Enzymatic Synthesis, Purification, Identification And Bioactivity Of Theaflavins

Theaflavins are orange or orange-red in color and possess a benzotropolone skeleton that is formed from cooxidation of selected pairs of catechins,one with a victrihydroxyphenyl moiety,and the other with an orthodihydroxyphenyl structure.It is known that theaflavins,which account for 0.5-3%of the dry weight of solids in black tea,contribute to astringency and briskness of black tea.theaflavin 3,3'-digallate has the most astringency taste(6.4 times higher than theaflavin).Thus, theaflavin composition has been regarded as an important factor that can affect color and briskness of black tea.Recently,theaflavins have attracted considerable interest because of their potential benefits for human health,including antimutagenicity, suppression of cytochrome P450 1A1 in cell culture,anticlastogenic effects in bone marrow cells of mice,suppression of extracellular signals and cell proliferation,and anti-inflammatory and cancer chemopreventive action.Theaflavins also scavenge H₂O₂ and have been shown to inhibit lipid oxidation,LDL oxidation,DNA oxidative damage,and xanthine oxidase activity.Researching and developing theaflavins has important theoretical value and wonderful future of industrial production,especially in natural medicine,health care products,makeup and so on.However,the separation of theaflavins from black tea is both times consuming and tedious.In the face of the limited information available on the biological activities of individual theaflavins,we have devised a simple method for synthesizing theaflavins from tea catechins mixtures using enzymatic oxidation methods.The resulting extracts were subjected to to column chromatography(polyamide),preparative HPLC and HSCCC.After successive chromatography,it was expected to recover theaflavins monomers.We then examined the theaflavins so created for anti-hyperlipemia,anti-inflammatory and anti-cancer activities.1 Selection of polyphenol oxidaseThe isoenzymes of polyphenol oxidase of different source(pear,apple,tea, mushroom and fungal laccase)were studied by means of the polyacrylamide gel vertical slab electrophoresis.Similar bands were observed in different source,but the number and width of the bands & Rf value and molecular weight were different.11 bands of PPO activity were visible in pear extract.4 bands of PPO activity were visible in apple extract.5,5 and 1 bands were present in tea,mushroom extract and laccase,respectively.Rf value of PPO isoenzymes was between 0.40 and 0.70. Molecular weight of tea PPO isoenzymes was more than 94 000,while that of pear,mushroom,apple and laccase PPO isoenzymes ranged from 45 000 to 94 000.An in vitro model fermentation system,containing tea catechins and crude pear polyphenol oxidase from different source has been used to determine the effect of PPO activity and isoenzyme forms on the formation of theaflavins.The ability to synthesis of theaflavins was in the order:pear PPO,tea PPO,microorganism PPO, apple PPO,laccase and mushroom PPO.The bands of fengshui pear were more than that of other cultivars and its PPO activity is the highest among pear cultivars.So its ability to synthesis of theaflavins was the highest among pear cultivars.2 Enzymatic synthesis of theaflavinsAn in vitro model fermentation system,containing tea catechins and crude pear polyphenol oxidase from pear fruits has been used to determine the effect of tea catechin mixtures of different proportions & physico-chemical conditions on the formation of theaflavin under single factor and orthogonal experimental design.We could synthesize theafalvins complex of different proportions by oxidizing tea catechins mixture of different proportions.The optimum temperature for theaflavins formation is 30℃and the pH optima for theaflavin formation were 5.5.The concentration of tea catechins mixture(EGC>200mg/g,EGCG>200 mg/g,total catechins>500 mg/g)for theaflavins formation is 5 mg/ml and the concentration of enzyme for theaflavins formation is 49162.50U.pH and temperature in the model fermentation system are the most important factors(P<0.05).By applying the stepwise linear regression,the synthetical effects of preparing parameters of theaflavins complex from tea catechins mixture using