Inhibitory Mechanism of Apigenin on Î±-Glucosidase and Synergy

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Inhibitory Mechanism of Apigenin on #Glucosidase and Synergy Analysis of Flavonoids Li Zeng, Guowen Zhang, Suyun Lin, and Deming Gong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02314 • Publication Date (Web): 01 Sep 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Journal of Agricultural and Food Chemistry

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Inhibitory Mechanism of Apigenin on α-Glucosidase and

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Synergy Analysis of Flavonoids

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Li Zeng a, Guowen Zhang a,*, Suyun Lin a, Deming Gong b

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a

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State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

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School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand

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Running title: Inhibition of Apigenin on α-Glucosidase

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________________________

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*

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Corresponding author: Professor Guowen Zhang, Ph.D., Tel: +86-791-88305234, fax: +86-791-88304347. E–mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Inhibition of α-glucosidase activity may suppress the postprandial hyperglycemia.

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The inhibition kinetic analysis showed that apigenin reversibly inhibited

24

α-glucosidase activity with the IC50 value of (10.5 ± 0.05) × 10−6 mol L−1, and the

25

inhibition was in a non-competitive manner through a monophasic kinetic process.

26

The fluorescence quenching and conformational changes determined by fluorescence

27

and circular dichroism were due to the formation of α-glucosidase–apigenin complex,

28

and the binding was mainly driven by hydrophobic interactions and hydrogen bonding.

29

The molecular simulation showed that apigenin bound to a site close to the active site

30

of α-glucosidase, which may induce the channel closure to prevent the access of

31

substrate, eventually leading to the inhibition of α-glucosidase. The isobolographic

32

analysis of the interaction between myricetin and apigenin or morin showed that both

33

of them exhibited synergistic effects at low concentrations, and tended to exhibit

34

additive or antagonistic interaction at high concentrations.

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KEYWORDS:

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Isobologram

α-Glucosidase;

Apigenin;

Inhibition

38 39 40 41 42 43 2


mechanism;

Synergy;

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INTRODUCTION

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α-Glucosidase is widespread in various organisms including bacteria, yeasts, fungi,

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archaea, plants and animals.1 In humans, salivary and pancreatic α-amylase and four

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intestinal mucosal α-glucosidase activities are involved in the generation of dietary

48

glucose from starchy foods. The α-glucosidase activities are associated with

49

maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI).2 MGAM and SI located

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in the brush border of the small intestine, hydrolyze linear α-1→4 and branched

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α-1→6 linkage of oligosaccharides from the non–reducing end to release glucose.3

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One of the effective methods to suppress the postprandial hyperglycemia is to control

53

the release of glucose by inhibiting α-glucosidase.4 α-Glucosidase inhibitors can

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reduce the digestion rate of carbohydrate.5 Two α-glucosidase inhibitors, acarbose and

55

miglitol, have been used clinically to control the postprandial hyperglycemia, but

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these drugs usually have many side effects, such as abdominal distention, flatulence,

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diarrhea and meteorism.6 Therefore, it is necessary to find plant-based foods or

58

supplements as alternatives for α-glucosidase inhibitors due to their low cost and

59

being relatively safe.7

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Flavonoids are a large group of low molecular weight phenolic compounds

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possessing benzo–γ–pyrone structures, and secondary metabolites that are

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ubiquitously distributed in fruits, vegetables, nuts, tea, red wine and herbs8,9 The

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interest in flavonoids as a dietary component has been stimulated by their association

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with low incidence of chronic diseases, including cardiovascular diseases,

65

neurodegenerative diseases, type 2 diabetes mellitus and possibly cancers.9 In recent 3



66

years, many flavonoids have been reported to be potential antidiabetic agents.10 Li et

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al. extracted some flavonoids from hawthorn leaves, and they were identified as

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α-glucosidase inhibitors.11 Iio et al. found that most of 16 flavonoids inhibited

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α-glucosidase.12 Apigenin (4’, 5, 7–trihydroxyflavone, Figure 1A), a bioflavonoid, is

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widely distributed in a variety of plants, such as, tea, onions, thyme, sweet red pepper

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and especially celery.13 Modern pharmacological investigations found that apigenin

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has multiple bioactivities, including anti–inflammatory, antitumor, antioxidative,

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angiogenesis and hepatoprotective effects.14 Besides, apigenin has been reported to

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possess an inhibition activity on α-glucosidase.12 However, to our knowledge, the

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inhibitory mechanism of apigenin on α-glucosidase is still unclear, and the influence

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of the interaction between these flavonoids on α-glucosidase has not been

77

investigated.

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It is common that two or more drugs can produce overtly similar effects on the

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ingestion of food or supplements. The main pharmacotherapy goal of using a

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combination of different drugs is to improve efficacy without either increasing

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adverse effects or decreasing efficacy.15 It is vital to determine the combination of two

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drugs that produce increased effects. Isobolograms are a convenient and popular way

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of displaying the results of drug combination graphically as the paired values of

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experimental points falling below or above the line (additive isobole) indicate supra–

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or sub–additive combination.16

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The main objectives of this study were to determine the inhibition kinetics and

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inhibition mechanism of apigenin against α-glucosidase, and explore the inhibitory

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effect of combinations of flavonoids (between myricetin and apigenin or morin) on

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the enzyme. This study may provide new insights into the application of the

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nutrimental dietary sustenance which would be beneficial for the patients with

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postprandial hyperglycemia.

