巴西酒神菊化学成分及其体外抑制MDA-MB-231细胞增殖作用
2022-12-28黄璐琦刘宏栋闵建新朱金华周小青
李 影, 黄璐琦, 刘宏栋, 闵建新, 朱金华, 周小青, 李 斌*
(1.江西中医药大学院士工作站,江西 南昌 330004;2.中国中医科学院,北京 100700;3.江西中医药大学生命科学学院,江西 南昌 330004)
巴西酒神菊Baccharistrimera(Less.) DC为菊科酒神菊属植物,广泛分布于南美洲[1],其地上部位在巴西民间应用历史悠久,具有保肝、抗炎等药理作用,用于治疗肝病、糖尿病、痛风等[2-3]。随着我国中医药事业的深入发展,中药资源的种类和数量日益减少,故有效开展外来资源的“中药化”“本土化”研究意义重大。本实验对巴西酒神菊进行系统的化学成分和药理活性研究,从中分离得到25个化合物,其中化合物1~2、21为首次从酒神菊属植物中分离得到,3~7、12~15、23~24为首次从巴西酒神菊中分离得到,15~16具有体外抑制MDA-MB-231乳腺癌细胞增殖的作用。
1 材料
R-1001-VN型核磁共振波谱仪(瑞士Bruker公司);Triple ESI 5600+ 型高分辨飞行时间质谱联用仪(美国AB SCIEX公司);LC-20AT 型高效液相色谱仪(日本岛津公司);Waters 515 制备型液相色谱仪(美国Waters公司);ODS反相色谱填料(日本YMC公司);Sephadex LH-20凝胶(美国Amersham PharmaciaBiotech公司);中压制备液相色谱仪(瑞士Buchi公司);QUINTI电子分析天平(德国赛多利斯公司)。薄层色谱、柱色谱用硅胶(青岛海洋化工有限公司)。其他试剂均为市售分析纯和色谱纯。
药材于2018年12月采自巴西亚马逊支流城市北科伦斯,经安徽中医药大学药学院彭华胜教授鉴定为菊科植物巴西酒神菊Baccharistrimera(Less.) DC.的花和枝干。
2 提取与分离
取干燥巴西酒神菊10 kg,95%乙醇加热回流提取2次,每次2 h,合并提取液,浓缩干燥,得浸膏900.0 g,经硅胶柱,依次用石油醚、二氯甲烷、乙酸乙酯、甲醇洗脱,减压回收溶剂,得石油醚部位0.3 g、二氯甲烷部位400.0 g、乙酸乙酯部位147.6 g、甲醇部位351.8 g。
二氯甲烷部位(400.0 g)经大孔树脂,乙醇-水梯度洗脱,得30%乙醇部位(5.3 g)、50%乙醇部位(90.9 g)、70%乙醇部位(85.3 g)、95%乙醇部位(153.9 g),即Fr.1~Fr.4。Fr.2(90.9 g)经聚酰胺柱,乙醇-水梯度洗脱,得10%乙醇部位(20.1 g)、20%乙醇部位(22.2 g)、30%乙醇部位(9.8 g)、35%乙醇部位(10.9 g)、45%乙醇部位(11.0 g)、50%乙醇部位(16.1 g),即Fr.2-1~Fr.2-6。Fr.2-3(950.0 mg)经硅胶柱,二氯甲烷-甲醇(150∶1~ 0∶1)洗脱,ODS柱乙醇-水(0∶100~100∶0)、制备HPLC(甲醇-水)分离,得化合物4(6.0 mg)、5(2.8 mg)、6(2.5 mg)、7(3.0 mg)、8(50.0 mg)、9(2.8 mg)、10(20.0 mg)、11(2.8 mg)、24(6.0 mg)。Fr.2-4(5.3 g)经硅胶柱,二氯甲烷-甲醇(250∶1~ 0∶1)洗脱,重结晶分离,得化合物14(15.0 mg)。Fr.2-5(1.2 g)经硅胶柱,二氯甲烷-甲醇(100∶1~0∶1)洗脱,重结晶分离,得化合物17(2.8 mg)、19(5.0 mg)、23(2.1 mg)。Fr.3(85.3 g)经硅胶柱,石油醚-二氯甲烷(10∶1~ 0∶1)、二氯甲烷-甲醇(200∶1~ 0∶1)梯度洗脱,得Fr.3-1~Fr.3-8,Fr.3-1(3.6 g)经ODS柱,乙醇-水(0∶100~100∶0)洗脱,重结晶分离,得化合物16(2.0 g);Fr.3-4(43.9 g)经ODS柱,乙醇-水(0∶100~100∶0)洗脱,制备HPLC(甲醇-水)分离,得化合物12(5.0 mg)、13(3.0 mg);Fr.3-5(22.7 g)经硅胶柱,二氯甲烷-甲醇(200∶1~ 0∶1)洗脱,重结晶分离,得化合物15(40.0 mg)、18(3.0 mg)。Fr.4(150.0 g)经硅胶柱,石油醚-乙酸乙酯(300∶1~ 0∶1)洗脱,得Fr.4-1~Fr.4-10,其中Fr.4-1(3.1 g)重结晶分离,得化合物21(5.0 mg);Fr.4-3(3.1 g)经硅胶柱,石油醚-乙酸乙酯(250∶1~ 0∶1)洗脱,制备HPLC(70%甲醇-水)纯化,得化合物2(4.2 mg);Fr.4-4(1.2 g)经硅胶柱,石油醚-乙酸乙酯(300∶1~ 0∶1)洗脱,重结晶分离,得化合物1(2.8 mg);Fr.4-6(1.2 g)分别经硅胶柱,石油醚-乙酸乙酯(100∶1~0∶1)洗脱,以及凝胶柱,二氯甲烷-甲醇(2∶1)洗脱,重结晶分离,得化合物20(2.0 mg)、22(3.0 mg);Fr.4-7(1.8 g)分别经硅胶柱,石油醚-二氯甲烷-(50∶1~0∶1)、二氯甲烷-甲醇(300∶1~ 0∶1)洗脱,以及凝胶柱,二氯甲烷-甲醇(2∶1)洗脱,制备HPLC(甲醇-水)、重结晶纯化,得化合物3(3.0 mg)、25(3.0 mg)。
