LC-MS-based metabolomics reveals the chemical changes of

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Omics Technologies Applied to Agriculture and Food

LC-MS-based metabolomics reveals the chemical changes of polyphenols during high-temperature roasting of large-leaf yellow tea Jie Zhou, You Wu, Piaopiao Long, Chi-Tang Ho, Yijun Wang, Zhipeng Kan, Luting Cao, Liang Zhang, and Xiaochun Wan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05062 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Journal of Agricultural and Food Chemistry

LC-MS-based metabolomics reveals the chemical changes of polyphenols during high-temperature roasting of large-leaf yellow tea Jie Zhou, † ,‡,# You Wu, † ,‡,#, Piaopiao Long, † ,‡, Chi-Tang Ho, ‡ ,§, Yijun Wang, † ,‡, Zhipeng Kan,†,‡, Luting Cao,†,‡, Liang Zhang,*,†,‡ and Xiaochun Wan*,†,‡ †

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

University, 130 West Changjiang Road, Hefei 230036, China ‡

International Joint Laboratory on Tea Chemistry and Health Effects, Anhui

Agricultural University, 130 West Changjiang Road, Hefei 230036, China. §Department

#

of Food Science, Rutgers University, New Brunswick, NJ, USA.

These authors contributed equally.

*Corresponding author: Tel./Fax: +86-551-65786765, E-mail: [email protected] (L. Zhang); [email protected] (XC. Wan). Declarations of conflict of interest: none

1

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1

ABSTRACT

2

Large-leaf yellow tea (LYT) is made from mature tea leaves with stems and has

3

unique sensory characteristics different from other teas. To study the chemical

4

changes of LYT during processing, samples were collected from each step for

5

quantitative and qualitative analyses by high performance liquid chromatography

6

(HPLC) and liquid chromatography-mass spectrometry (LC-MS). LC-MS based

7

non-targeted and targeted metabolomics analyses revealed that the tea sample after

8

roasting was markedly different from samples before roasting, with the levels of

9

epi-catechins and free amino acids significantly decreased, but the epimerized

10

catechins increased dramatically. After accounting for common compounds in tea,

11

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols were found to be the marker

12

compounds responsible for the classification of all samples, as they rapidly rose with

13

increasing processing temperature. These findings suggested that the predominant

14

changes in the tea constituents during large-leaf yellow tea roasting were the

15

thermally induced degradation and epimerization of catechins and the formation of

16

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols from L-theanine.

17

Keywords:

18

(-)-Epigallocatechin gallate

Large-leaf

yellow

tea;

Roasting;

19

2


Metabolomics;

L-theanine;

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20

INTRODUCTION

21

Large-leaf yellow tea (LYT) is a special variety of tea in China mainly produced in

22

the Dabie mountain area.1 To make LYT, the mature fresh tea leaves with stems are

23

harvested mechanically in the summer and autumn and then processed through six

24

steps to yield the final tea product. The main processes are fixation, rolling, first

25

drying, yellowing, second drying and roasting (Figure 1). The three initial steps are

26

similar to those used to manufacture green tea. Fixation, rolling, and the first drying

27

deactivate the endogenous enzymes such as polyphenol oxidases and peroxidases in

28

fresh tea leaves and reduce the moisture content to about 10%. After first drying, the

29

tea leaves are piled for a yellowing step (light fermentation) for a week, and then

30

dried under 120-140 ℃ for several minutes (second drying). The last step is roasting

31

under high temperature (140-150 ℃), which brings forth the brown color and lightly

32

astringent taste of the final yellow tea product.

33

Roasting is a critical step in the shaping of quality and sensory characteristics of

34

products such as coffee and cocoa bean, but is seldom used in the processing of tea

35

products.2, 3 Only a few tea products are produced by a roasting process, such as Wuyi

36

Rock Tea and LYT. During LYT manufacturing, the processing temperature increases

37

gradually with time, resulting in LYT having a darker color than the semi-fermented

38

teas. The polymerization, oxidation and degradation of tea polyphenols is thought to

39

happen during the high-temperature step.4-6 The main bioactive component of tea,

40

(-)-epigallocatechin gallate (EGCG) and its dimers can be decomposed to gallic acid

41

and

polymerized

into

pigments

at

high

temperature.7

3


Furthermore,

the


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42

high-temperature treatment leads to the roasted and caramel aromas and flavors of

43

LYT due to the Maillard reactions of sugars and amino acids in the tea.8

44

The different processes used to make teas lead to the distinctive chemical

45

profiles, aromas, tastes, colors and biological activities of the various teas.9,10 The

46

main compounds in fresh tea leaves are flavan-3-ols, alkaloids, flavonoids and other

47

phenolic acids. These secondary metabolites change with the manufacture process.

