In Vitro Regio- and Stereoselective Oxidation of β-Ionone by Human Liver Microsomes

Article abstract of DOI:10.1055/s-0042-112128

The metabolism of the norisoprenoid β-ionone was investigated in vitro using human liver microsomes and 11 different recombinant cytochrome P450 enzymes expressed in Trichoplusia ni cells. β-Ionone was found to be oxidized via 4S-hydroxylation by CYP2B6 i

Full text of DOI:10.1055/s-0042-112128

Original Papers
In Vitro Regio- and Stereoselective Oxidation of
β-Ionone by Human Liver Microsomes
1
2
3
4
2,5
Authors
Shinsuke Marumoto , Ryoyu Shimizu , Genzoh Tanabe , Yoshiharu Okuno , Mitsuo Miyazawa
Affiliations
The affiliations are listed at the end of the article
Key words
Abstract
Vmax for oxidation of β-ionone by CYP1A2 and
CYP2B6 was 107.9 ± 36.0 µM and 3200.3 ±
323.0 nmol/min/nmol P450 and 5.6 ± 1.2 µM and
572.8 ± 29.8 nmol/min/nmol P450, respectively.
The reaction rates observed using human liver
microsomes and recombinant CYP2B6 were very
high compared with those of other CYP2B6 sub-
strates reported thus far. These results suggest
that β-ionone, a norisoprenoid present in nature,
is one of the effective substrates for CYP2B en-
zymes in human liver microsomes. To the best of
our knowledge, this is the first time that 4-
hydroxy β-ionone has been described as a human
metabolite of β-ionone.
"
l β‑ionone
!
"
l human liver microsome
The metabolism of the norisoprenoid β-ionone
was investigated in vitro using human liver mi-
crosomes and 11 different recombinant cyto-
chrome P450 enzymes expressed in Trichoplusia
ni cells. β-Ionone was found to be oxidized via
"
l cytochrome P450
"
l regio‑ and stereoselective
oxidation
l 4‑hydroxy‑β‑ionone
"
4
S-hydroxylation by CYP2B6 in human liver mi-
crosomes. CYP1A2 also regioselectively catalyzed
the hydroxylation of β-ionone to yield 4-hydrox-
ylation; this conversion was not stereoselective.
Further kinetic analysis revealed that CYP2B6 ex-
hibited the highest activity for β-ionone 4-hy-
droxylation. Kinetic analysis showed that Km and
Introduction
terns of metabolite and intermediate formation,
and kinetics are often studied using CYP human
liver microsomes (HLMs). Studies of HLM metab-
olism indicate that CYP is a major enzyme in the
metabolism of monoterpenes 1,8-cineole, (+)-
and (−)-limonenes, (+)-fenchone, and (−)-cam-
phor [12–15]. The major pathways used for the
metabolism of these compounds involve CYP-
dependent hydroxylation or oxidation.
Although β-ionone is contained in foods con-
sumed daily and is widely used in cosmetic prod-
ucts, no studies describe its metabolism by HLMs.
Investigation of β-ionone metabolism by HLMs
will provide a better understanding of the metab-
olism and toxicity of related naturally occurring
volatile compounds in the human body.
!
The ionone derivatives, α-, β- and γ-, mainly occur
in plants containing β-carotene. β-Ionone is found
in a variety of foods including carrots, raspberries,
pumpkin, and many other fruits and herbs [1–3].
These plants generally share the woody-floral,
violet scent characteristics of natural compounds
and also have odors. Humans are also exposed to
β-ionone through the daily dietary intake of β-
ionone-rich food or the passive transfer of pasture
species alfalfa (lucerne) through the consumption
of dairy products [4].
