Resveratroloside Alleviates Postprandial Hyperglycemia in Diabetic

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Bioactive Constituents, Metabolites, and Functions

Resveratroloside Alleviates Postprandial Hyperglycemia in Diabetic Mice by Competitively Inhibiting a-Glucosidase Xiaohui Zhao, Jihong Tao, Ting Zhang, Sirong Jiang, Wei Wei, Hongping Han, Yun Shao, Guoying Zhou, and Huilan Yue J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00455 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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Journal of Agricultural and Food Chemistry

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Resveratroloside Alleviates Postprandial Hyperglycemia in Diabetic Mice by

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Competitively Inhibiting a-Glucosidase

3

Xiaohui Zhao†, Jihong Tao†, Ting Zhang†, Sirong Jiang†, Wei Wei†‡, Hongping Han§,

4

Yun Shao†, Guoyin Zhou*†, Huilan Yue *†

5

†

Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau

6

Biology, Chinese Academy of Sciences and Qinghai Provincial Key Laboratory of

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Tibetan Medicine Research, Qinghai 810008, China.

8 9

‡

School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu

273165, Shandong, China

10

§

11

Plateau in Qinghai Province, Xining 810008, China

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AUTHOR INFORMATION

13

Corresponding Authors

14

* Tel: +86 13897243464. Fax: 86-0971-6143282. E-mail: [email protected]

15

* Tel: +86 18797379366. Fax: 86-0971-6143282. E-mail: [email protected]

Key Laboratory of Medicinal Animal and Plant Resources in Qinghai-Tibetan

1

ACS Paragon Plus Environment


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ABSTRACT

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The regulation of postprandial blood glucose (PBG) levels is an effective

18

therapeutic method to treat diabetes and prevent diabetes-related complications.

19

Resveratroloside is a mono-glucosylated form of stilbene that is present in red wine,

20

grapes and several traditional medicinal plants. In our study, the effect of

21

resveratroloside on reducing PBG was studied in vitro and in vivo. Compared with the

22

starch treatment alone, the oral administration of resveratroloside-starch complexes

23

significantly inhibited the PBG increase in a dose-dependent pattern in normal and

24

diabetic mice. PBG level treated with resveratrol (30 mg/kg) was not lower than that

25

of resveratroloside. Further analyses demonstrated that resveratroloside strongly and

26

effectively inhibited a-glucosidase, with an IC50 value of 22.9 ± 0.17 µM, and its

27

inhibition was significantly stronger than those of acarbose and resveratrol (264 ±

28

3.27 µM and 108 ± 2.13 µM). Moreover, a competitive inhibition mechanism of

29

resveratroloside on a-glucosidase was determined by enzyme kinetic assays and

30

molecular docking experiments. The molecular docking of resveratroloside with

31

α-glucosidase demostrated the competitive inhibitory effect of resveratroloside, which

32

occupies the catalytic site and forms strong hydrogen bonds with the residues of

33

α-glucosidase. Resveratrol was also determined to be competitive inhibition

34

mechanism on a-glucosidase by enzyme kinetic assays and molecular docking

35

experiments. This study suggested that resveratroloside had the ability to regulate

36

PBG levels and can be considered a potential agent for the treatment of diabetes

37

mellitus. 2


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KEYWORDS: a-glucosidase, postprandial blood glucose, diabetes mellitus,

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resveratroloside, competitive inhibition.

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40

INTRODUCTION

41

Diabetes mellitus (DM) is a chronic, metabolic disease that has been recognized as

42

one of the most serious public health problems in our society1. The International

43

Diabetes Federation (IDF) estimated that 425 million people worldwide have diabetes,

44

and this figure is projected to reach nearly 700 million by the year 2045.

45

Hyperglycemia (high blood glucose levels) is a major phenotype of DM and leads to

46

severe diabetic complications over time, including coronary artery disease, stroke,

47

peripheral artery disease, retinopathy, nephropathy, and neuropathy2. Therefore,

48

controlling postprandial high blood glucose is important to treat DM and prevent the

49

complications caused by DM.