48

For example, in rolled and crushed tea leaves, the polyphenol oxidases (PPO) convert

49

the simple catechins into theaflavins, whereas the microbial post-fermentation of

50

ripened pu-erh tea increases the gallic acid level by hydrolysis of galloylated

51

catechins.11,

52

induce other chemical transformation of polyphenols.13-15 Recently, some novel

53

compounds, especially catechin derivatives, have been identified in ripened pu-erh

54

tea, black tea, white tea and in certain varieties of Camellia sinensis.16-20 Among these

55

compounds,

56

Strecker-degradation product of L-theanine at the C-8 or C-6 position (high

57

nucleophilic centers) of the A-ring of flavan-3-ols.21 About eighteen N-ethyl

58

pyrrolidinone-substituted flavan-3-ols have been isolated and identified from tea

59

products

60

pyrrolidinone-substituted EGCG and ECG. While these compounds occur at low

61

levels, they often impart the specific and desirable characteristics of some special teas.

62

It is clear that some of these unique flavoalkaloids found in ripened pu-erh tea are

63

created during microbial post-fermentation.16 However, it remains unknown the

12

Furthermore, the pH, presence of metal ions, and the temperature

flavoalkaloids

and

C.

are

sinensis

derived

varieties,

from

most

4


the

of

substitution

which

are

of

the

N-ethyl

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64

formation of these compounds during processing of other types of tea.

65

Tea has been widely reported to have many health benefits. We have found that,

66

compared with other teas, LYT possesses a competitive hypoglycemic effect,1 and

67

suppressing liver toxicity induced by carbon tetrachloride in rats.22 LYT also has the

68

ability to regulate postprandial blood glucose and attenuates macrophage-related

69

chronic inflammation and metabolic syndrome in mice fed a high-fat diet and in db/db

70

mice.23, 24 However, the active compounds in LYT have not been identified. So far, it

71

is still unknown if the main chemical compounds of LYT are different from other

72

teas. While LYT likely contains the same compounds, found in various other teas,

73

such as caffeine, simple catechins and flavonoid glycosides. Therefore, a

74

high-throughput tool is required to find the unique compounds present in LYT.

75

Metabolomics is a potent tool for clarifying the differences of teas grown under

76

different geographies and shading treatments and processed using different

77

techniques.25-27 Non-targeted metabolomics assists in finding compounds that can be

78

used as markers of different samples, but its results are usually distorted by the high

79

levels of main chemical compounds, making it hard to see constituents that occur at

80

lower amounts. For example, the main tea polyphenols, e.g. EGCG and other

81

catechins, are the main variable compounds between tea samples. Targeted

82

metabolomics is more suitable for detection of minor or trace secondary metabolites.

83

The aim of the present study was to ascertain the changes in the main tea polyphenols

84

found in LYT during processing and to explore the process-related chemical markers

85

by both non-targeted and targeted metabolomics. 5



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86

MATERIALS AND METHODS

87

Samples and Chemicals

88

All the large-leaf yellow tea samples were produced by Huibinyi Tea Co, Ltd.

89

(Huoshan, Anhui, China). Fresh, mature tea leaves with stems (Camellia sinensis (L.)

90

O. Kuntze) were harvested in the summer (mid-June). Samples (about 1000 g) were

91

taken after each of the seven consecutive processes, namely fresh leaves (FL), fixation

92

(F), rolling (RL), first drying (FD), yellowing (Y), second drying (SD), and roasting

93

(RS, the final LYT product). Three tea samples for each processing step were

94

collected, immediately freeze-dried, and stored at -30 C before analysis.

95

Standards

for

DL-4-Chlorophenylalanine

(>98%

purity),

Gallic

acid

96

(GA, >98%), eighteen kinds of amino acids (including L-theanine, >99%), caffeine

97

(CAF, >98%), theobromine (THB, >98%), (+)-catechin (C, >98%), (-)-epicatechin

98

(EC, >98%), (-)-gallocatechin (GC, >98%), (-)-epigallocatechin (EGC, >98%),

99

(-)-gallocatechin gallate (GCG, >98%), (-)-epigallocatechin gallate (EGCG, >98%)

100

and (-)-epicatechin gallate (ECG, >98%) were purchased from Yuanye Biotechnology

101

Company (Shanghai, China). HPLC and LC/MS-grade acetonitrile, methanol and

102

water were purchased from Thermo Fisher Scientific Co. (Fair Lawn, NJ, USA).

