Article

Cite This: J. Agric. Food Chem. 2018, 66, 7209−7218 pubs.acs.org/JAFC

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

pyrrolidinone-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*,†
†
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
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

§
Zhejiang Provincial Center for Disease Control and Prevention, 3399 Binsheng Road, Hangzhou, Zhejiang 310051, People’s
Republic of China
*
S Supporting Information
Downloaded via DURHAM UNIV on July 13, 2018 at 09:55:43 (UTC).

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-3-
ols 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 environment-
controlled 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

1. INTRODUCTION storage at 20 °C for 6 months. Ning et al.11 investigated the

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
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 character-
ization 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 compre-
hensive 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−quad-
rupole time-of-flight mass spectrometry (UHPLC−QTOF/
MS)-based metabolomics approach to investigate the
variations in the non-volatile compound profiles of white tea
Received: April 19, 2018
Revised: May 23, 2018
Accepted: June 19, 2018
Published: June 19, 2018

© 2018 American Chemical Society 7209 DOI: 10.1021/acs.jafc.8b02038

J. Agric. Food Chem. 2018, 66, 7209−7218
Journal of Agricultural and Food Chemistry
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 3-
galactoside (>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 theaflavin-
3,3′-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%), theobro-
mine (>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 non-
volatile 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 (<0.15 mm) and incubated
at 70 °C for 30 min to extract non-volatile compounds from white tea.
Then, 1.5 mL of the solution was centrifuged at 10000g (Centrifuge
5810R, Eppendorf) for 10 min, and the supernatants were passed
through a 0.22 μm membrane. Each tea sample was prepared in
duplicate. The obtained solution was then analyzed by UHPLC−
QTOF/MS. Three internal standards of flumequine, sulfafurazole,
and sulfacetamide (0.2 μg/mL) were spiked into each tea sample to
evaluate the stability during the LC−MS analytical process. In
addition, quality control (QC) samples prepared by mixing equal
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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 N-
ethyl-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
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Figure 1. PCA of the compound patterns of (A) BHYZ and (B) BMD white teas with different storage durations. PLS-DA of the compound
patterns of (C) BHYZ and (D) BMD white teas with different storage durations.
of stereoisomeric EPSFs, 21,22 they were identified as 5′′′S-
epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (S-EGCG-
cThea) and 5′′′R-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone
(R-EGCG-cThea), respectively. 5′′′S-Epicatechin gallate-8-C N-ethyl-
2-pyrrolidinone (S-ECG-cThea) and 5′′′R-epicatechin gallate-8-C N-
ethyl-2-pyrrolidinone (R-ECG-cThea) were found in the reaction
solution containing theanine and ECG, but only R-ECG-cThea was
detected in the stored white teas. Therefore, compound 3 was
identified as R-ECG-cThea. Both compounds 4 and 5 were found in
the reaction solution containing theanine and EGC but not in the
reaction solution containing theanine and GC. Both compounds 6
and 7 could be found in the reaction solution containing theanine and
EC but not in the reaction solution containing theanine and C. In
combination with the reported elution orders of stereoisomeric
EPSFs,21,22 the chemical structures of compounds 4−7 were
identified as 5″S-epigallocatechin-8-C N-ethyl-2-pyrrolidinone (S-
EGC-cThea), 5″R-epigallocatechin-8-C N-ethyl-2-pyrrolidinone (R-
EGC-cThea), 5″S-epicatechin-8-C N-ethyl-2-pyrrolidinone (S-EC-
cThea), and 5″R-epicatechin-8-C N-ethyl-2-pyrrolidinone (R-EC-
cThea), respectively. The MS2 spectra and putative fragmentation
pathways of compounds 1−7 are shown in Figures S1−S4 of the
Supporting Information. Post-fermented pu-erh dark tea was also
analyzed to confirm the compound identifications, and only
compounds 4−7 were detected in pu-erh tea, which agreed with
the report by Wang et al.21
The contents of the EPSFs (compounds 1−7) in white teas were
quantified using a purified 8-C N-ethyl-2-pyrrolidinone-substituted
EGCG standard by UHPLC−QTOF/MS. Standard solutions of 0.2,
0.5, 1.0, 5.0, and 20.0 μg/mL were used to establish the calibration
curve (r2 = 0.9996).
To investigate whether EPSFs are formed during the compound
extraction process, a methanol/water solution (70:30, v/v) containing
5 g/L EGCG and 2 g/L theanine and a freeze-dried fresh tea leave
sample were heated at 70 °C for 30 min, respectively, and then
analyzed by UHPLC−QTOF/MS. Both results showed that there is
no formation of N-ethyl-2-pyrrolidinone-substituted EGCG, which
indicated that the EPSFs are not formed during the compound
extraction process.
2.7. Validation of EPSFs Changes in Stored White Teas. To
validate that EPSFs were formed during the white tea storage and
positively related with the storage duration, two experiments were
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designed: (1) During the synthesis of compounds 1−7 from
standards of flavan-3-ols (EGCG, EGC, ECG, or EC) and theanine,
50 μL of the reaction solution was sampled at 0, 1, 2, 3, 4, 5, and 12
days and the samples were immediately stored at −20 °C pending
LC−MS analysis. (2) BMD white tea (5 kg) was stored in an
environment-controlled cabinet at 45 °C and 35% humidity and was
sampled at 0, 0.5, 1, 1.5, 2, and 2.5 months. The samples were
immediately stored at −20 °C prior to analysis by UHPLC−QTOF/
MS. Each sample was prepared in quintuplicate.
2.8. Statistical Analysis. Principal component analysis (PCA),
partial least squares discriminant analysis (PLS-DA), and partial least
squares (PLS) regression analysis were performed using Simca-P 11.5
software (Umetrics AB, Umeå, Sweden) after weight normalization
and Pareto scaling to investigate the overall tea compound profile
variation among the 1, 2−4, and >4 year BHYZ and BMD white teas
and to screen the characteristic compounds in stored white teas. Heat-
map analysis and clustering analysis were performed by MultiExperi-
ment 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 reproduci-
bility 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
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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-
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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
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Figure 3. Heat map of the compound contents in BMD white tea samples with different storage durations.

