Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

Inhibitory potential of phenylpropanoid glycosides from Ligustrum purpurascens Kudingcha against α‐glucosidase and α‐amylase in vitro

The leaves of Ligustrum purpurascens are used in a Chinese traditional tea called small‐leaved kudingcha, which is rich in phenylpropanoid glycosides (PPGs) and has many beneficial properties. Two critical exoacting glycoside hydrolase enzymes (glucosidases) involved in carbohydrate digestion are α‐glucosidase and α‐amylase. We investigated the properties of PPGs from L. purpurascens for inhibiting α‐amylase and α‐glucosidase activity in vitro and found IC₅₀ values of 1.02 and 0.73 mg mL^?1, respectively. The patterns of inhibiting both α‐amylase and α‐glucosidase were mixed‐inhibition type. Multispectroscopy and molecular docking studies indicated that the interaction between PPGs and α‐amylase and α‐glucosidase altered the conformation of enzymes, with binding at the site close to the active site of enzymes resulting in changed enzyme activity. Our studies may help in the further health use of small‐leaved kudingcha.     

The detection of α‐dicarbonyl compounds in wine by formation of quinoxaline derivatives

A method to determine the most abundant α‐dicarbonyl compounds in wine was developed by reaction with 2,3‐diaminobenzene. Products such as quinoxaline derivatives were detected by high‐performance liquid chromatography (HPLC) or by gas chromatography with a mass‐selective detector (GC–MS) or a thermoionic detector (GC–NPD). HPLC and detection with a spectrophotometer (313 nm) were used for routine quantitative analysis of wines. The method is sensitive, linear and has good repeatability. Diacetyl, pentane‐2,3‐dione, glyoxal and methylglyoxal were quantified in a single run; these compounds are always encountered in wines, but levels vary with different types of wine and also during fermentation and maturation processes. A new dicarbonyl compound, phenylglyoxal, was found in wine. The evolution of dicarbonyl compounds during fermentation is reported in this paper. © 2000 Society of Chemical Industry     

α‐Glucosidase and α‐amylase inhibitory activities of phloroglucinal derivatives from edible marine brown alga,Ecklonia cava

BACKGROUND: Diabetes mellitus (DM) is a chronic metabolic disorder characterized by defects in insulin secretion and action, which can lead to damaged blood vessels and nerves. With respect to effective therapeutic approaches to treatment of DM, much effort has being made to investigate potential inhibitors against α‐glucosidase and α‐amylase from natural products. The edible marine brown alga Ecklonia cava has been reported to possess various interesting bioactivities, which are studied here. RESULTS: In this study, five phloroglucinal derivatives were isolated from Ecklonia cava: fucodiphloroethol G ( 1 ), dieckol ( 2 ), 6,6′‐bieckol ( 3 ), 7‐phloroeckol ( 4 ) and phlorofucofuroeckol A ( 5 ); compounds 1, 3 and 4 were obtained from this genus for the first time and with higher yield. The structural elucidation of these derivatives was completely assigned by comprehensive analysis of nuclear magnetic spectral data. The anti‐diabetic activities of these derivatives were also assessed using an enzymatic inhibitory assay against rat intestinal α‐glucosidase and porcine pancreatic α‐amylase. Most of these phlorotannins showed significant inhibitory activities in a dose‐dependent manner, responding to both enzymes, especially compound 2 , with the lowest IC₅₀ values at 10.8 µmol L^?1 (α‐glucosidase) and 124.9 µmol L^?1 (α‐amylase), respectively. Further study of compound 2 revealed a non‐competitive inhibitory activity against α‐glucosidase using Lineweaver‐Burk plots. CONCLUSION: These results suggested that Ecklonia cava can be used for nutritious, nutraceutical and functional foods in diabetes as well as for related symptoms. Copyright © 2009 Society of Chemical Industry     

Inhibitory activities of kaempferol,galangin, carnosic acid and polydatin against glycation and α‐amylase and α‐glucosidase enzymes