Human metabolism of naturally occurring vola-
tile compounds is interesting because their me-
tabolites are important determinants of food
safety and biological activity. A variety of compo-
nents of numerous plant species are oxidized by
cytochrome P450 (CYP) in laboratory animals
and humans [5–7]. The CYP enzymes detoxify
and/or toxify these compounds to more polar
and sometimes more reactive metabolites [7–
11]. In mammals, the liver plays a major role in
the metabolism and systemic elimination of
xenobiotics after exposure. For this reason, inves-
tigations of β-ionone metabolic pathways, pat-
received
revised
accepted
March 23, 2016
June 22, 2016
July 6, 2016
Bibliography
DOI http://dx.doi.org/
0.1055/s-0042-112128
1
Published online
Planta Med © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Mitsuo Miyazawa
Department of Applied
Chemistry
Faculty of Science and
Engineering
Kinki (Kindai) University
Kowakae, Higashiosaka-shi
Osaka 577–8502
The present study uses HLMs to investigate the
metabolism of β-ionone, including identification
of metabolites, elucidation of CYP-catalyzed me-
tabolism by 11 types of recombinant human CYP
enzymes, and determination of enzyme kinetics.
Japan
Phone: + 81667212332
Fax: + 81667272024
miyazawa@apch.kindai.ac.jp
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
Fig. 1 GC‑MS analysis of β-ionone and its trans-
formation products, 4-oxo β-ionone and 4-hydroxy
β-ionone. Panel A shows the GC‑MS chromatogram
of β-ionone with HLMs in the presence of an
NADPH-generating system (1) and without HLMs
(
4
2). Panel B shows the full scan mass spectrum of
-oxo β-ionone. Panel C shows the full scan mass
spectrum of 4-hydroxy β-ionone.
Fig. 2 Panel A shows the GC‑MS chromatogram of
4
-hydroxy β-ionone with HLMs in the presence of an
NADPH-generating system (1) and without HLMs
2). Panel B shows the GC‑MS chromatogram
(
of 4-oxo β-ionone with HLMs in the presence
of an NADPH-generating system (1) and without
HLMs (2).
Results
!
synthesis. In addition to chromatographic behavior, panel C
(l Fig. 1) shows spectral data indicating the presence of a 4-
"
The metabolism of β-ionone was examined using HLMs (HF207)
in the presence of an NADPH-generating system. Using GC‑MS
analysis, we identified the two metabolites as 4-hydroxy-β-ion-
one and 4-oxo-β-ionone (l Fig. 1). Component identification
was made based on mass spectral fragmentation and comparison
of retention time to an authentic sample obtained by organic
hydroxylated metabolite. The molecular mass of the metabolite
increased by from 192 to 208 upon the introduction of an oxygen
atom. Although the 4-oxo metabolite was generated by HLMs
from 4-hydroxy-β-ionone, the production of two metabolites via
HLMs indicated that the 4-oxo metabolite was formed by the
"
"
auto-oxidation of 4-hydroxy-β-ionone (l Fig. 2). The chirality of
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
the metabolite was determined using chiral HPLC. The metabo-
lism of β-ionone by HLM (HFC207) produced isomers of (S)- and
(
(
R)-4-hydroxylated metabolites at
l Figs. 3 and 4). Recombinant human CYPs expressed in T. ni
a
ratio of isomers 2:1
"
cells were tested for their ability to catalyze β-ionone oxidation.
CYP1A2 catalyzed the oxidation of (+)-(S)-4-hydroxy- and
(
−)-(R)-4-hydroxy-β-ionones. In contrast, CYP2B6 catalyzed the
enantioselective oxidation of (+)-(S)-4-hydroxy-β-ionone
"
(
l Fig. 5). All other CYP enzymes had very low activity or activity
below the limit of detection.
To further assess whether the β-ionone metabolic pathways in-
volved catalysis by CYP1A2 and CYP2B6, we examined the effects
of chemical inhibitors of CYP1A2 (α-naphthoflavone) and
CYP2B6 (ticlopidine) on β-ionone metabolism by the HLMs
HFC205 and recombinant CYP enzymes. HLMs contained rela-
tively high CYP1A2 and CYP2B6 activity. The concentrations of
α-naphthoflavone and ticlopidine needed to effectively inhibit
oxidation of β-ionone were determined using recombinant
"
"
CYP1A2 (l Fig. 6A) and CYP2B6 (l Fig. 6C). In assays of the re-
combinants, α-naphthoflavone and ticlopidine strongly inhibited
the oxidation of β-ionone by CYP1A2 and CYP2B6, respectively.
