, Volume 35, Issue 4, pp 1435–1447

A Novel Combination of Methotrexate and Epigallocatechin Attenuates the Overexpression of Pro-inflammatory Cartilage Cytokines and Modulates Antioxidant Status in Adjuvant Arthritic Rats

  • Souvik Roy
  • Santanu Sannigrahi
  • Ram Prasad Vaddepalli
  • Balaram Ghosh
  • Priyanka Pusp


The present study was designed to evaluate the combinatory effect of methotrexate (MTX) and epigallocatechin (EGCG) on the progression of adjuvant-induced arthritis in rats. Adjuvant arthritis (AA) was induced by a single intradermal injection of Freund’s complete adjuvant. AA rats were treated with methotrexate (0.3 mg/kg) thrice a week, EGCG (100 mg/kg) daily, and combination of MTX and EGCG thrice a week for a period of 28 days. Paw swelling changes and histopathological and radiographic analysis was assessed to evaluate the antiarthritic effect. Lipid peroxidation and antioxidant enzyme activities in joint tissue homogenate were performed to observe the modulation of antioxidant status along the expression of different pro-inflammatory cartilage cytokines like TNF-α and IL-6. MTX and EGCG combination potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect (SOD, GSH, and catalase) as well as suppression of lipid peroxidation. Combination therapy of MTX and EGCG significantly inhibited the development phase of arthritis, which is supported by histopathological, radiographical, and attenuation of overexpression of cartilage cytokines. EGCG act as potent antioxidant and immunomodulator, suggesting that combined administration of MTX along with EGCG suppressed the development phase of arthritic progression in rats.


methotrexate epigallocatechin adjuvant arthritis cytokines antioxidant 


  1. 1.
    Firestein, G.S. 2003. Evolving concept of rheumatoid arthritis. Nature 423: 356–361. doi:10.1038/nature01661. PMID: 12748655.PubMedCrossRefGoogle Scholar
  2. 2.
    Mathew, A.J., J. Antony, S. Eremenco, B.V. Paul, B. Jayakumar, and J. Philip. 2009. Health-related quality of life in rheumatoid arthritis patients in South India. Singapore Medical Journal 50: 800–803. PMID: 19710980.PubMedGoogle Scholar
  3. 3.
    Wortmann, R.L. 1991. Pathogenesis of rheumatoid arthritis: Basis for future therapies. Seminars in Arthritis and Rheumatism 21(suppl): 35–39. PMID: 1749947.PubMedCrossRefGoogle Scholar
  4. 4.
    Zvaifler, N.J. 1988. New perspectives on the pathogenesis of rheumatoid arthritis. American Journal of Medicine 85(suppl): 12–17. PMID: 3052052.PubMedCrossRefGoogle Scholar
  5. 5.
    Hassan, M.Q., R.A. Hadi, Z.S. Al-Rawi, V.A. Padron, and S.J. Stohs. 2001. The glutathione defense system in the pathogenesis of rheumatoid arthritis. Journal of Applied Toxicology 21: 69–73. doi:10.1002/jat.736. PMID: 11180282.PubMedCrossRefGoogle Scholar
  6. 6.
    Heliovaara, M., P. Knekt, K. Aho, R.K. Aaran, G. Alfthan, and A. Aromaa. 1994. Serum antioxidants and risk of rheumatoid arthritis. Annals of the Rheumatic Diseases 53: 51–53. PMID: 8311556.PubMedCrossRefGoogle Scholar
  7. 7.
    Jaswal, S., H.C. Mehta, A.K. Sood, and J. Kaur. 2003. Antioxidant status in rheumatoid arthritis and role of antioxidant therapy. Clinica Chimica Acta 338: 123–129. PMID: 14637276.CrossRefGoogle Scholar
  8. 8.
    Feldmann, M., F.M. Brennan, and R.N. Maini. 1996. Role of cytokines in rheumatoid arthritis. Annual Review of Immunology 14: 397–440. doi:10.1146/annurev.immunol.14.1.397. PMID: 8717520.PubMedCrossRefGoogle Scholar
  9. 9.
    Chu, C.Q., M. Field, M. Feldmann, and R.N. Maini. 1991. Localization of tumor necrosis factor alpha in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis. Arthritis and Rheumatism 34: 1125–1232. PMID: 1930331.PubMedCrossRefGoogle Scholar
  10. 10.
    Farahat, M.M., G. Yanni, R. Poston, and G.S. Panayi. 1993. Cytokine expression in synovial membrane of patients with rheumatoid arthritis & osteoarthritis. Annals of the Rheumatic Diseases 52: 870–875. PMID: 8311538.PubMedCrossRefGoogle Scholar
  11. 11.
