【作者】 刘江；

【导师】 陈兴福；

【作者基本信息】 四川农业大学 ， 药用植物学， 2010， 硕士

【摘要】 《中国药典》(2005年版)收载的麦冬药材为百合科(Liliaceae)沿阶草属(Ophiopogon)多年生草本植物麦冬Ophiopogon japonicus(Thunb.)Ker-Gawl.的干燥块根,其主要分布于我国四川、浙江等地。本研究搜集了四川丘陵区、盆周山区、川西平原区及重庆地区等26个不同来源的野生麦冬资源,建立起了四川盆地麦冬种质资源的色谱指纹图谱,并开展了模式识别研究；在麦冬种质资源的形态指标,大类成分、氨基酸含量,指纹图谱等研究的基础上,建立了麦冬多层次的质量评价体系,对四川盆地不同地区野生麦冬资源进行了综合评价分析；初步筛选出了多个优质的麦冬品种资源,为麦冬化学计量学分类及其质量的科学评价、有效控制及品种选育工作提供了有效参考。主要研究结果如下：1.麦冬种质资源形态性状研究对引种的26份四川盆地麦冬种质资源样品的13个形态性状进行研究,结果表明,麦冬资源的形态特征存在广泛变异,其中,块根鲜重、干重及茎叶鲜重、干重的变异系数最大(≥58.45%),根长和叶片长的变异最小,变异系数分别为14.39%和25.89%；各形态性状间存在相关性,且多数达到0.01显著水平；主成分分析及聚类分析结果表明,第1主成分反映麦冬植株的数量性状特征,第2主成分反映茎叶的大小特征,第3主成分反映块根的大小特征,都是造成麦冬形态变异的主要因素；26个不同资源样品被划分为4大类,其类别间各主成分性状的大小差异明显,可根据不同需要从众多野生麦冬资源中选择特异性种质资源加以开发利用。2.麦冬种质资源大类成分研究采用分光光度法测定了四川盆地不同麦冬种质资源各大类成分(皂甙、黄酮、可溶性多糖)的含量。不同来源麦冬样品的皂甙平均含量为2.55%,资阳市简阳市的麦冬中皂甙含量最高,达到了6.172%；广安市武胜县的麦冬中皂甙含量最低,含量仅为0.442%,样品间相对标准偏差为60.47%,差异性较大。不同来源麦冬样品的黄酮平均含量为0.12%,乐山市中区的麦冬中黄酮含量最高,达到了0.23%；泸洲市中区的麦冬中黄酮含量最低,含量仅为0.04%,样品间相对标准偏差为38.55%,差异性较大。不同来源麦冬样品的可溶性多糖平均含量为16.68%,泸州市市中区Ⅰ样品中可溶性多糖含量最高,达到了40.33%；广安市市中区样品中可溶性多糖含量最低,含量仅为0.04%,样品间相对标准偏差为57.34%,差异性较大。不同来源野生麦冬资源间大类成分含量的差异性均较大,变异明显。3.麦冬种质资源氨基酸含量及其评价研究使用日立L-8800全自动氨基酸分析仪测定了四川盆地不同麦冬种质资源的氨基酸含量及分析讨论了其分布特征,结果表明,麦冬中含有多种氨基酸,不同麦冬资源间氨基酸种类不尽相同；其总氨基酸、必需氨基酸含量均大于常见蔬菜、水果；多数样品中各必需氨基酸的含量均低于FAO/WHO评分模式中同种氨基酸的含量；脯氨酸、谷氨酸、苯丙氨酸、蛋氨酸等具有特殊功效、呈味效果敏感的氨基酸在麦冬中的含量较高。麦冬不适合作为常规食品直接食用,宜作为营养补充剂、保鲜剂,天然食品添加剂、调味剂的天然原料加以利用；四川盆地麦冬种质资源丰富,样品间氨基酸分布差异较大,可根据实际需要,从中筛选出不同用途的麦冬资源,并加以综合利用。4.麦冬种质资源色谱指纹图谱研究利用乙醇作为溶剂进行超声提取,以麦冬脂溶性成分作为考察主体,采用反相高效液相色谱法(RP-HPLC)和气相色谱质谱(GC-MS)联用法,连续测定了四川盆地不同来源野生麦冬资源的色谱图,分别建立了川产麦冬种质资源的液相特征图谱和气相特征图谱。26批野生川麦冬样品共有28个HPLC特征峰，13个GC特征峰,其中24批样品图谱与所建立的特征图谱间的相似度均达到90%。建立的麦冬色谱特征图谱具有相似性,重现性良好,其样品处理方法简单、快速、可连续使用,色谱方法简便、准确,结果可靠,特征图谱直观明确,特征明显。综合利用了RP-HPLC与GC-MS的图谱信息,对麦冬种质资源进行了系统聚类分析,不同批次麦冬样品被聚为不同层次的5类,结合麦冬药材HPLC、GC中共有特征峰的信息,可为麦冬的资源分类、质量评价提供参考。5.麦冬种质资源综合评价对四川盆地不同麦冬种质资源的主要化学成分及产量性状等质量指标进行主成分聚类分析,综合评价了四川盆地麦冬种质资源的质量,结果表明,资阳市市中区、乐山市市中区等样品的综合质量评价值较高,其中以资阳市市中区样品对川产麦冬种质资源的贡献度最大,达州市大竹县样品的贡献度最小,原始数据的聚类分析结果亦呈现类似规律；综合HPLC及GC指纹图谱共有特征峰的相对峰面积量化数据,分别进行直接系统聚类分析及“主成分—聚类”分析,结果表明,“主成分—聚类”分析结果更符合指纹图谱的整体情况,适合进行整体定性鉴别,而直接聚类分析结果更加符合样品间各具体化合成分的含量情况,适合进行局部定量比较。质量指标主成分综合评价与色谱指纹图谱综合评价从不同角度对麦冬质量进行了综合比较,色谱指纹图谱综合评价可用以从整体上比较鉴别麦冬样品,质量指标主成分综合评价可进一步全面、系统地比较麦冬样品质量；四川盆地野生麦冬资源丰富,其质量存在较大差异,通过主成分分析、聚类分析对麦冬质量进行综合评价的方法,具有可靠性,可为麦冬优质资源选择提供参考。更多还原

【Abstract】 The medicinal materials of O. japonicus is the dry root of Ophiopogon japonicus(Thunb.)Ker-Gawl. in Chinese Pharmacopoeia (2005 Edition), which is mainly distributed in Sichuan, Zhejiang and other places of China. In this study, we have collected wild resources of O. japonicus which were came from 26 different areas in hilly area and basin mountain of Sichuan, western Sichuan plain, and the Chongqing region. We have established the chromatographic fingerprints on wild germplasm resource of 0. japonicus and developed the study of chemical pattern recognition. At the same time, we created multi-level quality evaluation system for O. japonicus integrate with morphological parameter, content of major categories compositions and amino acid, fingerprints and other multi-dimensional information, to conducted a comprehensive evaluation analysis on wild resources of O. japonicus in different parts of Sichuan Basin. Now we have preliminarily