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

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Omics Technologies Applied to Agriculture and Food

Metabolomics investigation reveals 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02038 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of Agricultural and Food Chemistry

1

Metabolomics investigation reveals 8-C N-ethyl-2-pyrrolidinone

2

substituted flavan-3-ols are potential marker compounds of

3

stored white teas

4 5

Weidong Dai1, Junfeng Tan1, Meiling Lu2, Yin Zhu1, Pengliang Li1, Qunhua Peng1, Li

6

Guo1, Yue Zhang1, Dongchao Xie1, Zhengyan Hu3, **, Zhi Lin1, *

7 8

1

9

Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 Meiling South

Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture,

10

Road, Hangzhou, Zhejiang 310008, PR China

11

2

12

Beijing 100102, PR China

13

3

14

Hangzhou, Zhejiang 310051, PR China

Agilent Technologies (China) Limited, 3 Wangjing North Road, Chaoyang District,

Zhejiang Provincial Center for Disease Control and Prevention, 3399 Binsheng Road,

1

ACS Paragon Plus Environment


15

Abstract

16

White teas of different stored ages have varied flavor, bioactivity, and commercial

17

value. In this study, liquid chromatography-mass spectrometry (LC-MS)-based

18

metabolomics investigation revealed that there are distinct differences among the

19

compound patterns of Baihaoyinzhen (BHYZ) and Baimudan (BMD) white teas with

20

various storage durations. The levels of flavan-3-ols, procyanidins, theasinensins,

21

theaflavins, flavonol-O-glycosides, flavone-C-glycosides, and most of the amino acids

22

were reduced after long-term (>4 years) storage. More importantly, 8-C

23

N-ethyl-2-pyrrolidinone substituted flavan-3-ols (EPSFs), including 7 novel

24

compounds discovered in white teas for the first time, were formed from theanine and

25

flavan-3-ols during storage, and their contents were positively correlated with the

26

storage duration. These findings were further confirmed by the linearly increasing

27

formation of EPSFs in reaction solution and in BMD white teas stored in an

28

environment-controlled cabinet. In conclusion, EPSFs were detected in white teas for

29

the first time and were discovered as marker compounds and potential indicators for

30

long-term storage of white tea.

31 32

Keywords: white tea, storage, metabolomics, LC-MS, theanine, flavan-3-ol

2


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1 Introduction Tea is one of mostly consumed beverages in the world due to its health benefits and 1-3

35

satisfactory sensory experience

36

most popular teas, white tea is a rare form that undergoes the least amount of

37

processing (only withering and drying process are involved) and can be generally

38

classed into three types according to the quality of plucked fresh tea leaves:

39

Baihaoyinzhen (BHYZ, bud only, also called silver needle), Baimudan (BMD, a bud

40

with one or two leaves, also called white peony), and Shoumei (more than two leaves,

41

with or without a bud) 4.

42

. Compared with green tea and black tea, the two

Storage is crucial for the quality of teas and could change the aroma

5,6

, taste 7,

43

bioactivities, and chemical components of teas. White tea and pu-erh tea stored for

44

long periods are considered to have higher quality and commercial value. By contrast,

45

green tea stored for long periods is considered not fresh and umami. Some

46

bioactivities of teas were also found to change during storage. Nekvapil et al.

47

reported that the antioxidant capacity of beverages containing black, green, and white

48

tea extracts decreased during storage. He et al. 9 found that the anti-bacterial effect in

49

latest white tea was the best and decreased along with the extension of the storage

50

time.

51

8

Some investigations on chemical changes in stored teas have been carried out. 10

52

Friedman

studied the stability of green tea catechins and found that the average

53

overall decrease in the total catechin concentrations of 8 teas was 32% after storage at

54

20 ºC for 6 months. Ning et al.

11

investigated the changes in gallic acid, caffeine, 3



55

catechins, and amino acids in Shoumei white teas under different storage times using

56

ultra performance liquid chromatography coupled with a triple quadrupole mass

57

spectrometer (UPLC-QQQ-MS/MS) and found that the contents of catechins and

58

amino acids decreased with increased storage time, while the content of gallic acid

59

increased. These studies focused only on the major compounds or determined the total

60

contents of amino acids and flavonoids in teas. Therefore, a comprehensive

61

characterization of white tea metabolome during the white tea storage is urgently

62

needed. In addition, there is a lack of a survey on possible novel compounds that are

63

formed during storage.