50

One class of drugs to treat DM is a-glucosidase inhibitors, which can prevent the

51

carbohydrates digestion and consequently defer glucose absorption and suppress the

52

postprandial hyperglycemia3,4. Therefore the inhibitors of a-glucosidase have been

53

recommended as first-line agents for the treatment of DM. They can be used alone or

54

in combination with other antidiabetic agents for the treatment of type 2 DM and can

55

also be used for patients with type 1 DM 5. Inhibitors of a-glucosidase, such as

56

voglibose, miglitol and acarbose, are currently used clinically, but their use may be

57

limited due to side effects, such as diarrhea, abdominal cramping, flatulence, and

58

vomiting6,7. Therefore, much effort has been devoted to developing a lead compound

59

by searching for effective α-glucosidase inhibitors from natural sources.

60 61

Resveratroloside

(trans-3,5,4’-trihydroxystilbene-4’-O-β-D-glucopy-ranoside),

which is known to be present in red wine, grapes and several traditional medicinal 4


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plants, including Rheum tanguticum, Rheum rhaponticum, Polygonum multiflorum

63

and Polygonum cuspidatum, is a mono-glucosylated form of stilbene and has attracted

64

great interest for its solubility in water8-12. Resveratroloside has been shown to

65

possess antioxidant13, antiaging14, anti-inflammatory15 and antimicrobial activities16, 17

66

and has neuroprotective and cardioprotective effects18, 19. Moreover, it has also been

67

demonstrated to have more potent antiallergic and phosphodiesterase (PDE)

68

inhibitory activities than those of the corresponding aglycone18, 20. Several reports

69

have shown that the stilbenes and stilbene glycosides in Pterocarpus marsupium,

70

Rheum undulatum, Rheum palmaturn, and Rumex bucepahlophorus possess

71

antihyperglycemic activity in vivo and in vitro21-24. Stilbenes such as resveratrol and

72

piceatannol have also been reported to delay the absorption of carbohydrates and

73

lower

74

hyperglycemic mice25. In addition, stilbene glycosides, including desoxyrhaponticin,

75

could reduce glucose uptake in the intestinal and renal membrane vesicles26. However,

76

studies on the antidiabetic activity of resveratroloside are lacking.

postprandial

blood

glucose

concentrations

in

high-fat

diet-induced

77

To the best of our current knowledge, this is the first report demonstrating that

78

resveratroloside inhibits α-glucosidase activity in vitro and in vivo, leading to

79

decreased postprandial blood glucose in normal and alloxan-induced diabetic mice. In

80

addition, the underlying mechanism was determined by enzyme kinetic experiments

81

and molecular docking assays.

82

MATERIALS AND METHODS

83

Chemicals. Resveratrol (>98.9%) was obtained from MCE (MedChemExpress Inc., 5



84

Monmouth Junction, NJ, USA). α-Glucosidase from Saccharomyces cerevisiae (EC

85

3.2.1.20), acarbose, and 4-Nitrophenyl α-D-glucopyranoside (pNPG) were obtained

86

from Sigma-Aldrich (St. Louis, MO, USA). All solvents and chemicals were of

87

reagent grade unless otherwise stated.

88

Resveratroloside preparation. The powdered root (5kg) of Rheum tanguticum

89

was extracted three times and each for 2h using 50% ethanol under reflux. The extract

90

was concentrated and then extracted with ethyl acetate (2.0 L), and n-butanol (2.0 L),

91

respectively. The fraction of n-butanol was concentrated to generate 300 g n-butanol

92

extract. The n-butanol extract was loaded into a glass column (100 cm × 12 cm,

93

containing 3000 g D101 macroporous resin), which then were eluted with different

94

proportions of water and ethanol (100:0, and 80:20v/v; 10L for each proportion). The

95

water and ethanol (80:20) fraction (110g) was further separated to produce 435mg

96

resveratroloside by HPLC with a Megresss C18 column (4.6mm × 250mm, 10um).

97

The detection wavelength was 310 nm and the flow rate was controlled at 15.0

98

mL/min. Acetonitrile-water as the mobile phase was applied with a gradient program

99

as follow: 0-60 min, 10-30% acetonitrile.

100

Resveratroloside (pale brown needle, 97.3%), 1H-NMR (MeOD, 400 MHz): δ =

101

3.50–3.42 (4 H, m, sugar-H); 3.73 (1 H, dd, J = 12.0 Hz, J = 5.2 Hz, sugar-H); 3.92 (1

102

H, dd, J = 12.0 Hz, J = 1.6 Hz, sugar-H); 4.91 (1 H, d, J = 7.3 Hz, sugar-H); 6.20 (1 H,

103

d, J = 2.4 Hz, H-4’); 6.49 (1 H, d, J = 2.4 Hz, H-2’, 6’); 6.89 (1 H, d, J = 16.4 Hz,

104

olefinic H); 7.01 (1 H, d, J = 16.4 Hz, olefinic H); 7.10 (2 H, d, J = 8.8 Hz, H-3, 5);

105

7.47 (2 H, d, J = 8.8 Hz, H-2, 6).

13

C-NMR (MeOD, 100 MHz) δ = 158.2, 157.3, 6


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139.6, 131.8, 127.5, 127.2, 127.1, 116.5, 104.5, 101.5, 100.8, 76.8, 76.6, 73.5, 70.0,

107

61.1. The 1H-NMR and

108

in the literature10. The chemical structures and HPLC chromatograms of

109

resveratroloside was shown in figure 1.

13

C-NMR data were in line with resveratroloside data found

110

Experimental animals. Male Kunming mice (18~22 g) were obtained from the

111

Lanzhou Medical University SPF Experimental Animal Center (Lanzhou, China). The

112

animals were housed in a controlled environment with free access to water and a

113

commercial stock diet (Crude protein ≥ 18.0%, Crude ash ≤ 7.0%, Crude fiber ≤ 5.0%,

114

Crude fat ≥ 4.0%, Phosphorus 0.6-1.0%, Calcium 1.0-1.6%, Vitamin D ≥ 800 IU/kg,

115

Vitamin A ≥ 7000 IU/Kg, Moisture ≤ 8.0%,) at a temperature of 22 ±2 °C with a 12 h

116

light/dark cycle. After one week of adaptation, diabetes was induced by intravenous

117

injection of alloxan (60 mg/kg body weight). The fasting blood glucose (FBG) level

118

was determined 72 h later. The animals with blood glucose levels between 11.0 mM

119

(198 mg/dL) and 20.0 mM (360 mg/dL) were considered diabetic mice for the

120

postprandial blood glucose (PBG) evaluation. All procedures applied in our study

121

were allowed by the Animal Ethics Committee of the Chinese Academy of Sciences.

122

Oral starch tolerance test in normal and diabetic mice. Normal and diabetic

123

mice were fasted overnight and were randomly assigned into 5 groups of 10 mice:

124

Group 1: normal or diabetic mice treated with starch at 6 g/kg; Group 2: normal or

125

diabetic mice treated with a low dose of resveratroloside (10 mg/kg) and starch (6

126

g/kg); Group 3: normal or diabetic mice treated with a medium dose of

127

resveratroloside (30 mg/kg) and starch (6 g/kg); Group 4: normal or diabetic mice 7



128

treated with a high dose of resveratroloside (50 mg/kg) and starch (6 g/kg); and Group

129

5: normal or diabetic mice treated with acarbose (10 mg/kg) and starch (6 g/kg).

130

Blood samples from the tail vein were collected at 0, 30, 60 and 120 min, and the

131

blood glucose concentration was determined by a glucometer (Roche Diagnostics

132

GmbH, China). The area under the curve (AUC) was counted on the basis of the

133

following formula:

134

AUC = 1/4 (PBG0 + PBG30) + 1/4 (PBG30 + PBG60) + 1/4 (PBG60 + PBG120)

135

PBG0, PBG30, PBG60, and PBG120 are the postprandial blood glucose levels at 0,

136

30, 60 and 120 min, respectively.

