A rapid assay to screen adenosine deaminase inhibitors from Ligustri lucidi fructus against metabolism of cordycepin utilizing ultra-high performance liquid chromatography-tandem mass spectrometry.

Cordycepin has recently received extensive attention for its abundant pharmacological activity. However, as adenosine deaminase (ADA) is widely distributed in mammal blood and tissues, cordycepin can be quickly metabolized after entering the body and become inactive metabolite 3'-deoxyinosine, leading to exert its effects difficultly alone. Therefore, we presented a novel UHPLC-MS/MS method, which was developed for screening ADA inhibitors against metabolism of cordycepin. Cordycepin and 3'-deoxyinosine were chosen as substrate and product. A proper separation was achieved for all analytes within 3 min. 3'-Deoxyinosine was quantified in presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product and it was not interfered by endogenous substance and the ADA inhibitors added. After optimizing the enzymatic incubation and UHPLC-MS/MS conditions, the ADA deamination of cordycepin with Km value was 95.18 ± 7.85 μM and Vmax value was 363.90 ± 12.16 μmol/min/unit. The oleanolic acid and ursolic acid were selected out as ADA inhibitors from Ligustri lucidi fructus with IC50 values of 21.82 ± 0.39 and 18.41 ± 0.14 μM. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.