【作者】 罗学军；

【导师】 黄艳萍；

【作者基本信息】 天津医科大学 ， 药物分析， 2011， 硕士

【摘要】 大叶药Piper laetispicum C.DC.,为胡椒科胡椒属植物,主要分布在我国广东、广西、云南及海南省。民间用于活血、消肿、止痛,主治跌打损伤、瘀血肿痛。近期研究发现大叶药具有较强的抗抑郁作用,其活性和一系列酰胺类化合物有大。为进一步研究大叶药抗抑郁物质基础及其分析方法,服务于新药开发,本课题采用反向研发模式,即从药材入血成分出发,进行入血成分分析、制备及结构鉴定,然后对得到的单体化合物进行抗抑郁活性筛选,确定大叶药抗抑郁主要活性成分。最后,本课题开发了一种大叶药多成分分析方法,可以为大叶药活性成分的进一步开发提供技术支持,也可以作为将来大叶药中间体及制剂的含量测定方法。具体内容包括以下四个方面：1、入血成分分析利用体内抗抑郁动物模型对大叶药进行抗抑郁活性验证,结果表明大叶药提取物显示较好的抗抑郁活性。采用肠外翻囊模型进行大叶药入血成分的分析,结果显示大叶药醇提取物中除个别化合物外,其余化合物均能够在肠吸收透过液中被检测到,以母液19个色谱峰计,在十二指肠吸收液中检测到16个,结肠和空肠中分别检测到15个,直肠中检测到10个。这说明大叶药醇提物中大多数化合物均可以入血。2、入血成分制备和结构确认以大叶药入血成分的HPLC色谱图为参照,采用大孔树脂富集,硅胶柱分离,Flash快速制备等手段,得到14个单体化合物,其中12个为酰胺类化合物,化合物G13-1(1-[(2E,4E,7E)-9-(?)(3,4－次甲二氧基苯)九碳二烯酰]四氢吡咯)和G13-2(1-[(2E,7E)-N-7-(3,4-次甲二氧基苯)九碳二烯酰]四氢吡咯)为新化合物,G9((2E,4E)-N-异丁基-9-(3,4-次甲二氧基苯)七碳二烯酰胺)、G12(5’-甲氧基-3’,4’-次甲二氧基桂皮酸四氢吡咯)和G14(1-[(2E,4E)-9-(3,4-次甲二氧基苯)七碳二烯酰]四氢吡咯)为大叶蒟中首次分离的化合物。经HPLC-PDA测定,12个酰胺类化合物纯度均能达到98%以上,可以在后续的研究中作为对照品使用。3、活性研究采用小鼠尾悬吊和小鼠强迫游泳抗抑郁模型对部分化合物进行活性研究,结果表明化合物G5、G6和G11均具有明显的抗抑郁活性,其中G5和G6与空白对照组比较具有显著性差异,强于相同剂量的阳性药氟西汀组。G6为大叶药素,其活性研究结果和文献报道的基本吻合,表明大叶药酰胺类生物碱是大叶药抗抑郁活性的物质基础。4、分析方法研究建立了能同时测定大叶药中12个酰胺类生物碱的HPLC分析方法。该方法能够很好的分离12个生物碱并且有效避免了其他杂质的干扰。方法中色谱柱采用了C18色谱柱、流动相：乙腈-水(0 min,55:45；20 min,60:40；30min,90:10；32 min,55:45；37 min,55:45,),流速为1 ml/min,DAD检测器。该方法简单、快速、专属性强,稳定性好,可以用于大叶药药材及相关产品的质量控制。本实验还探索了一测多评技术在大叶药酰胺类化合物含量测定中的应用,结果表明该方法校正因子在不同条件下相对稳定,但是色谱峰的定位方面存在较大误差,由含量测定的结果看,与标准曲线法计算结果偏差很小。因此在规定仪器和色谱柱型号的条件的该方法可以用于大叶药中12个酰胺类化合物的含量测定。更多还原

