Effects of Eupatilin and Jaceosidin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

Eupatilin and jaceosidin are bioactive flavones found in the medicinal herbs of the genus Artemisia. These bioactive flavones exhibit various antioxidant, antiinflammatory, antiallergic, and antitumor activities. The inhibitory potentials of eupatilin and jaceosidin on the activities of seven major human cytochrome P450 enzymes in human liver microsomes were investigated using a cocktail probe assay. Eupatilin and jaceosidin potently inhibited CYP1A2-catalyzed phenacetin O-deethylation with 50% inhibitory concentration (IC50) values of 9.4 μM and 5.3 μM, respectively, and CYP2C9-catalyzed diclofenac 4-hydroxylation with IC50 values of 4.1 μM and 10.2 μM, respectively. Eupatilin and jaceosidin were also found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation, CYP2D6-catalyzed bufuralol 1'-hydroxylation, and CYP2C8-catalyzed amodiaquine N-deethylation. Kinetic analysis of human liver microsomes showed that eupatilin is a competitive inhibitor of CYP1A2 with a Ki value of 2.3 μM and a mixed-type inhibitor of CYP2C9 with a Ki value of 1.6 μM. Jaceosidin was shown to be a competitive inhibitor of CYP1A2 with a Ki value of 3.8 μM and a mixed-type inhibitor of CYP2C9 with Ki value of 6.4 μM in human liver microsomes. These in vitro results suggest that eupatilin and jaceosidin should be further examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.

The use of botanical drugs to prevent or alleviate common illnesses has increased dramatically in Asian, North American and European countries [26]. Because the botanical drugs share the same metabolic and transport proteins including cytochrome P450 (CYP) enzymes, UDPglucuronosyltransferase (UGT) enzymes, and drug transporters such as P-glycoprotein with commonly used drugs, the potential for drug-botanical interaction is substantial. FDA guidance has addressed the concern that the interactions between botanical drugs and commonly used drugs and/or dietary supplements should be investigated [27]. The laboratory animal and human studies on druginteractions of common botanical drugs, including black cohosh, Echinacea, garlic, Ginko biloba, green tea, kava, milk thistle, and St. John's wort have been reviewed [28].
To our knowledge, no previous study has reported the effect of eupatilin and jaceosidin on human CYP enzymes. In this study, the effects of eupatilin and jaceosidin on the activities of seven major human CYPs were examined using pooled human liver microsomes to evaluate the possibility of eupatilin and jaceosidin-drug interactions.

Results and Discussion
The inhibitory effects of eupatilin and jaceosidin toward seven major human CYP isoforms were evaluated by using a cocktail of CYP probe substrates in human liver microsomes. Eupatilin and jaceosidin were found to potently inhibit CYP1A2-dependent phenacetin O-deethylation with IC 50 values of 9.4 μM and 5.3 μM, respectively, and CYP2C9-dependent diclofenac 4-hydroxylation with IC 50 values of 4.1 μM and 10.2 μM, respectively. Eupatilin and jaceosidin were found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation with IC 50 values of 48.1 μM and 64.9 μM, respectively, and CYP2D6-catalyzed bufuralol 1′-hydroxylation (IC 50 values of 58.7 μM and 79.1 μM, respectively). Eupatilin and jaceosidin were found to weakly inhibit CYP2C8-dependent amodiaquine N-deethylation with IC 50 values of 104.9 μM and 106.4 μM, respectively (Table 1). Eupatilin and jaceosidin showed negligible inhibition of CYP2A6-catalyzed coumarin 7-hydroxylation and CYP3A4-catalyzed midazolam 1′-hydroxylation at the highest concentration tested (100 μM). The inhibitory potencies of eupatilin and jaceosidin were not affected significantly after a 30 min preincubation with microsomes in the presence of NADPH ( Table 2), indicating that there is no mechanism-based inhibition.  In an inhibition study, it was found that the apparent K i value is a more appropriate parameter for defining the interaction of the inhibitor with a particular enzyme. The K i values and inhibition types (competitive, noncompetitive, uncompetitive, or mixed) were determined for eupatilin and jaceosidin using Lineweaver plots, Dixon plots, and secondary reciprocal plots. The results are summarized in Table 2. Eupatilin showed the mixed-type inhibition for CYP2C9-catalyzed diclofenac 4-hydroxylation (K i , 1.6 μM; α, 10.3) and CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation (K i , 28.7 μM; α, 8.7) (Figure 1, Table 2). Eupatilin was found to competitively inhibit CYP1A2catalyzed phenacetin O-deethylation (K i , 2.3 μM), CYP2C8-dependent amodiaquine N-deethylation (K i , 101.9 μM), and CYP2D6-catalyzed bufuralol 1′-hydroxylation (K i , 94.6 μM) ( Figure 1, Table 2). Jaceosidin was found to exhibit competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation (K i , 3.8 μM), CYP2C8-dependent amodiaquine N-deethylation (K i , 109.4 μM), CYP2C9-catalyzed diclofenac 4-hydroxylation (K i , 6.4 μM), and CYP2D6-catalyzed bufuralol 1′-hydroxylation (K i , 57.8 μM). Jaceosidin was found to exhibit the mixed type of inhibition for CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation with a K i of 45.1 μM (Figure 2, Table 2).  Eupatilin and jaceosidin were found to be potent competitive inhibitors of CYP1A2 with K i values of 2.3 and 3.8 μM, respectively, indicating that eupatilin and jaceosidin should be used carefully with drugs metabolized by CYP1A2 such as acetaminophen, caffeine, phenacetin, ropivacaine, tacrine, theophylline, and tizanidine to avoid drug interactions [29]. Some natural compounds including oltipraz, trans-resveratrol, tanshinone I, tanshinone IIA, and cryptotanshinone have been shown to potently inhibit CYP1A2 [29,30]. These results support the report that the antimutagenic activity of eupatilin and jaceosidin against Trp-P-2 in TA98 is a result of the inhibition of CYP1A1-catalyzed ethoxyresorufin deethylase activity in rat liver S9 fractions [12]. Eupatilin and jaceosidin were shown to be potent competitive inhibitors of CYP2C9 with K i values of 1.6 and 6.4 μM, respectively, suggesting that eupatilin and jaceosidin should be used carefully with drugs metabolized by CYP2C9 such as celecoxib, diclofenac, glyburide, losartan, tolbutamide, torasemide, and S-warfarin to avoid drug interactions [31]. The herbal preparations and medicinal herbs containing eupatilin and jaceosidin may affect CYP1A2 and CYP2C9 activity. These in vitro results suggest that eupatilin and jaceosidin should be examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.
To evaluate the effect of preincubation on inhibitory potency, eupatilin and jaceosidin (0.1−100 μM) were preincubated for 30 min with NADPH, buffer, and microsomes before the addition of the CYP probe substrates. The reaction was started by addition of the cocktail of probe substrates.

