Protective effect of bioactive compounds from Echinophora cinerea against cisplatin-induced oxidative stress and apoptosis in the PC12 cell line

Document Type : Original Article


1 Pharmaceutical Sciences Research Center, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran

2 Student Research Committee, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran


Objective(s): The present study aims to evaluate the protective effect of the compounds isolated from Echinophora cinerea (E. cinerea) against oxidative stress and apoptosis induced by cisplatin (CIS) in PC12 cells.
Materials and Methods: Six compounds were isolated as quercetrin-3-O-β-D-glucopyranoside (QUE), osthol (OST), verbenone-5-O-β-D-glycopyranoside (VER), Isoimperatorin (ISO), kaempferol-3-O-β-D-glucopyranoside (KAM), and echinophorin B (ECH). For this study, we used MTT reduction assay for detection of protective effects of isolated compounds on CIS-induced cytotoxicity in PC12 cells. The effects of isolated compounds against apoptosis induced by CIS were investigated through the measurement of mitochondrial membrane potential (MMP), Bax and Bcl2 mRNA expression, and caspase-3 activation. We also assessed oxidative stress by measuring reactive oxygen species (ROS) generation with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA).
Results: Treatment of cells with QUE and OST before exposure to the CIS increased cell viability, i.e., these compounds protected the cells against CIS -induced cytotoxicity. In addition, pre-treatment with QUE and OST decreased CIS-induced apoptosis through up-regulation of Bcl-2, inhibition of caspase-3 activity, and mitochondrial membrane potential (MMP) increase. OST decreased ROS generation induced by CIS, as well.
Conclusion: Our in vitro experiment showed that QUE and OST are apoptotic inhibitors that effectively block CIS-induced neurotoxicity predicting their therapeutic potential in the prevention of chemotherapy-induced neurotoxicity.


