马兰Kalimeris indica (L.) Sch. -Bip. 属于菊科马兰草属一年生草药,其资源丰富,亦常作野菜食用,主要分布在我国的湖北、江苏、浙江、黑龙江等地。马兰具有清热利湿、凉血解毒、活血散瘀的功效。近期研究表明,马兰中主要含有三萜类、黄酮类、生物碱类、苯酚衍生物类以及挥发油等成分[1, 2]。其中含有的4-羟基3-[1-(甲氧基羰基)-乙烯基]-苯甲酸和5-(1-羧基乙烯氧基)-2-羟基苯甲酸对肝癌细胞和表皮癌细胞有一定的抑制作用,槲皮素具有抗氧化活性,马兰的乙醇提取液具有抗炎、镇痛和镇咳作用[3, 4]。本实验对马兰全草进行了化学成分研究,并对分离得到的化合物进行了抑菌活性研究,从中得到15个化合物,分别鉴定为木栓酮(friedelin,1)、表木栓醇(epifriedelanol,2)、豆甾醇(stimasterol,3)、β-谷甾醇(β-sitosterol,4)、α-菠甾醇(α-spinasterol,5)、古柯二醇(erythodiol,6)、十八烷酸(octadecanoic acid,7)、梣酮(fraxinellone,8)、β-胡萝卜苷(β-daucosterol,9)、二十六烷醇(hexacosanol,10)、麦角甾醇(ergoaterol,11)、牛防风素(sphondin,12)、邻苯二甲酸二丁酯(dibntyl phthalate,13)、羽扇豆醇乙酸酯(oleanic acid,14)、达玛二烯醇乙酸酯(dammerdienly acetate,15)。其中化合物5、8~13均为该植物中首次分离得到。对化合物1、3~6、8、10~12进行了植物致病菌的抑菌活性筛选,其中化合物1、3、5、8、12对苹果腐烂病菌和水稻纹枯病菌均有抑制作用。
1 仪器与材料美国Varian Unity—plus-500(600 MHz)核磁共振仪,Waters 2489高效液相色谱系统,FTIR—4200 Shimadzu红外光谱仪;Yanako熔点仪。除高效液相色谱使用的溶剂为色谱纯外,其他溶剂均为分析纯。苹果腐烂病菌Cytospora sp.、水稻纹枯病菌Rhizoctonia solani,菌种均购于中国农业大学,葡萄糖购于天津市凯通化学试剂有限公司,琼脂购于北京博兴生物技术有限责任公司。
实验用植物2012年8月采自齐齐哈尔嫩江边。经齐齐哈尔大学生命学院杨晓杰教授鉴定为菊科植物马兰Kalimeris indica (L.) Sch. -Bip.。
2 提取与分离取干燥的马兰全草20.0 kg,用95%乙醇浸泡回流提取4次,合并提取液减压回收溶剂,加适量水分散溶解,分别用正己烷、醋酸乙酯和正丁醇进行萃取。对正己烷萃取部分进行柱色谱分离,用正己烷-醋酸乙酯(10∶1→0∶10)梯度洗脱,得到6个流分。第1流分经硅胶柱色谱,用正己烷-醋酸乙酯(100∶1→1∶1)梯度洗脱,得化合物7(11.2 mg)、10(2.3 mg);第3流分经硅胶柱色谱,用正己烷-醋酸乙酯(30∶1→0∶1)梯度洗脱,得化合物1(30.2 mg),2(5.3 mg);第4流分经硅胶柱色谱,用正己烷-醋酸乙酯(20∶1→1∶1)梯度洗脱,得化合物4(14.3 mg);第5流分经硅胶柱色谱,用正己烷-醋酸乙酯(15∶1→0∶1)梯度洗脱,得化合物3(22.5 mg)、5(42.7 mg);第6流分合并经硅胶柱色谱,用正己烷-醋酸乙酯(7∶3→0∶1)梯度洗脱,得化合物6(15.5 mg)、8(17.6 mg)。醋酸乙酯部分,用正己烷-醋酸乙酯(10∶1→0∶10)梯度洗脱,得到4个流分。第1流分先用正己烷-醋酸乙酯(10∶1→0∶10)梯度洗脱,再经HPLC,以正己烷-醋酸乙酯(5∶1、4∶1、6∶2、7∶3)纯化,得化合物11(15.9 mg)、12(17.4 mg)、13(19.8 mg);第2流分先用正己烷-醋酸乙酯(4∶1→0∶1)分离后,再用HPLC,以正己烷-醋酸乙酯(3∶1、7∶3、1∶1)分离纯化,得化合物9(12.2 mg)、14(15.5 mg)、15(24.6 mg)。
