傣药“芽害巴”(珠子草)化学成分研究

2019-03-21 02:54李晓花杨文玉
亚太传统医药 2019年2期
关键词:分子式珠子乙酸乙酯

李晓花,杨文玉,王 剑*

(1.滇西应用技术大学 傣医药学院;2.西双版纳版纳药业有限责任公司,云南 景洪 666100)

珠子草(PhyllanthusniriruL.) 为大戟科叶下珠属植物,是我国和印度的一种常用传统民间草药[1]。珠子草傣语称为芽害巴,入风、水塔,具有清热解毒、利湿通淋的功能,临床用于黄疸肋痛、泻利水肿、小便不利的治疗,是傣药国药准字号品种珠子肝泰胶囊的主要组方药材[2]。近年来,国内外学者对珠子草的化学成分和药理活性开展了大量研究[3]。根据文献报道,珠子草的化学成分包括生物碱、花色苷、绿原酸、香豆素、黄酮、木脂素、酚酸、单宁、三萜、皂苷等多种类型;现代药理学研究显示,珠子草具有抗乙肝病毒、降血糖、排泌尿系统结石、抗肿瘤、肝保护、降血脂、抑菌、止痛、抗溃疡和胃保护作用、抗疟疾、肾保护作用等多种活性。

傣医将珠子草打粉或提取,全株入药。结合临床应用,为了寻找珠子草抗乙肝病毒的活性成分,本文根据傣医的临床使用方法,对珠子草提取物的各个极性部位进行了系统的化学成分研究,共分离鉴定了14个单体化合物,根据波谱数据和质谱数据,查阅文献,鉴定了他们的结构(图1)。并根据不同类型化合物的文献报道,推测珠子草抗乙肝病毒活性可能与木脂素类成分和多酚类成分有关。

图1 珠子草中化合物的结构

1 仪器与材料

Bruker 600M AVACE Ⅲ核磁共振仪(TMS 为内标)(德国Bruker公司);Agilent 1260-6120LC/MS (美国Agilent科技公司);R210型旋转蒸发仪(瑞士Buchi公司);C18反相硅胶(YMC ODS-A,50μm)(日本YMC公司);正相硅胶板(GF254)(青岛海洋化工厂);反相硅胶板(TLC Silica Gel RP-18 F254s)(德国Merk公司);氘代试剂(Sigma公司);Waters Prep150 型高效液相色谱仪(检测器2489UV / Visible Detector,沃特世科技有限公司);制备色谱柱YMC ODS-A柱(250mm×10mm,5μm,YMC科技有限公司);柱色谱硅胶(100~200目,200~300 目,青岛海洋化工有限公司),薄层色谱硅胶(GF-254,青岛海洋化工有限公司); Sephadex LH-20(北京慧德义科技发展有限公司)。

珠子草药材采自西双版纳版纳药业有限公司栽培基地,经公司王剑总经理鉴定为珠子草PhyllanthusniruriL.(芽害巴)。

2 提取分离

药材自然干燥,切2~3cm小段,取5.0kg,放置适当容器中,加8倍量95%乙醇室温冷浸提取3次,每次24h,合并提取液并减压浓缩,得到浸膏约0.8kg。浸膏加适量水悬浮,分别用石油醚、乙酸乙酯和正丁醇萃取,每次萃取至颜色较淡。合并各萃取部分,减压浓缩至浸膏,得到石油醚部位(101g)、乙酸乙酯部位(285g)、正丁醇部位(320g)和水部位。

石油醚部位采用硅胶柱色谱分离,石油醚-乙酸乙酯梯度洗脱(10∶0~1∶1),收集流分,每份500mL,薄层色谱检测合并主点相同部位,共得到10个组分(Fr11~10);乙酸乙酯部位用硅胶柱色谱进行粗分,氯仿-甲醇梯度洗脱(10∶0~1∶1),收集流分,每份500mL,薄层色谱检测合并主点相同部位,得到8个组分(Fr21~8);正丁醇部位用硅胶柱色谱进行粗分,二氯甲烷-甲醇梯度洗脱(10∶0~1∶1),收集流分,每份500mL,薄层色谱检测合并主点相同部位,共10个组分(Fr31~10)。选取提取物组分中主点明确的部分,采用Sephadex LH-20凝胶色谱柱(甲醇洗脱)、ODS柱色谱和制备液相色谱(甲醇-水系统),结合重结晶等方法,从石油醚部位分离得到化合物β-谷甾醇(1)、木栓酮(2)、珠子草脂素(3)、珠子草次素(4)、珠子草素(5),乙酸乙酯部位分离得到化合物β-胡萝卜苷(6)、羽扇豆醇(7)、芦丁(8)、山奈酚-3-O-芸香糖苷(9)、短叶苏木酚(10);正丁醇部位分离得到化合物没食子酸(11)、鞣花酸(12)、柯里拉京(13)、老鹳草素(14)等4个多酚类成分。

