Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 391, Issue 3, pp 285–297 | Cite as

Betulinic acid alleviates dextran sulfate sodium-induced colitis and visceral pain in mice

  • Jaspreet Kalra
  • Madhu Cholenahalli Lingaraju
  • Karikalan Mathesh
  • Dhirendra Kumar
  • Subhashree Parida
  • Thakur Uttam Singh
  • Anil Kumar Sharma
  • Dinesh Kumar
  • Surendra Kumar Tandan
Original Article


Betulinic acid (BA) exhibits many biological effects including anti-inflammatory and anti-oxidant activities. Free radicals and pro-inflammatory mediators play an important role in the pathology of inflammatory bowel disease (IBD) and associated pain. We, therefore, examined the anti-oxidant, anti-inflammatory, and anti-nociceptive potential of BA in colitis. Colitis was induced with 3% (w/v) dextran sulfate sodium (DSS) in drinking water in mice for 1to7 days. BA (3, 10 and 30 mg/kg) was given orally for 0 to 7 days. BA was also tested for its efficacy in acetic acid and mustard oil-induced visceral nociception in mice at same doses. BA significantly prevented diarrhea; bleeding and colonic pathological changes induced by DSS. Further, BA reduced the colon nitrite, malondialdehyde, myeloperoxidase, and lipid hydroperoxide levels and restored the superoxide dismutase, catalase and reduced glutathione levels to normalize the redox balance in DSS-exposed mice. Inflammatory mediators like matrix metalloproteinase-9 and prostaglandin E2 levels were also significantly attenuated by BA in colitis mice. Additionally, BA reduced acetic acid and mustard oil-induced visceral pain in mice. In conclusion, the results of the present study suggest that BA possesses good anti-nociceptive activity and the anti-IBD effects of BA are due to its anti-oxidant and anti-inflammatory potential.


Betulinic acid Inflammatory bowel disease Colitis Visceral pain Anti-inflammatory Anti-oxidant 



The authors thank the Indian Council of Agricultural Research, New Delhi, India, for providing financial support to conduct this study.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest in this work.