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MATERIALS AND METHODS

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Chemicals. Since it is not easy to acquire a pure mammalian α-glucosidase,

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another alternative model, yeast α-glucosidase was used in this study. α-Glucosidase

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(EC 3.2.1.20, from Saccharomyces cerevisiae, 23.2U/mg) purchased from

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Sigma-Aldrich Co. (St. Louis, MO, USA), acarbose and p-nitrophenyl-α-D-

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glucopyranosi de (pNPG) from Aladdin Chemistry Co. (Shanghai, China) were

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prepared by using 0.1 mol L−1 sodium phosphate buffer (pH 6.8). The stock solutions

99

of α-glucosidase and pNPG (9.1 × 10−3 mol L−1) were freshly made just before the

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experiments. Apigenin (98%), obtained from the National Institute for the Control of

101

Pharmaceutical and Biological Products (Beijing, China) was dissolved in absolute

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ethyl alcohol to a concentration of 4.0 × 10−3 mol L−1. Myricetin (≥98%) and morin

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(≥98%) purchased from Aladdin Chemistry Co. (Shanghai, China) were dissolved in a

104

portion of absolute ethanol and dimethyl sulfoxide (DMSO), respectively, then both

105

were diluted with the ultrapure water to make stock solutions (5.0 × 10−3 mol L−1).

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The final concentrations of both ethanol and DMSO in the experiments were below

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1% (v/v), which had no effect on the structure and activity of α-glucosidase. All the

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stock solutions were stored at 0–4 ºC, and all other reagents were analytical reagent

109

grade. Ultrapure water was used throughout the study. 5



110

Enzyme Activity Assay. The inhibitory activity of inhibitors against α-glucosidase

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was evaluated by using the method adapted from Ohta et al.17 In brief, a series of

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reaction solutions, including a fixed amount of α-glucosidase, different concentrations

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of inhibitors and specific volume of sodium phosphate buffer, were incubated at 37 ºC

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for 2 h. To initiate the reaction, pNPG (4.55 × 10−4 mol L−1, used as a substrate), was

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added into the pre-incubated mixtures and the final volume of reaction system was

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kept at 2.0 mL. p-Nitrophenol released from pNPG substrate was used as the target

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substance to quantify the enzymatic activity. The absorbance of p-nitrophenol was

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monitored at 405 nm every 5 s by a double beam UV−vis spectrophotometer

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(TU−1901 ,Persee, Beijing, China) with a 1.0 cm quartz cell. The enzymatic activity

120

in the absence of an inhibitor was defined as 100%. The concentration of an inhibitor

121

that reduces the enzyme activity by 50% was determined by the plot of relative

122

enzymatic activity against the corresponding inhibitor concentration. The relative

123

enzymatic activity (%) = (slope of reaction kinetics equation obtained by the reaction

124

with inhibitor)/(slope of reaction kinetics equation obtained by the reaction without

125

inhibitor) × 100.18 Acarbose was used as a positive control.

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Kinetic Analysis of Inhibitory Type. The same method as enzyme activity assay

127

was used to determine the kinetic mode of apigenin against α-glucosidase. The mode

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of inhibition was analyzed on the basis of the inhibitory effect of five different

129

apigenin concentrations with four different substrate concentrations on the kinetic

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parameters, namely the Michaelis−Menten constant (Km) and maximal rate (Vmax) of

131

the enzyme.19 The parameters can be determined by Lineweaver–Burk plots, which is

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the double reciprocal plot of enzyme reaction velocity (ν) against the concentration of

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substrate (pNPG) namely 1/ν versus 1/ [pNPG]. The secondary plot slope against

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[Apigenin] was also determined.

135

Fluorescence Measurement. Fluorescence spectra at three different temperatures

136

(298, 304 and 310 K) were conducted on a spectrofluorimeter (Model F−7000,

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Hitachi, Tokyo, Japan) equipped with a 1.0 cm path−length quartz cell and thermostat

138

bath. The steady-state fluorescence emission spectra were recorded between 300 nm

139

and 450 nm at the excitation wavelength of 280 nm, and both the excitation and

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emission slits were set at 2.5 nm. Apigenin at different concentrations (from 0 to 2.40

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× 10−5 mol L−1) were added into the 3.0 mL solution containing a fixed amount of

142

α-glucosidase (8.33 × 10−7 mol L−1). All the mixtures were held for 5 min to

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equilibrate before measurements. The fluorescence spectra of buffer were subtracted

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as the background fluorescence. The synchronous fluorescence spectra of

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α-glucosidase in the absence and presence of apigenin were scanned by setting the

146

excitation and emission wavelength interval (∆λ) at 15 and 60 nm, respectively.

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To eliminate the probability of re-absorption and inner filter effects in UV

148

absorption, all the fluorescence data were corrected for absorption of exciting light

149

and emitted light based on the following relationship: 20

150

Fc = Fm e ( A1 +A 2 )/2

(1)

151

Fc and Fm are the corrected and measured fluorescence. A1 and A2 represent the

152

absorbance of apigenin at the excitation and emission wavelengths, respectively.

153

Circular Dichroism (CD) Measurement. All the CD spectra were scanned on a

7



154

CD spectrometer (Bio-Logic MOS 450, Claix, France). The CD spectra in far−UV

155

region (190−250 nm) were measured by using a 1.0 mm path length cuvette under

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constant nitrogen flush. The concentration of α-glucosidase was maintained at 2.0 ×

157

10−6 mol L−1, and the molar ratios of apigenin to α-glucosidase were 0:1, 1:1, 2:1 and

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4:1, respectively. All the data were expressed in mean residue ellipticity [θ] (deg cm2

159

dmol−1). The CD spectra of the buffer were subtracted for the sake of baseline

160

correction. The CD spectra data were analyzed by the online SELCON3 program

161

(http://dichroweb.cryst.bbk.ac.uk/html/home.shtml)21 to determine the contents of

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different secondary structures of α-glucosidase.

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Molecular Docking. The docking study was used to investigate the binding details

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in the receptor−ligand complex.22 The blind docking was performed on AutoDock 4.2.

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The crystal structure of α-glucosidase (PDB ID: 3A4A) was downloaded from the

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Protein Data Bank (http://www.rcsb.org/pdb),23 and then checked for any error.

