Silybin

The cells were counted by Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA) and the cell suspension containing cell density 10⁵ cells/mL was split into the 96-well plate. The plates were then incubated for 24 h at 37 °C in humidified atmosphere of 5% CO₂. Then, the tested compounds were added. To assess the effect of silibinin, cells were pre-treated with silibinin at the given concentrations 2 h prior to mycotoxin exposure. After 72 h incubation, the cell viability was tested by standard resazurin assay [114 (link)]. Briefly, the cells were washed three times with 100 µL of PBS and incubated with 100 μL of resazurin solution (0.025 mg/mL) for 3 h. Finally, the fluorescence was measured by a SpectraMax i3x microplate reader (Molecular Devices, UK) at a wavelength of 560 nm excitation/590 nm emission.

Silymarin was obtained from the following suppliers: Sigma-Aldrich (SM 1; St. Louis, MO, USA, batch No. BCBJ0393V), Liaoning Senrong Pharmaceuticals (SM 2; Panjin, China, batch No. 120501), INDENA (SM 3; Settala, Italy, batch No. 32621/M5), Panjin Huacheng Pharmaceutical Company (SM 4; with an additive for better solubility in water, Panjin, China, batch No. E5S66), Takeda (SM 5; Konstanz, Germany, Flavobion^® coated tablets, batch No. 383036), Panjin Huacheng Pharmaceutical Company (SM 6; supplied in August 2019 without batch No., Panjin, China). Flavobion^® coated tablets (25 tablets, 11.360 g) were powdered and subjected to Soxhlet extraction with acetone (450 mL) for 2 h, the extract was evaporated in vacuo to yield the sample SM 5 (126 mg of dry extract). The extract was stored at −80 °C. Standards of silybin A, silybin B, 2,3-cis-silybin A, 2,3-cis-silybin B, 10,11-cis-silybin A, 10,11-cis-silybin A, silychristin A, silychristin B, isosilychristin, silydianin, isosilybin A, isosilybin B, silyhermin, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B, 2,3-dehydrosilychristin A, 2,3-dehydrosilychristin B, 2,3-dehydroisosilybin, and 2,3-dehydrosilydianin were prepared and fully characterized in the Laboratory of Biotransformation, Institute of Microbiology, Prague, CZ [11 (link),21 (link),22 (link),23 (link),24 (link),25 (link)]. Taxifolin was purchased from Amagro (Prague, Czech Republic) and coniferyl alcohol from Sigma-Aldrich (Merck, Kenilworth, NJ, USA). All standard solutions for calibration curves were prepared in dimethyl sulfoxide in volumetric flasks. The silymarin preparations SM 1–SM 6 were also dissolved in dimethyl sulfoxide and their concentrations were 10.3, 6.5, 13.3, 8.3, 15.5, and 23.1 mg/mL, respectively. All substances dissolved in dimethyl sulfoxide were stable during the measurement; no new peaks appeared in repeated measurements even after several weeks. The concentrations of the flavanonol, flavonolignans, and 2,3-dehydroflavonolignans were calculated using seven-point calibration curves.
Silymarin fraction containing concentrated 2,3-dehydroderivatives of flavonolignans was obtained from silymarin (SM 2 preparation) as described previously [23 (link)] using Sephadex LH-20 glass column XK 50 (100 × 5 cm, bead size 25–100 μm, GE Healthcare Life Sciences, Pittsburgh, PA, USA) equipped with a thermostatic jacket (23 °C). Isocratic elution with methanol, flow rate 3 mL/min, volume of each fraction was 30 mL, UV detection at 254 nm, run-time 28 h [23 (link)]. Briefly, 6 g of “silybin free” silymarin (see Appendix B) was loaded onto the column and eluted with methanol to obtain a fraction enriched in 2,3-dehydroderivatives of flavonolignans (typically 0.8 g).
Acetonitrile, methanol, formic acid, dimethyl sulfoxide (Avantor, Radnor, PA, USA), and deionized water were of LC-MS grade.

Silibinin, trans-chalcone (benzylideneacetophenone), thioflavin S, and Congo red were purchased from Sigma (USA). Regular insulin was purchased from EXIR Pharmaceutical Company (Iran). Chloroform, monopotassium phosphate (KH₂PO₄), potassium phosphate (KH₂PO), and dimethyl sulfoxide (DMSO) were purchased from Merck (Germany).

Initially, insulin solution was prepared with a final concentration of 0.5 mg/mL in phosphate buffer (50 mM, pH 7.4) in the presence or absence of 1 mM concentration of silibinin and trans-chalcone. The vials containing the insulin samples were then incubated for 24 h at 37°C on a stirrer rotating at 100 rpm (10 (link)). Since the Congo red marker binds specifically to amyloid fibrils, resulting in an increase in λ_m as well as in absorbance levels, Congo red absorption rate of incubated protein samples was assayed in the absence and presence of silibinin and trans-chalcone (1 mM). To do so, 500 μL of Congo red solution (20 μM in 5 mM potassium phosphate and 150 mM sodium chloride, pH 7.4) plus 25 μL of the sample was poured into a 0.5 mL microtube and filtered using a 2-μM pore syringe filter, then stirred and placed in the laboratory for 10 min until color stabilization. The Congo red absorption spectrum was then recorded at 600-400 nm using the Shimadzu UV-1800 spectrophotometer (11 (link)).

The brain was removed for histological examination and placed in fixative formalin for 24 h. Next, during the dehydration and clarification steps, the samples were embedded into molten paraffin. Using a microtome (Cambridge Medical Instruments, United Kingdom), 6-micron-thick sections were prepared. The samples were placed on a slide, and then half of the samples were stained with hematoxylin-eosin and the other half with thioflavin S.
The main goal of preparing sections of the hippocampus and staining them was to investigate possible changes in brain tissue and to quantitatively examine the amyloid fibrils formed in the absence and presence of silibinin and trans-chalcone in each section. Five tissue sections in each group were randomly examined microscopically. Since we observed that silibinin performed better than trans-chalcone in the initial tests, both DG and CA1 regions were checked for the former and DG for the latter.