Two Pairs of Isomerically New Phenylpropanoidated Epicatechin

Subscriber access provided by UNIV OF LOUISIANA

Bioactive Constituents, Metabolites, and Functions

Two Pairs of Isomerically New Phenylpropanoidated Epicatechin Gallates with Neuroprotective Effects on H2O2-Injured SHSY5Y Cells from Zijuan Green Tea and Their Changes in Fresh Tea Leaves Collected from Different Months and Final Product Jia-Ping Ke, Wen-Ting Dai, Wen-Jun Zheng, Hao-Yue Wu, Fang Hua, Fenglin Hu, Gang-Xiu Chu, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01365 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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 33

Journal of Agricultural and Food Chemistry

Two

Pairs

Epicatechin

of

Isomerically

Gallates

with

New

Phenylpropanoidated

Neuroprotective

Effects

on

H2O2-Injured SH-SY5Y Cells from Zijuan Green Tea and Their Changes in Fresh Tea Leaves Collected from Different Months and Final Product Jia-Ping Ke,1,† Wen-Ting Dai,2,† Wen-Jun Zheng,1,† Hao-Yue Wu,1 Fang Hua,1 Feng-Lin Hu,3 Gang-Xiu Chu,1,* Guan-Hu Bao1,* 1Natural

Products Laboratory, State Key Laboratory of Tea Plant Biology and

Utilization, Anhui Agricultural University, Hefei, Anhui Province 230036, China 2Department

of Pharmacy, the Second People’s Hospital of Hefei, Hefei 230011,

China 3Research

Center on Entomogenous Fungi, Anhui Agricultural University, 130 West

Changjiang Road, Hefei, Anhui Province 230036, China

*Corresponding author. Phone: +86-551-65786401. Fax: +86-551-65786765; E-mail: [email protected] (G.-H. Bao). *Corresponding author. Phone: +86-551-65786217. Fax: +86-551-65786765. E-mail: [email protected] (G.-X. Chu).

1

ACS Paragon Plus Environment


2

Abstract: Zijuan tea (Camellia sinensis var. assamica), an anthocyanin-rich cultivar

3

with purple leaves, is a kind of valuable material for manufacturing tea with unique

4

color and flavor. In this paper, four new phenylpropanoid substituted epicatechin

5

gallates (pECGs), Zijuanins A−D (1−4), were isolated from Zijuan green tea by

6

different column chromatography. Their structures were identified by extensive high

7

resolution mass spectroscopy (HR-MS), nuclear magnetic resonance (NMR),

8

experimental and calculated circular dichroism (CD) spectroscopic analyses.

9

Detection of the changes in fresh tea leaves collected from April to September and the

10

final processed product by high performance liquid chromatography (HPLC) -HRMS

11

suggested that production of compounds 1 and 2 may be enhanced by the processing

12

procedure of Zijuan green tea. Additionally, 1-4 were proposed to be synthesized

13

through interaction between the abundant secondary metabolite ECG and phenolic

14

acids from tea leaves by two key steps of phenol-dienone tautomerism. 1 and 2

15

showed impressive activity in protecting SH-SY5Y cells against H2O2-induced

16

damage at the concentration of 1.0 μM.

17

Keywords: Camellia sinensis, Zijuan green tea, phenylpropanoidated epicatechin

18

gallates, neuroprotective, SH-SY5Y cells.

2


Page 2 of 33

Page 3 of 33


20

INTRODUCTION

21

Tea is a natural, daily drink all over the world, offering a large number of health

22

benefits. Depending on different manufacturing processes, tea can be classified into

23

six major types, green, white, yellow, oolong, black, and dark tea.1,2 Green tea, a

24

representative of non-fermented tea, is produced through steaming and drying the

25

fresh leaves which stops the enzymatic oxidation, keeping the components more

26

original to those of fresh leaves. i.e., it contains the largest amount of catechins.2,3

27

Catechins are considered as the most beneficial compounds in tea for human health

28

due to the diversity of bioactivities, such as antioxidant properties,4,5 wound-healing

29

effects,6 antihyperglycemic activity,7,8 and antihypertensive function.9

30

Zijuan tea is a characteristic variety in Yunnan province, China. The stem, bud, and

31

leaf of Zijuan tea are purple ascribed to its rich in anthocyanins.10 The aqueous and

32

ethyl acetate extracts of Zijuan tea were reported effective in anti-inflammation,

33

anti-proliferation, and methylglyoxal-trapping.11 Recently, our group reported the

34

isolation and identification of 22 compounds from Zijuan green tea including three

35

phenylpropanoidated catechins with impressive acetylcholinesterase inhibitory

36

activity.12 The neuroprotective effect of the caffeoylated epicatechin (ECC) was

37

further reported with the possible mechanism through interaction with neutrophil

38

gelatinase-associated lipocalin.13 Besides, the biosynthetic pathway of ECC was

39

initially proposed and studied in the paper.12 Inspired by the impressive activity of

40

ECC, we continuingly studied the constituents of Zijuan green tea and discovered

41

more phenylpropanoidated catechins.

