Gs 15r

The water absorption capacity was determined according to Delatte et al., and Torbica, Belović and Tomić [31 (link),32 (link)] with modifications. A total of 40 mL of a 1% (d.b.) starch suspension was prepared in distilled water at 30 °C. The suspension was heated at a rate of 1.5 °C/min until it reached 60, 70, 80 or 90 °C and was kept at these temperatures for 30 min with constant stirring. It was allowed to cool to room temperature and centrifuged at 2500 rpm for 15 min, in a GS-15R centrifuge (Beckman Instruments, Inc. Brea, CA, USA). The resulting gel was weighed. The water absorption capacity for each temperature was calculated as the weight (g) of the gel per g of dry sample. The WAI was calculated and expressed as in Equation (5): $$\mathrm{WAI}=\frac{\mathrm{Weight} \mathrm{of} \mathrm{sedminet} \left(\mathrm{g}\right)}{\mathrm{Weight} \mathrm{of} \mathrm{sample} \left(\mathrm{g}\right)}\times 100\%$$

Dried leaf material (10 g) was powder and extracted by ultrasound assisted extraction (Branson 3800 MH) for 40 min each and three cycles with H₂O:CH₃OH (1:1) solution (260 mL). The flask was centrifuged at 4800 rpm (Beckman, GS-15R) for 10 min at 4 °C. Subsequently, after centrifugation, the extract was filtered on Whatman paper and concentrated under vacuum, obtaining a dried crude extract (3.78 g).
The dried crude extract dissolved in H₂O was chromatographed on Amberlite XAD-4, eluting first with water and then with methanol. The enriched organic fraction (696.1 mg) was chromatographed through Flash CC SiO₂, eluting with the lower phase of CHCl₃/CH₃OH/H₂O (13:7:4), to afford three fractions (MC_A–MC_C)
Fraction MC_A (208.2 mg) was chromatographed on Sephadex LH-20 column, using Hexane: CHCl₃: CH₃OH (5:1:1) as mobile phase, in turn to have three fractions MC_A1, MC_A2, MC_A3.
Fraction MC_B (92.8 mg), subjected to HPLC using H₂O:CH₃OH (1:1) as mobile phase, gave pure compounds 1 (5 mg), 2 (1.1 mg), 3 (7.2 mg), 4 (1.7 mg) and 5 (2.4 mg).
Fraction MC_C chromatographed on silica gel TLC (1 mm) eluting with the organic phase of biphasic solution CHCl₃: MeOH: Me₂CO: H₂O 13:7:1:3) gave two spots identified as compounds 6 (40 mg) and 7 (7 mg).

Livers were extracted from mice, following transcardial whole-body perfusion with ice-cold PBS, were preserved by snap-freezing in liquid nitrogen, and were stored at −80°C. For homogenization, 500 μl of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA; 89901) with 100× Halt™ Protease Inhibitor Cocktail (1:100, Thermo Fisher Scientific, 78429) was used per 10 mg of snap-frozen liver tissue. Liver tissue was allowed to sit in RIPA buffer for 10 min at 4°C and was then disrupted by mechanical homogenization for 3 min, at 1,500 rpm and 4°C (POTTER S, B. Braun, 8533032). Samples were centrifuged for 15 min, at 14,000 rpm and 4°C (Beckman Coulter, GS-15R). Supernatants were aliquoted and frozen at −80°C.

A colony of either Z. obscura LS31012019 or A. pullulans ATCC 15233 was transferred from PDA plates to 250 mL Erlenmeyer flasks containing 50 mL of semi-synthetic medium (50 g/L sucrose, 2.0 g/L yeast extract, 5.0 g/L KH₂PO₄, 0.2 g/L MgSO₄ × 7 H₂O, 1.0 g/L NaCl, and 0.01 g/L FeSO₄ × 7 H₂O) and chloramphenicol (50 µL of a 30 mg/mL of ethanol solution). Then, each culture was incubated for 5 days at 25 °C. The yeast cells were harvested by centrifugation at 15,000×g for 20 min at 4 °C. The lyophilized washed cells (obtained as described above in paragraph 2.2) were ground to a fine powder in a mortar and pestle; then, 5 mL of hexane were added, and the suspension was transferred in a fresh Falcon tube and incubated at room temperature for 30 min. The supernatant was moved into a new 50 mL test tube after centrifugation at 4800 rpm for 10 min in a Beckman centrifuge GS-15R. Five millilitres of absolute ethanol was added to the pellet, dried under a hood, and centrifuged again after 30 min of incubation. The extraction with ethanol/1% acetic acid (v/v) 80:20 was performed as a last extraction following the upper procedure. The supernatants collected from hexane, absolute ethanol, and acid ethanol extraction were dried under a vacuum and were subjected to image analysis.

The powdered leaves (30 g) were sequentially and exhaustively extracted by ultrasound assisted extraction (Branson 3800 MH) for 40 min each (2 cycles × 3.0 L) with chloroform, MeOH, and MeOH:H₂O (1:1). After centrifugation at 4800 rpm (Beckman, GS-15R) at 4 °C for 10 min, the solutions were concentrated under vacuum, giving three crude extracts: chloroform (PF1, 2.2 g), MeOH (PF2, 4 g), and MeOH:H₂O (PF3, 1.6 g).

This property was determined according to AACC method 88-04 [7 ]. Approximate water absorption capacity was first determined by weighing out 0.1 g (d.b.) of sample, adding water until saturation (approximately 5 mL), and centrifuging at 2000 ×g for 10 min in a Beckman GS-15R centrifuge. Excess water was discarded and the residue was weighed. Approximate water absorption capacity was calculated by dividing the increase in sample weight (g) by the quantity of water needed to complete original sample weight to 15 g. Water absorption capacity (WA_bC) was then determined by placing samples in four tubes, adding different quantities of water to bracket the measurement (1.5 and 0.5 mL water above original weight and 1.5 and 0.5 mL water below; one in each tube), agitating vigorously in a vortex for 2 min, and centrifuging at 2000 ×g for 10 min in a Beckman GS-15R centrifuge. The supernatant was discarded and the residue was weighed. Average water absorbed was calculated and the WA_bC was calculated, expressed as g water absorbed per g of sample.