The inhibition by various concentrations of chemical inhibitors
"
on the metabolism of β-ionone by HLMs is shown in l Fig. 6B,D.
Fig. 3 Representative chromatographic traces for 4-hydroxylation of β-
The formation of (S)- and (R)-4-hydroxy β-ionones in the pres-
ence of 50-donor pool HLMs, recombinant CYP1A2 and CYP2B6
with an NADPH-generating system varied depending on incuba-
ionone in HLMs, CYP1A2, and CYP2B6 incubates.
tion time, CYP concentration, and substrate concentration
"
(
l Fig. 7A–I, respectively). Using different incubation times, we
found a linearity of the β-ionone 4-hydroxylation activities cata-
lyzed by the enzymes up to 40 min. Increasing of the CYP enzyme
concentration or the β-ionone concentration led to increasing 4-
hydroxy β-ionone formation. In the absence of the CYP enzymes,
we detected no β-ionone hydroxylation at all. Kinetic analysis of
β-ionone oxidation activity was carried out using recombinant
CYP1A2, CYP2B6, and 50-donor pool HLMs of human samples
"
(
(
l Table 1). Kinetic analysis showed that the Vmax/Km values for
+)-(S)- and (−)-(R)-4-hydroxy-β-ionone, catalyzed by HLMs,
were 0.3 and 0.1 µL/min/mg protein, respectively. Recombinant
CYP1A2 and CYP2B6 catalyzed the production of (+)-(S)-4-
hydroxy-β-ionone. The Vmax/Km values were 29.4 and 102.1 µM/
min, respectively. The Vmax/Km value for (−)-(R)-4-hydroxy-β-
ionone, catalyzed by CYP1A2, was 11.8 µM/min.
Fig. 4 Metabolism of β-ionone by human CYP1A2/CYP2B6 and in vitro.
Our results suggest that CYP1A2 and CYP2B6 are important en-
"
zymes in β-ionone oxidation by HLMs. l Fig. 8 depicts the rela-
tionship between the oxidation activity of β-ionone and specific
activities of CYP1A2 and CYP2B6 in eight different HLMs, indicat-
ing a good correlation between (+)-(S)-4-hydroxy-β-ionone and
CYP2B6 activity (r = 0.854). The formation rates of (+)-(S)- and
CYP1A2 in catalyzing β-ionone using HLMs. Ticlopidine, a specific
CYP2B6 inhibitor, significantly suppressed the activity of (S)-4-
hydroxylation β-ionone catalyzed by HLMs. β-Ionone (S)-4 oxida-
tion activity was found to correlate with CYP2B6, but not
CYP1A2, present in eight types of HLMs. The reason that CYP2B6
is more active than CYP1A2 in the stereoselective oxidation of β-
ionone by HLMs may be related to the higher content of this CYP
species in the human liver. Our data suggested that the mean lev-
els of CYP2B6 in HLMs of eight human samples were approxi-
mately fivefold higher than those of CYP1A2, probably resulting
in a more important role of CYP2B6 in the catalysis of β-ionone
oxidation by HLMs.
(
−)-(R)-4-hydroxy-β-ionone correlated slightly with the activity
of CYP1A2. There was no correlation between CYP2B6 activity
and the formation of (−)-(R)-4-hydroxy-β-ionone.
Discussion
!
In this study, we found that β-ionone was selectively oxidized to
its respective 4-hydroxylation metabolites by CYP1A2 and
CYP2B6 in HLMs. This activity preceded stereoselective oxidation
Species differences have been reported in the metabolism of β-
ionone by CYPs, and CYP101B1 has been shown to produce two
oxidized metabolites: 3-hydroxy-β-ionone and 4-hydroxy-β-ion-
one [16]. Similarly, CYP102A1 and P450 SU1, SU2, and SOY have
been shown to metabolize 4-hydroxy-β-ionone [17]. In HLMs,
β-ionone is converted to 4-hydroxy-β-ionone. Therefore, our re-
"
(
(
S form) by CYP2B6 (l Fig. 4). Although the enzyme efficiency
Vmax/Km ratio) of CYP2B6 was much higher than that of CYP1A2
for the formation of (S)-4-hydroxy β-ionone from the substrate
by recombinant CYP, CYP2B6 was found to be more active than
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
sults, together with those of previous studies, show that the me-
tabolism of β-ionone by CYPs proceeds via preferential oxidation
at the 4-position (allylic position). Allylic hydroxylation of β-ion-
one at position 4, which contains conjugated π-bonded carbon
atoms, producing allylic alcohols, has been reported as a common
CYP-mediated reaction [18]. Accordingly, allylic alcohols have
been identified as enzymatic products of other monoterpenes,
3
3
such as limonene, 1,8-cineole, and Δ -carene [19,20]. Δ -Carene
is also hydroxylated by CYP2B6. Allylic hydroxylation of mono-
terpenes is catalyzed by other CYPs, such as CYP2C9, 2C19 2D6,
and CYP3A4.
