目的 研究黄杨木Buxus microphylla茎枝中的化学成分及其体外抗肝癌活性。方法 利用硅胶、反相C₁₈、羟丙基葡聚糖凝胶、高效液相、薄层色谱等方法系统分离纯化，采用核磁共振、质谱等光谱、波谱技术鉴定结构，使用MTT法评价化合物对人肝癌细胞HepG2和Hep3B的细胞毒性，同时采用流式细胞术检测其对细胞凋亡的影响；Western blotting检测凋亡相关蛋白表达。结果 从黄杨木茎枝70%乙醇提取物中分离到18个化合物，分别鉴定为常春藤皂苷元（1）、3-O-β-D-glucopyranosylspinasterol（2）、fagraeanolide（3）、rel-(2α,3β)-7-O-methylcedrusin（4）、caruilignan D（5）、(7'S,8R,8'S)-异落叶松素脂素（6）、3-羟基-1,2-二甲氧基𠮿酮（7）、1-甲氧基-2,3-亚甲二氧基𠮿酮（8）、1,2,3-三甲氧基𠮿酮（9）、丁香醛（10）、丁香酸（11）、香草酸（12）、对羟基苯甲酸（13）、远志糖醇（14）、9-二十五烷酸（15）、rhamnakoside B（16）、羽扇豆醇（17）和白桦脂醇（18）。MTT毒性测试表明，化合物2、3、16～18对HepG2和Hep3B细胞显示出不同程度的细胞毒性，化合物2具有明显的细胞毒性。初步作用机制研究表明，在10 μmol/L浓度下化合物2显著增加凋亡细胞的比例，并上调促凋亡蛋白Bcl-2关联X蛋白（recombinant Bcl-2 associated x protein，Bax）、下调抗凋亡蛋白B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）的表达，与对照组比较均具有统计学意义（P＜0.001）。结论 化合物1、17～18为三萜类化合物，化合物2为甾体类化合物，化合物3～6为木脂素类化合物，化合物7～9为𠮿酮类化合物，化合物10～13为酚酸类化合物，化合物14～16为其他类化合物，其中化合物2～5、7～9和14～16为首次从黄杨属植物中分离得到。体外抗肝癌活性表明，化合物2具有明显的细胞毒活性，其对HepG2细胞和Hep3B细胞的半数抑制浓度（median inhibition concentration，IC₅₀）值分别为5.87、6.42 μnol/L，可以有效诱导HepG2细胞凋亡。

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.

R284.1

国家自然科学基金资助项目（82274154）；山东省重点研发计划项目（2021CXGC010508）