Separation and antioxidant activities of new acetylated EGCG compounds.

Acetylation could improve the bioavailability of (-)-Epigallocatechin-3-Gallate (EGCG), but the relationship of substitution degree and antioxidant capacity of acetylated EGCG was unclear. The acetylated EGCG products were separated by preparation high performance liquid chromatography (HPLC). Two mono substituted acetylated EGCG, three substituted acetylated EGCG (T-AcE), eight substituted acetylated EGCG (E-AcE) and (-)-Epigallocatechin gallate (EGCG) were isolated. The 7-acetyl-EGCG (S7-ACEGCG) and 7-acetyl-EGCG (T-AcE) were identified for the first time. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of EGCG decreased significantly after acetylation modification. The more EGCG acetylation modification sites, the lower the total antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of 5-acetyl-EGCG (S5-ACE) were higher than 7-acetyl-EGCG (S7-AcE). Combining all the results in this and previous studies, acetylation modification is not conducive to the performance of EGCG antioxidant capacity.

Bitterness quantification and simulated taste mechanism of theasinensin A from tea.

Theasinensin A is an important quality chemical component in tea, but its taste characteristics and the related mechanism are still unclear. The bitterness quantification and simulated taste mechanism of theasinensin A were researched. The results showed that theasinensin A was significantly correlated with the bitterness of tea. The bitterness threshold of theasinensin A was identified as 65 µmol/L for the first time. The dose-over-threshold (DOT) value of theasinensin A was significantly higher than that of caffeine in black tea soup. The concentration-bitterness curve and time-intensity curve of theasinensin A were constructed. The bitterness contribution of theasinensin A in black tea was higher than in oolong and green tea. Theasinensin A had the highest affinity with bitterness receptor protein TAS2R16, which was compared to TAS2R13 and TAS2R14. Theasinensin A was mainly bound to a half-open cavity at the N-terminal of TAS2R13, TAS2R14, and TAS2R16. The different binding capacity, hydrogen bond, and hydrophobic accumulation effect of theasinensin A and bitterness receptor proteins might be the reason why theasinensin A presented different bitterness senses in human oral cavity.

Inhibitory Activities of Samples on Tyrosinases Were Affected by Enzyme Species and Sample Addition Methods.

The inhibition of tyrosinase (TYR) activity is an effective measure to inhibit melanin synthesis. At present, there are many methods with discrepant details that study the TYR inhibitory activity of samples. Under the same experimental conditions, this paper systematically studies whether enzyme species and sample addition methods are the key factors that determine the TYR inhibitory activity of samples. TYRs extracted from B16F10 cells, apple and mushroom, called BTYR, ATYR and MTYR, respectively, were selected to implement this study. Results showed that TYR inhibitory activities of samples were obviously affected by the above two factors. It was necessary to select the appropriate enzyme according to the problems to be explained. It was speculated that indirectly inhibitory activity reflected the comprehensive effects of samples on TYR catalytic activity and intracellular TYR synthesis pathway, while directly inhibitory activity reflected the effects of samples on TYR catalytic activity. Additionally, kojic acid could be used as a positive control for both B16F10 cells and MTYR models. The TYR inhibitory activity of ß-arbutin was complicated and fickle, while that of epigallocatechin gallate (EGCG) was universal and stable, which is to say, EGCG always inhibited TYR activity in a dose-dependent manner. In conclusion, the TYR inhibitory activities of samples were affected by enzyme species and sample addition methods. Compared with the unstable ß-arbutin, EGCG was more valuable for clinical research.

Kojic Acid Showed Consistent Inhibitory Activity on Tyrosinase from Mushroom and in Cultured B16F10 Cells Compared with Arbutins.

Kojic acid, ß-arbutin, α-arbutin, and deoxyarbutin have been reported as tyrosinase inhibitors in many articles, but some contradictions exist in their differing results. In order to provide some explanations for these contradictions and to find the most suitable compound as a positive control for screening potential tyrosinase inhibitors, the activity and inhibition type of the aforementioned compounds on monophenolase and diphenolase of mushroom tyrosinase (MTYR) were studied. Their effects on B16F10 cells melanin content, tyrosinase (BTYR) activity, and cell viability were also exposed. Results indicated that α-arbutin competitively inhibited monophenolase activity, whereas they uncompetitively activated diphenolase activity of MTYR. ß-arbutin noncompetitively and competitively inhibited monophenolase activity at high molarity (4000 µM) and moderate molarity (250-1000 µM) respectively, whereas it activated the diphenolase activity of MTYR. Deoxyarbutin competitively inhibited diphenolase activity, but could not inhibit monophenolase activity and only extended the lag time. Kojic acid competitively inhibited monophenolase activity and competitive-noncompetitive mixed-type inhibited diphenolase activity of MTYR. In a cellular experiment, deoxyarbutin effectively inhibited BTYR activity and reduced melanin content, but it also potently decreased cell viability. α-arbutin and ß-arbutin dose-dependently inhibited BTYR activity, reduced melanin content, and increased cell viability. Kojic acid did not affect cell viability at 43.8-700 µM, but inhibited BTYR activity and reduced melanin content in a dose-dependent manner. Therefore, kojic acid was considered as the most suitable positive control among these four compounds, because it could inhibit both monophenolase and diphenolase activity of MTYR and reduce intercellular melanin content by inhibiting BTYR activity without cytotoxicity. Some explanations for the contradictions in the reported articles were provided.

