Metabolomics Investigation Reveals That 8-C N-Ethyl-2-pyrrolidinone

Article Cite This: J. Agric. Food Chem. 2018, 66, 7209−7218

pubs.acs.org/JAFC

Metabolomics Investigation Reveals That 8‑C N‑Ethyl-2pyrrolidinone-Substituted Flavan-3-ols Are Potential Marker Compounds of Stored White Teas Weidong Dai,† Junfeng Tan,† Meiling Lu,‡ Yin Zhu,† Pengliang Li,† Qunhua Peng,† Li Guo,† Yue Zhang,† Dongchao Xie,† Zhengyan Hu,*,§ and Zhi Lin*,†

Downloaded via DURHAM UNIV on July 13, 2018 at 09:55:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 Meiling South Road, Hangzhou, Zhejiang 310008, People’s Republic of China ‡ Agilent Technologies (China), Limited, 3 Wangjing North Road, Chaoyang, Beijing 100102, People’s Republic of China § Zhejiang Provincial Center for Disease Control and Prevention, 3399 Binsheng Road, Hangzhou, Zhejiang 310051, People’s Republic of China S Supporting Information *

ABSTRACT: 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-3ols 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 environmentcontrolled 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. KEYWORDS: white tea, storage, metabolomics, LC−MS, theanine, flavan-3-ol storage at 20 °C for 6 months. Ning et al.11 investigated the changes in gallic acid, caffeine, catechins, and amino acids in Shoumei white teas under different storage times using ultra performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC−QQQ−MS/ MS) and found that the contents of catechins and amino acids decreased with increased storage time, while the content of gallic acid increased. These studies focused only on the major compounds or determined the total contents of amino acids and flavonoids in teas. Therefore, a comprehensive characterization of white tea metabolome during the white tea storage is urgently needed. In addition, there is a lack of a survey on possible novel compounds that are formed during storage. Metabolomics enables the measurement of hundreds of endogenous compounds simultaneously, providing a comprehensive view of the chemical compositions, and has been widely used in food chemical research.12,13 In this study, we used an ultrahigh-performance liquid chromatography−quadrupole time-of-flight mass spectrometry (UHPLC−QTOF/ MS)-based metabolomics approach to investigate the variations in the non-volatile compound profiles of white tea

1. INTRODUCTION Tea is one of most consumed beverages in the world as a result of its health benefits and satisfactory sensory experience.1−3 In comparison to green tea and black tea, the two most popular teas, white tea is a rare form that undergoes the least amount of processing (only withering and drying processes are involved) and can be generally classed into three types according to the quality of plucked fresh tea leaves: Baihaoyinzhen (BHYZ, bud only, also called silver needle), Baimudan (BMD, a bud with one or two leaves, also called white peony), and Shoumei (more than two leaves, with or without a bud).4 Storage is crucial for the quality of teas and could change the aroma,5,6 taste,7 bioactivities, and chemical components of teas. White tea and pu-erh tea stored for long periods are considered to have higher quality and commercial value. In contrast, green tea stored for long periods is considered not fresh and umami. Some bioactivities of teas were also found to change during storage. Nekvapil et al.8 reported that the antioxidant capacity of beverages containing black, green, and white tea extracts decreased during storage. He et al.9 found that the antibacterial effect in the latest white tea was the best and decreased along with the extension of the storage time. Some investigations on chemical changes in stored teas have been carried out. Friedman et al.10 studied the stability of green tea catechins and found that the average overall decrease in the total catechin concentrations of eight teas was 32% after © 2018 American Chemical Society

Received: Revised: Accepted: Published: 7209

April 19, 2018 May 23, 2018 June 19, 2018 June 19, 2018 DOI: 10.1021/acs.jafc.8b02038 J. Agric. Food Chem. 2018, 66, 7209−7218

