The effect of oxidation on berberine-mediated CYP1 inhibition: Oxidation behavior and metabolite-mediated inhibition

Article abstract of DOI:10.1124/dmd.115.063966

The protoberberine alkaloid berberine carries methylenedioxy moiety and exerts a variety of pharmacological effects, such as antiinflammation and lipid-lowering effects. Berberine causes potent CYP1B1 inhibition, whereas CYP1A2 shows resistance to the inh

Full text of DOI:10.1124/dmd.115.063966

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2015/05/07/dmd.115.063966.DC1.html
1
D
521-009X/43/7/1100–1107$25.00
RUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.115.063966
Drug Metab Dispos 43:1100–1107, July 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
The Effect of Oxidation on Berberine-Mediated CYP1 Inhibition:
s
Oxidation Behavior and Metabolite-Mediated Inhibition
Sheng-Nan Lo, Chien-Chang Shen, Chia-Yu Chang, Keng-Chang Tsai, Chiung-Chiao Huang,
Tian-Shung Wu, and Yune-Fang Ueng
Divisions of Basic Chinese Medicine (S.-N.L., C.-Y.C., C.-C.H., Y.-F.U.), Chinese Medicinal Chemistry (C.-C.S.), and Chinese
Materia Medica Development (K.-C.T.), National Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China; Institute
of Biopharmaceutical Sciences, School of Life Science (S.-N.L., Y.-F.U.) and Department of Pharmacology, School of
Medicine (Y.-F.U.), National Yang-Ming University, Taipei, Taiwan, Republic of China; Institute of Medical Sciences, Taipei
Medical University, Taipei, Taiwan, Republic of China (C.-Y.C., Y.-F.U.); and Department of Chemistry, National Chung-Kung
University, Tainan, Taiwan, Republic of China (T.-S.W.)
Received February 24, 2015; accepted May 7, 2015
ABSTRACT
The protoberberine alkaloid berberine carries methylenedioxy moiety substrate was present. In contrast, the demethylenation by
and exerts a variety of pharmacological effects, such as anti- CYP1B1 showed the property of substrate inhibition. CYP1B1-
inflammation and lipid-lowering effects. Berberine causes potent catalyzed berberine oxidation had low K
m
values, but it had Vmax
CYP1B1 inhibition, whereas CYP1A2 shows resistance to the values less than 8% of those of CYP1A1. The dissociation
inhibition. To reveal the influence of oxidative metabolism on CYP1 constants generated from the binding spectrum and fluorescence
inhibition by berberine, berberine oxidation and the metabolite- quenching suggested that the low K values of CYP1B1-catalyzed
m
mediated inhibition were determined. After NADPH-fortified preincu- oxidation might include more than the rate constants describing
bation of berberine with P450, the inhibition of CYP1A1 and CYP1B1 berberine binding. The natural protoberberine/berberine fmetabolites
variants (CYP1B1.1, CYP1B1.3, and CYP1B1.4) by berberine was not with methylenedioxy ring-opening (palmatine, jatrorrhizine, and
enhanced, and CYP1A2 remained resistant. Demethyleneberberine demethyleneberberine) and the demethylation (thalifendine and
was identified as the most abundant metabolite of CYP1A1- and berberrubine) caused weak CYP1 inhibition. These results
CYP1B1-catalyzed oxidations, and thalifendine was generated at demonstrated that berberine was not efficiently oxidized by
a relatively low rate. CYP1A1-catalyzed berberine oxidation had the CYP1B1, and metabolism-dependent irreversible inactivation
highest maximal velocity (Vmax) and exhibited positive cooperativity, was minimal. Metabolites of berberine caused a relatively weak
suggesting the assistance of substrate binding when the first inhibition of CYP1.
Introduction
the metabolites, demethyleneberberine and thalifendine, showed
the inductive effect of berberine on the mRNA levels of either the insulin
receptor or the low-density lipoprotein receptor, but to a lesser extent
Protoberberine alkaloids, including berberine, palmatine, and
jatrorrhizine, have been found in several medicinal plants, including
Coptis chinensis (Huang-Lian), Berberis aristata (Indian barberry),
and Hydrastis canadensis (goldenseal) (Grycova et al., 2007; Kulkarni
and Dhir, 2010). Berberine appeared to be the most abundant
protoberberines of Huang-Lian and goldenseal, and berberine and its
metabolites were the main contributors to their pharmacological
effects (Abourashed and Khan, 2001; Tang et al., 2009; Cao et al.,
(
Li et al., 2011). The 24-hour exposure to 15 mM berberrubine, jatrorrhizine,
and thalifendine decreased the cellular triglyceride level in HepG2 cells
Cao et al., 2013). Unlike berberine, palmatine and jatrorrhizine do not
(
have the methylenedioxyl substitute. Palmatine and jatrorrhizine were
oxidized by liver microsomal enzymes or P450s to form the
hydroxylation and/or demethylation products (Yang et al., 2009; Zhou
et al., 2013). The clinical impact of their metabolites remained unclear.
