Inhibitory effects of Clematis orientalis aqueous ethanol extract and fractions on inflammatory markers in complete Freund's adjuvant-induced arthritis in Sprague–Dawley rats

  • Umme Habiba Hasan
  • AlamgeerEmail author
  • Muhammad Shahzad
  • Shah Jahan
  • Zahid Rasul Niazi
  • Ishfaq Ali Bukhari
  • Asaad Mohamed Assiri
  • Hamayun Riaz
Original Article


Clematis orientalis Linn has long been used as ethnopharmacy for the treatment of arthritis. This study is intended to evaluate the curative efficacy of Clematis orientalis in treating polyarthritis in rats. Aqueous ethanolic extract and fractions (hexane, butanol and aqueous) were administered orally at 200 mg/kg for 28 days after CFA immunization. Paw swelling, paw diameter, arthritic score, body weight, hematological parameters, radiographic and histological analysis of ankle joints were evaluated. Moreover, levels of various inflammatory markers through RT-PCR and ELISA were measured. DPPH and reducing power assays were used to appraise antioxidant capacity. Qualitative phytochemical analysis, determination of total phenolic and flavonoid contents were also carried out. Aqueous ethanolic extract and fractions significantly (p < 0.001) reduced paw volume, paw thickness and arthritic score and considerably prevented decrease in body weight along with anomalous alterations in hematological parameters in comparison with arthritic control. X-ray and histological examination revealed no significant structural changes in ankle joints of treated rats. Expression levels of IL-1β, TNF-α, IL-6, COX-2 and NF-Kβ were significantly (p < 0.05–0.001) suppressed as well as noteworthy increase in the levels of IL-4 and IL-10 among treated animals has been detected. Overproduction of TNF-α and PGE2 was substantially prevented in animals given different treatments. Aqueous ethanol extract and its fractions demonstrated significant and concentration-dependent antioxidant potential. In general, among fractions aqueous fraction exhibited a greater anti-arthritic effect. Phytochemical analysis of aqueous fraction confirmed the presence of flavonoids and glycosides, 215.29 mgGAE/ml phenolic content and 633.03 μgQE/ml flavonoid content. Thus, we suggest Clematis orientalis as a potent strategy for the treatment of rheumatoid arthritis.


Rheumatoid arthritis Clematis orientalis Complete freunds adjuvant Real-time polymerase chain reaction Enzyme-linked immunosorbent assay 



The authors extend their appreciation to the deanship scientific research at King Saud University for funding this research through # (RG-1439-002).

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to disclose.


