茶叶中甲基化EGCG的分析方法建立与资源化学研究
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摘要
甲基化EGCG是儿茶素类的一种,它主要包括EGCG3 " Me、EGCG4 " Me。EGCG3 " Me、EGCG4 " Me为白色粉末状物质,易溶于水和有机溶剂,它们是EGCG的两种甲基化衍生产物,分子结构分别为(-)-表没食子儿茶素3-O(3-O-甲基)没食子酸醋(C-1)和(-)表没食子儿茶素3-O(4-O-甲基)没食子酸醋(C-2),它们具有高效的抗过敏功效,尤其是抗花粉过敏。
本研究旨在建立一套准确、高效的茶叶中甲基化EGCG的HPLC检测方法,并利用此方法对种植在湖南地区的部分茶树品种资源进行含量分析,以期筛选出高含量甲基化儿茶素的特异茶树品种资源。本文还对影响甲基化EGCG含量的一些内外因子进行了深入的探讨,对甲基化EGCG的研究与利用具有重要的理论意义和实用价值。获得的主要研究结果如下：
(1)建立了茶叶中甲基化EGCG的HPLC分析方法,色谱分析条件为：流动相：0.02mol／L磷酸二氢钾溶液：乙腈,梯度洗脱,色谱柱：C18 5μm ID4.6*250mm；,流速：1.0ml／min,柱温：30℃,用磷酸调pH=2.5,检测波长278nm。该方法可以清晰分离EGCG3"Me和EGCG4"Me,且准确度、精确度好。
(2)本研究对117个不同来源的茶样进行了甲基化EGCG分析,其中只有16个的EGCG3"Me含量在检测限(0.01%)之下,占总数的13.7%,而高于0.1%的为40个,占总数的34.2%,含量在0.3%以上的有18个,占总数的15.4%,其中含量最高的为C48,达到了1.7%；含有EGCG4"Me的品种只有10个,只占总数的8.5%,其中含量最高的是C45,含量为0.55%,最低的是C69,只有0.036%。
(3)同一茶样,蒸青固样的茶叶EGCG3"Me的平均含量为0.120%,冷冻干燥固样的茶叶EGCG3"Me的平均含量为0.127%。方差分析结果表明,两者对茶叶中EGCG3"Me的含量影响不显著。
(4)不同季节采摘的茶叶中甲基化EGCG的HPLC分析发现,同一品种、不同季节的茶叶中EGCG3"Me含量,夏季叶含量相对最高,秋季叶次之,春季叶最低,且差异达到极显著水平。
(5)茶树不同叶位EGCG3"Me含量变化显著,表现为在第三叶、第四叶含量最高,其次是第五叶,而在茶梗中含量极低。
(6)绿茶加工过程中EGCG3"Me含量变化研究表明,不同加工工序对茶叶中EGCG3"Me的含量影响极显著,表现为摊放叶时含量最高,杀青叶时有所下降,揉捻叶时基本不变,最后到干燥叶时再度下降,但下降幅度很小。
O-Methylated catechin is a kind of catechin, It mainly includes EGCG3 "Me and EGCG4" Me. EGCG3 "Me and EGCG4" Me are a kind of white powder,which can be dissolved in water and organic solvents easily.They are derivatives of EGCg.The molecular structures are (-)-epicatechin-3-O-(3-O-Methyl) gallate(EGCG3 "Me) and (-)-epicatechin-3-O-(4-O-Methyl) gallate(EGCG4" Me) respectively.They have shown efficient anti-allergic effect, especially on anti-pollen-allergic action.
This research aims at establishing an accurate and efficient method of O-Methylated EGCG by the HPLC and using this method to analyzing the content of tea. Thus we can screen out some higher O-Methylated EGCG content tea plant varieties and resourses which grow in hunan province.We also discuss some factor which affect the content of methylated EGCG.This study has an important theoretical significance and practical value on O-Methylated EGCG research and utilization.The study has acquired the following conclusions:
1、Established the HPLC method to determining the content of O-Methylated EGCG(EGCG3 "Me and EGCG4" Me) in tea. The optimum condition was as follows:Kromasil C18 column;mobile phase:0.02mmol/L KH2PO4-H3PO4 buffer solution(pH=2.5) and acetonitrile;wavelength:278nm;flow rate:1.0ml/min;column temperature:30℃. This method showed good linearity for both EGCG3 "Me and EGCG4" Me.The result demonstrated that this method is precise,accurate and can be used for the determination of methylated EGCG. 2、This study analyzed 117 kinds of tea samples from different source. Among these tea samples,only 16 kinds of tea's content was under detection limit (0.01%), which accounted for 13.7%, the highest one is C48,whose content reached for 1.7%.Only 10 kinds of tea samples contained EGCG4" Me, which accounted for 8.5%,the highest one is C45,whose content reached for 0.55%, the lowest content is C69,whose content is only 0.036%.
3、We use two kinds of method(steaming fixing and freeze drying) to dispose the same tea sample.The EGCG3 "Me average content of steaming fixing sample is 0.120%,while the freeze drying sample is 0.127%,The result showed different fixing method has no definite effect on the content of EGCG3 "Me.
4、Through determination of tea leaves which picked in different season. We have found that the content of EGCG3" Me in the same variety also has definite difference. Summer is the highest and second in autumn, lowest in spring.
5、The content of EGCG3 "Me in different parts of tea leaves was also significantly different and has the highest in the fourth leaf and the third leaf, followed by the fifth leaf and the lowest content in tea stalk.