【Abstract】 Piper laetispicum C.DC. (Piperaceae), which enjoys vast uses for invigorating circulation and reducing stasis, detumescence and analgesic in the southern part of China, is popularly known in folk as a folk medicine, Present studies show that extracts of P. laetispicum C.DC. have distinctly antidepressant activity. In addition, its activity relates with a serial of amides.To further study on the antidepressant bioactive constituents and analytical method of Piper laetispicum, which can serve research and development of new drug, a new model called Reverse R&D was used. Based on the absorbed components analysis, preparation, structure identifying and bioactivity screening of absorbed components of Piper laetispicum C.DC., the antidepressant bioactive constituents were confirmed. Finally, a method for the simultaneous determination of multicomponent was developed, it is useful as a technical support for further study on the antidepressant bioactive constituents and a measure method for intermediate and preparation. The main contents were as follows:1.Analysis of components migrating into bloodAt the beginning, in vivo animal model of depression was used to verify antidepressant activity of Piper laetispicum C.DC..The results showed that it has distinctly anti-depressant activity. Then everted gut scas were used to analysis the intestinal components of the extractive Piper laetispicum C.DC. absorbed by the different intestinal section in vitro. It showed that most of compounds which can be detected by the HPLC were absorbed in the intestine.16 compounds in the duodenum, 15 compounds in the colon and jejunum respectively,10 compounds in the rectum were detected considering 19 compounds as total in solution. This indicates that most of the compounds of Piper laetispicum C.DC. can be absorbed into the blood.2.preparation and structure identifying of components migrating into bloodTake the HPLC chromatogram of constituents migrating to blood as the reference, 14 compounds including 12 amide alkaloids were separated and purified by the means of Macroporous resin, silica gel and flash rapid preparative chromatography. G13-1(1-[1-oxo-9(3,4-methylenedioxy-phenyl)-2E,4E,7E-nonatrienyl] pyrrolidine) and G13-2(1-[1-oxo-9(3,4-methylenedioxy-phenyl)-2E,7E-nonadienyl] pyrrolidine) are new compounds, G9(N-isobutyl-7(3,4-methylenedioxy-phenyl)-2E-4E-HeptaDie-namide)、G12 (1-[1-oxo-3(3,4-methylenedioxy-5-methoxy-phenyl)-2E-propenyl] pyrrolidine) and G14 (1-[1-oxo-7(3,4-methylenedioxy-phenyl)-2E,4E-heptadienyl] pyrrolidine) are separated initially from Piper laetispicum C.DC. The purity determined by HPLC-PDA of 12 amides alkaloids is more than 98% indicating these amides alkaloids can be used as reference substances in the following study.3.Study on antidepressant activityForced swimming test and tail suspension test ha been used in this part. The results demonstrated that compound G5, G6 and Gll have distinctly anti-depressant activity. In the one single dose test, the activity of G5 and G6, which was stronger than the same dose of positive drug fluoxetine group was significantly different from the control group, G6 is the laetispieine and its activity is consistent with the reported, which shows that amide alkaloids are material basis of antidepressant activity of Piper laetispicum C.DC.4.Study on Analysis MethodA simple, rapid, precise and accurate reversed-phase HPLC method had been developed and validated for the simultaneous determination of the 12 amide alkaloids in Piper laetispicum. The method had shown adequate separation for separating these 12 compounds from their associated main impurities. Separation was achieved on a 5μm,250mm×4.6mm i.d. column using a mobile phase consisting of acetonitrile-water (0 min,55:45;20 min,60:40;30min,90:10; 32 min,55:45;37 min,55:45,) at a flow rate of 1 ml/min and UV detection at 240 nm. Good linearity over the range 0.8-300μg/ml was observed, and the regression coefficient for the linear equations for the 12 compounds lay between 0.9995 and 1. The intra-day and inter-day precis-ions (CV) of analysis lay in the range of 0.22-1.86 and 0.55-1.90%, respectively, and the accuracy ranged from 85.46-116.36%. This method had been successfully applied to the analysis of 12 amide alkaloids in Piper laetispicum.更多还原