137

a-Glucosidase inhibition assay. The α-glucosidase inhibition assay was carried

138

out based on the method reported by Zhao et al27 with minor modifications. Different

139

concentrations of resveratroloside (13.0, 26.0, 52.0, 104, 208, 416 and 833 µM) and

140

50 μL a-glucosidase (0.6 U/mL) from Saccharomyces cerevisiae in 0.1 M phosphate

141

buffer (pH 6.5) were added to a 96-well microplate and incubated for 10 min at 37 °C.

142

Then 50 μL pNPG (0.5 mM) was added to the above mixed solution as a substrate to

143

initiate the reaction. The 96-well microplate was incubated at 37 °C for an additional

144

20 min, followed by adding 50 μL Na2CO3 (0.1 M) to stop the reaction. The

145

absorbance of the reaction mixture was detected at 405 nm by a microplate reader

146

(Siemens Healthineers, Germany).

147

Mechanism of a-glucosidase inhibition. The general operational steps of the

148

mechanism study are similar to that for the above inhibition experiments. Various

149

concentrations of a-glucosidase (0.4-4.0 U/mL) were incubated with two 8


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150

concentrations of resveratroloside for 10 min at 37 °C. Then, the reaction was

151

initiated by adding 0.8 mM pNPG to the above mixture. The absorbance of the

152

reaction mixture was monitored at 405 nm. All assays were carried out in triplicate.

153

Kinetic characterization of a-glucosidase inhibition. The general operational

154

steps for the kinetics of a-glucosidase were also similar to that for the above inhibition

155

experiments. Two concentrations of resveratroloside (16.0 and 32.0 µM) and 0.6

156

U/mL a-glucosidase in sodium phosphate buffer were added into a 96-well microplate

157

and incubated for 10 min at 37 °C. Then, the reaction was started by adding various

158

concentrations of the pNPG (1.8-0.6 mM) to the above mixture. The absorbance of

159

reaction mixture was monitored at 405 nm. All assays were carried out in triplicate.

160

The kinetics of a-glucosidase inhibition was analyzed by using Lineweaver-Burk

161

plots.

162

Molecular docking. The binding site and efficacy between resveratroloside and

163

α-glucosidase was estimated by molecular docking. The a-glucosidases from

164

Saccharomyces

165

oligo-1,6-glucosidase (isomaltase). The homology modeling of maltase was

166

constructed using Modeller 9.17 with 3A47 and 3AXH as templates. The isomaltase

167

crystal structure (PDB ID: 3A4A) was derived from the Protein Data Bank (PDB)

168

database. The initial structures of the receptor protein were prepared using AutoDock

169

Tools 1.5.6 for the subsequent molecular docking. The MOPAC program was used to

170

optimize the structures of the ligands (resveratroloside, resveratrol and isomaltose)

171

and to calculate the AM1 atomic charge. Then, the ligand structure was prepared

cerevisiae

contain

a-1,4-glucosidase

9


(maltase)

and


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172

using AutoDock Tools 1.5.6 for docking. Molecular docking was carried out using

173

AutoDock 4.2.6 with the following settings: number of docking runs = 100, maximum

174

number of energy evaluations = 25,000,000, and population size = 150. The number

175

of grids was 50 × 40 × 40 and 62 × 40 × 44 with a 0.375 Å grid spacing.

176

Data and statistical analysis. All date were presented as the mean ± SD/SEM. The

177

statistical analysis was carried out by the computer software GraphPad Prism 6.0. The

178

significant

179

ANOVA followed by Bonferroni post-hoc test. A value of P < 0.05 was considered

180

statistically significant. The calculation of the 50% inhibitory concentration (IC50) in

181

the enzyme activity was performed by a nonlinear regression.

