Psychotria vellosiana Benth: A cyclotide-rich species

Cyclic cystine knot proteins (cyclotides) are complex cyclic peptides that occur naturally in plants and have a peculiar cyclic structure, known as cyclic cystine motif (CCK). This topology confers them remarkable stability and wide range of biological and/or therapeutic applications, between them, anti-HIV,1 anthelmintic,2 insecticidal,3 molluscicidal,4 antimicrobial,5 uterotonic,6 cytotoxic,7 hemolytic8, trypsin inhibitory9, and immunosuppressive activities.10 In this context, both the scientific community and pharmaceutical industry have been a future view for using cyclotides in drug development. Thus, this work describes the diversity of this compound in stems and the identification of a novel cyclotide from Psychotria vellosiana. Dried and pulverized stems were extracted with MeOH/H2O (60:40, v/v) at room temperature. The extract was partitioned with CH2Cl2:MeOH:H2O and the aqueous phases was concentrated and eluted through solidphase extraction (SPE) cartridge. The fraction eluted with the mixture 80% buffer B (90% acetonitrile, 0.08% trifluoroacetic acid) in A (0.1% aqueous trifluoroacetic acid) was considered the peptide-rich fraction. This was submitted to MALDI-TOF mass spectrometry, to determine the molecular weight signals in the range between the 2500-4500 Da. It was observed the presence of many mass signals in this region, that indicate a great diversity of possible cyclotides. To separation and purification were employed HPLC chromatography that allowed to isolate a novel bracelet cyclotides from P. vellosiana with average mass m/z 2889. The cyclotide sequencing was performed by reduction, alkylation and enzymatic digest with endoproteinases GluC, trypsin, chymotrypsin and followed by MS/MS analysis.