Characterization and Antifungal Activity of a Hevein-Like Peptide-Containing Extract From Capsicum annuum Leaves...

This study is aimed at isolating and characterizing antimicrobial peptides (AMPs) from the leaves of Capsicum annuum (L.) cv. Carioquinha and investigating their antifungal activity and mechanism of action against phytopathogenic fungi. An initial extract was obtained from leaves through hydromethanolic extraction followed by partitioning with dichloromethane. The extract was resuspended in aqueous acetonitrile and centrifuged to obtain the soluble supernatant fraction (ExS). The ExS fraction was purified by reverse-phase high-performance liquid chromatography (RP-HPLC). Protein bands from the ExS fraction were excised from Tricine-SDS-PAGE gels and analyzed by LC-MS/MS. The obtained peptide sequences were compared with protein databases. Antifungal activity of the ExS fraction was evaluated through quantitative fungal growth inhibition assays against the phytopathogenic fungi Fusarium solani, Fusarium oxysporum, Colletotrichum gloeosporioides, and Colletotrichum scovillei, and tests were conducted to investigate the mechanism of action of the peptides present in the ExS fraction. RP-HPLC analysis of the ExS fraction revealed a single peak (P1) corresponding to a protein band of approximately 6.5 kDa, and all fragments obtained by LC-MS/MS were similar to peptides belonging to the hevein-like family. The highest inhibition was observed for C. gloeosporioides, with 78.3% growth inhibition at 200 μg mL^-1. Mechanistic studies revealed that the ExS fraction caused membrane permeabilization in C. gloeosporioides, induced reactive oxygen species (ROS) production, and reduced cell viability. The ExS fraction also caused vacuolization of hyphae in F. oxysporum. This study highlights the potential of AMPs isolated from C. annuum leaves to inhibit phytopathogenic fungi.