Food Chemistry 145 (2014) 205–211

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phytonutrients for controlling starch digestion: Evaluation of grape skin

extract
Ming Miao a,⇑, Huan Jiang a, Bo Jiang a, Tao Zhang a, Steve W. Cui a,b, Zhengyu Jin a
a
State Key Laboratory of Food Science & Technology, Ministry of Education, Key Laboratory of Carbohydrate Chemistry & Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi,
Jiangsu 214122, PR China
b
Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont. N1G 5C9, Canada

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this work was to evaluate the structure–function relationship between grape skin extract
Received 1 May 2013 and human a-amylase. The grape skin extract was characterised as resveratrol-3-O-glucoside by RP-
Received in revised form 19 July 2013 HPLC–ESI-MS, which showed strong inhibition towards a-amylase and the IC50 value was 1.35 mg/ml.
Accepted 14 August 2013
The kinetic results demonstrated grape skin extract obeyed the non-competitive mode against amylase.
Available online 27 August 2013
Fluorescence data revealed the ability of grape skin binding to amylase belonged to static quenching
mechanism with a complex formation and there was only one binding site in a-amylase for grape skin
Keywords:
extract. Docking study showed a best pose with total energy value of 118.3 kJ/mol and grape skin
Grape skin extract
Human a-amylase
extract interacted with side chain of Asp300 with hydrogen bonds and Van der Waals forces. This preli-
Inhibition minary observation provides the basis for further evaluation of the suitability of grape skin extract as nat-
Fluorescence quenching ural inhibitor with potential health benefits.
Docking Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction ibility and the corresponding postprandial glycaemic response,

Diabetes mellitus and obesity have become major public health
concerns worldwide, with the number of cases increasing expo-
nentially in recent years. The multi-factorial aetiology of this
worldwide epidemic, and the idea that dietary factor may contrib-
ute to it, is now well recognised (Aston, 2006; Coll, Farooqi, &
O’Rahilly, 2007; Englyst & Englyst, 2005; Sies, Stahl, & Sevanian,
2005). Compelling evidence from epidemiologic studies indicates
that the blood glucose from ingested carbohydrate sources is nec-
essary to reduce the complications and cost for controlling and
preventing the metabolic syndromes, including diabetes and pre-
diabetes, cardiovascular diseases, obesity and overweight (Ludwig,
2002; Semjonous et al., 2009; Sies et al., 2005). New developments
in food and nutritional science have led to the conclusion that
slowing down the rate of carbohydrate digestion helps to blunt
glycaemia, reduces insulin requirements, and causes satiety by
reducing the stress on regulatory systems related to glucose
homeostasis and energy metabolism (Aston, 2006; Coll, Farooqi,
& O’Rahilly, 2007; Englyst & Englyst, 2005; Miao, Jiang, & Zhang,
2009; Semjonous et al., 2009; Zhang & Hamaker, 2009).
Amongst food carbohydrates, starch occupies a unique position
based on the basic source of metabolic energy for the majority of
the world’s population. According to the rate and extent of digest-
⇑ Corresponding author. Tel.: +86 (0)510 853 27859; fax: +86 (0)510 859 19161.
E-mail addresses: miaoming@jiangnan.edu.cn, miao20@purdue.edu (M. Miao).
starch is generally classified into rapidly digestible starch (RDS),
slowly digestible starch (SDS), and resistant starch (RS) related to
its physiological effect after consumption (Englyst, Kingman, &
Cummings, 1992). RDS is rapidly digested and absorbed in the duo-
denum and proximal regions of the small intestine leading to a fast
elevation of blood glucose and insulin level, and RS is not digested
in the upper gastrointestinal tract, but its microbial fermentation
in the colon produces short chain fatty acids (SCFA) that is benefi-
cial to colonic health, whilst SDS is digested slowly throughout the
entire small intestine to provide sustained glucose release with a
low initial glycaemia and subsequently a slow and prolonged re-
lease of glucose, which is essential to regular physiological pro-
cesses and optimal health (Englyst & Englyst, 2005). Therefore,
improving food quality with higher amounts of SDS is becoming
a hot research filed for researchers from industry and academia.
There are numerous reports and patents on SDS preparation, but
there is no commercially available SDS or SDS-state foods in the
market (Miao, Zhang, Mu, & Jiang, 2010; Miao et al., 2009; Zhang
& Hamaker, 2009).
In the past decades, considerable research effort has been de-
voted to novel ways for achieving the physiological effects of SDS
for glycaemic control and the prevention of related diseases
(Björck, Liljeberg, & Östman, 2000; Ludwig, 2002). Compared to
the commercial inhibitors (i.e. Acarbose and Phase II), the interac-
tions of starch with different components present in the food sys-
tem have become an innovative target for improvement of

