【作者】 刘常青；

【导师】 赖小平；

【作者基本信息】 广州中医药大学 ， 中药学， 2012， 硕士

【摘要】 研究背景：青天葵源于兰科多年生宿根小草本植物毛唇芋兰Nervilia fordii (Hance) Schltr.,药用部位为叶或带块茎的全草,性凉,味甘,归心、肺、肝经；具有清热润肺止咳,解毒散瘀止痛的功效,主要用于治疗肺部疾病。在治疗哮喘、急性支气管炎、喘息型慢性支气管炎方面效果显著,对治疗小儿肺炎有特效。作为南方民间用药,其在临床上的应用越来越多,已经广泛被临床医师认可。在两广、港澳及东南亚等地较为常用,出口量较大,价格昂贵。主产于广西、广东和海南,四川、云南、泰国亦有分布,由于青天葵资源分布有限,自然繁殖率极低,加之乱采乱挖,野生资源日益枯竭,利用植物组培技术发展青天葵人工资源已成为其研究热点。在商品规格上,青天葵按叶片大小分为大叶、中叶、小叶三种,民间亦有用芋兰属植物毛叶芋兰Nervilia plicata (Andr.) Schltr.代替青天葵药用,称作毛叶青天葵。由于青天葵市场价格昂贵,所以其混、伪品也较多,常见伪品有车前草、番薯叶等,应用较混乱。目的：青天葵主要含有黄酮类、氨基酸类、挥发油类等。根据中药化学成分与疗效关系,黄酮类化合物具有清热解毒、化痰止咳作用,而青天葵常用临床功效恰好与之相符。结合本课题“青天葵对全身炎症反应综合征-急性肺损伤大鼠炎症影响机制研究(国家自然科学基金资助项-30472209)”已完成的药理药效研究表明,黄酮类化学成分是其抗炎镇痛、增强免疫、解热、止咳平喘的物质基础。结合青天葵的研究现状,本论文以青天葵中黄酮化合物为重点,从黄酮类化学成分的系统分离、黄酮HPLC特征指纹图谱、双指标黄酮成分定量测定、工艺研究四个方面对青天葵药材中黄酮成分进行研究,以期能够为青天葵黄酮资源的有效、合理开发和质量评价提供科学依据,促进青天葵的合理开发综合利用。方法：1.采用大孔树脂、聚酰氨树脂等柱层析方法富集青天葵黄酮部位,经真空浓缩等方法制备青天葵活性提取物,以HPLC等方法进行分析检测；2.结合青天葵药理实验结果,针对有效部位,采用现代色谱技术进行化学成分的单体分离,以质谱、紫外光谱、核磁共振谱来解析其化学结构。3.收集大、中、小共26批样品,以分离获得的黄酮单体成分为参照物,通过色谱条件单因素考察,建立药材HPLC指纹图谱,建立药材共有模式图,并进行相似度分析。4.建立HPLC液相条件,对药材中的两种特征成分同时进行方法学考察与含量测定,初步比较不同产地、批次26批间含量差异。5.建立总黄酮的含量测定方法,并采用星点设计-效应面法这一在国外被广泛采用的工艺研究方法对青天葵总黄酮提取工艺进行优化,验证；比较不同来源的总黄酮差异。结果：1.采用大孔树脂结合聚酰氨树脂等技术富集青天葵总黄酮,总黄酮得率约为1.14%,对产品进行紫外光谱扫描检测,结果显示其在266nm附近有较强的吸收；2.采用高效制备液相从青天葵总黄酮中分离得到几个黄酮类成分,经光谱鉴定,收率较高的两种分别是：沙苑子苷和鼠李柠檬素；以沙苑子苷为参照物,26批样品中多数样品HPLC指纹图谱相似度良好,个别样品相似度低,对其进行了不同时间区段比较分析,得出药材共有模式图。3.采用HPLC法建立青天葵中沙苑子苷和鼠李柠檬素含量测定方法,经方法学论证,线性关系良好；样品精密度和稳定性良好,加样回收率为98.43%和98.10%,该方法成功用于青天葵药材含量的测定,对多批青天葵进行含量测定,结果青天葵药材中这两个黄酮含量在0.9582～15.5032mg·g-1与0.3134～7.2406mg·g-1之间,表明不同产地、不同品种青天葵叶片中含量有比较显著的差异,可能地理、生态环境、贮存时间等对其有较大影响。暂定按药材干燥品计,沙苑子苷和鼠李柠檬素含量分别不低于2.0mg·g-1、0.5mg·g-1。不同采收期的青天葵药材中两种成分含量随月份不同而有一定差异,从七月到九月,随时间的递增,两种物质的积累总量呈递增趋势。4.以沙苑子苷为对照品,紫外法建立总黄酮含量测定方法,经方法学论证,线性关系良好；样品精密度和稳定性良好,加样回收率为95.51%±1.37%,该方法成功用于青天葵药材含量的测定,对多批青天葵进行含量测定,结果青天葵药材中总黄酮含量在18.88～47.44mg·g-1之间,表明不同产地、不同品种青天葵中总黄酮含量有比较显著的差异,说明地理、生态环境对其有较大影响。暂定青天葵药材黄酮含量限度为：按干燥品计,每克药材总量不得少于20.0mg。星点设计-效应面法用过单因素考察,提取次数暂定为两次,对提取时间、提取溶媒浓度、料液比进行五水平实验,得总黄酮较佳提取工艺范围：乙醇浓度：62%～72%,时间：60～77min,倍量：27～30倍。考虑到工业生产的实际情况,故实验确定提取青天葵中总黄酮的最优工艺为：30倍量65%乙醇回流提取2次,每次75min。结论：1.本文采用高效制备液相这一先进的分离化合物的方法对青天葵黄酮化学成分进行系统分离研究,丰富了青天葵化学成分研究方面的内容,解决了因化学对照品缺乏而限制其质量标准提高和进一步深入开发的关键环节。采取指纹图谱与双指标成分定量相结合的体系,同时体现了中药材中黄酮成分的整体特征和个体特征,得以更好的控制药材质量,同时采用星点设计-效应面法对总黄酮的提取工艺进行优化,可以为该药的后续质量标准研究和新药开发提供实验参考依据。2.体现了“选择疗效确切的中药为研究对象,通过在体动物试验模型所得的药理作用,基于药效作用跟踪的有效成分单体分离,以先进可靠的分析检测方法监控质量”这一科研思路。建立药材有效成分质量科学评价方法,为实现中药新药开发提供了科学依据。更多还原

【Abstract】 Background:Herba Nervilia Fordii, the leaves or the whole plant of Nervilia fordii (Hance) Schltr.(Orchidaceae), which is cool in nature, sweet in flavor, is a famous traditional Chinese medicine of removing heat and toxic material, relieving cough and asthma, removing blood statis and stopping pain.It is mainly used for the treatment of pulmonary diseases, especially in treating Children pneumonia. As the folk medicine in the south of China, it has been widely recognized by clinical physicians.It is mainly distributed in Southern China such as Guangdong, Guangxi, Hainan, and it can also be found in Sichuan, Yunnan province and Thailand. Because of its limited distributions and resources, low reproduction coefficient as well as unauthorized mining, resources of wild Nervilia fordii (Hance) Schltr. gradually exhausted.Using plant tissue technology to develop its artificial resource has become the