Dynamic Profiling of Phenolic Acids during Pu-erh Tea Fermentation

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Food and Beverage Chemistry/Biochemistry

Dynamic profiling of phenolic acids during Pu-erh tea fermentation using derivatization LC-MS approach Yahui Ge, Xiqing Bian, Baoqing Sun, Ming Zhao, Yan Ma, Yu-Ping Tang, Na Li, and Jian-Lin Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00789 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Journal of Agricultural and Food Chemistry

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Dynamic profiling of phenolic acids during Pu-erh tea fermentation using

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derivatization LC-MS approach

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Yahui Ge,†,ǁ Xiqing Bian,†,ǁ Baoqing Sun,‡ Ming Zhao,*,§ Yan Ma,§ Yuping Tang,ǂ Na

4

Li,*,† Jian-Lin Wu*,†

5

† State

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Applied Research in Medicine and Health, Macau University of Science and

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Technology, Avenida Wai Long, Taipa 999078, Macau, SAR China

8

‡

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Respiratory Diseases, Guangzhou Institute of Respiratory Diseases, First Affiliated

Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for

State Key Laboratory of Respiratory Disease, National Clinical Center for

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Hospital, Guangzhou Medical University, Guangzhou, Guangdong Province, China

11

§

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650201, China

13

ǂ

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Industrialization and College of Pharmacy, Shaanxi University of Chinese Medicine,

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Xianyang 712083, China

College of Longrun Pu-erh Tea, Yunnan Agricultural University, Kunming, Yunnan

Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources

16 17

Corresponding Authors

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*Tel.: +853-8897-2405; E-mail: [email protected] (N. Li)

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*Tel.: +86-871-6522-6565; E-mail: [email protected] (M. Zhao)

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*Tel.: +853-8897-2406; E-mail: [email protected] (J. L. Wu) 1

ACS Paragon Plus Environment


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ABSTRACT: Pu-erh tea, a famous traditional Chinese tea with multiple health

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benefits, is produced by microbial fermentation. It has been reported that major

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known bioactive compounds in green tea, e.g. epicatechin, epigallocatechin gallate

24

and theanine, decreased during fermentation. Then which components account for the

25

benefits of Pu-erh tea? Phenolic acids are aromatic secondary metabolites and possess

26

various biological properties. In this research, phenolic acids in Pu-erh tea were

27

investigated qualitatively and quantitatively to reveal the influence of fermentation

28

and

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derivatization-UHPLC-Q-TOF/MS approach. 33 phenolic acids were determined and

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most of them were detected in Pu-erh tea for the first time. Moreover, gallic acid and

31

theogallin were the major components in ripened and raw Pu-erh tea, respectively.

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Dynamic profiling revealed the increase of simple phenolic acids and the decrease of

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most of phenolic acid esters during Pu-erh tea fermentation. These results provided

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firm basis for practical fermentation and quality control of Pu-erh tea.

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KEYWORDS: phenolic acids, dynamic profiling, Pu-erh tea, fermentation,

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derivatization LC-MS

their

potential

effects

using

5-(diisopropylamino)amylamine

2


(DIAAA)

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

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Pu-erh tea, a famous traditional Chinese tea, is produced from the sun-dried leaves of

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Camellia sinensis (Linn.) var. assamica (Masters Kitamura) in Yunnan, China. It is

40

the most famous representative of dark teas.1 According to difference in preparation,

41

they could be classified as raw Pu-erh tea and ripened Pu-erh tea. Raw Pu-erh tea is

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simply prepared by pressing sun-dried green tea leaves into a disk or bowl shape, and

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ripened Pu-erh tea is produced from a post-fermentation of sun-dried green tea

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leaves.2 The latter is more popularly consumed than the former in the market.

INTRODUCTION

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Pu-erh tea, especially ripened Pu-erh tea, is famous as having a variety of

46

pharmacological activities, such as anti-hyperlipidemic, anti-diabetic, antioxidative,

47

antitumor, antimicrobial, anti-inflammatory, and anti-viral effects.3-4 It is widely

48

reported that epicatechin, epigallocatechin gallate (EGCG) and theanine are the main

49

bioactive components in green tea,5-6 and their high contents could provide many

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health benefits.1, 7-8 Recently, in our study, we also found that polyphenols and amino

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acids from the tea leaf pubescence of C. sinensis var. assamica cv. yunkang 10, a

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broad-leaf tea cultivar, are responsible for its good taste.9 While tea polyphenols have

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been reported to have multiple functions, their low oral-absorption rates limit the

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bioavailability in human bodies.10 In addition, modern technology has revealed that

55

the contents of these compounds in ripened Pu-erh tea are far less than that in green

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tea after a series of oxidation, polymerization, condensation and degradation reactions

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during fermentation.2 Due to the decrease of most known bioactive components 3



58

during fermentation, the bioactive constituents of Pu-erh tea remain unclear and need

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further study.

