Characterization of Cyclotides via LC-MS/MS from the Vietnamese Plant Viola annamensis Bak.f.: Insights into Antimicrobial and Larvicidal Activities.

Viola annamensis Bak.f., a Vietnamese endemic species, is reported here for the first time as a source of cyclotides. LC-MS and LC - MS/MS analyses identified 11 cyclotides (3.1-3.8 kDa) bearing the conserved cyclic cystine knot (CCK) motif, including four novel cyclotides designated Vian A, Vian B, Vian C, and Vian D. Fraction M1 contained eight cyclotides, including Vian A, whereas fractions M2 and M3 mainly contained Vian B/Vian C and Vian D, respectively. Vian D was successfully purified, sequenced, and evaluated for antimicrobial activity. The peptide mixtures M2 and Vian D showed antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis, and Listeria ivanovii. Minimum inhibitory concentration (MIC) values for M2 and Vian D ranged from 6.2 to 8.3 mg/mL, confirming the contribution of these cyclotides to the antimicrobial properties of V.annamensis. Cytotoxicity assessment using the Artemia salina assay showed low toxicity for the salt-precipitated sample, with an LC50 value of 1.042 mg/mL, whereas the purified peptide mixtures exhibited higher, concentration-dependent toxicity. Among them, M1 demonstrated greater potency, with an LC50 of 0.049 mg/mL, followed by M2 with an LC50 of 0.131 mg/mL. This study identifies V. annamensis as a novel source of new cyclotides with potential antimicrobial applications.