hot spot.There are three commercial species of it as Big-leaf, Middle-leaf and Small-leaf according to the leaf size.In some places, Nervilia plicata (Andr.) Schltr. was used as the substitute.Due to its expensive price, some other adultrants such as Plantago depreessa Willd., Centella asiatica(L) Urb.are usually used to replace it, which makes the application confused.Objective:According to the chemical composition and the effect relationship of Traditional Chinese medicine, Flavonoids have the role of removing heat and toxic material, relieving cough and asthma,which matches Herba Nervilia Fordii’s clinical efficacy.With the completed subject "Nervilia Fordii on the pharmacological effect of the systemic inflammatory response syndrome-Mechaiam of rats with acute lung injury inflammation National (The Natural Science Foundation funded project)" has showed that Flavonoids are material basis of anti-inflammatory, enhancing the immunity, fever, cough and asthma.In order to promote the comprehensive Utilization of its rational development, this thesis is focused on the Flavonoids in Nervilia Fordii, including four aspects:the separation of Flavonoids, HPLC fingerprint, quantitative determination of two flavonoids and extraction technology research.Methods:1.Flavonoids were enriched by macroporous resin column, polyamide resin and so on. After the concentration under the vacuum, the bioactive compounds was determined by the HPLC(with PDA detector).2. According to the results of the pharmacological experiment of previous study of this subject, the bioactive Flavonoid-chemicals were isolated by the technique of Preparative High Performance Liquid and the chemical structure were identified by the data of MS, UV and NMRC.3.Using the Separated flavonoid as the reference, this study established the HPLC fingerprint chromatographic conditions. Chromatographic fingerprints of26batches of Herba Nervilia Fordii were analysed and obtaineded the medicinal pattern.4. A HPLC method for the simultaneous determination of two characteristic Flavonoid ingredients was established. The contents of different batches of Herba Nervilia Fordii were compared preliminarily.5. A UV method for the total Flavonoids was developed, and the process of extracting Total flavonoids from Nervilia fordii by central composite design and response surface methodology was optimized.The contents of flavonoids from different sources were compared as followed.Results:1.Total Flavonoids (about1.14%of the total amount of Nervilia fordii) were enriched by macroporous resin column, polyamide resin and so on. After the concentration under the vacuum, the bioactive compounds was determined by the HPLC(with PDA detector) and UV absorption.The results shows that at the266nm it has the strong absorption.2.Several compounds of flavone were isolated from the total Flavonoids by high liquid preparation HPLC chromatogram.Using the Separated flavone-complanatuoside as the reference, this study established a HPLC fingerprinting chromatographic conditions.26batches of Herba Nervilia Fordii were analysed and obtaineded the HPLC fingerprint pattern, which was calculated by a software named "Similarity evaluation system for chromatographic fingerprint of TCM".Most samples showed high good similarity except for seven, from which