We have recently reported the metabolism of monoterpenes by
HLMs. Of these, (+)-fenchone, was most efficiency catalyzed by
human CYP2B6 (10-hydroxylation) and CYP2A6 (6-endo and
-
exo hydroxylations), with metabolic rates (Vmax/K ) of 175,
m
167, and 150 /nM/min, respectively. The Vmax/Km of β-ionone 4-
hydroxylation for CYP2B6 (S form) and CYP1A2 (S and R forms)
was determined as 102.1, 29.4, and 11.8 /µM/min, respectively.
Compared with the Vmax and Vmax/Km values of other monoter-
penes metabolized by CYP enzymes, the values described here,
particularly for CYP2B6, are high. CYP2B6 is expressed mainly in
human liver, although this enzyme has also been detected in var-
ious extra-hepatic tissues [21]. Considerable variability exists not
only in hepatic expression of CYP2B6 mRNA (280-fold) and pro-
tein (> 288-fold) but also in CYP2B6 enzyme activity (80-fold)
Fig. 5 Oxidation of β-ionone by 11 types of recombinant human P450
expressed in T. ni cells.
[
22–24]. The magnitude of CYP2B6 catalytic activity may be al-
tered as a result of enzyme inhibition by various synthetic drugs
25,26] and naturally occurring compounds such as curcuminoid
[
extract [27], phenethyl isothiocyanate [28], and citral [29]. Im-
portant CYP2B6 drug substrates include the alkylating anticancer
prodrug cyclophosphamide [30] and the tobacco use cessation
agent bupropion [31]. Bupropion and efavirenz are often admin-
istered in combination with several other drugs. For example,
tenofovir increases the plasma concentration of efavirenz, a sub-
strate of CYP2B6, under conditions of limited efavirenz metabo-
lism [32]. Since the rate of β-ionone metabolism by CYP2B6 is
high, itʼs possible that high amounts of these agents might alter
the bioavailability of drugs that are metabolized by CYP2B6;
however, in vitro inhibition of CYP2B6 activity does not necessa-
rily translate into in vivo drug interactions.
Synthesis of (± ±-4-hydroxy-β-ionone
(±)-4-Hydroxy-β-ionone was synthesized as described previ-
ously [33]. The epoxidation of α-ionone (1 g, 5.3 mmol) with m-
CPBA (1.4 equivalents) at 5°C for 1 h in 20 mL of CH Cl yielded
2
2
4,5-epoxide. The 4,5-epoxide was subjected to a base catalyzed
rearrangement by refluxing with K CO (3.0 equivalent) in meth-
2
3
anol for 4 h to yield the final compound. Most of the methanol
was then evaporated, and the residue was dissolved in ethyl ace-
tate. The organic phase was washed successively with water and
brine. The solution was evaporated and the residue was purified
by silica gel column chromatography (ethyl acetate-hexane
In summary, our results show that β-ionone is regioselectively
oxidized to its respective 4-hydroxy derivatives by CYP1A2 and
CYP2B6 in HLMs. CYP2B6 regio- and stereoselectively catalyzes
β-ionone metabolism. Our results also show that CYP2B6 plays a
more important role than CYP1A2 in catalyzing β-ionone oxida-
tion. The precise role of the CYP2B6 enzyme in β-ionone metab-
olism in response to human exposure to these chemicals is still
unknown.
