Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury DOI: https://doi.org/10.31351/vol30iss2pp208-213 208 Safranal Effect against Cyclophosphamide-Induced Liver Injury Victoria S. Kareem* and Munaf H. Zalzala**,1 * Ministry of Health and Environment, Babil, Iraq ** Department of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad ,Iraq. Abstract The liver is the primary organ for drug metabolism. Cyclophosphamid is a nitrogen mustard-based alkylating agents, its metabolized by the liver into two cytotoxic metabolites, causing liver toxicity by increasing reactive oxygen species. Safranal as the most abundant chemical in saffron essential oil, it has anti-oxidant, anti- inflammatory, antiapoptic and free radical scavenger activity. The aim of study is to assess the protective effects of safranal on the cyclophosphamide-induce liver toxicity in rat model. This is investigated by using five different groups of rats; normal control group received the vehicles; model group received a single dose of cyclophosamide (150 mg/kg, i.p). The other two groups received orally safranal at doses of 50 and 100 mg/kg/day, respectively, for 8 consecutive days prior cyclophosamide injection (at day fifth). The last group received only oral safranol at dose 100 mg/kg/day for 8 consecutive days. This study showed safrenal-pretreatment significantly ameliorated the deterioration of liver function and exerted significant anti-oxidant with a marked decline in malondialdehyde level, and increase in glutathione and nuclear factor erythroid 2-related factor 2 levels in a dose dependent-manner. Keywords: Liver toxicity, Cyclophosamide, Safranal, Glutathione, Malondialdehyde السمية الكبدية الناتجة من استخدام مادة السايكلوفوسامايد ضدالسافرنال تاثير 1**،همناف هاشم زلزلو *فكتوريا سعد كريم وزارة الصحة والبيئة ، دائرة صحة بابل، بابل، العراق . * اقكلية الصيدلة، جامعة بغداد، العرفرع االدوية والسموم، ** الخالصة التي تنشط بواسطة انزيمات الكبد هو العضو الرئيسي للعمليات االيضية للجسم ,السايكلوفوسامايد هو من اقدم انواع العالجات السرطانية ساسية من الكبد لتنتج مادة االكرولين عالية السمية والفوسفوامايد ماسترد لتزيد االكسدة داخل الجسم ,السافرانال هو مستخلص الزيوت العطرية اال تلقت المجموعة النموذجية جرعة ذي ,في هذه الدراسة تلقت مجموعة الكونتروال فقط الماء المغالزعفران ويعمل كمضاد اكسدة ومضاد التهابات , يوم، على /كغم /ملغم 100و 50تلقت المجموعتان األخريان السفرنال عن طريق الفم بجرعات (. كجم /مجم 150)واحدة من السيكلوفوساميد /مجم 100افرانول عن طريق الفم بجرعة تلقت المجموعة األخيرة فقط س(. في اليوم الخامس)أيام متتالية قبل حقن سيكلوفوساميد 8التوالي، لمدة أظهرت هذه الدراسة أن المعالجة المسبقة للسفرينال خففت بشكل كبير من تدهور وظائف الكبد وأظهرت ارتفاع في مضادات أكسدة مع . يوم /كجم .بطريقة تعتمد على الجرعة 2بط باإلريثرويد المرت 2انخفاض ملحوظ في مستوى مالونديلديهيد ، وزيادة في مستويات الجلوتاثيون والعامل النووي . مالونديلديهيد ، سافرنال، لسايكلوفوسامايد ، الكبدية السمية :الكلمات المفتاحية Introduction The liver is a critical organ for metabolism and maintain homeostasis in general body function. It also acts as a source of nutrients and a detoxifier of undesirable substances (1) . Cyclophosphamid (CP), is one of the earliest chemotherapy drugs that still used today to treat many forms of cancers, CP is a prodrug, it is converted in the liver into acrolein and phosphoramide mustard by the Cytochrome P450 system. Many previoes studies indicated that during its oxidative metabolism, CP produces reactive oxygen species (ROS), and suppresses the antioxidant protection mechanisms of the liver (2) . Acrolein could easily react with glutathione (GSH), a thiol-containing protein that serves many essential functions including