Inhibition of Human CYP2B6-Catalyzed Bupropion Hydroxylation
by Ginkgo biloba Extract: Effect of Terpene Trilactones
Aik Jiang Lau and Thomas K. H. Chang
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
Received April 18, 2009; accepted May 28, 2009
Cytochrome P450 2B6 (CYP2B6) is expressed predominantly in
human liver. It catalyzes the oxidative biotransformation of various
drugs, including bupropion, which is an antidepressant and a to-
bacco use cessation agent. Serious adverse effects of high dos-
ages of bupropion have been reported, including the onset of
seizure. As Ginkgo biloba extract may be consumed with bupro-
pion or another CYP2B6 drug substrate, potential exists for an
herb-drug interaction. Therefore, we investigated the effect of G.
biloba extract and some of its chemical constituents (terpene
trilactones and flavonols) on the in vitro catalytic activity of
CYP2B6 as assessed by the bupropion hydroxylation assay with
recombinant enzyme and hepatic microsomes. The amount of
hydroxybupropion was quantified by a novel and validated ultra-
performance liquid chromatography/mass spectrometry method.
Enzyme kinetic analysis indicated that G. biloba extract competi-
tively inhibited hepatic microsomal CYP2B6-catalyzed bupropion
hydroxylation (apparent Kiwas 162 ? 14 ?g/ml). Bilobalide and
ginkgolides A, B, C, and J were not responsible for the inhibition of
CYP2B6 catalytic activity by the extract. Whereas the 3-O-glu-
coside and 3-O-rutinoside of quercetin, kaempferol, and isorham-
netin had no effect, the corresponding aglycones (10 and 50 ?g/ml)
decreased hepatic microsomal bupropion hydroxylation. The inhi-
bition of CYP2B6 by kaempferol was competitive (apparent Kiwas
10 ? 1 ?g/ml). In summary, G. biloba extract and its flavonol
aglycones are naturally occurring inhibitors of in vitro CYP2B6
catalytic activity and bupropion hydroxylation. Future studies are
needed to investigate whether G. biloba extract interacts in vivo
with bupropion or other clinically important CYP2B6 drug sub-
Cytochrome P450 2B6 (CYP2B6) is expressed mainly in human
liver, although this enzyme has also been detected in various extra-
hepatic tissues (Gervot et al., 1999). Considerable variability exists
not only in hepatic expression of CYP2B6 mRNA (280-fold) (Chang
et al., 2003) and protein (?288-fold) (Hesse et al., 2004) but also
CYP2B6 enzyme activity (80-fold) (Faucette et al., 2000). The basis
for the interindividual variability may relate to pharmacogenetics
(Hofmann et al., 2008) and the fact that this enzyme is subject to
induction by various drugs and other chemicals in a mechanism that
involves transcription factors such as the constitutive androstane
receptor (Sueyoshi et al., 1999), which also exhibits large interindi-
vidual differences (240-fold) in hepatic expression (Chang et al.,
2003). The magnitude of CYP2B6 catalytic activity may also be
altered as a result of enzyme inhibition by various synthetic drugs
(Turpeinen et al., 2004; Walsky et al., 2006); naturally occurring
compounds, including phenethyl isothiocyanate (Nakajima et al.,
2001), ?-viniferin (Piver et al., 2003), and citral (Kim et al., 2008);
and herbal supplements, such as Woohwangcheongsimwon (Kim et
al., 2008), Andrographis paniculata extract (Pekthong et al., 2008),
and curcuminoid extract (Volak et al., 2008). Important CYP2B6 drug
substrates include the alkylating anticancer prodrug cyclophospha-
mide (Chang et al., 1993) and the tobacco use cessation agent bupro-
pion (Faucette et al., 2000; Hesse et al., 2000). The biotransformation
of bupropion to hydroxybupropion is catalyzed predominantly by
CYP2B6 in human liver (Faucette et al., 2000; Hesse et al., 2000). As
a result, hepatic microsomal bupropion hydroxylation is used as an
enzyme-selective catalytic marker for CYP2B6 in human liver (Tur-
peinen et al., 2004; Walsky et al., 2006).
Bupropion inhibits dopamine and noradrenaline reuptake, and it
acts as an antagonist of neuronal nicotinic acetylcholine receptor
(Dwoskin et al., 2006). This drug was available initially as an anti-
depressant. It is now widely used as a non-nicotine drug for smoking
cessation. However, the use of high dosages of bupropion is associ-
ated with serious adverse effects (e.g., seizure), particularly among
susceptible individuals (Beyens et al., 2008). In humans, bupropion
undergoes extensive hepatic biotransformation to form hydroxybu-
propion, threohydrobupropion, and erythrohydrobupropion, which are
pharmacologically active metabolites (Dwoskin et al., 2006). Given
that inhibition of bupropion biotransformation leads to greater plasma
drug concentrations and the potential for the onset of serious adverse
effects, it is therefore important to identify factors (e.g., concomitant
This work was supported by the Canadian Institutes of Health Research [Grant
informationcan be foundat
ABBREVIATIONS: UPLC, ultraperformance liquid chromatography; MS, mass spectrometry; DMSO, dimethylsulfoxide; LLOQ, lower limit of
quantification; QC, quality control.