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Phenolic acids are aromatic secondary metabolites and widely spread throughout

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the plant kingdom.11 Some phenolic acids, like gallic acid, have been paid

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considerable attention with powerful anti-radical, antioxidative, antitumor and

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antimicrobial properties.12-14 Especially, gallic acid in Pu-erh tea is also reported to

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have antiobesity activity and can prevent oxidation-related diseases, e.g.

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atherosclerosis and vascular diseases.15-16 In addition, some studies have found that

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catechins, especially EGCG, could be degraded into phenolic acids in Pu-erh tea with

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the help of some particular fungi during fermentation.17-18 So, we hypothesized that

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phenolic acids might account for the health promoting effects of Pu-erh tea. Several

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phenolic acids have been reported from Pu-erh tea.1 Moreover, the content of gallic

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acid changed dynamically during the processing of Pu-erh tea.19-20 However, few

71

studies focus on the transformation of other phenolic acids during the fermentation

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process of Pu-erh tea. Therefore, comprehensive investigation on the dynamic

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variations of the phenolic acids is needed to provide firm basis for quality control of

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Pu-erh tea.

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Many analytical methods like high-performance liquid chromatography (HPLC)

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coupled with diode array ultraviolet detector (DAD-UV),21 mass spectrometry (MS)22

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as well as gas chromatography-MS (GC-MS)23 have been developed for the

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determination of phenolic acids. Unfortunately, the structure similarities and 4


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complicated sample preparation procedures could not satisfy the analytical

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requirements in most situations. Nowadays, liquid chromatography-MS (LC-MS) has

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become a predominant analytical technique because of its high sensitivity, high

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separation efficiency and short analysis time with less sample preparation.24-25

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However, the structures similarities, high polarity, low molecular weight, and low

84

ionization efficiency still challenge the determination of phenolic acids using LC-MS

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approach.26-27

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In this study, our previously reported 5-(diisopropylamino)amylamine (DIAAA)

87

derivatization-ultra-high performance liquid chromatography-quadruple-time of flight

88

mass spectrometry (UHPLC-Q-TOF/MS) approach28 was applied to profile and

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quantify phenolic acids in Pu-erh tea samples for the first time. The dynamic

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variations of phenolic acids during fermentation were also determined. It would

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provide foundation for practical fermentation of Pu-erh tea from a new point of view.

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

MATERIALS AND METHODS

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Reagents and Tea Samples. The phenolic acid standards including gallic acid (>

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99%), vanillic acid (> 97%), protocatechuic acid (> 99%), 4-hydroxyphenylacetic

95

acid (> 98%), salicylic acid (> 99%), 3-hydroxybenzoic acid (> 99%),

96

3-(4-hydroxyphenyl)propionic acid (> 99%), caffeic acid (> 98%), ferulic acid (>

97

99%), isoferulic acid (> 98%), homoveratric acid (> 99%), p-coumaric acid (> 98%),

98

theogallin (> 90%), chlorogenic acid (CGA, > 95%) and tyrosine (> 98%) were

99

purchased

from

JK

Scientific

(Beijing,

China);

5


4-Cl-phenylalanine,


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5-(diisopropylamino)amylamine

101

1-hydroxybenzotriazole

102

O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

103

(HATU), formic acid (MS grade) and triethylamine (TEA) were purchased from

104

Sigma-Aldrich (St. Louis, MO). Acetonitrile (ACN, LC/MS grade) and methanol

105

(MeOH, HPLC grade) were obtained from Anaqua Chemicals Supply Inc., Ltd.

106

(Houston, TA). The deionized water was prepared with a Millipore water purification

107

system.

