Dietary Phenolic Compounds Selectively Inhibit the Individual

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Dietary phenolic compounds selectively inhibit activities of individual subunits of Maltase-Glucoamylase and Sucrase-Isomaltase for modulating glucose release Meric Simsek, Roberto Quezada-Calvillo, Mario G. Ferruzzi, Buford Nichols, and Bruce R. Hamaker J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 Mar 2015 Downloaded from http://pubs.acs.org on March 30, 2015

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Journal of Agricultural and Food Chemistry

Dietary phenolic compounds selectively inhibit the individual subunits of MaltaseGlucoamylase and Sucrase-Isomaltase with the potential of modulating glucose release

Meric Simsek1, Roberto Quezada-Calvillo2,3, Mario G. Ferruzzi1, Buford L. Nichols3, Bruce R. Hamaker1*

1

Whistler Center for Carbohydrate Research and Department of Food Science, Purdue

University, West Lafayette, IN 47907, USA 2

Department of Chemistry, Universidad Autonoma de San Luis Potosi, SLP, Mexico

3

USDA, Agricultural Research Service, Children’s Nutrition Research Center, Department of

Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

*Correspondence: Professor Bruce R. Hamaker, Phone: 765-494-5668, Fax: 765-494-7953, Email: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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In this study, we hypothesized that dietary phenolic compounds selectively inhibit the

3

individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal α-glucosidases,

4

Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI), for a modulated glycemic

5

carbohydrate digestion. The inhibition by chlorogenic acid, caffeic acid, gallic acid, (+)-catechin,

6

and (-)-epigallocatechin gallate (EGCG) on individual recombinant human Nt-MGAM and Nt-

7

SI, and mouse Ct-MGAM and Ct-SI, was assayed using maltose as the substrate. Inhibition

8

constants, inhibition mechanisms, and IC50 values for the each combination of phenolic

9

compound and enzymatic subunit were determined. EGCG and chlorogenic acid were found to

10

be more potent inhibitors for selectively inhibiting the two subunits with highest activity, Ct-

11

MGAM and Ct-SI. All compounds displayed non-competitive type inhibition. Inhibition of fast-

12

digesting Ct-MGAM and Ct-SI by EGCG and chlorogenic acid could lead to a slow, but

13

complete, digestion of starch for improved glycemic response of starchy foods with potential

14

health benefit.

15

Key words: α-glucosidases, inhibition, Maltase-Glucoamylase, phenolics, Sucrase-Isomaltase

16

17

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INTRODUCTION

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Starchy foods are a major component of the human diet and provide a large portion of

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energy intake. Starch is digested into the glucose by six different enzyme activities: salivary and

22

pancreatic α-amylase; and the α-glucosidases Maltase-Glucoamylase (MGAM) and Sucrase-

23

Isomaltase (SI), each composed of two active subunits located on the respective C and N

24

terminals (Ct, Nt) of their original protein. The first step in starch digestion is performed by

25

salivary and pancreatic α-amylases, with production of linear glucose oligomers and branched α-

26

limit dextrins, and very little free glucose.1 The α-amylase degradation products are further

27

hydrolyzed into free glucose by the mucosal α-1,4 exoglucosidases MGAM and SI, located on

28

the small intestinal brush border membrane.2

29

MGAM and SI enzyme complexes belong to the GH31 family of glucohydrolases. The

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Nt subunit of both MGAM and SI attach these enzyme complexes to the apical membrane of

31

small intestinal enterocytes through an O-glycosylated stalk domain.3 Although all four subunits

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have high α-1,4-exoglucosidic activity,4,5 each MGAM and SI subunit has different α-glucosidic

33

catalytic properties related to their independent active sites; therefore, their contribution to

34

glucose production from glycemic carbohydrates is different.1,6 Ct-SI and Nt-SI subunits display

35

distinctive sucrase and isomaltase activities, respectively. In MGAM, Ct and Nt subunits share

36

activities against linear glucose oligomers, but the higher activity of Ct-MGAM on longer

37

glucose oligomers led to the naming of this subunit as glucoamylase, while Nt-MGAM has been

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ascribed as the maltase subunit.1 Among the SI and MGAM subunits, Ct-MGAM recently has

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been shown to be the most active, with ability to digest whole, undigested starch molecules,7 and

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with the highest maltase activity compared to the other subunits.4,5 Although SI is the