DRUG METABOLISM AND DISPOSITION
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
DMD 37:1931–1937, 2009
Vol. 37, No. 9
Printed in U.S.A.
at ASPET Journals on October 27, 2015
administration of a drug or herb) that may interfere with the biotrans-
formation and clearance of bupropion.
Ginkgo biloba is an herbal medicine that contains terpene trilac-
tones (?6% w/w), such as bilobalide, ginkgolide A, ginkgolide B,
ginkgolide C, and ginkgolide J, and flavonols (?24% w/w), such as
the aglycone and glycosides of kaempferol, quercetin, and isorham-
netin (van Beek and Montoro, 2009). Various commercial prepara-
tions of G. biloba extract are available, and a common use of these
products by consumers is to improve cognitive performance (e.g., in
dementia) (Ramassamy et al., 2007). G. biloba is one of the most
popular herbal medicines. In a recent survey, 20% of the 4202
prescription and nonvitamin dietary supplement users reported con-
sumption of G. biloba (Gardiner et al., 2006). Given that G. biloba
may be ingested with bupropion or another CYP2B6 drug substrate,
potential exists for an herb-drug interaction, which may lead to
adverse drug effects, especially among susceptible individuals, such
as those with a chronic illness or compromised hepatic or renal
function. Currently, it is not known whether G. biloba extract inter-
feres with the biotransformation of bupropion or other CYP2B6
The current study was designed to 1) investigate in detail the effect
of G. biloba extract on bupropion hydroxylation catalyzed by recom-
binant CYP2B6 and human hepatic microsomes, as analyzed by a new
and validated ultraperformance liquid chromatography (UPLC)/tan-
dem mass spectrometry (MS) method and evaluated by enzyme ki-
netic analysis; 2) ascertain the role of bilobalide, ginkgolide A,
ginkgolide B, ginkgolide C, and ginkgolide J in the observed effect of
the extract; and 3) compare the effect of the aglycone, 3-O-glucoside
(a monoglycoside), and 3-O-rutinoside (a diglycoside) of kaempferol,
quercetin, and isorhamnetin on hepatic microsomal CYP2B6-medi-
ated bupropion hydroxylation. Our results show that G. biloba extract
and the aglycones of kaempferol, quercetin, and isorhamnetin are
naturally occurring inhibitors of CYP2B6 catalytic activity and bu-
propion hydroxylation in vitro.
Materials and Methods
G. biloba Extract. G. biloba extract was provided as dry powder by Indena
S.A. (Milan, Italy). Shown in Table 1 are levels of individual terpene trilac-
tones and flavonols in each of the five lots of G. biloba extract used in the
Chemicals. Bilobalide was provided by Indena S.A. Ginkgolide A, ginkgol-
ide B, and ginkgolide C were purchased from LKT Labs (St. Paul, MN), and
ginkgolide J was from ChromaDex (Irvine, CA). Kaempferol, isorhamnetin,
quercetin 3-O-glucoside, kaempferol 3-O-glucoside, isorhamnetin 3-O-glu-
coside, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamnetin
3-O-rutinoside were bought from Indofine Chemicals (Hillsborough, NJ).
Quercetin dihydrate, bupropion hydrochloride (?98% purity), triprolidine
hydrochloride (?99% purity), NADPH, dimethylsulfoxide (DMSO), and for-
mic acid (LC/MS grade) were obtained from Sigma-Aldrich (St. Louis, MO).
Authentic hydroxybupropion metabolite standard (95.2% purity) was pur-
chased from Toronto Research Chemicals Inc. (North York, ON, Canada). All
the other commercially available chemicals were of analytical or high-perfor-
mance liquid chromatographic grade.
Human Hepatic Microsomes and Recombinant CYP2B6 Enzyme. Hu-
man hepatic microsomes (lot 41207 pooled from 19 individual donors and lot
18888 pooled from 24 individual donors), microsomes from baculovirus-
infected insect cells (Supersomes) that coexpressed CYP2B6 and NADPH/
cytochrome P450 reductase, and control insect cell microsomes were pur-
chased from BD Gentest (Woburn, MA).
Bupropion Hydroxylation Assay. Unless specified otherwise, each stan-
dard 200-?l incubation mixture contained potassium phosphate buffer (50
mM, pH 7.4), EDTA (1 mM), magnesium chloride (3 mM), human hepatic
microsomes (100 ?g of protein) or recombinant CYP2B6 (5 pmol), NADPH
(1 mM), and bupropion (30 ?M in incubations containing recombinant
CYP2B6 and 50 ?M in incubations containing human hepatic microsomes).
Each incubation mixture was prewarmed for 2 min at 37°C in a shaking water
bath. Enzymatic reaction was initiated by the addition of NADPH and termi-
nated 15 min (recombinant CYP2B6) or 30 min (human hepatic microsomes)
later by the addition of 100 ?l of ice-cold acetonitrile containing triprolidine (1
?M final concentration; internal standard). Each sample was mixed on a vortex
and then centrifuged at 8000g for 10 min. Supernatant was transferred to an
autosampler vial or 96-well plate for metabolite analysis by UPLC/MS/MS. To
construct calibration curves, authentic hydroxybupropion (0.2–50 ?M) was
freshly prepared and added to the complete incubation mixture but with
heat-inactivated enzymes (inactivated at 65°C for 20 min) and processed as
described above. Enzyme kinetic analysis of bupropion hydroxylation was
performed at substrate concentrations from 10 to 500 ?M. The values of Vmax
and apparent Kmwere determined by nonlinear regression analysis of the
metabolite formation-substrate concentration data using the equation for the
one-component Michaelis-Menten model (Enzyme Kinetics Module, version
1.1; SPSS Inc., Chicago, IL).