(DIAAA),

dimethyl hydrate

sulfoxide

(DMSO), (HOBt),

hexafluorophosphate

108

There were two types of Pu-erh tea samples in this study. The first type covered

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four tea products, two raw teas purchased from Jingmai Mountain (RWJ) and Bulang

110

Mountain (RWB) as well as two ripened Pu-erh teas purchased from Menghai County

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(RPM) and Lincang (RPL), Yunnan Province, China. To investigate the dynamic

112

profiling of phenolic acids, 9 tea samples (D0-D8), including the sun-dried raw

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materials (D0) and 1st to 8th pile-turning samples of Pu-erh tea (D1-D8), were

114

collected from Yunnan D Tea Co., Ltd (Yunnan Province, China). They were

115

showered and mixed with water to the percentage of appropriate 40-50 kilograms per

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100 kilograms tea leaves, and then wet tea mass was piled up to the height of about 60

117

centimeters and covered with wet gunny cloth for the purpose of maintaining its

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moisture and temperature. The first pile-turning was conducted after 13 days and then

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the following pile-turnings were conducted every 7 days. Normally after the 5th or 6th

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pile-turning, the ripened Pu-erh tea was obtained. However, in this study, we 6


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continuously collected pile-turning samples from 1st to 8th pile-turnings.

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Sample Preparation. The mixture of phenolic acid standards was prepared in 60%

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methanol at the concentration of 10 g/mL and stored at −80 °C before use. Each tea

124

sample was freeze-dried and ground to 40-mesh size. About 100 mg was weighted

125

accurately and 5 L 4-Cl-phenylalanine (2.9 mg/mL) was added. After extracted

126

successively by 1 mL 80% MeOH, 1 mL 40% MeOH, and 1 mL distilled water in an

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ultrasonic bath (40 kHz) for 30 min, the extract was centrifuged at 15,890  g for 5

128

min, and the supernatants were mixed evenly. Two duplicated aliquots (30 L) of the

129

mixture and 50 L of 10 g/mL standard mixture were evaporated under a stream of

130

nitrogen, respectively. Next, the residue was derivatized as follows.28 The residue of

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samples or standards was sequentially mixed with 5 L of 20 mM HOBt in DMSO, 5

132

L of 100 mM DIAAA in DMSO containing 200 mM TEA and 5 L of 20 mM

133

HATU in DMSO. After reacting for 1 min, 35 µL ACN was added to make up to the

134

final volume of 50 L, while another residue was dissolved in 50 L of DMSO-ACN

135

(v:v/15:85) for non-derivatization analysis.

136

UHPLC-Q-TOF/MS Analysis. The LC separation was performed on an Agilent

137

1290 Infinity LC system (Santa Clara, CA) consisting of a thermostated autosampler,

138

a thermostated column compartment, a degasser, and a binary pump equipped with a

139

Waters ACQUITY UPLC HSS T3 column (2.1  100 mm, 1.8 m). The mobile phase

140

was composed of A (0.1% formic acid in H2O) and B (0.1% formic acid in ACN)

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with a gradient elution, 0–0.5 min, 5% B; 0.5–2.5 min, 5–6% B; 2.5–4.5 min, 6–7% B, 7



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4.5–5.5 min, 7–7.3% B; 5.5–7.5 min, 7.3–7.8% B; 7.5–11.0 min, 7.8–9.0% B; 11.0–

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13.0 min, 9.0–14.0% B; 13.0–18.0 min, 14.0–23.0% B; 18.0–19.0 min, 23.0–25.0% B;

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19.0–23.0 min, 25.0–47.0% B; 23.0–28.0 min, 47.0–60.0% B; 28.0–30.0 min, 60.0–

145

95.0% B; 30.0–32.9 min, 95.0% B; 33.0 min, 2.0% B and a post time of 3 min. The

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flow rate was 0.3 mL/min, and the injection volume was 1 L. The column

147

temperature was maintained at 35 C.

148

The mass spectrometry was acquired on an Agilent 6550 UHD accurate-mass

149

Q-TOF/MS system with a dual Jet stream electrospray ion source (dual AJS ESI). The

150

instrument was operated in positive and negative ion modes. The dry gas flow rate

151

was set at 15 L/min with a temperature of 250 C, and the nebulizer pressure was set

152

at 22 psi. The sheath gas flow rate was set at 11 L/min with a temperature of 300 C.

153

The capillary voltage was set at 5000 V and the nozzle voltage was set at 500 V for

154

positive ion mode and 1500 V for negative ion mode. The mass spectra were recorded

155

across the range of 100-1000 m/z for derivatized samples and 50-1000 m/z for

156

non-derivatized samples. For MS/MS acquisition, automated and targeted MS/MS

157

were applied and the collision cell energy was set at 30 eV.

