Insulin

Vero, A431, PANC-1, A172 cells were cultured in DMEM (PanEco, Moscow, Russia). BT-20, HepG2, Hs578T cells were cultured in EMEM (PanEco, Russia). BT-474 cells were cultured in RPMI (PanEco, Russia). All cell lines were cultured at 37 °C under 5% CO₂ in the presence of 10% fetal calf serum (BioWest, France), penicillin/streptomycin (PanEco, Russia). BT-474 and Hs578T cells were maintained in medium supplemented with 1 μg/mL insulin (PanEco, Russia). All cell lines were obtained from the All-Russian Collection of Vertebrate Cell Cultures (Institute of Cytology RAS, St.-Petersburg, Russia).

The cytotoxicity analysis of EOs on 10⁴ MRC-5 cells/well was performed in MEM-supplemented medium at 37 °C and 5% CO₂ seeded onto the test plates for 72 h. Cell viability was assessed fluorometrically after the addition of resazurin as described above. Cytotoxicity was also studied using MCF-10A cells cultured in medium supplemented with 5% donor horse serum (BioSera, Nuaille, France), 20 ng/mL epidermal growth factor (PanEco, Moscow, Russia), 0.5 µg/mL hydrocortisone (ChemCruz, Dallas, TX, USA), and 10 µg/mL insulin (PanEco) at 37 °C, 5% CO₂ and 80–85% humidity). Briefly, 60 × 10³ MCF-10A cells were seeded into 24-well plates in 900 μL of the medium, and then, plates were incubated for 24 h at 37 °C and 5% CO₂. Then, different concentrations of EOs were added, and the plates were incubated for 48 h under the same conditions. Cell viability was then measured by the MTT method as described above for cancer cell lines. Finally, in the model of PMM, 3 × 10⁵ cells/mL was treated with different concentrations of EOs over 48 h in the same conditions. Then, 15 μL of MTT solutions were added to each well, and, after 4 h of additional incubation in the same conditions, the supernatant was discarded, formazan crystals were dissolved with 100 μL of DMSO and the absorbance was obtained as described above.