Enzyme Inhibition Experiments. G. biloba extract, a terpene trilactone, a
flavonol, or the corresponding vehicle (assay buffer for G. biloba extract and
DMSO for the individual compounds) was added to the standard incubation
mixture as described in each figure legend. Unless specified otherwise, the
final concentration of DMSO was 0.1% v/v. A previous study showed that
DMSO concentrations of ?0.2% did not affect the enzyme kinetics of
CYP2B6-catalyzed bupropion hydroxylation (Vuppugalla et al., 2007). To
characterize the enzyme kinetics of CYP2B6 inhibition, multiple concentra-
tions of the inhibitor (as specified in the figure legends) and bupropion (25, 50,
100, and 200 ?M) were used. The apparent Kivalues and the mode of
inhibition were determined by nonlinear regression analysis of the metabolite
formation data collected at various substrate and inhibitor concentrations using
equations for competitive, noncompetitive, mixed, and uncompetitive inhibi-
tion (Enzyme Kinetics Module, version 1.1; SPSS Inc.). The Akaike informa-
tion criterion was used as a measure of goodness of fit. The mode of inhibition
was verified by visual inspection of Lineweaver-Burk plots and Dixon plots of
the enzyme kinetic data.
Quantification of Hydroxybupropion by UPLC/MS/MS. The amount of
hydroxybupropion was quantified by an UPLC/MS/MS method. UPLC was
performed using a Waters (Milford, MA) ACQUITY UPLC system equipped
with a binary solvent manager and an autosampler. Chromatography was
performed on a Waters ACQUITY UPLC BEH C18column (100 ? 2.1 mm
i.d., 1.7 ?m). The mobile phases were 0.1% formic acid in water (A) and 0.1%
formic acid in methanol (B). The elution conditions were optimized as follows:
isocratic at 2% B (0.0–1.5 min), linear gradient from 2% to 98% B (1.5–1.6
Content of terpene trilactones and flavonols in various lots of G. biloba extract
The content of terpene trilactones and flavonols were determined by gas chromatography
(Indena S.A.) and LC/MS (ChromaDex, Inc., Irvine, CA), respectively.
Content in G. biloba Extract
Lot ALot BLot CLot DLot E
Total terpene trilactones
Quercetin (sum of aglycone and
Kaempferol (sum of aglycone
Isorhamnetin (sum of aglycone
11.2 6.3N.A.N.A. N.A.
N.A., not available.
LAU AND CHANG
at ASPET Journals on October 27, 2015
min), isocratic at 98% B (1.6–4.0 min), linear gradient from 98% to 2% B
(4.0–4.1 min), and isocratic at 2% B (4.1–6.0 min). The total run time was 6
min. Column temperature was set at 30°C. The flow rate was 0.2 ml/min, and
the injection volume was 2 ?l. UPLC effluent was introduced directly (without
splitting) into the mass spectrometer interface from 2.5 to 4.0 min.
MS was performed using a Micromass Quattro Premier triple-quadrupole
mass spectrometer (Waters) with a Z-spray electrospray ion source. The mass
scale of the instrument was periodically calibrated using a solution of sodium
and cesium iodides. The mass spectrometer was operated in the positive
electrospray ionization mode. Nitrogen gas was used as the desolvation gas and
cone gas at a flow rate of 900 and 5 l/h, respectively. The mass spectrometer
tune parameters were optimized to give the highest product ion intensities
using full MS and daughter scans. The optimized parameters were as follows:
electrospray capillary, 3.5 kV; cone, 20 V; extractor, 3.0 V; radiofrequency
lens, 0.5 V; source temperature, 100°C; desolvation temperature, 300°C; mass
resolution (low mass 1, high mass 1, low mass 2, high mass 2), 15.0; ion
energy 1, 0.5; ion energy 2, 3.0; entrance, 0; exit, 3; and multiplier, 680 V.
MS/MS experiments were performed using nitrogen as the collision gas, and
the pressure in the collision cell was 4.3 ? 10?3mbar. The optimal collision
energy was determined to be 15 eV. Hydroxybupropion and triprolidine
(internal standard) were analyzed in the multiple reaction monitoring scan
mode using the transitions m/z 257.3 3 239.1 and m/z 280.5 3 208.8,
respectively (Fig. 1). The dwell time was 0.10 s, and the interscan delay was
0.10 s. The transitions were verified by daughter and parent scans. Data were
acquired and processed using MassLynx version 4.1 software with QuanLynx
application manager (Waters). A calibration curve was acquired before the
analysis of each set of samples. The amount of hydroxybupropion in each
sample was determined based on the calibration curve, which was constructed
using weighted (1/x2) linear least-squares regression analysis of the peak area
ratio of hydroxybupropion to triprolidine.
