Presence of free gallic acid and gallate moieties reduces auto-oxidative browning of epicatechin (EC) and epicatechin gallate (ECg).

Auto-oxidation of flavan-3-ols leads to browning and consequently loss of product quality during storage of ready-to-drink (RTD) green tea. The mechanisms and products of auto-oxidation of galloylated catechins, the major flavan-3-ols in green tea, are still largely unknown. Therefore, we investigated auto-oxidation of epicatechin gallate (ECg) in aqueous model systems. Oxidation products tentatively identified based on MS included δ- or γ-type dehydrodicatechins (DhC2s) as the main contributors to browning. Additionally, various colourless products were detected, including epicatechin (EC) and gallic acid (GA) from degalloylation, ether-linked ε-type DhC2s, and 6 new coupling products of ECg and GA possessing a lactone interflavanic linkage. Supported by density function theory (DFT) calculations, we provide a mechanistic explanation on how presence of gallate moieties (D-ring) and GA affect the reaction pathway. Overall, presence of gallate moieties and GA resulted in a different product profile and less intense auto-oxidative browning of ECg compared to EC.

Chemical characterization, antiproliferative and antifungal activities of Clinacanthus nutans.

Clinacanthus nutans Lindau (Family: Acanthaceae) is a medicinal herb widely distributed in the tropic and subtropic areas of Asia. C. nutans is traditionally consumed as vegetable or herbal tea, as well as a folk medicine for anticancer and antifungal activities. However, to date, chemical constituent responsible for observed health beneficial effects of this medicinal plant is not clear. In the current study, 32 compounds (1-32), including three new megastigmanes (1-3) were isolated from the aerial parts of C. nutans. Their structures were elucidated on the basis of comprehensive NMR, MS, and CD spectroscopic data analysis, as well as chemical hydrolysis. Among the isolates, cycloartane triterpenoids (9, 10, and 12) displayed moderate anti-proliferative effects against HepG2 cell growth with IC50 values ranging from 9.12 to 19.89 µM. Data obtained from flow cytometry analysis and western blotting assays revealed that compounds 9 and 12 induced apoptosis of HepG2 cells by modulating the expression of proteins associated to mitochondrial-mediated apoptotic pathway. Furthermore, megastigmanes 1, 2, 7, and 8 enhanced the anti-Candida albicans activity of amphotericin B (AmB), supporting the synergistic effects between megastigmanes and AmB. This is the first report of anticancer and antifungal potential of cycloartane triterpenoids and megastigmanes in C. nutans, which shed useful insights on the relationship between C. nutans's chemical constituent and its beneficial effects to health. Findings from this study support further development of this medicinal plant for potential pharmaceutical applications.

Browning of Epicatechin (EC) and Epigallocatechin (EGC) by Auto-Oxidation.

Green tea catechins are well known for their health benefits. However, these compounds can easily be oxidized, resulting in brown color formation, even in the absence of active oxidative enzymes. Browning of catechin-rich beverages, such as green tea, during their shelf life is undesired. The mechanisms of auto-oxidation of catechins and the brown products formed are still largely unknown. Therefore, we studied auto-oxidative browning of epicatechin (EC) and epigallocatechin (EGC) in model systems. Products of EC and EGC auto-oxidation were analyzed by reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled to mass spectrometry (RP-UHPLC-PDA-MS). In the EC model system, 11 δ-type dehydrodicatechins (DhC2s) and 18 δ-type dehydrotricatechins (DhC3s) that were related to browning could be tentatively identified by their MS2 signature fragments. In the EGC model system, auto-oxidation led to the formation of 13 dihydro-indene-carboxylic acid derivatives and 2 theaflagallins that were related to browning. Based on the products formed, we propose mechanisms for the auto-oxidative browning of EC and EGC. Furthermore, our results indicate that dimers and oligomers that possess a combination of an extended conjugated system, fused rings, and carbonyl groups are responsible for the brown color formation in the absence of oxidative enzymes.

A novel spatial-resolution targeted metabolomics method in a single leaf of the tea plant (Camellia sinensis).

