Ultrospec 2100 spectrophotometer

The inhibitory effect of W. filifera extracts on xanthine oxidase activity was determined spectrophotometrically by monitoring the formation of uric acid at 295 nm. XO activity was measured according to the method previously reported.^{5 (link)} The reaction mixture contained 879 μL of 100 mM phosphate buffer pH 7.5, 50 μL of an aqueous solution of XO (0.5 U mL⁻¹) and 10 μL of the extract sample solution or the control sample solution. After mixing, 61 μL of 0.82 mM xanthine solution was added, and the enzyme activity was determined at 295 nm for 3 min at 25 °C. Allopurinol was used as the standard XO inhibitor. The inhibition potency for ChEs and XO was expressed as the IC₅₀ values, which represent the inhibitor concentration need to cause 50% inhibition of enzyme activity. The IC₅₀ values were calculated by interpolation in dose–response curves. The IC₅₀ values displayed represented the mean ± standard deviation for the three independent assays.
Spectrophotometric determinations were made in an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, England) using 1 cm path cells and with a FLUOstar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany).