Assessment of thymoquinone effects on apoptotic and oxidative damage induced by morphine in mice heart

Cyrus Jalili; Maryam Sohrabi; Faramarz Jalili; Mohammad Reza Salahshoor

doi:10.14715/cmb/2018.64.9.5

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Vol. 64 No. 9: Issue 9

Issue Published : July 19, 2018

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Assessment of thymoquinone effects on apoptotic and oxidative damage induced by morphine in mice heart

https://doi.org/10.14715/cmb/2018.64.9.5

Cyrus Jalili

Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Maryam Sohrabi

Fertility and Infertility Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Faramarz Jalili

Students Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran

Mohammad Reza Salahshoor

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Corresponding Author(s) : Mohammad Reza Salahshoor

reza.salahshoor@yahoo.com

Cellular and Molecular Biology, Vol. 64 No. 9: Issue 9
Article Published : June 30, 2018

Abstract

Opioids bind to specific receptors that are located in the central nervous system (CNS) and many other organs such as cardiovascular tissue. Morphine binds to opioid receptors and can induce oxidative stress under some certain conditions. Thymoquinone (TQ) has shown many therapeutic effects such as anti-inflammatory, antioxidant and immunomodulatory ones. Considering the oxidative effects of morphine, antioxidant effects of TQ and effects of oxidative damage in various types of biomolecules, the present study was conducted to determine the effect of morphine plus TQ on the expression of apoptotic genes in the heart of male mice. Hence we used real-time PCR to identify alterations in mRNA expression of genes involved in apoptotic pathway, including p53, Bax and Bcl-2 between the morphine-treated and TQ plus morphine-treated mice. Serum nitric oxide (NO) (Griess assay) and total antioxidant capacity (TAC) were analyzed and compared. In the morphine group, compared to control group, a significant increase in P53 and Bax mRNA expression and a significant decrease in Bcl-2 mRNA expression were observed (p < 0.01). In TQ plus morphine groups, NO was decreased (P <0 .001) and TAC levels were increased significantly (P < .001). Interestingly, TQ (9 and 18 mg/kg) plus morphine caused a significant decrease in p53 and Bax and a significant increase in Bcl2 mRNA expression, compared to morphine-treated group (p < 0.01). Collectively, the results of this study indicated that TQ, as an antioxidant, can improve the apoptotic effects induced by morphine in the heart tissue of mice.

Keywords

Morphine Thymoquinone Apoptosis P53.

Jalili, C., Sohrabi, M., Jalili, F., & Salahshoor, M. R. (2018). Assessment of thymoquinone effects on apoptotic and oxidative damage induced by morphine in mice heart. Cellular and Molecular Biology, 64(9), 33–38. https://doi.org/10.14715/cmb/2018.64.9.5

