Optimization of cyclotide extraction parameters

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds, including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50% MeOH for 6 h at a plant material to solvent ratio of 0.5:10 (g/mL) represents an optimum extraction method.