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During the process, all water molecules were removed, and polar hydrogen atoms as

168

well as Kollman charges were added to the protein, then the Gasteiger charges were

169

computed. The 3D structures of apigenin and myricetin were portrayed in Chem3D

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Ultra 8.0. The size of grid box was 80 Å × 90 Å ×100 Å in the x, y and z dimensions

171

with a grid spacing of 0.375 Å. In the default parameters, Lamarckian Genetic

172

Algorithm (LGA) was chosen for docking calculations and the search parameters

173

were set at 100 genetic algorithm runs. Compared to the rigidity of protein, the ligand

174

remained flexible in the docking process. In the outcomes of docking, the docked

175

model with lowest docking energy and maximum docking number was selected to

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represent its most favorable binding mode predicted by this program.

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Interaction between Myricetin and Apigenin or Morin. To determine the

178

interaction between myricetin and agigenin or morin, isobolographic analyses were

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conducted according to a published protocol with slight modifications.24 The enzyme

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activity of α-glucosidase was measured with the same method as above. The IC30,

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IC50 and IC70 values (loss of 30%, 50% and 70% enzymatic activity, respectively) for

182

myricetin, apigenin and morin alone on enzyme activity were obtained from

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concentration–response curves for the purpose of constructing isobolograms. In this

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plot, each axis in Cartesian coordinate represented the content of one of the flavonoids.

185

The concentration of one compound was kept constant and the contents of the other

186

were varied to calculate the values for each compound in the combination of two

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agents on the specific effect. In the combination of myricetin and apigenin, the doses

188

of myricetin were fixed at 0.25, 0.75 or 1.25 × 10−5 mol L−1 and the concentration of

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apigenin was changed to achieve a 30% inhibition. Also, the concentrations of

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myricetin were kept at 0.25, 1.25 and 2.25 × 10−5 mol L−1 and the amount of apigenin

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was changed to achieve a 70% inhibition. As for 50% inhibition effect level, the doses

192

of myricetin were fixed at 0.25, 0.75 and 1.5 × 10−5 mol L−1 or apigenin at 2.0, 5.0

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and 9.0 × 10−6 mol L−1 and the other was changed. The concentrations of myricetin in

194

the combination of myricetin and morin were the same as the combination of

195

myricetin and apigenin, and 0.5, 1.5 and 3.5 × 10−6 mol L−1 of morin were chosen in

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the 50% inhibition effect level. The experimentally determined data points lying on,

197

below or above the corresponding isobole indicated additive, synergistic or

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antagonistic effects between the flavonoids.25

199

Statistical Analysis. All the data were measured three times and expressed as

200

means ± standard deviation (n = 3). Data were analysed by one-way analysis of

201

variance (ANOVA), followed by multiple tests using SAS statistical package (version

202

8.0, SAS Institute, Cary, NC, USA). A p value < 0.05 was considered statistically

203

significant.

204

RESULTS AND DISCUSSION

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Inhibition of α-Glucosidase in Vitro. The dose–effect plots were used to

206

determine the inhibition effects of the four inhibitors (apigenin, morin, myricetin and

207

acarbose) on the activity of α-glucosidase (Figure 2A). The inhibition percentage of

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all the three flavonoids (apigenin, morin and myricetin) on α-glucosidase activity was

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found to increase in a dose dependent manner. The IC30, IC50 and IC70 values of

210

apigenin on α-glucosidase were estimated to be (6.66 ± 0.06) × 10−6, (10.5 ± 0.05) ×

211

10−6 and (14.1 ± 0.08) × 10−6 mol L−1, respectively. Those of morin were (2.82 ± 0.03)

212

× 10−6 (IC30), (4.48 ± 0.04) × 10−6 (IC50) and (6.57 ± 0.08) (IC70) × 10−6 mol L−1,

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while the values of myricetin were (1.68± 0.03) × 10−6 (IC30), (2.25 ± 0.05) × 10−6

214

(IC50) and (2.83 ± 0.07) (IC70) × 10−6 mol L−1. The IC50 value of apigenin in this work

215

was similar to a previous report (IC50 = 2.185 × 10−5 mol L−1).

216

flavonoids were more effective than acarbose (IC50 = (3.04 ± 0.04) × 10−4 mol L−1) in

217

the inhibition on α-glucosidase, and obviously the inhibitory potential of myricetin

218

was the highest among them. The structure of myricetin was similar to morin, which

219

had three hydroxyl groups at the position of 3’, 4’ and 5’ on B ring, while morin just 10


13

All the three

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had two hydroxyl groups at 2’ and 4’. The ortho–position hydroxyl groups might be

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responsible for the higher inhibition activity which was in favor of the interaction

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between α-glucosidase and flavonoid, while the meta–position hydroxyl groups on B

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ring would decrease the electron cloud density of band I resulting in a lower

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inhibitory activity.21 Besides, the increased number of hydroxyl groups may enhance

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the inhibition which was consistent with a previous report. 26 Compared to myricetin

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and morin, the IC50 value of apigenin was the lowest in the three flavonoids which

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may mainly be due to the lack of 3–hydroxyl groups on C ring.7 These results were in

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accordance with a previous report showing that both the increase of hydroxyl group

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on B ring and the hydroxylation at 3 position on C ring enhanced the inhibitory

230

effect.26

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Reversibility. The plots of ν vs. [α-glucosidase] at different concentrations of

232

apigenin are shown in Figure 2B. All the straight lines passed through the origin point

233

of the coordinate, and the increase in the concentration of apigenin led to the decrease

234

of the slope. The results showed that apigenin reversibly inhibited α-glucosidase

235

activity. If the inhibition had been irreversible, the enzyme was inactivated by the

236

covalent binding between inhibitor and enzyme and the formation of stable complex.

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If so, the plots of ν versus enzyme concentration would not pass through the origin.27

238

Type of α-Glucosidase Inhibition. To further explore the type of inhibition

239

characterized by apigenin against α-glucosidase, the Lineweaver−Burk double

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reciprocal plots were generated. For noncompetitive inhibition, the Lineweaver−Burk

241

equations can be described as follows: 28

11



242

1 Km [I ] 1 1 [I ] [1 + ] [1 + ] = + v v max K i [S ] v max aKi

243

and secondary plot can by plotted from:

244

Slope =

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(2)

K m K m [I ] + V max V max K i

(3)

245

where ν is the enzyme reaction rate in the absence and presence of apigenin, Ki and

246

Km represent the inhibition constant and Michaelis–Menten constant, respectively. [I]

247

[S] is the concentration of inhibitor or substrate. α denotes the ratio of the

248

non-competitive inhibition constant to competitive inhibition constant, and in the

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noncompetitive inhibition the value of α is 1. The secondary re-plot of slope vs. [I]

250

was linearly fitted, implying a single inhibition site or a single class of inhibition sites.