detoxification. When GSH was depleted, acrolein could react with cellular nucleophiles, such as the thiol groups of cysteine residues in the proteins and the atoms of nitrogen in lysine and histidine, causing protein loss, thus inducing oxidative stress and eventually leading to catastrophic hepatocyte effects (3) .CP interferes with liver oxidant/antioxidant balance, which contributes to reactive oxygen (ROS) accumulation, influencing lipid peroxidation and signaling of cytotoxicity pathways (4) .Free radicals-induced lipid peroxidation change membrane structure and function. they are also involved in cell defects including mutation and cell death.(5) Oxidative stress triggers multiple intracellular signals leading to pro- inflammatory cytokine up-regulation.(6). Moreover, many recent studies found that the oxidative stress, inflammation, and apoptosis are the main pathways leading to CP hepatotoxicity (7). 1Corresponding author E-mail: munafzalzala@copharm.uobaghdad.edu.iq Received: 6/3/2021 Accepted: 6/6/2021 Published Online First: 2021-12-11 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol30iss2pp208-213 Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury 209 Glutathione is a thiol-containing tripeptide that helps to minimize oxidative stress by providing a major non-enzymatic endogenous antioxidant. The liver has high glutathione levels because of its active synthesis (8). Nuclear related factor 2 (Nrf2), is a transcription factor, which is a highly preserved archaic protein that can orchestrate and balance an intracellular oxidative stress response. Several studies have shown that the fundamental mechanism of activation Nrf2 is based on their negative regulator of kelch-like ECH-associated protein 1. Nrf2 binds to the antioxidant cisactant (ARE) reaction element that controls the expression of detoxifying and antioxidant enzymes. Nrf2 -stress dependent induction results in induction in the transcription of many antioxidant enzymes directly (9), 10) . Most abundant chemicals in saffron plant oils is Safranal (2,6,6-trimethyle 1, 3- cyclohexadien-1-carbox-aaldehyde). that is composed of 60–70% of dry saffron's volatile fraction. (11), Various research groups have documented medicinal properties of safranal with anti-oxidant, anticonvulsant, cardioprotective, anti- nociceptive, anti-cancer, anti-inflammatory effect and others. The majority of these therapies may be attributed to their ability to quench ROS and to minimize the body's oxidative stress.(12) Safranal modulates the expression of antioxidant genes and controls mitochondrial antioxidant genes, which produces a less radical mitochondrial oxygen capacity. Inflammatory and immune system disease could be effectively affected by safranal, modulating pro-inflammatory cytokines, stress oxidative and immune factors. (13, 14) further, safranal lowered inflammation and decreased TNF-α level, possibly due to its high antioxidant and anti-apoptotic capacity (15, 16) . Aim of study The study designed to assess the defensive impact of safranal against liver toxicity induced by cyclophosamide Materials and Methods Cyclophosphamide was purchased from Baxter Oncology (Frankfurt, Germany). MDA and glutathione rat Elisa kit were purchased from SunLong Biotech,NRF2elisa kit purchased from mybiosource co. ALT and AST kits were purchased from Randox laboratories (united kingdom),safranal purchased from sigma Aldrich (india) Animals Rats of Wistar were picked from the animal house of the Pharmacology and Toxicology Department of the Collage of Pharmacy at University of Baghdad. Adult albino male rat'sweighing (150-250) gm. Animals