Validation of the UPLC/MS/MS Method. To determine the lower limit of
quantification (LLOQ), varying amounts of hydroxybupropion were added to
the standard incubation mixture for the bupropion hydroxylation assay, except
that heat-inactivated microsomes (100 ?g of protein) were used. The LLOQ
was established based on a signal/noise ratio of 5:1, a precision of ?20%, and
an accuracy of ?20%. To determine intraday and interday accuracy and
precision of the assay, quality control (QC) samples were prepared in six
replicates at low (0.5 ?M), mid (20 ?M), and high (80 ?M) concentrations of
authentic hydroxybupropion in the incubation mixture. The 18 QC samples
were prepared independently on three separate days and quantified using the
calibration curve constructed on each day. Accuracy was assessed based on the
percentage bias of the measured concentration relative to the nominal concen-
tration. Precision (% CV) was calculated by dividing the S.D. by the group
mean of each set of QC samples and multiplied by 100. Matrix interference
was investigated by comparing the MS response of authentic hydroxybupro-
pion (0.2 ?M) in the complete incubation mixture containing heat-inactivated
enzyme (human hepatic microsomes or recombinant CYP2B6) with that in the
complete incubation mixture without enzyme.
Statistical Analysis. Data were analyzed by one-way analysis of variance,
followed by the Student Newman-Keuls multiple comparison test where ap-
propriate (GraphPad Prism 3.0; GraphPad Software Inc., San Diego, CA). The
level of statistical significance was set a priori at p ? 0.05.
Analytical Method Development and Validation. A new UPLC/
MS/MS method was developed for the quantification of hydroxybu-
propion. The blank sample (incubations containing heat-inactivated
microsomes but without analyte) did not display peaks at the m/z
transition corresponding to hydroxybupropion or triprolidine (data not
shown), thereby showing specificity and lack of interference by the
matrix. The peak area ratio of the analyte (0.2 ?M) to the internal
standard in incubations containing heat-inactivated hepatic micro-
somes (0.091 ? 0.007, mean ? S.E.M.; n ? 4) or recombinant
CYP2B6 enzyme (0.095 ? 0.004; n ? 4) was not significantly
different (p ? 0.11) from that obtained in incubations without enzyme
(0.074 ? 0.008; n ? 4), indicating that the matrix did not interfere
with the magnitude of the MS response. The LLOQ for hydroxybu-
propion was 0.27 pmol. The calibration curve was linear from 0.2 to
100 ?M as assessed by the coefficient of determination (r2? 0.99)
and visual inspection of the regression line and residuals. The mea-
sured concentration of each of the standards was within 15% of the
nominal concentration. Determination of intraday (n ? 6) and inter-
day (n ? 3) accuracy of low, mid, and high analyte concentrations
showed a bias of ?10.2%, whereas the intraday and interday precision
was ?6.9% for low, mid, and high analyte concentrations (Table 2).
Optimization of the Bupropion Hydroxylation Assay. Initial
experiments were performed to optimize the conditions of the enzyme
assay. The bupropion hydroxylation assay was linear with respect to
the amount of enzyme (10–200 ?g of human hepatic microsomal
protein; 0.25–10 pmol of recombinant CYP2B6) and incubation time
(up to 20 min for recombinant CYP2B6 and 40 min for human hepatic
microsomes). All the subsequent bupropion hydroxylation assays
were performed with 100 ?g of microsomal protein (or 5 pmol of
recombinant CYP2B6) and incubation time of 20 min (recombinant
CYP2B6) or 30 min (human hepatic microsomes). Enzyme kinetic
analysis indicated that the apparent Kmvalues were 34 ? 5 ?M and
59 ? 1 ?M for bupropion hydroxylation catalyzed by recombinant
FIG. 1. Total ion chromatograms of hydroxybupropion and triprolidine (internal
standard). Pooled human hepatic microsomes (100 ?g of protein) were incubated
with bupropion (50 ?M) for 30 min at 37°C in 50 mM potassium phosphate buffer,
pH 7.4, containing 1 mM EDTA and 3 mM magnesium chloride. The internal
standard (1 ?M triprolidine in ice-cold acetonitrile) was added to terminate the
reaction. Shown are total ion chromatograms of hydroxybupropion and triprolidine
as analyzed by UPLC/MS/MS in the multiple reaction monitoring scan mode using
the transitions m/z 257.30 3 239.10 and m/z 280.50 3 208.80, respectively.
Intraday and interday accuracy and precision in the UPLC/MS/MS analysis of
Low, mid, and high concentrations of authentic hydroxybupropion were added to the
complete incubation mixture containing heat-inactivated microsomes and quantified by
UPLC/MS/MS. Intraday (n ? 6) and interday (n ? 3) accuracy and precision were
G. biloba AND CYP2B6 CATALYTIC ACTIVITY
at ASPET Journals on October 27, 2015
CYP2B6 and human hepatic microsomes, respectively, whereas the
Vmaxvalues were 17 ? 2 pmol/min/pmol CYP2B6 and 473 ? 57
pmol/min/mg protein when the assay was conducted with recombi-
nant CYP2B6 and human hepatic microsomes, respectively. These
values are comparable with those reported previously (Faucette et al.,
2000; Hesse et al., 2000; Walsky and Obach, 2004).
