Hot-Water Extracts from Roots of Vitis thunbergii var. taiwaniana and

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Hot-Water Extracts from Roots of Vitis thunbergii var. taiwaniana and Identified #-Viniferin Improve Obesity in High-Fat Diet-Induced Mice Yeh-Lin Lu, Shyr-Yi Lin, Sheng-Uei Fang, Ying-Ying Hsieh, ChiyRong Chen, Chi Luan Wen, Chi-I Chang, and Wen-Chi Hou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00327 • Publication Date (Web): 12 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Journal of Agricultural and Food Chemistry

Hot-Water Extracts from Roots of Vitis thunbergii var. taiwaniana and Identified ε-Viniferin Improve Obesity in High-Fat Diet-Induced Mice

Yeh-Lin Lu,▓,# Shyr-Yi Lin,§,▼,# Sheng-Uei Fang,▽,⊗ Ying-Ying Hsieh,† Chiy-Rong Chen,△ Chi-Luan Wen,‡ Chi-I Chang,┴,* and Wen-Chi Hou†,* School of Pharmacy, †Graduate Institute of Pharmacognosy, College of Pharmacy, §Department of

▓

General Medicine, ⊗Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan ┴

Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Department of Life Science, National Taitung University, Taitung 950, Taiwan

△

‡

Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Taichung 426, Taiwan

▼

Department of Primary Care Medicine,

▽

Division of Gastroenterology and Hepatology,

Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan #

These authors contributed equally to this study.

Running title: Anti-obesity activity of VTT-R-HW and ε-viniferin

1

ACS Paragon Plus Environment


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ABSTRACT 1

In this study, hot-water extracts (HW) from roots of Vitis thunbergii var. taiwaniana (VTT-R) were

2

shown significantly (P < 0.01 or 0.001) to lower lipid accumulations compared to the control in

3

3T3-L1 adipocytes. The VTT-R-HW (40 mg/kg) interventions concurrent with a high-fat (HF) diet

4

in C57BL/6 mice over a 5-week period were shown significantly (P < 0.05) to reduce body weights

5

compared to those fed with the HF diet under the same food-intake regimen. The (+)-ε-viniferin

6

isolated from VTT-R-HW was shown significantly to reduce lipid deposits compared to the control

7

(P < 0.05 or 0.001) in 3T3-L1 adipocytes and dose-dependent 3-hydroxy-3-methylglutaryl-CoA

8

(HMG-CoA) reductase inhibitions which the 50% inhibitory concentration was calculated to be 96

9

µM. The two-stage (+)-ε-viniferin interventions (10 mg/kg, day 1 to day 38; 25 mg/kg, day 39 to

10

day 58) were shown significantly (P < 0.05 or 0.001) to lower mice body weights, the weight ratio

11

of mesenteric fat, blood glucose, total cholesterol, and low-density lipoprotein compared to the HF

12

group under the same food-intake regimen but without concurrent VTT-R-HW interventions. It

13

might be possible to use VTT-R-HW or (+)-ε-viniferin as ingredients to develop functional foods

14

for weight management and will need further investigation.

15 16 17

KEYWORDS: high-fat (HF) diet, Vitis thunbergii var. taiwaniana, obesity, 3T3-L1 adipocyte,

18

(+)-ε-viniferin

19 20 21

2


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22

█ INTRODUCTION

23

The prevalence of obesity worldwide is increasing quickly. According to the parameter of body

24

mass index (BMI, kg/m2), the obesity prevalence in 2014 was estimated to have more than doubled

25

since 1980. WHO estimated that over 1.9 billion adults (age ≥18) were overweight (BMI ≥ 25) in

26

2014, and among those, over 600 million adults were obese (BMI ≥ 30).1 Therefore, 39% of adults

27

were overweight, and about 13% of the adult population was obese in 2014.1 A sedentary lifestyle,

28

alcohol, stress, high-fat/energy-dense diets, and universal urbanization were all identified as causes

29

of obesity’s increasing prevalence,2, 3 and the direct healthcare costs from the overweight condition

30

and obesity were expected to reach an estimated US$957 billion by 2030.2 The recent report

