【作者】 王晓雪；

【导师】 再帕尔·阿不力孜；

【作者基本信息】 北京协和医学院 ， 药物分析学， 2011， 博士

【摘要】 本论文重点运用质谱分析方法开展了珍稀、濒危野生福建金线莲中主要成分黄酮类及芳香酸类化合物的快速分析鉴定,以及野生与生物技术栽培品种相关成分及其含量比较分析研究。首先,运用正、负离子检测模式相结合的RRLC-MS/MS及MSE方法对福建金线莲中所含化学成分进行了系统分析,建立了适合于快速鉴定福建金线莲粗提物中黄酮及芳香酸类成分的质谱分析方法。进一步采用RRLC-MS与1H NMR的平行动态谱(NMR/RRLC-MS PDS)和异相关谱(NMR/RRLC-MS HCS)方法,开展了该提取物中多组分无需完全分离的同步结构鉴定研究,建立了针对混合物组分群中黄酮及芳香酸等不同类型成分的NMR/RRLC-MS PDS和HCS谱学分析方法,拓展了这二种新型谱学分析方法的应用范围；同时结合RRLC-MS/MS等手段,实现了混合物组分群中不同类型成分无需完全分离的快速结构鉴定。此外,利用RRLC-MS/MS方法,开展了野生与生物技术栽培的福建金线莲中主要成分及其含量的比较分析研究。本项研究结果为福建金线莲的可持续利用以及筛选与评价其人工培育品种及技术工艺研究提供了重要分析依据。本论文的研究内容主要包括以下三个部分：1.福建金线莲粗提物多组分的RRLC-MS/MS及MSE快速鉴定方法研究为了建立适用于福建金线莲粗提物多组分的RRLC-MS/MS分析方法,首先系统开展了液相色谱条件与质谱参数的优化,获得了适宜的色谱和质谱条件,成功地建立了用于95%乙醇提取物中常量与微量成分结构鉴定的正、负离子检测模式相结合的RRLC-MS/MS及MSE分析方法。采用上述分析方法,开展了福建金线莲95%乙醇提取物中化学成分的系统分析研究,同时结合黄酮苷类化合物的裂解规律及相关文献报道,共分析推断出17个化合物的结构。其中包括4个芳香酸类化合物,5个黄酮苷元、5个黄酮O-单糖苷类,2个黄酮O-双糖苷类化合物和对羟基苯甲醛,其中3个化合物为首次从该植物中发现的化学成分。本研究表明,采用以RRLC-MS/MS方法为主,并结合MSE技术的分析思路,可以实现福建金线莲中黄酮类、芳香酸类及其它化学成分的简便、快速的结构鉴定。2.福建金线莲提取物组分群的NMR与RRLC-MS平行动态谱和异相关谱分析方法研究以福建金线莲95%乙醇提取物为研究对象,利用反相制备型高效液相色谱技术获取含量呈连续动态变化的系列混合物,并分别进行RRLC-MS和1H NMR谱测定。然后采用MATLAB软件编写的数据处理程序,对原始数据进行处理与分析,构成了NMR/RRLC-MS PDS和NMR/RRLC-MS HCS谱。通过综合分析,获取多组分混合物中同一成分的质荷比(m/z)和化学位移值(6)等相关性信息,利用这些关键信息并结合RRLC-MS/MS分析方法,对系列流份中的黄酮类及芳香酸类等相关成分进行了分析鉴定研究。实现了该提取物中13个成分的同步结构鉴定,其中包括8个黄酮类化合物,4个芳香酸类化合物和对羟基苯甲醛,其中5个化合物为首次从该属植物中发现的化学成分。因此,运用本研究方法实现了混合物中不同类型成分无需完全分离的快速结构鉴定,并拓展了这二种新型谱学分析方法的应用范围。3.野生与生物技术栽培的福建金线莲主要成分及其含量的比较分析研究在上述研究基础上,采用RRLC-MS/MS方法,进一步开展了野生与生物技术栽培的福建金线莲中相关成分及其含量的比较分析研究。首先分别从精密度、准确度等方面对该分析方法进行了细致、系统的方法学考察,结果显示其灵敏度高、准确度高、精密度良好。采用建立的RRLC-MS/MS方法,针对野生(JO)与9种不同生物技术栽培品种(J1-J9)获得的金线莲95%乙醇提取物进行了化学成分比较分析研究,结果发现在生物技术栽培获得的品种J1和J5中化学成分种类和数目与野生品种极为相近。进一步对于野生与生物技术栽培品种中20个主要成分进行了含量分析,同时结合标准品对6个黄酮类成分进行了定量分析。结果发现与野生金线莲主要成分及含量近似的2个生物技术栽培品种(J5,J6)；以及与野生品种主要成分及含量差异较大的品种(J2)。本项研究建立了金线莲生物技术栽培品种的含量评价方法,并为筛选与评价福建金线莲人工培育品种以及大量繁育工艺技术研究提供了重要分析依据。更多还原