In this study, a novel spatial-resolution targeted metabolomics method was developed for a single leaf on the basis of microscale sample preparation and dansylation derivatization coupled with ultraperformance liquid chromatography-tandem mass spectrometry with a spatial resolution of 1.7 × 1.7 mm2. The practicability of this method was demonstrated by providing absolute quantitative within-leaf distribution information for 56 endogenous metabolites (including 8 catechins, 2 alkaloids, theanine, 4 theaflavins, 14 flavonol/flavone and their glycosides, 21 amino acids, and 6 phenolic acids). An application of this method in a mechanically pierced tea leaf indicated that astringent catechins, quercetin, and quercetin glycosides may be involved in the tea plant response to the wounding. In conclusion, the proposed novel method offered richest information on the within-leaf distribution of metabolites in tea to date and will be greatly helpful in understanding the defensive responses of tea plants against biotic and abiotic stresses.

Nontargeted metabolomics predicts the storage duration of white teas with 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols as marker compounds.

Storage has a dramatic influence on the chemical composition, sensory qualities, biological activity, and therefore the commercial value of white tea. In this study, the metabolites in white teas stored for 1, 3, 7, and ≥ 10 years were comprehensively compared by a nontargeted metabolomics investigation. Most metabolites, including catechins, flavonol/flavone glycosides, amino acids, nucleosides, organic acids, aroma precursors, lipids, and carbohydrates, decreased with increasing storage duration, while 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) and pyroglutamic acid increased. The absolute quantifications of 24 storage-related compounds combined with linear regression analysis showed that a panel of 5 indexes based on EPSFs has a good predictive ability for the storage duration of white teas (correlation coefficients were 0.9294 and 0.8812 in the model and test sets, respectively). The errors between the predicted and the actual storage durations ranged from -1.75 to 1.84 years for the white teas stored for <10 years.

Identification and quantification of key odorants in the world's four most famous black teas.

Keemun, Assam, Darjeeling and Ceylon black teas are honored as the world's four most famous black teas, and their excellent aroma qualities are well received by people around the world. In this study, aroma components in these four types of teas were analyzed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) and gas chromatography-olfactometry (GC-O) technologies. A total of 42 aroma-active compounds were ultimately identified, especially benzeneacetaldehyde, geraniol, (Z)-3-hexen-1-yl hexanoate, trans-ß-ionone, cis-linalool oxide (pyranoid), hotrienol, and methyl salicylate presented the strongest aroma strengths with pleasant scents in all tested teas. The quantification results indicated that 19 compounds including (Z)-3-hexenol, 1-octen-3-ol, linalool, phenylethyl alcohol, hexanal, benzeneacetaldehyde, limonene, heptanoic acid, (Z)-3-hexen-1-ol, acetate, benzyl alcohol, trans-linalool oxide (furanoid), hotrienol, 1-octen-3-one, 2-nonanone, (E)-2-octenal, nonanal, ß-myrcene, 2-pentylfuran, and methylpyrazine were identified as the key compounds with odor activity values (OAVs) higher than 1.0 in the world's four most famous black teas. Notably, the comparison of GC-O and OAV calculation results showed that methyl salicylate (Ceylon), (E)-2-octenal (Assam), benzeneacetaldehyde (Keemun) and linalool and trans-linalool oxide (furanoid) (Darjeeling) might be the most definitive odorants in the corresponding tea categories.

Variation patterns in the content of glycosides during green tea manufacturing by a modification-specific metabolomics approach: Enzymatic reaction promoting an increase in the glycosidically bound volatiles at the pan firing stage.

The glycosides are presumed to influence the quality of green tea but the molecular mechanism behind remains unclear. To elucidate the contribution of glycosides to the flavor formation of green tea, changes of both glycosidically bound non-volatiles (GBNVs) and glycosidically bound volatiles (GBVs) during the manufacturing of green tea were investigated using a modification-specific metabolomics method. A total of 64 glycosides (47 GBNVs and 17 GBVs) were identified and their contents mainly changed during the pan firing and drying stages of green tea manufacturing. Notably, the contents of GBVs significantly increased by 1.12-4.46-fold during pan firing. Correlation analysis showed that the GBVs contents were negatively related to the contents of volatiles and glucose. Model experiments revealed that enzymatic synthesis contributed to the increase in the content of GBVs during the pan firing. This comprehensive study on the glycosides changes revealed the molecular bases for GBVs increments during the pan firing.