Effect of G. biloba Extract on CYP2B6 Catalytic Activity. To
determine the effect of G. biloba extract on the catalytic activity of
CYP2B6, the bupropion hydroxylation assay was performed on five
different lots of the extract (300 ?g/ml) obtained from the same
manufacturer. Each lot of the extract decreased bupropion hydroxy-
lation catalyzed by recombinant CYP2B6 (Fig. 2). The magnitude of
the inhibitory effect was similar among the five lots of the extract. In
all the subsequent experiments, lot A was used.
Dose-Response Relationship in CYP2B6 Inhibition by G. biloba
Extract. G. biloba extract (10, 100, 200, 300, 400, 600, or 800 ?g/ml)
or assay buffer (vehicle) was incubated with bupropion and recombi-
nant CYP2B6 or human hepatic microsomes. As shown in Fig. 3, a
similar dose-response relationship was obtained in the inhibition of
bupropion hydroxylation catalyzed by recombinant CYP2B6 and hu-
man hepatic microsomes. A log-linear decrease in bupropion hy-
droxylation activity was evident at extract concentrations of 100 to
Enzyme Kinetic Analysis of Inhibition of CYP2B6-Mediated
Hepatic Microsomal Bupropion Hydroxylation by G. biloba Ex-
tract. The bupropion hydroxylation assay was performed with vary-
ing concentrations of G. biloba extract (0, 200, 300, or 400 ?g/ml)
and bupropion (25, 50, 100, or 200 ?M). Shown in Fig. 4 is a
Lineweaver-Burk plot of the inhibition of hepatic microsomal
CYP2B6-mediated bupropion hydroxylation by G. biloba extract. As
determined by nonlinear regression analysis of the enzyme kinetic
data and judged by Akaike information criterion and graphical plots of
the enzyme kinetic data (Lineweaver-Burk plot and Dixon plot), the
mode of inhibition was best described as competitive. The apparent Ki
was 162 ? 14 ?g/ml.
Role of Terpene Trilactones in the Inhibition of CYP2B6-
Catalyzed Bupropion Hydroxylation Activity by G. biloba Ex-
tract. The next experiment was performed to determine whether a
terpene trilactone was responsible for the inhibition of hepatic micro-
somal CYP2B6 enzyme activity by G. biloba extract. Therefore, the
bupropion hydroxylation assay was performed with human hepatic
microsomes in the presence of ginkgolide A (5.4 ?g/ml), ginkgolide
B (1.8 ?g/ml), ginkgolide C (9 ?g/ml), ginkgolide J (3.6 ?g/ml),
bilobalide (17 ?g/ml), or a mixture of these five terpene trilactones.
The concentration of each of these individual chemicals was chosen to
reflect the level present in a selected concentration (600 ?g/ml) of the
extract (lot A, Table 1). The results showed that at the concentrations
indicated above, none of these chemicals, either individually or as a
mixture, decreased hepatic microsomal bupropion hydroxylation
when compared with the vehicle-treated control group (data not
shown). A lack of an effect by the terpene trilactones was also
obtained when the enzymatic incubations were performed with re-
combinant CYP2B6 enzyme (data not shown).
Effect of Flavonol Glycosides and Aglycones on CYP2B6 Cat-
alytic Activity. The flavonols in G. biloba extract are present primar-
ily as a mixture of monoglycosides, diglycosides, and more complex
FIG. 2. Effect of different lots of G. biloba extracts on bupropion hydroxylation
catalyzed by recombinant CYP2B6. The assay was conducted with recombinant
CYP2B6 (5 pmol), bupropion (30 ?M), and G. biloba extract (0 or 300 ?g/ml). The
mixture was incubated for 15 min at 37°C. The amount of hydroxybupropion was
quantified by UPLC/MS/MS as described under Materials and Methods. Results are
expressed as mean ? S.E.M. for five independent experiments. ?, significantly
different from the control group (p ? 0.05).
FIG. 3. Dose-response relationship on the inhibition of CYP2B6 catalytic activity
by G. biloba extract. Pooled human hepatic microsomes (100 ?g of protein) or
recombinant CYP2B6 (5 pmol) was incubated with bupropion (30 ?M for CYP2B6
and 50 ?M for hepatic microsomes) and G. biloba extract (0, 10, 100, 200, 300, 400,
600, or 800 ?g/ml) at 37°C. Enzymatic reaction was initiated with 1 mM NADPH
and terminated after 15 min (recombinant CYP2B6) or 30 min (hepatic micro-
somes). The amount of hydroxybupropion was quantified by UPLC/MS/MS as
described under Materials and Methods. Results are expressed as mean ? S.E.M.
for four independent experiments. Bupropion hydroxylation in the control group
was 8.8 ? 1.1 pmol/min/pmol CYP2B6 in incubations containing recombinant
CYP2B6 and 268 ? 48 pmol/min/mg protein in incubations containing human
FIG. 4. Lineweaver-Burk plot for the competitive inhibition of human hepatic
microsomal CYP2B6-mediated bupropion hydroxylation by G. biloba extract.
