药学学报  2014, Vol. 49 Issue (8): 1150-1154   PDF    
生附子的化学成分研究
张晶, 孙桂波, 雷崎方, 李广志, 王钧篪, 斯建勇     
中国医学科学院、北京协和医学院药用植物研究所, 北京 100193
摘要:为研究中药附子强心活性的物质基础,对其化学成分进行了研究。采用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、MCI、ODS等柱色谱及制备液相色谱等手段从附子90%乙醇提取物中分离得到10个化合物,根据化合物的理化性质和波谱数据鉴定了其结构,分别为5-羟基-2-苯甲酰胺-苯甲酸甲酯 (1)、和厚朴酚 (2)、松脂醇 (3)、水杨酸 (4)、对羟基桂皮酸 (5)、松果灵 (6)、多根乌头碱 (7)、新乌头碱 (8)、次乌头碱 (9) 和苯甲酰次乌头碱 (10)。化合物1为一个新的化合物,命名为附子酰胺(aconitamide),并通过核磁、质谱、紫外、红外及单晶衍射数据确定了其结构。化合物2~5为首次从附子中分离得到。
关键词附子     化学成分     附子酰胺    
Chemical constituents of lateral roots of Aconitum carmichaelii Debx
ZHANG Jing, SUN Gui-bo, LEI Qi-fang, LI Guang-zhi, WANG Jun-chi, SI Jian-yong     
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
Abstract: In order to find the cardiotonic constituents of lateral roots of Aconitum carmichaelii Debx., the investigation was carried out. Silica gel column chromatography, Sephadex LH-20, medium-pressure MCI and reverse phase ODS column chromatography were used to separate the 90% EtOH extract of the lateral roots of Aconitum carmichaelii Debx. The structures of the isolated compounds have been identified by chemical properties and spectroscopic analyses. Ten compounds were isolated and their structures were elucidated as benzoic acid-5-hydroxy-2-benzoyl-amino methyl ester (1), honokiol (2), pinoresinol (3), salicylic acid (4), p-hydroxy-cinnamic acid (5), songorine (6), karakoline (7), mesaconitine (8), hypaconitine (9) and 14-benzoylhypaconitine (10), separetely. Compound 1 is a new compound and its structure has been established by NMR, HR-ESI-MS, UV, IR and X-Ray. Compound 2-5 are isolated from the lateral roots of Aconitum carmichaelii Debx. for the first time.
Key words: lateral roots of Aconitum carmichaelii Debx.     constituent     aconitamide    

附子 (lateral roots of Aconitum carmichaelii Debx.) 为毛茛科植物川乌 (Aconitum carmichaelii Debx.) 的侧生子根及加工品,为四川道地药材之一,根据加工方法不同,分为盐附子、黑顺片及白附片等。本品性辛、甘,有毒,具有回阳救逆、补火助阳、散寒止痛等作用[1]。现代药理研究证明了附子具有强心、抗炎、镇痛、镇静等作用,一般认为附子的活性和毒性成分为二萜类生物碱,附子在炮制过程中二萜类生物碱发生转化,剧毒成分的双酯型生物碱含量降低,单酯型生物碱含量增加[2,3]。除此之外还有一些其他类型化合物,如木脂素、脂肪酸、黄酮、皂苷、糖类等,但这些化学成分研究较少。为了揭示附子药效和毒性的物质基础,本实验对生附子活性部位进行了化学成分的研究,从中分离得到了10个化合物,它们分别是附子酰胺 (1)、和厚朴酚 (2)、松脂醇 (3)、水杨酸 (4)、对羟基桂皮酸 (5)、松果灵 (6)、多根乌头碱 (7)、新乌头碱 (8)、次乌头碱 (9) 和苯甲酰次乌头碱 (10),其中1为新化合物,25为首次从附子中分离得到。

