樹豆葉二苯乙烯類成分的甲醚化和體外抗腫瘤活性

李恩念, 周娟, 譚海波, 沈小玲, 胡英杰*

?

樹豆葉二苯乙烯類成分的甲醚化和體外抗腫瘤活性

李恩念1, 周娟1, 譚海波2, 沈小玲1, 胡英杰1*

(1. 廣州中醫(yī)藥大學(xué)熱帶醫(yī)學(xué)研究所中藥新藥發(fā)現(xiàn)實(shí)驗(yàn)室, 廣州 510405; 2. 中國科學(xué)院華南植物園, 中國科學(xué)院植物資源保護(hù)與可持續(xù)利用重點(diǎn)實(shí)驗(yàn)室, 廣州 510650)

為獲得具有抗腫瘤活性的甲醚化先導(dǎo)化合物，對(duì)樹豆()葉中的二苯乙烯類成分進(jìn)行甲醚化，并測(cè)定其對(duì)人腫瘤細(xì)胞的細(xì)胞毒性。將木豆素C、樹豆酮酸A、Cajanotone和木豆素與碘甲烷-碳酸鉀反應(yīng)制備-甲基產(chǎn)物，通過波譜數(shù)據(jù)分析產(chǎn)物結(jié)構(gòu)分別鑒定為：2-異戊烯基-3,5-二甲氧基二苯乙烯 (1)、樹豆酮酸A甲醚(2)、5--methyl cajanotone (3)和3--甲基木豆素(4)，其中化合物3是新化合物。腫瘤細(xì)胞增殖抑制實(shí)驗(yàn)(CCK-8法)結(jié)果表明，木豆素C對(duì)乳腺癌MDA-MB- 231、宮頸癌HeLa、肝癌HepG2、結(jié)腸癌SW480及3種非小細(xì)胞肺癌細(xì)胞(A549, NCI-H460和NCI-H1299)的半數(shù)抑制濃度IC50分別為14.4、16.1、19.6、17.4和25.7~29.6mol L–1；木豆素對(duì)宮頸癌和結(jié)腸癌之外的5種腫瘤細(xì)胞有弱抑制作用，IC50為44.9~78.3mol L–1；而對(duì)照的3,4′,5-三甲氧基二苯乙烯(三--甲基白藜蘆醇)對(duì)乳腺癌MDA-MB-231、宮頸癌HeLa、結(jié)腸癌SW480和肝癌HepG2細(xì)胞有較強(qiáng)抑制作用(IC50為3.0~14.5mol L–1)。樹豆葉二苯乙烯甲醚化衍生物1~4對(duì)7種腫瘤細(xì)胞系無明顯細(xì)胞毒活性。

樹豆；二苯乙烯；甲醚化；細(xì)胞毒活性；化學(xué)成分

癌癥是嚴(yán)重威脅人類生命和健康的重大疾病。據(jù)統(tǒng)計(jì)，2014年全國惡性腫瘤估計(jì)死亡病例229.6萬例，腫瘤死亡率0.17%，肺癌和乳腺癌分別位居男女性發(fā)病的第1位[1]。尋找新的抗腫瘤先導(dǎo)化合物具有重要意義。一些酚類物質(zhì)在甲醚化后抗腫瘤活性有所提高，例如:黃酮類化合物白楊素甲醚化后生成5,7-二甲氧基黃酮，能夠降低急性淋巴細(xì)胞白血病細(xì)胞增殖水平[2]。二苯乙烯類植物多酚白藜蘆醇(3,5,4′-三羥基二苯乙烯)及其類似物的生物活性,可因結(jié)構(gòu)中甲氧基及其取代位置的不同而變化。Weng等[3–4]報(bào)道白藜蘆醇及其甲氧基類似物如三甲氧基白藜蘆醇可抑制肝癌細(xì)胞的轉(zhuǎn)移和浸潤(rùn)性質(zhì), 還對(duì)非小細(xì)胞肺癌A549細(xì)胞增殖有明顯抑制作用，而白藜蘆醇對(duì)A549細(xì)胞無效；白藜蘆醇和三甲氧基白藜蘆醇在25mol L–1工作濃度下對(duì)乳腺癌MDA-MB-231細(xì)胞的生長(zhǎng)有明顯抑制作用[5]。