Article

Journal of Agricultural and Food Chemistry

amounts of each tea sample were also used to evaluate the LC−MS analysis. 2.3. Metabolomics Analysis. The metabolomics measurements of white tea samples were conducted following the procedures that we developed previously.14−16 Briefly, a UHPLC system (Infinity 1290, Agilent Technologies, Santa Clara, CA, U.S.A.) coupled to a QTOF mass spectrometer (6540, Agilent Technologies, Santa Clara, CA, U.S.A.) was applied for the LC−MS analysis. Chromatographic separation of the white tea compounds was performed on a Zorbax Eclipse Plus C18 column (150 × 3.0 mm, 1.8 μm, Agilent Technologies, Little Falls, DE, U.S.A.). The column was maintained at 40 °C. Binary mobile phases were used for gradient elution with the flow rate of 0.4 mL/min, where phase A was water containing 0.1% (v/v) formic acid and phase B was methanol. The linear gradient elution program was as follows: 0 min, 10% B; 4 min, 15% B; 7 min, 25% B; 9 min, 32% B; 16 min, 40% B; 22 min, 55% B; 28 min, 95% B; and 30 min, 95% B. A total of 4 min was allowed for column equilibration between two consecutive injections. The injection volume was 3 μL. The QTOF mass spectrometer with an assembled electrospray ionization (ESI) source was operated in positive mode. The major MS parameters were the same as those in previous reports.14,16 The mass scan range of m/z 100−1000 was applied for the full-scan analysis. Reference ions with m/z 121.0509 (purine) and 922.0098 [hexakis(phosphazene)] were continuously infused via the reference sprayer during data acquisition for online calibration to ensure the MS accuracy. The compounds were identified according to authentic standards, accurate masses, MS2 spectra, metabolomics databases, and our previous works.14−18 2.4. Metabolomics Data Processing. Raw data files acquired by LC−MS analysis were first processed by Profinder software (Agilent Technologies, Santa Clara, CA, U.S.A.) for compound feature extraction and then imported into Mass Profiler Professional software (version 13.0, Agilent Technologies, Santa Clara, CA, U.S.A.) for peak alignment. Ions with a relative standard deviation (RSD) less than 30% in the QC samples were used for further univariate and multivariate statistics.19,20 2.5. Synthesis of 8-C N-Ethyl-2-pyrrolidinone-Substituted Flavan-3-ols (EPSFs). Theanine (2 g) and EGCG (2 g) were dissolved in 15 mL of methanol/formic acid/water solution (40:1:60, v/v/v) and then heated at 50 °C for 12 days. The solution was separated into 20 fractions by reversed-phase column chromatography with a gradient elution of methanol solution (from 15 to 80%). The fractions 10−12 were combined and then separated into 20 fractions by reversed-phase column chromatography again to obtain 8-C Nethyl-2-pyrrolidinone-substituted EGCG. The purity was 93% tested by a third-party laboratory using an ultra performance liquid chromatography coupled with ultraviolet (UPLC−UV) method at 280 nm. 1H NMR (600 MHz, DMSO-d6): δ 6.77 (d, J = 23.4 Hz, 2H), 6.40 (d, J = 19.0 Hz, 1H), 6.26 (d, J = 53.2 Hz, 1H), 6.02 (d, J = 22.1 Hz, 1H), 5.42−5.25 (m, 1H), 5.17 (d, J = 27.1 Hz, 1H), 5.00 (d, J = 54.3 Hz, 1H), 3.64 (t, J = 7.6 Hz, 1H), 3.06−2.95 (m, 1H), 2.81− 2.60 (m, 1H), 2.39 (s, 1H), 2.24 (s, 2H), 2.02 (t, J = 7.7 Hz, 1H), 1.72−1.57 (m, 1H), 0.98−0.77 (m, 3H). 13C NMR (151 MHz, DMSO-d6): δ 174.20, 168.29, 158.84, 158.53, 148.6, 148.3, 135.18, 131.31, 122.48, 111.74, 108.57, 108.50, 79.68, 70.75, 46.42, 36.3, 35.64, 29.02, 26.25, 15.45. Flavan-3-ol standards of EGC, ECG, EC, GCG, GC, CG, and C were also used to react with theanine in methanol/formic acid/water solution (40:1:60, v/v/v). The solutions were subjected to LC−MS and LC−MS/MS analyses without purification. 2.6. Identification and Quantification of EPSFs in Stored White Tea. Compounds 1−7 were preliminarily identified from flavan-3-ols and theanine according to accurate mass, MS2 spectrum, and ref 21. Then, the syntheses of compounds 1−7 in reaction solution containing standards of theanine and flavan-3-ol (EGCG, EGC, ECG, EC, GCG, GC, CG, and C) were carried out for the confirmation of chemical structures. Compounds 1 and 2 were found in the reaction solution containing theanine and EGCG but not in the reaction solution containing theanine and GCG. In combination with the reported elution orders

samples with various storage durations and then surveyed novel potential marker compounds related to the stored white tea.

