Enantiomers of naringenin as pleiotropic, stereoselective inhibitors of cytochrome P450 isoforms

Article abstract of DOI:10.1002/chir.21005

Interactions between naringenin and the cytochrome P450 (CYP) system have been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The effects of naringenin on other CYP isoforms have been less investigated. In addition

Full text of DOI:10.1002/chir.21005

CHIRALITY 23:891–896 (2011)
Enantiomers of Naringenin as Pleiotropic, Stereoselective Inhibitors
of Cytochrome P450 Isoforms
1
2
1
1
2
WENJIE JESSIE LU, * VALENTINA FERLITO, CONG XU, DAVID ALASTAIR FLOCKHART, AND SALVATORE CACCAMESE
1
Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, Indiana Institute for Personalized Medicine, Indiana University
School of Medicine, Indianapolis, Indiana 46202
2
Dipartimento di Scienze Chimiche, Universit a` di Catania, viale A. Doria 6, 95125 Catania, Italy
ABSTRACT
Interactions between naringenin and the cytochrome P450 (CYP) system have
been of interest since the first demonstration that grapefruit juice reduced CYP3A activity. The
effects of naringenin on other CYP isoforms have been less investigated. In addition, it is well
known that interactions with enzymes are often stereospecific, but due to the lack of readily
available pure naringenin enantiomers, the enantioselectivity of its effects has not been charac-
terized. We isolated pure naringenin enantiomers by chiral high-performance liquid chromatog-
raphy and tested the ability of (R)-,(S)- and rac-naringenin to inhibit several important drug-
metabolizing CYP isoforms using recombinant enzymes and pooled human liver microsomes.
Naringenin was able to inhibit CYP19, CYP2C9, and CYP2C19 with IC50 values below 5 lM. No
appreciable inhibition of CYP2B6 or CYP2D6 was observed at concentrations up to 10 lM.
Whereas (S)-naringenin was 2-fold more potent as an inhibitor of CYP19 and CYP2C19 than
(
R)-naringenin, (R)-naringenin was 2-fold more potent for CYP2C9 and CYP3A. Chiral flava-
nones like naringenin are difficult to separate into their enantiomeric forms, but enantioselec-
tive effects may be observed that ultimately impact clinical effects. Inhibition of specific drug
metabolizing enzymes by naringenin observed in vitro may be exploited to understand pharma-
cokinetic changes seen in vivo. Chirality 23:891–896, 2011. VV C 2011 Wiley-Liss, Inc.
KEY WORDS: flavonoid; drug metabolism; CYP19; CYP2C9; CYP2C19; CYP3A; enantioselectivity;
food–drug interaction
INTRODUCTION
well documented to exibit enantioselective inhibition of
1
1–13
P450-mediated metabolism.
Difficulties in separating
Naringenin (Fig. 1, Structure 1) is a chiral flavanone
enantiomers and the resulting absence of readily available
pure enantiomers have limited research in this area, until
now. In one report, inhibition of recombinant CYP3A4 by
specific components of grapefruit juice was described for sin-
gle enantiomers of naringenin (50 lM), but details on the
separation of the enantiomers and their purity were not
belonging to the flavonoid class that has potential health
related properties. It is contained in some Citrus species, in
particular grapefruit, that contain large amounts of its 7-O-
neohesperidoside, naringin (Fig. 1, Structure 2). Upon inges-
tion, naringin undergoes cleavage of the sugar moiety, leav-
ing the free aglycone naringenin in the gastrointestinal
1
4
1
given.
tract. In a seminal article, naringenin was found to inhibit
The inhibition of human drug metabolism by flavonoids
like naringenin may be of medical importance. Since the
demonstration that grapefruit juice can inhibit the metabo-
the cytochrome P450 (CYP) 3A4-mediated oxidation of two
dihydropyridine cardiodepressive drugs, more markedly
2
than naringin. Later, naringenin was found inter alia to in-
1
5
lism of the CYP3A substrate felodipine, it has become
widely appreciated that the ingestion of a number of fruit jui-
hibit the activity of CYP isoforms that activate the potent
environmental carcinogen, nicotine-derived nitrosamine ke-
1
6
3
ces can slow drug metabolism, and thereby increase the
concentrations of important medications such as cyclospo-
tone. Naringenin also inhibited quinine 3-hydroxylation
mediated by CYP3A4, although it is recognized that other
1
7
18
4
rine and methadone. The biochemical basis for these
interactions involves the interaction of flavonoids with spe-
cific CYP isoforms. Although CYP3A has been most fre-
quently studied, a number of different isoforms may interact
components of grapefruit juice are more active. It may also
act as a potent immunomodulator in mice with pulmonary fi-
5
brosis, and the antioxidant properties of naringenin and
6
other polyphenols have been well studied.
