Accumulation Profile And Condensation Mechanism Of Proanthocyanidins In The Tea Plant [Camellia Sinensis (L.) O. Kuntze]

There are abundant polyphenol compounds in the tea plant[Camellia sinensis(L.)], collectively referred to as tea polyphenols, including flavanol, flavonoids, flavonols, anthocyanosides, proanthocyanidins and phenolic acids, etc. Phenolic compounds in tea planst play a crucial role in dominating tea flavor and possess a number of key pharmacological benefits on human health.Based on separation and purification and molecular biological techniques, the identification and tissue-specific, development-dependent accumulation of phenolic compounds included PAs in tea plants were studied, the main research results are as follows.1. Tissue-specific, development-dependent phenolic compounds accumulation profile and gene expression pattern in tea plantA total of 50 phenolic compounds were identified qualitatively using liquid chromatography in tandem mass spectrometry technology(LC-TOF-MS 、UPLC-QQQ-MS/MS). Of which 29 phenolic compounds were quantified based on their fragmentation behaviors(MRM). Most of the phenolic compounds were higher in young leaves than that in stems and roots, whereas the total amount of proanthocyanidins were unexpectedly higher in the root. The results have changed previous view in tea academic circles that the root of tea plant only contains a small amount of phenolic compounds.The expression patterns of 63 structural and regulator genes involved in the shikimic acid, phenylpropanoid, and flavonoid pathways were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR) and cluster analysis. Based on the similarity of their expression patterns, the genes were classified into two main groups: C1 and C2; and the genes in group C1 had high relative expression level in the root or low in the bud and leaves. The expression patterns of genes in C2-2-1 and C2-2-2-1 groups were probably responsible for the development-dependent accumulation of phenolic compounds in the leaves. Enzymatic analysis suggested that the accumulation of catechins was influenced simultaneously by catabolism and anabolism.2. Purification and accumulation pattern of proanthocyanidins in different organs of tea plantsIn this paper, we systematically optimize those parameters which affected proanthocyanidins extracting rate. The best extraction conditions were selected as follows: 80% acetone, extraction for 35 min at 50°C with 0.25% acid additives. PAs in roots of tea plants were purified and identified to facilitate structural characterization of PAs with 1H-NMR assay. The results suggested linkages of polymeric PAs in roots of tea plants were C4-C6 or C4-C8, and the main monomeric catechins units were di-hydroxyl, cis- and nongalloylated flavan-3-ols. Cyanidin pigment was generated from PAs in roots of tea plants by butanol-HCl pyrohydrolysis. Followed by separation and purification, the cyanidin pigment with more than 98% purity was obtained.The proanthocyanidins in different organs of tea plants were qualitatively and quantitatively identified combining pyrohydrolysis and thiolysis assays, NP-HPLC, HPLC-ESI-MS, MALDI-TOF-MS and 13C-NMR techniques. The results showed that in leaves, the tri-hydroxyl, cis- and galloylated flavan-3-ols were the main monomeric catechins units, and(epi)catechin was found to be the major unit of polymeric flavan-3-ols when the degree of polymerization was greater than five. In roots, the PAs were abundant, and epicatechin formed the predominant extension unit of oligomeric and polymeric PAs.The detection conditions of PAs in tea plant with Vanillin method and Dimethylaminocinnamaldhyde method were also systematically optimized, and the results showed that the optimal conditions of Vanillin method were as follows: 0-25°C, reaction for 15 min, 50% H2SO4 and 10 g/L vanillin; while the optimal conditions of DMACA method were as follows: 0°C, reaction for 5 min, 1.2 mol / L HCl, and 2 g / L DMACA. To validate the sensitivity of different methods, epicatechin(monomeric PAs), B2(dimeric PAs), and polymeric PAs(the mixture) were used as standard substance. The flavan-3-ols presented different levels of sensitivity to Vanillin-H2SO4, DMACA-HCl, and butanol-HCl reagents. When purified root PAs were used as standard, the PAs content was detected to be 121.77 mg/g, 104.32 mg/g, and 85.06 mg/g by Vanillin-H2SO4, DMACA-HCl, and butanol-HCl protocols, respectively. By comparison, The content of PAs of in roots was detected to be 81mg/g by purification. The result suggested the butanol-HCl assay closely matched the results of the purification method.3. Preliminary study on the condensation mechanism of proanthocyanidins in tea plantsIn order to understand the mechanism of proanthocyanidins polymerization, auto-condensation of the flavan-3-ols was investigated. Whether in acid or alkaline solution, catechins could be condensed to form multiple dimers under mild conditions in vitro(25°C). Perplexing, the dimers formed in vitro did not match with those found in tea plants, It could be speculated that the dimers formed from commercial catechins in vitro may be the oxidation products, which do not exist in fresh tea plants.While the substrates used were either procyanidin B2 and monomeric flavan-3-ols(epicatechin or catechin), or only procyanidin B2, the same trimers(m/z 865) were detected in the extracts of tea plants and in the non-enzymatic in vitro assay, in weak acid as well as weak alkaline solutions at room temperature. This suggested that procyanidin B2 not only released carbocation as electrophilic upper units, but also could be used as nucleophilic lower units directly itself, to form the procyanidin trimer in vitro or in vivo.