64

Metabolomics enables the measurement of hundreds of endogenous compounds

65

simultaneously, providing a comprehensive view of the chemical compositions, and

66

has been widely used in food chemical research

67

ultrahigh performance liquid chromatography−quadrupole time-of-flight mass

68

spectrometry (UHPLC-Q-TOF/MS)-based metabolomics approach to investigate the

69

variations in the non-volatile compound profiles of white tea samples with various

70

storage durations and then surveyed novel potential marker compounds related to the

71

stored white tea.

12,13

. In this study, we used an

72 73

2 Materials and Methods

74

Chemicals

75

LC–MS-grade methanol was purchased from Merck (Darmstadt, Germany). Formic

76

acid (purity >96.0%), (-)-epigallocatechin (EGC, >95.0%), (+)-catechin (C, >98.0%), 4


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(-)-epigallocatechin gallate (EGCG, >95.0%), (-)-epicatechin gallate (ECG, >98.0%),

78

(-)-epicatechin

79

(+)-gallocatechin (GC, >95.0%), (-)-catechin gallate (CG, >98.0%), kaempferol

80

3-glucoside (>97.0%), kaempferol 3-galactoside (>90.0%), kaempferol 3-rutinoside

81

(>98.0%), vitexin (>95.0%), isovitexin (>98.0%), luteolin-8-C-glucoside (>97.0%),

82

quercetin 3-glucoside (>98.0%), tryptophan (>98.0%), glutamic acid (>99.0%),

83

pyroglutamic acid (>99.0%), proline (>99.0%), glutamine (>99.0%), aspartic acid

84

(>98.0%), γ-aminobutyric acid (GABA, >99.0%), leucine (>98.0%), isoleucine

85

(>99.0%), threonine (>98.0%), lysine (>98.0%), histidine (>99.0%), valine (>98.0%),

86

arginine (>99.0%), and adenine (>99.0%) were obtained from Sigma (St. Louis, MO,

87

USA). Myricetin 3-galactoside (>98.0%), procyanidin B1 (>98.0%), procyanidin B2

88

(>98.0%), theaflavin (TF, >98.0%), theaflavin-3-gallate (TF-3-g, >98.0%), and

89

theaflavin-3,3’-digallate (TF-3,3’-dg, >98.0%) were purchased from ChemFaces

90

(Wuhan, Hubei, China). Theanine (>99.0%), quercetin 3-galactoside (>97.0%),

91

quercetin 3-rhamnoglucoside (rutin, >98.0%), theobromine (>98.0%), tyrosine

92

(>99.0%), and phenylalanine (>99.0%) were obtained from J&K Scientific Ltd.

93

(Beijing, China). Caffeine (>99.0%) was purchased from Enzo Life Sciences Inc.

94

(Farmingdale, NY, USA). Epiafzelechin (>98.0%), strictinin (>95.0%), benzyl

95

glucoside, and phenylethyl glucoside were purchased from Yuanye Bio-Technology

96

Co., Ltd. (Shanghai, China). Deionized water was produced by a Milli-Q water

97

purification system (Millipore, Billerica, Massachusetts, USA).

98

Stored white tea sample collection and treatment

(EC,

>98.0%),

(-)-gallocatechin

5


gallate

(GCG,

>98.0%),


99

Two types of white tea samples (BHYZ and BMD, Camellia sinensis (L.) O.

100

Kuntze cv. Fuding Dabaicha) of various produced years (ranged from 2000 to 2015)

101

were collected in 2016 in this study. BHYZ and BMD white tea samples were

102

obtained from Fujian Pinpinxiang Tea Co., Ltd. (Fuding, Fujian, China) and Fujian

103

Yuda Tea Co., Ltd. (Fuding, Fujian, China), respectively (Table S1). BHYZ is

104

produced from only one bud, and BMD is produced from one bud with two leaves.

105

The raw fresh leaves were picked up from Fuding city of Fujian province in April and

106

were processed by skillful workers according to a typical white tea manufacturing

107

procedure which only includes withering and drying process (Dai et al., 2017). The

108

white tea products were naturally preserved in storehouse maintained at 15-25 °C and

109

25-50% humidity. Detailed information of the stored white tea samples was included

110

in Table S1. To investigate the variations in non-volatile compounds, BHYZ and

111

BMD tea samples were divided into 3 groups according to their storage durations: “1

112

year” group (1.0 ± 0.0 year (average ± SD) for the BHYZ and BMD samples), “2-4

113

year” group (2.9 ± 0.8 and 2.8 ± 0.8 years for the BHYZ and BMD samples,

114

respectively), and “> 4 year” group (6.2 ± 1.1 and 10.1 ± 2.8 years for the BHYZ and

115

BMD samples, respectively).