豆科植物樹豆[(Linn.)Mill.](又名木豆)葉有多方面的藥理作用, 文獻(xiàn)報(bào)道，12-羥基木豆菧酸具有良好的自由基清除活性和體外抗腫瘤活性[6]；木豆素體外作用于乳腺癌細(xì)胞可引起細(xì)胞周期停滯并誘導(dǎo)細(xì)胞凋亡[7]；木豆菧H具有體外誘導(dǎo)人骨髓間充質(zhì)細(xì)胞分化為成骨細(xì)胞的作用，并對(duì)多種腫瘤細(xì)胞表現(xiàn)出中等強(qiáng)度抑制作用[8]；木豆素C對(duì)多種人源腫瘤細(xì)胞株的增殖表現(xiàn)出明顯抑制作用[9–10]；樹豆酮酸A經(jīng)動(dòng)物試驗(yàn)，具有顯著降低糖尿病大鼠的血糖、血脂的作用[11]。樹豆二苯乙烯類成分的結(jié)構(gòu)與白藜蘆醇相似，其甲氧基類似物有可能也具有抗腫瘤生物活性，所以我們對(duì)源自于樹豆葉的4個(gè)二苯乙烯類成分木豆素C、樹豆酮酸A、Cajanotone和木豆素進(jìn)行甲醚化反應(yīng)[12]，反應(yīng)產(chǎn)物經(jīng)硅膠柱層析純化和波譜分析確定結(jié)構(gòu)。以白藜蘆醇及其甲氧基衍生物為對(duì)照，檢測(cè)相關(guān)化合物的體外抗腫瘤活性。本文報(bào)道這類甲醚化衍生物的制備、結(jié)構(gòu)鑒定，以及體外抗腫瘤活性的測(cè)試結(jié)果。

1 材料和方法

1.1 儀器、試劑和耗材

DRX-400核磁共振儀、EQUINOX55型紅外波譜儀(德國Bruker公司)；TU-1901雙光束紫外可見分光光度計(jì)(北京普析通用儀器有限公司)；C506- Triple TOF 5600系統(tǒng)(美國AB SCIEX公司)；LC6000型高效制備液相色譜儀、LC3000型高效制備液相色譜儀(北京創(chuàng)新通恒有限公司)；UV D-170U、Chro- meleon色譜工作站高效液相色譜儀(美國Dionex公司)；SHIM-PACK色譜柱(日本SHIMADZU公司); 硅膠(200~300目)、硅膠TLC薄層預(yù)制板(青島海洋化工廠分廠)；Sephadex LH-20葡聚糖凝膠(美國GE Healthcare公司)；HPLC分析，使用Merck公司甲醇; 其他溶劑和試劑均為國產(chǎn)分析純產(chǎn)品。TLC顯色劑為6%香蘭素乙醇液-12%高氯酸溶液等體積混合液。

細(xì)胞培養(yǎng)基RPMI 1640 (德國Biological Industries公司，批號(hào)0012118)；胎牛血清(FBS)(澳大利亞Gibco公司，批號(hào)1704206)。

CO2細(xì)胞培養(yǎng)箱(上海力申科學(xué)儀器有限公司);垂直超凈工作臺(tái)(上海智城分析儀器制造有限公司);倒置光學(xué)顯微鏡(日本Olympus公司)；低速離心機(jī)(科大創(chuàng)新股份有限公司中佳分公司)；MK3型酶標(biāo)儀(美國Thermo fisher公司)；Cell Counting Kit-8 (CCK-8)細(xì)胞增殖-毒性檢測(cè)試劑盒(日本同仁化學(xué)研究所)。

1.2 反應(yīng)原料

木豆素C，樹豆酮酸A，Cajanotone和木豆素均從樹豆葉分離得到[8,10,13]。

1.3 細(xì)胞株

人乳腺癌MDA-MB-231細(xì)胞、人宮頸癌HeLa細(xì)胞、人肝癌HepG2細(xì)胞、人結(jié)腸癌SW480細(xì)胞、人非小細(xì)胞肺癌A549、NCI-H1299和NCI-H460均來自中國科學(xué)院上海生命科學(xué)研究院細(xì)胞資源中心。