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predominant molecule in the apical membrane of human small intestinal epithelial cells, MGAM

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compensates its relative low abundance with its higher hydrolytic activity.8

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A possible strategy to control of the rate of free glucose release from the digestion of

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starch is to inhibit the hydrolytic activity of salivary and pancreatic α-amylases, or preferably of

45

the intestinal α-glucosidases where glucose is actually generated. Through selective inhibition of

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the faster digesting α-glucosidase Ct subunits, while leaving the slower digesting Nt subunits

47

active, one could expect to achieve a slower starch digestion effect. Such selective inhibition of

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intestinal α-glucosidases may have multiple positive health implications related to control of the

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glycemic response profile of starchy foods, and perhaps eliciting the ileal brake and gut-brain

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axis response to reduce appetite and food intake.9 We recently showed that diverse synthetic

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compounds can selectively inhibit the most active α-glucosidases, Ct-MGAM and Ct-SI, causing

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slower digestion of the α-amylase degradation products of starch.10 The inhibition of α-

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glucosidic activity of specific individual subunits of MGAM and SI can be considered as a key

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approach for controlling glucose release, because the α-glucosidase catalytic activities are

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responsible for producing virtually all the free glucose available for intestinal absorption.

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A variety of chemical compounds have been shown to have inhibitory effect on

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mammalian intestinal α-glucosidases. These compounds are derived either from chemical

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synthesis,11,12 or from natural sources such as fruits13,14 or plants15-17. Naturally-occurring plant

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phenolic compounds are one group of chemical metabolites reported to have inhibitory effect on

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the α-glucosidases. For instance, caffeic and chlorogenic acids are typical hydroxycinnamic acids

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found in high concentration in potatoes18 and coffee beans.19,20 Chlorogenic acids are esters of

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caffeic acids and quinic acids.21 It was identified as the main α-glucosidase inhibitor found in the

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leaf extract of Nerium indicum, causing reduced postprandial rise in blood glucose after ingestion 4 ACS Paragon Plus Environment

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of 2 g maltose or sucrose/kg body weight in rats administered either the leaf extract or pure

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chlorogenic acid (25 mg/kg body weight).22 A single serving of coffee can have 70-350 mg of

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chlorogenic acid and a regular coffee drinker can take up to 1 g of chlorogenic acids per day.23

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Caffeic acid was shown to be one of the most potent rat intestinal α-glucosidase inhibitors among

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different cinnamic acid derivatives.24 The oxidized form of gallic acid caused nearly 40%

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inhibition of rat brush border sucrase and maltase activities.25,26 (-)-Epigallocatechin gallate

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(EGCG), the main catechin found in green tea,27 was also observed to be a potent inhibitor of

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maltase activity in rat brush border membrane vesicles,28 rat intestinal acetone powder,29 and

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recombinant human intestinal maltase.30 It has been also shown that tea catechins, especially

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EGCG, may have potential for treating Type 2 diabetes.31,32 Subjects who consumed more than 6

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cups of green tea daily were at less risk for Type 2 diabetes compared to subjects who drank less

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than a cup per week.33

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Overall, this information suggests that natural phenolic compounds present as normal

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dietary components may exert selective and differential inhibition of the individual subunits of

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MGAM and SI, for the potential modulation of digestion of starchy and other glycemic

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carbohydrate rich foods. Although there are multiple publications describing inhibition of α-

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glucosidases by phenolic compounds, the possibility of subunit selectivity for modulating

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glucose release has not been studied. In the present study, we analyzed the inhibitory properties

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and potential for the selective inhibition of the individual Ct and Nt subunits of MGAM and SI

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using five phenolic and polyphenolic compounds [chlorogenic acid, caffeic acid, gallic acid, (+)-

84

catechin, EGCG] commonly found in Western diets, and whose α-glucosidase inhibition

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properties have been previously described.

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MATERIALS AND METHODS

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Materials

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All

reagents

including

phenolic

standards

of

chlorogenic

acid

(3-(3,4-

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dihydroxycinnamoyl)quinic acid), caffeic acid, gallic acid, (+)-catechin, and (-)- epigallocatechin

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gallate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Enzyme preparation

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Cloning, expression, and purification of recombinant mouse Ct-MGAM (Glucoamylase

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subunit; spliceform N20), recombinant mouse Ct-SI (Sucrase subunit),4 recombinant human Nt-

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MGAM (Maltase subunit),34 and human recombinant Nt-SI35 (Isomaltase subunit) were reported

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previously. Mouse Ct-MGAM and Ct-SI were generated by recombinant expression in a

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baculovirus-Sf9 insect cell system.4 Human Nt-MGAM and Nt-SI were expressed in Drosophila

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S2 cells.34,35 Nickel-Sepharose resin was used to isolate the secreted proteins from the cell media

100

that were further purified using anion exchange chromatography.