Pooled human hepatic microsomes (100 ?g of protein) were incubated with bupro-
pion (25, 50, 100, or 200 ?M) and G. biloba extract (0, 200, 300, or 400 ?g/ml) for
30 min at 37°C. The amount of hydroxybupropion was quantified by UPLC/MS/MS
as described under Materials and Methods. Results are expressed as mean ? S.E.M.
for four independent experiments.
LAU AND CHANG
at ASPET Journals on October 27, 2015
glycosides of kaempferol, quercetin, and isorhamnetin (van Beek and
Montoro, 2009). In the present study, the identity and levels of the
individual flavonol glycosides and aglycones in our G. biloba extracts
are not known (Table 1). Therefore, it was not possible to determine
which flavonol glycoside(s) or aglycone(s) was responsible for the
inhibition of CYP2B6 catalytic activity by the extract. However, as a
proof-of-principle experiment to determine whether a flavonol glyco-
side is capable of inhibiting CYP2B6 catalytic activity, the bupropion
hydroxylation assay was conducted with recombinant CYP2B6 and a
flavonol monoglycoside or diglycoside. The concentrations of the
monoglycosides [i.e., quercetin 3-O-glucoside (87 ?g/ml), kaempferol
3-O-glucoside (90 ?g/ml), and isorhamnetin 3-O-glucoside (18 ?g/
ml)] and diglycosides [i.e., quercetin 3-O-rutinoside (87 ?g/ml),
kaempferol 3-O-rutinoside (90 ?g/ml), and isorhamnetin 3-O-rutino-
side (18 ?g/ml)] were chosen to reflect the levels present in an
800-?g/ml concentration of G. biloba extract, with the assumption
that the glucoside or rutinoside accounted for the entire amount of
each flavonol for lot A (Table 1). However, even at those concentra-
tions, none of these flavonol glycosides affected CYP2B6 catalytic
activity (data not shown).
To determine whether flavonol aglycones are capable of inhibiting
CYP2B6 catalytic activity, we performed a dose-response experiment
to investigate the effect of quercetin, kaempferol, and isorhamnetin on
hepatic microsomal CYP2B6-mediated bupropion hydroxylation. As
shown in Fig. 5, each of these flavonol aglycones at concentrations up
to 2 ?g/ml had little or no effect on hepatic microsomal bupropion
hydroxylation, whereas decreases were evident at concentrations of 10
and 50 ?g/ml.
Enzyme Kinetic Analysis of Inhibition of Hepatic Microsomal
CYP2B6-Mediated Bupropion Hydroxylation by Kaempferol.
The bupropion hydroxylation assay was performed in incubations
containing kaempferol (0, 5, 10, or 20 ?g/ml), bupropion (25, 50, 100,
or 200 ?M), and human hepatic microsomes. Figure 6 is a Line-
weaver-Burk plot showing competitive inhibition of hepatic microso-
mal bupropion hydroxylation by kaempferol. The apparent Kiwas
10 ? 1 ?g/ml (35 ? 3 ?M). Enzyme kinetic experiment was not
performed with quercetin or isorhamnetin because of the limited
solubility of these chemicals at higher concentrations (?50 ?g/ml).
Relatively little scientific information is available on the effect of
herbal medicines and naturally occurring compounds on the catalytic
activity of CYP2B6, which plays an important role in the biotrans-
formation of specific drugs and other chemicals. In the present study,
we identified G. biloba extract as a novel inhibitor of CYP2B6
catalytic activity as assessed with recombinant enzymes and human
hepatic microsomes. The extract competitively inhibited hepatic mi-
crosomal CYP2B6-mediated bupropion hydroxylation with an appar-
ent Kiof 162 ? 14 ?g/ml (IC50estimated to be 284 ? 10 ?g/ml when
the assay was performed at a substrate concentration of 50 ?M). The
potency in the inhibition of CYP2B6 catalytic activity by G. biloba
extract appears to be in the same order of magnitude as that reported
for another herbal medicine known as Woohwangcheongsimwon
(Kim et al., 2008), which is a suspension of 29 herbs (mainly Calculus
bovis, Moschus, Borneolum syntheticum, Radix ginseng, and Rhizoma
dioscoreae). The apparent Kiwas not determined in that study, al-
though the reported IC50was 110 ?g/ml for the inhibition of hepatic
microsomal bupropion hydroxylation (at a substrate concentration of
50 ?M). Inhibition of CYP2B6 catalytic activity has also been shown
for three other herbal supplements: 1) a herbal cold remedy containing
a mixture of eight different herbal extracts (maltodextrin, lonicera,
forsythia, Chinese vitex, ginger, schizonepeta, isatis root, and echina-
cea) and nine vitamins and minerals (identity not reported) (Foti et al.,
2007); 2) A. paniculata extract, which contained andrographolide and
deoxyandrographolide (Pekthong et al., 2008); and 3) curcuminoid
extract, which contained the principal constituents curcumin, deme-
thoxycurcumin, and bisdemethoxycurcumin (Volak et al., 2008).
However, a direct comparison of their CYP2B6 inhibitory potency
with that of G. biloba extract is not possible because either the
apparent Ki(or IC50) was not determined (Foti et al., 2007) or the IC50
was expressed in terms of the molar concentration of a principal
constituent in the extract (Pekthong et al., 2008; Volak et al., 2008).