    Nishimoto, N. 2006. Interleukin-6 in rheumatoid arthritis. Current Opinion in Rheumatology 18: 277–281. PMID: 16582692.PubMedCrossRefGoogle Scholar
  12. 12.
    Akira, S., T. Taga, and T. Kishimoto. 1993. Interleukin-6 in biology and medicine. Advances in Immunology 54: 1–78. PMID: 8379461.PubMedCrossRefGoogle Scholar
  13. 13.
    Kishimoto, T. 2005. IL-6: From laboratory to bedside. Clinical Reviews in Allergy & Immunology 28: 177–186. doi:10.1385/CRIAI:28:3:177. PMID: 16129902.CrossRefGoogle Scholar
  14. 14.
    Benslay, D.N., and A.M. Bendele. 1991. Development of a rapid screen for detecting and differentiating immunomodulatory vs anti-inflammatory compounds in rats. Agents and Actions 34: 254–256. PMID: 1793040.PubMedCrossRefGoogle Scholar
  15. 15.
    Halloran, M.M., J.M. Woods, Z. Szekanecz, N. Barquin, G.K. Haines, A. Walz, and A.E. Koch. 1999. The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis. Journal of Immunology 162: 7492–7500. PMID: 10358204.Google Scholar
  16. 16.
    Billiau, A., and P. Matthys. 2001. Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases. Journal of Leukocyte Biology 70: 849–860. PMID: 11739546.PubMedGoogle Scholar
  17. 17.
    Braun, J., and R. Rau. 2009. An update on methotrexate. Current Opinion in Rheumatology 21: 216–223. PMID: 19373092.PubMedCrossRefGoogle Scholar
  18. 18.
    Cronstein, B.N. 2005. Low-dose methotrexate: A mainstay in the treatment of rheumatoid arthritis. Pharmacological Reviews 57: 163–172. doi:10.1124/pr.57.2.3. PMID: 15914465.PubMedCrossRefGoogle Scholar
  19. 19.
    Joannides, A.J., H. El-Shaboury, R. Guirguis, J.J. Fasler, and V. Rajagopal. 2011. Methotrexate-associated lymphoproliferative disorder presenting as disseminated malignancy. Rheumatology 50: 1927–1928. doi:10.1093/rheumatology/ker214. PMID: 21719419.PubMedCrossRefGoogle Scholar
  20. 20.
    MCkendry, R.J., and P. Dale. 1993. Adverse effects of low dose methotrexate therapy in rheumatoid arthritis. Journal of Rheumatology 20: 1850–1856. PMID: 8308769.PubMedGoogle Scholar
  21. 21.
    Smolen, J.S., D. Aletaha, J.W. Bijlsma, F.C. Breedveld, D. Boumpas, G. Burmester, B. Combe, M. Cutolo, M. de Wit, M. Dougados, P. Emery, A. Gibofsky, J.J. Gomez-Reino, B. Haraoui, J. Kalden, E.C. Keystone, T.K. Kvien, I. McInnes, E. Martin-Mola, C. Montecucco, M. Schoels, and D. van der Heijde. 2010. Treating rheumatoid arthritis to target: Recommendations of an international task force. Annals of the Rheumatic Diseases 69: 631–637. doi:10.1136/ard.2009.123919. PMID: 20215140.PubMedCrossRefGoogle Scholar
  22. 22.
    Weinblatt, M.E., J.M. Kremer, A.D. Bankhurst, K.J. Bulpitt, R.M. Fleischmann, R.I. Fox, C.G. Jackson, M. Lange, and D.J. Burge. 1999. A trial of etanercept, a recombinant tumor necrosis factor receptor: Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. The New England Journal of Medicine 340: 253–259. PMID: 9920948.PubMedCrossRefGoogle Scholar
  23. 23.
    Trevisanato, S.I., and Y.I. Kim. 2000. Tea and health. Nutrition Reviews 58: 1–10. PMID: 10697388.PubMedCrossRefGoogle Scholar
  24. 24.
    Yang, C.S., and Z.Y. Wang. 1993. Tea and cancer. Journal of the National Cancer Institute 85: 1038–1049. doi:10.1093/jnci/85.13.1038. PMID: 8515490.PubMedCrossRefGoogle Scholar
  25. 25.
    Katiyar, S.K., and C.A. Elmets. 2001. Green tea polyphenolic antioxidants and skin photo protection. International Journal of Oncology 18: 1307–1313. PMID: 11351267.PubMedGoogle Scholar
  26. 26.
    Lin, Y.L., and J.K. Lin. 1997. Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by downregulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB. Molecular Pharmacology 52: 465–472. PMID: 9281609.PubMedGoogle Scholar
  27. 27.
    Sachinidis, A., R.A. Skach, C. Seul, Y. Ko, J. Hescheler, H.Y. Ahn, and J. Fingerle. 2002. Inhibition of the PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins. The FASEB Journal 16: 893–895. PMID: 12039871.Google Scholar
  28. 28.