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Determination of protein concentration

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Protein concentration of solutions was determined by the Bio-Rad Protein Assay kit

103

(Hercules, CA, USA ), using 10 µl of the protein solution and 200 µl of Bradford dye reagent,

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and let sit for at least 5 min. Absorbance was read at 595 nm using a Synergy HT microplate

105

reader (BioTek; Winooski, VT, USA). Standards of bovine serum albumin were used in the

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concentration range of 31.25 to 500 µg/ml.

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Enzyme and inhibition assays 6 ACS Paragon Plus Environment

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Glucose release was measured by the Tris Glucose Oxidase (TGO) method36 modified for

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microplate wells. Maltose solution (10 µl) at final concentrations ranging from 2.1 to 16.7 mM,

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(approximately 1 to 8 times the Km for maltose of Ct subunits and 0.3 to 3 times Km for maltose

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of Nt subunits) was mixed with 10 µl of the enzyme solution containing 0.2 mU of activity for

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Ct-MGAM, Ct-SI, and Nt-MGAM; and 0.1 mU of activity for Nt-SI. One unit (U) of enzyme

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activity was defined as the activity that cleaves one µmol of maltose per min. The mixtures were

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incubated for 1 h at 37 °C and then TGO (180 µl) was added and incubated for an additional 45

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min.36 Absorbance was read at 450 nm in the microplate reader.

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For inhibition assays, 10 µl of the phenolic inhibitor solutions were added to the above

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reaction mixtures to attain final concentrations ranging from 0 to 666.7 µM for the gallic acid,

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caffeic acid and (+)-catechin; while chlorogenic acid and EGCG were used at a final

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concentration of 0-83.3 µM for C terminals of both MGAM and SI, or 0-666.7 µM for N

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terminals of MGAM and SI. Analyses were conducted in triplicate. In order to correct for the

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scavenger effect of phenolics on the oxidative intermediates generated during the glucose

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oxidase-peroxidase reaction, which can lead to possible misleading readings of optical density

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(OD),37 curves of glucose concentration vs OD in the presence of different concentrations of

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each individual phenolic compound were constructed and used to adjust the OD obtained during

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the assay of inhibition of the phenolics on the α-glucosidase enzymes. At the selected

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concentrations, phenolics caused less than 20% inhibition of the glucose oxidase-peroxidase

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developing reaction.

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Determination of inhibition constant and inhibition mechanism

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The inhibition mechanism and the respective inhibition constant (Ki) of each enzyme

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subunit-phenolic combination were determined by the best fit of non-linear regression analysis 7 ACS Paragon Plus Environment


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with the Levenberg–Marquardt algorithm, (Sigma Plot; Systat Software Inc.; San Jose, CA.,

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USA) and the standard mathematic equations describing the inhibition mechanisms for

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competitive, uncompetitive, non-competitive homogeneous, and non-competitive heterogeneous

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inhibition.38 The mathematical fit with the best parameters of quality (highest r2, lowest p, lowest

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residuals; p ≤ 0.05 for the statistical significance) for each enzyme-inhibitor combination was

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chosen as the most probable inhibition mechanism in each case. For the calculations of kinetic

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parameters we choose nonlinear regression methods instead of linear transformations and linear

138

regression, to avoid the additional error introduced by the latter procedures.39, 40

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Determination of IC50 values

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The concentration of each polyphenolic compound needed to cause 50% inhibition of the

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activity of the individual enzyme subunits was defined as the IC50 value. It was calculated by

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linear interpolation of the semi-log plot of (log [I]) versus the relative activity (v/Vmax = 0.5) at

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16.67 mM maltose concentration (Minitab 14; State College, Pennsylvania, USA).

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RESULTS

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Kinetics and mechanism of inhibition of the recombinant Ct-MGAM, Nt-MGAM, Ct-SI

146

and Nt-SI

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The inhibition capacity, the inhibition mechanism and corresponding inhibition constants

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were determined for each phenolic compound against each recombinant α-glucosidase subunit.

