molecules
Article
Effects of Flavonoids in Lysimachia clethroides Duby
on the Activities of Cytochrome P450 CYP2E1 and
CYP3A4 in Rat Liver Microsomes
Zhi-Juan Zhang 1 , Zhao-Yang Xia 1 , Jin-Mei Wang 1,2 , Xue-Ting Song 3 , Jin-Feng Wei 1,2,3, * and
Wen-Yi Kang 1,2, *
1
2
3
*
Institute of Chinese Materia Medica, Henan University, Kaifeng 475004, China;
[email protected] (Z.-J.Z.); [email protected] (Z.-Y.X.); [email protected] (J.-M.W.)
Kaifeng Key Laboratory of Functional Components in Health Food, Kaifeng 475004, China
Minsheng College, Henan University, Kaifeng 475004, China; [email protected]
Correspondence: [email protected] (J.-F.W.); [email protected] (W.-Y.K.);
Tel.: +86-371-2388-0680 (J.-F.W. & W.-Y.K.)
Academic Editor: Isabel C. F. R. Ferreira
Received: 3 May 2016; Accepted: 1 June 2016; Published: 14 June 2016
Abstract: Incubation systems were established to investigate the effects of quercetin, kaempferol,
isoquercitrin and astragalin in Lysimachia clethroides Duby on the activities of CYP2E1 and CYP3A4 in
rat liver microsomes in vitro. Probe substrates of 4-nitrophenol and testosterone as well as flavonoids
at different concentrations were added to the incubation systems. After incubation, a validated
high performance liquid chromatography (HPLC) method was applied to separate and determine
the relevant metabolites. The results suggested that kaempferol exhibited a weak inhibition of
CYP2E1 activity with an IC50 of 60.26 ˘ 2.54 µM, while quercetin and kaempferol caused a moderate
inhibition of CYP3A4 activity with IC50 values of 18.77 ˘ 1.69 µM and 32.65 ˘ 1.32 µM, respectively.
Isoquercitrin and astragalin had no effects on the activities of either CYP2E1 or CYP3A4. It could
be speculated from these results that the inhibitory effects of quercetin and kaempferol on the
activities of CYP2E1 and CYP3A4 could be the mechanisms underlying the hepatoprotective effects
of L. clethroides.
Keywords: Lysimachia clethroides Duby; flavonoids; HPLC; CYP2E1; CYP3A4; liver microsomes
1. Introduction
Lysimachia clethroides Duby (L. clethroides), belonging to the family Primulaceae, is found in mild
regions of northeast China, the southwest and other eastern provinces [1]. Phytochemical research
has showed that flavonoids and glycosides, triterpenes and organic acids are the main compounds in
L. clethroides [2–5]. Pharmacological investigation showed that L. clethroides possesses antitumor [6,7],
antioxidant [8], hypoglycemic [9] and hepatoprotective [10] effects. The flavonoids are a group of the
most important active compounds in L. clethroides.
Glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) are the main
non-specific functional enzymes in the liver [11,12], their activities reflect the degree of hepatocyte
damage to a certain extent. Malondialdehyde (MDA) and superoxide dismutase (SOD) are the key
indicators of the severity of lipid peroxidation [13]. Our previous pharmacodynamic studies showed
that the levels of both GOT and GPT in serum in response to carbon tetrachloride (CCl4 )-induced
acute liver injury in mice were significantly decreased by intragastric administration of extracts of
L. clethroides, and that the content of MDA in liver was significantly decreased while the level of SOD
was significantly increased in vivo [14]. These results indicated that L. clethroides had hepatoprotective
effect on CCl4 -induced acute liver injury and its antioxidant effect was the one of the mechanisms for
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Molecules 2016, 21, 738
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the hepatoprotective effect of L. clethroides. However, the hepatoprotective mechanisms for the active
compounds in L. clethroides are complex and no studies have been reported yet.
It has been shown by studies in the literature that the enzyme CYP2E1 which mediates the
hepatocyte damage caused by a variety of compounds, such as ethanol and CCl4 , is considered
to be an important determinant of human susceptibility to toxicity and carcinogenicity caused by
the industrial and environmental chemicals [15]. CYP3A4, a major enzyme that is expressed in
adult liver, has been considered to be an important factor affecting drug absorption. CYP2E1 and
CYP3A4 are not only the metabolic enzymes directly involved in hepatic oxidative damage, but
they also are two important targets of the oxidation mechanism of acute liver injury [16]. Quercetin,
kaempferol, isoquercitrin, astragalin and other flavonoids have strong antioxidant activities [9,17],
therefore, we speculated that the flavonoids might be active compounds that can reduce the contents
of liver-damaging substances by inhibiting the activities of CYP450 enzymes, which might be one of
the hepatoprotective mechanisms of L. clethroides.
On the basis of previous studies, the impacts of quercetin, kaempferol, isoquercitrin and astragalin
on the activities of enzymes CYP2E1 and CYP3A4 in rat liver microsomes were examined, aiming to
investigate the correlation between the hepatoprotective effects of L. clethroides and the antioxidant
activities of these four compounds. These studies could provide an important basis for the development
of L. clethroides as a liver-protective drug and guide the rational use of these drugs in the clinic.
2. Results
2.1. Method Validation
2.1.1. Standard Curves and the Linearity
Different concentrations of the standard solutions mentioned in Section 4.6 were accurately
injected into the chromatographic instrument for the construction of calibration curves. Then the
standard curves were constructed by plotting the ratios of peak areas of metabolites and phenacetin
(internal standard) versus the concentrations of their metabolites. The results are listed in Table 1.
Within the selected concentration ranges, good linearities were obtained, with r values of 0.9999 and
0.9998, respectively.
Table 1. Analytical performances.