251

It is obvious that all the lines intersected on the horizontal axis, and the value of

252

−1/Km (horizontal axis intercept) didn’t change while the value of Vmax deceased along

253

with the increase of apigenin concentration (Figure 2C). Therefore, it was concluded

254

that the inhibition of apigenin was non–competitive, and the inhibitor and substrate

255

bound simultaneously with the enzyme.29 The replot (slope versus [apigenin]) was

256

linearly fitted, suggesting only one inhibition site or a single class of inhibition sites

257

on α-glucosidase for apigenin. Additionally, the value of Ki was calculated to be (1.07

258

± 0.07) × 10−5 mol L–1 from equations (2) and (3).

259

Kinetic Time–courses in the Presence of Apigenin. The time course of enzyme

260

activity in the presence of apigenin at four different concentrations was measured to

261

determine the kinetics and rate constants (Figure 3). The results showed that the

262

catalytic activity of α-glucosidase declined gradually along with time until reaching a

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263

stable state in the presence of increasing concentrations of apigenin. The subsequent

264

analyses based on semi-logarithmic plots found the first–order inactivation kinetics of

265

α-glucosidase. The partial inactivation of α-glucosidase induced by apigenin followed

266

a monophasic process without the formation of intermediates, and the inactivation rate

267

constants (k) were calculated from the right plots of Figure 3 and summarized in Table

268

1. The transition free-energy change (∆∆G°) can be calculated by ∆∆G° = –RTlnk,

269

and the changes of ∆∆G° resulted in the inactivation of α-glucosidase.30

270

Analysis of Fluorescence Quenching of α-Glucosidase with Apigenin. To

271

further investigate the interaction between apigenin and α-glucosidase, fluorescence

272

spectroscopy was conducted. Figure 4A shows the fluorescence emission spectra

273

acquired from α-glucosidase at λex = 280nm with and without apigenin. α-Glucosidase

274

gave a strong emission peak at 346 nm, as the addition of non-fluorescent apigenin

275

under the same conditions, the fluorescence intensity of α-glucosidase decreased

276

progressively, with no obvious shift of the emission peak. The phenomenon was a

277

direct evidence for the interaction between α-glucosidase and apigenin.

278

These results verified the strong fluorescence quenching of α-glucosidase by

279

apigenin but did not elucidate the mechanism of quenching. To shed light on the

280

interaction mechanism, the Stern-Volmer equation was used to analyze the

281

fluorescence data: 31

282

F0 = 1 + K SV [Q ] = 1 + K qt 0 [Q ] F

(4)

283

where F0 and F denote the steady-state fluorescence intensities before and after the

284

addition of quencher, respectively. [Q] is the concentration of quencher. KSV, 13



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285

determined by the linear regression of the plot F0/F against [Q], represents the

286

Stern-Volmer quenching constant. Kq is the bimolecular quenching constant, and τ0

287

(10−8 s) 31 means the average lifetime of fluorophore in the absence of quencher. The

288

Stern-Volmer plot showed good linearity at different temperatures, suggesting that the

289

quenching mechanism may be either dynamic or static procedure. The KSV values

290

[(6.02 ± 0.02) × 104 (298 K), (5.89 ± 0.03) × 104 (304 K) and (5.41 ± 0.03) × 104 L

291

mol−1 (310 K)] were found to decrease gradually as the temperature rose, indicating

292

the fluorescence quenching induced by the formation of enzyme-apigenin complex

293

(static type). The higher quenching constants than the maximal scatter collision

294

quenching constant (2.0 × 1010 L mol−1s−1) also corroborated the above point that the

295

quenching mechanism may be static.32

296 297

To determine the binding constant (Ka) and number of binding sites (n), the double-logarithm regression plot was built according to the following relationship: 33

log 298

F0 - F 1 = n log K a - n log (F - F )[Pt ] F [Q t ] - 0 F0

(5)

299

[Qt] and [Pt] represent the total concentrations of apigenin and α-glucosidase,

300

respectively. As shown in Figure 4B, all the curves presented a good linear correlation,

301

and the values of Ka and n were calculated from the intercept and slope (Table 2). The

302

values of Ka were in the order of 104 L mol−1, indicating a moderate binding affinity

303

of apigenin for α-glucosidase. Furthermore, the value of Ka was inversely correlated

304

with temperature, suggesting that the stability of the apigenin-α-glucosidase complex

305

decreased at a higher temperature.34 In addition, the value of n (approximately 1)

14


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306

further confirmed the existence of only one binding site for apigenin on

307

α-glucosidase.

308

Thermodynamic Parameters. The thermodynamic parameters were calculated to

309

ascertain the main forces contributing to ligand-protein stability. In this experiment,

310

the effect of temperature on enthalpy change (∆H°) was pretty small, so it could be

311

regarded as a constant. Therefore, enthalpy change, entropy changes (∆S°) and free

312

energy change (∆G°) could be determined from the van’t Hoff equation: 35

∆H ° ∆S ° + 2.303RT 2.303R

313

log K a = −

314

∆G° = ∆H ° − T∆S °

(6) (7)

315

here Ka is the binding constant obtained from equation (5) at the corresponding

316

temperature, and R is the gas constant (8.314 J mol−1 K−1). The values of ∆H° and ∆S°

317

were obtained from the linearly fitted plot of logKa vs 1/T (Table 2). The negative

318

value of ∆G° suggested that the binding process was thermodynamically favorable

319

and occurred spontaneously. The exothermic nature of the binding reaction was

320

revealed by the negative value of ∆H° (−10.98 ± 0.2 kJ mol−1). The value of ∆S° was

