For a glucosidase assay Dimethyl sulfoxide Reagent 995 Sodium phosphate

For a glucosidase assay dimethyl sulfoxide reagent

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For a-glucosidase assay: Dimethyl sulfoxide (Reagent ≥ 99.5%), Sodium phosphate monobasic (≥99.0%). Sodium Phosphate dibasic (≥ 99.0%), Acarbose (≥95%), a-glucosidase (from Saccharomyces cerevisiae, Lyophphilized powder), and 4-Nitrophenyl a-D- glucopyranoside were purchased from Sigma- Aldrich. Plants preparation The collected botanical parts were laid down and dried at room temperature. Each plants sample was grinded to powder using electric coffee grinder. Each sample was packed in plastic bag, given a number and stored in fridge (°C). Sample preparation To prepare the dried extract, different solvents Hexane, Methanol, Acetone, chloroform, Ethylacetate, Ethanol distilled water (both Hot and cold) were used to find the most active fraction exhibit a-glucosidase activity. 1gm of powdered plants of each sample was mixed with 10 ml of different solvents. The mixture was then shaken vigorously for 1
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Methodology and Result 4 minute, sonicated for 30 minutes using Grant ultrasonic bath and macerated for 24 hours. After filtration with qualitative filter paper using Fisher, each sample extract of hexane, acetone, ethyl acetate, methanol, chloroform the supernatant was left to dry at room temperature. Water sample extract was freeze-dried in order to evaporate the solvent. The dried extract was then dissolved in DMSO and buffered. A-glucosidase enzyme inhibition activity The a glucosidase enzyme inhibition activity was determined by an assay modified from (Yuan et al., 2012) A-glucosidase assayed by using 50ul of sample extract (dissolved DMSO), 100ul a-glucosidase enzyme solution (1U/ml) in 0.1 M phosphate buffer (pH 6.9) then incubated for in 96 well plates at 25c for 10 minutes. After pre incubation 50ul of the (p- nitrophenyl-a-D-glucopyranoside (PnPG) solution in 0.1 M phosphate buffer (6.9 pH) was added to each well. The reaction mixture then was incubated at 25c for 5 minutes before (at 0 minute) and after (at 5 minute) the incubation, the absorbance was taken at 405nm using TECAN infinite® 200 PRO microplate reader and compared to control that had 50ml of (buffer and DMSO). Sample results were also compared with acarbose as positive control (100, 10, 1, 0.1, 0.01, and 0.001) mole per millilitre. The a-glucosdase inhibitory activity was expressed as percentage inhibition and was calculated by: % inhibition = (absorbance of control-absorbance of extract / absorbance of control) x 100
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