Metabolite
4-Nitrocatechol
6β-Hydroxytestosterone
Regressive equation
r
Linear range
Y = 0.0184X ´ 0.0042
0.9999
0.8–51.2 µM
Y = 0.0880X ´ 0.0062
0.9998
0.1–25.6 µg¨ mL´1
Precision (RSD, %)
High concentration
Moderate concentration
Low concentration
0.25
0.35
3.93
0.22
0.46
0.96
Stability (RSD, %)
High concentration
Moderate concentration
Low concentration
0.28
0.80
2.63
0.075
0.17
1.39
2.1.2. Precision and Stability
The precision and stability of the used methods were determined by the relative standard
deviation (RSD). The precision was obtained by analyzing the standard solutions at three different
concentrations six consecutive times, respectively. The stability was obtained by analyzing the standard
solutions at three different concentrations six times in one day. Every experiment was performed in
triplicate. The results are shown in Table 1.
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2.1.3. Recovery
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Different concentrations of 4-nitrocatechol (at final concentrations of 5, 10 and 15 µM) and
2.1.3. Recovery
6β-hydroxytestosterone
(final concentrations of 0.15, 0.30 and 0.45 µg¨ mL´1 ) were added to the
incubation
systemsconcentrations
in vitro, respectively.
Then spiked
were prepared
according
Different
of 4-nitrocatechol
(at samples
final concentrations
of 5,and
10 measured
and 15 μM)
and
to the
methods
described
in
Section
4.5.
The
results,
presented
in
Table
2,
demonstrate
that
the
−1
6β-hydroxytestosterone (final concentrations of 0.15, 0.30 and 0.45 μg·mL ) were added tomethods
the
wereincubation
reasonablesystems
and feasible.
in vitro, respectively. Then spiked samples were prepared and measured
according to the methods described in Section 4.5. The results, presented in Table 2, demonstrate that
Table 2. Recoveries of the metabolites.
the methods were reasonable and feasible.
Metabolite
Metabolite
4-Nitrocatechol (µM)
4-Nitrocatechol (μM)
Original
Added
Found
Table 2. Recoveries of the metabolites.
10.9
5
15.4 ˘ 0.11
Original
Added
Found
10.9
10
19.7 ˘ 0.09
10.9
5
15.4 ± 0.11
10.9
15
25.5 ˘ 0.17
10.9
10.9
´1 )
6β-Hydroxytestosterone (µg¨ mL0.296
6β-Hydroxytestosterone (μg·mL−1)
0.296
0.296
10
0.29615
0.296
0.15
0.30
0.296
19.7 ± 0.09
0.15 25.50.43
˘ 0.01
± 0.17
0.30 0.430.55
˘ 0.01
± 0.01
± 0.01
0.45 0.550.69
˘ 0.01
0.45
0.69 ± 0.01
Recovery (%)
89.4 ˘ 2.27
Recovery (%)
87.1 ˘ 0.85
89.4 ± 2.27
96.8 ˘ 1.12
87.1 ± 0.85
91.5
˘ 4.77
96.8
± 1.12
85.4
˘ 1.01
91.5
± 4.77
85.4
± 1.01
86.9
˘ 2.46
86.9 ± 2.46
RSD (%)
RSD (%)
5.02
5.02
4.40
4.40
2.2. The Optimal Conditions of Incubation System in Vitro
2.2. The Optimal Conditions of Incubation System in Vitro
TheThe
optimal
incubation
times
andand
protein
concentrations
were
determined
according
to to
thethe
linear
optimal
incubation
times
protein
concentrations
were
determined
according
relationship
between incubation
time or concentration
of protein
concentration
of metabolite,
linear relationship
between incubation
time or concentration
of and
protein
and concentration
of
respectively
(Figures
1
and
2).
The
optimal
incubation
times
for
CYP2E1
and
CYP3A4
60 and
metabolite, respectively (Figures 1 and 2). The optimal incubation times for CYP2E1 and were
CYP3A4
45 min,
optimalThe
protein
concentrations
for CYP2E1
and CYP3A4
werewere
0.5 and
wererespectively.
60 and 45 min,The
respectively.
optimal
protein concentrations
for CYP2E1
and CYP3A4
´1 , mg·mL
−1, respectively.
0.75 0.5
mg¨
mL0.75
respectively.
and
Figure 1. Influences of incubation times on the production rates of 4-nitrocatechol (A) and
Figure 1. Influences of incubation times on the production rates of 4-nitrocatechol (A) and
6β-hydroxytestosterone (B).
6β-hydroxytestosterone (B).
The apparent Km (Michaelis constant) value was calculated according to Michaelis-Menten
The apparent
Km (Michaelis constant)
value3).was
Michaelis-Menten
equation
and Lineweaver-Burk
plots (Figure
Thecalculated
regressiveaccording
equations toobtained
by the
equation
and
Lineweaver-Burk
plots
(Figure
3).
The
regressive
equations
obtained
Lineweaver-Burk method for CYP2E1 and CYP3A4 were Y = 835.9883X + 9.1943 (r = 0.9864) and by
Y = the
2388.0110X + 14.1598
(r =for
0.9961)
respetively.
The Kmwere
valuesY calculated
were+ 90.92
and
μM, and
Lineweaver-Burk
method
CYP2E1
and CYP3A4
= 835.9883X
9.1943
(r 168.65
= 0.9864)
respectively.+The
concentration
of probe
substrate
system should
exceed
the Km µM,
Y = 2388.0110X
14.1598
(r = 0.9961)
respetively.
TheinKincubation
werenot
90.92
and 168.65
m values calculated
values [18],The
thus,
90 and 160 μM
4-nitrophenol
testosterone system
were chosen
in this
respectively.
concentration
offor
probe
substrateand
in incubation
should
notstudy.
exceed the Km
values [18], thus, 90 and 160 µM for 4-nitrophenol and testosterone were chosen in this study.
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Figure
2.Influences
Influences of protein concentrations
concentrations on
the
production
rates
ofof4-nitrocatechol
(A) and
Figure
2. 2.
on the
the production
productionrates
ratesof
4-nitrocatechol
and
Figure
Influencesofofprotein
protein concentrations on
4-nitrocatechol
(A)(A)
and
6β-hydroxytestosterone
(B).
6β-hydroxytestosterone
(B).
6β-hydroxytestosterone (B).
Figure 3. Lineweaver-Burk plot of 4-nitrocatechol (A) and 6β-hydroxytestosterone (B).
Figure 3. Lineweaver-Burk plot of 4-nitrocatechol (A) and 6β-hydroxytestosterone (B).