Despite a volume of research over many years, the rele-
vance of stereochemistry at the C-2 stereogenic center of nar-
ingenin has not been carefully evaluated. It is well known
that interactions between an enzyme system and a substrate
All the authors declare that they have no conflict of interest.
Contract grant sponsor: National Institutes of Health National Center for
Research Resources; Contract grant number: K24RR020815. (DAF)
Contract grant sponsor: National Institute for General Medical Sciences;
Contract grant number: T32GM008425, U01GM061373. (DAF)
Contract grant sponsor: Department of Defense Breast Cancer Research Pro-
gram; Contract grant number: W81XWH-11-1-0016. (WJL)
7
are frequently stereospecific, and often influence the po-
8
tency and the response to single enantiomer. For example,
quinidine is a clinically relevant and potent inhibitor of
*
Correspondence to: Wenjie Jessie Lu, Address: 1001 West 10th Street
9
CYP2D6, while its diastereomer quinine is not, (S)-lansopra-
W7123, Myers Building, Indianapolis, Indiana 46202. E-mail: lu20@iupui.edu
Received for publication 31 May 2011; Accepted 28 June 2011
DOI: 10.1002/chir.21005
Published online 22 September 2011 in Wiley Online Library
(wileyonlinelibrary.com).
zole is a more potent inhibitor of CYP2C9, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4-mediated hydroxylation than
1
0
(
R)-lansoprazole, and many other chiral drugs have been
VV C 2011 Wiley-Liss, Inc.

8
92
LU ET AL.
3
-cyano-7-hydroxycoumarin. CYP2D6 activity was determined using the
metabolism of 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-meth-
ylcoumarin (AMMC) to 3-[2-(N,N-diethylamino)ethyl]-7-methoxy-4-meth-
ylcoumarin. Experimental procedures were consistent with the pub-
21
lished methodology. All incubations were performed using incubation
times and protein concentrations that were within the linear range for
reaction velocity. Substrates and inhibitors were prepared in acetonitrile.
A series of concentrations of inhibitor in a volume of 4 ll were mixed
with 96 ll of NADPH-Cofactor Mix (16.3 lM NADP, 828 lM glucose-6-
2
phosphate, 828 lM MgCl , and 0.4 U/ml glucose 6-phosphate dehydro-
genase), and prewarmed for 10 min at 378C. Enzyme/Substrate Mix was
prepared with fluorometric substrate, recombinant human CYP450 iso-
form and 0.1 M potassium phosphate buffer (pH 7.4). Reactions were ini-
tiated by adding 100 ll of Enzyme/Substrate Mix to bring the incubation
volume to 200 ll. The optimal final recombinant enzyme concentrations,
substrate concentrations, and incubation times were: 7.5 nM CYP19 1
Fig. 1. Structures of naringenin (1, R 5 H) and naringin (2, R 5 neohes-
peridosyl).
in this way. It is therefore important to study interactions of
naringenin with multiple CYP isoforms to be aware of any
potential clinically relevant interactions between naringenin
and medications.
For these reasons, we set out to develop an efficient proce-
dure for the isolation of single naringenin enantiomers (R
and S) in sizeable amounts. We used rac-naringenin and the
purified (R)- and (S)-naringenin preparations to test the abil-
ity of naringenin to inhibit a series of key drug metabolizing
CYP isoforms in vitro, with a focus on the enantioselectivity
of the interaction.