0308-8146/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.08.056
206 M. Miao et al. / Food Chemistry 145 (2014) 205–211
postprandial hyperglycaemia with fewer gastrointestinal side ef-
fects (Obiro, Zhang, & Jiang, 2008; Sies et al., 2005). Besides two
most important interactions between starch and protein or lipid,
the soluble fibres (guar gum, psyllium, b-glucan or pectin), anti-
nutrients (tannin, phytate, saponin or lectin), phenolic compounds
(luteolin, myricetin or quercetin) and organic acids (lactic acid,
propionate or vinegar) have been used to improve overall glycae-
mic control by inhibition of amylolytic enzymes (Gonçalves, Ma-
teus, & de Freitas, 2011; McDougall, Kulkarni, & Stewart, 2008;
Zhang & Hamaker, 2009). Recent studies have shown that grape
is a natural source of notable bioactive compounds, such as flavo-
nols, anthocyanins and procyanidins, which are can positively
influence risk factors associated with cardiovascular health, cancer,
diabetes, inflammation, neurodegenerative disease, and age-re-
lated cognitive decline (Chuang et al., 2012; Hogan et al., 2011;
Vislocky & Fernandez, 2010; Zunino, 2009). Also, resveratrol is a
unique component of grapes and has anti-aging, anti-carcinogenic,
anti-inflammatory, and anti-oxidant properties that might be rele-
vant to chronic diseases and/or longevity in humans (Smoliga,
Baur, & Hausenblas, 2011). In particular, the stilbene resveratrol
has shown potential for reducing hyperglycaemia, improving insu-
lin sensitivity, and protecting against b-cell loss (Brasnyó et al.,
2011; Lagouge et al., 2006; Zunino, 2009). However, very little
information exists regarding grape skin extract related to control
starch digestion. In the current investigation, the structure–
function relationship between grape skin extract and human
a-amylase was elucidated with in vitro assays and in silico
modelling, which is important for practical application in making
tailor-made carbohydrate foods with low glycaemic index.
2. Materials and methods
2.1. Materials
The grape skin extract sample was purchased from Riotto
Botanicals Co., Ltd. (Shaanxi, China). Normal maize starch was ob-
tained from Changchun Dacheng Industrial Group Co. Ltd. (Changc-
hun, Jilin, China). Alpha-amylase (Cat. No. A-9972, P100 units/mg
protein) from human pancreas and piceid (Cat. No. 15721, P95%)
were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO).
All chemicals were reagent grade and were obtained from Sinop-
harm Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Polyphenols assays
The grape skin extract sample (approximate 1 mg) was dis-
solved in 10 ml of methanol and then centrifuged (5000g for
10 min). The supernatant was filtered through a 0.22 lm mem-
brane filter and then injected into a Waters 2690 HPLC with a
Micromass ZMD mass spectrometer, and a Waters 996 diode array
detector (Waters Corp., Milford, MA, USA). The acquisition and pro-
cessing data were performed by the version 3.1 MassLynx soft-
ware. An analytical reverse phase C18 column (250  4.6 mm,
PurospherÒ STAR, 5 lm, Merck Millipore International) was used
in the analysis. The mobile phase was composed of 1% acetic acid
in water (v/v, A) and methanol (B). The linear gradient conditions
were as follows: from 0% to 100% B in 40 min flow at a flow rate
of 0.3 ml/min and the column temperature set at 35 °C. The poly-
phenol was detected by monitoring the elution at 520 nm. The
electrospray ionization mass spectrometry parameters were as fol-
lows: positive mode, capillary voltage 3.8 kV, cone voltage 30 V,
extractor voltage 5 V, source block temperature 120 °C, desolvation
temperature 300 °C and gas flow of N2 9 L/min, nebulizer pressure
60 psi, scan range from m/z 100–900 with scan time 1 s and inter-
