The effect of flavonoid aglycones on the CYP1A2, CYP2A6, CYP2C8 and CYP2D6 enzymes activity

Article abstract of DOI:10.3390/molecules24173174

Cytochromes P450 are major metabolic enzymes involved in the biotransformation of xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are exposed through everyday diet. In the previous study, selected flavonoid aglycones showed inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose, the O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated metabolites were monitored by high-performance liquid chromatography coupled with diode array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.

Full text of DOI:10.3390/molecules24173174

molecules
Article
The Effect of Flavonoid Aglycones on the CYP1A2,
CYP2A6, CYP2C8 and CYP2D6 Enzymes Activity
3
Mirza Bojic´ 1,*, Martin Kondža 2 , Hrvoje Rimac 1, Goran Benkovic´ and Željan Maleš 4
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb,
A. Kovacˇic´a 1, 10000 Zagreb, Croatia
Matice hrvatske, Faculty of Pharmacy, University of Mostar, 88000 Mostar, Bosnia and Herzegovina
Agency for Medicinal Products and Medical Devices, Ksaverska cesta 4, 10000 Zagreb, Croatia
Department of Pharmaceutical Botany, Faculty of Pharmacy and Biochemistry, University of Zagreb,
2
3
4
Schrottova 39, 10000 Zagreb, Croatia
*
Correspondence: mbojic@pharma.hr; Tel.: +385-1-4818-304
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 August 2019; Accepted: 31 August 2019; Published: 1 September 2019
Abstract: Cytochromes P450 are major metabolic enzymes involved in the biotransformation of
xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels
of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are
exposed through everyday diet. In the previous study, selected flavonoid aglycones showed
inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids
inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose,
the O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin
7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme
activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated
metabolites were monitored by high-performance liquid chromatography coupled with diode
array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2
activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin,
chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed
inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and
CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most
prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.
Keywords: flavonoids; CYP1A2; CYP2A6; CYP2C8; CYP2D6; inhibition
1. Introduction
Cytochrome P450 (CYP) enzymes are the most significant enzymes for the metabolism of substances
foreign to the human body, including drugs [1]. These enzymes are hemoproteins containing heme,
which is bound to the apoprotein part. The iron ion is linked to the heme by four coordinative
covalent bonds, while the fifth coordinative covalent bond links iron to the cysteine residue of the
apoprotein. Functionally cytochromes P450 are monooxygenases that incorporate one atom of oxygen
from molecular oxygen into the substrate [1].
The reactions catalyzed by cytochromes P450 are not limited to one type of reaction or one
substrate. Instead, each cytochrome P450 enzyme has numerous substrates and catalyzes chemically
diverse reactions. Beside oxidation reactions (e.g., hydroxylations, dealkylations), cytochromes catalyze
reduction, desaturation, ester cleavage, ring expansion, ring formation, aldehyde scission, dehydration,
ipso attack, one-electron oxidation, coupling reactions, rearrangement of fatty acid, and prostaglandin
hydroperoxides [2].
Molecules 2019, 24, 3174; doi:10.3390/molecules24173174
www.mdpi.com/journal/molecules

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The major site of cytochromes P450 activity within the human organism is the liver. The liver
cytochromes P450 involved in the metabolism of most drugs are: CYP3A4/5 (30%), CYP2D6 (20%),
CYP2C9 (13%), CYP1A2 (9%), and others [3]. As nearly 94% of oxidation reactions of medicinal drugs
are catalyzed by cytochromes P450, the involvement of these enzymes in the pharmacokinetic of the
drugs is significant.
Drugs and other xenobiotics can be substrates of cytochromes P450, but they can also act as
inhibitors. Two substrates competing for the same active site of the cytochrome P450 enzyme present the
most common observed interaction, e.g., ketoconazole is a competitive CYP3A4 inhibitor that can reduce
terfenadine metabolism, which is almost exclusively metabolized by CYP3A4 [4]. The aforementioned
interaction is reversible, i.e., discontinuation of the ketoconazole application normalizes terfenadine
metabolism and pharmacokinetic.