The activities of four natural phenolics, kaempferol, galangin, carnosic acid and polydatin in scavenging free radicals, inhibiting advanced glycation end‐product (AGE) formation, α‐amylase and α‐glucosidase and trapping methylglyoxal (MGO), were evaluated in this study. Carnosic acid and galangin had the highest activity in scavenging free radicals. Kaempferol and galangin had the greatest activity in preventing bovine serum albumin (BSA) against glycation and reducing glycated proteins. Polydatin had the greatest performance in trapping MGO to reduce glycation reaction. However, there was no significant difference for kaempferol, galangin and carnosic acid in inhibiting AGE formation by BSA‐MGO reaction. Kaempferol, galangin and carnosic acid were the competitive inhibitors for α‐amylase, while kaempferol and carnosic acid were noncompetitive inhibitors for α‐glucosidase. However, polydatin showed as a mixed noncompetitive inhibitor for both α‐amylase and α‐glucosidase. The results indicated that the four natural phenolics have potential in inhibiting AGE production and the digestive enzymatic activity with different mechanisms.     

Validation of analytical method for α‐dicarbonyl compounds using gas chromatography–nitrogen phosphorous detector and their levels in alcoholic beverages

This study aimed to establish an analytical method for α‐dicarbonyl compounds (α‐DCs) including glyoxal, methylglyoxal and diacetyl, to determine the content of α‐DCs in 101 various alcoholic beverages using gas chromatography–nitrogen phosphorous detector (GC‐NPD) and to perform exposure assessment. The limit of detection and limit of quantification for α‐DCs were 0.05–0.22 and 0.15–0.70 μg g^?1, respectively. The accuracy and precision were validated in five matrices. The raspberry fruit wine had the highest value at 139.74 μg g^?1 total α‐DCs. The lowest α‐DC concentration among the beverages was detected in rice wine (Makgeolli) at 1.59 μg g^?1. The levels of α‐DCs in various samples were detected as follows: 1.59–56.68 μg g^?1 in rice wine (Makgeolli), 2.73–16.77 μg g^?1 in beer, 8.22–139.74 μg g^?1 in fruit wine and 8.17–91.56 μg g^?1 in rice wine (Cheongju). The estimated daily intake of α‐DCs in the intake‐only group and population group was calculated as 4.22–97.94 μg kg^?1 bw day^?1 and 0.28–7.13 μg kg^?1 bw day^?1, respectively.     

In vitro inhibition of α‐chymotryptic activity by phenolic compounds

α‐Chymotrypsin was modified by covalent attachment of selected phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, quinic acid, m‐/o‐/p‐dihydroxybenzene and p‐benzoquinone) at pH 9. The derivatives formed were characterised in terms of their activity and selected physicochemical properties. In vitro experiments showed that the proteolytic digestion of food proteins with α‐chymotrypsin derivatives was adversely affected. This decrease depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The derivatisation was accompanied by a reduction in the amount of free lysine and tryptophan residues. Moreover, the solubility of the derivatives decreased over a broad pH range, with a parallel increase in the hydrophobicity. The isoelectric point was shifted to a lower pH value, and formation of high‐molecular‐weight fractions was documented by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). © 2001 Society of Chemical Industry     

Screening grape seeds recovered from winemaking by‐products as sources of reducing agents and mammalian α‐glucosidase and α‐amylase inhibitors

Grape seeds collected from vinification of various grape varieties were extracted by supercritical CO₂ for oil recovery. The defatted residues thus obtained were considered as a re‐utilisable co‐product and assessed for phenolic content, reducing capacity and inhibitory activities against mammalian α‐amylase and α‐glucosidase enzymes. Supercritical CO₂ treatment led to higher recovery of anthocyanins. Reducing capacity of phenolic extracts reached up to ~2200 mmolFe(II) kg^?1, much higher than that of various natural phenolic sources. The anthocyanin‐rich extracts showed the highest inhibitory effectiveness towards α‐glucosidase (I₅₀ value equal to ~40 μg gallic acid equivalents (GAE)/mL ~ half than acarbose). Inhibitory effectiveness towards α‐amylase activity was similar among grape varieties, with I₅₀ values comparable to that of acarbose and correlated with proanthocyanidin contents. These results could pave the way for an efficient processing of grapes, including cascade processes, namely: winemaking, oil extraction from recovered grape seeds and phenolic extraction from defatted grape seeds as potential cost‐effective nutraceuticals.     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

Artificial simulated gastrointestinal digestion of four carbohydrates containing beta‐d‐1 → 4 linkages and new GC‐TQ/MS‐MS method for characterising released monosaccharides

