Dietary Flavonoids and Acarbose Synergistically Inhibit Î±-Glucosidase

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Dietary Flavonoids and Acarbose Synergistically Inhibit alpha-Glucosidase and Lower Postprandial Blood Glucose Bowei Zhang, Xia Li, Wenlong Sun, Yan Xing, Zhilong Xiu, Chunlin Zhuang, and Yuesheng Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02531 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Journal of Agricultural and Food Chemistry

Dietary Flavonoids and Acarbose Synergistically Inhibit α-Glucosidase and Lower Postprandial Blood Glucose

1 2 3 4 5 6

Bo-wei Zhanga, Xia Lia, Wen-long Suna, Yan Xinga, Zhi-long Xiua, Chun-lin Zhuang*b, Yue-sheng

7

Dong*a

8

a

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China

9 10

b

School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China

11 12 13

Corresponding author：：

14

Yue-sheng Dong, Ph. D.

15

School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,

16

Dalian 116024. China; E-mail: [email protected]; Tel./Fax: +86-411-84706344

Associate Professor

17 18

Chun-lin Zhuang, Ph. D. Associate Professor

19

School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,

20

China; E-mail: [email protected]; Tel: 86-21-81871258-804

21 22

Running title:

23

Synergistic inhibition of α-glucosidase by baicalein and acarbose. 1

ACS Paragon Plus Environment


24

Abstract

25

The inhibition of porcine pancreatic α-amylase and mammalian α-glucosidase by sixteen

26

individual flavonoids was determined. The IC50 values for baicalein, (+)-catechin, quercetin, and

27

luteolin were 74.1 ± 5.6, 175.1 ± 9.1, 281.2 ± 19.2, and 339.4 ± 16.3 µM, respectively, against

28

α-glucosidase. The IC50 values for apigenin and baicalein were 146.8 ± 7.1 and 446.4 ± 23.9 µM,

29

respectively, against α-amylase. The combination of baicalein, quercetin or luteolin with acarbose

30

showed synergistic inhibition, and the combination of (+)-catechin with acarbose showed

31

antagonistic inhibition of α-glucosidase. The combination of baicalein or apigenin with acarbose

32

showed additive inhibition of α-amylase at lower concentrations and antagonistic inhibition at a

33

higher concentration. Kinetic studies of α-glucosidase activity revealed that baicalein alone, acarbose

34

alone, and the combination showed non-competitive, competitive, and mixed-type inhibition,

35

respectively. Molecular modeling revealed that baicalein had higher affinity to the non-competitive

36

binding site of maltase, glucoamylase, and isomaltase subunits of α-glucosidase, with glide scores of

37

-7.64, -6.98, and -6.88, respectively. (+)-Catechin had higher affinity to the active sites of maltase

38

and glucoamylase and to the non-competitive site of isomaltase. After sucrose loading, baicalein

39

dose-dependently reduced the postprandial blood glucose (PBG) level in mice. The combination of

40

80 mg/kg baicalein and 1 mg/kg acarbose synergistically lowered the level of PBG, and the

41

hypoglycemic effect was comparable to 8 mg/kg acarbose. The results indicated that baicalein could

42

be used as a supplemental drug or dietary supplement in dietary therapy for diabetes mellitus.

43 44

Key words：baicalein, acarbose, synergistic inhibition, postprandial blood glucose, α-glucosidase

45 2


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Introduction

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Diabetes is one of the most serious global health emergencies of the 21st century. The international

48

diabetes federation estimated that approximately 415 million people suffered from diabetes and that

49

there were 318 million people with impaired glucose tolerance in 2015. Diabetes is associated with

50

metabolic disorders that occur when the body cannot produce enough insulin or cannot use insulin,

51

and it is diagnosed by observing the level of fasting blood glucose (FBG) or PBG. Consistently high

52

levels of blood glucose in diabetes patients can lead to serious complications that affect the heart,

53

blood vessels, kidneys, nerves, and eyes.1 One therapeutic approach to treat diabetes is to delay the

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glucose absorption by inhibiting digestive enzymes such as α-amylase and α-glucosidases.2

55

Dietary carbohydrates are hydrolyzed into disaccharides and polysaccharides by α-amylase, which

56

is secreted by the salivary glands and pancreas.3 The polysaccharides and disaccharides are then

57

processed

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maltase-glucoamylase and sucrase-isomaltase, each of which is composed of two active subunits

59

located on the C- and N-terminus of their original proteins, respectively.5 All four subunits of the

60

α-glucosidases can hydrolyze maltose. However, only the C-terminal subunit of sucrase-isomaltase

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can hydrolyze sucrose.6, 7 It has been reported that inhibiting the digestion of dietary carbohydrates

62

can stop the progression of diabetes mellitus.8 Acarbose, an α-glucosidase inhibitor, can suppress the

63

digestion of carbohydrates and consequently delay the absorption of glucose and reduce the

64

postprandial hyperglycemia.9 However, the mechanism of the hypoglycemic effect of acarbose is

65

limited to the inhibition of digestive enzymes, and acarbose was found to be less effective than

66

metformin and sulfonylureas in long-term treatment of diabetes.10, 11

67

into

monosaccharides

by

α-glucosidases.4

α-Glucosidases

are

divided

into

Growing evidence suggested that the combination of anti-diabetic natural products with acarbose 3



68

can enhance the efficacy of acarbose and/or extend the simple mechanism of its hypoglycemic

69

activity. The combination of berry extracts with acarbose showed synergistic inhibition of α-amylase

70

and α-glucosidases.12 The combination of black tea extracts and acarbose exhibited a synergistic

71

inhibition against intestinal α-glucosidase and a hypoglycemic effect.13 Cyanidin derivatives in

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combination with acarbose synergistically alleviate postprandial hyperglycemia in rats.14 Our

73

previous research suggested that the co-administration of Oroxylum indicum seed extracts with

74

acarbose could extend the mechanism of the hypoglycemic effect of acarbose and enhance the

75

efficacy of acarbose by up to 5-fold in a 8-week treatment of diabetes in mice.15 In addition,

76

individual inhibitions of α-amylase and α-glucosidases by natural plant extracts16 or phenolic

77

compounds such as kaempferol,17 caffeic acid,18 and chlorogenic acid5 have been reported. These

78

data suggested that, either administered individually or in combination, dietary constituents can be

79

alternative agents to prevent and treat diabetes.

