Regioselective hydroxylation of phloretin, a bioactive compound from apples, by human cytochrome p450 enzymes

Article abstract of DOI:10.3390/ph13110330

Phloretin, the major polyphenol compound in apples and apple products, is interesting because it shows beneficial effects on human health. It is mainly found as a form of glucoside, phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore, identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450 (P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin oxidation were examined using human liver microsomes and recombinant human liver P450s. One major metabolite of phloretin in human liver microsomes was 3‐OH phloretin, which is the same product of a bacterial CYP102A1‐catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed kcat values of 3.1 and 5.8 min^?1, respectively. However, CYP3A4 has a 3.3‐fold lower Km value than CYP2C19. The catalytic efficiency of a CYP3A4‐catalyzed reaction is 1.8‐fold higher than a reaction catalyzed by CYP2C19. Whole‐cell biotransformation with CYP3A4 was achieved 0.16 mM h^?1 productivity for 3‐OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent inhibitor of CYP3A4‐catalyzed testosterone 6β‐hydroxylation activity. Antibodies against CYP3A4 inhibited up to 90% of the microsomal activity of phloretin 3‐hydroxylation. The immunoinhibition effect of anti‐2C19 is much lower than that of anti‐CYP3A4. Thus, CYP3A4 majorly contributes to the human liver microsomal phloretin 3‐hydroxylation, and CYP2C19 has a minor role.

Full text of DOI:10.3390/ph13110330

pharmaceuticals
Article
Regioselective Hydroxylation of Phloretin, a Bioactive
Compound from Apples, by Human Cytochrome
P450 Enzymes
1
1
1
1
2
Ngoc Anh Nguyen , Ngoc Tan Cao , Thi Huong Ha Nguyen , Thien-Kim Le , Gun Su Cha ,
3
3
1,4
1,4
1,4,
Soo-Keun Choi , Jae-Gu Pan , Soo-Jin Yeom , Hyung-Sik Kang
and Chul-Ho Yun
*
1
School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University,
Yongbong-ro 77, Gwangju 61186, Korea; ngocanh61093@gmail.com (N.A.N.);
caongoctan93@gmail.com (N.T.C.); huongha0207@gmail.com (T.H.H.N.);
thienkim.1611@gmail.com (T.-K.L.); soojin258@chonnam.ac.kr (S.-J.Y.); kanghs@jnu.ac.kr (H.-S.K.)
Namhae Garlic Research Institute, 2465-8 Namhaedaero, Gyeongsangnamdo 52430, Korea;
gscha450@gmail.com
Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Daejeon 34141, Korea;
sookeun@kribb.re.kr (S.-K.C.); jgpan@kribb.re.kr (J.-G.P.)
School of Biological Sciences and Technology, Chonnam National University, Yongbong-ro 77,
Gwangju 61186, Korea
2
3
4
*
Correspondence: chyun@jnu.ac.kr; Tel.: +82-62-530-2194
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 29 September 2020; Accepted: 21 October 2020; Published: 22 October 2020
Abstract: Phloretin, the major polyphenol compound in apples and apple products, is interesting
because it shows beneficial effects on human health. It is mainly found as a form of glucoside,
phlorizin. However, the metabolic pathway of phloretin in humans has not been reported. Therefore,
identifying phloretin metabolites made in human liver microsomes and the human cytochrome P450
(
P450) enzymes to make them is interesting. In this study, the roles of human liver P450s for phloretin
oxidation were examined using human liver microsomes and recombinant human liver P450s. One
major metabolite of phloretin in human liver microsomes was 3-OH phloretin, which is the same
product of a bacterial CYP102A1-catalyzed reaction of phloretin. CYP3A4 and CYP2C19 showed
−1
k
cat values of 3.1 and 5.8 min , respectively. However, CYP3A4 has a 3.3-fold lower K
m
value than
CYP2C19. The catalytic efficiency of a CYP3A4-catalyzed reaction is 1.8-fold higher than a reaction
−1
catalyzed by CYP2C19. Whole-cell biotransformation with CYP3A4 was achieved 0.16 mM h
productivity for 3-OH phlorein from 8 mM phloretin at optimal condition. Phloretin was a potent
inhibitor of CYP3A4-catalyzed testosterone 6 -hydroxylation activity. Antibodies against CYP3A4
β
inhibited up to 90% of the microsomal activity of phloretin 3-hydroxylation. The immunoinhibition
effect of anti-2C19 is much lower than that of anti-CYP3A4. Thus, CYP3A4 majorly contributes to the
human liver microsomal phloretin 3-hydroxylation, and CYP2C19 has a minor role.