182

RESULTS AND DISCUSSION

differences

among

the

groups

were

evaluated

by

one-way

183

Hypoglycemic effects of resveratroloside. The hypoglycemic effects of

184

resveratroloside and acarbose were evaluated through a starch tolerance experiment in

185

normal mice. The time points for the levels PBG were detected before and at 30, 60,

186

90, and 120 min after starch loading (6 g/kg) (Figure 2A and Figure 2B). The blood

187

glucose level of the control group peaked at 30 min and then decreased. Compared

188

with the control group, the mice in the 10 mg/kg resveratroloside group and acarbose

189

group displayed a reduction in the PBG levels, but the differences were not significant

190

among the three groups. Compared with the control group, resveratroloside at 30 and

191

50 mg/kg significantly decreased the PBG levels at 30 min after starch loading. This

192

result indicated that resveratroloside has the ability to control the elevation of

193

starch-associated PBG levels. The normal mice treated with resveratroloside showed a 10


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194

23.4%, 24.1%, and 15.0% reduction in blood glucose reduction at doses of 50, 30 and

195

10 mg/kg, respectively (Figure 2B).

196

A similar experiment was carried out in alloxan-induced diabetic mice (Figure 2C

197

and Figure 2D). In this experiment, after starch loading, the PBG level diabetic

198

control group was increased up to 996 mg/dL at 30 min. In the test groups, after starch

199

loading along with resveratroloside at doses of 50, 30 and 10 mg/kg or acarbose, the

200

average blood glucose levels were significantly decreased compared to that of the

201

diabetic control group. The diabetic mice treated with resveratroloside showed a

202

50.8%, 47.1%, and 24.9% reduction in blood glucose at doses of 50, 30 and 10 mg/kg,

203

respectively (Figure 2D). The results strongly confirm the ability of resveratroloside

204

to reduce starch-mediated PBG levels.

205

The hypoglycemic effect of resveratrol was also evaluated through starch tolerance

206

test in normal (Figure 3A and 3B). Compared with the control group, the PBG levels

207

after the administration of acarbose (10 mg/kg), resveratrol (30 mg/kg), and

208

resveratroloside (30 mg/kg) were reduced at 30 min after starch loading. This result

209

showed that resveratroloside, resveratrol and acarbose have the ability to control the

210

elevation of starch-associated PBG levels. Similar test was carried out in diabetic

211

mice (Figure 3C and 3D). Compared with the control group, the acarbose-,

212

resveratrol-, and resveratroloside administered groups shown decrease in PBG level.

213

The PBG levels after the administration of resveratroloside (30 mg/kg) and acarbose

214

(10 mg/kg) were significantly decreased at 30 and 60 min after starch loading. PBG

215

level after the administration of resveratrol (30 mg/kg) was not lower than those of 11



216

resveratroloside and acarbose. The result revealed that hypoglycemic effect of

217

resveratroloside is stronger than that of its aglycone. However, the equal efficacy

218

could be achieved by simply increasing the dose of resveratrol, and this could be

219

accomplished in the absence of toxicity.

220

In vivo experiment shown that the hypoglycemic activity of resveratrol was not

221

stronger than that of resveratroloside under the same dosage of 30 mg/kg. Since the

222

molecular weight resveratroloside (M = 390) is 1.7-fold higher than that of resveratrol

223

(M = 228), the absolute amount of glycoside was 1.7-fold lower than that of aglycone.

224

It is possible that resveratroloside has higher bioavailability in the animal organism as

225

compared to resveratrol, since the presence of a saccharide usually enhance the

226

solubility of substances in water.

227

a-Glucosidase inhibition of resveratroloside. The a-glucosidase inhibition

228

activity of resveratroloside, resveratrol and acarbose was investigated, and the results

229

are presented in Figure 4. The IC50 values for acarbose and resveratrol were 264 ±

230

3.27 and 108 ± 2.13 µM, respectively. Mathews et al reported that the IC50 values of

231

acarbose and resveratrol on yeast a-glucosidase were 377 µM and 399 µM

232

respectively25. Resveratroloside showed a strong effective inhibition with an IC50

233

value of 22.9 ± 0.17 µM, and its inhibition was significantly stronger than those of

234

acarbose and resveratrol.

235

Mechanism characterization. The mechanism of inhibition of resveratroloside

236

and resveratrol on a-glucosidase was all studied. The relationship between the enzyme

237

capacity and various enzyme concentrations at different concentrations of 12


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238

resveratroloside and resveratrol is shown in Figure 5A and Figure 5B. A set of straight

239

lines in different slopes was constituted, and all the lines passed though the origin.