3 结构鉴定化合物1:无色针状晶体(醋酸乙酯),mp 254~256 ℃。1H-NMR (600 MHz,CDCl3) δ: 0.72 (3H,s,H-24),0.87 (3H,d,J = 6.4 Hz,H-25),0.95 (3H,s,H-29),1.02 (3H,s,H-30),1.01 (3H,s,H-26),1.05 (3H,s,H-27),1.18 (3H,s,H-28);13C-NMR (150 MHz,CDCl3) δ: 22.2 (C-1),41.5 (C-2),213.3 (C-3),58.2 (C-4),42.1 (C-5),41.2 (C-6),18.2 (C-7),53.0 (C-8),37.4 (C-9),59.4 (C-10),35.6 (C-11),30.6 (C-12),39.7 (C-13),38.3 (C-14),32.4 (C-15),36.0 (C-16),30.1 (C-17),42.8 (C-18),35.3 (C-19),28.2 (C-20),32.7 (C-21),39.2 (C-22),6.8 (C-23),14.6 (C-24),17.9 (C-25),20.2 (C-26),18.6 (C-27),32.0 (C-28),35.0 (C-29),37.1 (C-30)。以上数据与文献报道基本一致[5],故鉴定化合物1为木栓酮。
化合物2:无色晶体(正己烷-醋酸乙酯),mp 263~265 ℃。1H-NMR (600 MHz,CDCl3) δ: 3.74 (1H,brs,H-3),1.17 (3H,s,H-28),1.01 (3H,s,H-27),1.00 (3H,s,H-29),0.99 (3H,s,H-26),0.96 (3H,s,H-30),0.94 (3H,s,H-24),0.93 (3H,s,H-23),0.86 (3H,s,H-25);13C-NMR (150 MHz,CDCl3) δ: 15.8 (C-1),36.1 (C-2),72.8 (C-3),49.2 (C-4),39.3 (C-5),41.8 (C-6),17.6 (C-7),53.2 (C-8),37.1 (C-9),61.4 (C-10),35.4 (C-11),30.7 (C-12),37.8 (C-13),38.4 (C-14),32.4 (C-15),35.6 (C-16),30.0 (C-17),42.8 (C-18),35.2 (C-19),28.2 (C-20),32.8 (C-21),39.7 (C-22),11.6 (C-23),16.4 (C-24),18.2 (C-25),18.7 (C-26),20.1 (C-27),32.1 (C-28),31.8 (C-29),35.0 (C-30)。以上数据与文献报道一致[6],故鉴定化合物2为表木栓醇。
化合物3:无色针晶(正己烷-醋酸乙酯),mp 167~168 ℃。1H-NMR (600 MHz,CDCl3) δ: 5.14 (1H,m,H-6),5.02 (1H,dd,J = 8.4,15.0 Hz,H-22),5.01 (1H,dd,J = 15.0,8.4 Hz,H-23),3.57 (1H,m,H-3),1.03 (3H,s,H-21),1.01 (3H,s,H-19),0.81 (3H,t,J = 2.8 Hz,H-29),0.79 (3H,s,H-27),0.54 (3H,s,H-18);13C-NMR (150 MHz,CDCl3) δ: 37.1 (C-1),31.9 (C-2),71.8 (C-3),43.3 (C-4),121.7 (C-5),140.1 (C-6),40.2 (C-7),31.9 (C-8),50.2 (C-9),36.4 (C-10),21.1 (C-11),39.8 (C-12),42.2 (C-13),32.1 (C-14),25.4 (C-15),28.5 (C-16),55.9 (C-17),12.4 (C-18),19.2 (C-19),40.2 (C-20),21.5 (C-21),138.2 (C-22),129.4 (C-23),51.2 (C-24),31.8 (C-25),21.4 (C-26),19.2 (C-27),25.3 (C-28),12.2 (C-29)。以上数据与文献报道基本一致[7],故鉴定化合物3为豆甾醇。
化合物4:白色针晶(正己烷-醋酸乙酯),mp 169~171 ℃。