2. MATERIALS AND METHODS 2.1. Chemicals. Liquid chromatography−mass spectrometry (LC−MS)-grade methanol was purchased from Merck (Darmstadt, Germany). Formic acid (purity of >96.0%), (−)-epigallocatechin (EGC, >95.0%), (+)-catechin (C, >98.0%), (−)-epigallocatechin gallate (EGCG, >95.0%), (−)-epicatechin gallate (ECG, >98.0%), (−)-epicatechin (EC, >98.0%), (−)-gallocatechin gallate (GCG, >98.0%), (+)-gallocatechin (GC, >95.0%), (−)-catechin gallate (CG, >98.0%), kaempferol 3-glucoside (>97.0%), kaempferol 3galactoside (>90.0%), kaempferol 3-rutinoside (>98.0%), vitexin (>95.0%), isovitexin (>98.0%), luteolin-8-C-glucoside (>97.0%), quercetin 3-glucoside (>98.0%), tryptophan (>98.0%), glutamic acid (>99.0%), pyroglutamic acid (>99.0%), proline (>99.0%), glutamine (>99.0%), aspartic acid (>98.0%), γ-aminobutyric acid (GABA, >99.0%), leucine (>98.0%), isoleucine (>99.0%), threonine (>98.0%), lysine (>98.0%), histidine (>99.0%), valine (>98.0%), arginine (>99.0%), and adenine (>99.0%) were obtained from Sigma (St. Louis, MO, U.S.A.). Myricetin 3-galactoside (>98.0%), procyanidin B1 (>98.0%), procyanidin B2 (>98.0%), theaflavin (TF, >98.0%), theaflavin-3-gallate (TF-3-g, >98.0%), and theaflavin3,3′-digallate (TF-3,3′-dg, >98.0%) were purchased from ChemFaces (Wuhan, Hubei, China). Theanine (>99.0%), quercetin 3-galactoside (>97.0%), quercetin 3-rhamnoglucoside (rutin, >98.0%), theobromine (>98.0%), tyrosine (>99.0%), and phenylalanine (>99.0%) were obtained from J&K Scientific, Ltd. (Beijing, China). Caffeine (>99.0%) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, U.S.A.). Epiafzelechin (>98.0%), strictinin (>95.0%), benzyl glucoside, and phenylethyl glucoside were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Deionized water was produced by a Milli-Q water purification system (Millipore, Billerica, MA, U.S.A.). 2.2. Stored White Tea Sample Collection and Treatment. Two types of white tea samples [BHYZ and BMD, Camellia sinensis (L.) O. Kuntze cv. Fuding Dabaicha] of various produced years (ranging from 2000 to 2015) were collected in 2016 in this study. BHYZ and BMD white tea samples were obtained from Fujian Pinpinxiang Tea Co., Ltd. (Fuding, Fujian, China) and Fujian Yuda Tea Co., Ltd. (Fuding, Fujian, China), respectively (Table S1 of the Supporting Information). BHYZ is produced from only one bud, and BMD is produced from one bud with two leaves. The raw fresh leaves were picked up from Fuding of Fujian, China, in April and were processed by skillful workers according to a typical white tea manufacturing procedure, which only includes withering and drying processes.16 The white tea products were naturally preserved in a storehouse maintained at 15−25 °C and 25−50% humidity. Detailed information on the stored white tea samples was included in Table S1 of the Supporting Information. To investigate the variations in nonvolatile compounds, BHYZ and BMD tea samples were divided into three groups according to their storage durations: “1 year” group {1.0 ± 0.0 year [average ± standard deviation (SD)] for the BHYZ and BMD samples}, “2−4 year” group (2.9 ± 0.8 and 2.8 ± 0.8 years for the BHYZ and BMD samples, respectively), and “>4 year” group (6.2 ± 1.1 and 10.1 ± 2.8 years for the BHYZ and BMD samples, respectively). A total of 40 mL of a 70 °C methanol/water solution (70:30, v/v) was added to 0.3 g of ground tea powder (4 year BHYZ and BMD white teas and to screen the characteristic compounds in stored white teas. Heatmap analysis and clustering analysis were performed by MultiExperiment Viewer software (version 4.8.1) to illustrate the compound content differences among white tea samples after data autoscaling. Student’s t test and analysis of variance (ANOVA) were performed using PASWstat software (version 18.0, Chicago, IL, U.S.A.).