240

The slope decreased with increasing inhibitor concentration. These results indicated

241

that the inhibitory actions of resveratroloside and resveratrol on a-glucosidase were

242

all reversible.

243

Kinetic characterization. Lineweaver-Burk double reciprocal plots was further

244

applied to study the kinetic behavior of resveratroloside and resveratrol against

245

a-glucosidase. The a-glucosidase inhibitory activity in the presence of various pNPG

246

concentrations at different concentrations of resveratrolosides and resveratrols is

247

shown in Figure 5C and Figure 5D. A set of straight lines with different slopes was

248

constituted, and all the lines intersected at a certain point of the Y axis. As the

249

inhibitor concentration increased, the slope increased. The maximum rate of the

250

a-glucosidase-catalyzed reaction was not changed and the Km was increased at

251

different concentrations of resveratroloside and resveratrol. This result indicated that

252

the a-glucosidase inhibitory actions of resveratroloside and resveratrol were all based

253

on competition.

254

Molecular docking. For a-1,4-glucosidase (maltase), the docking results indicated

255

that the resveratroloside was inserted into the active site of the maltase in an "in-line"

256

shape (Figure. 6A), and the free energy of binding was -7.42 kcal/mol.

257

Resveratroloside has a lower binding energy than do resveratrol and isomaltose

258

(Table 1, -7.42 vs -6.82 and -5.79 kcal/mol). From the perspective of energy, it can be

259

speculated that resveratroloside acts as an inhibitor and has a strong interaction with 13



260

maltase than does resveratrol. To further explore the competitive inhibition

261

mechanism of resveratroloside on maltase, the interaction between the two was

262

further analyzed. As shown in Figure 6A, the ligand resveratroloside can enter the

263

active site, and the meta-diphenol group forms strong hydrogen bonds with Asp214

264

and Glu276, with bond lengths of 1.7 and 2.5 Å, respectively (Asp214 and Glu276

265

catalytic residues play a key role in the catalytic process.). Simultaneously, the

266

meta-diphenol group also forms hydrogen bonds with Asp68, Arg212 and Arg439.

267

The bond lengths are 2.1, 2.1, and 2.0 Å, respectively. In addition, the glucopyranose

268

hydroxyl groups of resveratroloside form four hydrogen bonds with Lys155, His239,

269

Ser308, and Pro309, and the hydrogen bond lengths are 1.9, 2.7, 2.2, and 2.0 Å,

270

respectively. These strong hydrogen bonds could further strengthen the stable position

271

of resveratroloside in the active site. Resveratrol can also enter the maltase catalytic

272

site and constitute hydrogen bonds with Asp68, Gln181, His245 and Asp349. The

273

hydrogen bond length is 1.8-2.5 Å. In addition, resveratrol has a π-π stacking

274

interaction with Phe157 (Figure 6B).

275

For oligo-1,6-glucosidase (isomaltase, 3A4A), the binding free energy of

276

resveratroloside with 3A4A was -7.41 kcal/mol. The binding free energy of

277

resveratrol and isomaltose with 3A4A was -6.45 and -5.25 kcal/mol, respectively

278

(Table 1). Resveratroloside enters the catalytic site of isomaltase and forms hydrogen

279

bonds with the residues Asp69, Arg213, Asp242, Arg315, and His351. The bond

280

length is 2.0-3.0 Å. In addition, resveratroloside also has a π-π stacking action with

281

Tyr158, further stabilizing the binding of the ligand (Figure 7A). Resveratrol enter the 14


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282

catalytic site of isomaltase and form hydrogen bonds with residues Asp69, Arg213,

283

Asp215, Asp307, Arg442, respectively, with a bond length of 1.8-2.5 Å. Meanwhile,

284

resveratrol has a π-π stacking interaction with Phe303 (Figure 7B).