103

Other reagents were of analytical grade.

104

Sample Preparation

105

All he tea samples were milled with a pulverizer (A11, IKA, Staufen, Germany). A

106

100 mg aliquot of each sample was weighed into a 5-mL centrifuge tube before the

107

addition of 3 mL of 70% methanol in water solution (v/v). Samples were extracted 6


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108

thrice by ultrasound at room temperature for 10 min. The extracts were centrifuged at

109

5000 ×g for 10 min. Three separate extracts per processing step were combined and

110

diluted with 70% methanol to 10 mL. Each sample was prepared in six replicates. The

111

supernatants were filtered through a 0.22-µm Millipore filter into a centrifuge tube,

112

and then diluted to 2.5 mg/mL for HPLC and UPLC-Q-TOF/MS analysis, with 5 µL

113

of DL-4-chlorophenylalanine (1 mg/mL) added as the internal standard. A quality

114

control (QC) sample was prepared by mixing an equal volume of each test sample (20

115

μL), which was injected after every twelve tea samples.

116

Analyses of Free Amino Acids

117

To prepare the sample for the analyses of free amino acids and L-theanine, 1.5 g of

118

tea sample was mixed with 220 mL of water under boiling water bath for 45 min. This

119

mixture was transferred into a 250-mL volumetric flask and water was added to 250

120

mL line. Pre-column derivatization of free amino acids and L-theanine was performed

121

using the AccQ-Fluor Reagent Kit according to the manufacturer’s specifications. The

122

contents of free amino acids and L-theanine were determined on a Waters ACQUITY

123

UPLC H-Class system (Waters, Milford, MA, USA) equipped with a binary solvent

124

delivery pump, an auto sampler, and a photodiode array detector (PDA) and

125

controlled by the Empower-II software.

126

Separation was performed on an HPLC system equipped with a Waters AccQ

127

Tag reversed-phase HPLC column (150 mm × 3.9 mm, 4 μm), according to the

128

manufacturer’s specifications, with slight modifications. Briefly, mobile phase A

129

consisted of AccQ Tag Eluent A Concentrate in deionized water (1:10, v/v), mobile 7



130

phase B was acetonitrile, and mobile phase C was deionized water. A gradient

131

program was used for the separation of amino acids: 0–17 min, 100% A; 17–24min,

132

linear gradients from 100% to 91% A, 0% to 5% B, and 0%-4% C; 17–24 min, linear

133

gradients from 91% to 80% A, 5% to 17% B, and 4%-3% C; 24–32 min, linear

134

gradients from 80% to 68% A, 17% to 20% B, and 3%-12% C; 32–34 min, a constant

135

ratio of 68% A, 20% B, and 12% C; 34–35 min, linear gradients from 68% to 0% A,

136

20% to 40% B, and 12%-60% C; 35–37 min, a constant ratio of 0% A, linear

137

gradients from 40% to 60% B, and 60%-40% C; 37–38 min, linear gradients from

138

0%-100% A, 60% to 0% B, and 40%-0% C; and 38-45 min, a constant ratio of 100%

139

A, 0% B, 0% C. The sample injection volume was 5 μL. Flow rate was 1.0 mL/min.

140

Column temperature was set at 30 ℃. Amino acids were detected at 254 nm.

141

Determination of Tea Polyphenols and Purine Alkaloids

142

An Agilent 1260 Infinity HPLC system (Agilent Technologies, Palo Alto, CA, USA),

143

consisting of an Infinity binary pump, integrated vacuum degasser, auto sampler,

144

thermostated column compartment, and diode array detector (DAD), was used to

145

determine tea polyphenols and purine alkaloids. The analytical column used was an

146

Agilent SB-Aq C18 reversed phase column (250 mm × 4.6 mm i.d., 5 μm) protected

147

with a Phenomenex C18 guard column (10 mm × 4.6 mm, 5 μm; Phenomenex,

148

Torrance, CA, USA). The chromatographic conditions were the same as previously

149

reported,28 and the contents of each analyte were calculated with a regression

150

equation.