3. RESULTS AND DISCUSSION A total of 2584 compound ions were obtained after peak alignment and used for unsupervised PCA. The QC samples were clustered in the center of the PCA score plot (Figure S5 of the Supporting Information), indicating good reproducibility of the compound extraction and LC−MS analysis in the metabolomics investigations. The BHYZ and BMD samples were clearly separated, which indicated that the type of white tea has a larger influence on the non-volatile chemical constituents than the storage (Figure S5 of the Supporting Information). Therefore, the influence of storage on BHYZ and BMD were investigated separately. 3.1. Influence of Storage on the White Tea Compounds. PCA and PLS-DA models were constructed to investigate the influence of storage on the white tea compounds (Figure 1), and a 50 times permutation test was applied for the validation of the PLS-DA models. The 7211

DOI: 10.1021/acs.jafc.8b02038 J. Agric. Food Chem. 2018, 66, 7209−7218

Article


Figure 2. Heat map of the compound contents in BHYZ white tea samples with different storage durations.

permutation test showed that y intercepts for R2 and Q2 were 0.381 and −0.267, respectively, for the BHYZ PLS-DA model and were 0.328 and −0.271, respectively, for the BMD PLS-

DA model, which indicated that these two PLS-DA models were not overfitted. Distinct compound patterns of white tea samples stored for 1, 2−4, and >4 years were observed in the 7212


Article


Figure 3. Heat map of the compound contents in BMD white tea samples with different storage durations.

including flavan-3-ols, dimeric flavan-3-ols (theaflavins, theasinensins, and procyanidins), alkaloids, flavonol/flavone glycosides, amino acids, phenolic acids, nucleosides, organic

two-dimensional score plots of PCA and PLS-DA (Figure 1). These patterns indicated that the white tea compound patterns obviously changed during storage. A total of 125 compounds, 7213


Article


Figure 4. (A) Extracted ion chromatograms (EICs) of EPSFs in stored white teas and (B) Pearson correlation coefficients (R = 0.714−0.850) between compounds 1−7 and storage periods in BMD white teas.

year white tea samples and then decreased the in >4 year white tea samples (Figure S6 of the Supporting Information). These phenomena were observed in both BHYZ and BMD. These results do not totally agree with the findings by Ning et al.11 potentially as a result of the different types of white tea used (BHYZ and BMD versus Shoumei) and the different years of the white tea samples collected in the study. After long-term (>4 years) storage, flavan-3-ols of EGCG, EC, ECG, EGC, C, and epiafzelechin in white tea were significantly reduced in

acids, lipids, and carbohydrates, were identified to characterize the compound variations (Figures 2 and 3). Flavan-3-ols (catechins) are one group of characteristics and the most abundant compounds in teas, which account for up to 10% of the content and are considered the major bioactive constituents in white tea.4 Ning et al.11 reported that the total catechin, EGCG, EGC, ECG, and EC contents in “Shoumei” white tea significantly decreased with the lengthening of the storage duration. In this study, flavan-3-ols, including EGCG, ECG, EGC, GC, and GCG, were slightly increased in the 2−4 7214


Article


Figure 5. Contents of EPSFs in (A) BHYZ and (B) BMD white teas. The significance of the compound difference among groups was tested using ANOVA. (C) Correlation coefficients between EPSFs and theanine and between EPSFs and flavan-3-ols in white teas.

arginine, and tyrosine) decreased in a stepwise manner in the stored white teas (Figure S9 of the Supporting Information). This might be induced by the Maillard reaction of amino acids with reducing sugars, which commonly occurs during thermal processing and long storage of foods and leads to the browning of foods.27 The content of glutamic acid decreased in the stored BMD white teas but increased in the 2−4 year BHYZ white teas. The level of threonine also increased during the storage of BHYZ white teas. Pyroglutamic acid, a cyclized derivative of glutamic acid that can be formed non-enzymatically from glutamate and glutamine, was increased significantly in the 2−4 and >4 year BHYZ white teas as well as in the >4 year BMD white teas. This indicated that a non-enzymatic cyclization reaction of glutamic acid or glutamine in white tea may occur during storage (Figure S9 of the Supporting Information). Aroma primeverosides are regarded as aroma precursors, which release aroma compounds during the tea manufacturing and brewing processes.2 In this study, the levels of all aroma primeverosides, including benzyl primeveroside, phenylethyl primeveroside, linalool primeveroside, and linalool oxide primeveroside, were highest in the 2−4 year BHYZ and BMD white teas and lowest in the 1 year BHYZ and BMD white teas (Figure S10 of the Supporting Information). Here, it should be noted that the contents of some metabolites (e.g., flavan-3-ol) in teas are susceptible to the climate factors, such as sunlight explosion, rainfall, temperature, etc., and can vary from year to year and also from season to season.17,28 The initial metabolite contents in white teas can affect the results. 3.2. EPSFs Are Potential Marker Compounds of Stored White Tea. In addition to the compounds discussed above, consisting of flavan-3-ols, dimeric flavan-3-ols, flavonol/ flavone glycosides, amino acids, phenolic acids, nucleosides, organic acids, lipids, and carbohydrates (Figures 2 and 3), we also found that several compound features showed strong positive correlations with the storage duration. As shown in Figure 4, the correlation coefficients between compounds 1−7 and the storage duration ranged from 0.714 to 0.850 in the BMD white teas and ranged from 0.565 to 0.659 in the BHYZ white teas. In addition, in a PLS regression analysis of the BHYZ and BMD white teas (the compound contents and the