80

Flavonoids are widely distributed in plants and are present in considerable quantities in human

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diets, including in vegetables, fruits, tea, and red wine, and are regarded as the major effective

82

constituent.19, 20 Several clinical and basic research studies have shown that flavonoids have positive

83

effects for the treatment of multiple diseases, including diabetes,20 obesity,21 cardiovascular

84

diseases,22 and cancer.23 As a class of α-glucosidase inhibitors,24,

85

PBG-controlling effects.26 A previous study and our previous research have shown that flavonoids or

86

flavonoid-containing natural products exhibited synergistic PBG-lowering and α-glucosidase

87

inhibitory effects with acarbose.14,

88

includes at least six subgroups (including flavones, flavonols, flavanones, isoflavones, flavans-3-ol

89

and anthocyanins).28 The synergistic PBG-lowering effect of the combination of flavonoids other

27

25

the flavonoids also display

However, flavonoids have a rich structural diversity that

4


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than cyanidin derivatives and acarbose is still unclear, and the mechanism of the synergistic effect of

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flavonoids and acarbose needs to be revealed.

92

In the present study, the inhibition of α-glucosidases and α-amylase by dietary flavonoids and the

93

combination with acarbose was screened. The individual and combined PBG-lowering effects of the

94

selected compounds were evaluated in vivo. The mechanism of the synergism was analyzed and

95

evaluated through in vitro and molecular docking studies.

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Materials and methods

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Chemicals

98

Acarbose was purchased from Bayer Healthcare Co., Ltd (Berlin, Germany). All flavonoid

99

standards were purchased from Must Biotechnology Co., Ltd (Chengdu, China). Starch, maltose and

100

sucrose were purchased from Solarbio Co., Ltd (Beijing, China). Mammalian α-glucosidase was

101

prepared by our lab. Porcine pancreatic α-amylase was purchased from Sigma Chemical Co (St.

102

Louis, MO, USA). The glucose test kit (ACCU-CHEK) used for the in vivo assays was purchased

103

from Roche Diagnostics GmbH (Germany). The glucose assay kit used for the in vitro assays was

104

purchased from Nanjing Jiancheng Bioengineering Institute. (Jiangsu, China).

105

α-Amylase assay

106

α-Amylase (EC 3.2.1.1) activity was determined following the method previously reported with

107

slight modifications.29 Briefly, baicalein or acarbose and 0.1 U/mL porcine pancreatic α-amylase

108

were pre-incubated in phosphate-buffered saline (pH 7.0) for 15 min at 37°C. Fifty microliters (50

109

µL) of 0.4% (w/v) starch as the substrate was added to the reaction mixture for a total volume of 200

110

µL, and the reaction mixture was incubated at 37°C for 10 min. After the incubation, the reaction was

111

stopped by the addition of 1 mL dinitrosalicylic (DNS) reagent (1% 3, 5-dinitrosalicylic acid, 0.2% 5



112

phenol, 0.05% Na2SO3, and 1% NaOH in aqueous solution) to the reaction mixture. The mixtures

113

were then heated for 10 min at 100°C. After the mixture was cooled to room temperature, the

114

absorbance was read at 540 nm with a spectrophotometer. Acarbose was used as a positive control.

115

α-Glucosidase assay

116

The α-glucosidase solution was prepared as previously reported with minor modifications.30 The

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rats were sacrificed after a 16-h starvation period. The small intestinal tissues were excised and

118

homogenized in 100 mM phosphate buffer saline with 14 mM NaCl (1:10 dilution; w/v). After a

119

centrifugation for 15 min at 12000 g, the supernatant was collected and used as the enzyme solution.

120

The α-glucosidase activity (maltase: EC 3.2.1.20, sucrase: EC 3.2.1.48) was determined following

121

the method of a previous report.29 Briefly, 100 µL of the enzyme solution containing 0.002 U/mL of

122

α-glucosidase activity and 100 µL of the sample (baicalein or acarbose) were pre-incubated for 15

123

min at 37°C. Then, 100 µL of 27 mM sucrose or 9 mM maltose was added to the reaction mixture as

124

the substrate. The mixture was then incubated for 30 min at 37°C. After the incubation, the reaction

125

was terminated by incubating the reaction mixture at 100°C for 10 min. The amount of the liberated

126

glucose in the supernatant was determined with a commercial assay kit based on the glucose oxidase

127

method. Except for a change in the concentration of sucrose, experiments to determine the type of

128

inhibition by baicalein on mammalian α-glucosidase activity were carried out in the same manner.

129

The type of inhibition was determined by Lineweaver-Burk plots and further confirmed by a

130

nonlinear regression analysis with the Levenberg-Marquardt algorithm (Sigma Plot; Systat Software

131

Inc., San Jose, CA, USA).5 The nonlinear regression method, which could avoid additional errors

132

introduced by linear regression, was also used to calculate the kinetic parameters.

133

Evaluation of synergy in the α-glycosidase inhibition assay 6


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The inhibitors were added individually or in combination at different concentrations in the

135

α-glycosidase assay. The combination index (CI), which is a quantification of the degree of inhibitor

136

interactions based on the median-effect principle developed by Chou and Talalay, were determined

137

using the Compusyn software.31

138

The equation for the median-effect principle is as follows:

139

(1)

140

where fa is the fraction affected by dose D, fu is the unaffected fraction (fu = 1 - fa), m is the

141

coefficient signifying the shape of the dose-effect curve, D is the dose of the inhibitor, and Dm is the

142

median-effect dose (IC50 in this article).