151

Non-targeted Metabolomics Analysis 8


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152

Non-targeted metabolomics analysis was performed on a UHPLC-ESI-MS system

153

consisting of an Agilent 6545 tandem Quadrupole time-of-flight mass spectrometer

154

(Q-TOF-MS) (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent

155

1290 series HPLC system (Agilent Technologies) equipped with an auto-injector and

156

a binary solvent delivery system. Separation was carried out on an Acquity UPLC

157

shield RP-18 column (50 mm × 2.1 mm, 1.7 μm) equipped with an Acquity UPLC

158

C18 guard column (Waters, Milford, MA, USA) at a flow rate of 0.3 mL/min, a

159

column temperature of 30 ℃, and a detection wavelength of 278 nm. The mobile

160

phase consisted of 0.1% formic acid/water (v/v, A) and acetonitrile (B). The gradient

161

elution was 0-5 min, 5-15% B; 5-8 min, 15-30% B; 8-13 min, 30-30% B; 13-23 min,

162

30-88% B; 23-28 min, 88-95% B; 28-30 min, 95-95% B; 30-33 min, 95-5% B; and

163

33-35 min, 5-5% B. The injection volume was 2 μL. During the analysis, the

164

instrument parameters were set as follows: capillary voltage, 3500 V for the positive

165

ion polarity mode; gas temperature, 320 ℃; sheath gas temperature, 350 ℃; sheath

166

gas flow, 11 L/min; gas flow, 8 L/min; and nebulizer, 35 psi. The mass scan range

167

was m/z 100-1500 in negative ionization mode.

168

Data Processing

169

All of the MS raw data files were imported into MS-DIAL (version 2.74) for data

170

processing, including retention time (min), mass-to-charge ratio (m/z) value, and MS

171

intensity of each feature. Principal Component Analysis (PCA) of the entire data set

172

was performed by SIMCA-P (version 14.1, Umetrics, Umeå, Sweden). The data

173

within a 95% confidence interval was accepted. The results obtained give a rough 9



174

classification of all tea samples. Then, the data was autofitted by supervised Partial

175

Least Squares Analysis (PLS) and Orthogonal Least Squares Analysis (OPLS). The

176

obtained results were subjected to cluster analysis and discriminant analysis.

177

Hierarchical Cluster Analysis (HCA) classified the similarities and differences among

178

these LYT processing samples, with Orthonormal Partial Least Squares Discriminant

179

Analysis (OPLS-DA) applied to classify samples of solely Y variables. Based on the

180

analysis of the results of all the models, several of the most important marker

181

compounds were obtained.

182

Targeted Metabolomics Study

183

Targeted metabolomics analysis was performed on a UHPLC-ESI-MS system

184

consisting of an Agilent 6545 tandem Quadrupole time-of-flight mass spectrometer

185

(Q-TOF-MS) (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent

186

1290 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with

187

an auto-injector and a binary solvent delivery system. Separation was achieved using

188

an Acquity UPLC shield RP-18 column (50 mm × 2.1 mm, 1.7 μm) equipped with an

189

Acquity UPLC C18 guard column (Waters, Milford, MA, USA) at a flow rate of 0.3

190

mL/min and a column temperature of 30 ℃, while the detection wavelength was set at

191

278 nm. The mobile phase consisted of 0.1% formic acid in water (v/v) (A) and

192

acetonitrile (B), with the gradient elution at 0-10 min: 5-15% B, 10-20 min: 15-35%

193

B, 20-23 min: 35-35% B, 23-26 min: 35-88% B, 26-28 min: 88-95% B, 28-30 min:

194

95-95% B, 30-33 min: 95-5% B, 33-35 min: and 5% B. During the analysis, the

195

instrument parameters were set as follows: capillary voltage, 3500 V for the positive 10


Page 10 of 33

Page 11 of 33


196

ion polarity mode; gas temperature, 320 ℃; sheath gas temperature, 350 ℃; sheath

197

gas flow, 11 L/min; gas flow, 8 L/min; and nebulizer, 35 psi. LC-Q-TOF-MS/MS

198

mode was employed to detect the targeted compounds. The collision energies (CE)

199

were set up for the parent ion at m/z 400.14 (9.1 min, CE 37 mV), 400.14 (9.65 min,

