Revealing the variances in color formation and bioactivities of seven catechin monomers throughout the enzymatic reaction by colorimetric and mass spectrometry.

The capacity differences of seven catechin monomers to produce colors after treating with catechin-free extract were investigated. After 240-min reaction, only (-)-epicatechin (EC) and (+)-catechin (C) presented obvious luminous red color with L* values of 63.32-71.73, a* values of 37.13-46.44, and b* values of 65.64-69.99. Meanwhile, the decrease rate of EC and C was 43.52 %-50.35 %, which were significantly lower than those of other catechin monomers (85.91 %-100 %). The oxidized products of catechin monomers were analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry coupled with diode array detector, wherein dehydro-dimers and -trimers (oxidative coupling products of catechins' A-B ring) were found to be the major chromogenic compounds of EC and C. Additionally, the antioxidant capacity of catechin monomers only decreased after 30-min reaction, while along with further enzymatic reaction, catechin monomers presented comparable oxyradical scavenging ability (e.g., the DPPH inhibitory rates of catechin monomers were in the range of 24.42 %-50.77 %) to vitamin C (positive control, DPPH inhibitory rate was 27.66 %). Meanwhile, the inhibitory effects of most catechin monomers on α-glucosidase were enhanced in different degrees. These results provided basis for the development of enzymatically-oxidized catechin monomers as functional food color additives.

The effects of tea plant age on the color, taste, and chemical characteristics of Yunnan Congou black tea by multi-spectral omics insight.

The present study comprehensively used integrated multi-spectral omics combined with sensory evaluation analysis to investigate the quality of three types of Yunnan Congou black teas from different tree ages (decades, DB; hundreds, HB; a thousand years, TB). TB infusion presented the highest scores of sweetness and umami, higher brightness, and yellow hue. Eighty-four marker metabolites were identified, including Amadori rearrangement products, catechin oxidation products, flavonoid glycosides, and organic acids, which are simultaneously related to tea infusions' color and taste. Moreover, the content of some characteristic flavonoid glycosides and organic acids was determined. Our finding implied trans-4-O-p-coumaroylquinic acid and quercetin 3-O-rutinoside contributed to bitterness and astringency, while dehydro theanine-glucose Amadori product and xylopyranosyl-glucopyranose resulted in umami and sweetness. These results provided quantitative and qualitative information for deciphering differences among black teas with different tea plant ages, conducing to the further utilization of ancient tea plants in Southwest China.

Chemical, sensory and biological variations of black tea under different drying temperatures.

As the final processing step, drying temperature between 90 and 140 â„ƒ is usually applied to terminate enzymatic activities and improve sensory characteristics of black tea. Liquid chromatography tandem mass spectrometry (LC-MS) based non-targeted and targeted metabolomics analyses combined in vitro biological assays were adopted to investigate the chemical and biological variations after drying. Fifty-nine differentially expressed metabolites including several hydroxycinnamic acid derivatives and pyroglutamic acid-glucose Amadori rearrangement products (ARPs) were identified, the latter of which was correspondingly accumulated with increasing temperature. The levels of theaflavins (TFs), thearubigins (TRs), monosaccharides and free amino acids gradually decreased with increasing temperature. Furthermore, the bioassays of black tea showed that drying under 110 â„ƒ provided the highest antioxidant capacities, but the inhibitory effects on α-glucosidase and α-amylase were decreasing along with increasing drying temperature. These results are valuable for optimizing drying process to obtain superior sensory properties and preserve bioactivities of black tea.

Novel Flavonol Alkaloids in Green Tea: Synthesis, Detection, and Anti-Alzheimer's Disease Effect in a Transgenic Caenorhabditis elegans CL4176 Model.

Novel N-ethy-2-pyrrolidinone-substituted flavonols, myricetin alkaloids A-C (1-3), quercetin alkaloids A-C (4a, 4b, and 5), and kaempferol alkaloids A and B (6 and 7), were prepared from thermal reaction products of myricetin, quercetin, kaempferol─l-theanine, respectively. We used HPLC-ESI-HRMS/MS to detect 1-7 in 14 cultivars of green tea and found that they were all present in "Shuchazao," "Longjing 43", "Fudingdabai", and "Zhongcha 108" green teas. The structures of 1-4 and 6 were determined by extensive 1D and 2D NMR spectroscopies. These flavonol alkaloids along with their skeletal flavonols were assessed for anti-Alzheimer's disease effect based on molecular docking, acetylcholinesterase inhibition, and the transgenic Caenorhabditis elegans CL4176 model. Compound 7 strongly binds to the protein amyloid ß (Aß1-42) through hydrogen bonds (BE: -9.5 kcal/mol, Ki: 114.3 nM). Compound 3 (100 µM) is the strongest one in significantly extending the mean lifespan (13.4 ± 0.5 d, 43.0% promotion), delaying the Aß1-42-induced paralysis (PT50: 40.7 ± 1.9 h, 17.1% promotion), enhancing the locomotion (140.0% promotion at 48 h), and alleviating glutamic acid (Glu)-induced neurotoxicity (153.5% promotion at 48 h) of CL4176 worms (p < 0.0001).