Inhibitions that can have more significant consequences are irreversible. These are usually caused
by inactivation of cytochromes P450 by a reactive intermediar that covalently binds to the apoprotein
or the heme part of cytochrome P450. A good example is the antihypertensive drug mibefradil, which
irreversibly inhibits CYP3A4. Mibefradil is converted to a reactive intermediar in the catalytic cycle
of CYP3A4. The generated intermediar covalently binds to the heme part of CYP3A4, causing the
inactivation of the enzyme [5]. The enzyme activity is lost and can only be regained by the expression
of an additional protein (enzyme), which takes days to weeks [6].
Except drugs, other xenobiotics like flavonoids can interact with cytochrome P450. Flavonoids are
natural polyphenols abundantly present in higher plants, including fruits, vegetables, and plant-derived
products, such as wine and propolis [7–9]. Flavonoids are regarded as vitamins that are important in the
regulation of oxidative stress and act as antioxidants. Consequently, flavonoids have many beneficial
health effects such as antiallergic, anti-inflammatory, antioxidative, antimicrobial, antitumorigenic,
and antimutagenic effects, thus preventing cancer, heart disease, bone loss, and a number of other
diseases [8,10–13].
Flavonoids, in nature, mainly come bound to a sugar moiety as glycosides. Glycosides are
susceptible to hydrolysis, and aglycones are liberated and absorbed in the gut [6]. In the liver, aglycones
are metabolized by the cytochromes P450. The major metabolic reactions to which flavonoids are
susceptible are aromatic hydroxylations and O-dealkylations, which are catalyzed by cytochromes
P450 [14
,
15]. The major cytochromes P450 involved in the metabolism of flavonoid aglycones are
15]. Flavonoids can also act as inhibitors of metabolic enzymes,
CYP1A2, CYP3A4, and CYP2D6 [14
,
causing clinically significant interactions [16–21].
In the previous study [22], it was shown that some flavonoid aglycones can cause the inhibition
of CYP3A4 metabolic activity. These flavonoids were: Hesperetin, pinocembrin, acacetin, chrysin,
chrysin-dimethylether, flavone, tangeretin, galangin, isorhamnetin, morin, and tamarixetin (statistical
significance p < 0.1) (Figure 1). Thus, the aim of this study was to assess the inhibitory effect of the
aforementioned flavonoids on the CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes activity.

Molecules 2019, 24, 3174
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O
O
CH3
CH3
HO
O
O
HO
O
O
HO
O
O
OH
hesperetin
pinocembrin
acacetin
OH
OH
OH
CH3
O
HO
O
O
O
O
O
O
chrysin
chrysin-dimethylether
flavone
CH3
OH
O
O
H3C
O
CH3
O
OH
CH3
CH3
O
O
HO
O
O
HO
O
O
H3C
O
OH
OH
isorhamnetin
tangeretin
galangin
O
O
OH
OH
H3
C
OH
HO
OH
O
CH3
HO
O
O
HO
O
O
OH
OH
tamarixetin
morin
OH
OH
Figure 1. Structural characteristics of flavonoids used in this study. Flavanones are marked in red,
flavones in green, and flavanols in blue.
2. Results and Discussion
To test the enzyme activity of each individual cytochrome P450, marker substrates/reactions
were used, namely phenacetin O-deethylation for monitoring CYP1A2 enzyme activity, coumarin
7-hydroxylation for monitoring CYP2A6 enzyme activity, paclitaxel 6-α-hydroxylation for monitoring
CYP2C8 enzyme activity, and dextromethorphan O-demethylation for monitoring CYP2D6 enzyme
activity (Figure 2). All generated products were determined using high-performance liquid
chromatography coupled with a diode-array detector (HPLC-DAD) with the appropriate method of
analysis for each individual reaction (vide infra).