3



42

Phenylpropanoidated catechins are a distinctive class of secondary metabolites with

43

the typical flavonoid skeleton substituted with a C6-C3 unit structure at the electron

44

rich positions C-6 or/and C-8 of the A ring or esterized with the 3-hydroxyl group at

45

C ring. Four new phenylpropanoid-substituted flavan-3-ols were first isolated from

46

the bark of Cinchona succirubra.14 From tea, two known together with other four new

47

ones were reported from the famous dark tea, Pu-er tea.15,16 Although present

48

literatures described phenylpropanoidated catechins, phenylpropanoidated catechin

49

gallates have not been reported so far. In this study, we isolated and identified two

50

pairs of isomerically new phenylpropanoidated epicatechin gallates (pECGs), named

51

Zijuanins A-D (1-4), through repeated column chromatography (CC) and semi

52

preparative high performance liquid chromatography (HPLC). The structures of

53

compounds 1-4 were elucidated on the basis of detailed spectroscopic analyses,

54

including 1D and 2D NMR, HR-MS, and CD spectra.

55

Human neuroblastoma cell line SH-SY5Y, subcloned from the SK-N-SH cell line,

56

exhibits neuronal properties and is a classic in vitro model for screening

57

neuroprotective agents used in Parkinson’s or Alzheimer’s disease (AD).17-19 AD is

58

caused by mutifactors and oxidative stress is one of the leading causes. The famous

59

anti-AD drug Huperzine A (hup A) and the major tea polyphenols esp. ester type tea

60

catechins (ETC) have been reported to display neuroprotective effects against H2O2

61

induced injuries on the SH-SY5Y cells.13,18 In this paper, the isolated pECGs were

62

assayed on the same cell line and compounds 1 and 2 showed promising protective

63

effects.

4


Page 4 of 33

Page 5 of 33


64

MATERIALS AND METHODS

65

Chemicals. Pure distilled water was purchased from Watson’s Food & Beverage

66

Co., Ltd. (Guangzhou, China), HPLC grade organic reagent including methanol

67

(MeOH), formic acid, acetonitrile (CH3CN), and acetic acid were bought from

68

DUKSAN (Ansansi, Korea). Analytical grade petroleum ether (PE), ethyl acetate

69

(EtOAc), methanol, dichloromethane (CH2Cl2) were purchased from Kelong

70

Chemical Reagent Co., Ltd (Chengdu, China). Dimethyl sulfoxide (DMSO) was

71

bought from Shanghai Sinopharm Chemical Reagent Co., Ltd (China). Sephadex

72

LH-20 (GE Healthcare Bio-Sciences AB, Sweden), Silica gel (Jiangyou Silicon

73

Development Co., Ltd., Shandong, China), MCI-Gel CHP20P (Mitsubishi Ltd.,

74

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

75

ODS C-18 (Fuji Silysia Chemical Ltd., Japan) as column materials were filled in open

76

column chromatography (CC) to purify compounds. SH-SY5Y cells (Jingke Co., Ltd,

77

Anhui China), Fetal bovine serum (FBS) (Excell, Uruguay), Dulbecco's modified

78

eagle

79

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Solarbio,

80

China) were used in the cell assay experiments.

81

1D (1H and

82

spectra were recorded in dimethyl-d6 sulfoxide (DMSO-d6) with Agilent DD2 600

83

MHz. The Agilent 1100 HPLC system was used to detect samples with a photodiode

84

array detector (PAD), time of flight (TOF) mass spectrometer, and an ESI source. IR

85

spectrum was measured on an FTIR-650 spectrometer purchased from GangDong Sci.

medium

13C

(DMEM)

(HyClone,

America),

NMR) and 2D (1H–1HCOSY, HSQC, HMBC and NOESY) NMR

5



86

& Tech. Development Co., Ltd (Tianjin, China). MCP 100 modular circular

87

polarimeter (Anton Paar GmbH, Graz, Austria) was used to measure Optical rotation.

88

Circular dichroism (CD) spectra were detected with a JASCO J-815 spectrometer

89

(JASCO, Tokyo, Japan).

90

Tea Materials. Zijuan green tea (Camellia sinensis var. assamica cv. Zijuan) for

91

research from a natural products approach (produced in 2016) was purchased from

92

Menghai, Yunnan Province, China. Zijuan tea for LC-MS detection was collected

93

from different months from the Guohe Tea Garden of Anhui Agricultural University

94

at Guohe, Hefei, Anhui Province, China.

