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.     

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.     

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     

Physicochemical characterisation and α‐amylase inhibitory activity of tea polysaccharides under simulated salivary,gastric and intestinal conditions

Tea polysaccharides (TPS) are one of the main components of tea with various bioactivities. Digestion could result in the changes of the physicochemical properties and bioactivities of polysaccharides. The objective of this study was to determine the changes in physicochemical properties and α‐amylase inhibitory activities of TPS after simulated digestion. The structure of TPS determined by UV, IR and SEM showed that no obvious changes after salivary digestion, but significant changes after gastrointestinal digestion were observed. After intestinal digestion, the contents of reducing sugar were increased from 0.650 ± 0.011 to 1.594 ± 0.078 mm , and the centre molecular weight was decreased from 540.57 to 375.35 KDa. The molar ratio of monosaccharide was changed after digestion. And α‐amylase inhibition rate was significantly increased (P < 0.05). The results could provide information on the digestibility of TPSin vitro and contribute to the development of related functional foods or medicines.     

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.     

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.     

α‐Amylase inhibitors from an Indonesian medicinal herb,Phyllanthus urinaria

BACKGROUND: Diabetes mellitus and associated diseases are an increasing problem around the world. One of the hyperglycemic remedies is glucose absorption reduction by suppressing carbohydrate digestion due to utilization of α‐amylase inhibitors. RESULTS: Prospective herbs were analyzed by in vitro enzyme assay to evaluate their inhibitory activity against porcine pancreatic amylase (PPA), and it was found that Phyllanthus urinaria and three other herbs to showed a potent inhibitory activity. A 50% aqueous methanol‐soluble extract of the leaves of P. urinaria was chromatographed using a silica gel column. The active fractions were further purified by preparative high‐performance liquid chromatography to isolate active principles against PPA. Structural determination revealed that these isolated compounds were gallic acid, corilagin, and macatannin B, and showed mild activity against PPA (activity in 1 mmol L^?1 concentration: 23%, 21%, 33%, respectively). CONCLUSION: P. urinaria extracts show inhibitory activity against PPA. This activity, together with the information on isolated compounds, may benefit further exploration of P. urinaria utilization in the management of borderline diabetes patients. Copyright © 2011 Society of Chemical Industry     

Hypolipidemic Activity of Peony Seed Oil Rich in α‐Linolenic,is Mediated Through Inhibition of Lipogenesis and Upregulation of Fatty Acid β‐Oxidation

Peony seed oil (PSO) is a new resource food rich in α‐Linolenic Acid(ALA) (38.66%). The objective of this study was to assess the modulatory effect of PSO on lipid metabolism. Lard oil, safflower oil (SFO), and PSO were fed to wistar rats with 1% cholesterol in the diet for 60 d. Serum and liver lipids showed significant decrease in total cholesterol (TC), triglyceride (TG), and low density lipoprotein‐cholesterol (LDL‐C) levels in PSO fed rats compared to lard oil and SFO fed rats. ALA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), contents were significantly increased, whereas linoleic acid (LA), arachidonic acid (AA) levels decreased in serum and liver of PSO fed rats. Feeding PSO increased ALA level and decreased n‐6 to n‐3 polyunsaturated fatty acid (PUFA) ratio. The hypolipidemic result of PSO indicated that PSO participated in the regulation of plasma lipid concentration and cholesterol metabolism in liver. The decreased expression of sterol regulatory element‐binding proteins 1C (SREBP‐1c), acetyl‐CoA carboxylase (ACC), and fatty acid synthase (FAS)‐reduced lipid synthesis; Activation of peroxisome proliferator–activator receptor (PPARα) accompanied by increase of uncoupling protein2 (UP2) and acyl‐CoA oxidase (AOX) stimulated lipid metabolism and exerted an antiobesity effect via increasing energy expenditure for prevention of obesity.     

Isolation of a Recombinant Bacillus sp. TS‐23 α‐Amylase by Adsorption‐Elution on Raw Starch

A Bacillus sp. TS‐23 α‐amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCl buffer (pH 8.5). The adsorption‐elution procedure resulted in a yield of 53% α‐amylase activity and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α‐amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption‐ elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption‐elution cycles. These results suggest that the raw starch adsorption‐elution technique has a great potential in the isolation of Bacillus sp. TS‐23 α‐amylase from the culture broth of recombinant E. coli.     