Keywords: human cytochrome P450; human liver microsomes; human metabolite; phloretin;
polyphenol; regioselective hydroxylation
1
. Introduction
Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one] is a natural polyphenol
compound found mainly in most parts of apple trees, including in apple skin and apple pomace [1]
(Figure 1). It is a dihydrochalcone among flavonoids and is mostly found as the glucoside phlorizin
in plants [2]. Similar to many polyphenol compounds, phloretin displays antioxidant properties
and interferes with the growth of several types of cancer cells [
3]. Recently, we found that bacterial
Pharmaceuticals 2020, 13, 330; doi:10.3390/ph13110330
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CYP102A1 regioselectively hydroxylates phloretin to produce 3-OH phloretin and that 3-OH phloretin
dramatically inhibits differentiating 3T3-L1 preadipocytes into adipocytes and lipid accumulation [ ].
4
Phloretin and phlorizin are currently accepted as health-beneficial polyphenols from apples useful in
treating hyperglycemia [5].
Figure 1. Chemical structures of phlorizin, phloretin, and 3-OH phloretin. (
glucoside form of phloretin. ( ) Phloretin’s conversion to its product is catalyzed by human cytochrome
P450 (P450) in the presence of reduced -nicotinamide adenine dinucleotide phosphate (NADPH). A
A) Phlorizin is a major
B
β
star (*) marks the hydroxylation site on phloretin catalyzed by P450.
Phlorizin (phloretin 2’-O-glucose) is the major form of phloretin found in apples. Phlorizin
is absorbed in the small intestine and transported via sodium–glucose transporters (SGLTs) with
subsequent deglycosylation by β-glucosidase activity of lactate-phlorizin hydrolase [6,7]. Phlorizin is
known to be a specific competitive inhibitor of SGLTs in intestines (SGLT-1) and kidneys (SGLT-2),
and it seems to show beneficial effects on obesity and diabetes [8]. The major cytochrome P450 (P450
or CYP) enzyme involved in the metabolic reactions of flavonoids is CYP1A2, whereas other human
liver P450s (such as CYP3A4, CYP2D6, CYP2E1, CYP2C19, and CYP2E1) contributed as minor roles to
flavonoid metabolism when the metabolisms of 30 flavonoid aglycones were screened using human
liver P450 enzymes [9]. The main oxidation reaction is hydroxylation at the carbon in the phenyl ring.
However, the metabolic pathway of phloretin in humans has not been reported.
Phloretin shows a potent chemopreventive effect against aflatoxin B1 via its inhibitory effect on
CYP3A4 and CYP1A2. On the other hand, it has an inductive effect on glutathione S-transferase
activity [10]. Phloretin can also inhibit the catalytic activity of human CYP1A1 [11]. Phloretin’s
inhibitory effect on P450-catalyzed reactions may suggest that phloretin can be a substrate to human
P450 enzymes. However, to the best of our knowledge, no reports exist on phloretin metabolism
in humans.
This study aimed to determine major phloretin metabolites in the human liver and find the
contributing roles of particular P450 enzyme(s) involved in forming the metabolites. For these aims,
we performed catalytic activity assays of phloretin 3-hydroxylation with human liver microsomes
(HLMs) and recombinant enzymes. We found 3-OH phloretin as the major metabolite in HLMs,
and that CYP3A4 contributes as a major enzyme to produce 3-OH phloretin. Such information can
have a considerable clinical impact regarding potential drug–drug and food–drug interactions and
interindividual variations of drug metabolism.
2
. Results
2
.1. Phloretin Metabolism by Human Liver Microsomes and Identification of the Major Metabolite
First, to determine HLMs’ ability to oxidize phloretin, the catalytic activity of HLMs toward
◦
phloretin was measured via a 200-
µ
M substrate for 60 min at 37 C. Figure 2A shows the
high-performance liquid chromatography (HPLC) profiles of phloretin and its metabolites via HLMs.
One major metabolite appeared at a retention time of 32.4 min. The major product of HLMs in the
presence NADPH showed the same retention time of 3-OH phloretin, which is phloretin’s major

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product produced by CYP102A1 enzymes [
4
]. The turnover number for 3-OH phloretin formation was
−1
0.052 min under this experimental condition (Figure 2). To confirm the product’s chemical structure,
we did an analysis via liquid chromatography-mass spectrometry (LC-MS) (Figure 3 and Figure S1).
A full-scan chromatogram of the reaction of a mixture of phloretin with HLMs appears in Figure 3.
When the mixture was compared with the matrix control, phloretin shows a major metabolite—M1.