化合物1 白色针状结晶,易溶于甲醇。mp 179~180 ℃。HR-ESI-MS给出其准分子离子峰为m/z 294.075 1 [M+Na]+,确定分子式为C15H13NO4 (Calcd. for C15H13NO4,294.074 3),不饱和度为10。UV λmax (MeOH) 338.5 nm和279.5 nm处有最大吸收,提示结构中含有芳环等共轭系统。IR (KBr) 显示有苯环的骨架振动 (1 602、1 505 cm -1)、共轭羰基 (1 658、1 680 cm-1) 及羟基 (3 321 cm-1) 吸收峰。13C NMR (150 MHz,MeOD) 共给出了13个碳信号 (表 1),包括1个甲氧基信号 (δC 52.9)、2个共轭羰基信号 (δC 169.8、167.1)、4个芳香季碳信号 (δC 119.0、134.3、136.0、154.4) 和6个芳香叔碳信号 (δC 117.6、122.3、123.6、128.1、129.8、133.0); 化合物1有10个不饱和度,扣除2个羰基,结构中应含有2个苯环。1H NMR (600 MHz,MeOD) (表 1) 给出了1个甲氧基δH 4.00 (3H,s,-OCH3) 信号,一组ABX自旋系统的芳质子信号δH 8.56 (1H,d,J = 9.0 Hz,H-3)、7.54 (1H,d,J = 3.0 Hz,H-6)、7.12 (1H,dd,J = 3.0,9.0 Hz,H-4),一组苯环单取代结构信号δH8.03 (2H,d,J = 7.2 Hz,H-2',H-6')、7.65 (1H,t,J = 7.2 Hz,H-4')、7.59 (2H,t,J = 7.2 Hz,H-3',H-5')。综合以上信息,推断其含有2个羰基、1个甲氧基、1个酚羟基、1个单取代苯环和1个形成ABX系统的苯环。从分子式分析,扣除上述基团的碳、氢,还剩1个NH基团,即还有1个NH取代基。各取代基连接位置通过分析HMBC谱确定。HMBC谱 (图 1) 中单取代苯环中的δH 8.03 (H-2',6') 与 δC 167.1 (CO-7') 相关,表明该苯环与1个羰基相连。δH 4.00 (-OCH3) 只与δC 169.8 (CO-7) 相关,表明甲氧基接在羰基上,为羧酸甲酯; δH 7.54 (H-6) 与δC 169.8 (CO-7)、122.3 (C-4)、154.4 (C-5) 相关; δH 8.56 (H-3) 与δC 119.0 (C-1)、134.3 (C-2)、154.4 (C-5) 及169.8 (CO-7) 相关; 根据上述数据可以推出化合物1中三取代苯环的结构为2-胺基-5-羟基苯甲酸甲酯或2-羟基-5-胺基苯甲酸甲酯,单取代的苯甲酰基只能与胺基相连。因此,化合物1的结构为5-羟基-2-苯甲酰胺-</ span>苯甲酸甲酯或2-羟基-5-苯甲酰胺-苯甲酸甲酯,为确证化合物1的结构,对化合物1进行单晶衍射 (图 2),单晶衍射数据见表 2,确定化合物的结构为5-羟基-2-苯甲酰胺-苯甲酸甲酯,为一新化合物,命名为附子酰胺。

Table 1 1H and 13C NMR data of compound 1 (MeOD)

Table 2 The X-ray crystal data (Cu Kα) of compound 1

Figure 1 Structure and key HMBC correlations of compound 1

Figure 2 X-ray crystallographic structure of compound 1
实验部分

Bruker AM-600核磁共振仪 (德国Bruker公司); LTQ Orbitrap XL Spectometer (美国Thermo Fisher Scientific公司); 薄层色谱硅胶GF254 (烟台化学工业研究所); 凝胶Sephadex LH-20 (美国GE公司); 柱色谱硅胶 (青岛海洋化工厂); MCI (日本三菱公司); 大孔阳离子交换树脂D001 (浙江争光实业股份有限公司); 中压液相色谱仪 (美国Waters公司); 分析纯试剂均购自北京化工厂; 色谱纯试剂均购自Karl Fischer公司。

