Many diseases are associated with oxidative stress caused by free radicals. Fungal endophytes are microbes that inhabit host plants without causing disease and have been recognized as potential sources of pharmaceutically valuable compounds. The extract of the endophytic fungi, Phialophora lignicola found in roots of Pinus sylvestris (Scots pine) seedlings has revealed protective bioactivity on human retinal pigment epithelial cells (hRPE) against oxidative stress which can lead to obtain compounds for the treatment or prevention of Age-related macular degeneration (AMD). The current research was directed towards finding the naturally-occurring antioxidant(s) in P. lignicola and to characterize and identify them by NMR spectroscopy and MS analysis.
The bioactive aqueous extract of P. lignicola was fractionated by preparative HPLC and the antioxidant activity of each sample was evaluated by H2O2 scavenging test and bioassay with model cells of hRPE cells. The bioactive fractions were characterized by 500 Mhz NMR spectroscopy, performing both one- and two-dimensional NMR experiments, including 1H NMR, COSY, TOCSY, HSQC, ROESY, 31P-NMR and 31P-1H correlation. ESI-TOF MS in the positive mode was used for the determination of molecular weights found in the fractions. Additionally, LC-MSn was used for the separation of compounds and mass analysis to complement the information given by ESI-TOF MS.
The present study provides evidence that two bioactive fractions of P. lignicola possessed the presence of antioxidative activity. The NMR experiments suggested arginine and polysaccharides. ESI-TOF MS results established the presence of arginine, a hexose-arginine conjugate and other compounds that based on NMR and mass spectrometry literature they could be chitin oligomers. Furthermore, LC-MSn identified fragments typical of L-arginine. For future research, some drawbacks of this study such as concentration of samples and chromatographic purification method should be improved to optimize the NMR and mass spectrometry analysis.
