Food Chemistry 145 (2014) 205–211
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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
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Article history: Received 1 May 2013 Received in revised form 19 July 2013 Accepted 14 August 2013 Available online 27 August 2013 Keywords: Grape skin extract Human a-amylase Inhibition Fluorescence quenching Docking
a b s t r a c t The objective of this work was to evaluate the structure–function relationship between grape skin extract and human a-amylase. The grape skin extract was characterised as resveratrol-3-O-glucoside by RPHPLC–ESI-MS, which showed strong inhibition towards a-amylase and the IC50 value was 1.35 mg/ml. The kinetic results demonstrated grape skin extract obeyed the non-competitive mode against amylase. 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 extract. Docking study showed a best pose with total energy value of 118.3 kJ/mol and grape skin extract interacted with side chain of Asp300 with hydrogen bonds and Van der Waals forces. This preliminary observation provides the basis for further evaluation of the suitability of grape skin extract as natural inhibitor with potential health benefits. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus and obesity have become major public health concerns worldwide, with the number of cases increasing exponentially in recent years. The multi-factorial aetiology of this worldwide epidemic, and the idea that dietary factor may contribute 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 necessary to reduce the complications and cost for controlling and preventing the metabolic syndromes, including diabetes and prediabetes, 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.
ibility and the corresponding postprandial glycaemic response, 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 duodenum 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 beneficial 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 release of glucose, which is essential to regular physiological processes 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 devoted 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 interactions of starch with different components present in the food system have become an innovative target for improvement of
E-mail addresses: [email protected], [email protected] (M. Miao). 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
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postprandial hyperglycaemia with fewer gastrointestinal side effects (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), antinutrients (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 glycaemic control by inhibition of amylolytic enzymes (Gonçalves, Mateus, & 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 flavonols, anthocyanins and procyanidins, which are can positively influence risk factors associated with cardiovascular health, cancer, diabetes, inflammation, neurodegenerative disease, and age-related 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 relevant to chronic diseases and/or longevity in humans (Smoliga, Baur, & Hausenblas, 2011). In particular, the stilbene resveratrol has shown potential for reducing hyperglycaemia, improving insulin 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 obtained from Changchun Dacheng Industrial Group Co. Ltd. (Changchun, 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 Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Polyphenols assays The grape skin extract sample (approximate 1 mg) was dissolved in 10 ml of methanol and then centrifuged (5000g for 10 min). The supernatant was filtered through a 0.22 lm membrane 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 processing data were performed by the version 3.1 MassLynx software. 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 polyphenol was detected by monitoring the elution at 520 nm. The electrospray ionization mass spectrometry parameters were as follows: 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 interscan 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 composed solvent A (10% water, v/v) and solvent B (60% acetonitrile, v/v); injection volume 5 ll; flow rate 1.0 ml/min; and quantification 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. Enzyme solution was prepared by suspending human pancreatic aamylase (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 supernatant into a beaker. The maize starch sample (200 mg) was dissolved 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 prepared by replacing the inhibitor solution with phosphate buffer. Percentage of pancreatic a-amylase inhibition was calculated according to the equation below:
%Inhibition ¼
ðAcontrol Acontrolblank Þ ðAsample Asampleblank Þ 100 Acontrol Acontrolblank
where Acontrol, Acontrolblank, Asample and Asampleblank refer to the absorbance 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% inhibition 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 spectrometer (Model 650–60, Hitachi, Tokyo, Japan) was used for Fluorescence 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 fluorescence intensities were obtained at different grape skin extract concentrations 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 constant Ka were obtained using the slopes and intercept of these
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linear plots (F0/F vs Q or log (F0/F 1) vs log Q), where F0 and F were the fluorescence intensities before and after the addition of the grape skin extract, respectively, Q was the concentration of the grape skin extract.
and Van der Waals forces interactions were also obtained from the docking results. 2.6. Statistical analysis
2.5. Docking studies
All results were analysed by the Duncan test using the statistical analysis system (SAS Institute, Cary, NC). A level of 0.05 was set to determine statistical significance.