Kinetic analysis
For the determination of K i values, human liver microsomes (0.2 mg/mL) were incubated with various concentrations of substrates (10−100 μM phenacetin for CYP1A2, 0.5−5 μM amodiaquine for CYP2C8, 1−50 μM diclofenac for CYP2C9, 10−100 μM [S]-mephenytoin for CYP2C19, and 0.5−5 μM bufuralol for CYP2D6), 1 mM NADPH, 10 mM MgCl 2 , and various concentrations of jaceosidin or eupatilin in 50 mM potassium phosphate buffer (pH 7.4) in a total incubation volume of 200 μL. The reactions were initiated by addition of NADPH at 37 ºC and stopped after 15 min by placing the incubation tubes on ice and adding 100 μL of ice-cold methanol containing an internal standard. The incubation mixtures were centrifuged at 10,000× g for 5 min, and aliquots (5 μL) of the supernatants were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS).

LC/MS/MS analysis
All seven metabolites produced from the cocktail of CYP isoform-specific substrates were simultaneously determined by LC/MS/MS. The system consisted of a tandem quadrupole mass spectrometer (TSQ Quantum Access, ThermoFisher Scientific, CA, USA) coupled with a Nanospace SI-2 LC system. The separation was performed on an Atlantis dC 18 column (5 μm, 2.1 mm i.d. × 100 mm, Waters, Waltham, MA, USA) using the gradient elution of a mixture of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.25 mL/min: 10% mobile phase B for 1 min, 10% to 95% mobile phase B for 1 min, 95% mobile phase B for 5 min. The column and autosampler temperatures were 50 ºC and 4 ºC, respectively. After 1.5 min, the LC eluant was diverted from waste to the mass spectrometer that was fitted with an electrospray ionization (ESI) source and operated in the positive ion mode. The ESI source settings for the ionization of the metabolites were as follows: electrospray voltage, 5.0 kV; vaporizer temperature, 420 ºC; capillary temperature 360 ºC; sheath gas pressure, 35 psi; auxiliary gas pressure, 10 psi. Quantification was performed by selected reaction monitoring (SRM) of the [M+H] + ion and the related product ion for each metabolite. SRM transitions for the metabolites and internal standards are summarized in Table 3. The analytical data were processed using Xcalibur ® software (ThermoFisher Scientific).

Data analysis
The IC 50 values (concentration of inhibitor causing 50% inhibition of the original enzyme activity) were calculated using WinNonlin software, a non-linear regression analysis program (Pharsight, Mountain View, CA, USA). The apparent kinetic parameters for inhibitory potential (K i values) were estimated from the fitted curves using Enzyme Kinetics Ver. 1.3 program (Systat Software Inc., San Jose, CA, USA).

Conclusions
By screening the inhibitory effects of eupatilin and jaceosidin on the activities of seven CYP isoforms in human liver microsomes, eupatilin and jaceosidin were shown to be the potent inhibitors of CYP1A2 and CYP2C9 and moderate inhibitors of CYP2C8, CYP2C19, and CYP2D6. Eupatilin and jaceosidin are pharmacologically active components in medicinal herbs such as Artemisia spp. as well as Eupatorium spp. These results suggest that the use of eupatilin, jaceosidin, and herbal preparations thereof may cause interactions with drugs metabolized by CYP1A2 and CYP2C9 in some individuals. However, it is notable that in vitro inhibition of CYP1A2 and CYP2C9 activities does not necessarily result in drug interactions in clinical situations.