Research on the Influences of Five Food-Borne Polyphenols on In

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Research on the influences of five food-borne polyphenols on invitro slow starch digestion and the mechanism of action Shuncheng Ren, Keke Li, and Zelong Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01724 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 20, 2019

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Journal of Agricultural and Food Chemistry

Research on the influences of five food-borne polyphenols on in vitro slow starch digestion and the mechanism of action

Shuncheng Ren*，Keke Li, and Zelong Liu

School of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, P R China

*Corresponding author. Tel: +86-371-68883238; Fax: +86-371-67789817; E-mail: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT

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Inhibiting starch digestion can effectively control postprandial blood sugar level. In this study, the in

3

vitro digestion differences among the mixtures of five polyphenols (i.e., procyanidins [PAs], catechin

4

[CA], tannic acid [TA], rutin [RU], and quercetin [QU]) and starch were analyzed through an in vitro

5

simulation test of starch digestion. The interaction characteristics of these five polyphenols with

6

α-amylase and α-glucosidase were investigated in terms of the inhibition effect, dynamics,

7

fluorescence quenching, and circular dichroism (CD). The results revealed that the rapidly digestible

8

starch (RDS) contents decreased, while the resistant starch (RS) contents increased. All five

9

polyphenols inhibited the α-amylase activity through the noncompetitive approach but inhibited the

10

α-glucosidase activity through the competitive approach. Five polyphenols combined with α-amylase

11

spontaneously by using hydrophobic effect. The interaction of PAs and QU with α-glucosidase were

12

recognized as van der Waals force and H bond, whereas CA and TA interacted with α-glucosidase

13

through the hydrophobic effect. All five polyphenols can cause conformational change in enzymes.

14 15

KEYDORDS: polyphenols, α-amylase, α-glucosidase, inhibition kinetics, inhibition mechanism

2


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INTRODUCTION

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The population of patients with diabetes is increasing at an unexpected rate worldwide and

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increased from 0.151 billion in 2000 to 0.425 billion in 2017. On the average, 1 out of 11 adults

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suffer from diabetes. The population of patients with diabetes may reach 0.629 billion in 2045.1

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Hyperglycemia is a typical characteristic of diabetes, and the best approach for treatment is to inhibit

21

the activity of starch digestive enzymes (i.e., α-amylase and α-glucosidase) to delay glucose

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absorption.2, 3 α-Amylase and α-glucosidase are two starch digestive enzymes receiving considerable

23

attention in research; the former can hydrolyze α-1,4-glucosidic bonds in starches into maltose,

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oligosaccharide, and few glucoses. Then, the intestinal α-glucosidase catalyzes disaccharide

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decomposition to release glucose in blood circulation. Thus, glucose can be carried by a specific

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transporter into cells through the intestinal tract. Therefore, inhibiting α-amylase and α-glucosidase

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has become an effective method to control postprandial hyperglycemia.4-6 Acarbose, which is widely

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used to treat postprandial hyperglycemia, easily causes gastrointestinal disorders, thereby further

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leading to untoward effects of abdominal distension, stomachache, and diarrhea.7,

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studying new inhibitors of starch digestive enzymes is important.

8

Therefore,

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Polyphenols, which is the secondary plant metabolite, has extensive distributions in plant tissues

32

and is an important component of human diet. Polyphenols have attracted considerable attention

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from researchers and food manufacturers as an antioxidant, an antimutagen, and a free radical

34

scavenger.9 Meanwhile, polyphenols can combine with macromolecular substances such as

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carbohydrates and digestive enzymes to relieve amylolysis and reduce glycemic index. Hence,

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polyphenols have become a topic receiving considerable research attention in the prevention of

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metabolic diseases (e.g., diabetes) at present.10, 11 The rapidly digestible starch (RDS) content in corn

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starch decreases significantly, and the resistant starch (RS) content increases dramatically after

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adding blue maize extract that is rich in anthocyanin.12 CA in sorghum can decrease RDS, while 3



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tannic acid (TA) decreased slowly digestible starch (SDS); CA and TA both increase the RS