3 结构鉴定
化合物1:白色针晶(二氯甲烷),mp 167~168 ℃;HR-ESI-MSm/z:457.404 8[M+H]+,分子式C31H52O2。1H-NMR(600 MHz,CDCl3)δ:0.98(3H,s,H-18),0.94(3H,d,J=0.8 Hz,H-19),1.17(3H,s,H-21),0.92(3H,d,J=6.8 Hz,H-26),0.84(3H,d,J=6.9 Hz,H-27),1.08(3H,s,H-28),1.04(3H,s,H-29),0.87(3H,d,J=1.0 Hz,H-30),1.11(3H,s,H-31);13C-NMR(150 MHz,CDCl3)δ:40.1(C-1),34.3(C-2),218.4(C-3),47.6(C-4),55.5(C-5),19.8(C-6),34.7(C-7),40.4(C-8),50.1(C-9),37.0(C-10),22.1(C-11),25.7(C-12),43.4(C-13),50.2(C-14),31.2(C-15),27.0(C-16),50.5(C-17),15.4(C-18),16.2(C-19),85.9(C-20),25.2(C-21),37.3(C-22),36.2(C-23),85.4(C-24),38.2(C-25),17.7(C-26),18.9(C-27),26.9(C-28),21.2(C-29),16.4(C-30),23.1(C-31)。以上数据与文献[4]报道基本一致,故鉴定为alnincanone。
化合物2:白色粉末;HR-ESI-MSm/z:459.418 6[M+H]+,分子式C31H54O2。1H-NMR(600 MHz,CDCl3)δ:3.20(1H,dd,J=11.5,4.8 Hz,H-3),0.94(3H,s,H-18),0.84(3H,s,H-19),1.16(3H,s,H-21),0.92(3H,d,J=6.8 Hz,H-26),0.84(6H,d,J=6.9 Hz,H-27),0.97(3H,s,H-28),0.77(3H,s,H-29),0.86(3H,s,H-30),1.10(3H,s,H-31);13C-NMR(150 MHz,CDCl3)δ:39.2(C-1),27.5(C-2),79.1(C-3),39.1(C-4),56(C-5),18.4(C-6),35.4(C-7),40.4(C-8),50.8(C-9),37.3(C-10),21.6(C-11),25.7(C-12),43.3(C-13),50.2(C-14),31.2(C-15),26.9(C-16),50.5(C-17),15.6(C-18),16.3(C-19),85.9(C-20),25.1(C-21),37.3(C-22),36.1(C-23),85.3(C-24),38.1(C-25),17.7(C-26),18.9(C-27),28.1(C-28),15.5(C-29),16.5(C-30),23.1(C-31)。以上数据与文献[4]报道基本一致,故鉴定为 β-alnincanyl。
化合物3:白色粉末;HR-ESI-MSm/z:459.419 3[M+H]+,分子式C31H54O2。1H-NMR(600 MHz,CDCl3)δ:3.20(1H,dd,J=11.5,4.7 Hz,H-3),0.90(3H,s,H-18),0.85(3H,s,H-19),1.16(3H,s,H-21),1.06(3H,s,H-26),1.03(3H,d,J=3.6 Hz,H-27),0.97(3H,s,H-28),0.77(3H,s,H-29),0.96(3H,s,H-30),4.74(1H,s,H-31α),4.68(1H,s,H-31β);13C-NMR(150 MHz,CDCl3)δ:39.2(C-1),27.6(C-2),79.1(C-3),39.1(C-4),56.0(C-5),18.4(C-6),35.4(C-7),40.5(C-8),50.8(C-9),37.3(C-10),21.7(C-11),24.9(C-12),42.5(C-13),50.5(C-14),31.4(C-15),27.7(C-16),49.9(C-17),16.6(C-18),16.4(C-19),75.5(C-20),25.5(C-21),39. 5(C-22),28.5(C-23),156.7(C-24),34.2(C-25),22.1(C-26),22.1(C-27),28.1(C-28),15.5(C-29),15.6(C-30),106.3(C-31)。以上数据与文献[5]报道基本一致,故鉴定为 foliasalacins A4。
