Journal of Chromatography B, 1029 (2016) 145–156

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Comparison of the in vitro metabolism of psoralidin among different

species and characterization of its inhibitory effect against UDP-
glucuronosyltransferase (UGT) or cytochrome p450 (CYP450)
enzymes
Xianbao Shi a,1 , Gang Zhang a,b,1 , Brianna Mackie b , Shuman Yang c , Jian Wang d ,
Lina Shan a,∗
a
Department of Pharmacy, The First Affiliated Hospital of Liaoning Medical University, Jinzhou, China
b
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA
c
Department of Internal Medicine/Community Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada
d
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, China

a r t i c l e i n f o a b s t r a c t

Article history: Psoralidin has shown a variety of biological and pharmacological activities such as anti-tumor anti-
Received 8 April 2016 oxidant, anti-bacterial, anti-depressant and anti-inflammatory activities. Herein, we reported the
Received in revised form 17 June 2016 metabolism of psoralidin among different species and its inhibitory effect against UGTs and CYP450s.
Accepted 18 June 2016
Liquid chromatography was used to investigate the inhibitory activity of psoralidin against ten different
Available online 30 June 2016
UGTs and eight distinct CYP450 isoforms. In addition, we characterized the CYP450 isoforms involved in
the psoralidin metabolism on the basis of chemical inhibition studies and screening assays with recom-
Keywords:
binant human cytochrome P450s. In vitro metabolic profiles and metabolites of psoralidin from varying
Psoralidin
Cytochrome P450
liver microsomes obtained from human (HLMs), monkey (MLMs), rat (RLMs), dog (DLMs), minipig (PLMs)
Glucuronosyltransferase (UGT) and rabbit (RAMs) were determined by LC–MS/MS. In vivo pharmacokinetic profiles were investigated by
Drug-drug interactions injecting psoralidin (2 mg/kg) into the tail vein of Wistar rats. Molecular modeling studies were carried
LC–MS/MS out in order to assess the binding profile and recognition motif between psoralidin and the enzymes.
Psoralidin showed potent and noncompetitive inhibition against UGT1A1, UGT1A7, CYP1A2 and CYP2C8
with IC50 values of 6.12, 0.38, 1.81, 0.28 ␮M, respectively. The metabolism of psoraldin exhibited signifi-
cant differences among humans, monkeys, dogs, minipigs, rabbits and rats; however, monkeys showed
the highest similarity to humans. Furthermore, eleven metabolites were observed among these species
and their structures were characterized by LC–MS/MS. CYP2C19 played a key role in the metabolism of
psorslidin in human liver microsomes. These findings could be used to advance the understanding of
psoralidin.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Psoraleacorylifolia is a plant widely used in traditional Chinese
medicine, which exhibits anti-oxidative, anti-microbial, anti-
inflammatory and anti-tumor activities [1–3]. One of the major
components isolated from the seed of psoraleacorylifolia is psora-
lidin (Fig. 1), which is extensively used by itself or along with other
∗ Corresponding author at: The First Affiliated Hospital of Liaoning Medical Uni-
versity, No. 5 Renmin Street, Jinzhou 121001, China.
E-mail address: shanlina123@163.com (L. Shan).
1 withdrawal of drugs already on the market [14,15]. Psoralen and
These authors have contributed equally to this work.
drugs to treat various ailments [4–7]. Previous studies have demon-
strated biological and pharmacological activities of psoralidin, but
pharmacokinetic evaluation has never been reported. Pharmacoki-
netic information could be critical to further understand the clinical
efficiency and safety of psoralidin. Cytochrome p450 (CYP450)
and UDP-glucuronosyltransferase (UGT) are the most important
enzyme systems of detoxication, and approximately 70%–80% of
the known Phase I and II metabolisms are attributed to them [8–10].
Inhibition of CYP450s and UGTs might increase the concentra-
tion of co-administered drugs in the blood or therapeutic targets
and result in adverse effects [11–13]. These severe adverse effects
have resulted in costly and late failures of drug development, and

