茶黄素形成的生化调控和分离纯化技术研究
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摘要
茶黄素作为红茶的主要活性物质，具有多种药理功能与保健功效，在某些方面甚至优于儿茶素。茶黄素的分离纯化方法和制备工艺也一直是茶叶化学工程的研究重点。茶黄素研究与开发具有重要的理论价值和广阔的产业化前景。
本文通过研究4种茶黄素组分的色谱行为、流动相的组成、梯度洗脱条件、检测波长和柱温对茶黄素分离的影响，建立了茶黄素分析的高效液相色谱法。该方法简便、快速、准确，样品的总分析时间在33分钟之内。色谱柱为C_(18) Shim-pack VP-ODS；流动相A为2％醋酸(HAc)，流动相B为乙腈(ACN)；乙酸乙酯(EtOAc)=21：3(v／v)，梯度洗脱；柱温40℃；紫外检测波长280nm。被测组分与峰面积有良好的线性关系(r=0.9990-0.9992)；加标回收率在80.25％-89.79％；方法的精密度良好(平均RSD＜1.5％)。
采用体外模拟发酵，对大叶种原料提制的儿茶素(以下简称大叶儿茶素，其中儿茶素含量为85.47％)进行酶促氧化，加入纯化的多酚氧化酶，通入氧气，并定时用高效液相色谱检测茶黄素的含量变化。结果表明，发酵80分钟时，茶黄素的生成量最高(为15.05％)。
以K_3Fe(CN)_6／NaHCO_3为氧化剂，系统研究了儿茶素组成、儿茶素浓度、氧化剂比例对茶黄素形成的影响，通过分光光度法与HPLC法相结合对氧化产物的组成分析，结果表明：以大叶儿茶素为材料，浓度为10mg／ml，氧化剂的比例为2：3：1(儿茶素：K_3Fe(CN)_6：NaHCO_3)时，有利于茶黄素类物质的形成。
本文以有机溶剂萃取制备的茶黄素粗品为试验材料，进行了大孔吸附树脂色谱法制备高含量茶黄素(茶黄素总量＞15％)的研究。选用8种物理和化学性质各不相同的树脂为吸附剂，应用色谱理论对静态和动态吸附过程中茶黄素含量与组分的变化作了系统研究。结果表明：以茶黄素粗品为原料，NKA为柱填充料，收集解吸液一定区段的馏分，真空浓缩、冷冻干燥，即可制得高含量茶黄素制品，得率为48.04％，茶黄素总量大于15％。此法纯化效果好，操作简便，为茶黄素的制备与分离提供了一条新的途径，也为高含量茶黄素的工业化生产提供了新的工艺技术。
As the major active constituents of black tea, theaflavins (TFs) is of many kinds of pharmaceutical effects and health-keeping functions, which is even more powerful than catechins in some areas. The isolation, purification and industrial preparation of TFs are always a very important research branch in tea chemical engineering. Researching and developing Teaflavins has important theoretical value and wonderful future of industrial production.
Through researches on the chromatosraphy action of 4 kinds of theaflavin, the effects of mobile constituents, gradient elution conditions, detection wavelength & column temperature on isolation of TFs, the HPLC analysis method of TFs was established. This method is relatively simple, rapid and accurate, which just need 33 minutes to finish the determination. Shim-pack VP-ODS is used as column, ACN: EtOAc(21:3, V/V) as mobile B, and aqueous acetic acid (50:1,V/V) as mobile A, linear gradient elution is adopted. Column temperature is 40C and detection wavelength is 280nm. There was a good linear relationship between the component and its peak area for TFs, with the correlation coefficients of 0.9990-0.9992. The average recoveries (n=3) were 80.25%~ 89.79%. The average relative standard derivation was less than 1.5%.
By adopting the in vitro simulated fermentation, tea catechins extracted from big leaf tea were treated with polyphenol oxidase (PPO) and O^. The dynamic variations of TFs during fermentation were monitored by HPLC together with UV spectro-photometric detection. It was shown that the highest content of TFs (15.05%) was formed at 80 min fermentation with PPO.
The influences of catechins constituent, catechins concentration, the ratio of oxidants on the formation of TFs was systematically carried out by using the K^Fe (CN)6/NaHCO3 as the oxidants. According to the analysis on the components of oxidant products with HPLC and UV-VIS method, the optimum condition for the formation of TFs is Big leaf catechins with concentration 10 mg/ml and the ratio of oxidants 2:3:1 (catechins: K3Fe(CN)6: NaHCO3).
Using crude TFs produced by organic solvent extraction as raw materials, the feasibility of preparing high content TFs (theaflavins>15%) with macroporous adsorption resin by column chromatography was studied in this paper. 8 kinds of resin which is of different physical and chemical properties were choose as adsorbents, the variation of TFs content and constituents during the process of static and dynamic adsorption were
systematically tested by HPLC. Results showed that the high content of TFs could be obtained by collection the fractions of special zones of column chromatography when crude TFs used as raw material, NAK as adsorbent and the elutes were concentrated by vacuum evaporator and freeze-dried. The production rate was 48.04% and the content of TFs is higher than 15%. This process was convenient and has good effect of purification. This research provides a new way for isolation and preparation of TFs as well as new technology of industrial production of high purity TFs in the future.