化合物4:白色晶体(二氯甲烷),mp 130~132 ℃;HR-ESI-MSm/z:331.190 5[M+H]+,分子式C20H26O4。1H-NMR(600 MHz,CDCl3)δ:6.76(1H,dd,J=7.5,2.2 Hz,H-3),5.87(1H,t,J=1.7 Hz,H-14),4.76(2H,d,J=1.8 Hz,H-16),0.86(3H,d,J=6.6 Hz,H-17),4.31(1H,d,J=8.1 Hz,H-19α),3.93(1H,dd,J=8.1,2.2 Hz,H-19β),0.66(3H,s,H-20);13C-NMR(150 MHz,CDCl3)δ:19.8(C-1),27.8(C-2),135.5(C-3),138.7(C-4),45.7(C-5),34.6(C-6),27.8(C-7),36.8(C-8),38.9(C-9),48.2(C-10),34.7(C-11),22.2(C-12),169.9(C-13),115.6(C-14),173.8(C-15),71.8(C-16),15.8(C-17),169.2(C-18),73.1(C-19),17.8(C-20)。以上数据与文献[6]报道基本一致,故鉴定4为desoxyarticulyn。
化合物5:无色油状物;HR-ESI-MSm/z:325.183 0[M+H]+,分子式C20H36O3。1H-NMR(600 MHz,CDCl3)δ:5.53(1H,t,J=3.4 Hz,H-3),4.03(1H,q,J=3.3 Hz,H-7),3.71~3.65(2H,m,H-15),0.91(3H,d,J=6.6 Hz,H-16),1.01(3H,d,J=7.2 Hz,H-17),4.15~4.12(2H,m,H-18),1.35(3H,s,H-19),0.99(3H,s,H-20);13C-NMR(150 MHz,CDCl3)δ:17.9(C-1),26.6(C-2),121.9(C-3),148.8(C-4),37.4(C-5),42.4(C-6),73.8(C-7),39.2(C-8),38.1(C-9),46.4(C-10),36.7(C-11),30.2(C-12),30.2(C-13),40.0(C-14),61.4(C-15),20.0(C-16),12.6(C-17),63.2(C-18),23.5(C-19),20.3(C-20)。以上数据与文献[7]报道基本一致,故鉴定为7α,15,18-trihydroxy-ent-cleroda-3-ene。
化合物6:无色油状物;HR-ESI-MSm/z:447.271 5[M+Na]+,分子式C24H40O6。1H-NMR(600 MHz,CDCl3)δ:5.53(1H,s,H-3),4.01(1H,d,J=2.7 Hz,H-7),4.02(2H,s,H-15),4.12(2H,m,H-16),1.00(3H,s,H-17),4.13(2H,s,H-18),1.35(3H,s,H-19),1.00(3H,s,H-20),2.05(6H,s,2OCOCH3);13C-NMR(150 MHz,CDCl3)δ:18.0(C-1),26.6(C-2),121.8(C-3),148.7(C-4),37.4(C-5),42.4(C-6),73.6(C-7),39.2(C-8),38.2(C-9),46.4(C-10),36.0(C-11),24.0(C-12),35.2(C-13),30.6(C-14),66.8(C-15),62.6(C-16),12.6(C-17),63.2(C-18),23.4(C-19),20.3(C-20),21.1(-CH3CO),21.1(-CH3CO),171.2(-CH3CO),171.3(-CH3CO)。以上数据与文献[7]报道基本一致,故鉴定为 15,16-diacetoxy-7α,18-dihydroxy-ent-cleroda-3-ene。
化合物7:无色油状物;HR-ESI-MSm/z:359.219 8[M+Na]+,分子式C20H32O4。1H-NMR(600 MHz,CDCl3)δ:5.52(1H,t,J=3.5 Hz,H-3),4.02(1H,q,J=3.4 Hz,H-7),2.44(1H,p,J=7.6 Hz,H-13),2.17~2.12(1H,m,H-14α),2.65(1H,dd,J=17.4,8.4 Hz,H-14β),3.90(1H,dd,J=9.0,7.2 Hz,H-16α),4.43(1H,dd,J=8.9,7.4 Hz,H-16β),1.01(6H,d,J=5.4 Hz,H-17,H-20),4.16~4.07(2H,m,H-18),1.35(3H,s,H-19);13C-NMR(150 MHz,CDCl3)δ:18.0(C-1),26.5(C-2),121.7(C-3),148.6(C-4),37.3(C-5),42.4(C-6),73.4(C-7),39.2(C-8),38.1(C-9),46.4(C-10),37.2(C-11),26.9(C-12),36.3(C-13),34.8(C-14),177.2(C-15),73.6(C-16),12.6(C-17),63.1(C-18),23.4(C-19),20.0(C-20)。以上数据与文献[7]报道基本一致,故鉴定为15,16-epoxy-7α,18-dihydroxy-15-oxo-ent-cleroda-3-ene。
