毛果鱼藤化学成分研究

毛果鱼藤化学成分研究

毛果鱼条肝科植物区系的物种。花期6~7月,果期9月至翌年1月。生于山地疏林中,海拔1200~1400m。主要分布在广西、云南一带,别名为土甘草、鸡血藤、藤甘草等［1］。其根入药能补血、润肠,用于发烧胸闷、咳嗽、咽喉痛,藤茎具有利尿除湿、镇咳化痰之功效,民间常用来治疗肾炎、膀胱炎、尿道炎、咳嗽等症［2-4］。文献曾对其化学成分进行预试研究,推测该植物中含有氨基酸、皂苷、黄酮、蒽醌、三萜等化合物。目前毛果鱼藤中已报道的化学成分仅三萜类化合物［6］,其他类型的化学成分尚未见报道。为了进一步了解毛果鱼藤化学成分,以及研究其中是否含有抗结核分枝杆菌活性成分,本实验对毛果鱼藤化学成分进行了系统的研究,从中分离得到9个化合物,其中除化合物1、6外,其他化合物均为首次从该植物中分离得到。1显微熔点测定VarianINOVA-400MHz核磁共振波谱仪(TMS内标);HP-5973型质谱仪(EI);HP1100-MSD型液相色谱质谱联用仪(ESI);XT-4型显微熔点测定仪(温度计未校正,北京泰克仪器有限公司)。默克反相硅胶,柱层析硅胶(40~80目、200~300目和300~400目)和薄层层析硅胶(GF254)均为青岛海洋化工有限公司生产,SephadexLH-20为瑞典Amersham公司生产。毛果鱼藤药材采自贵州省兴义市,经贵阳市中医学院陈德媛教授鉴定为豆科鱼藤属植物毛果鱼藤DerriseriocarpaHow,样本保存在贵州省中国科学院天然产物化学重点实验室。2化合物24的合成36kg毛果鱼藤干燥全草粉碎后,分别用95%乙醇充分浸泡提取1次,75%乙醇充分浸泡提取2次,合并提取液,减压蒸馏回收乙醇至无醇味,浸膏加水溶解,分别用石油醚、乙酸乙酯和正丁醇各萃取3次,得到石油醚部位浸膏482g,乙酸乙酯部位浸膏311g,正丁醇部位浸膏537g。石油醚部位浸膏进行硅胶柱色谱(200~300目)分离,用石油醚-乙酸乙酯(100∶1~0∶1)进行梯度洗脱,经TLC检测合并相同流分,得到7个组分(Fr.1~Fr.7)。Fr.3经硅胶柱色谱(石油醚-乙酸乙酯50∶1)分离得到化合物1。Fr.4反复运用硅胶柱色谱(石油醚-乙酸乙酯、石油醚-丙酮系统)及SephadexLH-20(三氯甲烷-甲醇1∶1)分离纯化,得到化合物2~4。乙酸乙酯部位浸膏进行硅胶柱色谱(200~300目)分离,以石油醚-丙酮(50∶1~0∶1)为洗脱剂进行梯度洗脱,经TLC检测合并了相同流分,得到6个组分(Fr.1~Fr.6)。Fr.2经硅胶柱色谱(石油醚-丙酮系统)和重结晶分离纯化,得到化合物5~6。Fr.4以石油醚-丙酮(10∶1)为洗脱剂进行硅胶柱层析,分离得到化合物7。Fr.6经反相硅胶柱色谱(甲醇-水5∶95-100∶0)梯度洗脱析出无色晶体,用SephadexLH-20(三氯甲烷-甲醇1∶1)及重结晶纯化,得到化合物8~9。3化合物3的合成化合物1:白色粉末(乙酸乙酯),mp197~199℃。EI-MSm/z426[M]+,411,218,203,189。1H-NMR(CDCl3,400MHz)δ:5.18(1H,t,J=3.6Hz,H-12),3.21(1H,t,J=4.8Hz,H-3),1.14(3H,s,H-27),1.03(3H,s,H-23),1.00(3H,s,H-25),0.97(3H,s,H-24),0.93(3H,s,H-29),0.86(3H,s,H-30),0.84(3H,s,H-28),0.78(3H,s,H-26)。13C-NMR(CDCl3,100MHz),δ:38.6(C-1),28.0(C-2),78.6(C-3),38.9(C-4),53.0(C-5),18.6(C-6),32.8(C-7),40.1(C-8),47.8(C-9),37.6(C-10),23.7(C-11),121.9(C-12),143.2(C-13),42.0(C-14),28.4(C-15),27.0(C-16),32.8(C-17),37.3(C-18),35.8(C-19),31.9(C-20),35.2(C-21),36.1(C-22),28.9(C-23),17.5(C-24),15.8(C-25),17.9(C-26),27.6(C-27),28.9(C-28),33.8(C-29),24.6(C-30)。