Novel Oxidation Oligomer of Chlorogenic Acid and (-)-Epigallocatechin and Its Quantitative Analysis during the Processing of Keemun Black Tea.

Not only do flavan-3-ols participate in the formation of chromogenic oxidation products such as theaflavins, but chlorogenic acid (3-caffeoylquinic acid, CQA) is also involved in the enzymatic oxidation during black tea processing. The critical oxidation product of CQA and (-)-epigallocatechin (EGC) were identified as an adduct containing benzobicyclo[3.2.2]nonenone structure, which was named as the dichlorogeniccatechin (DCGC) oligomer. It was composed of two molecules of CQA and one molecule of EGC. The effects of the initial reactant ratio and reaction time on the generation of DCGC were also analyzed. A high proportion of CQA promoted the production of DCGC, but a high proportion of EGC inhibited the DCGC formation. In addition, the content of DCGC in Keemun black tea during processing was determined. The content of DCGC highly increased after withering but decreased after drying. This study provides a new perspective for the investigation of other oxidation oligomers in black tea.

Formation Mechanism of Di-N-ethyl-2-pyrrolidinone-Substituted Epigallocatechin Gallate during High-Temperature Roasting of Tea.

Four di-N-ethyl-2-pyrrolidinone-substituted epigallocatechin gallate (EGCG) and two di-N-ethyl-2-pyrrolidinone-substituted gallocatechin gallate (GCG) flavan-3-ols (di-EPSFs) were prepared by the thermal simulation reaction. The effects of reaction temperature and time, initial reactant ratios, and pH values on the content of di-EPSFs were studied. The formation of six di-EPSFs was most favored when the initial reactant ratio of EGCG and theanine was 1:2 and heated under 130 °C at pH 10 for 120 min. The contents of di-EPSF1, di-EPSF2, and di-EPSF5 in large-leaf yellow tea (LYT) increased with the increase of roasting degree. Through quantitative analysis, it was found that EGCG would interact with the Strecker degradation products of theanine to form EPSFs, which further combined with the Strecker degradation products of theanine to form di-EPSFs. This study further improved the understanding of the transformation pathways of EGCG and theanine during tea processing and contributed to exploring the flavor characteristics and health benefits of di-EPSFs.

Widely targeted metabolomics using UPLC-QTRAP-MS/MS reveals chemical changes during the processing of black tea from the cultivar Camellia sinensis (L.) O. Kuntze cv. Huangjinya.

Huangjinya is a light-sensitive mutant tea cultivar that produces fresh leaves with a yellow phenotype, and the leaves also be used to produce black tea with special sensory characteristics. To thoroughly explore the chemical changes that occur during the processing of Huangjinya black tea, tea samples were collected from each processing step to perform quantitative and qualitative analyses by high-performance liquid chromatography and ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Compared to fresh tea leaves, only approximately 20% of the catechins remained at the end of processing, while theaflavins levels peaked at the rolling step and were slightly reduced in the fermentation and drying processes. The levels of amino acids derived from protein hydrolysis increased significantly in the withering and rolling processes. Altogether, 620 differential metabolites were identified from 11 subclasses using widely targeted metabolomics based on UPLC-HRMS for the four steps used to process Huangjinya black tea. Flavonoids, phenolic acids, and lipids were the three major classes of differential metabolites, accounting for 52.4% of the differential compounds. The greatest changes in the metabolite profile occurred during the rolling step, with 292 metabolites showing increases or decreases. Two glycoconjugates of the amino acid were first identified in tea, which was sharply increased in the drying stage. The present study provides comprehensive information on the chemical changes during the processing of Huangjinya black tea, and this information is valuable for optimizing manufacturing process and utilization of the Huangjinya tea plant.

Amadori Reaction Products of Theanine and Glucose: Formation, Structure, and Analysis in Tea.