3 结构鉴定

化合物(1): 白色结晶(甲醇),薄层色谱浓硫酸显色呈紫红色。ESI-MS m/z 415 [M+H]+,分子式C29H50O。1H-NMR (500 MHz,CDCl3) δ :0.65 (3H,s,H-18),1.00 (3H,s,H-19),0.90 (3H,m,H-21),0.82 (3H,m,H-26),0.84 (3H,s,H-27),0.89 (3H,s,H-29),3.50 (1H,m,H-3),5.33 (1H,d,J=2.4 Hz,H-6)。13C-NMR (150 MHz,CDCl3) δ:37.8 (C-1),31.6 (C-2),72.0 (C-3),42.1 (C-4),141.0 (C-5),121.8 (C-6),32.4 (C-7),32.8 (C-8),50.5 (C-9),38.0 (C-10),22.0 (C-11),42.3 (C-12),42.7 (C-13),59.4 (C-14),25.3 (C-15),30.0 (C-16),55.6 (C-17),11.5 (C-18),20.2 (C-19),36.7 (C-20),18.8 (C-21),36.2 (C-22),27.3 (C-23),44.8 (C-24),29.9 (C-25),17.6 (C-26),19.6 (C-27),22.9 (C-28),11.3 (C-29)。以上数据与文献报道[4-5]基本-致,因此鉴定化合物1为β-谷甾醇(β-sitosterol)。

化合物(2): 白色针状结晶(乙酸乙酯)。ESI-MS m/z: 425[M-H]-,分子式为C30H50O。1H-NMR (500 MHz,CDCl3) δ: 1.17 (3H,s,H-28),1.05 (3H,s,H-27),1.00 (3H,s,H-26),0.98 (3H,s,H-30),0.90 (3H,s,H-29) 0.88 (3H,d,J = 6.8 Hz,H-23),0.87 (3H,s,H-25),0.72 (3H,s,H-24);13C-NMR (125 MHz,CDCl3) δ: 22.2(C-1),41.3 (C-2),213.2(C-3),58.2 (C-4),42.0 (C-5),41.2 (C-6),18.1 (C-7),53.0 (C-8),37.3 (C-9),59.5 (C-10),35.6 (C-11),30.3 (C-12),39.5 (C-13),38.3 (C-14),32.3 (C-15),36.0 (C-16),30.0 (C-17),42.7 (C-18),35.4(C-19),28.0 (C-20),32.5 (C-21),39.3 (C-22),6.8 (C-23),14.7(C-24),8.0(C-25),20.11(C-26),18.7 (C-27),32.5(C-28),35.0 (C-29),31.8 (C-30)。以上数据与文献报道[6-7]一致,鉴定化合物2为木栓酮(Friedelin)。

化合物(3): 无色块状晶体(乙酸乙酯)。ESI-MS m/z 419 [M+H]+,分子式C24H34O6。1H-NMR (500 MHz,CDCl3) δ: 6.77 (1H,d,J = 8.0 Hz ,H-5,19),6.64(1H,dd,J = 8.0,1.8 Hz,H-6,18) ,6.60 (1H,d,J = 1.8 Hz,H-2,22),3.88 (3H,s,H-8,24),3.76 (3H,s,H-7,23),3.35~3.33 (1H,m,H-11,14),3.28 (3H,s,H-12,13),3.26~3.23 ( 1H,m,H-11,14),2.70 ~ 2.56 (2H,m,H-9,16),2.09~1.97 (1H,m,H-10,15);13C-NMR (150 MHz,CDCl3) δ: 148.8 (C-3,21),147.2(C-4,20),133.5 (C-1,17),121.0 (C-6,18),112.1 (C-2,22),109.5 (C-5,19),72.7 (C-11,14),58.9 (C-12,13),56.0 (C-7,23),55.3 (C-8,24),40.8 (C-10,15),35.0 (C-9,16)。与文献[8- 9]对照,鉴定该化合物为叶下珠脂素 (Phyllanthin)。