以上数据与文献报道的β-香树脂醇的数据基本一致。化合物2:淡黄色油状物(乙酸乙酯),ESI-MSm/z319[M+Na]+。1H-NMR(CDCl3,400MHz)δ:10.60(1H,brs,-OH),5.35-5.45(2H,m,H-10,11),2.35(2H,t,J=7.6Hz,H-2),2.00(4H,m,H-9,12),1.63(2H,m,H-3),1.25-1.30(22H,m,H-4,5,6,7,8,13,14,15,16,17,18),0.88(3H,t,J=6.4Hz,H-19)。13C-NMR(CDCl3,100MHz)δ:179.7(C-1),34.0(C-2),24.7(C-3),29.1(C-4),29.1(C-5),29.5(C-6),29.7(C-7),29.4(C-8),27.2(C-9),129.7(C-10),130.0(C-11),27.2(C-12),29.4(C-13),29.8(C-14),29.6(C-15),29.2(C-16),31.9(C-17),22.7(C-18),14.1(C-19)。以上数据与文献报道的顺式-10-十九碳烯酸的数据一致。化合物3:无色针晶(丙酮),mp166~168℃。EI-MSm/z412[M]+,398,370,289,271,245,229,124,107,95,69,65。1H-NMR(CDCl3,400MHz)δ:5.72(1H,s,H-4),2.44~2.23(4H,m,H-2,6),2.06~1.98(3H,m,H-1,12),1.88~1.80(2H,m,H-7,15),1.74~1.65(2H,m,H-1,24),1.65~1.46(3H,m,H-8,11,16),1.39~1.09(10H,m,H-11,15,16,17,20,22,23,28),1.05~0.98(3H,m,H-7,14,22),0.98~0.88(2H,m,H-9,25),1.18(3H,s,19-CH3),0.93(3H,d,J=6.4Hz,21-CH3),0.84(3H,t,J=7.2Hz,29-CH3),0.82(3H,d,J=1.6Hz,26-CH3),0.80(3H,d,J=7.6Hz,27-CH3),0.71(3H,s,18-CH3)。13C-NMR(CDCl3,100MHz).δ:35.6(C-1),33.9(C-2),199.7(C-3),123.7(C-4),171.7(C-5),32.9(C-6),32.0(C-7),35.6(C-8),53.8(C-9),38.6(C-10),21.0(C-11),39.6(C-12),42.3(C-13),55.8(C-14),28.2(C-15),24.1(C-16),55.9(C-17),11.9(C-18),17.3(C-19),36.1(C-20),18.7(C-21),33.8(C-22),26.0(C-23),29.1(C-24),45.8(C-25),19.8(C-26),19.0(C-27),23.0(C-28),11.9(C-29)。以上数据与文献报道的豆甾-4-烯-3-酮的数据基本一致。化合物4:无色针晶(丙酮),mp165~167℃。EI-MSm/z:424[M]+,409,313,245,218,205,189,149,109,95。1H-NMR(CDCl3,400MHz)δ:4.69(1H,d,J=2.4Hz,H-20α),4.57(1H,d,J=2.4Hz,H-20β),1.71(3H,s,H-30),1.07(3H,s,H-28),1.07(3H,s,H-27),1.03(3H,s,H-26),0.96(3H,s,H-25),0.93(3H,s,H-24),0.80(3H,s,H-23)。13C-NMR(CDCl3,100MHz),δ:38.1(C-1),35.5(C-2),218.3(C-3),47.9(C-4),54.9(C-5),19.6(C-6),33.5(C-7),43.0(C-8),48.2(C-9),36.9(C-10),21.4(C-11),25.1(C-12),39.6(C-13),40.7(C-14),27.4(C-15),34.1(C-16),42.9(C-17),49.8(C-18),47.3(C-19),150.9(C-20),29.8(C-21),40.0(C-22),26.6(C-23),21.0(C-24),18.0(C-25),15.8(C-26),14.5(C-27),16.0(C-28),109.4(C-29),19.3(C-30)。