【作者】 张军；

【导师】 赖小平；

【作者基本信息】 广州中医药大学 ， 中药学， 2008， 博士

【摘要】 隔山香为伞形科山芹属植物隔山香Ostericum citriodorum（Hance）Yuan etShan的根及全株,始载于《植物名实图考》,《中华本草》、《中药大辞典》及诸多地方本草著作如《广西药植名录》、《广西实用中草药新选》、《江西草药》、《浙江民间常用草药》、广州部队《常用中草药手册》都有收载。隔山香药效物质基础研究仅见从其根部石油醚部位分离得到反式异莳萝脑、异莳萝脑乙二醇、β—谷甾醇和一种新化合物的报道。国家中药部颁标准品种“护心胶囊”是以隔山香为君药的岭南特色中成药,疗效可靠确切,但隔山香薄弱的药效物质基础研究成为制约其“二次开发”的瓶颈。开展我国特有物种隔山香成分分离工作,丰富其药效物质基础研究,可阐释其本草应用的科学内涵;在此基础上,建立药材质量控制体系,有利于药材质量的一致性和临床疗效的重复性,亦为隔山香的新药研究和护心胶囊”二次开发”打下基础。本论文以隔山香药效物质基础及其复方制剂“二次开发”的关键工艺为研究课题,进行了化学成分、质量控制方法、有效部位精制工艺研究,以及含有隔山香药材的中药复方的提取精制工艺研究。通过色谱技术,从隔山香乙醇提取物中分离鉴定了8个化合物,其中1个成分是新化合物,有7个化合物系首次从隔山香中分离得到。首次以两种专属性成分作为指标,建立了药材的TLC定性鉴别和HPLC定量分析方法。开展护心复方药液的大孔吸附树脂精制工艺,分别以淫羊藿苷标定的总黄酮、复方药液指纹图谱主峰保留率、酚类成分的颜色检视反应为指标,筛选确定了最大上样量、洗脱溶媒种类及用量等工艺参数;提出“谱效结合”的工艺评价方法,以护心复方药液HPLC图谱主峰保留率和抗急性心肌缺血的药效指标对其大孔吸附树脂精制工艺合理性进行评价。本课题将基础研究与应用开发有机结合,将民间草药药效物质基础研究的“科学内涵”与其特色成药二次开发的“技术含量”相结合,力图促进民间草药文化的传承与发展。一、隔山香药效物质基础及质量控制的研究（一）隔山香挥发油的GC/MS分析利用气相色谱/质谱（GC/MS）对隔山香挥发油进行分析鉴定,共鉴定出47种成分,主要成分有芹菜脑、人参炔醇、肉豆蔻醚、异榄香脂素、丁香烯、棕榈酸、十六烷酸、亚油酸等。部分成分如人参炔醇、肉豆蔻醚、异榄香脂素、丁香烯的药理报道与药材“疏风清热,止咳化痰,活血散瘀,行气止痛”功能相关。（二）隔山香醇提浸膏的成分分离与结构鉴定通过硅胶柱色谱对隔山香醇提浸膏的石油醚部位和乙酸乙酯部位进行成分分离,共分离化合物12个,对其中8个化合物进行结构鉴定。其中隔山香素（Ⅰ）为新化合物;其余分别为异芹菜脑（Ⅱ）、芹菜醛（Ⅲ）、methoxylatifolone（Ⅸ）、6,22-何帕二醇（Ⅴ）、正二十二烷（Ⅵ）、豆甾醇（Ⅶ）、正十六碳酸α-甘油酯（Ⅷ）,上述化合物为首次从该植物分离得到。已鉴定的化合物类型包括苯丙素类、三萜类、甾醇类、脂肪酸类等,其中三个属于苯丙素类化合物。文献报道的反式-异莳萝脑与异芹菜脑（Ⅱ）为同分异构体。药材中更多苯丙素类和其它化合物的发现可进一步丰富其药效物质基础研究。（三）以专属成分为指标,建立隔山香TLC鉴别方法根据隔山香成分分离及药理研究基础、临床应用经验,其TLC鉴别实验围绕挥发油和水溶性成分开展。1.隔山香挥发油TLC鉴别方法:照薄层色谱法（《中国药典》（一部）附录Ⅵ）,以异芹菜脑为对照品,以石油醚—丙酮（9:0.5）为展开剂,建立了隔山香挥发油的TLC鉴别方法。2.隔山香水溶性成分TLC鉴别方法:照薄层色谱法（《中国药典》（一部）附录Ⅵ）,以隔山香素为对照品,以甲苯-甲酸乙酯-甲醇-甲酸（7:2:1:0.3）为展开剂,建立了隔山香水溶性成分的TLC鉴别方法。（四）以专属成分为指标,建立隔山香HPLC含量测定方法按照中药质量标准研究的共性要求,结合隔山香成分分离及其药理研究基础、临床应用经验,本课题建立了隔山香药材中异芹菜脑和隔山香素的HPLC测定方法,上述两对照品可分别作为挥发油和水溶性部位的指标成分。1.隔山香中异芹菜脑含量测定方法:以Kromasil C₁₈色谱柱（250mm×4.6mm,5μm）为固定相,甲醇—水（70:30）溶液为流动相,流速为1.0ml/min,检测波长为280nm;异芹菜脑线性范围为0.2016～3.6288μg（r=0.9999）,平均回收率为99.06%,RSD为1.98%（n=6）。2隔山香中隔山香素含量测定方法:以Kromasil C₁₈柱（250mm×4.6mm,5μm）为固定相,以乙腈—水（45:55）溶液为流动相,流速为1.0ml/min,检测波长为320nm;隔山香素线性范围为0.02032～0.2032μg（r=1）,平均回收率为98.68%,RSD为2.54%（n=6）。测定三批药材,其中异芹菜脑含量为6.5mg/g以上,隔山香素含量为0.25mg/g以上。（五）建立隔山香药材HPLC色谱方法以Kromasil C₁₈柱（250mm×4.6mm,5μm）为固定相;以乙腈—0.3%磷酸溶液梯度洗脱为流动相,流速为1.0ml/min;检测波长为320nm;柱温为20℃。以50%乙醇提取药材制备供试品溶液,以异芹菜脑为对照品。供试品溶液制备方法和流动相梯度洗脱系统兼顾了药材高、中、低极性成分的提取和检测。4批药材的HPLC色谱测定结果表明,其HPLC色谱峰峰位基本一致,色谱峰所代表的成分含量有所差异。鉴于药材批次较少,且所分析药材的生长地点及年限、采收加工、贮存诸多信息无法溯源,暂不拟定其标准指纹图谱。（六）隔山香药液大孔吸附树脂精制工艺研究以隔山香HPLC色谱中主峰保留率为指标,通过优化树脂用量、洗脱溶媒种类等参数,所得醇洗液HPLC图谱与上柱液基本一致,醇洗液中主峰平均保留率为76.7%,表明隔山香药液大孔吸附树脂精制工艺基本保留了其药材HPLC图谱主峰所代表的化学成分。二、护心复方药液大孔吸附树脂精制工艺的研究护心复方以隔山香为君药,组方具有鲜明的岭南特色,疗效确切。鉴于处方药材隔山香、毛麝香、石菖蒲、吴茱萸含有较多挥发油（相当于全方生药量0.5%以上）,且挥发油具有与处方功能密切相关的药理活性,采用固体分散的滴丸制剂既可使挥发油固化,在制剂中稳定,又有利于挥发油的快速溶出,迅速起效。方中药材水溶性成分亦具有心血管活性,但提取后得膏率较高,导致滴丸服用量过大。护心复方提取药液的精制工艺成为其“二次开发”的技术关键。基于对方中单味药材的大孔树脂精制工艺研究结果,我们采用大孔树脂对护心复方药液进行精制。分别以淫羊藿苷标定的总黄酮、复方药液HPLC图谱主峰保留率、酚类成分的颜色检视反应为指标,筛选优化了最大上样量、洗脱溶媒种类及用量等工艺参数。（一）护心复方药液过AB—8柱的动态吸附曲线研究以淫羊藿苷标定的总黄酮为指标,结合HPLC指纹图谱,考察了复方药液的动态吸附曲线。结果表明最大上样量（以5%泄漏点计）为164.61mg总黄酮/g干树脂,在此上样量,过柱液HPLC图谱中未见主峰成分明显泄漏。（二）护心复方药液过AB—8柱的洗脱溶媒种类研究考察不同溶媒种类对淫羊藿苷标定的总黄酮洗脱率,结合HPLC图谱和单味药材精制工艺的前期研究,洗脱溶媒种类确定为95%乙醇。（三）护心复方药液过AB—8柱的洗脱溶媒用量研究考察不同用量95%乙醇对淫羊藿苷标定的总黄酮洗脱率,结合HPLC图谱和单味药材精制工艺的前期研究,洗脱溶媒用量确定为以95%乙醇洗脱2倍柱体积。（四）酚类成分颜色反应对护心复方药液过AB—8柱工艺条件的验证以三氯化铁（2%）—铁氰化钾（1%）试液标示的酚类成分颜色反应,对上述工艺参数进行考察。