scan delay 0.1 s.
The resveratrol derivative was analysed using an Agilent 1100
HPLC system (Agilent Technologies, USA) equipped with a reversed
phase symmetry C18 column (250  4.6 mm, Waters, USA). The
chromatographic conditions were as follows: mobile phase com-
posed solvent A (10% water, v/v) and solvent B (60% acetonitrile,
v/v); injection volume 5 ll; flow rate 1.0 ml/min; and quantifica-
tion of resveratrol at 304 nm. The piceid was used as a standard
sample for HPLC test.
2.3. Alpha-amylase assays
The digestibility of each starch was analysed according to the
procedure of Englyst et al. (1992) with a slight modification. En-
zyme solution was prepared by suspending human pancreatic a-
amylase (12.0 g) in phosphate buffer (100 ml, 0.2 M, pH 5.2) with
magnetic stirring for 10 min, centrifuging the mixture for 10 min
at 1500 g, and then transferring a portion (50 ml) of the superna-
tant into a beaker. The maize starch sample (200 mg) was dis-
solved in 15 ml of phosphate buffer (0.2 M, pH 5.2) by heating at
95 °C for 10 min. After the solution was cooled and equilibrated
at 37 °C for 5 min, enzyme solution (5.0 ml) and grape skin extract
(10%, based on starch) were added. Then, the samples were shaken
in a 37 °C water bath at 150 rpm. Aliquots of hydrolysed solution
(0.5 ml) were taken at different time intervals and mixed with
4 ml of absolute ethanol to deactivate the enzymes. The reducing
sugar content was determined with the Nelson-Somogyi method
by measuring the absorbance at 540 nm. A control vial was pre-
pared by replacing the inhibitor solution with phosphate buffer.
Percentage of pancreatic a-amylase inhibition was calculated
according to the equation below:
ðAcontrol  Acontrolblank Þ  ðAsample  Asampleblank Þ
%Inhibition ¼  100
Acontrol  Acontrolblank
where Acontrol, Acontrolblank, Asample and Asampleblank refer to the absor-
bance value of reaction vial containing live enzyme and buffer, dead
enzyme and buffer, live enzyme and inhibitor and dead enzyme and
inhibitor respectively. Substrate was present in all these vials. IC50
value (concentration of inhibitor required to produce a 50% inhibi-
tion of the initial rate of reaction, mg/ml) was obtained graphically
by an inhibition curve.
The Michaelis–Menten kinetic model was employed to evaluate
the effect of grape skin extract on starch hydrolysis. The amount of
glucose liberated under different starch concentrations (5, 10, 15,
20, 25 mg/ml) of cooked maize starch in the presence of grape skin
extract (0, 1, 4 mg/ml) was used to measure the type of inhibition.
A Lineweaver–Burk plot between 1/[S] (starch concentration) and
1/[V] (reaction rate) was used to examine the action type of grape
skin extract on the starch hydrolysis.
2.4. Fluorescence measurements
The quenching effect of grape skin extract on human a-amylase
fluorescence was assayed as described in the literature with some
modification (Lakowicz, 2006). A HITACHI fluorescence spectrome-
ter (Model 650–60, Hitachi, Tokyo, Japan) was used for Fluores-
cence quenching assays. The sample (1 ml) was excited at
280 nm, with 1 nm excitation and emission slits, and spectra were
recorded between 300 and 500 nm at 0.1 nm resolution. A stock
solution of a-amylase and the quenchers of grape skin extract were
prepared by dissolving in phosphate buffer (pH 5.2). The fluores-
cence intensities were obtained at different grape skin extract con-
centrations and plotted according to the Stern–Volmer equation.
The quenching constant Ksv, the quenching rate constant Kq, the
number of binding sites n and apparent associative binding con-
stant Ka were obtained using the slopes and intercept of these
 M. Miao et al. / Food Chemistry 145 (2014) 205–211 207