In humans and rodents, the bioavailability of berberine was low,
and about half was eliminated in the form of metabolites (Ma and Ma,
2
013). In human urine, the glucuronide/sulfate conjugates of
demethyleneberberine (demethylenation), thalifendine (10-demethylation),
berberrubine (9-demethylation), and jatrorrhizine (ring breakage)
have been identified (Liu et al., 2009) (Scheme 1). In HepG2 cells,
2
013; Spinozzi et al., 2014). In participants taking 15 mg/kg berberine
chloride for 3 months, the mean steady-state plasma concentrations of
the unconjugated forms of berberine, berberrubine, demethyleneber-
berine, and jatrorrhizine were 1.7–6.7 nM (Spinozzi et al., 2014). Zuo
et al. (2006) reported that plasma concentrations of berberrubine,
demethyleneberberine, jatrorrhizine, and thalifendine were generally
higher than the plasma berberine concentration in rats treated orally
with 40 mg/kg berberine. The area under the curve of the
concentration versus time (AUC)0-t values of plasma berberine
This work was supported by Ministry of Science and Technology, Taiwan
[
Grants NSC101-2320-B-077-001-MY3 and MM10211-0226] and National Re-
search Institute of Chinese Medicine, Taiwan [Grants D123101].
S.-N.L. and C.-C.S. contributed equally to this work.
dx.doi.org/10.1124/dmd.115.063966.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: AUC, area under the curve; EROD, 7-ethoxyresorufin O-deethylation; HPLC, high-performance liquid chromatography.
100
1

Influence of Oxidation on Berberine-Mediated CYP1 Inhibition
1101
roles, such as the oxidation of carcinogen benzo(a)pyrene in target
organs, including the breast, lung, and ovary (Shimada and Fujii-
Kuriyama, 2004). Estradiol is metabolized by CYP1A1 to generate
mainly the 2-hydroxylation metabolite. CYP1B1 is predominantly
expressed in steroidogenic tissues and preferentially generates the
genotoxic 4-hydroxyestradiol (Lee et al., 2003), which has been
noticed as a marker of mammary tumors (Liehr and Ricci, 1996).
Recently, CYP1A1 and CYP1B1 have also been identified as being
involved in the oxidation of arachidonic acid (Choudhary et al., 2004).
CYP1B1 was considered to be involved in angiotensin II-induced
hypertension (Jennings et al., 2010). Using 7-ethoxyresorufin as
a substrate, our previous report demonstrates that berberine potently
inhibits CYP1B1, and CYP1A2 shows resistance to berberine-
mediated inhibition (Lo et al., 2013). To elucidate the influence of
metabolism on the berberine-mediated inhibition of human CYP1s,
berberine oxidation and the CYP1 inhibition by oxidation metabolites
were determined in recombinant enzyme systems.
Materials and Methods
Chemicals and Enzymes. Berberine chloride, glucose-6-phosphate,
+
glucose-6-phosphate dehydrogenase (yeast), and b-NADP sodium salt were
purchased from Sigma-Aldrich (St. Louis, MO). Palmatine and jatrorrhizine were
isolated and purified from Fibraurea tinctoria by T. S. Wu (Department of Chemistry,
National Chung-Kung University, Tainan, Taiwan, ROC) following the method as
previously described (Su et al., 2007). Berberrubine, demethyleneberberine, and
thalifendine were synthesized by C. C. Sheng (Division of Chinese Medicinal
Chemistry, National Research Institute of Chinese Medicine, Taipei, Taiwan,
ROC) following the methods reported by Liu et al. (2009, 2010). The structures of
1
isolated natural products and synthetic metabolites were identified by H-NMR
and C-NMR analyses (Su et al., 2007; Bodiwala et al., 2011). The purities of
synthetic metabolites were $98% based on the NMR and high-performance liquid
chromatography (HPLC) analyses.
Scheme 1. Structures of palmatine and jatrorrhizine (A) and the metabolism of
berberine to form demethyleneberberine, thalifendine, and berberrubine (B).
13
metabolites (in the unconjugated and conjugated forms) were 2–50
times greater than that of berberine. In rat livers, the AUC0-t value of
demethyleneberberine was comparable to that of berberine in rats
treated orally with 100 mg/kg berberine (Liu et al., 2010). At 4 hours
after oral administration, the hepatic berberine and demethyleneber-
berine concentrations were about 0.30 mM. In the heart and kidney,
the AUC0-t value of demethyleneberberine was also comparable to that
of berberine. Besides the extensive hepatic metabolism of berberine,
Preparation of Bacterial Membranes Expressing Human CYP1
Enzymes and CYP1B1 Variants. The expression constructs of CYP1A1*1,
CYP1A2*1, and CYP1B1*3 with N-terminal modification were generously
provided by F. Peter Guengerich (Nashville, TN) (Shimada et al., 1998b). The
construct for expressing CYP1B1.1 (Leu432) was prepared as described before
(
Lo et al., 2013). Bicistronic human constructs consisting of the coding
sequence of P450 followed by that of NADPH-P450 reductase were
transformed to Escherichia coli DH5a by electroporation (Gene Pulser II;
Bio-Rad, Hercules, CA). The construct of CYP1B1*4 was prepared by
high tissue distribution might also be one of the factors leading to the introducing the 4390A.G substitution to CYP1B1*1 using the primer-directed
low plasma concentration of berberine (Liu et al., 2010). The AUC0-t enzymatic amplification method, according to the manufacturer’s instructions
values of unconjugated berberine in the liver, kidney, lung, and spleen for the QuickChange Lightning site-directed mutagenesis kit (Stratagene, an
Agilent Technologies Company, La Jolla, CA). The oligonucleotide primer sets
were 40–7530% greater than that in plasma. The AUC0-t values of the
used for mutagenesis were described in a previous report (Lo et al., 2013). P450
unconjugated metabolites, such as demethyleneberberine, were also
expression and membrane preparation were performed following the methods
higher in the liver and heart than in the plasma. The high tissue-
of Shimada et al. (1998a) and Parikh et al. (1997), respectively. The P450
distribution level may cause significant biochemical influence not only
content of the bacterial membrane fraction was determined following the
in the liver, but also in the extrahepatic tissues.
