止瀉木子二氯甲烷部位的化學(xué)成分研究

歐陽(yáng)勝，王興瑞，蘭亞平，施 旻，楊武亮

江西中醫(yī)藥大學(xué)，南昌 330004

止瀉木子(Semenholarrhenae)是夾竹桃科(Apocynaceae)植物止瀉木(HolarrhenaantidysentericaWall.ex A.DC.)的干燥種子，目前從止瀉木屬中分離得到了生物堿類等化學(xué)成分[1]。其常與連翹、地梢瓜組合成復(fù)方藥物[2]。止瀉木子具有清熱、利膽、止瀉的作用，用于治療赤巴病、肝膽病、胃腸熱病、腹瀉、痢疾等[3]。且進(jìn)一步研究發(fā)現(xiàn)，錐絲堿具有較好的乙酰膽堿酯酶抑制活性，在殺滅寄生蟲、抗菌等方面也具有較好的藥理活性[4-9]。

現(xiàn)對(duì)止瀉木子的化學(xué)成分進(jìn)行了研究，從中分離得到了11個(gè)化合物，通過理化性質(zhì)及現(xiàn)代波譜分析，分別鑒定為羽扇豆醇乙酸酯(1)，羽扇豆醇硬脂酸酯(2)，羽扇豆醇棕櫚酸酯(3)，α-香樹脂酮(4)，α-香樹醇乙酸酯(5)，α-香樹醇棕櫚酸酯(6)，β-谷甾醇棕櫚酸酯(7)，5-Methyl-5H-pyrido[4,3-b]indole(8)，(Z)-10-Eicosenoic acid(9)，十七碳酸甲酯(10)，鄰苯二甲酸二正戊酯(11)?；衔?～11均為首次從止瀉木子中分離得到。

1 儀器與試藥

1.1 儀器

RE-5203型旋轉(zhuǎn)蒸發(fā)器(上海亞榮生化儀器廠)；Q-300B型超聲波清洗器(昆山市超聲儀器有限公司)。

1.2 試藥

止瀉木子(產(chǎn)地尼泊爾)，由楊武亮教授鑒定為止瀉木的干燥種子(Semenholarrhenae)；柱色譜硅膠(青島海洋化工廠)；其他試藥均為分析純。

2 提取與分離

將6.738 kg的止瀉木子油渣全部放入預(yù)先準(zhǔn)備好的滲漉桶中，以大約10倍量體積分?jǐn)?shù)為95%乙醇滲漉提取，減壓濃縮至無醇味。將濃縮液用石油醚溶解，用一定體積分?jǐn)?shù)的酸性乙醇(pH2～3)萃取4次左右，直至醇部位澄清透亮。分別得到醇部位和石油醚部位。石油醚不能溶解的浸膏用蒸餾水溶解，加入2 mol·L-1的鹽酸，進(jìn)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，再向酸化后的體系中加入25 mL·L-1的氨水，使溶液堿化至pH10左右，再向堿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無色。得到二氯甲烷部位Ⅰ、二氯甲烷部位Ⅱ、堿水層。通過硅膠柱層析及重結(jié)晶等方法，從二氯甲烷部位Ⅰ(84.3 g)中分離得到以下11個(gè)化合物。

先用石油醚沖洗硅膠柱。洗脫液首先采用石油醚-二氯甲烷(1∶1)開始洗脫，之后分別用二氯甲烷、二氯甲烷-甲醇不同比例洗脫，洗脫梯度逐漸降低。柱體積每1/3收集一次流份，濃縮，得到各個(gè)流份，用薄層色譜法(TLC)進(jìn)行檢測(cè)，相同斑點(diǎn)合并。最后得到10個(gè)組分。其中組分1～4合并后的樣品繼續(xù)用TLC摸索下一步柱層析條件，再用硅膠柱層析法進(jìn)一步分離，直到TLC檢測(cè)為單一點(diǎn)，且換過3種不同的展開系統(tǒng)也保持單點(diǎn)。

組分1(3.6 g)用石油醚等度洗脫，得到的組分1～1(0.8 g)繼續(xù)用石油醚-二氯甲烷(12∶1)為洗脫劑等度洗脫，分別得到化合物1、化合物3和化合物5。組分2(0.8 g)用石油醚-二氯甲烷(9∶1)等度洗脫，得到化合物11。組分3(6.8 g)分別用不同比例的石油醚：二氯甲烷(20∶1,10∶1,5∶1,1∶1)、二氯甲烷梯度洗脫，分別得到組分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)等度洗脫，得到化合物10。3～2(1.2 g)用石油醚-二氯甲烷(8∶1)等度洗脫，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)等度洗脫，得到了化合物2、化合物6和化合物7。組分4(2.5 g)用二氯甲烷-甲醇(20∶1)等度洗脫，其中的組分4～1(0.6 g)以二氯甲烷-甲醇(10∶1)為展開劑，通過薄層色譜法，得到化合物8。

3 結(jié)構(gòu)鑒定

化合物1分子式C32H52O2，白色針狀結(jié)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)。以上數(shù)據(jù)與文獻(xiàn)[10]報(bào)道基本一致，故鑒定化合物1為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′)。以上數(shù)據(jù)與文獻(xiàn)[11]報(bào)道基本一致，故鑒定化合物2為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′)。以上數(shù)據(jù)與文獻(xiàn)[12]報(bào)道基本一致，故鑒定化合物3為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)。分析以上波譜數(shù)據(jù)，與文獻(xiàn)[13]數(shù)據(jù)基本一致，故鑒定化合物4為α-香樹脂酮。

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)。碳譜及氫譜的數(shù)據(jù)與文獻(xiàn)[14]報(bào)道的一致，故鑒定化合物5為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′)。以上數(shù)據(jù)與文獻(xiàn)[15]報(bào)道一致，故鑒定化合物6為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)。以上數(shù)據(jù)與文獻(xiàn)[16]報(bào)道基本一致，故鑒定化合物7為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。以上數(shù)據(jù)與文獻(xiàn)[17]報(bào)道基本一致，故鑒定化合物8為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數(shù)據(jù)與文獻(xiàn)[18]報(bào)道的基本一致，故鑒定化合物9為(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的質(zhì)子信號(hào)。13C-NMR(500 MHz, CDCl3) δ174.3表示C=O的碳信號(hào),δ51.4為甲氧基的碳信號(hào),δ14.1為甲基碳信號(hào),δ34.2,31.9,29.7,29.7,29.5,29.4,29.3,29.2,25.0,22.7為-CH2碳信號(hào)。1H-NMR和13C-NMR數(shù)據(jù)與文獻(xiàn)[19]報(bào)道的基本一致，故鑒定化合物10為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′)。以上數(shù)據(jù)與文獻(xiàn)[20]報(bào)道基本一致，故鑒定化合物11為dipentyl phthalate。