茶葉兒茶素之代謝機制與生物活性

Abstract

茶葉中主要之保健成分-兒茶素及其聚合物具有不同保健功效，因此了解兒茶素於加工及貯存過程中之代謝與其生理活性，可以做為日後開發不同機能性茶飲產品之參考依據。 兒茶素類水溶液不穩定且易氧化，因此兒茶素類的化學轉變，在茶飲料加工上是一項很重要的議題。本研究第一部分以層析、質譜等方法研究兒茶素水合物在鹼性環境下照射藍光的變化。兒茶素水合物溶液為無色透明，在鹼性環境下照藍光轉為黃色，黃色溶液經鑑定是兒茶素二聚體(catechin dimer)屬原花青素類的物質，在兒茶素水合物光反應時添加抗壞血酸，能降低二聚體的形成，表示抗壞血酸能抑制兒茶素的光敏化氧化作用。兒茶素水合物在鹼性環境下照藍光，經光敏化氧化作用，產生超氧陰離子自由基，能加強鮑氏不動桿菌及多重抗藥菌株的失活，可提供對環境微生物失活的安全方法。 第二部分探討加工對茶葉兒茶素代謝之影響及活性評估。以同一批茶菁原料，加工成不同發酵程度茶葉，以層析、質譜等方法進行研究，並分析不同發酵程度茶葉之抗氧化能力與兒茶素代謝之相關性。結果顯示：隨發酵程度增加，兒茶素代謝反應增加，兒茶素隨之減少，取而代之的是烏龍茶質(theasinensins) TSB、TSC及茶黃質(theaflavins) TF、TF3G與沒食子酸(gallic acid)含量增加。綠茶(不發酵茶）、包種茶（輕發酵茶）及烏龍茶（重發酵茶）由於兒茶素含量較高，有較佳之DPPH自由基清除能力、還原力與ABTS+清除力，紅茶(全發酵茶)具有較多之茶黃質、烏龍茶質及沒食子酸，相對應有較佳螯合亞鐵活性。 利用液相層析串聯質譜儀針對三種不同程度發酵茶葉進行組成分析，以碰撞誘發解離 (collision-induced dissociation, CID) 技術對分子身份進行鑑定，共有46種以上分子被鑑定。茶葉經發酵後於分子組成具有大幅度變動，兒茶素與黃酮醇醣苷(flavonol glycoside)等分子顯著降解，同時生成了許多有機酸及烏龍茶質。此外，於綠茶中，也檢測到大量兒茶素二聚體且與兒茶素一樣，隨著發酵程度增加而有所減少。 第三部分探討貯存對茶葉兒茶素代謝之影響與活性評估。以不同年份生產之宜蘭茶區條形部分發酵茶為試驗材料，除分析茶葉化學成分，並利用脂多醣(LPS )刺激巨噬細胞 RAW 264.7，使產生發炎前驅物質 NO，來研究茶葉抑制發炎反應的機制。結果顯示：隨貯藏時間增加，茶葉總多元酚及總兒茶素、總兒茶素二聚體及總烏龍茶質含量下降，但並未產生茶黃質，顯示貯存過程之兒茶素代謝機制與發酵加工過程之兒茶素代謝機制有所不同。兒茶素代謝產物沒食子酸及其他有機酸消長之綜合表現，造成茶葉pH值呈現先降後升的趨勢。在茶葉生理活性方面，隨貯存時間及施用濃度增加，茶葉抗發炎能力越好，與貯存時間具正相關的沒食子酸、鄰苯三酚及咖啡酰酒石酸衍生物及奎寧酸衍生物、楊梅黃酮醣苷、山奈酚醣苷與鞣花酸等，均與抗發炎活性有很好的相關性。

Catechins and its polymers offer various health benefits. Therefore, it is very important to realize catechin metabolic process and its physiological activity during manufacturing and storage process. This result will support for future various functional tea production. Catechin is unstable and easily oxidized in water solution. Non-enzymatic browning of tea was due to photooxidation, temperature and pH variation. Hence catechin chemical transformation at different environmental conditions is an important issue in tea drink manufacture. In this study, first, we try to figure out the change of catechin under blue light irradiation in alkaline solution by chromatography and mass spectrometry techniques. Catechin is colorless in water solution. However, it was turn into yellow color under blue light irradiation in alkaline condition. The yellow substance was characterized as catechin dimer (proanthocyanidin) and it can be prevented by ascorbic acid addition. It showed that ascorbic acid will inhibit catechin photooxidation. A superoxide anion was produced through catechin photooxidation under blue light irradiation in alkaline condition. This free radical can cause inactivation of multidrug-resistant Acinetobacter baumannii and support as solution for environmental microbial safety. Furthermore, different fermented tea from the same resource of tea leaves was produced and was analyzed by chromatography and mass spectrometry techniques to understand correlation of catechin metabolic pathway during different fermented process. As the degree of fermentation (oxidation) increases, metabolic reaction of catechin was increased and catechin was decreased followed by increasing of theasinensins (TSB and TSC), theaflavins (TF and TF3G) and gallic acid. Non-fermented tea (green tea) and semi-fermented tea (pouchong and oolong tea) have high DPPH scavenging effect, reducing power and ABTS free radical scavenging ability, owing to high catechin concentration. Completely fermented tea (black tea) has high ferrous ion chelating ability because of high theasinensins, theaflavin and gallic acid concentration. Forty-six compounds were characterized for three different fermented tea by analytical liquid chromatograph tandem mass spectrometer with collision-induced dissociation (CID). It showed that variation of molecular composition after tea fermentation. Catechins and flavonol aglycones were significantly decreased followed by formation of organic acid, theasinensins and theaflavin. In addition, a lot amount of catechin dimers were detected in green tea, and decreasing when fermentation. Chemical composition was analyzed from different age of strip type partial fermented tea to explore the effects of storage on the metabolism of catechins. The mechanism of tea inhibited inflammatory response was discussed by immune response was stimulated by foreign substances lipopolysaccharide (LPS) in macrophages RAW 264.7 and trigger nitric oxide (NO) generate. As the results, as the time go by, total polyphenol, total catechin, total catechin dimer and total theasinensin were reduced without theaflavin production. It showed that metabolic mechanism is difference between manufacture process and tea storage. The phenomenon of pH value first drops and then rise of tea was cause by combination of catechin metabolic products “gallic acid” and other organic acids change. Anti-inflammatory ability of tea was enhanced with rising of storage time and test dose. The bioactivity test of tea storage time indicating a positive correlation between metabolic compounds: gallic acid, pyrogallol, caffeoyl tartaric acid derivative, quinine derivative, bayberry flavonoid glycoside, kaempferol and ellagic acid and anti-inflammatory ability.