31

showed positive correlations between body fat (BMI of 25 to 29.9, classified as overweight; BMI ≥

32

30, classified as obese) and several cancer risks, with greater correlations in those with greater

33

amounts of fat as compared to those with normal BMI (18.5 to 24.9).4

34

Obesity is termed an excess of fat accumulation beneath the skin or deposited around the

35

organs, which causes dyslipidemia, hypertension, impaired insulin sensitivity, and, later, elevated

36

blood glucose levels, which are classified as metabolic risk criteria for cardiovascular diseases and

37

atherosclerosis.5,6 Orlistat (Xenical®), the only FDA-approved drug in 1998 available for long-term

38

weight control,7–10 is shown to retard caloric intake by inhibiting pancreatic lipase-mediated fat

39

hydrolysis and absorption.10 However, its unacceptable effects of diarrhea, fecal urgency, oily

40

spotting, bloating, and dyspepsia, affect its broad use.8, 10 Liraglutide (Saxenda®), the injectable

41

drug for weight management approved by the FDA in Dec. 2014, acts as an anorectic agent and

42

GLP1 receptor agonist in the hypothalamus, and another use for this drug was approved in 2010

43

(Victoza®) for type 2 diabetes mellitus treatment.7,10 Anti-obesity drug development and clinical

44

trials are currently ongoing.7 However, researchers still seek anti-obesity compounds or extracts

45

from natural resources worldwide that have acceptable, mild side effects.9, 11 Generally, high-fat 3



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(HF) diets in rodent models are used to evaluate the biological activity of drugs, extracts, or purified

47

compounds following two protocols. One is the obesity pre-induction by HF diets followed by

48

sample interventions concurrent with HF diets.12–14 The other protocol is the use of sample

49

interventions along with HF diets.15–18

50

Several resveratrol derivatives, including vitisinols A-G, (+)-ε-viniferin, (-)-viniferal,

51

ampelopsin C, miyabenol A, (+)-vitisin A and (+)-vitisin C, have been isolated from ethanolic

52

extracts of Vitis thunbergii roots.19, 20 The purified resveratrol derivatives from ethanolic extracts of

53

Vitis thunbergii have been reported to have ABTS radical scavenging activities and inhibitions

54

against platelet aggregation,19 and the fractionated fraction with the major components of (+)-vitisin

55

A, (-)-vitisin B, and ampelopsin C from ethanolic extracts of Vitis thunbergii has been reported to

56

have preventive capacities against bone loss in ovariectomized mice.21 A variety of Vitis thunbergii,

57

the so-called Taiwan small-leaf grape (Vitis thunbergii var. taiwaniana, VTT), with smaller leaves

58

and fruits compared to its related grape (Vitis vinifera). In Taiwan, the dried whole VTT is also used

59

as a tea material. The ethanol extracts of VTT have been reported to exhibit vasodilating effects and

60

antihypertensive activities;22, 23 the active components may include (+)-vitisin A, ampelopsin C, and

61

(+)-ε-viniferin. The VTT ethanol extracts from the stems and leaves have been reported to improve

62

the impaired glucose tolerance and systolic blood pressure of pre-induced obese Wistar rats

63

compared to obese rats without intervention.13 The resveratrol tetramer of (+)-vitisin A from VTT

64

showed weight-lowering effects. Intervention with (+)-hopeaphenol, (+)-vitisin A and (-)-vitisin B

65

showed improved cardiovascular risk parameters of TC, TG, LDL and free fatty acid in

66

HF-diet-induced obese mice compared to those without intervention.24 Besides the biological

67

activities of VTT ethanol extracts, the extracts of hot water (HW) from portions of stem and root

68

(SR) of VTT also showed preventive activities against lipopolysaccharide (LPS)-induced

69

prostaglandin E2 productions in primary human chondrocytes and acute inflammatory arthritis in

70

LPS-induced rabbits.25 The VTT-SR contained resveratrol, hopeaphenol, and (+)-ɛ-viniferin 4


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71

analyzed by HPLC.25 In this study, VTT-R-HW and the isolated (+)-ɛ-viniferin were investigated

72

with

73

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitory activity, and anti-obesity in

