Protective effect of thymoquinone, the active constituent of Nigella sativa fixed oil, against ethanol toxicity in rats

Document Type: Original Article


1 Department of Pharmacodynamy and Toxicology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Pharmaceutical Research Center, Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

4 Medical Toxicology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Food Control Laboratory, Food and Drug Administration, Shiraz University of Medical Sciences, Shiraz, Iran

6 Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Long term consumption of ethanol may induce damage to many organs. Ethanol induces its noxious effects through reactive oxygen species production, and lipid peroxidation and apoptosis induction in different tissues and cell types. Previous experiments have indicated the antioxidant characteristics of thymoquinone, the active constituent of Nigella sativa fixed oil, against biologically dangerous reactive oxygen species. This experiment was planned to evaluate the protective effect of thymoquinone against subchronic ethanol toxicity in rats.
Materials and Methods: Experiments were performed on six groups. Each group consisted of six animals, including control group (saline, gavage), ethanol-receiving group (3 g/kg/day, gavage), thymoquinone (2.5, 5, 10 mg/Kg/day, intraperitoneally (IP)) plus ethanol and thymoquinone (10 mg/Kg/day, IP) groups. Treatments were carried out in four weeks.
Results: Thymoquinone reduced the ethanol-induced increase in the lipid peroxidation and severity of histopathological alteration in liver and kidney tissues. In addition it improved the levels of proinflammatory cytokines in liver tissue. Furthermore, thymoquinone corrected the liver enzymes level including alanine transaminase, aspartate transaminase and alkaline phosphatase in serum and glutathione content in liver and kidney tissues. Other experiments such as Western blot analysis and quantitative real-time RT-PCR revealed that thymoquinone suppressed the expression of Bax/Bcl-2 ratio (both protein and mRNA level), and caspases activation pursuant to ethanol toxicity.
Conclusion: This study indicates that thymoquinone may have preventive effects against ethanol toxicity in the liver and kidney tissue through reduction in lipid peroxidation and inflammation, and also interrupting apoptosis.


1. Ghosh S, Guria S, Das M, editors. Alcohol as a risk factor for cancer burden: a review. Proc Zool Soc 2015; 69:32–37

2. Bondy SC. Ethanol toxicity and oxidative stress. Toxicol Lett 1992; 63:231-241.

3. Lieber CS. Hepatic and metabolic effects of ethanol: pathogenesis and prevention. Ann Med 1994; 26: 325-330.

4. Flier JS, Underhill LH, Lieber CS. Medical disorders of alcoholism. N Engl J Med 1995; 333: 1058-1065.

5. Yang H-Y, Lin H-S, Chao JC, Chien Y-W, Peng H-C, Chen J-R. Effects of soy protein on alcoholic liver disease in rats undergoing ethanol withdrawal. J Nutr Biochem 2012; 23: 679-684.

6. Darwish HA, Raboh NRA, Mahdy A. Camel’s milk alleviates alcohol-induced liver injury in rats. Food Chem Toxicol 2012; 50: 1377-1383.

7. Hosseinzadeh H, Parvardeh S, Asl MN, Sadeghnia HR, Ziaee T. Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus. Phytomedicine 2007; 14:621-627.

8. Arslan SO, Gelir E, Armutcu F, Coskun O, Gurel A, Sayan H, et al. The protective effect of thymoquinone on ethanol-induced acute gastric damage in the rat. Nutr Res 2005; 25: 673-680.

9. Darakhshan S, Pour AB, Colagar AH, Sisakhtnezhad S. Thymoquinone and its therapeutic potentials. Pharmacol Res 2015; 95: 138-158.

10. Mollazadeh H, Hosseinzadeh H. The protective effect of Nigella sativa against liver injury: a review. Iran J Basic Med Sci 2014; 17: 958-966.

11. Razavi B, Hosseinzadeh H. A review of the effects of Nigella sativa L. and its constituent, thymoquinone, in metabolic syndrome. J Endocrinol Invest 2014; 37: 1031-1040.

12. Amin B, Taheri M, Hosseinzadeh H. Effects of intraperitoneal thymoquinone on chronic neuropathic pain in rats. Planta Med 2014; 80: 1269-1277.

13. Havakhah S, Sadeghnia HR, Mosa-Al-Reza Hajzadeh NM, Roshan SS, Hosseinzadeh H, Mohareri N, et al. Effect of Nigella sativa on ischemia-reperfusion induced rat kidney damage. Iran J Basic Med Sci 2014;17:986-992.