6、During the course of processing green tea,different processing procedure has a definite effect on the content of EGCG3 "Me.The content of EGCG3" Me was higher in spreaded leaves,droped in fixing leaves,keeped constant in rolled leaves and droped in dried leaves again.but the range is small.
本研究旨在建立一套准确、高效的茶叶中甲基化EGCG的HPLC检测方法,并利用此方法对种植在湖南地区的部分茶树品种资源进行含量分析,以期筛选出高含量甲基化儿茶素的特异茶树品种资源。本文还对影响甲基化EGCG含量的一些内外因子进行了深入的探讨,对甲基化EGCG的研究与利用具有重要的理论意义和实用价值。获得的主要研究结果如下：
(1)建立了茶叶中甲基化EGCG的HPLC分析方法,色谱分析条件为：流动相：0.02mol／L磷酸二氢钾溶液：乙腈,梯度洗脱,色谱柱：C18 5μm ID4.6*250mm；,流速：1.0ml／min,柱温：30℃,用磷酸调pH=2.5,检测波长278nm。该方法可以清晰分离EGCG3"Me和EGCG4"Me,且准确度、精确度好。
(2)本研究对117个不同来源的茶样进行了甲基化EGCG分析,其中只有16个的EGCG3"Me含量在检测限(0.01%)之下,占总数的13.7%,而高于0.1%的为40个,占总数的34.2%,含量在0.3%以上的有18个,占总数的15.4%,其中含量最高的为C48,达到了1.7%；含有EGCG4"Me的品种只有10个,只占总数的8.5%,其中含量最高的是C45,含量为0.55%,最低的是C69,只有0.036%。
(3)同一茶样,蒸青固样的茶叶EGCG3"Me的平均含量为0.120%,冷冻干燥固样的茶叶EGCG3"Me的平均含量为0.127%。方差分析结果表明,两者对茶叶中EGCG3"Me的含量影响不显著。
(4)不同季节采摘的茶叶中甲基化EGCG的HPLC分析发现,同一品种、不同季节的茶叶中EGCG3"Me含量,夏季叶含量相对最高,秋季叶次之,春季叶最低,且差异达到极显著水平。
(5)茶树不同叶位EGCG3"Me含量变化显著,表现为在第三叶、第四叶含量最高,其次是第五叶,而在茶梗中含量极低。
(6)绿茶加工过程中EGCG3"Me含量变化研究表明,不同加工工序对茶叶中EGCG3"Me的含量影响极显著,表现为摊放叶时含量最高,杀青叶时有所下降,揉捻叶时基本不变,最后到干燥叶时再度下降,但下降幅度很小。
O-Methylated catechin is a kind of catechin, It mainly includes EGCG3 "Me and EGCG4" Me. EGCG3 "Me and EGCG4" Me are a kind of white powder,which can be dissolved in water and organic solvents easily.They are derivatives of EGCg.The molecular structures are (-)-epicatechin-3-O-(3-O-Methyl) gallate(EGCG3 "Me) and (-)-epicatechin-3-O-(4-O-Methyl) gallate(EGCG4" Me) respectively.They have shown efficient anti-allergic effect, especially on anti-pollen-allergic action.
This research aims at establishing an accurate and efficient method of O-Methylated EGCG by the HPLC and using this method to analyzing the content of tea. Thus we can screen out some higher O-Methylated EGCG content tea plant varieties and resourses which grow in hunan province.We also discuss some factor which affect the content of methylated EGCG.This study has an important theoretical significance and practical value on O-Methylated EGCG research and utilization.The study has acquired the following conclusions:
1、Established the HPLC method to determining the content of O-Methylated EGCG(EGCG3 "Me and EGCG4" Me) in tea. The optimum condition was as follows:Kromasil C18 column;mobile phase:0.02mmol/L KH2PO4-H3PO4 buffer solution(pH=2.5) and acetonitrile;wavelength:278nm;flow rate:1.0ml/min;column temperature:30℃. This method showed good linearity for both EGCG3 "Me and EGCG4" Me.The result demonstrated that this method is precise,accurate and can be used for the determination of methylated EGCG. 2、This study analyzed 117 kinds of tea samples from different source. Among these tea samples,only 16 kinds of tea's content was under detection limit (0.01%), which accounted for 13.7%, the highest one is C48,whose content reached for 1.7%.Only 10 kinds of tea samples contained EGCG4" Me, which accounted for 8.5%,the highest one is C45,whose content reached for 0.55%, the lowest content is C69,whose content is only 0.036%.
3、We use two kinds of method(steaming fixing and freeze drying) to dispose the same tea sample.The EGCG3 "Me average content of steaming fixing sample is 0.120%,while the freeze drying sample is 0.127%,The result showed different fixing method has no definite effect on the content of EGCG3 "Me.
4、Through determination of tea leaves which picked in different season. We have found that the content of EGCG3" Me in the same variety also has definite difference. Summer is the highest and second in autumn, lowest in spring.
5、The content of EGCG3 "Me in different parts of tea leaves was also significantly different and has the highest in the fourth leaf and the third leaf, followed by the fifth leaf and the lowest content in tea stalk.
6、During the course of processing green tea,different processing procedure has a definite effect on the content of EGCG3 "Me.The content of EGCG3" Me was higher in spreaded leaves,droped in fixing leaves,keeped constant in rolled leaves and droped in dried leaves again.but the range is small.
引文
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