【作者】 郭秋平；

【导师】 李卫民；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 本文以百合科植物卷丹Lilium lancifolium Thunb.、百合Lilium brownii F.E.Brown var.viridulum Baker.、细叶百合Lilium pumilum DC.为研究对象,在文献研究的基础上,进行了百合药材质量标准研究、总皂苷提取纯化工艺研究、百合药材HPLC指纹图谱研究等内容,并以卷丹为对象,对其部位进行了抗抑郁的药效筛选,并进一步对筛选得到的有效部位深入进行了抗抑郁的机制研究。目的在于建立完善的百合药材的质量标准,建立三种不同来源的百合药材及中间体百合总皂苷的指纹图谱信息库,从而有效地控制药材及其制剂的质量;对其部位进行抗抑郁的药效筛选,找出其抗抑郁的有效部位,并对该部位进行深入的抗抑郁的机制研究,找出其抗抑郁的主要作用途径,为充分开发这一药用资源提供理论依据。质量标准研究方面,百合药材在05版药典中收载的药材标准中仅有性状鉴别和薄层鉴别项,质量标准不完善,没有含量测定项。本文对其质量标准进一步完善。收集了三种来源的百合药材,卷丹10批,百合10批,细叶百合20批,其他产地的百合7批。对三种不同来源的百合进行了显微鉴别:按药典条件进行了薄层鉴别;按照药典方法,进行了杂质、水分、灰分和酸不溶性灰分的测定,确定了药材中杂质、水分、灰分和酸不溶性灰分的限度;对水溶性浸出物及醇溶性浸出物进行了测定,确定了水溶性浸出物、醇溶性浸出物的限度;以薯蓣皂苷为对照品,采用用紫外—可见分光光度法,测定了三种不同来源的百合药材中总皂苷含量,确定了药材中总皂苷含量的限度;采用高效液相色谱法(HPLC法),测定了三种不同来源百合药材中薯蓣皂苷的含量,确定了百合药材中薯蓣皂苷含量的限度。较05版药典增加了显微鉴别项、检查项,以及含量测定项,完善了百合药材的质量标准,提高了百合药材的质量控制水平。对百合总皂苷的提取纯化工艺进行了研究,分别进行了单因素考察和正交考察。单因素考察进行了不同乙醇浓度、溶媒倍量、提取时间、提取次数和提取温度对总皂苷提取率和纯度的影响的考察;建立了四因素三水平的正交试验,最终确立了最佳的提取工艺为:10倍量80%乙醇70℃水浴回流提取3次,每次3小时。用大孔吸附树脂对总皂苷粗提物进行了纯化处理,筛选了不同型号的树脂,考察了树脂的静态、动态吸附性能,并对上样液浓度和流速,洗脱溶剂浓度和体积等相关因素进行了优化,确立了最佳的纯化工艺为:AB-8型大孔吸附树脂,上样液中药材与湿树脂质量比1:1,依次用10BV蒸馏水、20BV80%乙醇以1-3ml/min的洗脱速度洗脱,收集80%乙醇部分,减压浓缩至干。在此工艺下得到的百合总皂苷纯度达67.95%。对三种来源的百合药材和相应的百合总皂苷提取物进行了HPLC指纹图谱研究,通过色谱条件的优化建立了各自的指纹图谱及检测方法,分别标定了卷丹药材16个共有峰,百合药材13个共有峰,细叶百合药材18个共有峰;卷丹总皂苷10个共有峰,百合总皂苷10个共有峰,细叶百合总皂苷15个共有峰,以控制药材的质量。参照国家药监局颁布的《中药注射剂指纹图谱研究的技术要求(暂行)》及运用国家药典委员会中药色谱指纹图谱相似度评价系统(2004年A版)对指纹图谱进行了评价,结果显示,10批卷丹药材、11批百合药材、10批细叶百合药材和相应的卷丹总皂苷提取物、百合总皂苷提取物、细叶百合总皂苷提取物的相似度都在0.9～1.0之间。对三种来源的百合药材指纹图谱进行分区比较,卷丹药材在一区的表观强度较大,百合药材在二区的表观强度较大,细叶百合药材在三区的表观强度较大,五区三者表现一致。对百合药材和总皂苷提取物指纹图谱进行分区比较,二者主要差异在一区,提示百合药材经过提取纯化以后,总皂苷成分基本保留。采用聚类分析的方法对三种来源的百合TLC及指纹图谱进行聚类分析,分别以薄层鉴别的Rf值、指纹图谱的RRT值为指标,进行聚类分析,结果显示,三种不同来源的百合药材分别属于三种不同的类别。可见三种来源的百合药材及总皂苷是确实存在着一定的差异,这种差异可以通过数理统计的方法来区分,进一步说明本课题建立的指纹图谱信息存在着实用价值。三种来源百合药材及总皂苷指纹图谱信息的建立,有利于进一步控制百合药材及百合总皂苷的质量。抗抑郁的药效筛选方面,进行了百合有效部位抗抑郁药效筛选,分别富集了百合皂苷、百合多糖,提取了百合水液。另设了对照组及阳性药氟西汀组。分别进行了小鼠悬尾实验、小鼠利血平拮抗实验、小鼠强迫游泳实验,结果得出,百合皂苷为抗抑郁的主要有效成分之一。抗抑郁的机制研究方面,对百合皂苷进行了抗抑郁药效研究,制备了大鼠慢性心理应激抑郁模型,分别观察了百合皂苷对抑郁模型大鼠行为学的影响,包括抑郁模型大鼠体重、耗食量、糖水偏嗜度及敞箱实验的的检测;百合皂苷对抑郁模型大鼠COR、ACTH的影响;百合皂苷对抑郁模型大鼠CRFmRNA、GR、MRmRNA的影响;百合皂苷对了单胺类神经递质,如DA、5-HT、NE、5-HIAA的影响。结果显示百合皂苷可改善抑郁模型大鼠的体重减轻、耗食量减少及快感缺失等症状,可以改善抑郁模型大鼠的活动能力,改善抑郁模型大鼠的行为迟缓;百合皂苷可提高血液COR、ACTH的含量;百合皂苷可减少抑郁模型大鼠下丘脑CRFmRNA的表达,增加海马GR、MRmRNA的表达;百合皂苷体提高抑郁模型大鼠脑内神经递质的含量,包括DA、5-HT。得出以下结论:百合皂苷有抗抑郁作用,其作用机制可能是(1)增高抑郁模型大鼠大脑皮层的单胺类神经递质的含量;(2)抑制抑郁模型大鼠亢进的HPA。更多还原

【Abstract】 The study object of this article was mainly about the dried bulb of Lilium lancifolium Thunb.,L.brownii F.E.Brown var.viridulum Baker.,and L.pumilum DC.On the basis of the literature research about Lilium Brownii,the quality standard study,the extraction and purification process of total Saponins, and fingerprint study of Lilium Brownii material were researched. Antidepression effect filtration study was also performed with the dried bulb of Lilium lancifolium Thunb.,and to make it clear,the antidression mechanism of the saponins was reaearched.The objection was to establish the integrated quality standard,to collect the fingerprint information of the three different souce of Lilium Brownii,so the quality of Lilium Brownii can be under good control.The antidepression filtration was done to find the effective part of Lilium Brownii.The antidepression mechanism study was done to find the antidepression effective approach of the effective part.All these research can help to establish