The effects of casticin and myricetin on liver damage induced by methotrexate in rats

Document Type : Original Article


1 Department of Nutrition and Dietetics, Health School, Ağrı İbrahim Çeçen University, Ağrı, Turkey

2 Department of Pathology, Faculty of Veterinary, Atatürk University, Erzurum, Turkey

3 Department of Biochemistry, Faculty of Veterinary, Atatürk University, Erzurum, Turkey

4 Department of Chemistry, Faculty of Science, Atatürk University, Erzurum, Turkey

5 Department of Animal Health, School of Eleşkirt Celal Oruç, Ağrı İbrahim Çeçen University, Ağrı, Turkey


Objective(s): In this study, we evaluated the therapeutic effects of casticin and myricetin on liver  damage induced by methotrexate in rats.
Materials and Methods: Thirty-six male rats were used for the study and divided into 6 groups: control, methotrexate, casticin, myricetin, casticin+methotrexate, and myricetin+methotrexate. It was performed by methotrexate (20 mg/kg single dose, IP) in methotrexate, casticin+methotrexate and myricetin+methotrexate groups. Casticin 200 mg/kg dose was given to casticin and casticin+methotrexate groups. Myricetin 50 mg/kg dose was given to myricetin and myriceytin+methotrexate groups. At the end of the experiment, liver tissues were removed for the purpose of histopathological, biochemical and immunohistochemical assessments.
Results: In our study, we have detected that MDA levels increased and activities of antioxidant enzymes SOD, CAT, and GPX decreased in the methotrexate group compared to the other groups, but the level of MDA decreased and activities of these enzymes increased in casticin+methotrexate and myricetin+methotrexate groups compared to the methotrexate group. In immunohistochemical examinations of control, casticin and myricetin groups in liver tissues no caspase-3 and 8-OHdG expressions were observed. In the MTX group, caspase-3 and 8-OHdG expressions were seen at the severe levels. Caspase-3 and 8-OHdG expressions were mild in hepatocytes in the casticin+methotrexate and myricetin+methotrexate groups. When the liver tissues of the rats in the methotrexate group were examined, severe pathological damage was detected both in the parietal region and in the portal region.
Conclusion: By looking at these results, we can say that casticin and myricetin are effective against liver damage induced by methotrexate.


