Protective effect of bioactive compounds from Lonicera japonica Thunb. against H2O2-induced cytotoxicity using neonatal rat cardiomyocytes

Document Type : Original Article


1 Department of Medical market, Cangzhou Central Hospital, Hebei Province, 061001, China

2 Department of Cardiology, Cangzhou Cardiovascular Research Institute, Cangzhou Central Hospital, Hebei Province, 061001, China

3 Department of Pharmacy, General Hospital of Jixi Mining Industry Group, Heilongjiang Province, 158100, China

4 Department of Pharmacy, Harbin Medical University, Heilongjiang Province, 150086, China


Objective(s):Pharmacological studies showed that the extracts of Jin Yin Hua and its active constituents have lipid lowering, antipyretic, hepatoprotective, cytoprotective, antimicrobial, antibiotic, antioxidative, antiviral, and anti-inflammatory effects. The purpose of the present study was to investigate the protective effects of caffeoylquinic acids (CQAs) from Jin Yin Hua against hydrogen peroxide (H2O2)-induced and hypoxia-induced cytotoxicity using neonatal rat cardiomyocytes.
Materials and Methods:Seven CQAs (C1 to C7) isolated and identified from Jin Yin Hua were used to examine the effects of H2O2-induced and hypoxia-induced cytotoxicity. We studied C4 and C6 as preventative bioactive compounds of the reactive oxygen species (ROS) production, apoptotic pathway, and apoptosis-related gene expression.
Results:C4 and C6 were screened as bioactive compounds to exert a cytoprotective effect against oxidative injury. Pretreatment with C4 and C6, dose-dependently attenuated hypoxia-induced ROS production and reduced the ratio of GSSG/GStotal. Western blot data revealed that the inhibitory effect of C4 on H2O2-induced up and down-regulation of Bcl-2, Bax, caspase-3, and cleaved caspase-3. Apoptosis was evaluated by detection of DNA fragmentation using TUNEL assay, and quantified with Annexin V/PI staining.
Conclusion: In vitro experiments revealed that both C4 and C6 protect cardiomyocytes from necrosis and apoptosis during H2O2-induced injury, via inhibiting the generation of ROS and activation of caspase-3 apoptotic pathway. These results demonstrated that CQAs might be a class of compounds which possess potent myocardial protective activity against the ischemic heart diseases related to oxidative stress.