116

Forty milliliters of a 70 °C methanol/water solution (70/30: v/v) was added into 0.3

117

g of ground tea powder (< 0.15 mm) and incubated at 70 °C for 30 min to extract

118

nonvolatile compounds from white tea. Then, 1.5 mL of the solution was centrifuged

119

at 10,000 g (Centrifuge 5810R, Eppendorf) for 10 min and the supernatants were

120

passed through a 0.22 µm membrane. Each tea sample was prepared in duplicate. The 6


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121

obtained solution was then analyzed by UHPLC-QTOF/MS. Three internal standards

122

of flumequine, sulfafurazole, and sulfacetamide (0.2 µg/mL) were spiked into each tea

123

sample to evaluate the stability during the LC-MS analytical process. In addition,

124

quality control (QC) samples prepared by mixing equal amounts of each tea sample

125

were also used to evaluate the LC-MS analysis.

126

Metabolomics analysis

127

The metabolomics measurements of white tea samples were conducted following 14-16

128

the procedures that we developed previously

. Briefly, a UHPLC system (Infinity

129

1290, Agilent Tech., Santa Clara, CA, USA) coupled to a Q-TOF mass spectrometer

130

(6540, Agilent Tech., Santa Clara, CA, USA) was applied for the LC-MS analysis.

131

Chromatographic separation of the white tea compounds was performed on a Zorbax

132

Eclipse Plus C18 column (150 × 3.0 mm, 1.8 µm, Agilent Tech., Little Falls, DE,

133

USA). The column was maintained at 40 °C. Binary mobile phases were used for

134

gradient elution with the flow rate of 0.4 mL/min, where phase A was water

135

containing 0.1% (v/v) formic acid and phase B was methanol. The linear gradient

136

elution program was as follows: 0 min, 10% B; 4 min, 15% B; 7 min, 25% B; 9 min,

137

32% B; 16 min, 40% B; 22 min, 55% B; 28 min, 95% B; and 30 min, 95% B. 4 min

138

was allowed for column equilibration between two consecutive injections. The

139

injection volume was 3 µL. The Q-TOF mass spectrometer assembled an electrospray

140

ionization (ESI) source was operated in positive mode. The major MS parameters

141

were the same as those in previous reports 14,16. The mass scan range of m/z 100–1000

142

was applied for the full scan analysis. Reference ions with m/z 121.0509 (purine) and 7



143

922.0098 (hexakis phosphazene) were continuously infused via the reference sprayer

144

during data acquisition for online calibration to ensure the MS accuracy. The

145

compounds were identified according to authentic standards, accurate masses, MS2

146

spectra, metabolomics databases, and our previous works 14-18.

147

Metabolomics data processing

148

Raw data files acquired by LC-MS analysis were first processed by Profinder

149

software (Agilent Tech., Santa Clara, CA) for compound features extraction, and then

150

imported into Mass Profiler Professional software (Version 13.0, Agilent Tech., Santa

151

Clara, CA) for peak alignment. Ions with relative standard deviation (RSD) less than

152

30% in the QC samples were used for further univariate and multivariate statistics

153

19,20

154

Synthesis of 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols (EPSFs)

.

155

Theanine (2 g) and EGCG (2 g) were dissolved in 15 mL of methanol/formic

156

acid/water solution (40/1/60: v/v/v) and then heated at 50 °C for 12 days. The solution

157

was separated into 20 fractions by revered-phase column chromatography with a

158

gradient elution of methanol solution (from 15% to 80%). The fraction 10-12 were

159

combined and then separated into 20 fractions by revered-phase column

160

chromatography again to obtain 8-C N-ethyl-2-pyrrolidinone substituted EGCG. The

161

purity was 93% tested by a third-party laboratory using a UPLC-UV method at 280

162

nm. 1H NMR (600 MHz, DMSO-d6) δ 6.77 (d, J = 23.4 Hz, 2H), 6.40 (d, J = 19.0 Hz,

163

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,

164

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, 8


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165

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 –

166

1.57 (m, 1H), 0.98 – 0.77 (m, 3H). 13C NMR (151 MHz, DMSO-d6) δ 174.20, 168.29,

167

158.84, 158.53, 148.6, 148.3, 135.18, 131.31, 122.48, 111.74, 108.57, 108.50, 79.68,

168

70.75, 46.42, 36.3, 35.64, 29.02, 26.25, 15.45.