Accuracy results for molecular weight and an elemental analysis indicated that M1 (m/z 289) was a
monohydroxylated metabolite when compared to the substrate of phloretin (m/z 273). This result
indicates that P450s in HLMs catalyze phloretin’s regioselective hydroxylation to produce a catechol
product. A minor metabolite (t = 33.5 min) was also observed (Figure 2A). However, we could not
R
characterize this compound because any apparent mass data with LC-MS analysis were not observed.
Figure 2. HPLC chromatograms of phloretin and its metabolites via human liver microsomes. (
A)
Peaks of the reaction mixtures were identified by comparing their retention times (t ) with those of
R
standard compounds: (
0.1 min).
B) 3-OH phloretin (tR = 32.4 min), phloretin (tR = 34.6 min), and phlorizin (tR =
3
Next, we examined each human P450’s possible contributions to the phloretin 3-hydroxylation.
We examined the catalytic activity of several human CYPs, such as CYP3A4, CYP1B1, CYP1A2,
CYP2D6, CYP2A6, CYP2E1, and CYP2C19. Only CYP2C19 and CYP3A4 have apparent phloretin
3
-hydroxylation activity. The same product, 3-OH phloretin, was detected in phloretin metabolized
via CYP2C19 and CYP3A4 in the presence of NADPH (Figure S2). To identify phloretin metabolites
generated by CYP2C19 and CYP3A4, analyses of HPLC (Figure S2) and LC-MS were done (Figures
+
S3 and S4). The mass spectrum of phloretin showed a protonated molecular ion ([M-H] ) at m/z 273.
The metabolites had m/z 289, so these results indicate that all HLMs, CYP3A4, and CYP2C19 samples
produce only one major product, a 3-OH product.

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Figure 3. LC-MS analyses of the phloretin metabolite via human liver microsomes (HLMs). LC-MS
chromatogram of phloretin metabolites catalyzed by HLMs in the presence ( ) of NADPH. The MS
spectra show that the protonated molecular ions of 3-OH phloretin ( ) and phloretin ( ) were 289 and
73, respectively.
A
B
C
2
2
.2. Kinetics Parameters and Total Turnover Numbers (TTNs) of Phloretin Hydroxylation via HLMs and
Recombinant Human CYPs
Table 1 and Figure S5 show the steady-state kinetics of 3-OH phloretin formation via HLMs and
−
1
the two recombinants of CYP3A4 and CYP2C19. HLMs had a lower kcat value (0.094 min ) and
an intermediate K value (120 M) compared to CYP3A4 and CYP2C19. Although CYP2C19 had
the highest kcat value of 5.8 min , it showed the highest K
m
µ
−
1
m
value of 208
had a lower kcat value of 3.1 min compared to CYP2C19, it showed a much lower K
M. Therefore, the CYP3A4-catalyzed reaction’s efficiency was 1.8-fold higher than that of CYP2C19.
µ
M. Although CYP3A4
−
1
m
value of
63 µ
This result suggests CYP3A4 is the major contributor toward the production of 3-OH phloretin in
human livers.
Table 1. Kinetic parameters of phloretin hydroxylation via human liver microsomes (HLMs), CYP3A4,
and CYP 2C19.
−1
−1
−1
Enzymes
kcat (min
)
Km (µM)
kcat/Km (min µM )
HLMs
CYP3A4
CYP2C19
0.094 ± 0.006
3.1 ± 2.9
120 ± 36
63 ± 11
0.00083 ± 0.00025
0.049 ± 0.023
5.8 ± 0.6
208 ± 50
0.028 ± 0.007
When the time profile of 3-OH phloretin formation by HLMs ranged from 10 to 180 min, the
formation of 3-OH phloretin gradually increased with the reaction time. The total turnover number
(TTN, nmol product/nmol enzyme) was 17.3 at 180 min into the reaction (Figure 4).
It is well known that cytochrome b (b ) plays an important role in liver P450-derived substrate
5
5
metabolism. The effect of b on the phloretin 3-hydroxylation activity of CYP3A4 and CYP2C19 was
5

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examined. The b increased the activities of CYP3A4 and CYP2C19 by 52% and 41%, respectively
5
(
Figure S6). It is known that the stimulatory effects of b on both enzymes are very diverse [12].
5
Figure 4. Time profiles of 3-OH phloretin formation via HLMs. Total turnover numbers (TTNs) for
◦
HLMs were determined via HPLC after accomplishing the reactions at indicated times at 37 C. The
-OH phloretin was measured in the reaction mixtures containing P450 (0.40 M of HLMs), an NADPH
3
µ
regeneration system, and phloretin (0.50 mM) for a total volume of 0.25 mL.