Figure 3. Lineweaver-Burk plot of 4-nitrocatechol (A) and 6β-hydroxytestosterone (B).
2.3. Effects of Flavonoids in L. clethroides on CYP450 Activity
2.3. Effects of Flavonoids in L. clethroides on CYP450 Activity
2.3. Effects of Flavonoids in L. clethroides on CYP450 Activity
2.3.1. Effects on CYP2E1 Activity
2.3.1. Effects on CYP2E1 Activity
2.3.1. Effects
on CYP2E1 Activity
The effects of quercetin, kaempferol, isoquercitrin and astragalin in L. clethroides and
The effects of quercetin, kaempferol, isoquercitrin and astragalin in L. clethroides and
clomethiazole
(positive
control)
on CYP2E1
activity areand
shown
in Table
Figure 4.
Clomethiazole
The effects of
quercetin,
kaempferol,
isoquercitrin
astragalin
in3L.and
clethroides
and
clomethiazole
clomethiazole (positive control) on CYP2E1 activity are shown in Table 3 and Figure 4. Clomethiazole
inhibited
the
production
of
4-nitrocatechol
with
an
IC
50
of
1.07
±
0.01
μM.
The
effects
ofinhibited
quercetin,
(positive
control)
on
CYP2E1
activity
are
shown
in
Table
3
and
Figure
4.
Clomethiazole
inhibited the production of 4-nitrocatechol with an IC50 of 1.07 ± 0.01 μM. The effects of quercetin,the
kaempferol,
isoquercitrin and astragalin
onofCYP2E1
acivity
were
very
weak
and their IC
50 values
production
of 4-nitrocatechol
an IC50on
1.07 ˘ 0.01
µM.
Thevery
effects
of quercetin,
kaempferol,
kaempferol,
isoquercitrin andwith
astragalin
CYP2E1
acivity
were
weak
and their IC
50 values
isoquercitrin and astragalin on CYP2E1 acivity were very weak and their IC50 values could not be
could not be calculated. When its concentration was gradually increased to 100 μM [19], kaempferol
could not be calculated. When its concentration was gradually increased to 100 μM [19], kaempferol
inhibited the production of 4-nitrocatechol with an IC50 of 60.26 ± 2.54 μM (Figure 5), but at this
inhibited the production of 4-nitrocatechol with an IC50 of 60.26 ± 2.54 μM (Figure 5), but at this
concentration, the other three flavonoids still could not cause 50% inhibition of the enzyme activity.
concentration, the other three flavonoids still could not cause 50% inhibition of the enzyme activity.
Table 3. Effects of flavonoids on CYP2E1 isozyme activities (⎯x ± s, n = 3).
Table 3. Effects of flavonoids on CYP2E1 isozyme activities (⎯x ± s, n = 3).
Molecules 2016,
21, 738
Concentration
5 of 12
Inhibition Rate (%)
Inhibition Rate (%)
Concentration
(μM)
Clomethiazole
Quercetin
Kaempferol
Isoquercitrin
Astragalin
(μM)
Clomethiazole
Quercetin
Kaempferol
Isoquercitrin
Astragalin
4
85.41 ± 4.78
12.54 ± 0.81
16.49 ± 1.23
21.12 ± 1.56
16.16 ± 1.13
4
85.41 ± 4.78
12.54 ± 0.81
16.49 ± 1.23
21.12 ± 1.56
16.16 ± 1.13
2
68.15 ± 4.97
9.99 ± 0.71
7.12 ± 0.50
22.06 ± 1.12
15.58 ± 1.02
2
68.15 ± 4.97 was 9.99
± 0.71
7.12 ± 0.50to 100 µM
22.06[19],
± 1.12 kaempferol
15.58 ± 1.02
calculated. When
its concentration
gradually
increased
inhibited the
1
48.70 ± 2.19
7.21 ± 0.43
5.50 ± 0.29
18.91 ± 1.43
13.97 ± 0.81
1
48.70 ± 2.19
7.21 ± 0.43
5.50 ± 0.29
18.91 ± 1.43
13.97 ± 0.81
0.5
25.99 ±with
1.27 an IC5.46
±
0.33
5.09
±
0.37
16.27
±
0.83
6.14
±
0.45
production of 4-nitrocatechol
of
60.26
˘
2.54
µM
(Figure
5),
but
at
this
concentration,
the
50 ± 0.33
0.5
25.99 ± 1.27
5.46
5.09 ± 0.37
16.27 ± 0.83
6.14 ± 0.45
0.25
18.49 ± 0.59
3.68 ± 0.30
0.97 ± 0.80
16.02 ± 1.05
1.73 ± 0.13
0.25
± 0.59not cause
3.68 ±50%
0.30 inhibition
0.97 ± 0.80
16.02 ± 1.05
other three flavonoids
still18.49
could
of the enzyme
activity. 1.73 ± 0.13
Figure 4. Effects of flavonoids on CYP2E1 isozyme activities.
Figure
4. Effects
flavonoids on
activities.
Figure
4. Effects
ofofflavonoids
on CYP2E1
CYP2E1isozyme
isozyme
activities.
Figure 5. Effect of kaempferol on CYP2E1 isozyme activity.
Figure 5. Effect of kaempferol on CYP2E1 isozyme activity.
Figure 5. Effect of kaempferol on CYP2E1 isozyme activity.
2.3.2. Effects on CYP3A4 Activity
2.3.2. Effects on CYP3A4 Activity
Tableof
3. Effects
of flavonoids
on CYP2E1
isozyme
(x ˘ins, nL.= 3).
