The porcine α-Amylase inhibitory activity of the samples was evaluated using the 3,5-dinitrosalicylic acid (DNSA) method described by Kwon et al. [2008] (link). The samples and standard (acarbose) were dissolved using phosphate buffer (0.02 M, pH 6.9 containing 6 mM NaCl) to a final concentration of 200-500 µg/mL and 1-9 µg/mL for the samples and standard, respectively. α-Amylase (purchased from Sigma-Aldrich (St. Louis, MO, USA) was also dissolved in phosphate buffer (0.02 M, pH 6.9 containing 6 mM NaCl) to a final concentration of 1 mg/mL. One hundred microliters of the samples or the standard were mixed with 100 µL of α-Amylase (200 units/mg) in a reaction tube and pre-incubated to 25 o C. Two hundred microliters of a starch solution (1%, prepared in 0.02 M phosphate buffer, pH 6.9 containing 6 mM NaCl) were added to the assay mixture and incubated for 10 min at room temperature. The reactions were halted after the mixture was incubated in boiling water for 5 min. Then, 1000 µL DNSA was added and the mixture was cooled. Two hundred microliters of the mixture were pipetted into the 96-well microplate after diluting to the ratio of 1:5 with double distilled water. The absorbance was then measured with a microplate reader at 540 nm. The percentage inhibition was then determined using Equation ( 7):
α amylase
Manufactured by Merck Group
Sourced in United States, Germany, China, Italy, Poland, United Kingdom, India, Spain, France, Switzerland, Singapore, Sao Tome and Principe, Canada, Sweden, Belgium, Australia
α-amylase is an enzyme commonly used in laboratory settings. It functions by catalyzing the hydrolysis of starch, glycogen, and related polysaccharides into smaller carbohydrate units such as maltose and glucose.
Automatically generated - may contain errors
Lab products found in correlation
α glucosidase, by Merck Group (109 mentions)
Pepsin, by Merck Group (99 mentions)
Acarbose, by Merck Group (79 mentions)
Pancreatin, by Merck Group (64 mentions)
Gallic acid, by Merck Group (57 mentions)
Amyloglucosidase, by Merck Group (46 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (46 mentions)
Quercetin, by Merck Group (44 mentions)
Bile salt, by Merck Group (37 mentions)
Lipase, by Merck Group (33 mentions)
Methanol, by Merck Group (31 mentions)
Bovine serum albumin (bsa), by Merck Group (30 mentions)
Folin ciocalteu reagent, by Merck Group (29 mentions)
Trypsin, by Merck Group (28 mentions)
Ascorbic acid, by Merck Group (25 mentions)
P nitrophenyl α d glucopyranoside, by Merck Group (24 mentions)
Hydrochloric acid (hcl), by Merck Group (23 mentions)
Sodium hydroxide (naoh), by Merck Group (23 mentions)
3 5 dinitrosalicylic acid, by Merck Group (23 mentions)
Sodium carbonate, by Merck Group (21 mentions)
493 protocols using α amylase
1
Porcine α-Amylase Inhibitory Activity Evaluation
2
Glycogen Analysis by Amylase Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described previously (1 (link)), α-amylase (Sigma-Aldrich, catalog number A4551) was reconstituted in PBS at 0.5 mg/mL. The glycogen fractions that were extracted from SC5314 and gsy1Δ/Δ or aliquots of commercial bovine liver or oyster glycogen (Sigma-Aldrich) were reconstituted in PBS and treated with α-amylase or PBS. Samples were incubated at 37°C for 60 min on a rotary shaker (20 rpm). Aliquots (200 μL) were stained with iodine solution pre- and post-amylase treatment, as described above, and were then measured at OD600.
3
Measuring α-Amylase Inhibitory Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The measurement of α-amylase inhibitory activity was carried out via the iodine reaction method as described by Wilson et al. with a slight modification [16 (link)]. Briefly, α-amylase (Sigma-Aldrich, St. Louis, MO, USA) derived from human saliva was dissolved in phosphate-buffered saline (PBS) at a concentration of 20 unit/mL. As a substrate for α-amylase, soluble starch was dissolved in PBS at a concentration of 1%. To measure the inhibitory activity against α-amylase, 290 μL of PBS, 10 μL of α-amylase solution (20 unit/mL), and 50 μL of the test substance were mixed and preincubation was performed at 37 °C for 10 min. After preincubation, 350 μL of 1% soluble starch as substrate was added and reacted at 37 °C for 30 min. To determine the amount of soluble starch remaining after the reaction, 300 μL of iodine solution (0.1% KI + 0.01% I2/0.05 N HCl) was added to the reaction solution and the absorbance was measured at 620 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Infinite 200 Pro; Tecan Austria GmBH, Grödig, Austria).