1.4 樹豆菧類成分的甲醚化

木豆素C (500 mg, 1.7 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(400 mg, 2.9 mmol)，于室溫(28℃~32℃)下攪拌20 min，緩慢滴入200L碘甲烷(460 mg, 3.2 mmol)，攪拌3 h (經(jīng)TLC檢測(cè)反應(yīng)至終點(diǎn))，加水5 mL淬滅反應(yīng)；加乙酸乙酯15 mL萃取反應(yīng)液3次，合并乙酸乙酯溶液，減壓蒸干，得粗產(chǎn)物；粗產(chǎn)物經(jīng)硅膠(200~300目)柱層析，以正己烷-乙酸乙酯(50∶1)洗脫，得產(chǎn)物1 (317 mg，圖1)，收率60.6%。

樹豆酮酸A (93 mg, 0.26 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(120 mg, 0.87 mmol)，緩慢滴入20L碘甲烷(46 mg, 0.32 mmol)，后續(xù)處理過程與制備化合物1的相同，硅膠柱層析流動(dòng)相為正己烷-乙酸乙酯(20∶1~10∶1)，得化合物2 (35.6 mg，圖1)，收率36.8%。

以Cajanotone (40 mg, 0.13 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(60 mg, 0.44 mmol)，緩慢滴入10L碘甲烷(23 mg, 0.16 mmol)，后續(xù)處理過程與制備化合物1的相同，硅膠柱層析流動(dòng)相為正己烷-乙酸乙酯(20∶1~10∶1)，得化合物3 (14.3 mg，圖1)，收率34.2%。

木豆素(236 mg, 0.70 mmol)溶于乙腈(5 mL)，加入無水碳酸鉀(300 mg, 2.17 mmol)，緩慢滴入80L碘甲烷(184 mg, 1.28 mmol)，后續(xù)處理過程與制備化合物1的相同，硅膠柱層析流動(dòng)相為正己烷-乙酸乙酯(50∶1~20∶1)，得化合物4 (35 mg，圖1)，收率14.8%。

1.5 結(jié)構(gòu)鑒定

化合物1 白色粉末，ESI-MS: 308.9 [M + H]+。1H NMR (400 MHz, CDCl3):7.55 (2H, d,= 7.6 Hz, H-2′, H-6′), 7.42 (2H, m, H-3′, H-5′), 7.41 (1H, m, H-4′), 7.30 (2H, d,= 12 Hz, H-7, H-8), 6.79 (1H, d,= 2.4 Hz, H-4), 6.46 (1H, d,= 2 Hz, H-6), 5.16 (1H, t,= 6.4 Hz, H-2′′), 3.95 (3H, s, 3-OCH3), 3.88 (3H, s, 5-OCH3), 3.48 (2H, d,= 9.2 Hz, H-1′′), 1.84 (3H, s, H-4′′), 1.71 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):158.5 (C, C-3), 158.3 (C, C-5), 137.5 (C, C-1, C-1′), 130.9 (CH, C-3′′), 130.3 (CH, C-7), 128.6 (CH, C-3′, C-5′), 127.6 (CH, C-4′′), 126.8 (CH, C-8), 126.5 (CH, C-2′, C-6′), 123.4 (CH, C-2′′), 121.1 (C, C-2), 101.4 (CH, C-4), 98.1 (CH, C-6), 55.6 (CH3, 3-OCH3), 55.3 (CH3, 5- OCH3), 25.9 (CH3, C-4′′), 24.4 (CH2, C-1′′), 17.9 (CH3, C-5′′)。以上數(shù)據(jù)與5--methyl-longistilin C一致[14]。

化合物2 白色粉末，ESI-MS: (neg.) 367.1 [M - H]–。1H NMR (400 MHz, CDCl3):11.59 (1H, s, H-COOH), 8.06 (2H, s,= 7.6 Hz, H-2′, H-6′), 7.61 (1H, t,= 7.2 Hz, H-4′), 7.52 (2H, t,= 7.6 Hz, H-3′, H-5′), 6.46 (1H, s, H-3), 4.95 (1H, t,= 7.2 Hz, H-7), 3.85 (3H, s, 4-OCH3), 3.48 (3H, s, 2-OCH3), 3.27 (2H, d,= 6 Hz, H-1′′), 1.61 (3H, s, H-4′′), 1.59 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):197.1 (C, C-8), 171.2 (C, 1-COOH), 163.6 (C, C-4), 162.5 (C, C-2), 137.0 (C, C-1′), 136.7 (C, C-6), 133.0 (C, C-4′), 131.6 (C, C-3′′), 128.7 (CH, C-3′, C-5′), 123.1 (C, C-5), 122.7 (CH, C-2′′), 105.2 (C, C-1), 98.6 (CH, C-3), 55.7 (CH3, 4-OCH3), 51.9 (CH3, 2-OCH3), 41.6 (CH2, C-7), 25.6 (CH3, C-5′′), 24.9 (CH2, C-1′′), 17.8 (CH3, C-4′′)。以上數(shù)據(jù)與5--methyl-cajanonic acid A的一致[15]。