Another objective of the current study was to determine whether
any of the terpene trilactones present in G. biloba was responsible for
the inhibition of CYP2B6 enzyme activity by the extract. Therefore,
experiments were performed in which the enzymatic incubation con-
tained bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, or
ginkgolide J at a concentration that reflected the level present in a
specific concentration of the extract. However, none of the terpene
trilactones, either individually or as a mixture, decreased bupropion
hydroxylation catalyzed by recombinant CYP2B6 or human hepatic
microsomes. These terpene trilactones are also not responsible for the
inhibition of the catalytic activity of human CYP1A1, CYP1A2, or
FIG. 5. Dose-response effect of flavonol aglycones on human hepatic microsomal
bupropion hydroxylation. Pooled human hepatic microsomes (100 ?g of protein)
were incubated with bupropion (50 ?M) in the presence of quercetin, kaempferol,
isorhamnetin (5, 10, or 20 ?g/ml), or DMSO (0.1% v/v final concentration; vehicle
control) for 30 min at 37°C. The amount of hydroxybupropion was quantified by
UPLC/MS/MS as described under Materials and Methods. Results are expressed as
mean ? S.E.M. for four independent experiments. Bupropion hydroxylation in the
control group was 190 ? 8 pmol/min/mg protein.
FIG. 6. Lineweaver-Burk plot for the competitive inhibition of human hepatic
microsomal CYP2B6-mediated bupropion hydroxylation by kaempferol. Pooled
human hepatic microsomes (100 ?g of protein) were incubated with bupropion (25,
50, 100, or 200 ?M) and kaempferol (5, 10, or 20 ?g/ml) or DMSO (0.1% v/v final
concentration; vehicle control) for 30 min at 37°C. The amount of hydroxybupro-
pion was quantified by UPLC/MS/MS as described under Materials and Methods.
Results are expressed as mean ? S.E.M. for four independent experiments.
G. biloba AND CYP2B6 CATALYTIC ACTIVITY
at ASPET Journals on October 27, 2015
CYP1B1 by G. biloba extract as reported previously (Chang et al.,
2006). Data from experiments with human recombinant enzymes
support the notion that ginkgolides are not inhibitory toward cyto-
chrome P450 enzymes because ginkgolide A, ginkgolide B, and
ginkgolide C at a concentration of 200 ?M do not affect the catalytic
activity of human recombinant CYP1A2, CYP2C9, CYP2C19,
CYP2D6, or CYP3A4 (Zou et al., 2002). In contrast, bilobalide
inhibits only CYP2D6 (IC50? 11 ?M) (Zou et al., 2002).
Flavonol is another class of chemicals found in G. biloba; however,
they are present mainly as glycosides rather than as aglycones (van
Beek and Montoro, 2009). Flavonol glycosides are hydrolyzed to the
corresponding aglycones by the ?-glucosidases present in intestinal
microflora, and the resulting aglycones are absorbed (Cermak and
Wolffram, 2006). Kaempferol and quercetin aglycones appear to be
more potent than the corresponding glycosides in various biological
activities; for example, antioxidant activity (Bedir et al., 2002). In the
present study, 3-O-glucoside (a monoglycoside) and 3-O-rutinoside (a
diglycoside) of kaempferol, quercetin, and isorhamnetin did not affect
CYP2B6 catalytic activity. Structural studies have shown that
CYP2B6 substrates are nonplanar, neutral or weakly basic, and fairly
hydrophobic with one or two hydrogen bond acceptors (Lewis, 2000).
Therefore, a plausible explanation for our findings is that the bulky
and hydrophilic glycoside groups of kaempferol, quercetin, and isorh-
amnetin may hinder their interactions with the CYP2B6 enzyme
Kaempferol and quercetin aglycones have been reported to inhibit
the in vitro catalytic activity of various human cytochrome P450
enzymes, for example, CYP1A2 and CYP3A (von Moltke et al.,
2004), but the effect on CYP2B6 catalytic activity has not been
investigated. As shown in the present study, kaempferol, quercetin,
and isorhamnetin are capable of inhibiting hepatic microsomal
CYP2B6-mediated bupropion hydroxylation. Consistent with our data
for quercetin, a previous screening experiment reported inhibition of
human recombinant CYP2B6 catalytic activity by quercetin when
studied at a single concentration of 30 ?M (9 ?g/ml) (Walsky et al.,
2006). As shown in Table 3, the apparent Kivalue for the inhibition
of hepatic microsomal CYP2B6 by kaempferol is comparable with
that reported for most of the other naturally occurring compounds.
The most potent naturally occurring CYP2B6 inhibitors reported to
date appear to be phenethyl isothiocyanate and ?-viniferin, with
apparent Kivalues of 2 and 3 ?M, respectively, as analyzed with
7-benzyloxyresorufin as the substrate (Nakajima et al., 2001; Piver et
al., 2003). By comparison, the most potent CYP2B6 inhibitor reported
to date is the synthetic drug ticlopidine (apparent Ki? 0.2 ?M)
(Turpeinen et al., 2004).
It is not known whether the in vitro inhibition of CYP2B6 catalytic
activity by G. biloba extract is of in vivo significance. The reason is
that the chemical constituent(s) directly responsible for the in vitro
inhibitory effect of the extract remains to be conclusively identified.