74

C57BL/6 mice induced by HF diets in vivo.

respect

to

their

effects

on

lipid

accumulations

in

3T3-L1

adipocytes,

75 76

█ MATERIALS AND METHODS

77

Chemicals. The HF diet (5.24 Kcal/gm, the 773.85 g providing 4057 Kcal, and 60% total

78

calories were provided by fats) was a product (D12492) of Research Diets, Inc. (NJ, USA) for

79

obesity induction. The normal diet (3.04 Kcal/gm) was a product (Prolab RMH2500, 5P14 Diet)

80

from PMI Nutrition International (MO, USA). Other chemicals were obtained from Sigma

81

Chemical Co. (St. Louis, MO, USA). Optical rotation was measured with a JASCO DIP-180 digital

82

spectropolarimeter. UV spectrum was obtained in MeOH using on a Shimadzu UV-1700

83

spectrophotometer. NMR spectra were recorded on a Varian Mercury plus 400 NMR spectrometer.

84

1

85

acetone-d6 solvent resonance. The Finnigan/Thermo Quest MAT 95XL spectrometer was used to

86

record FAB-MS spectrum. Silica gel (230–400 mesh ASTM, Merck) was used for column

87

chromatography. TLC was carried out on silica gel 60 F254 plates (Merck, Germany). HPLC

88

(Hitachi L-7000 chromatograph, D-7000, Merck Hitachi, Tokyo, Japan) composed an L-7100

89

Binary pump and L-7420 UV-Vis detector, equipped with a column oven (waters, USA).

90

HSM-7000 software was used for data acquisition and processing. Two Betasil C18 HPLC columns

91

(particle size, 5 µM; 250 × 10 mm; 250 × 4.6 mm) were used for the semi-preparation of compound

92

and the analysis of crude extract, respectively.

H- and

13

C-NMR spectra were measured at room temperature and reported in ppm by using the

93

5



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The VTT Extracts Preparation and (+)-εε-Viniferin Isolation and Purification. The

95

preparation of VTT-HW from the leaf (L) and stem (S) was previously reported.26 For VTT-R-HW

96

preparation, the fresh roots were dried at 50°C oven for 3 days, and then cut into pieces and ground

97

mechanically to powders. The root powders (1.5 kg) were extracted with 7.5 L of boiling water

98

under refluxing for 3 hours, and the extract was filtered through filter paper (Whatman No. 1) after

99

cooling. A brown crude residue was obtained after evaporation by a rotary evaporator under

100

reduced pressure at 45°C to yield VTT-R-HW. For fingerprinting analysis, 10 mg of the

101

VTT-R-HW was dissolved in 1 mL of dimethyl sulfoxide and filtered prior to analysis, and the

102

analytical HPLC was performed using a Hitachi L-7000 chromatography system. The mobile phase

103

consisted of 0.1% formic acid aqueous solution (solution A) and acetonitrile (solution B). A

104

gradient elution program was set as follows: solution A, 95% (0–5 min), 95–76% (5–35 min),

105

76–60% (35–70 min). The column temperature was maintained at 25°C. The mobile phase was

106

pumped at the flow rate of 1.0 mL/min with 20 µL injection volume, and the wavelength was set at

107

280 nm for monitoring. For pure compound isolation, the VTT-R-HW (65.8 g) was suspended in

108

H2O, and then partitioned in sequences by ethyl acetate (EA) and n-butanol to generate EA fraction

109

and n-butanol fraction, respectively. The EA fraction (16.2 g) was subjected to silica gel column

110

chromatography (5×75cm) using solvent mixtures of n-hexane and EA with increasing polarity as

111

eluents to yield eleven fractions. Fraction 11 (6.6 g) was loaded on a RP-18 column (3.5×60 cm)

112

and eluted with water/methanol (1:1 to 0:1) to yield nine fractions (100 mL/fraction), naming fr.

113

11(A) to fr. 11(I). The fr. 11(F) was separated by semi-preparative HPLC column, and eluted with

114

water/acetonitrile (8:2 to 1:1) in gradients (flow rate, 2 mL/min), to yield (+)-ɛ-viniferin (16.1 mg,

115

tR = 53.7 min). The collection of the appropriate sample amount required in in vivo study was

116

obtained by repeated HPLC injections under the aforementioned conditions.