enzymatic oxidation methods were investigated.The parameter model were set up,Y=-23.758+21.359X₁-0.683 X₂-1.841X3.The optimum of preparing parameter of theaflavins complex was as same as above optimum parameter.3 Separation,purification and structural identification of theaflavins A preparative high-speed counter-current chromatography(HSCCC)method for the isolation and purification of theaflavins from the crude extract was successfully established by using hexane-ethyl acetate-methanol-water-acetic acid as the two-phase solvent system at a flow rate of 2ml/min and a revolution speed of 700rpm.The upper phase of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25,v/v/v/v/v)was used as the stationary phase of HSCCC.The mobile phase of HSCCC was the lower phase of hexane-ethyl acetate-methanol-water -acetic acid(1:5:1:5:0.25,v/v/v/v/v).Four kinds of theaflavins including theaflavin(TF),theaflavin 3-gallate(TF3G),theaflavin 3'-gallate(TF3'G)and theaflavin 3,3'-digallate(TFDG)were obtained from crude extract,with the purity of 94.2%,95.8%,93.7%and 96.4%,respectively,as determined by high performance liquid chromatography(HPLC).The structures of the isolated compounds were positively confirmed by ¹H NMR and ¹³C NMR,UV,IR,and MS analysis.A preparative reversed phase liquid chromatography(PRPLC)combined with polyamide column chromatography was used to isolate the chemical constituents from the theaflavins extract.The separation condition was optimized as follows.the column was PRC-ODSC18(20.0mm×250mm i.d.,10um).The mobile phase for the HPLC consisted of acetonitrile,ethylacetate,and 2%acetic acid at the ratio:21:3:76.The flow rate was set at 15ml/min.The detection wavelength and sample size were set at 280nm and 400μl(30mg/ml).Under the optimized conditions,four compounds (theaflavin,theaflavin 3-gallate,theaflavin 3-gallate and theaflavin 3,3'-digallate) were isolated with the purity of 96.9%,98.8%,95.9%and 98.9%,respectively,as determined by high performance liquid chromatography.Their chemical structures were identified as theaflavin,theaflavin 3-gallate,theaflavin 3-gallate and theaflavin 3,3'-digallate by spectroscopic methods including ultraviolet,infrared,electrospay mass spectrometry and nuclear magnetic resonance(NMR),as well as by comparison with published spectral data.High-performance Thin layer chromatography of theaflavins was performed on polyamide with chloroform-methanol 1:1(two fold development).TLC of EtOAc phase was performed on polyamide with acetic acid-methanol 1:1 and methanol-acetone-formic acid 2:1:1(two fold development).The following constituents were identified on the basis of their Rf value and absorbance spectrum,included epigallocatechin gallate,epicatechin gallate,epicatechin,theaflavins and their various gallate derivatives. 4 studying the anti-hyperlipemia,anti-inflammation and anti-cancer of theaflavins by High Throughput Screening(HTS)MethodHTS models of PPARs,FXR,LXR,TNFa and IL-1 were used to evaluate the pharmacological activity of theaflavins.TFs exhibited the highest inhibitory activity on LXR.The active times was 1.55.The second active one was TFDG.The results were in the following order:TFs>TFDG>TF3G≈TF>TF3'G TFs and TF,TF3G, TF3'G and TFDG had inhibition on FXR,in which TFDG exhibited the highest inhibitory activity.The active value is 2.35.The second active one was TF3'G which active value was 2.22.The results were in the following order:TFDG>TF3'G≈TF3G>TF>TFs.The results showed that the effects of theaflavins(TFs,TF,TF3G, TF3'G and TFDG)on TNFa and IL-1 were insignificant.The inhibition effects of theaflavins complex(TFs),theaflavin, theaflavin-3-gallate,theaflavin-3'-gallate,and theaflavin-3-3l-digallate on the growth of human liver cancer HepG2 cells,gastric cancer SGC-7901 cells,lung cancer A549 cells and acute promyelocytic leukemia K562 cells were investigated.The effects of theaflavins(TFs,TF,TF3G,TF3'G and TFDG)on SGC-7901 and A549 were significant at the concentration of 100μg/ml.TF3G exhibited inhibitory effect against SGC-7901,K562,A549 and HepG2 at the concentration of 50μg/ml and TF exhibited little inhibitory effect against HepG2 at the concentration of 100μg/ml.It implied that TF3G had more multifaceted bioactivity functions whereas TFs,TF, TF3'G and TFDG had less.