linear plots (F0/F vs Q or log (F0/F  1) vs log Q), where F0 and F were and Van der Waals forces interactions were also obtained from
the fluorescence intensities before and after the addition of the the docking results.
grape skin extract, respectively, Q was the concentration of the
grape skin extract.
2.6. Statistical analysis

2.5. Docking studies

The three-dimensional structure of human pancreatic a-amy-
lase was imported from the Protein Data Bank (1HNY). The struc-
ture of grape skin extract (resveratrol-3-O-glucoside) was
generated with the Cambridge Soft ChemBioDraw Ultra (Version
12.0) and energy minimised with the MM2 calculations using a
conjugate gradient. Before the docking procedure, water molecules
were removed from the enzyme crystal structure using Accelrys
Discovery Studio 3.0 software. Automated molecular docking stud-
ies of the inhibitory ligand at the amylase-binding site was per-
formed with the AutoDock 4.2 package, in the presence of
cofactors (calcium and chloride ions). The Binding Site tool was
used to determine the active site. The docking runs were per-
formed with a radius of 9 Å with coordinates x: 11.563, y:
46.792, and z: 44.400. The evaluation procedure of the molecular
docking was performed according to the scores of several scoring
functions. According to the scores and binding-energy value, the
best pose for grape skin extract was obtained. Hydrogen bonds
All results were analysed by the Duncan test using the statisti-
cal analysis system (SAS Institute, Cary, NC). A level of 0.05 was set
to determine statistical significance.
3. Results and discussion
3.1. Polyphenol assays
The grape skin extract was directly analysed by HPLC–ESI/MS
chromatogram as shown in Fig. 1. According to the report of Yawa-
dio, Tanimori, and Morita (2007), HPLC coupled to mass spectrom-
etry was extremely useful for peak assignment and
characterisation of individual compounds, due to the product ions
produced from the fragmentation of a selected precursor ion.
Therefore, the identification of polyphenol was based on the com-
parison of UV–vis absorption maxima (kmax) and mass spectral
analysis with data reported in previous studies. As shown in
Fig. 1(A), a single peak was monitor by an HPLC chromatogram

20111117-7 3: Diode Array

A 14.15 300
Range: 3.009
2.5

2.0
AU

1.5

1.0

5.0e-1

0.0
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

20111117-7 833 (14.419) 1: TOF MS ES-

227.0 1.60e4
100
B
%

228.0

229.0
185.0
0 m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Fig. 1. HPLC–ESI/MS chromatogram of polyphenol in grape skin extract. (A) HPLC profile, (B) Mass fragmentation pattern, (C) Structure of identified polyphenol.
208 M. Miao et al. / Food Chemistry 145 (2014) 205–211

Fig. 2. HPLC chromatograms of piceid standard (A) and grape skin extract (B).

