Establishment and Optimization of an In-vitro Method for Screening Anti-hyperuricemic Peptides
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Abstract:
Because UV spectrophotometry, NBT/PMS colorimetry, fluorescence analysis, MTS/PMS reduction and other xanthine oxidase (XO) inhibitory activity assays had limitations for the detection of peptides, a method for the screening and determination of anti-hyperuricemic peptides in vitro was established and optimized in this study. The in vitro xanthineoxidase inhibitory activity of peptides was determined by double enzymecoupling method combined with high performance liquid chromatography (HPLC). The optimal reaction conditions were determined by double-enzyme coupling method. The optimum reaction conditions were as follows: 0.22 mmol/L xanthine, 0.52 u/mL xanthine oxidase (XO), Tris-HCl pH 8.0, reaction temperature 30℃, and reaction time 20 min for catalytic reaction. Meanwhile, the optimal conditions for the determination of uric acid by HPLC were determined as follows: 95% 15 mmol/L NH4H2PO4 and 5% methanol at pH 6.5, 1 mL/min of flow rate, column temperature 25℃, and detection wavelength 290 nm. The results of double-enzyme coupling assay combing with HPLC method showed a good stability and higher accuracy than single HPLC and double-enzyme coupling detection method with the accuracy more than 95%. Double-enzyme coupling method combined with HPLC could effectively and accurately detect the activity of peptide-reduced uric acid. This novel detection method demonstrated that reduced glutathione (GSH) and oxidized glutathione (GSSG) had xanthine oxidase inhibitory activity which could be used as uric acidlowering peptides.
