Inflammation Research

, Volume 62, Issue 1, pp 115–126 | Cite as

Anti-arthritic activity of the Indian leafy vegetable Cardiospermum halicacabum in Wistar rats and UPLC–QTOF–MS/MS identification of the putative active phenolic components

  • Ramachandran Jeyadevi
  • Thilagar Sivasudha
  • Angappan Rameshkumar
  • Lakshmanan Dinesh Kumar
Original Research Paper



The present work was carried out to investigate the free radical scavenging activity of the ethanol extract of C. halicacabum leaves (EECH), to study its antioxidant properties and anti-rheumatic effects in Wistar rats with CFA-induced arthritis, and to profile the phenolic components thereof by LC–MS/MS.


The free radical scavenging activities of the extract was evaluated by NO and superoxide anion scavenging assays. Arthritis was induced to the albino Wistar rats by CFA. Fifteen days after CFA induction, arthritic rats received EECH orally at the doses of 250 and 500 mg/kg daily for 20 days. Diclofenac sodium was used as reference standard. EECH is subjected to LC–MS/MS analysis for the identification of phenolic compounds.


The IC50 value of the EECH to scavenge the NO and superoxide radicals are 83 and 60 μg/ml respectively. Ultrasonography and histology images of hind limb in EECH treated groups confirmed the complete cartilage regeneration. The LC/MS/MS analysis indicated the presence of anti-inflammatory compounds luteolin-7-O-glucuronide, apigenin-7-O-glucuronide and chrysoeriol.


These findings lend pharmacological support to the reported folkloric use of C. halicacabum in the treatment and management of painful, arthritic inflammatory conditions.


Cardiospermum halicacabum LC–MS/MS Complete Freund’s adjuvant Anti-arthritis 



Complete Freund’s adjuvant




Nitric oxide


White blood cells


Erythrocyte sedimentation rate


Red blood cells


Superoxide dismutase


Reduced glutathione




C-reactive protein


Rheumatoid factor



We thank University Grants Commission, New Delhi, India for the financial support. The mass spectrometry work was supported by Waters Corporation, Bangalore, India. We also thank Dr. J. Senthilkumar, Veterinary Surgeon and Dr. R. Banumathi for providing the Ultrasonography facility and interpretation of the same and histological results.