at 280 nm. Most phenolic compounds absorb at 280 nm because Table 1

they have at least one aromatic ring in their structure, giving a Summary of inhibiting, quenching and docking parameters of grape skin extract
nonspecific chromatogram (Lee et al., 2009). The peak was identi- against human a-amylase.
fied by the comparison with HPLC retention time, mass spectral Grape skin extracta Parameters
data and relevant literatures (Lee et al., 2009; Yawadio et al.,
% Inhibition 70.81 ± 2.16
2007). The MS spectrum of the grape skin extract is present in IC50 (mg/ml) 1.35 ± 0.24
Fig. 1(B), whereas Fig. 1(C) shows the structure of identified poly- Type Non-competitive
phenol. The MS analysis of peak showed an [M]+ ion at m/z 390 and Vmax (mg/ml  min)b 0.47 ± 0.02
a major fragmentation in MS2 at m/z 227 (163 amu), which would km (mg/ml) 33.85 ± 1.32
Ksv (mol/L) 1.63 ± 0.07
correspond to the loss of a glucose moiety. The MS2 fragmentation Kq (1011 L/mol  s) 6.97 ± 1.04
of the ion at m/z 227 would be corresponding to the resveratrol. n 1.3 ± 0.0
Thus, this peak was tentatively identified as resveratrol-3-O-gluco- Ka (mol/L) 3786 ± 2.0
side, which was in agreement with the other published literatures Total energy (kJ/mol) 118.3 ± 2.5
Hydrogen bonds energy (kJ/mol) 31.2 ± 0.8
(Ali et al., 2004; Zhou, Chen, & Zhong, 2007). The RP-HPLC chro-
Van der Waals energy (kJ/mol) 87.1 ± 1.3
matograms of piceid standard and grape skin extract are illustrated Hydrogen bonds contact residue Gln-302, Gly-304, Ala-310, Ile-312,
in Fig. 2. There was only one peak and its retention time was Arg-346, Asn-352
16.10 min based on a liquid chromatography analysis, which indi- Van der Waals forces contact residues Gln-302, Arg-303, Gly-304, Gly-309,
cated that resveratrol-3-O-glucoside was the most abundant com- Ala-310, Ile-312, Phe-348, Gly-351,
Asn-352
ponent in grape skin extract as detected by RP-HPLC–ESI/MS.
a
IC50, concerntration of inhibitor required to produce a 50% inhibition of the
initial rate of reaction, Vmax, maximum enzyme reaction rate, km, Michaelis–Menten
3.2. Alpha-amylase inhibition studies
constant, Ksv, Stern–Volmer quenching constant, Kq, quenching rate constant, n,
number of binding sites, Ka, apparent associative binding constant.
Alpha-amylase is a main enzyme involved in starch digestion b
Vmax was determine at 1 mg/ml of grape skin extract.
for hydrolysis of a-1,4-glycosidic internal linkages, which has been
suggested as a typical in vitro model for studying the effect grape
skin extract of on enzyme inhibitory activity and mode (Englyst inhibiting a-glucosidase. A dietary supplementation study also
et al., 1992). Inhibitory potency of grape skin extract was deter- demonstrated a significant beneficial effect of antioxidant-rich
mined, and % inhibition and IC50 are listed in Table 1. The percent- grape skin extract in improving glycaemic control and inflamma-
age of inhibition against a-amylase and concentration of grape tion in obese mice induced by a typical Western high fat diet (Ho-
skin extract for 50% inhibition were 70.81% and 1.35 mg/ml, gan et al., 2011). In rodent models of diet-induced obesity,
respectively. Zhang et al. (2011) reported that Norton grape skin resveratrol improves insulin sensitivity and lowers body weight,
extract at 14.3 lg/ml exerted significantly stronger inhibition than which has lead to much speculation about its potential as an
a commercial inhibitor acarbose at 285.7 lg/ml. The IC50 of Norton anti-diabetic in humans (Smoliga et al., 2011). Akkarachiyasit,
grape skin extract was 32-fold more effective than acarbose in Charoenlertkul, Yibchok-anun, and Adisakwattana (2010) reported
 M. Miao et al. / Food Chemistry 145 (2014) 205–211
that cyanidin-3-glucoside (IC50 = 0.3 mM) showed highest inhibi-
tion against pancreatic a-amylase than cyanidins and cyanidin-3-
galactoside and cyanidin-3,5-diglucoside, which indicated that gly-
cosylation of hydroxyl group improved the inhibitory effect against
a-amylase. According to Cer, Mudunuri, Stephens, and Lebeda
(2009), IC50 of an inhibitor was dependent on enzyme concentra-
tion and origin, substrate type and concentration along with other