以上数据与文献报道的羽扇-20(29)-烯-3-酮的数据一致。化合物5:白色针晶(三氯甲烷),mp284~286℃。EI-MSm/z:456[M]+,423,248,189,175。1H-NMR(CDCl3,400MHz)δ:12.08(1H,s,brs,28-COOH),4.69(1H,d,J=2.8Hz,H-29a),4.56(1H,d,J=2.8Hz,H-29b),3.13(1H,dd,J=1.6,2.0Hz,H-3),3.02(1H,dd,J=5.2,9.6Hz,H-19),1.66(3H,s,H-30),0.97(3H,s,H-27),0.96(3H,s,H-26),0.93(3H,s,H-25),0.83(3H,s,H-24),0.72(3H,s,H-23)。13C-NMR(CDCl3,100MHz)δ:37.6(C-1),28.0(C-2),79.5(C-3),39.0(C-4),56.1(C-5),19.0(C-6),35.2(C-7),41.8(C-8),51.8(C-9),38.1(C-10),21.4(C-11),26.4(C-12),37.0(C-13),42.8(C-14),28.9(C-15),33.3(C-16),56.9(C-17),48.6(C-18),47.8(C-19),152.7(C-20),31.8(C-21),38.6(C-22),28.2(C-23),15.6(C-24),16.6(C-25),16.5(C-26),15.4(C-27),181.8(C-28),108.5(C-29),19.5(C-30)。以上数据与文献报道的白桦脂酸的数据一致。化合物6:白色针晶(丙酮),mp138~140℃。与β-谷甾醇对照品TLC对比,Rf值一致,且混合溶点不降低,故鉴定为β-谷甾醇。化合物7:无色针晶(丙酮),mp254~256℃。ESI-MSm/z:481[M+Na]+。1H-NMR(CDCl3,400MHz)δ:5.25(1H,brs,H-12),4.21(1H,d,J=11.2Hz,H-24a),3.44(2H,m,H-3,22),3.35(1H,d,J=10.4Hz,H-24b),1.25(3H,s,H-27),1.11(3H,s,H-23),1.04(3H,s,H-28),0.95(3H,s,H-),0.90(3H,s,H-),0.87(3H,s,H-29),0.84(3H,s,H-30)。13C-NMR(CDCl3,100MHz)δ:38.4(C-1),27.6(C-2),80.8(C-3),41.4(C-4),55.8(C-5),18.4(C-6),33.1(C-7),39.6(C-8),47.7(C-9),37.4(C-10),23.7(C-11),122.3(C-12),143.9(C-13),42.0(C-14),25.8(C-15),28.2(C-16),36.6(C-17),44.7(C-18),46.1(C-19),30.5(C-20),42.7(C-21),76.6(C-22),22.4(C-23),64.5(C-24),16.1(C-25),16.8(C-26),25.4(C-27),29.7(C-28),32.8(C-29),20.0(C-30)。以上数据与文献[12-13]报道的soyasapogenolB的数据基本一致。化合物8:无色针晶(丙酮),mp212~213℃。EI-MSm/z198[M]+,183,127,109。1H-NMR(CDCl3,400MHz)δ:12.61(1H,s,-COOH),9.21(1H,s,-OH),7.20(2H,s,H-2,6),3.80(6H,s,-OCH3×2)。13C-NMR(CDCl3,100MHz)δ:120.4(C-1),106.9(C-2),147.5(C-3),140.3(C-4),147.5(C-5),106.