南葶苈子化学成分与质量研究

【作者】 王爱芹；

【导师】 王秀坤；

【作者基本信息】 北京中医药大学 ， 中药鉴定学， 2003， 硕士

【摘要】 葶苈子为常用中药，始载于《神农本草经》，具有泻肺平喘、利水消肿之功效。2000年版《中国药典》规定其来源为十字花科植物独行菜Lepidium apetalum Willd和播娘蒿Descurainia sophia (L.)Webb.ex Prantl的干燥成熟种子，前者称为“北葶苈子”、后者称为“南葶苈子”。但目前仍存在葶苈子药材品种混乱、质量低劣问题。为了对葶苈子的质量进行有效地控制，通过调查，了解到当前葶苈子药材的主流品种为南葶苈子，本论文结合文献和药理实验，针对南葶苈子的70%乙醇提取物进行了化学成分研究。确定了南葶苈子中定性和定量对照品，继而用HPLC法对收集到的大量样品进行含量测定及各样品的指纹图谱的测定，给出了南、北葶苈子的指纹图谱。制订了南葶苈子药材的质量控制标准。首先进行葶苈子商品药材调查，经对全国范围内收集的30份葶苈子药材进行分析研究，结果表明目前葶苈子商品药材主要为南葶苈子、北葶苈子或二者的混合品，其中南葶苈子为主流品种。南葶苈子化学成分的研究：由于南葶苈子为主流商品，故选定南葶苈子进行深入地研究。从其75%乙醇提取物中，采用硅胶、聚酰胺、sephadex LH20和C18柱层析分离纯化获得单体成分，共分离得到了15种化合物。采用化学和谱学方法（UV、1HNMR、13CNMR、HSQC、HMBC、TOCSY、FAB-MS、EI-MS、ESI-MS），鉴定了其中的12种化合物，分别为：DS1，槲皮素-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷；DS2，山萘酚-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷；DS3，异鼠李素-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷；DS4，槲皮素-7-O-β-龙胆双糖苷；DS5，山萘酚-7-O-β-龙胆双糖苷；DS6，异鼠李素-7-O-β-龙胆双糖苷；DS7，槲皮素-3、7-二-O-β-D-吡喃葡萄糖苷；DS8，山萘酚-3、7-二-O-β-D-吡喃葡萄糖苷；DS9，异鼠李素-3、7-二-O-β-D-吡喃葡萄糖苷；DS10，山萘酚-3-O-β-D-吡喃葡萄糖基-7-O-β-D-[2-芥子酰基-β-D-吡喃葡萄糖基（1→6）]-吡喃葡萄糖苷；DS11，芥子酸乙酯；DS12，4-甲氧基芥子酸。其中化合物DS6、DS10为新化合物，DS4、DS5、DS7、DS8、DS9为首次自十字花科植物中分离得到，DS1、DS2、DS3为首次自播娘蒿属植物中分离得到，DS11、DS12为首次自该植物中分离得到，DS4，DS5中糖的连接方式为首次明确确定。南葶苈子化学成分研究为科学使用葶苈子提供了理论基础。确定以及制备对照品：DS1在南葶苈子中含量较高，是自然界中极少存在的化合物，从文献研究和实验研究显示，DS1为南葶苈子的专属性特征成分，并且在一定程度上反映了南葶苈子的药效，故选定DS1为南葶苈子的定性、定量用对照品，制备了210mg，纯度为98%以上。黄酮苷成分DS1的水溶性强，不溶于无水甲醇、无水乙醇等有机溶剂，分离制备较困难。经反复试验及优化，确定了经济、简便、可重复的对照品制备工艺。南葶苈子定性研究：本实验确定DS1为专属性对照品，采用聚酰胺薄膜法，以乙酸乙酯/甲醇/水（7：2：1）为展开剂，取得较好的分离效果，经大量样品验证，证明该方法具有良好的重现性。南葶苈子定量研究：根据南葶苈子的临床疗效而设计的抗血小板激活因子（PAF）的药<WP=5>理筛选实验，证明南葶苈子总黄酮成分为其有效部位。再结合文献报道及对南葶苈子系统的化学研究结果，表明以槲皮素、山萘酚及异鼠李素为母核的黄酮为南葶苈子的有效成分。故采用HPLC法对南葶苈子中含量较高DS1、DS2和DS3进行了含量测定，并通过方法学考察及全国各地收集的30份葶苈子样品的测定，结果发现葶苈子中黄酮类成分的含量因产地、品种及储存时间的不同而不同，其中南葶苈子中含有这三种黄酮苷，而北葶苈子中不含。通过本项研究确定以黄酮苷DS1、DS2和DS3的含量可作为评价南葶苈子质量的依据，建立了南葶苈子的质量标准。指纹图谱：初步给出了南、北葶苈子的HPLC指纹图谱，对25份葶苈子（22种南葶苈子和3种北葶苈子）药材进行测定，分别记录了1hr. 的色谱图，确定了南葶苈子的共有峰，对其中3个特征峰进行了化学结构的标定。并且指出了南、北葶苈子的主要区别。综合文献资料及本论文的实验结果，得出以下结论：1 化学研究：自南葶苈子药材中分离鉴定了12种黄酮苷和有机酸衍生物类化合物。分别为：槲皮素-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷（DS1）；山萘酚-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷（DS2）；异鼠李素-3-O-β-D-吡喃葡萄糖基-7-O-β-龙胆双糖苷（DS3）；槲皮素-7-O-β-龙胆双糖苷（DS4）；山萘酚-7-O-β-龙胆双糖苷（DS5）；异鼠李素-7-O-β-龙胆双糖苷（DS6）；槲皮素-3、7-二-O-β-D-吡喃葡萄糖苷（DS7）；山萘酚-3、7-二-O-β-D-吡喃葡萄糖苷（DS8）；异鼠李素-3、7-二-O-β-D-吡喃葡萄糖苷（DS9）；山萘酚-3-O-β-D-吡喃葡萄糖基-7-O-β-D-[2-芥子酰基-β-D-吡喃葡萄糖基（1→6）]-吡喃葡萄糖苷（DS10）；芥子酸乙酯（DS11）；4-甲氧基芥子酸（DS12）。其中化合物DS6、DS10为两种新化合物，DS4、DS5、DS7、DS8、DS9为5种首次自十字花科植物中分离得到的黄酮苷，DS1、DS2、DS3为3种首次自播娘蒿属植物中分离得到的黄酮苷，DS11、DS12为2种首次自该植更多还原