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References

Reference:
Jacoby E, Bouhelal R, Gerspacher M, Seuwen K. The 7 TM G"protein"coupled receptor target family. ChemMedChem 2006; 1(8): 760-782.
Insel P, Snead A, Murray F et al. GPCR expression in tissues and cells: are the optimal receptors being used as drug targets? British journal of pharmacology 2012; 165(6): 1613-1616.
Sobanski P, Krajnik M, Shaqura M, Bloch-Boguslawska E, Schäfer M, Mousa SA. The presence of mu-, delta-, and kappa-opioid receptors in human heart tissue. Heart and vessels 2014; 29(6): 855.
Chen A, Ashburn MA. Cardiac effects of opioid therapy. Pain Medicine 2015; 16(suppl_1): S27-S31.
Stanley TH, Gray NH, Stanford W, Armstrong R. The Effects of High–dose Morphine on Fluid and Blood Requirements in Open–heart Operations. Anesthesiology 1973; 38(6): 536-541.
Skrabalova J, Drastichova Z, Novotny J. Morphine as a potential oxidative stress-causing agent. Mini-reviews in organic chemistry 2013; 10(4): 367-372.
Caldwell JR, Rapoport RJ, Davis JC et al. Efficacy and safety of a once-daily morphine formulation in chronic, moderate-to-severe osteoarthritis pain: results from a randomized, placebo-controlled, double-blind trial and an open-label extension trial. Journal of pain and symptom management 2002; 23(4): 278-291.
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The international journal of biochemistry & cell biology 2007; 39(1): 44-84.
Patel VP, Chu CT. Nuclear transport, oxidative stress, and neurodegeneration. International journal of clinical and experimental pathology 2011; 4(3): 215.
Zhu H, Jia Z, Misra H, Li YR. Oxidative stress and redox signaling mechanisms of alcoholic liver disease: updated experimental and clinical evidence. Journal of digestive diseases 2012; 13(3): 133-142.
Jimenez-Del-Rio M, Velez-Pardo C. The bad, the good, and the ugly about oxidative stress. Oxidative medicine and cellular longevity 2012; 2012.
Rezazadeh H, Kahnouei MH, Hassanshahi G et al. Regulatory effects of chronic low-dose morphine on nitric oxide level along with baroreflex sensitivity in two-kidney one-clip hypertensive rats. Iranian journal of kidney diseases 2014; 8(3): 194.
Zhang YT, Zheng QS, Pan J, Zheng RL. Oxidative damage of biomolecules in mouse liver induced by morphine and protected by antioxidants. Basic & clinical pharmacology & toxicology 2004; 95(2): 53-58.
í–zmen ć°, Nazıroğlu M, Alici HA, Åžahin F, Cengiz M, Eren I. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochemical research 2007; 32(1): 19-25.
Schinzel A, Kaufmann T, Borner C. Bcl-2 family members: integrators of survival and death signals in physiology and pathology [corrected]. Biochimica et biophysica acta 2004; 1644(2-3): 95-105.
Sharpe JC, Arnoult D, Youle RJ. Control of mitochondrial permeability by Bcl-2 family members. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2004; 1644(2): 107-113.
Donovan M, Cotter TG. Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2004; 1644(2): 133-147.
Toshiyuki M, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995; 80(2): 293-299.
Li GY, Xie P, Li HY, Hao L, Xiong Q, Qiu T. Involment of p53, Bax, and Bcl"2 pathway in microcystins"induced apoptosis in rat testis. Environmental toxicology 2011; 26(2): 111-117.
Ali B, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytotherapy Research 2003; 17(4): 299-305.
Norouzi F, Abareshi A, Asgharzadeh F et al. The effect of Nigella sativa on inflammation-induced myocardial fibrosis in male rats. Research in pharmaceutical sciences 2017; 12(1): 74.
Iddamaldeniya SS, Thabrew M, Wickramasinghe S, Ratnatunge N, Thammitiyagodage MG. A long-term investigation of the anti-hepatocarcinogenic potential of an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra. Journal of carcinogenesis 2006; 5(1): 11.
Gali-Muhtasib HU, Kheir WGA, Kheir LA, Darwiche N, Crooks PA. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anti-cancer drugs 2004; 15(4): 389-399.
Gali-Muhtasib H, Diab-Assaf M, Boltze C et al. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. International journal of oncology 2004; 25(4): 857-866.
Shoieb AM, Elgayyar M, Dudrick PS, Bell JL, Tithof PK. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. International journal of oncology 2003; 22(1): 107-113.
Elbarbry F, Ragheb A, Marfleet T, Shoker A. Modulation of hepatic drug metabolizing enzymes by dietary doses of thymoquinone in female New Zealand White rabbits. Phytotherapy research 2012; 26(11): 1726-1730.
El"Mahdy MA, Zhu Q, Wang QE, Wani G, Wani AA. Thymoquinone induces apoptosis through activation of caspase"8 and mitochondrial events in p53"null myeloblastic leukemia HL"60 cells. International journal of cancer 2005; 117(3): 409-417.
