茶氨酸的分离机理与定量分析及抗衰老功能研究
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摘要
茶氨酸是茶叶中的非蛋白氨基酸,占茶叶游离氨基酸的50%。茶氨酸具有特殊的鲜爽味,能缓解茶叶的苦涩味,是评价绿茶品质的重要指标。由于具有保护神经等一系列药理功能,茶氨酸作为食品添加剂和保健品倍受人们关注。为了为茶氨酸的有效开发利用提供科学理论支撑,本论文在前人基础上对茶氨酸检测方法、离子交换色谱对茶氨酸的吸附机理、茶氨酸抗衰老以及与衰老相关的疾病等药理功能等方面进行了系统研究。主要研究结果如下：
(1)建立了一种反相离子对高效液相色谱法快速测定茶叶中茶氨酸的检测方法。茶叶样品制备只需超声10min。该方法以全氟辛酸(PDFOA,2mmol/L)为离子对试剂、乙腈-水(12%：82%)为流动相,检测波长为200nm,进样体积为10μL。在此分离条件下,茶氨酸的最低定量限、最低检测限分别为15.6ng与5.1ng。抗干扰试验表明,茶叶中其它成分对茶氨酸的测定并无干扰。所建立方法的样品制备方法简单,只需超声提取10min。该测定方法准确,灵敏,并具高回收率,适用于茶叶中茶氨酸的快速检测。
(2)为了采用离子交换树脂对茶氨酸进行吸附分离提供理论依据,研究了离子交换树脂对茶氨酸的吸附行为。结果表明,pH对茶氨酸在732阳离子交换树脂、717阴离子交换树脂和HD-8大孔强酸性阳离子交换树脂三种离子交换树脂上的吸附量影响都不大；阳离子树脂的吸附量比阴离子交换树脂的大10倍以上,大孔强酸性树脂的吸附量比普通凝胶型阳离子交换的吸附树脂量稍大；在303-323K与研究浓度范围内,HD-8大孔强酸性树脂对茶氨酸的吸附行为很好地符合Freundlich吸附等温方程；动力学研究表明HD-8大孔强酸性树脂对茶氨酸的吸附速率受液膜扩散和化学反应等因素的共同控制。
(3)衰老以及与衰老相关的疾病已经成为日趋严重的医学甚至社会问题。以秀丽线虫作为模式动物研究茶氨酸的抗衰老作用。研究结果表明,茶氨酸不能延长正常条件下线虫的寿命,但可以显著延长热应激条件下秀丽线虫寿命的12.8%。通过检测茶氨酸对带有绿色荧光蛋白的秀丽线虫CL2070在热应激下产生的热休克蛋白表达影响,发现茶氨酸能上调热应激后热休克蛋白HSP-16.2的表达。采用蛋白印迹技术检测野生型秀丽线虫N2中HSP-16.2的表达水平,也得到了一致的研究效果。这样从蛋白水平验证了茶氨酸能延长热应激条件下秀丽线虫寿命的原因。
(4)以秀丽线虫CL2006为研究模型,研究了茶氨酸对阿尔茨海默病的保护作用。研究表明茶氨酸能减轻CL2006中Aβ1-42介导的瘫痪行为,下调CL2006体内的Aβ1-42mRNA表达量,并能减轻由此引起的氧化应激水平,降低体内活性氧的产生。
Theanine is an unique nonproteinous amino acid comprising 50%of total free amino acids in tea(Camellia sinensis). Owing to its delicate taste and the ability to alleviate the astringency, theanine plays an important role in determining the quality and characteristics of green tea. Furthermore, various favorable physiological functions of theanine such as neuroprotective effects have been reported. The demand for theanine has increased greatly in the industry of natural health care products and functional drinks, not only as a taste-enhancing additive, but also as a supplement for improving and maintaining human health. In order to provide effective scientific theory support for the development and utilization of theanine, the studies focus on four aspects in this paper as follows:(1) Measurement of theanine in tea; (2) The adsorption behaviors of theanine on ion exchange resin; (3) The anti-senescence effect of theanine; (4) The protective effect of theanine on Alzheimer's disease.
(1) An ion-pair reversed-phase liquid chromatographic method for the analysis of theanine has been developed. The sample can be analyzed directly after sonication for 10 min. 2 mmol/L pentadecafluorooctanoic acid aqueous solution (ion pair reagent) and acetonitrile (82:18, v/v) were used as the mobile phase.The wavelength was kept at 200 nm. The LOD and LOQ were 5.1ng and 15.6ng respectively. The purity identification showed other ingredients in tea do not interfere with the analysis of theanine. This method is accurate with high recovery and can be applied for the analysis of theanine in commercial tea and theanine products.
(2) In order to provide the theory basis for adsorption and separation of theanine ion exchange resin, the adsorption behaviors of theanine on ion exchange resin were studied. Effect of pH on adsorption of theanine was not obvious; The adsorption data of theanine on HD-8 macroporous anion resin fit well into the Freundlich isotherm equation within temperature range of 303-323K and investigative concentration range; The rate of adsorption was under control of some factors simultaneously such as film diffusion and chemical reaction.
(3) Senescence and related disease has become increasingly serious medical even social problems. The anti-senescence effect of theanine in vivo biological model of C.elegans(Caenorhabditis elegans) had been investigated. The results showed that theanine can extend C.elegans lifespan under heat stress. But it is difficult to find this effect in normal animal without stress. Using methods including GFP expression detection in transgenic worm line CL2006 and west-blotting in wide-type C. elegans N2, It was found that theanine can up-regulate the expression of heat shock protein HSP-16.2.