Mechanisms of the Release of Bound β‐Amylase

The aim was to identify the mechanism(s) responsible for the release of bound β‐amylase in soluble forms in vitro and during the germination of barley. Preparations of bound β‐amylase, from barley, were treated with different agents including papain, thiols, starchy endosperm extract and reagents with amphipathic characteristics. The time courses of the release of the bound enzyme were compared using the different releasing agents. All the reagents tested caused some release of bound β‐amylase. Probably a very complex combination of mechanisms is involved in the release of bound β‐amylase as barley germinates. Initial release may be by cleavage of disulphide bonds which bind β‐amylase to insoluble protein. Material having amphipathic characteristics may break hydrophobic associations between β‐amylase and insoluble protein(s). The larger molecular weight isoforms are proteolysed to form the lower molecular weight materials present in malt. Limited proteolysis may also occur before the release of bound enzyme. Whether or not proteolysis is directly responsible for the release of bound enzyme remains uncertain.     

α‐Amylase immobilization on functionalized nano CaCO3 by covalent attachment

In this study, α‐amylase was immobilized on glutaraldehyde activated silanized calcium carbonate nanoparticles by a using covalent binding method. The surface modified nano calcium carbonate (CaCO₃) were characterized using FTIR and SEM. Immobilization yield was found as 199.43 mg/g of calcium carbonate nanoparticles. The maximum activity was observed at pH 6.5. The immobilized enzyme had a higher activity at elevated temperature (50–90°C) than the free one. Reuse studies demonstrated that the immobilized enzyme could reuse 25 times while retaining 18.2% of its activity. Free enzyme lost its activity completely within 15 days. V_max values for the free and immobilized enzymes were calculated as 10 and 0.35 mg/mL/min, respectively.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

Malting of Sorghum: Further Studies on Factors influencing α‐Amylase Activity

The effects of steep regime, nature of alkaline steeping agent, and kilning condition on α‐amylase development were studied for four Nigerian sorghum cultivars. Malt α‐amylase activity was highly significantly (p<.001) influenced by all the four factors as well as their various assortments of interaction. Generally malts from the Local Red (LR) variety produced the highest a‐amylase values, followed by those of SK 5912, Local White and KSV 8 in the above sequence. The presence/absence of air‐rest processes in steep regimes was a significant factor (p<.001) influencing malt α‐amylase response to final warm steeping as well as to the other factors under study. Similarly, the nature of the steeping agent was a very significant determinant of malt α‐amylase response to kilning condition and regime of steeping. Of significant interest was the observation that Ca (OH)₂ steeps enhanced malt α‐amylase activity at the higher temperature of kilning. The significantly lower α‐amylase values given under similar conditions by the other alkaline liquors suggest a possible increase in malt thermostability due to steeping in Ca (OH)₂. Additionally, the fact that the extent of enhancement of malt α‐amylase activity by Ca (OH)₂, at 50°C Kiln temperature, was regime‐dependent, suggests that the latter was an important modulator of sorghum germination physiology.     

Response surface optimization and characteristics of Indica rice starch‐based fat substitute prepared by α‐amylase

Response surface methodology (RSM) was used to study the effect of enzyme to substrate ratio (11.8–23.6 U α‐amylase/g rice starch), hydrolysis temperature (90–100°C) and pH value (6.0–6.6) on the gel strength of rice starches‐based fat substitute using α‐amylase hydrolysis. The optimum conditions obtained from response surface analysis was 16.52 U/g enzyme dosage, 92°C hydrolysis temperature while the influence of pH was found insignificant in the range tested. Under these optimum conditions, the gel strength of this fat substitute was 113 g/cm², very close to the gel strength of butter of 114 g/cm², while the solubility of the substitute was 1.33 ± 0.01% and the swelling power 4.85 ± 0.02. There were no observable differences in the granular size distribution between the untreated rice starch and the hydrolyzed rice starch. Rheological properties of this rice starch‐based fat substitute implied that it is easier for the substitute to form three‐dimensional networks under 34°C.     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.     

Purification of α‐amylase from human saliva by superparamagnetic particles

BACKGROUND: Several studies of magnetic carrier technology have focused on the application of separation technology, because the magnetic support can separate the target from the reaction medium by application of a magnetic field and because the magnetic carrier can be easily recovered. In the present study, superparamagnetic iron oxide (SPIO) modified by epichlorohydrin was employed as a support whose surface could be coated with starch. The starch‐coated support was used for isolating human salivary amylases. RESULTS: The results showed that the starch‐SPIO support could isolate amylases from human saliva with 91.1% recovery and 3.5‐fold purification to high specific activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the purifed amylase comprised two isoamylases with estimated molecular weights of 55 and 59 kDa. The activities of crude and purified amylases showed optimal pH values of 6–7 and 7 and optimal temperatures of 40 and 30 °C respectively. The thermal stability range for both crude and purified amylases was between 20 and 40 °C. CONCLUSION: The attachment of substrates to SPIO could offer a novel and efficient method for purifying enzymes either as an initial step prior to further purification or as a final step for diagnostic usage. Copyright © 2009 Society of Chemical Industry