143

The equation for the CI is expressed as follows: (2)

144

145

where (D)1 and (D)2 are the doses of inhibitors that produce a certain level of inhibition in the

146

combination system, and (Dx)1 and (Dx)2 are the doses of inhibitors added alone that lead to the

147

same level of inhibition.

148

The combined inhibition was divided into synergistic (CI1.1) inhibition, according to the CI values.

150

Molecular docking

151

Molecular docking was performed to predict the binding site and efficacy between the

152

α-glucosidase proteins and flavonoids using the Schrodinger Maestro 10.2 software package.

153

N-terminal sucrase-isomaltase (PDB: 3LPP), N-terminal maltase-glucoamylase (PDB: 2QMJ) and

154

C-terminal maltase-glucoamylase (PDB: 3TOP) were examined in this study. All proteins were 7



155

subjected to cavity analysis and ranked based on their expanded van der Waals volume for binding

156

evaluation using Sitemap following the software instructions.32 For each protein structure, the four

157

best ranked cavities were selected with SiteScores > 0.9 and volumes > 60. The protein preparation

158

was performed following a previous report.33 The docking grid (32 × 32 × 32 Å) was generated

159

based on the chosen binding sites. The original ligand within the PDB structure was then re-docked

160

into the corresponding protein structure without any constraints. The reliability of the docking

161

procedure was evaluated by comparing the CαRMSD between the positions of heavy atoms of the

162

ligand in the calculated crystal structure. This procedure re-docked acarbose, miglitol, salacinol, and

163

kotalanol with low CαRMSD values (Figure S1) and was used in the subsequent in silico evaluation.

164

The 5 best conformations were generated based on the empirical glide score (kcal/mol). The

165

interactions between the subunits of the α-glucosidase and flavonoids were predicted. Blind docking

166

was also carried out using Autodock 4.2 to cross-check the docking results for active site binding,34

167

with the following settings: maximum number of 25,000,000 energy evaluation, number of

168

generations = 27,000, mutation rate = 0.02, maximum number of iterations = 300, and number of

169

docking runs = 100. The grid size was 120 × 120 × 120 Å with a spacing of 0.375 Å centered on the

170

active site for each protein.

171

Animals

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Kunming mice (18~22 g, 6 weeks old) and SD rats (180~220 g) were purchased from Dalian

173

Medical University SPF experimental animal center (Dalian, China). The animals were provided a

174

standard rodent diet and free access to water and were maintained at a temperature of 20~22°C. The

175

animals and protocols used in this study were approved by the Animal Care Committee of Dalian

176

Medical University. 8


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Oral sugar tolerance test

178

The oral sugar tolerance test was performed according to a previous report.35 Kunming mice that

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had been fasted for 16 h were randomly divided into several groups. A baicalein-loaded

180

self-microemulsifying drug delivery system was prepared according to previous reports.36 The final

181

concentrations of ethyl oleate, PEG 400 and tween-80 used in the oral sugar tolerance test were 2%,

182

2% and 4% (w/w), respectively. The vehicle (control group); baicalein at 40, 80, and 240 mg/kg; or

183

acarbose at 0.3, 1 and 8 mg/kg was orally administered to the mice. Sucrose or glucose was orally

184

administered at 2 g/kg body weight simultaneously with the drugs. Blood samples were collected

185

from the tail vein at 0, 30, 60 and 120 min, and the levels of blood glucose were measured with a

186

glucose test kit.

187

Statistical Analysis

188

The statistical analysis was performed using SPSS 17.0. All the results were expressed as the mean

189

± standard deviation. Comparisons between groups were analysed using one-way ANOVA followed

190

by Fisher LSD test. A p value less than 0.05 was judged as statistically significant. The concentration

191

of the inhibitors that caused 50% inhibition (IC50) of the enzyme activity was calculated by nonlinear

192

regression.

193

Results and Discussion

194

Individual inhibition of mammalian α-glucosidase and porcine pancreatic α-amylase by

195

flavonoids

196

Sixteen dietary flavonoids, which have various hydroxylation and glycosylation patterns across the

197

A, B, or C rings and belong to 5 subgroups of flavonoids, including flavones, flavonols, flavanones,

198

isoflavones, and flavans-3-ol (Figure 1), were selected and tested for their inhibition of mammalian 9



199

α-glucosidase and porcine pancreatic α-amylase.

200

For the maltose-hydrolyzing activity, the IC50 value of acarbose was 0.4 ± 0.1 µM, which was

201

similar to that reported previously.37 Baicalein and (+)-catechin showed the strongest inhibition

202

among the tested flavonoids with IC50 values of 74.1 ± 5.6 µM and 175.1 ± 9.1 µM (Table 1, Figure

203

S2), respectively. The IC50 value for baicalein was similar to that in a previous report.38 Quercetin

204

and luteolin showed weaker inhibition than baicalein, with IC50 values of 281.2 ± 19.2 µM and 339.4

205

± 16.3 µM, respectively. The IC50 values for the other flavonoids were greater than 400 µM (Table 1).

206

Comparison of IC50 values showed that the inhibition of maltase by baicalein was 1/200 of that by

207

acarbose.

208

For the sucrose-hydrolyzing activity, the IC50 value for acarbose was 1.2 ± 0.3 µM. Baicalein

209

showed the strongest inhibition among the tested flavonoids. The IC50 value for baicalein was 14.6 ±

210

2.7 µM (Table 1), which was 1/12 of that of acarbose. The relative inhibition intensity of baicalein to

211

acarbose showed that baicalein exhibited a stronger inhibition against sucrase than maltase. The

212

inhibitory activities of all the other tested flavonoids were weak at 400 µM (Table 1).