117

6


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Cell Culture, Differentiation and Oil Red O Stains. The mouse pre-adipocyte cell line,

119

3T3-L1, was purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium

120

(DMEM) containing 10% bovine calf serum in a humidified 37ºC CO2 incubator, with 5% CO2 in

121

the 48-well plate. For differentiation experiments, cells were cultured to 100% confluence, and the

122

culture media was changed to differentiation medium [DMEM containing 10% fetal bovine serum

123

(Gibco)] containing the 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/mL insulin, and 1 µM

124

dexamethasone (day 0) for 2 days.28 The cells were then maintained for 2 days in differentiation

125

medium containing 10 µg/mL insulin and were then further cultured by changing the differentiation

126

medium every 2 days from day 4 to day 8. The hot-water extracts (250 and 500 µg/mL in DMSO,

127

final concentration, 0.25%) from the stem (VTT-S-HW), the leaf (VTT-L-HW), and the root

128

(VTT-R-HW), and isolated (+)-ε-viniferin (2.5, 5, and 10 µM in DMSO, final concentration,

129

0.25%) were added for the entire differentiation steps, from the onset of differentiation induction

130

and were renewed each time the medium was changed. For the control experiment, the 0.25%

131

DMSO (final concentration) was used instead of hot-water extracts or (+)-ε-viniferin for the

132

parallel experiments. For Oil Red O stainings,29 the medium was removed and the cells were fixed

133

by 4% neutralized paraformaldehyde at room temperature for 30 minutes. After PBS wash and a

134

quick rinse with 60% isopropanol, the staining solution (1.8 mg/mL of Oil Red O in 60%

135

isopropanol) was applied for 20 minutes and removed, and then washed by running tap water for 10

136

minutes. After air-drying overnight, the Oil Red O was extracted with isopropanol, and the

137

absorbance was determined by a microplate reader at a wavelength of 510 nm, and expressed

138

relative to the control (100%) for the lipid accumulation.

139 140

HMG-CoA Reductase Inhibitory Assays. The effects of ε-viniferin or resveratrol on

141

HMG-CoA reductase activities were performed according to the instructions of the HMG-CoA 7



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142

reductase activity assay kit (ab204701, Abcam Inc., MA, USA). The 340 nm absorbance was

143

measured for the expense of NADPH in the reduction of HMG-CoA to mevalonate catalyzed by

144

HMG-CoA reductase and expressed as HMG-CoA reductase inhibition (%).

145 146

Effects of VTT-R-HW or ε-Viniferin Interventions on Body Weights of C57BL/6 Mice in

147

the state of HF-Diet-Induced Obesity. The C57BL/6 mice (male, from National Laboratory

148

Animal Center, Taipei, Taiwan) were purchased for animal experiments. All protocols for animal

149

experiments were reviewed and approved by IACUC of Taipei Medical University (LAC-99-0136

150

and LAC-2014-0349). For evaluation of the effects of the VTT-R-HW on body weights of C57BL/6

151

mice in the state of HF-diet-induced obesity, after one week acclimation, the C57BL/6J mice

152

(three-week-old, N=36) were divided randomly into groups (N=12 per group), naming the normal

153

diet group, the HF diet group, and the VTT-R-HW group concurrent with the HF diet for 5-week

154

experiments. For VTT-R-HW interventions, 40 mg/kg were orally administered once a day by oral

155

gavage. For evaluation of the isolated (+)-ε-viniferin on reductions of body weights in HF-induced

156

obese mice, after one week acclimation, C57BL/6 (the five-week-old, N=24) were divided

157

randomly into groups (N=8 per group), naming the normal diet group, the HF diet group, and the

158

(+)-ε-viniferin intervention concurrent with the HF diet group, for successive two-stage

159

experiments (stage 1, day 1 to day 38; stage 2, day 39 to day 58). For (+)-ε-viniferin interventions,

160

10 mg/kg (stage 1) and 25 mg/kg (stage 2) were orally administered once a day by oral gavage. The

