4 nitrophenyl α d glucopyranoside

The solvent for extractions, LC-MS and NMR analysis, Folin-Ciocalteu phenol reagent, 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), vitamin C, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate (K₂S₂O₈), phosphate-buffered saline (PBS) solution, Trolox, α-glucosidase enzyme (from Saccharomyces cerevisiae), and 4-nitrophenyl α-D-glucopyranoside (PNPG), were purchased from Sigma Aldrich (Milano, Italy).

Rat intestinal acetone powder, purchase from Sigma Aldrich, was used to obtain partially purified α-glucosidase. Enzymatic immobilization was done according to Hano et al. [32 (link)] on CNBr-activated sepharose 4B. Hano et al. [32 (link)] proposed chromogenic method was employed to evaluate enzymatic activity using end capped 0.45 μm polyethylene filter column. Briefly, intestinal fluid (1mL) was used to perform assay containing 4-nitrophenyl-α-D-glucopyranoside (5mM, 4NPG; Sigma). After half hour incubation at room temperature, solution was column filtered to stop reaction and sodium carbonate (1M) solution was added in equal volume. Increase in absorbance value compared to blank at 405 nm was used to determine enzymatic activity. The % enzyme inhibition for each sample was calculated by subtraction of absorbance values in the presence and absence of calli extracts.

Nicotinamide (N0636), streptozotocin (S0130), ethylenediaminetetraacetic acid (EDS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (H3375), imidazole (I0250), sodium dodecyl sulfate (L3771), ascorbic acid (A7506), malachite green (M6880), Tween^® 20 (P1379), Tris-HCl (T3253), MgCl₂ (M9272), glucose 6-phosphate (G7879), fructose 1,6-bisphosphate (F6803), adenosine 5′-monophosphate (A2252), 4-nitrophenyl α-D-glucopyranoside (N1377), and intestinal acetone powders from rat (I1630) were purchased from Sigma-Aldrich (Steinheim, Germany). Ammonium molybdate (AT0330-5) was bought from Tecsiquim (Mexico City, Mexico).

α-Glucosidase and 4-nitrophenyl α-D-glucopyranoside were obtained from Sigma Aldrich, USA. DNSA (3,5-dinitrosalicylic acid) was obtained from SRL Pvt. Ltd. (Mumbai, India). Copper sulphate, dipotassium hydrogen phosphate (K₂HPO₄), potassium dihydrogen phosphate (KH₂PO₄), methanol, sodium potassium tartarate, and sodium hydroxide (NaOH) were procured from Qualigens, Mumbai, India. Porcine pancreatic α-amylase and sodium chloride (NaCl) were obtained from HiMedia Laboratories, Mumbai, India. Acarbose was obtained from Bayer Pharmaceuticals Pvt. Ltd. (Mumbai, India). All the chemicals and reagents procured were of AR grade.

The reaction mixture contained 10 µL of extract (a minimum of five extract concentrations were tested in order to calculate the IC₅₀) and 30 µL of α-glucosidase (0.1 U/mL, G5003-100UN, Sigma-Aldrich, Merck, Darmstadt, Germany). It was incubated for 15 min at 37 °C in a microplate reader (SPECTROstar Nano Microplate Reader, BMG LABTECH, Ortenberg, Germany). Afterwards, 25 µL of 1 mM 4-nitrophenyl-α-D-glucopyranoside (N 1377, Sigma-Aldrich, Merck, Darmstadt, Germany) was added. The reaction mixture was then shaken and incubated at 37 °C for 10 min. The reaction was terminated by adding 60 µL of 0.2 M sodium carbonate solution. Blanks were prepared by adding the extract after the termination of the reaction. The absorbance at 405 nm was measured using a microplate reader. Enzyme without inhibitor was used as a negative control. The α-glucosidase inhibition percentage of blank corrected data was assessed using the following formula (2):
The results are expressed as concentration of extract (IC₅₀) in mg/mL that inhibited 50% of α-glucosidase.