213

For the α-amylase activity, the IC50 value for acarbose was 5.3 ± 3.1 µM (Table 1), which is

214

consistent with the previously reported value of 6.46 µM.35 Apigenin showed the strongest inhibition

215

(Table 1). Luteolin, baicalein, quercetin and kaempferol showed weaker inhibition than apigenin.

216

The IC50 values for apigenin and baicalein were 146.8 ± 7.1 µM and 446.4 ± 23.9 µM. Comparison

217

of IC50 values suggested that the inhibitory activity of apigenin and baicalein were 1/28 and 1/84 of

218

that of acarbose, respectively.

219

The inhibition of individual flavonoids against α-glucosidase and α-amylase revealed a

220

structure-activity relationship. Generally, the tested flavonoids showed relatively stronger individual 10


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inhibition of the maltose-hydrolyzing activity than sucrose-hydrolyzing activity. For the

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maltose-hydrolyzing activity, the fact that baicalein showed stronger inhibition than chrysin

223

suggested that the 6-hydroxyl group is important for exerting inhibition (Figure 1, Table 1), which is

224

consistent with a previous report.38 The importance of the 3'- and 4'-hydroxyl groups can be shown

225

by comparing the inhibition of luteolin and apigenin, scutellarin and baicalin, quercetin and

226

kaempferol, respectively. The importance of the 6-hydroxyl group was greater than that of the

227

4'-hydroxyl group as baicalein showed a stronger inhibition than apigenin. The introduction of

228

7-glucuronic acid group decreased the inhibition as baicalein showed a stronger inhibitory activity

229

than baicalin. Genistein showed very weak inhibition compared with apigenin, suggesting that the

230

linkage of the B-ring at the 2-position is important for the inhibitory activity. (+)-Catechin showed

231

stronger inhibition than quercetin, suggesting that the lack of a 4-carbonyl group in the C-ring can

232

enhance the inhibition.

233

For α-amylase activity, comparisons of the inhibition by apigenin vs chrysin and scutellarin vs

234

baicalin, showed that the 4'-hydroxyl group were important (Table 1). The fact that baicalein showed

235

stronger inhibition than chrysin suggested that the 6-hydroxyl group is important for the inhibitory

236

activity. The inhibition by apigenin was greater than that of naringenin, suggesting that the nearly

237

planar structure of the C-ring is important for the inhibitory activity. Apigenin exhibited stronger

238

inhibition than genistein, suggesting that linkage of the B-ring at the 2-position can enhance the

239

inhibitory activity.

240

Combined inhibition of mammalian α-glucosidase and porcine pancreatic α-amylase by

241

flavonoids and acarbose

242

The combined inhibition of flavonoids with acarbose against α-glucosidase and α-amylase were 11



243

determined. CI values, a quantitative measurement for the evaluation of combined effect, were used

244

to assess whether combinations provided more (synergistic, CI1.1) inhibitions against α-glucosidase and α-amylase than

246

those of flavonoids and acarbose alone.31

247

For the maltose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced

248

the inhibition compared with individual compounds. For example, the combination of baicalein at

249

17.5 µM and acarbose at 0.194 µM caused 62.4% inhibition of the activity of α-glucosidase (Figure

250

2A), which is higher than the inhibition caused by baicalein alone at 35.0 µM or acarbose alone at

251

0.387 µM (twice the concentration of each compound). The calculated CI values were less than 0.41

252

at all dosages, suggesting a strongly synergistic interaction between baicalein and acarbose. The CI

253

values for the combinations of either quercetin or luteolin with acarbose were between 0.51 and 0.83,

254

which suggests moderate synergism (Figure 2B and 2C). For the combination of (+)-catechin and

255

acarbose, the CI values were less than 0.9 at low concentrations but were higher than 1.1 at the

256

highest concentration, which suggests antagonism (Figure 2D). No obvious enhancement was found

257

when acarbose was combined with the flavonoids that displayed weak or no individual inhibition

258

(data not shown), suggesting that inhibitory activity of each compounds in the combination was

259

necessary for exerting synergistic inhibition.

260

For the sucrose-hydrolyzing activity, the combination of baicalein and acarbose strongly enhanced

261

the inhibition compared with the individual compounds (Figure 3). The CI values were between 0.61

262

and 0.70 at lower concentrations (3.19, 6.39, and 10.7 µM for baicalein), suggesting synergism. And

263

the CI value was 0.39 at a higher concentration (14.8 µM for baicalein), suggesting strong synergism.

264

These data indicated that the synergistic inhibition on sucrase became stronger at higher 12


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concentration of baicalein and acarbose.

266

For the α-amylase activity, the combinations of flavonoids with acarbose did not show any

267

obvious enhancement of the inhibition, compared with that observed for acarbose or the various

268

flavonoids added individually. For example, the combination of baicalein at 148 µM and acarbose at

269

1.86 µM caused 35.1% inhibition of the activity of α-amylase, which is lower than the inhibition

270

caused by baicalein alone at 296 µM and close to the inhibition caused by acarbose at 3.72 µM

271

(twice the concentration of each compound). Most calculated CI values were higher than 0.9,

272

suggesting additive inhibition or antagonistic inhibition. The only exception was the combination of

273

apigenin at 46.3 µM and acarbose at 0.93 µM, with a CI value of 0.71, suggesting weak synergistic

274

inhibition. (Figure 4).

275

Because the combined effect cannot be evaluated by a simple arithmetic sum of the inhibition

276

produced by individual inhibitors, it is difficult to differentiate the synergistic, antagonistic and

277

additive effects, until Chou and Talalay introduced CI value to distinguish the three types of

278

inhibition. This method have been widely used for the evaluation of drug combination.39 Using this

279

method, we determined the synergistic inhibition of α-glucosidase by the combination of baicalein,

280

luteolin, and quercetin with acarbose. Boath et al has reported a synergistic effect of acarbose and

281

black currant extracts.40 However, no synergistic inhibitory interaction of flavones or flavonol

282

compounds with acarbose has been reported. Interestingly, (+)-catechin showed antagonistic but not

283

synergistic effect at higher concentrations in our present work. Gao et al has reported that the

284

combination of epigallocatechin gallate and acarbose showed synergistic inhibition at lower

285

concentrations and antagonistic inhibition at higher concentrations, and that the CI values increased

286

with the concentrations of the inhibitors.41 Although these authors used yeast α-glucosidase in their 13



287

studies, their results were comparable to our observation.