化合物8:白色结晶(二氯甲烷),mp 203~205 ℃;HR-ESI-MSm/z:371.183 2[M+Na]+,分子式C20H28O5。1H-NMR(600 MHz,CDCl3)δ:1.32(1H,m,H-1α),1.40(1H,s,H-1β),2.34(1H,dd,J=14.2,2.4 Hz,H-2α),2.17(1H,m,H-2β),6.71(1H,dd,J=7.5,2.1 Hz,H-3),4.10(1H,d,J=3.2 Hz,H-7),2.49(1H,m,H-13),2.68(1H,dd,J=17.3,8.4 Hz,H-14α),2.17(1H,m,H-14β),4.47(1H,m,H-16α),3.93(1H,dd,J=9.1,7.1 Hz,H-16β),1.03(3H,d,J=7.1 Hz,H-17),0.86(3H,s,H-18),5.30(1H,d,J=7.6 Hz,H-19α),3.90(1H,dd,J=7.7,2.1 Hz,H-19β);13C-NMR(150 MHz,CDCl3)δ:19.5(C-1),27.8(C-2),135.1(C-3),139.2(C-4),45.0(C-5),40.6(C-6),72.6(C-7),40.5(C-8),38.5(C-9),48.3(C-10),34.8(C-11),26.7(C-12),36.3(C-13),36.3(C-14),176.8(C-15),73. 3(C-16),12.1(C-17),169.9(C-18),72.8(C-19),19.4(C-20)。以上数据与文献[8]报道基本一致,故鉴定为 7α-hydroxy-3,13-clerodadiene-16,15:18,19-diolide。
化合物9:白色晶体(二氯甲烷),mp 151~153 ℃;HR-ESI-MSm/z:333.206 2[M+H]+,分子式C20H28O4。1H-NMR(600 MHz,CDCl3)δ:6.75(1H,dd,J=7.5,2.2 Hz,H-3),2.67(1H,dd,J=17.3,8.4 Hz,H-14α),2.17(1H,dd,J=17.3,8.0 Hz,H-14β),4.46(1H,dd,J=9.0,7.3 Hz,H-16α),3.93(1H,d,J=1.9 Hz,H-16β),0.82(3H,d,J=8.0 Hz,H-17),4.29(1H,d,J=1.8 Hz,H-19α),3.90(1H,d,J=2.1 Hz,H-19β),0.59(3H,s,H-20);13C-NMR(150 MHz,CDCl3)δ:19.8(C-1),27.8(C-2),135.6(C-3),138.7(C-4),45.7(C-5),34.5(C-6),27.8(C-7),36.7(C-8),38.8(C-9),48.1(C-10),34.8(C-11),26.6(C-12),35.4(C-13),36.3(C-14),176.9(C-15),71.8(C-16),15.7(C-17),169.4(C-18),73.4(C-19),17.9(C-20)。以上数据与文献[9]报道基本一致,故鉴定为6,6,7,8,9,10-hexahydro-7,8-dimethyl-7-[2-(tetrahydro-5-oxo-3-furanyl)ethyl]-1H-naphtho[1,8-c]furan-3(5H)-one。
化合物10:白色晶体(二氯甲烷),mp 195~196 ℃;HR-ESI-MSm/z:347.185 7[M+H]+,分子式C20H26O5。1H-NMR(600 MHz,CDCl3)δ:1.54~1.79(6H,m,H-1,H-6,H-11),6.73(1H,dd,J=7.5,2.1 Hz,H-3),4.13(1H,d,J=3.1 Hz,H-7),1.97~2.48(6H,m,H-2,H-8,H-10,H-12),5.88(1H,t,J=1.7 Hz,H-14),1.06(3H,d,J=7.1 Hz,H-17),3.92(1H,dd,J=7.7,2.1 Hz,H-19α),5.30(1H,d,J=7.6 Hz,H-19β);13C-NMR(150 MHz,CDCl3)δ:19.4(C-1),27.8(C-2),135.0(C-3),139.2(C-4),45.0(C-5),40.6(C-6),72.4(C-7),40.6(C-8),38.6(C-9),48.5(C-10),35.6(C-11),22.3(C-12),169.8(C-13),115.6(C-14),173.8(C-15),73.1(C-16),12.2(C-17),19.3(C-18),72.7(C-19),169.9(C-20)。以上数据与文献[9]报道基本一致,故鉴定为7-[2-(2,5-dihydro-5-oxo-3-furanyl) ethyl]-6,6,7,8,9,10-hexahydro-9-hydroxy-7,8-dimethyl-1H-naphtho[1,8-c]furan-3(5H)-one。
