Cytokine release assay

The effect of the bioactive isolates on the synthesis of inflammatory cytokines, TNF-α, and IL-1β in mononuclear cells was measured by ELISA. LPS (1 μg/mL) and isolated compounds (3.125–100 μg/mL) or dexamethasone (0.0005–5 μg/mL) were incubated with PBMC suspension for 12 hours at 37°C, 5% CO₂. The compounds and standards were dissolved in DMSO and diluted in cell culture medium to the required concentrations. Then, they were centrifuged at 300× g for 10 minutes at 4°C, and the supernatant was transferred to 96-well plates. The assessment of cytokine levels in the supernatant of human blood cell cultures was accomplished using the appropriate ELISA kit. The cytokine concentration in the sample was determined from appropriate calibration curves.26