Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid

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A Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid Accumulation in 3T3-L1 Cells from Lu'an GuaPian Tea and Quantification of Flavonoid Glycosides in Six Major Processing Types of Tea Running title: A Novel Acylated Flavonol Tetraglycoside Against Lipid Accumulation from Lu'an GuaPian Tea Wu-Xia Bai, Chao Wang, Yijun Wang, Wen-Jun Zheng, Wei Wang, Xiaochun Wan, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00239 • Publication Date (Web): 24 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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Journal of Agricultural and Food Chemistry

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A Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid

2

Accumulation in 3T3-L1 Cells from Lu'an GuaPian Tea and Quantification of

3

Flavonoid Glycosides in Six Major Processing Types of Tea

4

Wu-Xia Bai⊥, Chao Wang⊥, Yi-Jun Wang, Wen-Jun Zheng, Wei Wang, Xiao-Chun

5

Wan, Guan-Hu Bao *

6

Tea natural product laboratory of International Joint Lab of Tea Chemistry and Health

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

8

University, Hefei, P.R. China

9

⊥

These two authors contribute equally to the paper

10

* Corresponding Author

11

Professor Guan-Hu Bao

12

Tea natural product laboratory of International Joint Lab of Tea Chemistry and Health

13

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

14

University

15

Office Phone: 0551-65786401

16

Office Fax: 0551-65786765

17

[email protected]

ACS Paragon Plus Environment


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ABSTRACT: A novel acylated flavonol tetraglycoside, kaempferol 3-O-[(E)-p-

19

coumaroyl-(1→2)][α-L-arabinopyranosyl-(1→3)][β-D-glucopyranosyl

20

rhamnopyranosyl (1→6)]-β-D-glucopyranoside (camellikaempferoside C, 1), together

21

with two flavonol and eighteen flavone and flavonol glycosides (FGs) (2-21) was

22

isolated from the green tea Lu'an GuaPian (Camellia sinensis L.O. Kuntze). Their

23

structures were identified by spectroscopic and chemical methods. Four acylated FGs

24

(1, 7, 8, 9) were found to inhibit the proliferation and differentiation of 3T3-L1

25

preadipocytes at the concentrations 25, 50, 100 µM (P < 0.05). Furthermore, we

26

established a rapid UPLC method to quantify nine FGs in six major processing types

27

of tea. The results showed that dark tea had the highest amount of 20 (0.70 ± 0.017

28

mg/g) and black tea had the highest amount of 8 (0.09 ± 0.012 mg/g) while the

29

amounts of 10 and 16 basically decreased with the increasing degree of fermentation

30

and could contribute to the discrimination of different processing types of tea.

31

KEYWORDS: Flavone and flavonol glycosides (FGs), camellikaempferoside C,

32

Lu'an GuaPian, 3T3-L1 cells, Camellia sinensis


(1→3)-α-L-

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INTRODUCTION

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Tea (Camellia sinensis), a traditional beverage originated in China, has a wide range

35

of consumers among different ages, places, cultures, societies, etc. due to its unique

36

flavor and healthy benefits. According to the different manufacturing processes, tea is

37

generally divided into six major types as green, yellow, white, oolong, black and dark

38

tea.1,2 The manufacturing processes basically determine the flavor, type and content of

39

components of tea.3 Lu'an GuaPian tea, a type of baked green tea produced in Lu'an,

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Anhui province, is one of the top ten tea consumed in China. It is well-known for its

41

unique quality and processing methods. Only fresh leaves without buds or stems of

42

Camellia sinensis (C. sinensis) are used as the material and high baking temperature is

43

needed during its drying step. However, few studies have been conducted on

44

systematic purification and structural identification of chemical constituents from this

45

unique processed green tea.

46

Recently, flavone and flavonol glycosides (FGs) have drawn increasing attention due

47

to their contribution to tea taste and bioactivities. Tea FGs was firstly studied over 50

48

years ago.4 Major types of flavonoids presented in tea are kaempferol, quercetin,

49

myricetin and apigenin, conjugated with glycosides through hydroxyl groups in the

50

case of flavonoid O-glycosides or bound directly to the carbon atoms as flavonoid

51

C-glycosides.5 The glycosides usually are glucoside, galactoside, rhamnoside and

52

arabinoside and the number of the glycosides substituted on the aglycone varies from

53

one to four. Researches showed that FGs affected the astringent taste of tea at a very

54

low threshold concentration in tea, and also FGs can enhance bitterness by



55

augmenting the bitterness of caffeine in tea infusions.6,7 Furthermore, the aqueous

56

solution of FGs has a yellow-green color, which is a key factor to the appearance of

57

green tea soup.8 Besides the main bitterness contributing components of tea, FGs also

58

have various biological activities including anti-oxidant, anti-obesity, anti-cancer and

59

hypoglycemic effects.9-12

60

In this study, we purified and identified the structures of FGs in Lu’an GuaPian green

61

tea. The cytotoxicity and inhibitory effect of four acylated FGs (1, 7, 8, 9) against lipid

62

differentiation and accumulation in 3T3-L1 cells were tested as well. Furthermore, a

63

rapid UPLC method was also established to quantify nine FGs conjugated with sugar

64

units from one to four including four acylated FGs in six major processing types of

65

tea prepared from the same tea leaves.

66 67

MATERIALS AND METHODS

68

Chemicals. HPLC grade acetonitrile, methanol, formic acid and acetic acid were

69

purchased from Duksan (Ansansi, Korea). The standard sugar of L-rhamnose (≥ 99%)

70

was purchased from Shanghai Hushi Laboratorial Equipment Co.Ltd. (Shanghai,

71

China). D-glucose (≥ 99.5%), L-arabinose (98%), and trimethylsilylimidazole (98%)

72

were purchased from Shanghai Energy chemical (Shanghai, China). 3T3-L1 cells

73

were provided by Jiangsu KeyGEN BioTECH Corp., Ltd. Dulbecco’s modified

74

Eagle’s medium (DMEM) were purchased from GIBCO (Invitrogen Co. Ltd., USA).