  1. Abbas Q, Khan SW, Khatoon S, Hussain SA, Hassan SN, Hussain A, Qureshi R, Hussain I (2014) Floristic biodiversıty and traditional uses of medicinal plants of Haramosh Valley Central Karakoram National Park of Gilgit district, Gilgit-Baltistan. Pak J Bio Env Sci 5:75–86Google Scholar
  2. Alamgeer Uttra AM, Hasan UH (2017) Anti-arthritic activity of aqueous-methanolic extract and various fractions of Berberis orthobotrys Bien ex Aitch. BMC Complement Altern Med 17:371CrossRefGoogle Scholar
  3. Alamgeer Hasan UH, Uttra AM, Rasool S (2015) Evaluation of in vitro and in vivo anti-arthritic potential of Berberis calliobotrys. Bangladesh J Pharmacol 10:807–819CrossRefGoogle Scholar
  4. Alamgeer Niazi SG, Uttra AM, Qaiser MN, Ahsan H (2017) Appraisal of anti-arthritic and nephroprotective potential of Cuscuta reflexa. Pharm Biol 55:792–798CrossRefGoogle Scholar
  5. Amaeze OU, Ayoola GA, Sofidiya MO, Adepoju-Bello AA, Adegoke AO, Coker HAB (2011) Evaluation of antioxidant activity of tetracarpidium conophorum (M¨ull. Arg) Hutch and Dalziel Leaves. Oxid Med Cell Longev. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder Ronald L, Hunder GG (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthr Rheum 31:315–324CrossRefGoogle Scholar
  7. Arulpriya P, Lalitha P, Hemalatha S (2010) In vitro antioxidant testing of the extracts of Samanea saman (Jacq.) Merr. Der Chem Sin 1:73–79Google Scholar
  8. Bischoff SC (2008) Quercetin: potentials in the prevention and therapy of disease. Curr Opin Clin Nutr Metab Care 11:733–740CrossRefGoogle Scholar
  9. Chaouche TM, Haddouchi F, Ksouri R, Atik-Bekkara F (2014) Evaluation of antioxidant activity of hydromethanolic extracts of some medicinal species from South Algeria. J Chin Med Assoc 77:302–307CrossRefGoogle Scholar
  10. Connolly KM, Stecher VJ, Danis E, Pruden DJ, LaBrie T (1988) Alteration of interleukin-1 activity and the acute phase response in adjuvant arthritic rats treated with disease modifying antirheumatic drugs. Agents Actions 25:94–105CrossRefGoogle Scholar
  11. Fattahi MJ, Mirshafiey A (2012) Prostaglandins and rheumatoid arthritis. Arthritis. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Ghasemzadeh A, Ghasemzadeh N (2011) Flavonoids and phenolic acids: role and biochemical activity in plants and human. J Med Plant Res 5:6697–6703Google Scholar
  13. Goekoop-Ruiterman YP, de Vries-Bouwstra JK, Allaart CF, van Zeben D, Kerstens PJ, Hazes JM, Zwinderman AH, Ronday HK, Han KH, Westedt ML, Gerards AH, van Groenendael JH, Lems WF, van Krugten MV, Breedveld FC, Dijkmans BA (2005) Clinical and radiographic outcomes of four different treatment strategies in patients with early rheumatoid arthritis (the BeSt study): a randomized, controlled trial. Arthritis Rheum 52:3381–3390CrossRefGoogle Scholar
  14. Gomaa A, Elshenawy M, Afifi N, Mohammed E, Thabit R (2009) Enhancement of the anti-inflammatory and anti-arthritic effects of theophylline by a low dose of a nitric oxide donor or non-specific nitric oxide synthase inhibitor. British J Pharmacol 158:1835–1847CrossRefGoogle Scholar
  15. Gorman CL, Cope AP (2008) Immune-mediated pathways in chronic inflammatory arthritis. Best Pract Res Clin Rheumatol 22:221–238CrossRefGoogle Scholar
  16. Han W, Xiong Y, Li Y, Fang W, Ma Y, Liu L, Li F, Zhu X (2013) Anti-arthritic effects of clematichinenoside (AR-6) on PI3 K/Akt signaling pathway and TNF-α associated with collagen-induced arthritis. Pharm Biol 51(1):13–22CrossRefGoogle Scholar
  17. Hasan UH, Alamgeer (2018) anti-arthritic efficacy of Clematis orientalis. Bangladesh J Pharmacol 13:142–148CrossRefGoogle Scholar
  18. Irmler I, Bräuer R (2007) Paradoxical role of interferon-gamma in arthritis. Rheumatol 66:591–594Google Scholar
