【作者】 张良；

【导师】 李玉锋；

【作者基本信息】 西华大学 ， 食品科学， 2008， 硕士

【摘要】 贝母Frritillaira为百合科Liliaceae贝母属Fritillaria L.植物的干燥鳞茎,是常见的中药材之一,收录于《中国药典》2005版一部。贝母在我国有悠久的使用历史,有清热润肺,化痰止咳之功效,主治肺热燥咳、咯痰带血、痰多胸闷等症。本论文首先研究了川贝母中有效成分生物碱和皂苷的最佳提取工艺。以贝母总生物碱为指标,采用单因素考察了药材粒度,再用正交试验考察了提取时间、提取次数、料液比对贝母类药材总生物碱提取的影响,最终确定最佳提取工艺为:时间24小时、料液比40ml、提取次数2次;采用超临界CO₂流体提取技术提取川贝母游离生物碱,选取萃取压力、萃取温度、萃取时间和夹带剂用量作为考察条件进行单因素试验,在单因素试验的基础上,再通过正交试验。实验结果显示CO₂萃取川贝母游离生物碱的最佳萃取条件为:萃取压力20MPa,乙醇用量300ml,萃取时间2h,萃取温度45℃;采用乙醇（甲醇）索式法、乙醚（丙酮）沉淀法提取贝母总皂苷。选取药材粒度、提取时间、甲醇浓度三因素,通过正交实验确定了提取总皂苷最佳工艺,结果显示最佳提取条件为提取时间为8小时,粒度为100目,甲醇浓度100%。本论文还对川贝母的质量标准进行了探讨,分别测定了贝母类药材的微量元素,采用二极管阵列-高效液相色谱的方法初步构建贝母类药材的指纹图谱,并利用分子生物学标记技术探索了川贝母的质量标准。实验中利用火焰（石墨炉）原子吸收分光光度仪和定碳、定硫仪对10种不同产地、品种的贝母中16种微量元素进行了测定。以测定结果含量为指标,提取特征因子,再利用特征因子作聚类分析。结果表明微量元素,与中药质量标准有相关性;实验还对8种不同品种、产地的贝母作了高效液相色谱分析,通过检测其主要成分生物碱的含量来评价其质量。以贝母素甲为标准品,采用外标法,并对8种贝母药材进行柱前衍生化处理,检测贝母中生物碱并做出分析。本研究初步建立了川贝母的指纹图谱,为川贝母的品种鉴定和质量评价提供一定的参考;采用用随机片段多态性DNA（RAPD）的方法扩增了8个贝母样品总DNA,根据电泳后获得的DNA片段图谱,在同一位置上,重复性的DNA扩增条带“有”记为“1”,“没有”记为“0”,然后在Rapdistance软件上进行分析,并进行聚类。得到的聚类图基本上反映了这些贝母的亲缘关系,由此表明,RAPD的方法可进行川贝母药材的评价与鉴定。更多还原

【Abstract】 The dried bulb of Frritillaira, as a usual Chinese medicine is registered in Pharmacopoeia of People’s Republic of China（2005 edition）volum 1. The Frritillaira has the glorious use history in China. Frritillaira has alleviates fever Run the lung, reduces phlegm and stops coughing effect. Mainly treats the lung hotly to cough dry, the expectorate belt blood, the phlegm multi-chests stuffily and so on sickness.An optimum method was established in this paper to extract and purify the effective fraction in Frritillaira. Adopting the method of orthogonal test and using the orthogonal table of L9（ 3⁴）, take the content of total alkaloid and the ratio of dry extraction as target, investigating the effect of extractive time, extractive number of times, fluid concentration on the extraction results, and then the optimum conditions for the extraction of total alkaloid was determined, which was adding 40% fluid, refluxing and extracting for 24h, totally extracting 2 times. Adopting the method of supercritical CO₂ fluid extraction technology extraction Frritillaira dissociation alkaloid, the selection extract pressure, the extract temperature, the extract time and the entrainer amount used carries on the single factor experiment as the inspection condition, in single factor experiment foundation, again through orthogonal test. The optimum conditions for the extraction of total alkaloid was determined, which was: extract pressure 20MPa, ethyl alcohol amount used 300ml, extract time 2h, extract temperature 45℃; Uses the ethyl alcohol （methyl alcohol） the rope type, the ether （acetone） the precipitation method extraction Frritillaira total sopanin. Adopting the method of orthogonal test and using the orthogonal table of L9（ 3⁴）, investigating the effect of alcohol concentration, alcohol amount, extractive time on the extraction results,and then the optimum extraction condition for the extraction time is 8 hours, the granularity is 100 items, the methyl alcohol density 100%.This paper has also carried on the discussion to Frritillaira ’s quality specification, has determined the trace element of Frritillaira separately. Uses the method of DAD-HPLC preliminary construction Frritillaira ’s fingerprint. And uses the molecular biology labeling technique exploring Frritillaira ’s quality specification. Uses the method of ASS to determine 16 kind of trace elements from 10 different of samples. Take determines the result content as the target, the extraction characteristic factor, makes the cluster analysis using the characteristic factor. The result indicates the trace element, has the relevance with the traditional Chinese medicine quality specification. The experiment uses the method of DAD-HPLC to examines its principal constituent alkaloid to appraise its quality. Take the permine as the standard, uses the external standard method, and carries on processing of derivative to 8 kind of Frritillaira, examines in the alkaloid and makes the analysis. This research has established the Frritillaira fingerprint atlas initially, provides certain reference for the its appraisal and the quality rating; The experiment uses the method of RAPD to increase the total DNA of samples, and obtains DNA fragment atlas. In the identical position, the duplicated DNA banding "has" records as "1", "does not have" to record as "0", then carries on the analysis on the Rapdistance software, and cluster. Obtains the cluster chart had basically reflected these Frritillaira’s sibship, indicated from this, the RAPD method may be able to appraise Frritillaira’s quality specification.更多还原