75

Standard FGs (compounds 1, 7, 8, 9, 10, 16, 17, 20, 21) were isolated from Lu’an

76

GuaPian tea in our tea natural product laboratory and the purity of these compounds


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was ≥ 98% confirmed by UPLC analysis.

78

HPLC purification was carried out with a Waters e2695 separation module

79

accompanied with a Waters 2998 photodiode detector array (PDA) detector (Waters,

80

USA). The column used for separation was a semipreparative X Bridge Prep C18

81

Column (10 × 250 mm, 5 µm, Waters, Ireland) at 30 °C. Samples for LC-MS analysis

82

were performed on a Agilent 1100 HPLC with a PDA combined with a 6210

83

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

84

negative mode (Agilent Technologies, USA). A SunFire™ C18 column (4.6 × 150

85

mm i.d., 3.5 µm, Waters, USA) was applied for analyzing at 30 °C. GC-MS were

86

performed on a HP-5MS column (length = 30 m, i.d. = 0.25 µm, Agilent Technologies,

87

USA) with GCMS-QP2010S (Shimadzu Corp., Japan). 1H NMR,

88

COSY, HMQC and HMBC spectrum were performed in dimethyl-d6 sulfoxide

89

(DMSO-d6) with a Agilent DD2 spectrometer (600 MHz, Agilent Technologies, USA).

90

IR spectrum was obtained with an iS50 FI-IR spectrometer (Thermo, USA). MTT and

91

oil-red O assays were applied on a Tunable Microplate Reader (EL-x800, BioTek

92

Instruments, USA). UPLC analysis for quantification of FGs was performed on

93

Waters ACQUITY UPLCTM HClass UPLC system equipped with a 2489 UV detector

94

on a Waters C18 column (2.1 × 150 mm, 1.7 µm, Waters, Ireland) at 350 nm (Waters,

95

USA).

96

Tea Material and Extraction for UPLC Analysis. Lu’an GuaPian tea used for

97

isolation (produced in 2014) was purchased from Anhui Lu’an GuaPian Tea Company

98

(Anhui, China). Six processing types of tea samples (green, yellow, white, Oolong,


13

C NMR, 1H-1H


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99

black and dark tea) used for UPLC analysis were processed using same material

100

plucked from the cultivars Longjingchangye (C. sinensis var. sinensis) with the

101

corresponding tea manufacturing processes (Figure 1)

102

plucked in May 2015 from the tea base of Anhui Agricultural University (Hefei,

103

Anhui, China). 0.25 g of ground tea powder were stored in 10 mL 70% aqueous

104

methanol which we habitually used in analysis of chemical constituents of tea samples,

105

and extracted twice within 12 h (15 min each time) by ultrasonic extraction. Before

106

UPLC analysis, the tea infusion extracts were centrifuged at 10,000 rpm for 10 min.

107

Then the supernatant was saved after running through a 0.22 µm filter.

108

Extraction and Isolation. Thinking of the large volume of solvent and the low

109

boiling point of acetone easily being dry as well as without apparent difference of the

110

FGs profile between 70% methanol and 80% acetone extraction (Figure S1), Lu’an

111

GuaPian tea (9 kg) was ground and extracted with 80% aqueous acetone for three

112

times at room temperature and concentrated to a water-soluble extract. The water

113

extract

114

dichloromethane-soluble fraction (400 g) and a water-soluble fraction. The

115

water-soluble fraction was further extracted by ethyl acetate (1:1, v/v) to provide ethyl

116

acetate-soluble fraction (760 g) and residue water-soluble fraction (630 g). The

117

water-soluble phase of Lu’an GuaPian tea (400 g) was subjected to MCI-Gel CHP20P

118

gel column chromatography (CC) with methanol:water (1:0 to 0:1, v/v) in a gradient

119

elution, giving five fractions A1-A5. Fraction A2 (40 g) was applied to Sephadex

120

LH-20 CC to get fractions B1-B13. Fraction B13 was eluted with methanol:water (3:7,

was

then

mixed

with

dichloromethane

1-3

. The tea leaves were

(1:1,


v/v)

to

provide

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121

v/v) on ODS CC to give compound 2 (10 mg). Fraction B12 (107 mg) was eluted with

122

30% aqueous methanol on ODS CC and Sephadex LH-20 CC with methanol to get

123

fraction C. Fraction C was separated by HPLC (water:acetonitrile = 8:2) to give

124

compound 3 (3.7 mg) and compound 4 (3.5 mg). Fraction A5 (1 g) was eluted with

125

dichloromethane methanol solution with increasing polarity (50:1 to 0:1, v/v) applied

126

on silica gel CC to get compound 5 (43 mg). Fraction A4 (1 g) was applied to silica

127

gel CC with dichloromethane:methanol mixture with increasing polarity (40:1 to 0:1,

128

v/v) and with methanol on Sephadex LH-20 CC to get compound 6 (22 mg). Silica gel

129

CC was applied on fraction A3 (1.6 g) eluted with dichloromethane methanol solution

130

from 20:1 to 0:1, (v/v), and Sephadex LH-20 CC with methanol to get fractions

131

D1-D2. Fraction D1 was subjected to toyopearl CC for purifying to give compound 7

