Varioskan lux multimode microplate reader esw version 1

The α-amylase inhibition assay was carried out following the work of Yao et al. (2013 (link)) and Telagari and Hullatti (2015 (link)). Different concentrations (20, 40, 60, 80, 100, and 120 μg/mL) of standard acarbose and the sample AAHY extract were prepared. 50 μL of sodium phosphate buffer (100 mM, pH 6.8) was mixed with 10 µL α-amylase (2U/mL) soluble starch (1%). After 30 min of incubation at 37°C, 20 µL substrate, 1% soluble starch prepared in phosphate buffer 100 mM (pH: 6.8) was added and further re-incubated at 37°C for 30 min. The reaction was terminated by adding 100 µL dinitrosalicylic acid reagent solution and boiling for 10 min. The enzyme activity was estimated by measuring the absorbance at 540 nm using a microplate reader (Varioskan LUX multimode microplate reader, ESW version 1.00.38) from Thermo Fisher Scientific. The inhibition assay was performed thrice, and the inhibition percentage was calculated as follows: $\mathrm{\alpha }-\mathrm{A}\mathrm{m}\mathrm{y}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{y}\left(\%\right)=\left(1-\frac{Asample}{Acontrol}\right)\times 100,$ where Asample is the absorbance in the presence of both α-glucosidase and sample and Acontrol is the absorbance of the reaction mixture containing the same volume of buffer solution instead of the sample.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, described by Mosmann, was used to assess the anticancer effect of the extracts [75 (link)]. Cultured cells were harvested, and 5 × 10³ cells were seeded in a 96-well plate (final volumes of 100 µL per well) and further incubated for 24 h at 37 °C in a CO₂ incubator. Increasing concentrations (0–500 µg/mL) of the extracts were treated on the cells and further incubated for 48 h. A 15 µL volume of MTT solution (5 mg/mL) was added to each of the 96-well plates and incubated for 4 h at 37 °C. A 100 µg/mL amount of DMSO was added to each well to solubilize the water-insoluble purple formazan crystals formed. Absorbance was read at 570 nm using a microplate reader (Varioskan LUX multimode microplate reader, ESW version 1.00.38) from Thermo Fisher Scientific (Waltham, MA, USA). The generated dosage response curve was used to calculate the IC₅₀ and the percentage of cell growth inhibition.