结果表明:在上述工艺条件下,过柱液中酚类成分的泄漏远未达到上柱液的5%;95%乙醇洗脱2倍柱体积后,后续洗脱液中酚类成分的浓度远低于上柱液浓度的5%。提示以酚类成分颜色反应定性评价所确定的树脂工艺参数,亦具有合理性。该工艺条件下,得膏率由过柱前的16%降低为过柱后的6.5%,为制备辅料用量较大的滴丸和分散片提供了可行性。三、“谱效结合”对护心复方药液大孔吸附树脂精制工艺的评价研究护心复方药液的大孔吸附树脂精制工艺,显著降低了得膏率,却面临是否保留药效活性的质疑。尤其护心复方面临药效物质基础和成分协同研究基础薄弱的背景,如方中主药隔山香、毛冬青、毛麝香都未有明确的有效部位共识,若采用文献常用的单一的指标成分或笼统的有效部位评价其过柱精制工艺合理性,显得苍白无力。这也是大多数中药复方提取、精制工艺研究面临的普遍背景,即“黑箱”因素的存在。本课题以护心复方药液的大孔吸附树脂精制工艺为载体,提出“谱效结合”的评价思路,即以“指纹图谱”主峰所代表的化学成分保留率与关键“药效学”代表的生物活性对复方精制工艺进行评价。护心复方药液经大孔吸附树脂精制后,醇洗液中隔山香、毛冬青、毛麝香、淫羊藿主峰保留率达到90%以上,水洗液未见主峰明显泄漏,上柱液与醇洗液HPLC图谱基本一致,表明护心复方药液经AB—8大孔吸附树脂精制,很好地保留了HPLC图谱主峰所代表的化学成分,包括已知成分淫羊藿苷和异芹菜脑。护心复方药液过柱前后的抗心肌缺血药效学试验结果显示,上柱液及醇洗液可明显改善急性心肌缺血症状,表现为抑制Ⅱ导心电图ST段的升高,缩小梗死范围,抑制血清心肌酶的升高;过柱及水洗液药效作用微弱,在部分指标如梗死区占危险区体积百分数、血清心肌酶AST、LDH未显示药效活性。抗心肌缺血活性强弱的综合比较:醇洗液（0.4g/kg/d）)上柱液（0.4g/kg/d）)过柱及水洗液（0.4g/kg/d）。表明护心复方药液经AB—8大孔吸附树脂精制,醇洗液保留了护心复方药液抗心肌缺血活性。“谱效结合”的评价指标综合考虑了物质基础及其效应,适合中药制剂药物作用的物质基础不是完全清晰的研究背景,尊重中药制剂多成分作用的特点。即使药效物质基础在我们指纹图谱视野之外,药效学指标的复核也会避免工艺路线的差错。采用“谱效结合”的评价指标,有助于我们在尚未完全认识药材或复方药效物质基础的现有状况下,减少因药效物质基础及协同作用的“黑箱”因素所导致工艺研究出现差错的概率;同时“谱效结合”的工艺评价过程所积累的不同工艺产物的多批“谱”与“效”的相关性研究又为复方药效物质基础及成分协同作用研究提供了线索,为“谱效学”或“组效学”的研究提供了素材。本项目的研究特色在于以传承和发展民间草药为主线,将隔山香基础研究与护心胶囊“二次开发”紧密结合,以新工艺的运用和新剂型的开发提高产品“技术含量”,将产品的“技术含量”与民间草药基础研究的“科学内涵”结合起来,是传承和发展民间草药可行技术路线的具体实施。本项目的创新点在于从隔山香药材中分离获得新化合物1个,首次从该药材分离化合物7个,丰富其药效物质基础研究;首次以两种专属性成分为指标,建立了包括药材TLC鉴别和HPLC含量测定、HPLC图谱的质量控制体系;对护心胶囊“二次开发”的关键工艺——大孔吸附树脂精制工艺进行了多指标结合的优化筛选。本项目的创新点还在于提出并实施“谱效结合”的工艺评价方法,以指纹图谱主峰所代表的化学成分保留率与关键药效学实验所代表的生物活性对中药制剂新工艺进行评价,丰富和发展了中药药剂学的工艺评价方法。更多还原

【Abstract】 Folk herbal medicine is the treasure of Chinese nation and the mutual wealth of human beings.However,the precious experience of herbal medicine and affluent medical resources are in the danger of disappearance,due to the economic development of the modern society,the destruction of ecological environment,and the deficiency of herbal medicine records—limited studies of chemical constituents and restriction of clinical application.So,it is urgent to protect and utilize them.By applying modern technical method,we can use the treasury to establish the bridge between the folk medicine and modern technology sufficiently.It has an actual sense of not only spreading the culture of traditional medicine,but also promoting the investigation and development,serving the human beings and transforming it into commodity’s advantages,which consequently advance economy development rapidly in local region.According to the aforementioned,we selected the effective substance of Lemonfragrant Angelica Root and the critical process of Huxin extract as the studying topic,combined the fundamental research and application to inherit and develop the culture of Chinese medicine.1.Research on chemical constituents and quality control of Lemonfragrant Angelica RootLemonfragrant Angelica is a species endemic to China.Its root and the whole plant are used as crude drag recorded in Iconographia Plantarum Chinese Materia Medica Chinese Materia Medica dictionary and many local medical books,such as Guangxi herbal medicine Jiangxi herbal medicin Zhejing common folk herbal medicine and common herbal medicine manual from Guangzhou troops.The main components were always wroten as starch,flavonoid glycoside,essential oil,organic acid and carbohydrate,common herbal medicine manual