameliorative effect of different doses of WSAq and methotrexate on C-reactive protein (CRP) in CIA (n ¼ 6 per group) rats. *Significantly different from control; asignificantly different from induced group (p < 0.05).

of FRAP which was effectively increased after treatment with WSAq at all three doses and the results were comparable to that of MTX-treated rats (Figure 6(D)). The GSH levels were significantly decreased and GST activity was increased in CIA rats as compared to control rats. Treatment with WSAq at different doses caused a reduction in GST activity with a concomitant increase in the GSH levels. These results were similar to that of MTXtreated rats (Figure 6(B) and 6(C)).

Discussion RA is a chronic, autoimmune joint disease of unknown cause affecting about 1 % of the population worldwide. RA can have a severe impact on health and socio-economic status and if left untreated it may lead to premature death. Optimal management of RA involves arresting or controlling the progression of the disease through early diagnosis and treatment. Allopathic drugs which are normally used for the

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Figure 6: Ameliorative effect of different doses of WSAq and methotrexate on lipid peroxidation (MDA, A), glutathione content (GSH, B), glutathione-S-transferase activity (GST, C), ferric ability of plasma (FRAP, D) in CIA (n ¼ 6 per group) rats. *Significantly different from control; asignificantly different from induced group (p < 0.05).

treatment of RA have severe side effects. Owing to the several side effects and the high cost of allopathic drugs, many patients look for complementary and alternative medicine (CAM) options in coping with this debilitating disease. Research has indicated that people suffering from chronic pain, as in RA, and those dissatisfied with current treatment are very likely to seek alternative treatments, and an estimated 60–90 % of persons with arthritis use CAM [14]. This increase in the interest in use of alternative herbal products indicates the necessity for more thorough investigation into the safety and efficacy of CAM. Although WS is reported to have antioxidant, antiinflammatory, immunomodulatory and antitumor activities, there are practically no reports on the effect of WS root extracts on biomarkers of RA. Hence, the present study was designed to evaluate the antioxidant, anti-inflammatory and anti-arthritic effect of WSAq at different doses in CIA rats. CIA is the most commonly used model for study of the mechanism underlying immune responses to an autoantigen potentially involved in human disease [15] and the symptoms of this model are analogous to those seen in human RA [16]. The important clinical significance of the model lies in the fact that CII is the major constituent protein of the cartilage in the joints, the main site which is most badly affected in RA. Hence, for evaluating the efficacy of herbal drugs and pathogenesis of disease, CIA model is mostly used [15]. AI represents the severity of arthritis due to the infiltration of mononuclear cells causing inflammation and

multiplication of synoviocytes leading to pannus formation at the joints and is used to evaluate the efficacy of treatment. Treatment with WSAq caused a decrease in AI suggesting the anti-arthritic activity of WSAq (Figure 3). Clinically, loss of body mass is associated with RA [17] and is used as a nonspecific symptomatic marker to assess the ameliorating effect of treatment. Our result shows that the reversal of body weight loss in treated rats is probably due to a decrease in inflammation and may be effective in the relief of arthritis-associated weight loss and its consequences (Figure 2). CRP is produced under conditions of inflammation in cartilage and bone and is a useful biomarker in the evaluation of disease progression and response to therapeutic intervention in a number of inflammatory disorders, including RA. Presence of high concentration of CRP indicates increased joint damage in arthritis [18]. Decrease in CRP levels in the present study has shown that treatment with WSAq reversed the inflammatory damage in joints when compared with CIA rats. Our findings are consistent with previous studies [19, 20] that reported the ant-inflammatory activity of WS root in CIA rats (Figure 5). This decrease in inflammation is possibly due to a decrease in the production of free radicals and subsequent damage. There is a great deal of evidence indicating the important role of oxidative stress in the physiopathology of RA [21] and oxygen-free radicals have been implicated

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Khan et al.: Anti-arthritic activity of aqueous extract of Withania somnifera roots