14. Hosseinzadeh H, Taiari S, Nassiri-Asl M. Effect of thymoquinone, a constituent of Nigella sativa L., on ischemia–reperfusion in rat skeletal muscle. Naunyn Schmiedebergs Arch Pharmacol 2012; 385: 503-508.

15. Hosseinzadeh H, Parvardeh S, Nassiri-Asl M, Mansouri M-T. Intracerebroventricular administration of thymoquinone, the major constituent of Nigella sativa seeds, suppresses epileptic seizures in rats. Med Sci Monit Basic Res 2005;11:BR106-BR110.

16. Pourbakhsh H, Taghiabadi E, Abnous K, Hariri AT, Hosseini SM, Hosseinzadeh H. Effect of Nigella sativa fixed oil on ethanol toxicity in rats. Iran J Basic Med Sci 2014;17:1020-1031.

17. Amin B, Hosseinzadeh H. Black cumin (Nigella sativa) and its active constituent, thymoquinone: an overview on the analgesic and anti-inflammatory effects. Planta Med 2016;82:8-16.

18. Javidi S, Razavi BM, Hosseinzadeh H. A review of neuropharmacology effects of Nigella sativa and its main component, thymoquinone. Phytother Res 2016; 30:1219-1229.

19. Forouzanfar F, Bazzaz BSF, Hosseinzadeh H. Black cumin (Nigella sativa) and its constituent (thymoquinone): a review on antimicrobial effects. Iran J Basic Med Sci 2014;17:929-938.

20. Ziaee T, Moharreri N, Hosseinzadeh H. Review of pharmacological and toxicological effects of Nigella sativa and its active constituents. J Med Plant Res 2012;2:16-42.

21. Hosseinzadeh H, Fazly Bazzaz B, Haghi MM. Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in mice. Pharmacologyonline 2007;2:429-435.

22. Parvardeh S, Nassiri-Asl M, Mansouri M, Hosseinzadeh H. Study on the anti-convulsant activity of thymoquinone, the major constituent of Nigella sativa L. seeds, through intracerebroventricular injection. J Med Plant Res 2005; 2: 45-52.

23. Hosseinzadeh H, Eskandari M, Ziaee T. Anti-tussive effect of thymoquinone, a constituent of Nigella sativa seeds, in guinea pigs. Pharmacologyonline 2008; 2: 480-484.

24. Woo CC, Kumar AP, Sethi G, Tan KHB. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharmacol 2012; 83: 443-451.

25. Pourgholamhossein F, Sharififar F, Rasooli R, Pourgholi L, Nakhaeipour F, Samareh-Fekri H, et al. Thymoquinone effectively alleviates lung fibrosis induced by paraquat herbicide through down-regulation of pro-fibrotic genes and inhibition of oxidative stress. Environ Toxicol Pharmacol 2016; 45: 340-345.

26. Aycan İÖ, Tüfek A, Tokgöz O, Evliyaoğlu O, Fırat U, Kavak GÖ, et al. Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats. Int J Surg 2014; 12: 213-218.

27. Ismail M, Al-Naqeep G, Chan KW. Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Radic Biol Med 2010; 48: 664-672.

28. Kanter M, Coskun O, Uysal H. The antioxidative and antihistaminic effect of Nigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage. Arch Toxicol 2006; 80: 217-224.

29. El Mezayen R, El Gazzar M, Nicolls MR, Marecki JC, Dreskin SC, Nomiyama H. Effect of thymoquinone on cyclooxygenase expression and prostaglandin production in a mouse model of allergic airway inflammation. Immunol Lett 2006;106:72-81.

30. Tavakkoli A, Ahmadi A, Razavi BM, Hosseinzadeh H. Black seed (Nigella sativa) and its constituent thymoquinone as an antidote or a protective agent against natural or chemical toxicities. Iran J Pharmac Res 2017; 16: 2-23.

31. Mehri S, Shahi M, Razavi BM, Hassani FV, Hosseinzadeh H. Neuroprotective effect of thymoquinone in acrylamide-induced neurotoxicity in Wistar rats. IJBMS. 2014;17:1007-1011.

32. Niehaus W, Samuelsson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-130.

33. Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979; 582: 67-78.

34. Hosseinzadeh L, Behravan J, Mosaffa F, Bahrami G, Bahrami A, Karimi G. Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species. Food Chem Toxicol 2011; 49: 1102-1109.

35. Mallikarjuna K, Chetan PS, Reddy KS, Rajendra W. Ethanol toxicity: Rehabilitation of hepatic antioxidant defense system with dietary ginger. Fitoterapia 2008; 79: 174-178.

