Discovery of insecticidal cyclotides from the African plants Allexis batangae and Allexis obanensis

Cyclotides are plant-derived cyclic peptides recognized for their crucial role in host defense against insects and fungal pathogens. They are characterized by an ultra-stable head-to-tail cyclic backbone and a cystine knot formed by three conserved disulfide bonds, and are considered to be a promising group of molecules for a diverse array of agricultural and pharmaceutical applications. Cyclotides have been identified in five major plant families, with the Violaceae being a particularly rich source. Although cyclotides are ubiquitous in the Violaceae, many genera within this family remain unexplored. In the current study, the presence of cyclotides in Allexis batangae and Allexis obanensis from the Allexis genus in the Violaceae family was investigated. Five bracelet cyclotides, including three novel cyclotides, Alba A-C, were isolated and characterized using MS-MS analysis and NMR spectroscopy. The cytotoxic activity of these cyclotides was evaluated against Spodoptera frugiperda (Sf9) insect cells and demonstrated high potency as insecticidal agents, with Alba C exhibiting the highest activity (CC 50 = 0.18 ± 0.02 µM). The insecticidal effect of the discovered cyclotides was further evaluated in a feeding study using Helicoverpa armigera L3 larvae. Both a plant extract and individual cyclotides effectively inhibited larval growth. The A. batangae extract showed the greatest effect, followed by Alba B and the A. obanensis extract, achieving approximately 70 % and 50 % greater inhibition than the known insecticidal cyclotide kalata B2. Moreover, ecotoxicological evaluation using Daphnia magna revealed no acute toxicity of the tested cyclotides, underscoring their safety for non-target aquatic organisms. These findings underscore the potential of cyclotides as potent bioinsecticide agents and highlight their promising applications in agricultural pest management. • Five distinct cyclotides were isolated from Allexis batangae and Allexis obanensis . • De-novo sequencing and NMR analysis confirmed sequences and CCK structural motifs. • The novel cyclotides exhibit cytotoxicity against Sf9 insect cells. • Plant extracts and a novel cyclotide inhibit the growth of Helicoverpa armigera L3. • The activity of cyclotides suggests their potential use as natural bioinsecticides.