化合物3 白色粉末，ESI-MS/: (pos.) 347.0 [M + Na]+。1H NMR (400 MHz, CDCl3):8.01 (2H, d,= 7.6 Hz, H-2′, H-6′), 7.57 (1H, t,= 7.2 Hz, H-4′), 7.46 (2H, t,= 7.2 Hz, H-3′, H-5′), 6.41 (1H, d,= 2 Hz, H-6), 5.00 (1H, t,= 6.4 Hz, H-2′′), 4.26 (2H, s, H-7), 3.78 (3H, s, 3-OCH3), 3.74 (3H, s, 5- OCH3), 3.26 (2H, d,= 6.4 Hz, H-1′′), 1.59 (3H, s, H- 4′′), 1.51 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):197.8 (C, C-8), 158.4 (C, C-3, C-5), 136.7 (C, C-1′), 134.9 (C, C-1), 133.1 (CH, C-4′), 131.2 (C, C-3′′), 128.6 (CH, C-2′, C-6′), 123.2 (CH, C-2′′), 121.1 (C, C-2), 106.6 (CH, C-4), 97.5 (CH, C-6), 55.6 (CH3, 3- OCH3), 55.2 (CH3, 5-OCH3), 43.2 (CH2, C-7), 25.6 (CH3, C-5′′), 24.8 (CH2, C-1′′), 17.8 (CH3, C-4′′)。因此，化合物3的結(jié)構(gòu)確定為5--methyl-cajanotone (表1，圖2)[16]。

表1 Cajanotone與化合物3的核磁數(shù)據(jù)(溶劑為CDCl3)

化合物4 白色粉末，ESI-MS: (neg.) 351.0 [M - H]–。1H NMR (400 MHz, CDCl3):11.74 (1H, s, COO), 7.75 (1H, d,= 16 Hz, H-8), 7.53 (2H, t,= 7.6 Hz, H-2′, H-6′), 7.39 (2H, t,= 7.6 Hz, H-3′, H-5′), 6.81 (1H, d,= 16 Hz, H-7), 6.62 (1H, s, H-5), 5.23 (1H, t, H-2′′), 3.94 (3H, s, 4-OCH3), 3.93 (3H, s, 2-OCH3), 3.40 (2H, d,= 8 Hz, H-1′′), 1.81 (3H, s, H-4′′), 1.69 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):171.9 (C, 1-COOH), 161.4 (C, C-4), 161.3 (C, C-2), 140.3 (C, C- 6), 137.4 (C, C-1′), 131.9 (C, C- 3′′), 130.6 (CH, C-8), 128.7(CH, C-3′, C-5′), 127.7 (CH, C-7), 126.5 (CH, C-2', C-6'), 122.0 (CH, C-2′′), 116.6 (C, C-3), 104.4 (C, C-1), 102.8 (CH, C-5), 55.6 (CH3, 4-OCH3), 52.3 (CH3, 2-OCH3), 25.8 (CH3, C-4′′), 22.1 (CH2, C-1′′), 17.8 (CH3, C-5′′)。以上數(shù)據(jù)與5-- methyl-cajaninstilbene acid的一致[17]。

1.6 體外抗腫瘤活性研究

樣品對(duì)HeLa、SW480、HepG2、A549、NCI- H460、NCI-H1299、MDA-MB-231細(xì)胞的增殖抑制試驗(yàn)(CCK-8法)：胰酶消化收集生長(zhǎng)良好的細(xì)胞, 用新鮮培養(yǎng)液配成5×104個(gè)mL–1的單細(xì)胞懸液，按每孔100L接種于96孔板中，置于孵育箱中培養(yǎng)24 h使細(xì)胞貼壁。將樣品用細(xì)胞培養(yǎng)液稀釋至需要的百分濃度，按每孔100L替換原有的細(xì)胞培養(yǎng)液，每個(gè)濃度設(shè)3個(gè)復(fù)孔。試驗(yàn)設(shè)置不含藥物的陰性對(duì)照，并對(duì)每個(gè)藥物作用濃度設(shè)置不含細(xì)胞的藥物空白對(duì)照。將細(xì)胞板置于孵育箱孵育72 h后取出, 每孔加入10L CCK-8，再置于孵育箱孵育1.5 h。取出細(xì)胞培養(yǎng)板，于450 nm檢測(cè)每孔吸收值A(chǔ)。樣品對(duì)腫瘤細(xì)胞增殖的抑制率(%)=100×(A陰性-A藥物)/ (A陰性-A空白)，并求得其對(duì)細(xì)胞增殖的半數(shù)抑制濃度IC50，即細(xì)胞增殖抑制率為50%時(shí)的作用濃度。