【Abstract】 Mass spectrometric analytical method was used to identify main constituents including flavonoids and organic acids in the rare endangered plant Anoectochilus roxburghii (A. roxburghii), and to compare constituents and their content in the wild and artificial ones bred with biotechnologies. First of all, constituents in the crude extract of A. roxburghii were identified using RRLC-MS/MS and RRLC-MSE in both positive and negative ion modes. Consequently, combined with RRLC-MS/MS, the NMR/RRLC-MS PDS and NMR/RRLC-MS HCS were used to identify the main constituents in the crude extract. Three types of natural products were identified successfully, which exploited the application of correlation spectroscopy of NMR/RRLC-MS. In addition, comparison of constituents and their content between the wild and artificial breeding A. roxburghii were implemented using RRLC-MS/MS. Overall, the results of this paper have provided important basis for sustainable utilization of A. roxburghii and evalution of the cultivated species.1. Fast identification of constituents in the crude extract of Anoectochilus roxburghii using RRLC-MS/MS and MSEIn order to establish RRLC-MS/MS and MSE analytical methods to identify the constituents in the crude extract of A. roxburghii, the analytical condition of liquid chromatography and mass spectrometry analysis have been investigated systematically. RRLC-MS/MS and RRLC-MSE analytical methods were then established and allowed the identification of seventeen constituents, including four aromatic acids, five flavonoid aglycones, five flavonoid mono-O-glycosides, two aromatic acids and p-hydroxybenzaldehyde, of which three constituents were discovered in the plant for the first time. The results indicated that rapid identification of flavonoids, aromatic acids and other constituents in A. roxburghii could be achieved using RRLC-MS/MS and RRLC-MSE2. Study of NMR/RRLC-MS PDS and NMR/RRLC-MS HCS on crude extract of Anoectochilus roxburghiiBased on the incomplete separation analysis strategy, the crude extract of A. roxburghii was separated into a series of fractions with the concentration of constituent dynamic variation using reversed-phase preparative chromatography. The RRLC-MS and 1H NMR data of series of fractions were acquired and then processed by MATLAB to form NMR/RRLC-MS PDS and NMR/RRLC-MS HCS. The correlation relationship between 1H NMR and the extracted ion chromatogram (XIC) signals deriving from the same individual constituent could be correlated and extracted simultaneous from the mixture spectra. Combined with RRLC-MS/MS spectra, three kinds of natural products were then successfully identified, including eight flavonoids, four aromatic acids and p-hydroxybenzaldehyde, five of which have not previously been reported in A. roxburghii. The results highly proved that this approach should be of benefit in unequivocal structural determination of a variety of classes of compounds from an extremely complex mixture, such as herbs and their active extracts.3. Comparison on the main constituents and their contents between the wild Anoectochilus roxburghii and the artificial breeding speciesAn RRLC-MS/MS method was established to compare the wild A. roxburghii with the artificial breeding species on main constituents and their contents. First of all, sensitivity, precision, calibration curves and accuracy of the methods have been studied systematically. The results indicated that RRLC-(-)ESI-MS/MS analysis methods in negative ion mode could quantify the main constituents accurately and exactly. Nine artificial breeding species (J1-J9) were then compared with wild A. roxburghii (JO)on constituents using this method, and it was found that Jl and J5 is almost the same with JO on constituents. Furthermore, qualitiation of twenty constituents and quantification of six constituents in wild and artificial breeding A. roxburghii were implemented, and the results indicated J5 and J6 were found to have almost the same constituents and their constants with JO, while J2 showed great difference from that of JO. In all, analytical method for content assessment of artificial breeding species was established, and the results provided important scientific basis for screening and evaluation of artificial cultivation technologies.更多还原