2
.3. Inhibition of Phloretin Hydroxylation Activity by Antibodies in HLMs
To determine to what extent CYP2C19 and CYP3A4 contribute to phloretin hydroxylation,
immunoinhibition studies with anti-CYP2C19 and anti-CYP3A4 were conducted. Anti-CYP2C19 and
anti-CYP3A4 inhibited phloretin hydroxylation activity as the antibodies’ concentration increased
(
5
Figure 5). Anti-CYP2C19 inhibited 10% and 13% of the phloretin hydroxylation activity when 2 and
mg of Immunoglobulin G (IgG) per nmol of P450 were added, respectively. Anti-CYP3A4 showed
a much higher inhibitory effect of 70% and 87% when 2 and 5 mg of IgG per nmol of P450 were
added, respectively. This result indicates that CYP3A4 and CYP2C19 are a major and a minor CYP for
phloretin 3-hydroxylation reaction, respectively. These immunoinhibition studies also suggest that
CYP3A4 and CYP2C19 cause almost all the phloretin oxidation in HLMs.
Figure 5. Immunoinhibition of antibodies on phloretin 3- hydroxylation activity catalyzed by HLMs.
The effects of anti-CYP3A4 (
activity in HLMs were measured. Values represent the means ± SD of three determinations.
•
), anti-CYP2C19 (
#
), and preimmune IgG (
ꢀ
) on phloretin 3- hydroxylation

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2
.4. Inhibitory Effect of Phloretin on Testosterone 6β-Hydroxylation Catalyzed by CYP3A4
CYP3A4 was the major P450 for phloretin 3-hydroxylation activity in HLMs, so we examined
phloretin’s effect on the CYP3A4 catalytic activity using testosterone as a substrate. Testosterone
β-hydroxylation is a widely used marker of CYP3A4’s enzyme activity [13].
Phloretin inhibited testosterone 6 -hydroxylation catalyzed by CYP3A4 and HLMs as the
concentration of phloretin increases (Figure 6). It showed a high inhibitory effect on CYP3A4-catalyzed
testosterone 6 -hydroxylation. When 10 M and 50 M of phloretin were preincubated with CYP3A4,
0% and 91% of the CYP3A4 activity was inhibited, respectively. Phloretin’s inhibitory effect on the
activity of HLMs was much lower than on CYP3A4. When 50 M of phloretin was preincubated
with HLMs, 63% of activity was inhibited. The half maximal inhibitory concentration (IC ) values
6
β
β
µ
µ
4
µ
50
of testosterone 6
respectively. Thus, phloretin can be a substrate of CYP3A4 with a high affinity, and other P450(s) in
addition to CYP3A4 may be involved in testosterone 6 -hydroxylation in HLMs. It is known that
β-hydroxylation via phloretin against CYP3A4 and HLMs were 13 µM and 92 µM,
β
human CYP2C9 and CYP2C19 have the catalytic activity of testosterone 6β-hydroxylation in addition
to CYP3A4 [14].
100
CYP3A4
HLMs
50
0
0
20
40
60
80
100
Phloretin ( M)
Figure 6. Phloretin’s inhibitory effect on the testosterone 6
β-hydroxylation by CYP3A4 and HLMs.
After preincubating phloretin with CYP3A4 (
testosterone (0.50 mM) was added to start the reaction. The reactions were terminated after 30 min.
#
) and HLMs (
•
) in the presence of NADPH for 5 min,
The formation of 6
three determinations.
β
-OH testosterone was measured by HPLC. Values represent the means
±
SD of
2
.5. Whole-Cell Biotransformation of Phloretin
In this study, we developed CYP3A4 expressing whole-cell biocatalyst to produce 3-OH phloretin
for industrial applications. To find the best host Escherichia coli cells of recombinant human CYP3A4 for
whole-cell biotransformation, six different types of competent E. coli cells (Shuffle T7, Rosetta, MG1655,
DH5
α-F’IQ, BL21, and JM109) were tested for CYP3A4 expression in a Luria–Bertani medium [15].
The optimum temperature and culture time were determined by measuring enzyme activity from
◦
◦
2
0 C to 30 C for a set of culture times of 10, 20, 22, 24, and 26 h. Results show DH5
α
-F’IQ was the
◦
best strain for CYP3A4 expression after a 22 h culture at 30 C. The expression level of CYP3A4 was
0
.20 µM. Thus, the DH5α-F’IQ strain was selected for the whole-cell biotransformation experiments.