95

Extraction and Isolation. Zijuan green tea (4.5 kg) was ground and extracted

96

with PE, EtOAc, and MeOH for three times at room temperature, respectively.12 The

97

tea leaves were entirely soaked by the solvent during the extraction process. The

98

extracted solution was concentrated under reduced pressure to afford three remains:

99

PE fraction (FrA, 18 g), EtOAc fraction (FrB, 50 g), and MeOH fraction (FrC, 600 g).

100

Part of FrC (400 g) was dissolved in water and extracted with CH2Cl2. The remaining

101

aqueous fraction of FrC was applied to a Sephadex LH-20 gel CC (60 cm × 7 cm,

102

length × internal diameter, abbreviated as 60 × 7 cm) eluted with MeOH-H2O (0:1 to

103

1:0), 8000 mL per gradient, yielding seven fractions (C-1 to C-7). Fractions C-2 to

104

C-6 were combined (FrD) and subjected to MCI gel CC (50 × 6 cm) eluted with

105

MeOH-H2O (0:1 to 1:0), 4500 mL per gradient, yielding seven fractions (D-1 to D-7).

106

Fraction E (D-3 to D-6) was applied to a silica gel CC (15 × 5 cm) using

107

CH2Cl2-MeOH (50:1 to 1:1) as the eluant, 1000 mL per gradient, yielding fractions

6


Page 6 of 33

Page 7 of 33


108

(E-1 to E-6). Fraction F (E-4 to E-5) was subjected to a Toyopearl HW-40S gel CC

109

(45 × 3 cm) eluted with MeOH-H2O (20:80 to 100:0), 1000 mL per gradient, yielding

110

five fractions (F-1 to F-5). Fraction G (F-3 to F-5) was subjected to a Sephadex

111

LH-20 gel CC (65 × 1.4 cm) eluted with methanol (300 mL) to yield seven fractions

112

(G-1 to G-7). Fraction H (G-3 to G-6) was applied to a Toyopearl HW-40S gel CC

113

(50 × 1.3 cm) eluted with methanol (200 mL) to get fractions H-1 and H-2. HPLC

114

separation of compounds 1 (3.1 mg) and 2 (4.2 mg) from H-1 was carried out using an

115

SP ODS-A C18 column (250 mm × 4.6 mm i.d., 5 μm). Column temperature was set

116

at 25 °C. The eluent was composed of mobile phase A (water) and mobile phase B

117

(acetonitrile). The isocratic mobile phase A was as follows (Table S1): 0–30 min,

118

72%. The flow rate was 1.0 mL/min. The injection volume was 30 μL. The UV

119

detection wavelength was monitored at 210 and 280 nm during the HPLC separation

120

(Figure S1). Compound 3 (5.1 mg) and 4 (5.6 mg) from H-2 were separated with

121

eluting system as follows (Table S2): 0–5 min, 77% 5–15 min, from 77% to 73%;

122

then kept at 73% for 10 min; 25–26 min, from 73% to 77%, then kept at 77% for 10

123

min. The flow rate was 1.0 mL/min. The injection volume was 30 μL. The UV

124

detection wavelength was monitored at 210 and 280 nm during the HPLC separation

125

(Figure S2). Compounds 2-4 were acquired with the purity ≥ 96% by HPLC analysis

126

(detection methods shown at Table S3, purity check at Figure S3).

127

Zijuanin A (1), colourless powder. [α]25D

= -5500 (c 0.08, 70% aqueous

128

CH3CN); CD (80% aqueous CH3CN), 229 (Δε - 33.20) , 250 (Δε - 5.50) , 274 (Δε -

129

16.80). UV λmax (MeOH) nm (log ε): 194.1 (2.6), 276.8 (2.4); IR (KBr) νmax 3384,

7



Page 8 of 33

130

1708, 1612, 1522, 1451, 1286, 1212, 1106, 1007, 948, 821, 767 cm-1. HR-ESI-TOF

131

m/z 633.1249 [M-H]- (C32H25O14, calcd 633.1244). 1H and 13C NMR data see Table 1.

132

Zijuanin B (2), colourless powder. [α]25D = -15278 (c 0.04, 70% aqueous CH3CN);

133

CD (80% aqueous CH3CN), 227 (Δε - 31.69) , 249 (Δε - 6.50), 286 (Δε - 14.70). UV

134

λmax (MeOH) nm (log ε): 198.8 (2.5), 276.8 (2.2). IR (KBr) νmax 3385, 1707, 1613,

135

1509, 1449, 1286, 1213, 1104, 1037, 971, 823, 767 cm-1. HR-ESI-TOF m/z 633.1260

136

[M-H]- (C32H25O14, calcd 633.1244). 1H and 13C NMR data see Table 1.

