Comparison of cytotoxicity evaluation of chlorogenic acid extract between Real-time cell analysis and CCK-8 method

  • Ping Xu College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, China
Keywords: inflammatory, chlorogenic acid, bovine, bovine mammary epithelial cells, cytotoxicity


Critical cytotoxicity evaluation of pharmaceuticals is necessary for the clinical practice of chemotherapy. To quantitatively evaluate cell viability, currently there are two main types of sensitive methods including real-time cell analysis (RTCA) and CCK-8 assay, in which RTCA records electrochemical signal changes around an incubated cell, whereas CCK-8 is based on the colorimetric method. Despite the different detection principles adopted for the cytotoxicity assessment, the comparison of the two methods in terms of the application scope is lacking. In order to compare and determine the best experimental method for the study of the toxicity of chlorogenic acid extract from taraxacum officinale on dairy cow mammary epithelial cells. The real time cell analysis (RTCA) and CCK-8 method were used to analyze the cytotoxicity of chlorogenic acid extract to BMEC and calculate its IC50. The results of the real time cell analysis method and the CCK-8 method showed that different concentrations of chlorogenic acid extract reduced the viability of dairy cow mammary epithelial cells, and the decrease was most obvious at 400 ug/mL. The IC50 of the two analysis methods were 326.8 and 320.4 ug/mL, respectively. In contrast, the CCK-8 method had limitations in fixed-point determination. However, the real time cell analysis method can monitor the dynamic biological response process of cell growth and proliferation in real time. Therefore, the real time cell analysis method can observe cell growth more intuitively and accurately, compensate for the shortcomings of the CCK-8 method, and it is a new experimental method for studying cytotoxicity.


Braun, K., Stürzel, C. M., Biskupek, J., Kaiser, U., Kirchhoff, F., & Linden, M. (2018). Comparison of different cytotoxicity assays for in vitro evaluation of mesoporous silica nanoparticles. Toxi-col in Vitro, 52, 214–221. doi: 10.1016/j.tiv.2018.06.019.

Budzianowski, J. (1997). Coumarins, caffeoyltartaric acids and their artifactual methyl esters from Taraxacum officinale leaves. Planta medica, 63(3), 288. doi: 10.1055/s-2006-957681.

Elisia, I., Popovich, D. G., Hu, C., & Kitts, D. D. (2008). Evaluation of Viability Assays for Anthocyanins in Cultured Cells. Phyto-chem Anal, 19(6), 479–486. doi: 10.1002/pca.1069.

Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., & Kissel, T. (2003). In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials, 24(7), 1121–1131. doi: 10.1016/S0142-9612(02)00445-3.

Gao, R., Yang, H., Jing, S., Liu, B., Wei, M., He, P., & Zhang, N. (2018). Protective effect of chlorogenic acid on lipopolysaccha-ride-induced inflammatory response in dairy mammary epitheli-al cells. Microb Pathog, 124, 178–182. doi: 10.1016/j.micpath.2018.07.030.

Hudec, J., Burdova, M., Komora, L., Komora, L., Macho, V., Kogan, G., Turianca, I., Kochanova, R., Lozek, O., Haban, M., & Chlebo, P. (2007). Antioxidant capacity changes and phenol-ic profile of Echinaccea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators. J Agric Food Chem, 55(14), 5689–5696. doi: 10.1021/jf070777c.

Kitts, D. D., & Hu, C. (2005). Dandelion (Taraxacum officinale) flower extract suppresses both reaction oxygen species and ni-tric oxide and prevents lipid oxidation in vitro. Phytomedicine, 12(5), 588–597. doi: 10.1016/j. phymed.2003.12.012.

Ling, Y., & Xu, Y. (1998). Studies on the chemical constituents of Taraxacum officinale. China J China Mat Med, 23(4), 232–233.

Liang, N., & Kitts, D. D. (2015). Role of chlorogenic acids in con-trolling oxidative and inflammatory stress conditions. Nutrients, 8(1), 16. doi: 10.3390/nu8010016.

Liao, J., Zheng, H., Fei, Z., Lu, B., Zheng, H., Li, D., Xiong, X., & Yi, Y. (2018). Tumor-targeting and pH-responsive nanoparti-cles from hyaluronic acid for the enhanced delivery of doxoru-bicin. Int J Biol Macromol, 113, 737–747. doi: 10.1016/j.ijbiomac.2018.03.004.

Liu, M., Chang, Y., Yang, J., You, Y., He, R., Chen, T., & Zhou, C. (2016). Functionalized halloysite nanotube by chitosan graft-ing for drug delivery of curcumin to achieve enhanced anti-cancer efficacy. J Mater Chem B, 4, 2253–2263. doi: 10.1039/C5TB02725J.