288

When combined with acarbose, baicalein showed a lower CI value against maltose-hydrolysis than

289

those of luteolin and quercetin, indicating stronger synergistic inhibition. This result suggests that

290

either the introduction of 6-hydroxyl group or the removal of 3'-, 4'-hydroxyl groups is important for

291

exerting synergism (Figure 1). The fact that quercetin showed stronger synergistic inhibition with

292

acarbose than that of (+)-catechin suggested that the introduction of a 4-carbonyl group in the C-ring

293

could enhance the synergism.

294

Type of inhibition by baicalein and acarbose on mammalian α-glucosidase

295

The types of inhibition by baicalein and its combination with acarbose against mammalian

296

α-glucosidase were determined by both Lineweaver-Burk and Michaelis-Menton plots. The

297

Lineweaver-Burk plots of acarbose generated straight lines which had intersections on the Y-axis,

298

suggesting competitive inhibition (Figure 5A). This data was consistent with the previous report.42

299

The straight lines generated by baicalein alone and the combination had intersections on the X-axis

300

and in the second quadrant, suggesting non-competitive and mixed-type inhibitions, respectively.

301

The types of inhibition were further confirmed by the Michaelis-Menton plots using the best fit of

302

nonlinear regression analysis with the Levenberg-Marquardt algorithm. Compared with the control,

303

the addition of acarbose significantly increased the Michaelis constant (Km), but the maximum

304

reaction velocity (Vmax) was not significantly changed; the addition of baicalein significantly reduced

305

the Vmax, but the Km was not significantly changed (Figure 5B, Figure 5C, and Table 2). When a

306

combination of baicalein and acarbose was added in a fixed ratio, the Km value was significantly

307

higher than those of both acarbose alone and the control, and the Vmax was significantly lower than

308

that of the control (Figure 5D and Table 2). The values of Km, Vmax, and their patterns confirmed 14


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that baicalein alone, acarbose alone, and the combination exhibited non-competitive, competitive,

310

and mixed-type inhibition, respectively. Besides, the significantly higher Km value of the

311

combination than that of acarbose alone also indicated that the combination caused greater reduction

312

of the affinity of enzyme for substrate. This result indicated that, when used in combination, the

313

inhibitors had stronger binding capacity at the competitive binding site, i.e. active site or

314

substrate-binding site. The fact that baicalein alone did not change the Km value suggested that

315

baicalein, a non-competitive inhibitor, cannot bind to the competitive binding site and thereby reduce

316

the affinity for substrate. Thus, baicalein is expected to bind to the non-competitive binding site and

317

enhance the affinity of enzyme for acarbose, and finally reduce the affinity of enzyme for substrate.

318

This speculation was supported by a previous study, in which black tea extract was found to be a

319

non-competitive inhibitor against α-glucosidases and it exhibited synergism with acarbose.

320

Furthermore, black tea extract also reduced the affinity of enzyme for substrate.13 However, further

321

study is still needed to clarify the exact change in the α-glucosidases caused by flavonoid binding.

322

On the other hand, Adisakwattana et al also reported that cyanidin-3-rutinoside, another

323

non-competitive inhibitor of α-glucosidase, showed synergistic inhibition with acarbose.14 Taking

324

these data into consideration, binding at the non-competitive site of α-glucosidase might be the basic

325

requirement for an inhibitor that could exert synergistic inhibition with acarbose.

326

Molecular docking

327

To identify the binding sites for the flavonoids in the intestinal glucosidases, we performed

328

molecular docking for baicalein, (+)-catechin, quercetin, luteolin, and acarbose with α-glucosidases

329

using Schrodinger software. As no crystal structure of rat α-glucosidase is currently available, crystal

330

structures

of

three

available

human

α-glucosidase

subunits,

15


2QMJ

(N-terminal

of


331

maltase-glucoamylase), 3TOP (C-terminal of maltase-glucoamylase) and 3LPP (N-terminal of

332

sucrase-isomaltase), were used. The sequence homology of the three subunits to their corresponding

333

human counterpart is 92%, 90%, and 86%, respectively, within 10 Å around the predicted binding

334

sites (Figure S3-S5) of baicalein. Four binding sites in each protein were chosen according to the

335

site score values that were calculated based on their expanded van der Waals volume for binding

336

evaluation and ranked using the sitemap panel (Figure 6), which was similar to those previously

337

reported.32 For each protein, the active site was included in the chosen sites.

338

For N-terminal maltase-glucoamylase (2QMJ), the glide score for acarbose in the active site (site 2)

339

was -7.34 kcal/mol. Baicalein was predicted to bind to site 1, with a better glide score of -7.64

340

kcal/mol compared to the interaction with the active site (site 2, -5.50 kcal/mol). Three major

341

interactions were observed to contribute to the maintenance of the binding: (a) hydrogen bonds

342

between Phe535 and Ser521 and the hydroxyl group in the A-ring (Figure 7); (b) a hydrogen bond

343

between Lys776 and the carbonyl group in the C-ring; and (c) hydrophobic interactions in a large

344

pocket (Val779, Leu286, Ala780, Pro287 and Ile523). (+)-Catechin was predicted to bind to the

345

active site (site 2), with a glide score of -6.55 kcal/mol. Like acarbose, (+)-catechin bound to the -1

346

and +1 sugar-binding site residues Asp327, Asp443 and Asp542 through hydrogen bonds with the

347

hydroxyl groups in the B- and C-rings. Luteolin and quercetin were predicted to bind to site 1, with

348

glide scores of -8.15 and -7.05 kcal/mol, respectively. The H-bonding interactions of hydroxyl and

349

carbonyl groups with Val779, Asp777, Lys776, Phe535, and Ser521 for luteolin and Asp777, Lys776,

350

Phe535, and Ser521 for quercetin were predicted to be the predominant interactions (Figure S6A,

351

S6B).