Main Subjects

1. Kane D, Gogarty M, O’Leary J, Silva I, Bermingham N, Bresnihan B, et al. Reduction of synovial sublining layer inflammation and proinflammatory cytokine expression in psoriatic arthritis treated with methotrexate. Arthritis Rheum 2004; 50:3286-3295.
2. Norris RE, Adamson PC. Clinical potency of methotrexate, aminopterin, talotrexin and pemetrexed in childhood leukemias. Cancer Chemoth Pharm 2010; 65:1125-1130.
3.    Widemann BC, Balis FM, Kempf-Bielack B, Bielack S, Pratt CB, Ferrari S, et al. High-dose methotrexate-induced nephrotoxicity in patients with osteosarcoma - Incidence, treatment, and outcome. Cancer-Am Cancer Soc. 2004; 100:2222-2232.
4.    Jahovic N, Cevik H, Sehirli AO, Yegen BC, Sener G. Melatonin prevents methotrexate-induced hepatorenal oxidative injury in rats. J Pineal Res. 2003; 34:282-287.
5.    Pesce C, Mansi C, Bogliolo G, Tobia F, Pannacciulli I. Pulmonary Toxicity in Mice after High-Dose Methotrexate Administration with and without Leucovorin Rescue. Eur J Cancer Clin On 1985; 21:875.
6.    Gouveia LDV, Cardoso CA, de Oliveira GMM, Rosa G, Moreira ASB. Effects of the Intake of Sesame Seeds (Sesamum indicum L.) and Derivatives on Oxidative Stress: A Systematic Review. J Med Food 2016; 19:337-345.
7.    Moghadam AR, Tutunchi S, Namvaran-Abbas-Abad A, Yazdi M, Bonyadi F, Mohajeri D, et al. Pre-administration of turmeric prevents methotrexate-induced liver toxicity and oxidative stress. Bmc Complem Altern M 2015; 15.
8.    Hemeida RA, Mohafez OM. Curcumin attenuates methotraxate-induced hepatic oxidative damage in rats. Journal of the Egyptian National Cancer Institute 2008; 20:141-148.
9.    Hafez HM, Ibrahim MA, Ibrahim SA, Amin EE, Goma W, Abdelrahman AM. Potential protective effect of etanercept and aminoguanidine in methotrexate-induced hepatotoxicity and nephrotoxicity in rats. Eur J Pharmacol 2015; 768:1-12.
10. Kandemir FM, Kucukler S, Caglayan C, Gur C, Batil AA, Gulcin I. Therapeutic effects of silymarin and naringin on methotrexate-induced nephrotoxicity in rats: Biochemical evaluation of anti-inflammatory, antiapoptotic, and antiautophagic properties. J Food Biochem 2017; 41:e12398.
11. Kobayakawa J, Sato-Nishimori F, Moriyasu M, Matsukawa Y. G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Viticis Fructus (Vitex rotundifolia Linne fil.). Cancer Lett 2004; 208:59-64.
12. He LH, Yang XH, Cao XC, Liu F, Quan MF, Cao JG. Casticin induces growth suppression and cell cycle arrest through activation of FOXO3a in hepatocellular carcinoma. Oncol Rep 2013; 29:103-108.
13. Lee H, Jung KH, Lee H, Park S, Choi W, Bae H. Casticin, an active compound isolated from Vitex Fructus, ameliorates the cigarette smoke-induced acute lung inflammatory response in a murine model. Int Immunopharmacol 2015; 28:1097-1101.
14. Xu Y, Xie Q, Wu SH, Yi D, Yu Y, Liu SB, et al. Myricetin induces apoptosis via endoplasmic reticulum stress and DNA double-strand breaks in human ovarian cancer cells. Mol Med Rep 2016; 13:2094-2100.
15. Yang C, Lim W, Bazer FW, Song G. Myricetin suppresses invasion and promotes cell death in human placental choriocarcinoma cells through induction of oxidative stress. Cancer Lett 2017; 399:10-19.
16. Arslan Ö. Investigation Of The Effect Of Trans-3- Hydroxycinnamic Acid Against Liver Damage Induced-Methotrexate In Rats. Enstitute of Science: Ağrı İbrahim Çeçen University 2016.
17. Ling Y, Zhu JY, Fan MS, Wu B, Qin LP, Huang CG. Metabolism studies of casticin in rats using HPLC-ESI-MSn. Biomed Chromatogr 2012; 26:1502-1508.
18. Sun Y, Lian M, Lin Y, Xu B, Li Y, Wen J, et al. Role of p-MKK7 in myricetin-induced protection against intestinal ischemia/reperfusion injury. Pharmacol Res 2018; 129:432-442.
19. Placer ZA, Cushmanni LL, Johnson BC. Estimation of products of lipid peroxidation (as malondialdehyde) in biochemical systems. Analytic Biochem 1966;16:359-64.
20. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide-dismutase. Clin Chem 1988; 34:497-500.
21. Aebi H. Catalase Invitro. Method Enzymol 1984; 105:121-126.
22. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968; 24:192-205.
23. Lawrence RA, Burk RF. Glutathione Peroxidase Activity In Selenium-Deficient Rat Liver. 1976. Biochem Biophys Res Commun 2012; 425:503-509.
24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. The Journal of biological chemistry 1951; 193:265-275.
25. Devrim E, Ccedil;etin R, Kilicoglu B, Erguder BI, Avci A, Durak I. Methotrexate causes oxidative stress in rat kidney tissues. Renal Failure 2005; 27:771-773.
26. Uz E, Oktem F, Yilmaz H, Uzar E, Ozguner F. The activities of purine-catabolizing enzymes and the level of nitric oxide in rat kidneys subjected to methotrexate: Protective effect of caffeic acid phenethyl ester. Mol Cell Biochem 2005; 277:165-170.
27. Gulcin I. Antioxidant activity of food constituents: an overview. Arch Toxicol 2012; 86:345-391.