The three-dimensional structure of human pancreatic a-amylase was imported from the Protein Data Bank (1HNY). The structure 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 studies of the inhibitory ligand at the amylase-binding site was performed 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 performed 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
20111117-7
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 Yawadio, Tanimori, and Morita (2007), HPLC coupled to mass spectrometry 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 comparison 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
A
3: Diode Array 300 Range: 3.009
14.15
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AU
2.0 1.5 1.0 5.0e-1 0.0 2.00
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20111117-7 833 (14.419)
1: TOF MS ES1.60e4
227.0
B
%
100
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185.0
0
m/z 60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
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C
Fig. 1. HPLC–ESI/MS chromatogram of polyphenol in grape skin extract. (A) HPLC profile, (B) Mass fragmentation pattern, (C) Structure of identified polyphenol.
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Fig. 2. HPLC chromatograms of piceid standard (A) and grape skin extract (B).
at 280 nm. Most phenolic compounds absorb at 280 nm because they have at least one aromatic ring in their structure, giving a nonspecific chromatogram (Lee et al., 2009). The peak was identified by the comparison with HPLC retention time, mass spectral data and relevant literatures (Lee et al., 2009; Yawadio et al., 2007). The MS spectrum of the grape skin extract is present in Fig. 1(B), whereas Fig. 1(C) shows the structure of identified polyphenol. The MS analysis of peak showed an [M]+ ion at m/z 390 and a major fragmentation in MS2 at m/z 227 (163 amu), which would correspond to the loss of a glucose moiety. The MS2 fragmentation of the ion at m/z 227 would be corresponding to the resveratrol. Thus, this peak was tentatively identified as resveratrol-3-O-glucoside, which was in agreement with the other published literatures (Ali et al., 2004; Zhou, Chen, & Zhong, 2007). The RP-HPLC chromatograms of piceid standard and grape skin extract are illustrated in Fig. 2. There was only one peak and its retention time was 16.10 min based on a liquid chromatography analysis, which indicated that resveratrol-3-O-glucoside was the most abundant component in grape skin extract as detected by RP-HPLC–ESI/MS. 3.2. Alpha-amylase inhibition studies Alpha-amylase is a main enzyme involved in starch digestion 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 et al., 1992). Inhibitory potency of grape skin extract was determined, and % inhibition and IC50 are listed in Table 1. The percentage of inhibition against a-amylase and concentration of grape skin extract for 50% inhibition were 70.81% and 1.35 mg/ml, respectively. Zhang et al. (2011) reported that Norton grape skin extract at 14.3 lg/ml exerted significantly stronger inhibition than a commercial inhibitor acarbose at 285.7 lg/ml. The IC50 of Norton grape skin extract was 32-fold more effective than acarbose in
Table 1 Summary of inhibiting, quenching and docking parameters of grape skin extract against human a-amylase. Grape skin extracta
Parameters
% Inhibition IC50 (mg/ml) Type Vmax (mg/ml min)b km (mg/ml) Ksv (mol/L) Kq (1011 L/mol s) n Ka (mol/L) Total energy (kJ/mol) Hydrogen bonds energy (kJ/mol) Van der Waals energy (kJ/mol) Hydrogen bonds contact residue
70.81 ± 2.16 1.35 ± 0.24 Non-competitive 0.47 ± 0.02 33.85 ± 1.32 1.63 ± 0.07 6.97 ± 1.04 1.3 ± 0.0 3786 ± 2.0 118.3 ± 2.5 31.2 ± 0.8 87.1 ± 1.3 Gln-302, Gly-304, Ala-310, Ile-312, Arg-346, Asn-352 Gln-302, Arg-303, Gly-304, Gly-309, Ala-310, Ile-312, Phe-348, Gly-351, Asn-352
Van der Waals forces contact residues
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 constant, Ksv, Stern–Volmer quenching constant, Kq, quenching rate constant, n, number of binding sites, Ka, apparent associative binding constant. b Vmax was determine at 1 mg/ml of grape skin extract.