169

Flavan-3-ol standards of EGC, ECG, EC, GCG, GC, CG, and C were also used to

170

react with theanine in methanol/formic acid/water solution (40/1/60: v/v/v),

171

respectively. The solutions were subjected to LC-MS and LC-MS/MS analyses

172

without purification.

173

Identification and quantification of EPSFs in stored white tea

174

Compounds 1-7 were preliminarily identified formed from flavan-3-ols and

175

theanine according to accurate mass, MS2 spectrum, and reference

21

176

syntheses of compounds 1-7 in reaction solution containing standards of theanine and

177

flavan-3-ol (EGCG, EGC, ECG, EC, GCG, GC, CG, and C, respectively) were

178

carried out for the confirmation of chemical structures.

. Then, the

179

Compound 1 and 2 were found in the reaction solution containing theanine and

180

EGCG but not in the reaction solution containing theanine and GCG. Combining the

181

reported elution orders of stereoisomeric EPSFs

182

5’’S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (S-EGCG-cThea) and

183

5’’R-epigallocatechin

184

respectively. 5’’S-epicatechin gallate-8-C N-ethyl-2-pyrrolidinone (S-ECG-cThea)

185

and 5’’R-epicatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-ECG-cThea) were found

186

in the reaction solution containing theanine and ECG, but only R-ECG-cThea was

gallate-8-C

21,22

, they were identified as

N-ethyl-2-pyrrolidinone

9


(R-EGCG-cThea),


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187

detected in the stored white teas. Therefore, compound 3 was identified as

188

R-ECG-cThea. Both compounds 4 and 5 were found in the reaction solution

189

containing theanine and EGC but not in the reaction solution containing theanine and

190

GC. Both compounds 6 and 7 could be found in the reaction solution containing

191

theanine and EC but not in the reaction solution containing theanine and C.

192

Combining the reported elution orders of stereoisomeric EPSFs

193

structures of compounds 4-7 were identified as

5’’S-epigallocatechin-8-C

194


5’’R-epigallocatechin-8-C

195


196


197

N-ethyl-2-pyrrolidinone (R-EC-cThea), respectively. The MS2 spectra and putative

198

fragmentation pathways of compounds 1-7 are shown in Figure S1-S4.

199

Post-fermented pu-erh dark tea was also analyzed to confirm the compound

200

identifications, and only compounds 4-7 were detected in pu-erh tea which agreed

201

with Wang’s report 21.

202

(S-EGC-cThea), (R-EGC-cThea), (S-EC-cThea),

21,22

, the chemical

5’’S-epicatechin-8-C and

5’’R-epicatechin-8-C

The contents of the EPSFs (compounds 1-7) in white teas were quantified using a

203

purified

8-C


substituted

EGCG

standard

by

204

UHPLC-QTOF/MS. Standard solutions of 0.2, 0.5, 1.0, 5.0, and 20.0 µg/mL were

205

used to establish the calibration curve (r2 = 0.9996).

206

To investigate whether EPSFs are formed during the compound extraction process,

207

a methanol/water solution (70/30: v/v) containing 5 g/L EGCG and 2 g/L theanine

208

and a freeze-dried fresh tea leave sample were respectively heated at 70 °C for 30 min, 10


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209

and then were analyzed by UHPLC-QTOF/MS. Both results showed that there is no

210

formation of N-ethyl-2-pyrrolidinone substituted EGCG, which indicated that the

211

EPSFs are not formed during the compound extraction process.

212

Validation of EPSFs changes in stored white teas

213

To validate that the EPSFs were formed during the white tea storage and positively

214

related with the storage duration, two experiments were designed: (1) During the

215

synthesis of compounds 1-7 from standards of flavan-3-ols (EGCG, EGC, ECG, or

216

EC) and theanine, 50 µL of the reaction solution was sampled at 0, 1, 2, 3, 4, 5, and

217

12 days and the samples were immediately stored at -20 °C pending LC-MS analysis.