药材生附子购自四川江油中坝附子科技发展有限公司,采挖时间为2009年6月30日。经中国医学科学院药用植物研究所张本刚研究员鉴定为毛茛科乌头属植物乌头 (Aconitum carmichaeli Debx.) 的干燥子根。

1 提取分离

取20 kg生附子,粉碎,用90% 乙醇回流提取3次,每次2 h,得浸膏8 kg,充分溶解于1% HCl溶液中,过滤得到的酸水液通过D001大孔阳离子交换树脂,用去离子水将树脂冲至中性,用10% 氨水碱化,用氯仿提取,减压蒸干后得到氯仿部位78.5 g,采用100~200目硅胶柱色谱,正己烷-丙酮 (100∶1→1∶1→丙酮) 系统进行梯度洗脱,合并得到9个流分 (Fr.1~Fr.9)。Fr.3经反复硅胶柱色谱,采用氯仿-甲醇系统进行洗脱,分离得到化合物8 (24 mg)、9 (50 mg)、10 (30 mg); Fr.4经300~400目硅胶柱色谱,采用氯仿-甲醇 (50∶1→1∶1→甲醇) 系统洗脱,分离得到化合物6 (11 mg)、7 (19 mg); Fr.5经MCI柱色谱分离,甲醇-水系统梯度洗脱,制备液相色谱 (ODS柱) 分离得到化合物1 (3 mg)、5 (9 mg); Fr.6经MCI柱色谱分离,甲醇-水系统梯度洗脱,制备液相 (ODS柱) 分离得到化合物2 (10 mg)、3 (5 mg)、4 (10 mg)。

2 结构鉴定

化合物1 白色针状结晶 (甲醇)。mp 179~ 180 ℃。HR-ESI-MS: m/z 294.075 1 [M+Na]+。NMR数据及单晶数据见表 1表 2

化合物2 棕红色粉末 (氯仿)。ESI-MS: m/z 267 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.23 (1H,dd,J = 8.4,2.4 Hz,H-4),7.21 (1H,d,J = 2.4 Hz,H-6),7.05 (1H,dd,J = 8.4,2.4 Hz,H-2'),7.02 (1H,d,J = 2.4 Hz,H-6'),6.92 (1H,d,J = 8.4 Hz,H-3),6.90 (1H,d,J = 8.4 Hz,H-3'),6.00 (2H,m,H-8,8'),5.09 (4H,m,H-9,9'),3.46 (2H,d,J = 6.6 Hz,H-7),3.35 (2H,d,J = 6.6 Hz,H-7'); 13C NMR (150 MHz,CDCl3) δ 154.3 (C-2),151.1 (C-4'),138.1 (C-8'),136.3 (C-8),132.5 (C-5'),131.4 (C-6),130.5 (C-6'),130.0 (C-1'),129.2 (C-2'),128.9 (C-4),128.0 (C-1),126.7 (C-5),117.2 (C-9),116.9 (C-3),115.9 (C-3'),115.8 (C-9'),39.7 (C-7),35.4 (C-7')。以上数据与文献[4]报道数据基本一致,鉴定为和厚朴酚 (honokiol)。

化合物3 无色针晶 (甲醇)。ESI-MS: m/z 359 [M+H]+1H NMR (600 MHz,MeOD) δ 6.96 (2H,s,H-2',2''),6.83 (2H,d,J = 8.4 Hz,H-6',6''),6.79 (2H,d,J = 8.4 Hz,H-5',5''),4.73 (2H,d,J = 6.4 Hz,H-2,6),4.25 (4H,m,H-4,8),3.87 (6H,s,2×-OMe),3.15 (2H,m,H-1,5); 13C NMR (150 MHz,MeOD) δ 149.0 (C-3',3''),147.2 (C-4',4''),133.7 (C-1',1''),119.9 (C-6',6''),115.9 (C-5',5''),110.9 (C-2',2''),87.4 (C-2,6),72.5 (C-4,8),56.3 (2×-OMe),55.2 (C-1,5)。以上数据与文献[5]报道数据基本一致,鉴定为松脂醇(pinoresinol)。