screening out a number of high-quality species resources of 0. japonicus and provided a valid reference for the works of stoichiometric classification, scientific quality evaluation, effective quality control and breeding of new varietyThe specific results of these studies are as follow:1. Morphological characteristics of germplasm resources of 0. japonicus in Sichuan basin.The results showed that there are extensive variations in morphology, the most significant variance lay in the fresh root weight, dry root weight, and fresh leaf weight, dry leaf weigh, the least significant variance lay in the root length and leaf length; Meanwhile, morphological characteristics of O. japonicus have significant relationships; The results of principal component analysis and cluster analysis showed that the first principal component reflected the characteristics of plant quantity, the second principal component reflected the characteristics of stem leaf, and the third principal component reflected the characteristics of earthnut, all of these were main factors which resulted in morphological variation of O. japonicus. cluster analysis based on the morphological characters showed that the 26 germplasm resources of O. japonicus in Sichuan basin were divided into 4 types. The differences of principal component among the types of O. japonicus were obvious. It is able to select the specific samples from the wide resources of wild 0. japonicus according to the different needs of development and utilization.2. Content of main components in the germplasm resources of O. japonicus in Sichuan basin.To determine the content of main components(such as saponin, flavonoid and soluble polysaccharide) in different germplasm resources of O. japonicus in Sichuan basin with spectrophotometric determination. The results showed that, to the samples of O. japonicus from different areas, their average content of saponin is 2.55%, the content in the sample which was came from Ziyang Jianyang is the highest, it is reached to 6.17%; The content in the sample which was came from Guang’an Wusheng is the lowest, it is merely 6.17%; The relative standard deviation between the samples is 60.47%, the difference is enormous. The average content of flavonoid is 0.12%, the content in the sample which was came from Leshan Shizhong is the highest, it is reached to 0.23%; The content in the sample which was came from Luzhou Shizhong is the lowest, it is merely 0.04%; The relative standard deviation between the samples is 60.47%, the difference is enormous. The average content of soluble polysaccharide is 16.68%, the content in the sample which was came from Luzhou Shizhong a is the highest, it is reached to 40.33%; The content in the sample which was came from Guang’an Shizhong is the lowest, it is merely 0.04%; The relative standard deviation between the samples is 57.34%, the difference is enormous. There are large differences for the contents of major categories compositions among the samples of O. japonicus from different areas, the quality variation for the wild resources of O. japonicus is evident.3. Contents of amino acids in the germplasm resources of O. japonicus in Sichuan basin.The contents of amino