α-glucosidase inhibitory activity of compounds 1 –3 and 5 -8 was evaluated according to the literature experimental method (Ma et al., 2014 (link)). A mixture including 25 μl of different compounds (final concentrations of 0.0625, 0.125, 0.25, 0.5, and 1.0 mM), 25 μl of α-glucosidase (0.2 U/ml, from baker’s yeast, Sigma), and 175 μl phosphate buffer (pH 6.8) was left to stand for 10 min at room temperature in a 96-well plate, and then 25 μl of 23.2 mM p-nitrophenyl α-D-glucopyranoside (Sigma-Aldrich) was added and further incubated at 37°C for 15 min. Finally, the absorbance was measured at 405 nm to determine the amount of p-nitrophenol cleaved by the enzyme using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.). The control was prepared by adding phosphate buffer instead of the sample in the same way as the test. The blank was prepared by adding phosphate buffer instead of α-glucosidase using the same method. The inhibition rates (%) = [(ODcontrol − ODcontrol blank) − (ODsample – ODsample blank)]/(ODcontrol − ODcontrol blank) × 100%. Acarbose was used as the positive control with an IC50 value of 0.72 ± 0.01 mM.
P nitrophenyl α d glucopyranoside
Manufactured by Merck Group
Sourced in United States, Germany, Australia, China, Italy, France, India, United Kingdom, Canada
P-nitrophenyl-α-D-glucopyranoside is a chemical compound used as a substrate in various laboratory techniques. It is a colorless, crystalline solid that is soluble in water and organic solvents. The compound is commonly used as a reporter molecule to detect and quantify enzymatic activity, particularly in the study of carbohydrate-metabolizing enzymes.
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Lab products found in correlation
Acarbose, by Merck Group (58 mentions)
α glucosidase, by Merck Group (49 mentions)
Gallic acid, by Merck Group (24 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (24 mentions)
Quercetin, by Merck Group (20 mentions)
α amylase, by Merck Group (20 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (18 mentions)
Methanol, by Merck Group (16 mentions)
3 5 dinitrosalicylic acid, by Merck Group (15 mentions)
Ascorbic acid, by Merck Group (13 mentions)
Formic acid, by Merck Group (13 mentions)
Yeast α glucosidase, by Merck Group (12 mentions)
Folin ciocalteu reagent, by Merck Group (12 mentions)
Acetonitrile, by Merck Group (12 mentions)
Sodium carbonate, by Merck Group (11 mentions)
Hydrochloric acid (hcl), by Merck Group (9 mentions)
Acetic acid, by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Soluble starch, by Merck Group (7 mentions)
Rutin, by Merck Group (7 mentions)
149 protocols using p nitrophenyl α d glucopyranoside
1
Evaluation of α-Glucosidase Inhibitory Activity
2
Porcine Pancreatic α-Amylase Inhibition
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The α-amylase inhibitory activity was assayed in the same way (Watanabe et al. 1997 (link)) as described for the α-glucosidase inhibitory assay, except that porcine pancreatic amylase (100 U, Sigma, St. Louis, MO) and blocked. p-Nitrophenyl-α-d -glucopyranoside (Sigma, St Louis, MO, USA) were used as enzyme and substrate, respectively.
3
Phytochemical Analysis of Botanical Extracts
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Folin & Ciocalteu’s phenol reagent, sodium carbonate (≥99.5%), 2,2′-diphenyl-1-picrylhydrazyl (95%) (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (97%; trolox), vanillin (99%), (+)-catechin hydrate (>98%), methanol (≥99.9%), hydrochloric acid (fuming 37%), aluminum chloride (99%), sodium nitrite (≥99%), α-glucosidase from intestinal acetone powders from rat, p-nitrophenyl-α-D-glucopyranoside (≥99%; p-PNG), acarbose (≥95%), potassium phosphate monobasic (≥99%), formic acid (≥98%), quercetin-3-O-glucoside (≥90%; Q-3-G), theobromine (≥98.5%), caffeine (≥98.5%), and quercetin (≥98.5%) were provided by Sigma-Aldrich (Milan, Italy). Potassium phosphate dibasic (≥ 98%) was acquired from Carlo Erba (Milan, Italy). Gallic acid, ethanol (≥99.9%), sodium hydroxide (1 M), (-)-epicatechin (>90%), procyanidin B1 (≥98.5%; PCB1), procyanidin B2 (≥98.5%; PCB2), protocatechuic acid (>97%), caffeic acid (≥95%), and vanillic acid (≥99%) were supplied by Fluka (Milan, Italy). Ultrapure water was prepared in a Milli-Q filter system (Millipore, Milan, Italy).
4
Quantification of Phenolic Compounds
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The following standards were used for the quantification of phenolic compounds: (−)-epicatechin; (+)-catechin; procyanidins B2, B3, C1; chlorogenic acid; and cryptochlorogenic acid from Extrasynthese (Genay, France). UPLC-grade water, prepared by using an HLP SMART 1000 s system (Hydrolab, Gdańsk, Poland), was additionally filtered through a 0.22 µm membrane filter immediately before use. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), phloroglucinol, hydrochloric acid, acetic acid, formic acid, sulfuric acid, ascorbic acid, acetonitrile, methanol for UPLC (gradient grade), sodium acetate, and sodium hydroxide, 3,5-dinitrosalicylic acid, potassium sodium tartrate tetrahydrate, sodium phosphate monobasic, starch from potato, α-amylase from porcine pancreas (type VI-8), dipotassium hydrogen orthophosphate dihydrogen, p-nitrophenyl-α-d -glucopyranoside, soybean lipoxygenase (type V), linoleic acid, xylenol orange and α-glucosidase from Saccharomyces cerevisiae (type I) were purchased from Sigma-Aldrich (Steinheim, Germany).