158

Data Processing. All the data were in triplicate. The raw data were acquired and

159

processed with Agilent MassHunter Qualitative Analysis B.06.00 software (Agilent

160

Technology). Some constituents were identified based on the comparison of retention

161

times, MS, and MS/MS spectra with corresponding standards. The compounds

162

without corresponding standards were determined as follows. Firstly, the DIAAA 8


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derivatives were determined from the MS and characteristic MS/MS fragmentations.

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Then the molecular formula of the corresponding compounds was calculated by

165

subtracting the chemical formula of DIAAA and adding H2O. Finally, the compounds

166

were identified by searching mass spectrometry information in METLIN

167

(https://metlin.scripps.edu/index.php). The contents were relatively quantified by the

168

peak area ratios between phenolic acid derivatives and 4-Cl-phenylalanine in

169

DIAAA-derivatization and were further processed using GraphPad Prism 5.0.

170



RESULTS AND DISCUSSION

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Identification of Phenolic Acids in Pu-erh Tea. It is well known that phenolic

172

acids have high hydrophilicity and are difficult to retain on C18 column. In order to

173

enhance its retention, DIAAA derivatization-UHPLC-Q-TOF/MS approach was

174

firstly developed for determination of phenolic acids in Pu-erh tea. At the same time,

175

LC-MS chromatogram of phenolic acids using non-derivatization method was also

176

acquired for comparison (Figure 1A). It was found that the retention times for

177

DIAAA-derivatives significantly increased compared to that of corresponding

178

phenolic acids (Figure 1B and 1C). Moreover, the ionization efficiency was also

179

greatly enhanced. For example, 4-hydroxyphenylacetic acid (Peak 8) could be

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detected obviously using DIAAA-derivatization approach (Figure 1C), while the peak

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was merged in the noise using non-derivatization approach (Figure 1B). Similar

182

phenomenon was also observed for other phenolic acids.

9



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Figure 1. Total compound chromatograms of Pu-erh tea determined by

185

non-derivatization

186

chromatograms of phenolic aicds (B) and DIAAA-derivatized phenolic acids (C) in

187

Pu-erh tea.

and

DIAAA-derivatization

method

(A).

Extracted

ion

188

Using derivatization method, phenolic acids in Pu-erh tea were determined from the

189

characteristic MS/MS fragmentation patterns and retention times by comparison with 10


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that of authentic standards, as well as MS with that in the database, such as METLIN,

191

as follows.

192

The MS/MS characterization of DIAAA-derivatized phenolic acids was first

193

investigated using the standards. The characteristic MS/MS fragmentation ions of

194

[M+H42]+, [M+H84]+ and/or [M+H101]+ derived from the neutral loss of one or

195

two propene and/or diisopropylamine as well as m/z 128 and 86 assigned to the ions

196

of N-isopropyl-N-vinylpropan-2-aminium and N-vinylpropan-2-aminium were related

197

to the derivatization reagent DIAAA. Thus, the compound with these fragmentation

198

patterns might contain the carboxyl group before DIAAA-derivatization. As a

199

consequence, the molecular formula of DIAAA-derivative could be determined from

200

the exact m/z and the distribution of molecular and isotopic ions, and the molecular

201

formula of the corresponding phenolic acid could be calculated by subtracting

202

C11H26N2 and adding H2O. Although the intensities of other fragmentation ions were

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relatively low, they still could provide important information of other functional

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groups for identification. For example, the fragmentation ions of theogallin (Peak 5)

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at m/z 153.0190, 361.2684, and three pairs of diagnostic ions (m/z 301.2111/283.2001,

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242.1380/224.1277, 224.1277/206.1190) indicated the presence of trihydroxybenzoyl,

207

quinic acid and three hydroxyl groups (Figure S1A). Similarly, the existence of

208

fragmentation ions at m/z 136.0772 and 107.0499 in MS/MS spectrum contributed to

209

the structure identification of 4-hydroxyphenylacetic acid (Figure S1B). In the same

210

way, the fragmentation ions at m/z 190.0567, 206.1193, 231.0682, and 259.0615 11



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could also be used to identify the DIAAA-derivative of teadenol (Peak 32) (Figure

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S1C).

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To sum up, based on retention times, MS, and MS/MS spectra, 33 phenolic acids

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were identified from Pu-erh tea using DIAAA-derivatization approach (Table 1),

215

while only 10 were observed without derivatization.

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Difference of Phenolic Acids between Commercial Raw and Ripened Pu-erh

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Tea Products. Next, DIAAA-derivatization was applied to investigate the difference

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of phenolic acids in two raw Pu-erh tea products (RWJ and RWB) and two ripened

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Pu-erh tea products (RPM and RPL). Considering it’s impossible to obtain all

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phenolic acid standards, a relative quantification approach was established by

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comparing to the internal standard (4-Cl-phenylalanine) (Figure 2 and Figure S2).

