Isolation of N-Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography.

N-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5'''R- and 5'''S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-EGCG-cThea and S-EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) and 2. N-hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v/v/v/v) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio KD values, selectivity factors α, and resolution factors RS. After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R-EGCG-cThea and 2.9 mg of S-EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R- and S-EGCG-cThea possessed in vitro inhibition effects on α-glucosidase with IC50 of 70.3 and 161.7 µM, respectively.

Investigations of the highly efficient processing technique, chemical constituents, and anti-inflammatory effect of N-ethyl-2-pyrrolidinone-substituted flavan-3-ol (EPSF)-enriched white tea.

N-Ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) are a newly discovered compound class in tea with various bioactivities. This study aimed to develop a novel processing technique to enhance EPSF contents in white tea efficiently. Using optimal processing parameters of 125 °C and 30 min in a high-temperature sterilizing oven, total EPSF content significantly increased by 1.42-18.80-fold to 1.57-6.22 mg/g without impacting sensory characteristics. Metabolomics analysis revealed elevated levels of nucleosides, nucleotides, bases, theaflavins, flavonol aglycones, EPSFs, and most flavone-C-glycosides, as well as decreased levels of amino acids, procyanidins, theasinensins, several flavanols, and flavonol-O-glycosides after EPSF-enrichment treatment. Furthermore, the EPSF-enriched white tea exhibited notable anti-inflammatory effects, mitigating xylene-induced ear edema in mice and carrageenan-induced paw edema and cotton ball-induced granulomas in rats. This study developed a new processing technique for highly efficient enhancement of EPSFs in white tea and demonstrated that EPSF-enriched white tea has a potential to serve as effective anti-inflammatory dietary supplement.

High-throughput screening and investigation of the inhibitory mechanism of α-glucosidase inhibitors in teas using an affinity selection-mass spectrometry method.

An affinity selection-mass spectrometry method was applied for high-throughput screening of α-glucosidase (AGH) inhibitors from teas. Fourteen out of nineteen screened AGH inhibitor candidates were clustered as galloylated polyphenols (GPs). "AGH-GPs" interaction studies, including enzyme kinetics, fluorescence spectroscopy, circular dichroism, and molecular docking, jointly suggested that GPs noncompetitively inhibit AGH activity by interacting with amino acid residues near the active site of AGH and inducing changes in AGH secondary structure. Representative GPs and white tea extract (WTE) showed comparable AGH inhibition effects in Caco2 cells and postprandial hypoglycemic efficacy in diabetic mice as acarbose. The area under the curve of oral sucrose tolerance test was lower by 8.16%, 6.17%, and 7.37% than control group in 15 mg/kg EGCG, 15 mg/kg strictinin, and 150 mg/kg WTE group, respectively. Our study presents a high-efficiency approach to discover novel AGH inhibitors and elucidates a potential mechanism by which tea decreases diabetes risks.

Metabolomics combined with proteomics provides a novel interpretation of the compound differences among Chinese tea cultivars (Camellia sinensis var. sinensis) with different manufacturing suitabilities.

Different tea cultivars differ in their manufacturing suitability. In this study, metabolomics and proteomics were applied to investigate the metabolite and protein differences in fresh leaves from 23 Chinese tea cultivars suitable for manufacturing green, white, oolong, and black teas. The combined analysis revealed 115 differential metabolites and significant differences in the biosynthesis pathways for amino acids, phenylpropanoids, flavonoids, and terpenoids, and in the peroxidases abundances among these four groups. Green tea cultivars had higher abundances of amino acids and amino acids biosynthesis-related enzymes but lower abundances of flavanols and flavonoids biosynthesis-related enzymes. Black tea cultivars presented higher abundances of flavanols, flavanol-O-glycosides, flavonoids biosynthesis-related enzymes, and peroxidases. Oolong tea cultivars showed higher abundances of enzymes involved in terpenoids biosynthesis. Our study provides a novel interpretation of the manufacturing suitability of tea cultivars from the perspective of both metabolites and proteins and will be helpful for cultivar breeding.

Metabolomic characterization of the chemical compositions of Dracocephalum rupestre Hance.

Maojian tea (MJT) is a traditional Chinese herbal tea beverage manufactured from the leaves of the Dracocephalum rupestre Hance plant. In this study, a nontargeted metabolomics approach combined with absolute quantifications was applied to comprehensively investigate the chemical compositions of MJT and to determine the effects of the processing methods on compounds. Flavones (apigenin and luteolin, 0.06-1.35 mg/g), flavanones (eriodictyol and naringenin, 0.1-2.3 mg/g), flavone 7-O-glycosides (0.15-5.98 mg/g), flavanone 7-O-glycosides (0.28-19.41 mg/g), and triterpenoids were presumed to be characteristic components of MJT. Applying imitative green and black tea processing methods to MJT led to increases in flavone/flavanone aglycones, lipids, and triterpenoids and decreases in flavone/flavanone glycosides, amino acids, organic acids, and most phenolic acids. This study offers novel insights into the chemical compositions and the influences of processing methods on MJT and will be utilized for the quality control of MJT.