Amadori rearrangement products (ARPs) derived from the Maillard reaction between theanine and glucose (ARP 1), as well as pyroglutamic acid and glucose (ARP 2), were identified by liquid chromatograph tandem mass spectroscopy methods. The effects of initial reactant ratio, temperature, pH, and heating time on ARP generation were analyzed. The formation of both ARPs was most favored under 100 °C, while an alkaline environment slightly promoted the generation of ARP 1 and acidic conditions contributed more to ARP 2 formation. The decomposition of ARP 1 was suggested to be the predominant formation mechanism of ARP 2. Preparation, purification, and structure identification of ARP 1 were conducted, with its structure confirmed as 1-deoxy-1-l-theanino-d-fructose. The contents of ARP 1 in green, black, dark, white, yellow, and Oolong teas were quantitatively determined, of which black teas contained the highest levels of ARP 1, possibly due to the high glucose content and processing techniques.

Comparison of the chemical composition and antioxidant, anti-inflammatory, α-amylase and α-glycosidase inhibitory activities of the supernatant and cream from black tea infusion.

Tea cream is a kind of turbid substance commonly existing in tea infusion and tea beverage upon cooling. Herein, a comparative study was conducted on the supernatant and cream from black tea infusion in terms of antioxidant, anti-inflammatory and enzyme inhibitory activities, and chemical composition. Ultraviolet-visible (UV-vis) spectrometry and high-performance liquid chromatography (HPLC) analysis showed that the contents of protein, polyphenols, theaflavins, thearubigins, theabrownins, and caffeine in cream were significantly higher than those in the supernatant. The contents of Al, Ca, Cu, and Fe elements in cream were higher than those in the supernatant. However, higher levels of monosaccharides and free amino acids were detected in the supernatant compared with cream. The ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) based metabolomics analysis revealed that the main marker compounds between the supernatant and the cream were organic acids, phenolic acids, and flavan-3-ols and their oxidation products, flavonol glycosides and amino acids. The cream showed better antioxidant and anti-inflammatory, as well as α-amylase and α-glycosidase inhibitory activities than the supernatant, because it contained higher contents of polyphenols than the supernatant. The present study expanded the new vision towards the cream of black tea infusion.

The Oxidation Mechanism of Flavan-3-ols by an Enzymatic Reaction Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics Combined with Captured o-Quinone Intermediates of Flavan-3-ols by o-Phenylenediamine.

During the enzymatic oxidation of black tea, flavan-3-ols undergo a complicated chemical transformation and generate theaflavins and thearubigins. So far, the oxidation mechanism of flavan-3-ols has not been clarified. Liquid chromatography-tandem mass spectrometry-based metabolomics combined with o-quinone intermediates captured by o-phenylenediamine was developed and successfully applied in the liquid incubation of fresh tea homogenates. During the oxidation, the contents of catechins continuously decreased, while theaflavins increased first but decreased subsequently at the end of incubation. Meanwhile, the content of thearubigins greatly increased at the late stage of incubation. Dehydrotheasinensins were accumulated at the end of oxidation along with the decrease of theasinensins. Through o-phenylenediamine derivation, several adducts of (-)-epigallocatechin gallate, (-)-epigallocatechin, theasinensins A, B, C, and D, and corresponding dehydrotheasinensins were identified, which were considered as the substrates of thearubigins. These results suggested that theaflavins and these oxidation products contributed to the formation of thearubigins.

Chemical Variation of Chenpi (Citrus Peels) and Corresponding Correlated Bioactive Compounds by LC-MS Metabolomics and Multibioassay Analysis.

The peel of Citrus reticulata "Chachi" (CP) possesses various health-promoting benefits and is not only one of the most famous Chinese herbal medicine, but also an ingredient in fermented foods. In the present study, the effects of storage years (1-, 3-, 4-, 5-, 6-, and 11-years) on the chemical profiling and potential bioactive compounds of CP were compared by metabolomics and in vitro bioactivity analysis. With the increase of storage time, the content of hesperidin significantly decreased, but nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin were increased. Meanwhile, the antioxidant activity of CP was enhanced. Phenolic acids, flavonol glycosides, fatty acids, and alkyl glycosides were marker compounds that were responsible for distinguishing the storage time of CP. Correlation analysis suggested that some polyphenols including quercetin-glucoside, quinic acid, trihydroxydimethoxyflavone, and rutin were potential antioxidant compounds in CP. The dichloromethane and n-butanol fractions showed the better antioxidant capacity and inhibitory effects on glucose-hydrolysis enzymes. They mainly contained ferulic acid, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, kaempferol, and hesperidin.

