江南紫金牛为紫金牛科(Myrsinaceae)紫金牛属Ardisia Swartz植物江南紫金牛Ardisia faberi Hemsl. 的根或全株。其味苦、辛,性平,归肺、胃经;具有疏风散热、解毒利咽、消肿的功能;主治风热感冒、咳嗽、咽肿、跌打损伤[1];别名月月红、白毛金刚[2]、木步走马胎[3]、毛青杠[4]、毛矮地茶、毛青松[1]等。江南紫金牛常绿矮小灌木,生于海拔1 000~1 300 m的山谷疏林下阴湿处或岩石缝中,分布于我国西南及湘西、湖北、广东、广西等地[4],是我国南方少数民族常用药物之一。目前,文献报道从江南紫金牛中分离得到的化合物有鲍尔烯醇、槲皮素、岩白菜素、紫金牛苷等[5]。在前期研究中,本课题组对江南紫金牛进行人肺癌A549细胞毒活性检测,结果表明江南紫金牛在质量浓度为100μg/mL时对人肺癌A549细胞的增殖有显著抑制作用[6],为探究其抗肺癌的有效物质基础,本课题组对该植物的化学成分进行系统研究。从江南紫金牛全草的80%乙醇渗漉液中分离得到11个化合物,分别鉴定为鲍尔烯醇(elucidateasbauerenol,1)、油酸(oleic acid,2)、(Z)-10-heneicosenoic acid(3)、蚱蜢酮(grasshopper ketone,4)、岩白菜素(bergenin,5)、mallonanoside A(6)、没食子酸乙酯(ethyl gallate,7)、落新妇苷(astilbin,8)、槲皮苷(quercitrin,9)、百两金皂苷B(ardisiacrispin B,10)、ardicrenin(11)。其中,化合物2、7、9、10、11为首次从该植物中分离得到,化合物3、4、6、8为首次从该属植物中分离得到。
1 仪器与材料Bruker AM-400/600型核磁共振仪(德国Bruker公司);Waters Synapt G2高分辨质谱仪;X-4显微测定熔点仪(未校正);优普UPT系列超纯水器(成都优普电子产品有限公司);十万分之一电子天平(瑞士奥豪斯DV-215-CD);KQ-600DE型数控超声波清洗器(40 kHz,600 W);ZF-90型暗箱式紫外透射仪(上海顾村电光仪器厂);100-A自动部份收集器(上海沪西分析仪器厂有限公司);(Hanbon Sci & Tech.)NU3000 Serials UV/VIS 检测器、NP 7000 Serials 泵)。Sephadex LH-20(美国Pharmacia公司);MCI gel CHP-20P(75~150 μm,日本三菱化学公司柱色谱硅胶)、ODS反相填料(YMC gel ODS-A,50 μm,日本YMC公司);薄层色谱硅胶GF254、柱色谱硅胶(200~300目)、制备薄层预制板均为青岛海洋化工厂生产;试剂均为分析纯(成都科龙化工试剂厂);Dubhe C18(250 mm×20 mm,10 μm,江苏汉邦科技有限公司);YMC pack ODS-A(250 mm×10 mm,5 μm,日本YMC公司)。
植物样品采自四川省峨眉山,由成都中医药大学峨嵋学院祝世杰教授鉴定为紫金牛科紫金牛属植物江南紫金牛Ardisia faberi Hemsl;标本(20130520)存放于成都中医药大学药学院中药学实验室。
2 提取与分离自然阴干的药材全草(1.5 kg),切段粉碎,用80%乙醇渗漉提取得渗漉液,将其减压干燥后得到的浸膏用水分散,依次用醋酸乙酯、水饱和正丁醇萃取,萃取液减压干燥得浸膏醋酸乙酯部位24.27 g,正丁醇部位84 g。醋酸乙酯部位采用正相硅胶柱色谱,氯仿-甲醇(100∶0、98∶2、96∶4、92∶8、90∶10、85∶15、70∶30)、纯甲醇梯度洗脱后合并为12个部分Fr. 1~Fr. 12。