from Guangzhou troops introduced its actions:root,circulating the blood and eliminating stasis,promoting the flow of qi to relieve pain,stoping coughing and eliminating phlegm,applied to angina pectoris,gastralgia,chronic cough and also venomous snake biting.The effect of Lemonfragrant Angelica Root on cardiovascular diseases is reliable and clear that it is used as a monarch drug in the well-known Chinese medical preparation "HUXIN Capsule".But the lack of researches on chemical components of Lemonfragrant Angelica Root make the investigation just focus on isodillapiol,isodillapiolglycol andβ-sitosterol isolated in petroleum ether part from the root,there was no more records about the chemical constituents in the literature at home and abroad.（1）.GC-MS analysis of essential oil from Lemonfragrant Angelica RootOur study initially used GC-MS method to analyse and identify essential oil from Lemonfragrant Angelica Root,47 compounds were identified including apiol, panaxynol,myristicin,isoelemicine,caryophyllene,hexadecanoic acid,hexadecanoic acid, linoleic,etc.The pharmacologic action of partial components was related to the action of Lemonfragrant Angelica Roots.（2）.Chemical constituents from Roots of Lemonfragrant AngelicaLemonfragrant Angelica roots（10kg）was extracted with 95%ethanol and filtered. The filtrate was concentrated by rotary evaporation,the resulting residue was partitioned between H₂O and petroleum ether,then by EtOAc,n-butanol.The petroleum ether fraction and EtOAc fraction were subjected to silica gel column,then gradiently eluted with Petroleum eher-EtOAc respectively.Similar fractions were combined with TLC plates screening,then repeated to the silica gel column and recrystallized to gain purified compounds.12 compounds were isolated and 8 compounds were identified by the the IR,MS,1H and 13C-NMR spectrograph.One of them is a new compound and the rest 7 compounds are obtained from this plant from the first time.（3）.Founded a new TLC method for identifying two types of components from Lemonfragrant Angelica RootⅠ.TLC method for the essential oil from Lemonfragrant Angelica RootThe coarse powder of plant material（20g）was extracted by distillating for 4h to give the volatile oil,and then the oil was dissolved in 10ml ether to prepare the sample solution, when 1,3-Benzodioxole,4,7-dimethoxy-5-（1E）-1-propenyl were served as standard. According to the thin layer chromatography ruled by Chinese Pharmacopoeia,took 10μl the sample solution and 10ul standard solution to have small spot on the same thin layer plate;used petroleum ether-acetone（9:0.5）as the developer;took out and volatilized the solvent when the developer moved up to the leading edge,and finally sprayed Phospho-molybdic acid,heating for visualizing the spots.There was the same spot on the same position of the sample comparing standard solution.