both BHYZ and BMD. These results are consistent with the findings by Ning et al. in Shoumei white teas.11 The decreased flavan-3-ols were not ultimately converted to dimeric flavan-3-ols (theaflavins, theasinensins, and procyanidins) in the stored white teas. The levels of theaflavins [TF, TF-3-g, theaflavins-3-gallate (TF-3′-g), and TF-3,3′-dg] decreased in a stepwise manner during the storage process (Figure S7 of the Supporting Information). The contents of procyanidins [procyanidin B1, procyanidin B2, EC-(4 → 8)ECG, and EC-(4 → 8)-EGCG] were slightly increased in the 2−4 year white teas and significantly decreased in the >4 year white teas (Figure S7 of the Supporting Information). Flavonol-O-glycosides and flavone-C-glycosides are also major phenolic compounds in teas with a series of bioactivities18,23 and low astringent taste thresholds.24,25 In this study, the contents of six kaempferol-O-glycosides (kaempferol 3-glucoside, kaempferol 3-galactoside, kaempferol 3-rutinoside, kaempferol 3-glucosylrutinoside, kaempferol 3galactosylrutinoside, and kaempferol 3-arabinoside), seven quercetin-O-glycosides (quercetin 3-glucoside, quercetin 3galactoside, quercetin 3-rutinoside, quercetin 3-glucosylrutinoside, quercetin 3-galactosylrutinoside, quercetin diglucoside, and quercetin triglucoside), and two myricetin-O-glycosides (myricetin 3-glucoside and myricetin 3-galactoside) decreased during white tea storage (Figure S8 of the Supporting Information). Except kaempferol 3-dicoumaroylglucoside, the contents of these flavonol-O-glycosides were lowest in the >4 year white teas (both BHYZ and BMD). The levels of flavoneC-glycosides, including apigenin-6,8-C-diglucoside, apigenin-6C-arabinoside-8-C-glucoside, apigenin-6-C-glucosyl-8-C-arabinoside, vitexin, isovitexin, and luteolin-8-C-glucoside, also showed decreased trends in the 2−4 and >4 year white teas (Figure S8 of the Supporting Information). These results did not agree with the findings by Zhou et al.26 that the content of flavonoids increased during white tea storage and a significantly high content of flavonoids was observed in white tea stored for 20 years. This disagreement may be because a colorimetric method for the quantification of total flavonoids was used in the study by Zhou et al., while a LC−MS method was applied in this work. The levels of most amino acids (theanine, phenylalanine, valine, leucine, isoleucine, asparagine, proline, tryptophan, 7215


Article


Figure 6. (A) Chemical structures of compounds 1−7 and (B) proposed route for the formation of EPSFs.

storage durations were set as the x and y variables, respectively), compounds 1−7 were among the most important contributors (Figure S11 of the Supporting Information). Variable importance in projection (VIP) was also used to evaluate the contribution of compounds 1−7 to the total variance in the PLS-DA model (panels C and D of Figure 1). VIP values of them ranged from 2.53 to 7.65 and from 2.29 to 8.40 in BHYZ and BMD PLS-DA models, respectively (Table S3 of the Supporting Information), which indicated that the contents of compounds 1−7 distinctly differ in white teas of different storage periods. Particularly, compounds 1−7 showed significantly higher contents in BHYZ (Figure 5A) and BMD (Figure 5B) white teas stored for a long period. Results of non-parametric Mann−Whitney U tests of EPSFs between each two groups also indicated that the contents of compounds 1−7 in the >4 year group were significantly higher (Table S2 of the Supporting Information). Compounds 1−7 showed accurate masses of m/z 570.1609 (compounds 1 and 2), 554.1663 (compound 3), 418.1500 (compounds 4 and 5), and 402.1555 (compounds 6 and 7) in positive ionization mode, which indicated possible formulas of C28H27NO12, C28H27NO11, C21H23NO8, and C21H23NO7, respectively. Compounds 1 and 2, 4 and 5, as well as 6 and 7 had nearly identical MS2 spectra, respectively (Figures S1− S4 of the Supporting Information). In addition, compounds 1−7 had common MS2 fragment ions of m/z 262.1074, 250.1074, and 205.0490, which indicated that these compounds have the same structural skeleton. The accurate masses (m/z 418.1500 and 402.1555) and MS2 spectra of compounds

