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Phenolic acids are widely found in fruits and vegetables. The inhibitory effect of phenolic acids on α-amylase, a key enzyme for starch digestion, has attracted the attention of researchers. To further investigate the effects of different substituents on the benzene ring of phenolic acid on the inhibition of α-amylase activity, in vitro experiments and molecular docking were used. The structure-activity relationships of 17 phenolic acids with benzoic acid as the parent nucleus were analyzed by determining their half inhibitory concentration (IC50) toward α-amylase. The results showed that 2,3,4-trihydroxybenzoic acid had the strongest inhibitory effect on α-amylase with an IC50 value of 17.30 ± 0.73 mM. According to the structure-activity analysis, the hydroxyl group at the 2-position on the benzene ring had a strong positive effect on the inhibitory activity of α-amylase, while methoxylation at the 2-position and hydroxylation at the 5-position had a negative effect. Molecular docking revealed that hydrogen bonding and hydrophobic interactions were involved in the inhibition, with hydrogen bonding being the primary force. These findings provide a more comprehensive understanding of phenolic acids as inhibitors of α-amylase and provide new ideas for the design of dietary formulations for diabetic patients. [ABSTRACT FROM AUTHOR]

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In this study, the structure-activity relationship and interaction mechanism of nine flavonoids and porcine pancreatic α-amylase were studied through enzyme kinetics, multispectral analysis and molecular simulation. The analysis of the structure-activity relationship illustrated that hydroxylation at C4′ and C5′ position of flavonoids increased the inhibitory activity against α-amylase while the methylation at the corresponding position decreased it. According to the fluorescence quenching assay, all flavonoids could efficiently quench the intrinsic fluorescence of amylase by static mechanism and the thermodynamic parameters of representative Luteolin and Quercetin indicated the key role of hydrogen bonds and van der Waals interactions. The multispectral study suggested that the binding of flavonoids alters the secondary structure of α-amylase, leading to significant inhibition. Moreover, these findings were further confirmed by molecular docking which demonstrating the importance of hydrogen bonding and hydrophobic forces in flavonoid and porcine pancreatic α-amylase interactions.

The inhibitory mechanism of chlorogenic acid (CHA) against porcine pancreatic α-amylase (PPA) was examined by enzyme kinetic analysis, circular dichroism, fluorescence quenching and molecular docking. As a result, CHA showed a mixed-type inhibitory action on PPA, with the IC50 value of 0.498 ± 0.013 mg/mL. Analysis of fluorescence and circular dichroism spectra confirmed that CHA altered the secondary structure of PPA, by interacting with the amino acid residues around or distant from the catalytic site of PPA, mainly through hydrogen bonds, and this interaction was closely associated with the enzyme’s activity. Molecular docking indicated that the best pose between CHA and PPA was achieved with the binding energy of −7.8 and −7.2 kcal/mol at the active site and inactive site of PPA, respectively. The performed study reveals that CHA has the potential to inhibit the activity of α-amylase, thereby representing a novel idea to delay the digestion of starch.

Highlights • Binding of young apple polyphenols with α-amylase is an exothermal process. • Interaction constant values are in the similar orders for pure polyphenols. • α-Amylase inhibition by young apple polyphenols results from binding with the enzyme. • Young apple polyphenols can partially unfold the α-amylase structure. • The in-vitro and in-silico methods can be combined to reveal the interaction mechanism. Abstract The binding interactions between young apple polyphenols and porcine pancreatic α-amylase were investigated through isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and molecular docking. The results obtained were compared with those obtained through inhibition kinetics and fluorescence quenching. It was found that binding of tannic acid, chlorogenic acid, caffeic acid and epicatechin with α-amylase is an exothermal process, with the binding constants in the order of tannic acid > chlorogenic acid > caffeic acid > epicatechin. This is consistent with the orders of reciprocal of competitive inhibition constant and fluorescence quenching constant. The binding energy obtained through molecular docking showed the same order, except for epicatechin. These results are consistent with the inhibition of α-amylase being caused by the binding of the polyphenols with the enzyme. In addition, from the fluorescence quenching and DSC data, total polyphenols, tannic acid, chlorogenic acid and caffeic acid were found to partially unfold the enzyme structure. [ABSTRACT FROM AUTHOR]