Ⅱ.Identified method for the water-solubility part from Lemonfragrant Angelica RootThe coarse powder of plant material（2.5g）was extracted with CH₃OH（25ml）by reflux for 1h.After filtered and concentrated the filtrate,the residule was dissolved with water（20ml）to subject to AB-8 type of macroporous resin column（1.5cm×8cm）.The followed process was:firstly eluted by water,then by 95%EtOH,collected the 95%EtOH eluted solution and recovered EtOH,the residue was dissolved with 2ml MeOH and using citriodorumin as standard.According to the thin layer chromatography ruled by Chinese Pharmacopoeia took 10μl sample solution and 10ul standard solution to have small spot on the same thin layer plate;used Methyl benzene-Ethyl formate-MeOH-Formic acid（（7:2:1:0.3））as the developer and pre-saturated for 15min;took out and volatilized the solvent when the developer moved up to the leading edge,and finally sprayed aluminium trichloride solution,inspecting in the UV lamp（365nm）.There was the same spot on the same position of the sample compared with standard solution.（4）.Initially developed fingerprint HPLC spctrum for Lemonfragrant Angelica RootⅠ.Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18（250mm×4.6mm,5μm）column as stationary phase,when acetonitrile（A）and 0.3%hydrogenphosphate（B）were served as gradient elution in the flow rate of 1.0ml/min.Detective wavelength was set at 320nm, simultaneously,the column temperature was maintained at 20℃.Ⅱ.Preparation of standard solutionUsing 5.0mg Bdp as standard,weighed accurately,then dissolved in 25ml MeOH and mixed for injecting.Ⅲ.Preparation of sample solutionWeighed 1.0g coarse powder of plant material accurately;dissolved with 50ml 50%EtOH;weighed and extracted by reflux for 1h,weighed again to make up the lose weight with 50%EtOH.The extracted solution was filtered through 0.45μm syringe filter for HPLC injecting. Ⅳ.Determination methodTook 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.（5）Founded firstly HPLC quantity determination method of two Phenylpropanoid componens from Lemonfragrant Angelica RootⅠ.Quantity determination method of Bdp from Lemonfragrant Angelica Roota.Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18（250mm×4.6mm,5μm）column as stationary phase,when MeOH（A）and H₂O（B）were served as elution in the proportion of 70:30.Detective wavelength was set at 280nm.b.Preparation of standard solutionAn accurately weighed standard Bdp was dissolved in MeOH to prepare the standard solution with a concentration of 200μg/ml.c.Preparation of sample solutionWeighed 0.5g coarse powder of plant material accurately;dissolved with 50ml MeOH and then extracted by ultrasound for 30min;weighed again to make up the lose weight with 50%MeOH after descending to room temperature;finally filtered and took the filtrate as sample solution.d.determination methodTook 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.Ⅱ.Quantity determination method of citriodorumina.Chromatographic conditions and system suitability testThe assaying was performed by Kromasil C18（250mm×4.6mm,5μm）column as stationary phase,when acetonitrile（A）and H2O（B）were served as elution in the proportion of 45:55.Detective wavelength was set at 320nm.b.Preparation of standard solutionAn accurately weighed citriodorumin standard was dissolved in MeOH to prepare the standard solution with a concentration of 10μg/ml.c.Preparation of sample solutionWeighed 0.5g coarse powder of plant material accurately;dissolved with 50ml MeOH and then extracted by ultrasound for 30min;weighed again to make up the lose weight with 50%MeOH after descending to room temperature;finally filtered and took the filtrate as sample solution.d.determination method Took 10μl standard solution and 10μl sample solution accurately and injected into the chromatographic system for determination.