ACE Inhibitory Peptides from Bellamya bengalensis Protein Hydrolysates: In Vitro and In Silico Molecular Assessment

Bellamya bengalensis muscle meat is known for ethnopharmacological benefits. The present study focuses on the identification of ACE inhibitory peptides from the proteolytic digests of muscle protein of Bellamya bengalensis and its underlying mechanism. After ultrafiltration of 120 min alcalase hydrolysates (BBPHA120) to isolate the small peptide fraction (<3 kDa), in vitro ACE inhibitory activity was analyzed. The IC50 value of the 120 min hydrolysate ultrafiltered fraction was 86.74 +/- 0.575 mu g/mL, while the IC50 of lisinopril was 0.31 +/- 0.07 mu g/mL. This fraction was assessed in a MALDI-ToF mass spectrometer and five peptides were identified from the mass spectrum based on their intensity (>1 x 10(4) A.U.). These peptides were sequenced via de novo sequencing. Based on the apparent hydrophobicity (%), the IIAPTPVPAAH peptide was selected for further analysis. The sequence was commercially synthesized by solid-phase standard Fmoc chemistry (purity 95-99.9%; by HPLC). The synthetic peptide (IC50 value 8.52 +/- 0.779 mu g/mL) was used to understand the thermodynamics of the inhibition by checking the binding affinity of the peptide to ACE by isothermal titration calorimetry compared with lisinopril, and the results were further substantiated by in silico site-specific molecular docking analysis. The results demonstrate that this peptide sequence (IIAPTPVPAAH) can be used as a nutraceutical with potent ACE inhibition.