Four types of carbohydrates, including Dendrobium officinale polysaccharide, Dendrobium aphyllum polysaccharide and β‐glucans from yeast and barley, were examined, and their structures were found to mainly contain 1,4‐linked‐β‐d ‐Glcp. Artificially simulated gastrointestinal digestion was conducted to characterise the changes of molecular weight, reducing sugars and released free monosaccharides by high‐performance liquid chromatography, kits and the newly developed gas chromatography (GC)‐mass spectrometry (MS)/MS analysis, which indicated that high molecular weight and complex spatial structures contributed to delayed monosaccharide release following exposure to digestive solution. The spatial structures of carbohydrates were changed during gastric digestion, but their primary structures were destroyed during intestinal digestion. Additionally, for the developed 7890A/7000 GC‐TQ/MS‐MS, the new analytical method was successfully used to analyse very low concentrations of monosaccharides in the simulated gastrointestinal digestive system.     

Concentration‐dependent antioxidant activities of conjugated linoleic acid and α‐tocopherol in corn oil

BACKGROUND: Antioxidants prevent rancidity (lipid peroxidation) and natural antioxidants, e.g., α‐tocopherol, likely provide additional value to oil‐based food products because of their health benefits. Conjugated linoleic acid (CLA) has potential health benefits and may exhibit antioxidant properties. The main aim of this study was to compare the antioxidant efficacy of α‐tocopherol, trans‐10, cis‐12‐CLA and cis‐9, trans‐11‐CLA (in graded concentrations) added to antioxidant‐stripped corn oil. RESULTS: As compared to α‐tocopherol, both CLA isomers displayed significant inhibition of corn oil lipid peroxidation induced by copper. Inhibition of thiobarbituric acid reactive substances (TBARS) were CLA concentration dependent for both isomers but with significant inhibition occurring at 0.1 and 1 ppm of CLA isomers or α‐tocopherol, respectively (P < 0.05). Graded concentrations of α‐tocopherol, and for both CLA isomers and time, had significant effects on TBARS formation (P < 0.0001). There were significant effects in interactions between graded concentrations and time for both CLA isomers (P < 0.0001) but not for α‐tocopherol (P > 0.05). CONCLUSION: CLA compounds could serve as useful food antioxidants and provide additional value because of their potential bioactivity in disease prevention. Copyright © 2007 Society of Chemical Industry     

Glycoalkaloids in potato tubers: the effect of variety and drought stress on the α‐solanine and α‐chaconine contents of potatoes

Six varieties of Solanum tuberosum L potato grown in the Bolivian highlands under drought stress, with and without irrigation, were analysed for their content of glycoalkaloids (GAs). The plant material consisted of three drought‐tolerant varieties from a local breeding programme (PROINPA), Potosina, Chapaquita and Pampeña, and three control cultivated varieties, Malcacho, Sani Imilla and Desiree, either susceptible or relatively tolerant to drought. α‐Solanine and α‐chaconine were quantified in both the peel and flesh of the tubers. A significant increase in GA concentration (α‐solanine + α‐chaconine) was observed under drought stress conditions in most varieties; average concentration increases of 43 and 50% were registered in the improved and control cultivars respectively. In all tested cultivars, however, the GA concentration remained lower than the recommended food safety level (200 mg kg⁻¹ fresh tubers). It ranged from 52.4 to 100 mg kg⁻¹ fresh tubers in the improved cultivars and from 55.6 to 122.3 mg kg⁻¹ fresh tubers in the controls. In the improved and control varieties the α‐solanine content averaged 42.6 and 35.4% of the total potato GAs respectively and was not significantly affected by drought stress, except in Desiree. In all conditions the peel contained the greatest proportion of total GAs. The hybrid variety Pampeña (new drought‐tolerant variety) contained the lowest amounts of GAs, which were lower than those of the control varieties, with and without irrigation. © 2000 Society of Chemical Industry     

α‐Glucosidase Inhibitory Activities of Fatty Acids Purified from the Internal Organ of Sea Cucumber Stichopus japonicas