1.  Gulec M,Oral E, Dursun OB,Yucel A,Hacimuftuoglu A,Akcay F. Mirtazapine protects against cisplatin‐induced oxidative stress and DNA damage in the rat brain. Physical CiNeuro Sci 2013;67 : 50-8.
2. Cavaletti G , PezzoniG, Pisano C, Oggioni N, Sala, F, Zoia C. Cisplatin-induced peripheral neurotoxicity in rats reduces the circulating levels of nerve growth factor. Neuro Sci Lett 2002;322:103-106.
3. Podratz JL,Knight AM, Ta LE,Staff NP, Gass JM, GenelinK. Cisplatin induced mitochondrial DNA damage in dorsal root ganglion neurons. Neuro  Biol  Dis 2011;41: 661-668.
4. Park SA,Choi KS,Bang JH, Huh K, Kim SU. Cisplatin‐Induced Apoptotic Cell Death in Mouse Hybrid Neurons Is Blocked by Antioxidants Through Suppression of Cisplatin‐Mediated Accumulation of p53 but Not of Fas/Fas Ligand. J Neuro  Chem 2000;75 : 946-953.
5. da Silva Machado C, Mendonca LM, de Paula Venancio V,Bianchi MLP, Antunes, LMG. Coenzyme Q10 protects Pc12 cells from cisplatin-induced DNA damage and neurotoxicity. Neuro  Toxicol 2013;36:10-16.
6. Karimi G, Ramezani M, Tahoonian Z, Cisplatin nephrotoxicity and protection by milk thistle extract in rats. Evid-BasComplementary Alt  Med 2005; 2:383-386.
7. Shokoohinia Y, Hosseinzadeh L, Moieni-Arya M, Mostafaie A, Mohammadi-Motlagh, H-R. Osthole Attenuates Doxorubicin-Induced Apoptosis in PC12 Cells through Inhibition of Mitochondrial Dysfunction and ROS Production. Bio Med Res Int 2014;383-386.
8. Mojarrab M, Mehrabi M, Ahmadi F, Hosseinzadeh L, Protective effects of fractions from Artemisia biennis hydroethanolic extract against doxorubicin- induced oxidative stress and apoptosis in PC12 cells. Iran J Basic Med  Sci 2016;19:803-810.
9. Mojarrab M, Nasseri S, Hosseinzadeh L, Farahani F, Evaluation of antioxidant and cytoprotective activities of Artemisia ciniformis extracts on PC12 cells.  Iran  J  Basic  Med Sci 2016; 19: 430-438.
10. Roleira FM, Tavares-da-Silva EJ, Varela CL, Costa SC, Silva T, Garrido J, Borges F, Plant derived and dietary phenolic antioxidants: anticancer properties. Food Chem 2015;183: 235-258.
11. Sajjadi SE, Ghannad A, Composition of the Essential Oil of Echinophora cinerea (Boiss.) Hedge et Lamond. J Essen Oil Res 2002;14:114-15.
12. GhasemiPirbalout,i A, Gholipou,r Z,Chemical composition, antimicrobial and antioxidant activities of essential oil from Echinophora cinerea harvested at two phenological stages. J Essent Oil Res 2016; 1-11.
13. Gokbulut I, Bilenler T, Karabulut, I. Determination of chemical composition, total phenolic, antimicrobial, and antioxidant activities of Echinophora tenuifolia essential oil. IntJ  Food Prop2013; 16:1442-1451.
14. Sharafati-chaleshtoriR, Rafieian-kopaei M,  MortezaeiS, Sharafati-chaleshtori A,Amini E, Antioxidant and antibacterial activity of the extracts of Echinophora platyloba DC. Afr. J Pharm Pharmacol 2012;6 :2692-5.
15. Shafee-Zadeh F, Medicinal plants of Lorestan (1st ed.): Lorestan University of Med Sci 2002.
16. Shokoohinia, Rashidi M,  Hosseinzadeh L,   Jelodarian Z. Quercetin-3-O-β-D-glucopyranoside,     a dietary flavonoid, protects PC12 cells from H2O2-induced cytotoxicity through inhibition of reactive oxygen species. Food  Chem 2015;167:162-7.
17. Yoo Y, Hong J,   Hur KC,   Oh E-S,  Chung J. Iron enhances NGF-induced neurite outgrowth in PC12 cells  Mol Cells2004;17:340-6.
18. Jelodarian Z, Shokoohinia Y, Rashidi M, Ghiasvand N, Hosseinzadeh L, Iranshahi M, New polyacetylenes from Echinophora cinerea (Boiss.) Hedge et Lamond..Nat Prod Res 2017:1-8.
19. Mosmann T, "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays". J Immunol Meth 1983; 65 : 55–63.
20. Mojarrab M, Jamshidi M, Ahmadi F, AlizadehE,  Hosseinzadeh L. Extracts of Artemisia ciniformis protect cytotoxicity induced by hydrogen peroxide in H9c2 cardiac muscle cells through the inhibition of reactive oxygen species. Adv  Pharm  Sci 2013;36:10-16.
21. Karlsson M,  Kurz T,  Brunk B,  Nilsson S E, Frennesson C.I, “What does the commonly used DCF test for oxidative stress really show?” Biochem J 2010;428:183–190.
22. ChandraG, DianaS, Beattie S. Coupling between electron transfer and proton pumping in respiratory chain complexes II+III of Rat Liver Mitochondria". ChinBio ChemJ 1995;11 :200-206.
23. ZhangL, Cheng, X-R Juan-JuanH, Lan S,  Guan-Hua D. Neuroprotective effects of hyperoside on sodium azide-induced apoptosis in PC12 cells. Chin  J Nat Med 2011;9 :450-455.
24. Zeiss CJ, The apoptosis-necrosis continuum: Insights from genetically altered mice. Vet Pathol 2003;40 :481-495.
25. Salakou S1, Kardamakis D, TsamandasAC, Zolota V, Apostolakis E, Tzelepi, V, PapathanasopoulosP, Bonikos DS, Papapetropoulos T,  Petsas T, Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis. In Vivo 2007;21 :123-32.
26. Shokoohinia Y, Hosseinzadeh L, Alipour M, Mostafaei A, Mohammadi Motlagh HR. Comparative evaluation of cytotoxic and apoptogenic effects of several coumarins on human cancer cell lines: osthole induces apoptosis in p53-deficient H1299 cells. Adv Pharmacol Sci 2014.
27. 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.
28. DW Li, SunJY, Wang K,  Zhang S, HouYJ, Yang, MF. Attenuation of Cisplatin-Induced Neurotoxicity by Cyanidin, a Natural Inhibitor of ROS-Mediated Apoptosis in PC12. Cells Mol  Neur Bio 2015; 35:995-1001.
29. Chao X, Zhou J, Chen T, Liu W, Dong, W, Qu Y. Neuroprotective effect of osthole against acute ischemic stroke on middle cerebral ischemia occlusion in rats. Brain  Res 2010;1363:206-211.
30. Hollman PC, van Trijp JM, Buysman MN, vd Gaag MS, Mengelers MJ, de Vries JH, et al. Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Let 1997; 418:152-156.
31. Lei X1,  Chao H,  Zhang Z,  LvJ,  LiS,  Wei H1, Xue R1,  Li F1, Li Z Neuroprotective effects of quercetin in a mouse model of brain ischemic/reperfusion injury via anti-apoptotic mechanisms based on the Akt pathway. Mol Med Rep 2015; 12:3688-3696.
32. Vuković L, Puhović J, Erhardt J, Oršolić N. Neuroprotective effect of quercetin against hydrogen peroxide-induced oxidative injury in P19 neurons. J Mol Neuro Sci 2012; 47:286-299.
33. Chen TJ1, Jeng JY,  Lin CW, Wu CY, Chen YC. Quercetin inhibition of ROS-dependent and -independent apoptosis in rat glioma C6 cells. Toxicol2006;223 :113-126.
34. He Y, Qu S, Wang J, He X, Lin W, Zhen H. Neuroprotective effects of osthole pretreatment against traumatic brain injury in rats. Brain Res2012;1433:127-136.
35. Hai-Jie Ji, Jin-Feng Hu, Yong-Hui Wang, Xiao-Yu Chen, Ran Zhou, Nai-Hong Chen. Osthole improves chronic cerebral hypo-perfusion induced cognitive deficits and neuronal damage in hippocampus. Eur J Pharmacol2010; 636: 96–101.