Zhengzhu Zhang

Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds.

BackgroundTea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.ResultsUsing high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR).ConclusionsAn extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.

New triterpenoids and other constituents from a special microbial-fermented tea-Fuzhuan brick tea.

Fuzhuan brick tea, a special microbial-fermented tea prepared from the leaves of Camellia sinensis var. sinensis, is a traditional beverage used in China throughout history. Phytochemical investigation of this material led to the identification of three new triterpenoids, 3beta,6alpha,13beta-trihydroxyolean-7-one (1), 3beta-acetoxy-6alpha,13beta-dihydroxyolean-7-one (2), and 3beta-O-(8-hydroxyoctanoyl)-12-oleanene (3), together with 11 known compounds, friedelin (4), beta-amyrone (5), beta-amyrin (6), alpha-spinasterone (7), alpha-spinasterol (8), 22,23-dihydro-alpha-spinasterone (9), 22,23-dihydro-alpha-spinasterol (10), alpha-phytol (11), alpha-tocopherol (12), alpha-tocoquinone (13), and caffeine (14). The structures of 1-13 were determined by spectroscopic and chemical methods. Compounds 1 and 2 are the first two examples of triterpenoids possessing a 6-hydroxy-7-one function. All of the compounds, except 6, 8, 10, 11, and 14, were isolated from tea and Camellia spp. for the first time. The antibacterial activities of 1 were assessed against some enteric pathogenic microbes. Compound 1 showed no cytotoxic activity against A-549, Bel-7402, and HCT-8 cell lines.

Green tea polyphenols alleviate obesity in broiler chickens through the regulation of lipid-metabolism-related genes and transcription factor expression.

The current study investigated the effects of green tea polyphenols (GTPs) on lipid metabolism and its mechanisms using broiler chickens (Gallus gallus domesticus). A total of 36 male chickens (35 days old) had been subjected to an oral administration of GTPs at a dosage of 0, 50 (low), and 100 (high) mg/kg of body weight for 20 days. Our results showed that GTPs significantly decreased the abdominal and subcutaneous fat masses of broilers and reduced the serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels compared to those of the control. Furthermore, the expression levels for lipid anabolism genes were significantly downregulated, while the expression levels of fat transportation and catabolism-related genes, carnitine palmitoyl transferase I (CPT-I), acyl-CoA oxidase 1 (ACOX1), and peroxisome proliferator-activated receptor-α (PPARα) in liver, adipose triglyceride lipase (ATGL) in abdominal fat, and lipoprotein lipase (LPL) in skeletal muscles, were notably upregulated. Our data have revealed that GTPs alleviate obesity and serum lipid levels in broiler chickens by suppressing fatty acid synthesis and stimulating lipolysis.

Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea

HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed that the level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) dropped increasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discovery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C-F (1-4) together with three known BRFCs (5-7), six known catechins (8-13), five simple phenols (14-18), seven flavones and flavone glycosides (19-25), two alkaloids (26, 27), three triterpenoids (28-30) and one steroid (31). The structures were elucidated by spectroscopic methods including 1D and 2D NMR, LC-HR-ESI-MS, IR, and CD spectra. Five compounds (16-18, 28, 29) were reported for the first time in tea. Possible pathways for the degradation of major tea catechins and the generation of BRFCs were also provided.

Antiseptic activity and phenolic constituents of the aerial parts of Vitex negundo var. cannabifolia.

Four phenolics, salviaplebeiaside (1), γ-tocopherol (2), chrysosplenol-D (4), and isovitexin (5), along with α-tocoquinone (3) and β-sitosterol (6) were isolated from the aerial parts of Vitex negundo var. cannabifolia. The isolation was performed using bio-assay tracking experiments. The structures of compounds 1-5 were established by spectroscopic means. The antibacterial activities of the compounds were assessed against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus and Pseudomonas fluorescens. Chrysosplenol-D (4) exhibited activities against all the four spoilage microorganisms.

Simultaneous Distillation Extraction of Some Volatile Flavor Components from Pu-erh Tea Samples—Comparison with Steam Distillation-Liquid/Liquid Extraction and Soxhlet Extraction

A simutaneous distillation extraction (SDE) combined GC method was constructed for determination of volatile flavor components in Pu-erh tea samples. Dichloromethane and ethyl decylate was employed as organic phase in SDE and internal standard in determination, respectively. Weakly polar DB-5 column was used to separate the volatile flavor components in GC, 10 of the components were quantitatively analyzed, and further confirmed by GC-MS. The recovery covered from 66.4%–109%, and repeatability expressed as RSD was in range of 1.44%–12.6%. SDE was most suitable for the extraction of the anlytes by comparing with steam distillation-liquid/liquid extraction and Soxhlet extraction. Commercially available Pu-erh tea samples, including Pu-erh raw tea and ripe tea, were analyzed by the constructed method. the high-volatile components, such as benzyl alcohol, linalool oxide, and linalool, were greatly rich in Pu-erh raw teas, while the contents of 1,2,3-Trimethoxylbenzene and 1,2,4-Trimethoxylbenzene were much high in Pu-erh ripe teas.