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content.13 Previous research also demonstrated that quercetin (QU), isoquercetin, and rutin (RU) are

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the effective inhibitors of α-glucosidase and α-amylase, and QU showed the strongest inhibitory

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activity to α-glucosidase and α-amylase.14, 15 Procyanidins (PAs) in grape seed extract could inhibit

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the α-amylase activity.16 Further fluorescence quenching test has proven the strong interactions of

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CA, epicatechin, RU, and QU with bovine serum albumin.17 The interactions of RU and QU with

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bovine serum albumin were also proven by fluorescence and UV-visible absorption spectra,18 and

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QU shows the strongest bonding force. A study pointed out that TA is not only the major quenching

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molecule of bovine serum albumin and α-amylase in human saliva, but also a natural α-glucosidase

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inhibitor, with an IC50 value of 0.44 μg/mL (Xiao et al., 2015).19, 20 Most existing studies focused on

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the interaction between the crude extract of polyphenols and starch digestive enzymes, that is,

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α-amylase and α-glucosidase.21 However, few studies were conducted focusing on the monomeric

52

compounds of polyphenols.

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In this study, we analyzed the influences of five common food-borne polyphenols (Figure 1) on

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the in vitro slow starch digestion of α-amylase and α-glucosidase through an in vitro simulation test.

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The inhibition mode of the five polyphenols with α-amylase and α-glucosidase were analyzed via

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enzyme activity detection and kinematic analysis, respectively. The influences of five polyphenols on

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the secondary structures of α-amylase and α-glucosidase were studied by CD. The bonding types of

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these polyphenols with α-amylase and α-glucosidase were studied by fluorescence spectra, and their

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thermodynamic parameters were determined. The findings of this study can provide new references

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in screening starch enzyme inhibitors and lay theoretical foundations for the clinical use of the five

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polyphenols as inhibitors of α-amylase and α-glucosidase and for the development of special foods

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for patients with diabetes.

63

MATERIALS AND METHODS 4


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Materials. High-amylose corn starch (HACS, amylose content=63.13%) was obtained from

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Shanghai Naijin Industrial Co., Ltd. (Shanghai, China). PAs (dimer, >98%), CA (>98%), and TA

66

(>96%) were bought from Nanjing Longyuan Natural Polyphenols Compound Factory (Nanjing,

67

Jiangsu Province, China). RU (>95%) and QU (>95%) were brought from Shanghai Yuanye Biotech

68

Co., Ltd. (Shanghai, China).

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Chemical reagents. Soluble starch was obtained from Luoyang Haohua Chemical Reagent Co.,

70

Ltd. (Luoyang, Henan province, China). p-Nitrophenyl-α-D-glucopyranoside (pNPG) was bought

71

from Beijing Suolaibao Technological Co., Ltd. (Beijing, China). Pig pancreatic α-amylase was

72

bought from Sigma Chemical Co. (St. Louis, MO, USA), and yeast α-glucosidase was obtained from

73

Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). The remaining reagents were all purchased

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from Zhengzhou Xinfeng Assay Device Co., Ltd. (Zhengzhou, Henan, China).

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In vitro starch digestion. 3, 5-Dinitrosalicylic acid (DNS) colorimetric method was performed as

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follows. A total of 0.1 g starch samples with 2.5% polyphenols were collected accurately and placed

77

in a 15 mL test tube (mixed evenly through eddy oscillation), and 7.5 mL of natrium aceticum buffer

78

solution (0.2 M, pH of 5.2) was added evenly. Afterward, 5 mL of the digestive enzyme mixing

79

solution containing pig pancreatic α-amylase (300 U/mL) and α-glucosidase (60 U/mL) was added.