12


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Figure 2. Contents of representative phenolic acids and phenolic acid esters in raw

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(RWJ and RWB) and ripened Pu-erh tea products (RPM and RPL).

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The most abundant phenolic acid in ripened Pu-erh tea was gallic acid (Peak 2),

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and its content increased four to six times than that in raw tea. As usually known,

227

gallic acid has many kinds of bioactivities, including antioxidant, antibacteria,

228

antitumor and antiobesity, so the significant accumulation of gallic acid after

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fermentation should take great part in health care effects of ripened Pu-erh tea on

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oxidative damage, inflammation, human liver cancer and lipid metabolism.29-32 Other 13



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benzoic acid derivatives, for example, vanillic acid (Peak 3), protocatechuic acid

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(Peak 4), dimethoxybenzoic acid isomers (Peak 6, 7), dihydroxy-methoxy-benzoic

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acid (Peak 9), salicylic acid (Peak 11), and 3-hydroxybenzoic acid (Peak 12), also

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improved sharply in ripened Pu-erh tea. Many researches have revealed that these

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constituents

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chemopreventive activity on cancer.33-35 In addition, protocatechuic acid is reported to

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take positive effects on internal secretion, digestive tract, liver, cardiovascular system,

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nervous system and reproductive system.36-37 Vanillic acid, salicylic acid and

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3-hydroxybenzoic acid are reported to present partial effects of protocatechuic acid.

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Therefore, the improvement of their contents after fermentation might be related to

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the attenuation of Pu-erh tea on diabetes, blood pressure, oxidative stress, as well as

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irritation to the stomach.34,

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effects of gallic acid should make important contribution to functional effects of

244

Pu-erh tea.

also

show

antimicrobial

36, 38

and

insulin-like

effect

as

well

as

Thus, these compounds compensating for multiple

245

4-Hydroxyphenylacetic acid (Peak 8), hydroxyl-dimethoxy-phenylacetic acid (Peak

246

10), homoveratric acid (Peak 24) and its isomer (Peak 16) belonging to phenylacetic

247

acid were identified in our study. The content of 4-hydroxyphenylacetic acid

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strikingly ascended after fermentation, and it was the third highest simple phenolic

249

acid in the fermented teas. According to the previous studies, 4-hydroxyphenylacetic

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acid shows important antioxidative, antiradical activities, and is also active against

251

two nematodes to a certain extent.18, 39 14


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Nine propionic acid derivatives, including tyrosine (Peak 1), 3-(4-hydroxyphenyl)

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propionic acid (Peak 13) and its isomer (Peak 27), caffeic acid (Peak 17) and its

254

isomer (Peak 14), ferulic acid (Peak 19), p-coumaric acid (Peak 25) and its isomer

255

(Peak 23) and isoferulic acid (Peak 28), were identified. Except tyrosine, all had

256

escalating trend after fermentation. p-Coumaric acid has anti-inflammatory and

257

antioxidative activities.40 Caffeic acid is recognized to have biochemical,

258

antibacterial, antioxidative, and antiviral properties.41-42

259

Dihydroxyphenylvaleric acid (Peak 18) and hydroxylphenylvaleric acid (Peak 29)

260

classified as phenylvaleric acid derivatives had upwards tendency after fermentation.

261

The tendencies were consistent with most phenolic acids. Teadenol (Peak 32) and its

262

isomer (Peak 33) were also detected in Pu-erh tea. Teadenol is reported to be

263

biosynthesized from EGCG in the fermentation process of tea leaves and has the

264

antioxidant activity.43 This kind of phenolic acid is first identified in fermented

265

Camellia sinensis leaves by some Japanese scientists in 2011.44

266

Compared to simple phenolic acids, the contents of the determined phenolic acid

267

esters, including theogallin (Peak 5), CGA (Peak 20), CGA isomers (Peak 15, 22),

268

coumaroylquinic acid isomers (Peak 21, 26 and 30), all descended after fermentation.

269

Theogallin and CGA are reported to be related to antioxidative, and anti-inflammatory

270

activities. 43, 45-46

271

However, as usually known, except for fermentation, other factors such as

272

collection season, tea leave maturity, growing location, processing method and 15



273

harvest time also have influence on the types and contents of components. In order to

274

focus on the effects of fermentation, the Pu-erh tea samples processed by different

275

pile-turnings were further investigated.