were randomly allocated into five groups (6 rats each). These rats are housed in a temperature- regulated room at 25 ± 2 Cº, 50-70 percent humidity and 12 hours of lighting period under probable hygienic conditions. All rats are fed during the experiments with standard pellets and tap water. Animals are weighed and their initial weights were registered after 2 weeks of acclimatization, and then weighted every 2 days. The rats are divided into five group as the following: Group I (Control):Six rats received liquid paraffin orally for 8 days and normal saline intraperitoneal (I.P) in the fifth day(3 days before the end of the experiment). Group II (Model group): six rats received cyclophosphamide 150 mg/kg intraperitoneal (I.P) in the fifth day (3 days before the end of the experiment). Group III: six rats received safranal orally in a dose of 50 mg/kg/day consecutively for 8 days and cyclophosphamide 150 mg/kg intraperitoneal (I.P) in the fifth day. Group IV: six rats received safranal orally in a dose of 100 mg/kg/day consecutively for 8 days and cyclophosphamide 150 mg/kg intra peritoneal (I.P) in the fifth day. Group V: six rats received safranal orally in a dose of 100 mg/kg/day consecutively for 8 days Biochemical evaluation Blood sample is obtained from animal by heart puncture after anesthetized using diethyl ether, the sample left half hour at room temperature then take serum after centrifuging for10minunt at 10000 rpm and stored at -20 °C for determination of liver enzyme, Alanine transaminase activity was measured by colorimetric method Determination of NRF2, MDA, Glutathione level in homogenizing tissue A 0.1gm of liver tissue was homogenate using cold phosphate buffer solution (10%w\v) after centrifuging then keep the supernatant at-20 °. Estimation of NRF2, MDA ,glutathione levels in rat tissues by using the enzyme-linked immuno-sorbent assay (ELISA) kit according to Sandwich-ELISA principle, at 37°C and Read the Optical Density (O.D.) at 450 nm. Statistical analysis All the values are presented as means ±standard error of the means (SEM) of all experiments. Comparisons between different groups were carried out using one-way analysis of variance (ANOVA) The difference was considered significant when P ˂ 0.05. SPSS software was used to carry out these statistical tests. Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury 210 Results Effects of safranal on serum liver microsomal enzymes in CP-induced hepatotoxicity in rats. Cyclophosphamide (CP, 150 mg/kg, I.P.) resulted in acute liver damage in rats as evidenced by the significant elevation of serum alanine transaminase (ALT) and aspartate transaminase (AST), as compared to the normal control group. (Table 1 and 2 ,respectively). Table 1. level of ALT level among various groups Groups Serum ALT level (U/L) Control 18.96 ± 2.16 CP 40.42 ± 3.97 * CP/Safranal 50mg/kg 36.21 ± 5.27 * CP/Safranal 100mg/kg 23.14 ± 2.09 # safranal 100mg/kg 19.06 ± 1.35 # All values are expressed as mean ±SD * compared with control group, significant different (p<0.05). # compared with model CP group significant different, (p<0.05). Table 2. AST level among various groups Groups Serum AST level (U/L) Control 77.78 ± 22.09 CP 169.96 ± 47.39 * CP/Safranal 50mg/kg 122.66 ± 34.35 CP/Safranal 100mg/kg 80.78 ± 12.88 # safranal 100mg/kg 78.83 ± 16.49 # All values are expressed as mean ±SD * compared with control group, significant different (p<0.05). # compared with model CP group significant different, (p<0.05). Effect of safranal on hepatic NRF2 and glutathione level in CP-induced hepatotoxicity in rats. CP induced acute liver damage