Research On High-Throughput Screening And The Function Of Active Compounds Of Fuzhuan Tea

With the rapid developing of global economy and improving of people's life, theincidence of disease of hyperlipidemia and adiposity increase everyday. They havebeen one of the first healthy problems that people facing. Preventing and curing thesekinds of disease have been the focus of global healthy industry. Searching natural,high effect, safe medicine and healthy food for therapy hyperlipidemia and adiposityhave been the project that global science and technology field and product field caremostly.It is centuries-old that tea has been used as medicine. With the improvement ofscience technique and the deep research of tea's medicine function, it has been provedthat tea has the function of therapy hylipidemia and reducing cardiovascular diseases.Hunan is the main product area of Fuzhuan Tea, which is the choice of the frontierminority and necessary daily drinking. The modern isolation and purificationtechnique was applied step by step to isolate the active compounds. Combining withHigh-Throughput Screening(HTS) technique, metabolish enzyme activities testingand animal experiments, the function of Fuzhuan Tea as natural therapyhyperIipidemia was researched systematically. The achievements were as follows:1. The active compounds were isolated and identified from Fuzhuan Tea for thefirst timeCombining modern separating techniques and High-Throughput Screening(HTS)techniques, the main active compounds in Fuzhuan Tea were isolated and identified. 8compounds were separated from Fuzhuan Tea for the first time. By using IR,UV, MS,NMR, HPTLC and HPLC, etc. techniques, the compounds were identified as follows:gallic acid, gallocatechin (GC), 3- methoxy- 4,5-dihydroxy-benzoic acid,3,4-dihyddroxy-benzoic acid, epigallocatechin (EGC), epigallocatechingallate(EGCG), epicatechin gallate(ECG) and rutin.2. The function of therapy of hyperlipidemia and adiposity of Fuzhuan Tea were tested by HTS and animal experiments.The water extracts of Fuzhuan Tea was extracted by chloroform, 1-butanol andethyl acetate step by step. The tea residue was extracted by ethanol. HTS was the firsttime to research the function of therapy hyperlipidemia and adiposity of Fuzhuan Teaby PPARα,PPARσ,PPARγ,LXR,FXR and 3T3-L1.The results showed that thefunctions of restraining 3T3-L1 and activating PPARαof all samples were not evident,which were active to FXR. The active effect of ethanol extraction was the mostevident. The active value was 2.313. The second active one was ethyl acetate extract.The active value was 2.083.The most strong one which restrained FXR was the layerof water sample. The second one was the 1-butanol extract. 1-butanol extract couldactivate LXR. Chloroform extract and water layer sample could activate PPARγstrongly. Among all samples, ethyl acetate extract was the best one. Its active valuesto PPARδ, PPARγ, LXR and FXR were all more than 1.80.5 compounds separated from ethyl acetate extract were tested by HTS. The resultsshowed that EGCG was very active to FXR on the concentration of 10μg/mL and 50μg/mL. When the concentration was 50μg/mL, the active value reached 6.000. Whenthe concentration of ECG was 50μg/mL, the active value reached 3.220. EGC wasactive to FXR, PPARδ, PPARγand LXR. The active value to PPARγwas 1.619. 3-methoxy-4,5-dihydroxy-benzoic acid was effective to FXR restraining, PPARγandLXR activating. The active value of 3- methoxy- 4,5-dihydroxy-benzoic acid toPPARγwas 1.734. The restraining multiple to FXR of 3,4-dihyddroxy-benzoic acidwas 3.641,while that of CK CDCA was 6.435. All the results showed that all thecompounds were functional ones.Wistar rats were fed on different dose of 0.085 g/kg·bw, 0.170 g/kg·bw and 0.510g/kg·bw of Fuzhuan Tea extracts by 30 days. The results showed that the middle doseFuzhuan Tea could decrease TC and TG apparently (p＜0.01) and decrease the weightand HDL-C not apparently (P＞0.05).3. The mechanism of therapy of hyperlipidemia and adiposity of Fuzhuan Teawas reported.Fuzhuan Tea could activate PPARδtarget gene to decrease weight, make bodyslim and improve blood lipid, stave atherosclerosis, reverse cholesterol sediment in foam cells when atherosclerosis damage. It could activate PPARγto adjust lipocytesignal transduction, induce lipocyte special gene produce, reduce cardiovasculardiseases being correlative to metabolize syndrome and increase fatty acid metabolizein bone muscle. It could activate LXR to increase cholesterol expelling from liver,decrease cholesterol being absorbed in small intestine and adjust the level of bloodlipid, and activate FXR to increase the concentration of triglyceride in VLDL andCM, reduce VLDL and LDL, increase the synthesize of HDL and decrease thetriglyceride in plasma and restrain FXR to enhance cholesterol change to bile acidand decrease the concentration of cholesterol in small intestine and blood to adjustblood lipid. In one words, the results indicated that Fuzhuan Tea was helpful ofadjusting cholesterol metabolish balance, making body slim, improving blood lipidand keep healthy blood lipid level.The research results showed that different extracts of Fuzhuan Tea by chloroform,1-butanol and ethyl acetate separately had different effect on pancreatic lipaseactivity. Chloroform extract could restrain pancreatic lipase a little. The water extract,ethyl acetate extract and 1-butanol extract had evident activation, that of 1-butanolextract was the most active. The multiple was 2.54.All extracts apart from chloroform extract of Fuzhuan Tea were active toα-amylase, among them, ethyl acetate extract was the most active. Its multiple was4.09. Water extract sample was the second one and its multiple was 2.99. That of thelayer of water sample was the last one and its multiple was 1.20. The samplesseparated from ethyl acetate extract and 1-butanol extract were active to a-amylaseand pancreatic lipase respectively.4. The Variations of the components of Fuzhuan Tea during fungus growingThe character of Fuzhuan Tea is different from dark green tea because of the step offungus growing. The variations of several main components' concentration ofFuzhuan Tea during process were tested. The results showed that the concentration ofall kinds of components were decreased. The decreases of catechins, flavones andpolyphenols were separately 63.64%, 51.52% and 54.62%. The decrement of aminoacids and caffeine were 36.84% and 10.85% separately, that of soluble sugar andwater extract were not too much. The components decreased fastest during fungus growing. The unique character of Fuzhuan Tea was because of Eurotium Cristatumetc. fungus.5. Fuzhuan Tea was proved to have no poison by animal experiment.Fuzhuan Tea was researched about the toxicity experiments to evaluate its safety.The results showed that LD₅₀ was 14700 mg/kg·bw to male mouse through mouth andwas 19600 mg/kg·bw to female mouse (The limit reliability of 95% was 10600-36000). The experiments of Ames and making mouse semen catface were negative.The rats were fed on feedingstuff mixed with Fuzhuan Tea of the concentration of0.85%, 1.25%,1.70% separately. Each group of animals grew well during theexperiment. The rate of liver to body weight was increased markedly but has nobiology signification. All doses had no effects on the hemogram of male and femalerats' biochemical index of male rats triglyceride, Urea anhydride, blood muscleanhydride and blood sugar of male rats and the rate spleen to body weight, kidney tobody weight and the organism of pathology of female rats(p＜0.05). The resultsshowed that the samples had no evident toxin side-effect to rats during the 30 feedingdays. Fuzhuan Tea had no poison.