Identification of aroma-active compounds responsible for the floral and sweet odors of Congou black teas using gas chromatography-mass spectrometry/olfactometry, odor activity value, and chemometrics.

BACKGROUND: Floral and sweet odors are two typical characteristic aromas of Congou black tea, but their aroma-active compounds are still unclear. Characterizing the key aroma-active compounds can provide a theoretical foundation for the practical aroma quality evaluation of Congou black tea and directional processing technology of high-quality black tea with floral or sweet odors. Gas chromatography-olfactometry (GC-O) combined with odor activity value (OAV) is often used to screen key aroma-active substances, but the interaction between aroma components and their impact on the overall sensory quality is ignored. Therefore, in this study, OAV combined with variable importance in projection (VIP) and Spearman correlation analysis (SCA) were used to characterize the aroma-active components of Congou black teas with floral and sweet odors. RESULTS: Eighty-five volatiles were identified in these samples using gas chromatography-mass spectrometry (GC-MS). Twenty-three compounds were identified as potential markers for the floral and sweet odors of Congou black teas from orthogonal partial least squares discriminant analysis (OPLS-DA). Eighteen compounds were selected as candidate aroma compounds based on GC-O analysis and OAV calculations. In addition, 26 compounds were screened as crucial aroma compounds based on SCA. Finally, 19 compounds were evaluated as key aroma compounds by the comprehensive evaluation of VIP, OAV, and SCA. Terpenoids are the main active compounds that contribute to the floral odor of Congou black tea, whereas aldehydes are the key compounds for the sweet odor. CONCLUSION: The proposed method can effectively screen the aroma-active compounds and can be used for comprehensive quality control of products. © 2022 Society of Chemical Industry.

Effect of Active Groups and Oxidative Dimerization on the Antimelanogenic Activity of Catechins and Their Dimeric Oxidation Products.

Some catechins and their dimeric oxidation products are well known to possess antimelanogenic activity, which could be influenced by their structures and oxidative dimerization. This study compared the antimelanogenic activity of different catechins and dimeric oxidation products and clarified the mechanism using an α-MSH-stimulated B16F10 cell model. It was found that 100 µg/mL (-)-gallocatechin gallate, (-)-epigallocatechin gallate, theasinensin A, and theaflavine-3,3'-digallate could significantly inhibit melanin synthesis without cytotoxicity. The tyrosinase (TYR) activities were 26.24 ± 4.97, 31.57 ± 5.37, 66.10 ± 9.62, and 78.19 ± 5.14%, respectively, and the melanin contents were 38.29 ± 3.50, 41.21 ± 7.62, 62.13 ± 9.80, and 68.82 ± 11.62%, respectively. These compounds inhibit melanin production by attenuating the mRNA levels of TYR, TRP1, and TRP2 gene. The structure-activity relationship showed that geometrical isomerism was not the key factor affecting catechins' antimelanogenic activity. Compared with the catechol, catechins with B-ring pyrogallol inhibited melanin synthesis more effectively. The number of galloyl groups was positively correlated with antimelanogenic activity. Compared with 3-galloyl, 3'-galloyl was a stronger active group in antimelanogenesis. Interestingly, the contribution of B-ring pyrogallol to the antimelanogenic activity was significantly stronger than that of 3-galloyl in catechins. Additionally, the antimelanogenic activity of the dimeric oxidation product at 100 µM was more than or equal to that of individual substrate-catechin, while being significantly less than that of the substrate-catechin mixture. Results indicated that pyrogallol and galloyl were the active groups inhibiting melanin synthesis. The oxidative dimerization weakened the antimelanogenic activity of the substrate-catechin mixture.