1H-NMR (600 MHz,CDCl3) δ: 0.68及1.02 (3H,s,H-18,19),0.92 (3H,d,J = 6.8 Hz,H-21),0.81 (3H,d,J = 6.8 Hz,H-27),0.82 (3H,d,J = 6.8 Hz,H-26),0.84 (3H,t,J = 7.3 Hz,H-29),3.52 (1H,dd,J = 8.5,8.5 Hz,H-3),5.35 (1H,dd,J = 5.0,2.0 Hz,H-6);13C-NMR (150 MHz,CDCl3) δ: 37.2 (C-1),31.6 (C-2),71.8 (C-3),42.3 (C-4),140.7 (C-5),121.7 (C-6),31.9 (C-7),31.9 (C-8),50.1 (C-9),36.5 (C-10),21.8 (C-11),39.7 (C-12),42.3 (C-13),56.7 (C-14),24.3 (C-15),26.0 (C-16),56.1 (C-17),11.8 (C-18),19.3 (C-19),36.1 (C-20),18.8 (C-21),33.9 (C-22),28.2 (C-23),45.4 (C-24),29.1 (C-25),19.8 (C-26),19.0 (C-27),23.1 (C-28),11.9 (C-29)。以上数据与文献报道一致[8],故鉴定化合物4为β-谷甾醇。
化合物5:无色针晶(正己烷-醋酸乙酯),mp169~171 ℃。1H-NMR (600 MHz,CDCl3) δ: 4.97 (1H,m,H-7),5.18 (1H,dd,J = 15.0,8.5 Hz,H-23),5.35 (1H,dd,J = 15.0,8.5 Hz,H-23),3.53 (1H,m,H-3);13C-NMR (150 MHz,CDCl3) δ: 37.3 (C-1),31.6 (C-2),71.1 (C-3),38.22 (C-4),40.4 (C-5),29.8 (C-6),177.5 (C-7),139.6 (C-8),49.7 (C-9),34.3 (C-10),21.7 (C-11),39.6 (C-12),43.4 (C-13),55.2 (C-14),23.1 (C-15),28.4 (C-16),56.2 (C-17),12.1 (C-18),13.1 (C-19),40.7 (C-20),21.2 (C-21),138.1 (C-22),129.7 (C-23),51.3 (C-24),31.9 (C-25),19.2 (C-26),21.4 (C-27),25.4 (C-28),12.2 (C-29)。以上数据与文献报道一致[9],故鉴定化合物5为α-菠甾醇。
化合物6:白色针晶(正己烷-醋酸乙酯),mp 230~231 ℃。1H-NMR (600 MHz,CDCl3) δ: 5.19 (1H,t,J = 3.6 Hz,H-12),3.56 (1H,d,J = 11.2 Hz,H-28),3.23 (1H,t,J = 6.0 Hz,H-3),3.21 (1H,d,J = 11.2 Hz,H-28),1.16 (3H,s,H-27),0.99 (3H,s,H-24),0.94 (3H,s,H-26),0.93 (3H,s,H-25),0.88 (3H,s,H-30),0.86 (3H,s,H-29),0.78 (3H,s,H-23);13C-NMR (150 MHz,CDCl3) δ: 23.2 (C-30),33.1 (C-29),69.8 (C-28),25.9 (C-27),16.8 (C-26),15.5 (C-25),15.6 (C-24),28.0 (C-23),31.2 (C-22),34.1 (C-21),30.9 (C-20),46.4 (C-19),42.3 (C-18),47.5 (C-17),21.8 (C-16),25.4 (C-15),41.6 (C-14),144.2 (C-13),122.4 (C-12),23.5 (C-11),36.9 (C-10),47.5 (C-9),39.7 (C-8),32.5 (C-7),18.3 (C-6),55.1 (C-5),38.7 (C-4),79.3 (C-3),27.1 (C-2),38.5 (C-1)。以上数据与文献报道一致[10],故鉴定化合物6为古柯二醇。
化合物7:白色粉末。1H-NMR (600 MHz,CDCl3) δ: 2.34 (1H,t,J = 7.5 Hz,H-2),1.62 (2H,m,H-3),1.26 (28H,m,H-4~17),0.88 (3H,t,J = 6.5 Hz,H-18)。以上数据与文献报道一致[11],故鉴定化合物7为十八烷酸。
化合物8:白色结晶(正己烷-醋酸乙酯),mp 115~116 ℃。1H-NMR (600 MHz,CDCl3) δ: 7.47 (1H,brs,H-2′),7.43 (1H,brs,H-5′),6.34 (1H,s,H-4′),4.87 (1H,s,H-9),1.85 (3H,s,H-8),0.88 (3H,s,H-9);13C-NMR (150 MHz CDCl3) δ: 169.8 (C-1),83.4 (C-3),43.0 (C-4),20.3 (C-5),31.7 (C-6),148.5 (C-7),127.4 (C-8),32.1 (C-9),139.8 (C-2′),120.6 (C-3′),108.6 (C-4′),143.4 (C-5′),18.2 (7-CH3),18.5 (9-CH3)。以上数据与文献报道一致[12],故鉴定化合物8为梣酮。
