茶叶中有效生化成分高效液相色谱检测法的建立及其在凤凰乌龙茶检测中的应用
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摘要
茶叶是数千年来上世界最受欢迎的饮料之一,中国是茶叶生产和消费大国。茶叶中含有多种有益人体健康的成分,如多酚类物质、氨基酸、生物碱等,正是这些有效生化成分通过多种途径发挥着多种有益作用,如抗氧化、抗突变、抗癌和抗过敏等。因此,针对茶叶中的有效生化成分建立快速、高效、准确的定性定量检测方法成为研究热点。
近年来,产于中国广东省潮安县凤凰镇的凤凰乌龙茶,因为其香气类似天然花香并具有特殊韵味,引起国内众多学者的兴趣。目前针对凤凰乌龙茶的研究主要集中于其香气形成原因、抗氧化作用和生产管理方面,对其主要有效生化成分进行系统全面的检测分析还未见报道。
因此,本实验建立了对茶叶中10种儿茶素单体、没食子酸、可可碱、咖啡碱和茶碱在15mmin内的高效液相色谱(HPLC)检测法,以及同时对茶叶中17种游离氨基酸的HPLC检测法；并将建立的方法应用于凤凰乌龙茶的检测分析。主要研究结果如下：
1.通过实验室已有的儿茶素标品和甲基化儿茶素高含量茶——日本Benifuji茶,采用大孔树脂HP-20对Benifuji绿茶茶样富集得到粗茶多酚后,运用中压层析系统,80%乙醇洗脱Toyopearl HW-40S柱,对不同组分分别收集再次以同样的条件分离纯化后,得到了纯度分别为98.4%和95.3%的甲基化儿茶素EGCG3"Me和ECG3'Me(所得甲基化儿茶素的结构经1H NMR、ESI-TOF-MS分析,被证明分别与文献报道的EGCG3"Me和ECG3'Me的1H-NMR数据一致),以及纯度>97%的儿茶素单体EC、EGC、ECG和EGCG。
2.将得到的较高浓度儿茶素单体EC、EGC、EGCG和ECG,加热异构化后由半制备色谱柱Zorbax SB-C18,采用AKTA purifier分离得到纯度均>98%儿茶素单体C、GC、GCG和CG。所得单体结构也均经过1H-NMR、ESI-TOF-MS分析证明。
3.采用OPA(邻苯二甲醛)柱前衍生化HPLC检测,建立了17种游离氨基酸检测的定性和定量方法；采用SPE(固相萃取)柱处理茶汤,吸附了茶汤中大部分多酚类物质,从而去除了其对氨基酸检测时的影响,并设计实验证明了SPE处理法的可行性和有效性。
4.通过已经建立的HPLC法对11种凤凰乌龙茶中10种儿茶素、咖啡碱、可可碱、茶碱和没食子酸进行定量分析。发现凤凰乌龙茶中没食子酸、可可碱和茶碱的含量较低；咖啡碱含量比较高,与凤凰茶的生长环境有关；所有凤凰乌龙茶中的酯型儿茶素含量都很高,从侧面说明了凤凰乌龙茶的优秀品质形成条件；采用SPE处理,OPA柱前衍生化对11种凤凰乌龙茶中的17种游离氨基酸进行了定量分析。发现虽然11种茶样的氨基酸总量都比较低,所含种类和含量有较大差异,但天冬氨酸Asp、谷氨酸Glu、丝氨酸Ser和茶氨酸Thea仍为所有茶样中的主要氨基酸。
For thousands of years, tea is one of the most popular drinks in the world. There are various components which have benefits for people's health in the tea, such as polyphenols, amino acids, and purine alkaloids. A lot of biological functions, such as anti-oxidation, anti-cancer and anti-allergy, have been attributed to the presence of the effective biological components from tea. Therefore, the development of these components' determination, which are fast, high-effective and accurate, has been studied a lot.
Recently, Fenghuang Oolong Tea, which is only produced in Fenghuang Tawn, Chao An Country, Guang Dong Province, China, has gained several domestic researchers' interest, for whose aroma is similar to the aroma of natural flowers and taste is very special. Now, the study of Fenghuang Oolong Tea are focusing on the formation of the special aroma、the function of anti-oxidation and the management of the produce, however, the systemic determination of the major effective biological components in Fenghuang Oolong Tea has not been reported yet.