化合物(4): 无色针状结晶(乙酸乙酯)。ESI-MS m/z 431 [M+H]+,分子式C24H30O7。1H-NMR (500 MHz,CDCl3) δ: 6.74 (1H,d,J = 8.1 Hz,H-19),6.70 (1H,d,J = 2.0 Hz,H-22),6.66 (1H,dd,J = 8.0,1.8 Hz,H-18),6.46 (1H,s,H-10),5.73 (1H,d,J = 1.4 Hz,H-16),5.45 (1H,d,J = 1.4 Hz,H-16),4.00 (1H,d,J = 7.8 Hz,H-1),3.90 (3H,s,H-15),3.86 (3H,s,H-24) ,3.70 (3H,s,H-23),3.42~3.40 (2H,m,H-6),3.33 (3H,s,H-5),3.31 (3H,s,H-4),3.31~3.20 (2H,m,H-3),2.67~2.85 (2H,m,H-8),1.90~2.00 (1H,m,H-7),1.80~1.84 (1H,m,H-2);13C-NMR (150 MHz,CDCl3) δ: 148.5 (C-21),147.2 (C-20),147.0 ( C-13),142.2 (C-11),138.1(C-17),133.4 (C-12),132.0 (C-9),120.3 (C-18),115.2 (C-14),111.9 (C-22),110.99 (C-19),106.6 (C-10),100.8 (C-16),75.8 (C-6),71.9 (C-3),59.0 (C-5),58.8 (C-4),56.5 (C-15),56.0 (C-24),55.9 (C-23),45.5 (C-2),42.0 (C-1),36.8 (C-7),33.3 (C-8) 。以上数据与文献[9-10]报道的hypophyllanthin 数据基本-致,化合物4 被确定为叶下珠次素(Hypophyllanthin)。

化合物(5): 无色针状晶体(乙酸乙酯)。ESI-MS m/z 433 [M+H]+,分子式C24H32O7。1H-NMR (500 MHz,CDCl3) δ: 6.77 (1H,d,J = 8.0 Hz,H-15),6.70 (1H,d,J = 8.0Hz,H-14),6.60 (1H,s,H-19),6.31 (1H,s,H-3),6.25 (1H,s,H-21),5.90 (2H,s,H-1),3.86 (3H,s,H-17),3.84(3H,s,H-24),3.82 (3H,s,H-20),3.30 (6H ,H-8,9),3.28 (4H ,H-7,10),2.68~2.62 (2H,m,H-12),2.61~2.59 (2H,m,H-5),2.02 (1H,H-11),2.01 (1H,H-6);13C-NMR (125 MHz,CDCl3) δ: 101.1 (C-1),149.0 (C-2),102.30 (C-3),135.9 (C-4),34.8 (C-5),41.0 (C-6),72.8 (C-7), 59.0 ( C-8),58.8 (C-9),73.1 (C-10),40.9 (C-11),34.8 (C-12),133.7 (C-13),121.6 (C-14), 110.8 (C-15),147.30 (C-16),55.8 (C-17),148.9 (C-18),112.0 (C-19),55.9 (C-20),108.3 (C-21),43.5 (C-22),133.4 (C-23),56.6 (C-24) 。数据与文献[9-11]报道的珠子草素数据基本-致,化合物5被确定为珠子草素(Niranthin)。