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Chlorogenic acid. Chlorogenic acid had higher inhibition binding affinities and correspondingly

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lower inhibition constants for Ct-MGAM (3 µM) and Ct-SI (1.8 and 28 µM) compared to caffeic

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acid, gallic acid, and (+)-catechin (Table 1). The binding affinity of chlorogenic acid was


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approximately 56 and 15 times greater for Ct-SI and Ct-MGAM, than for Nt-SI and Nt-MGAM,

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respectively. Chlorogenic acid showed non-competitive homogeneous inhibition for Ct-MGAM

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and Nt-MGAM, while Ct-SI and Nt-SI were inhibited in a non-competitive heterogeneous way.

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(-)-Epigallocatechin gallate. Ct-MGAM was also the most strongly inhibited subunit by EGCG,

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with Ki1 and Ki2 values of 1.7 and 6.5 µM, respectively (Table 1). Figure 1 illustrates the change

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in the relative activities (v/Vmax) of Ct-MGAM, Nt-MGAM, Ct-SI, and Nt-SI with the different

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concentrations (0-666.7 µM) of EGCG in the presence of different maltose concentrations (2.1,

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4.2, 8.3 and 16.7 mM). It is notable the difference in the sensitivity to inhibition by EGCG

160

observed between the Ct-subunits and Nt-subunits, the former showing a Ki two orders of

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magnitudes smaller than the latter. Thus, EGCG was a comparatively potent inhibitor for Ct-

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MGAM and Ct-SI (Figure 1a and b). EGCG inhibited Ct-MGAM, Nt-MGAM, and Nt-SI by a

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non-competitive

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homogeneous inhibition on Ct-SI.

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(+)-Catechin. (+)-Catechin showed similar inhibition potency towards all four subunits (Figure

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2). Affinities or inhibition constants of (+)-catechin for Ct-MGAM, Nt-MGAM and Ct-SI were

167

in the range of 20.4 to 97.2 µM (Table 1). The value of 33.7 µM (Table 1) for the Ki on Nt-SI

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indicated a higher inhibitory effect on this subunit compared to the other phenols. (+)-Catechin

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showed non-competitive heterogeneous inhibition on Ct-MGAM, Nt-MGAM, and Ct-SI, while it

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showed non-competitive homogeneous inhibition on Nt-SI.

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Caffeic acid. Caffeic acid had higher binding affinities (lower inhibition constant) for Ct-

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MGAM and Ct-SI, than for the two N terminal enzyme subunits tested. The inhibition constants,

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Ki1 and Ki2 for Ct-MGAM were 56.5 and 191.8 µM, while they were 58.2 and 323 µM for Ct-SI

heterogeneous

inhibition

mechanism,

and

caused

non-competitive



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(Table 1). Caffeic acid showed non-competitive heterogeneous inhibition for Ct-MGAM, Nt-

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MGAM and Ct-SI, while it inhibited Nt-SI in a non-competitive homogeneous manner.

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Gallic acid. As with caffeic acid, Ct-MGAM and Ct-SI were the most sensitive to inhibition by

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gallic acid, with values for Ct-MGAM of 24.9 and 50 µM for Ki1 and Ki2, and 31.3 and 142.8

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µM for Ct-SI Ki1 and Ki2, respectively (Table 1). Gallic acid was inhibited by a non-competitive

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heterogeneous mechanism for all four subunits.

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Overall, inhibition by EGCG and chlorogenic acid showed marked preference toward the

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C terminals of MGAM and SI than for N-terminals, but they displayed notable differences in

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their selectivity to the two enzymes. Greatest inhibition of Ct-MGAM was caused by EGCG and

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Ct-SI by chlorogenic acid. With the notable exception of (+)-catechin, which showed

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comparably higher inhibition of Nt-SI, there was a general trend toward higher binding affinities

185

for Ct subunits over Nt.