化合物9:白色颗粒状结晶(甲醇),mp 277~279 ℃。1H-NMR (600 MHz,DMSO-d6) δ: 5.53 (1H,dd,J = 5.2,2.0 Hz,H-6),4.23 (1H,d,J = 8.0 Hz,Glc-H-1),3.52 (1H,dddd,J = 8.4,8.4,4.4,4.4 Hz,H-3),2.89 (1H,dd,J = 8.3,8.3 Hz,Glc-H-2),3.12 (1H,dd,J = 8.6,8.3 Hz,Glc-H-3),3.01 (1H,d,J = 8.6 Hz,Glc-H-4),3.06 (1H,m,Glc-H-5),3.47 (1H,m,Glc-H-6a),3.64 (1H,d,J = 10.2 Hz,Glc-H-6b),2.29 (1H,ddd,J = 13.0,4.5,2.0 Hz,H-4),1.26 (1H,m,H-23),0.96 (3H,s,H-19),0.92 (3H,d,J = 6.8 Hz,H-21),0.84 (3H,t,J = 7.2 Hz,H-29),0.83 (3H,d,J = 6.8 Hz,H-26),0.81 (3H,d,J = 6.8 Hz,H-27),0.65 (3H,s,H-18)。以上数据与文献报道一致[13],故鉴定化合物9为β-胡萝卜苷。
化合物10:白色鳞状结晶(正己烷-醋酸乙酯),mp 76~78 ℃。1H-NMR (600 MHz,CDCl3) δ: 3.64 (1H,t,J = 6.8 Hz,H-1),1.58 (2H,m,H-2),1.34~1.25 (46H,brs,H-3~25),0.88 (3H,t,J = 6.7 Hz,H-26);13C-NMR (150 MHz,CDCl3) δ: 62.2 (C-1),32.1 (C-2),14.2 (C-26)。以上数据与文献报道一致[14],故鉴定化合物10为二十六烷醇。
化合物11:白色粉末,mp 148~150 ℃。1H-NMR (600 MHz,CDCl3) δ: 0.63 (1H,s,H-18),0.82 (3H,d,J = 6.4 Hz,26-CH3),0.84 (3H,d,J = 6.4 Hz,27-CH3),0.92 (3H,d,J = 6.8 Hz,28-CH3),0.95 (3H,s,19-CH3),1.03 (3H,d,J = 6.8 Hz,21-CH3),3.96 (1H,m,H-30),5.14 (1H,dd,J = 15.0,7.8 Hz,H-23),5.22 (1H,dd,J = 15.0,7.8 Hz,H-22),6.23 (1H,m,H-6),6.51 (1H,m,H-7);13C-NMR (150 MHz,CDCl3) δ: 38.4 (C-1),28.3 (C-2),79.3 (C-3),33.1 (C-4),39.8 (C-5),19.6 (C-6),130.2 (C-7),135.3 (C-8),46.3 (C-9),37.1 (C-10),21.1 (C-11),39.1 (C-12),42.8 (C-13),54.6 (C-14),23.0 (C-15),28.2 (C-16),55.8 (C-17),12.1 (C-18),16.3 (C-19),40.4 (C-20),19.7 (C-21),132.3 (C-22),135.2 (C-23),42.8 (C-24),33.1 (C-25),20.0 (C-26),21.1 (C-27),17.6 (C-28)。以上数据与文献报道一致[15],故鉴定化合物11为麦角甾醇。
化合物12:淡黄色粉末,mp 188~190 ℃。1H-NMR (600 MHz,CDCl3) δ: 7.76 (1H,d,J = 9.6 Hz,H-4),7.71 (1H,d,J = 2.1 Hz,H-2′),7.15 (1H,d,J = 2.1 Hz,H-3′),6.77 (1H,s,H-5),6.37 (1H,d,J = 9.6 Hz,H-3),4.30 (3H,s,6-OCH3)。以上数据与文献报道一致[16],故鉴定化合物12为牛防风素。