65:35) to give the (±)-4-hydroxy-β-ionone white powder
1
(420 mg, 48%) with H NMR (CDCl , 800 MHz) δ 7.18 (1H, dq,
3
J = 16.4, 1.3 Hz, H-8), 6.11 (1H, d, J = 16.4 Hz, H-7), 4.01 (1H, t,
J = 4.9 Hz), 2.55–2.53 (1H, m, H-3), 2.35 (3H, s, H-11) 1.90–1.89
(1H, m, H-2), 1.80 (3H, d, J = 1.0 Hz, H-10), 1.19 (6H, s, H-12, 13);
1
3
C NMR (CDCl , 200 MHz) δ 198.5 (C, C-9), 142.7 (CH, C-7), 139.4
3
(C, C-6), 133.9 (C, C-5), 133.0 (C, C-8), 69.9 (CH, C-4), 35.5 (C, C-1),
34.6 (CH , C-2), 28.8 (CH , C-12 or − 13), 28.3 (CH , C-3), 27.5
2
3
2
(
2
CH , C-12 or − 13), 27.3 (CH , C-10), 18.4 (CH , C-11); EIMS m/z
08 [M] (21), 137 (17), 123 (25), 109 (79), 95 (18), 91 (20), 43
3 3 3
+
Materials and Methods
!
(100), and 41 (24).
Chemicals and reagents
Preparation of (−±-(R±-4-hydroxy-β-ionone
β-Ionone (purity > 99%) was purchased from Tokyo Chemical In-
(−)-(R)-4-Hydroxy-β-ionone enantiomer was prepared as de-
scribed previously [33]. A mixture of (±)-4-hydroxy-β-ionone
(200 mg, 1.0 mmol), dry lipase (100 mg), activated molecular
sieves 4 Å (150 mg), and vinyl acetate (0.5 equivalent) in 10 mL
of dry n-hexane-THF (9:1 v/v) was stirred at room temperature
for 18 h. The reaction was monitored by chiral HPLC and termi-
nated at ~ 50% conversion. The reaction mixture was filtered
and the filtrate was concentrated in vacuo. The residue was puri-
fied further by silica gel column chromatography. Elution with a
dustry Co., Ltd. α-Naphthoflavone (purity ≥ 98%) and ticlopidine
+
(
purity ≥ 99%), were purchased from Sigma-Aldrich. NADP , glu-
cose 6-phosphate, and glucose 6-phosphate dehydrogenase were
purchased from Oriental Yeast, Ltd. The other regents and chem-
icals used in this study were of the highest quality commercially
available and obtained from sources as described previously [12–
15].
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
mixture of ethyl acetate and hexane (65:35) yielded (−)-(R)-4-
hydroxy-β-ionone (103 mg; ee 95% as analyzed by chiral HPLC;
2
0.4
[α]
D
− 8.01° [CHCl , c 1.0]). The spectral data of enantiomer
3
were identical to those of racemate.
Synthesis of 4-oxo-β-ionone
Dess-Martin periodinane (1.0 equivalents) was added to a solu-
tion of (±)-4-hydroxy-β-ionone (200 mg, 1.0 mmol) in 10 mL of
CH Cl . The reaction mixture was stirred until the starting alco-
2
2
hol was consumed, as determined by TLC analysis. Once the reac-
tion was judged complete, the mixture was diluted with aqueous
NaHCO3 and CH Cl . The precipitated solids were filtered
2
2
through a bed of diatomaceous earth. The filter cake was rinsed
with CH Cl , and the biphasic filtrate was separated. The organic
2
2
layer was extracted and evaporated. The resulting residue was
eluted through a silica gel column with a ratio of ethyl acetate-
hexane (65:35) to obtain the corresponding 4-oxo-β-ionone
1
(
143 mg, 75%), with a pale yellow solid; H NMR (CDCl3,
800 MHz) δ 7.24 (1H, dq, J = 16.5, 1.0 Hz, H-8), 6.19 (1H, d,
J = 16.5 Hz, H-7), 2.55–2.53 (1H, m, H-3), 2.35 (3H, s, H-11) 1.90–
1
1
1
3
2
1
.89 (1H, m, H-2), 1.80 (3H, d, J = 1.0 Hz, H-10), 1.19 (6H, s, H-12,
3); 13C NMR (CDCl , 200 MHz) δ 198.6 (C, C-4), 197.4 (C, C-9),
3
57.7 (C, C-6), 140.3 (CH, C-7), 133.5 (CH, C-8), 131.4 (C, C-5),
7.3 (CH , C-2), 35.5 (C, C-1), 34.2 (CH , C-3), 27.9 (CH , C-11),
2
2
3
+
7.3 (CH , C-12, 13), 13.4 (CH , C-10); EIMS m/z 206 [M] (26),
3
3
64 (16), 163 (59), 135 (17), 122 (18), 121 (35), 43 (100), and 41
(26).