Aroma formation and dynamic changes during white tea processing.

The formation of and dynamic changes in aroma during white tea processing have not previously been systematically investigated. In this study, advanced comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry was employed to investigate the mechanism of white tea aroma formation. A total of 172 volatiles were identified and mainly comprising endogenous volatiles, which displayed diverse change trends during the withering period. In this process, free aroma precursor amino acids and glycosidically bound volatiles (GBVs) were found to contribute to the formation of white tea aroma, with the differential expression of aroma-related key genes accounting for various accumulation of endogenous volatiles and GBVs. In addition, the drying was also shown to play an important role in the formation of white tea aroma. Our study provides the first characterization of white tea aroma formation and establishes a theoretical basis for quality control during white tea processing operations.

Metabolomics Investigation Reveals That 8-C N-Ethyl-2-pyrrolidinone-Substituted Flavan-3-ols Are Potential Marker Compounds of Stored White Teas.

White teas of different stored ages have varied flavor, bioactivity, and commercial value. In this study, a liquid chromatography-mass spectrometry-based metabolomics investigation revealed that there are distinct differences among the compound patterns of Baihaoyinzhen (BHYZ) and Baimudan (BMD) white teas with various storage durations. The levels of flavan-3-ols, procyanidins, theasinensins, theaflavins, flavonol- O-glycosides, flavone- C-glycosides, and most of the amino acids were reduced after long-term (>4 years) storage. More importantly, 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs), including seven novel compounds discovered in white teas for the first time, were formed from theanine and flavan-3-ols during storage, and their contents were positively correlated with the storage duration. These findings were further confirmed by the linearly increasing formation of EPSFs in reaction solution and BMD white teas stored in an environment-controlled cabinet. In conclusion, EPSFs were detected in white teas for the first time and were discovered as marker compounds and potential indicators for long-term storage of white tea.

Identification of key odorants responsible for chestnut-like aroma quality of green teas.

A chestnut-like aroma is widely considered an important indicator of an excellent-quality green tea; however, the key odorants responsible for chestnut-like aroma have never been systematically studied and remain unknown. In this study, the aroma components of green teas and Chinese chestnuts were analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOFMS), and 58 compounds were identified as common aroma components among green teas, boiled Chinese chestnuts, roasted Chinese chestnuts and raw Chinese chestnuts. Subsequently, 17 volatiles, including 3-methylbutanal, (E)-3-penten-2-one, ethylbenzene, heptanal, benzaldehyde, 2-pentylfuran, octanal, benzeneacetaldehyde, (E)-2-octenal, (E,E)-3,5-octadien-2-one, linalool, nonanal, (E)-2-nonenal, decanal, (Z)-hex-3-en-1-yl hexanoate, trans-ß-ionone and (E)-nerolidol, were identified as the key odorants responsible for chestnut-like aroma based on the odor activity value (OAV) calculation method. Besides, the comparison of OAVs of key odorants between fresh tea leaves and finished teas indicated that all key odorants were present in fresh tea leaves and that their contents increased or decreased during tea processing. Moreover, the comparison between results of OAV and gas chromatography-olfactometry (GC-O) methods showed that ethylbenzene, heptanal, benzaldehyde, 2-pentylfuran, (E,E)-3,5-octadien-2-one, linalool, (Z)-hex-3-en-1-yl hexanoate and trans-ß-ionone were the common identified compounds between the two methods. The identification of chestnut-like aroma in green teas will provide a theoretical basis for further research on the directional adjustment and control of tea aroma quality.

Application of metabolomics profiling in the analysis of metabolites and taste quality in different subtypes of white tea.

Three subtypes of white tea, Silver Needle (SN), White Peony (WP), and Shou Mei (SM), differ in their taste, aroma, bioactivity, and commercial value. Here, a metabolomics investigation on the chemical compositions combining taste equivalent-quantification and dose-over-threshold (DoT) determination on the taste qualities were applied to comprehensively characterize the white tea subtypes for the first time. Significant differences in the contents of catechins, dimeric catechins, amino acids, phenolic acids, flavonol/flavone glycosides, and aroma precursors were observed among these 3 white teas. Metabolite content comparison and partial least-squares (PLS) analysis suggest that theanine, aspartic acid, asparagine, and AMP were positively correlated with the umami taste in white tea, and flavan-3-ols, theasinensins, procyanidin B3, and theobromine had positive correlations with higher bitterness and astringency tastes. In addition, puckering astringent (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and theogallin, bitter-tasting caffeine, and the mouth-drying/velvety-like astringent γ-aminobutyric acid (GABA) were identified as key taste compounds of white tea infusion by absolute quantification and DoT factor calculations. This work provided systematic and comprehensive knowledge on the chemical components, taste qualities, and sensory active metabolites for the subtypes of white tea.