化合物11:无色油状物;HR-ESI-MSm/z:337.237 2[M+H]+,分子式C20H32O4。1H-NMR(600 MHz,CDCl3)δ:2.34(1H,d,J=2.5 Hz,H-2α),2.16(1H,dd,J=4.9,2.2 Hz,H-2β),6.72(1H,dd,J=7.5,2.2 Hz,H-3),3.74~3.65(2H,m,H-15),0.93(3H,d,J=6.6 Hz,H-16),1.02(3H,d,J=7.2 Hz,H-17),5.27(1H,d,J=7.6 Hz,H-19α),3.90(1H,dd,J=7.6,2.2 Hz,H-19β),0.84(3H,s,H-20);13C-NMR(150 MHz,CDCl3)δ:19.4(C-1),29.9(C-2),135.3(C-3),139.3(C-4),45.1(C-5),40.0(C-6),73.0(C-7),40.0(C-8),38.4(C-9),48.3(C-10),35.9(C-11),27.8(C-12),30.2(C-13),40.5(C-14),61.3(C-15),19.9(C-16),19.6(C-17),170.1(C-18),73.0(C-19),12.0(C-20)。以上数据与文献[10]报道基本一致,故鉴定为7α-15-dihydroxy-ent-clerod-3-en-18,19-olide。
化合物12:黄色针晶(二氯甲烷),mp 188 ℃;HR-ESI-MSm/z:329.102 4[M+H]+,分子式C18H16O6。1H-NMR(600 MHz,CDCl3)δ:6.58(1H,s,H-3),6.55(1H,s,H-8),7.85(2H,d,J=6.8 Hz,H-2′,H-6′),7.02(2H,d,J=7.8 Hz,H-3′,H-5′),12.78(1H,s,5-OH),3.93(3H,s,6-OCH3),3.90(3H,s,7-OCH3),3.97(3H,s,4′-OCH3);13C-NMR(150 MHz,CDCl3)δ:164.2(C-2),104.3(C-3),182.8(C-4),153.4(C-5),132.7(C-6),158.9(C-7),90.7(C-8),153.2(C-9),106.3(C-10),123.7(C-1′),128.2(C-2′,C-6′),162.7(C-4′),114.7(C-3′,C-5′),61.0(6-OCH3),56.5(7-OCH3),55.7(4′-OCH3)。以上数据与文献[11]报道基本一致,故鉴定为鼠尾草素。
化合物13:黄色粉末;HR-ESI-MSm/z:329.101 4[M+H]+,分子式C18H16O6。1H-NMR(600 MHz,CDCl3)δ:6.59(1H,s,H-3),6.38(1H,s,H-6),6.50(1H,s,H-8),12.80(5-OH),7.34(1H,s,H-2′),6.98(1H,d,J=8.5 Hz,H-5′),7.53(1H,d,J=8.2 Hz,H-6′),3.98(3H,s,7-OCH3),3.89(3H,s,3′-OCH3),3.97(3H,s,4′-OCH3);13C-NMR(150 MHz,CDCl3)δ:165.6(C-2),104.8(C-3),182.5(C-4),157.8(C-5),98.2(C-6),164.1(C-7),92.8(C-8),162.3(C-9),105.7(C-10),123.9(C-1′),108.9(C-2′),152.4(C-3′),149.4(C-4′),111.3(C-5′),120.3(C-6′),56.0(7-OCH3),56.3(3′,4′-OCH3)。以上数据与文献[12]报道基本一致,故鉴定为5-羟基-7,3′,4′-三甲氧基黄酮。
化合物14:黄色粉末;HR-ESI-MSm/z:329.065 3[M-H]-,分子式C17H14O7。1H-NMR(600 MHz,CDCl3)δ:6.60(1H,s,H-3),6.56(1H,s,H-6),7.33(1H,d,J=1.8 Hz,H-2′),7.04(1H,d,J=8.5 Hz,H-5′),7.48(1H,dd,J=8.5,1.8 Hz,H-6′),13.08(1H,s,5-OH),4.01(3H,s,8-OCH3),4.05(3H,s,3′-OCH3);13C-NMR(150 MHz,CDCl3)δ:164.3(C-2),104.1(C-3),183.1(C-4),153.2(C-5),193.5(C-6),155.1(C-7),130.5(C-8),147.0(C-9),105.9(C-10),123.5(C-1′),108.4(C-2′),149.4(C-3′),152. 3(C-4′),115.1(C-5′),120. 9(C-6′),61.0(8-OCH3),56. 3(3′-OCH3)。以上数据与文献[13]报道基本一致,故鉴定为5,7,4′-三羟基-3′,8-二甲氧基黄酮。