4−7 perfectly matched with those of the EPSFs reported by Wang et al.21 These compounds were found in post-fermented dark teas and were formed from flavan-3-ols and theanine.21 In combination with the syntheses of compounds 1−7 in reaction solution containing standards of theanine and flavan-3-ol (EGCG, EGC, ECG, EC, GCG, GC, CG, and C), these seven compounds were finally identified as 8-C N-ethyl-2-pyrrolidinone-substituted EGCG (S-EGCG-cThea and R-EGCGcThea), 8-C N-ethyl-2-pyrrolidinone-substituted ECG (RECG-cThea), 8-C N-ethyl-2-pyrrolidinone-substituted EGC (S-EGC-cThea and R-EGC-cThea), and 8-C N-ethyl-2pyrrolidinone-substituted EC (S-EC-cThea and R-ECcThea), respectively (Figure 6A). The total contents of these seven compounds were 0.754 ± 0.080, 0.761 ± 0.253, and 1.340 ± 0.28 mg/g in the 1, 2−4, and >4 year BHYZ white teas, respectively, and 1.009 ± 0.060, 1.028 ± 0.152, and 1.930 ± 0.438 mg/g in the 1, 2−4, and >4 year BMD white teas, respectively (Figure 5A). To the best of our knowledge, SEGCG-cThea, R-EGCG-cThea, and R-ECG-cThea were discovered in teas for the first time, and these seven compounds were found in white teas for the first time. The proposed route for the formation of EPSFs in stored white tea is shown in Figure 6B. Theanine converts to 1-ethyl5-hydroxy-2-pyrrolidinone after Strecker degradation and cyclization,21,29 and this product then reacts with flavan-3-ols to form EPSFs. The correlation coefficients between EPSFs and theanine and between EPSFs and flavan-3-ols were calculated and are shown in Figure 5C. EPSFs displayed negative correlations with flavan-3-ols and theanine (R ranged 7216


Article


Figure 7. Linearly increasing formations of 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols in (A and B) reaction solution (containing standards of theanine and flavan-3-ols) within 12 days and (C and D) Baimudan white teas stored in an environment-controlled cabinet within 2.5 months (n = 5).

from −0.76 to 0.13). These results provide new insight into the interactions between primary compounds of catechins and theanine in teas during long-term storage. 8-C N-Ethyl-2pyrrolidinone-substituted GCG, CG, GC, and C were not found in the stored white teas potentially as a result of the much lower contents of GCG, CG, GC, and C than EGCG, ECG, EGC, and EC in the white teas. 3.3. Validation of EPSF Changes in Stored White Teas. To further validate that the EPSFs were formed during the white tea storage and that their contents were positively related to the storage duration, two experiments were designed. As shown in panels A and B of Figure 7, the contents of compounds 1−7 increased in a stepwise manner in the reaction solutions at 1, 2, 3, 4, 5, and 12 days. The linear correlation coefficients ranged from 0.955 to 0.999. In the white tea samples stored in an environment-controlled cabinet, the contents of compounds 1−7 also increased in a stepwise manner over 2.5 months. The linear correlation coefficients ranged from 0.929 to 0.999 for compounds 1−5 and were 0.747 and 0.728 for compounds 6 and 7, respectively (panels C and D of Figure 7). These results demonstrated that the formation of EPSFs from theanine and flavan-3-ols is linearly correlated to the storage duration (or reaction duration). These results indicated that EPSFs are marker compounds of stored white tea, which are potential markers for the discrimination of the storage duration of white teas. In addition, it was believed that aged white teas have stronger effects against flu and inflammation in folk medicine,26 and 8-C N-ethyl-2-pyrrolidinone-substituted EGC, GC, EC, and C were reported to have potential protective effects on human microvascular endothelial cell (HMEC) injury induced by hydrogen dioxide compared to other tea polyphenols.21 Therefore, bioactivity investigations of EPSFs will be carried out in the future. Furthermore, studies of