  1. Alzoghaibi MA (2013) Concepts of oxidative stress and antioxidant defense in Crohn’s disease. World J Gastroenterol 19(39):6540–6547. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Amrouche-Mekkioui I, Djerdjouri B (2012) N-acetylcysteine improves redox status, mitochondrial dysfunction, mucin-depleted crypts and epithelial hyperplasia in dextran sulfate sodium-induced oxidative colitis in mice. Eur J Pharmacol 691(1-3):209–217. CrossRefPubMedGoogle Scholar
  3. Beyak MJ, Vanner S (2005) Inflammation induced hyperexcitability of nociceptive gastrointestinal DRG neurones: the role of voltage gated ion channels. Neurogastroenterol Motil 17(2):175–186. CrossRefPubMedGoogle Scholar
  4. Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE (2014) Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev 94(2):329–354. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boismenu R, Chen Y (2000) Insights from mouse models of colitis. J Leukoc Biol 67(3):267–278CrossRefPubMedGoogle Scholar
  6. Brandhorst G, Weigand S, Eberle C, Raddatz D, Karaus M, Oellerich M, Walson PD (2013) CD41 immune response as a potential biomarker of patient reported inflammatory bowel disease (IBD) activity. Clin Chim Acta 421:31–33. CrossRefPubMedGoogle Scholar
  7. Buanne P, Di Carlo E, Caputi L, Brandolini L, Mosca M, Cattani F, Pellegrini L, Biordi L, Coletti G, Sorrentino C, Fedele G, Colotta F, Melillo G, Bertini R (2007) Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice. J Leukoc Biol 82(5):1239–1246. CrossRefPubMedGoogle Scholar
  8. Casagrande R, Georgetti SR, Verri WA Jr, Dorta DJ, dos Santos AC, Fonseca MJ (2006) Protective effect of topical formulations containing quercetin against UVB-induced oxidative stress in hairless mice. J Photochem Photobiol B 84(1):21–27. CrossRefPubMedGoogle Scholar
  9. Cooper HS, Murthy SN, Shah RS, Sedergran DJ (1993) Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Investig 69(2):238–249PubMedGoogle Scholar
  10. Cross RK, Wilson KT (2003) Nitric oxide in inflammatory bowel disease. Inflamm Bowel Dis 9(3):179–189. CrossRefPubMedGoogle Scholar
  11. Debnath T, Kim d H, Lim BO (2013) Natural products as a source of anti- inflammatory agents associated with inflammatory bowel disease. Molecules 18(6):7253–7270. CrossRefPubMedGoogle Scholar
  12. Gaudio E, Taddei G, Vetuschi A, Sferra R, Frieri G, Ricciardi G, Caprilli R (1999) Dextran sulfate sodium (DSS) colitis in rats: clinical, structural, and ultrastructural aspects. Dig Dis Sci 44(7):1458–1475. CrossRefPubMedGoogle Scholar
  13. Geboes K, Riddell R, Öst Ä, Jensfelt B, Persson T, Löfberg R (2000) A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut 47(3):404–409. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15 N] nitrate in biological fluids. Anal Biochem 126(1):131–138. CrossRefPubMedGoogle Scholar
  15. Halliwell B, Gutteridge JM (1990) The antioxidants of human extracellular fluids. Arch Biochem Biophys 280(1):1–8. CrossRefPubMedGoogle Scholar
  16. Hokari R, Kato S, Matsuzaki K, Kuroki M, Iwai A, Kawaguchi A, Nagao S, Miyahara T, Itoh K, Sekizuka E, Nagata H, Ishii H, Miura S (2001) Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis. Free Radic Biol Med 31(2):153–163. CrossRefPubMedGoogle Scholar
  17. Jahanshahi G, Motavasel V, Rezaie A, Hashtroudi AA, Daryani NE, Abdollahi M (2004) Alterations in antioxidant power and levels of epidermal growth factor and nitric oxide in saliva of patients with inflammatory bowel diseases. Dig Dis Sci 49(11-12):1752–1757. CrossRefPubMedGoogle Scholar
  18. Jalil J, Sabandar CW, Ahmat N (2015) Inhibitory effect of triterpenoids from Dillenia serrata (Dilleniaceae) on prostaglandin E2 production and quantitative HPLC analysis of its koetjapic acid and betulinic acid contents. Molecules 20(2):3206–3220. CrossRefPubMedGoogle Scholar
  19. Koike K, Moore FA, Moore EE (1992) Endotoxin after gut ischemia/reperfusion causes irreversible lung injury. J Surg Res 52(6):656–662. CrossRefPubMedGoogle Scholar
  20. Kono Y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186(1):189–195. CrossRefPubMedGoogle Scholar