218

(2) BMD white tea (5 kg) was stored in an environment-controlled cabinet at 45 °C

219

and 35% humidity, and was sampled at 0, 0.5, 1, 1.5, 2, and 2.5 months. The samples

220

were immediately stored at -20 °C prior to analysis by UHPLC-QTOF/MS. Each

221

sample was prepared in quintuplicate.

222

Statistical analysis

223

Principal component analysis (PCA), partial least squares discriminant analysis

224

(PLS-DA), and PLS regression analysis were performed using Simca-P 11.5 software

225

(Umetrics AB, Umeå, Sweden) after weight-normalization and Pareto scaling to

226

investigate the overall tea compound profile variation among the 1 year, 2-4 year, and >

227

4 year BHYZ and BMD white teas and to screen the characteristic compounds in

228

stored white teas. Heat-map analysis and clustering analysis were performed by

229

MultiExperiment Viewer software (version 4.8.1) to illustrate the compound content

230

differences among white tea samples after data auto-scaling. Student’s t-test and 11



231

ANOVA were performed using PASWstat software (version 18.0, Chicago, IL, USA).

232 233

3 Results and Discussion

234

A total of 2584 compound ions were obtained after peak alignment and used for

235

unsupervised PCA analysis. The QC samples were clustered in the center of the PCA

236

score plot (Figure S5), indicating good reproducibility of the compound extraction

237

and LC-MS analysis in the metabolomics investigations. The BHYZ samples and

238

BMD samples were clearly separated, which indicated that the type of white tea has

239

larger influence on the non-volatile chemical constituents than the storage (Figure S5).

240

Therefore, the influence of storage on BHYZ and BMD were investigated separately.

241

Influence of storage on the white tea compounds

242

PCA and PLS-DA models were constructed to investigate the influence of storage

243

on the white tea compounds (Figure 1) and 50 times permutation test was applied for

244

the validation of the PLS-DA models. The permutation test showed y-intercepts for R2

245

and Q2 were 0.381 and -0.267 respectively for BHYZ PLS-DA model and were 0.328

246

and -0.271 respectively for BMD PLS-DA model, which indicated these two PLS-DA

247

models were not over-fitted. Distinct compound patterns of white tea samples stored

248

for 1 year, 2-4 years, and >4 years were observed in the two-dimensional score plots

249

of PCA and PLS-DA (Figure 1). These patterns indicated that the white tea compound

250

patterns obviously changed during storage. A total of 125 compounds, including

251

flavan-3-ols, dimeric flavan-3-ols (theaflavins, theasinensins, and procyanidins),

252

alkaloids, flavonol/flavone glycosides, amino acids, phenolic acids, nucleosides, 12


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253

organic acids, lipids, and carbohydrates, were identified to characterize the compound

254

variations (Figures 2 and 3).

255

Flavan-3-ols (catechins) are one group of characteristic and the most abundant

256

compounds in teas, which account for up to 10% of the content and are considered the

257

major bioactive constituents in white tea 4. Ning et al.

258

catechins, EGCG, EGC, ECG, and EC contents in “Shoumei” white tea significantly

259

decreased with the lengthening of the storage duration. In this study, flavan-3-ols,

260

including EGCG, ECG, EGC, GC, and GCG were slightly increased in the 2-4 year

261

white tea samples and then decreased the in >4 year white tea samples (Figure S6).

262

These phenomena were observed in both BHYZ and BMD. These results do not

263

totally agree with Ning’s findings 11 potentially due to the different types of white tea

264

used (BHYZ and BMD vs. Shoumei) and the different years of the white tea samples

265

collected in the study. After long-term (>4 years) storage, flavan-3-ols of EGCG, EC,

266

ECG, EGC, C, and epiafzelechin in white tea were significantly reduced in both

267

BHYZ and BMD. These results are consistent with Ning’s findings in Shoumei white

268

teas 11.

