Cytotoxic effect of Cotinus coggygria extract on Hep3B cancer cell line

Abstract Cotinus coggygria Scop. is a medicinal plant which contains a variety of precious phytochemical compounds applicable in traditional medicine. The aim of this study is to investigate the cytotoxic effect of Cotinus coggygria extract on Hep3B cancer cells. The cytotoxic effect was measured by MTT assay. Based on IC50 values, the C. coggygria leaf extract showed cytotoxic effect on Hep3B cancer cells, which was found better at 24 h (IC50 = 3.642) then at 48 h (IC50 = 3.956). Also, the dose of C. coggygria extract showed negative correlation (p < 0.05) with cell viability of 24 h and 48 h and positive correlation (p < 0.05) with cytotoxic effect of 24 h and 48 h. The cytotoxic effects were concentration-time dependent. According to our study, C. coggygria extract had protective effect at 24 h on AML12 cell line and cytotoxic effect on the Hep3B cancer cell line, where it indicates that it may have an antitumorigenic effect. Graphical Abstract


Introduction
Cotinus coggygria plant of the family Anacardiaceae is one of the plants used in traditional medicine (Novakovic et al. 2007).C. coggygria had a usage in treating of the acute icteric infectious hepatitis as a herbal in traditional treatment (Mati c et al. 2011).Also C. coggygria syrup protects liver from any chemical damages, reduces tension in the common bile duct sphincter and increases the immunity of the body (Shen et al. 1991).C. coggygria extract also shows biological activities such as antioxidative (Westenburg et al. 2000) antimicrobial, anti-inflammatory (Mar ceti c et al. 2013) antibacterial, antiviral, antigenotoxic, anticancer and the hepatoprotective effects (Mati c et al. 2016).
The content of secondary metabolites of plants varies according to the chemical composition of the soil, the ecological situation in the planting area and the climatic conditions of the region (Asyakina et al. 2021).A few studies have been reported the anticancer effects of Cotinus coggygria Scop. on the different cancer cells (Mar ceti c et al. 2013;Antal et al. 2016;Gospodinova et al. 2021).In this study, it was aimed to investigate the anticancer effect on Hep3B cell line (tumorigenic), which has not been studied before according to our literature survey.

Results and discussion
Herbal medicines have been used in medical applications since ancient times.According to the results seen in Figure S1, C.coggygria extract (0.95, 1.9, 3.9, 7.9, 15.625, 31.25, 62.5, 125, 250, 500, 1000 ppb) of 24 h has been observed that it is effective in the proliferation of healthy liver cell lines (AML12).According to the MTT results seen in Figure S2, it was observed that the cytotoxic effect of 24 h was higher than the 48 h.The dose of C. coggygria extract showed negative correlation (p < 0.05) with cell viability of 24 h and 48 h, and positive correlation (p < 0.05) with the cytotoxic effect of 24 h and 48 h (Table S1).So, it was observed that with increasing dose, % viability decreased in AML12 (lower concentrations were better) while % cytotoxicity increased with increasing dose in Hep3B.In the other hand, it was observed that with increasing incubation time, % cell death increased in healthy cells at 48 h, and with the increasing incubation time, % cell death decreased in cancer cells (Table S2).
According to IC 50 values, the C.coggygria leaf extract did not showed toxicity on AML12 cell line at 24 h, but at 48 h it showed very low toxicity (IC 50 ¼ 9.830).On the Hep3B cell line, it was found that the C.coggygria leaf extract showed the cytotoxic effect at 24 h (IC 50 ¼ 3.642), which was better than 48 h (IC 50 ¼ 3.956) (Table S2).So, a better incubation time was found at 24 h.
The cytotoxic effect of C. coggygria may be related to its phenolic compounds such as gallic acid and ellagic acid (Westenburg et al. 2000), gallocatechin, methyl gallate, myricetin-3-O-a-rhamnoside, myricetin-3-O-b-galactoside, and 1, 2, 3, 4, 6-penta-O-galloyl-b-glucose ( € Ozbek et al. 2019).In the literature, there are cytotoxic effects of C. coggygria extracts on other different cancer cell lines A549, HCT116, MCF7, Hep-G2, TK6, U937, U87, U251, DBTRG-05MG, A2780, MDA-MB-231, HeLa, LS174 ( Savikin et al. 2009;Wang et al. 2015;Antal et al. 2016;Pollio et al. 2016) except Hep3B cell line.The strongest inhibitory effect has been shown against A2780 ovarian cancer cells (Gospodinova et al. 2017).In another work, authors reported the toxicological properties of C. coggygria growing in the Russian on the Hep-G2, MCF-7, A549, HCT116 cell lines (Sukhikh et al. 2021), but it is not reported any study on Hep3B cell line.Although Hep3B and HepG2 are two liver cancer cell lines, they have a wide range of differences in biology, genetics, ethnic origins and biochemistry.The Hep3B and HepG2 in the similar experimental conditions often show different and opposite results in response to the same pharmacological treatment (Qiu et al. 2015).Another difference is that HepG2 is hepatitis B virus negative and non-tumorigenic, but Hep3B is hepatitis B virus positive and tumorigenic (Knowles et al. 1980;Knasmuller et al. 1998).Some studies reported that some treatments have different effects (inhibiting cell growth, cytotoxic effect) on this two cell lines (Matsumoto et al. 2006;You et al. 2019).So, our study will be the first work in identifying anticancer effect of C. coggygria on Hep3B cell line, where we can say that the plant extract has the antitumorigenic effect.
These studies indicate that C. coggygria extracts have a potential as anticancer agent in the pharmaceutical industry.According to our results, it is also important that the C. coggygria extract shows the ability to protect healthy cells (AML12) while destroying cancer cells (Hep3B).

Conclusions
The results of this study showed that C. coggygria extract has a concentration-and timedependent cytotoxic effect on Hep3B cancer cells.Also, C. coggygria extract showed protective effect on AML12 healthy liver cells, at 24 h incubation time.These results seem to support the use of C. coggygria plant in liver related diseases in the folk medicine.Although this plant is a candidate to be a medicinal plant, further studies are also needed.