321

54.59 ± 0.1 J mol−1K−1, indicating the increased randomness caused by the binding of

322

apigenin. On account of |∆H°| < |T∆S°|, it was concluded that the reaction may be

323

mainly driven by the entropy change,36 and the negative ∆H° and positive ∆S° values

324

indicated that hydrophobic interactions and hydrogen bonding play major role in the

325

complex formation. 37

326

Energy Transfer between α-Glucosidase and Apigenin. If radiation energy

327

transfer happened, the fluorescence spectrum may be malformed, since the 15



Page 16 of 45

328

fluorescence spectrum (Figure 4A) was not distorted, thus non-radiation energy

329

transfer was hypothesized to occur in this reaction.35

330

The quantum yield (φx) of α-glucosidase was obtained by comparing fluorescence

331

intensity of human serum albumin (HSA) under identical conditions according to the

332

following relationship: 38

333

ϕ x = ϕ st .

Fx Ast . Fst Ax

(8)

334

here Fx and Fst represent the fluorescence intensities of α-glucosidase and HSA; Ax

335

and Ast denote the absorption values of α-glucosidase and HSA at excitation

336

wavelength of HSA; φx and φst (0.13) are the fluorescence quantum yields of

337

α-glucosidase and HSA, respectively. The quantum yield of α-glucosidase was 0.11

338

by equation (8). Based on Förster non-radiative energy transfer theory, the average

339

distance between acceptor and donor can be determined by the following equations: 39

F0 − F R6 = 6 0 6 F0 R0 + r

340

E=

341

R06 = 8.79 ×10−25 κ 2 Ν −4ϕ J

(10)

342

ΣF ( λ ) ε ( λ ) λ 4 ∆λ J= ΣF ( λ ) ∆λ

(11)

(9)

343

where E is the energy transfer efficiency, R0 is the critical distance when the transfer

344

efficiency is 50%; κ2 is the spatial orientation factor of the dipole; N is refractive

345

index of the medium; J represents the overlap integral of the fluorescence emission

346

spectra of α-glucosidase and the absorption spectra of apigenin; F(λ) and ε(λ) indicate

347

the fluorescence intensities of α-glucosidase and molar absorptivity of apigenin at the

348

wavelength of λ. Figure 4C displays the overlap between the fluorescence emission 16


Page 17 of 45


349

spectrum of α-glucosidase and the absorption spectrum of apigenin. In this case, κ2 =

350

2/3, N = 1.336 and φ = φx = 0.11.40 Based on equations (9)−(11), J = 1.96 × 10−14 cm3

351

L mol−1, R0 = 2.69 nm, r = 2.77 nm. Obviously, r < 8 nm and 0.5R0 < r < 1.5R0,

352

suggesting the high probability of energy transfer between α-glucosidase and apigenin.

353

Besides, r > R0 confirmed the static quenching mechanism inferred from steady-state

354

fluorescence studies.41

355

Synchronous Fluorescence. The conformational changes of α-glucosidase were

356

monitored by the record of synchronous fluorescence, which was characterized by the

357

simultaneous scanning of excitation and emission monochromators of a fluorimeter at

358

different scanning intervals (∆λ =λem – λex).42 When the ∆λ was stabilized at 15 or 60

359

nm, the synchronous fluorescence spectra would give information about the

360

microenvironment changes of tyrosine (Tyr) and tryptophan (Trp) residues,

361

respectively. As shown in Figure 5A (∆λ = 15 nm) and B (∆λ = 60 nm), the

362

synchronous fluorescence intensities of both Tyr and Trp decreased regularly as the

363

amount of apigenin increased. Simultaneously, the maximum emission wavelength of

364

Tyr had a blue shift (from 294 nm to 291 nm) while that of Trp exhibited an

365

inconspicuous red shift (from 285 nm to 286 nm). These results suggested that both

366

Tyr and Trp may play a crucial role in the binding of apigenin to α-glucosidase and

367

the microenvironment around Tyr and Trp may be changed. Meanwhile, the

368

hydrophobicity was increased around Tyr, but for Trp the polarity might have a slight

369

increase.43

370

Fluorescence Phase Diagram. The “Phase diagram” method of fluorescence was

17



Page 18 of 45

371

conduct to further investigate the conformational changes of α-glucosidase induced by

372

apigenin. It was first established to analyze the intrinsic fluorescence emission

373

intensity of protein molecules at a fixed emission wavelength.44 The fluorescence

374

phase diagram was made by recording the fluorescence emission intensities of

375

α-glucosidase I(λ1) and I(λ2) containing different concentrations of apigenin at two

376

specific emission wavelengths λ1 ( 320 nm) and λ2 (360 nm) which were expressed as

377

the following equations: 45

378

I (λ1 ) = a + bI (λ2 )

379

a = I 1 (λ1 ) −

380

b=

381

If the changes between two different conformations are an all–or–none transition,

382

the relationship in equation (12) will be fitted linearly. Stated differently, there is no

383

partial intermediate between the initial and final states of protein. Inversely, the

384

nonlinearity of this function is a sign of sequential structural transformation. Besides,

385

each linear portion of the plot depicts an individual all-or-none transition, suggesting

386

one or more folding intermediates may exist in the process of conformational change

387

of protein.45 The fluorescence phase diagram of α-glucosidase containing various

388

amounts of apigenin (Figure 5C) showed a good linearity (R2 = 0.9996), inferring that

389

no intermediates were formed in the process of apigenin-induced conformational

390

change of α-glucosidase. The conclusion was in line with that from inactivation

391

kinetics analysis.

(12)

I 2 (λ1 ) − I 1 (λ1 ) I 1 (λ 2 ) I 2 (λ 2 ) − I 1 (λ 2 )

(13)

I 2 (λ1 ) − I 1 (λ1 ) I 2 (λ 2 ) − I 1 (λ 2 )

(14)

18


Page 19 of 45


392

CD Spectra. CD spectroscopy is a sensitive spectroscopic method to monitor

393

conformational changes in chiral molecules which absorb light to different extents.