EPSFs during the storage of other teas, such as green, black, and dark teas, are also needed.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02038. Information of included white tea samples (Table S1), non-parametric Mann−Whitney U tests of EPSFs (Table S2), VIP values of EPSFs in PLS-DA models (Table S3), MS2 spectra and the putative fragmentation pathways of 8-C N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (Figures S1−S4), PCA score plot of white teas and QC samples (Figure S5), changes of catechins, dimeric catechins, flavone glycosides, flavonol glycosides, amino acids, and aroma glycosides during white tea storage (Figures S6−S10), and PLS regression analysis of white tea storage duration and compound contents (Figure S11) (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-571-87115272. E-mail: [email protected]. *Telephone: +86-571-86650617. E-mail: [email protected]. ORCID

Zhi Lin: 0000-0001-5976-1806 Funding

The authors appreciate the funding support from the Central Public-Interest Scientific Institution Basal Research Fund (1610212016008 and 1610212018009), the National Natural Science Foundation of China (31500561 and 31501565), and the Earmarked Fund for China Agricultural Research System (CARS-19). 7217


Article

Journal of Agricultural and Food Chemistry Notes

(19) Dai, W. D.; Wei, C.; Kong, H. W.; Jia, Z. H.; Han, J. K.; Zhang, F. X.; Wu, Z. M.; Gu, Y.; Chen, S. L.; Gu, Q.; Lu, X.; Wu, Y. L.; Xu, G. W. Effect of the traditional Chinese medicine tongxinluo on endothelial dysfunction rats studied by using urinary metabonomics based on liquid chromatography−mass spectrometry. J. Pharm. Biomed. Anal. 2011, 56, 86−92. (20) Dai, W.; Yin, P.; Zeng, Z.; Kong, H.; Tong, H.; Xu, Z.; Lu, X.; Lehmann, R.; Xu, G. Nontargeted modification-specific metabolomics study based on liquid chromatography−high-resolution mass spectrometry. Anal. Chem. 2014, 86, 9146−9153. (21) Wang, W.; Zhang, L.; Wang, S.; Shi, S.; Jiang, Y.; Li, N.; Tu, P. 8-C N-Ethyl-2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese dark teas formed in the post-fermentation process provide significant antioxidative activity. Food Chem. 2014, 152, 539−545. (22) Ren, D.-M.; Guo, H.-F.; Yu, W.-T.; Wang, S.-Q.; Ji, M.; Lou, H.-X. Stereochemistry of flavonoidal alkaloids from dracocephalum rupestre. Phytochemistry 2008, 69, 1425−1433. (23) Perez-Vizcaino, F.; Duarte, J. Flavonols and cardiovascular disease. Mol. Aspects Med. 2010, 31, 478−494. (24) Scharbert, S.; Holzmann, N.; Hofmann, T. Identification of the astringent taste compounds in black tea infusions by combining instrumental analysis and human bioresponse. J. Agric. Food Chem. 2004, 52, 3498−3508. (25) Scharbert, S.; Hofmann, T. Molecular definition of black tea taste by means of quantitative studies, taste reconstitution, and omission experiments. J. Agric. Food Chem. 2005, 53, 5377−5384. (26) Zhou, Q.; Sun, W.; Ye, Y.; Chen, X. Study on the main biochemical components of white tea stored at different years. Sci. Technol. Food Ind. 2014, 35, 351−359. (27) Gupta, R. K.; Gupta, K.; Sharma, A.; Das, M.; Ansari, I. A.; Dwivedi, P. D. Maillard reaction in food allergy: Pros and cons. Crit. Rev. Food Sci. Nutr. 2018, 58, 208−226. (28) Jayasekera, S.; Kaur, L.; Molan, A.-L.; Garg, M. L.; Moughan, P. J. Effects of season and plantation on phenolic content of unfermented and fermented Sri Lankan tea. Food Chem. 2014, 152, 546−551. (29) Tanaka, T.; Watarumi, S.; Fujieda, M.; Kouno, I. New black tea polyphenol having N-ethyl-2-pyrrolidinone moiety derived from tea amino acid theanine: Isolation, characterization and partial synthesis. Food Chem. 2005, 93, 81−87.

The authors declare no competing financial interest.

