Objective To investigate the chemical constituents of Buxus microphylla and their anti-hepatocarcinoma activity in vitro. Methods The compounds were isolated and purified by column chromatography of silica gel, reversed-phase C₁₈, Sephadex LH-20, semi-preparative HPLC and preparative TLC. Their structures were elucidated by physicochemical properties and spectral analyses. The cytotoxicity of the isolates against human hepatocellular carcinoma HepG2 and Hep3B cells was assessed using the MTT assay. Flow cytometry was employed to analyze cell apoptosis. Western blotting was used to detect the expression of proteins related to cell apoptosis. Results Eighteen compounds were isolated and identified from the 70% ethanol extract of B. microphylla, including hederagenin (1), 3-O-β-D-glucopyranosylspinasterol (2), fagraeanolide (3), rel-(2α,3β)-7-O-methylcedrusin (4), caruilignan D (5), (7′S,8R,8′S)-isolariciresinol (6), 3-hydroxy-1,2-dimethoxyanthrone (7), 1-methoxy-2,3-methylenedioxyanthrone (8), 1,2,3-trimethoxyanthrone (9), syringaldehyde (10), syringic acid (11), vanillic acid (12), p-hydroxybenzoic acid (13), polygital (14), 9-pentacosenoic acid (15), rhamnakoside B (16), lupeol (17), and betulin (18). The in vitro activity screening revealed that compounds 2, 3, 16—18 exhibited cytotoxicity in HepG2 and Hep3B cells, among which compound 2 was the most potent. compound 2 significantly increased the proportion of apoptotic cells and elevated the expression of apoptosis-related proteins such as Bax, decreased the expression of Bcl-2, all of which were statistically significant compared with control group (P < 0.001) at 10 μmol/L. Conclusion Compound 1 and 17—18 are triterpenoids; Compound 2 is a steroidal glycoside; compounds 3—6 are lignans, compounds 7—9 are anthrones, compounds 10—13 are phenolic acids, and compounds 14—16 are other types. Among them, compounds 2—5, 7—9, and 14—16 are isolated from the genus Buxus for the first time. In the anti-hepatocarcinoma activity assay, compound 2 demonstrated significant effects, with IC₅₀ values of 5.87 μnol/L and 6.42 μnol/L against HepG2 and Hep3B cells, respectively. Moreover, it effectively induced apoptosis in HepG2 cells.

Buxus microphylla Siebold & Zucc.  /  hepatocellular carcinoma  /  cytotoxic activity  /  apoptosis  /  3-O-β-D-glucopyranosyl- spinasterol  /  3-hydroxy-1,2-dimethoxyanthrone  /  polygital