2
5 lM MFC for 30 min, 15 nM CYP2C9 1 150 lM MFC for 45 min, 7.5
nM CYP2C19 1 25 lM CEC for 30 min, and 7.5 nM CYP2D6 1 1.5 lM
AMMC for 30 min. All reactions were stopped by adding 75 ll of acetoni-
trile/0.1 M tris base. The generation of fluorescent metabolites was
determined immediately by measuring fluorescent response using a Bio-
Tek (Winooski, VT) Synergy 2 fluorometric plate reader. Excitation–
emission wavelengths were 400–540 nm for the MFC metabolite or 400–
4
60 nm for the CEC and AMMC metabolites. Standard curves were con-
structed using the appropriate fluorescent metabolite standards. Quanti-
fication of samples was performed by applying the linear regression
equation of the standard curve to the fluorescence response. The limits
of quantification for the metabolites of MFC, CEC, and AMMC were 4
pmol, 0.1 pmol, and 0.8 pmol in a final volume of 200 ll, respectively,
with intra- and inter-assay coefficients of variation of less than 10%.
MATERIALS AND METHODS
Chemicals and Reagents
Rac-naringenin, testosterone, 6-b hydroxytestosterone, desmethyldia-
zepam, b-NADP, glucose-6-phosphate dehydrogenase, and glucose-6-
phosphate were purchased from Sigma-Aldrich (St. Louis, MO). Bupro-
pion, 4-hydroxybupropion, and nevirapine were purchased from Toronto
Research Chemicals (North York, ON, Canada). Magnesium chloride
was purchased from Fisher Scientific (Pittsburgh, PA). All drug solu-
tions were prepared by dissolving each compound in methanol or aceto-
nitrile, and were stored at 2208C. All high-performance liquid chroma-
tography (HPLC)-grade reagents and chemicals used for mobile phase
Inhibition of Specific CYP450 Isoforms Using Pooled
HLMs
A single isoform-specific substrate concentration at the respective K
value (10 lM testosterone or 100 lM bupropion) was incubated at 378C
in duplicate with pooled HLMs and the NADPH-generating system (1.3
2
mM NADP, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl , and 0.4 U/ml
m
glucose 6-phosphate dehydrogenase) in the absence or the presence of
a range of (R)-, (S)- or rac-naringenin (up to 50 lM). The activity of
CYP2B6 was determined by measuring the formation rate of 4-hydroxy-
bupropion from bupropion in pooled HLMs, while the activity of CYP3A
was determined by measuring the formation rate of 6b-hydroxytestoster-
one from testosterone. All incubations were performed using incubation
time and protein concentrations that were within the linear range for
reaction velocity. An incubation mixture that consisted of substrate
probes, HLMs, and 100 mM phosphate reaction buffer (pH 7.4) was pre-
warmed for 5 min at 378C. The reaction was initiated by the addition of
the NADPH-generating system, and incubated at 378C for 15 min. The
final protein concentration of pooled HLMs was 0.25 mg/ml. All reac-
tions were terminated by the addition of 500 ll of acetonitrile, followed
by immediate vortex and placement of the tubes on ice.
19
and buffers were obtained as previously described. Pooled human liver
microsomes (HLMs) and the CYP inhibitor screening kits for CYP19,
2C9, 2C19, and 2D6 were purchased from BD Biosciences (San Jose,
CA). All microsomal preparations were stored at 2808C.
Separation, Isolation, and Purification of Individual
Enantiomers of Naringenin
The HPLC apparatus used for separation of the (R)- and (S)-enantiom-
ers consisted of a Jasco pump PU 980 with Rheodyne 20 or 200-ll sam-
ple loops, a low pressure mixer LG-1580-02 and a line degasser 1550-54,
a Uvidec 100-IIII spectrophotometric detector operating at 292 nm
(
Jasco, Tokyo, Japan). Chromatograms were acquired and processed
using computer-based Jasco Borwin 2 software. Circular dichroism (CD)
spectra were recorded on a Jasco 810 spectropolarimeter using a 1-mm
cell. Chromatographic separations were performed on a Lux Cellulose 2
column, 250 mm 3 4.6 mm i.d., whose chiral selector was tris-3-chloro-4-
methylphenylcarbamate coated on 5-lm silica gel (Phenomenex Italia,
Bologna). A column in line filter with 0.5-lm stainless steel frit of 3 mm
diameter from Rheodyne was used to protect the HPLC column. Dispos-
able polytetrafluoroethylene (PTEF) filters of 0.2 lm pore size were
used for filtration of sample solutions. HPLC chromatographic parame-
Quantification of 4-Hydroxybupropion
and 6b-Hydroxytestosterone Formation
All samples were extracted immediately after the incubations were
performed. First, an internal standard was added to each sample. The
incubation mixture was then centrifuged at 14,000 rpm for 5 min at room
temperature. The supernatant layer was made alkaline by adding 500 ll
of 1 M glycine–NaOH buffer (pH 11.3) and extracted by adding 6 ml of
ethyl acetate. This mixture was vortex-mixed for 10 min and then centri-
fuged at 36,000 rpm for 15 min. The organic layer was transferred to 13
20
s
ters a and R were calculated according to the usual procedure.