In the NADPH-dependent oxidation of berberine by human liver
microsomal enzymes and the recombinant hepatic P450s, demthyle-
spectrophotometric method (Omura and Sato, 1964) using a spectral de-
termination buffer for dilution (Parikh et al., 1997).
CYP1 Activity and the Inhibitory Effect Determinations. The 7-
neberberine appeared to be the most abundant metabolite. To a lesser ethoxyresorufin O-deethylation (EROD) activities of CYP1A1/1A2/1B1
extent, thalifendine was also generated (Li et al., 2011) (Scheme 1). were determined using 5 pmol P450/ml. The generation of the metabolite
The formation of berberrubine and jatrorrhizine was below the resorufin was determined by measuring the increase of the fluorescence
intensity (excitation wavelength: 550 nm; emission wavelength: 585 nm)
detection limit. Among the main hepatic P450s, CYP2D6 had the
highest berberine oxidation activities. Although CYP1A2 had
berberine demethylenation and 10-demethylation activities of about
(
Pohl and Fouts, 1980). For the determination of the inhibitory effect, P450
activity was determined in the incubation with or without NADPH-fortified
preincubation. The NADPH-fortified preincubation consisted of P450,
increasing concentrations of protoberberines, and a NADPH-generating
system in 50 mM HEPES buffer, pH 7.85. The NADPH-generating system
5
0–60% of those of CYP2D6, the contribution of CYP1A2, CYP2D6,
and CYP3A4 to human liver microsomal demethylenation was
estimated to be almost equal, and the contribution of CYP1A2 to
the 10-demethylation was predominant (Li et al., 2011).
+
comprised 0.5 mM NADP , 5 mM glucose-6-phosphate, and 1.0 U/ml
glucose-6-phosphate dehydrogenase. After a 10-minute preincubation, the
Although the liver has the most abundant P450 enzymes, substrate 7-ethoxyresorufin (2 mM) was added into the reaction mixtures and
extrahepatic CYP1A1 and CYP1B1 play important toxicological the O-deethylation activity was determined, as described above.

1
102
Lo et al.
Berberine Oxidation. Berberine oxidation activity was determined using enzymes with increasing concentrations of berberine at ambient temperature. The
a method modified from the method reported by Li et al. (2011). Berberine absorbance was measured in a wavelength scan mode using a spectrophotometer
chloride was dissolved in dimethyl sulfoxide. Berberine oxidation activity was with a double beam (U3000; Hitachi, Tokyo, Japan). To prevent the absorbance
determined in an incubation mixture (0.5 ml) consisting of 50 mM potassium interference of berberine, tandem (tandem/divided chambers) cuvettes were used.
+
phosphate buffer, pH 7.4, 20 pmol/ml P450, 0.5 mM NADP , 5 mM glucose-6- Both reference (without binding) and sample (with binding) cuvettes contained
2
phosphate, 10 mM MgCl , and various concentrations of berberine chloride. The P450 and berberine in the buffer solution. Each side of a tandem cuvette
oxidation was initiated by the addition of 1.0 U/ml glucose-6-phosphate contained 1 mM P450 (in 0.1 M potassium phosphate buffer, pH 7.4) and various
dehydrogenase and incubated at 37ꢀC for 30 minutes. Reaction was terminated concentrations of berberine diluted in 0.1 M potassium phosphate buffer, pH 7.4.
by the addition of 1 ml methanol. After centrifugation at 13,800g for 10 minutes Baseline correction was carried out in the absence of berberine. After the addition
at 4ꢀC, the supernatant was subjected to HPLC analysis using a HPLC system of berberine in the buffer chamber, solutions in the two chambers remained
(
4
(
Hitachi High-Tech, Tokyo, Japan) equipped with a C18 column (Cosmosil, separate in the reference cuvette. In the sample cuvette, berberine and P450
.6 mm  25 cm, 5 mm; Nacalai Tesque, Kyoto, Japan) and an absorbance detector solutions on each chamber were completely mixed, and the spectrum was
Hitachi 7420, Tokyo, Japan). Metabolites were separated by 17.5% acetonitrile recorded. The absorbance difference ΔA372–425 nm and ΔA370–420 nm was
and 10% methanol in 20 mM sodium phosphate buffer, pH 3.0, at a flow rate of determined for the binding of berberine to CYP1A2 and CYP1B1, respectively.
ml/min. The production of demethyleneberberine and thalifendine was de- The spectral dissociation constant K was determined by nonolinear least-squares
termined by measuring the absorbance at 346 nm, and the quantification was + S),
regression without weight according to the equation: ΔA = ΔAmax  S/(K
performed using the synthetic metabolite standards. Based on the high berberine where S is the concentration of berberine or demethyleneberberine.
oxidation activity of CYP1A1, the oxidation products generated by CYP1A1 Data Analyses. The concentration of an inhibitor required for causing a 50%
1
s
s
were further identified using a liquid chromatography-mass spectrometry system. decrease of catalytic activities (IC50) was calculated by curve fitting (Grafit;
A HPLC system (Chromaster; Hitachi High-Tech) was equipped with an Erithacus Software, Staines, UK). The statistical significance of the differences
absorbance detector (SPD-20A; Shimadzu, Kyoto, Japan) and a mass spectrom- between the two groups of IC50 values was evaluated using Student’s t test.