394

The conformational changes of a biomolecule can be inferred from differences in the

395

absorbance of right and left circularly polarized light in the UV region.46 As shown in

396

Figure 5D, the far-UV CD spectra of α-glucosidase with various concentrations of

397

apigenin were characterized mainly by two negative bands at 210 nm and 222 nm

398

(typical feature of α–helix) caused by π → π* and n → π* transitions of amide groups

399

in the far ultraviolet wavelength region.47 The addition of apigenin caused a

400

noticeable and regular decrease in the intensity of both negative bands without any

401

significant changes in the peak position and shape. A similar case was reported in the

402

interaction of zinc oxide nanoparticles with bovine serum albumin.48 The CD spectra

403

were then analyzed by the SELCON3 program to determine the contents of different

404

secondary structures of α-glucosidase (Table 3). With the increase in molar ratios of

405

apigenin to α-glucosidase (from 0:1 to 4:1), the α–helix content decreased from 38.4%

406

to 31.7%, while the random coil content increased from 25.1% to 28.1%. These

407

results indicate that the binding of apigenin to α-glucosidase may induce a partial

408

unfolding of the constitutive polypeptides, then influence the conformation of

409

α-glucosidase, leading to inhibition of enzyme activity. Moreover, due to the lack of

410

major changes of the peak position and shape, it was concluded that the basic

411

structure of the enzyme was kept intact after binding with apigenin.49

412

Binding Sites of Apigenin on α-Glucosidase. The inhibition of apigenin to

413

α-glucosidase seemed to stem from its ability to interact with the specific regions on

19



414

the enzyme, and the molecular docking was thus performed to determine the preferred

415

orientation of the ligand in 3D space into the protein binding site. The 3D structure

416

information of α-glucosidase was still unknown although X–ray crystallographic

417

structures of α-glucosidase from some bacteria have been reported.50 Therefore,

418

homology modeling was conducted before the simulation docking to construct the 3D

419

structure of α-glucosidase. The crystallographic structure of isomaltase from

420

Saccharomyces cerevisiae (3A4A PDB) with the sequence identity of 73% was

421

selected as the template. The conformational clusters were obtained by using the

422

Lamarckian Genetic Algorithm and the cluster that possessed the most frequent locus

423

along with the lowest energy was chosen to be the most optimal cluster for subsequent

424

analysis. As shown in Figure 6A, myricetin was well accommodated inside the active

425

site of α-glucosidase while apigenin binding was near the active site. The distance

426

between oxygen on C ring of apigenin and Asp215 (catalytic nucleophile) was 21.3 Å

427

(Figure 6B).51 The phenomenon was consistent with the result from the inhibition

428

kinetics analysis. Apigenin mainly interacted with amino acid residues Ser311,

429

Pro312, Val319, Thr310, Gly309, Val308, Asp307, Phe321 and Pro320 (Figure 6C).

430

Besides, one hydrogen bonding was formed between the oxygen of hydroxyl group at

431

C−7 position on A ring of apigenin and Gly309 with a distance of 2.219Å, indicating

432

that hydrogen bonding played a role in the binding of apigenin to α-glucosidase.

433

Additionally, the 4’−hydroxyl group at B ring of apigenin inserted into the

434

hydrophobic region to interact with residues close to active site, such as Phe303,

435

Pro312 and Arg315, which might induce the channel closure to prevent the access of

20


Page 20 of 45

Page 21 of 45


436

substrate, eventually leading to the inhibition of α-glucosidase. These results

437

confirmed the interaction between apigenin and residues adjacent to the active site of

438

α-glucosidase.

439

Inhibitory Effect of Combinations of Flavonoids on α-Glucosidase and

440

Isoboles. To study the drug combinations, the isobolograms were built to distinguish

441

additive, synergistic and antagonistic interactions. When the individual dose-effect

442

curves exhibited a constant relative potency (R), namely, dose A/dose B = R at all

443

effect levels, then the expected isoboles were linear. On the contrary, the isoboles

444

would be nonlinear in a situation of variable potency ratio.52 For linear isoboles, the

445

relationship was commonly expressed in the following equation: 16

446

a b + =1 Ai B i

(0 ≤ a ≤ Ai, 0 ≤ b ≤ Bi)

(15)

447

where all points (a, b) on the line represent dose pairs that give the specified effect.

448

For flavonoid A and B, Ai and Bi denoted the doses described here, respectively. For

449

two flavonoids achieving the maximal effect but had different Hill coefficients, this

450

was a situation of a variable potency ratio which generated nonlinear isoboles.

451

Because the drug A (equivalent dose of drug B) added to dose a, is different from

452

adding the drug B (equivalent dose of drug A) to dose b, so two additive isoboles were

453

given by the following: 16

454

b = B 50 (

455

b = Bi -

Ai - a q / p ) A 50

(16)

B 50 A 50 q / p ( ) a

(17)

21



456

where p and q represent the Hill coefficients of the two drugs. The curves obtained

457

from equations (16) and (17) were supposed to be symmetry with respect to the point

458

(Ai/2, Bi/2). Each boundary curve denoted additive isobole, hence, the region between

459

the two curves represented a region of additivity.