Inhibition of Recombinant Human CYP450 Isoforms
3
100-mm glass culture tubes and evaporated to dryness. The resulting
The activity of each recombinant human CYP450 isoform was deter-
mined by measuring the conversion rate of a fluorometric substrate to
its fluorescent metabolite. CYP19 and CYP2C9 activities were deter-
mined using the metabolism of 7-methoxy-4-trifluoromethylcoumarin
residue was reconstituted in mobile phase. HPLC assays with ultraviolet
(UV) detection were developed for the quantification of 4-hydroxybupro-
pion and 6b-hydroxytestosterone. The HPLC system consisted of a Shi-
madzu LC-10AT pump, SIL-10AD auto-sampler, SCL-10A system control-
ler, and SPD-10A UV–vis detector (Shimadzu Scientific Instruments, Co-
lumbia, MD). (1) 4-hydroxybupropion and nevirapine (internal standard)
(
MFC) to 7-hydroxytrifluoromethylcoumarin. CYP2C19 activity was
determined using the metabolism of 3-cyano-7-ethoxycoumarin (CEC) to
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE CYP INHIBITION BY NARINGENIN
893
were separated using a Zorbax SB-C18 column (150 mm 3 4.6 mm, 3.5
lm particle size; Phenomenex, Torrance, CA), a Luna C18 Guard column
30 mm 3 4.6 mm, 5 lm; Phenomenex) and mobile phase consisting of
5% (v/v) 10 mM KH PO (pH adjusted to 3) and 15% acetonitrile (flow
rate, 1 ml/min). UV detection was set at 214 nm for 4-hydroxybupropion
retention time: 14.2 min) and 282 nm for nevirapine (retention time: 29
(
8
2
4
(
min). (2) 6b-hydroxytestosterone and desmethyldiazepam (internal
standard) were separated using the same columns, but with a mobile
phase consisting of 40% 30 mM ammonium acetate (pH adjusted to 6.3)
and 60% methanol (flow rate, 1 ml/min). UV detection was set at 254 nm
for 6b-hydroxytestosterone (retention time: 5 min) and desmethyldiaze-
pam (retention time: 10.4 min). Peak areas for each peak were obtained
from an integrator, and peak area ratios with internal standard were cal-
culated. Formation rate of metabolite from the respective probe sub-
strate was quantified by using the appropriate standard curve. Intra- and
inter-day coefficients of variation of the assays were less than 15%.
Kinetic Analysis
The rates of metabolite formation from substrate probes in the pres-
ence of the test inhibitors were compared with those in control in which
the inhibitor was replaced with vehicle. The extent of CYP450 inhibition
was expressed as percent enzyme activity remaining compared to con-
trol. IC50 values were determined as the inhibitor concentrations that
brought about a 50% reduction in enzyme activity by fitting all the data
to a one-site competition equation using Prism version 5.01 for Windows
(
GraphPad Software, San Diego, CA).
