A validated HPLC method for kallikrein activity assessment and its use in evaluating fruit extracts

The conventional ultraviolet-visible (UV-vis) spectrophotometric assay for kallikrein (KLK) activity, which monitors the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) at 253 nm, is prone to significant error due to spectral interference, especially in complex matrices. This study aimed to establish a robust high-performance liquid chromatography (HPLC) method to overcome this limitation. The method achieved complete separation of BAEE and its product, N-benzoyl-L-arginine (BA), with retention times of approximately 15.5 and 5.5 min, respectively. It demonstrated excellent linearity within 1-5 min of reaction time, along with high precision, repeatability and accuracy (mean recovery: 100.9%). The limit of detection (LOD) for BA was 0.25 mg/L. When applied to screen aqueous, ethanolic and pepsin-hydrolyzed extracts from eighteen fruits, the validated method revealed distinct modulatory effects. Ethanolic extracts tended to promote KLK activity, whereas aqueous extracts were predominantly inhibitory. Notably, pepsin hydrolysates exhibited the most potent and divergent effects, ranging from strong inhibition (e.g., kiwifruit) to marked activation (e.g., mango). These findings confirmed that the established HPLC method was a reliable tool for assessing KLK activity in complex samples and successfully identified specific fruit extracts as sources of novel KLK modulators.