Metabolomic analysis reveals the composition differences in 13 Chinese tea cultivars of different manufacturing suitabilities.

BACKGROUND: Green tea and black tea are manufactured using appropriate tea cultivars in China. However, the metabolite differences relating to the manufacturing suitability of tea cultivars are unclear. In the present study, we performed a non-targeted metabolomic analysis on 13 Chinese tea cultivars using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry to investigate comprehensively the metabolite differences between cultivars suitable for manufacturing green tea (GT cultivars) and cultivars suitable for manufacturing both green tea and black tea (G&BT cultivars). RESULTS: Multivariate statistical analysis and cluster analysis divided the 13 cultivars into two groups, namely GT cultivars and G&BT cultivars, which correlated with their manufacturing suitability. The GT cultivars contained higher levels of flavonoid glycosides, whereas the G&BT cultivars contained higher levels of catechins, dimeric catechins, phenolic acids and alkaloids. CONCLUSION: Metabolic pathway analysis revealed that the flavonoid pathway inclined toward the synthesis of flavonoid glycosides in GT cultivars, whereas it inclined toward the synthesis of catechins and phenolic acids in G&BT cultivars. The results of the present study will be helpful for discriminating the manufacturing suitability of tea cultivars and investigating their breeding. © 2017 Society of Chemical Industry.

Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted metabolomics approach.

White tea is considered the least processed form of tea and is reported to have a series of potent bioactivities, such as antioxidant, anti-inflammatory, anti-mutagenic, and anti-cancer activities. However, the chemical composition of white tea and the dynamic changes of the metabolites during the manufacturing process are far from clear. In this study, we applied a nontargeted metabolomics approach based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) to comprehensively profile the characteristic metabolites of white tea. There were significant differences in the content of amino acids, catechins, dimeric catechins, flavonol and flavone glycosides, and aroma precursors in white tea compared with green and black teas that were manufactured from the same fresh tea leaves. Furthermore, the dynamic changes of the metabolites in the tea samples with various withering durations of 0, 4, 8, 12, 16, 20, 24, 28, and 36 h were also profiled. This study offers a comprehensive characterization of the metabolites and their changes in white tea.

Enantiomeric and quantitative analysis of volatile terpenoids in different teas (Camellia sinensis).

Volatile terpenoids play important roles in the formation of tea aroma quality due to their pleasant scents and low odor thresholds. Most volatile terpenoids contain stereogenic centers, which results in various stereo distributions of their enantiomers and diastereoisomers in different types of tea. However, the distribution characteristics of terpenoid enantiomers in teas were still unclear, which poses an obstacle to the scientific understanding of tea aroma. In this work, a new and efficient analysis approach based on headspace solid phase microextraction (HS-SPME)-chiral gas chromatography-mass spectrometry (GC-MS) was established to analyze 12 pairs of familiar terpenoid enantiomers in different teas. The extraction efficiency of the HS-SPME method to extract volatile terpenoids in teas was the greatest when using CAR-DVB-PDMS (50/30µm) fibers and 1:10 proportions between tea and boiling water at a 50°C extraction temperature for 40min, and the stability observation of enantiomeric ratios of the terpenoids well proved the feasibility of the extraction method. The favorable limits of detection, limits of quantitation, repeatability, linearity, and concentration ranges of each terpenoid enantiomer demonstrated the repeatability and reliability of the analytical approach. The enantiomeric and quantitative analyses indicated that S-limonene, S-linalool, (2S, 5S)-linalool oxide A, (2S, 5R)-linalool oxide B, R-4-terpineol, (2S, 5R)-linalool oxide C, (2S, 5S)-linalool oxide D, S-α-terpineol, R-α-ionone, peak 1 of theaspirane A and peak 2 of theaspirane B were the major terpenoid components in most Chinese teas; instead, higher proportions of the opposite enantiomers of the above terpenoids were frequently detected in black teas with large leaf origin and Indonesia white teas. Besides, great diversities of enantiomeric ratios and concentrations among different teas were observed. Furthermore, partial least-squares discriminant analyses were performed to distinguish the concentration differences of the terpenoid enantiomers among different teas; the analysis results indicated that highly significant concentration differences existed between large and small leaf origins of black teas, and significant differences of the concentrations of linalool oxides A-C were observed between green, white and dark teas. The successful application of this chiral analysis technique of tea aroma will lay a scientific foundation for further quality assessment, botanical origin determination and authenticity assessment of teas.