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IC50 values of inhibitors

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IC50 values were also determined for the different phenolic compounds on the four

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individual α-glucosidase subunits. These values result from the overall inhibition effects of the

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different binding mechanisms that each compound may display and, therefore, are not

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necessarily expected to be of same ranking as the inhibition constants. In this context IC50

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represents the required concentration to inhibit 50% of the enzymatic activity and is conducted at

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one single substrate concentration. The general trends observed before for the inhibition

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constants held up. Among the compounds tested, chlorogenic acid and EGCG required the

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lowest concentration range (2.3-13.8 µM) to inhibit 50% of the activity of Ct-MGAM and Ct-SI,

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while a higher concentration range (39.6-159.4 µM) was required for Nt-MGAM and Nt-SI


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(Figure 3). (+)-Catechin showed 50% inhibition of Ct-SI and Nt-SI at a comparably lower

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concentration of 34.3 and 36 µM, respectively (Figure 3). Caffeic acid showed IC50 values in the

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range of 137.4 to 266 µM (Figure 3) for all of the α-glucosidase subunits; however, it did inhibit

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selectively Ct-MGAM and Nt-SI and with lower concentrations than the required for the other

200

two subunits (Figure 3). Gallic acid also showed stronger inhibition (lower IC50 values) on Ct-

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MGAM and Nt- SI than on the other two subunits.

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DISCUSSION

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Phenolic acids and polyphenols have been reported to inhibit the activities of the

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intestinal α-glucosidases, MGAM and SI.24-30 The use of phenolic compounds as modulators of

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the rate of glucose release from starchy foods has been proposed for the treatment of metabolic

206

diseases such as diabetes.41 Although there are known strong inhibitors of α-glucosidases, such

207

as acarbose,41 they substantially reduce digestion of starch to glucose, resulting in dumping of

208

starch and starch products into the large intestine to cause bloating and discomfort. The purpose

209

of this study was to explore the selective inhibition by selected phenolic and polyphenolic

210

compounds on individual MGAM and SI subunits. We propose this effect could produce a slow,

211

but still complete digestion in the small intestine of the main glycemic carbohydrates, starch, and

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sucrose. Additionally, slowly digested starch may trigger the ileal brake and gut-brain axis

213

mechanisms that respectively controls stomach emptying rate and influences hypothalamic

214

regulation of appetite, with potential impact on obesity and associated diseases.9,42,43

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Our findings reveal that the four subunits of the intestinal α-glucosidases, SI and MGAM,

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can be selectively and differentially inhibited by certain plant-derived phenolics. This introduces

217

the potential to induce different rates or profiles of digestion for starches, starch products, and



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sucrose. For instance, selective inhibition of the most active starch degrading subunit, Ct-

219

MGAM (or Glucoamylase), was achieved with EGCG and chlorogenic acid. This implies that

220

reducing activity of this enzyme could slow the rate of starch digestion, and reduce particularly

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the glycemic spike, as was proposed in a recent paper by our group.44 Chlorogenic acid had the

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highest binding affinity (Ki1) for Ct-SI (Sucrase). This suggests the possibility of slowing the

223

rate of digestion of sucrose rich foods. Thirdly, (+)-catechin showed higher inhibition for Nt-SI,

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the only subunit with endogenous α-1,6 branch hydrolyzing activity. The combination of (+)-

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catechin with a highly branched starch or starch products, such as a highly branched

226

maltodextrin,45 could obtain a slow glucose release effect. The results imply that the low

227

inhibitory effect of phenolics on the slower acting Nt-MGAM and SI subunits, may allow for the

228

slow digestion of starch in diets containing such phenolics, but avoiding the inhibition of all four

229

subunits caused by stronger inhibitors like acarbose which, although effectively reducing

230

glycemic carbohydrate digestion, cause the delivery of large quantities of carbohydrates into the

231

colon.

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The tested phenolics were shown to affect the ability of the substrate to bind the active

233

site of all enzyme subunits (Ki1); in some cases, phenolics may also bind already formed

234

enzyme-substrate complex, although this is with lesser affinity (Ki2). It has been reported that the

235

catalytic pockets of Ct-MGAM, Nt-MGAM and Nt-SI are surrounded by several aromatic amino

236

acids, which may comprise the substrate binding motifs of these enzymes.4 Therefore, it may be

237

speculated that phenolic compounds could interact and/or bind to these aromatic motifs in

238

proximity to the active site, impairing the binding of the substrate with a correct orientation. The

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substantially higher values for Ki1 of phenolics than those found for other competitive

240

inhibitors,10 make unlikely the possibility for the existence of a typical competitive binding 12 ACS Paragon Plus Environment

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component in the interaction and therefore discards the possibility of the existence of classic

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mixed type inhibition. Thus, the model of typical non-competitive inhibition was the one that