inhibiting a-glucosidase. A dietary supplementation study also demonstrated a significant beneficial effect of antioxidant-rich grape skin extract in improving glycaemic control and inflammation in obese mice induced by a typical Western high fat diet (Hogan et al., 2011). In rodent models of diet-induced obesity, resveratrol improves insulin sensitivity and lowers body weight, which has lead to much speculation about its potential as an anti-diabetic in humans (Smoliga et al., 2011). Akkarachiyasit, Charoenlertkul, Yibchok-anun, and Adisakwattana (2010) reported
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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 spectra 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 aamylase. 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 aamylase is composed of 496 amino acids, including 19 tryptophan (Trp), 19 tyrosine (Tyr) and 23 phenylalanine (Phe), which use
180 a
Flourescence intensity
that cyanidin-3-glucoside (IC50 = 0.3 mM) showed highest inhibition against pancreatic a-amylase than cyanidins and cyanidin-3galactoside and cyanidin-3,5-diglucoside, which indicated that glycosylation 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 concentration 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 inhibitors form grape skin extract was determined by the Michaelis– Menten kinetic mode. Fig. 3 shows the double-reciprocal (Lineweaver–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 mathematical equations for all of the inhibitors and the control differ in slopes and y-intercepts, their x-intercepts were nearly the same, indicating the type of enzyme inhibition belonged to the non-competitive inhibition. A decreased reaction rate without affecting the enzyme’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, & Malleshi, 2009). Yoon and Robyt (2003) also reported that both acarbose and the acarbose analogue (maltohexaosyl acarbose or maltododecaosyl acarbose) showed the non-competitive inhibition for porcine pancreatic a-amylase.
135
e
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 cyanidin3-glucoside quenched the tryptophan fluorescence of a-amylase and upon ligand binding a change in protein structure was observed 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 collision 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 lifetime (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 Table 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 human pancreatic a-amylase for grape skin extract.
40 2.5
30
2
F/F0
1/V (ml×min/mg
50
20 10
-0.05
0
1.5 1 0.5 0
0.05
0.15 1/Q (ml/mg)
0.25
Fig. 3. Lineweaver–Burk plots of grape skin extract for the a-amylase inhibitory activity. h: Control, r: 1 mg/ml, }: 4 mg/ml.
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.
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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 pancreatic a-amylase catalytic site showed a best pose with a total energy 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 domain 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 intermediate is formed and hydrolysed by acid/base catalysis via an oxocarbenium ion-like transition state (Maurus et al., 2008). During 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 fluorescence quenching studies (Table 1). Brayer et al. (2000) also reported 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 aamylase. 4. Conclusions This study showed that grape skin extract was identified as resveratrol-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 inhibitors 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 postprandial 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 Development Program of China (2013AA102102), the Science & Technology Pillar Program of Jiangsu Province (BY2012049, BE2012613). References Akkarachiyasit, S., Charoenlertkul, P., Yibchok-anun, S., & Adisakwattana, S. (2010). Inhibitory activities of cyanidin and its glycosides and synergistic effect with acarbose against intestinal a-glucosidase and pancreatic a-amylase. International Journal of Molecular Sciences, 11, 3387–3396. Ali, Z., Ito, T., Tanaka, T., Nakaya, K.