80

The mixture and two glass beads were oscillated in a 37 °C water bath (rotating speed=150 rpm) and

81

timed accurately. Next, 1 mL of hydrolysate was collected at 20 (G20) and 120 min (G120) of

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hydrolysis, in which 5 mL of absolute ethyl alcohol was added for enzyme deactivation. The mixture

83

was centrifuged for 10 min at the rate of 3000 rpm. Then, 1 mL of the supernate was collected and

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added with 1 mL of DNS, followed by subjecting in 5 min of boiled water bath and then to a

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constant volume of 25 mL. The absorbance was measured at 520 nm. The starch content was equal to

86

the value of glucose multiplied by the transforming factor 0.9. Each starch content was tested by

87

three times.22 5



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Rapid digestive starch: RDS(%)=(G20−FG)×0.9/TS

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Slow digestive starch: SDS(%)=(G120−G20)×0.9/TS

90

Resistant starch: RS(%)=(1−[RDS+SDS])

91

where G20 is the glucose content in hydrolysate at 20 min (mg), FG is the free glucose content (mg)

92

in starch before enzyme processing, G120 is the glucose content in hydrolysate at 120 min (mg), and

93

TS is the total starch content in the samples (mg).

94

Starch Digestive Enzyme Activity Test

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α-Amylase activity test. The soluble starch was used as the substrate for testing. A total of 1.0

96

g/100 mL soluble starch solution were prepared by dissolving starch in phosphate buffer (0.2 M, pH

97

6.8) and gelatinized by 30 min in 80 °C water bath kettle. The polyphenol solution concentrations

98

were 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL, which were achieved in the phosphate buffer solution:

99

DMSO ratio of 9:1. The different concentrations of 250 μL of polyphenol solutions and 250 μL of

100

α-amylase (300 U/mL), which were dissolved in phosphate buffer (0.2 M, pH 6.8), were added into a

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glass tube with plugs. The glass tube was placed in a 37 °C water bath for 10min of vibration

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reaction at the rate of 150 rpm, and then 500 μL1.0 g/100 mL starch solution was added. Next, 1.0

103

mL of DNS color-developing agent (28 mM 3,5-DNS, 0.6 M seignette salt and 2 M NaOH) was

104

added to terminate the reaction. Subsequently, the reaction products were heated in boiled water for 5

105

min and then cooled to room temperature, followed by constant volume processing to 25 mL by

106

deionized water. UV–visible spectrophotometer (UV-752, Shanghai Jinghua Technological

107

Instrument Co., Ltd., Shanghai, China) was used to test the absorbance at 540 nm.23The activity

108

inhibition of α-amylase under five concentrations was measured, and the IC50 value was calculated.

109

The calculation equation for the inhibition effect is as follows:

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α-Amylase inhibition rate (%)=[1−(Asample−Acontrol)/(Ablank−Ablank control)]×100% 6


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111

where Asample is the absorbance of the mixture of polyphenols, substrate, enzyme, and DNS

112

color-developing agent; Acontrol is the absorbance of the mixture of polyphenols, substrate, and DNS

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color-developing agent, in which enzyme was replaced by a phosphate buffer; Ablank is the absorbance

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of the mixture of substrate, enzyme, and DNS color-developing agent, in which polyphenol inhibitor

115

was replaced by buffer solution; and Ablank

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substrate and DNS color-developing agent.

control

is the absorbance of the mixture of enzyme-free

117

α-Glucosidase activity test. A total of 60 U/mL of α-glucosidase and pNPG were dissolved in

118

phosphate buffer (0.2 M, pH of 6.8). Different polyphenol concentrations (i.e., 0.2, 0.4, 0.6, 0.8, and

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1.0 mg/mL) were prepared in phosphate buffer (0.2 M, pH of 6.8, containing 10% DMSO). Next,

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100μL of different polyphenol solution concentrations and 50μL enzyme solution were added into a

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96-hole plate and mixed evenly, followed by 10 min water bath reaction at 37 °C. Afterward, 50 μL

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of 5.0 mM pNPG substrate solution was added and underwent reaction for 30 min at 37 °C. Then,

123

100 μL of 0.2 M Na2CO3 solution was added immediately to terminate the reaction, followed by

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5min oscillation in a shaker. Finally, the absorbance at 405 nm was measured by the 96-hole