460

Myricetin, a competitive inhibitor for α-glucosidase, bound in the active pocket

461

with a Hill coefficient of 1.17. The values of IC30, IC50 and IC70 were estimated to be

462

1.68 × 10−6, 2.25 × 10−6 and 2.83 × 10−6 mol L−1 from the dose–effect curve. All the

463

three flavonoids can completely inhibit the activity of α-glucosidase. Both apigenin

464

and morin bound near the active site, and the Hill coefficients were 1.17 and 1.33,

465

respectively. The values of IC30, IC50 and IC70 of apigenin were 6.66 × 10−6, 10.5 ×

466

10−6 and 14.1 × 10−6 mol L−1, and those of morin were 2.82 × 10−6, 4.48 × 10−6 and

467

6.57 × 10−6 mol L−1, respectively. For the combination of myricetin and apigenin in

468

the inhibition of α-glucosidase, the isoboles for the 30%, 50% and 70% inhibition

469

effect levels were linear since they shared the same Hill coefficient (Figure 7A). The

470

additive isoboles (based on equation 15) for several inhibition effect levels (30%, 50%

471

and 70%) were included, and parallelism between the lines was found. The results of

472

isobolographic analysis of all combinations of myricetin and apigenin for 30%

473

inhibition effect level suggested a synergistic interaction due to all the dots were

474

below the additive isobole. All the combinations to 50% inhibition effect level

475

revealed a super-additive interaction except two dose pairs (one was the concentration

476

of myricetin fixed at 1.5 × 10−5 mol L−1, and the other was the amount of apigenin

477

kept at 9.0 × 10−6 mol L−1) located in close proximity to the corresponding additive

22


Page 22 of 45

Page 23 of 45


478

isobole which may imply an additive interaction. In the case of 70% inhibition effect

479

level, two dots lay below the additive isobole indicating a synergistic interaction, but

480

when the concentration of myricetin was maintained at 2.25 × 10−5 mol L−1, it lay

481

above the additive isobole, indicating an antagonistic interaction. To determine the

482

effect of the combination of myricetin and morin on the inhibition of α-glucosidase,

483

the additive isoboles were constructed based on equations (16) and (17) since they had

484

different Hill coefficients (Figure 7B). The isobolographic analysis results were

485

similar to the combination of myricetin and apigenin. Almost all the combinations for

486

every effect level were synergistic interaction, only two combinations displayed

487

additive interaction. One of them was for the concentration of morin kept at 3.5 × 10−6

488

mol L−1 in 50% inhibition effect level, and the other was for the concentration of

489

myricetin maintained at 2.25 × 10−5 mol L−1. These results showed that the

490

combination of myricetin with apigenin or moirn exhibited synergistic effect at low

491

concentrations, while it tended to exhibit additive or antagonistic interaction with the

492

increase of concentration.

493

In summary, apigenin drastically and completely inhibited the activity of

494

α-glucosidase in a competitive manner. Apigenin quenched the fluorescence of

495

α-glucosidase by spontaneously forming the α-glucosidase–apigenin complex and the

496

main forces driving the interaction were hydrophobic interaction and hydrogen

497

bonding. The most likely binding site on α-glucosidase for apigenin was found to be

498

somewhere close to the active pocket, which mainly interacted with amino acid

499

residues Ser311, Pro312, Val319, Thr310, Gly309, Val308, Asp307, Phe321 and

23



500

Pro320, and the binding process induced a conformational change with no

501

intermediate to hinder the access of substrate. In addition, the isobolographic analysis

502

of myricetin combined with apigenin or morin showed mainly synergistic interaction

503

except a few dose pairs. This research provides new insights into the inhibitory

504

mechanism of apigenin on α-glucosidase and the valuable information for the

505

pharmacotherapy by a combination of different drugs.

506

ACKNOWLEDGMENTS

507

This work was financially supported by the National Natural Science Foundation of

508

China (Nos. 31460422 and 31060210), the Natural Science Foundation of Jiangxi

509

Province (20143ACB20006), the Foundation of Jiangxi Provincial Office of

510

Education (GJJ150187), and the Objective-Oriented Project of State Key Laboratory

511

of Food Science and Technology (SKLF-ZZA-201612).

512

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the protein–nanoparticle conjugates aqueous solution using circular dichroism

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spectroscopy. Anal. Chem. 2015, 87, 6455–6459.

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between

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Multispectroscopic analyses and docking simulations. Food Chem. 2015, 170,

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423–429.

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human serum albumin and its effect on protein conformation stability. Food

671

Chem. 2016, 192, 178–187.

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of novel α-glucosidase inhibitors based on the virtual screening with the

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homology-modeled protein structure. Bioorg. Med. Chem. 2008, 16, 284–292.

Kuznetsova, I. M.; Turoverov, K. K.; Uversky, V. N. Use of the phase

Ji, X.; Ma, X.; Bian, L. Spectral studies on the conformational transitions of

Ranjbar, B.; Gill, P. Circular dichroism techniques: Biomolecular and

Li, S.; Peng, Z.; Leblanc, R. M. Method to determine protein concentration in

Wu, D.; Yan, J.; Wang, J.; Wang, Q.; Li, H. Characterisation of interaction food

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Yamamoto, K.; Miyake, H.; Kusunoki, M.; Osaki, S. Crystal structures of

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isomaltase from Saccharomyces cerevisiae and in complex with its competitive

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inhibitor maltose. FEBS J. 2010, 277, 4205–4214.

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679

Ther. 2007, 113, 197–209.

Tallarida R J. Interactions between drugs and occupied receptors. Pharmacol.

680

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681

Figure captions

682

Figure 1. Structures of apigenin(A), morin(B), myricetin (C) and acarbose (D).

683

Figure 2. (A) Inhibitory effects of apigenin, myricetin and morin on α-glucosidase

684

(pH 6.8, T = 310 K). c(α-glucosidase) = 5.08 × 10–8 mol L–1 and c(pNPG) = 4.54 ×

685

10–4 mol L–1. (B) Plots of ν vs. [α-glucosidase]. c(apigenin) = 0, 0.4, 0.8, 1.2 and 1.4 ×

686

10–5 mol L–1 for curves a→e, respectively. (C) Lineweaver–Burk plots. c(apigenin) =

687

0, 0.2, 0.4, 0.8 and 1.0 × 10–5 mol L–1 for curves a→e, respectively. The secondary

688

plot represented slope vs. [apigenin].

689

Figure 3. Kinetic time-course for relative activity of α-glucosidase in the presence of

690

apigenin at concentrations of 0.2, 0.8, 1.0 and 1.2 × 10–5 mol L–1 for curves a→d,

691

respectively. c(α-glucosidase) = 5.08 × 10–8 mol L–1 and c(pNPG) = 4.54 × 10–4 mol

692

L–1. Semi-logarithmic plot analysis for apigenin at 0.2 × 10–5 mol L–1 (the upper right)

693

and 1.2 × 10–5 mol L–1 (the lower right), and the slope of the curve suggests the

694

inactivation rate constants.

