Novel Flavoalkaloids from White Tea with Inhibitory Activity against the

Subscriber access provided by UNIV OF DURHAM

Bioactive Constituents, Metabolites, and Functions

Novel Flavoalkaloids from White Tea with Inhibitory Activity Against Formation of Advanced Glycation End Products Xiao Li, Guang-Jin Liu, Wei Zhang, Yv-Long Zhou, Tie-Jun Ling, Xiao-Chun Wan, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00650 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

Novel Flavoalkaloids from White Tea with Inhibitory Activity against Formation of Advanced Glycation End Products Xiao Li†,§, Guang-Jing Liu†,§, Wei Zhang†, Yv-Long Zhou†, Tie-Jun Ling†, Xiao-Chun Wan†, Guan-Hu Bao*, † †

Natural Products Laboratory, International Joint Lab of Tea Chemistry and Health effects, State

Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 230036, Hefei, People’s Republic of China *

Phone: +86-551-65786401. Fax: +86-551-65786765. E-mail: [email protected].

1

ACS Paragon Plus Environment


1

Page 2 of 34

Abstract: Two novel flavoalkaloids, (−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-epigallocatechin

2

-O-gallate

(ester-type

catechins

pyrrolidinone

3

(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-O-gallate (etc-pyrrolidinone B，2) and

4

new

5

(−)-8-N-ethyl-2-pyrrolidinone-epigallocatechin-O-gallate (etc-pyrrolidinone C，3a and etc-

6

pyrrolidinone D，3b), were isolated from white tea (Camellia sinensis). Their structures were

7

identified by extensive NMR spectra. Absolute configuration of 1 and 2 was decided by

8

comprehensive CD spectroscopic analyses. The isolated flavoalkaloids together with

9

(−)-epigallocatechin-O-gallate (EGCG) were evaluated for their inhibition against the

10

formation of advanced glycation end products (AGEs) with IC50 values ranging from 10.3 to

11

25.3 µM. UPLC−DAD−ESI/MS detected these flavoalkaloids in both white tea and fresh tea

12

leaves, which demonstrated the existence of a corresponding biosynthetic pathway in tea

13

plant. Therefore, a possible pathway was proposed to involve deamination, decarboxylation,

14

and spontaneously cyclization of l-theanine, and then attachment of the product to EGCG to

15

form the flavoalkaloids.

naturally

A,

occurring

etc-pyrrolidinone

A,

1),

flavoalkaloids

16

Keywords: pyrrolidinone, epigallocatechin-O-gallate (EGCG), Camellia sinensis, absolute

17

configuration, advanced glycation end products (AGEs), l-theanine

2


Page 3 of 34


18

Introduction

19

White tea is one of the six traditional Chinese tea categories (green, white, yellow,

20

oolong, black, and dark tea), which is mostly produced in Fujian province, China. It is

21

a lightly fermented tea since its manufacture process has only two simple steps called

22

withering and drying.1 White tea can be ranked into four classes according to its

23

quality as the following sequence: Silver Needle (or Baihao-Yinzhen), White Peony

24

(or Bai-Mudan), Tribute Eyebrow (or Gong-Mei), and Longevity Eyebrow (Shou

25

-Mei).2 Among them, Bai-Mudan is a popular one which was prepared by one bud

26

with one or two fresh tea leaves (Camellia sinensis var. sinensis, C. sinensis var.

27

sinensis). Recent studies have indicated that white tea shows anti-oxidant,

28

anti-obesity, anti-cancer, and preventive effect on cardiovascular diseases and so

29

on.3-6 One study found that white tea was the richest in catechin derivatives associated

30

with the best intestinal bioaccessibility and bioavailability among all of the tea

31

samples tested (green, white, black tea).7 Another research suggested that white tea

32

had better antimutagenic activity than green tea in the Salmonella assay, which might

33

be related to the different levels of the nine major constituents, and these major

34

components may act synergistically with other minor constituents.8

35

The diverse functional properties of white tea can be attributed to its abundant active

36

components, whereas few studies have been conducted on systematic purification and

37

structural identification of chemical constituents from white tea. Recently, we have

38

found several minor catechin derivatives from both dark and green tea.9,10 One minor

39

catechin derivative (−)-epicatechin 3-O-caffeoate (ECC) showed the strongest

3



40

inhibitory effect against acetylcolinesterase and neutrophil gelatinase-associated

41

lipocalin (NGAL) among different catechins,10,11 which suggested minor new

42

catechins in tea materials are worthy of study. UPLC-MS analysis showed that

43

flavoakaloids may exist in the white tea Bai-Mudan (big white tea originated in

44

Fuding, Fujian province, cultivar: Fuding-Dabai, class: Bai-Mudan) (Figure S1),

45

which encouraged us to study the chemical constituents from this type of tea.

46

Flavoalkaloids are an unusual group of secondary metabolites from plant, which have

47

a unique molecular framework possessing a nitrogen ring system linked to a flavonoid

48

skeleton, as the name imply.12 (+)−Ficine and (+)−isoficine were the first two

49

flavoalkaloids isolated from the wild fig, Ficuspantoniana, Moraceae in 1965.13 Since

50

then, more than 100 flavoalkaloids have been found from different plants,

51

successively.14 Ethylpyrrolidinonyl theasinensin A was the first flavoalkaloid isolated

52

from black tea in 2005.15 In 2014, eight more flavoalkaloids, puerins I-VIII with

53

significant antioxidative activity, were reported in the Chinese dark tea.16 Recently,

54

due to their potential possibility of multiple biosynthetic pathways and pronounced

55

bioactivities, flavoalkaloids have gathered much more attention.17-19 Flavopiridol, a

56

semi-synthetic flavoalkaloid also known as alvocidib, was performed effectively for

57

the treatment of acute myeloid leukaemia in phase II clinical trials by alteration of

58

tyrosine phosphorylation of cyclin-dependent kinase (CDK) 1/2 and competitive

59

inhibition with adenosine triphosphate (ATP).20 Furthermore, flavoalkaloids were also

60

demonstrated as inhibitors against the formation of advanced glycation end products

61

(AGEs), which are the pathogenic factor of some chronic degenerative diseases such

4


Page 4 of 34

Page 5 of 34


62

as diabetic and neurodegenerative diseases.21 As it could be anticipated,

63

flavoalkaloids indicate promising potential for the discovery of new therapeutic

64

agents and deserve more research.

