ABSTRACT
Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1â¯mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cyclotides/pharmacology , Plant Extracts/pharmacology , Viola/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclotides/biosynthesis , Cyclotides/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Escherichia coli/drug effects , HEK293 Cells , Humans , Microbial Sensitivity Tests , Plant Extracts/biosynthesis , Plant Extracts/chemistry , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Viola/chemistry , Viola/embryologyABSTRACT
Cyclotides are cyclic cystine knotted macrocyclic plant peptides that have several promising applications. This study was undertaken to detect and identify known and new cyclotides in Viola odorata, a commercially important medicinal plant, from three geographical locations in India. The number of cyclotides in the plant varied with the tissue (leaves, petioles, flowers, runners, and roots) and with geographical locations in India. Using liquid chromatography coupled to Fourier transform mass spectrometry (FTMS), 166 cyclotide-like masses were observed to display cyclotide-diagnostic mass shifts following reduction, alkylation, and digestion, and 71 of these were positively identified based on automated spectrum matching. Of the remaining 95 putative cyclotides observed, de novo peptide sequencing of three new cyclotides, namely, vodo I1 (1), vodo I2 (2), and vodo I3 (3), was carried out with tandem mass spectrometry.