243

most closely described the effects of phenolics. Therefore, phenolic and polyphenolic inhibitors

244

bind to at least a second additional site. Mathematical fittings of the experimental data indicated

245

that only five (Nt-SI/caffeic acid; Nt-SI/(+)-catechin; Ct-MGAM/chlorogenic acid; Nt-

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MGAM/chlorogenic acid; and Ct-SI/EGCG) out of the twenty different inhibitor-subunit

247

combinations studied, showed the same values for Ki1 and Ki2. In these cases; therefore, the

248

binding affinity of phenolic compounds to the free enzyme and enzyme-substrate complex was

249

essentially the same, implicating that there was a single binding site for phenolics, and the

250

binding of substrate did not interfere with the binding of phenolics at their binding site. This also

251

implies the occurrence of conformational changes in the enzyme after binding of these phenolics,

252

with effect on their substrate binding capability or catalytic mechanism.38

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Another study, using rat intestinal powder, showed that chlorogenic acid has a non-

254

competitive inhibition behavior on maltase and sucrose activities, which agreed with our

255

results.22 However, the IC50 values of chlorogenic acid for maltase and sucrase were 2.99 and

256

2.18 mM, which are substantially higher than our calculated IC50 values (Figure 3).22 This

257

difference could be explained by binding multiple protein components present in the crude

258

preparations of rat mucosal proteins. In another study, EGCG was shown as a competitive

259

inhibitor and its IC50 was estimated as 20 µM for human intestinal maltase.30 Also, results of an

260

earlier study showed that caffeic acid had very low inhibitory effect with IC50 values of 0.74 and

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0.49 mM for maltase and sucrase, respectively.24 In our study, caffeic and gallic acid were not

262

nearly as strong inhibitors as chlorogenic acid and EGCG.



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Stability of phenolic compounds must be considered, for such a selective inhibition

264

concept to be pursued in a practical sense. In one study, chlorogenic acid and caffeic acid were

265

found to be stable after incubating with human gastric juice, duodenal fluid and ileostomy

266

effluent.21 Another study showed that most (97.9-99.2%) of orally administered chlorogenic acid

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(700 µmol/kg) and a small amount of caffeic acid (0.8-2.1%) were found in the rat small

268

intestine.46 Record and Lane47 tested the stability of gallic acid, caffeine, and EGCG in green tea

269

at acidic pH and at a slightly alkaline pH. Caffeine concentration did not change under the

270

exposure of either acidic (pH 2.0) or slightly alkaline pH (pH 7.5) conditions. Also, the

271

concentration of gallic acid was only slightly reduced at pH of 2.0 and at pH of 7.5. On the other

272

hand, although EGCG concentration slightly declined at pH of 2.0; its concentration was lower

273

with incubation for 1 h at pH of 7.5, decreasing from 1880 to 21 µmol/l in 1 g of green tea/50 ml

274

of water. Several studies also reported that while acidic pH similar to that of the stomach slightly

275

affected the concentration of catechins in green and black tea, the slightly alkaline pH (above pH

276

7.4) caused a significant decrease in their concentration.48-51 Those studies used alkaline pH

277

(above pH 7.4), however it may not reflect the actual pH of the lumen of the entire small

278

intestine since the pH measured in humans subjects was of 6.4-6.6 in the duodenum or proximal

279

small intestine and increased to 7.3-7.5 in the distal small intestine.52,53 This difference could be

280

important as the glycemic spike seen in the response profile arises from rapid proximal digestion

281

of glycemic carbohydrates, and at this lower pH the catechins could be found with good qualities

282

and high inhibition potential.

283

Of note, oxidative reactions in the gut lumen may lead to formation of complex dimers

284

including theasinesins.54 The ability of these larger phenolic forms to interact with enzymes at

285

the brush border is unknown.


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In conclusion, the finding of selective inhibition by plant-derived phenolic compounds of

287

the individual mucosal α-glucosidases implies that digestion rate of starch, starch products, and

288

sucrose can be differentially affected and modulated by these dietary components. The potential

289

to control glycemic response profiles or sucrose hydrolysis rate using this selective inhibition

290

concept with phenolic compounds warrants further study with an in vivo model.