S
eter (Expression CMS; Adivon, Ithaca, NY). The metabolites were separated Kinetic analysis of the pattern with positive cooperativity was performed
with a mobile phase of 35% methanol containing 0.1% formic acid at a flow rate following the nonlinear least-squares regression (SigmaPlot software) based on
n
n
n
of 0.2 ml/min. Ions were detected using the positive ion mode with an the equation: v = (Vmax  S )/(S50 + S ), where S50 is the substrate concentration
electrospray voltage of 3500 V and gas temperature at 250ꢀC. The generation of showing half of the maximal velocity (Vmax) and the Hill coefficient n is
demethyleneberberine and thalifendine was identified by a selected ion mode a measure of cooperativity. For substrate inhibition, the kinetics can be regarded
with the m/z range of 324–325 and 322–323, respectively.
as a form of uncompetitive inhibition by the substrate (Houston and Kenworthy,
2000). Values of velocities (v) at various substrate concentrations (S) were fitted
by nonlinear least-squares regression without weight according to the equation:
Intrinsic Fluorescence Measurements. The binding of berberine to P450 in
membrane fractions was monitored by determining the decrease of intrinsic
fluorescence intensity (fluorescence quenching) using a method modified from
the report of Liu et al. (2012). CYP1A2 and CYP1B1 variant-expressed
m i i
v = Vmax  S/{K + S[1 + (S/K )]}, where K was the inhibitor constant. The
10-demethylation activity of CYP1B1 was too low to be determined at a berberine
bacterial membrane fractions were diluted to 0.2 mM P450 using 0.1 M concentration less than 1 mM. Thus, the hyperbolic plot was analyzed following
potassium phosphate buffer (pH 7.4). The emission fluorescence spectra of
P450 were measured between 280 and 400 nm (excitation wavelength at
the Michaelis-Menten equation: v = Vmax  S/(K
m
+ S). Estimates of variance
(denoted by 6) are presented from the analysis of individual sets of data.
295 nm). The emission wavelength with maximal fluorescence intensity was used
for the following determination of fluorescence quenching. The fluorescence
intensity of bacterial membrane fractions expressing P450 was measured at
Results
3
36 nm. The reduction of fluorescence intensity by the addition of increasing
The Inhibition after NADPH-Fortified Preincubation of CYP1
with Protoberberines. Recombinant CYP1A1, CYP1A2, and
CYP1B1.1 had EROD activities of 77.9 6 5.5, 4.7 6 1.2, and 9.0
concentrations of berberine (I) was calculated. The dissociation constants (K
were estimated by nonlinear regression analysis (SigmaPlot 11.0; Jandel
Scientific, San Rafael, CA), according to the equation for a single binding site:
d
)
6
2.7 nmol/min/nmol P450, respectively. NADPH-fortified preincu-
F
0
2 F = DFmax  I/(K
before and after the addition of berberine, respectively. DFmax is the maximal
value of (F 2 F) (Sampedro et al., 2007). The estimates of variance (denoted by 6)
are presented from the analysis of individual sets of data.
d 0
+ I), where F and F are the fluorescence intensities
bation provides an incubation of the CYP1 system with protoberber-
ines in the presence of the NADPH-generating system. This
preincubation allows the preoxidation of protoberberines by CYP1
0
Binding Spectral Analysis. The spectral changes due to the binding of to be investigated regarding the influence of oxidative metabolism on
berberine to recombinant P450 were determined by the titration of CYP1 the CYP1 inhibition by themselves. The inhibition of CYP1A1 by
TABLE 1
Effect of preoxidation on the inhibition of 7-ethoxyresorufin O-deethylation activity of CYP1A1, CYP1A2, and
CYP1B1 variants by berberine, palmatine, and jatrorrhizine
IC50 values were calculated by graph-fitting software. Estimates of variance generated from the curve fitting of individual sets of data
were less than 15%. Data represent the mean 6 S.E. of three determinations.
IC50, mM
P450
NADPH-Fortified Preincubation
Berberine
Palmatine
Jatrorrhizine
CYP1A1
2
+
2
0.79 6 0.05
1.19 6 0.08*
.60
6.34 6 0.30
16.3 6 2.9*
.60
2.12 6 0.07
4.56 6 0.30*
.60
CYP1A2
+
2
+
2
+
2
+
.60
.60
.60
CYP1B1.1
CYP1B1.3
CYP1B1.4
0.09 6 0.00
0.15 6 0.01*
0.10 6 0.01
0.11 6 0.01
0.16 6 0.03
0.19 6 0.02
34.4 6 7.7
41.6 6 2.7
n.d.
2.75 6 0.30
5.09 6 0.29*
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d., Not determined.
*Values significantly different from the values calculated from the determinations without 10-minute preincubation.

