Assay Of Α-amylase Research Articles

A facile approach synthesis of benzoylaryl benzimidazole as potential α-amylase and α-glucosidase inhibitor with antioxidant activity

Synthetic routes to a series of benzoylarylbenzimidazol 3a-h have been derived from 3,4-diaminobenzophenone and an appropriate arylaldehyde in the presence of ammonium chloride or a mixture of ammonium chloride and sodium metabisulfite as catalyst. The antioxidant activity of targeted compounds 3a-h has been measured by four different methods and the overall antioxidant evaluation of the compounds indicated the significant MCA, FRAP, and (DPPH-SA) of the compounds except for the compound 3h. In vitro antidiabetic assay of α-amylase and α-glucosidase suggest a good to excellent activity for most tested compounds. The target benzimidazole 3f containing hydroxyl motif at para-position of phenyl revealed an important activity inhibitor against α- amylase (IC50 = 12.09 ± 0.38 µM) and α-glucosidase (IC50 = 11.02 ± 0.04 µM) comparable to the reference drug acarbose. The results of the anti hyperglycemic activity were supported by means of in silico molecular docking calculations showing strong binding affinity of compounds 3a-h with human pancreatic α-amylase (HPA) and human lysosomal acid-α-glucosidase (HLAG) active sites that confirm a good to excellent activity for most of tested compounds.

Hypoglycemic and antioxidant effects of green tomato ( Physalis ixocarpa Brot.) calyxes’ extracts

The aim of this study was to evaluate the hypoglycemic and antioxidant potential of green tomato (Physalis ixocarpa Brot.) calyxes' extracts. Three methods were used to obtain the extracts: maceration (M), ultrasound-assisted (US), and infusion. Regarding in vitro hypoglycemic evaluation, glucose diffusion assay and enzymatic inhibitory action of α-amylase and α-glucosidase were performed. Whereas, for in vivo assessment an oral starch tolerance test (OSTT) was tested with aqueous extracts [infusion (40mg/kg b. wt.), maceration (M) water (98mg/kg b. wt.), and US water (82.24mg/kg b. wt.)] on male Wistar rats. Additionally, in vitro antioxidant activity of P. ixocarpa calyxes' was evaluated through inhibition of scavenging radical assay and lipid peroxidation. Extracts decreased the glucose diffusion in a range of 18%-56% compared with the negative control. Additionally, extracts inhibited α-amylase (above 80%) and α-glucosidase enzymes (above 90%). All groups treated with P. ixocarpa calyxes' significantly reduced the glucose levels at 120min (infusion=13.3%, M Water=12.7%, and US Water=19.4%) in comparison with the negative control, and similar levels to acarbose at 120min (13.1%). Finally, extracts showed IC50 values in a range of 2.5-6.6µg/µl for radical scavenging, and 118-199µg/µl for lipid oxidation. Our results show that P. ixocarpa calyxes' extracts induce hypoglycemia and antioxidant effects in vitro and in vivo. PRACTICAL APPLICATIONS: The green tomato is usually consumed in Mexico, the United States, and Central America. This fruit grows inside a calyx, which is considered an agro-food waste. However, some regions of Latin America have a traditional medicine purpose for diabetes affections. To the best of our knowledge, there are no published data that supports its hypoglycemic action. The information provided will be useful to nutraceutical applications that allow value-added products and sustainable green tomato production.

Disposable Stochastic Sensors Based on Nanolayer Depositon(s) of Silver, and AgC Composite on Plastic for the Assay of α-Amylase in Whole Blood and Saliva

Nanolayer deposition(s) of Ag, and AgC composite on a plastic substrate was used to design disposable stochastic sensors. The first sensor was based on an Ag nanofilm deposited on plastic; for the second sensor, an AgC composite was deposited on plastic, and for the third sensor on the plastic was deposited first a nanofilm of Ag, and on top of it a nanofilm of the AgC composite. α-Cyclodextrin was used to modify the active surface of the sensor. Wide linear concentration ranges were obtained as follows: for de assay of α-amylase in whole blood: 1.00x10-7-1.00x103 U mL-1, and for the assay of α-amylase in saliva: 1.5x10-15 - 1.5x102 U mL-1. α-Amylase was reliably determined from whole blood and saliva samples using the proposed disposable stochastic sensors.

Disposable Stochastic Sensors Based on Nanolayer Deposition(s) of Silver and AgC Composite on Plastic for the Assay of α-amylase in Whole Blood and Saliva.

Nanolayer deposition(s) of Ag and AgC composite on a plastic substrate was used to design disposable stochastic sensors. Three shapes of nanocoatings were tested. The first shape was obtained by deposition of a nanofilm of Ag on the plastic material; the second shape was obtained by deposition of a composite AgC nanolayer on the plastic material; the third shape was obtained by nanolayer deposition of a composite material of AgC on the top of the Ag nanofilm deposited on the plastic material. α-Cyclodextrin was used to modify the active surface of the sensor. Wide linear concentration ranges were obtained as follows: for de-assay of α-amylase in whole blood: 1.00 × 10−7–1.00 × 103 U mL−1 and for the assay of α-amylase in saliva: 1.5 × 10−15–1.5 × 102 U mL−1. α-Amylase was reliably determined from whole blood and saliva samples using the proposed disposable stochastic sensors.