36. Husain K, Scott BR, Reddy SK, Somani SM. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 2001; 25: 89-97.

37. Jurczuk M, Brzóska MM, Moniuszko-Jakoniuk J, Gałażyn-Sidorczuk M, Kulikowska-Karpińska E. Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol. Food Chem Toxicol 2004; 42: 429-438.

38. Scott R, Reddy K, Husain K, Schlorff E, Rybak L, Somani S. Dose response of ethanol on antioxidant defense system of liver, lung, and kidney in rat. Pathophysiology 2000;7: 25-32.

39. Hoek JB, Pastorino JG. Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol 2002; 27: 63-68.

40. Yoon S-J, Koh E-J, Kim C-S, Zee O-P, Kwak J-H, Jeong W-J, et al. Agrimonia eupatoria protects against chronic ethanol-induced liver injury in rats. Food Chem Toxicol 2012; 50: 2335-2341.

41. El-Dakhakhny M, Barakat M, El-Halim MA, Aly S. Effects of Nigella sativa oil on gastric secretion and ethanol induced ulcer in rats. J Ethnopharmacol 2000;72:299-304.

42. Girish C, Koner BC, Jayanthi S, Ramachandra Rao K, Rajesh B, Pradhan SC. Hepatoprotective activity of picroliv, curcumin and ellagic acid compared to silymarin on paracetamol induced liver toxicity in mice. Fundam Clin Pharmacol 2009;23:735-745.

43. Lu Z-M, Tao W-Y, Zou X-L, Fu H-Z, Ao Z-H. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. J Ethnopharmacol 2007;110:160-164.

44. Iimuro Y, Gallucci RM, Luster MI, Kono H, Thurman RG. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 1997; 26:1530-1537.

45. Esposito E, Cuzzocrea S. TNF-alpha as a therapeutic target in inflammatory diseases, ischemia-reperfusion injury and trauma.   2009;16:3152-3167.

46. Wu D, Cederbaum AI. Ethanol induced apoptosis to stable HepG2 cell lines expressing human cytochrome P4502E1. Alcohol Clin Exp Res 1999;23:67-76.

47. Rossé T, Olivier R, Monney L, Rager M, Conus S, Fellay I, et al. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 1998;391:496-499.

48. El-Ghany R, Sharaf N, Kassem L, Mahran L, Heikal O. Thymoquinone triggers anti-apoptotic signaling targeting death ligand and apoptotic regulators in a model of hepatic ischemia reperfusion injury. Drug Discov Ther 2009;3:296-306.

49. Nadkarni K, Nadkarni A. Indian Materia Medica, vol. 1Popular Prakashan. Bombay, India. 1976:615-616.

50. Sayed MD. Traditional medicine in health care. J Ethnopharmacol. 1980;2:19-22.

51. Lautenbacher L-M. Schwarzkummelol: eine neue quelle ungesattigter fettsauren. Dtsch Apoth Ztg 1997;137:68-69.

52. Ali B, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 2003;17:299-305.

53. Houghton PJ, Zarka R, de las Heras B, Hoult J. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med 1995;61:33-36.

54. Marsik P, Kokoska L, Landa P, Nepovim A, Soudek P, Vanek T. In vitro inhibitory effects of thymol and quinones of Nigella sativa seeds on cyclooxygenase-1-and-2-catalyzed prostaglandin E2 biosyntheses. Planta Med 2005;71:739-742.

55. El Gazzar M, El Mezayen R, Marecki JC, Nicolls MR, Canastar A, Dreskin SC. Anti-inflammatory effect of thymoquinone in a mouse model of allergic lung inflammation. Int Immunopharmacol 2006;6:1135-1142.

56. Badary OA. Thymoquinone attenuates ifosfamide-induced Fanconi syndrome in rats and enhances its anti-tumor activity in mice. J Ethnopharmacol 1999;67:135-142.

57. Hosseinzadeh H, Parvardeh S. Anti-convulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine 2004;11:56-64.

58. Abdel-Fattah A-FM, Matsumoto K, Watanabe H. Anti-nociceptive effects of Nigella sativa oil and its major component, thymoquinone, in mice. Eur J Pharmacol 2000;400:89-97.

59. Halawani E. Anti-bacterial activity of thymoquinone and thymohydroquinone of Nigella sativa L. and their interaction with some anti-biotics. Adv Biol Res. 2009;3:148-52.

60. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res 2000;14:323-28.

61. Al-Ghamdi M. The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa. J Ethnopharmacol  2001;76:45-48.