Sil g 25

To extract secondary metabolites, A. alternata and A. oryzae strains were grown for 1 to 7 days on their respective production media at 28°C. Four disks from each plate were excised with the back of a blue pipette tip and extracted by shaking with 1 mL of ethyl acetate for 1 h. After centrifugation at 13,000 rpm for 10 min, 700 μL of supernatant containing secondary metabolites of each sample was token into a 1.5-mL microtube and dried at room temperature, followed by dissolving secondary metabolites again using 70 μL of ethyl acetate for further TLC analysis or dissolving in 1 mL ACN:H₂O (1:1, vol/vol) for further LC-HRMS analysis.
For the qualitative analysis of the A. alternata extracts, 10 μL of crude extract was put on TLC plates coated with 0.25-mm silica gel as stationary phase (precoated TLC plates SIL G-25, Macherey-Nagel, Düren, Germany). The mobile phase for separation of the metabolites consisted of 50% of toluol, 40% of ethyl acetate, and 10% of formic acid. The secondary metabolites were visualized using UV light at 254 nm.

Instrumentation included JEOL spectrometer with tetramethylsilane as an internal standard for ¹H (500 MHz) and ¹³C (125 MHz) NMR spectra. Materials used in chromatography; normal-phase silica gel for column chromatography (Fluka^®, St. Louis, Mo, USA, 230–400 mesh), pre-coated TLC-plates ALUGRAM Xtra SIL G/UV254 (MACHEREY-NAGEL^®, Düren, Germany, 0.2 mm) (normal phase), Sil G-25 unmodified standarad silica layers on glass for Preparative TLC, layer thickness 2 mm (MACHEREY-NAGEL^®, Düren, Germany) and Sephadex LH-20 (Sigma Aldrich^®, Darmstadt, Germany). Anisaldehyde–sulfuric acid was used as a spraying reagent.

The TAG fraction was isolated by thin layer chromatography (TLC), according to the method described by Cossignani et al. [24 (link)], from total fat of pumpkin seed samples using silica gel plates (SIL G-25, 0.25 mm, 20 cm × 20 cm; Macherey-Nagel, Germany) and petroleum ether/diethyl ether/formic acid (70:30:1, v/v/v) as a developing solvent. The TAG fraction was scraped off, extracted with hexane/diethyl ether (1:1, v/v), subjected to transesterification, and analyzed by high-resolution gas chromatography (HRGC) as reported in Section 2.6 to obtain the constituent fatty acid methyl esters (FAME). The obtained data represent the total composition of FA esterified in all 3 sn-positions of TAG, named A_t.