132

(9 mg) and compound 8 (5 mg). Fraction D2 was performed on toyopearl CC to give

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compound 1 (18.7 mg) and compound 9 (5.6 mg,). Fraction B2 to B7 (35 g) were

134

merged and subjected to silica gel CC eluting with ethyl acetate methanol solution

135

from 20:1 to 0:1 (v/v) to obtain ten fraction F1 to F10. Applying Sephadex LH-20 CC

136

on Fraction F9 with methanol and toyopearl CC with methanol gave fraction G1 to

137

G3. Fraction G2 was compound 10 (20 mg). Fraction G3 was subjected to polyamide

138

CC (ethyl acetate:methanol:water = 3:1 to 0:1, v/v ) to get fraction H1 (compound 11,

139

10 mg) and fraction H2. Fraction H2 was subjected to toyopearl CC eluting with

140

methanol to give three fractions, fraction I1 to I3. Fraction I3 was compound 12 (2.7

141

mg). Fraction I1 was separated by HPLC (water:acetonitrile = 85:15, v/v) to get

142

compound 15 (3.2 mg) and compound 16 (37.4 mg). Fraction I2 was separated by



143

HPLC (water:methanol = 6:4, v/v) to get compound 13 (9.6 mg) and compound 14

144

(3.2 mg). Fraction F4 and F5 were merged, and then subjected to a silica gel CC with

145

dichloromethane methanol solution from 100:1 to 0:1 (v/v), which step produced

146

eight fractions, fraction J1 to J8. Fraction J5 was subjected to toyopearl CC with

147

methanol to give three fractions, fraction K1 to K3. Fraction K1 was applied to ODS

148

CC with methanol:water (3:7, v/v) to give fraction L1 and fraction L2. Fraction L1

149

was applied to toyopearl CC with methanol to give compound 17 (43.5 mg). Fraction

150

L2 was applied to toyopearl CC with methanol to give ten fractions, fraction M1 to

151

M10. Fraction M10 was applied to toyopearl CC with methanol:water (3:7 to 7:3, 1:0,

152

v/v) and 50% aqueous methanol to give compound 19 (13.8 mg). Fraction K2 was

153

applied to toyopearl CC eluting with methanol:water (3:7 to 7:3, 1:0, v/v) and

154

methanol:water (1:1, v/v) to give compound 18 (17.0 mg). Fraction J6 was applied to

155

toyopearl CC eluting with 50% aqueous methanol to give compound 20 (10.5 mg).

156

Fraction J3 was performed on toyopearl CC eluting with methanol:water (3:7 to 7:3,

157

1:0, v/v) and 50% aqueous methanol to give compound 21 (6.0 mg).

158

Acid Hydrolysis and Sugar Analysis of Compound 1. The method was modified

159

according to reference.13 Briefly, compound 1 (0.8 mg) was melted in 2 M HCl (0.8

160

mL), and heated in water bath for 4 h at 80 °C. The reactants were extracted using

161

chloroform to get the supernatant and vacuum freeze-dried. The sample was melted in

162

0.4 mL of pyridine mixed with 10 mg/mL L-cysteine methyl ester hydrochloride at

163

60 °C for 2 h. The solution was then dried by vacuum freeze-drying, following with

164

the addition of 0.2 mL trimethylsilylimidazole to the solid. The mixture was then


Page 8 of 33

Page 9 of 33


165

heated for 1.5 h at 70 °C, and then partitioned between n-hexane and water. GC-MS

166

was carried out to analyze the n-hexane fraction with injector temperature at 280 °C.

167

The oven temperature firstly started at 160 °C for 1 min, and then rose up to 200 °C at

168

a speed of 6 °C/min , and then kept warming-up until a temperature of 280 °C at a

169

speed of 3 °C/min and held for 5 min. The sugar standards were analyzed under the

170

same condition. A comparison between the sugar standards and the sugar units of

171

compound 1 was made with peak and retention time (RT). As a result, D-glucose (RT

172

22.16 min), L-rhamnose (RT 18.55 min) and L-arabinose (RT 16.97 min) were

173

confirmed in a ratio of 2:1:1 for compound 1.

174

Cell Culture. 3T3-L1 cells were cultured in DMEM supplemented with 10% fetal

175

bovine serum (FBS) together with 1% penicillin/streptomycin antibiotics, stored in a

176

moisturized atmosphere with 5% CO2 at 37 °C .14

177

Cell

178

2H-tetrazolium bromide (MTT) assay, cell toxicity of the four acylated FGs (1, 7, 8, 9)

179

was determined.15 3T3-L1 cells were seeded on 96-well plates at a density of 3 × 104

180

cells/well. After 24 h, the media for each compound at three concentrations (25, 50,

181

and 100 µM) was removed and 100 µL of the media was added into the respective

182

wells containing the cells. The negative control was wells with untreated cells, and the

183

positive control was wells treated with taxol (10 µM). After cultured, the 20 µL 5

184

mg/mL MTT was added into every well. The cells culture medium was discarded after

185

incubation for 4 h and the purple precipitate attached to the bottom of the plates were

186

melted in DMSO for 10 min in the dark. The optical density (OD) of each well was

Viability

Assay.

By

using

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-



Page 10 of 33

187

measured at 490 nm.

188

3T3-L1 Cell Adipocyte Differentiation and Oil-red O staining. To stimulate the

189

differentiation, the confluent 3T3-L1 preadipocytes (day 0) were treated with 10

190

µg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 1 µM dexamethasone,

191

respectively.15 After 2 days, the medium was changed to DMEM supplemented with

192

10% FBS. 3T3-L1 preadipocytes were administrated with compound (0, 25, 50 and

193

100 µM) from day 0 to day 8, and the positive control resveratrol (88 µM). After then,

194

mature adipocytes needed leaching with phosphate buffered saline (PBS), and fixed

195

with 10% formalin, and then leached with 60% isopropanol. After air drying, 0.5%

196

oil-red O in isopropanol and water (3:2, w/v) was added to every well at room

197

temperature for 3-4 h. Then the solution was withdrawn and distilled water was used

198

for washing cells. Pictures of stained cells were saved. The oil-red O in triglyceride

199

droplets was extracted with 100% isopropanol and determined at OD510

200

quantification.