125

ELIASA (Multiskan FC, Samer Feishier Instrument Co., Ltd., Shanghai, China).24

126 127

α-Glucosidase inhibition rate (%)=[1−(Asample−Acontrol)/(Ablank−Ablank control)]×100%. Test of the Inhibition Kinetics of Starch Digestive Enzymes

128

Test of α-amylase inhibition kinetics. The α-amylase inhibition modes of different polyphenols

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were determined by Michaelis–Menten and Lineweaver–Burk models. The inhibition kinetics was

130

discussed in section 2.4.1. The difference was that the final polyphenol concentrations were 0.4 and

131

0.6 mg/mL, while the starch solution concentrations were 0.5%, 1%, 1.5%, and 2% (w/v). One test

132

tube from each concentration was taken every 5 min, and 1.0 mL of DNS color-developing agent was

133

added. The reactants were heated for 5min in boiled water and then cooled to room temperature 7



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134

before reaching constant volume of 25 mL. The absorbance at 540 nm was tested by the UV–visible

135

spectrophotometer.

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The Michaelis–Menten equation is as follows: V = Vmax ×

137

138

[S]

Km +[S]

. (1)

A diagram of 1/[S] relative to 1/V was created, and the Lineweaver–Burk equation is as follows: 1

139

V

=V

1

max

K

+V m × max

1 S

. (2)

140

where Kic and Kiu are calculated by the secondary drawing method, and the double reciprocal

141

straight lines under different Is are drawn, thereby obtaining the slope of one drawing under different

142

Is. The equation is as follows:25

143

Widg =

144

Y − intercept =

ꊸᐄ

1

V’max

+ =

ꊸᐄ

1

Vmax

. (3) + αK

1

iu Vmax

I. (4)

145

The secondary diagrams of slope or Y-intercept with I were drawn for linear fitting, where V is the

146

initial reaction speed, Vmax is the maximum initial reaction speed, I is the inhibitor concentration, α is

147

apparent coefficient, Km is the Michaelis constant, Kic is the competitive inhibition constant, and Kiu

148

is the noncompetitive inhibition constant.

149

Test of the α-glucosidase inhibition kinetics. α-Glucosidase inhibition kinetics was discussed by

150

the method introduced in section 2.4.2. The difference was that the polyphenol concentrations were

151

0.4 and 0.6 mg/mL, and pNPG the with concentration in the range of 0.5–5.0 mM was used as the

152

substrate. Enzyme activity was tested by adding different polyphenol concentrations. The absorbance

153

at 405 nm was tested every 5min by the 96-hole ELISA (Multiskan FC, Samer Feishier Instrument

154

Co., Ltd., Shanghai, China). 8


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Analysis of Starch Digestive Enzyme Fluorescence Quenching

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Analysis of pig pancreatic α-amylase fluorescence quenching. The fluorescence quenching

157

spectra of polyphenols on pig pancreatic α-amylase were tested by a fluorospectrophotometer

158

(G9800A, Agilent Technological Co., Ltd., Malaysia). A total of 300 U/mL of pig pancreatic

159

α-amylase were dissolved in phosphate buffer (0.2 M, pH of 6.8). Different polyphenol

160

concentrations (i.e., 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL) were configured in phosphate buffer

161

(0.2 M, pH of 6.8, containing 10% DMSO). Next, exactly 0.2 mL of different polyphenol solution

162

concentrations were added to 3 mL of pig pancreatic α-amylase solution, which was mixed evenly

163

through eddy oscillation for 3 min and then processed to a constant volume of 10 mL. The mixture

164

was oscillated in a constant temperature water bath at 30 °C and 37 °C for 30 min. The phosphate

165

buffer with 3 mL of enzyme solution was used as the control group. Equivalent phosphate buffer

166

solution was used as the blank control. Fluorescence emission spectra was scanned in the wavelength

167

range of 290–500 nm by the excitation wavelength of 278 nm, emission wavelength of 290 nm, and

168

slit width of 5 nm.26 Fluorescence quenching was described by the Stern–Volmer equation, as

169

follows: F0

170

F

= 1 + Kq τ0 [Q] = 1 + KSV [Q]. (5)

171

where F and F0 are fluorescence intensity with and without quenching agent, respectively; KSV is

172

the Stern–Volmer quenching constant; [Q] denotes the quenching agent concentration; Kq represents

173

the quenching constant of biomolecules; and τ0 reflects the life of the fluorophore, that is, τ0=2.97 ns

174

for α-amylase27 and τ0=10−8 s for α-glucosidase.