in rats as evidenced by an decrease in hepatic NRF2 and glutathione content as compared to the normal control group. Pretreatment of rats with safranal (50 and 100 mg/kg/day, oral) for five days prior CP injection significantly inhibit the reduction in hepatic NRF2 and glutathione in a dose dependent manner compared to the CP-control group, recording normal levels of NRF2 and glutathione (Table 3 and 4 ,respectively). Table 3. Impact of safranal on NRF2 level among various groups Groups Hepatic NRF2 level (ng/0.1gm) Control 112.95 ± 13.21 CP 66.28 ± 7.98 * CP/Safranal 50mg/kg 120.4 ± 26.78 # CP/Safranal 100mg/kg 138.82 ± 36.18 # safranal 100mg/kg 143.36 ± 20.06 # All values are expressed as mean ±SD * compared with control group, significant different (p<0.05). # compared with model CP group significant different, (p<0.05). Table 4. Effect of safranal on glutathione level among various groups Groups GSH level (ng /0.1g) Control 69.26 ± 14.43 CP 35.74 ± 11.0 * CP/Safranal 50mg/kg 73.7 ± 16.02 # CP/Safranal 100mg/kg 82.31 ± 27.77 # safranal 100mg/kg 71.84 ± 10.75 # All values are expressed as mean ±SD *Different significant variations relative to the control group (p<0.05). #Different significant variations relative to the model group (p<0.05) . Effect of safranal on hepatic MDA in CP-induced hepatotoxicity in rats. Cyclophosphamide (CP, 150 mg/kg, i.p.) resulted in acute liver damage in rats as evidenced by a significant increase in hepatic MDA as compared to the normal control group. Pretreatment of rats with safranal, at 50 and 100 mg/kg, significantly reduced the elevation in liver MDA in a dose-dependent manner compared to the CP control group, recording normal levels of MDA dose with insignificant difference from normal control group (Table 5). ). Compared to the control group, there is no statistical difference between the safranal group treated alone (P>0.05). Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury 211 Table 5 . Effect of safranal on MDA level among various groups Groups MDA level (ng/0.1g) Control 38.39 ± 8.06 CP 56.02 ± 9.14 * CP/Safranal 50mg/kg 51.56 ± 6.84 * CP/Safranal 100mg/kg 43.19 ± 3.16 # safranal 100mg/kg 45.79 ± 8.4 All values are expressed as mean ±SD *Different significant variations relative to the control group (p<0.05). #Different significant variations relative to the model group (p<0.05) . Discussion Cyclophosphamide (CP) is one of the universally used antineoplastic drugs due to its beneficial effects on various cases of cancer, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, CP is clinically limited because of adverse effects and toxicity.(18, 17) Even at low doses of CP can produce significant hepatotoxicity in humans which often stands as a barrier against its clinical use. CP-induced toxicity is known to have been associated with biochemical pathogenesis with inflammatory cascading and oxidative stress across generations of tissue inflammatory cytokines and free radicals.(19) The degree of CP induced hepatotoxicity has been correlated with elevated levels of AST and ALT in patients.(20-22). Consequently, It is quite important to develop alternative and safe natural products against liver damage caused by hepatotoxins such as CP. The hepatoprotective agent should restore the normal structure of the liver and protect the normal physiological mechanisms damaged by hepatotoxins. Several studies were conducted to define the biological and pharmacological activities of safranal. In the present study, the hepatoprotective, antioxidative and anti- inflammatory properties of safranal against oxidative stress and inflammation in CP induced liver damage were investigated. It was reported that safranal stabilized bio- membranes in biological systems and reduced unsaturated membrane lipids and reactive oxygen species, effect of safranal on enhanced serum enzymes prevent infiltration of intracellular enzymes.