    Mukhtar, H., and N. Ahmad. 2000. Tea Polyphenols: Prevention of cancer and optimizing health. American Journal of Clinical Nutrition 71: 1698S–1702S. PMID: 10837321.PubMedGoogle Scholar
  29. 29.
    Constantin, A., P. Loubet-lescoulie, N. Lambert, and A. Cantagrel. 1998. Anti-inflammatory and immunoregulatory action of methotrexate in the treatment of rheumatoid arthritis: Evidence of increased interleukin-4 and interleukin-10 gene expression demonstrated in vitro by competitive reverse transcriptase-polymerase chain reaction. Arthritis and Rheumatism 41: 48–57. doi:10.1002/1529-0131(199801)41. PMID: 9433869.PubMedCrossRefGoogle Scholar
  30. 30.
    Chesbrough, M., and J. McArthur. 1972. Laboratory manual of rural tropical hospitals. Churchil: The English Language Book Society.Google Scholar
  31. 31.
    David, G., and A.J. Sykes. 1951. Westergren and Wintrobe methods of estimating ESR compared. British Medical Journal 2: 1496–1497. PMID: 14886634.CrossRefGoogle Scholar
  32. 32.
    Balasubramaniam, P., and A. Malathi. 1992. Comparative study of hemoglobin estimated by Drabkin’s and Sahli’s methods. Journal of Postgraduate Medicine 38: 8–9. PMID: 1512732.PubMedGoogle Scholar
  33. 33.
    Okhawa, H., N. Oishi, and K. Yagi. 1979. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Analytical Biochemistry 95: 351–358. PMID: 36810.CrossRefGoogle Scholar
  34. 34.
    Ellman, G.L. 1959. Tissue sulphydril groups. Archives of Biochemistry and Biophysics 82: 70–77. PMID: 13650640.PubMedCrossRefGoogle Scholar
  35. 35.
    Kakkar, P., B. Das, and P.N. Viswanthan. 1984. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemistry & Biophysics 21: 130–132. PMID: 6490072.Google Scholar
  36. 36.
    Narendhirakannan, R.T., S. Subramanian, and M. Kandaswamy. 2005. Free radical scavenging activity of Cleome gynandra L. leaves on adjuvant induced arthritis in rats. Molecular and Cellular Biochemistry 276: 71–80. doi:10.1007/s11010-005-3234-6. PMID: 16132687.PubMedCrossRefGoogle Scholar
  37. 37.
    Hegen, M., J.C. Keith, M. Collins, and C.L. Nickerson-Nutter. 2008. Utility of animal models for identification of potential therapeutics for rheumatoid arthritis. Annals of the Rheumatic Diseases 67: 1505–1515. PMID: 18055474.PubMedCrossRefGoogle Scholar
  38. 38.
    Bevaart, L., M.J. Vervoordeldonk, and P.P. Tak. 2010. Evaluation of therapeutic targets in animal models of arthritis: How does it relate to rheumatoid arthritis? Arthritis and Rheumatism 62: 2192–2205. doi:10.1002/art.27503. PMID: 20506322.PubMedCrossRefGoogle Scholar
  39. 39.
    Quinones, M.P., S.K. Ahuja, F. Jimenez, J. Schaefer, E. Garavito, A. Rao, G. Chenaux, R.L. Reddick, W.A. Kuziel, and S.S. Ahuja. 2004. Experimental arthritis in CC chemokine receptor 2-null mice closely mimics severe human rheumatoid arthritis. The Journal of Clinical Investigation 113: 856–866. doi:10.1172/JCI20126. PMID: 15067318.PubMedGoogle Scholar
  40. 40.
    Altman, A., and F.J. Dixon. 1989. Immunomodifiers in vaccines. Advances in Veterinary Science and Comparative Medicine 33: 301–343. PMID: 2467538.PubMedGoogle Scholar
  41. 41.
    Crofford, L.J., H. Sano, K. Karalis, E.L. Webster, E.A. Goldmuntz, G.P. Chrousos, and R.L. Wilder. 1992. Local secretion of corticotrophin releasing hormone in the joints of Lewis rats with inflammatory arthritis. The Journal of Clinical Investigation 90: 2555–2564. doi:10.1172/JCI116150. PMID: 1281840.PubMedCrossRefGoogle Scholar
  42. 42.
    Linblad, E.B. 2000. Freund’s adjuvants. In Methods in molecular medicine: Vaccine adjuvants—preparation methods and research protocols, ed. D.T. O’ Hagan, 49–63. Totowa: Humana Press.Google Scholar
  43. 43.