Yin D, Mufson RA, Wang R, Shi Y. Fas-mediated cell death promoted by opioids. Nature 1999; 397(6716): 218-218.
Karkhah A, Ataee R, Ataie A. Morphine pre-and post-conditioning exacerbates apoptosis in rat hippocampus cells in a model of homocysteine-induced oxidative stress. Biomedical Reports 2017; 7(4): 309-313.
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(6): 296-306.
Jalili C, Salahshoor MR, Hoseini M, Roshankhah S, Sohrabi M, Shabanizadeh A. Protective Effect of Thymoquinone Against Morphine Injuries to Kidneys of Mice. Iranian Journal of Kidney Diseases 2017; 11(2).
Jalili C, Tabatabaei H, Kakaberiei S, Roshankhah S, Salahshoor MR. Protective role of Crocin against nicotine-induced damages on male mice liver. International journal of preventive medicine 2015; 6.
Stoops WW, Hatton KW, Lofwall MR, Nuzzo PA, Walsh SL. Intravenous oxycodone, hydrocodone, and morphine in recreational opioid users: abuse potential and relative potencies. Psychopharmacology 2010; 212(2): 193-203.
Flemming K. The use of morphine to treat cancer-related pain: a synthesis of quantitative and qualitative research. Journal of pain and symptom management 2010; 39(1): 139-154.
Asgary S, Sarrafzadegan N, Naderi G-A, Rozbehani R. Effect of opium addiction on new and traditional cardiovascular risk factors: do duration of addiction and route of administration matter? Lipids in health and disease 2008; 7(1): 42.
Miaskowski C. A review of the incidence, causes, consequences, and management of gastrointestinal effects associated with postoperative opioid administration. Journal of PeriAnesthesia Nursing 2009; 24(4): 222-228.
Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology: The Journal of the American Society of Anesthesiologists 2011; 115(6): 1363-1381.
Seiri L, Mokri A, Dezhakam H, Noroozi A. Using tincture of opium for treatment of opiate abusers in Iran. Drug & Alcohol Dependence 2014; 140: e200.
Sharifipour M, Izadpanah E, Nikkhoo B et al. A new pharmacological role for donepezil: attenuation of morphine-induced tolerance and apoptosis in rat central nervous system. Journal of biomedical science 2014; 21(1): 6.
Hassanzadeh K, Roshangar L, Habibi-asl B et al. Riluzole prevents morphine-induced apoptosis in rat cerebral cortex. Pharmacological Reports 2011; 63(3): 697-707.
Liu L-W, Lu J, Wang X-H, Fu S-K, Li Q, Lin F-Q. Neuronal apoptosis in morphine addiction and its molecular mechanism. International journal of clinical and experimental medicine 2013; 6(7): 540.
Hu S, Sheng WS, Lokensgard JR, Peterson PK. Morphine induces apoptosis of human microglia and neurons. Neuropharmacology 2002; 42(6): 829-836.
Bhat RS, Bhaskaran M, Mongia A, Hitosugi N, Singhal PC. Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation. Journal of leukocyte biology 2004; 75(6): 1131-1138.
Lin X, Wang YJ, Li Q et al. Chronic high"dose morphine treatment promotes SH"SY5Y cell apoptosis via c"Jun N"terminal kinase"mediated activation of mitochondria"dependent pathway. The FEBS journal 2009; 276(7): 2022-2036.
Ohara T, Itoh T, Takahashi M. Immunosuppression by morphine-induced lymphocyte apoptosis: is it a real issue? Anesthesia & Analgesia 2005; 101(4): 1117-1122.
Li R, Wong GT, Wong TM, Zhang Y, Xia Z, Irwin MG. Intrathecal morphine preconditioning induces cardioprotection via activation of delta, kappa, and mu opioid receptors in rats. Anesthesia & Analgesia 2009; 108(1): 23-29.
Salahshoor MR. Protective effect of crocin on liver toxicity induced by morphine. Research in pharmaceutical sciences 2016; 11(2): 120.
Jalili C, Ahmadi S, Roshankhah S, Salahshoor M. Effect of Genistein on reproductive parameter and serum nitric oxide levels in morphine-treated mice. International Journal of Reproductive BioMedicine 2016; 14(2): 95.
Gali-Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: a promising anti-cancer drug from natural sources. The international journal of biochemistry & cell biology 2006; 38(8): 1249-1253.
Nutten S, Philippe D, Mercenier A, Duncker S. Opioid receptors stimulating compounds (thymoquinone, nigella sativa) and food allergy. Google Patents; 2010.
Mahmoud AM, Ahmed OM, Galaly SR. Thymoquinone and curcumin attenuate gentamicin-induced renal oxidative stress, inflammation and apoptosis in rats. EXCLI journal 2014; 13: 98.
Alenzi F, El-Bolkiny YE-S, Salem M. Protective effects of Nigella sativa oil and thymoquinone against toxicity induced by the anticancer drug cyclophosphamide. British journal of biomedical science 2010; 67(1): 20-28.