(13) This is consistent with the commonly accepted view that, with the recovery of the hepatic parenchyma and hepatocyte regeneration, serum transaminase levels return to normal.(23) CP therapy lowers the amount of nuclear Nrf2 as well as NQO-1 and HO-1 (heme oxygenase-1) expression. The decreased in the level of nuclear Nrf2 and the subsequent reduction in enzymes NQO-1 and HO-1 that may be due to an increase in the level of MAP kinase p38(24) . MAPK P38 can phosphorylate Nrf2 has been reported to promote the interaction between Nrf2 and Keap1 proteins, potentially inhibiting Nrf2 nuclear translocation and ultimately preventing HO-1 expression.(24) Further research found that safranal inhibited kelch- like ECH-associated protein, 1 (Keap1) expression and promoted elevation in Nrf2 translocation. Meanwhile, downstream Nrf2 antioxidant enzyme genes including glutathione S transferase (GST), glutamate-cysteine ligase catalytic subunit (GCLc), NADPH-quinone oxidoreductase 1 (NQO1) and glutathione S transferase (GST), NADPH-quinone oxidoreductase 1 (NQO1) were also induced by safranal.(26) CyP is activated by cytochrome P450, yielding phosphoramide mustard and acrolein. Acrolein inhibits P-450 by alkylating the groups of sulf- hydryl. Acrolein is metabolized mainly by rapid modification of the groups of glutathione sulfhydryl (GSG), forming mer-capturic acid that is removed in the urine. Acrolein directly raises cellular oxidative stress through this process by reducing glutathione levels.(27, 28) CP-induced GSH depletion. Not only does Acrolein interact with GSH, but also with cysteine, one of GSH's constituent amino acids.(29) Safranal has been reported to modulate the expression of antioxidant genes and regulate mitochondrial antioxidant genes, providing a lower radical potential for mitochondrial oxygen.(13) The increase in the cellular GSH content of, which may increase the GSH/GSSG ratio and decrease lipid peroxidation due to the effects of safranal.(30) The CP oxidative product responsible for the induction of LPO and the ROS under inflammation which will attack normal tissue and disrupt redox cycle, raising LPO(31) and significantly enhance the degree of lipid peroxidation in experimental animal livers with MDA as the most popular breakdown product.(32) Moreover, safranal exhibited concentration dependent radical scavenging property as proved by its action of donating a hydrogen atom to the diphenylpicryl hydrazine (DPPH) radical (33) It also protected cell membrane polyunsaturated fatty acids from damage and promote hepatic cell regeneration. This is revealed by their impact on the LPO terminal product.(34) Conclusion In conclusion, safranal is a phytochemical known for its antioxidative properties. This study showed that cyclophosphamide led to the development of or increased lipid peroxidation, free radicals, resulting in hepatic injury. Safrenal- pretreatment significantly ameliorate the deterioration of liver function and exerted significant anti-oxidant with a marked decline in Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury 212 malondialdehyde level, and increase in glutathione and nuclear factor erythroid 2-related factor 2 levels in a dose dependent-manner. References 1. K. Gulati, M. R. Reshi, N. Rai, and A. Ray, “Hepatotoxicity : Its Mechanisms , Experimental Evaluation and Protective Strategies,” Am. J. Pharmacol.,2018; vol. 1, no. 1, pp. 1–9. 