65

In spite of studies on the flavoalkaloids from black and dark tea, there is less attention

66

to flavoalkaloids from other tea categories. Therefore, in this research, we

67

systematically studied flavoalkaloids from white tea and successfully obtained two

68

novel together with new naturally occurring flavoalkaloids from white tea. The

69

isolation, structural elucidation, AGEs inhibition, and possible biosynthetic pathway

70

of the flavoalkaloids were presented in this study.

71

Materials and Methods

72

Chemicals. HPLC grade acetonitrile, methanol, and formic acid were purchased from

73

Duksan pure chemicals Co., Ltd (Ulsan, Korea). Bovine serum albumin was

74

purchased from Nanjing Duly Biotech Co., Ltd (Nanjing, China). Penicillin was

75

obtained from Harbin Pharmaceutical Group Co., Ltd (Harbin, China). Phosphate

76

buffered

77

Aminoguanidine hydrochloride was bought from Shanghai TCI Development Co.,

78

Ltd (Shanghai, China). (−)-Epigallocatechin-3-O-gallate (EGCG) was isolated from

79

tea plants in our laboratory and the purity was ≥ 98% confirmed by HPLC analysis.

80

The purification materials filled in column chromatography in this study included

81

MCI-Gel CHP20P (Mitsubishi Ltd., Tokyo, Japan), Sephadex LH-20 (GE Healthcare

82

Bio-Sciences AB, Stockholm, Sweden), ODS C-18 (ODS, Fuji Silysia Chemical Ltd.,

83

Kasugai, Japan), Toyopearl HW-40F (Tosoh Bioscience Shanghai Co., Ltd., Shanghai,

saline

(PBS)

was

purchased

from

Solar-bio

5


(Beijing,

China).


84

China), DIAION HP20SS gel (Mitsubishi Ltd., Tokyo, Japan), and silica gel (Yantai

85

jiangyou silicon development co., Ltd., Shandong, China).

86

IR spectrum was measured on an FTIR-650 spectrometer purchased from Tianjin

87

GangDong Sci. & Tech. Development Co., Ltd (Tianjin, China). Optical rotation was

88

measured on MCP 100 Modular Circular Polarimeter (Anton Paar GmbH, Graz,

89

Austria). 1H and 13C NMR, 1H-1H COSY, ROESY, HSQC, and HMBC spectra were

90

recorded with a DD2 (600 MHz) spectrometer in methanol-d4 or dimethylsulfoxide

91

(DMSO)-d6 (Agilent Inc., Santa Clara, CA, USA). CD spectra were obtained with a

92

Jasco-810-CD apparatus (Jasco, Tokyo, Japan). Mass spectra were performed on

93

Agilent 1290 UPLC with a photodiode detector array (PDA) coupled to a 6545

94

time-of-flight (TOF) mass spectrometer with electrospray ionization (ESI) source in

95

negative mode (Agilent Inc., Santa Clara, CA, USA). HPLC semi-preparation was

96

performed on a Waters e2695 combined with a Waters 2998 PDA detector (Waters,

97

Milford, Massachusetts, USA). The semi-preparative column was X Bridge Prep C18

98

(10 × 250 mm i.d., 5 µm) (Waters, Wexford, Ireland). The fluorescence of the

99

samples was measured on a SpectraMax M2e ELIASA (Molecular Devices, Santa

100

Clara, CA, USA). The melting point (mp) was measured on SGWX-4 Micromelting

101

point apparatus purchased from Beijing century science instruments Co., Ltd (Beijing,

102

China).

103

Tea Materials. The commercial white tea Bai-Mudan was purchased from Fujian

104

Pinpinxiang Tea Co., Ltd (Fujian, China) in 2014 and it belongs to Fuding-Dabai tea

105

cultivar.

6


Page 6 of 34

Page 7 of 34


106

Extraction and Isolation. Bai-Mudan tea (4 kg) was ground and extracted with 80%

107

acetone/water for three times at room temperature and then concentrated under

108

reduced pressure to produce a water-soluble extract.22 The aqueous extract was then

109

mixed into dichloromethane (1:1, v/v) to provide dichloromethane-soluble fraction

110

(300 g) and an aqueous phase. The aqueous phase was further extracted with ethyl

111

acetate (1:1, v/v), and n-butanol, successively to provide ethyl acetate-soluble fraction

112

(680 g), n-butanol soluble fraction (200 g), and residue water-soluble fraction (630 g).

113

The n-butanol soluble fraction was subjected to Sephadex LH-20 column

114

chromatography (CC), eluting with water/methanol (1:0−0:1), to get fractions A1-A5,

115

and then the fraction A4 was subjected to MCI-Gel CHP20P gel CC (water/methanol

116

= 1:0−0:1) to obtain fractions B1-B20. Fraction B19 was subjected to Toyopearl CC

117

and eluted with methanol/water (1:1, v/v), yielding ten fractions (C1 to C10). Fraction

118

C1 was then performed on the semi-preparative HPLC eluted with gradient

119

acetonitrile/water (The gradient elution of acetonitrile was set as follows: 0−6 min,

120

18%; 6−8 min, from 18% to 20%; 8−13 min, 20%; 13−13.5 min, from 20% to 18%;

121

13.5−22 min, 18%) to get compound 1 (10 mg), 2 (5 mg), 3a and 3b (15 mg),

122

respectively (Figure 1). Fraction C5 was subjected to Toyopearl CC (water/methanol

123

= 8:2−1:0) to give compound 15 (74 mg). Fraction B4 was applied to Sephadex

124

LH-20 CC with H2O containing increasing proportion of methanol to get fractions

125

D1-D20. Fraction D7 was eluted with water/methanol (6:4) on Toyopearl CC to get

126

compound 4 (12 mg). Fraction D10 was eluted with methanol on Toyopearl CC to

127

obtain compound 5 (8 mg). Fraction D9 was separated by Toyopearl CC

7



128

(methanol/water = 8:2) to give compound 6 (11 mg) and compound 9 (9 mg). Fraction

129

D12 was purified by Sephadex LH-20 CC with 95% ethanol, followed by Toyopearl

130

CC with methanol to afford compound 7 (7 mg) and compound 10 (15 mg). Use of

131

Sephadex LH-20 CC on fraction D15 with methanol and then Toyopearl CC with

132

methanol gave fractions E1-E6. Fraction E2 was subjected to Sephadex LH-20 CC

133

with methanol to obtain compound 11 (11 mg). Fraction D18 was eluted with

134

methanol on Sephadex LH-20 CC and Toyopearl CC to give compound 12 (15 mg).