Enzymatic dough improvers (DIs) are increasingly used as baking co-adjuvants. Herein, an array of techniques, including Western blotting, PCR, electrophoresis-based and shotgun proteomics, was addressed to identify the enzymes in six commercial DI preparations. In particular, this work sought to exclude the possible undeclared use of amylolytic enzymes from porcine (or other animal origin) pancreas in DIs. PCR-amplified mitochondrial cytochrome b (mt cyt b ) gene region and porcine pancreatic α-amylase were the targets of DNA-based and protein methods, respectively, both assuring a limit of detection lower than 0.5–0.1% (w/w). Aspergillum oryzae α-amylase and Hordeum vulgare (barley) β-amylase were the most represented enzymes in all DI samples. Although one sample was PCR-positive, none among the DIs contained porcine pancreatic enzymes. Comparative gas chromatographic analysis of fatty acids suggested that the porcine contamination might arise from hard fats of porcine origin (lard), emphasizing the need of performing analyses at the protein level when the targets are enzymes or proteins. [ABSTRACT FROM AUTHOR]

The starch from waxy/amylose extender (wx/ae) double-mutant rice is almost indigestible by porcine pancreatic α-amylase (PPA) and behaves like resistant starch, even though PPA digests waxy mutant (wx) and wild-type (WT) rice starches easily. To clarify the relationship between binding ability and susceptibility to PPA hydrolysis, we studied the PPA adsorption capacity of wx/ae starch. Results showed that the wx/ae starch adsorbed 80% more PPA than wx and WT starches, consistent with the ratio of their specific surface areas. This suggests that PPA adsorption capacity is determined by the specific surface area of starch granules. Thus, the resistance to digestion of the wx/ ae starch is not due to adsorption capacity, but to the higher-order nanostructure of the starch granules. The morphological features of digested wx/ae starch granules observed by scanning electron microscopy (SEM) suggest that the higher-order nanostructure of wx/ae starch is similar to that of resistant starches, such as high-amylose rice and potato starches. [ABSTRACT FROM AUTHOR]

Mesoporous silica particles (MSPs) can be used as food additives, clinically for therapeutic applications, or as oral delivery vehicles. It has also been discussed to be used for a number of novel applications including treatment for diabetes and obesity. However, a major question for their possible usage has been if these particles persist structurally and retain their effect when passing through the gastrointestinal tract (GIT). A substantial breaking down of the particles could reduce function and be clinically problematic for safety issues. Hence, we investigated the biostability of MSPs of the SBA-15 kind prepared at large scales (100 and 1000 L). The MSPs were orally administered in a murine model and clinically in humans. A joint extraction and calcination method was developed to recover the MSPs from fecal mass, and the MSPs were characterized physically, structurally, morphologically, and functionally before and after GIT passage. Analyses with N 2 adsorption, X-ray diffraction, electron microscopy, and as a proxy for general function, adsorption of the enzyme α-amylase, were conducted. The adsorption capacity of α-amylase on extracted MSPs was not reduced as compared to the pristine and control MSPs, and adsorption of up to 17% (w/w) was measured. It was demonstrated that the particles did not break down to any substantial degree and retained their function after passing through the GITs of the murine model and in humans. The fact the particles were not absorbed into the body was ascribed to that they were micron-sized and ingested as agglomerates and too big to pass the intestinal barrier. The results strongly suggest that orally ingested MSPs can be used for a number of clinical applications.