acids were determined by HITICHI L-8800 automatic analysis instrument. The results showed that, there are so many kinds of amino acids in the O. japonicus, the sort of amino acids among such resources were different, their contents of amino acids and essential amino acids were higher than common vegetables and Fruits. The contents of essential amino acids in most samples were lower than the FAO/WHO recommended pattern. The contents of amino acids with special effects and sensitive flavor effects, such as Pro, Glu, Phe, Met, were higher than other amino acids. It is unfavorable to eat directly as commen foodstuff, it is favorable to use as natural raw materials on nutritional supplements, fresh-keeping agents, food additives, flavoring agent; There were plentiful variations among the germplasm resource of O. japonicus in Sichuan Basin, the differences of amino acids distribution among different samples were huge, it can be able to select resources of O. japonicus with kinds of uses, and to be utilized synthetically. 4. Chromatographic fingerprints of the germplasm resources of 0. japonicus in Sichuan basin.To used ethanol as a solvent for ultrasonic extraction and defined the fat-soluble components as main examination, the chromatographic fingerprint was established for evaluating and controlling the quality of germplasm resource of 0. japonicus in Sichuan Basin by reversed-phase high performance liquid chromatographic combined with gas chromatographic mass spectrometry. The results showed that each sample was characterized by the peak area of 28 HPLC peaks and 13 GC peaks in each program and these peaks were employed for hierarchical cluster analysis. Furthermore, the discrimination of the sample from different regions was achieved by hierarchical cluster analysis via recognizing the 16x41 datamatrix. This was the first report of hierarchical cluster analysis of wild germplasm resource of O. japonicus according to their Chemical fingerprint. Thus, the results proved it is a simple, rapid and accurate method suitable for the quality control of the traditional Chinese medicines.5. Synthetical evaluation for the germplasm resources of 0. japonicus in Sichuan basin.According to the Main contents and Yield traits, the wild germplasm resources of 0. japonicus from different areas of Sichuan basin were comprehensively compared by the SPSS 17.0 software with principal component analysis and cluster analysis. The results showed that, The six samples of Ziyang, Jianyang, Leshan, Yibin,Chongqing, Mianyang, their comprehensive evaluation value of quality were higher than others, and the sample of Ziyang have the best quality, the sample of Dazhou have the worst quality, the results of the cluster analysis to raw data were also shown a similar law as principal component analysis. The wild resources of 0. japonicus in Sichuan Basin is rich, there were much differences among their quality; the method, through principal component analysis to study the comprehensive evaluation of the 0. japonicus quality,was reliability and the results of cluster analysis was also support their Conclusions，it can be able to provide a reference to Select high-quality resources of O.japonicu.更多还原