135

Fraction A1 was subjected to Sephadex LH-20 CC eluted by methanol/water with

136

increasing polarity (1:9 to 10:0) to obtain fraction F1-F5. Fraction F4 was separated

137

by silica gel CC with ethyl acetate/methanol (40:1) and Toyopearl CC with methanol

138

to gain compound 8 (9 mg). Fraction A3 was separated into G1−G4 fractions by

139

Sephadex LH-20 CC with methanol. Fraction G2 was successively subjected to ODS

140

CC (water/methanol = 9:1) and Sephadex LH-20 CC (water/methanol = 1:0−0:1) to

141

obtain ten fractions (H1−H10). DIAION HP20SS gel CC eluting with water/methanol

142

(7:3) was used to separate fraction H4 to yield Fractions I1-I3. Fraction I3 was further

143

separated by Toyopearl CC (water/methanol = 6:4) to afford six subfractions (J1−J6).

144

Fraction H6 and J5 were merged and separated by Sephadex LH-20 CC, eluted with

145

water/methanol (1:0−0:1) in a gradient elution to afford fraction K6 which was further

146

purified by ODS CC (water/methanol = 1:9) to get compound 14 (79 mg). Fraction I2

147

was then purified by Toyopearl CC (water/methanol = 1:0−0:1), ODS CC

148

(water/methanol = 1:0−0:1), followed by Sephadex LH-20 CC (water/methanol =

149

1:0−0:1) to get compound 13 (10 mg).

8


Page 8 of 34

Page 9 of 34


150

UPLC−DAD−ESI/MS Analysis. UPLC−MS analysis was performed on an Agilent

151

1290 UPLC instrument with a PDA coupled to a 6545 TOF mass spectrometer with

152

ESI source in negative mode. The analysis was carried out using an ACQUITY

153

UPLC® BEH Shield RP18 column (2.1 × 150 mm, i.d., 1.7 µm) and Agilent

154

qualitative analysis software for data acquisition. The mobile phase A was 0.1%

155

aqueous formic acid and mobile phase B was 0.1% formic acid acetonitrile. The

156

gradient elution of mobile phase B was set as follows: 0−1.5 min, 6%; 1.5−4 min,

157

from 6 to 12%; 4−8 min, from 12 to 25%; 8−10 min, from 25 to 35%; 10−14 min,

158

from 35 to 90%; then kept at 90% for 5 min and return to 6% in 1 min and kept at 6%

159

for 3 min. The flow rate was 0.22 mL/min under the wavelength of 280 nm and the

160

injection volume was 2 µL. The tea sample was prepared by ultrasonic extracting 0.25

161

g of ground tea powder in 10 mL of 80% aqueous acetone three times within 12 h (15

162

min each time) for UPLC−DAD−ESI/MS analysis. Mass spectra were acquired in

163

negative and full scan mode from m/z 100 to 1700.

164

Determination of AGEs Formation. Determination of the AGEs formation was

165

based on a former reported method with modification.23 The buffer with 0.02%

166

penicillin to prevent degradation. The reaction mixture contained bovine serum

167

albumin 10 mg/mL in PBS with penicillin to prevent bacterial growth, and added with

168

36 mg/mL fructose and glucose. The reaction mixture was then mixed with

169

compounds (EGCG, 1, 2 and 3a, 3b) or positive control aminoguanidine. After

170

incubating at 37 °C for 14 d, the fluorescent reaction products were assayed on

171

ELISA at the excitation and emission maximum of 350 nm and 450 nm. Then a series

9



172

of gradient concentrations of the compounds were measured, all samples were

173

prepared in triplicate.

174

Statistical Analysis. All assay experiments were done in triplicate and the values

175

were presented as mean ± SD. We used GraphPad Prism software (version 6.0) for

176

statistical analysis and IC50 calculation.

177

Results and Discussion

178

Isolation and Identification of Compounds 1, 2 and 3a, 3b. For the phytochemical

179

investigation of white tea, the 80% aqueous acetone extract of white tea was

180

concentrated. The aqueous residue was successively extracted with dichloromethane,

181

ethyl acetate, and n-butanol. And the n-butanol soluble fraction was fractionated by

182

repeated column chromatography (CC) and semi-preparative HPLC to provide four

183

flavoalkaloids (Figure 2), two novel pure compounds (1, 2) and a mixture of two

184

isomers (3a, 3b). At the same time, 12 known compounds were obtained from the

185

Chinese commercial white tea including gallicin (4),24 (+)-catechin (5),25

186

(−)-epicatechin (6),25 gallic acid (7),26 kaempferol (8),27 procyanidin B2 (9),28

187

kaempferol-3-7-O-β-D-dirhamnoside (10),29 epigallocatechin-(4β-8)-epicatechin-3-O

188

-gallate (11),30 EGCG (12),31 1,6-di-O-galloyl-β-D-glucose (13),32 1-O-galloyl-4,6-

189

(−)-hexahydroxydiphenoyl-β-D-glucose (14),30 1,4,6-tri-O-galloyl-β-D-glucose (15).

