Comparative analysis of different dietary antioxidants on oxidative stress pathway genes in L6 myotubes under oxidative stress

Original Article


Enhanced oxidative stress plays an important role in the progression and onset of diabetes and its complications. Strategies or efforts meant to reduce the oxidative stress are needed which may mitigate these pathogenic processes. The present study aims to investigate the in vitro ameliorative potential of nine antioxidant molecules in L6 myotubes under oxidative stress condition induced by 4-hydroxy-2-nonenal and also to comprehend the gene expression patterns of oxidative stress genes upon the supplementation of different antioxidants in induced stress condition. The study results demonstrated a marked increase in the level of malondialdehyde and protein carbonyl content with a subsequent increase in the free radicals that was reversed by the pretreatment of different dietary antioxidant. From the expression analysis of the oxidative stress genes, it is evident that the expression of these genes is modulated by the presence of antioxidants. The highest expression was found in the cells treated with Insulin in conjugation with an antioxidant. Resveratrol is the most potent modulator followed by Mangiferin, Estragole, and Capsaicin. This comparative analysis ascertains the potency of Resveratrol along with Insulin in scavenging the reactive oxygen species (ROS) generated under induced stress conditions through antioxidant defense mechanism against excessive ROS production, contributing to the prevention of oxidative damage in L6 myotubes.


T2D Oxidative stress ROS HNE Antioxidants Myotubes 



The authors are grateful to Department of Biotechnology, Government of India for the research grant (BT/362/NE/TBP/2012) extended towards completion of this work. The authors thank Dr. Bidyut Kumar Sharma, Director, DBT-AAU Centre, Assam Agricultural University for providing instrumental support. The authors also thank Gunajit Goswami, Research Scholar, Assam agricultural University for extending his help in executing this research work. The authors would like to thank Prof. S.S. Ghosh and Anil Bidkar from IIT Guwahati for the help extended in the study.

Authors’ contributions

PS and AB performed the experimental work, and compilation of data. PS drafted the manuscript. SB designed the study, facilitated infrastructural and financial support to carry out the experiments. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest.

Supplementary material

10616_2018_209_MOESM1_ESM.doc (550 kb)
Fig. 1 a–b Activities of SOD, CAT in control and treatment groups of L6 myotubes. Results are expressed as means and standard deviations of the control and treated cells from triplicate measurements (n = 3) of three biological replicates. Data were subjected to one-way ANOVA and the significance of differences between means was calculated by Tukey’s Multiple Comparison Test using Graph pad Prism Software and significance was accepted at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001 versus control and #P < 0.05; ##P < 0.01; ###P < 0.001 versus HNE treated (DOC 549 kb)