-I., Murata, J., Darnaedi, D., et al. (2004). Acetophenone C-glucosides and stilbene O-glucosides in Upuna borneensis. Phytochemistry, 65, 2141–2146. Aston, L. M. (2006). Glycaemic index and metabolic disease risk. Proceedings of the Nutrition Society, 65, 125–134. Björck, I., Liljeberg, H., & Östman, E. (2000). Low glycaemic-index foods. British Journal of Nutrition, 83, 149S–155S. Brasnyó, P., Molnár, G. A., Mohás, M., Markó, L., Laczy, B., Cseh, J., et al. (2011). Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the Akt pathway in type 2 diabetic patients. British Journal of Nutrition, 106, 383–389. Brayer, G. D., Luo, Y., & Withers, S. G. (1995). The structure of human pancreatic aamylase at 1.8 Å resolution and comparisons with related enzymes. Protein Science, 4, 1730–1742. Brayer, G. D., Sidhu, G., Maurus, R., Rydberg, E. H., Braun, C., Wang, Y., et al. (2000). Subsite mapping of the human pancreatic a-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry, 39, 4778–4791. Cer, R. Z., Mudunuri, U., Stephens, R., & Lebeda, F. J. (2009). IC50-to-Ki: A web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding. Nucleic Acids Research, 37, 441–445. Chuang, C.-C., Shen, W., Chen, H., Xie, G., Jia, W., Chung, S., et al. (2012). Differential effects of grape powder and its extract on glucose tolerance and chronic
M. Miao et al. / Food Chemistry 145 (2014) 205–211 inflammation in high-fat-fed obese mice. Journal of Agricultural and Food Chemistry, 60, 12458–12468. Coll, A. P., Farooqi, I. S., & O’Rahilly, S. (2007). The hormonal control of food intake. Cell, 129, 251–262. Englyst, K. N., & Englyst, H. N. (2005). Carbohydrate bioavailability. British Journal of Nutrition, 94, 1–11. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, 30S–50S. Gonçalves, R., Mateus, N., & de Freitas, V. (2011). Inhibition of a-amylase activity by condensed tannins. Food Chemistry, 125, 665–672. Hogan, S., Canning, C., Sun, S., Sun, X., Kadouh, H., & Zhou, K. (2011). Dietary supplementation of grape skin extract improves glycemia and inflammation in diet-induced obese mice fed a western high fat diet. Journal of Agricultural and Food Chemistry, 59, 3035–3041. Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F., et al. (2006). Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell, 127, 1109–1122. Lakowicz, R. J. (2006). Principles of fluorescence spectroscopy (3rd ed.). New York: Springer. Lee, J. H., Kang, N. S., Shin, S. O., Shin, S. H., Lim, S. G., Suh, D. Y., et al. (2009). Characterisation of anthocyanins in the black soybean (Glycine max L.) by HPLCDAD–ESI/MS analysis. Food Chemistry, 112, 226–231. Ludwig, D. S. (2002). The glycemic index: Physiological mechanisms relating to obesity, diabetes, and cardiovascular disease. Journal of the American Medical Association, 287, 2414–2423. Maurus, R., Begum, A., Williams, L. K., Fredriksen, J. R., Zhang, R., Withers, S. G., et al. (2008). Alternative catalytic anions differentially modulate human alphaamylase activity and specificity. Biochemistry, 47, 3332–3344. McDougall, G. J., Kulkarni, N. N., & Stewart, D. (2008). Current developments on the inhibitory effects of berry polyphenols on digestive enzymes. BioFactors, 34, 73–80. Miao, M., Jiang, B., & Zhang, T. (2009). Effect of pullulanase debranching and recrystallization on structure and digestibility of waxy maize starch. Carbohydrate Polymers, 76, 214–221. Miao, M., Zhang, T., Mu, W., & Jiang, B. (2010). Effect of controlled gelatinization in excess water on digestibility of waxy maize starch. Food Chemistry, 119, 41–48. Obiro, W. C., Zhang, T., & Jiang, B. (2008). The nutraceutical role of the Phaseolus vulgaris a-amylase inhibitor. British Journal of Nutrition, 100, 1–12.