the theory foundation of medicinal resource development.In quality studies,the study established the criteria of quality control of Liliu（?） Brownii,for it was not consummated in the former criterion which had only morphological identification and TLC qualitative test,the quality standard was incomplete which had no content determination presented in pharmacopoeia（2005）.So the quality standard had to be integrated.Materials of three different source of Lilium Brownii were collected,including ten samples of Lilium lancifolium Thunb.,ten samples of L.brownii F.E.Brown var.viridulum Baker.,twenty samples of L.pumilum DC.,And the other samples from seven different habitats.The microstructure identification was done with the three different souce of Lilium Brownii.The TLC qualitative test was conducted after the pharmacopoeia;following the method of pharmacopoeia（2005）,The content of impurity,moisture,total ash,acid -insouble of Liliun Brownii were tested,and confirmed the limits of impurity, moisture,total ash,acid-insoluble ash;The content of water souble lixivium and ethanol souble lixivium were tested,and confirmed the limits of water souble lixivium and ethanol souble lixivium;The ultra-voilet spectrophotometry method was adopted to test the content of total Saponins in Lilium Brownii using dioscin as reference substance,and confirmed the limits of the total Saponins content;The RP-HPLC method was adopted to test the content of diosin using dioscin as reference substance,and confirmed the limits of the diosin content of Lilium Brownii.Compared to the standard quality of Lilium Brownii in pharmacopoeia（2005）,The microstructure identification, the examination item including impurity,moisture,total ash,acid-insoluble, water souble lixivium and ethanol souble lixivium,and the content test of total Saponins and diosin were added.The Lilium Brownii material quality could be under better control.Studies were carried on the extraction and purification process of total saponins by reviewing the impact of ethanol concentration,ratio of herb to solvent,extraction time and times,and extration temperature.Set up the L₉（3⁴）orthogonal test and confirmed the best extraction process as 10BV 80 %ethanol distilling three times,3h per time.Extract of total saponins was purified with macroeticular resins.Different kinds of resins were applied to review the static and dynamic adsorption ability,to optimize the concentration,volume and flow rate of eluant.It was confirmed that the best purification process was AB-8 macroeticular resins,material to wet resins being 1:1,eluted by 20BV 80%ethanol after 10BV water with flow rate of 1-3ml/min.80%ethanol part was collected and dried under vacuum resulting in the total saponins content reaching 67.95%.Studies of HPLC fingerprint was conducted to three different sources of Lilium Brownii,and their total saponins extract（intermediate）.Their fingerprints were built up by optimizing chromatogram condition,to characterize 16 and 14 common peaks of Lilium lancifolium Thunb.And its saponins extra,13 and 10 common peaks of L.brownii F.E.Brown var.viridulum Baker.and its saponins extra,18 and 16 common peaks of L.pumilum DC.and its saponins extra,so that the quality of medicinal materials can be controlled.Referring to "Technical Specifications of Fingerprint Study of Chinese Traditional Medicine Injection（Interim