*2.3. Enzyme Activity Assay*

Lanosterol 14α-demethylase activity of human CYP51A1 in the presence of flavonoids was determined in the reconstituted system. Only luteolin 7,3- -disulfate can inhibit the activity of the CYP51A1 (Table 2). Surprisingly, luteolin, being a more hydrophobic molecule compared to its sulfated derivative, does not have a similar effect. The apparent IC50 for luteolin 7,3- -disulfate is greater than 25 μM. At the same time, the level of inhibition by ketoconazole (94.6% at a concentration of compound of 5 μM) significantly exceeds the effect of luteolin 7,3- -disulfate (50.1% at a concentration of compound of 25 μM). Overall, the inhibition of CYP51A1 utilizing highly hydrophobic substrate by the water-soluble luteolin 7,3- -disulfate could not be predicted. This observation suggests a different mode of binding in the active site. To visualize the binding of luteolin and its disulfate in the active site we performed molecular docking.

**Table 2.** Effect of compounds on catalytic activity of human CYP51A1 (lanosterol 14α-demethylase) in the reconstituted system in vitro.


The final concentrations of CYP51A1 and cytochrome P450 reductase (CPR) were 0.5 and 2.0 μM, respectively. The final concentration of lanosterol was 50 μM.
