Studies On Biochemical Control Of Theaflavins Formation And Technics Of Isolation & Purification

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 weresystematically 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.