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止泻木子二氯甲烷部位的化学成分研究

2022-02-17欧阳胜王兴瑞兰亚平杨武亮

西北药学杂志 2022年6期
关键词:分子式木子二氯甲烷

欧阳胜,王兴瑞,兰亚平,施 旻,杨武亮

江西中医药大学,南昌 330004

止泻木子(Semenholarrhenae)是夹竹桃科(Apocynaceae)植物止泻木(HolarrhenaantidysentericaWall.ex A.DC.)的干燥种子,目前从止泻木属中分离得到了生物碱类等化学成分[1]。其常与连翘、地梢瓜组合成复方药物[2]。止泻木子具有清热、利胆、止泻的作用,用于治疗赤巴病、肝胆病、胃肠热病、腹泻、痢疾等[3]。且进一步研究发现,锥丝碱具有较好的乙酰胆碱酯酶抑制活性,在杀灭寄生虫、抗菌等方面也具有较好的药理活性[4-9]。

现对止泻木子的化学成分进行了研究,从中分离得到了11个化合物,通过理化性质及现代波谱分析,分别鉴定为羽扇豆醇乙酸酯(1),羽扇豆醇硬脂酸酯(2),羽扇豆醇棕榈酸酯(3),α-香树脂酮(4),α-香树醇乙酸酯(5),α-香树醇棕榈酸酯(6),β-谷甾醇棕榈酸酯(7),5-Methyl-5H-pyrido[4,3-b]indole(8),(Z)-10-Eicosenoic acid(9),十七碳酸甲酯(10),邻苯二甲酸二正戊酯(11)。化合物1~11均为首次从止泻木子中分离得到。

1 仪器与试药

1.1 仪器

RE-5203型旋转蒸发器(上海亚荣生化仪器厂);Q-300B型超声波清洗器(昆山市超声仪器有限公司)。

1.2 试药

止泻木子(产地尼泊尔),由杨武亮教授鉴定为止泻木的干燥种子(Semenholarrhenae);柱色谱硅胶(青岛海洋化工厂);其他试药均为分析纯。

2 提取与分离

将6.738 kg的止泻木子油渣全部放入预先准备好的渗漉桶中,以大约10倍量体积分数为95%乙醇渗漉提取,减压浓缩至无醇味。将浓缩液用石油醚溶解,用一定体积分数的酸性乙醇(pH2~3)萃取4次左右,直至醇部位澄清透亮。分别得到醇部位和石油醚部位。石油醚不能溶解的浸膏用蒸馏水溶解,加入2 mol·L-1的盐酸,进行酸化,使溶液酸化至pH3~4左右,加入二氯甲烷萃取3次左右,再向酸化后的体系中加入25 mL·L-1的氨水,使溶液碱化至pH10左右,再向碱化后的溶液中加入二氯甲烷萃取,多次萃取直至二氯甲烷萃取液无色。得到二氯甲烷部位Ⅰ、二氯甲烷部位Ⅱ、碱水层。通过硅胶柱层析及重结晶等方法,从二氯甲烷部位Ⅰ(84.3 g)中分离得到以下11个化合物。

先用石油醚冲洗硅胶柱。洗脱液首先采用石油醚-二氯甲烷(1∶1)开始洗脱,之后分别用二氯甲烷、二氯甲烷-甲醇不同比例洗脱,洗脱梯度逐渐降低。柱体积每1/3收集一次流份,浓缩,得到各个流份,用薄层色谱法(TLC)进行检测,相同斑点合并。最后得到10个组分。其中组分1~4合并后的样品继续用TLC摸索下一步柱层析条件,再用硅胶柱层析法进一步分离,直到TLC检测为单一点,且换过3种不同的展开系统也保持单点。

组分1(3.6 g)用石油醚等度洗脱,得到的组分1~1(0.8 g)继续用石油醚-二氯甲烷(12∶1)为洗脱剂等度洗脱,分别得到化合物1、化合物3和化合物5。组分2(0.8 g)用石油醚-二氯甲烷(9∶1)等度洗脱,得到化合物11。组分3(6.8 g)分别用不同比例的石油醚:二氯甲烷(20∶1,10∶1,5∶1,1∶1)、二氯甲烷梯度洗脱,分别得到组分3~1,3~2,3~3,3~4。其中3~1(1.6 g)用石油醚-二氯甲烷(10∶1)等度洗脱,得到化合物10。3~2(1.2 g)用石油醚-二氯甲烷(8∶1)等度洗脱,得到化合物4和化合物9。3~4(2.2 g)用石油醚-二氯甲烷(4∶1)等度洗脱,得到了化合物2、化合物6和化合物7。组分4(2.5 g)用二氯甲烷-甲醇(20∶1)等度洗脱,其中的组分4~1(0.6 g)以二氯甲烷-甲醇(10∶1)为展开剂,通过薄层色谱法,得到化合物8。