(a)
O
O
NH
NH
CH3
CH3
CYP1A2
O
HO
H3C
phenacetine
acetaminophen
(b)
CYP2A6
O
O
HO
O
O
coumarin
7-hydroxycoumarin
(c)
O
O
CH3
CH3
O
O
H3C
H3C
O
O
O
O
H3
C
H3
C
CH3
CH3
O
OH
CYP2C8
O
OH
OH
H3C
H3C
HN
HN
OH
OH
HO
HO
O
O
O
O
O
O
O
O
O
H3
O
H3
O
O
C
C
paclitaxel
6-hydroxypaclitaxel
(d)
CH3
O
HO
CYP2D6
N
N
CH3
CH3
dextromethorphan
dextrorphan
Figure 2. Marker substrates and reactions used for monitoring enzyme activity: (
O-deethylation for the CYP1A2 enzyme, ( ) coumarin 7-hydroxylation for the CYP2A6 enzyme,
) paclitaxel 6- -hydroxylation for the CYP2C8 enzyme, and ( ) dextromethorphan O-demethylation
for the CYP2D6 enzyme. The sites of reaction are marked in blue.
a) Phenacetin
b
(
c
α
d

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CYP1A2 activity was inhibited by five of the tested flavonoids, namely chrysin, hesperetin,
isorhamnetin, morin, and pinocembrin (Figure 3).
Figure 3. Residual CYP1A2 enzyme activity after incubation with 1 µM flavonoid in the experiments
for the determination of metabolism dependent inhibition (MDI), time dependent inhibition (TDI),
and direct inhibition (DI). * denotes that observed inhibition is statistically significant (p < 0.05) when
compared to the control (without addition of flavonoid).
Chrysin showed inhibition of CYP1A2 in all inhibition assays: Metabolism (MDI) and time
dependent (TDI), as well as in the direct inhibition (DI) assay. As direct inhibition and metabolism
inhibition assay are of comparable residual activities (23
±
6% (p = 0.001) and 18
±
4% (p = 0.004),
respectively), it can be concluded that inhibition is reversible. This is in accordance with previously
published data. Lee et al. [23] tested 21 flavonoids, out of which chrysin was shown to be the strongest
inhibitor of CYP1A2. The enzyme activity was tested with N3-demethylation of caffeine as a marker
reaction, and chrysin showed reversible, competitive inhibition. On rat liver microsomes, Siess et al. [24]
showed that chrysin inhibited ethoxy- and pentoxy-resorufin dealkylation and characterized the
inhibition as mix type reversible. Phenacetin O-deethylation and caffeine N3-demethylation are marker
reactions of CYP1A2, ethoxy- and pentoxy-resorufin, while dealkylation was used to characterize
the overall contribution of all cytochromes. The inhibition observed in the study performed by
Siess et al. [24] can be consequence of the inhibition of other cytochromes P450, rather than CYP1A2.
However, Kim et al. [25] confirmed the reversible mix inhibition of CYP1A2 using the ethoxyresorufin
O-deethylation reaction on a recombinant cytochrome P450 system. Mixed inhibition is in accordance
with observed results of the TDI assay that showed complete inhibition of CYP1A2 (Figure 3). In the
TDI assay, sufficient time is given by preincubating chrysin with the enzyme before starting the catalytic
marker reactions.
Hesperetin showed a less extensive inhibition of CYP1A2 under the same conditions compared
to other flavonoid inhibitors of CYP1A2. As residual activity increased in the order of DI, TDI, and
MDI assays, it can be concluded that hesperetin showed reversible, as well as irreversible, inhibition of
CYP1A2, although the later one was not statistically significant (Figure 3). Doostdar et al. [26] showed
that hesperetin is an inhibitor of CYP1B1, while residual CYP1A2 enzyme activity was not statistically
different to control in the analyzed range of concentrations, including the one used in the present study
(1 µM). This difference could be attributed to different marker reaction used by Doostdar et al. [26],
i.e., ethoxyresorufin O-dealkylation. The significance of hesperetin CYP1A2 inhibition was relevant for
the pharmacokinetic and metabolism of rasagiline mesylate in Wistar rats, explaining the observed
drug-hesperetin interaction [27].
