Background Cyclotides are macrocyclic peptides characterized by a head-to-tail cyclized backbone and a cyclic cystine knot motif, and are found in the petals of butterfly pea ( Clitoria ternatea ). Objective Clitoria ternatea cv. “Theparatpailin 63” is a newly established butterfly pea cultivar with large petals rich in anthocyanins. This study aimed to identify, quantify, and evaluate the biological activities of cyclotides present in this cultivar. Methods Dried petals and sepals of C. ternatea cv. “Theparatpailin 63” were ground in liquid nitrogen and extracted with methanol/dichloromethane. Following filtration, the extracts were fractionated by column chromatography using Amberlite XAD7-100G and OASIS HLB, and further purified by preparative reversed-phase high-performance liquid chromatography. The molecular masses of the isolated compounds were determined by MALDI-TOF mass spectrometry. Nucleotide sequences of individual cyclotides were analyzed using cyclotide-specific primers with genomic DNA and total RNA isolated from flowers as templates. Biological activities were assessed by examining the effects of purified cyclotides on the growth of HL-60 cells and Escherichia coli . Results Transcripts encoding cyclotides: cliotide T1 (cT1), cT2, cT3, cT4, and cT5 were detected in flowers. Mass spectrometric analysis revealed that cT3, cT4, cT5, and a methionine-oxidized form of cT3 cT3(O) were predominantly present in petals, whereas cT3 and cT3(O) were mainly detected in sepals. Purified cT5 and cT3(O) slightly promoted the growth of E. coli , while no significant effect was observed for cT3 and cT4. In contrast, the growth of HL-60 cells was slightly inhibited by the purified cyclotide mixture. Conclusion The major cyclotides present in the flowers of C. ternatea cv. “Theparatpailin 63” are cT3, cT4, cT5, and the methionine-oxidized form of cT3. These cyclotides exhibit modest and differential biological activities toward bacterial and human leukemia cells.
Ikeda et al. (Fri,) studied this question.