Adsorption Equilibrium and Thermodynamics of Tea Theasinensins on HP20-A High-Efficiency Macroporous Adsorption Resin.

The separation and preparation of theasinensins have been hot spots in the field of tea chemistry in recent years. However, information about the mechanism of efficient adsorption of tea theasinensins by resin has been limited. In this study, the adsorption equilibrium and thermodynamics of tea theasinensins by a high-efficiency macroporous adsorption HP20 resin were evaluated. The adsorption of theasinensin A, theasinensin B, and theasinensin C on HP20 resin were spontaneous physical reaction processes. Adsorption processes were exothermic processes, and lowering the temperature was beneficial to the adsorption. The Freundlich model was more suitable to describe the adsorption of tea theasinensins. The adsorption equilibrium constant and maximum adsorption capacity of theasinensin A were significantly higher than theasinensin B and theasinensin C, which indicated that the adsorption affinity of theasinensin A was stronger than that of theasinensin B and theasinensin C. The phenolic hydroxyl groups and intramolecular hydrogen bonds of theasinensin A were more than those of theasinensin B and theasinensin C, which might be the key to the resin's higher adsorption capacity for theasinensin A. The HP20 resin was very suitable for efficient adsorption of theasinensin A.

Tea chemistry - What do and what don't we know? - A micro review.

Progress in analytical tools have led to a deeper insight into the chemical constitution and reaction pathways during the tea manufacture. However, the challenges have also changed as "new" teas are traded internationally which makes the authentication much more complicated. This micro-review demonstrates that despite all the achievements in the field of validated methods, authenticity, non-targeted methods we still have some gaps. New reactions products have been detected and those might be useful for authenticity purposes. As regards definitions of certain types of tea it makes sense to combine compositional data generated by validated targeted methods with non-targeted work to get a clearer view. Some more work seems to be necessary to get e.g. a deeper insight in the fate of proanthocyanidins during different types of processing and to develop a concept to quantify the thearubigins. There was progress in our knowledge of the thearubigin fraction in the last decade, however, there are still concepts to develop.

Determination of flavonol glycosides in green tea, oolong tea and black tea by UHPLC compared to HPLC.

An UHPLC method for the determination of flavonol glycosides (FOG) from green and oolong tea vs. black tea has been developed for the first time. Sample clean-up method by means of polyamide column chromatography was optimized with multiple-step elution. Using UHPLC and HPLC with gradient elution and photodiode array detection, eighteen FOG compounds were determined with the aid of electrospray tandem mass spectrometry. These FOG compounds were qualified on both UHPLC and HPLC, and this UHPLC method successfully separated rutin (quercetin-3-O-rutinoside) and K-grg (kaempferol-3-O-glucorhamnoglucoside) while conventional HPLC method did not. The total amounts of FOG compounds in the tea samples were 2.32-5.67g/kg dry weight (calculated as aglycones), and there is no significant difference for the total FOG content among green tea, oolong tea and black tea. However, kaempferol glycosides are more abundant in green teas, while oolong tea has more quercetin and myricetin glycosides. In black tea quercetin glycosides were most abundant.

Development of a multi-toxin method for investigating the carryover of zearalenone, deoxynivalenol and their metabolites into milk of dairy cows.

A dose-response study was carried out to examine the carryover of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites into bovine milk. Therefore, a feeding trial with 30 dairy cows fed with three different levels of Fusarium (FUS) toxin-contaminated maize was performed. A control group (0.02 mg ZEN kg(-1) dry matter (DM) and 0.07 DON kg(-1) DM) was compared with two groups fed contaminated diets. The first diet contained 0.33 mg ZEN kg(-1) DM and 2.62 mg DON kg(-1) DM (group FUS-50) and the second diet contained 0.66 mg ZEN kg(-1) DM and 5.24 mg DON kg(-1) DM (group FUS-100). For milk sample analysis, a new cost-efficient sample preparation method was developed for the simultaneous determination of ZEN, DON and their metabolites. The method comprised the separation of the milk fat followed by an SPE clean-up on Oasis HLB and a LC-MS/MS measurement. The less toxic metabolite de-epoxy-DON had the highest detected concentration (5.6 ng ml(-1) milk) in the milk samples obtained from the feeding trial. Additionally, ZEN (up to 0.29 ng ml(-1)), α-zearalenol (up to 0.17 ng ml(-1)), ß-zearalenol (up to 0.95 ng ml(-1)) and DON (up to 2.5 ng ml(-1)) were detected in these samples. The milk toxin concentrations of cows fed the control diet were significantly lower compared with cows fed the contaminated diet. The calculated carryover rates ranged between 0 and 0.0075 for ZEN and metabolites and between 0 and 0.0017 for DON independent of exposure. It can be concluded that dietary toxin concentrations in the feed below or close to the current guidance values do not pose a risk for consumers due to negligible carryover rates.