    Cannella, A.C., and J.R. O’Dell. 2003. Is there still a role for traditional disease modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis? Current Opinion in Rheumatology 15: 185–192. PMID: 12707569.PubMedCrossRefGoogle Scholar
  44. 44.
    Swierkot, J., and J. Szechinski. 2006. Methotrexate in rheumatoid arthritis. Pharmacological Reports 58: 473–492. PMID: 16963793.PubMedGoogle Scholar
  45. 45.
    Rovensky, J., M. Stancikova, E. Rovenska, S. Stvrtina, V. Stvrtinova, and K. Svik. 2009. Treatment of rat adjuvant arthritis with flavonoid (Detralex), methotrexate, and their combination. Annals of the New York Academy of Sciences 1173: 798–804. doi:10.1111/j.1749-6632.2009.04618.x. PMID: 19758231.PubMedCrossRefGoogle Scholar
  46. 46.
    Alastair, G., and M.B. Mowat. 1971. Hematological abnormalities in rheumatoid arthritis. Seminars in Arthritis and Rheumatism 1: 195–219. doi:10.1016/0049-0172(72)90001-7. PMID: 4343443.Google Scholar
  47. 47.
    Sotnikova, R., S. Ponist, J. Navarova, D. Mihalova, V. Tomekova, M. Strosova, and K. Bauerova. 2009. Effects of sesame oil in the model of adjuvant arthritis. Neuro Endocrinology Letters 30: 22–24. PMID: 20027138.PubMedGoogle Scholar
  48. 48.
    Ekambaram, S.P., S.S. Perumal, and V. Subramanian. 2011. Assessment of the in-vivo and in-vitro antioxidant potential of Strychnos potatorum Linn. seeds in Freund’s adjuvant induced arthritic rats. Journal of Medicinal Plants Research 5: 4780–4787.Google Scholar
  49. 49.
    Karatas, F., I. Ozates, H. Canatan, I. Halifeoglu, M. Karatepe, and R. Colakt. 2003. Antioxidant status and lipid peroxidation in patients with rheumatoid arthritis. Indian Journal of Medical Research 118: 178–181. PMID:14700353.PubMedGoogle Scholar
  50. 50.
    Bauerová, K., S. Poništ, D. Mihalová, F. Dráfi, and V. Kuncírová. 2011. Utilization of adjuvant arthritis model for evaluation of new approaches in rheumatoid arthritis therapy focused on regulation of immune processes and oxidative stress. Interdisciplinary Toxicology 4: 33–39. doi:10.2478/v10102-011-0007-9. PMID: 21577282.PubMedCrossRefGoogle Scholar
  51. 51.
    Cong, X. D., M. J. Ding, D. Z. Dai, Y. Wu, Y. Zhang, and Y. Dai. 2011. ER Stress, P66shc, and P-Akt/Akt mediate adjuvant-induced inflammation, which is blunted by argirein, a supermolecule and rhein in Rats. Inflammation PMID: 22095404. doi:10.1007/s10753-011-9407-4.
  52. 52.
    Guerne, P.A., B.L. Zuraw, J.H. Vaughan, D.A. Carson, and M. Lotz. 1989. Synovium as a source of interleukin 6 in vitro: Contribution to local and systemic manifestations of arthritis. The Journal of Clinical Investigation 83: 585–592. doi:10.1172/JCI113921. PMID: 2464001.PubMedCrossRefGoogle Scholar
  53. 53.
    Nietfeld, J.J., B. Wilbrink, M. Helle, J.L.A.M. Vanroy, R.W. Denotter, A.J.G. Swaak, and O. Huber-Bruning. 1990. Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage. Arthritis and Rheumatism 33: 1695–1701. PMID: 2242066.PubMedCrossRefGoogle Scholar
  54. 54.
    Deleuran, B.W., C.Q. Chu, M. Field, F.M. Brennan, P. Katsiki, M. Feldmann, and R.N. Maini. 1992. Localization of interleukin-1 a, type 1 interleukin-1 receptor and interleukin-1 receptor antagonist in the synovial membrane and cartilage/pannus junction in rheumatoid arthritis. British Journal of Rheumatology 31: 809–811. doi:10.1093/rheumatology/31.12.801. PMID: 1458286.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Souvik Roy
    • 1
  • Santanu Sannigrahi
    • 1
  • Ram Prasad Vaddepalli
    • 2
  • Balaram Ghosh
    • 3
  • Priyanka Pusp
    • 4
  1. 1.Department of Pharmaceutical TechnologyNSHM Knowledge CampusKolkataIndia
  2. 2.St. Peter’s Institute of Pharmaceutical SciencesWarangalIndia
  3. 3.Department of PharmacologyMedical College KolkataKolkataIndia
  4. 4.Galaxy Concept Pvt. Ltd.KolkataIndia

Personalised recommendations