  19. Ishtiaq M, Mumtaz AS, Hussain T, Ghani A (2012) Medicinal plant diversity in the flora of Leepa Valley, Muzaffarabad (AJK). Pak Afr J Biotechnol 11:3087–3098Google Scholar
  20. Jin F (2012) The pharmaceutical potential of compounds from Tasmanian Clematis speciesGoogle Scholar
  21. Kavitha KS, Satish S (2013) Evaluation of antimicrobial and antioxidant activities from Toona ciliata Roemer. J Anal Sci Technol 4:23CrossRefGoogle Scholar
  22. Klareskog L, Catrina A, Paget S (2009) Rheumatoid arthritis. Lancet 373:659–672CrossRefGoogle Scholar
  23. Kroes BH, van den Berg AJ, Quarles van Ufford HC, Van Dijk H, Labadie RP (1992) Anti-inflammatory activity of gallic acid. Planta Med 58:499–504CrossRefGoogle Scholar
  24. Li Y, Yao J, Han C, Yang J, Chaudhry M, Wang S, Liu H, Yin Y (2016) Quercetin, inflammation and immunity. Nutrients 8(3):167CrossRefGoogle Scholar
  25. Liang N, Kitts DD (2016) Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lin B, Zhang H, Zhao X, Rahman K, Wang Y, Maa X, Zheng C, Zhang Q, Han T, Lu-Ping Q (2013) Inhibitory effects of the root extract of Litsea cubeba (lour.) pers.on adjuvant arthritis in rats. J Ethnopharmacol 147:327–334CrossRefGoogle Scholar
  27. Liu M, Ma W, Guan H, Li L, Wei B, Li P (2011) Effects of taurochenodeoxycholic acid on adjuvant arthritis in rats. Int J Immunopharmacol 11:2150–2158CrossRefGoogle Scholar
  28. Lü SW, Wang QS, Li GY, Sun S, Guo YY, Kuang HX (2015) The treatment of rheumatoid arthritis using Chinese medicinal plants: from pharmacology to potential molecular mechanisms. J Ethnopharmacol 176:177–206CrossRefGoogle Scholar
  29. Makrov SS (2001) NF-κB in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 3:200–206CrossRefGoogle Scholar
  30. Manach C, Scalbert A, Morand C, Remesy C, Jimenez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79:727–747CrossRefGoogle Scholar
  31. NRC (1996) Guide for the care and use of laboratory animals. National Academy Press, Washington DCGoogle Scholar
  32. Pan T, Cheng T, Jia Y, Li P, Li F (2017) Anti-rheumatoid arthritis effects of traditional Chinese herb couple in adjuvant-induced arthritis in rats. J Ethnopharmacol 205:1–7CrossRefGoogle Scholar
  33. Perveen K, Hanif F, Jawed H, Simjee SU (2013) Protective efficacy of N-(2-Hydroxyphenyl) acetamide against adjuvant-induced arthritis in rats. Biomed Res Int. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Perveen K, Hanif F, Jawed H, Jamall S, Simjee SU (2014) N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats. Mol Cell Biochem. CrossRefPubMedGoogle Scholar
  35. Pitsiou G, Kyriazis G, Hatzizisi O, Argyropoulou P, Mavrofridis E, Patakas D (2002) Tumor necrosis factor-alpha serum levels, weight loss and tissue oxygen at ionin chronic obstructive pulmonary disease. Respir Med 96:594–598CrossRefGoogle Scholar
  36. Rakesh T, Parashant T (2012) Effect of Cocculus hirsutus leaves extract on Freunds complete adjuvant and formaldehyde-induced arthritis. Int Res J Pharm 3:267–290Google Scholar
  37. Rio DC, Ares M, Hannon GJ, Nilsen TW (2010) Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010(6):pdb.prot5439CrossRefGoogle Scholar
  38. Rosloniec EF, Latham K, Guedez YB (2002) Paradoxical roles of IFN-γ in models of Th1-mediated autoimmunity. Arthritis Res 4:333–336CrossRefGoogle Scholar
  39. Sahoo S, Ghosh G, Das D, Nayak S (2013) Phytochemical investigation and in vitro antioxidant activity of an indigenous medicinal plant Alpinia nigra B.L. Burtt Asian Pac J Trop Biomed 3:871–876CrossRefGoogle Scholar
  40. Schulze-Koops H, Kalden JR (2001) The balance of Th1/Th2 cytokines in rheumatoid arthritis. Best Pract Res Clin Rheumatol 15:677–691CrossRefGoogle Scholar
  41. Scott DL, Wolfe F, Huizinga TW (2010) Rheumatoid arthritis. Lancet 376:1094–1108CrossRefGoogle Scholar