Production of 3-OH phloretin, the major metabolite, from the substrate phloretin was optimized
by varying the cell mass and the substrate concentrations (Figure 7A,B). Production of 3-OH phloretin
−
1
was increased up to 30 g cell L (Figure 7A) and 8 mM phloretin (Figure 7B). However, above
this concentration, the production plateaued. These results indicate that the optimal concentrations
−
1
of CYP3A4 harboring DH5
respectively. The production of 3-OH phloretin increased when substrate concentrations increase up to
α-F’IQ cells and phloretin were 30 g of cell L and 8 mM of phloretin,

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8
mM. E. coli cells expressing the CYP3A4 produced 0.16 mM of 3-OH phloretin after 60 min reaction,
−1
showing 0.16 mM h productivity when 8 mM of phloretin was used.
Figure 7. Whole-cell biotransformation of phloretin by a recombinant E. coli cell expressing CYP3A4.
) Effect of cell concentration on the conversion of phloretin into 3-OH phloretin by whole cells
(
A
−
1
expressing CYP3A4. The reactions were performed with cell concentrations from 5 to 45 g L and
1
◦
0 mM of phloretin at 37 C for 60 min. (
B) The effect of phloretin concentration on the production
of 3-OH phloretin by recombinant whole cells expressing CYP3A4. The reactions were performed in
−1
◦
a range from 1 to 15 mM with 30 g L cells at 37 C for 60 min. (
C) Time courses of phloretin into
3
3
-OH phloretin by recombinant E. coli cells expressing CYP3A4. The reactions were performed with
−
1
◦
0 g L cells and 8 mM of phloretin at 37 C for 180 min. Data are shown as the means
±
the SD of
triplicate determinations.
Finally, we demonstrated that high biotransformation of 8 mM phloretin into 3-OH phloretin
−
1
◦
was achieved by 30 g L CYP3A4 expressing whole cells at 37 C for 180 min (Figure 7C). The 3-OH
phloretin production gradually increased with increasing time up to 60 min when it was saturated.
−1
The productivity for 60 min reaction with CYP3A4 whole-cell bioconversion was 0.16 mM h . These
results provide CYP3A4 expressing whole-cells, which are useful key biocatalysts for 3-OH phloretin
production. In addition, high cell density culture and whole-cell immobilization of these whole-cell
biocatalysts should be further studied for industrial application.
3
. Discussion
Consuming polyphenol-rich foods are associated with health benefits, especially regarding chronic
diseases, such as cancer and obesity [16]. It is generally accepted that apples have diverse beneficial
effects on blood pressure, lipids, vascular function, hyperglycemia, inflammation, and even cancer. The
cardioprotective effect of apples may be associated with polyphenols [17
,18]. A well-known bioactive
polyphenol in apples is phloretin, which is mainly found as a form of glucoside: phlorizin [19
,
20].
Phloretin is a main compound, which belongs to the dihydrochalcone class among flavonoids and
also exists in strawberries and pears. Phloretin has a wide range of biological effects, including
antioxidation, anti-inflammatory, and anti-tumor properties, and it reduces vascular endothelial
dysfunction and liver injury [21
considered health-beneficial polyphenols from apples [
–
23]. At present, phloretin and its glucoside phlorizin are generally
]. In this study, we found that 3-OH phloretin
5
was a major metabolite in HLMs. Recently, we found that 3-OH phloretin, a monohydroxylated product
of phloretin catalyzed by bacterial CYP102A1, inhibits the differentiation of 3T3-L1 preadipocytes into
adipocytes. We also reported that lipid accumulation was dramatically inhibited by 3-OH phloretin
but increased by phloretin. Furthermore, after oral administration of Lithocarpus polystachyus Rehd to

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in vivo rat models, which has several flavonoids including phlorizin, 3-OH phloretin, and its conjugates
with sulfate were identified in plasma, urine, and feces [24]. This result suggests that 3-OH phloretin,
a human metabolite of phloretin, may show higher beneficial effects than those of phloretin itself.
Further studies with 3-OH phloretin are necessary to find its effects on human health.
Human P450s have a dominant contribution to metabolizing clinical drugs and other foreign
chemicals, including dietary natural polyphenols from fruits and vegetables [25,26]. CYP3A4 and
CYP2C19 are the principal catalysts for metabolizing phloretin into 3-OH phloretin. CYP3A4 is most
abundant in livers and small intestines, and it contributes to the metabolism of over 50% of clinical
medicines [27]. CYP3A4 shows a very broad substrate specificity, and it can also metabolize diverse
molecules with different sizes and chemical structures via diverse oxygenation reactions [28–30].