References

Reference:

Jacoby E, Bouhelal R, Gerspacher M, Seuwen K. The 7 TM G"protein"coupled receptor target family. ChemMedChem 2006; 1(8): 760-782.

Insel P, Snead A, Murray F et al. GPCR expression in tissues and cells: are the optimal receptors being used as drug targets? British journal of pharmacology 2012; 165(6): 1613-1616.

Sobanski P, Krajnik M, Shaqura M, Bloch-Boguslawska E, Schäfer M, Mousa SA. The presence of mu-, delta-, and kappa-opioid receptors in human heart tissue. Heart and vessels 2014; 29(6): 855.

Chen A, Ashburn MA. Cardiac effects of opioid therapy. Pain Medicine 2015; 16(suppl_1): S27-S31.

Stanley TH, Gray NH, Stanford W, Armstrong R. The Effects of High–dose Morphine on Fluid and Blood Requirements in Open–heart Operations. Anesthesiology 1973; 38(6): 536-541.

Skrabalova J, Drastichova Z, Novotny J. Morphine as a potential oxidative stress-causing agent. Mini-reviews in organic chemistry 2013; 10(4): 367-372.

Caldwell JR, Rapoport RJ, Davis JC et al. Efficacy and safety of a once-daily morphine formulation in chronic, moderate-to-severe osteoarthritis pain: results from a randomized, placebo-controlled, double-blind trial and an open-label extension trial. Journal of pain and symptom management 2002; 23(4): 278-291.

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The international journal of biochemistry & cell biology 2007; 39(1): 44-84.

Patel VP, Chu CT. Nuclear transport, oxidative stress, and neurodegeneration. International journal of clinical and experimental pathology 2011; 4(3): 215.

Zhu H, Jia Z, Misra H, Li YR. Oxidative stress and redox signaling mechanisms of alcoholic liver disease: updated experimental and clinical evidence. Journal of digestive diseases 2012; 13(3): 133-142.

Jimenez-Del-Rio M, Velez-Pardo C. The bad, the good, and the ugly about oxidative stress. Oxidative medicine and cellular longevity 2012; 2012.

Rezazadeh H, Kahnouei MH, Hassanshahi G et al. Regulatory effects of chronic low-dose morphine on nitric oxide level along with baroreflex sensitivity in two-kidney one-clip hypertensive rats. Iranian journal of kidney diseases 2014; 8(3): 194.

Zhang YT, Zheng QS, Pan J, Zheng RL. Oxidative damage of biomolecules in mouse liver induced by morphine and protected by antioxidants. Basic & clinical pharmacology & toxicology 2004; 95(2): 53-58.

í–zmen ć°, Nazıroğlu M, Alici HA, Åžahin F, Cengiz M, Eren I. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochemical research 2007; 32(1): 19-25.

Schinzel A, Kaufmann T, Borner C. Bcl-2 family members: integrators of survival and death signals in physiology and pathology [corrected]. Biochimica et biophysica acta 2004; 1644(2-3): 95-105.

Sharpe JC, Arnoult D, Youle RJ. Control of mitochondrial permeability by Bcl-2 family members. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2004; 1644(2): 107-113.

Donovan M, Cotter TG. Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 2004; 1644(2): 133-147.

Toshiyuki M, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995; 80(2): 293-299.

Li GY, Xie P, Li HY, Hao L, Xiong Q, Qiu T. Involment of p53, Bax, and Bcl"2 pathway in microcystins"induced apoptosis in rat testis. Environmental toxicology 2011; 26(2): 111-117.

Ali B, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytotherapy Research 2003; 17(4): 299-305.

Norouzi F, Abareshi A, Asgharzadeh F et al. The effect of Nigella sativa on inflammation-induced myocardial fibrosis in male rats. Research in pharmaceutical sciences 2017; 12(1): 74.

Iddamaldeniya SS, Thabrew M, Wickramasinghe S, Ratnatunge N, Thammitiyagodage MG. A long-term investigation of the anti-hepatocarcinogenic potential of an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra. Journal of carcinogenesis 2006; 5(1): 11.

Gali-Muhtasib HU, Kheir WGA, Kheir LA, Darwiche N, Crooks PA. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anti-cancer drugs 2004; 15(4): 389-399.

Gali-Muhtasib H, Diab-Assaf M, Boltze C et al. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. International journal of oncology 2004; 25(4): 857-866.

Shoieb AM, Elgayyar M, Dudrick PS, Bell JL, Tithof PK. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. International journal of oncology 2003; 22(1): 107-113.

Elbarbry F, Ragheb A, Marfleet T, Shoker A. Modulation of hepatic drug metabolizing enzymes by dietary doses of thymoquinone in female New Zealand White rabbits. Phytotherapy research 2012; 26(11): 1726-1730.

El"Mahdy MA, Zhu Q, Wang QE, Wani G, Wani AA. Thymoquinone induces apoptosis through activation of caspase"8 and mitochondrial events in p53"null myeloblastic leukemia HL"60 cells. International journal of cancer 2005; 117(3): 409-417.

Yin D, Mufson RA, Wang R, Shi Y. Fas-mediated cell death promoted by opioids. Nature 1999; 397(6716): 218-218.

Karkhah A, Ataee R, Ataie A. Morphine pre-and post-conditioning exacerbates apoptosis in rat hippocampus cells in a model of homocysteine-induced oxidative stress. Biomedical Reports 2017; 7(4): 309-313.

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(6): 296-306.

