Separation And Anylysis Of Representative Catechins And Theaflavins,and Their Attenuation Effects Of Metabolic Syndrome In Female Db/db Mice

Chinese tea production ranked the most in the world, for its greatest varieties and comprehensive categories. Tea industry has made great contribution to Chinese economy. Tea is the second most popular drink after water in the world. The habit of tea drinking has a long history. Recently, increasing study has shown that drinking tea is health-promoting, such as prevention of obesity, anti-cancer activity, and attenuation of cardiovascular and neurodegenerative diseases. The most frequently reported functional ingredients are catechins in green tea and theaflavins in black tea. However, most study applied tea extract for treatment, there still lacks a systematic procedure for large quantity purification of catechins and theaflavins for future molecular mechanisms investigation. As a result, this thesis investigated the purification of non-galloyl catechins from green tea and large quantity preparation of HPLC grade TF and EGC from theaflavins mixture. Then a UHPLC methodology for simultaneous determination of 15 representative tea constituents in 6 major types of Chinese teas was developed and distribution of the representative tea constituents in 243 Chinese tea samples was carried out based this method. Finally, metabolic syndrome attenuation effect of 0.1% catechins and theaflavins extract in female db/db mice was briefly explored.(1) The major catechins in green tea are EGCG and ECG, however, rare catechins EGC and EC are relatively expensive. After decaffeination, green tea catechins extract (CE) was prepared from green tea. After tannase-mediated biotransformation, TE was converted into non-galloyl catechins extract (NCE). The content of rare catechins EGC and EC increased to 390 mg/g and 170 mg/g. Applying HSCCC separation with N-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v) as the bilayer solvent system, a mixture of 100 mg CE and100 mg NCE produced 27.2 mg 98.8% EGCG, 14.1 mg 97.7% EGC, and 9.3 mg 98.9% EC under forward mode, while under reverse mode, a mixture of 80 mg CE and 80 mg NCE prepared 19.9 mg 95.0% EGCG,5.8 mg 95.7% ECG,8.9 mg 96.4% EC.(2) The contents of 4 individual theaflavin in theaflavins extract (TE) are low, and the remaining catechin contents are high together with other unidentified impurities. It is of great practical value to purify large quantity of TF and EGC in a single operation. After tannase-mediated biotransformation and decaffeination, TE was converted into tannased-mediated biotransformation product (TBP), in which the contents of TF and EGC were increased approximately 3 folds to 323 mg/g and 152 mg/g, respectively. Under the optimized HSCCC separation parameters:N-hexane/ethyl acetate/methanol/water (1:8:1:8, v/v/v/v) as the bilayer solvent system with a injection volume of 500 mg TBP, purification of 180.8 mg 97.3%TF,87.5 mg 95.7% EGC and 15.1 mg 96.2% EC in 2.5 hours was accomplished. Compared with HSCCC, prep HPLC, employing 55.5% acetonitrile as the eluent, separated 11.2 mg 94.9% TF and 7.7 mg 92.7% EGC from 120 mg TBP. Therefore, combination of HSCCC and tannase-mediated biotransformation for separation of TF and EGC are more advantageous.(3) To investigate the distribution of 15 major tea constituents in 6 types of Chinese teas, a standardized profiling method based on ultra high performance liquid chromatography (UHPLC) was established. The composition and content of 15 tea constituents were analyzed, including 8 catechins,4 theaflavins, phenolic acid (gallic acid) and purine alkaloids (theobromine and caffeine) in 30 minutes. Each tea variety had a unique chemical profile. The compositions of catechins were higher in the unfermented green tea and white tea, while the content of theaflavins was the highest in black tea, followed by half-fermented oolong tea and minor-fermented yellow tea. The content of caffeine in all the studied tea samples was relatively stable while the gallic acid levels were highly variable, lowest in oolong tea, and highest in pu-erh tea. The developed HPLC method and its wide applicability for simultaneous analysis of catechins, theaflavins, caffeine, theobromine, and gallic acid in 6 major kinds of Chinese teas made it a rapid and accurate method for analysis.(4) By applying the well established UHPLC method,15 major constituents in 243 tea samples from 5 selected types of Chinese tea were analyzed and determined. Compared with other large scale Chinese tea survey, catechins contents in white tea and green tea did not change greatly. The major catechins in green tea, black, and oolong tea are EGCG, EGC, ECG and EC. The caffeine contents in all samples did not differentiate much, while the gallic acid content changed a lot among different types of tea, especially in full-fermented black tea. The theaflavins contents in black tea were larger than oolong tea, while yellow tea (Jun Mountain Needle Tea) contains considerable amount of theaflavins which is interesting and requires further study to confirm this trend. To discriminate tea classification among 4 types of Chinese teas, principle component analysis (PCA) was performed to extract 3 major factors, which covered 85% data characters. After plotting 3D scatter figure to visualize the distribution patterns of the survey, clear discrimination among 4 types of Chinese teas was achieved.(5) Metabolic syndrome attenuation effect of 0.1% catechins (PPE) and theaflavin extract (TF) on 36 female db/db mice was studied for 6 weeks.0.1% PPE and TF had no significant alteration on body weight, water consumption, and food consumption in female db/db mice. PPE treatment decreased blood glucose level, serum lipids content, and serum cholesterol content, while TF slightly attenuated the 3 MetS characteristics. PPE increased the insulin level in serum and alleviated insulin resistance. The drug kinetics and metabolites distribution of TF in organs were studied. There was no TF detected in serum, urine, and liver, except for feces. The TF recovery reached 26% in feces sample, which indicated that TF were extensively biotransformed in the gut by microbiota. However, no TF and catechins metabolites were detected in brain, demonstrating that they were not permeable through blood brain barrier (BBB).