α‐Glucosidase inhibitory activities of the various solvent fractions (n‐hexane, CHCl₃, EtOAc, BuOH, and water) of sea cucumber internal organ were investigated. 1,3‐Dipalmitolein (1) and cis‐9‐octadecenoic acid (2) with potent α‐glucosidase inhibitory activity were purified from the n‐hexane fraction of sea cucumber internal organ. IC₅₀ values of compounds 1 and 2 were 4.45 and 14.87 μM against Saccharomyces cerevisiae α‐glucosidase. These compounds mildly inhibited rat‐intestinal α‐glucosidase. In addition, both compounds showed a mixed competitive inhibition against S. cerevisiae α‐glucosidase and were very stable at pH 2 up to 60 min. The K_I values of compounds 1 and 2 were 0.48 and 1.24 μM, respectively. Therefore, the internal organ of sea cucumber might be a potential new source of α‐glucosidase inhibitors suitably used for prevention of obesity and diabetes mellitus.     

Bioaccessibility,cellular uptake and transepithelial transport of α‐tocopherol and retinol from a range of supplemented foodstuffs assessed using the caco‐2 cell model

The bioaccessibility, or amount of a nutrient available for gastrointestinal absorption, can be determined using an in vitro digestion model, the addition of the resultant digestate to a caco‐2 transwell model system yields an approximation of nutrient bioavailability. The objective of the present study was to compare the bioaccessibility and bioavailability of α‐tocopherol and retinol from a range of digested foodstuffs. Minced pork, beef and turkey and apple sauce, bread and mayonnaise were supplemented with α‐tocopherol‐acetate and retinol‐acetate prior to being subjected to an in vitro digestion procedure. The aqueous fraction of each of the digested foodstuffs was then added to a caco‐2 transwell model and the transepithelial transport was determined. The findings of the present study indicate that α‐tocopherol and retinol are more bioaccessible from supplemented meat products than from supplemented apple sauce, bread and mayonnaise. It was found that turkey meat facilitated the highest bioaccessibility and subsequent cellular uptake and transport of retinol. The cellular uptake and secretion of α‐tocopherol was similar for all samples.     

Light‐induced glycoalkaloid accumulation of potato tubers (Solanum tuberosum L)

The total glycoalkaloid concentration of non‐sprouted potato tubers, cvs Marfona, Fianna, Maris Piper, Kestrel and Golden Wonder, was measured following 15 days of continuous illumination (250 µmol m⁻² s⁻¹ photosynthetically active radiation) using high‐performance liquid chromatography. Comparisons were made of the influence of four sources of illumination: fluorescent tube‐type warm white, high‐pressure sodium, high‐pressure mercury types MB/U and MBFR/U. Irrespective of cultivar, glycoalkaloid concentrations of tubers placed in the dark remained relatively constant from day 0 to day 15 and did not exceed food safety values of 200 mg kg⁻¹ FW. A highly significant cultivar, light and cultivar × light (P < 0.01) interaction was recorded. Cultivar Kestrel was shown to be light‐sensitive with high rates of glycoalkaloid accumulation irrespective of light source, and cv Maris Piper was demonstrated to be light‐insensitive. In the majority of cultivars tested, maximal rates of glycoalkaloid accumulation were recorded following exposure of tubers to sodium or fluorescent light, and minimal rates of accumulation were recorded following exposure of tubers to mercury (MB/U and MBFR/U) illumination. In virtually all cases glycoalkaloid concentrations steadily increased with time during light exposure with no indication of cessation. In some instances the α‐chaconine/α‐solanine ratio decreased over 15 days of illumination (P < 0.05), indicating enhanced α‐solanine synthesis over that of α‐chaconine. The implications of these results in terms of consumer safety are discussed. © 1999 Society of Chemical Industry     

Visual Observation of Hydrolyzed Potato Starch Granules by α‐Amylase with Confocal Laser Scanning Microscopy

Porous starch was produced by digestion of freeze‐dried potato starch with α‐amylase from Bacillus sp. The surface structure of the granules became perforated and in the interior of the granules a capsule‐like cavity was formed, i.e. the hydrolyzed starch can be used as an encapsulant. The structure change of the granules was observed with confocal laser scanning microscopy and scanning electron microscopy. The degree of starch hydrolysis could be correlated with the Avrami equation. The activation energy of starch hydrolysis by α‐amylase was 83 kJ/mol.     