化合物(6): 白色粉末(甲醇),薄层检测硫酸乙醇显紫红色。ESI-MS m/z 575[M-H]-,分子式C35H60O6。1H-NMR (500 MHz,DMSO) δ: 5.31 (1H,brs,H-6),4.87 (1H,d,J=5.0 Hz,glucose-OH),4.83 (2H,d,2glucose-OH),4.42 (1H,t,J=5.5 Hz,6’-OH),4.20 (1H,d,J=7.5 Hz,H-1’),3.56 (1H,dd,J=16.5,6.0 Hz,H-6’a),3.37 (1H,m,H-3),3.41 (1H,m,H-6’b),3.10 (3H,m,H-3’,4’,5’),3.00 (1H,m,H-2’),0.95 (3H,s,Me-19),0.90 (3H,d,J=6.0Hz,Me-21),0.85 (9H,m,Me-26,27,29),0.63 (3H,s,Me-18);13C-NMR(125 MHz ,CDCl3) δ: 37.1 (C-1),29.6 (C-2),78.1 (C-3),37.6 (C-4),140.5 (C-5),121.1 (C-6),31.8 (C-7),31.8 (C-8),50.0 (C-9),36.0 (C-10),21.2 (C-11),39.0 (C-12),42.1 (C-13),56.5 (C-14),24.1 (C-15),28.5 (C-16),55.6 (C-17),12.5 (C-18),19.6 (C-19),35.0 (C-20),19.1 (C-21),33.6 (C-22),26.0 (C-23),45.5 (C-24),29.1 (C-25),20.2 (C-26),19.5 (C-27),23.1 (C-28),12.2 (C-29),101.5 (C-l’),73.9 (C-2’),77.6 (C-3’),70.6 (C-4’),77.2 (C-5’),61.6 (C-6’)。以上数据与文献[12-13]中的β-胡萝卜苷-致,故鉴定化合物6为β-胡萝卜苷(Daucosterol)。

化合物(7): 针状结晶(石油醚)。ESI-MS m/z 427[M+H]+,分子式C30H50O。1H-NMR (500MHz,CDCl3) δ: 0.76 (3H,8,H-24),0.79 (3H,s,H-28),0.83 (3H,s,H-25),0.94 (3H,s,H-27),0.97 (3H,s,H-23),1.03 (3H,8,H-26),1.68 (3H,s,H-30),2.38 (1H,ddd,J=5.6,5.6Hz,16.6Hz,H-19),3.19 (1H,dd,J=5.3,10.6Hz,3α-H),4.56 (1H,d,J=2.0Hz,H-29),4.69 (1H,d,J=2.0Hz,H-29);13C-NMR (125MHz,CDCl3) δ: 38.8 (C-1),27.4 (C-2),79.0 (C-3),38.3(C-4),55.3 (C-5),18.4 (C-6),34.3 (C-7),40.9 (C-8),50.5 (C-9),37.2 (C-10),21.0 (C-11),25.2 (C-12),38.1 (C-13),42.9 (C-14),27.5 (C-15),35.6 (C-16),43.0 (C-17),48.4 (C-18),48.0 (C-19),151.0 (C-20),29.9 (C-21),40.0 (C-22),28.0 (C-23),15.4 (C-24),16.1 (C-25),16.0 (C-26),14.6 (C-27),18.0 (C-28),109.3 (C-29),19.3 (C-30)。根据光谱及相关信息鉴定该化合物为羽扇豆醇(Lupeol),其光谱数据与文献[14-15]报道一致。

化合物(8): 黄色粉末(无水甲醇),365nm紫外灯下显黄色荧光。ESI-MS m/z 609 [M-H]-,分子式C27H30O16。1H-NMR (500 MHz,CD3OD) δ: 12.60 (1H,s,5-OH),10.80 (1H,s,7-OH),9.53 (1H,s,3’-OH),9.18 (1H,s,4’-OH),7.55 (2H,m,H-2’,6’),6.83 (1H,d,J=8Hz,H-5’),6.39 (1H,d,J=1.6Hz,H-8),6.22 (1H,d,J=2.0Hz,H-6),5.35 (1H,d,J=4.8Hz,H-1”),5.30~4.35 (6H,糖上6个羟基信号),4.88 (1H,d,J=4.2 Hz,H-1”’),1.00 (3H,d,J=7.2 Hz,CH3-6”’);13C-NMR (125 MHz,CD3OD) δ: 156.4 (C-2),133.4 (C-3),177.4 (C-4),161.3 (C-5),98.8 (C-6),164.2 (C-7),93.6 (C-8),156.6 (C-9),104.0 (C-10),121.2 (C-l’),115.3 (C-2’),144.8 (C-3’),148.5 (C-4’),116.3 (C-5’),121.6 (C-6’),101.2 (C-l”),74.1 (C-2”),76.5 (C-3’’),70.0 (C-4”),75.9 (C-5”),67.3 (C-6”),100.8 (C-1”’),70.6 (C·2”’),70.8 (C-3”’),71.9 (C-4”’), 68.3 (C-5”’),17.8 (C-6”’),与文献[16-17]对照,鉴定化合物8为芦丁(Rutin)。