Chang et al. [28] showed competitive inhibition of CYP1A2-mediated 7-ethoxyresorufin O-dealkylation
with an inhibition constant of 0.14 µM. This is in accordance with results of our study, in which
isorhamnetin was shown to be a reversible inhibitor of CYP1A2. Interestingly, if isorhamnetin is
preincubated with CYP1A2, the residual activity of CYP1A2 decreases to 8 ± 2% (p = 0.012).

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On rat liver microsomes, Siess et al. [24] showed that morin inhibited ethoxy- and pentoxy-resorufin
dealkylation, and characterized the inhibition as mix type reversible similarly to chrysin. However,
the inhibition constant for morin was 16-fold greater (5 µM). This is in accordance with the results of
our study, as patterns observed in MDI, TDI, and DI assays were the same for morin and chrysin, and
chrysin was a more potent inhibitor than morin. Sahu et al. [29] showed that the inhibition of CYP1A2
could influence the febuxostat metabolism (substrate of CYP1A2), causing drug-flavonoid interactions,
while Li et al. [30] confirmed interactions of etoposide and morin, although later one was attributed to
the other cytochromes P450 as well.
Pinocembrin inhibited CYP1A2 enzyme activity. The observation that pinocembrin acts as a
metabolism dependent, time dependent, and direct inhibitor of CYP1A2 has not been previously
reported. As in the case of the most of aforementioned flavonoid inhibitors of CYP1A2, time dependent
inhibition is the most prominent compared to other types. This indicates that these flavonoids require
time to interact with CYP1A2 for the inhibition to be observed, and could be attributed to allosteric,
nonspecific binding to the enzyme.
Out of 11 analyzed flavonoids, apigenin, galangin, isorhamnetin, and tangeretin inhibited CYP2A6
enzyme activity (Figure 4). Although TDI was an important type of CYP1A2 enzyme, this was not the
case with the CYP2A6 enzyme, as it was shown only to be significant in the case of isorhamnetin.
Figure 4. Residual CYP2A6 enzyme activity after incubation with 1 µM flavonoid in the experiments
for the determination of metabolism dependent inhibition (MDI), time dependent inhibition (TDI),
and direct inhibition (DI). * denotes that observed inhibition is statistically significant (p < 0.05) when
compared to the control (without addition of flavonoid).
Apigenin was reported to inhibit CYP2A6 activity in the metabolism dependent inhibition assay,
as well as the direct inhibition assay of CYP2A6. Boonruanga et al. [31] reported similar IC50 values in
metabolism dependent and direct inhibition assays of 0.77
Likewise, under the similar experimental conditions and 1
comparable values of residual CYP2A6 enzyme activity: 47
±
0.16
M concentration of apigenin, we observed
7% (p = 0.037) and 48 1% (p = 0.018).
This indicates that apigenin is a direct, reversible inhibitor of the CYP1A2 enzyme.
Galangin showed similar values of residual CYP2A6 activity in MDI and DI assays, indicating that
it is a reversible, direct inhibitor of CYP1A2. This is in accordance with the results of Tiong et al. 32],
µ
M and 0.90
±
0.07 µM, respectively.
µ
±
±
[
who conducted experiments using the recombinant enzyme and the same marker reaction as our study.
Isorhamnetin showed inhibition of CYP2A6 enzyme activity, which was most prominent in the
TDI assay: 16
±
5% (p = 0.025). (Figure 4). This indicates that isorhamnetin acts as a reversible, time
dependent inhibitor of CYP1A2. This could explain the observed interactions of the Ginkgo biloba
extract with valproic acid observed by Numa et al. [33].
Although it has been reported that tangeretin is metabolized by cytochromes P450, namely
CYP1A2, CYP3A4, and CYP2D6 [14,15], it is interesting to note that tangeretin acts as a direct inhibitor
of CYP2A6 enzyme activity.
Although the inhibition of CYP2C8 was reported for some flavonoids [34
,35], this is the first
report on the inhibition of CYP2C8 paclitaxel 6- -hydroxylation by chrysin, chrysin-dimethylether,
α

Molecules 2019, 24, 3174
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and galangin. In the TDI assay, the reduction of enzyme activity was not observed, and values of
residual CYP2C8 enzyme activity were lower in MDI compared to DI assay (Figure 5). Thus, it can
be concluded that chrysin, chrysin-dimethylether, and galangin are mixed reversible and irreversible
inhibitors of CYP2C8.