Investigation of processes in black tea manufacture through model fermentation (oxidation) experiments.

Flavanol depleted whole fresh green tea leaf powder, as reported in the literature, was used as matrix for a systematic study of the endogenous oxidative enzymatic conversion of selected flavanol combinations to theaflavins and thearubigins. The activity of the two crucial enzymes polyphenol oxidase (PPO) and peroxidase (POD) was controlled individually through addition of H2O2 and/or O2. Using the endogenous peroxidase only it was shown that (-)-epicatechin alone did not react with POD. According to these results it is possible that theaflavin formation occurs via reaction of a flavanol quinone with a nonquinone flavanol. It was confirmed that only a dihydroxy-B-ring flavanol with a trihydroxy-B-ring flavanol gave a theaflavin upon enzymatic oxidation. Use of horseradish peroxidase in the presence of a flavanol depleted tea leaf matrix led to significantly higher kinetics on theaflavin 3-gallate degradation compared to the absence of leaf matrix, suggesting a catalytic effect of the leaf matrix not reported before.

Purification and gas chromatography-combustion-isotope ratio mass spectrometry of aroma compounds from green tea products and comparison to bulk analysis.

A method for carbon isotope ratio (δ(13)C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ(13)C value from bulk isotope analyses. The δ(13)C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl ß-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl ß-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ(13)C analysis of volatile compounds. On the basis of the gas chromatography-combustion-isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (Z)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ(13)C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea.

A new method for the preparative isolation of chlorogenic acid lactones from coffee and model roasts of 5-caffeoylquinic acid.

Chlorogenic acid lactones (CQL) are known to contribute to the bitter taste of roasted coffee. CQL might also have beneficial biological activities. Until now, there is a lack of pure standard compounds for quantification and biological testing. Using high-speed countercurrent chromatography, milligram amounts of lactones could be isolated. The structures of 3-O-caffeoyl-γ-quinide, 4-O-caffeoyl-muco-γ-quinide, and 5-O-caffeoyl-epi-δ-quinide were confirmed by 1D and 2D NMR spectroscopy including (13)C NMR data, which were previously not available from the literature. An UHPLC method was developed that enabled the separation of the lactones from roasted coffee in significantly shorter time than conventional HPLC.

Bioavailability and antioxidant potential of rooibos flavonoids in humans following the consumption of different rooibos formulations.

In a complete crossover design, a human study with twelve healthy male volunteers has been conducted using a placebo and different rooibos drinks (rooibos tea and an isolated active fraction) from unfermented rooibos (Aspalathus linearis). Blood and urine samples were collected before and up to 24h after consumption of the drinks. By HPLC-MS/MS, seven metabolites of aspalathin and nothofagin were identified in urine samples, as well as intact aspalathin and nothofagin. Moreover, sulphated, glucuronidated, methylated, both glucuronidated and methylated aspalathin, and glucuronidates of the aglycones of aspalathin and nothofagin were detected. The main metabolite excreted was methylated aspalathin. Most of the metabolites were detected after administration of both rooibos formulations. In plasma samples characteristic unchanged flavonoids derived from unfermented rooibos (e.g. aspalathin) were detected in trace quantities this is due to the changes in Table 5 after ingestion of both rooibos formulations. On average a total of 0.76nmol of flavonoids were detected during their peak concentration after intake of the rooibos tea, accounting for 0.26% compared to the total amount of flavonoids ingested. Despite the comparable intake of total flavonoids, only an overall 0.41nmol of flavonoids could be detected after ingestion of the isolated active fraction. No significant increase in plasma antioxidant capacity was observed using the ORAC assay giving rise to the assumption that the effects of rooibos flavonoids have to be detected using other endpoints.

A new myricetin-rhamnodiglucoside from Camellia sinensis.

The isolation and structural elucidation of the novel myricetin-3-O-[beta-D-glucopyranosyl-(1 --> 3)-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside] from tea (Camellia sinensis (L.) O. Kuntze) by means of HPLC-MS/MS and various NMR techniques is described. The compound was not present in all tea samples analysed. In the case of white tea, it was detected in 20 out of 40 samples analysed (35-255 mg kg(-1)), in green tea it was detected in 18 out of 35 samples in the same concentration range (37-205 mg kg(-1)), while it was found only in 1 sample (51 mg kg(-1)) of 5 black teas analysed.