Acid Oleic acid 9,12-Octadecadienoic acid (Z,Z)Octadecanoic acid 9-Octadecenamide, (Z)Ergosta-4,7,22-trien-3b-olMolecular formula C7H14O6 C7H10N2O2 C16H32O2 C18H34O2 C18H32O2 C18H36O2 C18H35NO C28H44OMolecular weight 194.18 154.17 256.42 282.46 280.45 284.49 281.48 396.Region ( ) LR-SC 32.17 3.51 three.31 0.24 four.71 1.04 1.56 five.Region ( ) was determined determined by the TIC of LR-SC (Figure S5). Identification from the compounds was based on mass spectral evaluation. RT, retention time; ND, not detected. doi:ten.1371/journal.pone.0102509.tElectrophoretic analysis of proteinsSodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) from the extracts was carried out working with 16 (w/v) separating and 5 (w/v) stacking gels inside a vertical slab gel apparatus (C.B.S. Scientific Corporation, Inc., California, USA), as previously reported [21]. Bands had been visualised by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) and silver staining.processing, and interpretation have been carried out working with the AB SCIEX Analyst 1.5 and Advanced Chemistry Development, Inc., (ACD/Labs, Ontario, Canada) MS Processor application. MarkerView Software program (AB SCIEX, Massachusetts, USA) was utilized for principal element evaluation (PCA). The following parameters had been utilised for PCA: retention time (RT) variety: 0215 min, RT tolerance: 0.4-Methoxycarbonyl-3-methyl-benzoicacid Chemical name five min, mass variety: m/z 10021000, mass tolerance: 0.Price of Pyrazolo[1,5-a]pyridine-5-carboxaldehyde 01 Da, and noise threshold: 5.Chromatographic and mass-spectrometric analysesSELDI-TOF-MS. For the surface-enhanced-laser-desorptionionisation time-of-flight mass spectrometry (SELDI-TOF-MS) evaluation, extracts had been spotted around the reverse-phase or hydrophobic H50 ProteinChip arrays and analysed using a ProteinChip SELDI Method PSC 4000 (Bio-Rad Laboratories, Inc., California, USA), as previously described [9,21]. GC-MS. The gas chromatography-mass spectrometry (GCMS) analysis was performed working with a 6890 N gas chromatograph (Agilent Technologies, Inc.PMID:33653255 , California, USA) equipped using a 5975 Mass Selective Detector. The HP-5 MS (five phenylmethylsiloxane) capillary column (30.0 m625 mm625 mm) was initially set at 70uC, elevated to 300uC, and then held for ten min. Helium was utilised because the carrier gas at flow price of 1 ml/min. The total ion chromatogram (TIC) was auto-integrated by ChemStation. Chemical constituents were identified by comparison with the accompanying spectral database (NIST 2011, Mass Spectral Library, USA) and literature, where applicable. UHPLC-ESI-MS/MS. The analysis was performed using a Flexar FX15 ultra high-performance liquid chromatograph (UHPLC, PerkinElmer, Inc., Massachusetts, USA) coupled with an AB SCIEX 3200 QTrap hybrid linear ion trap triplequadruple mass spectrometer equipped with a turbo ion spray source. Chromatographic separation was accomplished on a Phenomenex Aqua C18 (5 mm, 50 mm62 mm) column. Mobile phase A was composed of water with 0.1 (v/v) formic acid and 5 mM ammonium formate, whereas the mobile phase B consisted of acetonitrile containing 0.1 (v/v) formic acid and five mM ammonium formate. Elution was performed by suggests of a linear gradient from 10290 B (028 min) held for three min, returned to 10 B in 0.1 min, after which re-equilibrated for four min prior to the following injection. Ionisation was achieved by means of electrospray ionisation around the AB Sciex Turbo V supply with an ionisation temperature of 500uC and purified nitrogen gas (99 ) as the collision gas via nebulisation. Collision energy was set at 35 eV for massfragmentation purposes. Full scan with MS/MS data collection analyses was performed i.