285

Based on the docking results, we speculate that resveratroloside enters the active

286

cavity and that the meta-diphenol group of resveratroloside occupies the catalytic site

287

by forming strong hydrogen bonds with the residues, thereby exerting a competitive

288

inhibitory effect. Compared with resveratroloside and isomaltose, resveratrol's ability

289

to bind two proteins is more strongly than isomaltose and is weaker than

290

resveratroloside.

291

Resveratrol as commercially available and important natural stilbene compound

292

possesses diverse pharmacological properties28. Various investigations have exhibited

293

that resveratrol could benefit T2DM treatment. This compound could enhance insulin

294

sensitivity in patients with T2DM and diabetic rats, reduce blood glucose levels in

295

animals and protect the pancreatic cells 29, 30. Resveratroloside as a mono-glucosylated

296

form of resveratrol has been shown to possess antioxidant13, antiaging14,

297

anti-inflammatory15 and antimicrobial activities16, 17 and neuroprotective effects18, 19.

298

It has also been demonstrated to have more potent antiallergic and phosphodiesterase

299

(PDE) inhibitory activities than those of the corresponding aglycone18, 20. In addition,

300

resveratroloside exhibited higher cardioprotective effect as compared to that of

301

resveratrol30. Up to now, studies on the antidiabetic activity of resveratroloside are

302

lacking.

303

To the best of our current knowledge, this is the first report demonstrating that 15



304

resveratroloside has the ability to inhibit yeast α-glucosidase activity in vitro and

305

decrease PBG levels in normal and diabetic mice. The mechanism study indicated that

306

the inhibition of resveratroloside on α-glucosidase was belonged to competitive

307

inhibition of reversible inhibition. In addition, the molecular docking of

308

resveratroloside with α-glucosidase (maltase and isomaltase) further demonstrated

309

that the competitive inhibitory effect of resveratroloside, which occupies the catalytic

310

site and forms strong hydrogen bonds with the residues of α-glucosidase. The current

311

findings reveal that resveratroloside is possibly a natural source for control of

312

hyperglycemia and may be beneficial to the health of consumers.

313

Resveratroloside was found in red wine, grapes and several traditional officinal

314

plants, including Rheum tanguticum, Rheum rhaponticum, Polygonum multiflorum

315

and Polygonum cuspidatum. The dose of resveratroloside in our mouse test was 30

316

mg/kg. Thus, the dose was assessed to be 3.3 mg/kg for a 60 kg individual. This may

317

be a comparatively safe dose, on the basis that treatment with resveratrol at 500 mg

318

three times one day for four weeks32 or 1 g/day for 45 days33 was found to be well

319

tolerated and safe in healthy volunteers.

320

The metabolism stability of resveratroloside in MLMs, RLMs and HLMs was also

321

studied (See supporting information, Table S1). Metabolic bioavailability (MF%) of

322

resveratroloside was all 100% in human liver microsomes (HLMs) and rat liver

323

microsomes (RLMs). Metabolic bioavailability (MF%) of resveratroloside was 71.9%

324

in mouse liver microsomes (MLMs). The result revealed that resveratroloside has

325

good metabolic stability in MLMs, RLMs and HLMs. 16


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Page 17 of 33

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ACKNOWLEDGMENTS

327

This research has been supported by Qinghai International Cooperation Project

328

(2017-HZ-806), Qinghai Natural Fund Project (2018-ZJ-913), Qinghai International

329

Cooperation Project (2018-HZ-806), Key Laboratory of Qinghai Medicinal Animal

330

and Plant (2017-ZJ-Y13), Key Laboratory of Qinghai Tibetan Medicine Research

331

(2017-ZJ-Y11), and Provincial Academic Cooperation Project of Sichuan province

332

(2018JZ0019).

333

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Figure captions

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Figure 1. The chemical structures and HPLC chromatograms of resveratroloside.

452

Figure 2. PBG-lowering effect of acarbose and different doses of resveratroloside in

453

starch loaded normal mice (A and B) and diabetic mice (C and D). The values

454

represent the means ± SEM (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ***p

Resveratroloside Alleviates Postprandial Hyperglycemia in Diabetic

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