276

Dynamic Variation of Phenolic Acids during Pu-erh Tea Fermentation. In

277

Pu-erh tea-processing industry, the fermentation product of sun dried leaves produced

278

from 5th to 6th pile-turning was usually harvested as ripened Pu-erh tea product. Based

279

on the above mentioned findings on phenolic acids in raw and ripened Pu-erh tea

280

samples, dynamic variation of the constituents during fermentation procedure from 1st

281

to 8th pile-turnings was further investigated in our study. The changing trends could

282

be mainly classified as three categories as follows (Figure 3, Figure S3 and S4).

16


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283 284

Figure 3. Contents variation of representative phenolic acids and phenolic acid esters

285

during Pu-erh tea fermentation.

286

Compared to the sun-dried raw tea sample (D0), all simple phenolic acids evidently

287

increased after fermentation by 1st pile-turning, especially gallic acid. Along with the

288

increase of the pile-turning times, the content of gallic acid slightly enhanced

289

followed by gradually decreased, while it was still higher than that in raw materials. 17



290

This was the first type of changing trends. Gallic acid was found to be the main

291

phenolic acid in the fermented samples. Other simple phenolic acids, e.g.

292

protocatechuic acid, 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid,

293

hydroxylphenylvaleric acid, and dihydroxyphenylvaleric acid, had the same changing

294

trend.

295

p-Coumaric acid had rising trends at 1st pile-turning, and then dropped off

296

gradually in the following steps. At the end of fermentation, its content was close to

297

that at the beginning (D0). It was assigned as the second type. In addition, other

298

simple phenolic acids (caffeic acid and its isomer, ferulic acid, coumaric acid isomer,

299

tyrosine, teadenol and its isomer, etc.) also had the same trends with p-coumaric acid.

300

Since their contents variations had important inflection point during fermentation, this

301

type of constituents could be used as potential markers for determination of ending

302

point of fermentation, especially teadenol due to its relative high content.

303

Most of phenolic acid esters were classified as the third type. The contents of these

304

phenolic acid esters declined from the very beginning of fermentation and dropped

305

along with the increase of pile-turning times. At the end, these constituents almost

306

vanished eventually. A latest research has revealed that CGA could react with EGCG

307

or EGC to form two novel compounds, EGCG-CGA and EGC-CGA in black tea.47

308

This finding can provide partial reason for the loss of CGA in Pu-erh tea after

309

fermentation. On the other hand, since the contents of caffeic acids go up during

310

fermentation, it is reasonable to believe that the fermentation also catalyzes the 18


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311

transformation of CGA in sun dried leaves into caffeic acid. Some reports say that

312

CGA has relative low absorption only about 33% in the human body.48 Thus, the

313

fermentation affords the ripened Pu-erh tea a good absorption, which is very

314

important for executing their bioactive effects.

315

The possible biosynthetic pathways of phenolic acids during fermentation were

316

proposed in Figure 4. Firstly, EGCG was degraded into gallic acid and

317

epigallocatechin (EGC) by microbial esterases,49 and theogallin could also be

318

metabolized into gallic acid. Then EGC was hydrolyzed to create a series of phenolic

319

acids. For example, EGC could be metabolized into dihydroxyphenylacetic acid via

320

ring fission of the C-ring in various ways, and this could be subsequently degraded

321

into 4-hydroxyphenylacetic acid and protocatechuic acid by dehydroxylation and loss

322

of carbon atoms successively from side chain. On the other hand, ten other phenolic

323

acids were metabolized from proanthocyanidins, which are known to be transformed

324

into dihydroxyphenylvaleric acid through C-ring fission and the lactone ring

325

opening.50

326

hydroxyphenylvaleric acid. In addition, hydroxyphenylvaleric acid was metabolized

327

to 3-(4-hydroxyphenyl)propionic acid and then degraded into coumaric acid.

Dihydroxyphenylvaleric

acid

might

also

19


be

dehydroxylated

to


328 329

Figure 4. Possible biosynthetic pathway of phenolic acids during fermentation.

330

References (a) Am. J. Clin. Nutr. 2013, 98, 1631; (b) J. Agric. Food Chem. 2010, 58,

331

1296; (c) Drug Metab Dispos. 2011, 39, 2338; (d) J. Pharm. Pharmacol. 2007, 59,

332

1131.