化合物15:黄色粉末;HR-ESI-MSm/z:345.096 5[M+H]+,分子式C18H16O7。1H-NMR(600 MHz,CDCl3)δ:6.61(1H,s,H-3),6.56(1H,s,H-8),7.35(1H,d,J=2.2 Hz,H-2′),6.99(1H,d,J=8.5 Hz,H-5′),7.53(1H,dd,J=8.5,2.1 Hz,H-6′),4.00(3H,s,-OCH3),3.98(3H,s,-OCH3),3.97(3H,s,-OCH3);13C-NMR(150 MHz,CDCl3)δ:164.1(C-2),104.6(C-3),182.8(C-4),153.4(C-5),132.7(C-6),158.9(C-7),90.7(C-8),153.2(C-9),106.3(C-10),123.9(C-1′),108.8(C-2′),149.4(C-3′),152.4(C-4′),111.2(C-5′),120.2(C-6′),61.0(OCH3),56.5(OCH3),56.3(OCH3)。以上数据与文献[14]报道基本一致,故鉴定为异泽兰黄素。
化合物16:黄色粉末;HR-ESI-MSm/z:359.112 7[M+H]+,分子式C19H18O7。1H-NMR(600 MHz,CDCl3)δ:6.56(1H,s,H-3),6.61(1H,s,H-8),7.34(1H,d,J=1.8 Hz,H-2′),6.98(1H,d,J=8.5 Hz,H-5′),7.53(1H,dd,J=8.5,1.8 Hz,H-6′),12.76(1H,s,5-OH),3.99(3H,s,6-OCH3),3.98(3H,s,7-OCH3),3.97(3H,s,3′-OCH3),3.93(3H,s,4′-OCH3);13C-NMR(150 MHz,CDCl3)δ:164.1(C-2),104.6(C-3),182.8(C-4),153.4(C-5),132. 8(C-6),158.9(C-7),90.7(C-8),153.2(C-9),106.3(C-10),123.9(C-1′),111.3(C-2′),149.5(C-3′),152.4(C-4′),108.9(C-5′),120.2(C-6′),61.0(6-OCH3),56.5(3′-OCH3,7-OCH3),56.3(4′-OCH3)。以上数据与文献[15]报道基本一致,故鉴定为5-羟基-6,7,3′,4′-四甲氧基黄酮。
化合物17:黄色粉末;HR-ESI-MSm/z:611.303 6[M+H]+,分子式C27H30O16。1H-NMR(600 MHz,CD3OD)δ:6.21(1H,d,J=2.1 Hz,H-6),6.40(1H,d,J=2.1 Hz,H-8),7.66(1H,d,J=2.2 Hz,H-2′),6.87(1H,d,J=8.4 Hz,H-5′),7.63(1H,dd,J=8.4,2.2 Hz,H-6′),5.11(1H,d,J=7.7 Hz,H-1″);13C-NMR(150 MHz,CD3OD)δ:158.53(C-2),135.6(C-3),179.4(C-4),163.0(C-5),99.9(C-6),166.1(C-7),94.8(C-8),159.3(C-9),105.6(C-10),123.1(C-1′),116.0(C-2′),145.8(C-3′),149.8(C-4′),117.7(C-5′),123.5(C-6′),104.7(C-1″),75.7(C-2″),78.2(C-3″),71.4(C-4″),77.2(C-5″),68.5(C-6″),102.4(C-1‴),72.2(C-2‴),72.1(C-3‴),73.9(C-4‴),69.7(C-5‴),17.9(C-6‴)。以上数据与文献[16]报道基本一致,故鉴定为芦丁。
化合物18:黄色粉末;HR-ESI-MSm/z:303.021 1[M+H]+,分子式C15H10O7。1H-NMR(600 MHz,CD3OD)δ:6.18(1H,d,J=2.1 Hz,H-6),6.38(1H,d,J=2.1 Hz,H-8),7.73(1H,d,J=2.2 Hz,H-2′),6.88(1H,d,J=8.5 Hz,H-5′),7.63(1H,dd,J=8.5,2.2 Hz,H-6′);13C-NMR(150 MHz,CD3OD)δ:148.0(C-2),137.2(C-3),177.3(C-4),158.2(C-5),99.2(C-6),165.5(C-7),94.4(C-8),162.5(C-9),104.5(C-10),124.1(C-1′),116.0(C-2′),146.2(C-3′),148.8(C-4′),116.2(C-5′),121.6(C-6′)。以上波谱数据与文献[17]报道基本一致,故鉴定为槲皮素。
