Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE‐UV

Abstract

Prolyl hydroxylase domain 2 (PHD2) is a key enzyme regulating the expression of hypoxia inducible factor (HIF). Its inhibitors can improve the expression of HIF and downstream genes, which can treat hypoxia-related diseases. Therefore, the establishment of a reliable PHD2 inhibitors screening method is of great significance for the drug development of hypoxia-related diseases. In this work, an accurate, rapid, and simple screening method for PHD2 inhibitors was introduced by capillary zone electrophoresis (CZE). In order to improve the detection sensitivity, the derivative reaction of α-ketoglutaric acid (α-OG) and 1,2diaminobenzene (OPD) was used to enhance the UV absorption of α-OG (the substrate in the enzymatic reaction). The CZE method selected 20 mM Na 2 B 4 O 7 buffer (pH 9.0) as the separation buffer, +25 kV as the separation voltage, 25 • C as the cartridge temperature, and 210 nm as the detection wavelength. Under this condition, the analysis of a single sample can be realized within 9 min. Compared with the existing reported methods, the present work can directly screen the PHD2 inhibitory activity of traditional Chinese medicine (TCM) extracts, which is of significance for the target-purification of bioactive individual compounds from TCMs. Under the optimal conditions, the PHD2 inhibitor screening platform was successfully established, and it was found that 70% methanol/water extracts of Astragali Radix and Codonopsis pilosula had good PHD2 inhibitory activity. Furthermore, the present work provides a novel approach for screening the PHD2 inhibitory activity of TCM extracts and the discovery of anti-hypoxia bioactive compounds.

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…The CE-based enzyme assays are performed in two modes: (i) as precapillary assays with offline incubation of enzyme and substrate (and inhibitor) followed by CE analysis of substrate or product(s) [164,252] and (ii) incapillary assays, where the enzymatic reaction and analyte separation are performed in the same capillary [246,253]. The latter mode is performed either as electrophoretically mediated microanalysis with enzyme, substrate and inhibitor mixing due to their different electromigration velocities [254,255] or via their transverse diffusion of laminar flow profiles of their short in-series introduced closely neighboring zones [246,256,257].…”