Screening for Cyclotides in Sri Lankan Medicinal Plants: Discovery, Characterization, and Bioactivity Screening of Cyclotides from Geophila repens

Cyclotides are an intriguing class of structurally stable circular miniproteins of plant origin with numerous potential pharmaceutical and agricultural applications. To investigate the occurrence of cyclotides in Sri Lankan flora, 50 medicinal plants were screened, leading to the identification of a suite of new cyclotides from Geophila repens of the family Rubiaceae. Cycloviolacin O2-like (cyO2-like) gere 1 and the known cyclotide kalata B7 (kB7) were among the cyclotides characterized at the peptide and/or transcript level together with several putative enzymes, likely involved in cyclotide biosynthesis. Five of the most abundant cyclotides were isolated, sequenced, structurally characterized, and screened in antimicrobial and cytotoxicity assays. All gere cyclotides showed cytotoxicity (IC50 of 2.0-10.2 mu M), but only gere 1 inhibited standard microbial strains at a minimum inhibitory concentration of 4-16 mu M. As shown by immunohistochemistry, large quantities of the cyclotides were localized in the epidermis of the leaves and petioles of G. repens. Taken together with the cytotoxicity and membrane permeabilizing activities, this implicates gere cyclotides as potential plant defense molecules. The presence of cyO2-like gere 1 in a plant in the Rubiaceae supports the notion that phylogenetically distant plants may have coevolved to express similar cytotoxic cyclotides for a specific functional role, most likely involving host defense.

Automated summary

Cyclotides are structurally stable circular miniproteins of plant origin that have potential pharmaceutical and agricultural applications. In this study, new cyclotides were identified in Sri Lankan flora, particularly in Geophila repens, and were found to exhibit cytotoxic and antimicrobial activities. The localization of cyclotides in the epidermis of the leaves and petioles suggests their potential role as plant defense molecules. The presence of similar cytotoxic cyclotides in phylogenetically distant plants supports the idea of coevolution for host defense.