201

UPLC Analysis for Quantification of the FGs. The gradient elution of mobile phase

202

B (acetonitrile) in UPLC method was set as follows: 0-2 min, 15%; 2-8 min, from

203

15% to 25%; 8-12 min, from 25% to 30%; 12-16 min, kept at 30%; 16-18 min, from

204

30% to 43%; 18-20 min, kept at 30%; 20-22 min, from 43% to 15%; then returned to

205

15% for 2 min. Mobile phase A was 0.17% aqueous acetic acid with a flow rate of

206

0.22 mL/min under the wavelength of 350 nm. The injection volume was 1 µL. Three

207

replicates were analyzed for each sample. To validate the method described above, we

208

examined the standard curve, linear range, precision, repeatability, stability, limit of


nm

for

Page 11 of 33


209

detection (LOD), limit of quantification (LOQ) and the recovery ratio (supplementary

210

data).

211

Statistical Analysis. All assay experiments were done in triplicate (n = 3) and the

212

values were presented as mean ± SD, unless otherwise specified. One-way ANOVA

213

with Tukey tests was applied to determine significant differences. GraphPad Prism

214

(version 6.0) software was applied for statistical analysis.

215 216

RESULTS AND DISCUSSION

217

Identification of Compound 1. The extract of Lu'an GuaPian tea was suspended in

218

aqueous solution and partitioned successively with dichloromethane and ethyl acetate

219

to provide dichloromethane-soluble fraction, ethyl acetate-soluble fraction, and the

220

residual water-soluble fraction. The water-soluble fraction was subjected to multiple

221

chromatographic purification steps to produce a novel acylated flavonol

222

tetraglycoside (1) and 20 known compounds (2-21) (Figure 2).

223

Compound 1 was observed as yellow amorphous powder with molecular formula of

224

C47H54O26 based on its HR-ESI-MS- (m/z 1033.28429 [M-H]-, calcd 1033.28251). The

225

IR spectrum of 1 indicated the absorption bands of hydroxyl (3394 cm-1), carbonyl

226

(1700 cm-1), double bonds (1604, 1511 cm-1) and O-glycosidic group (1078 cm-1).

227

The 1H NMR and 13C NMR data of compound 1 were shown in Table 1, which were

228

similar to those of compound 9, except for the differences in signals of the B-ring

229

moiety.16 The 1H NMR and 13C NMR spectrum of 1 presented two doublet signals at

230

δH 7.94 (2H, d, J = 9.0 Hz) and δH 6.87 (2H, d, J = 9.0 Hz), and δC 131.3 and δC 115.6,



Page 12 of 33

231

respectively, which suggested presence of a kaempferol aglycone. Furthermore, the

232

signals appeared at δH 7.55 (1H, d, J = 15.6 Hz), δH 7.50 (2H, d, J = 9.0 Hz), δH 6.77

233

(2H, d, J = 9.0 Hz), and δH 6.34 (1H, d, J =15.6 Hz) indicated the presence of

234

trans-4-hydroxycinnamic [(E)-p-coumaric] acid fragment. The presence of four

235

anomeric 1H signals and 13C signals in the NMR indicated compound 1 is conjugated

236

with four sugars. The absolute configurations of L-rhamnose, L-arabinose, and

237

D-glucose on compound 1 were confirmed by chemical and GC-MS analysis. The

238

HMBC spectrum (Figure 3) determined the linkages between kaempferol,

239

(E)-p-coumaroyl, arabinopyranosyl, rhamnopyranosyl and glucopyranosyl, which

240

showed long-range correlations as follows: δH 5.54 (H-1, Glc1) to δC 133.2 (C-3 of

241

kaempferol unit), δH 4.39 (H-1, Rha) to δC 67.9 (C-6, Glc1), δH 4.27 (H-1’, Glc2) to δC

242

82.2 (C-3, Rha), δH 4.33 (H-1, Ara) to δC 80.7 (C-3, Glc1), and δH 4.98 (H-2, Glc1) to

243

δC 166.0 (carbonyl carbon of E-p-coumaroyl). Thus, the structure of 1 was identified

244

as kaempferol 3-O-[(E)-p-coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)][β-D-

245

glucopyranosyl-(1→3)-α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside,

246

was named as camellikaempferoside C.

247

Compounds 2-21 were confirmed by comparison of their spectral data with those

248

reported literatures and Tea Metabolome Database (TMDB)17 as myricetin

249

3-O-galactoside (2),7 quercetin 3-O-galactoside (3),18 myricetin 3-O-glucoside (4),7

250

kaempferol

251

[α-L-arabinopyranosyl-(1→3)][α-L-rhamno-pyranosyl (1→6)]-β-D-glucopyranoside

252

(7),20 quercetin 3-O-[(E)-p-coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)][α-L-

(5),19

quercetin

(6),19

which

kaempferol-3-O-[(E)-p-coumaroyl-(1→2)]


Page 13 of 33


(8),16

253

rhamnopyranosyl(1→6)]-β-D-glucopyranoside

254

coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-

255

rhamnopyranosyl(1→6)]-β-D-glucopyranoside

256

rutinoside (10),7 kaempferol 3-O-galactosylrutinoside (11),7 apigenin 6, 8-C-di

257

glucoside

258

arabinosyl-8-C-glucoside (14),21 quercetin 3-O-galactosylrutinoside (15),7 quercetin

259

3-O-glucosylrutinoside

260

3-O-rhamnosylgalactoside (18),23

261

Vitexin-4’’-O-glucoside (20),24 and kaempferol 3-O-glucoside (21)18.