The effects
quercetin,
kaempferol,
isoquercitrin
andactivities
astragalin
clethroides and
The effects of quercetin, kaempferol, isoquercitrin and astragalin in L. clethroides and
ketoconazole (positive control) on CYP3A4 activity are shown in Table 4 and Figure 6. Ketoconazole
ketoconazole (positive control) on CYP3A4 activity
are shown
in Table 4 and Figure 6. Ketoconazole
Inhibition
Rate
strongly
inhibited the production of 6β-hydroxytestosterone
(IC(%)
50 = 0.25 ± 0.01 μM), but the effects of
Concentration
strongly
inhibited the production of 6β-hydroxytestosterone (IC50 = 0.25 ± 0.01 μM), but the effects of
Isoquercitrin
Astragalin
(µM)
the four flavonoids
in Clomethiazole
L. clethroides on Quercetin
CYP3A4 wereKaempferol
very weak and
their IC50 values
could not be
the four flavonoids in L. clethroides on CYP3A4 were very weak and their IC50 values could not be
4
85.41 ˘ 4.78
12.54
˘ four
0.81 flavonoids
16.49 ˘ 1.23
˘ 1.56 increased
16.16 ˘to
1.13
calculated. When
the concentrations
of the
were 21.12
gradually
100 μM,
calculated. When the concentrations of the four flavonoids were gradually increased to 100 μM,
68.15
˘ 4.97
0.71
7.12
0.50
22.06 ˘ 1.12
15.58 IC
˘ 50
1.02
quercetin and2 kaempferol
could
inhibit9.99
the˘production
of ˘
6β-hydroxytestosterone
with
values of
quercetin and1 kaempferol
could
inhibit7.21
the˘production
of ˘
6β-hydroxytestosterone
with
values of
48.70
˘ 2.19
0.43
5.50
0.29
18.91 ˘ 1.43
13.97IC
˘ 50
0.81
18.77 ± 1.69 μM and 32.65 ± 1.32 μM, respectively (Figure 7).
0.5 and 32.65
25.99
˘ 1.27
5.46 ˘ 0.33 (Figure
5.09 ˘
16.27 ˘ 0.83
6.14 ˘ 0.45
18.77 ± 1.69 μM
± 1.32
μM, respectively
7).0.37
0.25
18.49 ˘ 0.59
3.68 ˘ 0.30
0.97 ˘ 0.80
16.02 ˘ 1.05
1.73 ˘ 0.13
Table 4. Effects of flavonoids on CYP3A4 isozyme activities (⎯x ± s, n = 3).
Table 4. Effects of flavonoids on CYP3A4 isozyme activities (⎯x ± s, n = 3).
2.3.2. Effects
on CYP3A4 Activity
Concentration
Inhibition Rate (%)
Inhibition Rate (%)
Concentration
(μM)
Ketoconazole
Quercetin
Kaempferol
Isoquercitrin
Astragalin
(μM)
Ketoconazole
Quercetin
Kaempferol
Isoquercitrin
The effects5 of quercetin,
isoquercitrin
and±astragalin
in L.± 1.00
clethroidesAstragalin
and
ketoconazole
87.28kaempferol,
± 6.55
33.18
± 1.96
26.20
1.81
16.68
6.65
± 0.41
5
87.28 ± 6.55
33.18 ± 1.96
26.20 ± 1.81
16.68 ± 1.00
6.65 ± 0.41
1
76.11 ± 5.25
24.97 ± 1.77
20.00 ± 1.28
13.31 ± 0.63
2.57 ± 0.19
(positive control)
on CYP3A4
activity 24.97
are ±shown
in 20.00
Table
4 and Figure
6. Ketoconazole
strongly
1
76.11 ± 5.25
1.77
± 1.28
13.31 ± 0.63
2.57 ± 0.19
0.2
47.33 ± 3.08
22.41 ± 1.46
17.70 ± 0.80
13.04 ± 1.11
2.22 ± 0.14
0.2
47.33
± 3.08
22.41 ± 1.46
17.70=
± 0.80
13.04
± 1.11 but the2.22
± 0.14 of the four
inhibited the production
of
6β-hydroxytestosterone
(IC
0.25
˘
0.01
µM),
effects
0.1
22.95 ± 1.08
15.79 ± 1.23
14.12
10.62 ± 0.62
0.51 ± 0.04
50 ± 0.55
0.1
22.95 ± 1.08
15.79 ± 1.23
14.12 ± 0.55
10.62 ± 0.62
0.51 ± 0.04
0.02clethroides14.72
0.56
15.04 ± 0.83 weak
10.98
± 0.87
9.03
± 0.35
0.42
± 0.04
flavonoids in 0.02
L.
on ±±CYP3A4
were
and
their IC50
values
not
be calculated.
14.72
0.56
15.04 ±very
0.83
10.98
± 0.87
9.03
± 0.35 could0.42
± 0.04
When the concentrations of the four flavonoids were gradually increased to 100 µM, quercetin and
kaempferol could inhibit the production of 6β-hydroxytestosterone with IC50 values of 18.77 ˘ 1.69 µM
and 32.65 ˘ 1.32 µM, respectively (Figure 7).
Table 4. Effects of flavonoids on CYP3A4 isozyme activities (x ˘ s, n = 3).
Inhibition Rate (%)
Concentration
(µM)
Ketoconazole
Quercetin
Kaempferol
Isoquercitrin
Astragalin
5
1
0.2
0.1
0.02
87.28 ˘ 6.55
76.11 ˘ 5.25
47.33 ˘ 3.08
22.95 ˘ 1.08
14.72 ˘ 0.56
33.18 ˘ 1.96
24.97 ˘ 1.77
22.41 ˘ 1.46
15.79 ˘ 1.23
15.04 ˘ 0.83
26.20 ˘ 1.81
20.00 ˘ 1.28
17.70 ˘ 0.80
14.12 ˘ 0.55
10.98 ˘ 0.87
16.68 ˘ 1.00
13.31 ˘ 0.63
13.04 ˘ 1.11
10.62 ˘ 0.62
9.03 ˘ 0.35
6.65 ˘ 0.41
2.57 ˘ 0.19
2.22 ˘ 0.14
0.51 ˘ 0.04
0.42 ˘ 0.04
Molecules 2016, 21, 738
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Figure6.6.Effects
Effectsof
offlavonoids
flavonoids on
on CYP3A4
Figure
CYP3A4isozyme
isozymeactivities.
activities.
Figure
on CYP3A4
CYP3A4isozyme
isozymeactivities.
activities.
Figure7.7.Effects
Effectsofofquercetin
quercetin and
and kaempferol
kaempferol on
3. 3.Discussion
Discussion
CYP2E1and
and CYP3A4
CYP3A4 are
metabolic
enzymes
involved
in liverininjury.