Influence of Oxidation on Berberine-Mediated CYP1 Inhibition
1103
berberine, palmatine, and jatrorrhizine was decreased by the and thalifendine (10-demethylation), whereas berberrubine was not
preoxidation (Table 1). The inhibition of CYP1B1 by berberine and detected (Fig. 1A). The formation of demethyleneberberine and
jatrorrhizine was also decreased by the preoxidation. The IC50 values thalifendine has been further identified by liquid chromatography-
were increased by 51–157%, probably due to the metabolism of mass spectrometry analysis (Fig. 1B). In CYP1A1-catalyzed
protoberberines forming metabolites with fewer inhibitory effects than berberine demethylenation and 10-demethylation, the plots of
the parent chemicals. With the preoxidation for the same time period velocity versus berberine concentration were sigmoidal (Fig. 2,
(
10 minutes), berberine caused more potent inhibition of CYP1B1 upper panel). The Lineweaver-Burk plots showed a nonlinear pattern
than palmatine and jatrorrhizine did. Even with preoxidation, and the Eadie-Hofstee plot showed a convex curve, indicating
CYP1A2-catalyzed EROD activity was still resistant to the inhibition positive cooperativity. Nonlinear regression analysis of the plot of
by protoberberines. Among CYP1 enzymes, berberine preferentially velocity versus berberine concentration generated kinetic parameters
inhibited CYP1B1 activity in the enzyme assay with and without the for demethylenation and 10-demethylation with an r value of 0.99
NADPH-fortified preincubation of P450 with berberine (Table 1). (Table 2). In CYP1B1-catalyzed demethylenation, the velocity of
Unlike berberine, palmatine and jatrorrhizine with the methylenedioxy CYP1B1 decreased at berberine concentrations higher than 7.5 mM,
ring opening did not show preference in CYP1B1 inhibition. Because suggesting a substrate inhibition pattern (Fig. 2, bottom panel).
berberine caused potent inhibition of CYP1B1, the influence of Nonlinear regression analysis according to substrate inhibition
preoxidation on the inhibition of CYP1B1 variants by berberine was generated the parameters with r = 0.99 (Table 2). The plot of
further studied. CYP1B1.3 (10.3 6 1.5 nmol/min/nmol P450) and CYP1B1-catalyzed 10-demethylation velocity versus berberine
CYP1B1.4 (16.7 6 2.1 nmol/min/nmol P450) had EROD activities concentration was hyperbolic, and nonlinear regression analysis
similar to CYP1B1.1. The preoxidation with berberine did not cause following the Michaelis-Meten equation generated the parameters
obvious decreases in the IC50 values for the inhibition of CYP1B1.3 with r = 0.99. The maximal demethylenation and 10-demethylation
and CYP1B1.4 activities (Table 1).
Berberine Demethylenation and 10-Demethylation Catalyzed CYP1A1, respectively. The K
activities (Vmax) of CYP1B1 were only 5% and 8% of those of
values of demethylenation and
m
by CYP1 Enzymes. HPLC analysis revealed that CYP1A1-catalyzed 10-demethylation by CYP1B1 were 4% and 48% of those by CYP1A1,
berberine oxidation generated demethyleneberberine (demethylenation) respectively.
Fig. 1. The chromatrograms of HPLC (A) and liquid
chromatography-mass spectrometry (B) analyses of ber-
berine oxidation products generated by CYP1A1,
CYP1A2, and CYP1B1. In a 0.5 ml assay without
NADPH, the incubation mixture containing 2.7 mM
demethyleneberberine (1), 2.4 mM thalifendine (2),
2
.4 mM berberrubine (3), and 4.8 mM berberine (4) was
prepared following the assay method and used as the
external standards for peak identification and quantifica-
tion. The chromatograms of P450-generated berberine
oxidation products show the chromatograms obtained
from the reaction using 20 nM P450 and 10 mM
berberine. The berberine oxidation products generated
by CYP1A1 were further analyzed by liquid chromatography-
mass spectrometry. Berberine oxidation was performed
using 50 nM P450 and 10 mM berberine. After incubation
for 30 minutes, the reaction product was subjected to
liquid chromatography-mass spectrometry analysis. The
blank incubation was carried out in the absence of
NADPH. A selected ion mode was used to find the
ionized berberine (m/z = 336–337), demethyleneberberine
(m/z = 324–325), thalifendine (m/z = 322–323), and
jatrorrhizine (m/z = 338–339).

1
104
Lo et al.
The dissociation constant (K ) was calculated by determining the
d
fluorescence quenching, presumably reflecting the binding of berberine
to P450. The plots of fluorescence quenching versus berberine
concentrations were hyperbolic (plot not shown). The apparent K
values for the interaction of berberine with CYP1B1 variants were
4.6–15.2 mM (Table 4). The differences between the binding
d
1
affinities of berberine to CYP1B1 variants were minimal. However,
the K value for the interaction of berberine with CYP1A2 was 18.2 6
d
1
.3 mM, which was 20–25% higher than the values for CYP1B1
variants.
The Spectral Changes Due to the Binding of Berberine to
CYP1A2 and CYP1B1. In contrast to the inhibition resistance and
high oxidation rate of CYP1A2 when a high concentration of
berberine was present (Li et al., 2011), CYP1B1 was potently
inhibited by berberine and had a low berberine oxidation rate. To
examine the spin state changes in hemoproteins of CYP1A2 and
CYP1B1.1 (L432), spectral changes due to the binding of berberine to
P450 were studied. The absorbance spectrum as seen with berberine
was analyzed first. Berberine had a maximal absorbance at 420 nm,
and this spectral property interfered with the binding spectral analysis
Fig. 2. Kinetic analyses of berberine demethylenation and 10-demethylation by (Fig. 3A). By using a set of tandem cuvettes to measure the binding
human CYP1A1 and CYP1B1. The plots of velocity (v) versus berberine spectra, spectral analyses revealed that CYP1A2 and CYP1B1
concentration (S) of CYP1-catalyzed berberine demethylenation, the 1/v versus
generated a difference spectrum with the maximal absorbance at 372
1
/S (Lineweaver-Burk plot), and v versus v/S (Eadie-Hofstee plot) are shown in the
and 370 nm, respectively (Fig. 3, B and C). The spectral troughs were
right, middle, and left panels, respectively. Lines show the best fit as determined by
nonlinear least squares regression according to the cooperative oxidation and at 425 and 420 nm for the binding to CYP1A2 and CYP1B1,
substrate inhibition for CYP1A1 and CYP1B1, respectively. Data represent the
mean 6 S.E. of three to four determinations.