Effects of Different β‐D‐Glycosidases on Bound Aroma Compounds in Muscat Grape Determined by HS‐SPME and GC‐MS

Important “floral” aromas naturally occur in grapes predominantly as flavourless glycoconjugate precursors. Since these aroma compounds can be released by hydrolysis, different glycosidase enzymes can potentially contribute different aromas to wines. In this paper, we first established a procedure for profiling the free and bound volatile compounds in grape using GC‐MS combined with headspace solid‐phase microextraction (HS‐SPME). Comparison of the free and bound aroma compounds revealed that non‐volatile glycosides, known as aroma precursors, occur in high concentrations in musts. Among all compounds identified, 11 were fully quantified according to established standard calibration curves, while others were semi‐quantified. Using three different glycosidase enzymes, a total of 38 bound volatile compounds were identified in Muscat grape, including terpenes, higher alcohols, C‐6 compounds, and phenols, among others. The different enzymes had significant effects on the varietal aroma. Principal component analysis indicated that the characteristic aroma hydrolyzed by the commercial enzyme AR2000 was clearly different from that produced by other enzymes.     

A critical review of methodologies used in determination of relative bio‐availability ratio of RRR‐α‐tocopheryl acetate and all‐rac‐α‐tocopheryl acetate

Bio‐availability of different α‐tocopherol forms in livestock animals is measured by the increase in plasma or tissue concentrations of α‐tocopherol after oral administration. It is generally accepted that RRR‐α‐tocopheryl acetate (natural source vitamin E derived from vegetable oil) has a higher bio‐availability compared to all‐rac‐α‐tocopheryl acetate (synthetic vitamin E, i.e. α‐tocopherol produced by chemical synthesis). However, different bio‐availability ratios have been reported in the literature. The major reason for conflicting results in literature studies was the inability to separate the proportion of α‐tocopherol originating from test materials, from the proportion of α‐tocopherol originating from basal dietary ingredients and pre‐feeding. This causes significant variability. For bio‐availability determination, a baseline or control treatment is essential. The estimation of bio‐availability without correction for basal vitamin E status will lead to incorrect interpretation of the results. When using proper methodologies, it is possible to correct for the impact of α‐tocopherol intake from basal ingredients and α‐tocopherol originating from pre‐feeding, therefore yielding results reflecting the true relative bio‐availability of different α‐tocopherol substances. When reviewing literature data a critical evaluation of the method used in determination of relative bio‐availability is recommended. Copyright © 2010 Society of Chemical Industry     

Beneficial effect of the unsaponifiable matter from rice bran on oxidative stress in vitro compared with α‐tocopherol

Unsaponifiable matter (UM) was prepared from rice bran using n‐hexane extraction followed by removal of its fatty acid methyl ester with supercritical CO₂ under heat‐stable conditions. The UM was made up of 1% of vitamin E isomers, 28% of γ‐oryzanol and 71% of uncharacterized compounds. The aim of this study was to determine the antioxidant activities of the UM, using α‐tocopherol (α‐T) as a positive control, by measuring the Fe³⁺‐reducing antioxidant power (FRAP), 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) free‐radical‐scavenging property and lipid peroxidation in rat liver microsomes. In addition, the effects of the UM on the tert‐butyl hydroperoxide (t‐BOOH)‐induced cytotoxicity in cultured rat hepatocytes were also investigated. In FRAP assay and DPPH^? free‐radical‐scavenging assay, the results were expressed g^?1α‐T or g^?1 UM. The amount of UM used in lipid peroxidation assay and cytotoxicity assay was the amount required to have equal amounts of total vitamin E isomers in the sample and the control α‐T. The UM, as well as α‐T, exhibited significant antioxidant activities in FRAP, radical‐scavenging and membrane‐lipid oxidation. The FRAP value for total vitamin E isomers in the UM (TVEIUM) was 9.1 times higher than that for α‐T. In terms of their capacities to perform radical‐scavenging and lipid peroxidation, both TVEIUM and α‐T showed similar antioxidant activities. In experiments using cultured rat hepatocytes, the t‐BOOH‐induced lactate dehydrogenase release was significantly inhibited by the addition of 63.5 and 160 µg ml^?1 of TVEIUM treatments (84 and 89%, respectively), and that of 63.5 and 160 µg ml^?1 of α‐T treatment (88 and 93%, respectively). The antioxidant function against oxidative stress of the UM prepared from rice bran may extend its use to being a potential dietary supplement. Copyright © 2004 Society of Chemical Industry