262

These 21 compounds include the main four kinds of flavonoid aglycone glycosides in

263

tea: kaempferol, quercetin, myricetin and apigenin glycosides. The glycosides

264

substituted on the aglycone contain monosaccharides, disaccharides, trisaccharides

265

and tetrasaccharides. Among these FGs, four are acylated ones: 1, 7, 8, 9. Twelve

266

compounds are groups of isomers: 2 and 4, 10 and 11, 13 and 14, 15 and 16, 12, 17,

267

19 and 20.

268

Effect of Four Acylated FGs (1, 7, 8, 9) on 3T3-L1 Cells Viability. The four

269

acylated FGs (1, 7, 8, 9) were investigated on their cell viability in 3T3-L1 cells using

270

MTT cell assay (Table 2). The 3T3-L1 cells inhibition activities of 1, 7, 8, 9 with 100

271

µM were 6.72±0.79%, 7.88 ±1.26%, 8.57 ±1.33%, 9.10 ± 0.78%, respectively

272

(inhibition ratio of the positive control taxol was 90.86 ± 3.04% at 10 µM). It

273

indicated that the IC50 values of these four compounds for 3T3-L1 cells were all higher

274

than 100 µM. So we can conclude that 1, 7, 8, 9 are non-cytotoxic against 3T3-L1 cells.

(9),16

quercetin

kaempferol

(12),21 apigenin-6-C-glucosyl-8-C-arabinoside

(16),7

kaempferol

3-O-[(E)-p-

3-O-glucosyl

(13),21 apigenin-6-C-

3-O-rutinoside

(17),22

quercetin

kaempferol 3-O-rhamnosylgalactoside (19),23



275

Inhibitory Effects of Four Acylated FGs against Lipid Accumulation in 3T3-L1

276

Cells. In order to assess the effect of the four acylated FGs (1, 7, 8, 9) on the

277

proliferation and differentiation of 3T3-L1 preadipocyte, 3T3-L1 preadipocyte cells

278

were incubated with each compound (25, 50 and 100 µM) for 8 days after stimulation

279

of differentiation, and then Oil-red O staining was carried out (Figure 4). In Figure 4,

280

AI-II were normal 3T3-L1 adipocytes (negative control, lipid accumulation as 100%)

281

and adipocytes treated with resveratrol (positive control, 88 µM), which indicated that

282

there was lipid accumulation in adipocytes and resveratrol can successfully reduce the

283

lipid accumulation to 40.35 ± 2.13%. In Figure 4, BI-V microscopic observation

284

showed that the number of oil droplets in the adipocytes treated with 1, 7, 8, 9 (50 µM)

285

was reduced compared with that in the negative control. Figure 5 showed that the

286

four acylated FGs (1, 7, 8, 9) can significantly reduce the lipid accumulation in

287

3T3-L1 adipocytes compared with normal 3T3-L1 adipocytes (negative control) at

288

different concentrations (25, 50, 100 µM) (P < 0.05). Therefore, we can conclude that

289

1, 7, 8 and 9 show good suppression against lipid accumulation in 3T3-L1 adipocytes.

290

UPLC Method Validation. As the main component of tea, the content of FGs in tea

291

is between 1% and 3%. In this study, we established a UPLC method to detect FGs

292

and to quantify nine FGs including flavonol mono to tetraglycosides in 24 minutes in

293

the six major types of tea processed from the same material (Figure 6). Calibration

294

curves of nine FGs showed good linearity over the concentration range with

295

correlation coefficients (R2) ranged from 0.9970 to 0.9998 in a certain concentration

296

range (Supporting Table 2). Method validation details were shown in Supporting


Page 14 of 33

Page 15 of 33


297

Table 3. Relative standard deviation (RSD) of the precision, repeatability and stability

298

were all less than 5.36%. The value of LOD and LOQ tested in UPLC were less than

299

0.61 and 2.04 ng, respectively. And the value of recovery varied from 94.71% to

300

107.57% with RSD values less than 5.5% for the investigated FGs.

301

UPLC Analysis of FGs in Six Processing Types of Tea. Six different types of tea

302

samples were processed through the corresponding manufacturing processes (Figure

303

1). 1-3 Previous quantification of FGs in tea has tended to focus on FGs in different

304

varieties of tea. little is know on the concentration of FGs in the six major processing

305

types of tea prepared from the same material. In our study, the content of nine FGs

306

varied among the six types of tea. Kaempferol 3-O-glucosyl rutinoside (10) is the

307

highest one in all six types of tea and quercetin 3-O-glucosylrutinoside (16) is the

308

second one (Table 3). The amounts of both 10 and 16 basically decrease with the

309

degree of fermentation of processing tea. And the amounts of both 10 and 16 are high

310

enough to be easily detected, which could contribute as factors to discriminate the

311

different processing types of tea17. Dark tea was also found to have the highest

312

amount of the flavone C-diglycosides, Vitexin-4’’-O-glucoside (20) (0.70 ± 0.017

313

mg/g), and black tea was found to have the highest amount of the acylated flavonol

314

tetraglycoside, quercetin 3-O-[(E)-p-coumaroyl-(1→2)][α-L-arabinopyranosyl-(1→3)]

315

[α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside (8) (0.09 ± 0.012 mg/g). The

316

level of acylated flavonol triglycoside was lower than that of acylated flavonol

317

tetraglycosides and even lower than that of non-acylated flavonol triglycosides. The

318

amount of the kaempferol acylated triglycosides (7) in some tea is even



319

non-detectable.