352

For C-terminal maltase-glucoamylase (3TOP), the glide score for acarbose in the active site (site 3) 16


Page 16 of 40

Page 17 of 40


353

was -8.00 kcal/mol (Table 4). Baicalein was predicted to bind to site 1, with a glide score (-6.98

354

kcal/mol) better than that for its interaction with active site (site 3, -5.70 kcal/mol). The hydroxyl

355

group in the A-ring formed hydrogen bonds with Pro1327 and Glu1284 (Figure 8). The carbonyl

356

group in the C-ring formed hydrogen bonds with Leu1291. The B-ring formed hydrophobic

357

interactions with Pro1405 and Leu1401. (+)-Catechin was predicted to bind to the active site (site 3)

358

with a glide score of -7.70 kcal/mol. Like acarbose, (+)-catechin bound to the -1 and +1

359

sugar-binding site residues His1584, Asp1279, Asp1526, Arg1510, and Asp1157 through hydrogen

360

bonds. Luteolin and quercetin was predicted to bind to active site (site 3) with glide scores of -7.52

361

and -7.19 kcal/mol, respectively. The H-bonding interactions of hydroxyl and carbonyl groups with

362

His1584, Asp1526, Asp1279, and Asp1157 for both luteolin and quercetin were predicted to be the

363

predominant interactions (Figure S6C, S6D).

364

For N-terminal sucrase-isomaltase (3LPP), the glide score for acarbose in the active site (site 3)

365

was -7.13 kcal/mol (Table 4). Baicalein, luteolin, quercetin and (+)-catechin were predicted to bind

366

to site 2, with better glide scores of -6.88, -6.47, -5.97, and -8.79 kcal/mol, respectively, compared

367

with their interactions with the active site (site 3, -4.68, -4.98, -4.98, and -5.81 kcal/mol,

368

respectively). Asp806 formed hydrogen bonds with the hydroxyl group in the A-ring of baicalein

369

(Figure 9). The residues (His600, Ile552, and Gly562) formed hydrogen bonds with the hydroxyl

370

group of (+)-catechin. The B-rings of both baicalein and (+)-catechin formed hydrophobic

371

interactions in a large pocket (Phe551, Ile552, Ala565, and Ala566).

372

For all three subunits of the α-glucosidases, the non-competitive site was predicted to have a much

373

higher binding affinity to baicalein. Quercetin and luteolin were predicted to have higher affinities

374

for the non-competitive site of maltase (2QMJ) and isomaltase (3LPP) and the active site of 17



375

glucoamylase (3TOP). In contrast, (+)-catechin was predicted to have a higher affinity for the active

376

sites of maltase (2QMJ) and glucoamylase (3TOP) but a higher affinity for the non-competitive site

377

for isomaltase (3LPP). These results are consistent with the previous reports that (+)-catechin is a

378

non-competitive inhibitor of isomaltase (3LPP) but could bind to different binding sites of maltase

379

and glucoamylase.5 These observations, together with the facts that baicalein displayed stronger

380

synergism than quercetin and luteolin and (+)-catechin displayed antagonism at higher

381

concentrations in the enzyme assays, suggested that non-competitive binding in the α-glucosidases

382

would enhance the synergistic inhibition, whereas competitive binding at the active sites of

383

α-glucosidases at higher concentrations might lead to antagonistic inhibition between flavonoids and

384

acarbose. In addition, H-bonds were formed between the carbonyl group of baicalein and the

385

residues in the non-competitive sites when binding to maltase (2QMJ) and glucoamylase (3TOP) but

386

not isomaltase (3LPP), which suggested that the carbonyl group in the flavonoids may be important

387

in producing the non-competitive inhibition against maltase (2QMJ) and glucoamylase (3TOP).

388

Hypoglycemic effect of baicalein and co-administration of acarbose

389

The hypoglycemic effect of baicalein and co-administration of acarbose was evaluated through

390

sucrose tolerance test in mice, and the time courses for the levels of blood glucose were measured

391

before and 30, 60, 90, and 120 min after sucrose loading (2 g/kg) (Figure 10). The PBG level of the

392

control group increased and peaked at 30 minutes and then decreased. The baicalein- and

393

acarbose-administered groups exhibited dose-dependent decrease in the level of PBG compared with

394

the control group. Baicalein at 80 and 200 mg/kg and acarbose at 1 and 8 mg/kg decreased the levels

395

of PBG significantly 30 min after sucrose loading (Figure 10A and Figure 10B). The combination

396

of baicalein at 80 mg/kg and acarbose at 1 mg/kg significantly reduced the levels of PBG 30 and 60 18


Page 18 of 40

Page 19 of 40


397

min after sucrose loading compared with the control group. This reduction was greater than that

398

observed in the groups that were administered the individual agents, 80 mg/kg baicalein or 1 mg/kg

399

acarbose (Figure 10C). PBG levels after the co-administration of baicalein (80 mg/kg) and acarbose

400

(1 mg/kg) were not significantly different from those receiving 8 mg/kg acarbose alone. Our data

401

suggested that the co-administration of baicalein with acarbose generated a synergistic hypoglycemic

402

effect in vivo, which was consistent with the synergistic effects of combination in vitro. The effective

403

dose of acarbose could be reduced by 87.5%.