333

In the dynamic investigation on Pu-erh tea fermentation, the contents of most of

334

phenolic acids increased more or less after fermentation compared to the sun-dried

335

leaves. Previous investigations exhibit that some phenolic acids are degraded and

336

transferred from the macromolecules like EGCG and proanthocyanidins.49-50 Thus, it

337

is out of question that Pu-erh tea becomes much easier to be absorbed when the

338

macromolecules are transformed into small molecules by fermentation. Furthermore, 20


Page 20 of 32

Page 21 of 32


339

considering the bioactivities of phenolic acids, it is worthy of fermentation. The

340

fermentation times had significant influence on the phenolic acids, especially gallic

341

acid, p-coumaric acid, salicylic acid, theogallin, CGA, and so on. From the contents

342

and their variations during fermentation as well as the comparison with the

343

commercial products, it might be better that fermentation is controlled at 5th or 6th

344

pile-turning, which could provide more phenolic acids. More researches should be

345

conducted on the health promoting effects and taste in the near future.

346

In this study, DIAAA derivatization-UHPLC-Q-TOF/MS approach was established

347

for the determination of phenolic acids. The derivatization provided higher sensitivity

348

and better separation, which resulted in the identification of 33 phenolic acids. Most

349

of them were detected from Pu-erh tea for the first time. The difference between

350

commercial raw and ripened Pu-erh tea as well as the dynamic investigation during

351

fermentation revealed that simple phenolic acids usually increased and phenolic acid

352

esters decreased after fermentation. Gallic acid was the major component in ripened

353

Pu-erh tea, while its ester, theogallin, was the main compound in the raw samples.

354

These findings may be served for the following investigation on the quality control

355

and manufacture of Pu-erh tea.

356



357

Author Contributions

358

ǁY.G

359

authors.

AUTHOR INFORMATION

and X.B. contributed equally to this work and should be regarded as joint first

21



360

Funding

361

This work was supported by Macau Science and Technology Development Fund

362

[009/2017/A1], Open Project of State Key Laboratory of Respiratory Disease

363

(SKLRD-OP-201803) and The National Natural Science Foundation of China (Grant

364

No. 31560221 and 31760225).

365

Notes

366

The authors declare no competing financial interest.

367



368

Supporting Information

369

MS/MS spectra for theogallin (A), hydroxyphenylacetic acid (B) and teadenol (C)

370

derivatized with DIAAA (Figure S1). Contents of 33 phenolic acids in raw and

371

ripened Pu-erh tea products (Figure S2). Contents variation of 33 phenolic acids

372

during fermentation in Pu-erh tea (Figure S3). Contents variation of epigallocatechin

373

gallate (EGCG) and epigallocatechin (EGC) during pile-turning (Figure S4).

ASSOCIATED CONTENT

22


Page 22 of 32

Page 23 of 32


374



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533 534

28


Produced

Microbiota

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with and

Page 29 of 32

535


Table 1. Phenolic acids determined from Pu-erh tea using DIAAA derivatization approach. No.

Name

RT (min)

Formula

Derivative formula

1 2 3 4

Tyrosine Gallic acid Vanillic acid Protocatechuic acid

4.62 8.26 9.70 11.68

C9H11NO3 C7H6O5 C8H8O4 C7H6O4

5

Theogallin

12.40

6

Dimethoxybenzoic acid isomer A Dimethoxybenzoic acid isomer B 4-Hydroxyphenylaceti c acid Dihydroxy-methoxy-b enzoic acid Hydroxy-dimethoxy-p henylacetic acid Salicylic acid

7 8 9 10 11

Measured m/z

Error MS/MS (ppm)

C20H35N3O2 C18H30N2O4 C19H32N2O3 C18H30N2O3

Theoretical m/z [M+H]+ 350.2802 339.2278 337.2486 323.2329

350.2796 339.2265 337.2489 323.2319

1.89 3.87 -0.33 3.10

C14H16O10

C25H40N2O9

513.2807

513.2805

0.27

12.62

C9H10O4

C20H34N2O3

351.2642

351.2635

2.08

13.27

C9H10O4

C20H34N2O3

351.2642

351.2636

3.11

13.32

C8H8O3

C19H32N2O2

321.2537

321.2537

0.00

13.87

C8H8O5

C19H32N2O4

353.2435

353.2439

-0.86

14.05

C10H12O5

C21H36N2O4

381.2748

381.2735

3.08

14.10

C7H6O3

C18H30N2O2

307.238

307.2365

4.79

29


308, 291, 249, 204, 128, 86 297, 255, 238, 153, 128, 86 295, 253, 236, 151, 128, 86 281, 239, 222, 194, 166, 137, 109, 128, 86 471, 453, 411, 361, 343, 301, 283, 242, 224, 206, 153, 128, 86 309, 267, 250, 165, 128, 86 309, 267, 250, 166, 137, 128, 86 279, 237, 220, 128, 107, 86 311, 269, 252, 224, 210, 167, 139, 128, 86 339, 297, 280, 196, 128, 86 265, 223, 206, 128, 121, 93, 86