The results from the present study have allowed us to conclude that
bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J,
kaempferol 3-O-glucoside, quercetin 3-O-glucoside, isorhamnetin
3-O-glucoside, kaempferol 3-O-rutinoside, quercetin 3-O-rutinoside,
and isorhamnetin 3-O-rutinoside are not responsible for CYP2B6
inhibition by the extract. Although kaempferol, quercetin, and isorh-
amnetin are shown to decrease hepatic microsomal CYP2B6-medi-
ated bupropion hydroxylation, their in vitro inhibitory concentrations
are greater than the maximal plasma levels (0.02–0.1 ?M) achieved in
human subjects who ingest G. biloba extract (Wo ´jcicki et al., 1995).
Other than terpene trilactones and flavonol glycosides, which account
for approximately 30% of the chemical constituents in G. biloba
extract, chemicals present in the extract include alkylphenols, organic
acids, proanthocyanidins, catechins, biflavones, and nonflavonol gly-
cosides (van Beek and Montoro, 2009). Whether any of these com-
pounds affects CYP2B6 catalytic activity remains to be investigated.
Flavonols are not only present in various herbal medicines but also
in fruits, vegetables, and certain beverages (Manach et al., 2005). In
the case of quercetin, it is also available as a supplement in health food
stores and is taken at dosages ranging from 500 mg to several grams
per day. Median maximal plasma concentrations of 0.18, 0.30, and
0.43 ?M have been reported in healthy human volunteers who in-
gested quercetin daily for 2 weeks at dosages of 50, 100, and 150
mg/day, respectively (Egert et al., 2008). By comparison, ingestion of
shallots (Allium cepa L. var. aggregatum) that contain quercetin at a
level of 1.4 mg/kg b.wt. (Wiczkowski et al., 2008) or onions that yield
an equivalent of 100 mg of quercetin (Manach et al., 2005) has
produced plasma quercetin levels of 1 to 4 ?M and 8 ?M, respec-
tively. Although hepatic levels of the ingested quercetin are not
known, this compound accumulates in liver, as shown in an animal
model (Bieger et al., 2008). It remains to be determined whether
dietary exposure to quercetin or other flavonols has any influence on
CYP2B6-mediated drug biotransformation.
Inhibition of cytochrome P450 enzymes may occur by various
mechanisms, including reversible inhibition (also known as alternate
substrate inhibition) and mechanism-based inactivation (Wienkers
and Heath, 2005). Reversible inhibitors and mechanism-based inacti-
vators of CYP2B6 have been identified (Turpeinen et al., 2006). The
present study was designed only to investigate reversible inhibition.
Future studies should address the question of whether G. biloba and
its flavonols are mechanism-based inactivators of CYP2B6 because
Apparent Kivalues for the inhibition of CYP2B6 catalytic activity by naturally occurring compounds
InhibitorSubstrate Apparent Ki
Mode of Inhibition Reference
Nakajima et al., 2001
Piver et al., 2003
Kim et al., 2008
Seo et al., 2008
Kim et al., 2008
Seo et al., 2008
Appiah-Opong et al., 2007
Piver et al., 2003
aRecombinant CYP2B6 enzyme.
bIndividual human hepatic microsomes.
cPooled human hepatic microsomes.
LAU AND CHANG
at ASPET Journals on October 27, 2015
data from those in vitro studies may help in predicting the potential of
G. biloba and flavonols to inhibit CYP2B6-mediated drug biotrans-
formation in vivo. The use of in vitro data to predict in vivo inhibitory
effects in a given individual is complicated by many factors, including
genetic variation (Wienkers and Heath, 2005). For example, when
compared with the wild-type CYP2B6, the CYP2B6*4 and
CYP2B6*6 variants appear to be less prone to inhibition (Talakad et
In summary, our major findings indicate that 1) G. biloba extract
competitively inhibited human hepatic microsomal CYP2B6-cata-
lyzed bupropion hydroxylation, with an apparent Kiof 162 ?g/ml; 2)
bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide
J were not responsible for the inhibitory effect of the extract; and 3)
whereas a monoglycoside and a diglycoside of kaempferol, quercetin,
and isorhamnetin had no effect, the corresponding aglycones inhibited
hepatic microsomal CYP2B6-mediated enzyme activity. The discov-
ery of G. biloba and flavonols as in vitro inhibitors of CYP2B6
provides an impetus for future investigations to expand our under-
standing of the pharmacological and toxicological consequences of
CYP2B6 inhibition by these natural products. Interestingly, it has
been suggested that CYP2B6 inhibitors may be beneficial in prevent-
ing tamoxifen-mediated endometrial cancer (Stiborova ´ et al., 2002).
Acknowledgments. We thank Indena S. A. (Milan, Italy) for the G.
biloba extract and bilobalide. We also thank Andras Szeitz for tech-
nical assistance with the UPLC/MS instrumentation. T.K.H.C. re-
ceived a Senior Scholar Award from the Michael Smith Foundation
for Health Research.
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Address correspondence to: Thomas K. H. Chang, Faculty of Pharmaceuti-
cal Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC,
V6T 1Z3, Canada. E-mail: firstname.lastname@example.org
G. biloba AND CYP2B6 CATALYTIC ACTIVITY
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