Therefore, in this study, a high performance liquid chromatography(HPLC) method which could detect ten different kinds of catechins, gallic acid, theobromine, caffeine and theophylline in 15mins, had been developed; and a HPLC method of detecting 17 different kinds of free amino acids in the tea had also been built. At the meantime, the two developed methods had been used in the determinations of Fenghuang Oolong Tea. Major research results were followed:
1. On the basis of purchased catechins standars and Japanese Benifuji Tea which with high content of methylated-catechins, the catechins extraction from Benifuji Tea which was concentrated by HP-20 resin, was separated by medium-pressure Toyopearl HW-40S column which was eluted by 80% ethanol. Then, the different fractions were collected and separated by the same conditions, respectively. Two kinds of methylated-catechins EGCG3"Me and ECG3'Me with the concentrations of 98.4% and 95.3%, whose structures have been identified by the results of the analysis of 1H NMR、ESI-TOF-MS and the data in the references, have been gained, as well as catechins EC, EGC, ECG and EGCG, whose purity were all above 97%.
2. Epimers of EC, EGC, EGCG and ECG with higher concentration which were prepared before, were heat-isomerizated and separated by a semi-preparative HPLC column Zorbax SB-C18 and an AKTA purifier, respectively. Epimers of C、GC、GCG and CG whose purity were all 98% were gained, and their structures have all been identified by the data of the results of 1H NMR. ESI-TOF-MS.
3. By using precolumn derivatization with o-phthaladehyde combined with HPLC, the methods of determination and quantitation of 17 kinds of free amino acids have been developed;with the treatment of SPE(solid-phase extraction) of the tea infusions, the phenolic compounds in tea infusion have been absorbed and the influences of the separation and determination of free amino acids were removed;the experiments have been designed to demonstrated the feasibility and effectivity of the treatment of SPE.
4. Using the HPLC methods which have been developed,11 kinds of Fenghuang Oolong Tea were detected and the contents of ten different kinds of catechins, gallic acid, theobromine, caffeine and theophylline have been quantitated. The data showed that the content of gallic acid, theobromine and theophylline were pretty low when caffeine was much higher, which was related to the growing environment of Fenghuang Oolong Tea; the content of ester-catechins in Fenghuang Oolong Tea was very high, which showed the forming conditions of the excellent quality of this kind of tea; after the treatment of SPE,17 kinds of free amino acids in the tea infusion were determined and quantitated by using precolumn derivatization with o-phthaladehyde combined with HPLC. The results illustrated that the total contents of the amino acids in the tea infusions, the concentrations and kinds of amino acids in different infusions were various, but Asp, Glu, Ser and Thea were the major amino acids in all infusions.
近年来,产于中国广东省潮安县凤凰镇的凤凰乌龙茶,因为其香气类似天然花香并具有特殊韵味,引起国内众多学者的兴趣。目前针对凤凰乌龙茶的研究主要集中于其香气形成原因、抗氧化作用和生产管理方面,对其主要有效生化成分进行系统全面的检测分析还未见报道。
因此,本实验建立了对茶叶中10种儿茶素单体、没食子酸、可可碱、咖啡碱和茶碱在15mmin内的高效液相色谱(HPLC)检测法,以及同时对茶叶中17种游离氨基酸的HPLC检测法；并将建立的方法应用于凤凰乌龙茶的检测分析。主要研究结果如下：
1.通过实验室已有的儿茶素标品和甲基化儿茶素高含量茶——日本Benifuji茶,采用大孔树脂HP-20对Benifuji绿茶茶样富集得到粗茶多酚后,运用中压层析系统,80%乙醇洗脱Toyopearl HW-40S柱,对不同组分分别收集再次以同样的条件分离纯化后,得到了纯度分别为98.4%和95.3%的甲基化儿茶素EGCG3"Me和ECG3'Me(所得甲基化儿茶素的结构经1H NMR、ESI-TOF-MS分析,被证明分别与文献报道的EGCG3"Me和ECG3'Me的1H-NMR数据一致),以及纯度>97%的儿茶素单体EC、EGC、ECG和EGCG。
2.将得到的较高浓度儿茶素单体EC、EGC、EGCG和ECG,加热异构化后由半制备色谱柱Zorbax SB-C18,采用AKTA purifier分离得到纯度均>98%儿茶素单体C、GC、GCG和CG。所得单体结构也均经过1H-NMR、ESI-TOF-MS分析证明。
3.采用OPA(邻苯二甲醛)柱前衍生化HPLC检测,建立了17种游离氨基酸检测的定性和定量方法；采用SPE(固相萃取)柱处理茶汤,吸附了茶汤中大部分多酚类物质,从而去除了其对氨基酸检测时的影响,并设计实验证明了SPE处理法的可行性和有效性。
4.通过已经建立的HPLC法对11种凤凰乌龙茶中10种儿茶素、咖啡碱、可可碱、茶碱和没食子酸进行定量分析。发现凤凰乌龙茶中没食子酸、可可碱和茶碱的含量较低；咖啡碱含量比较高,与凤凰茶的生长环境有关；所有凤凰乌龙茶中的酯型儿茶素含量都很高,从侧面说明了凤凰乌龙茶的优秀品质形成条件；采用SPE处理,OPA柱前衍生化对11种凤凰乌龙茶中的17种游离氨基酸进行了定量分析。发现虽然11种茶样的氨基酸总量都比较低,所含种类和含量有较大差异,但天冬氨酸Asp、谷氨酸Glu、丝氨酸Ser和茶氨酸Thea仍为所有茶样中的主要氨基酸。