161

oral gavage was also used to deliver the aliquot of water in the groups of normal diet and the HF

162

diet for parallel experiments. The excess feeds were provided, and the residual feeds were weighted

163

every two days to calculate the feed intakes of each group. The body weights of each group were

164

recorded during experiments. The feed conversion was calculated as feed intake (g) divided by

165

mouse weight gain (g). At the end of the experiments, organs (liver, kidney, and heart) and fat

166

tissues (epididymal, perirenal, and mesenteric fat) in mice of each group were weighed for 8


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comparisons. The mouse blood was collected at either day 39 after overnight fasting or at the end

168

of the experiments for blood glucose, TC, TG, and LDL assays by the National Laboratory Animal

169

Center (Nangang, Taipei).

170 171

Statistics for Analysis of Experimental Data. The experimental data were expressed as

172

means ± SD. The multiple groups of body weight under the fixed time and accumulated feed

173

intakes in animal experiments were compared by one-way analysis of variance (ANOVA) and the

174

post hoc Tukey’s test which the same alphabet marked among groups showed no significantly

175

different (P > 0.05). The lipid deposits in cell culture, weight ratio of organ or fat tissue,

176

biochemical parameters between the intervened group and the control group were compared using

177

Student’s t-test [P < 0.05 (*), or P < 0.01 (**), or P < 0.001 (***)].The GraphPad Prism 5.0 (San

178

Diego, CA, USA) was used for Statistical analyses.

179 180 181

█ RESULTS VTT-R-HW

Exhibited

Anti-Lipid

Accumulations

in

3T3-L1

Adipocytes

and

182

Anti-Obesity Activities in HF-Diet-Induced Mice Models. The VTT-S-HW, VTT-L-HW, and

183

VTT-R-HW under the same concentration of 250 and 500 µg/mL were assayed for reduction of

184

lipid deposits in differentiated 3T3-L1 cells. The accumulation of lipid in the differentiated 3T3-L1

185

adipocytes was used as the control. After 8 days of treatment, the stained Oil Red O was extracted

186

to measure lipid accumulations and expressed as relative lipid accumulation (% the control) (Figure

187

1A). The VTT-R-HW treatment, but not VTT-S-HW or VTT-L-HW, was shown to lower lipid

188

accumulation in 3T3-L1 adipocytes, and it showed significant differences compared to the control

189

(250 µg/mL, P < 0.01; 500 µg/mL, P < 0.001). There was no apparent cytotoxicity of VTT-HW in

190

the tested concentration. Therefore, the VTT-R-HW interventions were used to investigate the 9



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191

anti-obesity effects in HF-diet-induced obesity of C57BL/6 mice. It was found that mice fed the HF

192

diet could increase body weights and showed significant differences (P < 0.05) compared to those

193

in the normal diet group from day 24. However, the VTT-R-HW interventions (40 mg/kg)

194

concurrent with HF diets kept the body weights of mice similar to those fed normal diets and

195

showed a significant difference compared to mice fed the HF diet from day 34 to day 36 (P < 0.05)

196

(Figure 1B). The accumulation feed intakes of mice in two HF diet groups showed similar and had

197

no significant difference between two HF diet groups (P > 0.05) (Figure 1C). It was revealed that

198

the anti-obesity effect of VTT-R-HW resulted from the metabolic controls to lower body weight

199

gains in the HF-diet-induced models.

200 201

The (+)-εε-Viniferin Isolation and Purification from VTT-R-HW, and Anti-Lipid

202

Accumulations in 3T3-L1 Adipocytes and Anti-HMG-CoA Reductase. One resveratrol dimer,

203

(+)-ɛ-viniferin, was purified from the EA fraction of VTT-R-HW. The chemical structure of

204

(+)-ɛ-viniferin (Figure 2A) was confirmed by comparing the spectral and physical data with that

205

= +36.2 (c = 0.51, MeOH); UV λ described in the literature,30 including brown solid; [α]25 D

206

(MeOH): 285 (3.98), 319 (4.10) nm; 1H-NMR (400 MHz, acetone-d6): δ 4.47 (1H, d, J = 5.6 Hz,