as mediators of joint tissue and cartilage damage [22]. They also serve as important intracellular signaling molecules that amplify the synovial inflammatory-proliferative response [23]. We therefore evaluated the LPO end product MDA, level of GSH, GST activity and FRAP in CIA rats. The increase in the level of MDA in the serum of CIA rats suggests the enhancement of LPO possibly due to an increase in the generation of ROS in CIA rats. LPO is a well-established marker of oxidative stress and possibly responsible for the cellular injury that occurs during arthritis. In our study we found that there was a significant increase in LPO and GST activity, depletion in GSH as well as FRAP levels in CIA rats. These results are in agreement with other studies [7]. Treatment with WSAq significantly decreased the MDA levels indicating the ameliorative effect of this plant product. The antioxidant defense system may be affected by ROS through a reduction in the intracellular concentration of antioxidants such as GSH. The decreased level of GSH in CIA rats could be due to the increased consumption of GSH for neutralizing the free radicals/ROS by GST. GSH is the major endogenous antioxidant produced by the cells. GSH is the co-substrate of GST participating directly in the neutralization of free radicals. The increase in GST activity supports the hypothesis that arthritis may be associated with increased ROS. Our results are in accordance with previously reported work [24, 25] which showed that increased activity of GST in response to the oxidative stress is to counteract the production of ROS and subsequent damage. The arthritic rats exhibited a significant decrease in the total antioxidant status as evidenced by a decrease in FRAP. After WSAq treatment, the alterations produced in CIA rats with respect to LPO and antioxidant concentration were modulated nearly to the normal levels (Figure 6). This may be due to the presence of compounds like withanolides, alkaloids and flavonoids in the aqueous extracts. These compounds have been shown to scavenge free radicals, including hydroxyl and super-oxide anions and reduce the levels of LPO in stress-induced animals [26]. Oxidative modification of proteins and other biologic molecules leads to the expression of neoantigens, a possible first step in the development of autoimmunity, which may herald the future onset of clinically relevant autoimmune disease [27]. The oxidation of amino acids by ROS increases the antigenicity of DNA, low-density lipoprotein (LDL) and immunoglobulin G (IgG), generating ligands for which autoantibodies show higher avidity [28]. DNA reaction products, in particular 8-oxo-7hydro-deoxyguanosine formed by the reaction of hydroxyl radicals (OH•) with deoxyguanosine, are elevated in

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leukocytes and sera of patients with RA [29], which is both mitogenic and cytotoxic. It considers the evidence for ROS-induced antigenicity arising as a consequence of failure to remove or repair ROS damaged biomolecules and suggests that an associated defect, probably in T-cell signal processing or antigen presentation, is required for the development of disease [28]. As RA is an autoimmune disorder, autoantibodies play an important role in pathogenesis of arthritis. A number of autoantibodies (RF, a-CCP) are specifically known to be associated with severity of RA and are used as markers to diagnose the disease. In general, a-CCPs are a better diagnostic guide than RF due to their higher sensitivity and specificity for RA [30]. Our data suggest that the anti-arthritic activity of WSAq, as evidenced by a decrease in the autoantibodies (RF, a-CCP, ANA, a-CII, Figure 4), could be due to a decrease in the production of ROS. Our results are in agreement with previous studies that showed the protective role of antioxidant supplementation in the treatment of experimentally induced arthritis [31, 32].

Conclusions The present study clearly demonstrates that administration of the WSAq modulate the biochemical changes observed in CIA rats as well as a decrease in the biomarkers of RA. This efficacy in suppression of autoimmunity might be due to the antioxidant, anti-inflammatory and immunomodulatory activities of WS. Results of the present study are in corroboration with the use of WS in folklore and traditional medicine for the treatment of various musculoskeletal disorders. In conclusion, it can be said that WS roots have the ability to reduce the severity of arthritis and ameliorate the symptoms and the results are comparable to that of MTX-treated rats. This activity has been attributed to the biologically active steroids of which Withaferin-A is a major component and reported to exhibit fairly potent anti-arthritic and anti-inflammatory activities [33]. Studies on the direct effect of WS on autoantibodies produced in RA have not been reported before. The data in this pilot study are relevant because they provide the first preliminary evidence of the direct anti-arthritic effect of the aqueous root extract of WS in CIA rats. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

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Research funding: The authors are thankful to the Department of Biotechnology (DBT), Ministry of Science & Technology (Govt. of India) for providing financial support (No.BT/PR4612/MED/30/812/2012). Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References 1. Hitchon CA, El-Gabalawy HS. Oxidation in rheumatoid arthritis. Arthritis Res Ther 2004;6:265–78. 2. Olsen NJ, Stein CM. New drugs for rheumatoid arthritis. N Engl J Med 2004;350:2167–79. 3. Naidu PS, Singh A, Kulkarni SK. Effect of Withania somnifera root extract on haloperidol-induced orofacial dyskinesia: possible mechanisms of action. J Med Food 2003;6: 107–14. 4. Rasool M, Varalakshmi P. Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation: an in vivo and in vitro study. Vascul Pharmacol 2006;44:406–10. 5. Mir BA, Khazir J, Mir NA, Tu H, Botanical KS. Chemical and pharmacological review of Withania somnifera (Indian ginseng): an Ayurvedic medicinal plant. Indian J Drugs Dis 2012;1:147–60. 6. Subramaneyaan M, Yasmeen S, Arora VK, Tripathi AK, Banerjee BD, Ahmed RS. Safety evaluation and therapeutic efficacy of habb-e-asgand, a commonly used antirheumatic polyherbal unani formulation. Altern Ther Health Med 2013;19:52–9. 7. Campo GM, Avenoso A, Campo S, Ferlazzo AM, Altavilla D, Calatroni A. Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats. Arthritis Res Ther 2003;5:122–31. 8. Holmdahl R, Carlsen S, Mikulowska A, Vestberg M, Brunsberg U, Hansson A, et al. Genetic analysis of murine models for rheumatoid arthritis. In: Adolpho KW, editor. Human genome methods. New York: CRC Press,1998:215. 9. Hultqvist M, Olofsson P, Gelderman KA, Holmberg J, Holmdahl R. A new arthritis therapy with oxidative burst inducers. PLoS Med 2006;3:e348. 10. Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90:37–43. 11. Tietze F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502–22. 12. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9.

13. Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 1999;299:15–27. 14. Rao JK, Mihaliak K, Kroenke K, Bradley J, Tierney WM, Weinberger M. Use of complementary therapies for arthritis among patients of rheumatologists. Ann Intern Med 1999;131:409–16. 15. Pandey S. Various techniques for the evaluation of anti arthritic activity in animal models. J Adv Pharm Technol Res 2010;1: 164–71. 16. Bendele A. Animal models of rheumatoid arthritis. J Musculoskelet Neuronal Interact 2001;1:377–85. 17. Jou IM, Shiau AL, Chen SY, Wang CR, Shieh DB, Tsai CS, et al. Thrombospondin 1 as an effective gene therapeutic strategy in collagen-induced arthritis. Arthritis Rheum 2005;52:39–44. 18. Rhodes B, Fürnrohr BG, Vyse TJ. C-reactive protein in rheumatology: biology and genetics. Nat Rev Rheumatol 2011;7:282–9. 19. Paval J, Kaitheri SK, Potu BK, Govindan S, Kumar RS, Narayanan SN, et al. Comparing the anti-arthritic activities of the plants Justicia gendarussa burm F. and Withania somnifera linn. Int J Green Pharm 2009;3:281–4. 20. Sabina EP, Chandal S, Rasool MK. Inhibition of monosodium urate crystal-induced inflammation by withaferin A. J Pharm Pharm Sci 2008;11:46–55. 21. Filippin LI, Vercelino R, Marroni NP, Xavier RM. Redox signalling and the inflammatory response in rheumatoid arthritis. Clin Exp Immunol 2008;152:415–22. 22. Karatas F, Ozates I, Canatan H, Halifeoglu I, Karatepe M, Colakt R. Antioxidant status & lipid peroxidation in patients with rheumatoid arthritis. Indian J Med Res 2003;118:178–81. 23. Leonavičienė L, Bradūnaitė R, Vaitkienė D, Vasiliauskas A, Keturkienė A. Collagen-induced arthritis and pro-/antioxidant status in Wistar and Lewis rats. Biologija 2008;54:290–300. 24. Macdonald IO, Olusola OJ, Osaigbovo UA. Effects of chronic ethanol administration on body weight, reduced glutathione (GSH), malondialdehyde (MDA) levels and glutathione-s-transferase activity (GST) in rats. N Y Sci J 2010; 3:39–47. 25. Subramaneyaan M, Jain S, Yadav C, Arora VK, Banerjee BD, Ahmed RS. Quinalphos induced oxidative stress and histoarchitectural alterations in adult male albino rats. Environ Toxicol Pharmacol 2012;34:673–8. 26. Dhuley JN. Adaptogenic and cardioprotective action of ashwagandha in rats and frogs. J Ethnopharmacol 2000;70:57–63. 27. Pattison DJ, Winyard PG. Dietary antioxidants in inflammatory arthritis: do they have any role in etiology or therapy? Nat Clin Pract Rheumatol 2008;4:590–6. 28. Griffiths HR. Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease? Autoimmun Rev 2008;7:544–9. 29. Bashir S, Harris G, Denman MA, Blake DR, Winyard PG. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann Rheum Dis 1993;52:659–66.

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30. Song YW, Kang EH. Autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies. QJM 2010;103:139–46. 31. Chamundeeswari D, Vaijayanthi V, Umamaheswari S, Gandhimathi C, Karna SL, Meignanam E, et al. Antioxidant activity of arthritin – a polyherbal formulation. Indian J Exp Biol 2006;44:403–7.

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32. De Bandt M, Grossin M, Driss F, Pincemail J, Babin-Chevaye C, Pasquier C. Vitamin E uncouples joint destruction and clinical inflammation in a transgenic mouse model of rheumatoid arthritis. Arthritis Rheum 2002;46: 522–32. 33. Khare CP. Indian medicinal plants – an illustrated dictionary. New Delhi, India: Springer (India) Pvt. Ltd, 2007.

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Effect of Withania somnifera (Ashwagandha) root extract on amelioration of oxidative stress and autoantibodies production in collagen-induced arthritic rats.

Rheumatoid arthritis is an inflammatory autoimmune disorder. Withania somnifera Dunal (Solanaceae) (WS), is a common medicinal plant used in tradition...
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