200

CE 37 mV), 400.14 (9.88 min, CE 35 mV), 400.14 (10.01 min, CE 37 mV), 400.14

201

(10.63 min, CE 37 mV), 416.13 (6.54 min, CE 38 mV), 416.13 (7.02 min, CE 37

202

mV), 416.13 (7.13 min, CE 37 mV), 416.13 (7.34 min, CE 37 mV), 416.13 (7.64 min,

203

CE 37 mV), 416.13 (7.999 min, CE 37 mV), 416.13 (8.64 min, CE 30 mV),

204

552.15(13.95 min, CE 25 mV), 552.15 (14.14 min, CE 25 mV), 552.15 (14.76 min,

205

CE 28 mV), 552.15 (15.01 min, CE 28 mV), 552.15(15.21 min, CE 25 mV), 568.14

206

(11.87 min, CE 25 mV) , 568.14 (12.16 min, CE 25 mV) , 568.14 (12.39 min, CE 25

207

mV) , 568.14 (12.84 min, CE 26 mV) , 568.14 (13.58 min, CE 25 mV) , 568.14

208

(14.07 min, CE 25 mV) , and 568.14 (14.47 min, CE 25 mV).

209

Statistical Analysis

210

Results were expressed as mean ± standard deviation. Statistical analysis was carried

211

out using One-Way ANOVA. Values in the same table and figure that were labeled

212

with different letters represent a significant difference (P < 0.05).

213 214

RESULTS AND DISCUSSION

215

Contents of Tea Polyphenols and Purine Alkaloids during Processing of LYT

216

Three types of compounds, namely gallic acid (GA), flavan-3-ols (EGC, EC, ECG,

217

GC, C and EGCG, GCG), and purine alkaloids (THB and CAF), were simultaneous 11



218

determined by HPLC (Table 1). In the fresh tea leaves, the main polyphenols were

219

EGC, EC, ECG and EGCG. Through fixation, rolling, and first drying, the contents of

220

these compounds did not change too much. This means that the main secondary

221

metabolites of fresh tea leaves remained after these processes. The largest change was

222

the appearance of GCG, which was not detected before first drying, but elevated to

223

1.34 ± 0.01 mg/g after this step.

224

After yellowing, the contents of the flavan-3-ols (EGCG, ECG, EGC, EC, C and

225

GC) were similar to the first drying sample, but the content of gallic acid was

226

increased. This yellowing was different from polyphenols oxidase fermentation or

227

microbial fermentation, because it did not involve in the rapid growth of

228

microorganisms under low moisture. It is possible that the hydrolysis of

229

galloylated catechins or tannins released gallic acid.

230

The last step, high-temperature roasting, played an important role in forming the

231

distinct chemical profile of LYT. After roasting, the levels of EGCG, ECG, EC and

232

EGC were dramatically decreased. Conversely, the epimerized catechins, GC and

233

GCG, were significantly increased by several folds. The contents of two purine

234

alkaloids, CAF and THB, were relatively stable during the whole process. This result

235

suggested that roasting is the process that results in the epimerization and

236

decomposition of tea catechins.

237

Changes in Free Amino Acids and L-theanine Levels during Processing

238

Free amino acids are important taste compounds for many foods and beverages. In

239

fresh tea leaves and unfermented teas, L-theanine is the predominant amino acid and 12


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Page 13 of 33


240

the main contributor to the umami taste of a tea infusion. However, after fermentation

241

or post-fermentation, which are critical steps in black tea and dark tea processing, the

242

contents of L-theanine are highly decreased or hardly detectable.29, 30 Therefore, it is

243

worthy to analyze the changes in L-theanine and other amino acid levels during the

244

processing of LYT.

245

In LYT, L-theanine is the main amino acid in fresh tea leaves through to the

246

second to last step of drying, during which its content remained relatively stable

247

(Table 2). The level of L-theanine was slightly decreased after the first and second

248

drying steps, but sharply decreased after high-temperature roasting. Therefore, in the

249

final product (after roasting), the level of L-theanine was scarcely detected. The

250

dramatic decrease of L-theanine after the final process may be due to Maillard

251

reaction and Strecker-degradation of L-theanine and may be related to the color and

252

flavor of the final LYT product. It has been reported that L-theanine contributes to the

253

formation of roasted and caramel odorants, such as pyrazines, through the Maillard

254

reaction.8

255

Non-targeted Metabolomics Analysis

256

To comprehensively understand the changes in the chemical profile during

257

manufacturing process of LYT, non-targeted metabolomics analysis using

258

UHPLC-Q-TOF/MS was applied to detect the changes of metabolites in the tea

259

samples from the seven processing steps. The raw data was processed by MS-DIAL,

260

and then analyzed by SIMCA-P 14.1 multivariate statistical software. The hierarchical

261

clustering clearly classified the samples following each different processing step 13



262

Page 14 of 33

(Figure 2).