291

292

ABBREVIATIONS

293

MGAM: Maltase-Glucoamylase

294

SI: Sucrase-Isomaltase

295

Ct: C terminal end of proteins

296

Nt: N terminal end of proteins

297

EGCG: (-)-epigallocatechin gallate

298

Vmax: maximal rate of reaction

299

Ki: inhibition constant

300

IC50: the required concentration to inhibit 50 % of the enzymatic activity

301

CI: confidence interval

302

303



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ACKNOWLEDGEMENTS

305

This work was funded by CONACYT, Mexico, projects 80448 and 173965. We thank the

306

Whistler Center for Carbohydrate Research at Purdue University for its partial support of the

307

project. The authors declared no conflict of interest for this work.

308

Supporting Information Available: Reaction mechanisms for each of the different inhibition

309

type and corresponding equations. This material is available free of charge via the Internet at

310

http://pubs.acs.org.

311 312 313


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REFERENCES

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Figure captions

473

Figure 1. Relative activity (v/Vmax) of a) Ct-MGAM, b) Ct-SI, c) Nt-MGAM and d) Nt-SI at

474

different maltose concentrations (2.1, 4.2, 8.3 and 16.7 mM) in the presence of EGCG at 0 µM,

475

0.67 µM, 3.3 µM, 16.7 µM, and 83.3 µM for Ct-MGAM and Ct-SI (panels a and b); and 0 µM,

476

83.3 µM, 166.7 µM, 333.3 µM and 666.7 µM for Nt-MGAM and Nt-SI (panels c and d).

477

Figure 2. Relative activity (v/Vmax) of a) Ct-MGAM, b) Ct-SI, c) Nt-MGAM, and d) Nt-SI at

478

different maltose concentrations (2.1, 4.2, 8.3 and 16.7 mM) in the presence of (+)-catechin at 0

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µM, 83.3 µM, 166.7 µM, 333.3 µM and 666.7 µM.

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Figure 3. IC50 values (µM) of caffeic acid, gallic acid, (+)-catechin, chlorogenic acid, and EGCG

481

for the inhibition of Ct-MGAM, Nt-MGAM, Ct-SI and Nt-SI. Vertical lines represent the

482

calculated 95% CI for the experimental data.

483 484 485 486 487 488 489 490



Table 1. Inhibition mechanism and inhibition constant (Ki) for each α-glucosidase subunit. Inhibition mechanism and inhibition constant (Ki; µM ± sd) Ct-MGAM Ct-SI Nt-MGAM Chlorogenic acid Type Non-competitive Non-competitive Non-competitive homogeneous heterogeneous homogenous Ki1 3 ± 0.3 1.8 ± 0.3 43.2 ± 3.5 Ki2 28 ± 8.8 r2 0.937 0.983 0.943 EGCG Type Non-competitive Non-competitive Non-competitive heterogeneous homogenous heterogeneous Ki1 1.7 ± 0.7 15.1 ± 1.6 9 ± 1.5 Ki2 6.5 ± 2.1 97.6 ± 49.3 2 r 0.941 0.939 0.967 (+)-Catechin Type Non-competitive Non-competitive Non-competitive heterogeneous heterogeneous heterogeneous Ki1 20.4 ± 8.2 21.6 ± 8.1 20.4 ± 3.8 Ki2 96.7 ± 21.7 45.4 ± 5.9 97.2 ± 26.9 r2 0.925 0.973 0.967 Caffeic acid Type Non-competitive Non-competitive Non-competitive heterogeneous heterogeneous heterogeneous Ki1 56.5 ± 25.4 58.2 ± 21.2 75.2 ± 13.3 Ki2 191.8 ± 39.6 323 ± 100.9 478.3 ± 167.1 2 r 0.918 0.902 0.968 Gallic acid Type Non-competitive Non-competitive Non-competitive heterogeneous heterogeneous heterogeneous Ki1 24.9 ± 8.6 31.3 ± 7.2 85.7 ± 13.9 Ki2 50 ± 5.7 142.8 ± 24.7 460.6 ± 133.2 2 r 0.978 0.969 0.971

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Nt-SI Non-competitive heterogeneous 101.8 ± 36.6 259.7 ± 114.5 0.93 Non-competitive heterogeneous 76.3 ± 31.5 140.4 ± 53.5 0.919 Non-competitive homogenous 33.7 ± 4.1 0.921 Non-competitive homogenous 103.3 ± 11.1 0.909 Non-competitive heterogeneous 44.6 ± 14.4 127.6 ± 28.7 0.93


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Dietary Phenolic Compounds Selectively Inhibit the Individual

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