目的 研究土當(dāng)歸Angelica gigas的化學(xué)成分，對(duì)分離得到的單體化合物進(jìn)行抗前列腺癌活性評(píng)價(jià)。方法 利用硅膠柱色譜、半制備高效液相色譜等方法對(duì)土當(dāng)歸醋酸乙酯萃取物進(jìn)行系統(tǒng)分離純化，綜合運(yùn)用核磁共振、高分辨質(zhì)譜等多種波譜手段對(duì)得到的單體化合物進(jìn)行結(jié)構(gòu)鑒定。采用CCK-8法測(cè)定化合物抗前列腺癌活性，利用網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)土當(dāng)歸中活性成分抗前列腺癌的靶點(diǎn)、功能及通路，通過(guò)蛋白質(zhì)印跡實(shí)驗(yàn)進(jìn)行驗(yàn)證。結(jié)果 從土當(dāng)歸醋酸乙酯部位分離得到33個(gè)化合物，依次鑒定為傘形花內(nèi)酯（1）、茵芋苷（2）、東莨菪素（3）、7-去甲基軟木花椒素（4）、6,7-二甲氧基香豆素（5）、devenyol（6）、佛手柑內(nèi)酯（7）、異歐前胡素（8）、蛇床素（9）、歐前胡素（10）、花椒毒酚（11）、花椒毒素（12）、紫花前胡苷元（13）、異紫花前胡內(nèi)酯（14）、紫花前胡苷（15）、異補(bǔ)骨脂素（16）、噢洛內(nèi)酯（17）、(±)-vaginidiol（18）、二氫山芹醇當(dāng)歸酸酯（19）、花椒內(nèi)酯（20）、紫花前胡醇（21）、紫花前胡素（22）、紫花前胡醇當(dāng)歸酯（23）、(＋)-lomatin（24）、(＋)-順式凱林內(nèi)酯（25）、兒茶素（26）、槲皮素（27）、楊梅素（28）、煙酸（29）、阿魏酸（30）、綠原酸（31）、5-羥甲基糠醛（32）、5,5¢-二丁氧基-2,2¢-雙環(huán)呋喃（33）。化合物1、4、6、8、12、13、19、21～24表現(xiàn)出抑制前列腺癌細(xì)胞增殖作用，進(jìn)一步對(duì)抑癌作用最顯著的5個(gè)化合物（1、4、21～23）進(jìn)行抗前列腺癌活性評(píng)價(jià)，其中化合物22、23抑制前列腺腫瘤作用最強(qiáng)。通過(guò)網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)獲得的土當(dāng)歸中主要活性成分（化合物1、4、21～23）抗前列腺癌核心靶點(diǎn)11個(gè)，包括蛋白激酶B（threonine kinase，AKT）、表皮生長(zhǎng)因子受體（epidermal growth factor receptor，EGFR）、細(xì)胞周期蛋白D1（cyclin D1，CCND1）、前列腺素內(nèi)過(guò)氧化物合成酶2（prostaglandin-endoperoxide synthase 2，PTGS2）等；KEGG通路富集篩選得到信號(hào)通路涉及癌癥通路、磷脂酰肌醇3-激酶/蛋白激酶B（phosphatidylinositol 3-kinase/protein kinase B，PI3K/AKT）通路、EGFR信號(hào)通路等。經(jīng)Western blotting驗(yàn)證土當(dāng)歸中活性成分通過(guò)抑制PI3K/AKT通路中關(guān)鍵蛋白AKT的表達(dá)發(fā)揮抗前列腺癌的作用。結(jié)論 化合物6、16、17、24、33為首次從該屬植物中發(fā)現(xiàn)，化合物18、25、32為首次從該種植物中發(fā)現(xiàn)。化合物1、4、21～23表現(xiàn)出較好的抑制前列腺癌細(xì)胞增殖作用，其中化合物22和23的抑制作用最強(qiáng)，其通過(guò)抑制PI3K/AKT通路中關(guān)鍵蛋白AKT的表達(dá)從而發(fā)揮抗前列腺癌的作用。

Objective To study the chemical constituents of of Angelica gigas, and evaluate the activity of the isolated monomer compounds against prostate cancer. Methods The ethyl acetate extract of A. gigas was systematically isolated and purified using silica gel column chromatography and semi-preparative HPLC. The structures of the isolated monomer compounds were characterized by a combination of spectroscopic methods, including nuclear magnetic resonance, high resolution mass spectrometry. The anti-prostate cancer activity of the compounds was evaluated using the CCK-8 assay, and network pharmacology was employed to predict the targets, functions, and pathways of the active ingredients of A. gigas against prostate cancer, which was further verified by Western blotting (WB) experiments. Results A total of 33 compounds were isolated from the ethyl acetate fraction of A. gigas and identified as umbelliferone (1), skimmin (2), scopoletin (3), 7-demethylsuberosine (4), 6,7-dimethoxycoumarin (5) devenyol (6), bergapten (7), isoimperatorin (8), cnidicin (9), imperatorin (10), xanthotoxol (11), xanthotoxin (12), nodakenetin (13), marmesin (14), nodakenin (15), isopsoralen (16), oroselol (17), (±)-vaginidiol (18), columbianadin (19), xanthyletin (20), decursinol (21), decursin (22), decursinol angelate (23), (＋)-lomatin (24) (＋)-cis-khellactone (25), catechin (26), quercetin (27), myricetin (28), nicotinic acid (29), ferulic acid (30), chlorogenic acid (31), 5-(hydroxymethyl)furfural (32), 5,5¢-dibutoxy-2,2¢-bifuran (33). Compounds 1, 4, 6, 8, 12, 13, 19, and 21—24 exhibited inhibition of prostate cancer cell proliferation. The five compounds with the most significant cancer inhibition effects (1, 4, 21—23) were further evaluated for their anti-prostate cancer activities at multiple concentrations. Among them, compounds 22 and 23 showed the strongest inhibition of prostate tumors. A total of 11 core anti-prostate cancer targets of the chemical components in A. gigas were identified through network pharmacology prediction, including threonine kinase (AKT), epidermal growth factor receptor (EGFR), cyclin D1 (CCND1), prostaglandin-endoperoxide synthase 2 (PTGS2), and others. KEGG pathway enrichment screening revealed signaling pathways involved in the cancer pathway, the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and the EGFR signaling pathway. Western blotting analysis confirmed that the active ingredients in A. gigas exerted anti-prostate cancer effects by inhibiting the expression of AKT, a key protein in the PI3K/AKT pathway. Conclusion Compounds 6, 16, 17, 24, and 33 were identified for the first time from this genus, while compounds 18, 25, and 32 were found for the first time from this species. Compounds 1, 4, 21—23 showed better inhibition of prostate cancer cell proliferation, with compounds 22 and 23 having the strongest inhibitory effect, which demonstrated their anti-prostate cancer effects by inhibiting the expression of AKT, a key protein in the PI3K/AKT pathway.

R284.1

廣東省自然科學(xué)基金項(xiàng)目（2021A1515011485）；遼寧省中醫(yī)藥多學(xué)科交叉創(chuàng)新團(tuán)隊(duì)項(xiàng)目（LNZYYCXTD-JCCX-002）