Liu, Z., Li, G., Long, C., Xu, J., Cen, J., & Yang, X. (2018). The antioxidant activity and genotoxicity of isogarcinol. Food Chem, 253, 5–12. doi: 10.1016/j.foodchem.2018.01.074.

Lou, Z., Wang, H., Zhu, S., Ma, C., & Wang, Z. (2011). Antibacteri-al activity and mechanism of action of chlorogenic acid. J Food Sci, 76, M398–403. doi: 10.1111/j.1750-3841.2011.02213.x.

Otero-Gonzalez, L., Sierra-Alvarez, R., Boitano, S., & Field, J. A. (2012). Application and Validation of an Impedance-Based Real Time Cell Analyzer to Measure the Toxicity of Nanoparticles Impacting Human Bronchial Epithelial Cells. Environ Sci Tech-nol, 46, 10271. doi: 10.1021/es301599f.

Rezaei, S. J. T., Sarbaz, L., & Niknejad, H. (2016). Folate-decorated redox/pH dual-responsive degradable prodrug micelles for tu-mor triggered targeted drug delivery. RSC Adv, 6: 62630–62639. doi: 10.1039/C6RA11824K.

Rui, L., Xie, M., Hu, B., Zhou, L., Saeeduddin, M., & Zeng, X. (2017). Enhanced solubility and antioxidant activity of chloro-genic acid-chitosan conjugates due to the conjugation of chi-tosan with chlorogenic acid. Carbohydr Polym, 170, 206–216. doi: 10.1016/j.carbpol.2017.04.076.

Schütz, K., Carle, R., & Schieber, A. (2006). Taraxacum-A review on its phytochemical and pharmacological profile. J Eth-nopharmacol, 107, 313–323. doi: 10.1016/j.jep.2006.07.021.

Shi, S., Zhao, Y., Zhang, Y., Huang, K., & Liu, S. (2008). Phe-nylpropanoids from Taraxacum mongolicum. Biochem Syst Ecol, 36(9), 716–718. doi: 10.1016/j.bse.2008.06.002.

Sriraman, S.K., Pan, J., Sarisozen, C., Luther, E., & Torchilin, V. (2016). Enhanced Cytotoxicity of Folic Acid-Targeted Lipo-somes Co-Loaded with C6 Ceramide and Doxorubicin: In Vitro Evaluation on HeLa, A2780-ADR, and H69-AR Cells. Mol Pharm, 13, 428–437. doi: 10.1021/acs.molpharmaceut.5b00663.

Xu, Z., Shi, X., Jiang, H., Song, Y., Zhang, L., Wang, F., Du, S., & Chen, J. (2017). A general method to regenerate arrayed gold microelectrodes for label-free cell assay. Anal Biochem, 516, 57–60. doi: 10.1016/j.ab.2016.10.012.

Xu, Z., Song, Y., Jiang, H., Kong, Y., Li, X., Chen, J., & Wu, Y. (2018). Regeneration of Arrayed Gold Microelectrodes Equipped for a Real Time Cell Analyzer. J Vis Exp, 133, 56250. doi: 10.3791/56250.

Xu, Z., Cai, L., Jiang, H., Wen, Y., Peng, L., Wu, Y., & Chen, J. (2019). Real-time cell analysis of the cytotoxicity of a pH-responsive drugdelivery matrix based on mesoporous silica ma-terials functionalized with ferrocenecarboxylic acid. Anal Chim Acta, 1051, 138–146. doi: 10.1016/j.aca.2018.11.017.

Yan, Y., Liu, N., Hou, N., Dong, L., & Li, J. (2017). Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo. J Nutr Biochem, 46, 68–73. doi: 10.1016/j.jnutbio.2017.04.007.

Zhou, S., Guo, P., Li, J., Meng, L., Gao, H., Yuan, X., & Wu, D. (2018). An electrochemical method for evaluation the cytotoxi-city of fluorene on reduced graphene oxide quantum dots modi-fied electrode. Sens Actuators, 255(3), 2595–2600. doi: 10.1016/j.snb.2017.09.066.

Zhu, M., Wong, P. Y., & Li, R. C. (1999). Effect of Taraxacum officinale on the bioavailability and disposition of ciprofloxacin in rats. J Pharm Sci-us, 88(6), 632–634. doi: 10.1021/js980367q.
How to Cite
Xu, P. (2021). Comparison of cytotoxicity evaluation of chlorogenic acid extract between Real-time cell analysis and CCK-8 method. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(2), 58-61.