Mn 615

Dried Lycium leaf samples (330 mg) were extracted for 4 h min at room temperature while being stirred, using a dynamic maceration with 20 mL of pure methanol (MAC 1) and methanol/water 50:50 v/v (MAC 2). The extract was filtered through paper filter (MN 615, Macherey–Nagel, Düren, Germany), collected in amber glass vials, and kept at −20 °C until further analysis. The extraction was repeated three times.

The extraction procedure was carried out following the conditions of previous papers [16 (link),17 (link)] with slight modifications. A dried pumpkin sample (1 g) was extracted with 20 mL of solvent (hexane:isopropanol, 60:40 v/v or hexane: acetone: ethanol, 50:25:25 v/v/v) for 15 min or 30 min at 45 °C using a closed vessel system microwave (Model Initiator 2.0, version 2.3, Biotage AB, Uppsala, Sweden) under controlled conditions. The temperature was the preferred controlled variable to avoid degradation of the target compounds and to achieve maximum efficiency. The other parameters were directly dependent on the temperature, such as the magnetron power (maximum 40 W) and pressure (maximum 5 bar). At the end of the treatment, the vessel used was cooled to room temperature. The extracts were filtered through a paper filter (MN 615, Macherey–Nagel, Düren, Germany), collected in amber glass vials, and kept at −20 °C until further analysis. The extraction was repeated three times.

Cynara scolymus L., Asteraceae, plant material was purchased from a local pharmacy as dried and chopped leaves (Redwood, Bamberg, Germany, LOT 01160-033). A 12.3 g sample of leaves was homogenized and extracted with 300 mL 60% methanol for 3 h with stirring. After filtration (MN 615, Macherey Nagel, Düren, Germany), the extract was dried in a vacuum, freeze-dried and homogenized. This procedure yielded 26.8% of artichoke leaf extract (ALE).

Prior to all treatment procedures (except the overliming), the pH of the PAC was adjusted to 6.5 using solid NaOH pellets. Afterwards the condensate was centrifuged at 4700×g for 10 min at room temperature and the supernatant was filtered through a paper filter (Macherey–Nagel, type MN615, cellulose) to obtain the PAC that was used as starting material for the detoxification experiments and that is henceforth referred to as the untreated control. The centrifugation conditions described in this section were used for all other centrifugation steps performed during the PAC treatment experiments.

Dried Lycium leaf samples (330 mg) were extracted with 20 mL of pure methanol (MAE 1) and methanol/water 50:50 v/v (MAE 2) for 30 min at 45 °C using a closed vessel system microwave (Model Initiator 2.0, version 2.3, Biotage AB, Uppsala, Sweden) under controlled conditions. The temperature was the preferred controlled variable to avoid degradation of the target compounds and to achieve the maximum efficiency. The other parameters were directly dependent on the temperature, such as the magnetron power (maximum 40 W) and pressure (maximum 5 bar). At the end of the treatment, the vessel used was cooled to room temperature. The extract was filtered through paper filter (MN 615, Macherey–Nagel, Düren, Germany), collected in amber glass vials, and kept at −20 °C until further analysis. The extraction was repeated three times.

Illudin M was produced by submerged fermentation in stirred tank bioreactors [15 (link)]. The culture broth was separated from the biomass by centrifugation at 15,000×g for 30 min using the Sorvall RC-5B Refrigerated Superspeed Centrifuge (DuPont Instruments) at room temperature using the fixed-angle rotor Fiberlite™ F9-4 × 1000y (Thermo Fischer, USA). After centrifugation, a layer of antifoam formed on the surface of the supernatant which could not be fully removed. To eliminate antifoam residues, the supernatant was vacuum filtered using a Büchner funnel with a double layer of ø150 mm paper filter MN 615 (Macherey–Nagel) and an additional 4 cm layer of cotton wool on top. This setup was sufficient to clear > 10 L supernatant from antifoam without clogging and no significant drop in performance. The filtrate was collected in glass bottles and stored at 4 °C in darkness until further usage.