Antidiabetic and Antioxidant Potential of GancidinW from Streptomyces Paradoxus VITALK03

Background:The aim of the present study was to analyse the antidiabetic and antioxidant potential of GancidinW (GW) extracted fromStreptomyces paradoxusVITALK03.Materials and Methods:Antidiabetic potential of GW was evaluated by assay of α-amylase and α-glucosidase inhibitory activity; haemoglobin glycosylation and yeast glucose uptake. The antioxidant potential of GW was assessed by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay and superoxide assay. The inhibition of α-amylase and α-glucosidase by GW was also studied byin Silicomolecular docking analysis.Results:GW (1mg/ml) showed 69.32% of α-amylase and 54.04% of α-glucosidase inhibitory activity. GW (1mg/ml) prevented haemoglobin glycosylation up to 30.92% and the glucose uptake by yeast cells was increased up to 64.38%. The binding interaction GW with α-amylase showed the least free binding energy of -6.09Kcal/mol and -7.53Kcal/mol with α-glucosidase by docking studies. GW also demonstrated moderate antioxidant activity in all the antioxidant assays performed.Conclusion:The results of this study suggests that the antidiabetic and antioxidant potential of GW can be probed further to develop GW as effective antidiabetic agent.

Novel assay procedures for the measurement of α-amylase in weather-damaged wheat.

The measurement of α-amylase (EC 3.2.1.1) in sprout-damaged grains is a crucial analysis yet a problematic one owing to the typically low α-amylase levels in ground wheat samples. A number of standardised methods such as the Falling Number method and the Ceralpha method exist which are routinely used for the assay of α-amylase. These methods, however, are either highly substrate-dependent or lack the required sensitivity to assess sprout damage. Novel colorimetric and fluorometric reagents have been prepared (Amylase HR, Amylase SD, BzCNPG7 reagent and BzMUG7 reagent) for the direct and specific assay of α-amylase activity in sprout-damaged wheat. Assays employing these reagents have been developed and optimised to include a decolourisation step using activated charcoal. When used in a convenient assay format, Amylase SD--containing EtNPG7 (II) as the colorimetric substrate and α-glucosidase as the ancillary enzyme--was found to be an excellent reagent for the assessment of sprout damage in wheat with incubation times as short as 5 min. The assay using Amylase SD is completely specific for α-amylase. The use of the Amylase SD assay represents a sensitive and valid alternative to the traditionally used Falling Number values for the assessment of sprout damage in wheat samples.

Synthetic Assembly of Bifluorescence-Labeled Glycopolymers as Substrates for Assaying α-Amylase by Resonance Energy Transfer.

To meet the need for a convenient substrate for sensitive and continuous assay for α-amylase, we developed a fluorescence resonance energy transfer (FRET)-based polymer substrate. Radical copolymerization of FRET-component monomers in different ratios of fluorogenic donor and acceptor was utilized to prepare such polymers. A glycomonomer as a fluorogenic donor was derived from naphthylmethylated maltotetraose, and a dansyl derivative monomer was used as an acceptor. Their mixture and acryl amide were copolymerized in a typical radical polymerization to yield a bifluorescence-labeled polymer in good yield. All of the polymers showed effective FRET and were used for the continuous assay of human salivary α-amylase. The time course of α-amylase reactions led to the apparent kinetic parameters of Km = 4 μM and Vmax = 0.29 nmol/min. The results strongly suggested that FRET-sensitive polymers are conveniently accessible and applicable for the sensitive determination of biochemical events.

Interference of thiol-compounds with dextrinizing activity assay of α-amylase by starch-iodine colour reaction: Modification of the method to eliminate this interference

Interference of Luria broth and other bacteriological media with the starch–iodine colour assay of the dextrinizing activity of α-amylase (Fuwa's method) was observed, complete bleaching occurring with 0.4ml of the Luria broth. The interference was found to be due to the thiol groups present in the medium which compete with starch for iodine. Among the various metal salts tested for counteracting the interference, ZnSO4 was found to be the best which reverted the colour to about 73–85% of that of the blank. A combination of hydrogen peroxide (10 μl of 30% solution) and CuSO4 · 5H2O (50 μl of 0.1 M solution) completely protected the starch–iodine reaction in the presence of even 0.5 ml of Luria broth and a modified assay was developed based on this finding. The colour intensity, however, was almost double than that obtained for the same amount of starch and iodine in the absence of these protective agents. Nevertheless, with different concentrations of starch as well as with varying amounts of enzymes, the modified method showed perfect linearity and could be effectively used for estimation of dextrinizing activity of α-amylase in the presence of thiol groups.

A new chromogenic substrate for assay and detection of α-amylase

A new soluble chromogenic substrate for α-amylase was prepared by coupling partially hydrolyzed starch with a dye, Ostazin brilliant red H-3B. The substrate is precipitable from buffered solutions with ethanol and is equally suitable for assay of α-amylase, detection of separated α-amylase isoenzymes in gels, and selection of microbial producers of the enzyme.