5
In Vitro Bioactivity Evaluation
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Soluble starch, D-glucose, α-glucosidase (type V from rice), p-nitrophenyl α-D-glucopyranoside, acarbose, 3,5-dinitrosalicylic acid (DNS), Folin-Ciocalteu reagent, gallic acid, quercetin, 6-hydroxy-2-5-7-8-tetramethylchroman-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulphate, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), sodium fluorescein, 2,4,6-tripyridyl-s-triazine (TPTZ), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA. α-amylase (Bacillus amyloliquefaciens) was purchased from Roche Diagnostics, USA. All the other chemicals and reagents were of analytical grade. All the analyses were carried out using high-throughput 96-well microplate readers (SpectraMax Plus384, Molecular Devices, USA and SPECTRAmax-Gemini EM, Molecular Devices Inc, USA).
6
Antioxidant and Enzyme Inhibitory Activities Evaluation
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Gallic acid, Folin-Ciocalteu phenol reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2 carboxylic acid (Trolox), 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazine (DPPH), 2, 2'-azobis (2-amidino-propane) dihydrochloride (AAPH), 2,4,6-tripyridyl-s-triazine (TPTZ), potassium persulfate, ferric chloride, fluorescein, p-nitrophenyl-α-D-glucopyranoside (PNPG), sodium acetate, DNS reagent, acarbose, sodium carbonate and alpha-glucosidase (type V from rice) enzyme were purchased from Sigma-Aldrich Inc., USA. alpha-amylase (Bacillus amyloliquefaciens) enzyme was purchased from Roche Diagnostics, USA. All other chemicals used were of analytical grade.
7
Isolation and Characterization of Enzymes
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The α-amylase and α-glucosidase enzymes were purchased from Megazyme. p-Nitrophenyl α-d -glucopyranoside, soluble starch, potassium sodium tartrate, 3,5-di-nitro salicylic acid (DNS), sodium hydroxide, dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene, linoleic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin C (ascorbic acid), vitamin E (α-tocopherol), Follin–Ciocalteau reagent, gallic acid and other chemicals and solvents of analytical grade were purchased from Sigma (MO, USA). Gravity flow chromatography columns were using Merck Kieselgel 60 F254 Art No 1.07734.1000 (63–200 µm) for fractionation process and 1.09385.1000 (40–63 µm) for isolation.
8
Antioxidant and Enzyme Inhibition Assays
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Caffeic acid, quercetin, quercetin-3-galactoside, gallic acid, p-coumaric acid, chlorogenic acid, rutin, luteolin, phloretin, epicatechin, kaempferol-3-glucoside, catechin, quercetin, procyanidin B1, pyrogallol, epicatechin 3′-O-gallate, 3-hydroxybenzoic acid, protocatechuic acid, benzoic acid, cinnamic acid, polydatin, Folin-Ciocalteu phenol reagent (FCR), α-amylase from aspergillus oryzae, porcine pepsin, acarbose, rat intestinal powder, ammonium molybdate, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), p-nitrophenyl-α-d-glucopyranoside, potassium persulfate, bile salts, LC-MS grade formic acid, and HPLC grade acetic acid were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Pancreatin was obtained from Alfa Aesar (Ward Hill, MA, USA). NaCl, Na2CO3, KCl, NaOH, HCl, NaHCO3, (NH4)2CO3, MgCl2(H2O)6, CaCl2, KH2PO4, K2HPO4, Na3PO4, HPLC grade methanol and acetonitrile were purchased from Chem-supply Pty Ltd. (Melbourne, VIC, Australia).
9
Comprehensive Enzyme Inhibition Assays
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Folin and Ciocalteu’s phenol reagent, 2-Aminoethyl diphenylborinate 97%, ABTS (2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), DPPH: 2,2-Diphenyl-1-picrylhydrazyl, Trolox®: (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, MOPS: 3-(N-Morpholino) propanesulfonic acid, 4-Morpholinepropanesulfonic acid (≥99.5%), Orlistat: tetrahydrolipstatin (≥98%), DMSO: Dimethyl sulfoxide, α-Amylase from porcine pancreas (EC3.2.1.1), α-Glucosidase from Saccharomyces cerevisiae (EC3.2.1.20), lipase from porcine pancreas (EC3.1.1.3), Dipeptidyl Peptidase IV human (EC 3.4.14.5), quercetin-3-d -galactoside (≥98%), delphinidin 3-O-glucoside (≥98%), malvidin 3-O-glucoside (≥98%) and cyanidin 3-O-glucoside (≥98%), acarbose (≥95%), 3,5-Dinitrosalicylic acid (≥98%), p-Nitrophenyl-α-d -glucopyranoside (≥99%), catechin (99%), rutin (94%), gallic acid (97.5%), vanillin (99%), sodium nitroprusside dihyrate (≥99%), sitagliptin, tributyrin, sodium taurodeoxycholate hydrate, 4-Nitrophenol, tert-butanol and Griess reagent, were purchased from Sigma Aldrich (St. Louis, MO, USA). DPP-IV GLO® protease assay kit G8351 was purchased from Promega (Madison, WI, USA). All chemicals were analytical grade.
10
Preparation of MRS Agar Medium
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The de Man, Rogosa and Sharpe (MRS) agar medium were purchased from Difco (USA). The agar medium, Bromocresol Purple (BCP), p-nitrophenyl-α-d -glucopyranoside, yeast α-glucosidase, and 3,4-dihydroxy-l -phenylalanine (DOPA) were purchased from Sigma-Aldrich (Sigma, MO, USA). All other chemicals or reagents used were of analytical grade.
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