化合物(9): 黄色粉末(甲醇)。ESI-MS m/z 593 [M-H]-,分子式C27H30O15。1H-NMR (500 MHz,DMSO-d6) δ: 12.56 (1H,brs,5-OH),10.85 (1H,brs,7-OH),10.17 (1H,brs,4’-OH),8.03 (2H,d,J=8.8Hz,2’,6’-H),6.85 (2H,d,J=8.8Hz,3’,5’-H),6.42 (1H,d,J=1.9Hz,8-H),6.20 (1H,d,J=1.8Hz,6-H),5.25 (1H,d,J=7.2Hz,1”-H),4.32(1H,d,J=1.2Hz,1”’-H),3.05~3.70(m,糖质子信号),0.95 (3H,d,J=6.0Hz,6”’-H);13C-NMR (125MHz,DMSO-d6) δ: 157.0 (C-2),133.4 (C-3),178.0 (C-4),161.5 (C-5),99.0 (C-6),164.3 (C-7),94.0 (C-8),156.9 (C-9),104.7 (C-10),121.1 (C-1’),131.0 (C-2’),115.9 (C-3’),160.3 (C-4’),115.5 (C-5’),131.2 (C-6’),101.5 (C-1”),74.5 (C-2”),76.8 (C-3”),70.1 (C-4”),75.9 (C-5”),67.5 (C-6”),101.0 (C-1”’),70.5 (C-2”’),70.8 (C-3”’),72.2 (C-4”’),68.5 (C-5”’),18.0 (C-6”’),与文献[16-17]对照,鉴定该化合物9为山奈酚-3-O-芸香糖苷(Kaempferol 3-rutinoside)。

化合物(10): 淡黄色针状结晶(甲醇)。ESI-MS m/z 247 [M-H]-,分子式C15H12O8。1H-NMR (500 MHz,DMSO-d6) δ: 7.26 (1H,s,H-7),3.35 (2H,t,J = 3.8Hz,H-10),3.20 (2H,d,J = 3.6 Hz,H-9);13C-NMR (125 MHz,DMSO-d6) δ: 147.4 (C-2),140.6 (C-3),115.9 (C-3a),144.7 (C-4),141.3 (C-5),146.8 (C-6),113.1 (C-7a),108.2 (C-7),161.0 (C-8),23.3 (C-9),195.5 (C-11),33.3 (C-10),。以上数据与文献报道-致[18-19],故鉴定化合物10为短叶苏木酚(Vifolin)。

化合物(11): 白色针状结晶(甲醇)。ESI-MS m/z 169 [M-H]-,分子式C7H6O5。1H-NMR (500 MHz,CD3OD) δ: 7.03 (2H,s,H-2,6);13C-NMR (125MHz,CD3OD) δ:121.8 (C-1),109.2 (C-2,6),145.4(C-3,5),138.5 (C-4),169.7 (C-7)。以上数据与文献[20-21]报道基本一致,鉴定化合物11为没食子酸(Gallic acid)。

化合物(12): 白色芳香粉末(乙酸乙酯)。ESI-MS m/z 301 [M-H]-,分子式C14H6O8。1H-NMR(500MHz,DMSO) δ: 7.51 (2H,s,H-5,5’);13C-NMR (125MHz,DMSO) δ: 106.4 (C-1,1’),136.1 (C-2,2’),140.3 (C-3,3’),148.0 (C-4,4’),109.6 (C-5,5’),113.6 (C-6,6’),160.0(C-7,7’)。波谱和质谱检测数据与文献[21]报道基本一致,鉴定该化合物12为鞣花酸(Ellagic acid)。