  1. 1.
    Mclnnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7:429–42.CrossRefGoogle Scholar
  2. 2.
    Yeom MJ, Lee HC, Kim GH, Lee HJ, Shim I, Oh SK, Kang SK, Hahm DH. Anti-arthritic effects of Ephedra sinica STAPF herb-acupuncture: inhibition of lipopolysaccharideinduced inflammation and adjuvant-induced polyarthritis. J Pharm Sci. 2006;100:41–50.CrossRefGoogle Scholar
  3. 3.
    Los M, Dröge W, Stricker K, Baeuerle PA, Schulze-Osthoff K. Hydrogen peroxide as a potent activator of T lymphocyte functions. Eur J Immunol. 1995;25:159–65.PubMedCrossRefGoogle Scholar
  4. 4.
    Tinker AC, Wallace AV. Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? Curr Top Med Chem. 2006;6:77–92.PubMedCrossRefGoogle Scholar
  5. 5.
    Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT, Gewertz BL. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. Am J Physiol. 1999;277:L1057–65.PubMedGoogle Scholar
  6. 6.
    Chua CC, Hamdy RC, Chua BH. Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells. Free Radic Biol Med. 1998;25(8):891–7.PubMedCrossRefGoogle Scholar
  7. 7.
    Simon AR, Rai U, Fanburg BL, Cochran BH. Activation of the JAK-STAT pathway by reactive oxygen species. Am J Physiol. 1998;275:C1640–52.PubMedGoogle Scholar
  8. 8.
    Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995;270:296–9.PubMedCrossRefGoogle Scholar
  9. 9.
    Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol. 1996;14:397–440.PubMedCrossRefGoogle Scholar
  10. 10.
    Ozkan Y, Yardỳm-Akaydỳn S, Sepici A, Keskin E, Sepici V, Simsek B. Oxidative status in rheumatoid arthritis. Clin Rheumatol. 2007;26:64–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Paolett F, Mocali A. Determination of superoxide dismutase activity by purely chemical system based on NAD (P) H oxidation. Methods Enzymol. 1990;186:209–20.CrossRefGoogle Scholar
  12. 12.
    Paredes S, Girona J, Hurt-Camejo E, Vallvé JC, Olivé S, Heras M, et al. Antioxidant vitamins and lipid peroxidation in patients with rheumatoid arthritis: association with inflammatory markers. J Rheumatol. 2002;29:2271–7.PubMedGoogle Scholar
  13. 13.
    Mamdani M, Juurlink DN, Lee DS, et al. Cyclo-oxygenase-2 inhibitors versus non-selective non-steroidal anti-inflammatory drugs and congestive heart failure outcomes in elderly patients: a population based cohort study. Lancet. 2004;363:1751–6.PubMedCrossRefGoogle Scholar
  14. 14.
    Santana-Sabagun E, Weisman MH. Nonsteroidal anti-inflammatory drugs. In: Ruddy S, Harris JED, Sledge CB, Budd RC, Sergent JS, editors. Kelly’s textbook of rheumatology, vol. 1. Philadelphia: Saunders; 2001. p. 799–822.Google Scholar
  15. 15.
    Newman NM, Ling RSM. Acetabular bone destruction related to non-steroidal anti-inflammatory drugs. Lancet. 1985;2:11–4.PubMedCrossRefGoogle Scholar
  16. 16.
    Anastassiades T, Chopra R, Law C, Wong E. In vitro suppression of transforming growth factor-b induced stimulation of glycosaminoglycan synthesis by acetylsalicylic acid and its reversal by misoprostol. J Rheumatol. 1998;25:1962–7.PubMedGoogle Scholar
  17. 17.
    Chopra RN. Glossary of indian medicinal plants. New Delhi: Council for Scientific and Industrial Research; 1980. p. 51–5.Google Scholar
  18. 18.
    Ganesan K, Sehgal PK, Mandal AB, Sayeed S. Protective effect of Withania somnifera and Cardiospermum halicacabum extracts against collagenolytic degradation of collagen. Appl Biochem Biotechnol. 2011;165(3–4):1075–91.PubMedCrossRefGoogle Scholar
  19. 19.
    Sadique J, Chandra T, Thenmozhi V, Elango V. Biochemical modes of action of Cassia occidentalis and Cardiospermum halicacabum in inflammation. J Ethnopharmacol. 1987;19(2):201–12.PubMedCrossRefGoogle Scholar
  20. 20.
    Gopalakrishnan C, Dhananjayan R, Kameswaran L. Studies on the pharmacological actions of Cardiospermum halicacabum. Indian J Physiol Pharmacol. 1976;20:203–6.PubMedGoogle Scholar
  21. 21.