Study on In Vitro Preparation and Taste Properties of N-Ethyl-2-Pyrrolidinone-Substituted Flavan-3-Ols.

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) were prepared by an in vitro model reaction, and the taste thresholds of EPSFs and their dose-over-threshold factors in large-leaf yellow tea (LYT) were investigated. The effects of initial reactant ratios, reaction temperatures and time, pH values, and water addition on the yield of EPSFs were explored. The contents of EPSFs during roasting were determined by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). When the initial ratio of (-)-epigallocatechin gallate (EGCG) to theanine was 1:2 and roasted under 120 °C for 120 min, the contents of EPSFs were the highest. The bitterness and astringency thresholds of four EPSF isomers were measured by the half-tongue method, of which EPSF2 and EPSF3 had higher thresholds than EGCG. In LYT, four EPSFs had lower bitterness and astringency dose-over-threshold factors than EGCG. This study suggested that the reduction of bitterness and astringency of tea after roasting may be mainly due to the formation of EPSFs.

Model Studies on the Reaction Products Formed at Roasting Temperatures from either Catechin or Tea Powder in the Presence of Glucose.

During tea processing, roasting significantly affects the transformation pathway of catechins. When (-)-epigallocatechin gallate (EGCG) and glucose were roasted at different pH values, the degree of degradation and isomerization of EGCG was the lowest at pH 7 and the highest at pH 8. Thirty-five products were found in the model reaction of EGCG and glucose under high temperatures, of which four EGCG-glucose adducts were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition, catechins, gallic acid, and theanine in tea with added glucose were significantly reduced during roasting. The contents of four EGCG-glucose adducts were increased significantly at 150 °C after 30 min and dropped gradually after 60 min. Therefore, based on the present study, EGCG could form crosslinks with glucose under high temperatures in a short time, which provides insight for tea processing and synthesis of catechin-sugar adducts.

Targeted and nontargeted metabolomics analysis for determining the effect of storage time on the metabolites and taste quality of keemun black tea.

The black tea could be stored for a long time, and subsequently affects the flavor characteristics. In the present study, the effects of storage years (1, 2, 3, 4, 5, 10, 17 and 20 years) on the chemical profiling and taste quality of keemun black tea (KBT) were compared by metabolomics and quantitative sensory evaluation. The main polyphenols were degraded during the storing, especially 10-year storage, but caffeine and theobromine were stable. The intensity of bitterness, astringency, umami was negatively correlated to storage years, with correlation coefficient at -0.95, -0.91 and -0.83 respectively, whereas sweetness had positive correlation coefficient at 0.74. Quinic acid, galloylated catechins, linolenic acid, linoleic acid, malic acid, palamitic acid, and theaflavin-3́-gallate were marker compounds which were responsible for distinguishing short and long time preserved KBT. The contents of fatty acids were positively correlated to storage time and sweet intensity.

Alteration of local and systemic amino acids metabolism for the inducible defense in tea plant (Camellia sinensis) in response to leaf herbivory by Ectropis oblique.

Leaf herbivory on tea plants (Camellia sinensis) by tea geometrids (Ectropis oblique) can cause severe yield loss and quality damage for tea. In previous work, we discovered that leaf herbivory triggered systemic carbon depletion in undamaged roots to enhance resource investment for local defense induced in damaged leaves. Here, we investigated the dynamics of amino acids in the local and systemic responses and the roles of nitrogen resource reallocation for the inducible defense in tea plants in response to leaf herbivory. The comparative analysis of the dynamics of flavonoids, caffeine, theanine and basic amino acids at metabolic and transcriptome levels revealed that leaf herbivory triggered the differential reconfiguration of these amino acid-derived defensive metabolites and nitrogenous primary metabolism between the local and systemic responses. The tight association of the metabolism and reallocation of amino acids with the activation of defensive secondary metabolism indicated that the systemic nitrogen reallocation played a potentially important role for the resource investment in tea plant resistance against leaf herbivory. This study provided an extended understanding of the role of systemic nitrogen reallocation for the interaction of tea plants and geometrids and the root-mediated resource-based resistance strategy employed by tea plants in response to leaf herbivory.