CYP2C19 also plays an important role in processing or metabolizing important therapeutic drugs.
In particular, the CYP2C19 gene is highly polymorphic [31]. Humans have a wide interindividual
variation in the expression of individual P450s [17]. Understanding human P450s, which participate in
the oxidation of phloretin, would help in assessing individual susceptibility to this potent inhibitor
against adipocyte differentiation and lipid accumulation [4]. Formation of 3-OH phloretin by P450
enzymes in the liver with the consumption of apples and apple products might cause the inhibition of
adipocyte differentiation and lipid accumulation. Increasing apple intake may provide health benefits
and potentially help weight loss.
P450 enzymes are the major contributing enzymes involved in the oxidation reactions of dietary
natural compounds obtained from food and fruits in humans [25,26]. With the same approach to the
issue of MIST (Metabolites in Safety Testing) for drugs [32], efficient production systems are required
to permit the large-scale production of human metabolites of dietary compounds obtained from food
and fruits. As chemical synthesis methods sometimes cannot synthesize these metabolites, biocatalytic
production of the metabolites using whole-cell biotransformation with a recombinant expression of
human P450s would be a promising approach to provide the metabolites for the assessment of efficacy
and safety of each dietary compound.
Polyphenol compounds are abundant in the human diet, including in vegetables and fruits [33].
In this study, we found that CYP3A4 was the major enzyme to metabolize phloretin in the liver and
that phloretin inhibited the CYP3A4 catalytic activity. This result suggests a potent interaction of
phloretin, a popular polyphenol in apples, with clinical drugs metabolized by CYP3A4. As CYP3A4 is
also essential to metabolize a large set of clinical drugs, phloretin treatment could affect the usage of
many clinical drugs or cause significant side effects. Popular dietary supplements and foods also have
a high risk for interacting with drugs metabolized by CYP3A4, the most popular drug-metabolizing
enzyme in human livers and small intestines. However, the potential effects of drugs’ sensitivity to
phloretin remain unknown.
4
. Materials and Methods
4
.1. Materials
Phloretin, the oxidized form of
β
-nicotinamide adenine dinucleotide phosphate (NADP+),
glucose-6-phosphate, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, acetonitrile,
methanol, and ethyl acetate were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other chemicals
used in this work were obtained with the highest grade commercially available, and they were used
without further purification.
Recombinant human P450s were heterologously expressed in E. coli with a pCW vector containing
human P450 cDNA (CYP3A4 or CYP2C19) and rat NADPH-P450 reductase (CPR) [34
expressing P450 and CPR were constructed in previous studies: CYP1A2, CYP3A4, CYP2C19, CYP1B1,
CYP2E1, CYP2D6, and CYP2A6 [34 35]. Membrane fractions expressing P450 and CPR were prepared,
as described previously [34,35], and were used for the catalytic activity assays.
,35]. pCW vectors
,

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HLMs (a human liver microsome pool) were purchased from ThermoFisher Scientific (Walthan,
MA, USA).
Antibodies against CYP3A4 and 2C19 were prepared in the previous work [36].
4
.2. Oxidation of Phloretin Catalyzed by Human Liver Microsomes
◦
Human liver microsomal reactions included incubation at 37 C in 0.25-mL incubation mixtures
containing P450 (0.4 µM) in a potassium phosphate buffer (100 mM, pH 7.4), an NADPH-generating
+
system (NGS, final concentration: 10 mM glucose-6-phosphate, 0.50 mM NADP , and 1.0 IU yeast
glucose-6-phosphate dehydrogenase per mL), and the reaction substrate (0.50 mM). Incubations were
performed generally for 60 min with or without the NADPH-generating system (NGS). Phloretin and
its metabolites were extracted twice with ethyl acetate, followed by centrifugation (1000
separate the organic and aqueous layers [37,38].
×
g, 20 min) to
After the organic layer was concentrated using a stream of nitrogen gas, the residue was dissolved
in 180 L of mobile A:B (6:4, v/v). Mobile phase A contained 0.5% (v/v) methanol, 0.1% (v/v) formic acid,
and 99.4% water. Mobile phase B was 100% acetonitrile. Product formation was analyzed by HPLC,
and samples (30 L) were injected onto a Gemini C18 column (4.6 mm 150 nm, 5 m; Phenomenex,
µ
µ
×
µ
Torrance, CA, USA). Increasing mobile phase B from 9 to 100% (v/v) over 37.5 min was performed
as follows: initial 11 min, 9% acetonitrile; 11–13 min, 15% acetonitrile; 13–20 min, 17% acetonitrile;
20–37 min, 60% acetonitrile; and 37–37.5 min, 100% acetonitrile with the flow rate of 1.0 mL. The
stepwise gradient was done using a gradient pump (LC-20AD; Shimadzu, Kyoto, Japan). The detection
of phloretin and its metabolites was performed at 285 nm.
