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

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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

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

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

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

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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

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Journal of Agricultural and Food Chemistry

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

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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

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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.

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Therefore,

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

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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

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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

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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.

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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

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

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

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Co., Ltd. (Shanghai, China).

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

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

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

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

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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

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

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

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

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

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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

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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

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

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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

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Resistant starch: RS(%)=(1−[RDS+SDS])

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

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

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

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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

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

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

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

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

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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

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

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

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

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

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

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

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

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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

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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

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

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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,

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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

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

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

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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

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

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

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

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

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

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spectrophotometer.

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

137

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[S]

Km +[S]

. (1)

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

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V

=V

1

max

K

+V m × max

1 S

. (2)

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

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

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Is. The equation is as follows:25

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Widg =

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Y − intercept =

ꊸᐄ

1

V’max

+ =

ꊸᐄ

1

Vmax

. (3) + αK

1

iu Vmax

I. (4)

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

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

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

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is the noncompetitive inhibition constant.

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

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

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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

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

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

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

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Journal of Agricultural and Food Chemistry

Analysis of Starch Digestive Enzyme Fluorescence Quenching

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

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

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

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

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

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

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

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

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

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

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

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

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

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follows: F0

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F

= 1 + Kq τ0 [Q] = 1 + KSV [Q]. (5)

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

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

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

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

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

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protein

interacting

similarly

to

the

quencher.

This

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

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plot

also

indicates

that

only

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

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

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

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follows:28 F0

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F

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

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

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

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

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Stern–Volmer equation,29 as follows: lg

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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.

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

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

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

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

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

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Thermodynamic parameter assessment. Micromolecules and biological macromolecules can

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

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

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

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on two temperatures, 30as follows: 10

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lnKa =−

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ΔH

ΔS

+ . (8) RT R

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

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(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)

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

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

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

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

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

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

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

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

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

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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