respectively. Unexpectedly, the binding of berberine to CYP1A2 and
CYP1B1 caused the same type of spectral change, and the binding
spectra showed the characteristics of type I spectrum. However,
another absorbance peak was observed at ;470 nm, which was not
present in a typical type I spectrum. The dissociation constant (K
Inhibition of the 7-Eoxyresorufin O-Deethylation Activity of
CYP1 by Demethyleneberberine, Thalifendine, and Berberrubine.
In the assays of CYP1 activity, the presence of demethyleneberberine,
thalifendine, and berberrubine all resulted in a preferential inhibition
of CYP1B1 with IC50 values between 8.0 and 26.7 mM (Table 3).
Thalifendine inhibited CYP1A1 activity with an IC50 value of 30.4 6
s
)
values were calculated from the absorbance difference of the ΔA372–425 nm
and ΔA370–420 nm for the binding to CYP1A2 and CYP1B1, respectively
s
(Fig. 3, B and C). The K values for the binding of berberine to
CYP1A2 and CYP1B1 were 10.1 6 2.5 and 7.4 6 1.3 mM,
respectively. CYP1B1 had a spectral dissociation constant 27% lower
than the value of CYP1A2.
1
.2 mM, whereas demethyleneberberine and berberrubine had IC50
values higher than 60 mM. Compared with the results with the parent
compound berberine (Table 1), inhibition of CYP1A1/CYP1B1 by
metabolites of berberine was relatively weak. The EROD activity of
CYP1A2 was not inhibited by demethyleneberberine, thalifendine,
and berberrubine at the concentration up to 60 mM.
Discussion
The methylenedioxy phenyl derivatives have been reported to
Quenching of the Fluorescence of CYP1A2 and CYP1B1 generate a complex of carbene metabolite intermediate and P450,
Variants with Berberine. To examine the interaction of CYP1A2 resulting in a potent and irreversible inhibition after preoxidation
and CYP1B1 variants with berberine, the quenching of tryptophan (Murray, 2000). However, in human liver microsomes, results of
fluorescence of the apoprotein of P450 by berberine was determined. spectral analysis indicated that no P450-MI complex was formed after
CYP1B1.3 and CYP1B1.4 carry mutations at positions away from the NADPH-fortified microsomal incubation with berberine (Chatterjee
berberine-binding site in the apoprotein of P450. Upon excitation at and Franklin, 2003). Our findings revealed that the IC50 values were
2
95 nm, bacterial membranes expressing recombinant CYP1B1 increased when berberine was preoxidized by CYP1A1/1B1, in-
variants had intrinsic fluorescence emission peaks at 336–343 nm. dicating that berberine was not a mechanism-based inhibitor of
TABLE 2
Kinetic parameters of berberine demethylenation and 10-demethylation by CYP1A1 and CYP1B1
Kinetic parameters were determined by the nonlinear regression analysis according to each kinetic pattern, as described in Materials and
Methods. Data represent the values and the estimated variances (denoted by 6).
P450
CYP1A1
Type
n
K
m
(S50), mM
V
max, nmol/min/nmol P450
i
K , mM
Demethylenation
0-Demethylation Positive cooperativity 4.1 6 0.6 6.89 6 0.27
CYP1B1
Demethylenation
0-Demethylation Hyperbolic pattern
Positive cooperativity 4.6 6 0.2 4.87 6 0.05
21.3 6 0.17
6.63 6 2.46
—
—
1
Substrate inhibition
—
0.18 6 0.07
1.03 6 0.10
0.51 6 0.04
19.0 6 7.6
—
1
0.8 6 0.1 3.32 6 0.72
—, No available value for this kinetic pattern.

Influence of Oxidation on Berberine-Mediated CYP1 Inhibition
1105
TABLE 3
more freely than the methylenedioxy ring (Supplemental Fig. 1, A and
B). Among the derivatives and metabolites of berberine, palmatine
caused the least inhibition of CYP1B1 activity. Using the ASP scoring
Inhibition of the 7-ethoxyresorufin O-deethylation activity of CYP1 by
demethyleneberberine, thalifendine, and berberrubine
Inhibition of CYP1-catalyzed 7-ethoxyresorufin O-deethylation activity was studied in the
assays without NADPH-fortified preincubation. IC50 value was calculated, as described in
Materials and Methods. Data show the estimates and estimates of variance (denoted by 6)
determined from the curve fit analysis of individual sets of data.