3 结构鉴定

化合物1分子式C32H52O2,白色针状结晶,1H-NMR (CDCl3,400 MHz)δ: 0.78(3H,s,H-28),0.83(3H,s,H-24),0.84(3H,s,H-23),0.85(3H,s,H-25),0.93(3H,s,H-27),1.03(3H,s,H-26),1.68(3H,s,30-CH3),2.04(3H,s,O=C-CH3),4.47(1H,m,H-3),4.56(1H,d,J=2.4 Hz,H-30),4.68(1H,d,J=2.4 Hz,H-3)。13C-NMR(CDCl3,100MHz)δ:14.5(C-27),16.0(C-26),16.2(C-25),16.5(C-24),18.0(C-28),18.2(C-6),19.3(C-29),20.9(C-11),21.4(C-31),23.7(C-2),25.1(C-12),27.4(C-15),28.0(C-23),29.8(C-21),34.2(C-7),35.6(C-16),37.1(C-10),37.8(C-4),38.0(C-1),38.4(C-13)40.0(C-22),40.8(C-8),42.8(C-14),43.0(C-17),48.0(C-18),48.3(C-19),50.3(C-9),55.4(C-5),81.0(C-3),109.4(C-30),151.0(C-20),171.1(C-32)。以上数据与文献[10]报道基本一致,故鉴定化合物1为lupeol acetate。

化合物2分子式C48H84O2,1H-NMR(400 MHz,CDCl3) δ: 4.57、4.68(1H,ABdd,J=2.4 Hz,2H-29),4.47(1H,dd,J=6.8 Hz,J=5.2 Hz,H-3),2.37(1H,S,H-19),1.68(3H,m,30-CH3),1.02、0.93、0.87、0.85、0.83、0.78(3H,m,CH3),0.76(3H,t,J=8.0Hz),1.2-1.32(28H,br),2.28(2H,t,J=6.2Hz),1.37(2H,m)。13C-NMR(400 MHz,CDCl3)δ: 38.4(C-1),23.8(C-2),80.6(C-3),37.8(C-4),55.4(C-5),18.2(C-6),34.2(C-7),40.8(C-8),50.3(C-9),37.1(C-10),20.9(C-11),25.1(C-12),38.0(C-13),42.8(C-14),27.4(C-15),35.6(C-16),43.0(C-17),48.3(C-18),48.0(C-19),151.0(C-20),29.7(C-21),40.0(C-22),28.0(C-23),16.6(C-24),16.2(C-25),16.0(C-26),14.5(C-27),18.0(C-28),109.4(C-29),20.9(C-30),173.7(C-1′),34.9(C-2′),31.9(C-16′),29.7(C-11′),29.7(C-10′),29.7(C-9′),29.7(C-8′),29.80(C-5′),29.7(C-14′),29.7(C-13′),29.7(C-12′),29.6(C-7′),29.5(C-15′),29.4(C-6′),29.3(C-5′),29.2(C-4′),25.2(C-3′),22.7(C-17′),14.2(C-18′)。以上数据与文献[11]报道基本一致,故鉴定化合物2为lupeol stearate。

化合物3分子式C46H80O2,1H-NMR(400 MHz,CDCl3) δ: 4.57、4.68(1H,ABdd,J=2.4 Hz,2H-29),4.47(1H,dd,J=6.8 Hz,J=5.2 Hz,H-3),2.38(1H,S,H-19),1.68(3H,m,30-CH3),1.03,0.94,0.85,0.84,0.83,0.78(3H,m,CH3),0.76(3H,t,J=8.0Hz),1.2-1.32(28H,br),2.28(2H,t,J=6.2 Hz)。13C-NMR(400 MHz,CDCl3)δ: 38.4(C-1),23.8(C-2),80.6(C-3),37.8(C-4),55.4(C-5),18.2(C-6),34.2(C-7),40.9(C-8),50.3(C-9),37.1(C-10),20.9(C-11),25.1(C-12),38.0(C-13),42.8(C-14),27.4(C-15),35.6(C-16),43.0(C-17),48.3(C-18),48.0(C-19),151.0(C-20),29.7(C-21),40.0(C-22),28.0(C-23),16.6(C-24),16.2(C-25),16.0(C-26),14.5(C-27),18.0(C-28),109.4(C-29),20.9(C-30),173.8(C-1′),34.9(C-2′),31.9(C-3′),29.7(C-4′),29.80(C-5′),29.7(C-6′),29.7(C-7′),29.7(C-8′),29.6(C-9′),29.5(C-10′),29.4(C-11′),29.3(C-12′),29.2(C-13′),25.2(C-14′),22.7(C-15′),14.2(C-16′)。以上数据与文献[12]报道基本一致,故鉴定化合物3为lupenyl palmitate。