CYP2E1
are two
twoimportant
important
metabolic
enzymes
involved
liver Total
injury.
alkaloids
of
Rubus
alceaefolius
Poir
could
significantly
reduce
the
levels
of
GOT
and
GPT,
protect
liver
Total alkaloids of Rubus alceaefolius Poir could significantly reduce the levels of GOT and GPT, protect
cells
from
injury,
andand
inhibit
thethe
mRNA
expressions
of CYP2E1
and and
CYP3A1
in liver
tissuetissue
[20]. [20].
It
liver
cells
from
injury,
inhibit
mRNA
expressions
of CYP2E1
CYP3A1
in liver
has been
been indicated
indicated that
that Gegen
liver
It has
Gegen powder
powderpossesses
possessestherapeutic
therapeuticeffects
effectsononacute
acutealcohol-induced
alcohol-induced
liver
injury,
by
increasing
the
content
of
CYP450
and
reducing
the
activity
of
CYP2E1
[21].
Radix
injury, by increasing the content of CYP450 and reducing the activity of CYP2E1 [21]. Radix Glycyrrhizae
Glycyrrhizae
could
protect
the
liver injury
causeddioscorea
by Rhizoma
dioscoreapossibly
bulbifera,
possibly
due to its of
could
protect the
liver
injury
caused
by Rhizoma
bulbifera,
due
to its induction
induction of activity of CYP2E1 and CYP3A4 and inhibition of the mRNA expression [22].
activity of CYP2E1 and CYP3A4 and inhibition of the mRNA expression [22].
Clomethiazole and ketoconazole act as the positive inhibitors for CYP2E1 and CYP3A4 can
Clomethiazole and ketoconazole act as the positive inhibitors for CYP2E1 and CYP3A4 can
significantly inhibit the formation of metabolites. The IC50 values of clomethiazole and ketoconazole
significantly inhibit the formation of metabolites. The IC50 values of clomethiazole and ketoconazole
were 1.07 ± 0.01 μM and 0.25 ± 0.01 μM, respectively, which are consistent with literature values [23,24],
were 1.07 ˘ 0.01 µM and 0.25 ˘ 0.01 µM, respectively, which are consistent with literature values [23,24],
suggesting that the incubation systems in vitro can meet the activities of measurement of CYP2E1 and
suggesting that the incubation systems in vitro can meet the activities of measurement of CYP2E1
CYP3A4.
and CYP3A4.
According to the literature [25,26], a strong inhibition is considered for a compound with an IC50
According
the and
literature
[25,26],
a strong inhibition is considered for a compound with an IC
value below 1toμM,
if the IC
50 value is higher than 50 μM, the compound is considered to be a 50
value
below
1
µM,
and
if
the
IC
value
is
50 µM, effect
the compound
is considered
to be aand
weak
50
weak inhibitor. Thus, kaempferol
has ahigher
weaklythan
inhibitory
on CYP2E1,
while quercetin
inhibitor.
Thus,
kaempferol
has
a
weakly
inhibitory
effect
on
CYP2E1,
while
quercetin
and
kaempferol
kaempferol have moderate inhibitory effects on CYP3A4.
have moderate
inhibitory
effects
on CYP3A4.
The results
of this study
showed
that the two flavone glycosides (isoquercitrin and astragalin)
The
results
of
this
study
showed
that
flavoneThis
glycosides
and astragalin)
had
had no effect on the activities of CYP2E1the
andtwo
CYP3A4.
might be(isoquercitrin
due to the reason
that there is
no aeffect
on quantitative
the activitiesstructure-activity
of CYP2E1 and CYP3A4.
Thisbetween
might bethe
due
to the reason
thereand
is a their
certain
certain
relationship
structure
of the that
flavones
bioactivities.
A previous study
had shown
that the
location
of flavonoids’
hydroxy
quantitative
structure-activity
relationship
between
thenumber
structureand
of the
flavones
and their bioactivities.
substituents
andhad
the shown
conjugated
system
all have
on hydroxy
the activities
of CYP450
A previous
study
that the
number
and significant
location ofinfluence
flavonoids’
substituents
and
enzymes
[27].
As
the
number
of
hydroxy
substitutions
was
increased,
the
inhibition
of
CYP450
the conjugated system all have significant influence on the activities of CYP450 enzymes [27]. As the
enzymes
activitiessubstitutions
tended to be progressively
enhanced.
The high
polarityenzymes
of the glycosides
also to
number
of hydroxy
was increased,
the inhibition
of CYP450
activitiesmay
tended
be progressively enhanced. The high polarity of the glycosides may also have interfered with their
Molecules 2016, 21, 738
7 of 12
interaction with the CYP450 enzymes [28]. Currently, few studies have been done on the structure of
the compounds and their inhibition effects and further studies are needed.
Cytochromes P450, especially CYP2E1 and CYP3A4, are responsible for metabolism of ethanol,
CCl4 and other solvents in the body. In the model of alcohol-induced liver injury, the expression
levels of CYP2E1 and CYP3A4 were enhanced [29]. Researchers have used CYP2E1 knockout mice to
evaluate the effect of CYP2E1 on ethanol-induced chronic liver injury, and the results showed that the
fatty liver production and ethanol-induced oxidative stress of wild-type mice were significantly higher
than those in CYP2E1 knockout mice [30], showing that CYP2E1 is involved in the oxidative stress of
the body, increasing the severity of liver damage.
As a typical poison, CC14 is activated by CYP2E1 and metabolized to a series of highly reactive
free radicals. These radicals can attack the cell membrane by hydrogen adsorption and induce lipid
peroxidation. The cellular calcium concentration is augmented along with radical reaction-induced
cell death [31]. Lipid peroxidation alters the integrity of cellular membranes and damages proteins
and DNA, decreases hepatic antioxidants such as glutathione (GSH), finally lead to hepatic oxidative
damage even canceration [32].
The occurrence of liver injury can be reduced or even suppressed by minimizing the activities
of CYP2E1 and CYP3A4, making the discovery of more compounds with anti-oxidative stress from
natural products of important significance.
