One-step Bio-guided Isolation of Secondary Metabolites from the Endophytic Fungus Penicillium crustosum Using High-resolution Semi-preparative HPLC

Abstract

Background: An endophytic fungal strain Penicillium crustosum was isolated from the seagrass Posidonia oceanica and investigated to identify its antimicrobial constituents and characterize its metabolome composition. The ethyl acetate extract of this fungus exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) as well as an anti-quorum sensing effect against Pseudomonas aeruginosa.

Methods: The crude extract was profiled by UHPLC-HRMS/MS, and the dereplication was assisted by feature-based molecular networking. As a result, more than twenty compounds were annotated in this fungus. To rapidly identify the active compounds, the enriched extract was fractionated by semipreparative HPLC-UV applying a chromatographic gradient transfer and dry load sample introduction to maximise resolution. The collected fractions were profiled by ¹H-NMR and UHPLC-HRMS.

Results: The use of molecular networking-assisted UHPLC-HRMS/MS dereplication allowed preliminary identification of over 20 compounds present in the ethyl acetate extract of P. crustosum. The chromatographic approach significantly accelerated the isolation of the majority of compounds present in the active extract. The one-step fractionation allowed the isolation and identification of eight compounds (1-8).

Conclusion: This study led to the unambiguous identification of eight known secondary metabolites as well as the determination of their antibacterial properties.

Keywords: Penicillium crustosum; Posidonia oceanica; UHPLC-HRMS/MS molecular networking; anti-quorum sensing assays.; antibacterial natural products; fungal endophytes; highresolution semi-preparative HPLC.