2 結(jié)果和討論

我們利用樹豆天然二苯乙烯類成分木豆素C、樹豆酮酸A、Cajanotone和木豆素作為原料，以碘甲烷為甲醚化反應(yīng)試劑，進(jìn)行甲醚化反應(yīng)，得到了2-異戊烯基-3,5-二甲氧基二苯乙烯 (1)、樹豆酮酸A--甲醚(2)、5--methyl cajanotone (3)和3--甲基木豆素 (4)。其中化合物3是新化合物。Zhang等報(bào)道,Cajanotone和木豆素對(duì)肺癌A549細(xì)胞的IC50分別為5.9和4.4mol L–1，對(duì)肝癌HepG2細(xì)胞的IC50為12.2~17.9mol L–1；木豆素C對(duì)這幾種腫瘤細(xì)胞呈顯著細(xì)胞毒活性(IC50為3.5~6.0mol L–1)[16]; 木豆素C的甲氧基衍生物對(duì)肝癌HepG2細(xì)胞的IC50為3.7mol L–1[14]。三甲氧基白藜蘆醇對(duì)MDA-MB- 231、HeLa、SW480和HepG2細(xì)胞具有較強(qiáng)細(xì)胞毒作用，IC50分別為3.0、9.2、14.5和13.0mol L–1。三甲氧基白藜蘆醇的體外抗腫瘤活性最強(qiáng)，4′-甲氧基白藜蘆醇次之，白藜蘆醇較弱。從表2可見，樹豆葉二苯乙烯類成分中，木豆素C對(duì)7種腫瘤細(xì)胞株均呈顯著的細(xì)胞增殖抑制作用(IC50為14.4~ 29.6mol L–1)，木豆素對(duì)除HeLa和SW480外的其余5個(gè)細(xì)胞株均呈較弱的細(xì)胞毒活性(IC50為44.9~ 78.3mol L–1)，但甲醚化產(chǎn)物1~4對(duì)參試7種腫瘤細(xì)胞株均無明顯細(xì)胞毒作用(IC50>100mol L–1)。這4種甲醚衍生物的甲氧基數(shù)目和取代位置與三甲氧基白藜蘆醇有較大差別。有報(bào)道，3,4,5,4′-四甲氧基二苯乙烯在體外對(duì)人宮頸癌HeLa細(xì)胞、人前列腺癌LNCaP細(xì)胞、人結(jié)腸腺癌HT-29細(xì)胞、人肝癌HepG2細(xì)胞的抑制作用顯著增強(qiáng)[18]。由此推測(cè)，甲氧基數(shù)目和取代位置對(duì)樹豆葉二苯乙烯類成分的抗腫瘤活性有顯著影響。我們的試驗(yàn)為樹豆葉二苯乙烯類成分通過結(jié)構(gòu)修飾改變體外抗腫瘤活性提供了參考依據(jù)。

表2 化合物1~4對(duì)人腫瘤細(xì)胞增殖的影響(IC50, μmol L–1)

[1] CHEN W Q, SUN K X, ZHENG R S, et al. Report of cancer incidence and mortality in different areas of China, 2014 [J]. China Cancer, 2018, 27(1): 1–14. doi: 10.11735/j.issn.1004-0242.2018.01.A001.陳萬青, 孫可欣, 鄭榮壽, 等. 2014年中國分地區(qū)惡性腫瘤發(fā)病和死亡分析[J]. 中國腫瘤, 2018, 27(1): 1–14. doi: 10.11735/j.issn.1004-0242.2018.01.A001.