28. Halliwell B. Free-Radicals, Antioxidants, and human-disease-curiosity, cause, or consequence. Lancet 1994; 344:721-724.
29. Asci H, Ozmen O, Ellidag HY, Aydin B, Bas E, Yilmaz N. The impact of gallic acid on the methotrexate-induced kidney damage in rats. J food drug anal 2017; 25:890-897.
30. Mehrzadi S, Fatemi I, Esmaeilizadeh M, Ghaznavi H, Kalantar H, Goudarzi M. Hepatoprotective effect of berberine against methotrexate induced liver toxicity in rats. Biomedi pharmacother 2017; 97:233-239.
31. Vardi N, Parlakpinar H, Ates B, Cetin A, Otlu A. Antiapoptotic and antioxidant effects of beta-carotene against methotrexate-induced testicular injury. Fertil steril. 2009;92:2028-2033.
32. Feng Y, Siu KY, Ye X, Wang N, Yuen MF, Leung CH, et al. Hepatoprotective effects of berberine on carbon tetrachloride-induced acute hepatotoxicity in rats. Chin med 2010; 5:33.
33. Gulcin I, Huyut Z, Elmastas M, Aboul-Enein HY. Radical scavenging and antioxidant activity of tannic acid. Arab J Chem 2010; 3:43-53.
34. Tohma HS, Gulcin I. Antioxidant and radical scavenging activity of aerial parts and roots of turkish liquorice (Glycyrrhiza glabra L.). Int J Food Prop 2010; 13:657-671.
35. Gulcin I. Antioxidant properties of resveratrol: A structure-activity insight. Innov Food Sci Emerg 2010; 11:210-218.
36. Gulcin I. Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa. Amino Acids 2007; 32:431-438.
37. Gulcin I, Elmastas M, Aboul-Enein HY. Determination of antioxidant and radical scavenging activity of basil (Ocimum basilicum L. family Lamiaceae) assayed by different methodologies. Phytother Res 2007; 21:354-361.
38. Circu ML, Aw TY. Redox biology of the intestine. Free Radical Res 2011; 45:1245-1266.
39. Ekinci-Akdemir FN, Gulcin I, Gursul C, Alwasel SH, Bayir Y. Effect of P-coumaric acid against oxidative stress induced by cisplatin in brain tissue of rats. J Anim Plant Sci 2017; 27:1560-1564.
40. Gul M, Kutay FZ, Temocin S, Hanninen O. Cellular and clinical implications of glutathione. Indian J Exp Biol 2000; 38:625-634.
41. Oktay M, Yildirim A, Bilaloglu V, Gulcin I. Antioxidant activity of different parts of isgin (Rheum ribes L.). Asian J Chem 2007; 19:3047-3055.
42. El-Sharaky AS, Newairy AA, Badreldeen MM, Eweda SM, Sheweita SA. Protective role of selenium against renal toxicity induced by cadmium in rats. Toxicol 2007; 235:185-193.
43. Khalifa MMA, Bakr AG, Osman AT. Protective effects of phloridzin against methotrexate-induced liver toxicity in rats. Biomed Pharmacother 2017; 95:529-535.
44. Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts. J Gastroen Hepatol 2011; 26:173-179.
45. Ali N, Rashid S, Nafees S, Hasan SK, Sultana S. Beneficial effects of chrysin against methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis. Mol Cell Biochem 2014; 385:215-223.
46. El-Sheikh AAK, Morsy MA, Abdalla AM, Hamouda AH, Alhaider IA. Mechanisms of thymoquinone hepatorenal protection in methotrexate-induced toxicity in rats. Mediat Inflamm 2015; 859383
47. Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science 1998; 281:1312-1316.
48. Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH. Screening and detection of apoptosis. J Surg Res 2007; 139:143-156.
49. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997; 91:479-489.
50. Chang CJ, Lin JF, Chang HH, Lee GA, Hung CF. Lutein protects against methotrexate-induced and reactive oxygen species-mediated apoptotic cell injury of IEC-6 cells. Plos One 2013; 8: e72553.
51. Ali N, Rashid S, Nafees S, Hasan SK, Shahid A, Majed P, et al. Protective effect of Chlorogenic acid against methotrexate induced oxidative stress, inflammation and apoptosis in rat liver: An experimental approach. Chem-Biol Interact 2017; 272:80-91.
52. Breen AP, Murphy JA. Reactions of oxyl radicals with DNA. Free Radical Bio Med 1995; 18:1033-1077.
53. Beckman KB, Ames BN. Oxidative decay of DNA. J Bio Chem 1997; 272:19633-19636.
54. Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G -> T and a -> C substitutions. J Bio Chem 1992; 267:166-172.
55. Sova H, Jukkola-Vuorinen A, Puistola U, Kauppila S, Karihtala P. 8-Hydroxydeoxyguanosine: a new potential independent prognostic factor in breast cancer. Brit J Cancer 2010; 102:1018-1023.
56. Radak Z, Boldogh I. 8-Oxo-7,8-dihydroguanine: Links to gene expression, aging, and defense against oxidative stress. Free Radical Bio Med 2010; 49:587-596.
57. Long JD, Matson WR, Juhl AR, Leavitt BR, Paulsen JS, Investigators P-H. 8OHdG as a marker for Huntington disease progression. Neurobiol Dis 2012; 46:625-634.
58. Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta 2004; 339:1-9.
59. Lindsay K, Fraser AD, Layton A, Goodfield M, Gruss H, Gough A. Liver fibrosis in patients with psoriasis and psoriatic arthritis on long-term, high cumulative dose methotrexate therapy. Rheumatology 2009; 48:569-72.