P nitrophenyl glucopyranoside

α-Glucosidase enzyme ((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate (p-nitrophenyl glucopyranoside) were purchased from Sigma-Aldrich. Enzyme was prepared in potassium phosphate buffer (pH 6.8, 50 mM), and ploy-substituted-2,4-diarylbenzo[4,5]imidazo[1,2-a]pyrimidines 3a–ag, 4a, and 6a was dissolved in DMSO (10% final concentration). The various concentrations of these compounds (20 mL), enzyme solution (20 mL) and potassium phosphate buffer (135 mL), were added in the 96-well plate and incubated at 37 °C for 10 min. Then, the substrate (25 mL, 4 mM) was added to the mentioned mixture and allowed to incubate at 37 °C for 20 min. Finally, the change in absorbance was measured at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America). DMSO (10% final concentration) and acarbose were used respectively as control and standard drug. The percentage of enzyme inhibition was calculated and IC₅₀ values were obtained from non-linear regression curve using the Logit method^{67 (link)}. The statistical analyses were provided using SigmaStat 14.0 (Systat Software, Inc | Tools For Science).

All chemicals such as acetone, sodium hydroxide, hydrochloric acid, ethylacetate, sodium
phosphate, potassium ferricyanide, 1,1-diphenyl-2-picryl-hydrazil, gallic acid, hydrogen
peroxide, ascorbic acid, dinitrosalicylic acid, and p-nitrophenylglucopyranoside were
bought from Sigma-Aldrich (St Louis, MO), while all the assay kits used were procured from
(Randox Laboratories, Antrim, UK.

Acarbose, ranirestat, porcine pancreatic α-amylase, rat intestine acetone powder, aldose reductase, extra pure starch, dinitrosalicylic acid (DNS), p-nitrophenyl glucopyranoside (pNPG) and 2-chloro-4-nitrophenyl α-d-maltotrioside were obtained from Sigma-Aldrich, St. Louis, MO, USA. All other chemicals and reagents used are of analytical grade. Carpobrotus edulis leaves collected from the Agricultural Research Council—Vegetables, Industrial and Medicinal Plants campus in Pretoria, South Africa with voucher specimen Mulaudzi RB# 200 deposited in Bews Herbarium, University of KwaZulu-Natal, as described by Mulaudzi et al. [14 (link)] were lyophilised and ground into fine powders using a rotor mill (Fritsh Pulverisette 14, Labotec, Midrand, South Africa).

α-glucosidase enzyme (EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate (p-nitrophenyl glucopyranoside) were purchased from Sigma-Aldrich. α-glucosidase was dissolved in potassium phosphate buffer (50 mM, pH = 6.8) to obtain the initial activity of 1 U/ml. Then, 20 µl of this enzyme solution was added to 135 µl of potassium phosphate buffer and 20 µl of test compound at various concentrations in DMSO. After 10 min incubation at 37 °C, 25 µl of the substrate at a final concentration of 4 mM was added to the mixture and allowed to incubate at 37 °C for 20 min. Then, the change in absorbance was measured at 405 nm spectroscopically. DMSO (10% final concentration) as control and acarbose as the standard inhibitor were used. The percentage of inhibition for each entry was calculated by using the following formula: $$\text{\%}\text{Inhibition}=\left[\left(\text{Abs control}-\text{Abs sample}\right)/\text{Abs control}\right]\times 100$$ IC₅₀ values were obtained from the nonlinear regression curve using the Logit method^{15 (link),29 (link)}.

Saccharomyces cerevisiae (S. cerevisiae) form of α-glucosidase (EC3.2.1.20, 20 U/mg) and p-nitrophenyl glucopyranoside (substrate) were purchased from Sigma-Aldrich. α-Glucosidase solution was produced in potassium phosphate buffer (PPB, pH 6.8, 50 mM) and phthalimide-phenoxy-1,2,3-triazole-N-phenyl (or benzyl) acetamides 11a–n were dissolved in DMSO (10% final concentration)^{18 (link)}. The various concentrations of the latter compounds 11a–n (20 µL), α-glucosidase solution (20 µL) and PPB (135 µL) were added in the 96-well plate and incubated at 37 °C for 10 min. Then, substrate (25 µL, 4 mM) was added to this plate and allowed to incubate at 37 °C for 20 min. The change in absorbance was measured at 405 nm using by a standard spectrophotometer (Gen5, Power wave xs2, BioTek, America). Acarbose and DMSO (10% final concentration) were used as positive and negative controls, respectively.

The chemical reagents of α-glucosidase from Saccharomyces cerevisiae, p-nitrophenyl glucopyranoside (pNPG), lipase type II from porcine pancreas, p-nitrophenyl butyrate (pNPB), vanillic acid, 4-aminoantipyrine, horseradish peroxidase, tyramine, MAO-A, galantamine, acetylthiocholine iodide (ATCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), Tris, acetylcholinesterase (AChE), levodopa (L-DOPA), and tyrosinase were acquired through Sigma-Aldrich (Madrid, Spain); clorgyline and α-kojic acid were from Cymit quimica (Barcelona, Spain); MgCl₂·6H₂O, HCl, NaCl, and potassium phosphate were from Panreac (Barcelona, Spain).