4
Extraction and Characterization of Rhodiola Polyphenols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rhodiola crenulata rhizomes were collected from Lhasa (Tibet, China). PAs extracted from Rhodiola crenulata rhizomes were named as RcPs. Saccharomyces cerevisiae α-glucosidase (type I, from Saccharomyces cerevisiae), α-amylase (from porcine pancreas), α-amylase (from human saliva), 6-hydroxy-2,5,7,8-tetramethylchromo-2-carboxylic acid (Trolox), 2,4,6-tripyridinyl-1,3,5-triazine (TPTZ), dimethyl sulfoxide (DMSO), 4-nitrophenyl-α-D-glucopyranoside (P-NPG), benzyl mercaptan, trifluoroacetic acid (TFA), catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, and gallic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Methanol of chromatographic purity was purchased from TEDIA (Fairfield, OH, USA). 2-choro-4-nitrophenyl-&-galactosyl-maltoside (Gal-g2-α-cnp) was purchased from Toyobo Co., Ltd., (Osaka, Japan). Neocuproine was purchased from Shanghai Aladdin Reagents Co., Ltd. (Shanghai, China). Vitamin C was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Sodium acetate, monobasic potassium phosphate, potassium diammonium phosphate, copper sulfate, methanol, ethanol, acetone, petroleum ether (60~90 °C boiling range), and other pure analytical reagents were purchased from Xilong Science Co., Ltd., (Guangdong, China).
5
Enzymatic Hydrolysis of Wasted Bread
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wasted bread was collected in local supermarket (Bari, Italy) and ground into small crumbs (<1 mm) with an Ika-Werke M20 laboratory grinder (GMBH, Staufen, Germany). The proximal composition of bread, made with wheat flour, was: moisture, 11.84%; proteins, 12.12%; fat, 1.38%; carbohydrates, 70.12%; and dietary fibers, 4.54%.
Pepsin (from porcine gastric mucosa, 400 U/mg of protein) (Sigma-Aldrich, United States), α-amylase (from porcine pancreas, 5 U/mg) (Sigma-Aldrich, United States), and the protease Veron PS (from Aspergillus oryzae, 227 U/g) (AB enzymes, Germany) were used for the treatment of the wasted bread; α-amylase and α-amyloglucosidase (from Aspergillus niger, 120 U/mg) (Sigma-Aldrich, USA) were used for the hydrolysis of the starch into glucose before quantification.
Pepsin (from porcine gastric mucosa, 400 U/mg of protein) (Sigma-Aldrich, United States), α-amylase (from porcine pancreas, 5 U/mg) (Sigma-Aldrich, United States), and the protease Veron PS (from Aspergillus oryzae, 227 U/g) (AB enzymes, Germany) were used for the treatment of the wasted bread; α-amylase and α-amyloglucosidase (from Aspergillus niger, 120 U/mg) (Sigma-Aldrich, USA) were used for the hydrolysis of the starch into glucose before quantification.
6
Purification of Hordeum vulgare Phosphatases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (HvPho1, HvPho1ΔL78 and HvPho1Asp383Ala) were produced in E. coli and purified to high homogeneity as described above. To ensure that no dextrin impurities are present in the reaction mix, the G1P stock (500 mM) was incubated overnight with α-amylase (Sigma A3403) and pullulanase (SigmaP2986). This mixture was then boiled at 100°C for 5 min and centrifuged at 22000 g to separate the proteins from the G1P solution. HvPho1 and HvPho1ΔL78 (5 ml of 50 μg∙ml-1) were dialyzed with 20 changes of dialysis buffer in 250 ml dialysis buffer (50 mM sodium citrate pH 6.5, 25 mM NaCl). The dialysis buffer was prepared according to the following procedure: 1 l of 250 mM sodium citrate, 125 mM NaCl pH 5.0 was incubated with 5 ml pullulanase (Sigma P2986) at 22°C for 1 h. After incubation, the pH was increased to pH 6.9 and 5 ml of α-amylase (Sigma A3403) were added and the mixture was incubated for an additional hour at 22°C. The pH of the buffer was then adjusted to pH 6.5 before it was diluted to 5 L and stored at 4°C. This dextrin free buffer was then used as dextrin free dialysis buffer.