4. Material and Methods
4.1. Chemicals
Chromatographic grade acetonitrile was purchased from Avantor Performance Materials, Inc.
(Center Valley, PA, USA). Chromatographic grade methanol was purchased from Tianjin Shield
Fine Chemicals Co., Ltd. (Tianjin, China) Analytical grade glacial acetic acid was purchased
from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China) The water was Wahaha pure water.
Coomassie brilliant blue was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). The isoquercitrin, astragalin, quercetin and kaempferol with purity greater than 98% were
purchased from Chengdu Pufei De Biotech Co., Ltd. (Sichuan, China). The 4-nitrocatechol and
4-nitrophenol were purchased from A Johnson Matthey Company (Royston, UK). Phenacetin and
6β-Hydroxytestosterone and clomethiazolewere purchased from Sigma (St. Louis, MO, USA).
Testosterone was purchased from Beijing J & K Technology Co., Ltd. (Beijing, China). NADPH was
purchased from Blue Chemical Technology Co., Ltd. (Shanghai, China). Ketoconazole was purchased
from Tokyo Chemical Industry (Tokyo, Japan).
4.2. Instruments
A LC-20AT high performance liquid chromatography system (Shimadzu, Kyoto, Japan), equipped
with a degasser, a quaternary gradient low pressure pump, the CTO-20A column oven, a SPD-M20A
UV-detector, a SIL-20A autosampler was used. The data were acquired and processed using
a LC-Solution chromatography data processing system. Chromatographic separations of target analytes
were performed on an InertSustain C18 column (4.6 mm ˆ 150 mm, 5 µm). A GRP-9270 water-jacket
thermostatic constant incubator purchased from Shanghai Sumsung Laboratory Instrument Co., Ltd.
(Shanghai, China) was used. A TGL-16gR high-speed freezing centrifuge was purchased from Shanghai
Anting Scientific Instrument Factory (Shanghai, China). The Power Gen 125 tissue homogenizing
machine was purchased from Fisher Scientific (Waltham, MA, USA).
4.3. Animals
Sprague Dawley (SD) rats (200–220 g) were obtained from the Experimental Animal Center of
Henan Province (Zhengzhou, Hennan, China). The animals were kept under standard conditions
(12 h light/dark cycle, 25 ˝ C and humidity 50% to 65%) and housed in polycarbonate cages with
Molecules 2016, 21, 738
8 of 12
standard rodent diet and water. All the animal procedures were approved by the local ethical
committee in accordance with the “Institute Ethical Committee Guidelines” for animal experimentation
Molecules
21, 738
8 of 12
and
care 2016,
(HNPR-2009-05003).
rodent
diet andofwater.
AllMicrosomes
the animal procedures were approved by the local ethical committee in
4.4.
Preparation
Rat Liver
accordance with the “Institute Ethical Committee Guidelines” for animal experimentation and care
The rats were fasted for 12 h with free access to water and then sacrificed by cervical dislocation.
(HNPR-2009-05003).
The liver was removed promptly and washed with ice-cold physiological saline solution to yellowish
brown. The liver tissue was cut into pieces as small as possible and the liver homogenate solution of
4.4. Preparation of Rat Liver Microsomes
25% with phosphate buffer (pH 7.4) was processed at 0 to 4 ˝ C by a tissue homogenizing machine.
´1 and supernatant
The rats
were fasted
for 12was
h with
free access
then
by cervical
dislocation.
The liver
homogenate
solution
centrifuged
at to
4 ˝water
C for and
20 min
atsacrificed
12,000 r¨ min
The liverwas
wasreserved.
removedThe
promptly
and washed
with ice-cold
physiological
solution
to yellowish
fraction
supernatant
per millilitre
was added
with 0.1 saline
mL CaCl
2 (88 mM), gently
˝
brown.
The
liver
tissue
was
cut
into
pieces
as
small
as
possible
and
the
liver
homogenate
of
shaken and kept at 0 to 4 C for 5 min. The mixed solution was transferred to centrifuge solution
tubes and
˝
´
1
25%
with
phosphate
buffer
(pH
7.4)
was
processed
at
0
to
4
°C
by
a
tissue
homogenizing
machine.
centrifuged at 4 C for 30 min at 15,000 r¨ min . Then the supernatant was removed completely, the
The liver homogenate
solution was
at 4 °C for buffer
20 minand
at 12,000
r·min−1atand
precipitation
was re-suspended
withcentrifuged
moderate phosphate
centrifuged
4 ˝ Csupernatant
for 30 min
´
1
fraction
was
reserved.
The
supernatant
per
millilitre
was
added
with
0.1
mL
CaCl
2
(88
mM),
at 15,000 r¨ min [33,34]. The pink precipitate obtained after centrifugation was used as thegently
liver
shaken and kept
0 tomicrosomes
4 °C for 5 min.
mixed solution
was transferred
centrifuge
tubes 20%
and
microsomes.
The at
liver
was The
resuspended
in moderate
phosphatetobuffer
containing
−1. Then the supernatant was removed
˝
centrifuged
at
4
°C
for
30
min
at
15,000
r·min
completely,
the
glycerol and divided into two parts. One part was packed and stored at ´80 C for further analysis,
precipitation
was
re-suspended
with
moderate
phosphate
buffer
and
centrifuged
at
4
°C
for
30
min
and another part was used for determination of protein concentration. Protein concentration of the
−1 [33,34]. The pink precipitate obtained after centrifugation was used as the liver
at 15,000 r·min
microsomes
was determined
by the method of Bradford.
microsomes. The liver microsomes was resuspended in moderate phosphate buffer containing 20%
4.5.
Cytochrome
P450 Probe
Substrate
glycerol
and divided
into two
parts.Assays
One part was packed and stored at −80 °C for further analysis,
and another part was used for determination of protein concentration. Protein concentration of the
4.5.1.
4-Nitrophenol
2-Hydroxylation
Assay for
CYP2E1
microsomes
was determined
by the method
of Bradford.