For thousands of years, tea is one of the most popular drinks in the world. There are various components which have benefits for people's health in the tea, such as polyphenols, amino acids, and purine alkaloids. A lot of biological functions, such as anti-oxidation, anti-cancer and anti-allergy, have been attributed to the presence of the effective biological components from tea. Therefore, the development of these components' determination, which are fast, high-effective and accurate, has been studied a lot.
Recently, Fenghuang Oolong Tea, which is only produced in Fenghuang Tawn, Chao An Country, Guang Dong Province, China, has gained several domestic researchers' interest, for whose aroma is similar to the aroma of natural flowers and taste is very special. Now, the study of Fenghuang Oolong Tea are focusing on the formation of the special aroma、the function of anti-oxidation and the management of the produce, however, the systemic determination of the major effective biological components in Fenghuang Oolong Tea has not been reported yet.
Therefore, in this study, a high performance liquid chromatography(HPLC) method which could detect ten different kinds of catechins, gallic acid, theobromine, caffeine and theophylline in 15mins, had been developed; and a HPLC method of detecting 17 different kinds of free amino acids in the tea had also been built. At the meantime, the two developed methods had been used in the determinations of Fenghuang Oolong Tea. Major research results were followed:
1. On the basis of purchased catechins standars and Japanese Benifuji Tea which with high content of methylated-catechins, the catechins extraction from Benifuji Tea which was concentrated by HP-20 resin, was separated by medium-pressure Toyopearl HW-40S column which was eluted by 80% ethanol. Then, the different fractions were collected and separated by the same conditions, respectively. Two kinds of methylated-catechins EGCG3"Me and ECG3'Me with the concentrations of 98.4% and 95.3%, whose structures have been identified by the results of the analysis of 1H NMR、ESI-TOF-MS and the data in the references, have been gained, as well as catechins EC, EGC, ECG and EGCG, whose purity were all above 97%.
2. Epimers of EC, EGC, EGCG and ECG with higher concentration which were prepared before, were heat-isomerizated and separated by a semi-preparative HPLC column Zorbax SB-C18 and an AKTA purifier, respectively. Epimers of C、GC、GCG and CG whose purity were all 98% were gained, and their structures have all been identified by the data of the results of 1H NMR. ESI-TOF-MS.
3. By using precolumn derivatization with o-phthaladehyde combined with HPLC, the methods of determination and quantitation of 17 kinds of free amino acids have been developed;with the treatment of SPE(solid-phase extraction) of the tea infusions, the phenolic compounds in tea infusion have been absorbed and the influences of the separation and determination of free amino acids were removed;the experiments have been designed to demonstrated the feasibility and effectivity of the treatment of SPE.
4. Using the HPLC methods which have been developed,11 kinds of Fenghuang Oolong Tea were detected and the contents of ten different kinds of catechins, gallic acid, theobromine, caffeine and theophylline have been quantitated. The data showed that the content of gallic acid, theobromine and theophylline were pretty low when caffeine was much higher, which was related to the growing environment of Fenghuang Oolong Tea; the content of ester-catechins in Fenghuang Oolong Tea was very high, which showed the forming conditions of the excellent quality of this kind of tea; after the treatment of SPE,17 kinds of free amino acids in the tea infusion were determined and quantitated by using precolumn derivatization with o-phthaladehyde combined with HPLC. The results illustrated that the total contents of the amino acids in the tea infusions, the concentrations and kinds of amino acids in different infusions were various, but Asp, Glu, Ser and Thea were the major amino acids in all infusions.
引文
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