190

30

191

Compounds 1, 2, 3a and 3b showed similar IR spectrum, which suggested the

192

presence of hydroxyl groups (broad peak around 3397 cm-1), carbonyl group (1695

193

cm-1), aromatic rings (1620, 1539 cm-1).33 Their ESI−HR−MS spectrum showed the

10


Page 10 of 34

Page 11 of 34


194

same deprotonated molecular ion peak at m/z 568.1475 [M-H]− (calcd for 568.1455)

195

corresponding to the molecular formula C28H27NO12 with 16 degrees of unsaturation.

196

The odd number of the molecular weight (569) suggested the presence of a nitrogen

197

atom in the molecule. The UV λmax (MeOH) peaks of compounds 1, 2, 3a and 3b are

198

at 209, 276 nm. The 1H and 13C NMR data of the isomers 1, 2, 3a and 3b were nearly

199

the same (Table 1). Above experimental results suggest that the four flavoalkaloids

200

are regioisomers.

201

Compound 1 was observed as white amorphous powder, mp: 200 °C, [α]25D -36.94 (c

202

0.11, methanol). The 1H and

203

are shown in table 1. The existence of an EGCG skeleton in the molecule could be

204

easily deduced from the 1H NMR spectrum recorded in DMSO-d6 compared with that

205

of EGCG (Figure 3). The typical proton signals for rings A, B and C are similar to

206

those of EGCG,31 at δH 5.00 (1H, s, H-2), δH 5.56 (1H, s, H−3), 3.03 (1H, m, H−4β),

207

2.84 (1H, m, H−4α) (ring C), δH 6.51 (2H, s, H−2', 6') (ring B), δH 6.07 (s, 1H) (ring

208

A), δH 6.95 (2H, s) (galloyl−H), respectively (Figure 3).31 A single proton signal at

209

A-ring suggested a substitute at C−6 or C−8 of the A−ring. Besides signals from

210

flavan-3-ol and galloyl unit, the 1H and

211

attributable to a methine (C−5''' ), two methylenes (C−3''', C−4''' ), a carbonyl (C−2''' )

212

and an ethyl group. The 1H−1H COSY correlations of these methine and methylenes

213

indicated the presence of a partial structure of −CH2−CH2−CH−. In the HMBC

214

spectrum, the methylene protons of the N-ethyl group were correlated with the

215

carbonyl carbon (δC 178.4, C−2''') and the methine carbon (δC 55.6, C−5'''). The

13

C NMR data of compound 1 recorded in methanol-d4

13

C NMR spectra also showed signals

11



Page 12 of 34

216

methylene protons (δH 2.20, H−3''') were also correlated with carbonyl carbon. These

217

1

218

N-ethyl-2-pyrrolidinone ring (Figure 4). The weak ROESY correlations of singal at

219

A−ring (δH 6.07, CH-8) with that of B−ring (δH 6.51, CH−2', 6'), C−ring (δH 5.00,

220

CH−2) and 7-OH proton (δH 9.44) (Figure S12) allowed the attachment of the

221

N-ethyl-2-pyrrolidinone group at C−6 position and the proton signal (δH 6.07) belongs

222

to C−8 position (Figure 4),34 which are proven with the corresponding HMBC

223

correlations from H−5''' (δH 5.43) to C−5 (δC 156.4) and C−7 (δC 158.5).

224

The absolute configurations at C−2/3 of four flavoalkaloids were confirmed as 2R, 3R

225

by comparing CD curves with those of EGCG (Figure S32). The configuration at

226

C−5''' can be distinguished by subtracting one CD spectrum from the other

227

stereoisomer with the same configuration at C−2/3.16,35 In this research, compounds 1

228

and 2 had the same skeleton as EGCG. In order to determine the configuration at

229

C−5''' of 1 and 2, the CD spectra of 1 and 2 were compared after subtracting the CD

230

spectrum from each other (Figure 5).16 For compound 1, the arithmetically isolated

231

CD curves of C−5''' showed a strong positive cotton effect at 213 nm (∆ε＋10.5) and

232

was determined to be of the 5''' S-configuration. Therefore, the structure of compound

233

1

234

epigallocatechin-3-O-gallate and named as etc-pyrrolidinone A.

235

Compound 2 were obtained as white amorphous powder, mp: 193 °C, [α]25D -143.21

236

(c 0.02, methanol). Comparing 1H and

237

(Table 1), compound 2 was determined to be an isomer of 1. The position of

H−1H

COSY

was

and

HMBC

determined

correlations

to

be

13

revealed

the

presence

of

an

(−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-

C NMR spectra between compound 1 and 2

12


Page 13 of 34


238

N-ethyl-2-pyrrolidinone in 2 was also determined to be at C−6 by the analysis of the

239

ROESY (Figure S18) and HMBC spectra. Meanwhile, compound 2 presented a

240

negative cotton effect at 213 nm (∆ε − 10.5) comparing with 1 by arithmetically CD

241

curves (Figure 5) and was determined to be 5''' R-configuration. Therefore, the

242

structure

243

(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate

244

etc-pyrrolidinone B.

245

For compounds 3a and 3b, we observed them as a mixture in the format of white

246

amorphous powder, [α]25D -78.37 (c 0.22, methanol). Their melting point is greater

247

than 300 °C. The 1H and

248

those of 1. But for 3a, the ROESY correlation of a proton (δH 6.06) with 5-OH and

249

7-OH proton (δH 9.38, δH 9.53) confirmed that this proton (δH 6.06) belongs to C-6

250

and the N-ethyl-2-pyrrolidinone group linked to the C-8 position (Figure S28). So,

251

compounds

252

(−)-8-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate. Furthermore, 3a and 3b

253

can be identified by 2D NMR spectroscopy. Compound 3a was determined as

254

(−)-8-(5'''S)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate

255

etc-pyrrolidinone

256

8-(5'''R)-N-ethyl-2-pyrrolidinone-epigallocatechin-3-O-gallate

257

etc-pyrrolidinone D. Although 3a and 3b had been synthesized and identified as a

258

mixture too,15 they were isolated as new natural products from tea in present study.