化合物4 无色针晶 (甲醇)。ESI-MS: m/z 139 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.77 (1H,dd,J = 7.8,1.2 Hz,H-6),7.41 (1H,td,J = 8.0,1.8 Hz,H-4),6.91 (1H,td,J = 8.0,1.8 Hz,H-5),6.89 (1H,dd,J = 7.8,1.8 Hz)。以上数据与文献[6]报道数据基本一致,鉴定为水杨酸(salicylic acid)。

化合物5 无色针晶 (甲醇)。ESI-MS: m/z 165 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.62 (1H,d,J = 16.2 Hz,H-7),7.53 (2H,d,J = 8.4 Hz,H-2,6),6.84 (2H,d,J = 8.4 Hz,H-3,5),6.65 (1H,d,J = 16.2 Hz,H-8)。13C NMR (150 MHz,MeOD) δ 175.0 (C-9),161.1(C-4),146.2 (C-7),131.4 (C-2,6),127.5 (C-1),116.8 (C-3,5),116.7 (C-8)。以上数据与文献[7,8]报道数据基本一致,鉴定为对羟基桂皮酸 (p-hydroxy-cinnamic acid)。

化合物6 白色粉末 (氯仿)。与改良碘化铋钾试剂显色呈正反应。ESI-MS: m/z 358 [M+H]+1H NMR (600 MHz,CDCl3) δ 5.30,5.25 (各1H,s,H-17),4.38 (1H,s,H-15),3.90 (1H,br s,H-1),3.13 (1H,s,H-3),2.79 (1H,s,H-20),1.18 (3H,t,-NCH2CH3),0.88 (3H,s,18-CH3); 13C NMR (150 MHz,CDCl3) δ 208.1 (C-12),149.8 (C-16),110.8 (C-17),75.0 (C-15),66.8 (C-20),64.5 (C-1),56.3 (C-19),53.2 (C-13),52.2 (C-8),47.3 (N-CH2CH3),46.4 (C-7),42.4 (C-5),41.0 (C-10),39.3 (C-14),39.2 (C-3),37.5 (C-4),36.3 (C-11),34.9 (C-9),30.8 (C-6),30.3 (C-2),25.0 (C-18),19.8 (N-CH2CH3)。以上数据与文献[9]报道数据基本一致,鉴定为松果灵 (songorine)。

化合物7 白色粉末 (氯仿)。与改良碘化铋钾试剂显色呈正反应。ESI-MS: m/z 378 [M+H]+1H NMR (600 MHz,CDCl3) δ 4.21 (1H,t,H-14),3.72 (1H,br s,H-1),3.38 (1H,dd,H-16),3.33 (3H,s,16-OCH3),3.00 (1H,br s,18-β-OH),2.78 (1H,s,H-7),1.21 (3H,t,-NCH2CH3),0.88 (3H,s,18-CH3); 13C NMR (150 MHz,CDCl3) δ 82.2 (C-16),76.2 (C-14),74.5 (C-8),73.0 (C-1),63.5 (C-17),60.5 (C-19),56.5 (16-OCH3),49.8 (C-11),48.8 (-NCH2CH3),47.1 (C-5),46.8 (C-9),45.4 (C-7),44.3 (C-10),42.5 (C-15),40.1 (C-13),33.3 (C-4),31.5 (C-3),30.0 (C-2),28.5 (C-12),27.9 (C-18),25.4 (C-6),13.5 (-NCH2CH3)。以上数据与文献[10]报道数据基本一致,鉴定为多根乌头碱 (karakoline)。