化合物4分子式C30H48O,1H-NMRδ:0.79(3H,brs,H-29),0.80(3H,s,H28),0.87(3H,s,H-30),0.88(3H,s,H-26),0.92(3H,s,H-24),0.98(3H,s,H-25),1.01(3H,s,H27),1.07(3H,s,H-23),5.15(1H,t,J=4.2, Hz,H12)。13C-NMR(150 MHz,CDCl3)δ:15.5(C-25),16.8(C-26),17.5(C-29),19.7(C-6),21.4(C-30),21.5(C-24),23.2(C-27),23.6(C-11),26.6(C-16),28.1(C-23),28.8(C-15),29.4(C-28),31.3(C-21),32.5(C-7),33.8(C-17),34.2(C-2),36.6(C-10),39.5(C-1),39.6(C19),39.7(C-20),40.0(C-8),41.5(C-22),42.2(C-14),46.9(C-9),47.5(C-4),55.3(C-5),59.1(C-18),218.0(C-3),124.2(C-12),139.7(C-13)。分析以上波谱数据,与文献[13]数据基本一致,故鉴定化合物4为α-香树脂酮。

化合物5分子式C32H52O2,1H-NMR(CDCl3,500 MHz)δ:5.13(1H,t,J=3.5 Hz,H-12),4.50(1H,dd,J=6.0,10.0 Hz,H-3),2.05(3H,s,H3-Ac),1.07(3H,s,H3-27),1.01(3H,s,H3-26),0.98(3H,s,H3-25),0.92(3H,d,J=6.5 Hz,H3-30),0.88(3H,s,H3-23),0.87(3H,s,H3-24),0.80(3H,s,H3-28),0.79(3H,d,J=4.5 Hz,H3-29)。13C-NMR(CDCl3,125 MHz)δ:38.5(C-1),23.6(C-2),81.0(C-3),37.7(C-4),55.3(C-5),18.2(C-6),32.9(C-7),40.0(C-8),47.6(C-9),36.8(C-10),23.4(C-11),124.3(C-12),139.6(C-13),42.1(C-14),26.6(C-15),28.1(C-16),33.8(C-17),59.1(C-18),39.6(C-19),39.7(C-20),31.3(C-21),41.5(C-22),28.1(C-23),16.8(C-24),15.8(C-25),16.9(C-26),23.2(C-27),28.1(C-28),17.5(C-29),21.4(C-30),171.0(Ac-CO),21.4(Ac-CH3)。碳谱及氢谱的数据与文献[14]报道的一致,故鉴定化合物5为α-amyrin acetate。

化合物6分子式C46H80O2,白色粉末,1H-NMR(CDCl3, 500 MHz)δ:5.12(1H,t,J=3.5 Hz,H-12),4.50(1H,dd,J=6.0,10.0 Hz,H-3),2.05(3H,s,H3-Ac),1.07(3H,s,H3-27),1.01(3H,s,H3-26),0.98(3H,s,H3-25),0.92(3H,d,J=6.5 Hz,H3-30),0.88(3H,s,H3-23),0.87(3H,s,H3-24),0.80(3H,s,H3-28),0.79(3H,d,J=4.5 Hz,H3-29)。13C-NMR δ:38.4(C-1),23.6(C-2),80.6(C-3),37.8(C-4),55.3(C-5),18.3(C-6),32.9(C-7),39.7(C-8),47.6(C-9),36.8(C-10),23.6(C-11),124.3(C-12),139.6(C-13),42.1(C-14),26.6(C-15),28.1(C-16),33.8(C-17),59.1(C-18),39.7(C-19),39.6(C-20),32.9(C-21),41.5(C-22),28.1(C-23),16.8(C-24),15.7(C-25),16.9(C-26),23.4(C-27),28.1(C-28),17.5(C-29),21.4(C-30),173.7(C-1′),34.9(C-2′),25.2(C-3′),29.2(C-4′),29.3(C-5′),29.4(C-6′),29.7(C-7′),29.7(C-8′),29.7(C-9′),29.7(C-10′),29.7(C-11′),29.7(C-12′),29.7(C-13′),32.0(C-14′),22.7(C-15′),14.2(C-16′)。以上数据与文献[15]报道一致,故鉴定化合物6为3β-palmitoyl-α-amyrin。