259

Naturally occurring flavoalkaloid tend to exist as isomers in the plant kingdom

of

compound

13

C.

determined and

to named

be as

C NMR spectra of 3a and 3b were also closely similar to

and

3a

was

2

were

3b

3b

was

determined

and

determined

13


to

named as

and

named

be

as (−)as


260

including tea and other food materials,14,16,21 which results in the difficulty to purify

261

and identify these compounds from plants.

262

UPLC−MS Analysis. In this study, the four flavoalkaloids (1, 2, 3a and 3b) were

263

used as standards to analyze Bai-Mudan tea and fresh tea leaves of Fuding-Dabai by

264

UPLC−PDA−ESI/MS analysis. The result showed that these four flavoalkaloids (1, 2,

265

3a and 3b) could be found in white tea Bai-Mudan (Figure 6A), as well as the fresh

266

tea leaves (material of the former) (Figure S1), which implies the original presence of

267

flavoalkaloids in tea as pyrrolidinonated ester-type catechins (such as EGCG) with

268

epi-configuration at C-2 and -3 position (2R, 3R) and also suggests the existence of a

269

related biosynthetic pathway in Camellia sinensis. The MS/MS spectrum gave the

270

fragmental peaks at 416, 398, 236, 169, 125 (Figure 6B) corresponding to the

271

elucidated fragmental structures as shown in Figure 6C, which further confirmed the

272

identified flavoalkaloid structure.16,36 The retention time of compound 1, 2, 3a and

273

3b are 11.07, 11.88, 10.86 min, respectively. The content of these compounds is

274

around several ppm/dry weights on the base of the amount of the valualbe

275

flavoalkaloids we isolated, further accumulation of these flavoalkaloids and

276

quantification of them in different tea leaves is highly warranted.

277

Inhibition of Compounds 1, 2 and 3 (3a and 3b) on Formation of AGEs

278

The effect of the compounds on the formation of AGEs was evaluated with different

279

concentrations (0.1, 1, 5, 10, 25, 50, 100 µM). Figure 7 shows that the four

280

compounds (1, 2, 3a and 3b) and EGCG can effectively reduce the formation of

281

AGEs with a dose-response inhibition. Among them, the mixture compounds of 3a

14


Page 14 of 34

Page 15 of 34


282

and 3b exhibited the most potential inhibitory activity against AGEs formation, with

283

the IC50 values of 10.3 µM. The new compounds (1, 2) and EGCG also showed a

284

promising activity with IC50 values at 25.3, 13.5, and 11.7 µM, respectively, better

285

than the well known glycation inhibitor, aminoguanidine with the IC50 value at 228.8

286

µM .

287

Flavoalkaloids represent a convergence of diverse array of structures resulted from

288

multiple biosynthetic pathways.14,37 To date, a dozen of flavoalkaloids including these

289

in the present study were detected and isolated from Camellia sinensis.15,16 The first

290

tea flavoalkaloid ethylpyrrolidinonyl theasinensin A was obtained from black tea. The

291

researchers posed that l-theanine was degraded to Strecker aldehyde and conjugated

292

with tea polyphenol A rings during the drying and enzyme deactivation stages of

293

black tea production.15 The later study demonstrated that puerins I-VIII were

294

biosynthesized from catechins and l-theanine through a kind of typical fungi

295

Aspergillus niger in the Chinese dark tea during the post-fermentation process.16 Both

296

above researches suggested that flavoalkaloids were formed through a Strecker

297

aldehyde during manufacture process of tea. In our study, the four flavoalkaloids (1, 2,

298

3a and 3b) were isolated from the Chinese white tea (Bai-Mudan), and identified as

299

etc-pyrrolidinones sharing the same EGCG skeleton (Figure 2), which are different

300

from those reported from dark tea.16 The flavoalkaloids from dark tea all lose the

301

galloyl group in the structure since galloyl group of EGCG tends to fall off during the

302

fermentation process.10,16 However, the four flavoalkaloids (Figure 2) isolated from

303

commercial white tea were also detected in fresh tea leaves (Figure S1), which

15



Page 16 of 34

304

suggested that manufacture process is not the only pathway for the formation of tea

305

flavoalkaloids. EGCG and epicatechin gallate (ECG) are the most abundant catechins

306

in tea. L-theanine is the characteristic amino acid accounting for about 50% of total

307

amino acids in C. sinensis. Flavoakaloids in tea may be the products from the

308

interaction between the above most abundant metabolites EGCG or ECG, and

309

l-theanine in tea plant. Most of the original biosynthetic products may be present in

310

tea plant as pyrrolidinonated EGCG or ECG. The flavoalkaloids in dark tea may be

311

derived from losing galloy group of these pyrrolidinonated EGCG or ECG and then

312

configuration changes during the process.16 Therefore, flavoalkaloids may also be

313

biosynthesized by the function of enzymes in the fresh leaves of tea plants, with

314

EGCG or ECG and l-theanine as the vital precursors. As such, a biosynthetic pathway

315

in tea plant for these flavoalkaloids was proposed in Figure 8. The biosynthesis may

316

be

317

N-ethyl-pyrrolidinone, followed by coupling of the product and EGCG. The key

318

enzymes such as glutamic acid dehydrogenase38 and glutamic acid decarboxylase39

319

may play important roles in the reaction.

320

Flavoalkaloids are not common but often rewarded with pronounced biological

321

activities.14,37 We evaluated the isolated flavoalkaloids for their effect on the

322

formation of AGEs and demonstrated them as effective inhibitor against the formation

323

of AGEs with IC50 values ranging from 10.3 to 25.3 µM compared with the positive

324

control aminoguanidine with the IC50 at 228.8 µM (Figure 7). This observation is

325

well consistent with a former report that two new flavoalkaloids isolated from the

completed

through

deamination,

decarboxylation

16


of

l-theanine

into

Page 17 of 34


326

roots of Actinidia arguta, which also showed stronger activity than that of

327

aminoguanidine.21 The unsubstituted carbons at A ring of these flavoalkaloids are the

328

active sites for trapping reactive dicarbonyl species according to previous reports,

329

which suggest that these flavonoids may prevent the development of diabetic

330

complications.40 However, they showed no big difference in the assay from that of

331

EGCG, which implied that more assays are needed for determination of the special

332

bioactivity of these tea flavoalkaloids since they shared a pyrrolidinone ring and

333

should have different effect in some assays from EGCG.