[2] GOTO H, YANAGIMACHI M, GOTO S, et al. Methylated chrysin reduced cell proliferation, but antagonized cytotoxicity of other anti- cancer drugs in acute lymphoblastic leukemia [J]. Anti-Cancer Drugs, 2012, 23(4): 417–425. doi: 10.1097/CAD.0b013e32834fb731.

[3] WENG J C, YANG Y T, HO T H, et al. Mechanisms of apoptotic effects induced by resveratrol, dibenzoylmethane, and their analogues on human lung carcinoma cells [J]. J Agric Food Chem, 2009, 57(12): 5235–5243. doi: 10.1021/jf900531m.

[4] WENG J C, WU C F, HUANG H W, et al. Evaluation of anti-invasion effect of resveratrol and related methoxy analogues on human hepato- carcinoma cells [J]. J Agric Food Chem, 2010, 58(5): 2886–2894. doi: 10.1021/jf904182y.

[5] HSIEH C T, WONG C, BENNETT J D, et al. Regulation of p53 and cell proliferation by resveratrol and its derivatives in breast cancer cells: An in silico and biochemical approach targeting integrinv3 [J]. Int J Cancer, 2011, 129(11): 2732–2743. doi: 10.1002/ijc.25930.

[6] FU Y J, YU P. Method for synthesizing anti-form stilbene compounds and application of method in preparing anti-tumor medicines: China, CN201010592686.X [P]. 2012-07-11.付玉杰, 余平. 一種合成反式芪類化合物的方法及其在制備抗腫瘤藥物中的應(yīng)用: 中國, CN201010592686.X [P]. 2012-07-11.

[7] FU Y J, KADIOGLU O, WIENCH B, et al. Cell cycle arrest and induction of apoptosis by cajanin stilbene acid fromin breast cancer cells [J]. Phytomedicine, 2015, 22(4): 462–468. doi: 10. 1016/j.phymed.2015.02.005.

[8] CAI J Z, TANG R, YE G F, et al. A halogen-containing stilbene derivative from the leaves ofthat induces osteogenic differentiation of human mesenchymal stem cells [J]. Molecules, 2015, 20(6): 10839–10847. doi: 10.3390/molecules200610839.

[9] ASHIDI J S, HOUGHTON P J, HYLANDS P J, et al. Ethnobotanical survey and cytotoxicity testing of plants of south-western Nigeria used to treat cancer, with isolation of cytotoxic constituents fromMill. leaves [J]. J Ethnopharmacol, 2010, 128(2): 501–512. doi: 10.1016/j.jep.2010.01.009.

[10] CAI J Z. Chemical constituents from leaves ofandand anticancer effects [D]. Guangzhou: Guangzhou University of Chinese Medicine, 2012: 83–100.蔡佳仲. 朱砂根和樹豆葉的化學(xué)成分及抗腫瘤作用研究[D]. 廣州: 廣州中醫(yī)藥大學(xué), 2012: 83–100.

[11] SHEN X L, WANG L, HU Y J, et al. Application of pigeon pea ketonic acid A in terms of preparation of medicines for accompanied diseases of diabetes mellitus and hyperlipidaemia: China, CN201210119665.5 [P]. 2012-09-19.沈小玲, 王璐, 胡英杰, 等. 樹豆酮酸A在制備糖尿病伴隨癥及高脂血癥藥物中的應(yīng)用: 中國, CN201210119665.5 [P]. 2012-09-19.

[12] GUO R X. Studies on structural modification of quercetin, isoalan- tolactone and alantolactone [D]. Shijiazhuang: Hebei Medical University, 2014: 15–17.郭瑞霞. 槲皮素、異土木香內(nèi)酯和土木香內(nèi)酯的結(jié)構(gòu)修飾研究[D]. 石家莊: 河北醫(yī)科大學(xué), 2014: 15–17.

[13] HU Y L, SHEN X L, CHEN M G, et al. Structural identification on the saponification products of raw cajanolactone A [J]. J Trop Subtrop Bot,2017, 25(1): 93–97. doi: 10.11926/jtsb.3617.胡陽亮, 沈小玲, 陳梅果, 等. 樹豆內(nèi)酯A粗品皂化產(chǎn)物的化學(xué)結(jié)構(gòu)鑒定[J]. 熱帶亞熱帶植物學(xué)報(bào), 2017, 25(1): 93–97. doi: 10. 11926/jtsb.3617.

[14] SHAN Y, HONG T, WANG Y F, et al. Synthesis and cytotoxicity of longistylin C derivatives [J]. Chin J Nat Med, 2015, 13(4): 311–315. doi: 10.1016/S1875-5364(15)30021-2.