320

Obesity is a serious metabolic syndrome that has association with many diseases, such

321

as type II diabetes mellitus, coronary heart disease, respiratory complications,

322

hypertension, hyperlipidemia and cancer.25 A surfeit of calories and dearth of energy

323

expenditure is the basic factor of obesity. Tea has been used as an anti-obesity therapy

324

to prevent lipid accumulation in Traditional Chinese Medicine and tea polyphenols

325

have been regarded as the main contributors.26 FGs are widely distributed in plants

326

with potential therapeutic effects. Recent studies have demonstrated that FGs have

327

anti-obesity effects, which suggests that FGs-enriched plants can be considered as

328

supplements or functional foods to prevent lipid accumulation.27,28 In our research, we

329

systematically investigated FGs in the Chinese famous green tea, Lu'an GuaPian.

330

From Lu'an GuaPian tea, we obtained 21 flavonol and FGs with a novel acylated

331

flavonol tetraglycoside named as camellikaempferoside C. Here, the four acylated

332

FGs we isolated displayed non-cytotoxic effect against 3T3-L1 cells and compounds 1,

333

7, 8, 9 exhibited good inhibition against lipid accumulation in 3T3-L1 adipocytes at

334

25, 50, and 100 µM. The average amount of daily tea intake can be around 10 g.29 The

335

amount of FGs could be around 1-3 mg/g dry tea (Table 3). 17-52% FGs can be

336

absorbed through dietary intake.30 Therefore, daily intake of FGs in tea (around

337

0.20-3.0 µM) is far below the amount we used here, which means extra addition of

338

FGs is needed for meeting anti-obesity effect besides the daily dietary intake. In

339

addition, a UPLC method to quantified FGs including acylated FGs and flavonol

340

triglycosides in six major processing types of tea prepared from the same material has


Page 16 of 33

Page 17 of 33


341

been developed for the first time. The relation between some of the FGs and tea types

342

were also observed. The result showed that acylated FGs can be found and quantified

343

in tea. The content of two main FGs (10 and 16) in all types of tea decreases basically

344

with the increasing degree of fermentation, which could be developed as contributors

345

to efficiently discriminate the different processing types of tea combined with other

346

factors. 31 Moreover, the UPLC method we established could serve as a reference

347

method for the determination of different flavonol glycosides with one to four sugar

348

units at the same time in six processing types of tea.

349 350

ACCOCIATED CONTENTS

351

Supplementary Information

352

Supplementary data can be found in the online version. These data include

353

spectroscopic and physical data of compound 1, cell toxicity and inhibitory effects on

354

lipid accumulation in 3T3-L1 cells of four acylated FGs (1, 7, 8, 9), and quantification

355

method validation of nine FGs in tea for UPLC analysis.

356 357

ACKNOWLEDGEMENT

358

Financial assistances were received with appreciation from National Natural Science

359

Foundation of China 81170654/H0507, Anhui Agricultural University Talents

360

Foundation (YJ2011-06), Nutrition and Quality & Safety of Agricultural Products,

361

National Modern Agriculture Technology System (CARS-23).

362



Page 18 of 33

363

REFERENCES

364

(1) Engelhardt, U. H. 3.23 Chemistry of Tea, Comprehensive Natural Products II

365

Chemistry and Biology, Volume 3: Development & Modification of Bioactivity.

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(2) Han, M.; Zhao, G.; Wang, Y.; Wang, D.; Sun, F.; Ning, J.; Wan, X.; Zhang, J.

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Safety and anti-hyperglycemic efficacy of various tea types in mice. Sci. Rep., 2016, 6,

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agriculture applied to the plant family theaceae to identify novel interventions for

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(6) Scharbert, S.; Hofmann, T. Molecular definition of black tea taste by means of

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quantitative studies, taste reconstitution, and omission experiments. J. Agric. Food.

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compounds in black tea Infusions by combining instrumental analysis and human

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bioresponse. J. Agric. Food. Chem. 2004, 52, 3498-3508.

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(9) Takuya, K.; Naoto, I.; Yasuhiro, K.; Yasuhiro, K.; Yoshimitsu, Y.; Kuninori,

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S.; Yosuke, Y. Antioxidant flavonol glycosides in mulberry (Morus alba L.) leaves

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isolated based on LDL antioxidant activity. Food Chem. 2006, 97, 25-36.

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(10) Morikawa, T.; Ninomiya, K.; Miyake, S.; Miki, Y.; Okamoto, M.; Yoshikawa,

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M.; Muraoka, O. Flavonol glycosides with lipid accumulation inhibitory activity and

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simultaneous quantitative analysis of 15 polyphenols and caffeine in the flower buds

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of Camellia sinensis from different regions by LCMS. Food Chem. 2013, 140,

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(11) Ahn, E. M.; Han, J. T.; Kwon, B. M.; Kim, S. H.; Baek, N. I. Anti-cancer activity

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constituents from chinese natural medicines. XXIV.1) hypoglycemic effects of

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Sinocrassula indica in sugar-loaded rats and genetically diabetic KK-Ay mice and

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Chem. Pharm. Bull. 2007, 55, 1308-1315.

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(13) Zong, J. F.; Wang, R. L.; Bao, G. H.; Ling, T. J.; Zhang, L.; Zhang, X. F.; Hou, R.

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Y. Novel triterpenoid saponins from residual seed cake of Camellia oleifera Abel.