■

REFERENCES

(1) Sharangi, A. Medicinal and therapeutic potentialities of tea (Camellia sinensis L.)A review. Food Res. Int. 2009, 42, 529−535. (2) Ho, C.-T.; Zheng, X.; Li, S. Tea aroma formation. Food Sci. Hum. Wellness 2015, 4, 9−27. (3) Namita, P.; Mukesh, R.; Vijay, K. J. Camellia sinensis (green tea): A review. Global J. Pharmacol. 2012, 6, 52−59. (4) Tan, J.; Engelhardt, U. H.; Lin, Z.; Kaiser, N.; Maiwald, B. Flavonoids, phenolic acids, alkaloids and theanine in different types of authentic Chinese white tea samples. J. Food Compos. Anal. 2017, 57, 8−15. (5) Kaack, K.; Christensen, L. P. Effect of packing materials and storage time on volatile compounds in tea processed from flowers of black elder (Sambucus nigra L.). Eur. Food Res. Technol. 2008, 227, 1259−1273. (6) Katsuno, T.; Kasuga, H.; Kusano, Y.; Yaguchi, Y.; Tomomura, M.; Cui, J.; Yang, Z.; Baldermann, S.; Nakamura, Y.; Ohnishi, T.; Mase, N.; Watanabe, N. Characterisation of odorant compounds and their biochemical formation in green tea with a low temperature storage process. Food Chem. 2014, 148, 388−395. (7) Wang, L. F.; Kim, D. M.; Lee, C. Y. Effects of heat processing and storage on flavanols and sensory qualities of green tea beverage. J. Agric. Food Chem. 2000, 48, 4227−4232. (8) Nekvapil, T.; Kopriva, V.; Boudny, V.; Hostovsky, M.; Dvorak, P.; Malota, L. Decrease in the antioxidant capacity in beverages containing tea extracts during storage. Sci. World J. 2012, 2012, 1−5. (9) He, S.; Li, X.; Luo, C.; Chunfang, G.; Yun, S. Study on antibacterial activities of white tea produced in different years. Sci. Technol. Food Ind. 2016, 37, 164−168. (10) Friedman, M.; Levin, C. E.; Lee, S. U.; Kozukue, N. Stability of green tea catechins in commercial tea leaves during storage for 6 months. J. Food Sci. 2009, 74, H47−H51. (11) Ning, J.-M.; Ding, D.; Song, Y.-S.; Zhang, Z.-Z.; Luo, X.; Wan, X.-C. Chemical constituents analysis of white tea of different qualities and different storage times. Eur. Food Res. Technol. 2016, 242, 2093− 2104. (12) Herrero, M.; Simó, C.; García-Cañas, V.; Ibáñez, E.; Cifuentes, A. Foodomics: MS-based strategies in modern food science and nutrition. Mass Spectrom. Rev. 2012, 31, 49−69. (13) Hu, C.; Xu, G. Mass-spectrometry-based metabolomics analysis for foodomics. TrAC, Trends Anal. Chem. 2013, 52, 36−46. (14) Tan, J.; Dai, W.; Lu, M.; Lv, H.; Guo, L.; Zhang, Y.; Zhu, Y.; Peng, Q.; Lin, Z. Study of the dynamic changes in the non-volatile chemical constituents of black tea during fermentation processing by a non-targeted metabolomics approach. Food Res. Int. 2016, 79, 106− 113. (15) Dai, W.; Tan, J.; Lu, M.; Xie, D.; Li, P.; Lv, H.; Zhu, Y.; Guo, L.; Zhang, Y.; Peng, Q.; Lin, Z. Nontargeted modification-specific metabolomics investigation of glycosylated secondary metabolites in tea (Camellia sinensis L.) based on liquid chromatography−highresolution mass spectrometry. J. Agric. Food Chem. 2016, 64, 6783− 6790. (16) Dai, W.; Xie, D.; Lu, M.; Li, P.; Lv, H.; Yang, C.; Peng, Q.; Zhu, Y.; Guo, L.; Zhang, Y.; Tan, J.; Lin, Z. Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted metabolomics approach. Food Res. Int. 2017, 96, 40−45. (17) Dai, W.; Qi, D.; Yang, T.; Lv, H.; Guo, L.; Zhang, Y.; Zhu, Y.; Peng, Q.; Xie, D.; Tan, J.; Lin, Z. 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.). J. Agric. Food Chem. 2015, 63, 9869−9878. (18) Lv, H. P.; Zhu, Y.; Tan, J. F.; Guo, L.; Dai, W. D.; Lin, Z. Bioactive compounds from Pu-erh tea with therapy for hyperlipidaemia. J. Funct. Foods 2015, 19, 194−203. 7218


Metabolomics Investigation Reveals That 8-C N-Ethyl-2-pyrrolidinone

Recommend Documents