The kinetic parameters for substrate oxidation via recombinant P450s and HLMs were estimated
using 0.40 µM of recombinant P450 or HLMs, an NGS and phloretin (10–1000 µM) in a potassium
phosphate buffer (100 mM, pH 7.4). The reaction was initiated by the addition of a NADPH regeneration
◦
system and continued at 37 C. Reaction times for recombinant P450a and HLMs were 10 min and
30 min, respectively. Quantifying the metabolites was performed by comparing peak areas of the
metabolites to the mean peak areas of the 3-OH phloretin standard compound, which was prepared as
previously described [4].
The kinetic parameters (K and kcat) were calculated via the nonlinear weakening study using
m
GraphPad Prism software (GraphPad, Software Inc., San Diego, CA, USA). The equation was applied
for Michaelis–Menten kinetics.
The effects of b on phloretin 3-hydroxylation catalyzed by CYP3A4 and CYP2C19 were performed
5
by incubating 0.40
µM of recombinant P450, 0.80
µM of recombinant NADPH-P450 reductase, 0.8
µM
of recombinant human b , an NGS, and phloretin (300
pH 7.4). The reaction was started by adding an NGS and continued at 37 C for 10 min [39].
µ
M) in a potassium phosphate buffer (100 mM,
5
◦
4
.3. Phloretin’s Inhibitory Effect on Testosterone 6β-Hydroxylation Catalyzed via HLMs and CYP3A4
To examine phloretin’s effect on testosterone 6
reaction mixtures were performed at 37 C in 0.25 mL incubation mixtures containing 0.40
β
-hydroxylation catalyzed via CYP3A4 and HLMs,
◦
µM of P450
in a potassium phosphate buffer (100 mM, pH 7.4) and an NGS. After preincubation with indicated
◦
phloretin concentrations (10–100
µ
M) for 5 min in the presence of NGS at 37 C, testosterone (0.50 mM)
was added to start the reaction. After reacting for 30 min, the products were stopped and extracted via
ethyl acetate, followed by centrifugation (1000 g, 20 min) to separate the organic and aqueous layers.
-OH testosterone was analyzed via HPLC. The organic layer was obtained and then concentrated
using a stream of nitrogen gas, and the residue of the organic layer was dissolved in 180 L of mobile
phase (60% methanol). Product formation was analyzed by HPLC, and samples (30 L) were injected
into a Gemini C18 column (4.6 mm 150 nm, 5 m; Phenomenex, Torrance, CA). The flow rate was
.0 mL using 60% methanol as a mobile phase, and the substrate and its products were detected at
85 nm.
×
6β
µ
µ
×
µ
1
2

Pharmaceuticals 2020, 13, 330
10 of 13
4
.4. Immunoinhibition of Antibodies on Phloretin 3-Hydroxylation
Inhibition studies using specific antibodies to CYP3A4 and CYP2C19 were performed by incubating
◦
HLMs with varying concentrations of anti-P450 IgG for 30 min at 23 C before adding other components,
including phloretin and an NGS, required for catalytic activity [40]. The control experiments with
varying concentrations of the preimmune IgG were done.
4
.5. LC-Mass Spectrometric Analysis of a Phloretin Metabolite
To investigate the major product’s mass, a CYP3A4 reaction mixture containing 0.40 µM of P450,
5
00 µM of phloretin, and NGS was used in 0.25 mL of a potassium phosphate buffer (100 mM, pH 7.4).
◦
After these reaction mixtures were incubated at 37 C for 30 min, injection samples were prepared as
described above. Analyses of the mass values of phloretin and its products were performed using a
TSQ Quantum
™ Access MAX Triple Quadrupole Mass Spectrometer on an Accela 1250 HPLC system
(ThermoFisher Scientific, Waltham, MA, USA). The samples were separated on a ZORBAX SB-C18
column (250 mm × 4.6 mm i.d. 5 µm; Agilent, Santa Clara, CA, USA) at a flow rate of 1 mL/min. The
mobile phases were (A) 0.1% (v/v) formic acid in water and (B) acetonitrile. The isocratic flow of the
mobile phase was (A) 60% and (B) 40% on HPLC. The injection volume was 10 µL. The mass spectra
were recorded via electrospray ionization in positive mode to characterize phloretin metabolites. The
collision energy and scan rate were 10 V and 0.5 spectra/s, respectively.