Formation of Furan from Linoleic Acid Thermal Oxidation: (E,E)-2,4-Decadienal as a Critical Intermediate Product.

The linoleic acid reaction models were set at 150 °C for 120 min, and its oxidation process was monitored by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Results showed that no furan was formed from linoleic acid without heating, while furan accumulated throughout the heating process. Linoleic acid ran out within 30 min, which indicated that furan was formed mainly from the intermediate oxidation products of linoleic acid after 30 min. It should be noticed that the content of (E,E)-2,4-decadienal reached maximum once the linoleic acid ran out and then decreased with the formation of furan. Multivariate statistical analysis suggested that (E,E)-2,4-decadienal was the most important aldehyde related to furan formation during linoleic acid oxidation. To prove this assumption, the variation of furan from (E,E)-2,4-decadienal reaction models heating at 150 °C for 60 min was also studied. Results showed that the content of furan increased with the oxidation of (E,E)-2,4-decadienal. Furthermore, NMR and GC-MS data proved that (E,E)-2,4-decadienal could be oxidized to 4,5-epoxy-(E)-2-decenal. In conclusion, our results supported (E,E)-2,4-decadienal and trans-4,5-epoxy-(E)-2-decenal as critical intermediate products of furan formation from linoleic acid oxidation.

Novel Camellia sinensis O-Methyltransferase Regulated by CsMADSL1 Specifically Methylates EGCG in Cultivar "GZMe4".

Epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4″Me) in Camellia sinensis possesses numerous beneficial biological activities. However, the germplasm rich in EGCG4″Me and the O-methyltransferase responsible for EGCG4″Me biosynthesis are poorly understood. Herein, the content of EGCG3″Me and EGCG4″Me in the shoots of 13 cultivars was analyzed to demonstrate that EGCG4″Me is characteristically accumulated in the "GZMe4" cultivar but not in the other 12 cultivars. A novel O-methyltransferase (CsOMTL1) was identified from "GZMe4" using RNA-Seq and correlation analysis. Using the recombinant enzyme, EGCG4″Me was synthesized in vitro. Overexpression of CsOMTL1 via Agrobacterium-mediated genetic transformation caused constitutive accumulation of EGCG4″Me in C. sinensis callus. Moreover, the transcription factor CsMADSL1 localized in the nucleus activated the transcription of CsOMTL1 and specifically interacted with its promoter. Hence, our study identified a novel O-methyltransferase that characteristically catalyzes the synthesis of EGCG4″Me and a positive regulator of EGCG4″Me synthesis in "GZMe4", which might provide a strategy for the breeding of a tea cultivar rich in EGCG4″Me.

Novel methylated flavoalkaloids from Echa 1 green tea inhibit fat accumulation and enhance stress resistance in Caenorhabditis elegans.

Methylation is a common structural modification of catechins in tea, which can improve the bioavailability of catechins. Flavoalkaloids are catechin derivatives with a nitrogen containing five-membered ring at the C-6 or C-8 position. Here we isolated three new methylated flavoalkaloids from Echa 1 green tea (Camellia sinensis cv. Echa 1) and synthesized another four new methylated flavoalkaloids. The structures of the new ester-type methylated catechins (etmc)-pyrrolidinone A-G (1-7) were elucidated by various spectroscopic techniques, including nuclear magnetic resonance (NMR), optical rotation, infrared, UV-vis, experimental and calculated circular dichroism (CD) spectra, and high-resolution mass. Among them, 6 and 7 showed the strongest α-glucosidase inhibitory activity and significantly lowered lipid content of Caenorhabditis elegans with 73.50 and 67.39% inhibition rate, respectively. Meanwhile, 6 and 7 also exhibited strong antioxidant activity in vitro and stress resistance to heat, oxidative stress, and UV irradiation in nematodes.

Two new catechins from Zijuan green tea enhance the fitness and lifespan of Caenorhabditis elegans via insulin-like signaling pathways.

