中草药  2014, Vol. 50 Issue (22): 3246-3250
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马兰化学成分及生物活性研究
徐菁, 高鸿悦, 马淑丽, 李慧慧, 白丽明     
齐齐哈尔大学化学与化工学院, 黑龙江 齐齐哈尔 161006
摘要目的 研究马兰Kalimeris indica全草的化学成分。方法 采用硅胶柱色谱、Sephadex LH-20柱色谱等方法分离纯化,根据理化性质和波谱数据进行结构鉴定。结果 从马兰全草95%乙醇中分离得到了15个化合物,分别鉴定为木栓酮(1)、表木栓醇(2)、豆甾醇(3)、β-谷甾醇(4)、α-菠甾醇(5)、古柯二醇(6)、十八烷酸(7)、梣酮(8)、β-胡萝卜苷(9)、二十六烷醇(10)、麦角甾醇(11)、牛防风素(12)、邻苯二甲酸二丁酯(13)、羽扇豆醇乙酸酯(14)、达玛二烯醇乙酸酯(15)。结论 化合物5813均为该植物中首次分离得到。化合物1358、12对苹果腐烂病菌和水稻纹枯病菌均有抑制作用。
关键词马兰     木栓酮     梣酮     麦角甾醇     牛防风素     邻苯二甲酸二丁酯     α-菠甾醇    
Chemical constituents and bioactivity of Kalimeris indica
XU Jing, GAO Hong-yue, MA Shu-li, LI Hui-hui, BAI Li-ming    
School of Chemistry and Chemical Engineering, Qiqihaer University, Qiqihaer 161006, China
Abstract: Objective To study the chemical constituents from Kalimeris indica. Methods Chemical constituents were isolated by column chromatography and semi-prepared HPLC, and the structures were elucidated by spectral data and physical-chemical properties. Results Fifthteen compounds were isolated and respectively identified as friedelin (1), epifriedelanol (2), stimasterol (3), β-sitosterol (4), α-spinasterol (5), erythodiol (6), octadecanoic acid (7), fraxinellone (8), β-daucosterol (9), hexacosanol (10), ergoaterol (11), sphondin (12), dibntyl phthalate (13), oleanic acid (14), and dammerdienly acetate (15). Conclusion Compounds 5, 813 are isolated from K. indica for the frist time. Compounds 1, 3, 5, 8, and 12 have inhibitory effects on Cytospora sp. and Rhizoctonia solani.
Key words: Kalimeris indica (L.) Sch. -Bip.     friedelin     fraxinellone     ergoaterol     sphondin     dibntyl phthalate     α-spinterol    

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5813均为该植物中首次分离得到。对化合物13681012进行了植物致病菌的抑菌活性筛选,其中化合物1358、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13681012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135812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612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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