RESULTS
Separation, Isolation, and Purification of Naringenin
Enantiomers
An improved separation of the enantiomers of naringenin
was accomplished by chiral HPLC on a polysaccharide-
2
2
derived chiral stationary phase (Lux Cellulose 2) using a
mobile phase n-hexane/ethanol with 0.5% of trifluoroacetic
acid (TFA) 80:20 at 1.2 ml/min. We obtained good values for
Fig. 2. Separation of naringenin enantiomers. (A) Typical HPLC separa-
tion of the (S) and (R) enantiomers of naringenin with retention times of 6.87
and 11.19 min, respectively. Conditions: chiral stationary phase Lux Cellulose
2
; mobile phase: n-hexane/ethanol with 0.5% TFA, 80:20 at 1.2 ml/min. (B)
enantioselectivity (a) and resolution factor (R ), of 1.97 and
CD spectra (ethanol, 228C) of the enantiomers of naringenin obtained from
the first (1) and the second (2) HPLC eluted peaks. The negative CD band
centred at 288 nm corresponds to the (S)-configuration. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
s
8
.3, respectively. This is a large improvement in the resolu-
tion factor with respect to previous enantioseparation
2
3
reported on a different chiral stationary phase. A typical
chromatogram is shown in Figure 2A with retention times of
6
.87 and 11.19 min for the (S)- and (R)-enantiomers, respec-
shown in Figure 2A, is the (S)-enantiomer and the second
eluted enantiomer is the (R)-enantiomer. Remarkably, the
elution order of the enantiomers is reversed with respect to
the elution order observed using different polysaccharide-
tively. Isolation of the single enantiomers of naringenin was
performed using a composition 70:30 of the same mobile
phase without TFA, at a flow rate 1.5 ml/min. The absence
of TFA avoided problems in the solvent elimination from the
eluates although some tailing of the peaks was present.
These conditions are a compromise between short elution
times (t 5 3.38, t 5 4.18 min) and adequate resolution fac-
2
3,24
based chiral stationary phases.
Using this efficient procedure the (R)- and (S)-enantiomers
of naringenin were isolated in sizeable amounts (15 mg) and
used for subsequent experiments.
1
2
tors. The isolation was accomplished by repeated injections
about 160) of 70 ll of a solution (5 mg/ml) of rac-naringenin
(
Inhibition of Cytochrome P450 Isoform Activity
in n-hexane/ethanol (1:1). Collection of the eluates after fil-
tration and rotoevaporation yielded ꢀ20 mg of each enan-
tiomer. The enantiomeric purity (e.p.) was checked using
the same analytical conditions and it was 98.0 and 96.5% for
the first and the second eluted enantiomer, respectively.
Naringenin was able to inhibit a number of CYP isoforms.
Specifically, dose-dependent inhibition of CYP19, CYP2C9,
CYP2C19, and CYP3A was demonstrated (Figs. 3–6), while
no appreciable inhibition of CYP2B6 or CYP2D6 was
observed at concentrations up to 10 lM (data not shown).
When the enantiomers of naringenin were tested, enantiose-
lective inhibition of CYP19, CYP2C9, CYP219, and CYP3A
was shown. While the (S)-enantiomer was approximately
2-fold more potent than the (R)-enantiomer as an inhibitor of
CYP19 and CYP2C19, the (R)-enantiomer was about 2-fold
more potent as an inhibitor of CYP2C9 and CYP3A (Figs. 3–
6, Table 1). In each case, the IC50 value for rac-naringenin
was between that for the (R)-enantiomer and that for the (S)-
enantiomer.
Because of the high values of a and R , the method can be
scaled up using a larger column with the same chiral selector
s
(
not available to us) for isolating a greater quantity of single
enantiomers with a much minor number of injections.
The CD spectra of the enantiomers were mirror images of
each other indicating the enantiomeric nature of the col-
lected peaks, as shown in Figure 2B. The absolute configura-
tion of the enantiomers was established taking into account
that the negative CD band centred at 288 nm corresponds to
2
3,24
the (S)-configuration.
Thus, the first eluted enantiomer,
Chirality DOI 10.1002/chir

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94
LU ET AL.
Fig. 3. Enantioselective inhibition of human CYP19 by naringenin. A
Fig. 5. Enantioselective inhibition of human CYP2C19 by naringenin. A
range of concentrations of rac-naringenin (open circle), (R)-naringenin (dark
square), or (S)-naringenin (dark triangle) was incubated with recombinant
human CYP2C19 (7.5 nM) and substrate CEC at 378C for 30 min and enzyme
activity was determined in three independent experiments. Individual points
represent the mean of duplicate incubations.
range of concentrations of rac-naringenin (open circle), (R)-naringenin (dark
square), or (S)-naringenin (dark triangle) was incubated with recombinant
human CYP19 (7.5 nM) and substrate MFC at 378C for 30 min and enzyme
activity was determined in three independent experiments. Individual points
represent the mean of duplicate incubations.