Nontargeted Modification-Specific Metabolomics Investigation of Glycosylated Secondary Metabolites in Tea (Camellia sinensis L.) Based on Liquid Chromatography-High-Resolution Mass Spectrometry.

Glycosylation on small molecular metabolites modulates a series of biological events in plants. However, a large number of glycosides have not been discovered and investigated using -omics approaches. Here, a general strategy named "nontargeted modification-specific metabolomics" was applied to map the glycosylation of metabolites. The key aspect of this method is to adopt in-source collision-induced dissociation to dissociate the glycosylated metabolite, causing a characteristic neutral loss pattern, which acts as an indicator for the glycosylation identification. In an exemplary application in green teas, 120 glucosylated/galactosylated, 38 rhamnosylated, 21 rutinosylated, and 23 primeverosylated metabolites were detected simultaneously. Among them, 61 glycosylated metabolites were putatively identified according to current tea metabolite databases. Thanks to the annotations of glycosyl moieties in advance, the method aids metabolite identifications. An additional 40 novel glycosylated metabolites were tentatively elucidated. This work provides a feasible strategy to discover and identify novel glycosylated metabolites in plants.

Nontargeted Analysis Using Ultraperformance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry Uncovers the Effects of Harvest Season on the Metabolites and Taste Quality of Tea (Camellia sinensis L.).

The chemical composition and taste quality of tea fluctuate seasonally. However, the compounds responsible for the seasonal variation of metabolic pattern and taste quality are far from clear. This study compared the metabolite profiles of green teas of nine varieties that were plucked in spring, summer, and autumn by using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) on a reversed phase column. A multivariate analysis indicated distinct differences among the metabolite phenotypes of teas harvested in different seasons. Heat-map analysis and metabolic pathway analysis demonstrated that flavan-3-ols, theasinensins, procyanidins, quercetin-O-glycosides, apigenin-C-glycosides, and amino acids exhibited sharp seasonal fluctuations. An equivalent quantification of tea tastes showed that in summer and autumn teas, the bitterness and astringency were significantly elevated, whereas umami declined. Metabolite content comparisons and partial least-squares analysis suggested that several flavonoids and amino acids are mainly responsible for the seasonal variations in taste quality.

Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity.

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) present in leaves of Camellia sinensis has many beneficial biological activities for human health. However, EGCG3"Me occurs naturally in tea leaves in extremely limited quantities. Finding an enzyme from C. sinensis to catalyze the synthesis of EGCG3"Me is an alternative method to make up for the scarcity of EGCG3"Me in natural situations. In the present study, a complementary DNA (cDNA) encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) gene were isolated from C. sinensis (designated CsCCoAOMT). Nucleotide sequence analysis of CsCCoAOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The full-length DNA sequence (2678 bp) contained five exons and four introns. The deduced amino acid sequence of CsCCoAOMT shared 92% identity with CCoAOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of CsCCoAOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate (EGCG) compounds (EGCG4"Me, EGCG3"Me, and EGCG3'Me) were produced by CsCCoAOMT with K(m) in the micromolar range. Real-time polymerase chain reaction (RT-PCR) experiments indicated that the CsCCoAOMT transcript was present at low levels during the early stages of leaf maturity (the first leaf and bud on a shoot) but the relative expression was augmented at advanced stages of leaf maturity (the third or fourth leaf on a shoot), which accorded well with changes in EGCG3"Me content in fresh leaves. Hence, we concluded that CsCCoAOMT catalyzes the syntheses of methylated EGCGs.