Figure 5. Residual CYP2C8 enzyme activity after incubation with 1 µM flavonoid in the experiments
for determination of metabolism dependent inhibition (MDI), time dependent inhibition (TDI), and
direct inhibition (DI). * denotes that observed inhibition is statistically significant (p < 0.05) when
compared to the control (without addition of flavonoid).
Interestingly, no inhibition of CYP2D6 was observed on the set of analyzed flavonoids, neither
reversible nor irreversible. Flavonoids are weak acids, having mainly hydroxyl groups, while typical
CYP2D6 substrates contain a nitrogen atom, which can be protonated at physiological pH [36].
This could explain why not even direct competitive inhibition was observed when dextromethorphan
was used as the marker substrate of CYP2D6 activity.
In our previous research, a significant number of irreversible flavonoid inhibitors of CYP3A4 was
reported [22]. However, flavonoids in this study mainly served as reversible inhibitors of CYP1A2 and
CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible).
To assess the reversible binding of flavonoids to cytochromes P450, a docking study of the most
potent reversible inhibitor, i.e., chrysin, to the CYP1A2 enzyme was conducted.
The redocking of alpha-naphthoflavone, both in presence and in absence of HOH 733, was performed
and compared with crystallographic data (Figure 6). The docked positions of alpha-naphthoflavone
with and without the water molecule were rotated by approximately 180◦ compared to each other.
Alpha-naphthoflavone was docked with the water molecule having the virtually same coordinates as
the crystallographic alpha-naphthoflavone. This was used as a confirmation that the docking of our
chrysin species with the HOH 733 water molecule was a valid approach, as the water molecule can
affect the ligand position in the active site of CYP1A2 [37,38].
Figure 6. Comparison of the docked alpha-naphthoflavone with all water molecules removed
(green), docked alpha-naphthoflavone in presence of HOH 733 (yellow), and crystallographic
alpha-naphthoflavone (brown) in vicinity of cytochrome P450 1A2 heme. Oxygen atoms are depicted
in red, nitrogen atoms in blue, hydrogen atoms in white, and heme iron in orange.

Molecules 2019, 24, 3174
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For both chrysin species, docking with and without the HOH 733 water molecule was done and
their positions were compared (Figure 7, Table 1).
Figure 7. Comparison of docked chrysin species in presence and in absence of the HOH 733 water
molecule (the results are identical in both cases). Anion species are depicted in blue (top-ranked docked
species) and pink (second top-ranked docked species), while molecule species are depicted in green
(top-ranked molecule species) and orange (third top-ranked molecule species in presence of HOH 733
and second top-ranked molecule species in absence of HOH 733). Oxygen atoms are depicted in red,
nitrogen atoms in blue, hydrogen atoms in white, and heme iron in orange.
Table 1. Summary of Chrysin Species Docking.
Chrysin Anion at Position 7
In Presence of HOH 733
In Absence of HOH 733
Cluster
Rank
Percentage Binding Energy
Inhibitory
Constant (µM)
Cluster
Rank
Percentage Binding Energy
Inhibitory
Constant (µM)
of Runs
(kcal/mol)
of Runs
(kcal/mol)
1.
2.
84
16
−6.91
−6.70
8.66
12.22
1.
2.
80
20
−6.94
−6.75
8.23
11.26
Chrysin Molecule
In Presence of HOH 733
In Absence of HOH 733
Cluster
Rank
Percentage Binding Energy
Inhibitory
Constant (µM)
Cluster
Rank
Percentage Binding Energy
Inhibitory
Constant (µM)
of Runs
(kcal/mol)
of Runs
(kcal/mol)
1.
2.
3.
81
17
2
−8.23
−8.13
−8.07
0.93
1.10
1.21
1.
2.