404

The glucose tolerance test was also performed to exclude the influences other than carbohydrate

405

digestion. Glucose was administered orally to the fasted mice. No significant difference in PBG

406

levels was observed among the baicalein (80 mg/kg), acarbose (8 mg/kg), and control groups

407

(Figure 10D). This result suggested that the PBG lowering effect of baicalein resulted from the

408

inhibition of carbohydrate digestion.

409

We reported that baicalein could enhance the efficacy of acarbose both in vitro and in vivo for the

410

first time. It is known that the hypoglycemic effect of acarbose is limited to the inhibition of

411

carbohydrate digestion. Long-term treatment with baicalein, on the other hand, has also been

412

reported to have an anti-diabetic effect through the promotion of islet β-cell function,43 the activation

413

of AMPKα/IRS/Akt pathway,44 the suppression of inflammatory responses,45 and the reduction of

414

oxidative stress.46 Thus, baicalein and acarbose can inhibit the α-glucosidase in the gut lumen

415

synergistically to prevent hyperglycemia. And the absorbed baicalein is expected to increase the

416

insulin resistance, improve the complications and restore the pancreatic function. Therefore,

417

baicalein might extend the simple mechanism of acarbose alone, and enhance the efficacy of

418

acarbose. Baicalein alone also showed PBG lowering effect in vivo. Thus, our present work is 19



419

expected to expand the usage of baicalein in diabetes treatment as a dietary supplement.

420

Flavonoids are widely distributed in plants and are found in considerable quantities in human diets,

421

such as fruit, vegetables, nuts, seeds, fruits, tea, and red wine. Baicalein was found in abundance in

422

several natural products such as Oroxylum indicum and Scutellaria baicalensis.45 The dose of

423

baicalein in our in vivo study was 80 mg/kg. Therefore, the dose for human administration was

424

estimated to be 8.8 mg/kg using body surface area model,47, 48 which corresponds to approximately 3

425

times the average daily human intake of flavonoids.49 This is a relatively safe dose, on the basis that

426

treatments with baicalein at 800 mg twice a day for 10 days50 or a single dose of 100-2800 mg51 were

427

reported to be safe and well tolerated by volunteers. Besides, all the adverse events reported,

428

including blurred vision and plasma fibrinogen decreased, were rated as “mild” and resolved without

429

further treatment. Therefore, baicalein alone or the combination could be regarded as a low-toxicity

430

agent.

431

In conclusion, the combination of baicalein, quercetin, or luteolin with acarbose synergistically

432

inhibited α-glucosidase in vitro, and baicalein lowered PBG levels in vivo. The mechanism of the

433

synergistic inhibition is the non-competitive inhibition of the α-glucosidases by the flavonoids and

434

acarbose. These results indicated the potential of baicalein for reducing the effective dose and

435

extending the simple mechanism of the hypoglycemic effect of acarbose. Also, baicalein may be

436

used as a supplemental drug or dietary supplement for diabetes mellitus.

437

Abbreviations Used

438

Postprandial blood glucose (PBG)

439

Fasting blood glucose (FBG)

440

Oroxylum indicum seed extracts (OISE) 20


Page 20 of 40

Page 21 of 40


441

Dinitrosalicylic (DNS)

442

Combination index (CI)

443

Funding

444

This work was supported by the National Natural Science Foundation of China (81172966 and

445

81502978), Shanghai “ChenGuang” project (16CG42), and Open Fund of Key Laboratory of

446

Biotechnology and Bioresources Utilization (Dalian Minzu University), State Ethnic Affairs

447

Commission & Ministry of Education, China.

448

Supporting information description

449

The re-docked acarbose, miglitol, salacinol and kotalanol and the CαRMSD values (Figure S1).

450

The Individual inhibition of α-amylase by acarbose (A) and baicalein (B) (Figure S2). The amino

451

acid sequence of human maltase (2QMJ) and the sequence homology compared with rat maltase

452

(Figure S3). The amino acid sequence of human glucoamylase (3TOP) and the sequence homology

453

compared with rat maltase (Figure S4). The amino acid sequence of human isomaltase (3LPP) and

454

the sequence homology compared with rat maltase (Figure S5). The binding positions and

455

interaction patterns of luteolin and quercetin with α-glucosidases (Figure S6).

21



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24


Page 24 of 40

Page 25 of 40


Tables Table 1. Individual inhibition of flavonoids against mammalian α-glucosidase and porcine pancreatic α-amylase Maltase

Sucrase

α-Amylase

Flavonoid IC50 (µM)

Inhibitiona (%)

IC50 (µM)

Inhibitiona (%)

IC50 (µM)

Inhibitiona (%)