（6）.Process of Lemonfragrant Angelica root extract with maeroporous resinHPLC fingerprint of Lemonfragrant Angelica Root was adopted as index to investigate the purified process of Lemonfragrant Angelica Root extract with AB-8 type of macroporous resin.The results indicated that the HPLC fingerprints of the extract before and after absorption by macroporous resin were similar.2.Research on purification process of Huxin Extract with macroporous resinThe prescription of"Huxin" which use Lemonfragrant Angelica Root as monarch drug has vivid "Lingnan" feature and practically curative effect.Respecting that most drugs of the prescription such as（Ostericum citriodorum,Adenosma glutinosum,Acori Gramineus and Evodiae）contain a large amount of essential oil（equal to more than 0.5%of crude drug in prescription）which is similar in activity to the whole prescription,we chose drop pill praeparatum which have the advantages of stabilizing the essential oil by solidification and dissolving quickly.The refining processes of the extracted solution was an important part of the drop pill preparation.Based on the research of macroreticular resin processes for single drug in the prescription,we selected macroreticular resin to purify the compound extraction.By the content of total flavonoids（marked by icariin）,the Retention ratio of main Components in HPLC fingerprints and the Phenols compounds inspected by color reaction,kinetic adsorption curve,the type and dosage of elution solvent were investigated. The results indicated that this refining process could reduce the yield of extract effectively and retain the main components of the HPLC fingerprints.（1）.Study on the kinetic adsorption process with AB-8 macroreticular resin for the purification of Huxin extractUsing icariin as index and combining HPLC fingerprints to study the refinement process with AB-8 macroreticular resin.The results showed that the maximum dosage（the leak point was 5%）was 164.61mg total flavonoids/g dry resin,and the main components in the HPLC fingerprints were retained.（2）.Study on the kind of elution solvent during the purification process with AB-8 macroreticular resin of Huxin extractAccording to the elution ratio,HPLC fingerprint and the literature on refinement process of single drug,95%EtOH was used as solvent.（3）.Study on the dosage of elution solvent during the purification process with AB-8 macroreticular resin of Huxin extractBased on the elution ratio,HPLC fingerprint and the literature on refinement process of single drug,we finally concluded that 2 folds of column volume of 95%EtOH should be used to elute.