7
Cascara Kombucha's α-Amylase Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The α-amylase inhibitory effect of the CFS derived from cascara kombucha was determined by adding 10 μL of pH-neutralized CFS or heat-treated CFS derived from cascara kombucha to a mixture of 90 μL of PBS and 50 μL of 4 μM α-amylase (Sigma-Aldrich) solution in a 96-well microplate (Gu et al., 2021 (link)). The control group received distilled water, whereas the group receiving acarbose was considered a positive control. The plate was then incubated at 37°C for 10 min. Then, 50 μL of a 0.5 mg/mL starch solution was added, followed by incubation at 37°C for 10 min. Subsequently, 10 μL of 1 M HCl was added and the color reaction was initiated by adding 100 μL of 3,5-dinitrosalicylic acid (DNS) (Sigma-Aldrich) color reagent and boiled for 20 min. The absorbance of each reaction was measured spectrophotometrically at a wavelength of 590 nm. The experiment was conducted in triplicate with three independent replications and changes in the reaction were calculated by comparison with the control groups.
8
α-Amylase Inhibition Assay: DNSA Method
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 3,5-dinitrosalicylic acid (DNSA) method was used to determine α-amylase inhibition activity [19 (link)] in which 200 μL each of extracts (20–360 μg/mL) and 3 unit/mL α-amylase (Sigma-Aldrich, USA) were mixed and incubated for 15 min at 37°C. An additional 5 min of incubation at 37°C was carried out after the addition of 200 μL of 1% starch solution. Then, a 200 μL of DNSA solution was added to it and the whole reaction mixture was heated for 10 min at 95°C. The final volume was adjusted to 5 mL with the addition of distilled water. The absorbance was measured at λ = 540 nm in a UV-visible spectrophotometer. The results were compared with the activities of acarbose and 1% dimethyl sulfoxide (DMSO) was used as a positive control.
9
Quantifying Plant Soluble Sugars and Starch
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Soluble sugars and starch were extracted from plants that were used for bulb respiration measurements, following the protocol of Landhausser et al. (2018) . Thirty mg of ground tissues were mixed with 1.5 mL of ethanol 80%, then heated at 90 °C for 10 minutes. A subsample (0.2 mL) of supernatant was set aside for soluble sugar determination. The pellet was washed twice with ethanol then kept under a fume hood overnight to evaporate the remaining ethanol. Starch in the pellet was digested with α-amylase (MilliporeSigma, Oakville, ON, Canada) at 85 °C for 30 minutes. Thereafter, supernatant containing maltose from starch was digested with amyloglucosidase (MilliporeSigma) at 55 °C for another 30 minutes. Glucose from starch was quantified colorimetrically at 415 nm following a reaction with p-hydroxybenzoic acid hydrazide (MilliporeSigma; Blakeney et al., 1980) .
Reducing sugars from the initial supernatant were quantified using the same method both before and after sucrose digestion with invertase, allowing us to calculate the quantity of sucrose along with that of reducing sugars.
Reducing sugars from the initial supernatant were quantified using the same method both before and after sucrose digestion with invertase, allowing us to calculate the quantity of sucrose along with that of reducing sugars.
10
In Vitro Digestion of Pulse Crops
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chickpeas (Cicer arietinum variety Kabuli), Yellow peas (Pisum sativum), Faba beans (Vicia faba), Red beans (Phaseolus vulgaris variety Kidney), green lentils (Lens culinaris) were generously donated by Pulse Canada (Manitoba, Canada). α-Glucosidase from Saccharomyces cerevisiae (≥100 units/mg protein), dipeptidyl peptidase IV human (recombinant expressed in Sf9 cells), α-amylase (from porcine pancreas, type VI-B, ≥5 units/mg solid), pancreatin (from porcine pancreas, 8xUSP specification), pepsin (from porcine gastric mucosa, ≥250 units/mg solid), Calcoflour white stain (calcofluor white M2R 1 g/L, evans blue, 0.5 g/L), Toluidine blue O, gallic acid, vanillin and catechin were purchased from Millipore Sigma (Burlington, MA, USA). Lactobacillus plantarum ATCC® 8014™ was purchased from Cedarlane (Burlington, ON, Canada). DeMan, Rogosa and Sharpe (MRS) broth, M17 broth and Bacteriological agar were purchased from Oxoid (Nepean, ON, Canada). DCTM Protein Assay Kit II and Ladder Precision Plus Protein dual color standards were purchased from BioRad (Mississauga, ON, Canada). GelCode blue safe protein stain was purchased from Fisher Scientific (Toronto, ON, Canada).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!