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Semjonous, N. M., Smith, K. L., Parkinson, J. R. C., Gunner, D. J. L., Liu, Y. L., Murphy, K. G., et al. (2009). Coordinated changes in energy intake and expenditure following hypothalamic administration of neuropeptides involved in energy balance. International Journal of Obesity, 33, 775–785. Shobana, S., Sreerama, Y. N., & Malleshi, N. G. (2009). Composition and enzyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: Mode of inhibition of a-glucosidase and pancreatic amylase. Food Chemistry, 115, 1268–1273. Sies, H., Stahl, W., & Sevanian, A. (2005). Nutritional, dietary and postprandial oxidative stress. Journal of Nutrition, 135, 5969–5972. Smoliga, J. M., Baur, J. A., & Hausenblas, H. A. (2011). Resveratrol and health – A comprehensive review of human clinical trials. Molecular Nutrition & Food Research, 55, 1129–1141. Vislocky, L. M., & Fernandez, M. L. (2010). Biomedical effects of grape products. Nutrition Reviews, 68, 656–670. Wiese, S., Gärtner, S., Rawel, H. M., Winterhalter, P., & Kulling, S. E. (2009). Protein interactions with cyanidin-3-glucoside and its influence on a-amylase activity. Journal of the Science of Food and Agriculture, 89, 33–40. Yawadio, R., Tanimori, S., & Morita, N. (2007). Identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities. Food Chemistry, 101, 1616–1625. Yoon, S.-H., & Robyt, J. F. (2003). Study of the inhibition of four alpha amylases by acarbose and its 4IV-a-maltohexaosyl and 4IV-a-maltododecaosyl analogues. Carbohydrate Research, 338, 1969–1980. Zhang, G., & Hamaker, B. R. (2009). Slowly digestible starch: concept, mechanism, and proposed extended glycemic index. Critical Review in Food Science and Nutrition, 49, 852–867. Zhang, L., Hogan, S., Li, J., Sun, S., Canning, C., Zheng, S. J., et al. (2011). Grape skin extract inhibits mammalian intestinal a-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice. Food Chemistry, 126, 466–471. Zhou, M., Chen, X., & Zhong, D. (2007). Simultaneous determination of transresveratrol-3-O-glucoside and its two metabolites in rat plasma using liquid chromatography with ultraviolet detection. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 854, 219–223. Zunino, S. J. (2009). Type 2 diabetes and glycemic response to grapes or grape products. Journal of Nutrition, 139, 1794S–1800S.
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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
Article history: Received 1 May 2013 Received in revised form 19 July 2013 Accepted 14 August 2013 Available online 27 August 2013 Keywords: Grape skin extract Human a-amylase Inhibition Fluorescence quenching Docking
a b s t r a c t The objective of this work was to evaluate the structure–function relationship between grape skin extract and human a-amylase. The grape skin extract was characterised as resveratrol-3-O-glucoside by RPHPLC–ESI-MS, which showed strong inhibition towards a-amylase and the IC50 value was 1.35 mg/ml. The kinetic results demonstrated grape skin extract obeyed the non-competitive mode against amylase. 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 extract. Docking study showed a best pose with total energy value of 118.3 kJ/mol and grape skin extract interacted with side chain of Asp300 with hydrogen bonds and Van der Waals forces. This preliminary observation provides the basis for further evaluation of the suitability of grape skin extract as natural inhibitor with potential health benefits. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus and obesity have become major public health concerns worldwide, with the number of cases increasing exponentially in recent years. The multi-factorial aetiology of this worldwide epidemic, and the idea that dietary factor may contribute 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 necessary to reduce the complications and cost for controlling and preventing the metabolic syndromes, including diabetes and prediabetes, 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.
ibility and the corresponding postprandial glycaemic response, 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 duodenum 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 beneficial 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 release of glucose, which is essential to regular physiological processes 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 devoted 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 interactions of starch with different components present in the food system have become an innovative target for improvement of
E-mail addresses: [email protected], [email protected] (M. Miao). 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