化合物(13): 芳香褐色粉末(甲醇)。ESI-MS m/z 593 [M-H]-,分子式C27H22O18。1H-NMR (500 MHz,CD3OD) δ: 7.10 (2H,s,H-23,27),6.81 (1H,s,H-3 ),6.68 (1H,s,H-12 ),6.38(1H,d,J = 2.0 Hz,H-20),4.93 (2H,m,H-16,18),4.84 (1H,m,H-17),4.53 (1H,m,H-15a),4.15 (1H,m,H-15b),4.05 (1H,d,J = 1.2Hz,H-19) ;13C NMR (125 MHz,CD3OD) δ: 168.5 (C-1),125.3 (C-2),110.0 (C-3),145.0 (C-4),138.1 (C-5),145.5 (C-6),117.0 (C-7),115.9 (C-8),144.7 (C-9),138.7 (C-10),146.0 (C-11),108.7 (C-12),125.5 (C-13),170.0 (C-14),65.0 (C-15),71.6 (C-16),62.3 (C-17),76.5(C-18),69.5 (C-19),95.0 (C-20),166.0 (C-21),120.7 (C-22),111.0 (C-23,27),146.8 (C-24,26),140.5(C-25) 。以上数据与柯里拉京(corilagin) 的波谱数据[22-23]基本一致,故鉴定化合物13为柯里拉京(Corilagin)。

化合物(14): 棕黄色芳香粉末(丙酮)。ESI-MS m/z 951 [M-H]-,分子式C41H28O27。1H-NMR (500 MHz,acetone-d6 ) δ: 4.30 (1 H,dd,J= 8.2,10.5 Hz,glc-C6-H),4.75 (1 H,m,glc-C5- H),4.91(1 H,t,J=11.0 Hz,glc- C6’- H),5.08 (1 H,s,C1-H),5.45,5.47,5.50 (各l H,brs,glc C4,3,2- H),6.50 (1 H,s,C3’- H),6.57(1 H,brs,g lc-Cl-H),6.51,7.10 (各1 H,s,HHDP-H ),7.16 (2 H,s,galloy- H),7.20 (1H,s,C3-H);13C-NMR (125MHz,acetone-d6) δ: Glucose:91.3 (C-1),70.1 (C--2),63.6 (C-3),66.0 (C-4),72.6 (C-5),63.9 (C-6);Galloyl-A环: 119.9 (C-1),110.7 (C-2,6),146.2 (C-3),140.4 (C-4),165.1 (C-7); HHDP-B环: 116.6 (C-1),124.2 (C-2),110.0 (C-3),145.0 (C-4),137.5 (C-5),145.5 (C-6),166.6 (C-7);C环: 114.9 (C-1),125.0 (C-2),107.9 (C-3),144.9 (C-4),135.9 (C-5),145.2 (C-6),168.9 (C-7);2,4-Acyl-D环: 115.6 (C-1),119.0 (C-2),113.8 (C-3),146.0 (C-4),139.0 (C-5),142.8 (C-6),165.9 (C-7); E环: 46.0 (C-1),154.4 (C-2),129.0 (C-3),191.8 (C-4),96.5 (C-5),96.8 (C-6),165.9 (C-7)。以上数据与文献[23-24]报道基本一致,故鉴定该化合物14为老鹳草素(Geraniin)。

4 讨论

通过系统分离,从傣药珠子草中共分离鉴定出了14个单体化合物,分为甾体和三萜类化合物、木脂素类、黄酮类和多酚类等。根据文献报道,珠子草具有肝保护活性的研究主要采用全草水提取物、鲜叶汁和甲醇提取物,开展肝保护活性研究的单体化合物主要有叶下珠脂素、叶下珠次素和珠子草素等[3],而抗病毒活性以珠子草的甲醇提取物活性最强[25-26]。通过对珠子草系统的化学成分研究,傣医用其治疗肝脏类疾病,主要是通过其含有的木脂素类成分发挥保肝作用,通过多酚类成分发挥抗病毒作用。

傣药珠子草是西双版纳地区常用的药材之一,资源丰富,临床使用广泛,尤其是在乙型肝炎的治疗中属于最常用的傣药材之一。其化学成分和药理活性已有一定的研究基础,且有研究表明其提取物毒性低。根据国内外文献报道,珠子草除了用于肝病的治疗,还有多种活性,因此具有广阔的研究开发前景。

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