334

In conclusion, fifteen compounds including two novel (1 and 2) and new naturally

335

occurring flavoalkaloids (3a and 3b) were isolated from the commercial Chinese

336

white tea. The absolute configuration at C−5''' of the pyrrolidinone ring of the novel

337

compounds were unambiguously assigned by CD analyses. These flavoalkaloids 1, 2,

338

and 3 showed inhibitory effect against the formation of AGEs with the IC50 values at

339

25.3, 13.5, and 10.3 µM, respectively.

340

Supplementary Data

341

1

342

and 3b (Figure S5-28). The optical rotation value, CD value, and infrared

343

spectrogram of compound 1, 2, 3a and 3b (Figure S29-32).

344

Author Contributions: X. Li and G. J. Liu contribute equally to the paper

345

Funding

346

Financial assistances were received with appreciation from Nutrition and Quality

347

& Safety of Agricultural Products, National Modern Agriculture Technology System

348

Grant CARS-23.

H,

13

C, COSY, HSQC, HMBC, ROESY and DEPT spectra of compounds 1, 2, 3a

17



349

Notes

350

Guan-Hu Bao, Xiao Li, Xue-Shi Liu declare patent applications (CN201810201244.4)

351

with Anhui Agricultural University for the use of the two new flavoalkaloids 1 & 2.

352

All the other authors declare no competing financial interest. All the other authors

353

declare no competing financial interest.

354

References

355

1. Ning, J. M.; Ding, D.; Song, Y. S.; Zhang, Z. Z.; Luo, X.; Wan, X. C. Chemical

356

constituents analysis of white tea of different qualities and different storage times. Eur.

357

Food. Res. Technol. 2016, 242, 2093−2104.

358

2. Tan, J.; Engelhardt, U. H.; Lin, Z.; Kaiser, N.; Maiwald, B. Flavonoids, phenolic

359

acids, alkaloids and theanine in different types of authentic Chinese white tea samples.

360

J. Food Compos. Anal. 2017, 57, 8−15.

361

3. Pastoriza, S.; Mesias, M.; Cabrera, C.; Rufian-Henares, J. A. Healthy properties of

362

green and white teas: an update. Food Funct. 2017, 8, 2650−2662.

363

4. Shukla, Y. Tea and cancer chemoprevention: a comprehensive review. Asian Pac. J.

364

Cancer Prev. 2007, 8,155−166.

365

5. Yen, W. J.; Chyau, C. C.; Lee, C. P.; Chu, H. L.; Chang, L. W.; Duh, P. D.

366

Cytoprotective effect of white tea against H2O2-induced oxidative stress in vitro. Food

367

Chem. 2013, 141, 4107−4114.

368

6. Venditti, E.; Bacchetti, T.; Tiano, L.; Carloni, P.; Greci, L.; Damiani, E. Hot vs.

369

Cold water steeping of different teas: do they affect antioxidant activity?. Food Chem.

370

2010, 119, 1597–1604.

18


Page 18 of 34

Page 19 of 34


371

7. Tenore, G. C.; Campiglia, P.; Giannetti, D.; Novellino, E. Simulated gastrointestinal

372

digestion, intestinal permeation and plasma protein interaction of white, green, and

373

black tea polyphenols. Food Chem. 2015, 169, 320−326.

374

8. Santana, R. G.; Orner, G. A.; Amantana, A.; Provost, C.; Wu, S. Y.; Dashwood, R. H.

375

Potent antimutagenic activity of white tea in comparison with green tea in the

376

Salmonella assay. Mutat. Res. 2001, 495, 61−74.

377

9. Wang, W.; Fu, X. W.; Dai, X. L.; Hua, F.; Chu, X. G.; Chu, M. J.; Hu, F. L.; Ling, T.

378

J. Gao, L. P.; Xie, Z. W.; Wan, X. C.; Bao, G. H. Novel acetylcholinesterase inhibitors

379

from Zijuan tea and biosynthetic pathway of caffeoylated catechin in tea plant. Food

380

Chem. 2017, 237, 1172−1178.

381

10. Zhu, Y. F.; Chen, J. J.; Ji, X. M.; Hu, X.; Ling, T. J.; Zhang, Z. Z.; Bao, G. H.; Wan,

382

X. C. Changes of major tea polyphenols and production of four new B-ring fission

383

metabolites of catechins from post-fermented Jing-Wei Fu brick tea. Food Chem.

384

2015, 170, 110−117.

385

11. Zhang, W.; Li, X.; Hua, F.; Chen, W.; Wang, W.; Chu, G. X.; Bao, G. H. Interaction

386

between ester-type tea catechins (ETC) and neutrophil gelatinase - associated

387

lipocalin (NGAL): inhibitory mechanism. J. Agric. Food Chem. 2018, 66, 1147-1156.

388

12. Houghton, P. J. Chromatography of the chromone and flavoalkaloids. J.

389

Chromatogr. A. 2002, 96, 775−784.

390

13. Johns, S. R.; Russel, J. H. Ficine, a novel flavonoidal alkaloid from Ficus

391

pantoniana. Tetrahedron. Lett. 1965, 24, 1987−1991.

392

14. Calvert, M. B.; Sperry, J. Flavoalkaloids-Isolation, Biological Activity, and Total

19



393

Synthesis. The Alkaloids. 2017, 77, 1−31.

394

15. Tanaka, T.; Watarumi, S.; Fujieda, M.; Kouno, I. New black tea polyphenol having

395

N-ethyl-2-pyrrolidinone moiety derived from tea amino acid theanine: isolation,

396

characterization and partial synthesis. Food Chem. 2005, 93, 81−87.

397

16. Wang, W.; Zhang, L.; Wang, S.; Shi, S.; Jiang, Y.; Li, N.; Tu, P. F. 8-C

398

N-ethyl-2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese

399

dark teas formed in the post-fermentation process provide significant antioxidative

400

activity. Food Chem. 2014, 152, 539−545.