(
Korb et al., 2009) function of the ligand-docking software, the
docking of palmatine to the putative binding site of CYP1B1 had the
lowest score of 26.4. The lowest score of palmatine was consistent
with its poor CYP1B1-inhibitory effect. The docking scores of
berberine, jatrorrhizin, demethyleneberberine, thalifendine, and ber-
berrubine were 38.2, 38.6, 37.8, 37.7, and 36.6, respectively. The
docking scores were in the trend consistent with their IC50 values for
CYP1B1 inhibition (berberine , jatrorrhizin , demethyleneberberine
IC50, mM
P450
Demethyleneberberine
Thalifendine
Berberrubine
CYP1A1
CYP1A2
CYP1B1.1
.60
.60
8.0 6 1.2
30.4 6 1.2
.60
10.0 6 0.9
.60
.60
26.7 6 1.4
ꢀ
thalifendine , berberrubine). The presence of a bump or clash
between heme of the P450 and the methoxyl substitution at C3 was
predicted by docking monitoring (Supplemental Fig. 1D). This
CYP1A1/1B1. Thus, carbene intermediate may not be generated interaction may be associated with the relatively weak inhibition by
during the demethylenation of berberine. Berberine oxidation by palmatine. Thus, besides the important hydrogen bond formation of
CYP1A1 and CYP1B1 generated demethyleneberberine and thalifen- P450 with methoxyl substitutes at C9 and C10, the methylenedioxyl
dine. Demethyleneberberine and thalifendine inhibited CYP1B1 with moiety of berberine provides the potential hydrophobic interaction
a potency less than berberine did. The increased IC50 values after (Lo et al., 2013) and a nonfree-rotating planar structure, resulting in the
preoxidation of inhibitors can be attributed at least in part to the potent inhibition of CYP1B1 by berberine. These criteria can be
oxidation forming inactivated metabolites. Palmatine and jatrorrhizine important in the further drug development of a CYP1B1 selective
do not carry the methylenedioxy moiety. Among the primary hepatic inhibitor for the potential hypotensive and chemopreventive effects
P450 forms, CYP1A2 showed the highest demethylation activity (Shimada and Fujii-Kuriyama, 2004; Jennings et al., 2010).
toward jatrorrhizine at a high concentration of 500 mM (Zhou et al.,
In a study of supersomes, CYP1A2-catalyzed berberine demethyle-
2
013). However, our findings revealed that jatrorrhizine at a concen- nation had Km and Vmax of 126 mM and 4.8 nmol/min/nmol P450,
tration up to 60 mM could not compete with a substrate 7- respectively (Li et al., 2011). The 10-demethylation had Km and Vmax
ethoxyresorufin to cause CYP1A2 inhibition. Like berberine, the values of 100 mM and 5.4 nmol/min/nmol P450, respectively.
preoxidation reduced the CYP1A1 and CYP1B1 inhibition by Compared with the velocities of CYP1A1 and CYP1A2, the Vmax of
jatrorrhizine and CYP1A1 inhibition by palmatine. The CYP1B1 CYP1B1-mediated berberine oxidation was very low. Due to the
inhibition by palmatine was less affected by the preoxidation. opposite position of methylenedioxyl and the 10-methoxyl groups of
Palmatine and jatrorrhizine were not a mechanism-based inhibitor of berberine, the generation of demethyleneberberine and thalifendine
CYP1A1/1B1, either.
requires two binding orientations of berberine. Results of docking
The ratios (in parentheses) of IC50 values for CYP1B1 to CYP1A1 analysis consistently supported the orientation preference in the
inhibition by the derivatives without the methylenedioxy ring were in demethylenation by CYP1B1. Although CYP1B1 exhibited a low Km
the order of palmatine (5.4) . jatrorrhizine (1.3) . demethylene- value for berberine oxidation and was potently inhibited by berberine,
berberine (, 0.13). The number (in parentheses) of methoxyl berberine was a poorly oxidized substrate of CYP1B1. CYP1A1 had
substitute added was in the order of palmatine (2) . jatrorrhizine a high Vmax and a Km value within a range between those of CYP1B1
(
1) . demethyleneberberine (0). A greater methoxyl addition to the and CYP1A2. Unexpectedly, the Vmax value of CYP1A1 was greater
dihydroisoquinolino moiety seems to diminish the preference in than that of CYP2D6 reported by Li et al. (2011). Although the
CYP1B1 inhibition. With the methylenedioxyl substitute at C2 and difference in the alternative expression systems can be an influencing
C3, the monodemethylation at 9-(thalifendine) and 10-(berberubin) factor of the Vmax value, CYP2D6 and CYP1A1 may play crucial roles
positions suppressed the CYP1A1 and CYP1B1 inhibitory effects, but in the berberine oxidation in respective high-expression tissues due to
remained the CYP1B1 preferential inhibition. Interestingly, none of their differential tissue distribution. Although CYP1A2 exhibited
the protoberberines or berberine metabolites inhibited CYP1A2. a relatively high Km value in berberine oxidation and showed
Based on our previous report of the docking of berberine to the resistance to the inhibition by berberine (Li et al., 2011; Lo et al.,
putative CYP1B1 active site (Lo et al., 2013), the binding of 2013), the maximal oxidation velocity by CYP1A2 was about five-
derivatives/metabolites of berberine to CYP1B1 was predicted. fold greater than that of CYP1B1. However, the intrinsic clearance
Results of the best docking showed that the two hydroxyl side chains (Kcat/K ) of berberine by CYP1A2 was relatively low. Thus, upon
m
at C2 and C3 of demethyleneberberine were expected to be rotated a high-exposure concentration of berberine in the liver, CYP1A2 may
appear to efficiently metabolize berberine. However, upon a low-
exposure concentration of berberine, CYP1A1- and CYP1B1-
TABLE 4
catalyzed oxidations may become important, especially in the
extrahepatic tissues. The plots of velocity versus berberine concen-
tration of CYP1A1-catalyzed berberine demethylenation and
10-demethylation activities showed a sigmoidal pattern, and the
Eadie-Hofstee plots of both activities showed a convex property,
suggesting that there may be two substrate binding sites or that the
presence of a substrate caused a conformational change of CYP1A1.