Abstract: High-amylose starches are attracting considerable attention because of their potential health benefits and industrial uses. Enzyme hydrolysis of starch is involved in many biological and industrial processes. In this paper, starches were isolated from mature grains of high-amylose transgenic rice line (TRS) and its wild type rice cultivar Te-qing (TQ). The morphological and structural changes of starch residues following porcine pancreatic α-amylase (PPA) hydrolysis were characterized by SEM, DSC, XRD, 13C CP/MAS NMR, and ATR-FTIR. TQ starch was hydrolyzed faster than TRS starch. PPA pitted the starch granule surface first, then penetrated into the interior and hydrolyzed the granule from the inside out. The peripheral region of subgranule and the surrounding band of TRS starch were more resistant to PPA hydrolysis than the external region of TQ starch. Both amorphous and crystalline (the long- and short-range ordered) structures were simultaneously hydrolyzed in TQ starch. A-type polymorph of TRS C-type starch was hydrolyzed faster than B-type polymorph. The long-range ordered structure was hydrolyzed faster than the amorphous structure, whereas the short-range ordered structure was hydrolyzed slower than the amorphous structure in TRS starch in the initial hydrolysis stage. The above results indicated that B-type crystallinity and short-range ordered structure in the peripheral region of subgranule and the surrounding band of TRS starch increased the resistance of TRS starch to PPA hydrolysis. [Copyright &y& Elsevier]

Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-αAI showed two polypeptide bands of ~35.8 and ~32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 °C. The finding and information obtained in the present investigation about novel isoforms of α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index. [ABSTRACT FROM AUTHOR]

Abstract: A series of (2-phenyl-4H-benzopyrimodo[2,1-b][1,3]thiazol-4-yliden-4-yliden)acetonitrile derivatives have been prepared by ring transformation reaction of 4-(methylthio)-2-oxo-6-aryl-2H-pyrane-3-carbonitriles. The yield of ring transformation product is moderate to good. Furthermore the glycosidase inhibitory activities were tested by using α-amylase and α-glucosidase pancreatic, intestinal and liver enzymes, responsible for hyperglycemia in type II diabetes. The results revealed that all compounds exhibit significant glycosidase inhibitory activity. [Copyright &y& Elsevier]

Abstract: Gelatinized barley starch was hydrolysed using porcine pancreatic α-amylase for various time intervals and the hydrolysate fractionated according to molecular weight distribution (low, medium and high) by gel permeation chromatography. The effects of hydrolysed starch fractions (2.5%, w/w) on the solution viscosity of low- (∼50%, w/w) and high- (∼88%, w/w) purity barley β-glucan (0.75%, w/w,) at different temperatures (20 and 37°C) were determined and compared to that of a control. The results indicated that none of the hydrolysed starch fractions significantly influenced the solution viscosity of high purity β-glucan. However, addition of the medium molecular weight fraction to low purity β-glucan significantly increased its viscosity when determined at low shear rates (1.29–12.9s−1). Marginal changes in viscosity were observed at shear rates exceeding 12.9s−1. This study suggested that a non-β-glucan component in the low purity β-glucan concentrate probably influences the solution viscosity of “β-glucan–hydrolysed starch” blends. [Copyright &y& Elsevier]

Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different α-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic α-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four α-amylases. The acarbose inhibition constants, Ki, for the four α-amylases were 270, 13, 1.27, and 0.80 μM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent α-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known α-amylase inhibitors. [Copyright &y& Elsevier]

[Display omitted] • Hydroxylation and methoxylation of flavonoids greatly influence the inhibition on PPA. • The structure of Luteolin exhibits potent inhibitory effect. • Hydrophobic interaction and hydrogen bonds are essential for the interaction. • Flavonoids could alter the secondary structure of PPA. In this study, the structure-activity relationship and interaction mechanism of nine flavonoids and porcine pancreatic α-amylase were studied through enzyme kinetics, multispectral analysis and molecular simulation. The analysis of the structure-activity relationship illustrated that hydroxylation at C4′ and C5′ position of flavonoids increased the inhibitory activity against α-amylase while the methylation at the corresponding position decreased it. According to the fluorescence quenching assay, all flavonoids could efficiently quench the intrinsic fluorescence of amylase by static mechanism and the thermodynamic parameters of representative Luteolin and Quercetin indicated the key role of hydrogen bonds and van der Waals interactions. The multispectral study suggested that the binding of flavonoids alters the secondary structure of α-amylase, leading to significant inhibition. Moreover, these findings were further confirmed by molecular docking which demonstrating the importance of hydrogen bonding and hydrophobic forces in flavonoid and porcine pancreatic α-amylase interactions. [ABSTRACT FROM AUTHOR]