207

H-8a), 5.42 (1H, d, J = 5.6 Hz, H-7a), 6.24 (each 1H, br s, H-10a, 12a, 14a), 6.33 (1H, d, J = 1.6

208

Hz, H-12b), 6.71 (1H, d, J = 16.4 Hz, H-8b), 6.73 (each 1H, d, J = 8.4 Hz, H-3b, 5b), 6.83 (2H, d, J

209

= 8.4 Hz, H-3a, 5a), 6.91 (1H, d, J = 16.4 Hz, H-7b), 7.17 (each 1H, d, J = 8.4 Hz, H-2b, 6b), 7.20

210

(each 1H, d, J = 8.4 Hz, H-2a, 6a);

211

C-7a), 96.9 (d, C-12b), 102.2 (d, C-12a), 104.3 (d, C-14b), 107.1 (d, C-10a, -14a), 116.3 (d, C-3a,

212

-5a), 116.4 (d, C-3b, -5b), 119.9 (s, C-10b), 123.5 (d, C-8b), 128.0 (d, C-2a, -6a), 128.8 (d, C-2b,

213

-6b), 130.0 (s, C-1b), 130.2 (d, C-7b), 133.9 (s, C-1a), 136.5 (s, C-9b), 147.5 (s, C-9a), 158.3 (s,

214

C-4a), 159.7 (s,C-13b), 160.0 (s, C-11a, -13a), 162.5 (s, C-11b); FAB-MS m/z: 454 [M]+. For

215

fingerprinting analysis of VTT-R-HW, (+)-ɛ-viniferin was indicated with a retention time of 49.37

13

max

C-NMR (100 MHz, acetone-d6): δ 57.2 (d, C-8a), 94.0 (d,

10


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216

min (Figure 2B). The (+)-ɛ-viniferin showed dose-dependent activity (2.5, 5, and 10 µM) to lower

217

lipid accumulation in 3T3-L1 adipocytes for 8-day treatments and showed significant differences

218

compared to the control (2.5 µM, P < 0.05; 5 and 10 µM, P < 0.001). For 2.5, 5, and 10 µM

219

treatments, the lipid accumulation was reduced from 100% to 96.4%, 93.4%, and 92.3%,

220

respectively. The resveratrol showed minor inhibitory HMG-CoA reductase inhibitory activity; for

221

80, 120, and 160 µM, respectively, with 8.3%, 14.8%, and 22.3% inhibitions. However, the

222

resveratrol dimer of (+)-ɛ-viniferin showed higher and dose-dependent inhibitory activities against

223

HMG-CoA reductase, for 80, 120, and 160 µM, respectively, with 34.5%, 68.6%, and 78.4%

224

inhibitions. The 50% inhibitory concentration (IC50) of (+)-ɛ-viniferin for HMG-CoA reductase

225

was calculated to be 96 µM.

226 227

Anti-Obesity Activities of (+)-ɛ-Viniferin Interventions in HF-Diet-Induced Mice Models.

228

The resveratrol dimer of (+)-ɛ-viniferin isolated from VTT-R-HW was used to investigate the

229

anti-obesity activity along with HF diets by two-stage interventions. Figure 3A shows the weight

230

changes of mice without or with (+)-ɛ-viniferin interventions along with HF diets, stage 1, 10

231

mg/kg, day 1 to day 38; stage 2, 25 mg/kg, day 39 to day 58. As shown in Figure 3A, it was found

232

that mice fed with the HF diet showed much higher body weights than those fed with the normal

233

diet (P < 0.05). Mice in the (+)-ɛ-viniferin intervention group along with HF diets showed

234

gradually increasing body weights but lighter than those fed with the HF diet (P < 0.05) during

235

stage 1 or stage 2 interventions. The calculated total feed intakes at the end of the experiments

236

(Figure 3B) showed no significant difference between groups of the HF diet and the (+)-ɛ-viniferin

237

intervention (P > 0.05) and were much lower compared to those fed with the normal diet (P < 0.05).