we can easily distinguish the Nervilia fordii.3.A HPLC assay was developed for determination of complanatuoside and rhamnocitrin in the Herba Nervilia Fordii.After the methodology validation, Both of them showed good linearity, while the recovery was98.43%and98.10%respectively.This method was successfully applied for determination of Herba Nervilia Fordii. The results suggested that the total amount of two compounds of flavone ranged from0.9582～15.5032mg·g-1and0.3134～7.2406mg.g-1. Two compounds of different origins, varieties and harvest period exist difference.The content of two compounds from Guangxi is higher than other regions, different harvest seasons in September by content is the highest.This may result from environment, ecological environment, Storage time and so on. On the basis of test results, the contents of complanatuoside and rhamnocitrin should not be less than2.0mg·g-1、0.5mg·g-1separately.4.Using the Separated flavonoid--complanatuoside as the reference, a UV method for the total flavonoids was developed. After the method validation, it shows good linearity, while the recovery was95.51%. This method was also successfully applied for determination of total flavonoids. The results suggested that the total amount of flavonoids ranged from18.88～47.44mg·g-1, which indicated the contents of total flavonoids from different origin, varieties and harvest period were different.On the basis of test results, the contents of the above markers should not be less than20.0mg·g-1.The process of extracting Total flavonoids from Nervilia fordii was optimized by central composite design and response surface methodology. Extraction time is tentatively set for two times and five levels experiment was designed using three facors-extraction time, extraction solvent concentration and solvent multiples.At last, the optimized conditions of extraction process were65%ethanol,75minutes for reflux,30fold solvent and two times for extraction.Conclusion:1.In this paper, the chemical constituents of flavonoids-compounds in Nervilia fordii were separated systematacially by the advanced method of high liquid preparation HPLC chromatogram, which enriched the study of chemical constituents and settled the problem that the lack of chemical reference substance which limited its further development.Taking fingerprint and double index component quantitative system, at the same time emboding the flavonoids’general characteristics and individual characteristics, achieving "Spectral efficiency", which can better control the quality of medicinal materials. At the same time, the extraction process of flavonoids was designed to optimize by central composite design and response surface methodology.It can provide the quality standard and reference for the subsequent research and development.2.This research embodies the scientific research idea that "Choosing the Chinese medicine with definite effect as the research object, obtaining pharmacological effects through in body animal testing model, basing on the therapeutic effects of tracking the effective components monomer separation, using advanced and reliable analytical method to monitor quality".Finally establishing a scientific evaluation method for the effective components, which provids a scientific basis for new drugs’development.更多还原