Nontargeted and targeted metabolomics analysis provides novel insight into nonvolatile metabolites in Jianghua Kucha tea germplasm (Camellia sinensis var. Assamica cv. Jianghua).

Jianghua Kucha (JHKC) is a special tea germplasm with high bitterness growing in China; however, the chemical characteristics of JHKC are not completely understood. In this study, 61 differential metabolites were identified between 11 wild JHKC individuals and 3 control cultivars of Fudingdabai, Yunkang 10, and Zhuyeqi using comprehensive nontargeted and targeted metabolomics approach. The JHKC accessions mainly possessed significantly higher levels of purine alkaloids of theacrine (12.06 ± 5.23 mg/g) and 1,3,7-trimethyluric acid, non-epi-form flavanols (catechin, gallocatechin, catechin gallate, and gallocatechin gallate), and methylated flavanols of epigallocatechin-3-O-(3″-O-methyl)-gallate (4.79 ± 1.45 mg/g) and epicatechin-3-O-(3″-O-methyl)-gallate (1.02 ± 0.34 mg/g), as well as significantly lower levels of flavonol glycosides, which indicated that caffeine metabolism, flavonoid biosynthesis, and flavonol and flavone biosynthesis are mostly differential metabolic pathways. Our study demonstrated that JHKC germplasm is a promising resource for breeding novel tea cultivars with high contents of theacrine, non-epi-form flavanols, and methylated flavanols.

New insights into the influences of baking and storage on the nonvolatile compounds in oolong tea: A nontargeted and targeted metabolomics study.

A nontargeted and targeted metabolomics method was applied to comprehensively investigate the influences of baking and storage on chemical constituents in fresh-, strong-, and aged-scent types of Foshou oolong teas. The contents of N-ethyl-2-pyrrolidone-substituted flavanols (EPSFs), flavone C-glycosides, gallic acid, and most lipids increased after baking and storage, while the contents of cis-flavanols, alkaloids, flavonol O-glycosides, and most amino acids decreased. Degradation, epimerization, and interaction with theanine were main pathways for the decrease in cis-flavanols. Approximately 20.7%, 12.8%, and 11.6% of epigallocatechin gallate were degraded, epimerized, and interacted with theanine after baking, respectively; 22.5% and 8.71% of epigallocatechin gallate were degraded and interacted with theanine after 10-year storage, respectively. Simulated reactions confirmed that the increases in EPSFs and apigenin C-glycosides were caused by interactions between theanine and flavanols and between apigenin aglycone and glucose, respectively. This study offers novel insights into chemical changes during baking and storage of oolong tea.

Metabolomics Analysis Reveals Four Novel N-Ethyl-2-pyrrolidinone-Substituted Theaflavins as Storage-Related Marker Compounds in Black Tea.

Tea market is currently oversupplied, and unsold tea often needs to be properly stored for a period of time. However, the chemical changes occurring in black tea during storage are limitedly understood. In this study, a comprehensive nontargeted and targeted metabolomics approach was used to investigate the dynamic changes in compounds in time-series (0-19 months)-stored black teas. The contents of flavanols, theaflavins (TFs), theasinensins, procyanidins, most phenolic acids, amino acids, quercetin-O-glycosides, and myricetin-O-glycosides decreased during storage, while the contents of N-ethyl-2-pyrrolidinone-substituted flavanols, flavone-C-glycosides, and most kaempferol-O-glycosides increased. More importantly, four novel compounds strongly positively correlated with storage duration (r = 0.922-0.969) were structurally assigned as N-ethyl-2-pyrrolidinone-substituted TFs and validated with synthetic reactions of TFs and theanine standards. The content of N-ethyl-2-pyrrolidinone-substituted TFs was 51.54 µg/g in black tea stored for 19 months. To the best of our knowledge, N-ethyl-2-pyrrolidinone-substituted TFs were discovered in tea for the first time.

N-Ethyl-2-Pyrrolidinone-Substituted Flavan-3-Ols with Anti-inflammatory Activity in Lipopolysaccharide-Stimulated Macrophages Are Storage-Related Marker Compounds for Green Tea.

Fresh green tea (GT) is commonly considered to have better sensory flavor and higher commercial value than long-term-stored GT; however, the chemical variations during storage are unclear. In this study, the chemical profiles of stored GT were surveyed among time-series samples from 0 to 19 months using a nontargeted metabolomics method. Seven N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) increased from 0.022 ± 0.019 to 3.212 ± 0.057 mg/g within 19 months (correlation coefficients with storage duration ranging from 0.936 to 0.965), and they were the most significantly increased compounds among the 127 identified compounds. Two representative EPSFs (R-EGCG-cThea and S-EGCG-cThea) possess potential anti-inflammatory properties by suppressing the expression, phosphorylation, and nuclear translocation of nuclear factor kappa-B (NF-κB) p65 in lipopolysaccharide-stimulated macrophages based on western blotting and immunofluorescence results. In conclusion, EPSFs were found to be marker compounds for stored GT and showed potential anti-inflammatory activity by regulating the NF-κB signaling pathway.