    Asha VV, Pushpangadan P. Antipyretic activity of Cardiospermum halicacabum. Indian J Exp Biol. 1999;37:411–4.PubMedGoogle Scholar
  22. 22.
    Sheeba MS, Asha VV. Effect of Cardiospermum halicacabum on ethanol-induced gastric ulcers in rats. J Ethnopharmacol. 2006;106:105–10.PubMedCrossRefGoogle Scholar
  23. 23.
    Veeramani C, Pushpavalli G, Pugalendi KV. Antihyperglycaemic effect of Cardiospermum halicacabum Linn. leaf extract on STZ induced diabetic rats. J Appl Biomed. 2008;6:19–26.Google Scholar
  24. 24.
    Venkatesh Babu KC, Krishnakumari S. Cardiospermum halicacabum suppresses the production of TNF-alpha and nitric oxide by human peripheral blood mononuclear cells. Afr J Biomed Research. 2006;9:95–99.Google Scholar
  25. 25.
    Chisholmm J, Hopkins CY. Fatty acids of the seed oil of Cardiospermum halicacabum. Can J Chem. 1958;36:1537–40.CrossRefGoogle Scholar
  26. 26.
    Rajesh Kumara G, Murugananthana K, Nandakumar B, Sahil Talwar B. Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum. Phytomedicine. 2011;18:219–23.Google Scholar
  27. 27.
    Yen GC, Lai HH, Chou HY. Nitric oxide-scavenging and antioxidant effects of Uraria crinita root. Food Chem. 2001;74:471–8.CrossRefGoogle Scholar
  28. 28.
    Nishimiki M, Appaji N, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine mehosulphate and molecular oxygen. Biochem Biophys Res Commun. 1972;46:849–54.CrossRefGoogle Scholar
  29. 29.
    Ridge SC, Ferguson KM, Rath N, Galivan J, Freisheim JH, Oronsky AL, Kerwar SS. Methotrexate suppresses passive adjuvant arthritis: studies on the metabolism of methotrexate in mononuclear cells derived from normal and adjuvant arthritic rats. J Rheumatol. 1988;15:1193–7.PubMedGoogle Scholar
  30. 30.
    Buege AJ, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302–10.PubMedCrossRefGoogle Scholar
  31. 31.
    McCord JM, Fridovich I. Superoxide dismutase enzyme function for erythrocuprein (hemocuprein). J Biol Chem. 1969;244:6049–56.PubMedGoogle Scholar
  32. 32.
    Aebi H. Catalase. Meth Enzymol. 1984;105:121–6.PubMedCrossRefGoogle Scholar
  33. 33.
    Moron MS, De Pierre JN, Mannervik V. Levels of glutathione glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta. 1979;582:67–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Nohl H, Kozlov AV, Gille L, Staniek K. Cell respiration and formation of reactive oxygen species: facts and artifacts. Biochem Soc Trans. 2003;31:1308–11.PubMedCrossRefGoogle Scholar
  35. 35.
    Smith SM, Grishsm MB, Nancy EA, Granger DA, Kvietys PR. Gastric mucosal injury in rat. Role of iron and xanthine oxidase. Gastroenterol. 1987;92:950–56.Google Scholar
  36. 36.
    Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford: Clarendon Press; 1989. p. 416.Google Scholar
  37. 37.
    Pérez-Magariño S, Revilla I, González-SanJosé ML, Beltrán S. Various applications of liquid chromatography–mass spectrometry to the analysis of phenolic compounds. J Chromatogr A. 1999;847(1–2):75–81.PubMedGoogle Scholar
  38. 38.
    Schoedl K, Forneck A, Sulyok M, Schuhmacher R. Optimization, in-house validation, and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the quantification of selected polyphenolic compounds in leaves of grapevine (Vitis vinifera L.). J Agric Food Chem. 2011;59(20):10787–94.PubMedCrossRefGoogle Scholar
  39. 39.
    Liu AH, Guo H, Ye M, Lin YH, Sun JH, Xu M, Guo DA. Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry. J Chromatogr A. 2007;1161(1–2):170–82.PubMedGoogle Scholar
  40. 40.
    Sánchez-Rabaneda F, Jáuregui O, Lamuela-Raventós RM, Bastida J, Viladomat F, Codina C. Identification of phenolic compounds in artichoke waste by high performance liquid chromatography–tandem mass spectrometry. J Chromatogr A. 2003;1008(1):57–72.PubMedCrossRefGoogle Scholar
  41. 41.
    Miron TL, Plaza M, Bahrim G, Ibáñez E, Herrero M. Chemical composition of bioactive pressurized extracts of Romanian aromatic plants. J Chromatogr A. 2011;1218(30):4918–27.PubMedCrossRefGoogle Scholar
