**3. Discussion**

Plant flavonoids have a variety of biological activities in animals. However, despite numerous studies in this field, the mechanism/s of action of flavonoids remain poorly understood. Using animal models, it was shown that some flavonoids, luteolin in particular, may mitigate the toxicity of drugs [13,39]. However, the protective effect of flavonoids in humans has not been reliably ascertained [13]. There have been a number of studies reporting the effect of flavonoids, mostly on xenobiotic transformation by CYP enzymes and drug–drug interactions. In particular, baicalein showed an inhibition activity to CYP1A, CYP2B and CYP3A4, with IC50 values in the range of 0.5–36 μM [12,40]. Both baicalein and luteolin inhibit diclofenac 4- -hydroxylase activity in the CYP2C9 RECO system, with baicalein acting as a competitive inhibitor of CYP2C9 [41]. The most effective luteolin inhibition was shown for CYP2C8, while its close homologs, CYP2C9 and CYP2C19, were less effectively inhibited [13]. The activity of CYP1A2, CYP3A, CYP2B6, CYP2E1 and CYP2D6 was also inhibited by luteolin, with an IC50 in the range of 1.6–132.6 μM [13,42]. Notably, luteolin selectively inhibits CYP2D6-mediated metabolism with different substrates. For example, O-demethylation of 3-[2-(N,N-diethyl-N-methyl-ammonium)ethyl]-7-methoxy-4-methylcoumarin was inhibited to 40% by the administration of 20 μM luteolin, while the same concentration of luteolin showed less than a 5% inhibition in reaction with dextromethorphan [14]. Overall, the baicalein and luteolin inhibitory concentration on drug metabolizing CYPs is in the micromolar range.

The inhibition effect of flavonoids was also shown for CYPs involved in the biosynthesis of steroid hormones, neurosteroids, prostaglandins, as well as other regulatory metabolites. The effect of different flavonoids was evaluated on cortisol production in human adrenocortical H295R cells, and the competitive mechanism of inhibition was established for CYP21B1 [43]. The inhibition effect of luteolin was shown for human aromatase CYP19 [44]. A synthetic analogue of dihydrodaidzein, NV-52, inhibited the thromboxane A2 synthase CYP5A1 [45], while isoflavonoids inhibited the oxidation of vitamin D3 by CYP24A1 [46].

Sulfation, methylation and glucuronidation, occurring in the enterocytes and liver, are major factors affecting flavonoid bioavailability and are crucial for their transport via the blood [47]. Non-conjugated flavonoids are generally not present in plasma, however, there is an indication that a small amount of non-conjugated flavonoids can be transported through the blood system [48]. To the best of our knowledge, no studies have been conducted on the inhibition of CYP activity by sulfated forms of baicalein and luteolin. The inhibitory potential of other sulfated derivatives was probed with drug-metabolizing CYPs. It was shown that sulfated derivatives of quercetin and chrysin can inhibit several CYPs in vitro. In particular, quercetin 3- -sulfate has a selective concentration-dependent inhibition activity to CYP2C19 and CYP3A4 up to 30 μM, but the inhibition effect did

not exceed 50% and overall was less than that of ticlopidine and ketoconazole (used as positive controls) [49]. Chrysin 7-sulfate has an IC50 value of 2.7 μM to CYP2C9, which is comparable to that of the positive control sulfaphenazole [50]. Additionally, chrysin 7-sulfate showed a slight inhibition effect on CYP2C19 and CYP3A4 [50].

CYP51A1 is considered as a potent target for cholesterol-lowering drugs [51]. There is an indication that the regulation of CYP51A1 function could be important to the treatment of oncological pathologies [11]. It was shown that anticancer drugs, abiraterone and galeterone, which are steroidal inhibitors of CYP17A1, can interact with human CYP51A1. However, their inhibition potential was not estimated. The Kdapp values determined for the abiraterone and galeterone, were 22 and 16 μM, respectively [52], and are significantly higher than those for baicalein and luteolin obtained in this work (8.2 and 5.1 μM, respectively). In contrast, non-steroidal pyridine derivative LK-935 [53] and azole inhibitors, ketoconazole and econazole [38], have an affinity in the submicromolar range due to direct coordination with heme iron. However, azole derivatives have a poor bioavailability and relatively low selectivity which might cause adverse reactions.

We showed the inhibition of CYP51A1 activity by the sulfated derivative of luteolin isolated from seagrass within the family *Zosteraceae* (*Zostera marina* and *Zostera asiatica)*. It was previously demonstrated that luteolin 7,3- -disulfate has a wide range of biological activities that might be linked to its higher bioavailability [33,54]. Considering that natural flavonoids and their biological activities are currently a subject of great interest and in light of our data, it is plausible to suggest that CYP51A1 activity could also be modulated by this group of compounds. Obtained data on inhibition by luteolin 7,3- -disulfate could be further explored for the development of a new class of CYP51A1 inhibitors.