Enzymes
HLMs HG18, HG64, HH37, HH519, HH581, HH715, HFC205,
HFH705, and 50-donor pool were obtained from Gentest Co.,
Inc. and were stored at − 80°C. HLMs were available for complete
catalytic assays of the major CYPs: phenacetin-O-deethylase
(
CYP1A2), coumarin 7-hydroxylase (CYP2A6), (S)-mephenytoin
N-demethylase (CYP2B6), paclitaxel 6α-hydroxylase (CYP2C8),
′-hydroxylase (CYP2C9), (S)-mephenytoin 4′-demethylase
CYP2C19), bufuralol 1′-hydroxyase (CYP2D6), chlorzoxazone 6-
Fig. 6 Effects of α-naphthoflavone (A and B) as an inhibitor of CYP1A2
and ticlopidine (C and D) as an inhibitor of CYP2B6 on the oxidation of β-
ionone to (+)-(S)-4-hydroxy-β-ionone (&) and (−)-(R)-4-hydroxy-β-ionone
4
(
(•) by HLMs.
hydroxylase (CYP2E1), testosterone 6β-hydroxylase (CYP3A4),
and lauric acid 12-hydroxylase (CYP4A). Recombinant human
CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4
expressed in T. ni cells infected with a baculovirus containing
CYP and NADPH-P450 reductase cDNA inserts were obtained
from Gentest. The CYP content was determined using these ma-
terials as described in the manufacturers protocols.
(30 m × 0.25 mm i.d; film thickness 0.25 µm) and a DB‑WAX po-
lar capillary column (15 m × 0.25 mm i.d; film thickness 0.25 µm)
using helium (at 1.5 mL/min) as the carrier gas. The column tem-
perature was programmed as isothermal at 80°C for 5 min, then
raised to 260°C at a rate of 8°C/min and held at 260°C for 5 min.
The injector temperature was maintained at 270°C. The effluent
of the GC column was introduced directly into the source via a
transfer line (280°C). The ion source temperature was set at
230°C. The electron impact (EI) ionization voltage was set to
70 eV and positively charged ions were analyzed in full scan
mode applying a scan of m/z 40–500 amu.
β-Ionone oxidation assays
β-Ionone oxidation by CYP enzymes was determined as follows:
Standard reaction mixtures contained HLMs (0.2 mg protein/ml)
or recombinant CYP (11 different recombinants; 20 pmol/ml)
with 200 µM β-ionone in a final volume of 0.50 mL 100 mM po-
tassium phosphate buffer (pH 7.4) containing an NADPH-gener-
+
ating system (0.5 mM NADP , 5 mM glucose 6-phosphate, and
0.5 units glucose-6-phosphate dehydrogenase/ml) [12–15]. Incu-
Inhibition experiments
bations were carried out at 37°C for 30 min and terminated by
the addition of 1.0 mL CH Cl followed by vigorous mixing. The
The inhibitory effects of known selective inhibitors of CYP on the
metabolism of β-ionone by HLMs and recombinant CYP were
evaluated to determine the specific CYP enzymes involved in
each metabolic pathway. The inhibitors included α-naphthofla-
vone (1–20 µM), a selective CYP1A2 inhibitor [34], ticlopidine
(1–20 µM), a selective CYP2B6 inhibitor [35]. The reaction mix-
ture contained 20 nM recombinant CYP, inhibitor, 100 mM phos-
phate buffer (pH 7.4), and an NADPH-generating system (0.5 mM
2
2
extracts (organic layer) were collected by centrifugation at
000 rpm for 10 min and transferred to an insert for analysis by
3
GC‑MS.