Green tea polyphenols show positive effects on human health and longevity. However, knowledge of the antiaging properties of green tea is limited to the major catechin epigallocatechin gallate (EGCG). The search for new ingredients in tea with strong antiaging activity deserves further study. Here we isolated and identified two new catechins from Zijuan green tea, named zijuanin E (1) and zijuanin F (2). Their structures were identified by extensive high-resolution mass spectroscopy (HR-MS), nuclear magnetic resonance (NMR), ultraviolet-vis (UV), infrared (IR) and circular dichroism (CD) spectroscopic analyses, and their 13C NMR and CD data were calculated. We used the nematode Caenorhabditis elegans (C. elegans) to analyze the health benefits and longevity effects of 1 and 2. Compounds 1 and 2 (100 µM) remarkably prolonged the lifespan of C. elegans by 67.2% and 56.0%, respectively, delaying the age-related decline of phenotypes, enhancing stress resistance, and reducing ROS and lipid accumulation. Furthermore, 1 and 2 did not affect the lifespan of daf-16, daf-2, sir-2.1, and skn-1 mutant worms, suggesting that they might work via the insulin/IGF and SKN-1/Nrf2 signaling pathways. Meanwhile, 1 and 2 also exhibited strong antioxidant activity in vitro. Surface plasmon resonance (SPR) evidence suggests that zijuanins E and F have strong human serum albumin (HSA) binding ability. Together, zijuanins E and F represent a new valuable class of tea components that promote healthspan and could be developed as potential dietary therapies against aging.

α-Glucosidase Inhibitory Activities and the Interaction Mechanism of Novel Spiro-Flavoalkaloids from YingDe Green Tea.

Flavoalkaloids are a unique class of compounds in tea, most of which have an N-ethyl-2-pyrrolidinone moiety substituted at the A ring of a catechin skeleton. 1-Ethyl-5-hydroxy-pyrrolidone, a decomposed product of theanine, was supposed to be the key intermediate to form tea flavoalkaloids. However, we have also detected another possible theanine intermediate, 1-ethyl-5-oxopyrrolidine-2-carboxylic acid, and speculated if there are related conjugated catechins. Herein, four novel spiro-flavoalkaloids with a spiro-γ-lactone structural moiety were isolated from Yingde green tea (Camellia sinensis var. assamica) in our continuing exploration of new chemical constituents from tea. The structures of the new compounds, spiro-flavoalkaloids A-D (1-4), were further elucidated by extensive nuclear magnetic resonance (NMR) spectroscopy together with the calculated 13C NMR, IR, UV-vis, high-resolution mass, optical rotation, experimental, and calculated circular dichroism spectra. We also provided an alternative pathway to produce these novel spiro-flavoalkaloids. Additionally, their α-glucosidase inhibitory activities were determined with IC50 values of 3.34 (1), 5.47 (2), 22.50 (3), and 15.38 (4) µM. Docking results revealed that compounds 1 and 2 mainly interacted with residues ASP-215, ARG-442, ASP-352, GLU-411, HIS-280, ARG-315, and ASN-415 of α-glucosidase through hydrogen bonds. The fluorescence intensity of α-glucosidase could be quenched by compounds 1 and 2 in a static style.

Discovery of Camellia sinensis catechins as SARS-CoV-2 3CL protease inhibitors through molecular docking, intra and extra cellular assays.

BACKGROUND AND PURPOSE: Previous studies suggest that major Camellia sinensis (tea) catechins can inhibit 3-chymotrypsin-like cysteine protease (3CLpro), inspiring us to study 3CLpro inhibition of the recently discovered catechins from tea by our group. METHODS: Autodock was used to dock 3CLpro and 16 tea catechins. Further, a 3CLpro activity detection system was used to test their intra and extra cellular 3CLpro inhibitory activity. Surface plasmon resonance (SPR) was used to analyze the dissociation constant (KD) between the catechins and 3CLpro. RESULTS: Docking data suggested that 3CLpro interacted with the selected 16 catechins with low binding energy through the key amino acid residues Thr24, Thr26, Asn142, Gly143, His163, and Gln189. The selected catechins other than zijuanin D (3) and (-)-8-(5''R)-N-ethyl-2-pyrrolidinone-3-O-cinnamoylepicatechin (11) can inhibit 3CLpro intracellularly. The extracellular 3CLpro IC50 values of (-)-epicatechin 3-O-caffeoate (EC-C, 1), zijuanin C (2), etc-pyrrolidinone C and D (6), etc-pyrrolidinone A (9), (+)-gallocatechin gallate (GCG), and (-)-epicatechin gallate (ECG) are 1.58 ± 0.21, 41.2 ± 3.56, 0.90 ± 0.03, 46.71 ± 10.50, 3.38 ± 0.48, and 71.78 ± 8.36 µM, respectively. The KD values of 1, 6, and GCG are 4.29, 3.46, and 3.36 µM, respectively. CONCLUSION: Together, EC-C (1), etc-pyrrolidinone C and D (6), and GCG are strong 3CLpro inhibitors. Our results suggest that structural modification of catechins could be conducted by esterificating the 3-OH as well as changing the configuration of C-3, C-3''' or C-5''' to discover strong SARS-CoV-2 inhibitors.