1. Hayes O. Fact sheet: cardiovascular disease (ICD-9 390-448) and women. Chronic Dis Can 1996; 17:28-30.
2. Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 2005; 115:500-508.
3. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 2010; 48:749-762. 
4. Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA, et al. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. Am J Physiol Heart Circ Physiol 2005; 288:H1900-908.
5. Fan H, Yang L, Fu F, Xu H, Meng Q, Zhu H, et al. Cardioprotective effects of salvianolic Acid a on myocardial ischemia-reperfusion injury in vivo and in vitro. Evid Based Complement Alternat Med 2012; 2012:508938.
6. Du CS, Yang RF, Song SW, Wang YP, Kang JH, Zhang R, et al. Magnesium Lithospermate B Protects cardiomyocytes from ischemic injury via inhibition of TAB1-p38 apoptosis signaling. Front Pharmacol 2010; 1:111.
7. Qiao Z, Ma J, Liu H. Evaluation of the antioxidant potential of Salvia miltiorrhiza ethanol extract in a rat model of ischemia-reperfusion injury. Molecules 2011; 16:10002-10012.
8. Wang F, Jiang YP, Wang XL, Lin S, Pu PB, Zhu CG, et al. Chemical constituents from flower buds of Lonicera japonica. Zhongguo Zhong Yao Za Zhi 2013; 38:1378-1385.
9. Peng LY, Mei SX, Jiang B, Zhou H, Sun HD. Constituents from Lonicera japonica. Fitoterapia 2000; 71:713-715.
10. Zheng ZF, Zhang QJ, Chen RY, Yu DQ. Four new N-contained iridoid glycosides from flower buds of Lonicera japonica. J Asian Nat Prod Res 2012; 14:729-737.
11. Xiong J, Li S, Wang W, Hong Y, Tang K, Luo Q. Screening and identification of the antibacterial bioactive compounds from Lonicera japonica Thunb. leaves. Food Chem 2013; 138:327-333.
12. Lin LM, Zhang XG, Zhu JJ, Gao HM, Wang ZM, Wang WH. Two new triterpenoid saponins from the flowers and buds of Lonicera japonica. J Asian Nat Prod Res 2008; 10:925-929.
13. Shang X, Pan H, Li M, Miao X, Ding H. Lonicera japonica Thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacol 2011; 138:1-21.
14. Weon JB, Yang HJ, Lee B, Yun BR, Ahn JH, Lee HY, et al. Neuroprotective activity of the methanolic extract of Lonicera japonica in glutamate-injured primary rat cortical cells. Pharmacogn Mag 2011; 7:284-288.
15. Park HS, Park KI, Lee DH, Kang SR, Nagappan A, Kim JA, et al. Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs. Food Chem Toxicol 2012; 50:2407-2416.
16. Tzeng TF, Liou SS, Chang CJ, Liu IM. The ethanol extract of Lonicera japonica (Japanese honeysuckle) attenuates diabetic nephropathy by inhibiting p-38 MAPK activity in streptozotocin-induced diabetic rats. Planta Med 2014; 80:121-129.
17. Hau DK-P, Zhu G-Y, Leung AK-M, Wong RS-M, Cheng GY-M, Lai PB-S, et al. In vivo anti-tumour activity of corilagin on Hep3B hepatocellular carcinoma. Phytomedicine 2010; 18:11-15.
18. Lin LC, Yang LL, Chou CJ. Constituents from the stems of Ecdysanthera rosea. J Chines Med 2002; 13:191-195.
19. Li J, Yu D. Chemical constituents from herbs of Erigeron breviscapus. Zhongguo Zhong Yao Za Zhi 2011; 36:1458-1462.
20. Zhu X, Zhang H, Lo R. Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities. J Agric Food Chem 2004; 52:7272-7278.
21. Mao Q, Cao D, Jia XS. Studies on the chemical constituents of Lonicera macranthoides Hand. -Mazz. Yao Xue Xue Bao 1993; 28:273-281.
22. Choi CW, Jung HA, Kang SS, Choi JS. Antioxidant constituents and a new triterpenoid glycoside from Flos Lonicerae. Arch Pharm Res 2007; 30:1-7.
23. Ma J, Li N, Li X. Caffeoylquinic acid derivatives from leaves of Lonicera japonica. Zhongguo Zhong Yao Za Zhi 2009; 34:2346-2348.
24. Tang D, Li HJ, Chen J, Guo CW, Li P. Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH-HPLC-DAD-TOF/MS. J Sep Sci 2008; 31:3519-3526.
25. Lin YL, Lu CK, Huang YJ, Chen HJ. Antioxidative caffeoylquinic acids and flavonoids from Hemerocallis fulva flowers. J Agric Food Chem 2011; 59:8789-8795.
26. Han J, Miyamae Y, Shigemori H, Isoda H. Neuroprotective effect of 3,5-di-O-caffeoylquinic acid on SH-SY5Y cells and senescence-accelerated-prone mice 8 through the up-regulation of phosphogly-cerate kinase-1. Neuroscience 2010; 169:1039-1045.
27. Kumar D, Jugdutt BI. Apoptosis and oxidants in the heart. J Lab Clin Med 2003; 142:288-297.
28. Eefting F, Rensing B, Wigman J, Pannekoek WJ, Liu WM, Cramer MJ, et al. Role of apoptosis in reperfusion injury. Cardiovasc Res 2004; 61:414-426.
29. Santos CX, Anilkumar N, Zhang M, Brewer AC, Shah AM. Redox signaling in cardiac myocytes. Free Radic Biol Med 2011; 50:777-793.
30. Schulz JB, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative stress and neurodegeneration. Eur J Biochem 2000; 267:4904-4911.
31. Sulpizi M, Rothlisberger U, Carloni P. Molecular dynamics studies of caspase-3. Biophys J 2003; 84:22072215.
32. Delchev SD, Georgieva KN, Koeva YA, Atanassova PK. Bcl-2/Bax ratio, mitochondrial membranes and aerobic enzyme activity in cardiomyocytes of rats after submaximal training. Folia Med (Plovdiv) 2006; 48:50-56.