263

The PCA and OPLS-DA score plots showed a clear classification of the different

264

samples. In brief, the HCA classified the seven samples into three types, with the

265

PCA and OPLS-DA showing similar clustering (Figure 2). The fresh leaves and the

266

samples after fixation, rolling and first drying clustered together, the samples after

267

second drying and yellowing clustered together, while the roasted sample was a third

268

type. These results suggested that out of the whole manufacturing process, yellowing

269

and roasting are the two critical steps that result in changes in the chemical

270

constituents. To identify the chemical responsible for the classification of the various

271

tea samples and which can serve as process markers, the S-plot was generated, and

272

some critical compounds were listed in Table 3.

273

Most of these VIP compounds are the typical secondary metabolites in Camellia

274

sinensis and in unfermented teas (Table 3). For example, some flavonoid glycosides

275

are used as markers to discriminate different processed samples, such as

276

quercetin-glucosyl-rhamnosyl-galactoside

277

kaempferol-glucosyl-rhamnosyl-galactoside. Flavonoid glycosides have very low

278

threshold values for imparting an astringent taste compared to catechins.31 After

279

roasting the LYT, the levels of these flavonoid glycosides were decreased more than

280

40%. Moreover, we also found some N-ethyl-2-pyrrolidinone-substituted flavan-3-ols,

281

tentatively identified by referring to the previous results, were increased by dozens of

282

folds after roasting. Among these compounds, N-ethyl-2-pyrrolidinone can be

283

condensed with non-galloylated or galloylated catechins. To explore the formation

and

14


Page 15 of 33


284

pathways of these compounds, a targeted metabolomics study was conducted.

285

Targeted Metabolomics Analysis

286

The non-targeted metabolomics results suggested a preliminary classification for

287

different steps of the LYT manufacture process. However, many of the identified

288

marker compounds are the same as those found in other metabolomics studies on tea.

289

This means a more specific metabolomics is needed, one focusing on the unique

290

compounds correlated with each tea processing step, such as roasting. One class worth

291

investigating are the N-ethyl-2-pyrrolidinone-substituted flavan-3-ols, identified with

292

reference to mass fragments data (Figure 3). Catechins and L-theanine would be the

293

precursors for the transformation and synthesis of these compounds, and showed huge

294

variations before and after processing, identified them as critical compounds. This

295

further suggested that N-ethyl-2-pyrrolidinone-substituted flavan-3-ols should be

296

newly generated during processing. Therefore, based on the expected fragmentation

297

patterns of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols, a series of these

298

compounds were identified in the roasted LYT product (Figure 3). To better

299

understand the metabolism of these N-ethyl-2-pyrrolidinone substituted flavan-3-ols

300

and their relationships with the main tea polyphenols and L-theanine, the targeted

301

metabolomic analyses were conducted for all processed tea samples. In a previous

302

study, eight N-ethyl-2-pyrrolidinone-substituted flavan-3-ols were isolated and

303

identified

304

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols identified in the LYT roasted

305

samples were the same as those reported.18, 20

in

ripened

pu-erh

teas.16

The

mass

15


fragment

ions

for


306

Tea contains both galloylated and non-galloylated types catechins. In the

307

targeted metabolomics, five N-ethyl-2-pyrrolidinone-substituted non-galloylated

308

catechins and four N-ethyl-2-pyrrolidinone-substituted galloylated catechins were

309

found. In the first four samples (fresh leaves to first drying), these compounds were

310

not detected, but after second drying and roasting, their contents were obviously

311

increased. Since EGCG is the predominant flavan-3-ols in fresh tea leaves, processing

312

gradually increased the levels of N-ethyl-2-pyrrolidinone-substituted EGCG, until the

313

last step of roasting, where the levels soared (Figure 4). A previous study suggested

314

that microbial fermentation was indispensable for the biotransformation of

315

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols.16 However, the substituted unit

316

1-ethyl-5-hydroxy-2-pyrrolidinone was recently isolated from the air-dried leaves of

317

Camellia sinensis var. pubilimba, which led to speculation that there might be some

318

endogenous enzymes or endophytes that promote the biotransformation and

319

biosynthesis of catechins and L-theanine.19 In this study, trace amount of

320

N-ethyl-2-pyrrolidinone-substituted EGCG were also detected in fresh leaves.