Discovery and Targeted Isolation of Phenylpropanoid-Substituted Ester-Catechins Using UPLC-Q/TOF-HRMS/MS-Based Molecular Networks: Implication of the Reaction Mechanism among Polyphenols during Green Tea Processing.

Tea is an important beverage source of dietary polyphenols and well known for containing phenolic structure diversity. A series of phenylpropanoid-substituted catechins, flavonols, flavan-3-hexoside, and proanthocyanidin are present in different herbs with various biological activities, inspiring our exploration of phenylpropanoid-substituted ester type of catechins (PSECs) due to the enrichment of galloylated catechins in tea. In this study, we used a guiding-screening-location-isolation integrated route including creating a hypothesized PSEC dataset, MS/MS data acquiring, construction of molecular networks, and traditional column chromatography and preliminarily identified 14 PSECs by MS/MS spectrum. Two of these PSECs were further purified and elucidated by NMR and CD spectra. Further MS detection in tea products and fresh leaves suggests that the production of the two new compounds was enhanced during tea processing. The synthesis mechanism was proposed to obtain these types of components for further investigation on their roles in human health protection. This study provides an example for the exploration of new functional ingredients from food sources guided by MS/MS data-based networking, and also new insights into the reaction mechanism to form new catechin conjugates among polyphenols in green tea.

Detection and quantification of flavoalkaloids in different tea cultivars and during tea processing using UPLC-TOF-MS/MS.

Flavoalkaloids have been found from tea. However, there is limited information about their content in different teas. Herein, 51 tea samples were screened for flavoalkaloid content. Twelve teas with relatively higher contents of flavoalkaloids were further quantified by UPLC-TOF-MS/MS. The cultivars Yiwu and Bulangshan had the highest levels, with total flavoalkaloid contents of 3063 and 2727 µg g-1, respectively. Each of the six flavoalkaloids were at levels > 198 µg g-1 in these cultivars. Of the flavoalkaloids, etc-pyrrolidinone A had the highest content in the teas, reaching 835 µg g-1 in Yiwu. The content of the flavoalkaloids varied among tea cultivars and with processing procedures, particularly heating. The potential of using flavoalkaloids to discriminate grades of Keemun black tea was studied and discussed. The teas identified in this work with high levels of flavoalkaloids can be used in the future to study the mechanisms by which flavoalkaloids are synthesized in tea.

Feature-Based Molecular Networking Analysis of the Metabolites Produced by In Vitro Solid-State Fermentation Reveals Pathways for the Bioconversion of Epigallocatechin Gallate.

Dark teas are prepared by a microbial fermentation process. Flavan-3-ol B-ring fission analogues (FBRFAs) are some of the key bioactive constituents that characterize dark teas. The precursors and the synthetic mechanism involved in the formation of FBRFAs are not known. Using a unique solid-state fermentation system with ß-cyclodextrin inclusion complexation as well as targeted chromatographic isolation, spectroscopic identification, and Feature-based Molecular Networking on the Global Natural Products Social Molecular Networking web platform, we reveal that dihydromyricetin and the FBRFAs, including teadenol A and fuzhuanin A, are derived from epigallocatechin gallate upon exposure to fungal strains isolated from Fuzhuan brick tea. In particular, the strains from subphylum Pezizomycotina were key drivers for these B-/C-ring oxidation transformations. These are the same transformations seen during the fermentation process of dark teas. These discoveries set the stage to enrich dark teas and other food products for these health-promoting constituents.

Novel Cinnamoylated Flavoalkaloids Identified in Tea with Acetylcholinesterase Inhibition Effect.