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(14) Li, K. K.; Wong, H. L.; Hu, T. Y.; Zhang, C.; Han, X. Q.; Ye, C. X.; Leung, P. C.;

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Cheng, B. H.; Ko, C. H. Impacts of Camellia kucha and its main chemical

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components on the lipid accumulation in 3T3-L1 adipocytes. Int. J. Food Sci. Tech.



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2016, 51, 2546-2555

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(15) Yang, Y.; Qiao, L. L.; Zhang, X.; Wu, Z.; Weng, P. Effect of methylated tea

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catechins from Chinese oolong tea on the proliferation and differentiation of 3T3-L1

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preadipocyte. Fitoterapia 2015, 104, 45-49.

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(16) Manir, M. M.; Kim, J. K.; Lee, B. G.; Moon, S. S. Tea catechins and flavonoids

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from the leaves of Camellia sinensis inhibit yeast alcohol dehydrogenase. Bioorg.

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Med. Chem. 2012, 20, 2376-2381.

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J.; Wang, X. G.; Zhang, Z. Z.; Xia, T.; Wan, X. C.; Bao, G. H.

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TMDB: A literature-curated database for small molecular compounds found from tea.

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BMC Plant Biol. 2014, 14, 1-8.

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from Bidens parviflora Willd. Molecules 2008, 13, 1931-41.

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H.; Wan, X. C.; Ling, T. J. A new anti-proliferative acylated flavonol glycoside from

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(22) Cheng, N. A. L.; Tako, M.; Hanashiro, I.; Tamaki, H. Antioxidant flavonoid

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glycosides from the leaves of Ficus pumila L. Food Chem. 2008, 109, 415-420.

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(23) Brasseur, T.; Angenot, L. Flavonol glycosides from leaves of Strychnos variabilis.

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Photochem. 1986, 25, 563-564.

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(24) Kwon, Y. S.; Kim, E. Y.; Kim, W. J.; Kim, K. W.; Kim, C. M. Antioxidant

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constituents from Setaria viridis. Arch Pharm Res. 2002, 2, 300-305.

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(25) Kopelman, P. G. Obesity as a medical problem. Nature 2000, 404, 635-643.

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(26) Wolfram, S.; Wang, Y.; Thielecke, F. Anti-obesity effects of green tea: from

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bedside to bench. Mol. Nutr. Food Res. 2006, 50, 176-187.

438

(27) Tadahiro, Y.; Akihiro, D.; Susumu, K. Flavonol acylglycosides from flower of

439

Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells.

440

Chem. Pharm. Bull. 2012, 60, 129-136.

441

(28) Takuya, K.; Masayuki, Y.; Kuninori, S.; Tomoko, I.; Ichiro, M.; Keiko,

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A.; Yukikazu, Y. Effect of flavonol glycoside in mulberry (Morus alba L.) leaf on

443

glucose metabolism and oxidative stress in liver in diet-induced obese mice. J. Sci.

444

Food Agric. 2010, 90, 2386-2392.

445

(29) Peng, C. Y.; Cai, H. M.; Zhu, X. H.; Li, D. X.; Yang, Y. Q.; Hou, R. Y.; Wan, X.

446

C. Analysis of naturally occurring fluoride in commercial teas and estimation of its

447

daily intake through tea consumption. J. Food Sci. 2016, 81(1), H235-H239.

448

(30) Hollman, P. C.; deVries, J. H.; vanLeeuwen S. D.; Mengelers, M. J.; Katan, M. B.

449

Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy

450

volunteers. Am. J. Clin. Nutr. 1995, 62(6), 1276-1282.



451

(31) Ning, J.; Li, D.; Luo, X.; Ding, D.; Song, Y.; Zhang, Z.; Wan, X. Food Anal.

452

Methods, 2016, 9(11), 3242-3250.


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Page 23 of 33


453

Figure captions

454

Figure 1. The six major manufacturing processes of tea

455

Figure 2. The structures of compounds 1-21

456

Figure 3. The Key HMBC correlations of compound 1

457

Figure 4. Effects of four acylated FGs (1, 7, 8, 9) on lipid accumulation in 3T3-L1

458

adipocytes. A: (I) 3T3-L1 adipocytes in negative control group; (II) 3T3-L1

459

adipocytes were treated with resveratrol (88 µM); B: (I-V) 3T3-L1 adipocytes were

460

treated with 1, 7, 8, 9 (50 µM), respectively. The oil droplets were stained with

461

Oil-Red O.

462

Figure 5. Effect of the resveratrol (positive control, 88 µM), four acylated FGs (1, 7, 8,

463

9) on lipid accumulation in 3T3-L1 adipocytes at different concentrations (25, 50 and

464

100 µM, results are expressed as mean ± SD of triplicate tests). The lipid

465

accumulation without being added any compound in cells (negative control) was

466

supposed to be 100%. Data with different letters are significantly different at P < 0.05

467

among different treatments.

468

Figure 6. The UPLC analysis of nine FGs in Lu'an GuaPian green tea at 350 nm. (The

469

above curve was Lu'an GuaPian green tea, the below was FG standards. Peak

470

numbers corresponded to Table 3, from left to right are compound 20, 16, 10, 17, 21,

471

9, 1, 8 and 7, respectively.)