137

Zijuanin C (3), colourless powder. [α]25D = -194 (c 0.18, 70% aqueous CH3CN);

138

CD (80% aqueous CH3CN), 229 (Δε - 55.85) , 252 (Δε - 1.82) , 280 (Δε - 10.88). UV

139

λmax (MeOH) nm (log ε): 202.3 (2.9), 279.2 (2.2). IR (KBr) νmax 3387, 1751, 1701,

140

1614, 1514, 1451, 1336, 1234, 1105, 1035, 970, 949, 870, 820, 768 cm-1.

141

HR-ESI-TOF m/z 587.1194 [M-H]- (C31H23O12, calcd 587.1190). 1H and

142

data see Table 2.

143

Zijuanin D (4), colourless powder. [α]25D = -148 (c 0.23, 70% aqueous CH3CN); CD

144

(80% aqueous CH3CN), 217 (Δε - 55.15) , 235 (Δε + 14.62) , 252 (Δε - 13.99), 280

145

(Δε - 13.03). UV λmax (MeOH) nm (log ε): 211.7 (2.6), 279.2 (2.3). IR (KBr) νmax

146

3385, 1756, 1705, 1614, 1514, 1451, 1338, 1232, 1106, 1022, 972, 870, 824, 767 cm-1.

147

HR-ESI-TOF m/z 587.1194 [M-H]- (C31H23O12, calcd 587.1190). 1H and

148

data see Table 2.

13C

13C

NMR

NMR

149

LC-MS Detection of Compounds 1-4 in Zijuan Green Tea Collected from

150

Different Months and the Final Product. Six successive months tea leaves (April to

151

September) and the processed green tea product were ground into fine powder. Tea

8


Page 9 of 33


152

extracts were prepared by ultrasonic extraction twice for 2 h each time within 12 h

153

after adding 100 mL of 70% methanol to 10 g tea powder in a 100 mL glass flask

154

volumetric at 25 °C. After centrifugation at 10,000 rpm for 10 min, supernatant was

155

filtered through 0.22 μm syringe filters for LC-MS analysis.

156

The tea samples were analyzed using an Agilent 1100 HPLC with a PAD coupled

157

to a 6210 TOF mass spectrometer with electrospray ionization (ESI) source. The

158

chromatographic separation was performed on a SunFire™ C18 (3.5 µm, 4.6 ×150

159

mm) at 25 °C, and the HPLC parameters were as follows: injection volume, 10 μL;

160

flow rate, 0.3 mL/min; The mobile phase was a mixture of (A) 0.1% formic acid in

161

water and (B) 0.1% formic acid in acetonitrile, and gradient elution was carried out:

162

6% B for 0–4 min, 6-14% B for 4-16 min, 14-15% B for 16-22 min, 15-18% B for

163

22-32 min 18-29% B for 32-37 min, 29-45% B for 37-45 min, 45% B for 45-50 min,

164

45-100% B for 50-51 min, 100% B for 51-65 min, and 100-6% B for 65-66 min, 6%

165

B for 66-70 min, and the eluate was monitored with a PAD at full-length scan ranging

166

from 190 to 500 nm. The mass spectrometer parameter settings used for the

167

measurement were as follows: negative ionization mode; nebulizer pressure, 45 psi;

168

gas temperature, 350 °C; drying gas, 12 L/min; fragment voltage, 175 V; capillary

169

voltage, 3500 V; skimmer voltage, 65 V; and OCT 1 RF, 250 V. Mass spectra were

170

achieved in full scan MS mode from m/z 100 to 1700. The detailed TOF mass

171

spectrometer parameter settings can be found in the literature.20

172

Assay for Protection against H2O2-Induced Neuronal Damage in SH-SY5Y

173

Cells. The SH-SY5Y cells were cultured in DMEM medium supplemented with 10%

9



Page 10 of 33

174

fetal bovine serum (FBS), 100 units/mL penicillin and streptomycin in a humid

175

atmosphere of 5% CO2 and 95% air at 37 °C. Media were changed every 2 days.

176

SH-SY5Y cells were seeded at a density of 2 × 104 cells per well in 96-well plates and

177

cultured for 24 h. After being incubated with 200 μM H2O2 and 1.0 μM compounds

178

for 24 h, 20 μL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT,

179

5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. The

180

supernatants were removed and added 150 μL DMSO to each well. The absorbance

181

was measured at 490 nm by SpectraMax 190 microplate reader. Hup A (1.0 μM) was

182

used as the positive control. Results are expressed as the mean ± SD. All experiments

183

were done in six independent experiments and repeated triplicate.18

184

Statistical Analysis. All assay experiments were obtained ≥ triplicate, and the

185

values were presented as the mean ± SEM. One way ANOVA with Turkey tests was

186

applied to determine significant differences (***P < 0.001 vs. control, #P < 0.05, ###P

Two Pairs of Isomerically New Phenylpropanoidated Epicatechin

Recommend Documents