  21. Laird JM, Martinez-Caro L, Garcia-Nicas E, Cervero F (2001) A new model of visceral pain and referred hyperalgesia in the mouse. Pain 92(3):335–342. CrossRefPubMedGoogle Scholar
  22. Lee IA, Park YJ, Yeo HK, Han MJ, Kim DH (2010) Soyasaponin I attenuates TNBS-induced colitis in mice by inhibiting NF-κB pathway. J Agric Food Chem 58(20):10929–10934. CrossRefPubMedGoogle Scholar
  23. Lingaraju MC, Pathak NN, Begum J, Balaganur V, Bhat RA, Ramachandra HD, Ayanur A, Ram M, Singh V, Kumar D, Kumar D, Tandan SK (2015a) Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice. Cytokine 71(1):101–108. CrossRefPubMedGoogle Scholar
  24. Lingaraju MC, Pathak NN, Begum J et al (2015b) Betulinic acid negates oxidative lung injury in surgical sepsis model. J Surg Res 193:856–867CrossRefPubMedGoogle Scholar
  25. Lingaraju MC, Pathak NN, Begum J, Balaganur V, Ramachandra HD, Bhat RA, Ram M, Singh V, Kandasamy K, Kumar D, Kumar D, Tandan SK (2015c) Betulinic acid attenuates renal oxidative stress and inflammation in experimental model of murine polymicrobial sepsis. Eur J Pharm Sci 70:12–21. CrossRefPubMedGoogle Scholar
  26. Liu H, Patel NR, Walter L, Ingersoll S, Sitaraman SV, Garg P (2013) Constitutive expression of MMP9 in intestinal epithelium worsens murine acute colitis and is associated with increased levels of proinflammatory cytokine Kc. Am J Physiol Gastrointest Liver Physiol 304(9):G793–G803. CrossRefPubMedGoogle Scholar
  27. Luck H (1965) In: methods of enzymatic analysis, 2nd edn. Academic Press, New York, pp 885–890CrossRefGoogle Scholar
  28. Marchioni BR, Kane S (2014) Current approaches to the management of new-onset ulcerative colitis. Clin Exp Gastroenterol 7:111–132Google Scholar
  29. Matos I, Bento AF, Marcon R, Claudino RF, Calixto JB (2013) Preventive and therapeutic oral administration of the pentacyclic triterpene α, β-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: the relevance of cannabinoid system. Mol Immunol 54(3-4):482–492. CrossRefPubMedGoogle Scholar
  30. McCartney SA, Mitchell JA, Fairclough PD, Farthing MJ, Warner TD (1999) Selective COX-2 inhibitors and human inflammatory bowel disease. Aliment Pharmacol Ther 13(8):1115–1117. CrossRefPubMedGoogle Scholar
  31. Mohammad FK, Al-Baggou BKH, Naser AS (2012) Antinociception by metoclopramide, ketamine and their combinations in mice. Pharmacol Rep 64(2):299–304. CrossRefPubMedGoogle Scholar
  32. Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolff SP (1994) Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine. Anal Biochem 220(2):403–409. CrossRefPubMedGoogle Scholar
  33. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R (1990) A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98(3):694–702. CrossRefPubMedGoogle Scholar
  34. Oliveira LGD, Cunha ALD, Duarte ALDAC, Castañon MC, Chebli JM, Aguiar JA (2014) Positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis. Arq Gastroenterol 51(2):107–112. CrossRefPubMedGoogle Scholar
  35. Oyebanji BO, Saba AB, Oridupa OA (2013) Studies on the anti-inflammatory, analgesic and antipyrexic activities of betulinic acid derived from Tetracera potatoria. Afr J Tradit Complement Altern Med 11:30–33PubMedPubMedCentralGoogle Scholar
  36. Piechota-Polanczyk A, Fichna J (2014) The role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases. Naunyn Schmiedeberg’s Arch Pharmacol 387(7):605–620. CrossRefGoogle Scholar
  37. Quan HY, Kim DY, Kim SJ, Jo HK, Kim GW, Chung SH (2013) Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway. Biochem Pharmacol 85(9):1330–1340. CrossRefPubMedGoogle Scholar
  38. Rehman S, Chandra O, Abdulla M (1995) Evaluation of malondialdehyde as an index of lead damage in rat brain homogenates. Biometals 8:275–279PubMedGoogle Scholar
  39. Roberts PJ, Morgan K, Miller R, Hunter JO, Middleton SJ (2001) Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease. Gut 48(4):468–472. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Rzeski W, Stepulak A, Szymański M, Sifringer M, Kaczor J, Wejksza K, Zdzisińska B, Kandefer-Szerszeń M (2006) Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells. Naunyn Schmiedberg’s Arch Pharmacol 374(1):11–20. CrossRefGoogle Scholar