The in vitro incubation system of CYP2E1 contained liver microsomes, MgCl2 (5 mM),
4.5. Cytochrome
P450 substrate)
Probe Substrate
Assays
4-nitrophenol
(probe
and phosphate
buffer (pH 7.4) in a final volume of 200 µL. The mixture
˝
was pre-incubated 5 min at 37 C thermostatic constant incubator, the reaction was initiated by adding
4.5.1. 4-Nitrophenol
2-Hydroxylation
CYP2E1
NADPH
(1 mM). After
incubation, 100Assay
µL offor
ice-cold
acetonitrile containing 50 µM phenacetin was
added
to
the
incubation
system
(phenacetin
was
used
as internal
standard). After
The in vitro incubation system of CYP2E1 contained
liver microsomes,
MgClblending,
2 (5 mM),the
4˝
mixture
was (probe
kept atsubstrate)
ice-water and
bathphosphate
to terminate
the (pH
reaction,
then
centrifuged
4 The
C for
20 min
at
nitrophenol
buffer
7.4) inand
a final
volume
of 200 at
μL.
mixture
was
12,000
r¨ min´15. min
Then,
wasconstant
filtrated incubator,
through a 0.22
µm microporous
membrane
and
pre-incubated
atthe
37 supernatant
°C thermostatic
the reaction
was initiated
by adding
the
subsequent
filtrate
was
collected.
The
concentration
of
organic
solvent
was
not
higher
than
1%.
NADPH (1 mM). After incubation, 100 μL of ice-cold acetonitrile containing 50 μM phenacetin was
Thetomobile
phase of system
chromatographic
separation
of methanol
0.1%
acetic acid
added
the incubation
(phenacetin
was usedconsisted
as internal
standard).and
After
blending,
the
´1 . The column temperature was set to 30 ˝ C. The UV
(33:67,
v/v),
at
a
flow
rate
of
1.0
mL¨
min
mixture was kept at ice-water bath to terminate the reaction, and then centrifuged at 4 °C for 20 min
−1. Then,
detection
wavelength
was
at 250 nm. was
All the
injection
volumes
were
µL. HPLC chromatograms
at 12,000 r·min
theset
supernatant
filtrated
through
a 0.22
μm10
microporous
membrane and
of
standard solution
andcollected.
sample are
shown
in Figureof
8. organic solvent was not higher than 1%.
thethe
subsequent
filtrate was
The
concentration
6000
Intensity (mV)
2
3
4000
1
2000
A
0
B
5
10
15
20
Time (min)
Figure 8. HPLC chromatograms of standard solution (A) and sample solution (B) 1: 4-nitrocatechol;
Figure 8. HPLC chromatograms of standard solution (A) and sample solution (B) 1: 4-nitrocatechol;
2: 4-nitrophenol;
4-nitrophenol; 3:
3: phenacetin.
phenacetin.
2:
The mobile phase of chromatographic separation consisted of methanol and 0.1% acetic acid
(33:67, v/v), at a flow rate of 1.0 mL·min−1. The column temperature was set to 30 °C. The UV detection
wavelength was set at 250 nm. All the injection volumes were 10 μL. HPLC chromatograms of the
standard solution and sample are shown in Figure 8.
Molecules2016,
2016,21,
21,738
738
Molecules
of 12
12
99 of
4.5.2. Testosterone 6β-hydroxylation Assay for CYP3A4
4.5.2. Testosterone 6β-Hydroxylation Assay for CYP3A4
The incubation conditions of CYP3A4 were basically the same as those described in Section
incubation
of CYP3A4
were500
basically
as those
described
in Section
4.5.1
4.5.1 The
but the
volume conditions
of incubation
mixture was
μL andthe
thesame
reactions
were
terminated
by adding
but
the
volume
of
incubation
mixture
was
500
µL
and
the
reactions
were
terminated
by
adding
700
µL
700 μL of ice-cold acetonitrile containing 25 μM phenacetin. The mobile phase for separating
of
ice-cold acetonitrile
containing 25 µM
The mobile
for separating
testosterone,
testosterone,
6β-hydroxytestosterone
andphenacetin.
phenacetin consisted
of phase
acetonitrile
(A), methanol
(B) and
6β-hydroxytestosterone
andgradient
phenacetin
consisted
of acetonitrile
(A), min,
methanol
and
0.1% acetic acid (C), and the
program
was set
as follows: 0–15
45% B,(B)
55%
C;0.1%
15–25acetic
min,
−1
acid
(C),
and
the
gradient
program
was
set
as
follows:
0–15
min,
45%
B,
55%
C;
15–25
min,
0%–35%
A,
0%–35% A, 45%–10% B, 55% C; 25–40 min, 35% A, 10% B, 55% C. The flow rate was 1.0 mL·min . The
´
1
45%–10%
B, 55% C; 25–40
55%detection
C. The flow
rate was 1.0
column temperature
was min,
set to35%
30 A,
°C.10%
TheB,UV
wavelength
wasmL¨
setmin
at 245. The
nm. column
All the
˝ C. The UV detection wavelength was set at 245 nm. All the injection
temperature
was
set
to
30
injection volumes were 20 μL. HPLC chromatograms of the standard solution and sample are shown
volumes
in Figurewere
9. 20 µL. HPLC chromatograms of the standard solution and sample are shown in Figure 9.
100000
3
Intensity (mV)
75000
50000
25000
2
1
A
B
0
5
10
15
20
25
30
35
40
Time (min)
Figure 9. HPLC chromatograms of standard solution (A) and sample solution (B) 1: phenacetin; 2:
Figure 9. HPLC chromatograms of standard solution (A) and sample solution (B) 1: phenacetin;
6β-hydroxytestosterone; 3: testosterone.
2: 6β-hydroxytestosterone; 3: testosterone.
4.6. Preparation of Standard Solutions
4.6. Preparation of Standard Solutions
Different concentrations of 4-nitrocatechol (0.8–51.2 μM) and 6β-hydroxytestosterone (0.1–25.6
Different concentrations of 4-nitrocatechol (0.8–51.2 µM) and 6β-hydroxytestosterone
μg·mL−1), respectively,
were added in vitro to the incubation systems. Other operational procedures
(0.1–25.6 µg¨ mL´1 ), respectively, were added in vitro to the incubation systems. Other operational
were basically the same as those described in Section 4.5, but the NADPH in the incubation system
procedures were basically the same as those described in Section 4.5, but the NADPH in the incubation
was replaced with an equal volume of phosphate buffer.
system was replaced with an equal volume of phosphate buffer.