238

It was clear that the (+)-ɛ-viniferin intervention with the ability to reduce body-weight gains of

239

mice in the HF-diet-induced models did not reduce feed intakes. Figure 3C shows the weight ratios 11



Page 12 of 29

240

(body weight %) of the organs (heart, liver, and kidney) and fat tissues (epididymal fat, perirenal

241

fat, and mesenteric fat) in the HF diet group and the (+)-ɛ-viniferin intervention group. The weight

242

ratios of the heart, liver, kidney, epididymal fat, and perirenal fat showed similar and had no

243

significant difference (P > 0.05) between the (+)-ɛ-viniferin intervention group and the HF diet

244

group; however, the weight ratio of mesenteric fat in the (+)-ɛ-viniferin intervention group showed

245

a significant lower

compared to that fed with the HF diet (P < 0.05).

246

Figure 4 shows the biochemical index (blood glucose, Glc; total cholesterol, T-CHO; total

247

triglyceride, TG; low-density lipoprotein, LDL) at stage 1 (Figure 4A) or stage 2 (Figure 4B) of

248

mice plasma in normal diet or HF diet with or without (+)-ɛ-viniferin interventions. It was clear

249

that mice in the HF diet group showed higher Glc, T-CHO, TG, and LDL at stage 1 (Figure 4A) or

250

stage 2 (Figure 4B) and had significant differences compared to those fed with the normal diet (P
0.05). The lipid

505

accumulations in 3T3-L1 adipocytes during differentiations between the treated group and the

506

control group were analyzed using Student’s t-test, and any difference was considered statistically

507

significant when P < 0.05 (*), or P < 0.01 (**), or P < 0.001 (***).

508 509

Figure 2. (A) The structure of (+)-ɛ-viniferin. (B) The fingerprinting analysis of R-HW. The

510

(+)-ɛ-viniferin was arrow-indicated with retention time of 49.37 min. (C) Effects of (+)-ɛ-viniferin

511

(2.5, 5, and 10 µM) treatments on anti-lipid accumulations during differentiation of 3T3-L1

512

adipocytes. For the control experiment, the 0.25% DMSO (final concentration) was used instead of

513

(+)-ε-viniferin for the parallel experiments. (D) Effects of resveratrol and (+)-ɛ-viniferin (80, 120,

514

and 160 µM) treatments on HMG-CoA reductase activities. The lipid deposits in 3T3-L1 23



Page 24 of 29

515

adipocytes during differentiations between the treated group and the control group were analyzed

516

using Student’s t-test, and any difference was considered statistically significant when P < 0.05 (*),

517

or P < 0.01 (**), or P < 0.001 (***).

518 519

Figure 3. (A) Effects of two-stage (+)-ɛ-viniferin interventions (stage 1, 10 mg/kg, day 1 to day 38;

520

stage 2, 25 mg/kg, day 39 to day 58) on HF diet-induced obesity in C57BL/6 mice. The

521

(+)-ɛ-viniferin was orally administered by the gavage once a day concurrent with HF diets during

522

the experiments. The standard mouse/rat chow was used as the normal diet (Prolab RMH2500,

523

5P14 Diet). The oral gavage was also used with the normal diet group and the HF diet group (the

524

control group) to deliver the aliquot of water in the parallel experiments. (B) The accumulation feed

525

intakes during experiments. (C) The weight ratios of organs and fat tissues. The multiple groups of

526

body weight under the fixed time and accumulated feed intakes in animal experiments were

527

compared by one-way ANOVA and the post hoc Tukey’s test which the same alphabet marked

528

among groups showed no significantly different (P > 0.05). The weight ratio of organ or fat tissue

529

between the treated group and the HF diet group were analyzed using Student’s t-test; and any

530

difference was considered statistically significant when P < 0.05 (*), or P < 0.01 (**), or P < 0.001

531

(***).

532 533

Figure 4. The biochemical parameters (blood glucose, Glc; total cholesterol, T-CHO; total

534

triglyceride, TG; low-density lipoprotein, LDL) at the stage 1 (A) or stage 2 (B) of mice plasma in

535

normal diet or HF diet with or without (+)-ɛ-viniferin interventions. The Student’s t-test was used to

536

analyze the biochemical parameters between the treated group and the HF diet group or and the

537

normal diet group and the HF diet group; and any difference in comparison with the HF diet group

538

was considered statistically significant when P < 0.05 (*), or P < 0.01 (**), or P < 0.001 (***).