  1. Abdulkadir AA, Thanoon IA (2012) Comparative effects of glibenclamide and metformin on C-reactive protein and oxidant/antioxidant status in patients with Type II diabetes mellitus. Sultan Qaboos Univ Med J 12:55–61CrossRefGoogle Scholar
  2. Abiko T, Abiko A, Clermont AC et al (2003) Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase C activation. Diabetes 352:829–837CrossRefGoogle Scholar
  3. Aderibigbe AO, Emudianughe TS, Lawal BA (1999) Antihyperglycemic effect of Mangifera indica in rat. Phytother Res 13:504–507CrossRefGoogle Scholar
  4. Arulselvan P, Subramanian S (2007) Effect of Murraya koenigii leaf extract on carbohydrate metabolism studied in streptozotocin induced diabetic rats. Int J Biol Chem 1:21–28CrossRefGoogle Scholar
  5. Berlett BS, Stadtman ER (1997) Protein oxidation in aging. Dis Oxid Stress 272:20313–20319Google Scholar
  6. Breinholt V, Lauridsen ST, Daneshvar B et al (2000) Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett 154:201–210CrossRefGoogle Scholar
  7. Can Baser KH (2008) Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils. Curr Pharm Des 14:3106–3119CrossRefGoogle Scholar
  8. Cho SY, Park JY, Park EM et al (2002) Alternation of hepatic antioxidant enzyme activities and lipid profile in streptozotocin-induced diabetic rats by supplementation of dandelion water extract. Clin Chim Acta 317:109–117CrossRefGoogle Scholar
  9. Choi MS, Do KM, Park YS et al (2001) Effect of naringin supplementation on cholesterol metabolism and antioxidant status in rats fed high cholesterol with different levels of vitamin E. Ann Nutr Metab 45:193–201CrossRefGoogle Scholar
  10. Dachani SR, Avanapu SR, Ananth PH (2012) In vitro antioxidant and glucose uptake effect of Trichodesma indicum in L-6 cell lines. J Pharm Bio Sci 3:810–819Google Scholar
  11. Day C (2001) The rising tide of type 2 diabetes. Br J Diabetes Vasc Dis 1:37–432CrossRefGoogle Scholar
  12. Deng W, Lu H, Teng J (2013) Carvacrol attenuates diabetes-associated cognitive deficits in rats. J Mol Neurosci 51:813–819CrossRefGoogle Scholar
  13. Di Mascio P, Kaiser S, Sies H (1989) Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys 274:532–538CrossRefGoogle Scholar
  14. Du C, Cao H, Sun H et al (2017) Protective effect of baicalein on oxLDL-induced oxidative stress and inflammation injury in endothelial cell. Int J Pharmacol 13:280–285CrossRefGoogle Scholar
  15. Erhardt JG, Meisner C, Bode JC et al (2003) Lycopene, Beta-carotene and colorectal adenomas. Am J Clin Nutr 78:1219–1224CrossRefGoogle Scholar
  16. Guimaraes AG, Oliveira GF, Melo MS et al (2010) Bioassay-guided evaluation of antioxidant and antinociceptive activities of Carvacrol. Basic Clin Pharmacol Toxicol 107:949–957CrossRefGoogle Scholar
  17. Halliwell B, Gutteridge JM (1999) Free radicals in biology and medicine. Oxford University Press, OxfordGoogle Scholar
  18. Hernandez-Munoz R, Olguin-Martinez M, Aguilar-Delfin I et al (2013) Oxidant status and lipid composition of erythrocyte membranes in patients with type 2 diabetes, chronic liver damage, and a combination of both pathologies. Oxid Med Cell Longev 2013:9CrossRefGoogle Scholar
  19. Hussein HK, Abu-Zinadah OA (2010) Antioxidant effect of curcumin extracts in induced diabetic wister rats. Int J Zool Res 6:266–276CrossRefGoogle Scholar
  20. Jagetia GC, Baliga MS (2003) Evaluation of the radioprotective effect of the leaf extract of Syzygium cumini (Jamun) in mice exposed to a lethal dose of gamma-irradiation. Mol Nutr Food Res 47:181–185Google Scholar
  21. Jeon HJ, Seo MJ, Choi HS et al (2014) Gelidium elegans, an edible red seaweed, and hesperidin inhibit lipid accumulation and production of reactive oxygen species and reactive nitrogen species in 3T3-L1 and RAW264. 7 cells. Phytother Res 28:1701–1709CrossRefGoogle Scholar
  22. Kakkar R, Kalra J, Mantha SV et al (1995) Lipid peroxidation and activity of antioxidant enzymes in diabetic rats. Mol Cell Biochem 151:113–119CrossRefGoogle Scholar
  23. Kochhar KP (2008) Dietary spices in health and diseases (II). Indian J Physiol Pharmacol 52:327–354Google Scholar
  24. Kolsi RB, Salah HB, Jardak N et al (2017) Effects of Cymodocea nodosa extract on metabolic disorders and oxidative stress in alloxan-diabetic rats. Biomed Pharmacother 89:257–267CrossRefGoogle Scholar
  25. Koparal AT, Zeytinoglu M (2003) Effects of carvacrol on a human non-small cell lung cancer (NSCLC) cell line A549. Cytotechnol 43(1–3):149–154CrossRefGoogle Scholar
  26. Kowluru RA, Kenned A (2001) Therapeutic potential of anti-oxidants and diabetic retinopathy. Expert Opin Invest Drugs 10:1665–1676CrossRefGoogle Scholar
  27. Kutuk O, Adli M, Poli G et al (2004) Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts. BioFactors 20:1–10CrossRefGoogle Scholar