化合物8 白色粉末 (氯仿)。与改良碘化铋钾试剂显色呈正反应。ESI-MS: m/z 632 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.89 (2H,m,H-3',5'),7.87 (1H,m,H-4),7.61 (2H,t,H-2',6'),4.94 (1H,d,H-14),4.43 (1H,d,OH-15),4.32 (1H,d,H-16),4.31 (1H,m,H-15),3.89 (1H,d,H-1),3.55,3.21,3.18,3.15 (各3H,s,-OCH3),2.14 (3H,s,-NCH3),1.35 (3H,s,OCOCH3); 13C NMR (150 MHz,CDCl3) δ 20.8 (OCOCH3),35.5 (C-12),37.4 (C-2),40.4 (C-10),42.5 (C-4),43.4 (-NCH3),43.9 (C-9),44.5 (C-7),44.9 (C-5),48.8 (C-19),49.1 (C-11),55.5,57.9,58.4,61.3 (-OCH3),62.0 (C-17),66.5 (C-13),71.6 (C-3),74.3 (C-18),78.3 (C-15),78.9 (C-14),82.9 (C-1),81.4 (C-6),91.0 (C-16),91.5 (C-8),129.0 (C-3',5'),129.3 (C-2',6'),129.5 (C-1'),133.8 (C-4'),165.3 (CO-Ar),171.6 (OCOCH3)。以上数据均与文献[11]的报道基本一致,鉴定化合物8为新乌头碱 (mesaconitine)。

化合物9 白色粉末 (氯仿)。与改良碘化铋钾试剂显色呈正反应。ESI-MS: m/z 616 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.99 (2H,m,H-3',5'),7.65 (1H,m,H-4),7.56 (2H,t,H-2',6'),4.85 (1H,d,H-14),4.71 (1H,s,OH-8),4.34 (1H,d,H-16),4.33 (1H,m,H-15),3.86 (1H,d,H-1),3.51,3.15,3.14,3.09 (各3H,s,OCH3),2.17 (3H,s,NCH3),1.33 (3H,s,OCOCH3); 13C NMR (150 MHz,CDCl3) δ 21.4 (OCOCH3),21.5 (C-2),26.4 (C-12),34.7 (C-3),37.9 (C-10),39.4 (C-4),40.6 (C-13),42.8 (N-CH3),43.9 (C-7),44.7 (C-9),48.3 (C-5),49.8 (C-11),55.8,58.2,59.3,61.6 (OCH3),56.2 (C-19),62.3 (C-17),78.9 (C-14),79.2 (C-15),79.8 (C-18),83.1 (C-6),84.3 (C-1),91.5 (C-16),91.6 (C-8),129.2 (C-3',5'),129.5 (C-2',6'),130.1 (C-1'),134.1 (C-4'),165.9 (CO-Ar),172.3 (OCOCH3)。以上数据与文献[11]报道的基本一致 ,鉴定化合物9为次乌头碱 (hypaconitine)。

化合物10 无色针晶 (氯仿)。与改良碘化铋钾试剂显色呈正反应。ESI-MS: m/z 574 [M+H]+1H NMR (600 MHz,CDCl3) δ 7.98 (2H,m,H-3',5'),7.62 (1H,m,H-4),7.52 (2H,t,H-2',6'),4.72 (1H,d,H-14),4.33 (1H,d,H-16),4.12 (1H,br s,OH-8),3.93 (1H,d,H-15),3.72 (1H,d,H-1),3.52,3.22,3.19,3.15 (各3H,s,OCH3),2.16 (3H,s,NCH3); 13C NMR (150 MHz,CDCl3) δ 21.4 (C-2),35.4 (C-3),37.3 (C-12),39.1 (C-4),39.8 (C-10),42.4 (C-7),43.4 (N-CH3),45.1 (C-9),48.3 (C-5),49.3 (C-11),55.5,58.2,61.1,61.6 (OCH3),57.4 (C-19),66.8 (C-17),71.9 (C-13),74.3 (C-8),76.9 (C-18),79.5 (C-14),80.7 (C-15),82.3 (C-6),83.2 (C-1),92.1 (C-16),128.5 (C-3',5'),129.3 (C-1'),130.3 (C-2',6'),132.7 (C-4'),165.4 (CO-Ar)。以上数据与文献[12]报道的基本一致,鉴定化合物10为苯甲酰次乌头碱 (14-benzoylhypaconitine)。

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