化合物19:黄色粉末;HR-ESI-MSm/z:287.050 1[M+H]+,分子式C15H10O6。1H-NMR(600 MHz,CD3OD)δ:6.18(1H,d,J=1.8 Hz,H-6),6.36(1H,d,J=1.8 Hz,H-8),8.08(2H,d,J=8.4 Hz,H-2′,H-6′),6.90(2H,d,J=8.5 Hz,H-3′,H-5′);13C-NMR(150 MHz,CD3OD)δ:147.9(C-2),137.2(C-3),177.4(C-4),162.4(C-5),99.9(C-6),167.2(C-7),94.9(C-8),158.4(C-9),104.2(C-10),123.9(C-1′),130.7(C-2′,C-6′),116.4(C-3′,C-5′),160.6(C-4′)。以上波谱数据与文献[18]报道基本一致,故鉴定为山柰酚。
化合物20:白色针晶(二氯甲烷),mp 161~170 ℃;HR-ESI-MSm/z:411.303 6[M-H]-,分子式C29H48O。1H-NMR(600 MHz,CDCl3)δ:3.52(1H,m,H-3),5.34(1H,d,J=4.0 Hz,H-6),0.72(3H,s,H-18),1.03(3H,s,H-19),1.04(3H,d,J=6.6 Hz,H-21),5.17(1H,dd,J=15.2,8.7 Hz,H-22),5.04(1H,dd,J=15.1,8.8 Hz,H-23),0.82(3H,d,J=7.4 Hz,H-26),0.87(3H,d,J=6.3 Hz,H-27),0.81(3H,t,J=7.7 Hz,H-29);13C-NMR(150 MHz,CDCl3)δ:37.4(C-1),32.0(C-2),72.0(C-3),42.4(C-4),140.9(C-5),121.9(C-6),29.8(C-7),31.8(C-8),50.3(C-9),36.2(C-10),21.3(C-11),39.8(C-12),42.4(C-13),57.0(C-14),24.5(C-15),29.1(C-16),56.1(C-17),12.2(C-18),19.6(C-19),40.7(C-20),21.4(C-21),138.5(C-22),129.4(C-23),51.4(C-24),31.3(C-25),21.2(C-26),19.1(C-27),25.6(C-28),12.4(C-29)。以上波谱数据与文献[19]报道基本一致,故鉴定为豆甾醇。
化合物21:白色粉末;HR-ESI-MSm/z:423.224 9[M-H]-,分子式C29H60O。1H-NMR(600 MHz,CDCl3)δ:0.88(3H,d,J=7.1 Hz),1.25(22H,s),1.40~1.44(2H,m),1.59(2H,s),1.25(22H,s),2.21(m,-CHOH),3.58(1H,s,-OH);13C-NMR(150 MHz,CDCl3)δ:14.1(-CH3),22.8(-CH2),25.8(-CH2),29.5(-CH2),29.5(-CH2),29.7(-CH2),29.8(-CH2),29.8(-CH2),29.9(-CH2),29.9(-CH2),32.1(-CH2),32.1(-CH2),37.6(-CH2),72.2(-CH)。以上数据与文献[20]报道基本一致,故鉴定为15-二十九烷醇。
化合物22:白色针晶(二氯甲烷),mp 147~148 ℃;HR-ESI-MSm/z:415.301 6[M+H]+,分子式C29H50O。1H-NMR(600 MHz,CDCl3)δ:3.52(1H,ddd,J=15.9,11.1,4.6 Hz,H-3),5.35(1H,d,J=5.2 Hz,H-6),0.68(3H,s,H-18),0.81(3H,d,J=6.8 Hz,H-19),0.83(3H,d,J=6.8 Hz,H-21),0.85(3H,d,J=7.4 Hz,H-26),0.92(3H,d,J=6.5 Hz,H-27),1.01(3H,s,H-29);13C-NMR(150 MHz,CDCl3)δ:37.4(C-1),19.2(C-2),72.0(C-3),42.4(C-4),140.9(C-5),121.9(C-6),32.0(C-7),32.1(C-8),50.3(C-9),36.7(C-10),21.2(C-11),39.9(C-12),42.5(C-13),56.9(C-14),24.5(C-15),28.4(C-16),56.2(C-17),12.0(C-18),18.9(C-19),36.3(C-20),31.8(C-21),34.1(C-22),26.2(C-23),46.0(C-24),29.3(C-25),20.0(C-26),19.6(C-27),23.2(C-28),12.1(C-29)。以上数据与文献[19]报道基本一致,故鉴定为 β-谷甾醇。
