Characterization and evaluation of cytotoxic and antimicrobial activities of cyclotides from Viola japonica.

Cyclotides are a type of defense peptide most commonly found in the Violaceae family of plants, exhibiting various biological activities. In this study, we focused on the Viola japonica as our research subject and conducted transcriptome sequencing and analysis using high-throughput transcriptomics techniques. During this process, we identified 61 cyclotides, among which 25 were previously documented, while the remaining 36 were designated as vija 1 to vija 36. Mass spectrometry detection showed that 21 putative cyclotides were found in the extract of V. japonica. Through isolation, purification and tandem mass spectrometry, we characterized and investigated the activities of five cyclotides. Our results demonstrated inhibitory effects of these cyclotides on the growth of Acinetobacter baumannii and Bacillus subtilis, with minimum inhibitory concentrations (MICs) of 4.2 μM and 2.1 μM, respectively. Furthermore, time killing kinetic assays revealed that cyclotides at concentration of 4 MICs achieved completely bactericidal effects within 2 h. Additionally, fluorescence staining experiments confirmed that cyclotides disrupt microbial membranes. Moreover, cytotoxicity studies showed that cyclotides possess cytotoxic effects, with IC50 values ranging from 0.1 to 3.5 μM. In summary, the discovery of new cyclotide sequences enhances our understanding of peptide diversity and the exploration of their activity lays the foundation for a deeper investigation into the mechanisms of action of cyclotides.