[15] WANG Y N, GE P, HU Y J, et al. Synthesis method of diethyl- stilbestrol compound methyl pigeon pea ketonic acid A: China, CN201410032181. 6 [P]. 2015-05-27.王亞農(nóng), 戈平, 胡英杰, 等. 一種菧類化合物甲基樹豆酮酸A的合成方法: 中國, CN201410032181.6 [P]. 2015-05-27.

[16] ZHANG N L, ZHU Y H, HUANG M R, et al. Two new stilbenoids from[J]. Z Naturforsch B, 2012, 67(12): 1314–1318. doi: 10.5560/znb.2012-0184.

[17] LI Z R, JI X Y, XUE S T, et al. Cajanin compound with similar structures, as well as preparation method and application thereof: China, CN201110439374.X [P]. 2013-06-26.李卓榮, 季興躍, 薛司徒, 等. 一組木豆素結(jié)構(gòu)類似化合物、制備方法和應(yīng)用: 中國, CN201110439374.X [P]. 2013-06-26.

[18] GOSSLAU A, CHEN M, HO C T, et al. A methoxy derivative of resveratrol analogue selectively induced activation of the mitochondrial apoptotic pathway in transformed fibroblasts [J]. Br J Cancer, 2005, 92(3): 513–521. doi: 10.1038/sj.bjc.6602300.

Methoxylation of the Stilbenes Originated from Leaves ofand Their Anti-tumor Activities

LI En-nian1, ZHOU Juan1, TAN Hai-bo2, SHEN Xiao-ling1, HU Ying-jie1*

(1. Laboratory of Herbal Drug Discovery, Tropical Medicine Institute, Guangzhou University of Chinese Medicine,Guangzhou 510405, China; 2. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China)

The aim was to obtain lead compounds with anticancer activity by-methylation of stilbene pheolic deriatives extracted from leaves of. Four derivatives, such as longistylin C, cajanonic acid A, cajanotone and cajaninstilbene acid, were treated with iodomethane and potassium carbohydrate under mild condition. The products were purified through silica gel column chromatography and their structures were identified by spectroscopic analysis as 2-isoprenyl-3,5-dimethoxy-stilbene (1),-methyl-cajanonic acid A (2), 5--methyl cajanotone (3) and 3--methyl cajaninstilbene acid (4), respectively. Among them, compound 3 was a new compound. Effects of 1–4 and their reactants, together with resveratrol, 4′--methyl resveratrol and 3,4′,5--trimethyl resveratrol using as positive controls, on the proliferation of the cell lines of human breast cancer (MDA-MB-231), cervical cancer (HeLa), liver cancer (HepG2), colon cancer (SW480), and non-small cell lung cancer (A549, NCI-H460 and NCI-H1299) were determined by CCK-8 assay. The results showed that longistilin C distinctly inhibited the proliferation of all the seven cell lines, with the half inhibitory concentrations (IC50) to MDA-MB-231, HeLa, HepG2, and SW480 at 14.4, 16.1, 19.6, and 17.4mol L–1(strong suppresion), with IC50values of 25.7–29.6mol L–1to other three cell lines of non-small cell lung cancer; cajaninstilbene acid had a weak inhibition to HepG2, A549, NCI-H460, NCI-H1299 and MDA-MB-231 cells with IC50as 44.9–78.3mol L–1; tri--methylated resveratrol showed significantly cytotoxity against MDA-MB-231, HeLa, SW480 and HepG2 cells with IC50of 16.1-17.4mol L–1;-methylated stilbenes 1-4 did not exhibited obvious cytotoxicity in the cell lines aboved.

; Stilbene; Methoxylation; Cytotoxicity; Chemical constituent

10.11926/jtsb.3938

2018-05-03

2018-06-15

廣東省教育廳重大項(xiàng)目(2014KZDXM026)；廣東省科技計(jì)劃項(xiàng)目(2016A020217015)資助

This work was supported by the Key Projects in Department of Education of Guangdong (Grant No. 2014KZDXM026), and the Plan Project for Science and technology in Guangdong (Grant No. 2016A020217015).

李恩念，男，碩士研究生，研究方向?yàn)橹兴幮滤幯邪l(fā)。E-mail: ennian_li@163.com

E-mail: yingjiehu@gzucm.edu.cn