4
.6. Optimal Expression of CYP3A4 in E. coli
Six different strains of competent E. coli cells (BL21, DH5α-F’IQ, MG1655, Shuffle T7, Rosetta, and
JM109) harboring the CYP3A4 plasmid were inoculated into Luria-Bertani (LB) broth supplemented
◦
with ampicillin (100
µ
g/mL). The strain was grown at 37 C with shaking at 180 rpm overnight. It was
then inoculated to the Terrific Broth (TB) medium and was grown to absorbance at 600 nm (OD600
0.6–0.8. After that, isopropyl– -D-thiogalactopyranoside (0.5 mM) and -aminolevulinic acid (1.0 mM)
were added for enzyme induction and functional expression. After the cultures were grown at 20,
)
~
β
δ
◦
2+
2+
2
5, and 30 C with shaking at 170 rpm, Fe
·
CO versus Fe difference (CO) spectra were measured
at indicated cultured times from 10 h up to 26 h. The P450 concentrations were quantified via the
CO-difference spectra measurement using an extinction molecular coefficient of ε = 91 mM/cm [41].
4
.7. Whole-Cell Biotransformations
E. coli DH5 F’-IQ cells expressing recombinant human CYP3A4 were obtained at the stationary
α
phase and were used for biotransformation experiments. The cells were collected by centrifugation at
◦
6
000
×
g and 4 C for 10 min. After the removal of the supernatant, the cell pellet was resuspended in a
potassium phosphate buffer (100 mM, pH 7.4) containing 6.0 mM of magnesium acetate and 10 mM
of dextrose. The optimal concentrations of E. coli cells and phloretin substrate for 3-OH phloretin
production were determined via E. coli cells ranging from 5 to 45 g per liter with 8.0 mM of phloretin
and via phloretin ranging from 1.0 to 15 mM with 30 g cells per liter for 1 h, respectively. Production
of 3-OH phloretin via recombinant E. coli cells expressing CYP3A4 was performed with a potassium
phosphate buffer (100 mM, pH 7.4) containing 30 g of E. coli cells per liter and 8.0 mM of phloretin at
◦
3
7 C for 60 min. The formation rate of 3-OH phloretin was quantified by HPLC, as described above.
5
. Conclusions
In this study, the roles of human liver P450s in phloretin oxidation were investigated as phloretin
is a popular polyphenol compound that is rich in apples. The major metabolite of phloretin is
-OH phloretin. We found that CYP3A4 was a major P450 and CYP2C19 was a minor P450 to
catalyze phloretin 3-hydroxylation in the human liver by using recombinant human P450 enzymes and
HLMs. Phloretin was a potent inhibitor of CYP3A4-catalyzed testosterone 6 -hydroxylation activity.
As CYP3A4 is the most popular drug-metabolizing enzyme in the human liver, possible interactions of
3
β

Pharmaceuticals 2020, 13, 330
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phloretin with clinical drugs metabolized by CYP3A4 should be considered to avoid any drug side
effects with apple consumption.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/11/330/s1:
Figure S1. LC-MS analyses of the phloretin metabolite by HLMs; Figure S2. HPLC chromatograms of phloretin
and its metabolites via HLMs, CYP3A4 and CYP2C19LC-MS analyses of the phloretin metabolite by CYP3A4;
Figure S3. LC-MS analyses of the phloretin metabolite by CYP3A4; Figure S4. LC-MS analyses of the phloretin
metabolite by CYP2C19; Figure S5. The kinetic parameters for 3-OH phloretin formation by human CYP3A4,
CYP2C19, and HLMs; Figure S6. Effects of cytochrome b on phloretin 3-hydroxylation by CYP3A4 and CYP2C19.
5
Author Contributions: Conceptualization, N.A.N., C.-H.Y., J.-G.P., and S.-K.C.; investigation, N.A.N., N.T.C.,
T.-K.L., T.H.H.N., S.M.W., and G.S.C.; writing—original draft preparation, N.A.N., S.-J.Y., and C.-H.Y.; supervision,
S.-J.Y., H.-S.K., and C.-H.Y.; funding acquisition, S.-K.C., H.-S.K. and C.-H.Y. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Next-Generation BioGreen 21 program (SSAC, grant no.: PJ01333101),
Rural Development Administration, and the National Research Foundation of Korea (NRF-2018R1A4A1023882
and NRF-2018M3A9F3079566), Republic of Korea.
Conflicts of Interest: The authors declare no conflict of interest.
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