DISCUSSION
concentrations of naringenin we observed all fall in the low
micromolar range (Table 1), consistent with previous studies
that have shown maximal plasma concentrations of narin-
In this study we describe an efficient and effective separa-
tion of the chiral enantiomers of naringenin. This has
allowed the testing of these enantiomers as inhibitors of CYP
isoforms, but their use need not be limited to this study.
Many other naturally occurring flavonoids and isoflavanoids
exist as stereoisometric mixtures, in which one enantiomer
predominates. The enantioselective separation and purifica-
tion of chiral flavonoids such as that described here should
allow researchers to address biological questions with
clarity, and to understand biochemical mechanisms with pre-
cision.
2
5
genin after ingestion of grapefruit juice to be ꢀ6 lM. These
data suggest that naringenin may have effects in vivo on the
activity of multiple enzymes. Such inhibitory effects may con-
tribute to the clinically observed food–drug interactions
2
6
brought about by grapefruit juice and other fruit products.
These data are consistent with those reported on inhibition
of CYP3A, but whether interactions with CYP19, CYP2C9, or
CYP2C19 carry similar clinical relevance must await further
research.
Studies presented here with purified (R)- and (S)-narin-
genin showed that the individual enantiomers were selective.
The data presented here demonstrate the ability of narin-
genin to inhibit multiple CYP450 isoforms in vitro. The IC50
Fig. 4. Enantioselective inhibition of human CYP2C9 by naringenin. A
range of concentrations of rac-naringenin (open circle), (R)-naringenin (dark
square), or (S)-naringenin (dark triangle) was incubated with recombinant
human CYP2C9 (15 nM) and substrate MFC at 378C for 45 min and enzyme
activity was determined in three independent experiments. Individual points
represent the mean of duplicate incubations.
Fig. 6. Enantioselective inhibition of human CYP3A by naringenin. A
range of concentrations of rac-naringenin (open circle), (R)-naringenin (dark
square), or (S)-naringenin (dark triangle) was incubated with pooled HLMs
(protein concentration 0.25 mg/ml) and probe substrate testosterone at 378C
for 15 min and enzyme activity was determined in two independent experi-
ments. Individual points represent the mean of duplicate incubations.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE CYP INHIBITION BY NARINGENIN
895
TABLE 1. IC50 values for enantioselective inhibition of multiple human CYP450 isoforms by naringenin
lM
CYP19
CYP2C9
CYP2C19
CYP3A
rac-Naringenin
2.0 (1.7, 2.4)
2.8 (2.4, 3.4)
1.4 (1.1, 1.7)
2.6 (2.0, 3.4)
1.6 (1.2, 2.1)
3.4 (2.6, 4.5)
2.6 (2.4, 2.9)
4.9 (4.4, 5.5)
1.2 (1.1, 1.4)
17.6 (14.8, 20.8)
9.7 (7.3, 12.8)
21.4 (18.5, 24.7)
(
(
R)-Naringenin
S)-Naringenin
IC50 values are expressed as mean (95% confidence interval).
While the (R)-enantiomer was a more potent inhibitor of
some CYP isoforms (e.g., CYP2C9 and CYP3A), remarkably
the (S)-enantiomer was more potent on others (CYP2C19
and CYP19). These data shed light on the enantioselective
preferences of specific CYP isoforms. While this enantiose-
lective inhibition is clear in vitro, the predominant enan-
tiomer of naringenin in natural fruits is unknown and so the
vulnerability of specific enzymes to naringenin-related drug
interactions in vivo cannot be determined. While a large liter-
ature exists on the effects of grapefruit and other juices on
Lastly, the enantioselective properties exhibited by narin-
genin here could provide insight into the use of naringenin
and other flavonoids as novel, selective therapeutic agents.
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3
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A limitation of this study is that inhibitory potencies for dif-
ferent CYP isoforms were obtained using different microso-
mal systems. Four microsomal preparations of different
recombinant CYP isoforms and one preparation of pooled
HLMs were used. As a result the observed IC50 values in any
individual enzyme system should not be directly compared
with those obtained in any other system. The question of
whether or not naringenin inhibits CYP19, CYP2C9, or
CYP2C19 clinically, as has been reported for CYP3A,
requires further investigation. Despite this limitation, it is
clear that the inhibition of these CYP enzymes by naringenin
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CYP2C9, CYP2C19, and CYP3A. The data indicate that inhi-
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