321

However, high-temperature roasting resulted in large-scale increase of the

322

N-ethyl-2-pyrrolidinone-substituted EGCG. These results indicated that temperature is

323

a crucial factor for the formation of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols

324

during tea processing.

325

In large-leaf yellow tea, both yellowing and roasting are critical for the formation

326

of the chemical profile of large-leaf yellow tea and its special aroma and taste. The

327

contents of epi-catechins were significantly decreased after roasting, but the 16


Page 16 of 33

Page 17 of 33


328

epimerized catechins were significantly increased. The non-targeted metabolomics

329

indicated that roasting greatly decreased the flavonoid glycosides, but dramatically

330

increased

331

glycosides

332

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols may also be potential bioactive

333

compounds.

334

acetylcholinesterase (AChE), oxidative damage and the formation of advanced

335

glycation end products (AGEs) in vitro.18, 20, 32 They also showed a therapeutic effect

336

on ApoE−/− mice with dyslipidemia and diabetes and a preventive effect on

337

age-related neurodegenerative disorders in senescence-accelerated mouse prone 8

338

(SAMP8) mice.32, 33

the

N-ethyl-2-pyrrolidinone-substituted are

These

low-threshold

compounds

were

catechins.

While

astringent

reported

to

flavonoid

compounds,

inhibit

α-glucosidase,

339

In the present study, a comprehensive chemical analysis on large-leaf yellow tea

340

samples after each manufacturing step showed that the predominant changes in the

341

main chemical constituents were the thermal-induced degradation and epimerization

342

of catechins and the formation of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols.

343

Further studies on the synthesis, bioactivities, and sensory contributions of

344

N-ethyl-2-pyrrolidinone-substituted

345

development and utilization of large-leaf yellow tea.

flavan-3-ols

are

needed

to

promote

the

346 347

FUNDING

348

This work was supported by the Young Elite Scientist Sponsorship Program by CAST

349

(2016QNRC001), the Anhui Provincial Natural Science Foundation (1708085MC73, 17



350

1508085MC59), the Key Research and Development Projects of Anhui Province

351

(1804b06020367) and the Earmarked Fund for the China Agriculture Research

352

System (CARS-19).

353 354

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355

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460 461 462

23



FIGURE CAPTIONS Figure 1 The main manufacturing process of large-leaf yellow tea. Samples were taken from all seven steps (or) from the six steps after harvesting. Figure 2 Score plots of principle component analysis (A), clustering analysis (B), partial least squares analysis (C) and S-plots of tea samples between roasting (RS, the final LYT product) and second drying (D). Figure 3 The mass spectrum and fragment ions of targeted marker compounds. Figure 4 The relative fold change of N-ethyl-2-pyrrolidinone-substituted flavan-3-ols during the manufacture processes of LYT.

24


Page 24 of 33

Page 25 of 33


Table 1. Contents of the Main Compounds in Large-leaf Yellow Tea during Processing. Proce

GA

GC

EGC

C

EC

EGCG

GCG

ECG

THB

CAF

ND

2.87±

40.27±

2.04±

9.70±0

80.48±

ND

15.94±

0.43±0

24.74±

0.12b

2.30a

0.09bc

.67ab

4.93a

0.70a

.00a

0.82a

3.22±

39.36±

2.40±

9.76±0

78.18±

15.65±

0.37±0

24.31±

0.10b

1.59a

0.08a

.21ab

3.26a

0.63a

.02c

0.40a

3.11±

37.20±

2.24±

9.33±0

75.17±

14.70±

0.35±0

23.09±

0.20b

0.69b

0.15a

.66b

1.76a

0.34a

.02c

0.55a

3.22±

39.36±

2.34±

10.24±

77.99±

1.34±

14.96±

0.37±0

23.23±

0.06b

0.97a

0.08a

0.12a

3.54a

0.01c

0.45a

.01c

0.82a

0.32±

2.91±

38.49±

1.85±

9.32±0

82.01±

1.50±

15.61±

0.42±0

24.41±

0.01b

0.11b

1.55ab

0.08c

.22b

2.70a

0.02b

0.55a

.02ab

1.14a

0.38±

2.97±

38.90±

1.90±

9.28±0

81.98±

1.59±

15.69±

0.41±0

24.67±

0.02b

0.09b

0.90ab

0.07bc

.17b

1.96a

0.02b

0.35a

.01ab

0.56a

1.97±

6.35±

6.52±0.