Effect of salt treatment on theanine biosynthesis in Camellia sinensis seedlings

Theanine synthetase (TS) is an enzyme involved in theanine biosynthesis in tea plants. Recent studies have revealed that theanine biosynthesis, derived from nitrogen metabolism in tea (Camellia sinensis L.) plants, could be influenced by salt treatment. We have characterized CsTS at the molecular and biochemical level. The expression pattern of CsTS protein was examined by western blot using a self-prepared polyclonal antibody with high specificity and sensitivity. The effect of salt treatment on the levels of theanine synthesis was investigated in this study. Levels of theanine and the total free amino acids were gradually increased in shoots, and reached the maximum on the 8th day after treatment (DAT). The immunoblotting analysis suggested the accumulation of CsTS protein had increased gently up to 8 DAT, and subsequently declined, both in roots and shoots, which is one of the main evidences that resulted in the variation of theanine concentration under salt treatment. Together, these data revealed that theanine synthesis takes place both in root and shoot and CsTS accumulation is positively affected by salt treatment.

TMDB: a literature-curated database for small molecular compounds found from tea.

BackgroundTea is one of the most consumed beverages worldwide. The healthy effects of tea are attributed to a wealthy of different chemical components from tea. Thousands of studies on the chemical constituents of tea had been reported. However, data from these individual reports have not been collected into a single database. The lack of a curated database of related information limits research in this field, and thus a cohesive database system should necessarily be constructed for data deposit and further application.DescriptionThe Tea Metabolome database (TMDB), a manually curated and web-accessible database, was developed to provide detailed, searchable descriptions of small molecular compounds found in Camellia spp. esp. in the plant Camellia sinensis and compounds in its manufactured products (different kinds of tea infusion). TMDB is currently the most complete and comprehensive curated collection of tea compounds data in the world. It contains records for more than 1393 constituents found in tea with information gathered from 364 published books, journal articles, and electronic databases. It also contains experimental 1H NMR and 13C NMR data collected from the purified reference compounds or collected from other database resources such as HMDB. TMDB interface allows users to retrieve tea compounds entries by keyword search using compound name, formula, occurrence, and CAS register number. Each entry in the TMDB contains an average of 24 separate data fields including its original plant species, compound structure, formula, molecular weight, name, CAS registry number, compound types, compound uses including healthy benefits, reference literatures, NMR, MS data, and the corresponding ID from databases such as HMDB and Pubmed. Users can also contribute novel regulatory entries by using a web-based submission page. The TMDB database is freely accessible from the URL of http://pcsb.ahau.edu.cn:8080/TCDB/index.jsp. The TMDB is designed to address the broad needs of tea biochemists, natural products chemists, nutritionists, and members of tea related research community.ConclusionThe TMDB database provides a solid platform for collection, standardization, and searching of compounds information found in tea. As such this database will be a comprehensive repository for tea biochemistry and tea health research community.

Fuzhuanins A and B: the B-ring fission lactones of flavan-3-ols from Fuzhuan brick-tea.

Fuzhuan brick-tea is a special dark tea prepared from the leaves of Camellia sinensis var. sinensis. Its production involves a fungal fermentation stage, which forms the unique flavors and functions by a series of biochemical reactions. Our phytochemical research of the material led to the isolation of two new B-ring fission lactones of flavan-3-ols, fuzhuanins A (1) and B (2). In addition, three other flavan-3-ol derivatives (3-5), three flavone C-glycosides (6-8), eight flavonoid O-glycosides (10-17), five simple phenolics (19-23), two norisoprenoid glycosides (24, 25), two sesquiterpenoids (26, 27), and theobromine (28), as well as two flavonoid anions (9 and 18), were also identified. The structures of these compounds were determined by spectroscopic methods. Compounds 4, 19, 20, 22-24, 26, and 27 were reported for the first time in Camellia spp. and tea. Furthermore, HPLC analysis method was performed to compare the chemical constituents of the before/after fungal fermentation Fuzhuan brick-teas. Compound 1 was indicated as one of the major characteristic constituents generated in the fungal fermentation process. The IC50 value of the antiproliferative activity of 2 on HeLa cells was assayed as 4.48 μM. None of the isolated compounds showed any inhibition activity against the enteric pathogenic microbes at 800 μg/mL by the hole plate diffusion method.

A New Norisoprenoid and Other Compounds from Fuzhuan Brick Tea

Fuzhuan brick tea, a kind of dark tea consumed mainly in the border regions of Southwestern and Northwestern China since the 1860s, is produced from the leaves of Camellia sinensis var. sinensis by microbial fermentation. From this special fermented tea, a new norisoprenoid, 3R,9R-oxido-5-megastigmene, was isolated, together with α-linolenic acid, strictin, isovitexin, astragalin, (+)-catechin, (−)-epicatechin, (−)-epicatechin gallate, (+)-gallocatechin, (−)-epigallocatechin, (−)-epigallocatechin gallate and gallic acid. The structures of the compounds were identified by spectroscopic means. The new compound didn’t show any inhibition activity against the tested enteric pathogenic microorganisms at a concentration of 800 μg/mL by the hole plate diffusion method.