Peptide Fractionation from Cissus quadrangularis by Freeze–Thaw Method and Its Antimicrobial Applications

This study aimed to isolate and characterize bioactive peptides from Cissus quadrangularis (L.) and evaluate their antimicrobial activity. Plant material was dried at 37°C, powdered, and stored at −20°C. Peptides were extracted using phosphate-buffered saline (pH 7.2) and Tris–NaCl buffer (pH 7.4), followed by homogenization and freeze–thaw cycles for effective cell disruption.The extract was clarified by centrifugation and filtration, and proteins were precipitated using 80% ammonium sulfate. The pellet was reconstituted, dialyzed (1–3 kDa cut-off), and lyophilized. Further purification by HPLC produced a distinct peak at 3.16 minutes. Peptide concentration, determined by the Bradford assay, increased during purification, reaching 80.7 µg/mL. SDS-PAGE analysis confirmed low-molecular-weight peptides (3–10 kDa).Antimicrobial activity was assessed using the agar well diffusion method against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and Citrobacter freundii. The peptides showed strong inhibition against Gram-positive bacteria, especially S. aureus and B. subtilis, with weaker effects on Gram-negative strains.These findings highlight Cissus quadrangularis as a promising natural source of antimicrobial peptides.