3.1. Plant Materials and Cyclotide Isolation

Hybanthus enneaspermus, Viola odorata, and Oldenlandia affinis were grown in the glasshouse at the University of Queensland. Crude plant extracts were prepared in 50% MeCN (with 1% formic acid), filtered, and lyophilized. The extracts were fractionated using a Strata C18-E solid phase extraction (SPE) cartridge (with 20%, 50%, and 80% MeCN); the fraction eluted with 50% MeCN was collected and lyophilized. Hyen D (H. enneaspermus), cyO2 and cyO9 (V. odorata), and kB5 (O. affinis) were isolated from the 50% MeCN fractions using preparative and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to >95% purity. The molecular integrity and purity of peptides were examined using LC/MS (LCMS-2020, Shimadzu, Kyoto, Japan).