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postprandial hyperglycaemia with fewer gastrointestinal side effects (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), antinutrients (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 glycaemic control by inhibition of amylolytic enzymes (Gonçalves, Mateus, & 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 flavonols, anthocyanins and procyanidins, which are can positively influence risk factors associated with cardiovascular health, cancer, diabetes, inflammation, neurodegenerative disease, and age-related 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 relevant to chronic diseases and/or longevity in humans (Smoliga, Baur, & Hausenblas, 2011). In particular, the stilbene resveratrol has shown potential for reducing hyperglycaemia, improving insulin 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 obtained from Changchun Dacheng Industrial Group Co. Ltd. (Changchun, 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 Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Polyphenols assays The grape skin extract sample (approximate 1 mg) was dissolved in 10 ml of methanol and then centrifuged (5000g for 10 min). The supernatant was filtered through a 0.22 lm membrane 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 processing data were performed by the version 3.1 MassLynx software. 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 polyphenol was detected by monitoring the elution at 520 nm. The electrospray ionization mass spectrometry parameters were as follows: 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 interscan 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 composed solvent A (10% water, v/v) and solvent B (60% acetonitrile, v/v); injection volume 5 ll; flow rate 1.0 ml/min; and quantification 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. Enzyme solution was prepared by suspending human pancreatic aamylase (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 supernatant into a beaker. The maize starch sample (200 mg) was dissolved 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 prepared by replacing the inhibitor solution with phosphate buffer. Percentage of pancreatic a-amylase inhibition was calculated according to the equation below:
%Inhibition ¼
ðAcontrol Acontrolblank Þ ðAsample Asampleblank Þ 100 Acontrol Acontrolblank
where Acontrol, Acontrolblank, Asample and Asampleblank refer to the absorbance 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% inhibition 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 spectrometer (Model 650–60, Hitachi, Tokyo, Japan) was used for Fluorescence 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 fluorescence intensities were obtained at different grape skin extract concentrations 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 constant Ka were obtained using the slopes and intercept of these
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linear plots (F0/F vs Q or log (F0/F 1) vs log Q), where F0 and F were the fluorescence intensities before and after the addition of the grape skin extract, respectively, Q was the concentration of the grape skin extract.
and Van der Waals forces interactions were also obtained from the docking results. 2.6. Statistical analysis
2.5. Docking studies
All results were analysed by the Duncan test using the statistical analysis system (SAS Institute, Cary, NC). A level of 0.05 was set to determine statistical significance.
The three-dimensional structure of human pancreatic a-amylase was imported from the Protein Data Bank (1HNY). The structure 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 studies of the inhibitory ligand at the amylase-binding site was performed 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 performed 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
20111117-7
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 Yawadio, Tanimori, and Morita (2007), HPLC coupled to mass spectrometry 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 comparison 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
A
3: Diode Array 300 Range: 3.009
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0
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80
100
120
140
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180
200
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C
Fig. 1. HPLC–ESI/MS chromatogram of polyphenol in grape skin extract. (A) HPLC profile, (B) Mass fragmentation pattern, (C) Structure of identified polyphenol.
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Fig. 2. HPLC chromatograms of piceid standard (A) and grape skin extract (B).
at 280 nm. Most phenolic compounds absorb at 280 nm because they have at least one aromatic ring in their structure, giving a nonspecific chromatogram (Lee et al., 2009). The peak was identified by the comparison with HPLC retention time, mass spectral data and relevant literatures (Lee et al., 2009; Yawadio et al., 2007). The MS spectrum of the grape skin extract is present in Fig. 1(B), whereas Fig. 1(C) shows the structure of identified polyphenol. The MS analysis of peak showed an [M]+ ion at m/z 390 and a major fragmentation in MS2 at m/z 227 (163 amu), which would correspond to the loss of a glucose moiety. The MS2 fragmentation of the ion at m/z 227 would be corresponding to the resveratrol. Thus, this peak was tentatively identified as resveratrol-3-O-glucoside, which was in agreement with the other published literatures (Ali et al., 2004; Zhou, Chen, & Zhong, 2007). The RP-HPLC chromatograms of piceid standard and grape skin extract are illustrated in Fig. 2. There was only one peak and its retention time was 16.10 min based on a liquid chromatography analysis, which indicated that resveratrol-3-O-glucoside was the most abundant component in grape skin extract as detected by RP-HPLC–ESI/MS. 3.2. Alpha-amylase inhibition studies Alpha-amylase is a main enzyme involved in starch digestion 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 et al., 1992). Inhibitory potency of grape skin extract was determined, and % inhibition and IC50 are listed in Table 1. The percentage of inhibition against a-amylase and concentration of grape skin extract for 50% inhibition were 70.81% and 1.35 mg/ml, respectively. Zhang et al. (2011) reported that Norton grape skin extract at 14.3 lg/ml exerted significantly stronger inhibition than a commercial inhibitor acarbose at 285.7 lg/ml. The IC50 of Norton grape skin extract was 32-fold more effective than acarbose in