Jalili C, Salahshoor MR, Hoseini M, Roshankhah S, Sohrabi M, Shabanizadeh A. Protective Effect of Thymoquinone Against Morphine Injuries to Kidneys of Mice. Iranian Journal of Kidney Diseases 2017; 11(2).

Jalili C, Tabatabaei H, Kakaberiei S, Roshankhah S, Salahshoor MR. Protective role of Crocin against nicotine-induced damages on male mice liver. International journal of preventive medicine 2015; 6.

Stoops WW, Hatton KW, Lofwall MR, Nuzzo PA, Walsh SL. Intravenous oxycodone, hydrocodone, and morphine in recreational opioid users: abuse potential and relative potencies. Psychopharmacology 2010; 212(2): 193-203.

Flemming K. The use of morphine to treat cancer-related pain: a synthesis of quantitative and qualitative research. Journal of pain and symptom management 2010; 39(1): 139-154.

Asgary S, Sarrafzadegan N, Naderi G-A, Rozbehani R. Effect of opium addiction on new and traditional cardiovascular risk factors: do duration of addiction and route of administration matter? Lipids in health and disease 2008; 7(1): 42.

Miaskowski C. A review of the incidence, causes, consequences, and management of gastrointestinal effects associated with postoperative opioid administration. Journal of PeriAnesthesia Nursing 2009; 24(4): 222-228.

Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology: The Journal of the American Society of Anesthesiologists 2011; 115(6): 1363-1381.

Seiri L, Mokri A, Dezhakam H, Noroozi A. Using tincture of opium for treatment of opiate abusers in Iran. Drug & Alcohol Dependence 2014; 140: e200.

Sharifipour M, Izadpanah E, Nikkhoo B et al. A new pharmacological role for donepezil: attenuation of morphine-induced tolerance and apoptosis in rat central nervous system. Journal of biomedical science 2014; 21(1): 6.

Hassanzadeh K, Roshangar L, Habibi-asl B et al. Riluzole prevents morphine-induced apoptosis in rat cerebral cortex. Pharmacological Reports 2011; 63(3): 697-707.

Liu L-W, Lu J, Wang X-H, Fu S-K, Li Q, Lin F-Q. Neuronal apoptosis in morphine addiction and its molecular mechanism. International journal of clinical and experimental medicine 2013; 6(7): 540.

Hu S, Sheng WS, Lokensgard JR, Peterson PK. Morphine induces apoptosis of human microglia and neurons. Neuropharmacology 2002; 42(6): 829-836.

Bhat RS, Bhaskaran M, Mongia A, Hitosugi N, Singhal PC. Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation. Journal of leukocyte biology 2004; 75(6): 1131-1138.

Lin X, Wang YJ, Li Q et al. Chronic high"dose morphine treatment promotes SH"SY5Y cell apoptosis via c"Jun N"terminal kinase"mediated activation of mitochondria"dependent pathway. The FEBS journal 2009; 276(7): 2022-2036.

Ohara T, Itoh T, Takahashi M. Immunosuppression by morphine-induced lymphocyte apoptosis: is it a real issue? Anesthesia & Analgesia 2005; 101(4): 1117-1122.

Li R, Wong GT, Wong TM, Zhang Y, Xia Z, Irwin MG. Intrathecal morphine preconditioning induces cardioprotection via activation of delta, kappa, and mu opioid receptors in rats. Anesthesia & Analgesia 2009; 108(1): 23-29.

Salahshoor MR. Protective effect of crocin on liver toxicity induced by morphine. Research in pharmaceutical sciences 2016; 11(2): 120.

Jalili C, Ahmadi S, Roshankhah S, Salahshoor M. Effect of Genistein on reproductive parameter and serum nitric oxide levels in morphine-treated mice. International Journal of Reproductive BioMedicine 2016; 14(2): 95.

Gali-Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: a promising anti-cancer drug from natural sources. The international journal of biochemistry & cell biology 2006; 38(8): 1249-1253.

Nutten S, Philippe D, Mercenier A, Duncker S. Opioid receptors stimulating compounds (thymoquinone, nigella sativa) and food allergy. Google Patents; 2010.

Mahmoud AM, Ahmed OM, Galaly SR. Thymoquinone and curcumin attenuate gentamicin-induced renal oxidative stress, inflammation and apoptosis in rats. EXCLI journal 2014; 13: 98.

Alenzi F, El-Bolkiny YE-S, Salem M. Protective effects of Nigella sativa oil and thymoquinone against toxicity induced by the anticancer drug cyclophosphamide. British journal of biomedical science 2010; 67(1): 20-28.