  42. Shabbir A, Shahzad M, Ali A, Zia-ur-Rehman M (2014) Anti-arthritic activityof N0-[(2,4-dihydroxyphenyl)methylidene]-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide. Eur J Pharmacol 738:263–272CrossRefGoogle Scholar
  43. Shen L, Wang P, Guo J, Du G (2013) Anti-arthritic activity of ethanol extract of Fagopyrum cymosum with adjuvant-induced arthritis in rats. Pharm Biol 51:783–789CrossRefGoogle Scholar
  44. Sindhu G, Ratheesh M, Shyni GL, Nambisan B, Helen A (2012) Anti-inflammatory and antioxidative effects of mucilage of Trigonella foenum graecum (Fenugreek) on adjuvant-induced arthritic rats. Int Immunopharmacol 12:205–211CrossRefGoogle Scholar
  45. Smolen JS, Steiner G (2003) Therapeutic strategies for rheumatoid arthritis. Nat Rev Drug Discov 2(6):473–488CrossRefGoogle Scholar
  46. Smolen JS, Aletaha D, Koeller M, Weisman MH, Emery P (2007) New therapies for treatment of rheumatoid arthritis. Lancet 370:1861–1874CrossRefGoogle Scholar
  47. Srikanth G, Babu SM, Kavitha CHN, Rao MB, Vijaykumar N, Pradeep CH (2010) Studies on in vitro antioxidant activities of Carica papaya aqueous leaf extract. Res J Pharm Biol Chem Sci 1:59–65Google Scholar
  48. Sukketsiri W, Chonpathompikunlert P, Tanasawet S, Choosri N, Wongtawatchai T (2016) Effects of apium graveolens extract on the oxidative stress in the liver of adjuvant-induced arthritic rats. Prev Nutr Food Sci 21:79–84CrossRefGoogle Scholar
  49. Sun X, Liu Y, Yang Y, Liu X, Xiang D (2016) Anti-arthritic effect of total saponins from Clematis henryi Oliv. on collagen-induced arthritis rats. Eur J Inflamm 14(2):71–77CrossRefGoogle Scholar
  50. Tayar JH, Suarez-Almazor ME (2010) New understanding and approaches to treatment in rheumatoid arthritis. Brit Med Bull 94(1):201–214CrossRefGoogle Scholar
  51. Tracey K, Cerami AVH (1989) Cachectin/tumornecrosisfactor. Lancet 167:1122–1225CrossRefGoogle Scholar
  52. Tracey KJ, Wei H, Manogue KR, Fong Y, Hesse DD, Nguyen HT, Kuo GC, Beutler B, Cotran RS, Cerami A, Lowry SF (1988) Cachectin/tumornecrosisfactor induces cachexia, anemia, and inflammation. J Exper Med 167:1211–1227CrossRefGoogle Scholar
  53. Wanasundara PK, Shahidi F (1996) Optimization of hexametaphosphate- assisted extraction of flaxseed proteins using response surface methodology. J Food Sci 61:604–607CrossRefGoogle Scholar
  54. Woode E, Boakey-Gyasi E, Danquah CA, Anash C, Duwiejua M (2009) Anti-arthritic effects of Palisota hirsuta K. Schum. Leaf extract in Freund’s adjuvant-induced arthritis in rats. Int J Pharmacol 5:181–190CrossRefGoogle Scholar
  55. Yuting C, Rongliang Z, Zhongjian J, Yong J (1990) Flavonoids as superoxide scavengers and antioxidants. Free Rad Biol Med 9:19–21CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Umme Habiba Hasan
    • 1
  • Alamgeer
    • 1
    Email author
  • Muhammad Shahzad
    • 2
  • Shah Jahan
    • 3
  • Zahid Rasul Niazi
    • 4
  • Ishfaq Ali Bukhari
    • 5
  • Asaad Mohamed Assiri
    • 6
  • Hamayun Riaz
    • 7
  1. 1.Laboratory of Cardiovascular Research and Integrative Pharmacology, College of PharmacyUniversity of SargodhaSargodhaPakistan
  2. 2.Department of PharmacologyUniversity of Health SciencesLahorePakistan
  3. 3.Department of Immunology University of Health SciencesLahorePakistan
  4. 4.Department of Biomedical Sciences, Faculty of PharmacyGomal UniversityDI KhanPakistan
  5. 5.Department of Pharmacology, College of MedicineKing Saud UniversityRiyadhSaudi Arabia
  6. 6.Prince Abdullah Ben Khaled Celiac Disease Research Chair, Department of Pediatrics, Faculty of MedicineKing Saud UniversityRiyadhSaudi Arabia
  7. 7.Laboratory Rashid Latif College of PharmacyLahorePakistan

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