Fr. 1经醋酸乙酯反复重结晶得到化合物1(20.5 mg);Fr. 3经Sephadex LH-20(氯仿-甲醇3∶2)、Pre-TLC(氯仿-甲醇9∶1)得化合物2(10.0 mg);Fr. 4经正相硅胶,石油醚-丙酮(50∶1→3∶1),Sephadex LH-20(氯仿-甲醇3∶2)纯化得到化合物3(15.8 mg);Fr. 5经Sephadex LH-20(甲醇),Pre-HPLC(甲醇-水33∶67)制备得化合物4(10.1 mg);Fr. 8通过Pre-HPLC(甲醇-水40∶60)制备、重结晶(甲醇)和Sephadex LH-20(甲醇)纯化得化合物5(1.78 g),Fr. 10经反相C8柱色谱(甲醇-水50∶50、90∶100)、Pre-HPLC(甲醇-水10∶100→100∶0)中压制备、半制备(甲醇-水35∶65)、Sephadex LH-20(甲醇-水70∶30)柱色谱得化合物6(15.2 mg)、7(10.4 mg)、8(15.3 mg)、9(13.8 mg);正丁醇部位经D101大孔树脂,水及10%、30%、50%、70%、95%乙醇梯度洗脱得7个部分,其中70%乙醇部位经反相C18柱色谱(60%甲醇)得化合物10(10.01 g);Fr. 11经MCI、反相C18和Pre-HPLC(甲醇-水85∶15)制备得化合物11(0.7 g)。
3 结构鉴定化合物1:无色针晶(氯仿),mp 207~209 ℃。1H-NMR (400 MHz,CDCl3) δ: 5.43 (1H,dd,J = 6.8,2.8 Hz,H-7),3.25 (1H,ddd,J = 12.3,8.1,4.5 Hz,H-3),1.07,1.05,1.00,0.95,0.92,0.87,0.75 (各3H,s,7×-CH3),0.97 (3H,d,J = 5.1 Hz,H-30);13C-NMR (100 MHz,CDCl3) δ: 37.9 (C-1),28.0 (C-2),79.5 (C-3),39.1 (C-4),50.6 (C-5),24.4 (C-6),116.7 (C-7),145.3 (C-8),48.5 (C-9),35.4 (C-10),17.0 (C-11),32.7 (C-12),38.2 (C-13),41.5 (C-14),29.5 (C-15),31.8 (C-16),32.3 (C-17),55.1 (C-18),35.6 (C-19),39.0 (C-20),29.1 (C-21),38.0 (C-22),27.8 (C-23),14.9 (C-24),13.2 (C-25),23.9 (C-26),22.8 (C-27),32.3 (C-28),22.9 (C-29),25.9 (C-30)。以上数据与文献报道基本一致[7],故鉴定化合物1为鲍尔烯醇。
化合物2:无色油状物,HR-ESI-MS m/z: 281.247 6 [M-H]-,分子式为C18H34O2。1H-NMR和13C-NMR谱显示出1组典型的不饱和脂肪酸信号。1H-NMR (400 MHz,CD3OD) δ: 5.32 (2H,m,H-9,10),2.26 (2H,t,J = 6.0 Hz,H-2),2.02 (4H,d,J = 5.5 Hz,H-8,11),1.58 (2H,m,H-3),1.23~1.36 (20H,m,H-4~7,12~17),0.88 (3H,t,J = 5.0 Hz,H-18);13C-NMR (100 MHz,CD3OD) δ: 180.2 (C-1),35.26 (C-2),26.3 (C-3),30.6~30.9 (C-4~7,12~15),30.4 (C-8),131.0 (C-9),130.9 (C-10),28.3 (C-11),33.2 (C-16),23.8 (C-17),14.6 (C-18)。以上数据与文献报道基本一致[8],故鉴定化合物2为油酸。
化合物3:浅黄色油状物。1H-NMR (400 MHz,CDCl3) δ: 5.36~5.27 (2H,m,H-10,11),2.31 (2H,t,J = 7.5 Hz,H-2),2.05 (4H,m,H-9,12),1.66 (2H,q,J = 7.3 Hz,H-3),1.25 (26H,m,H-3~7,13~20),0.85 (3H,t,J = 6.8 Hz,H-21);13C-NMR (100 MHz,CDCl3) δ: 180.3 (C-1),130.3 (C-10),130.0 (C-11),34.3 (C-2),32.3 (C-19),30.0~29.3 (C-4~8,13~18),27.4 (C-9,12)。以上数据与文献报道基本一致[9],故鉴定化合物3为 (Z)-10-heneicosenoic acid。