The binding of the first substrate may assist the binding of a second
substrate to CYP1A1. However, to date, there was no report
supporting that there were two substrate binding sites in CYP1A1.
In CYP1B1-catalyzed berberine demethylenation, the activity
The fluorescence quenching of CYP1A2 and CYP1B1 variants by the binding of
berberine in the recombinant enzyme systems
Data show the estimates and estimates of variance (denoted by 6) determined from the curve
fit analysis of individual sets of data.
P450
d
K , mM
a
CYP1B1.1
CYP1B1.3
CYP1B1.4
CYP1A2
14.7 6 1.5
14.6 6 1.8
15.2 6 1.7
18.2 6 1.3
a
Data were obtained from the previous study reported by Lo et al. (2013).

1
106
Lo et al.
Fig. 3. The spectral difference due to interactions of berberine
with CYP1A2 and CYP1B1. (A) shows the absorbance spectrum
of berberine. To prevent the interference of the absorbance
spectrum of berberine, tandem cuvettes were used for the
detection of the difference spectrum resulting from the interaction
of berberine with P450. The left panels of (B) and (C) show the
spectra recorded at room temperature after the addition of
increasing concentrations of berberine to diluted bacterial
membrane fractions containing 0.5 mM CYP1A2 and CYP1B1,
respectively. The same amount of berberine was added to the
buffer chamber in the reference cuvette. The right panels of (B)
and (C) show the plot of absorbance versus berberine concentra-
tion, and the solid line shows the best fit estimated from the curve
fitting.
decreased with the increased berberine concentration. Together with resistant CYP1A2 and sensitive CYP1B1 elicited the same pattern of
the potent inhibition of CYP1B1-catalyzed EROD activity by berberine the type I spectrum. The type I spectrum indicated that the binding of
(Lo et al., 2013), these results reveal that the second substrate binds to the berberine to CYP1A2 and CYP1B1 caused the shift in equilibrium to
inhibitor site, which is nonproductive. Consistent with the results reported a high-spin hemoprotein and that berberine tends to be the substrate of
by Li et al. (2011), our determination of CYP1A2-catalyzed berberine CYP1A2 and CYP1B1. Compared with the 20–27% differences in the
oxidation showed the hyperbolic property of Michaelis-Menten kinetics dissociation constants (K , K ) from the binding analysis, the difference
d
s
(
data not shown). These results demonstrate the differential kinetic in K
properties of berberine oxidation by CYP1 isoforms, which can be crucial large, especially in the demethylenation. The big difference in K
in the assessment of berberine oxidation in vivo. be attributed to factors beyond the rate constants describing berberine
m
values of berberine oxidation by CYP1B1 and CYP1A2 was
m
may
Due to the difference of CYP1A2 and CYP1B1 being substrate and binding, in which the formation of activated Michaelis complex and
inhibitor, the interactions of berberine with heme and the apoprotein of product release may be involved (Yun et al., 2000).
CYP1A2/1B1 were analyzed with regard to the binding spectrum and
In conclusion, our findings reveal that berberine is not a mechanism-
fluorescence quenching, respectively. As a methylenedioxy phenyl based inhibitor of human CYP1 and it is poorly oxidized by CYP1B1.
moiety-carrying natural product, gomisin caused a mechanism-based Among CYP1 isoforms, CYP1A1 had the highest berberine oxidation
inhibition of CYP3A4 and showed a marker type III binding spectrum activity comparable to that of hepatic CYP2D6. CYP1A1-mediated
with the characteristic absorbance peaks at 455 and 430 nm (Iwata oxidation can be important for the biologic effect of berberine, es-
et al., 2004). However, the amine oxidation of methylenedioxyme- pecially in extrahepatic tissues. None of the berberine metabolites
thamphetamine caused a mechanism-based inhibition of CYP2D and caused CYP1A1 and CYP1B1 inhibition as potently as berberine did,
showed the type I binding spectrum, in which the peak and through of indicating that berberine-mediated inhibition can be eliminated through
the spectrum appeared at wavelengths of about 390 and 420 nm, metabolism.
respectively (Delaforge et al., 1999). Structurally diverse methylene-
dioxyl derivatives caused differential binding spectra. Berberine was
Acknowledgments
not a mechanism-based inhibitor of CYP1B1, and it appeared to be an
We sincerely appreciate Dr. F. Peter Guengerich (Vanderbilt University,
inhibitor rather than an efficiently oxidized substrate. A ligand
Nashville, TN) for generously providing the expression constructs of human
showing the type II binding spectrum was suggested to be an inhibitor
P450s.
and exhibited low turnover rates (Chiba et al., 2001). The type II
binding spectrum was suggested to result from the potent coordination
to the heme, which leads to the shift of hemoprotein to a low-spin
Authorship Contributions
Participated in research design: Ueng.
dominant form. This spin state change may increase the redox
potential and lead to the relatively low oxidation rate of the ligand.
Both the spectral change due to the binding of berberine to inhibition-
Conducted experiments: Lo, Shen, Chang, Huang, Wu, Ueng.
Performed data analysis: Lo, Shen, Chang, Tsai, Huang, Wu, Ueng.
Wrote or contributed to the writing of the manuscript: Ueng, Tsai.

Influence of Oxidation on Berberine-Mediated CYP1 Inhibition
1107
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