98
2
−8.26
−8.09
0.89
1.18
The anion species at position 7 binds in the same position both in presence and in absence of the
HOH 733 water molecule, with the 7-O- group closest to the heme iron. For the docked molecule
species, the HOH 733 water molecule does not play a significant role. The top-ranked docking pose
in both cases was identical, with the 5-OH and 4-keto group being oriented toward the heme iron.
Meanwhile, in the less populated clusters, the group closest to the heme iron was the 7-OH group.
The second-ranked cluster of docked molecule species in presence of HOH 733 is not shown, but it was
orientated with the B ring facing the heme iron. The binding energy for the molecule species (Table 1)
was much lower than for the anion species, suggesting that the molecule species was responsible
for most of the chrysin inhibitory effects. Even though the exact values of energy were probably
overestimated due to incorrect energy calculations [39], their relative comparison is still possible
since we are comparing different poses of the same molecule. The inhibitory constants obtained by
computational studies are of similar range to those observed experimentally (micromolar).
In conclusion, out of 11 analyzed flavonoids, hesperetin, pinocembrin, chrysin, isorhamnetin,
and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited
CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the
analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible
inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible
and irreversible). The determined types of inhibition are important for the further assessment of

Molecules 2019, 24, 3174
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flavonoid-drug interactions. If flavonoid is a reversible inhibitor, flavonoid-rich foods or dietary
supplements could inhibit drug metabolism mediated by cytochromes P450 and dose adjustment if
needed. If flavonoid is an irreversible inhibitor of cytochrome P450 enzyme, combinations with drugs
that it can interact with should be avoided.
3. Materials and Methods
3.1. Materials
Eleven flavonoids were used in this study. Acacetin, hesperetin, pinocembrin, chrysin, flavone,
tangeretin, galangin, isorhamnetin, and morin were acquired commercially from TransMIT (Gießen,
Hessen, Germany), while chrysin-dimethylether and tamarixetin were obtained from Extrasynthèse
(Genay, Lyon, France). The marker substrates and their corresponding metabolites were purchased
from Sigma-Aldrich (St. Louis, MO, USA), except 7-hydroxycoumarin, which was purchased from
Extrasynthèse (Genay, Lyon, France).
The recombinant baculosomes with hyperexpressed cytochromes P450 (1A2, 2A6, 2C8, and 2D6)
and coexpressed NADPH cytochrome P450 reductase and cytochrome b5 were used as a source of
enzyme. Baculosomes were commercially obtained from Thermo Fisher Scientific (Waltham, MA,
USA), and all other chemicals from Sigma-Aldrich if not otherwise indicated. A 1.0 M solution
phosphate buffer of pH 7.4 was prepared in house. NADPH generating system was prepared of 0.1 M
glucose-6-phosphate, 10 mg mL−1 NADP+, and 1000 IU mL−1 glucose-6-phosphate dehydrogenase in
ratio 100:50:2 (v/v/v) [40].
3.2. Determination of CYP1A2 Enzyme Activity
Phenacetine O-deethylation was used as a marker reaction for monitoring CYP1A2 enzyme
activity (Figure 2). Incubations of 100
incubation mixture was set at 7.4 using a potassium phosphate buffer (final concentration 50 mM).
The total amount of CYP1A2 enzyme was 5 pmol, and the final concentration of substrate was 150 M.
Residual activity was determined by incubating the flavonoid (1 M) with phenacetine as a substrate
for 15 min, with or without preincubation, depending of the type of inhibition assay. Reactions were
started by the addition of 15 L of NADPH generating system, while the reactions were stopped by
µ
L were conducted at the temperature of 37 ◦C. The pH of the
µ
µ
µ
the addition of 1 mL 1% formic acid in acetonitrile. After centrifugation (10 min, 1900
solution was transferred to a vial for HPLC analysis [22].
×
g), the clear
HPLC analysis was performed on Agilent 1100 instrument (Santa Clara, CA, USA) coupled with
a diode array detector (DAD) on Luna C18 column (4.6 150 mm, 3 m). The gradient method was
×
µ
used for separation. Mobile phase A consisted of water, acetonitrile, and glacial acetic acid in the
volume ratio of 90:10:0.1, while mobile phase B contained the same solvents in the ratio 10:90:0.1.