175

Generally, a linear Stern–Volmer plot indicates the presence of a single class of fluorophore in the

176

protein

interacting

similarly

to

the

quencher.

This

177

one quenching mechanism (dynamic or static) occurs. Nevertheless, fluorophore and quenching 9


plot

also

indicates

that

only


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agents interact through the mixed dynamic and static quenching mechanisms when the quenching

179

degree is high. Under this circumstance, the F0/F relative to [Q] is an upward curve, which is

180

inclined toward the y-axis. The Stern–Volmer equation that describes such situation is modified as

181

follows:28 F0

182

F

= e(Ksv[Q]) . (6)

183

Calculating natural logarithms in the two sides of the equation to draw the relationship curve

184

between ln(F0/F) and [Q] resulted in a straight line. The slope of this straight line is the apparent

185

static constant KSV. The fluorescence quenching mechanism can be described by the improved

186

Stern–Volmer equation,29 as follows: lg

187 188 189

F0 −F F

= lgKa + nlg Q . (7)

where Ka expresses the binding constant of polyphenols with α-amylase and α-glucosidase, and n is the binding site number.

190

Analysis of α-glucosidase fluorescence quenching. The fluorescence quenching spectra of

191

food-borne polyphenols to α-glucosidase were tested by the same method, except 60 U/mL of

192

α-glucosidase solution was dissolved in a phosphate buffer (0.2 M, pH of 6.8). Fluorescence

193

emission spectra were scanned in the wavelength range of 290–450 nm by using the excitation

194

wavelength of 278 nm and the slit width of 5 nm.

195

Thermodynamic parameter assessment. Micromolecules and biological macromolecules can

196

interact through hydrophobic bond, electrostatic attraction, van der Waals force, and H bond. The

197

thermodynamic parameters of the standard enthalpy change ΔH and standard entropy change ΔS can

198

be determined according to the van’t Hoff equation, and the results from van't Hoff plots were based

199

on two temperatures, 30as follows: 10


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lnKa =−

200

ΔH

ΔS

+ . (8) RT R

201

where R is the atmospheric constant with the value of 8.314 J/mol·K, T is the reaction temperature

202

(303 and 310 K), and Ka is the binding constant. The free energy ΔG is calculated by Eq. (9), as

203

follows: ΔG = ΔH − TΔS. (9)

204 205 206

The ΔH and ΔS values are calculated from the slope and intercept of the linear relation curve between lnKa and 1/T.

207

Secondary structure test of the starch digestive enzymes. The secondary structural changes in

208

α-amylase and α-glucosidase were measured by CD (MOS-450, Bio-Logic Company, France) with

209

and without the existence of polyphenols. The parameters were set as follows: temperature=37 °C,

210

bandwidth=1nm, and path length of quartz cuvette=1.0 mm.31 The mixtures of polyphenols and

211

α-amylase and those of polyphenols and α-glucosidase were diluted to appropriate concentrations by

212

a phosphate buffer (0.2 M, pH of 6.8, containing 10%DMSO). The blank phosphate buffer (0.2 M,

213

pH of 6.8) was used to replace the polyphenol solution.

214

Statistical analysis. All data were analyzed by SPSS v. 22 statistical software, and the results

215

were expressed in mean ± standard deviation (SD) (n=3). PQU>RU>PAs>CA, which agreed with the results of previous studies on the inhibition of

287

α-amylase activity. Table 3 shows that ΔG

Research on the Influences of Five Food-Borne Polyphenols on In

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