695

Figure 4. (A) Fluorescence spectra of α-glucosidase in the presence of apigenin at

696

various concentrations (pH 6.8, T = 298 K, λex = 280 nm, λem = 346 nm).

697

c(α-glucosidase) = 8.33 × 10–7 mol L–1, and c(apigenin) = 0, 0.27, 0.53, 0.80, 1.07,

698

1.33, 1.60, 1.87, 2.13 and 2.40 × 10–5 mol L–1 for curves a→j, respectively. Curve m

699

shows the emission spectrum of apigenin only, c(apigenin) = 1.60 × 10–5 mol L–1. The

700

Stern–Volmer plots for the fluorescence quenching of α-glucosidase by apigenin at

701

different temperatures were inserted. (B) The plots of log[(F0–F)/F] vs.

702

log{[Qt–(F0–F)[Pt]/F0]} for the interaction of apigenin and α-glucosidase. (C)

33



703

Spectral overlaps of the fluorescence spectrum of α-glucosidase (a) with the

704

absorption spectrum of apigenin (b) c(α-glucosidase) = c(apigenin) = 8.33 × 10–7 mol

705

L–1.

706

Figure 5. Synchronous fluorescence spectra of α-glucosidase in the absence and

707

presence of apigenin (A) ∆λ = 15 nm, (B) ∆λ = 60 nm (pH 6.8, T = 298 K).

708

c(α-glucosidase) = 8.33 × 10–7 mol L–1. c(apigenin) = 0, 0.27, 0.53, 0.80, 1.07, 1.33,

709

1.60, 1.87, 2.13 and 2.40 × 10–5 mol L–1 for curves a→j, respectively. (C) The phase

710

diagram of fluorescence of α-glucosidase with different concentrations of apigenin at

711

pH 6.8 and T = 298 K. c(α-glucosidase) = 8.33 × 10–7 mol L–1. c(apigenin) = 0, 0.27,

712

0.53, 0.80, 1.07, 1.33, 1.60, 1.87, 2.13 and 2.40 × 10–5 mol L–1. (D) The far–UV CD

713

spectra of α-glucosidase in the presence of increasing concentrations of apigenin at

714

pH 6.8. c(α-glucosidase) = 2.0 × 10–6 mol L–1, and the molar ratios of apigenin to

715

α-glucosidase were 0:1, 1:1, 2:1 and 6:1 for curves a→d, respectively.

716

Figure 6. (A) Predicted best binding mode of apigenin docked with α-glucosidase on

717

molecular surface. The green areas represent the catalytic activity site of

718

α-glucosidase. (B) Showed the distance between Asp215 and apigenin (yellow stick

719

structure). (C) The corresponding secondary structures of α-glucosidase interact with

720

apigenin. The short dotted green line stands for hydrogen bonds.

721

Figure 7. Isoboles of additivity for several effects (30%, 50% and 70%) in the

722

combinations of two agents with a constant relative potency apigenin and

723

myricetin(A), two full agonists with a variable potency ratio morin and myricetin (B).

34


Page 34 of 45

Page 35 of 45


Table 1. Inactivation rate constants of α-glucosidase in the presence of apigenin.

–5

–1

Inactivation rate constants (×10–4 s–1)a

Apigenin (×10 mol L ) k

Transition free-energy change (kJ mol–1 s-–1)b

0.2

3.58 ± 0.04

20.45

0.8

3.04 ± 0.05

20.87

1.0

2.47 ± 0.02

21.41

1.2

1.68 ± 0.05

22.40

a

k is the first–order rate constant.

b

Transition free–energy change, ∆∆G°= –RTlnk, where k is a time constant of the

inactivation reaction. The values of k were significantly different (p < 0.05) from each other in the same column.

35



Page 36 of 45

Table 2. The quenching constants KSV, binding constants Ka and relative thermodynamic parameters for the interaction of apigenin with α-glucosidase at different temperatures. T(K)

KSV (×10 L mol−1)

Ra

Ka (×10 L mol−1)

n

Rb

298

6.02 ± 0.02

0.9991

5.94 ± 0.03

1.01 ± 0.01

0.9994

304

5.89 ± 0.03

0.9992

5.53 ± 0.02

1.09 ± 0.03

0.9997

310

5.41 ± 0.03

0.9970

5.00 ± 0.03

1.17 ± 0.02

0.9996

4

a

4

∆H° (kJ mol−1)

∆G° (kJ mol−1)

∆S° (J mol−1K−1)

−27.25 ± 0.3 −10.98 ± 0.2

−27.58 ± 0.2 −27.91 ± 0.2

R is the correlation coefficient for the KSV values. b R is the correlation coefficient for the Ka values.

The values of KSV and Ka were significantly different (p < 0.05) from each other in the same column.

36


54.59 ± 0.1

Page 37 of 45


Table 3. The contents of secondary structures of free α-glucosidase and apigenin-α-glucosidase systems at pH 6.8. Molar ratio [apigenin]:[α-glu]

α-Helix (%)

β-Sheet (%)

β-Turn (%)

Random coil (%)

0:1

38.4

20.1

16.4

25.1

1:1

36.1

21.6

15.9

26.4

2:1

33.4

23.4

15.8

27.4

4:1

31.7

24.5

15.7

28.1

37



Figure 1. (A)

(B)

Morin

Apigenin (D)

(C)

Myricetin

Acarbose

38


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Page 39 of 45


Figure 2. (A)

(B)

(C)

39



Figure 3.

40


Page 40 of 45

Page 41 of 45


Figure 4. (A)

(B)

(C)

41



Figure 5. (B)

(A)

(D)

(C)

42


Page 42 of 45

Page 43 of 45


Figure 6. (B)

(A)

(C)

43



Figure 7. (A)

(B)

44


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TOC Graphic

45


Inhibitory Mechanism of Apigenin on Î±-Glucosidase and Synergy

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