  41. Sakanaka T, Inoue T, Yorifuji N, Iguchi M, Fujiwara K, Narabayashi K, Kakimoto K, Nouda S, Okada T, Kuramoto T, Ishida K, Abe Y, Takeuchi T, Umegaki E, Akiba Y, Kaunitz JD, Higuchi K (2015) The effects of a TGR5 agonist and a dipeptidyl peptidase IV inhibitor on dextran sulfate sodium-induced colitis in mice. J Gastroenterol Hepatol 30:60–65. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sánchez-Fidalgo S, Cárdeno A, Sánchez-Hidalgo M, Aparicio-Soto M, de la Lastra CA (2013) Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice. J Nutr Biochem 24(7):1401–1413. CrossRefPubMedGoogle Scholar
  43. Sandborn WJ (2012) The future of inflammatory bowel disease therapy: where do we go from here? Dig Dis 30(s3):140–144. CrossRefPubMedGoogle Scholar
  44. Santana A, Medina C, Paz-Cabrera MC, Díaz-Gonzalez F, Farré E, Salas A, Radomski MW, Quintero E (2006) Attenuation of dextran sodium sulphate induced colitis in matrix metallopro-teinase-9 deficient mice. World J Gastroenterol 12(40):6464–6472. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Saria A, Lundberg JM (1983) Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues. J Neurosci Methods 8(1):41–49. CrossRefPubMedGoogle Scholar
  46. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 25(1):192–205. CrossRefPubMedGoogle Scholar
  47. Şener TE, Kardaş RC, Şehirli AO et al (2013) The effect of betulinic acid on TNBS-induced experimental colitis. Marmara. Pharm J 17:52–59Google Scholar
  48. Sharma A, Thakur R, Lingaraju MC, Kumar D, Mathesh K, Telang AG, Singh TU, Kumar D (2017) Betulinic acid attenuates renal fibrosis in rat chronic kidney disease model. Biomed Pharmacother 89:796–804. CrossRefPubMedGoogle Scholar
  49. Shiba Y, Kinoshita T, Chuman H, Taketani Y, Takeda E, Kato Y, Naito M, Kawabata K, Ishisaka A, Terao J, Kawai Y (2008) Flavonoids as substrates and inhibitors of myeloperoxidase: molecular actions of aglycone and metabolites. Chem Res Toxicol 21(8):1600–1609. CrossRefPubMedGoogle Scholar
  50. Sipos F, Muzes G, Galamb O, Spisák S, Krenács T, Tóth K, Tulassay Z, Molnár B (2010) The possible role of isolated lymphoid follicles in colonic mucosal repair. Pathol Oncol Res 16(1):11–18. CrossRefPubMedGoogle Scholar
  51. Viji V, Helen A, Luxmi VR (2011) Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E2 production in human peripheral blood mononuclear cells. Br J Pharmacol 162(6):1291–1303. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wirtz S, Neurath MF (2007) Mouse models of inflammatory bowel disease. Adv Drug Deliv Rev 59(11):1073–1083. CrossRefPubMedGoogle Scholar
  53. Yogeeswari P, Sriram D (2005) Betulinic acid and its derivatives: a review on their biological properties. Curr Med Chem 12(6):657–666. CrossRefPubMedGoogle Scholar
  54. Zhu H, Li YR (2012) Oxidative stress and redox signaling mechanisms of inflammatory bowel disease: updated experimental and clinical evidence. Exp Biol Med (Maywood) 237(5):474–480. CrossRefGoogle Scholar
  55. Zimmerman M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16(2):109–110. CrossRefGoogle Scholar
  56. Qu ZW, Liu TT, Ren C, Gan X, Qiu CY, Ren P, Rao Z, WP H (2015) 17β-estradiol enhances ASIC activity in primary sensory neurons to produce sex difference in acidosis-induced nociception. Endocrinology 156:4660–4671CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Jaspreet Kalra
    • 1
  • Madhu Cholenahalli Lingaraju
    • 1
  • Karikalan Mathesh
    • 2
  • Dhirendra Kumar
    • 1
  • Subhashree Parida
    • 1
  • Thakur Uttam Singh
    • 1
  • Anil Kumar Sharma
    • 3
  • Dinesh Kumar
    • 1
  • Surendra Kumar Tandan
    • 1
  1. 1.Division of Pharmacology & ToxicologyIndian Veterinary Research InstituteBareillyIndia
  2. 2.Centre for Wildlife Conservation Management and Disease SurveillanceBareillyIndia
  3. 3.Division of PathologyIndian Veterinary Research InstituteBareillyIndia

Personalised recommendations