绿茶的定量色谱指纹图谱研究

发布时间：2018-11-20 17:52

【摘要】：茶作为中国传统饮品已有千年历史。绿茶是我国产量最多的一种茶且倍受人们喜爱。近年来的研究表明,绿茶提取物具有抗氧化、抗肿瘤、抗炎、降低心血管疾病患病风险等功效。影响绿茶品质的主要因素有:茶树品种以及绿茶产地的地表温度、平均降水量、海拔高度和日照时长等。现行的绿茶国家标准存在标龄较长,分析手段落后且耗时较长,评价指标不够完善等问题。如何建立可全面表征绿茶中化学成分的分析方法,如何高效、快速、准确地评价绿茶的质量是当前绿茶研究中亟待解决的现实问题。本研究以云南和湖南的绿茶为研究对象,采用超高效液相色谱-高分辨质谱联用技术对绿茶的化学成分进行了详细研究,主要的研究内容包括以下几个方面:1.采用超高效液相色谱-高分辨质谱联用技术对采集自云南和湖南的共72批绿茶进行分析。本研究对不同的提取溶剂(纯水、纯甲醇、70%甲醇)和不同的绿茶样本用量(0.1g、0.5g、1.0g)进行了比较和优化。结果表明,采用70%甲醇提取0.5g绿茶样本的效果最佳。为获取更为丰富的质谱信息,对实验的质谱条件包括源内诱导裂解电压和高能碰撞诱导电压等进行了优化选择。为避免获取前体离子时产生过多的干扰碎片,源内诱导裂解电压选择为OeV。同时,为获取更多的二级碎片,高能碰撞诱导电压采用梯度电压(10eV、30eV、50eV)。最后,为保证实验仪器在分析过程中的稳定性,利用混标及质控样本进行了精密度和稳定性等方法学考察。结果表明,在整个实验过程中,实验仪器稳定、可靠。2.绿茶中化学成分的定性定量分析。采用MZmine质谱分析软件对获取的质谱数据进行提取及分析。为尽可能多的提取有效质谱,对质谱分析软件的参数设置进行优化,包括峰校正、峰检测、峰提取、解卷积、峰对齐等过程中的峰高、峰宽、噪音水平等参数进行选择优化。研究发现噪音水平是最为关键的参数。经综合分析,噪音水平选择在5.00E8时既可以排除背景干扰又可以最大限度保留有效质谱信息。在此基础上,利用标准物质、多个质谱数据库和化学数据库中的一级和二级质谱信息匹配,在两地绿茶中共定性出包括儿茶素类、黄酮类、氨基酸等75种化合物,其中33种化合物经标准品进行确认。本研究以这75种化合物为绿茶化学特征成分群,采用内标法进行半定量分析,并建立了两地绿茶的定量色谱指纹图谱。研究表明,尽管两地绿茶的化学指纹图谱极为相似,但各物质含量却存在一定差异。3.云南和湖南产绿茶的差异性分析。首先,采用聚类分析和主成分分析两种无监督模式识别方法对云南和湖南产绿茶进行区分。结果表明,这两种方法均能对该两地绿茶进行有效地区分。在此基础上,采用偏最小二乘-判别分析(PLS-DA)结合三种变量选择方法(VIP、coefficient β、Lasso)对云南和湖南绿茶进行判别分析。结果表明,PLS-DA结合VIP法建立的模型具有最佳的判别能力,能够很好地区分云南绿茶和湖南绿茶,判别正确率为90.28%。通过VIP筛选出的三个特征差异性成分是 Epicatechin-(4beta-8)-epigallocatechin 3-O-gallate,没食子儿茶素没食子酸酯(GCG),牡荆素。其中,云南和湖南绿茶中没食子儿茶素没食子酸酯的含量差异最为明显。
[Abstract]:Tea, as a traditional Chinese drink, has a thousand years of history. Green tea is one of the most productive teas in our country and is popular for people. The research in recent years has shown that the green tea extract has the effects of resisting oxidation, resisting tumor, resisting inflammation, and reducing the risk of cardiovascular disease. The main factors that affect the quality of green tea include the tea varieties and the surface temperature, the average precipitation, the altitude and the length of the green tea. The current national standard of green tea has the problems of long age, backward analysis method, long time consumption and insufficient evaluation index. How to establish an analytical method to fully characterize the chemical components in green tea, how to efficiently, quickly and accurately evaluate the quality of green tea is the real problem to be solved in the present study of green tea. The chemical constituents of green tea from Yunnan and Hunan were studied in detail. The main contents of this study were as follows: 1. A total of 72 green tea collected from Yunnan and Hunan were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry. In this study, different extraction solvents (pure water, pure methanol, 70% methanol) and different amounts of green tea samples (0.1g, 0.5g, 1.0g) were compared and optimized. The results showed that 0. 5g of green tea samples were extracted with 70% methanol. In order to obtain more abundant mass spectrum information, the optimized selection of the mass spectrum conditions of the experiment, including the induced cracking voltage in the source and the high-energy collision-induced voltage, was selected. In order to avoid excessive interference fragments generated at the time of obtaining the precursor ions, the induced cracking voltage in the source is selected to be OeV. At the same time, in order to obtain more secondary fragments, the high-energy collision induced voltage is a gradient voltage (10eV, 30eV, 50eV). Finally, in order to ensure the stability of the experimental apparatus during the analysis, the precision and the stability of the quality control samples were studied. The results show that the experimental apparatus is stable and reliable throughout the experiment. Qualitative and quantitative analysis of the chemical constituents in green tea. The obtained mass spectrum data was extracted and analyzed by using the MZmine mass spectrum analysis software. In order to extract the effective mass spectrum as much as possible, the parameter setting of the mass spectrum analysis software is optimized, including peak correction, peak detection, peak extraction, deconvolution, peak alignment, etc., and the parameters such as peak height, peak width and noise level are selected and optimized. The study found that the noise level is the most critical parameter. Through the comprehensive analysis, the noise level is selected to be 5.00E8, the background interference can be eliminated, and the effective mass spectrum information can be kept to the maximum extent. On this basis, 75 compounds, including catechins, flavonoids, amino acids and the like, were identified in green tea by using standard substance, mass spectrum database and primary and secondary mass spectrum information in the chemical database, and 33 of the compounds were confirmed by standard products. A quantitative chromatographic fingerprint of green tea in two places was established by using the 75 compounds as a group of chemical constituents of green tea, using internal standard method to carry out semi-quantitative analysis. The results show that although the chemical fingerprints of green tea in the two places are very similar, there are some differences in the content of each substance. The difference of green tea in yunnan and hunan. First, using cluster analysis and principal component analysis, two unsupervised pattern recognition methods were used to distinguish the green tea from Yunnan and Hunan. The results show that the two methods can be used to separate the green tea from the two places. On this basis, the discriminant analysis of green tea in Yunnan and Hunan was carried out by using partial least squares-discriminant analysis (PLS-DA) in combination with three variable selection methods (VIP, coeffciant, and Lasso). The results show that the model of the PLS-DA combined with the VIP method has the best judgment ability, and can be divided into Yunnan green tea and Hunan green tea in a good area, and the discrimination rate is 90. 28%. The three characteristic difference components screened by VIP are Epicatechin-(4beta-8)-epiallocatorin 3-O-gallate, epigallocatechin gallate (GCG), and vitexin. Among them, the content of gallocatechin gallate is most obvious in Yunnan and Hunan green tea.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS272.51;O657.63

【参考文献】