化合物13:无色油状液体。1H-NMR (600 MHz,CDCl3) δ: 7.30 (1H,dd,J = 5.3,3.5 Hz,H-4),7.54 (1H,dd,J = 5.3,3.5 Hz,H-3),4.32 (2H,t,J = 6.7 Hz,H-9),1.72 (4H,m,H-11),1.44 (4H,m,H-10),0.96 (6H,t,J = 7.4 Hz,H-12)。以上数据与文献报道一致[17],故鉴定化合物13为邻苯二甲酸丁酯。
化合物14:无色针状晶体(正己烷-醋酸乙酯),mp 213~215 ℃。1H-NMR (600 MHz,CDCl3) δ: 4.68 (1H,s,H-29),4.61 (1H,s,H-29),4.51 (1H,dd,J = 12.9,6.4 Hz,H-3),2.22 (3H,s,COCH3),1.67 (3H,s,H-26),1.05 (3H,s,H-30),0.99 (3H,s,H-27),0.93 (3H,s,H-22),0.88 (3H,s,H-23),0.87 (3H,s,H-25),0.86 (3H,s,H-24),0.85 (3H,s,H-28);13C-NMR (150 MHz,CDCl3) δ: 38.4 (C-1),23.3 (C-2),80.8 (C-3),37.7 (C-4),55.4 (C-5),18.2 (C-6),34.5 (C-7),39.3 (C-8),50.4 (C-9),37.0 (C-10),21.4 (C-11),25.5 (C-12),38.3 (C-13),42.8 (C-14),27.6 (C-15),36.3 (C-16),42.8 (C-17),48.7 (C-18),48.6 (C-19),150.5 (C-20),29.5 (C-21),40.8 (C-22),27.9 (C-23),16.3 (C-24),16.8 (C-25),16.5 (C-26),14.7 (C-27),18.2 (C-28),109.4 (C-29),19.2 (C-30),170.8 (CO),21.4 (CH3COO)。以上数据与文献报道一致[17],故鉴定化合物14为羽扇豆醇乙酸酯。
化合物15:无色针状晶体(正己烷-醋酸乙酯),mp 264~265 ℃。1H-NMR (600 MHz,CDCl3) δ: 5.11 (1H,t,J = 8.8 Hz,H-24),4.68 (1H,d,J = 9.0 Hz,H-21),4.48 (1H,m,H-3),2.03 (3H,m,H-23),1.67 (3H,s,H-26),1.62 (3H,s,H-27),0.99 (3H,s,H-30),0.93 (3H,s,H-23),0.87 (3H,s,H-22),0.86 (3H,s,H-28),0.84 (3H,s,H-29);13C-NMR (150 MHz,CDCl3) δ: 37.1 (C-1),23.7 (C-2),80.8 (C-3),55.3 (C-4),36.7 (C-5),55.4 (C-6),18.2 (C-7),34.5 (C-8),50.3 (C-9),38.8 (C-10),21.64 (C-11),27.0 (C-12),48.6 (C-13),48.7 (C-14),31.3 (C-15),27.9 (C-16),45.3 (C-17),16.1 (C-18),16.5 (C-19),152.1 (C-20),107.2 (C-21),34.1 (C-22),23.6 (C-23),124.3 (C-24),131.1 (C-25),25.6 (C-26),17.7 (C-27),27.9 (C-28),15.5 (C-29),15.9 (C-30),170.8 (C-31),21.6 (32- CH3COO)。以上数据与文献报道一致[18],故鉴定化合物15为达玛二烯醇乙酸酯。
4 生物活性筛选采用平板打孔法,测定化合物1、3~6、8、10~12对苹果腐烂病菌、水稻纹枯病菌的抑菌作用。选用内径为8 cm,外径为10 cm的培养皿,选用处于对数生长期的实验菌种,试验样品用丙酮试剂溶解。样品质量浓度为5 mg/mL。实验样品抑菌作用的强弱由其抑菌圈半径的大小体现,选用无样品为空白样进行对照。通过观察各种抑菌圈半径,发现化合物1、3、5、8、12对水稻纹枯病菌和苹果腐烂病菌的抑菌率达到60%以上;化合物6、12对苹果腐烂致病菌的抑菌率均达到65%以上;化合物10对水稻纹枯致病菌的抑菌率为71.2%。
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