Regulation）" decreed by SFDA and similarity evaluation system for chromatographic fingerprint of TCM（2004 A）set by China pharmacopoeia committee,the results showed the similarity of Lilium Brownii and their total saponins extract（intermediate）were both from0.9 to 1.0.Compared fingerprint of the three different source Lilium brownie,the most apparent intensity of Lilium lancifolium Thunb.was in the first block,the most apparent intensity of brownii F.E.Brown var.viridulum Baker.was in the second block,the most apparent intensity of L.pumilum DC. was in the third block;Compared the fingerprint of Lilium Brownii and its saponins,the most difference was in the first,which hinted that the saponins component could be retained after extraction and purification.Clustering analysis was done to collect more information of the three different sources of Lilium Brownii.The statistical target was the Rf value of TLC and the RRT value of fingerprint.The statistical result showed that three different sources of Lilium Brownii were classified into three different sorts.The result also showed that there was undoubted difference among the different sources of Lilium Brownii.The difference could be found by the maths-statistical method.The fingerprint information of three different sources of Lilium Brownii and its saponins was collocted to make better control of the quality of the Lilium Brownii material and it preparation.In antidpression effect filtration study,Lilium Brownii saponins and Lilium Brownii amylase were extracted and purified,the water extration of Lilium Brownii were also in this study.The mouse tail suspended experiment, anti-reserpine effect experiment,mouse forced swimming experiment were performed,the result showed that Lilium Brownii saponins was the main antidepression component.In antidepression mechanism study,the rats’ depression model was made by chronic psychological stimulation.The following targets were observed: （1）The influence of Lilium Brownii saponins on the behavior of the depression rats,including the weights changes、the food consume、the sweet water consume and the open-field test;（2） The influence of Lilium Brownii saponins on the blood COR and ACTH;（3） The influence of Lilium Brownii saponins on the CRFmRNA expression in hypothalamus,and GR、MRmRNA expression in hippocampi;（4）The influence of Lilium Brownii saponins on the monoamine neurotransmitter and their metabolites in the depression rats brain,including norepinephrine（NE）, dopamine（DA）,5-hydroxytryptamiHe（5-HT）,5-hydroxy-3-indoleacetic acid（5-HIAA）.The result showed that（1） Lilium Brownii saponins could improve the depression rats’ behavior,including improving the depression rats weight decrease,increasing the food consume,increasing the sweet water consume, and improving behavior stagnancy;（2） Lilium Brownii saponins could reduce the content of COR and ACTH in the depression rats blood;（3） Lilium Brownii saponins could decrease the CRFmRNA expression in hypothalamus,and increase the GR、MRmRNA in hippocampi;（4） Lilium Brownii saponins could increase the monoamine neurotransmitter content in the depression rats’ brain,including the content of DA、5-HT.The conclusion was that the Lilium Brownii saponins had the antidepression effect;the mechanism might be connected with（1） increasing the monoamine neurotransmitter content;（2） restraining the sthenic HPA axis.更多还原