http://dx.doi.org/10.1016/j.jchromb.2016.06.031
1570-0232/© 2016 Elsevier B.V. All rights reserved.
146 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Fig. 1. The structure of psoralidin.
isopsoralen, two important compounds of psoralea, have shown the
inhibition activity against -CYP3A4 in a concentration-dependent
manner [16]. However, the effect of psoralidin on CYP450 isoforms
has never been reported. In recent years, in vitro methods have been
used to predict in vivo aspects of humans such as drug metabolism
and pharmacokinetics and received satisfactory results [17,18].
Although all members of the CYP450 or UGT super family pos-
sess highly conserved regions of amino acid residues, there are
a few differences in the primary amino acid sequence of CYP450
and UGT across species. However, minor differences in the amino
acid sequences at the active sites of CYP450s can result in a wide
variation of substrate specificity and catalytic activity [19]. The
differences in CYP450 or UGT isoforms are a major cause of the dif-
ferences in drug metabolism across varying species [20]. Therefore,
understanding the differences in CYP450 or UGT function between
animals and humans is critical.
We sought to examine the impact of psoralidin against ten
UGTs and eight CYP450 isoforms. The plasma concentrations and
pharmacokinetic parameters were also evaluated after intravenous
administration of psoralidin. CYP450 isozymes involved in pso-
ralidin metabolism were confirmed by assays with recombinant
CYP450 isoforms and chemical inhibition experiments. In addi-
tion, We determined the psoralidin metabolism characteristics in
liver microsomes from six species, including rat, minipig, rabbit,
dog, monkey and human. This was to confirm which species best
resembles the drug-metabolism capabilities of humans. Addition-
ally, molecular docking was employed to further understand the
interactions between psoralidin and CYP450s.
2. Materials and methods
2.1. Chemicals and reagents
Psoralidin was purchased from Shifeng Corp in Shanghai, China,
and its purity was above 98%. The substrates, metabolites and pos-
itive inhibitors of CYP450 isoforms are listed in Table 1. Probe sub-
strates and positive inhibitors were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Metabolites of probe substrates were provided
by the Research Institute for Liver Disease Co. (Shanghai, China).
1-aminobenzotriazole (ABT), glucose-6-phosphate dehydroge-
nase, NADP+ , d-glucose-6-phosphate, 4-Methylumbelliferone(4-
MU), 4-methylumbelliferone-␤-d-glucuronide (4-MUG), Tris-HCl,
7-hydroxycoumarin and uridine 5 -diphosphoglucuronic acid
(UDPGA) (trisodium salt) werepurchased from Sigma-Aldrich.
Recombinant human supersomes (UGT1A1, UGT1A3, UGT1A6,
UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15,
CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) were
obtained from BD Gentest Corp. (Woburn, MA, USA). All other
reagents were of HPLC grade or of the highest grade commercially
available.
2.2. Preparation and characterization of liver microsomes
Liver microsomes from humans (HLMs), monkeys (MLMs), rab-
bits (RAMs), rats (RLMs), dogs (DLMs), and minipigs (PLMs) used
in this study were provided by the Research Institute for Liver Dis-
ease Co. (Shanghai, China). The HLMs was prepared from eleven
individual human donor livers. MLMs, RAMs, RLMs, DLMs, and
PLMs were prepared from ten individual animal livers. Protein
concentration and enzyme activities of CYP1A2, CYP2C9, CYP2C8,
CYP2A6, CYP2D6, CYP2E1, CYP3A4 and CYP2C19 had been previ-
ously characterized by the Research Institute for Liver Disease Co. In
brief, the concentration of protein was determined by bicinchoninic