5] Multienzyme membrane electrodes

Multienzyme membrane electrodes, in which more than two different types of enzymes are utilized, have been developed for the assay of α-amylase, lactose, maltose, sucrose, and creatinine. This chapter focuses on α-amylase sensors and creatinine sensors. It is most important in enzyme electrodes to combine a permselective immobilized enzyme membrane with a special electrode. The combination acts selectively toward substances, which interfere with the electrode reaction. The permselectivity toward products or reagents of the reaction, such as hydrogen peroxide, is the most important function of the membrane in an enzyme electrode. If a polarographic probe consisting of a silver cathode and a platinum anode are used as an electrochemical sensor for hydrogen peroxide, the membrane covering the probe needs to be permeable to hydrogen peroxide but must exclude other electrically oxidative substances such as uric acid and ascorbic acid. Additionally, the membrane needs to have mechanical strength. An asymmetrically structured cellulose acetate membrane has these properties. Moreover, the porous layer of the asymmetric membrane, with its large surface area, is well suited as a support for the immobilization of a large amount of enzyme per unit area.

Measurement of cereal α-amylase: A new assay procedure

A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.

Description of an automated assay for measurement of α-amylase in vitro from rat parotid gland slices

1. A modified continuous assay of α-amylase is detailed and its usefulness in measuring amylase secretion from rat parotid salivary gland described. 2. The α-amylase secretion from the rat parotid gland can be evoked by acetylcholine, substance P and isoprenaline. 3. The secretion of amylase was greatest following isoprenaline stimulation and was of a slower time course than with the other two agonists.

Ethylidene protected substrate for the assay of human α-amylase

Ethylidene protected substrate for the assay of human α-amylase

Assay of α-amylase in soil and river sediments: Its use to determine the effects of heavy metals on starch degradation

α-Amylase (1,4-α- d-glucan glucanohydrolase EC 3.2.1.1) activity was assayed and characterized in soil and in starch amended river sediment. Zero order reactions between initial activity; length of incubation; soil or sediment weight; and substrate concentration were demonstrated. Optimum pH for α-amylase activity in soil was 5.0 and in sediment 5.9–6.2, while the temperature optima were 37°C and 45°C respectively. The addition of starch to samples of sediment led to a marked increase in α-amylase and numbers of amylolytic bacteria. An example is also given of how the assay can be used to determine the effects of heavy metals on α-amylase activity in a river sediment.

Radiochemical methods for the assay of α-amylase, glycogen synthetase and phosphorylase in the human endometrium

Radiochemical methods are described for the assay of α-amylase (E.C.3.2.1.1.), glycogen synthetase (E.C.2.4.1.11.) and phosphorylase (E.C.2.4.1.1.) activities in homogenates of human endometrial biopsy specimens. Data are presented on the optimal experimental conditions to be used as well as on the reliability of the proposed methods in terms of precision, accuracy and specificity. Using the methods described, it is possible to assay the three enzyme activities in as little as 20 to 25 mg of endometrial tissue.

Reducing value methods for maltodextrins: I. Chain-length dependence of alkaline 3,5-dinitrosalicylate and chain-length independence of alkaline copper

Molar reducing values for maltodextrins (maltose-maltoheptaose) were obtained using alkaline 3,5-dinitrosalicylate and alkaline copper reagents. It was found that equimolar quantities of the maltodextrins gave absorbance values with alkaline 3,5-dintrosalicylate that increased with the number of glucose units in the dextrin. Calcium ion (40 m M), however, repressed the increase in the absorbance values but did not completely eliminate overoxidation. The alkaline copper reagent gave a constant absorbance value for equimolar quantities of the maltodextrins. Thus, alkaline copper reagent gives a stoichiometric relationship with the number of hemiacetal, reducing groups, and alkaline 3,5-dinitrosalicylate gives a nonstoichiometric relationship. These results have an important consequence on the measurement of carbohydrase activity. The assay of α-amylase by alkaline 3,5-dinitrosalicylate gives a curvilinear relationship with enzyme concentration, whereas the assay by alkaline copper gives a linear relationship. The use of dinitrosalicylate will give erroneously high estimates of α-amylase activity, and it is clear that the effect of calcium on dinitrosalicylate reducing values has misled some workers into concluding that this ion affects α-amylase action patterns.

GEL-DIFFUSION METHOD FOR THE ASSAY OF α-AMYLASE

GEL-DIFFUSION METHOD FOR THE ASSAY OF α-AMYLASE

A MODIFICATION OF THE SANDSTEDT, KNEEN AND BLISH ASSAY OF α-AMYLASE

The Sandstedt, Kneen & Blish method for the assay of α-amylase uses as a measure of enzyme activity the reciprocal of the time taken for a standard decrease to occur in the starch-iodine blue colour of an incubation mixture containing a large quantity of added β-amylase. This method has been modified so that a photoelectric colorimeter is employed in conjunction with a standard graph. The precision of the method is thereby Improved and the work needed for each assay reduced. The principles involved could easily be applied to other enzyme assays.