Isolation, Identification Of Polyphenols In Black Tea And Tea (Camellia Sinensis) Flower And Studies On Their Antioxidant Function And Mechanism

Part 1 Autioxidant activity and mechanism of phenolic compounds present in black teaTea is one of the most widely consumed beverages in the world. Green tea is more popular in China, Japan, Korea and some African countries, whereas black tea is preferred in India and the Western countries. During the past decade numerous in vitro and in vivo studies have suggested the possible protective effects of green tea and catechins in cancer and cardiovascular disease development. Whereas catechins are the most abundant polyphenols in green tea, the typical pigments in black tea are theaflavins (TF), thearubigins (TR) and theabrownins (TB), which are derived from the oxidation of catechins and their gallates during fermentation stage of black tea processing. Recently, the molecular mechanism and bioactivities of polyphenols in black tea have attracted considerable interest because of its beneficial health properties.In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The major theaflavins monomers were separated by high-speed countercurrent chromatography (HSCCC). The antioxidant activities of these products were evaluated in Fenton reaction system, 2,2-diphenyl-1- picrylhydrazyl (DPPH) system and H₂O₂-induced oxidative damage system in the HPF-1 cells. Their antioxidant mechanisms were discussed. The main results were showed as followings:1. Separation of theaflavins monomers.Three theaflavins monomers were separated from the oxidation product of polyphenols by immobilized polyphenol oxidase plate using HSCCC. The effects of solvent systems and NaHCO₃ on the separation were investigated. The result showed that NaHCO₃ was helpful to purify the theaflavins. Separation was performed with a two-phase solvent system composed of ethyl acetate - hexane -methanol-water (3:1: 1: 6, V/V) and (3: 1: 3: 4, V/V) by eluting the lower aqueous phase at 1.2 ml/min at a revolution speed of 880 rpm. 57 mg of 97.7% theaflavin, 26 mg of 61.2% theaflavin-3'-gallate, 64 mg of 84.5% theaflavin-3-3'-gallate and 33 mg of 92.3% theaflavin-3-gallate and theaflavin-3'-gallate complex were obtained from 250 mg of the crude TFs sample.2. The antibacterial, and antioxidant function and mechanism of black tea pigments.The effects of theaflavins and tea polyphenols on two major oral bacterium, namely S.mutans Ingbritt and S.sobrinus 6715 were investigated. It indicated that the two polyphenols had the potent inhibiting activity. It was no remarkable difference between them. Furthermore, the two bacterium had no medicine-resistant against the two polyphenols.In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The order of OH·and DPPH scavenging ability of these oxidation products was TF> TB> TR. These oxidized phenolic compounds showed protection against H₂O₂-induced damage in HPF-1 cells and suppressed the accumulation of intracellular reactive oxygen species in H₂O₂-induced damage HPF-1 cells. Interestingly, TB, as a further oxidative product from TF or TR, showed the potent activity followed TF in the above four systems.The protective effect of three major black tea polyphenols, namely theaflavin (TF1), theaflavin-3 (3')-gallate (TF2) and theaflavin-3,3'-digallate (TF3) on oxidative damage human fibroblast (HPF-1) cell were investigated by comparing with epigallocatechin gallate (EGCG). The order of OH·and DPPH scavenging ability were TF3 > TF2 > TF1 >EGCG and TF3>TF2>EGCG>TF1 respectively. TF1, TF2, and TF3 showed more effective than EGCG in protection against H₂O₂-induced damage in HPF-1 cells. Pretreated for 2h and eliminated from the cell, TF1 and TF3 still showed protection against H₂O₂-induced damage in HPF-1 cells. TF1, TF2 and TF3 suppressed the accumulation of intracellular reactive oxygen species (ROS) in H₂O₂-induced damage HPF-1 cells. Our results suggest, other than inhibition of ROS, the protective effect of TF1 and TF3 on oxidative damage HPF-1 cell may also be responsible for improvement of normal HPF-1 cell resistive ability toward radical-damage. TF2 may protect against H₂O₂-induced damage in HPF-1 cells, through enhancing HPF-1 cell proliferation.Part 2 Analysis of chemical compositons in tea (Camellia sinensis) flowers and their antioxidant mechanismWhile these properties of tea have been extensively studied, the less attention has been given to these properties of tea flower. This is due to the fact that people have been only picking the tender shoots from tea tree to make tea for a long history, but for tea flowers, the majority lets them run their courses. Compared with tea leaves, tea flowers have similar chemical compositions such as polyphenols and caffeine, polysaccharide and protein etc. The aim of the present study was to ascertain the phenolic profile of tea flower extracts, as well as to know the major active components responsible for the antioxidant activity of tea flowers. The antioxidant activity of the extracts was evaluated by DPPH assay. Phenolic compounds were separated using HPLC-DAD, and identified by means of LC-MS-ion trap.1. Evaluation of the antioxidant compounds of extracts from tea (Camellia sinensis) flower.The antioxidant activity of tea flower was investigated by the DPPH free radical-scavenging assay and the hydroxyl radical-induced luminol chemiluminescence assy. The tea flower was extracted with ethanol (EE) and separated by the order of chloroform, ethyl acetate (EEA), and n-butanol. The result pointed tea flower ethyl acetate extract (EEA) had the strongest antioxidant activity and the most contribution to the antioxidant activity of tea flower. The stronger scavenging abilities to free radicals were shown by EE and EEA, which might be due to the content of polyphenols contained. However, almost non free radical-scavenging activity was observed while the tea flower was extracted with water and its fractions of several organic solvents.2. Liquid chromatographic-mass spectrometric analysis of phenolics in tea flower extract. High performance liquid chromatography (HPLC) combined with diode array (DAD) and electrospray (ESI)-ion trap-MS detection was used to separate and identify the compounds present in EEA. Eight catechins, namely, catechin, epicatechin (EC), gallocatechin, epigallocatechin, gallocatechin gallate, epigallocatechin gallate (EGCG), catechin gallate and epicatechin gallate (ECG), catechins derivatives, namely, EC-3'-di- rhamnoside and catechins dimer, and flavonol glycosides, namely, quercetin, luteolin, kaempferide and kaempferol mono-, diglycosides (glucoside and rhamnoside) were identified. In addition, it is found that EGCG and ECG are the major active components responsible for the antioxidant activity of tea flowers by HPLC separation and DPPH assay.