化合物4:蜡状固体。1H-NMR (400 MHz,C5D5N) δ: 5.93 (1H,s,H-8),3.80 (1H,s,H-3),2.32 (1H,ddd,J = 12.5,3.9,1.9 Hz,H-4),2.27 (3H,s,10-CH3),2.04 (1H,ddd,J = 12.7,4.2,2.1 Hz,H-2a),1,48 (3H,s,13-CH3),1.29 (3H,s,11-CH3),1.38~1.47 (1H,m,H-4b),1.32~1.37 (1H,m,H-2b),1.14 (3H,s,12-CH3);13C-NMR (100 MHz,C5D5N) δ: 209.6 (C-7),197.8 (C-9),119.8 (C-6),100.2 (C-8),71.3 (C-5),63.1 (C-9),50.2 (C-2),50.6 (C-7),36.2 (C-1),31.8 (12-CH3),30.9 (13-CH3),29.1 (11-CH3),26.2 (10-CH3)。以上数据与文献报道基本一致[10],故鉴定化合物4为蚱蜢酮。
化合物5:白色粉末,mp 236~238 ℃;HR- ESI-MS m/z: 327.071 7 [M-H]-(C14H16O9,计算值328.079 4);分子式为C14H16O9。1H-NMR (600 MHz,CD3OD) δ: 7.10 (1H,s,H-4),4.97 (1H,d,J = 10.5 Hz,H-9),4.07 (1H,t,J = 9.9 Hz,H-14),4.03 (1H,dd,J = 10.7,2.5 Hz,H-16),3.91 (3H,s,OCH3),3.71 (1H,d,J = 7.0 Hz,H-16),3.81 (1H,dd,J = 15.2,6.2 Hz,H-13),3.69 (1H,s,H-11),3.44 (1H,t,J = 9.0 Hz,H-12);13C-NMR (150 MHz,CD3OD) δ: 165.9 (C-2),119.6 (C-3),111.2 (C-4),152.5 (C-5),142.4 (C-6),149.6 (C-7),117.5 (C-8),74.4 (C-9),83.2 (C-11),72.1 (C-12),75.8 (C-13),81.6 (C-14),61.0 (C-15),62.8 (C-16)。以上数据与文献报道基本一致[11],故鉴定化合物5为岩白菜素。
化合物6:白色无定形粉末,mp 196~197 ℃,HR-ESI-MS m/z: 327.070 1 [M-H2O-H]-,345.080 8[M-H]-(C14H18O10,计算值345.082 1),分子式为C14H18O10。1H-NMR (600 MHz,CD3OD) δ: 7.09 (1H,s,H-6),3.90 (3H,s,-OCH3),4.96 (1H,d,J = 10.5 Hz,Glc-H-1);13C-NMR (150 MHz,CD3OD) δ: 119.6 (C-1),117.5 (C-2),149.6 (C-3),142.4 (C-4),152.6 (C-5),111.2 (C-6),61.0 (-OCH3),166.0 (-COOH),74.4 (Glc-C-1),81.6 (Glc-C-2),75.8 (Glc- C-3),72.0 (Glc-C-4),83.2 (Glc-C-5),62.8 (Glc-C-6)。以上数据与文献报道基本一致[12],故鉴定化合物6为mallonanoside A。
化合物7:白色针状结晶(氯仿),mp 136~138 ℃。1H-NMR (600 MHz,CD3OD) δ: 6.96 (2H,s,H-2,6),4.19 (2H,q,J = 7.1 Hz,-OCH2-),1.26 (3H,t,J = 7.1 Hz,-CH3);13C-NMR (150 MHz,CD3OD) δ: 121.9 (C-1),110.1 (C-2,6),146.6 (C-3,5),139.8 (C-4),168.7 (C-7),61.8 (C-8),14.8 (C-9)。以上数据与文献报道基本一致[13],故鉴定化合物7为没食子酸乙酯。