The following gradient timetable was used (t/min, %B): (0, 0), (2, 0), (13, 55), (14, 0), (20, 0). The flow
rate was 1 mL/min, and chromatograms were recorded at 254 nm. Volume of incubation injected to the
column was 15 µL. Retention time of substrate was 12.2 min, while product was detected at 5.7 min.
The amount of generated product (acetaminophen) was determined as the area under the curve based
on the calibration curve of the standard.
3.3. Determination of CYP2A6 Enzyme Activity
The aromatic hydroxylation of coumarin at the position 7 was used as a marker reaction for
CYP2A6. The incubation conditions were the same as for CYP1A2 enzyme (vide supra) unless
otherwise stated. The final concentration of coumarin was 10
extended to 30 min.
µM, while the incubation time was
HPLC-DAD analysis was conducted on the same system as the CYP1A2 enzyme with the following
modifications: The gradient timetable (t/min, %B) was (0, 20), (1.5, 20), (10, 70), (11, 20), (17, 20), and
the detection wavelength was 330 nm. The retention time of the substrate (coumarin) was 7.5 min,

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while the product (7-hydroxycoumarin) was detected at 4.8 min. The amount of generated product
was determined as the area under the curve based on the calibration curve of the standard [22].
3.4. Determination of CYP2C8 Enzyme Activity
Paclitaxel 6
α-hydroxylation was used as a marker reaction for monitoring CYP2C8 enzyme
activity (Figure 2). The incubation conditions were the same as for CYP1A2 enzyme (vide supra) unless
otherwose stated. The final concentration of paclitaxel was 20
30 min.
µM, while the incubation time was set to
HPLC-DAD analysis was conducted on the same system as the CYP1A2 enzyme with the following
modifications: The gradient timetable (t/min, %B) was (0, 50), (2, 50), (10, 90), (11, 50), (17, 50), and the
detection wavelength was 227 nm. The retention time of the substrate (paclitaxel) was 6.1 min, while
the product (6α-hydroxypaclitaxel) was detected at 3.4 min. The amount of generated product was
determined as the area under the curve based on the calibration curve of the standard [22].
3.5. Determination of CYP2D6 Enzyme Activity
O-demethylation of dextromethorphan to dextrorphan was used as a marker reaction for
monitoring CYP2D6 enzyme activity (Figure 2). The incubation conditions were the same as the
CYP1A2 enzyme (vide supra) unless otherwise stated. The final concentration of the marker substrate
in the incubation was 100 µM, while the incubation time was set to 30 min.
HPLC-DAD analysis was conducted on the same system as for CYP1A2 enzyme with the following
modifications: The gradient timetable (t/min, %B) was (0, 18), (5, 18), (10, 40), (15, 18), and the detection
wavelength was set to 224 nm. The retention time of the substrate (dextromethorphan) was 7.1 min,
while the product (dextrorphan) was detected at 4.1 min. The amount of generated product was
determined as the area under the curve based on the calibration curve of the standard [22].
3.6. Determination of the Inhibition Type
Three types of experiments were conducted to determine metabolism dependent inhibition, time
dependent inhibition, and direct inhibition of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes [40].
To determine metabolism dependent inhibition (MDI assay), the flavonoid was first preincubated
with the enzyme with the addition of the generating system for 30 min, after which the marker substrate
was added to determine residual enzyme activity (as described above for each enzyme).
If metabolism dependent inhibition was determined, time dependent inhibition and direct
inhibition were tested. Time dependent inhibition (TDI assay) was assessed by preincubating the
flavonoid and enzyme, without the generating system, after which residual activity was determined by
adding the NADPH generating system along with the substrate (as described above for each enzyme).
The direct inhibition assay (DI assay) was conducted without preincubation, i.e., the NADPH
generating system was added to the incubation mixture containing flavonoid and substrate following
the experimental set-up described above for each individual cytochrome P450 enzyme [40].
