Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS2 in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism

Xuqiang Liu; Nan Jiang; Xiaoqing Xu; Cunyu Liu; Zhenhua Liu; Yan Zhang; Wenyi Kang

doi:10.3389/fphar.2020.01342

Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS² in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism

Front Pharmacol. 2020 Sep 9:11:1342. doi: 10.3389/fphar.2020.01342. eCollection 2020.

Authors

Xuqiang Liu^{1

2}, Nan Jiang², Xiaoqing Xu¹, Cunyu Liu¹, Zhenhua Liu^{1

3}, Yan Zhang^{4

5}, Wenyi Kang^{1

3}

Affiliations

¹ National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, China.
² School of Biomedical Sciences, Huaqiao University, Xiamen, China.
³ Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Henan University, Kaifeng, China.
⁴ Hebei Food Inspection and Research Institute, Shijiazhuang, China.
⁵ College of Forensic Medicine, Hebei Medical University, Shijiazhuang, China.

Abstract

Scheflera heptaphylla (L.)Frodin, a kind of Traditional Chinese Medicine, is commonly used in anti-inflammatory, analgesic, anti-viral, anti-tumor, and hemostasis. This study aimed to determine the anti-hepatoma components and its mechanism from the leaves of S. heptaphylla. The spectrum-effect relationships were analyzed by the method of Partial least squares, indicating that P1, P2, and P10 were positively correlated to inhibitory activity of Huh7 cells. Whereas others were negatively correlated. The technologies of component knock-out and UPLC-MS² were used to determine compounds as 3,4-Dicaffeoylquinic acid (P6), 3,5-Dicaffeoylquinic acid (P7), 3α-Hydroxy-lup-20(29)-ene-23,28-dioic acid (P10, named Compound A). The results forecasted that Compound A had the best correlation with inhibitory activity. The effects of Compound A on the activities of human hepatoma cells (Huh7, SMMC-7721, HepG 2) and normal hepatocytes (L0-2, Chang liver) were evaluated. Cell apoptosis was observed with inverted microscope and flow cytometer. In addition, the proteins, related to apoptosis, were detected by Western blot. The results showed that Compound A (400 nM) could significantly inhibit the activity of three hepatoma cells (P < 0.001) with slight toxicity to normal hepatocytes, and the IC₅₀ values were 285.3 and 315.1 nM, respectively, which were consistent with the prediction of spectrum-effect relationships. After treatment with Compound A, the number of hepatoma cells decreased significantly. And the apoptosis rate of Huh7 cells increased significantly (P < 0.001) in Compound A (200, 400 nM) groups, SMMC-7721 and HepG 2 were directly necrotic. Compound A groups could significantly improve the level of intracellular reactive oxygen species (ROS) (P < 0.05, P < 0.001) in Huh7 with no effect on normal hepatocytes. The content of apoptotic protein (Bax and Bim) in mitochondria was significantly increased in Compound A groups (P < 0.001). On the contrary, the content of anti-apoptotic protein (Bcl-xL and Mcl-1) decreased significantly (P < 0.001). These results demonstrated that Compound A was the main anti-hepatoma active component in the S. heptaphylla leaves. It achieved the effect of promoting apoptosis of Huh7 cells by regulating the levels of ROS and Bcl-2 family protein in mitochondrial apoptosis pathway.

Keywords: Huh7; S. octophylla; active ingredient; mechanism; spectrum-effect relationship.