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,
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including flavan-3-ols, dimeric flavan-3-ols (theaflavins, the-
asinensins, and procyanidins), alkaloids, flavonol/flavone
glycosides, amino acids, phenolic acids, nucleosides, organic
DOI: 10.1021/acs.jafc.8b02038
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Journal of Agricultural and Food Chemistry
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.
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
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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
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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.
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 procyani-
dins) 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 3-
galactosylrutinoside, and kaempferol 3-arabinoside), seven
quercetin-O-glycosides (quercetin 3-glucoside, quercetin 3-
galactoside, quercetin 3-rutinoside, quercetin 3-glucosylrutino-
side, 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 flavone-
C-glycosides, including apigenin-6,8-C-diglucoside, apigenin-6-
C-arabinoside-8-C-glucoside, apigenin-6-C-glucosyl-8-C-arabi-
noside, 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,
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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-enzymati-
cally 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, temper-
ature, 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
DOI: 10.1021/acs.jafc.8b02038
J. Agric. Food Chem. 2018, 66, 7209−7218
Journal of Agricultural and Food Chemistry
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 com-
pounds have the same structural skeleton. The accurate masses
(m/z 418.1500 and 402.1555) and MS2 spectra of compounds
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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-pyrrolidi-
none-substituted EGCG (S-EGCG-cThea and R-EGCG-
cThea), 8-C N-ethyl-2-pyrrolidinone-substituted ECG (R-
ECG-cThea), 8-C N-ethyl-2-pyrrolidinone-substituted EGC
(S-EGC-cThea and R-EGC-cThea), and 8-C N-ethyl-2-
pyrrolidinone-substituted EC (S-EC-cThea and R-EC-
cThea), 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, S-
EGCG-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-ethyl-
5-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
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Journal of Agricultural and Food Chemistry
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-2-
pyrrolidinone-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
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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: zhyhu@cdc.zj.cn.
*Telephone: +86-571-86650617. E-mail: linzhi@caas.cn.
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).
DOI: 10.1021/acs.jafc.8b02038
J. Agric. Food Chem. 2018, 66, 7209−7218
Journal of Agricultural and Food Chemistry Article

Notes (19) Dai, W. D.; Wei, C.; Kong, H. W.; Jia, Z. H.; Han, J. K.; Zhang,
The authors declare no competing financial interest. 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
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