GC‑MS analysis was performed using an Agilent 6890 N gas chro-
matograph (Agilent Technologies) equipped with an Agilent
5973 N quadrupole mass selective detector. The metabolites were
+
separated using an HP-5MS nonpolar capillary column
NADP , 5 mM glucose 6-phosphate, and 0.5 units of glucose
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
Fig. 7 β-Ionone was incubated with HLMs (A–C),
CYP1A2 (D–F), and CYP2B6 (G–I) in the presence of
an NADPH-generating system, and the (S)-4-hy-
droxy-β-ionone (&) and (R)-4-hydroxy-β-ionone (•)
formed were analyzed on a chiral HPLC system. In
A, D, and G, concentrations of β-ionone, HLMs, CY-
P1A2, and CYP2B6 were 0.2 mM, 0.2 mg/ml,
2
0 pmol/P450, and 20 pmol/P450, respectively.
In B, E, and H, the incubation time and substrate
concentration was 30 min and 0.2 mM, respective-
ly. In C, F, and I, the incubation time and concen-
trations of HLMs, CYP1A2, and CYP2B6 were
3
0 min, 0.2 mg/ml, 20 pmol/P450, and 20 pmol/
P450, respectively.
Table 1 Kinetic analysis of the (S)- and (R)-4-hydroxylation of β-ionone by CYPs.
Enzyme source
Oxidation of β-ionone
+±-(S±-4-hydroxy-β-ionone
(
(−±-(R±-4-hydroxy-β-ionone
Km(µM±
Vmax
Vmax/Km
Km(µM±
Vmax
Vmax/Km
HLMa,c
CYP1A2b,d
CYP2B6b,d
96.5 ± 27.9
107.9 ± 36.0
5.6 ± 1.2
33.5 ± 2.8
0.3
29.4
179.1 ± 55.6
111.7 ± 19.8
20.5 ± 2.2
1312.1 ± 71.1
0.1
3200.3 ± 323.0
572.8 ± 29.8
11.8
102.1
a
Vmax expressed in nmol/min/mg protein for HLMs; b Vmax expressed in nmol/min/nmol P450 for recombinant P450; c Intrinsic clearance (Vmax/Km) expressed in µL/min/mg protein
for HLMs; d Intrinsic clearance (Vmax/Km) expressed in/µM/min
6
1
-phosphate dehydrogenase/ml). To the reaction mixture,
00 µM of substrate was added. The incubation was carried out
Blank tests
Incubation was carried out at 37°C for 30 min with 200 µM of
substrate or metabolites; 4-hydroxy-β-ionone and 4-oxo-β-ion-
one in a final volume of 0.5 mL of 100 mM potassium phosphate
buffer (pH 7.4) in combination with the NADPH-generating sys-
tem, but without the addition of microsomes.
for 30 min at 37°C.
Kinetic analysis
The kinetic parameters (Vmax and K ) for β-ionone metabolite
m
formation by recombinant human CYP were estimated by using
a computer program designed (GraphPad Prism) for nonlinear
regression analysis.
Chiral HPLC method
A simple reversed-phase chiral HPLC method was developed and
validated for the direct separation of the (±)-4-hydroxy-β-ionone
enantiomers. The enzyme reaction mixtures were then applied
Marumoto S et al. In Vitro Regio-… Planta Med

Original Papers
Conflict of Interest
!
The authors declare no conflict of interest.
Affiliations
1
Joint Research Center, Kinki (Kindai) University, Kowakae, Higashiosaka-shi,
Osaka, Japan
2
3
4
5
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki
(
Kindai) University, Kowakae, Higashiosaka-shi, Osaka, Japan
Pharmaceutical Research and Technology Institute, Kinki (Kindai) University,
Higashi-osaka, Osaka, Japan
Department of Materials Science, Wakayama National College of Technology,
Gobo, Wakayama Japan
Graduate School of Materials Science Nara Institute of Science and
Technology, Ikoma, Nara, Japan
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