11

reported that the total

269

The decreased flavan-3-ols were not ultimately converted to dimeric flavan-3-ols

270

(theaflavins, theasinensins, and procyanidins) in the stored white teas. The levels of

271

theaflavins (TF, TF-3-g, theaflavins-3-gallate (TF-3’-g), and TF-3,3’-dg) decreased in

272

a stepwise manner during the storage process (Figure S7). The contents of

273

procyanidins

274

EC-(4->8)-EGCG) were slightly increased in the 2-4 year white teas and significantly

(procyanidin

B1,

procyanidin

B2,

13


EC-(4->8)-ECG,

and


275 276

Page 14 of 34

decreased in the >4 year white teas (Figure S7). Flavonol-O-glycosides and flavone-C-glycosides are also major phenolic 18,23

277

compounds in teas with a series of bioactivities

278

thresholds

279

3-glucoside,

280

3-glucosylrutinoside,

281

3-arabinoside),

282

3-galactoside, quercetin 3-rutinoside, quercetin 3-glucosylrutinoside, quercetin

283

3-galactosylrutinoside, quercetin diglucoside, and quercetin triglucoside), and 2

284

myricetin-O-glycosides

285

decreased

286

3-dicoumaroylglucoside, the contents of these flavonol-O-glycosides were lowest in

287

the >4 year white teas (both BHYZ and BMD). The levels of flavone-C-glycosides,

288

including

289

apigenin-6-C-glucosyl-8-C-arabinoside,

290

luteolin-8-C-glucoside also showed decreased trends in the 2-4 year and >4 year white

291

teas (Figure S8). These results did not agree with Zhou’s finding 26 that the content of

292

flavonoids increased during white tea storage and a significantly high content of

293

flavonoids was observed in white tea stored for 20 years. This disagreement may be

294

because a colorimetric method for the quantification of total flavonoids was used in

295

Zhou’s study while an LC-MS method was applied in this work.

296

and low astringent taste

24,25

. In this study, the contents of 6 kaempferol-O-glycosides (kaempferol kaempferol

7

during

3-galactoside,

kaempferol

kaempferol

3-galactosylrutinoside,

quercetin-O-glycosides

(myricetin

white

tea

3-rutinoside,

(quercetin

3-glucoside storage

apigenin-6,8-C-diglucoside,

and

(Figure

and

kaempferol

3-glucoside,

myricetin S8).

kaempferol

quercetin

3-galactoside)

Except

kaempferol

apigenin-6-C-arabinoside-8-C-glucoside, vitexin,

isovitexin,

and

The levels of most of amino acids (theanine, phenylalanine, valine, leucine, 14


Page 15 of 34


297

isoleucine, asparagine, proline, tryptophan, arginine, and tyrosine) decreased in a

298

stepwise manner in the stored white teas (Figure S9). This might be induced by the

299

Maillard reaction of amino acids with reducing sugars, which commonly occurs

300

during thermal processing and long storage of foods and leads to the browning of

301

foods

302

increased in the 2-4 year BHYZ white teas. The level of threonine also increased

303

during the storage of BHYZ white teas. Pyroglutamic acid, a cyclized derivative of

304

glutamic acid that can be formed nonenzymatically from glutamate and glutamine,

305

was increased significantly in the 2-4 year and >4 year BHYZ white teas as well as in

306

the >4 year BMD white teas. This indicated a nonenzymatic cyclization reaction of

307

glutamic acid or glutamine in white tea may occur during storage (Figure S9).

27

. The content of glutamic acid decreased in the stored BMD white teas, but

308

Aroma primeverosides are regarded as aroma precursors, which release aroma

309

compounds during the tea manufacturing and brewing processes 2. In this study, the

310

levels of all aroma primeverosides including benzyl primeveroside, phenylethyl

311

primeveroside, linalool primeveroside, and linalool oxide primeveroside were highest

312

in the 2-4 year BHYZ and BMD white teas and lowest in the 1 year BHYZ and BMD

313

white teas (Figure S10).

314

Here, it should be noted that the contents of some metabolites (e.g., flavan-3-ol) in

315

teas are susceptible to the climate factors, such as sunlight explosion, rainfall, and

316

temperature, etc., and can vary from year to year and also season to season

317

initial metabolite contents in white teas can affect the results.