化合物23:无色针晶(甲醇),mp 144~145 ℃;HR-ESI-MSm/z:177.057 2[M-H]-,分子式C10H10O3。1H-NMR(600 MHz,CD3OD)δ:7.57(1H,d,J=15.9 Hz,H-1′),7.39(2H,d,J=8.2 Hz,H-2,H-6),6.86(2H,d,J=8.6 Hz,H-3,H-5),6.27(1H,d,J=15.9 Hz,H-2′),3.72(3H,s,-COOCH3);13C-NMR(150 MHz,CD3OD)δ:169.7,161.1,146.4,131.1,127.0,116.7,114.8,52.0。以上数据与文献[21]报道基本一致,故鉴定为对羟基苯甲酸乙酯。
化合物24:无色块状结晶(二氯甲烷),mp 170~173 ℃;HR-ESI-MSm/z:417.153 8[M-H]-,分子式C22H26O8。1H-NMR(600 MHz,CDCl3)δ:3.10(2H,d,J=5.3 Hz,H-1,H-5),4.73(2H,d,J=3.0 Hz,H-2,H-6),3.92(2H,m,H-4α,H-8β),4.28(2H,dd,J=8.7,5.9 Hz,H-4′α,H-8′β),6.58(4H,s,H-1′,H-1″),3.88(12H,s,-OCH3×4);13C-NMR(150 MHz,CDCl3)δ:54.5(C-1,C-5),86.2(C-2,C-6),72.0(C-4,C-8),132.2(C-1′,C-1″),102.8(C-2′,C-6′,C-2″,C-6″),147.3(C-3′,C-5′,C-3″,C-5″),134.4(C-4′,C-4″),56.5(4OCH3)。以上数据与文献[22]报道基本一致,故鉴定为丁香树脂醇。
化合物25:白色粉末;HR-ESI-MSm/z:429.121 0[M+H]+,分子式C30H52O。1H-NMR(600 MHz,CDCl3)δ:3.72(1H,m,H-3),0.93(3H,s,H-23),0.96(3H,s,H-24),0.86(3H,d,J=0.9 Hz,H-25),1.00(3H,s,H-26),0.99(3H,s,H-27),1.17(3H,s,H-28),0.99(3H,s,H-29),0.94(3H,s,H-30);13C-NMR(150 MHz,CDCl3)δ:15.9(C-1),35.2(C-2),72.9(C-3),49.3(C-4),37.2(C-5),41.8(C-6),17.7(C-7),53.3(C-8),38.5(C-9),61.5(C-10),35.7(C-11),30.8(C-12),38.0(C-13),39.8(C-14),32.5(C-15),36.2(C-16),30.2(C-17),42.9(C-18),35. 5(C-19),28.3(C-20),32.9(C-21),39. 4(C-22),11. 8(C-23),16.5(C-24),18.4(C-25),18.8(C-26),20.3(C-27),31.9(C-28),35.3(C-29),32.2(C-30)。以上数据与文献[23]报道基本一致,故鉴定为表无羁萜醇。
4 体外抑制MDA-MB-231乳腺癌细胞增殖实验
采用MTT法测试化合物15~18对MDA-MB-231细胞增殖的抑制活性。取对数生长期的细胞接种于96孔板中,每孔1×104个、100 μL,置于5% CO2、37 ℃培养箱中培养12 h,吸出培养基,实验组每孔分别加入不同浓度(10、100 μmol/mL)的药物100 μL,对照组每孔加入100 μL DMSO溶液,另设置空白组,每组设2个平行孔,置于5% CO2、37 ℃培养箱中孵育46 h。孵育结束后,每孔加入5 g/L MTT溶液 10 μL,混匀,继续培养4 h,弃去上清液后加入150 μL甲醇至药物充分溶解,酶标仪在490 nm波长处检测吸光度A,计算细胞存活率,公式为存活率=[(A实验组-A空白组)/(A对照组-A空白组)]× 100%。
结果显示,化合物15~16在10 μmol/mL时细胞存活率分别为(32.50±2.66)%、(24.77±2.32)%,对MDA-MB-231细胞增殖显示抑制作用,而17~18无明显作用。
5 讨论
本实验运用多种色谱、波谱鉴定技术对巴西酒神菊95%乙醇提取物的化学成分进行系统研究,共分离鉴定25个化合物,包括达玛烷型三萜、新克罗烷型二萜、黄酮等,其中化合物1~2、21为首次从该属植物中分离得到,3~7、12~15、23~24为首次从该植物中分离得到。巴西酒神菊具有保肝和抗炎等作用,推测其可能具有抗肿瘤作用,因此,对化合物15~18进行了体外抑制MDA-MB-231乳腺癌细胞增殖活性实验,发现15~16显示抑制作用。前期报道,新克罗烷型二萜具有抗蛇毒蛋白水解、抗出血、保肝等多种作用,符合巴西酒神菊的临床用途,故课题组后续将对其萜类成分进行系统的药理活性研究,有助于丰富拓展我国药用资源,促进中医药事业发展。