  28. Laaksonen DE, Atalay M, Niskanen L et al (1998) Exercise and oxidative stress in diabetes mellitus. Pathophysiology 5(Suppl 1):112CrossRefGoogle Scholar
  29. Levine RL (2002) Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic Biol Med 32:790–796CrossRefGoogle Scholar
  30. Limaye PV, Raghuram N, Sivakami S (2003) Oxidative stress and gene expression of antioxidant enzymes in the renal cortex of streptozotocininduced diabetic rats. Mol Cell Biochem 243:147–152CrossRefGoogle Scholar
  31. Liu Y, Tang Q, Hu Z et al (2015) Lycopene attenuates angiotensin II induced oxidative stress in H9c2 cells. Zhonghua xin xue guan bing za zhi 43:341–346Google Scholar
  32. Maritim AC, Sanders A, Watkins JB (2003) Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol 17:24–38CrossRefGoogle Scholar
  33. MatEs JM, Perez-Gomez C, De Castro IN (1999) Antioxidant enzymes and human diseases. Clin Biochem 32:595–603CrossRefGoogle Scholar
  34. Mc Garry JD (2002) Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes. Diabetes 51:7–18CrossRefGoogle Scholar
  35. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  36. Murugan P, Pari L (2006) Effect of tetrahydrocurcumin on lipid peroxidation and lipids in streptozotocin–nicotinamide-induced diabetic rats. Basic Clin Pharmacol Toxicol 99:122–127CrossRefGoogle Scholar
  37. Nakhaee A, Bokaeian M, Saravani M et al (2009) Attenuation of oxidative stress in streptozotocin-induced diabetic rats by Eucalyptus globulus. Indian J Clin Biochem 24:419–425CrossRefGoogle Scholar
  38. Ramakrishna V, Jailkhani R (2008) Oxidative stress in non-insulin-dependent diabetes mellitus (NIDDM) patients. Acta Diabetol 45:41–46CrossRefGoogle Scholar
  39. Robertson RP (2004) Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem 279:42351–42354CrossRefGoogle Scholar
  40. Saha S, Sadhukhan P, Sinha K et al (2016) Mangiferin attenuates oxidative stress induced renal cell damage through activation of PI3K induced Akt and Nrf-2 mediated signaling pathways. Biochem Biophys Rep 5:313–327Google Scholar
  41. Sanchez GM, Re L, Giuliani A et al (2000) OS Protective effects of Mangifera indica L. extract, mangiferin and selected antioxidants against TPA-induced biomolecules oxidation and peritoneal macrophage activation in mice. Pharmacol Res 42:565–573CrossRefGoogle Scholar
  42. Shao W, Yu Z, Chiang Y et al (2012) Curcumin prevents high-fat diet-induced insulin resistance and obesity via attenuating lipogenesis in liver and inflammatory pathway in adipocytes. PLoS ONE 7:28784CrossRefGoogle Scholar
  43. Shirwaikar A, Prabhu KS, Punitha IS (2006) In vitro antioxidant studies of Sphaeranthus indicus (Linn). J Exp Biol 44:993–996Google Scholar
  44. Srinivas A, Menon VP, Periaswamy V et al (2003) Protection of pancreatic beta cell by the potential antioxidant bis-o-hydroxycinnamoyl methane, analogue of natural curcuminoid in experimental diabetes. J Pharm Pharm Sci 6:327–333Google Scholar
  45. Stadtman ER, Levine RL (2000) Protein oxidation. Ann NY Acad Sci 899:191–208CrossRefGoogle Scholar
  46. Tiwari BK, Pandey KB, Abidi AB et al (2013) Markers of oxidative stress during diabetes mellitus. J Biomark 2013:378790CrossRefGoogle Scholar
  47. Usatyuk PV, Parinandi NL, Natarajan V (2006) Redox regulation of 4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins. J Biol Chem 281:35554–35566CrossRefGoogle Scholar
  48. Wang Z, Zhang XM, Ribnicky DM et al (2004) Effect of a alcoholic extract of Artemisia dracunculus (Tarralin™) on glucose uptake in human skeletal muscle culture. Diabetes 53:A406–A407Google Scholar
  49. Wiernsperger NF (2003) Oxidative stress as a therapeutic target in diabetes: revisiting the controversy. Diabet Med 29:579–585Google Scholar
  50. Wu CH, Yeh CT, Yen GC (2010) Epigallocatechin gallate (EGCG) binds to low-density lipoproteins (LDL) and protects them from oxidation and glycation under high glucose conditions mimicking diabetes. Food Chem 121:639–644CrossRefGoogle Scholar
  51. Zatalia SR, Sanusi H (2013) The role of antioxidants in the pathophysiology, complications, and management of diabetes mellitus. Acta Med Indones 45:141–147Google Scholar
  52. Zhang LL, Liu DY, Ma LQ et al (2007) Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity. Circ Res 100:1063–1070CrossRefGoogle Scholar
  53. Zhang Y, Ye M, Chen L et al (2015) Role of the ubiquitin-proteasome system and autophagy in regulation of insulin sensitivity in serum-starved 3T3-L1 adipocytes. Endocr J 62:673–686CrossRefGoogle Scholar
  54. Zhao Y, Li H, Gao Z et al (2005) Effects of dietary baicalin supplementation on iron overload-induced mouse liver oxidative injury. Eur J Pharmacol 509:195–200CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Bioengineering and TechnologyGUIST, Gauhati UniversityGuwahatiIndia

Personalised recommendations