Anti-Plasmodial Activity and Mass Spectrometric Profiling of Peptide-enriched Methanol/Water Extract of Leaves from the Himalayan Viola canescens Wall

Background: Plants belonging to the genus Viola and family Violaceae are well known in Ayurvedic and Unani medicine for their pharmaceutical importance. Viola, commonly known as Banafsha, has been traditionally used as an anti-malarial. The goal of this study was to determine the anti-Plasmodial activity of the cyclotide-rich crude methanol/water extract of leaves from Viola canescens Wall and to analyze its constituents by mass spectrometry (MS). Materials and Methods: Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) involving quadrupole time-of-flight (Q-ToF) and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight-mass spectrometry (ToF-MS) were applied to investigate the cyclotides rich in methanol/water extract. The intact molecular masses were observed in the range of 2,800–3,400 Daltons, suggesting the presence of peptides. Since various head-to-tail cyclized and intramolecularly disulfide-bonded peptides known as cyclotides have been identified in the Violaceae family, we wanted to verify whether cyclotides are present in our extract. So, the intact masses inferred from the observed spectra were interrogated with Cybase ( www.cybase.org.au ), which is a database on plant cyclotides. Results: We observed promising anti-Plasmodial activity (IC50 44 µg/ml) in the cyclotide-rich methanol/water extract prepared from the leaves of the Himalayan V. canescens Wall. The output obtained from Cybase confirmed the presence of about 20 different cyclotides in our extracts. However, no sequence information could be obtained from Cybase for 14 intact molecular masses, which were inferred from the observed LC-ESI-MS data. Hence, these could correspond to novel peptide/cyclotide sequences, which might be characteristic of Himalayan V. canescens. Conclusion: This is the first report on the anti-Plasmodial activity of the cyclotide-enriched methanol/water crude extract of the leaves of V. canescens.