本文通过研究4种茶黄素组分的色谱行为、流动相的组成、梯度洗脱条件、检测波长和柱温对茶黄素分离的影响，建立了茶黄素分析的高效液相色谱法。该方法简便、快速、准确，样品的总分析时间在33分钟之内。色谱柱为C_(18) Shim-pack VP-ODS；流动相A为2％醋酸(HAc)，流动相B为乙腈(ACN)；乙酸乙酯(EtOAc)=21：3(v／v)，梯度洗脱；柱温40℃；紫外检测波长280nm。被测组分与峰面积有良好的线性关系(r=0.9990-0.9992)；加标回收率在80.25％-89.79％；方法的精密度良好(平均RSD＜1.5％)。
采用体外模拟发酵，对大叶种原料提制的儿茶素(以下简称大叶儿茶素，其中儿茶素含量为85.47％)进行酶促氧化，加入纯化的多酚氧化酶，通入氧气，并定时用高效液相色谱检测茶黄素的含量变化。结果表明，发酵80分钟时，茶黄素的生成量最高(为15.05％)。
以K_3Fe(CN)_6／NaHCO_3为氧化剂，系统研究了儿茶素组成、儿茶素浓度、氧化剂比例对茶黄素形成的影响，通过分光光度法与HPLC法相结合对氧化产物的组成分析，结果表明：以大叶儿茶素为材料，浓度为10mg／ml，氧化剂的比例为2：3：1(儿茶素：K_3Fe(CN)_6：NaHCO_3)时，有利于茶黄素类物质的形成。
本文以有机溶剂萃取制备的茶黄素粗品为试验材料，进行了大孔吸附树脂色谱法制备高含量茶黄素(茶黄素总量＞15％)的研究。选用8种物理和化学性质各不相同的树脂为吸附剂，应用色谱理论对静态和动态吸附过程中茶黄素含量与组分的变化作了系统研究。结果表明：以茶黄素粗品为原料，NKA为柱填充料，收集解吸液一定区段的馏分，真空浓缩、冷冻干燥，即可制得高含量茶黄素制品，得率为48.04％，茶黄素总量大于15％。此法纯化效果好，操作简便，为茶黄素的制备与分离提供了一条新的途径，也为高含量茶黄素的工业化生产提供了新的工艺技术。
As the major active constituents of black tea, theaflavins (TFs) is of many kinds of pharmaceutical effects and health-keeping functions, which is even more powerful than catechins in some areas. The isolation, purification and industrial preparation of TFs are always a very important research branch in tea chemical engineering. Researching and developing Teaflavins has important theoretical value and wonderful future of industrial production.
Through researches on the chromatosraphy action of 4 kinds of theaflavin, the effects of mobile constituents, gradient elution conditions, detection wavelength & column temperature on isolation of TFs, the HPLC analysis method of TFs was established. This method is relatively simple, rapid and accurate, which just need 33 minutes to finish the determination. Shim-pack VP-ODS is used as column, ACN: EtOAc(21:3, V/V) as mobile B, and aqueous acetic acid (50:1,V/V) as mobile A, linear gradient elution is adopted. Column temperature is 40C and detection wavelength is 280nm. There was a good linear relationship between the component and its peak area for TFs, with the correlation coefficients of 0.9990-0.9992. The average recoveries (n=3) were 80.25%~ 89.79%. The average relative standard derivation was less than 1.5%.
By adopting the in vitro simulated fermentation, tea catechins extracted from big leaf tea were treated with polyphenol oxidase (PPO) and O^. The dynamic variations of TFs during fermentation were monitored by HPLC together with UV spectro-photometric detection. It was shown that the highest content of TFs (15.05%) was formed at 80 min fermentation with PPO.
The influences of catechins constituent, catechins concentration, the ratio of oxidants on the formation of TFs was systematically carried out by using the K^Fe (CN)6/NaHCO3 as the oxidants. According to the analysis on the components of oxidant products with HPLC and UV-VIS method, the optimum condition for the formation of TFs is Big leaf catechins with concentration 10 mg/ml and the ratio of oxidants 2:3:1 (catechins: K3Fe(CN)6: NaHCO3).
Using crude TFs produced by organic solvent extraction as raw materials, the feasibility of preparing high content TFs (theaflavins>15%) with macroporous adsorption resin by column chromatography was studied in this paper. 8 kinds of resin which is of different physical and chemical properties were choose as adsorbents, the variation of TFs content and constituents during the process of static and dynamic adsorption were
systematically tested by HPLC. Results showed that the high content of TFs could be obtained by collection the fractions of special zones of column chromatography when crude TFs used as raw material, NAK as adsorbent and the elutes were concentrated by vacuum evaporator and freeze-dried. The production rate was 48.04% and the content of TFs is higher than 15%. This process was convenient and has good effect of purification. This research provides a new way for isolation and preparation of TFs as well as new technology of industrial production of high purity TFs in the future.
引文
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