If the flavonoid is an irreversible inhibitor of the cytochrome P450, it will decrease enzyme activity
in the MDI assay, while no reduction of enzyme activity will be observed in TDI and DI assays. Pure
time dependent inhibitors require time to interact with the enzyme. Thus, TDI inhibitors will show a
reduction of enzyme activity in assays with preincubations, i.e., the MDI and TDI assays, but not in the
DI assay (no preincubation). Pure direct inhibitors are reversible inhibitors that usually compete with
the substrate binding to the active site and will show a decrease of enzyme activity with or without
preincubation. DI inhibitors will reduce enzyme activity in all three assays (MDI, TDI, and DI).
3.7. Docking Studies
To examine the binding of chrysin to the active site of cytochrome P450 1A2, a docking study was
performed using AutoDock 4.2.6. (The Scripps Research Institute, La Jolla, CA, USA) [39], which used
dispersion, hydrogen bonds, and electrostatic and desolvatation energy components to determine the

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conformation of the most probable complex. The three-dimensional coordinates of the cytochrome
P450 1A2 molecule co-crystallized with alpha-naphthoflavone were obtained from the RCSB [41].
This crystal structure was chosen due to similarity of alpha-naphthoflavone with our ligand, and
the crystal structure had a satisfactory resolution of 1.95 Å. The protein molecule was prepared for
docking by adding the missing side-chain atoms and hydrogen atoms, all Lys, Arg, His, and Cys
side-chains were protonated, all Asp and Glu side-chains were deprotonated, and the amino and
carboxy termini were charged. Since there was a presence of a water molecule (HOH 733) in the
vicinity of alpha-naphthoflavone molecule in the active site, the docking study was performed both
with all water molecules removed, as well as with all water molecules removed except HOH 733.
The three-dimensional forms of the ligands were drawn, and their initial geometries were minimized
in HyperChem 8.0 (Hypercube, Inc., Gainesville, FL, USA). Their charge was set to represent the most
abundant species at pH 7.4, calculated at chemicalize.com. At pH 7.4, chrysin-7-anion represents
65.49% and chrysin molecule represents 11.44% of all chrysin species. Both species were docked due
to the fact that the percentage of the molecule species sharply increases with lowering of the pH,
as happens in intrahepatic conditions [42]. Partial charges for flavonoid ligands were set according
to Ionescu et al. [43]. In AutoDock grid maps of size 70
×
70
×
70 Å were generated with 0.375 Å
spacing centered in the CYP1A2 active site cavity (4.0, 12.0, 23.0) by the AutoGrid program [39] and
Lamarckian genetic algorithm (LGA) [44] was applied. The receptor molecule was regarded as rigid,
while all ligand single bonds were allowed to rotate freely during the Monte Carlo simulated annealing
procedure. Ligand flexible docking simulations were performed with 100 runs, population size of 150,
2.5
×
107 energy evaluations, 27,000 numbers of generations, rate of gene mutation of 0.02, and rate
of crossover 0.08. A root-mean-square-deviation (RMSD) of 2.0 Å was used as a criterion for cluster
analysis of the docking results (in order to determine if two docked conformations were similar enough
to be included in the same cluster). First, the docking of alpha-naphthoflavone in presence and in
absence of HOH 733 was conducted in order to assess the appropriateness of the system. Afterward,
the docking of chrysin species was conducted, also in the presence and absence of HOH 733.
3.8. Statistical Analysis
All incubations were conducted in triplicate. The results are expressed as the residual activity of
the enzyme, i.e., the percentage of product generated in incubation with the addition of flavonoid in
ratio to the control without flavonoid. The statistical significance was tested with Student’s t-test in the
program R (The R Project for Statistical Computing, Vienna, Austria).
Author Contributions: M.B. designed the study; M.K., H.R. and G.B. performed incubation assays and HPLC-DAD
analysis; H.R. conducted computational study; Ž.M. and M.B. analyzed the data and wrote the paper.
Funding: This research was funded by Croatian Science Foundation, grant number UIP-2014-09-5704.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors and commercially.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).