4.7. Optimization
Optimization of
of Incubation
Incubation Conditions
Conditions in
in Vitro
Vitro
4.7.
4.7.1. Incubation
Incubation Time
Time
4.7.1.
The concentrations
concentrationsof of
protein
substrate
of incubation
system
were
fixed, the
The
protein
and and
probeprobe
substrate
of incubation
system were
fixed,
the incubation
incubation
were
90min,
and 120
min, respectively.
were preparated
according
times
were times
15, 30,
45, 15,
60, 30,
90 45,
and60,
120
respectively.
SamplesSamples
were preparated
according
to the
to
the
methods
described
in
Section
4.5.
The
concentrations
of
probe
substrate
metabolites
methods described in Section 4.5. The concentrations of probe substrate metabolites 4-nitrocatechol and
4-nitrocatechol and 6β-hydroxytestosterone
were calculated
referring
to standard
curves.times
The
6β-hydroxytestosterone
were calculated by referring
to standardby
curves.
The optimal
incubation
optimal
incubation
times were
determined
according between
to the linear
relationship
concentration
were
determined
according
to the
linear relationship
concentration
ofbetween
metabolite
and time.
of metabolite and time.
4.7.2. The Concentration of Protein
4.7.2. The concentration of Protein
The incubation time and probe substrate concentration of incubation system were constant, the
protein
were
0.5,
0.75, 1.0,
1.5 and 2.0 mg¨
mL´1 , respectively.
were
Theconcentrations
incubation time
and0.25,
probe
substrate
concentration
of incubation
system wereSamples
constant,
the
−1, respectively.
preparated
and the metabolite
concentrations
determined
according
to the methods
described
in
protein concentrations
were 0.25,
0.5, 0.75, were
1.0, 1.5
and 2.0 mg·mL
Samples
were
Section
4.5. The
concentrations
of protein
were
determined
by the linear
between
preparated
and optimal
the metabolite
concentrations
were
determined
according
to therelationship
methods described
concentration
metabolite
protein.
in Section 4.5.ofThe
optimaland
concentrations
of protein were determined by the linear relationship
between concentration of metabolite and protein.
4.7.3. The Concentration of Probe Substrate
4.7.3.AThe
Concentration
of ProbeofSubstrate
series
of concentrations
4-nitrophenol (20–500 µM) and testosterone (10–320 µM) were
added
the incubation
system in
to determine
the optimal
concentrations
probe μM)
substrates
A to
series
of concentrations
of order
4-nitrophenol
(20–500
μM) and
testosterone of
(10–320
were
added to the incubation system in order to determine the optimal concentrations of probe substrates
Molecules 2016, 21, 738
10 of 12
in vitro. The apparent Km (Michaelis constant) value was calculated according to the Michaelis-Menten
equation and Lineweaver-Burk plot.
4.8. Effects of Flavonoids in L. clethroides on CYP450 Activity
4.8.1. CYP2E1 Assay
To evaluate whether quercetin, kaempferol, isoquercitrin and astragalin affect the activity of
CYP2E1, the studies were carried out in three groups. Group 1 (normal control) was added with
phosphate buffer, group 2 (flavonoids reagent group) were added with flavonoids, and group 3 was
given clomethiazole as positive control. Groups 2 and 3 experiments were performed at concentrations
of 0.25, 0.5, 1, 2 and 4 µM, and all the the experiments were performed in triplicate. The concentrations
of probe substrate metabolites were calculated from the standard curves. The percentage of inhibition
was calculated using the formula: %Inhibition = ((A0 ´ A1 )/A0 ) ˆ 100%, in which A0 was the
concentration of probe substrate metabolite of group 1 and A1 was the concentration of probe substrate
metabolite of group 2 and 3. The IC50 values were calculated based on the concentration-effect linear
regression curve.
4.8.2. CYP3A4 Assay
The effects of flavonoids in L. clethroides on CYP3A4 activity were basically the same as
CYP2E1. The positive control was ketoconazole, and Groups 2 and 3 experiments were performed at
concentrations of 0.02, 0.1, 0.2, 1 and 5 µM [35].
5. Conclusions
In vitro rat liver microsomes incubation assay methods were adopted for determining the effects
of quercetin, kaempferol, isoquercitrin and astragalin in L. clethroides on the activities of CYP2E1
and CYP3A4. In conclusion, kaempferol has a weakly inhibitory effect on CYP2E1, while quercetin
and kaempferol had moderate inhibitory effects on CYP3A4 activity. Based on these results, it can
be speculated that the active hepatoprotective ingredients of L. clethroides include quercetin and
kaempferol, which confer the inhibitory effects on the activities of CYP2E1 and CYP3A4. The reduction
of CYP2E1 and CYP3A4 activities can mitigate the biotransformation of CC14 and prevent the
production of liver damaging substances to exert a liver protective role. Of course, the in vivo and
vitro activities may be different, a further verification of the above speculation needs to be done by
in vivo experiments.
Acknowledgments: This work was supported by Henan Province University Science and Technology Innovation
Team (16IRTSTHN019), Kaifeng City Science and Technology Innovation Talent (1509010), Basic and Advance
Project in Science and Technology Agency of Henan Province (142300410123 and 152300410064), National
Cooperation Project of Henan Province (2015GH12), Key Project in Education Department Item of Henan
Province (15A360014 and 16A360008), and College Students Innovation and Entrepreneurship Training Program
of Minsheng College, Henan University (MSCXCY2015013).
Author Contributions: Zhi-juan Zhang, Jin-feng Wei and Wen-yi Kang conceived and designed the experiments.
Zhi-juan Zhang and Zhao-yang Xia performed the experiments. Jin-mei Wang and Xue-ting Song made substantial
contributions to interpretation of data. Zhi-juan Zhang wrote the first draft of the manuscript. Jin-feng Wei and
Wen-yi Kang revised the draft and approved the version submitted.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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