Table 1 Summary of inhibiting, quenching and docking parameters of grape skin extract against human a-amylase. Grape skin extracta
Parameters
% Inhibition IC50 (mg/ml) Type Vmax (mg/ml min)b km (mg/ml) Ksv (mol/L) Kq (1011 L/mol s) n Ka (mol/L) Total energy (kJ/mol) Hydrogen bonds energy (kJ/mol) Van der Waals energy (kJ/mol) Hydrogen bonds contact residue
70.81 ± 2.16 1.35 ± 0.24 Non-competitive 0.47 ± 0.02 33.85 ± 1.32 1.63 ± 0.07 6.97 ± 1.04 1.3 ± 0.0 3786 ± 2.0 118.3 ± 2.5 31.2 ± 0.8 87.1 ± 1.3 Gln-302, Gly-304, Ala-310, Ile-312, Arg-346, Asn-352 Gln-302, Arg-303, Gly-304, Gly-309, Ala-310, Ile-312, Phe-348, Gly-351, Asn-352
Van der Waals forces contact residues
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 constant, Ksv, Stern–Volmer quenching constant, Kq, quenching rate constant, n, number of binding sites, Ka, apparent associative binding constant. b Vmax was determine at 1 mg/ml of grape skin extract.
inhibiting a-glucosidase. A dietary supplementation study also demonstrated a significant beneficial effect of antioxidant-rich grape skin extract in improving glycaemic control and inflammation in obese mice induced by a typical Western high fat diet (Hogan et al., 2011). In rodent models of diet-induced obesity, resveratrol improves insulin sensitivity and lowers body weight, which has lead to much speculation about its potential as an anti-diabetic in humans (Smoliga et al., 2011). Akkarachiyasit, Charoenlertkul, Yibchok-anun, and Adisakwattana (2010) reported
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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 spectra 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 aamylase. 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 aamylase is composed of 496 amino acids, including 19 tryptophan (Trp), 19 tyrosine (Tyr) and 23 phenylalanine (Phe), which use
180 a
Flourescence intensity
that cyanidin-3-glucoside (IC50 = 0.3 mM) showed highest inhibition against pancreatic a-amylase than cyanidins and cyanidin-3galactoside and cyanidin-3,5-diglucoside, which indicated that glycosylation 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 concentration 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 inhibitors form grape skin extract was determined by the Michaelis– Menten kinetic mode. Fig. 3 shows the double-reciprocal (Lineweaver–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 mathematical equations for all of the inhibitors and the control differ in slopes and y-intercepts, their x-intercepts were nearly the same, indicating the type of enzyme inhibition belonged to the non-competitive inhibition. A decreased reaction rate without affecting the enzyme’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, & Malleshi, 2009). Yoon and Robyt (2003) also reported that both acarbose and the acarbose analogue (maltohexaosyl acarbose or maltododecaosyl acarbose) showed the non-competitive inhibition for porcine pancreatic a-amylase.
135
e
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 cyanidin3-glucoside quenched the tryptophan fluorescence of a-amylase and upon ligand binding a change in protein structure was observed 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 collision 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 lifetime (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 Table 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 human pancreatic a-amylase for grape skin extract.
40 2.5
30
2
F/F0
1/V (ml×min/mg
50
20 10
-0.05
0
1.5 1 0.5 0
0.05
0.15 1/Q (ml/mg)
0.25
Fig. 3. Lineweaver–Burk plots of grape skin extract for the a-amylase inhibitory activity. h: Control, r: 1 mg/ml, }: 4 mg/ml.
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.
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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 pancreatic a-amylase catalytic site showed a best pose with a total energy 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 domain 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 intermediate is formed and hydrolysed by acid/base catalysis via an oxocarbenium ion-like transition state (Maurus et al., 2008). During 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 fluorescence quenching studies (Table 1). Brayer et al. (2000) also reported 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 aamylase. 4. Conclusions This study showed that grape skin extract was identified as resveratrol-3-O-glucoside. This type of polyphenol was an effective non-competitive inhibitor of human pancreatic a-amylase, which
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