  42. 42.
    Simirgiotis MJ, Silva M, Becerra J, Schmeda-Hirschmann G. Direct characterisation of phenolic antioxidants in infusions from four Mapuche medicinal plants by liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionisation tandem mass spectrometry (HPLC-ESI–MS). Food Chem. 2012;131:318–27.CrossRefGoogle Scholar
  43. 43.
    Mullen W, Marks SC, Crozier A. Evaluation of phenolic compounds in commercial fruit juices and fruit drinks. J Agric Food Chem. 2007;55(8):3148–57.PubMedCrossRefGoogle Scholar
  44. 44.
    Bravo L, Goya L, Lecumberri E. LC/MS characterization of phenolic constituents of mate (Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to commonly consumed beverages. Food Res Int. 2007;40:393–405.CrossRefGoogle Scholar
  45. 45.
    Fernandes A, Sousa A, Mateus N, Cabral M, De Freitas V. Analysis of phenolic compounds in cork from Quercus suber L. by HPLC–DAD/ESI–MS. Food Chem. 2011;125:1398–405.Google Scholar
  46. 46.
    Prakken BJ, Roord S, Ronaghy A, Wauben M, Albani S, van Eden W. Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis. Semin Immunopathol. 2003;25:47–63.CrossRefGoogle Scholar
  47. 47.
    Shah SU, Ashraf N, Soomro ZH, Shah MR, Kabir N, Simjee SU. The anti-arthritic and anti-oxidative effect of NBD (6-nitro-1,3-benzodioxane) in adjuvant-induced arthritis (AIA) in rats. Inflamm Res. 2012;. doi: 10.1007/s00011-012-0480-4.Google Scholar
  48. 48.
    Singh S, Majumdar DK. Effect of fixed oil of Ocimum sanctum against experimentally induced arthritis and joint edema in laboratory animals. Int J Pharmacol. 1996;34(3):218–22.CrossRefGoogle Scholar
  49. 49.
    Conner EM, Grisham MB. Inflammation, free radicals and antioxidants. Nutrition. 1996;12:274–7.PubMedCrossRefGoogle Scholar
  50. 50.
    Abolfathi AA, Mohajeri D, Rezaie A, Nazeri M. Protective effects of green tea extract against hepatic tissue injury in streptozotocin-induced diabetic rats. Evi Bas Com Alt Med. 2012;. doi: 10.1155/2012/740671.Google Scholar
  51. 51.
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthr Rheum. 1988;31:315–24.CrossRefGoogle Scholar
  52. 52.
    Scott DL. Prognostic factors in early rheumatoid arthritis. Rheumatology (Oxford). 2000;39:24–9.CrossRefGoogle Scholar
  53. 53.
    Gölbasi Z, Uçar O, Keles T, Sahin A, Cagli K, Camsari A, Diker E, Aydogdu S. Increased levels of high sensitive C-reactive protein in patients with chronic rheumatic valve disease: evidence of ongoing inflammation. Eur J Heart Fail. 2002;4(5):593–5.PubMedCrossRefGoogle Scholar
  54. 54.
    Nair MP, Mahajan S, Reynolds JL, Aalinkeel R, Nair H, Schwartz SA, Kandaswami C. The flavonoid quercetin inhibits proinflammatory cytokine (tumor necrosis factor alpha) gene expression in normal peripheral blood mononuclear cells via modulation of the NF-kb system. Clin Vaccine Immunol. 2006;13(3):319–28.PubMedCrossRefGoogle Scholar
  55. 55.
    Kang OH, Lee JH, Kwon DY. Apigenin inhibits release of inflammatory mediators by blocking the NF-κB activation pathways in the HMC-1 cells. Immunopharm Immunotoxic. 2011;33(3):473–9.CrossRefGoogle Scholar
  56. 56.
    Ueda H, Yamazaki C, Yamazaki M. Luteolin as an anti-inflammatory and anti-allergic constituent of Perilla frutescens. Biol Pharm Bull. 2002;25(9):1197–202.PubMedCrossRefGoogle Scholar
  57. 57.
    López-Lázaro M. Distribution and biological activities of the flavonoid luteolin. Mini Rev Med Chem. 2009;9(1):31–59.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2012

Authors and Affiliations

  • Ramachandran Jeyadevi
    • 1
  • Thilagar Sivasudha
    • 1
  • Angappan Rameshkumar
    • 1
  • Lakshmanan Dinesh Kumar
    • 2
  1. 1.Department of Environmental BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Department of BiotechnologyBharathidasan UniversityTiruchirappalliIndia

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