化合物8:白色无定形粉末。1H-NMR (600 MHz,CD3OD) δ: 5.22 (1H,d,J = 12.5 Hz,H-2),4.97 (1H,d,J = 10.7 Hz,H-3),5.80 (1H,d,J = 2.1 Hz,H-6),5.82 (1H,d,J = 2.1 Hz,H-8),6.86 (1H,d,J = 1.7 Hz,H-2′),6.71 (1H,d,J = 3.0 Hz,H-5′),6.74 (1H,d,J = 1.8 Hz,H-6′),4.09 (1H,d,J = 2.4 Hz,Rha-H-1),1.09 (3H,d,J = 6.2 Hz,Rha-CH3);13C-NMR (150 MHz,CD3OD) δ: 84.1 (C-2),78.7 (C-3),196.1 (C-4),165.6 (C-5),97.6 (C-6),168.8 (C-7),96.4 (C-8),164.2 (C-9),102.6 (C-10),129.3 (C-1′),116.5 (C-2′),146.7 (C-3′),147.5 (C-4′),115.6 (C-5′),120.6 (C-6′),102.3 (Rha-C-1),71.9 (Rha-C-2),72.3 (Rha-C-3),74.0 (Rha-C-4),70.7 (Rha-C-5),18.0 (Rha-C-6)。以上数据与文献报道基本一致[14],故鉴定化合物8为落新妇苷。
化合物9:黄色粉末,mp 183~185 ℃。1H-NMR (600 MHz,CD3OD) δ: 6.12 (1H,d,J = 1.8 Hz,H-6),6.29 (1H,d,J = 1.6 Hz,H-8),7.22 (1H,d,J = 1.9 Hz,H-2′),6.82 (1H,d,J = 8.3 Hz,H-5′),7.25 (1H,d,J = 1.8 Hz,H-6′),5.26 (1H,s,Rha-H-1),0.85 (3H,d,J = 6.2 Hz,Rha-CH3);13C-NMR (150 MHz,CD3OD) δ: 158.7 (C-2),136.4 (C-3),179.8 (C-4),159.5 (C-5),99.9 (C-6),166.1 (C-7),94.9 (C-8),163.4 (C-9),106.0 (C-10),123.0 (C-1′),116.5 (C-2′),146.6 (C-3′),149.9 (C-4′),117.1 (C-5′),123.0 (C-6′),103.7 (Rha-C-1),72.2 (Rha-C-2),72.3 (Rha-C-3),73.4 (Rha-C-4),72.1 (Rha-C-5),17.8 (Rha-C-6)。以上数据与文献报道基本一致[15],故鉴定化合物9为槲皮苷。
化合物10:白色粉末,mp 232~234 ℃,分子式为C53H86O22。1H-NMR (600 MHz,C5D5N) δ: 0.76 (3H,s,H-25),0.95 (6H,d,J = 6.8 Hz,H-24,29),1.10 (3H,s,H-23),1.21 (3H,s,H-26),1.47 (3H,s,H-27),1.74 (3H,d,J = 6.1 Hz,Rha-CH3),3.09 (1H,d,J = 7.6 Hz,H-28a),3.11 (1H,dd,J = 11.5,9.5 Hz,H-3),3.49 (1H,d,J = 7.4 Hz,H-28b),4.88 (1H,s,Ara-H-1),5.19 (1H,d,J = 7.6 Hz,inner Glc-H-1),5.31 (1H,d,J = 7.3 Hz,terminal Glc-H-1),6.35 (1H,s,Rha-H-1),9.56 (1H,s,H-30);13C-NMR (150 MHz,C5D5N) δ: 