Baicalein

74.1 ± 5.6

81.3 ± 4.1

14.6 ± 2.7

80.0 ± 6.2

446.4 ± 23.9

47.2 ± 3.8

Chrysin

> 400

4.2 ± 2.6

> 400

5.5 ± 2.1

> 400

24.5 ± 3.0

Luteolin

339.4 ± 16.3

53.9 ± 3.8

> 400

16.0 ± 0.9

> 400

54.3 ± 4.4

Apigenin

> 400

24.6 ± 2.9

> 400

8.9 ± 3.7

146.8 ± 7.1

79.3 ± 2.9

Scutellarin

> 400

44.1 ± 3.4

> 400

25.2 ± 1.4

> 400

-7.4 ± 1.0

Baicalin

> 400

4.8 ± 2.0

> 400

26.6 ± 4.2

> 400

-4.9 ± 2.2

Quercetin

281.2 ± 19.2

58.7 ± 4.8

> 400

16.0 ± 2.5

> 400

45.7 ± 4.1

Rutin

> 400

41.2 ± 5.1

> 400

-3.9 ± 3.1

> 400

-5.4 ± 2.6

Kaempferol

> 400

5.1 ± 1.2

> 400

16.5 ± 2.2

> 400

41.8 ± 2.3

Dihydromyricetin

> 400

27.6 ± 3.1

> 400

0.5 ± 0.2

> 400

4.1 ± 0.7

Hesperetin

> 400

7.4 ± 2.2

> 400

3.1 ± 1.1

> 400

3.2 ± 1.1

Naringenin

> 400

7.2 ± 2.4

> 400

16.0 ± 1.9

> 400

20.6 ± 2.8

Puerarin

> 400

28.1 ± 3.6

> 400

15.3 ± 2.4

394.2 ± 17.1

52.1 ± 4.6

Daidzein

> 400

-6.8 ± 2.4

> 400

14.7 ± 3.0

> 400

21.4 ± 2.6

Genistein

> 400

2.4 ± 1.2

> 400

-14.1 ± 2.3

> 400

32.4 ± 2.0

175.1 ± 9.1

71.7 ± 4.7

> 400

23.9 ± 2.5

> 400

-11.1 ± 1.9

0.4 ± 0.1

96.5 ± 7.4

1.2 ± 0.3

90.1 ± 5.6

5.3 ± 3.1

86.2 ± 6.1

Flavone

Flavonol

Flavanone

Isoflavone

Flavans-3-ol (+)-Catechin Acarbose a

Inhibition by 400 µM flavonoids. The values shown are the averages of triplicate assays. 25



Table 2. Kinetic parameters for mammalian sucrase Km (mM)

Vmax (µM/min)

Control

15.2 ± 1.9

387 ± 40.8

Baicalein

14.1 ± 2.2

275 ± 34.9#

Acarbose

29.5 ± 2.3#

388 ± 31.7

Acarbose + Baicalein (1:9.56)

43.6 ± 4.1#†

247 ± 20.4#

The values shown are the averages of triplicate assays. #p < 0.01 compared with the control group, and †p < 0.01 compared with the acarbose alone group.

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Table 3. The binding glide scores of acarbose, baicalein, luteolin, quercetin, and (+)-catechin on α-glucosidase proteins Docking glide score (kcal/mol) Binding Site a

Site Score

baicalein

luteolin

quercetin

(+)-catechin

acarbose

2QMJ (maltase)

1 2 (active site) 3 4

1.06 0.96 0.95 0.95

-7.64 -5.50 -6.16 -4.54

-8.15 -5.41 -7.10 -6.48

-7.05 -5.39 -7.03 -6.59

-6.01 -6.55 -6.04 -6.12

-6.73 -7.34 -5.37 -5.13

3TOP (glucoamylase)

1 2 3 (active site) 4

1.12 1.05 1.03 0.96

-5.08 -6.98 -5.70 -6.11

-7.40 -6.73 -7.52 -5.39

-7.15 -6.93 -7.19 -5.88

-6.86 -6.96 -7.70 -7.19

-5.71 -5.98 -8.00 -5.12

1 2 3 (active site)

1.11 1.08 1.00

-5.91 -6.88 -4.68

-5.93 -6.47 -4.95

-5.05 -5.97 -4.98

-5.93 -8.79 -5.81

N.C.b -4.97 -7.13

4

0.92

-6.49

-6.24

-5.92

-5.93

-5.54

3LPP (isomaltase)

a

The binding site underlined was predicted to have the strongest affinity with baicalein.

b

No binding conformation was produced.

27



Figure legends Figure 1. The structures of the flavonoids and the structure-activity relationships of flavonoids on the inhibition of mammalian α-glucosidase. Figure 2. The inhibitory effect of the combination of acarbose and flavonoids against mammalian α-glucosidase. The inhibitory effect of the combination of acarbose with baicalein (A), quercetin (B), luteolin (C) or (+)-catechin (D), using maltose as substrate. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error. Figure 3. The inhibitory effect of the combination of acarbose and baicalein against mammalian α-glucosidase, using sucrose as substrate. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the means of triplicate assays ± standard error. Figure 4. The inhibitory effect of the combination of acarbose with flavonoids against porcine pancreatic α-amylase and mammalian α-glucosidase. The inhibition of the combination of acarbose with apigenin (A) or baicalein (B) against porcine pancreatic α-amylase. CI values above data points were calculated by CompuSyn software. CI1.1 indicate synergism, additive effect, and antagonism, respectively. The values shown are the averages of triplicate assays ± standard error. Figure 5. Lineweaver-Burk plots of the combination (A) and Michaelis-Menton plots of acarbose alone (B), baicalein alone (C), the combination of baicalein and acarbose (D), against mammalian α-glucosidase, using sucrose as substrate. The values shown are the means of triplicate assays ± standard error. 28


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Figure 6. The selected binding sites of maltase (2QMJ), glucoamylase (3TOP), and isomaltase (3LPP). Figure 7. Binding sites for acarbose, baicalein, and (+)-catechin in the N-terminal maltase-glucoamylase (2QMJ) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure

8.

Binding sites of acarbose, baicalein, and (+)-catechin

in the C-terminal

maltase-glucoamylase (3TOP) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure

9. Binding sites of acarbose, baicalein, and (+)-catechin in the

N-terminal

sucrase-iasomaltase (3LPP) protein. The atoms of the protein are colored as follows: carbon – green, oxygen – red, nitrogen – blue. The carbon atoms of acarbose are colored light blue; the carbon atoms of baicalein are colored orange; the carbon atoms of (+)-catechin are colored purple. The hydrogen bonds are shown by yellow dashed lines. Figure 10. PBG-lowering effect of baicalein or acarbose alone or their combination. Baicalein and/or acarbose and sucrose (A, B and C) or glucose (D) were orally administered to the mice. The values represent the means ± SEM (n=10; *p < 0.05, #p < 0.01 compared with control group.).

29



Figures

Figure 1. 30


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Figure 2.

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Figure 3.

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Figure 4.

33



Figure 5.

34


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Figure 6.

35



Figure 7.

36


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Figure 8.

37



Figure 9.

38


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Figure 10.

39



Graphic abstract for table of contents

40


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Dietary Flavonoids and Acarbose Synergistically Inhibit Î±-Glucosidase

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