2.1. Extraction of secondary metabolites from P. setosum

Colony agar plug and culture broth extraction methods were implemented to understand the effectiveness of the extraction process, for the identification of the secondary metabolites of P. setosum. For the colony agar plug method, the fungus was inoculated on Czapek yeast autolysate (CYA) agar, and incubated for 7 days at 25 °C in the dark. Five agar plugs from the incubated fungal culture plate were expurgated using a sterile 7 mm cork borer. They were transferred to a glass scintillation vial containing a mixture of dichloromethane, ethyl acetate and methanol (3:2:1) (v/v/v) with 1% (v/v) formic acid. The vial was vortexed for 30 s and sonicated for 30 min at room temperature (Visagie et al., 2014, 2016). Then the solvent extract was filtered, dried, and dissolved in 1mL methanol. For the culture broth extraction method, the fungus was inoculated in CYA broth and incubated at 25 °C for 10 days at 100 rpm. After incubation, fungal biomass was discarded using Whatman no. 1 filter paper. Cell free culture broth and an equal volume of ethyl acetate were taken in separatory funnel, the mixture was shaken vigorously for 30 min and the solvent part was collected. The step was repeated three times, pooled and dried using a rotary drum evaporator at 45 ± 2 °C. The crude extract was reconstituted in 1 mL of methanol. For spectral analyses, samples were filtered through an ultra-membrane filter (pore size 0.45 μm) and collected in 1.5 mL amber HPLC glass vials (Frisvad et al., 2008).