Page 24 of 33

Table 1. NMR spectroscopic data of compound 1a Position

δH

(J in Hz)

δC

HMBC (1H to 13C)

Position

δH

(J in Hz)

δC

HMBC (1H to 13C)

2

157.4

5'

3.07 br s

76.9

C-Glc-(4', 6')

3

133.2

6'a

3.55 dd (1.8, 10.8)

61.1

C-Glc-(4', 5')

4

177.4

6'b

3.41 dd (3.6, 11.4)

5

161.6

Rha 1

4.39 br s

101.5

C-Glc-6, Rha-3

2

3.70 br s

69.7

C-Rha-1

3

3.29 m

b

82.2

C-Rha-4

4

3.25 t (9.0)

71.1

C-Rha-(3, 6)

68.2

C-Rha-1

6

6.14 d (1.8)

7

99.3

C-5, 7, 8, 10

164.7

8

6.34 d (1.8)

94.3

C-6, 7, 9, 10

b

C-Glc-(4', 5')

9

156.9

5

3.30 m

10

104.4

6

0.93 d (6.0)

18.0

C-Rha-(4, 5)

1'

121.1

Ara 1

4.33 d (5.4)

2'

7.94 d (9.0)

3'

6.87 d (9.0)

4'

131.3 115.6

C-2, 4', 6' C-1', 4', 5'

160.4

103.1

C-Glc-3, Ara-(3, 5)

3.31 m

b

70.6

C-Ara-(1, 3)

3

3.31 m

b

72.6

C-Ara-2

4

3.61 br s

67.2

C-Ara-5

65.1

C-Ara-(1, 4, 3)

2

5'

6.87 d (9.0)

115.6

C-1', 3', 4'

5a

3.74 dd (5.4, 12.0)

6'

7.94 d (9.0)

131.3

C-2, 4', 2'

5b

3.40 dd (3.0, 10.2)

Glc 1

5.54 d (7.8)

99.1

C-3

p-Cou

2

4.98 dd (9.0, 8.4)

73.2

C-Glc-(1, 3), Cou-1

1

－

166.0

3

3.82 t (9.6)

80.7

C-Glc-(2, 4), Ara-1

2

6.34 d (15.6)

114.7

C-Cou-1, 1'

4

3.23 m

69.3

C-Glc-(3, 5)

3

7.55 d (15.6)

145.3

C-Cou-1, 2, 2'

5

3.50 t (9.0)

75.6

C-Glc-(1, 4)

1'

6a

3.69 d (10.8)

67.9

C-Rha-1

2'

7.50 d (9.0)

130.7

C-Cou-3, 4', 6'

C-Rha-1

3'

6.77 d (9.0)

116.2

C-Cou-1', 4', 5'

b

C-Ara-(1, 4, 3)

125.6

6b

3.31 m

Glc 1'

4.27 d (7.8)

104.8

C-Rha-3, Glc-3'

4'

2'

3.00 dd (8.0, 9.0)

74.5

C-Glc-(1', 3', 4')

5'

6.77 d (9.0)

116.2

C-Cou-1', 3', 4'

3'

3.17 m

76.4

C-Glc-(2', 4')

6'

7.50 d (9.0)

130.7

C-Cou-3, 4, 2'

4'

3.09 d (9.6)

70.0

C-Glc-(2', 3')

a

1

b

signals were overlapped.

160.2

H at 600 MHz and 13 C NMR at 150 MHz in DMSO-d6.

Glc: glucopyranosyl, Ara: arabinopyranosyl, Rha: rhamnopyranosyl, p-Cou: E-p-hydroxycoumaroyl


Page 25 of 33


Table 2. Cytotoxicity of four acylated FGs (1, 7, 8, 9) on 3T3-L1 cell compounds 100 µM IC50 (µM)

1 6.72 ± 0.79% > 100 µM

7

8

9

7.88 ± 1.26% 8.57 ± 1.33% 9.10 ± 0.78% > 100 µM

> 100 µM


> 100 µM


Page 26 of 33

Table 3. The content of nine FGs in different tea (mg/g, n = 3, mean ± SD) FGs

Green tea

White tea b

20

0.57±0.015

0.37±0.036

16

2.10±0.008a

10

Yellow tea c

b

Oolong tea c

Black tea

Dark tea d

0.27±0.027

0.70±0.017a

0.60±0.025

0.40±0.008

1.86±0.161b

1.84±0.079b

1.23±0.003c

1.09±0.011c

1.17±0.025c

3.44±0.045a

2.75±0.177bc

3.02±0.115b

2.67±0.071c

2.50±0.232c

1.95±0.315d

17

0.30±0.014a

0.27±0.034a

0.28±0.026a

0.28±0.005a

0.26±0.032a

0.17±0.003b

21

0.08±0.001a

0.06±0.005c

0.07±0.003b

0.06±0.001c

0.06±0.003c

0.07±0b

9

0.26±0.008a

0.25±0.019a

0.22±0.01bc

0.24±0.013ab

0.19±0.019bc

0.20±0.003c

1

0.07±0c

0.08±0.001c

0.08±0.009ab

0.08±0.004bc

0.07±0.007a

0.09±0.013a

8

0.02±0.004d

0.04±0.003c

0.02±0d

0.05±0.003b

0.09±0.012a

0.02±0.002d

7

0.01±0a

0.01±0a

0.01±0.001a

ND

ND

ND

20: Vitexin 4''-O-Glc, 16: Q 3-O-Glc-Rha-Glc, 10: K 3-O-Glc-Rha-Glc, 17: K 3-O-Glc-Rha, 21: K 3-O -Glc, 9: Q 3-O-p-Cou-Glc-Ara-Rha-Glc, 1: K 3-O-p-Cou-Glc-Ara-Rha-Glc, 8: Q 3-O-p-Cou-Glc-RhaGlc, 7: K 3-O-p-Cou-Glc-Rha-Glc. ND: not detected. One-way ANOVA with Tukey tests was applied to determine significant differences. Different superscripts show significant differences (P < 0.05) for different processing types of tea sample.


Page 27 of 33


Figure 1



Figure 2


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Page 29 of 33


Figure 3



Figure 4


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Figure 5



Figure 6


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The TOC graphic


Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid

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