2. E. R. Delay, S. H. Socia, J. L. Girardin, B. C. Jewkes, J. H. King, and R. J. Delay, “Cyclophosphamide and the taste system: Effects of dose fractionation and amifostine on taste cell renewal,” PLoS One, 2019; vol. 14, no. 4, pp. 1–23. 3. Zhu, H., Long, MH., Wu, J. et al. Ginseng alleviates cyclophosphamide-induced hepatotoxicity via reversing disordered homeostasis of glutathione and bile acid. Sci Rep 5, 17536 (2015). 4. S. R. Fahmy, A. I. Amien, F. M. Abd-Elgleel, and S. M. Elaskalany, “Antihepatotoxic efficacy of Mangifera indica L. polysaccharides against cyclophosphamide in rats,” Chem. Biol. Interact.,2015; vol. 244, pp. 113–120, 2016. 5. E. Olayinka, A. Ore, O. Ola, and O. Adeyemo, “Ameliorative Effect of Gallic Acid on Cyclophosphamide-Induced Oxidative Injury and Hepatic Dysfunction in Rats,” Med. Sci.,2015; vol. 3, no. 3, pp. 78–92. 6. S. H. Aladaileh et al., “Galangin activates Nrf2 signaling and attenuates oxidative damage, inflammation, and apoptosis in a rat model of cyclophosphamide-induced hepatotoxicity,” Biomolecules,2019; vol. 9, no. 8, pp. 1–19. 7. H. Hosseinzadeh and H. R. Sadeghnia, “Safranal, a constituent of Crocus sativus (saffron), attenuated cerebral ischemia induced oxidative damage in rat hippocampus,” J. Pharm. Pharm. Sci.,2005;vol. 8, no. 3, pp. 394– 399. 8. Shang Y, Siow YL, Isaak CK, O K. Downregulation of Glutathione Biosynthesis Contributes to Oxidative Stress and Liver Dysfunction in Acute Kidney Injury. Oxid Med Cell Longev. 2016;2016:9707292. 9. Z. Zeng et al., “Nuclear factor erythroid 2 ( NF- E2 ) -related factor 2 ( Nrf2 ) in non-small cell lung cancer,”2020; vol. 254. 10. W. Y. Abdelzaher, A. H. S. AboBakr Ali, and N. F. G. El-Tahawy, “Mast cell stabilizer modulates Sirt1/Nrf2/TNF pathway and inhibits oxidative stress, inflammation, and apoptosis in rat model of cyclophosphamide hepatotoxicity,” Immunopharmacol. Immunotoxicol.,2020; vol. 42, no. 2, pp. 101– 109. 11. S. I. Bukhari, M. Manzoor, and M. K. Dhar, “A comprehensive review of the pharmacological potential of Crocus sativus and its bioactive apocarotenoids,” Biomed. Pharmacother.,2018; vol. 98, no. December 2017, pp. 733–745. 12. Y. Xue et al., “Safranal, an active constituent of saffron, ameliorates myocardial ischemia via reduction of oxidative stress and regulation of Ca2+ homeostasis,” J. Pharmacol. Sci.,2020; no. xxxx, pp. 0–8. 13. Alayunt ÖN, Aksoy L, Karafakioğlu YS, Sevimli S. Assessment of Anti-inflammatory and Antioxidant Properties of Safranal on CCI4-Induced Oxidative Stress and Inflammation in Rats. An Acad Bras Cienc. 2019 May 23;91(2):e20181235. 14. P. A. SOUTHORN and G. POWIS, “Free Radicals in Medicine. I. Chemical Nature and Biologic Reactions,” Mayo Clin. Proc.,1988; vol. 63, no. 4, pp. 381–389. 15. H. R. Sadeghnia, H. Shaterzadeh, F. Forouzanfar, and H. Hosseinzadeh, “Neuroprotective effect of safranal, an active ingredient of Crocus sativus, in a rat model of transient cerebral ischemia,” Folia Neuropathol.,2017; vol. 55, no. 3, pp. 206–213. 16. C. Zhang et al., “Neuroprotective effects of safranal in a rat model of traumatic injury to the spinal cord by anti-apoptotic, anti- inflammatory and edema-attenuating,” Tissue Cell, 2015; vol. 47, no. 3, pp. 291–300. 17. W. Jiang et al., “Magnesium isoglycyrrhizinate shows hepatoprotective effects in a cyclophosphamide-induced model of hepatic injury,” Oncotarget, 2017; vol. 8, no. 20, p. 33252. 18. M. Shokrzadeh, A. Ahmadi, F. Naghshvar, A. Chabra, and M. Jafarinejhad, "Prophylactic Efficacy of Melatonin on Cyclophosphamide- Induced Liver Toxicity in Mice", BioMed Research International, vol. 2014. 