Identifying Antihepatocellular Carcinoma Compounds in Gansui Banxia Decoction Using Live Cell Adsorption

Jiahao Huang,1,* Hongyu Mao,1,2,* Weidong Wu,3 Siliang Jiang,1 Lanru Chen,1 Tianyu Ma,1 Chunmei Zhai,3 Yonghai Meng3 1Key Laboratory of Basic and Application Research of Beiyao, Ministry of Education, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, People’s Republic of China; 2Sinopharm Harbin General Hospital, Harbin, Heilongjiang, People’s Republic of China; 3Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chunmei Zhai, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, 150040, People’s Republic of China, Email [email protected] Yonghai Meng, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, 150040, People’s Republic of China, Email [email protected]: Gansui Banxia Decoction (GSBXD) is a traditional Chinese medicine formula for the treatment of hepatocellular carcinoma (HCC). Preliminary studies have identified the constituents and anticancer efficacy of GSBXD, but there is a gap in the screening of its lead compounds. Using live cell affinity combined with solid-phase extraction (LCA-SPE) and virtual screening and in vitro activity assays, we obtained 14 reliable potential lead compounds.Methods: Coculturing H22 mouse HCC cells with GSBXD ethanol extract, isolating and purifying the bioactive fractions using LCA-SPE, and identifying the unknown bioactive components by ultraperformance liquid chromatography coupled with quadrupole time-of-flight full information tandem mass spectrometry (UPLC-QTOF-MSE) with the UNIFI information processing platform were performed. Network pharmacology and molecular docking techniques predicted the potential mechanisms of these compounds against HCC. The enzyme-linked immunosorbent assay was used to examine the effects of core compounds on the expression of p53 and Bcl-2 in vitro.Results: Fourteen compounds screened from GSBXD using SPE-LCA-UPLC-QTOF-MSE may be the main bioactive components. Network pharmacology predictions suggested that protein kinases regulate Bcl-2 and p53 expression to trigger the apoptosis in cancer cells. Molecular docking identified three core compounds, namely, lactiflorin, schaftoside, and violanthin, which showed high affinities for the relevant proteins. Experimental verification confirmed their superior anticancer activity in vitro. Their anti-HCC effects likely involved in the upregulation of p53 and downregulation of Bcl-2 expression at the cellular level.Conclusion: We developed a stable and accurate SPE-LCA method, which successfully isolated and characterized 14 potentially active compounds from GSBXD. They may improve HCC by promoting p53 expression and reducing Bcl-2 expression. This work lays a foundation for discovering lead compounds and exploring potential mechanisms in traditional Chinese medicine formulas.Plain Language Summary: The search for bioactive compounds from herbal remedies remains one of the most challenging tasks in natural products chemistry. Herein, we developed an SPE-LCA-UPLC-QTOF-MSE-based analytical procedure centered on the classic Chinese herbal formula Gansui Banxia decoction (GSBXD). This method used live mouse hepatocellular carcinoma (H22) cells as a stationary phase to screen for target compounds, which were enriched through a modified solid-phase extraction process and subsequently analyzed by LC-MS on the UNIFI platform. Using network pharmacology and molecular docking techniques, we further explored the potential antihepatocellular carcinoma (HCC) mechanisms of these compounds. This approach isolated and characterized 14 potentially active small molecules from the four GSBXD herbs. Preliminary validation indicated that their anti-HCC effects might be associated with the upregulation of p53 and downregulation of Bcl-2 at the cellular level. This method provides a reliable approach for screening lead compounds in complex herbal formulas, critical for advancing LCA-based analytical methods. Keywords: Gansui Banxia decoction, UNIFI platform, UPLC-QTOF-MSE, solid-phase extraction, biospecific live cell-based isolation