Assay Method Information

Assay Name:  Determination of Xanthine Oxidase Inhibitory Activity of Aryl Benzofuran Amidated Derivatives and Reagents (Amino Derivatives)
Description:  11.1 Preparation of Reagents and Standard Solutions(1) 75 mM phosphate buffer (PB, pH 7.4): containing KH2PO4 0.0956 g, K2HPO4 0.6946 g, EDTA 1.862 mg, dilute to 50 mL with pure water, to be used to dilute samples and other reagents;(2) XOD solution: Take 25 U/2.6 mL of XOD, dilute to 0.08 U/mL of XOD working solution with 75 mM PB solution, mix well with a pipette, store on ice for future use;(3) Substrate preparation: accurately weigh the appropriate amount of xanthine (XA) into 5 mL of 0.1 N NaOH solution, dissolve it ultrasonically, and add 95 mL of 75 mM PB solution to prepare a substrate mother liquor with a final concentration of 0.48 mM, perform vortex mixing for 1 min; it is to be freshly prepared before each experiment;(4) Preparation of the test drug: accurately weigh the appropriate amount of the test drug, dissolve it in DMSO to prepare a 10 mM stock solution, and store at −20 C. in the dark. Use DB to dilute to different concentrations (0-100 mM) before the experiment, and the DMSO content is less than 0.1%.11.2 Steps(1) Add 100 μL of different concentrations of the sample solution to be tested onto a 96-well plate, add 0.08 U/mL XOD 50 μL, and use the same volume of PB as a blank control and allopurinol as a positive control; incubate for 3 min at 37 C. on a microplate reader, and set up 4 replicate wells in parallel for each group.(2) Add the substrate 0.48 mM XA 50 μL to start the reaction, read every 15 s at a wavelength of 295 nm and record the absorbance for a total of 7 min. Data processing: Data is processed by using Excel analysis and half-inhibitory concentration (IC50) is calculated by using GraphPad Prism 6.0.2.(3) Measurement results are shown in Table 1, which indicate that most of the synthesized benzofuran amidated derivatives have significantly xanthine oxidase inhibitory activity.
Affinity data for this assay
 

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