化合物7C45H80O2,1H-NMR(400 MHz,CDCl3) δ:5.36(1H,d,J=5.2 Hz,H-6),3.53(1H,m,H-24),2.35(2H,t,J=7.2 Hz,H-4),2.24(2H,m,H-2′),1.00(3H,s,H-19),0.92(3H,m,H-21),0.87(3H,m,H-16′),0.84(3H,s,H-26),0.82(3H,m,H-29),0.81(3H,s,H-27),0.68(3H,s,H-18)。13C-NMR(100 MHz,CDCl3) δ: 177.7(C-1′),140.7(C-5),121.8(C-6),71.8(C-3),56.8(C-14),56.0(C-17),50.1(C-9),45.8(C-24),42.3(C-4,C-13),39.8(C-12),37.3(C-1),36.5(C-10),36.2(C-20),33.9(C-2′),33.7(C-22),31.9(C-7),31.9(C-8,C-14′),31.7(C-2),29.7(C-9′,C-10′,C-11′,C-12′,C-13′),29.6(C-8′),29.5(C-7′),29.4(C-6′),29.3(C-5′),29.1(C-25,C-4′),28.3(C-16),26.0(C-23),24.7(C-3′),24.3(C-15),23.1(C-28),22.7(C-15′),21.1(C-11),19.8(C-26),19.4(C-19),19.0(C-27),18.8(C-21),14.1(C-16′),12.0(C-29),11.9(C-18)。以上数据与文献[16]报道基本一致,故鉴定化合物7为β-sitosterol palmitate。

化合物8分子式C12H10N2,白色粉末,1H-NMR(CDCl3) δ 3.80(s,3H),7.28(br s,1H),7.32(dt,J=7.8,0.9 Hz,1H),7.42(d,J=8.1 Hz,1H),7.53(dt,J=8.1,0.9 Hz,1H),8.13(d,J=7.8 Hz,1H),8.60(br s,1H),9.30(br s,1H)。13C-NMR(CDCl3) δ 29.7,106.2,111.3,120.2,120.7,121.1,121.4,127.2,139.3,142.0,143.9,144.0。以上数据与文献[17]报道基本一致,故鉴定化合物8为5-methyl-5H-pyrido[4,3-b]indole。

化合物9分子式C20H38O2,白色粉末,1H-NMR(600 MHz,CDCl3)δ:5.43(1H,m,H-10),5.35(1H,d,J=4.9 Hz,H-11),2.21(2H,t,J=7.6 Hz,H-2),2.01(4H,q,J=6.4 Hz,H-9,12),1.62(2H,m,H-3),1.25(24H,m,H-4~8,13~18),0.88(3H,t,J=6.7 Hz,H-19)。13C-NMR(151 MHz,CDCl3)δ:175.7(C-1),35.9(C-2),25.5(C-3),29.5(C-4),29.5(C-5),29.5(C-6),29.3(C-7),29.3(C-8),29.2(C-9),129.9(C-10),129.9(C-11),27.2(C-12),29.6(C-13),29.6(C-14),29.6(C-15),29.7(C-16),29.8(C-17),31.9(C-18),22.7(C-19),14.1(C-20)。1H-NMR和13C-NMR数据与文献[18]报道的基本一致,故鉴定化合物9为(Z)-10-eicosenoic acid。

化合物10分子式C18H36O2,1H-NMR(500 MHz,CDCl3) δ0.89(3H,t,J=6.8 Hz) δ3.66(3H,s) δ2.30(2H,t,J=7.5 Hz)和1.61(2H,m,J=8.2 Hz) δ1.29(28H,m)表示-CH2的质子信号。13C-NMR(500 MHz, CDCl3) δ174.3表示C=O的碳信号,δ51.4为甲氧基的碳信号,δ14.1为甲基碳信号,δ34.2,31.9,29.7,29.7,29.5,29.4,29.3,29.2,25.0,22.7为-CH2碳信号。1H-NMR和13C-NMR数据与文献[19]报道的基本一致,故鉴定化合物10为methyl heptadecanoate。

化合物11分子式C18H26O4,1H-NMR(400 MHz,CDCl3)δ:7.71(2H,dd,J=3.2,5.6 Hz,H-3,6),7.52(2H,dd,J=3.2,5.6 Hz,H-4,5),4.30(4H,t,J=6.8 Hz,H-8,8′),1.44(4H,m,H-10,10′),1.71(4H,m,H-9,9′),1.25(4H,s,H-11,11′),0.96(6H,t,J=7.2 Hz,H-12,12′)。13C-NMR(100 MHz,CDCl3)δ:132.3(C-1,2),128.8(C-3,6),130.9(C-4,5),167.7(C-7,7′),65.6(C-8,8′),30.6(C-9,9′),29.7(C-10,10′),19.2(C-11,11′),13.8(C-12,12′)。以上数据与文献[20]报道基本一致,故鉴定化合物11为dipentyl phthalate。

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