(4) Base on the beta-amyloid transgenic model of C.elegans worm CL2006, The neuroprotective effect of theanine on Alzheimer's disease had been investigated. The studies suggested that theanine can decrease paralysis behavior of the CL2006. This behavior was caused by the down-regulation of Aβ1-42 mRNA and the level of reactive oxygen specises.
(1)建立了一种反相离子对高效液相色谱法快速测定茶叶中茶氨酸的检测方法。茶叶样品制备只需超声10min。该方法以全氟辛酸(PDFOA,2mmol/L)为离子对试剂、乙腈-水(12%：82%)为流动相,检测波长为200nm,进样体积为10μL。在此分离条件下,茶氨酸的最低定量限、最低检测限分别为15.6ng与5.1ng。抗干扰试验表明,茶叶中其它成分对茶氨酸的测定并无干扰。所建立方法的样品制备方法简单,只需超声提取10min。该测定方法准确,灵敏,并具高回收率,适用于茶叶中茶氨酸的快速检测。
(2)为了采用离子交换树脂对茶氨酸进行吸附分离提供理论依据,研究了离子交换树脂对茶氨酸的吸附行为。结果表明,pH对茶氨酸在732阳离子交换树脂、717阴离子交换树脂和HD-8大孔强酸性阳离子交换树脂三种离子交换树脂上的吸附量影响都不大；阳离子树脂的吸附量比阴离子交换树脂的大10倍以上,大孔强酸性树脂的吸附量比普通凝胶型阳离子交换的吸附树脂量稍大；在303-323K与研究浓度范围内,HD-8大孔强酸性树脂对茶氨酸的吸附行为很好地符合Freundlich吸附等温方程；动力学研究表明HD-8大孔强酸性树脂对茶氨酸的吸附速率受液膜扩散和化学反应等因素的共同控制。
(3)衰老以及与衰老相关的疾病已经成为日趋严重的医学甚至社会问题。以秀丽线虫作为模式动物研究茶氨酸的抗衰老作用。研究结果表明,茶氨酸不能延长正常条件下线虫的寿命,但可以显著延长热应激条件下秀丽线虫寿命的12.8%。通过检测茶氨酸对带有绿色荧光蛋白的秀丽线虫CL2070在热应激下产生的热休克蛋白表达影响,发现茶氨酸能上调热应激后热休克蛋白HSP-16.2的表达。采用蛋白印迹技术检测野生型秀丽线虫N2中HSP-16.2的表达水平,也得到了一致的研究效果。这样从蛋白水平验证了茶氨酸能延长热应激条件下秀丽线虫寿命的原因。
(4)以秀丽线虫CL2006为研究模型,研究了茶氨酸对阿尔茨海默病的保护作用。研究表明茶氨酸能减轻CL2006中Aβ1-42介导的瘫痪行为,下调CL2006体内的Aβ1-42mRNA表达量,并能减轻由此引起的氧化应激水平,降低体内活性氧的产生。
Theanine is an unique nonproteinous amino acid comprising 50%of total free amino acids in tea(Camellia sinensis). Owing to its delicate taste and the ability to alleviate the astringency, theanine plays an important role in determining the quality and characteristics of green tea. Furthermore, various favorable physiological functions of theanine such as neuroprotective effects have been reported. The demand for theanine has increased greatly in the industry of natural health care products and functional drinks, not only as a taste-enhancing additive, but also as a supplement for improving and maintaining human health. In order to provide effective scientific theory support for the development and utilization of theanine, the studies focus on four aspects in this paper as follows:(1) Measurement of theanine in tea; (2) The adsorption behaviors of theanine on ion exchange resin; (3) The anti-senescence effect of theanine; (4) The protective effect of theanine on Alzheimer's disease.
(1) An ion-pair reversed-phase liquid chromatographic method for the analysis of theanine has been developed. The sample can be analyzed directly after sonication for 10 min. 2 mmol/L pentadecafluorooctanoic acid aqueous solution (ion pair reagent) and acetonitrile (82:18, v/v) were used as the mobile phase.The wavelength was kept at 200 nm. The LOD and LOQ were 5.1ng and 15.6ng respectively. The purity identification showed other ingredients in tea do not interfere with the analysis of theanine. This method is accurate with high recovery and can be applied for the analysis of theanine in commercial tea and theanine products.
(2) In order to provide the theory basis for adsorption and separation of theanine ion exchange resin, the adsorption behaviors of theanine on ion exchange resin were studied. Effect of pH on adsorption of theanine was not obvious; The adsorption data of theanine on HD-8 macroporous anion resin fit well into the Freundlich isotherm equation within temperature range of 303-323K and investigative concentration range; The rate of adsorption was under control of some factors simultaneously such as film diffusion and chemical reaction.
(3) Senescence and related disease has become increasingly serious medical even social problems. The anti-senescence effect of theanine in vivo biological model of C.elegans(Caenorhabditis elegans) had been investigated. The results showed that theanine can extend C.elegans lifespan under heat stress. But it is difficult to find this effect in normal animal without stress. Using methods including GFP expression detection in transgenic worm line CL2006 and west-blotting in wide-type C. elegans N2, It was found that theanine can up-regulate the expression of heat shock protein HSP-16.2.
(4) Base on the beta-amyloid transgenic model of C.elegans worm CL2006, The neuroprotective effect of theanine on Alzheimer's disease had been investigated. The studies suggested that theanine can decrease paralysis behavior of the CL2006. This behavior was caused by the down-regulation of Aβ1-42 mRNA and the level of reactive oxygen specises.
引文
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