3-O-Cinnamoylepicatechin (1) was synthesized along with four flavoalkaloids, (-)-6-(5‴S)-N-ethyl-2-pyrrolidinone-3-O-cinnamoylepicatechin (2), (-)-6-(5‴R)-N-ethyl-2-pyrrolidinone-3-O-cinnamoylepicatechin (3), (-)-8-(5‴S)-N-ethyl-2-pyrrolidinone-3-O-cinnamoylepicatechin (4), and (-)-8-(5‴R)-N-ethyl-2-pyrrolidinone-3-O-cinnamoylepicatechin (5) via esterification of epicatechin followed by phenolic Mannich reaction of 1 with theanine in the presence of heat. The new compounds 1-5 were detected in leaves of three tea cultivars, Fuding-Dabai, Huangjingui, and Zimudan with the help of ultra-performance liquid chromatography hyphenated with a photodiode array detector and electrospray ionization high-resolution mass spectrometry (UPLC-PDA-ESI-HRMS), suggesting that they are naturally occurring in tea leaves. The structures of the novel natural products were characterized by one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) and mass spectroscopy. Compounds 1-5 were then evaluated for their acetylcholinesterase (AChE) inhibitory effect (IC50 = 0.12-1.02 µM). The availability of the synthesized epicatechin derivatives 1-5 via a synthetic route enabled the first unequivocal identification of these derivatives as tea secondary metabolites and made it possible to determine their content in the tea material as well as the diverse bioactivities.

Discovery of Neolignan Glycosides with Acetylcolinesterase Inhibitory Activity from Huangjinya Green Tea Guided by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Data and Global Natural Product Social Molecular Networking.

Global Natural Product Social feature-based networking was applied to follow the phytochemicals, including nine flavonoid glycosides, six catechins, and three flavonols in Huangjinya green tea. Further, a new 8-O-4'-type neolignan glycoside, camellignanoside A (1), and 15 known compounds (2-16) were isolated through a variety of column chromatographies, and the structure was elucidated extensively by ultra performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry, 1H and 13C nuclear magnetic resonance, heteronuclear single-quantum correlation, heteronuclear multiple-bond correlation, 1H-1H correlation spectroscopy, rotating frame nuclear Overhauser effect spectroscopy, and Nuclear Overhauser effect spectroscopy, and circular dichroism spectroscopies. Compounds 1 and 2 showed acetylcolinesterase inhibition activity, with IC50 = 0.75 and 0.18 µM, respectively.

Two Pairs of Isomerically New Phenylpropanoidated Epicatechin Gallates with Neuroprotective Effects on H2O2-Injured SH-SY5Y Cells from Zijuan Green Tea and Their Changes in Fresh Tea Leaves Collected from Different Months and Final Product.

Zijuan tea ( Camellia sinensis var. assamica), an anthocyanin-rich cultivar with purple leaves, is a valuable material for manufacturing tea with unique color and flavor. In this paper, four new phenylpropanoid substituted epicatechin gallates (pECGs), Zijuanins A-D (1-4), were isolated from Zijuan green tea by different column chromatography. Their structures were identified by extensive high resolution mass spectroscopy (HR-MS), nuclear magnetic resonance (NMR), and experimental and calculated circular dichroism (CD) spectroscopic analyses. Detection of the changes in fresh tea leaves collected from April to September and the final processed product by high performance liquid chromatography (HPLC)-HRMS suggested that production of compounds 1 and 2 may be enhanced by the processing procedure of Zijuan green tea. Additionally, 1-4 were proposed to be synthesized through interaction between the abundant secondary metabolite ECG and phenolic acids from tea leaves by two key steps of phenol-dienone tautomerism. 1 and 2 showed impressive activity in protecting SH-SY5Y cells against H2O2-induced damage at the concentration of 1.0 µM.

Flavoalkaloids with a Pyrrolidinone Ring from Chinese Ancient Cultivated Tea Xi-Gui.

Chinese Xi-Gui tea is one ancient cultivated variety of Camellia sinensis var. assamica. At present, it is used for producing expensive and elite tea in China. Five new flavoalkaloids, (-)-6-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (ester-type catechins pyrrolidinone E, etc-pyrrolidinone E, 1), (-)-6-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone F, 2) (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone G, 3a), (-)-8-(5''' S)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone I, 4a), (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-catechin-3- O-gallate (etc-pyrrolidinone J, 4b), and one new naturally occurring natural product (-)-8-(5''' R)- N-ethyl-2-pyrrolidinone-epicatechin-3- O-gallate (etc-pyrrolidinone H, 3b) together with the known flavoalkaloids etc-pyrrolidinones A-D (5, 6, 7a, and 7b) were detected and isolated from Xi-Gui green tea. Their structures were identified by comprehensive NMR spectroscopic analyses. Absolute configurations of 1-3 were established by comparison of the CD analyses with epicatechin-3- O-gallate (ECG). Compounds 1-4 were evaluated for their protection against high glucose induced cell senescence on human umbilical vein endothelia cells (HUVECs) and showed significant protection effects ( p < 0.01) at both 1.0 and 10 µM. A discussion on the possible evolution of tea plants divergent from related food plants on the basis of phytochemical view is also provided.