Flavomyces fulophazii, D.G.Knapp, Kovács, J.Z.Groenew. & Crous

Berek-Nagy, Peter Janos, Gergo Toth, Szilvia Bosze, Lilla Borbala Horvath, Andras Darcsi, Sandor Csikos, Daniel G. Knapp, Gabor M. Kovacs & Imre Boldizsar, 2021, The grass root endophytic fungus Flavomyces fulophazii: An abundant source of tetramic acid and chlorinated azaphilone derivatives, Phytochemistry (112851) 190, pp. 1-11 : 8-9

4.2. Preparation of F. fulophazii culture extracts for analysis and isolation

Each isolate was grown in three replicates (labeled as A, B, C) in Petri dishes (60 mm × 15 mm) on Potato dextrose agar medium (VWR, Hungary) at room temperature in dark for 30 days. The voucher specimens of all isolates are available in the Department of Plant Anatomy, E¨otvos ¨Lor´and University, Budapest, Hungary and two isolates were also deposited in the CBS collection (as indicated in Table 1 ). Complete in vitro cultures containing culture medium with the fungal mycelium grown on it were lyophilized and pulverized. The NMR solvents chloroform- d, dimethyl sulfoxide- d 6 (DMSO d 6) and methanol- d 4 were purchased from Sigma-Aldrich, Hungary. The other materials and reagents applied in the analysis and isolation of fungal metabolites, such as acetonitrile, distilled water, formic acid, methanol (Reanal, Hungary) were all of analytical reagent grade of the highest purity available.

1–5

literature data, obtained from Lajko´et al. (2018); Kiss et al. (2019); Baranyai et al. (2017); Tripodi et al. (2020); Orb´an et al. (2011), respectively.

a Daunomycin (Dau, against all cells) and 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (Sal, against the HepG2, U87 and MonoMac-6 cells) were used as positive control.

Extracts for analysis: Aliquots of the powdered cultures (10.0 mg) were extracted with 5 mL of methanol at 60 ◦ C, via a reflux condenser, for 30 min. The insoluble, centrifuged material was subsequently reextracted in the same way. The supernatants were combined and these combined solutions were dried by a rotary vacuum evaporator at 40 ◦ C. Before analysis, these dried extracts were dissolved in methanol.

Extracts for isolation: Procedure was the same as described above, except for the amounts of lyophilized and pulverized cultures. To isolate vermelhotin, hydroxyvermelhotin and flavochlorine G, total amount of six unified cultures (HF-3A, HF–3B, HF–3C, MF-7A, MF-7B, MF-7C) were extracted while to isolate flavochlorine A and flavochlorine G three unified cultures (MF-3B, HF-1A, HF–9B) were extracted. Compounds of these extracts were separated by preparative HPLC.