401

17. Ma, Q.; Xie, H.; Li, S.; Zhang, R.; Zhang, M.; Wei, X. Flavonoids from the

402

pericarps of Litchi chinensis. J. Agric. Food Chem. 2014, 62, 1073−1078.

403

18. Nguyen, T. B.; Lozach, O.; Surpateanu, G.; Wang, Q.; Retailleau, P.; Iorga, B. I.;

404

Meijer, L.; Gueritte, F. Synthesis, biological evaluation, and molecular modeling of

405

natural and unnatural flavonoidal alkaloids, inhibitors of kinases. J. Med. Chem. 2012,

406

55, 2811−2819.

407

19. Wang, L.; Wang, S.; Yang, S.; Guo, X.; Lou, H.; Ren, D. Phenolic alkaloids from

408

the aerial parts of Dracocephalum heterophyllum. Phytochemistry. 2012, 82,

409

166−171.

410

20. Christian, B. A.; Grever, M. R.; Byrd, J. C.; Lin, T. S. Flavopiridol in chronic

411

lymphocytic leukemia: a concise review. Clin. Lymphoma. Myeloma. 2009, 9,

412

179−185.

413

21. Jang, D. S.; Lee, G. Y.; Lee, Y. M.; Kim, Y. S.; Sun, H.; Kim, D. H.; Kim, J. S.

414

Flavan-3-ols having a γ-Lactam from the roots of Actinidia arguta inhibit the

20


Page 20 of 34

Page 21 of 34


415

formation of advanced glycation end products in Vitro. Chem. Pham. Bul. 2009, 57,

416

397−400.

417

22. Bai, W. X.; Wang, C.; Wang, Y. J.; Zheng, W. J.; Wang, W.; Wan, X. C.; Bao, G. H.

418

Novel acylated flavonol tetraglycoside with inhibitory effect on lipid accumulation in

419

3T3-L1 cells from Lu’an GuaPian tea and quantification of flavonoid glycosides in

420

six major processing types of tea. J. Agric. Food Chem. 2017, 65(14), 2999−3005.

421

23. Vinson, J. A.; Howard, T. B. Inhibition of protein glycation and advanced

422

glycation end products by ascorbic acid and other vitamins and nutrients. J. Nutr.

423

Biochem. 1996, 7, 659−663.

424

24. Gonzlez, A. G.; Bermejo, J.; Mansilla, H.; Galindo, A.; Amaro, J. M.; Massanet, G.

425

M. Chemistry of the compositae. Part 38. Structure and absolute configuration of

426

gallicin, a new germacranolide from Artemisia. J. Chem. Soc. Perkin. Trans. 1. 1978,

427

10, 1243−1246.

428

25. Abd-El-razek M. H. NMR Assignments of Four Catechin Epimers. Asian J. Chem.

429

2007, 19, 4867−4872.

430

26. Chanwitheesuk, A.; Teerawutgulrag, A.; Kilburnb, J. D.; Rakariyatham, N.

431

Antimicrobial gallic acid from Caesalpinia mimosoides Lamk. Food Chem. 2007, 100,

432

1044−1048.

433

27. Li, Y. L.; Li, J.; Wang, N. L.; Yao, X. S. Flavonoids and a new polyacetylene from

434

Bidens parviflora Willd. Molecules. 2008, 13, 1931−1941.

435

28. Lokman, K. M.; Haslam, E.; Williamson, M. P. Structure and Conformation of the

436

Procyanidin B-2 Dimer. Magn. Reson. Chem. 1997, 35, 854−858.

21



Page 22 of 34

437

29. Dao, TTH.; Kim, H. K.; Linthorst, HJM.; Choi, Y. H.; Verpoorte, R. Identification

438

of metabolites in Arabidopsis thaliana. Plant Physiol. Biochem. 2009, 47, 146−152.

439

30. Nonaka, G.; Sakai, R.; Nishioka, I. Hydrolysable Tannins and Proanthocyanidins

440

from Green Tea. Phytochemistry. 1984, 23, 1753−1755.

441

31. Davis, A. L.; Cai, Y.; Davies, A. P.; Lewis, J. R. 1H and 13C NMR assignments of

442

some green tea polyphenols. Magn. Reson. Chem. 1996, 34, 887−890.

443

32. Nonaka, G.; Nishioka, I. Tannins and related compounds. X. Rhubarb(2): Isolation

444

and structures of a glycerol gallate, gallic acid glucoside gallates, galloylglucoses and

445

isolindleyin. Chem. Pharm. Bull. 1982, 31, 1652−1658.

446

33. Li, N.; Shao, L.; Zhang, C. F.; Zhang, M.; Two new flavonoid alkaloids from

447

Senecio argunensis. J. Asian. Nat. Prod. Res. 2008, 10, 1143−1146.

448

34. Ilkei, V.; Spaits, A.; Prechl, A.; Müller, J.; Könczöl, Á.; Lévai, S.; Riethmüller,

449

E.; Szigetvári, Á.; Béni, Z.; Dékány, M.; Martins, A.; Hunyadi, A.; Antus, S.;

450

Szántay, J. C.; Tibor, B. G.; Kalaus, G.; Bölcskei, H.; Hazai, L. C8-selective

451

biomimetic transformation of 5,7-dihydroxylated flavonoids by an acid-catalysed

452

phenolic Mannich reaction: Synthesis of flavonoid alkaloids with quercetin and

453

(e)-epicatechin skeletons. Tetrahedron. 2017, 73, 1503−1510.

454

35. Ren, D. M.; Guo, H. F.; Yu, W. T.; Wang, S. Q.; Ji, M.; Lou, H. X.

455

Stereochemistry

456

Phytochemistry. 2008, 69, 1425−1433.

457

36. Umehara, M.; Yanae, K.; Maruki-Uchida, H.; Sai, M. Investigation of

458

epigallocatechin-3-O-caffeoate and epigallocatechin-3-O-pcoumaroate in tea leaves

of

flavonoidal

alkaloids

from

22


Dracocephalum

rupestre.