1.40±

1.96±0

35.59±

7.44±

8.10±0

0.40±0

24.49±

0.10a

0.33a

47c

0.05d

.05c

1.90b

0.32a

.39b

.02b

1.37a

ssing

FL F RL FD Y SD RS

ND ND ND

ND ND

Mean ± SD, n=6, mg/g. Values in the same column labeled with different letters differ significantly (P < 0.05). Gallic acid (GA), caffeine (CAF), theobromine (THB), (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-gallocatechin gallate (GCG) and (-)-epicatechin gallate (ECG).

25



Page 26 of 33

Table 2. Contents of Amino Acids in Large-leaf Yellow Tea during Processing. Amino acid (mg/g) Asp Ser Glu Gly His Arg Thr Ala Pro L-theanine Cys Tyr Val Met Lys Ile Leu Phe

Content (Mean ± SD) FL

F

RL

FD

Y

SD

RS

0.7674±

1.2392±

0.9889±

1.2480±

1.1384±

0.9584±

0.5219±

0.0257

0.0147

0.1942

0.0117

0.1491

0.1014

0.5123±

0.6704±

0.5664±

0.4269±

0.6513±

0.6250 ±

0.0183*** 0.2844±

0.04253

0.0076

0.0265

0.1984

0.0169

0.0476

0.7833±

1.0413±

0.9060±

1.1537±

0.9954±

0.8549±

0.0233

0.0249

0.1446

0.0556

0.0982

0.0820

0.2786±

0.2913±

0.2862±

0.2820±

0.2907±

0.2895±

0.0039*** 0.2792±

0.0141

0.0022

0.0005

0.0095

0.0003

0.0028

0.0007

1.4178±

1.6880±

1.3827±

1.1545±

1.3059±

1.2061±

0.3790±

0.0483

0.1493

0.0703

0.0496

0.0348

0.0957

0.8863±

0.8006±

0.7140±

0.5854±

0.7734±

0.7416±

0.0003*** 0.4744±

0.0426

0.0076

0.0137

0.1722

0.0119

0.0302

0.8149±

0.7127±

0.6538±

0.5847±

0.6910±

0.6565±

0.0126

0.0006

0.0172

0.0746

0.0104

0.0193

0.3658±

0.2465±

0.2119±

0.2520±

0.2413±

0.2166±

0.0065

0.0043

0.0226

0.0376

0.0082

0.0213

1.0452±

0.6563±

0.5851±

0.4284±

0.6222±

0.5704±

0.0036*** 0.4551±

0.0281

0.0033

0.0609

0.0361

0.0426

0.0400

0.0175

5.4255±

5.1689±

4.3917±

4.9379±

5.4714±

4.2659±

0.1171

0.0287

0.1808

0.0727

0.0785

0.6361

0.2652±

0.2017±

0.1923±

0.1524±

0.1952±

0.1909±

0.0006*** 0.4108±

0.0049** 0.4807± 0.0005** 0.1164±

ND 0.1942±

0.0200a

0.0012

0.0078

0.0056

0.0096

0.0163

0.0105

0.7811±

0.6524±

0.6278±

0.5469±

0.6392±

0.6363±

0.5330±

0.0110

0.0006

0.0074

0.0077

0.0032

0.0023

0.4823±

0.4284±

0.3973±

0.3298±

0.4191±

0.4095±

0.0014* 0.3234±

0.0058

0.0007

0.0095

0.0035

0.0055

0.0138

0.5768±

0.6309±

0.5454±

0.4460±

0.6374±

0.5492±

0.0010* 0.6476±

0.0060

0.0012

0.0605

0.0368

0.0559

0.0334

0.0375

0.3720±

0.3769±

0.3763±

0.3752±

0.3761±

0.3764±

0.3774±

0.0125

0.0008

0.0021

0.0049

0.0024

0.0033

0.0032

0.5237±

0.5838±

0.5209±

0.5033±

0.5395±

0.5364±

0.4775±

0.0326

0.0028

0.0702

0.0237

0.0743

0.0841

0.0684

0.4078±

0.3833±

0.3509±

0.2856±

0.3693±

0.3583±

0.2825±

0.0032

0.0003

0.0098

0.0133

0.0093

0.0128

0.6254±

0.5241±

0.5107±

0.4496±

0.5401±

0.5439±

0.0006 * 0.4508±

0.0212

0.0007

0.0047

0.0227

0.0015

0.0038

0.0006*

*P

LC-MS-based metabolomics reveals the chemical changes of

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