318

EPSFs are potential marker compounds of stored white tea 15


17,28

. The


319

In addition to the compounds discussed above, consisting of flavan-3-ols, dimeric

320

flavan-3-ols, flavonol/flavone glycosides, amino acids, phenolic acids, nucleosides,

321

organic acids, lipids, and carbohydrates (Figures 2 and 3), we also found that several

322

compound features showed strong positive correlations with the storage duration. As

323

shown in Figure 4, the correlation coefficients between compounds 1-7 and the

324

storage duration ranged from 0.714 to 0.850 in the BMD white teas and ranged from

325

0.565 to 0.659 in the BHYZ white teas. In addition, in a PLS regression analysis of

326

the BHYZ and BMD white teas (the compound contents and the storage durations

327

were set as the X-variables and the Y-variable, respectively), compounds 1-7 were

328

among the most important contributors (Figure S11). Variable important for the

329

projection (VIP) was also used to evaluate the contribution of compounds 1-7 to the

330

total variance in the PLS-DA model (Figure 1-C and D). VIP values of them ranged

331

from 2.53 to 7.65 and from 2.29 to 8.40 in BHYZ and BMD PLS-DA model,

332

respectively (Table S3), which indicated that the content of compounds 1-7 distinctly

333

differ in white teas of different storage periods. Particularly, compounds 1-7 showed

334

significantly higher contents in BHYZ (Figure 5-A) and BMD (Figure 5-B) white teas

335

stored for a long period. Results of nonparametric Mann-Whitney U tests of EPSFs

336

between each two groups also indicated the contents of compounds 1-7 in > 4 years

337

group were significantly higher (Table S2).

338

Compounds 1-7 showed accurate masses of m/z 570.1609 (compounds 1 and 2),

339

554.1663(compound 3), 418.1500 (compounds 4 and 5), and 402.1555 (compounds 6

340

and 7) in positive ionization mode, which indicated possible formulas of C28H27NO12, 16


Page 16 of 34

Page 17 of 34


341

C28H27NO11, C21H23NO8, and C21H23NO7, respectively. Compound 1 and 2, 4 and 5,

342

as well as 6 and 7 had nearly identical MS2 spectra, respectively (Figure S1-S4). In

343

addition, compounds 1-7 had common MS2 fragment ions of m/z 262.1074, 250.1074,

344

and 205.0490, which indicated that these compounds have the same structural

345

skeleton. The accurate masses (m/z 418.1500 and 402.1555) and MS2 spectra of

346

compounds 4-7 perfectly matched with those of the EPSFs reported by Wang et al. 21.

347

These compounds were found in post-fermented dark teas and were formed from

348

flavan-3-ols and theanine

349

solution containing standards of theanine and flavan-3-ol (EGCG, EGC, ECG, EC,

350

GCG, GC, CG, and C, respectively), these 7 compounds were finally identified as 8-C

351

N-ethyl-2-pyrrolidinone substituted EGCG (S-EGCG-cThea and R-EGCG-cThea),

352

8-C

353

N-ethyl-2-pyrrolidinone substituted EGC (S-EGC-cThea and R-EGC-cThea), and 8-C

354

N-ethyl-2-pyrrolidinone substituted EC (S-EC-cThea and R-EC-cThea), respectively

355

(Figure 6-A). The total contents of these 7 compounds were 0.754 ± 0.080, 0.761 ±

356

0.253, and 1.340 ± 0.28 mg/g in the 1 year, 2-4 year, and >4 year BHYZ white teas,

357

respectively, and 1.009 ± 0.060, 1.028 ± 0.152, and 1.930 ± 0.438 mg/g in the 1 year,

358

2-4 year, and >4 year BMD white teas, respectively (Figure 5-A). To the best of our

359

knowledge, S-EGCG-cThea, R-EGCG-cThea, and R-ECG-cThea were discovered in

360

teas for the first time, and these 7 compounds were found in white teas for the first

361

time.

362

21

. Combining the syntheses of compounds 1-7 in reaction


substituted

ECG

(R-ECG-cThea),

8-C

The proposed route for the formation of EPSFs in stored white tea is shown in 17



Page 18 of 34

363

Figure 6-B. Theanine converts to 1-ethyl-5-hydroxy-2-pyrrolidinone after Strecker

364

degradation and cyclization 21,29, and this product then reacts with flavan-3-ols to form

365

EPSFs. The correlation coefficients between EPSFs and theanine and between EPSFs

366

and flavan-3-ols were calculated and are shown in Figure 5-C. EPSFs displayed

367

negative correlations with flavan-3-ols and theanine (R ranged from -0.76 to 0.13).

368

These results provide a new insight into the interactions between primary compounds

369

of

370

N-ethyl-2-pyrrolidinone substituted GCG, CG, GC and C were not found in the stored

371

white teas potentially due to the much lower contents of GCG, CG, GC and C than

372

EGCG, ECG, EGC and EC in the white teas.

373

Validation of EPSF changes in stored white teas

catechins

and

theanine

in

teas

during

long-term

storage.