Page 23 of 34


459

by LC/MS-MS analysis. Food Res. Int. 2017, 102, 77–83.

460

37. Khadem, S.; Marles R. J. Chromone and flavoalkaloids: occurrence and

461

bioactivity. Molecules. 2012, 17, 191−206.

462

38. Smith, T. J. Green tea polyphenols in drug discovery: a success or failure. Expert

463

Opin. Drug. Discov. 2011, 6, 589−595.

464

39. Fenalti, G.; Law, R. H.; Buckle, A. M.; Langendorf, C.; Tuck, K.; Rosado, C. J.;

465

Faux, N. G.; Mahmood, K.; Hampe, C. S.; Banga, J. P.; Wilce, M.; Schmidberger,

466

J.; Rossjohn, J.; El-Kabbani, O.; Pike, R. N.; Smith, A. I.; Mackay, I. R.; Rowley, M.

467

J.; Whisstock, J. C. Green tea polyphenols in drug discovery: a success or failure? Nat.

468

Struct. Mol. Biol. 2007, 14, 280−286.

469

40. Huang, Q.; Wang, P.; Zhu, Y.; Lv, L. & Sang. S. Additive capacity of [6]-shogaol

470

and epicatechin to trap methylglyoxal. J. Agric. Food Chem. 2017, 65, 8356−8362.

471

23



472

Figure Captions

473

Figure 1. The semi-preparative chromatogram and ultraviolet absorption spectrogram

474

of four flavoalkaloids by HPLC.

475

Figure 2. The structure of four flavoalkaloids isolated from white tea.

476

Figure 3. 1H NMR spectra of (−)-epigallocatechin-O-gallate (EGCG) and compound

477

1 in dimethylsulfoxide (DMSO)-d6.

478

Figure 4. Selected two dimensional nuclear magnetic resonance (2D NMR)

479

correlations including the key 1H-1H COSY (heavy solid line)，HMBC (solid single

480

arrowhead line), ROESY (dashed double arrowhead line) correlations of 1 and 3a, 3b.

481

Figure 5. The configuration of compounds 1 and 2 was determined by arithmetically

482

CD curves subtracted each other for a couple of stereoisomers.

483

Figure 6. A: LC-MS total (TIC) and extracted (EIC at 568) ion chromatograms of the

484

80% aqueous acetone extract of Bai-Mudan tea together with the TIC of compounds 1

485

2, and 3. B: fragmental peaks of compounds 1-3 from MS/MS spectrum. C: the

486

deduced structures of the fragment peaks of compounds 1-3.

487

Figure 7. Does-response inhibition curves and IC50 of compounds 1, 2, 3, and

488

(−)-epigallocatechin -O-gallate (EGCG) against AGEs, with the IC50 of the positive

489

control aminoguanidine (AG).

490

Figure 8. A possible biosynthetic route to flavoalkaloids in tea plant.

24


Page 24 of 34

Page 25 of 34


Table 1. NMR Spectroscopic Data of Compound 1, 2, 3a, 3ba position 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 1'' 2'' 3'' 4'' 5'' 6'' 7'' 1''' 2''' 3''' 4''' 5''' 6''' 7''' a1

compound 1 δH (J, δc Hz) 5.00 s 79.3 5.56 s 70.4 3.03 m 28.2 2.84 m 156.4 109.0 158.5 6.07 s 98.2 157.3 100.7 131.4 6.51 s 107.7 147.5 134.7 147.5 6.51 s 107.7 122.2 6.95 s 111.1 147.1 140.7 147.1 6.95 s 111.1 168.4

compound 2 ∆H (J, δc Hz) 4.98 s 79.5 5.56 s 70.6 3.00 m 28.5 2.86 m 156.8 109.3 158.8 6.07 s 98.5 157.5 101.0 131.6 6.50 s 108.0 147.8 135.0 147.8 6.50 s 108.0 122.4 6.94 s 111.3 147.4 141.0 147.4 6.94 s 111.3 168.7

178.4 25.3 33.4

178.7 25.6 33.7

2.20 m 2.44 m 2.66 m 5.43 dd (5.2,9.2) 2.66 m 3.49 m 1.00 m

55.6 37.2 13.4

2.15 m 2.39 m 2.68 m 5.45 dd (4.8,9) 2.65 m 3.50 m 0.99 m

55.7 37.5 13.7

compound 3a δH (J, Hz) δc 5.00 s 5.53 s 2.99 m 2.88 m 6.06 s

6.52 s

6.52 s 6.95 s

6.95 s

2.20 m 2.39 m 2.65 m 5.55 m 2.90 m 3.56 m 1.14 m

79.8 70.5 27.6 157.3 97.0 158.3 106.4 155.9 99.9 131.6 107.7 147.6 134.8 147.6 107.7 122.5 111.2 147.2 140.8 147.2 111.2 168.4 178.4 25.3 33.5 55.1 37.8 14.0

compound 3b δH (J, Hz) δc 4.91 s 5.47 s 2.95 m 2.85 m 6.03 s

6.44 s

6.44 s 6.92 s

6.94 s

2.18 m 2.34 m 2.59 m 5.46 m 2.72 m 3.38 m 0.97 m

80.1 70.3 27.9 157.3 97.8 158.3 106.7 155.9 100.9 131.3 108.2 147.6 135.0 147.6 108.2 122.5 111.2 147.2 140.8 147.2 111.2 168.6 178.1 25.3 33.1 55.1 37.3 13.6

H at 600 MHz and 13 C NMR at 150 MHz in methanol-d4. s: single peak; d: double

peaks; m:multipeaks.

25



TOC Graphic

26


Page 26 of 34

Page 27 of 34


Figure 1 90x97mm (600 x 600 DPI)



73x64mm (600 x 600 DPI)


Page 28 of 34

Page 29 of 34







Page 30 of 34

Page 31 of 34







Page 32 of 34

Page 33 of 34


61x45mm (600 x 600 DPI)



291x117mm (300 x 300 DPI)


Page 34 of 34

Novel Flavoalkaloids from White Tea with Inhibitory Activity against the

Recommend Documents