39.0 (C-1),26.3 (C-2),88.9 (C-3),39.4 (C-4),55.4 (C-5),17.7 (C-6),34.1 (C-7),42.3 (C-8),50.2 (C-9),36.6 (C-10),18.9 (C-11),32.4 (C-12),86.1 (C-13),44.4 (C-14),36.6 (C-15),76.7 (C-16),43.8 (C-17),53.1 (C-18),33.2 (C-19),48.1 (C-20),30.3 (C-21),32.1 (C-22),27.8 (C-23),16.2 (C-24),16.1 (C-25),18.3 (C-26),19.5 (C-27),77.4 (C-28),23.9 (C-29),207.3 (C-30);Ara: 104.2 (C-1),80.1 (C-2),72.1 (C-3),74.4 (C-4),63.2 (C-5);inner-Glc: 102.8 (C-1),77.1 (C-2),79.3 (C-3),71.6 (C-4),78.1 (C-5),62.4 (C-6);Rha: 101.3 (C-1),72.1 (C-2),72.5 (C-3),74.6 (C-4),69.2 (C-5),18.7 (C-6);terminal-Glc: 105.3 (C-1),76.1 (C-2),77.8 (C-3),71.6 (C-4),77.8 (C-5),62.7 (C-6)。以上数据与文献报道基本一致[16],故鉴定化合物10为百两金皂苷B。
化合物11:白色晶体(甲醇),mp 239~240 ℃。1H-NMR (600 MHz,C5D5N) δ: 0.83 (3H,s,H-25),1.01 (3H,s,H-24),1.02 (3H,s,H-29),1.16 (3H,s,H-23),1.28 (3H,s,H-26),1.54 (3H,s,H-27),1.81 (3H,d,J = 6.1 Hz,Rha-H-6),4.96 (1H,s,Ara-H-1),5.25 (1H,d,J = 7.0 Hz,inner Glc-H-1 ),5.38 (1H,d,J = 7.6 Hz,terminal Glc-H-1),6.42 (1H,s,Rha-H-1),9.63 (1H,s,H-30);13C-NMR (150 MHz,C5D5N) δ: 39.5 (C-1),26.8 (C-2),89.5 (C-3),39.9 (C-4),55.9 (C-5),18.2 (C-6),34.7 (C-7),42.9 (C-8),50.7 (C-9),37.2 (C-10),19.5 (C-11),33.7 (C-12),86.7 (C-13),44.9 (C-14),37.1 (C-15),77.3 (C-16),44.4 (C-17),53.6 (C-18),33.0 (C-19),48.6 (C-20),30.8 (C-21),32.7 (C-22),28.4 (C-23),16.8 (C-24),16.7 (C-25),18.9 (C-26),20.1 (C-27),77.6 (C-28),24.4 (C-29),207.9 (C-30);Ara: 104.7 (C-1),79.9 (C-2),72.2 (C-3),78.7 (C-4),63.2 (C-5);inner-Glc: 105.7 (C-1),74.1 (C-2),79.2 (C-3),78.0 (C-4),77.7 (C-5),62.6 (C-6);Rha: 101.9 (C-1),72.7 (C-2),73.0 (C-3),75.0 (C-4),69.8 (C-5),19.3 (C-6);terminal-Glc: 103.4 (C-1),76.7 (C-2),78.4 (C-3),72.1 (C-4),78.4 (C-5),63.0 (C-6)。以上数据与文献报道基本一致[17],故鉴定化合物11为ardicrenin。
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