8-C

374

To further validate that the EPSFs were formed during the white tea storage and

375

that their contents were positively related to the storage duration, two experiments

376

were designed. As shown in Figure 7-A and B, the contents of compounds 1-7

377

increased in a stepwise manner in the reaction solutions at 1, 2, 3, 4, 5, and 12 days.

378

The linear correlation coefficients ranged from 0.955 to 0.999. In the white tea

379

samples stored in an environment-controlled cabinet, the contents of compounds 1-7

380

also increased in a stepwise manner over 2.5 months. The linear correlation

381

coefficients ranged from 0.929 to 0.999 for compounds 1-5 and were 0.747 and 0.728

382

for compounds 6 and 7, respectively (Figure 7-C, D). These results demonstrated that

383

the formation of EPSFs from theanine and flavan-3-ols is linearly correlated to the

384

storage duration (or reaction duration). These results indicated that EPSFs are marker 18


Page 19 of 34


385

compounds of stored white tea, which are potential markers for the discrimination of

386

the storage duration of white teas.

387

In addition, it was believed that aged white teas have stronger effects against flu 26

388

and inflammation in folk medicine

, and 8-C N-ethyl-2-pyrrolidinone substituted

389

EGC, GC, EC, and C were reported to have potential protective effects on human

390

microvascular endothelial cells (HMEC) injury induced by hydrogen dioxide

391

compared to other tea polyphenols

392

will be carried out in the future. Furthermore, studies of EPSFs during the storage of

393

other teas such as green, black, and dark teas are also needed.

21

. Therefore, bioactivity investigations of EPSFs

394 395 396

Supporting Information description

397

Information of included white tea samples (Table S1); Nonparametric Mann-Whitney

398

U tests of EPSFs (Table S2); VIP values of EPSFs in PLS-DA models (Table S3);

399

MS2 spectra and the putative fragmentation pathways of 8-C N-ethyl-2-pyrrolidinone

400

substituted flavan-3-ols (Figure S1-S4); The PCA score plot of white teas and QC

401

samples (Figure S5); Changes of catechins, dimeric catechins, flavone glycosides,

402

flavonol glycosides, amino acids, and aroma glycosides during white tea storing

403

(Figure S6-S10); PLS regression analysis of white tea storage duration and compound

404

contents (Figure S11). These materials are available free of charge via the Internet at

405

http://pubs.acs.org.

406 19



407

Corresponding Author

408

*(Z. L.) Phone: +86 571 86650617. Email: [email protected].

409

**(Z. H.) Phone: +86 571 87115272. Email: [email protected].

410 411

Funding

412

The authors appreciate the funding support from the Central Public-Interest Scientific

413

Institution Basal Research Fund (No. 1610212016008 & 1610212018009), the

414

National Natural Science Foundation of China (No. 31500561 & 31501565), and the

415

Earmarked Fund for China Agricultural Research System (No. CARS-19).

416 417

Notes

418

The authors declare no competing financial interest.

20


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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 21



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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. (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. Pharmaceut. 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 23



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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

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(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.

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Figure captions Figure 1. PCA analysis of the compound patterns of BHYZ (A) and BMD (B) white teas with different storage durations; PLS-DA analysis of the compound patterns of BHYZ (C) and BMD (D) white teas with different storage durations. Figure 2. Heat-map of the compound contents in BHYZ white tea samples with different storage durations. Figure 3. Heat-map of the compound contents in BMD white tea samples with different storage durations. Figure 4. (A) Extracted ion chromatograms (EICs) of EPSFs in stored white teas; (B) the Pearson correlation coefficients (R = 0.685-0.846) between compound 1-7 and storage periods in BMD white teas. Figure 5. The contents of EPSFs in BHYZ (A) and BMD (B) 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. Figure 6. (A) Chemical structures of compounds 1-7; (B) Proposed route for the formation of EPSFs. Figure 7. Linearly increasing formations of 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols in reaction solution (containing standards of theanine and flavan-3-ols) within 12 days (A and B) and in Baimudan white teas stored in an environment-controlled cabinet within 2.5 months (C and D, n = 5).

26


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Figure 1

27



Figure 2 28


Page 28 of 34

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Figure 3

29



Figure 4

30


Page 30 of 34

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Figure 5

31



Figure 6

32


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Page 33 of 34


Figure 7

33



Graphic for table of contents

34


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

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