【Abstract】 Semen Descurainiae and Semen Lepidii, or Tinglizi in Chinese, is a well-known Chinese medicine recorded first in the famous Shen Nong’s Herbal as a lower grade drug. It has the effect of removing heat from the lung and relieving asthma, inducing diuresis and diminishing swelling, according to the theory of Traditional Chinese Medicine.The pharmacopoeia of P. R. China (2000 revision) specifies Tinglizi as the ripe dried seeds of Descurainia sophia (L.) Webb ex Prantl and Lepidium apetalum Willd. However, seeds of other plants from Cruciferae and even some fakes are also commercialized under the same name Tinglizi. Therefore, a quality control of this drug is in great demand, which is the aim of this dissertation.In the first part of the research, a herb market investigation was held. 26 crude drug samples collected from 16 provinces all over the country were carefully analyzed. The result shows that Descurainia sophia (L.) Webb ex Prantl is the main sources of Tinglizi at present.TLC and HPLC were tried to identify Semen Descuraininae. According to the results, it is found that different species of Tinglizi and fakes have their own significant features. Semen Descurainiae though from different provinces has its own stable characteristics, which can be used to distinguish from the other similar species rapidly and accurately.Chemical constituents of the 75% ethyl alcohol extract of Semen Descurainiae were investigated. 15 compounds were isolated by polyamide, silica gel, sephadex LH20 and reverse silica gel C18 column chromatography. The structures of 12 compounds were identified by means of mp., UV, NMR and MS, including 10 flavonoids and 2 organic acid derivatives. They are kaempferol-3-O-β-D-glucopyranosyl-7-O-[（2-O-trans-sinnapoyl）-β-D-glucopyranosyl（1→6）]-β-D-glucopyran -oside, quercetin-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside, kaempferol-3-O-β-D-glucopyran -osyl-7-O-β-gentiobioside, isorhamnetin-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside, quercetin -7-O-β-gentiobioside, kaempferol-7-O-β-gentiobioside, isorhamnetin-7-O-β-gentiobioside, quercetin-3，7-di-O-β-D-glucopyranoside, kaempferol-3，7-di-O-β-D-glucopyranoside, isorham -netin-3，7-di-O-β-D-glucopyranoside, sinapic acid ethyl ester, 4-methoxyl sinapic acid. Among them, there are some first reports such as : kaempferol-3-O-β-D-glucopyranosyl-7-O- [（2-O-trans-sinnapoyl）-β-D-glucopyranosyl（1→6）]-β-D- glucopyranoside and isorhamnetin-7-O-β-gentiobioside from natural plants, quercetin-7-O-β-gentiobioside, kaempferol-7-O-β-gentiobi -oside, quercetin-3，7-di-O-β-D-glucopyranoside, kaempferol-3，7-di-O-β-D-glucopyranoside and isorhamnetin-3，7-di-O-β-D-glucopyranoside from Cruciferae, quercetin-3-O-β-D-glucopyranosyl -7-O-β-gentiobioside, kaempferol-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside and isorham -netin-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside from genus Descurainiae, sinapic acid ethyl ester and 4-methoxyl sinapic acid from Descurainia sophia (L.) Webb ex Prantl. The linkage of glucoses in compounds quercetin-7-O-β-gentiobioside and kaempferol-7-O-β-gentiobioside were determined definitely by high resolving power NMR.Preliminary pharmacological experiments in vitro were designed and carried out according to the<WP=7>clinical effects of Tinglizi. It is suggested that total flavonoids existing in 75% EtOH extract of Semen Descurainiae has the anti-PAF activities. The above results can in a way be considered as the pharmacological mechanism of Tinglizi’s relieving asthma to the body. Meanwhile, quercetin-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside can be considered as one of the important active ingredients in Semen Descurainiae due to its great amount.The characteristic ingredient quercetin-3-O-β-D-glucopyranosyl-7-O-β-gentiobioside, which exists only in Semen Descurainiae, is chosen as the reference substance for qualitative and quantitative analysis of the drug, and 210mg is prepared with purity of no less than 98%. Furthermore, an economical, simple and reproducible preparation pr更多还原