24


Page 25 of 29

Figure 1.

Relative lipid accumulation (% the Control)

150

***

(A) 140

*

250 µg/mL 500 µg/mL

130 120

*

110

**

100 90

***

80 30 20 10 0 Control

S-HW

L-HW

R-HW

(B)

27

Normal diet HF diet HF diet + R-HW (40 mg/kg)

26

b

b

b

b

25 b

24

b a

23 22 b

21 a

20

a

19

a

b a

b ab a

a a a

a

a

a a

a a

a a a

a a a

a a a

a a

a

a

ab ab a

a

a

a

a a

a a

18 17

Accumulated feed intakes (g)/mouse

28

Weight (g)

539


160

(C) Normal diet HF diet HF diet + R-HW (40 mg/kg)

140

b

120 100 80

a a

60 40 20 0

0 0

2

4 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

0

2 4

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Day

Day

25



Page 26 of 29

Figure 2.

(A)

(B) 3a

HO

2a

H

5a

HO

6a

7a

10a

8a

O 10b

H 12a

12b

14a

(+)-εε-viniferin

OH 14b

8b 7b

OH

2b

6b

3b

5b

OH (+)-ε-viniferin

90

(C) 105

*

100

*** ***

95 90 85

10

HMG-CoA reductase inhibition (%)

Relative lipid accumulation (% the control)

110

(D)

80 70 60 50

resveratrol ε-viniferin

40 30 20 10 0

0 Control

2.5

5

10

01070

80

ε-viniferin (µ µM)

90

100

110

120

130

140

150

160

170

Concentration (µ µM)

26


Page 27 of 29


Figure 3. HF diet or normal diet

(A)

Weight (g)

ε-viniferin (10 mg/kg) day 1 to day 38

33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18

ε-viniferin (25 mg/kg) day 39 to day 58

blood sampling c normal diet HF diet HF + ε-viniferin

c c c

c

c

c

b

c c c

c b c c c

b

b

c

b

b b c

a a a

a a

b b

a b

b

a

a

a

a b

b

a

b

b

b

b

b a

b b b

a a

a a a a a

a

a

a

a a

a

a a

a a

-2 0 2 4 6 8 101214161820222426283032343638

44 46 48 50 52 54 56 58 60 62

300

7

(B)

275

Normal diet

250

HF diet

b

HF +ε-viniferin

225 200

a a

175 150 125 100 75 50

Weight ratio (% to body weight )

Accumulated feed intakes (g)/mouse

Day

(C) HF diet HF + ε-viniferin

6 5

P > 0.05 P > 0.05

4 3

P > 0.05

2 1

P > 0.05

P < 0.05

P > 0.05

25 0

0 0

4

8

12 16 20 24 28 32 36

Day

44

48

52

56

ar he

t

er l iv

t y fat fat fa ne al al r ic n k id m e e t n dy r ir se idi Pe Me Ep

27



Page 28 of 29

Figure 4.

400

450

(A) P < 0.001

Normal diet HF diet HF + ε-viniferin

P < 0.05

350 300

(B) Normal diet HF diet HF + ε-viniferin

P < 0.001 P < 0.001

250

350 300

P < 0.001 P < 0.001

200

400

P < 0.001 P < 0.001

200 100 150

P < 0.001 P > 0.05 P < 0.001 P > 0.05 P < 0.001 P < 0.001

P < 0.001 P < 0.001

50

100 50

0

B lood biochem ical index (m g/gL)

B lood biochem ical index (m g/gL)

450

0 Glc

T-CHO

TG

LDL

Glc

T-CHO

TG

LDL

28


Page 29 of 29


Hot-Water Extracts of Vitis thunbergii var. taiwaniana and Identified ε-Viniferin Improve Obesity in High-Fat Diet-Induced Mice

TOC

29


Hot-Water Extracts from Roots of Vitis thunbergii var. taiwaniana and

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