12

Page 30 of 32

3-Hydroxybenzoic acid 3-(4-Hydroxyphenyl)p ropionic acid Caffeic acid isomer Chlorogenic acid isomer A

14.80

C7H6O3

C18H30N2O2

307.238

307.2367

4.12

265, 223, 206, 128, 121, 86

14.90

C9H10O3

C20H34N2O2

335.2693

335.2689

0.85

15.10 15.27

C9H8O4 C16H18O9

C20H32N2O3 C27H42N2O8

349.2486 523.3014

349.2494 523.3008

-3.36 0.98

Homoveratric acid isomer Caffeic acid Dihydroxyphenylvaler ic acid Ferulic acid Chlorogenic acid

15.35

C10H12O4

C21H36N2O3

365.2799

365.2802

-1.00

15.64 15.82

C9H8O4 C11H14O4

C20H32N2O3 C22H38N2O3

349.2486 379.2955

349.2478 379.2945

1.95 2.44

16.04 16.24

C10H10O4 C16H18O9

C21H34N2O3 C27H42N2O8

363.2642 523.3014

363.2624 523.3008

5.07 1.04

16.39

C16H18O8

C27H42N2O7

507.3065

507.3068

-0.42

16.54

C16H18O9

C27H42N2O8

523.3014

523.3007

1.85

23 24 25

Coumaroylquinic acid isomer A Chlorogenic acid isomer B Coumaric acid isomer Homoveratric acid p-Coumaric acid

16.74 16.77 16.96

C9H8O3 C10H12O4 C9H8O3

C20H32N2O2 C21H36N2O3 C20H32N2O2

333.2537 365.2799 333.2537

333.2534 365.2789 333.2531

0.69 2.59 1.62

26

Coumaroylquinic acid 17.32

C16H18O8

C27H42N2O7

507.3065

507.3069

-0.47

293, 251, 234, 165, 149, 128, 86 307, 265, 248, 128, 86 481, 464, 422, 404, 361, 319, 301, 283, 260, 242, 163, 128, 86 323, 281, 264, 208, 179, 128, 86 307, 265, 247, 230, 128, 86 337, 295, 278, 236, 193, 128, 86 321, 262, 177, 128, 86 481, 439, 422, 387, 337, 128, 86 465, 423, 406, 350, 289, 244, 147, 128, 86 481, 422, 393, 319, 301, 260, 242, 163, 128, 86 291, 249, 232, 147, 128, 86 323, 281, 264, 179, 128, 86 291, 232, 147, 128, 119, 93, 86 465, 406, 361, 343, 301,

13 14 15

16 17 18 19 20 21 22

30


Page 31 of 32


32

isomer B Hydroxyphenylpropio nic acid isomer Isoferulic acid Hydroxyphenylvaleric acid Coumaroylquinic acid isomer C Dihydroxyphenylvaler ic acid isomer Teadenol

33

Teadenol isomer

27 28 29 30 31

17.37

C9H10O3

C20H34N2O2

335.2693

335.2681

3.55

17.51 17.57

C10H10O4 C11H14O3

C21H34N2O3 C22H38N2O2

363.2642 363.3006

363.2622 363.3

6.07 1.40

17.81

C16H18O8

C27H42N2O7

507.3065

507.3065

0.14

17.87

C11H14O4

C22H38N2O3

379.2955

379.2941

2.80

18.99

C14H12O6

C25H36N2O5

445.2699

445.2699

-0.41

20.89

C14H12O6

C25H36N2O5

445.2699

445.2692

1.35

536 537

31


283, 147, 128, 119, 86 293, 251, 234, 165, 128, 86 321, 262, 177, 128, 86 321, 262, 205, 177, 149, 135, 128, 86 465, 406, 361, 343, 301, 283, 242, 147, 128, 119, 86 337, 295, 278, 193, 128, 86 403, 361, 344, 288, 259, 231, 203, 128, 86 403, 344, 231, 190, 128, 86


538 539

For Table of Contents Only

540 541

32


Page 32 of 32

Dynamic Profiling of Phenolic Acids during Pu-erh Tea Fermentation

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