（4）.Verification to the refinement process parameters by the color reaction of the Phenols compoundsThe processing parameters were confirmed respecting to the color reactions of the Phenols compounds marked by the chloride ferric（2%）-ferricyanatum Kalium（1%）solution. The results suggested that the Phenols compounds leak far less than 5%,compared with that of initial solution;after eluting 2 folds of column volume of 95%EtOH,the concentration of phenols compounds in the subsequent eluted solution was far less than 5%, compared with that of initial solution.As the case stands,we can retain phenols compounds during the refinement process.3.Evaluation on Huxin extract with macroporous resin using "Bioactivity and FingerPrint"as an indexThis experiment kept the product in the retention time in the main fingerprints and retained its bioactivity when compared with the initial extracts:the dia-column liquor, water eluant and the ethanol eluant respectively,which have a representative action of resisting myocardial ischemia injury.When using "Bioactivity and FingerPrint" as an index, this method also establishes a reasonable refined process of Huxin extract with macroporous resin.The retention time of the main peaks in HPLC chromatogram after the treatment of resin remained more than 90%without apparent leakage of the main peaks in water eluent, moreover,the HPLC chromatograms of the initial physic liquor and the ethanol eluant were similar,both indicating that the quantity of Lemonfragrant Angelica Root,Ilex pubescem, Adenosma glutinosum and Epimedii in the "Huxin" compound extract was reserved, including icariin and Bdp.The biological results of protecting myocardial ischemia after the experimental rats intragastically administrated with initial extract,the dia-column liquor,water eluant and the ethanol eluant,show as follows:the initial extract and the ethanol eluant can significantly reduce myocardial ischemia induced injury,which inhibit the upgrade of the ST segment in ECG and the myocardial enzyme in serum,and diminish the infarction area.However, dia-column liquor and water eluant has a weak action to attenuate myocardial ischemia injury;neither effect on volume percentage of infarction area occupied the risk area,nor decrease myocardial enzyme AST and LDH.The ability of different eluants to protect against myocardial ischemia injury was:the ethanol eluant（0.4g/kg/d）＞the initial extract （0.4g/kg/d）＞the water eluant（0.4g/kg/d）.It demonstrated that the active compounds were principally enriched in the ethanol eluant,and the process of refinement with macroporous resin remained the bioactivity of the initial extract.This project not only discovers a new compound,but also identifies seven compounds from Lemonfragrant Angelica roots.We establish a system to standardize the quality of the plant material which includes the identification of TLC,the quantitive determination and chromatogram of HPLC.The outcomes can be applied in the "Secondary Development" of "Huxin Capsule".Without application of new technologies,there will be no innovation of traditional chinese medicine products.There always exists a doubt that if the functions of the drug remains while the amount of formula seems less after the treatment with new technology.This topic puts forward and carries out evaluation index of "Bioactivity and Fingerprint".In other words,according to the retention time of main components represented by main peak of "HPLC fingerprints" and the bioactivity represented by the key pharmacodynamics experiment,it is possible and applicable to make evaluations on new technology of medicament preparation.更多还原