experimental conditions including reaction duration, temperature
and pH.
The type of inhibitive mode of the pancreatic a-amylase inhib-
itors form grape skin extract was determined by the Michaelis–
Menten kinetic mode. Fig. 3 shows the double-reciprocal (Linewe-
aver–Burk) plots. These plots deduced inhibitive linear equation
for control as Y = 39.42X + 1.19 (R2 = 0.98), for 1 mg/ml grape skin
extract as Y = 72.05X + 2.14 (R2 = 0.97) and for 4 mg/ml grape skin
extract as Y = 175.79X + 5.18 (R2 = 0.99). Although the mathemati-
cal equations for all of the inhibitors and the control differ in slopes
and y-intercepts, their x-intercepts were nearly the same, indicat-
ing the type of enzyme inhibition belonged to the non-competitive
inhibition. A decreased reaction rate without affecting the en-
zyme’s affinity for substrate (Km = 33.85 mg/ml) was observed
(Fig. 3 and Table 1). Similar behaviour is reported in the literature
for enzyme kinetic studies of millet phenolics, which indicated that
the Michaelis–Menton constant remained constant (1% starch) but
the maximal velocity decreased, revealing a non-competitive type
of inhibition on pancreatic a-amylase (Shobana, Sreerama, & Mal-
leshi, 2009). Yoon and Robyt (2003) also reported that both acar-
bose and the acarbose analogue (maltohexaosyl acarbose or
maltododecaosyl acarbose) showed the non-competitive inhibition
for porcine pancreatic a-amylase.
3.3. Fluorescence quenching studies
Fluorescence quenching as a technique for measuring binding
affinities, refers to the decrease of fluorescence intensity from a
variety of molecular interactions. The fluorescence emission spec-
tra of pancreatic a-amylase at various concentrations of grape skin
extract following the excitation at 280 nm is present in Fig. 4. With
gradual increase in concentration of grape skin extract, a-amylase
showed a significant reduction of fluorescence intensity, which
was caused by the interaction between grape skin extract and a-
amylase. Meanwhile, a faint red shift of the maximum emission
wavelength from 348 to 351 nm was observed, which meant that
the polarity of enzyme environment was higher than that of the
pure solution. The result indicated that grape skin extract could
bind to a-amylase and quench the intrinsic fluorescence intensity.
According to Brayer, Luo, and Withers (1995), human pancreatic a-
amylase is composed of 496 amino acids, including 19 tryptophan
(Trp), 19 tyrosine (Tyr) and 23 phenylalanine (Phe), which use
50
40
1/V (ml×min/mg
30
20
10
0 0
-0.05 0.05 0.15 0.25
1/Q (ml/mg)
Fig. 3. Lineweaver–Burk plots of grape skin extract for the a-amylase inhibitory
activity. h: Control, r: 1 mg/ml, }: 4 mg/ml.
209
180
a
135 e
Flourescence intensity
90
45
0
300 350 400 450 500
Wavelength (nm)
Fig. 4. The fluorescence spectra of a-amylase at different concentrations of grape
skin extract: (a) 0, (b) 0.25, (c) 0.5, (d) 1.25, (e) 2 mM measured in phosphate buffer,
pH 5.2, kex = 280 nm.
excitation wave length at 280 nm and the intrinsic fluorescence
emission of a-amylase can be detected. The bimolecular binding
between grape skin extract and a-amylase caused changes in the
microenvironment of enzyme, hence quenching the fluorescence
intensity as suggested by Gonçalves et al. (2011). Wiese, Gärtner,
Rawel, Winterhalter, and Kulling (2009) reported that cyanidin-
3-glucoside quenched the tryptophan fluorescence of a-amylase
and upon ligand binding a change in protein structure was ob-
served related to the corresponding decrease in the a-amylase
activity. The association constants of 25 to 77  103 L/mol were
calculated for different proteins, indicating weak interactions of
non-covalent nature.
The fluorescence quenching process can be classified as a colli-
sion process, dynamic quenching mechanism, or a formation of a
ground-state complex between quencher and fluorophore, static
quenching mechanism. Both mechanisms can be distinguished by
their different dependence on the temperature and excited life-
time (Lakowicz, 2006). As shown in Fig. 5, the Stern–Volmer plots
were linear within the studied concentrations and the slope (Ksv)