Dexamethasone release from natural γ-cyclodextrin (γCD) complexes was investigated in presence of porcine pancreatic α-amylase (PPA). The phase-solubility of dexamethasone in aqueous γCD solutions was determined, PPA degradation of γCD was investigated, and permeation studies were performed in simulated tear fluid. The phase-solubility profile was of B s type and the stability constant (K 1:1) of the dexamethasone/γCD complex determined from the initial linear section of the profile was relatively high or 12887 M−1. The high K 1:1 value indicates that dexamethasone has high affinity for γCD under the test condition. From the PPA catalyzed γCD degradation studies the Michaelis-Menten constant (K m) and V max were determined to be 3.24 mM and 9.79 × 10−3 mM/min, respectively. The permeation studies performed at low γCD concentrations, showed that dexamethasone is released from the complex solutions at faster rate when PPA was present than when no PPA was present. [ABSTRACT FROM AUTHOR]

• Chlorogenic acid showed a mixed-type inhibitory action on α-amylase. • Chlorogenic acid altered the secondary structure of α-amylase through hydrogen bonds. • Chlorogenic acid interacted with α-amylase active site or far from its catalytic site. • Chlorogenic acid is a potential inhibitor of α-amylase. The inhibitory mechanism of chlorogenic acid (CHA) against porcine pancreatic α-amylase (PPA) was examined by enzyme kinetic analysis, circular dichroism, fluorescence quenching and molecular docking. As a result, CHA showed a mixed-type inhibitory action on PPA, with the IC 50 value of 0.498 ± 0.013 mg/mL. Analysis of fluorescence and circular dichroism spectra confirmed that CHA altered the secondary structure of PPA, by interacting with the amino acid residues around or distant from the catalytic site of PPA, mainly through hydrogen bonds, and this interaction was closely associated with the enzyme's activity. Molecular docking indicated that the best pose between CHA and PPA was achieved with the binding energy of −7.8 and −7.2 kcal/mol at the active site and inactive site of PPA, respectively. The performed study reveals that CHA has the potential to inhibit the activity of α-amylase, thereby representing a novel idea to delay the digestion of starch. [ABSTRACT FROM AUTHOR]

Native starches were isolated from mature grains of high-amylose transgenic rice TRS and its wild-type rice TQ and treated with 0.1% and 0.4% NaOH for 7 and 14 days at 35 °C. Alkali-treated starches were characterised for structural and functional properties using various physical methods. The 0.1% NaOH treatment had no significant effect on structural and functional properties of starches except that it markedly increased the hydrolysis of starch by amylolytic enzymes. The 0.4% NaOH treatment resulted in some changes in structural and functional properties of starches. The alkali treatment affected granule morphology and decreased the electron density between crystalline and amorphous lamellae of starch. The effect of alkali on the crystalline structure including long- and short-range ordered structure was not pronounced. Compared with control starch, alkali-treated TRS starches had lower amylose content, higher onset and peak gelatinisation temperatures, and faster hydrolysis of starch by HCl and amylolytic enzymes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study investigated the structural and functional properties of C-type starches from pea seeds, faba bean seeds, yam rhizomes and water chestnut corms. These starches were mostly oval in shape with significantly different sizes and contents of amylose, damaged starch and phosphorus. Pea, faba bean and water chestnut starches had central hila, and yam starch had eccentric hilum. Water chestnut and yam starches had higher amylopectin short and long chain, respectively. Water chestnut and faba bean starches showed CA-type crystallinities, and pea and yam starches had C-type crystallinities. Water chestnut starch had the highest swelling power, granule swelling and pasting viscosity, lowest gelatinization temperatures and enthalpy. Faba bean starch had the lowest pasting viscosity, whereas yam starch had the highest gelatinization temperatures. Water chestnut and yam starches possessed significantly higher and lower susceptibility to acid and enzyme hydrolysis, the highest and lowest RDS contents, and the lowest and highest RS contents, respectively., (Copyright © 2013 Elsevier Ltd. All rights reserved.)