19. Y. Song et al., “Ferulic Acid against Cyclophosphamide-Induced Heart Toxicity in Mice by Inhibiting NF- k B Pathway,” 2016. 20. M. Zarei and T. Shivanandappa, “Amelioration of cyclophosphamide-induced hepatotoxicity by the root extract of Decalepis hamiltonii in mice,” Food Chem. Toxicol., 2013; vol. 57, pp. 179–184. 21. H. A. Ghafor, “Low-dose cyclophosphamide- induced acute hepatotoxicity,” 2013; pp. 345– 349. 22. M. L. Bajt, J. A. Lawson, S. L. Vonderfecht, J. S. Gujral, and H. Jaeschke, “Protection against Fas Receptor – Mediated Apoptosis in Hepatocytes and Nonparenchymal Cells by a Caspase-8 Inhibitor in Vivo : Evidence for a Postmitochondrial Processing of Caspase-8,” 2000; vol. 117, pp. 109–117. 23. M. I. Thabrew, P. D. T. M. Joice, and W. Rajatissa, “A Comparative Study of the Efficacy of Pavetta indica and Osbeckia Iraqi J Pharm Sci, Vol.30(2) 2021 Safranal effect on cyclophosphamide liver injury 213 octandra in the Treatment of Liver Dysfunction,” 1986; pp. 239–241. 24. Y. Keum, S. Yu, and P. P. Chang, “Mechanism of Action of Sulforaphane : Inhibition of p38 Mitogen-Activated Protein Kinase Isoforms Contributing to the Induction of Antioxidant Response Element − Mediated Heme Oxygenase-1 in Human Hepatoma HepG2 Cells Mechanism of Action of Sulforaphane :,” 2006. 25. W. Tu, H. Wang, S. Li, Q. Liu, and H. Sha, “The Anti-Inflammatory and Anti-Oxidant Mechanisms of the Keap1 / Nrf2 / ARE Signaling Pathway in Chronic Diseases,” 2019; vol. 10, no. 3, pp. 637–651. 26. X. Wen,L-Y. Qiao2#, X-N Wen Safranal prevents rotenone-induced oxidative stress and apoptosis in an in vitro model of Parkinson's disease through regulating Keap1/Nrf2 signaling pathway “Cellular and Molecular Biology,” 2016; vol. 62, no. 65, pp. 11–17. 27. M. K. Mohammad, D. Avila, J. Zhang, S. Barve, and G. Arteel, “Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress , mitochondrial dysfunction and oxidative stress,” Toxicol. Appl. Pharmacol., 2012 vol. 265, no. 1, pp. 73–82. 28. G. Cuce et al., “Chemico-Biological Interactions Chemoprotective effect of vitamin E in cyclophosphamide-induced hepatotoxicity in rats,” Chem. Biol. Interact., 2015; vol. 232, pp. 7–11. 29. Kehrer JP, Biswal SS. The molecular effects of acrolein. Toxicol Sci. 2000 Sep;57(1):6-15.. 30. S. Samarghandian, R. Afshari, and A. Sadati, “Evaluation of lung and bronchoalveolar lavage fluid oxidative stress indices for assessing the preventing effects of safranal on respiratory distress in diabetic rats,” Sci. World J., 2014. 31. J.E. Biaglow, M.E. Varnes, E.R. Epp, E.P. Clark, Antioxidant and redox enzymes in radioprotection, Pharmacology & Therapeutics, Volume 39, Issues 1–3, 1988, Pages 275-286. 32. Haque R, Bin-Hafeez B, Parvez S, Pandey S, Sayeed I, Ali M, Raisuddin S. Aqueous extract of walnut (Juglans regia L.) protects mice against cyclophosphamide-induced biochemical toxicity. Hum Exp Toxicol. 2003 Sep;22(9):473-80. 33. Assimopoulou AN, Sinakos Z, Papageorgiou VP. Radical scavenging activity of Crocus sativus L. extract and its bioactive constituents. Phytother Res. 2005 Nov;19(11):997-1000. 34. Hussein RM, El-Hussieny EA, Hassanin LA, El-Sayed WM (2017) ‘The Prophylactic and Therapeutic Effects of Safranal and Selenite on Liver Damage Induced by Thyrotoxicosis in Adult Male Rats. Int J Clin Pharmacol Toxicol. 6(3), 270-279. Baghdad Iraqi Journal Pharmaceutical Sciences by bijps is licensed under a Creative Commons Attribution 4.0 International License. Copyrights© 2015 College of Pharmacy - University of Baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/