decreased with increasing temperature, which indicated that only
static quenching mechanism occurred with a complex formation.
The calculated values of Ksv, Kq, n and Ka at 298 K are present in Ta-
ble 1. The value of Kq was 6.97  1011 L/(mol  s) and higher than
the maximum scatter collision quenching constant for quenchers
[2.0  1010 L/(mol  s)]. The value of n was approximately equal
to 1, which suggested that there was only one binding site in hu-
man pancreatic a-amylase for grape skin extract.
2.5
2
1.5
F/F0
1
0.5
0 0.4 0.8 1.2 1.6 2
Q (mM)
Fig. 5. Stern–Volmer plots for quenching of a-amylase by grape skin extract at 298
and 313 K, kex = 280 nm. j T = 313 K, d T = 298 K.
210 M. Miao et al. / Food Chemistry 145 (2014) 205–211
Fig. 6. Details of the interaction between a-amylase and grape skin extract: (a) general overview, and (b) best docked conformation.
3.4. Docking studies
The docking study of the grape skin extract at the human pan-
creatic a-amylase catalytic site showed a best pose with a total en-
ergy value of 118.3 kJ/mol, which was stabilised by hydrogen
bonds and Van der Waals forces interaction (Fig. 6 and Table 1).
As shown in Fig. 6B, the resveratrol-3-O-glucoside was surrounded
by Gln-302, Arg-303, Gly-304, Gly-309, Ala-310, Ile-312, Arg-346,
Phe-348, Gly-351 and Asn-352. Brayer et al. (2000) reported that
human pancreatic a-amylase belongs to the glycosyl hydrolase
family 13, which contain a characteristic (b/a)8-barrel catalytic do-
main and a variable number of other domains. There are three
structural domains, domain A (residues 1–99 and 169–404), forms
the eight-stranded parallel b-barrel on which are located the three
putative active site residues Asp197, Glu233 and Asp300, Domain
B (residues 100–168) forms a calcium binding site next to the wall
of the b-barrel of domain A, whilst domain C (residues 405–496) is
only loosely associated with the other two domains. As for the
hydrolysis of starch, this enzyme utilises a double displacement
catalytic mechanism in which a covalent b-glycosyl enzyme inter-
mediate is formed and hydrolysed by acid/base catalysis via an
oxocarbenium ion-like transition state (Maurus et al., 2008). Dur-
ing the reaction, Asp197 is likely nucleophile in catalysis, whilst
Glu233 and Asp300 act in the role of acid/base catalyst. It could
be concluded that grape skin extract occupied the binding site,
interacted with the side chain of Asp300, and formed hydrogen
bonds and Van der Waals forces with residues of the catalytic site,
which was in agreement with the only one binding site in fluores-
cence quenching studies (Table 1). Brayer et al. (2000) also re-
ported that substitution of the side chain of Asp300 lead to as
much as a 103 fold decrease in catalytic activity. According to
the study of Maurus et al. (2008), acarbose was anchored at the
catalytic centre and interacted with the side chain of Asp197,
Glu233 and Asp300, resulting in strong inhibition for human a-
amylase.
4. Conclusions
This study showed that grape skin extract was identified as res-
veratrol-3-O-glucoside. This type of polyphenol was an effective
non-competitive inhibitor of human pancreatic a-amylase, which
occupied one of binding sites, interacted with the side chain of
Asp300, and formed hydrogen bonds and Van der Waals forces
with residues of the catalytic site. The grape skin extract might
act as an antinutritional factor, in terms of its potential to inhibit
the activities of carbohydrate-hydrolysing enzymes. Further work
is underway to evaluate the possible side effect of natural inhibi-
tors through in vivo assays. The experimental results gained would
help discovery and development processes for designing novel
functional foods for controlling the digestion of starch and post-
prandial hyperglycaemia.
Acknowledgements
The research was financially supported by the Program of the
National Natural Science Foundation of China (31000764,
31230057), the National High Technology Research and Develop-
ment Program of China (2013AA102102), the Science & Technology
Pillar Program of Jiangsu Province (BY2012049, BE2012613).
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