acid (BCA) Protein Assay Kit, which was previously described by
Smith et al. [21]. The characterization of above CYP450s was per-
formed by detecting the metabolites of their probe substrates
using LC–MS/MS method, the procedure of which was described
in Section 2.5. All the probe substrates, their metabolites and the
inhibitors used as positive control were presented in Table 1.
2.3. Analytical instruments and conditions
The HPLC system (Shimadzu, Kyoto, Japan) consisted of a CBM-
20Alite system, two LC-20AB pumps, and an SPD-20A ultraviolet
light (UV) detector. The chromatographic separation was achieved
using an Inertsil ODS-3 C18 column (4.6 mm × 150 mm, 5 ␮m,
Shimadzu-GL Sciences Inc., Kyoto, Japan). The mobile phases con-
sisted of LC grade water containing 0.1% formic acid (A) and
LC grade acetonitrile (B), with the following gradient profile:
0–12 min, 20%–90% B; 12–17 min, 90% B; 17–17.30 min, 90%–20%
B; 17.30–22.00 min, 20% B. The flow rate was 1 mL/min and col-
umn temperature was set to 40 ◦ C. Psoralidin and its metabolites
were detected at 347 nm. Due to the absence of authentic standards
for metabolites of psoralidin, the standard curve for psoralidin was
used to quantify P3 in the incubation mixtures. Method validation
was provided in supporting materials (Tables S1–S2, Fig. S1). The
performance parameters including specificity, linearity, accuracy,
precision, decision limit, quantification limit and stability showed
the validated method was qualified for the determination of pso-
ralidin and its metabolites.
LC–MS/MS analysis was performed on Ultra Performance Liquid
Chromatograph (UPLC) equipped with a Q-TOF SYNAPT G2-Si High
Definition Mass Spectrometry (Waters Corporation, Milford, MA).
The UPLC system was fitted with an Acquity UPLC BEH C18 column
(2.1mm × 100 mm, 1.7 ␮m; Waters Co., Ltd., USA). Mass detection
was performed in negative mode from m/z 100 to 1000. The optimal
parameters were as follows: Collision gas is Argon; trap collision
energies were 6 V (low energy) and 20–50 V (high energy); capil-
lary and cone voltages were 2 kV and 40 V; source and desolvation
temperatures were 120 and 600 ◦ C; the cone and desolvation gas
flows were 50 and 800 L h−1 , respectively.
2.4. CYP450 probe substrate assays
The CYP450s activities were characterized based on their probe
reactions: CYP1A2 (phenacetin o-deethylation), CYP2A6 (coumarin
7-hydroxylation), CYP2C8 (paclitaxel 6␣-hydroxylation), CYP2C9
(diclofenac 4 -hydroxylation), CYP2D6 (dextromethorphan o-
demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4
(testosterone 6␤-hydroxylation) and CYP2C19 (S-mephenytoin4 -
hydroxylation) [22].
2.5. Incubation systems with liver microsomes or recombinant
CYP450 supersomes
The optimal incubation conditions of microsomes have been
reported [23,24]. In brief, the typical incubation system contained
 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Table 1
The substrates, inhibitors and metabolites of CYP450.
CYP isoforms Substrates
CYP1A2 phenacetin
CYP2C9 diclofenac
CYP2C8 paclitaxel
CYP2A6 coumarin
CYP2D6 dextromethorphan
CYP2E1 chlorzoxazone
CYP3A4 testosterone
CYP2C19 S-mephenytoin
100 mM potassium phosphate buffer (pH 7.4), NADPH-generating
system (1 mM NADP+ , 10 mM glucose-6-phosphate, 1 unit/mL
of glucose-6-phosphate dehydrogenase, and 4 mM MgCL2 ), the
appropriate concentration of liver microsomes or recombinant
CYP450s supersomes, the corresponding probe substrate and pso-
ralidin (or positive inhibitor for different probe reactions) in a final
volume of 200 ␮L. According to preliminary experiments (data not
shown), the final protein concentration of 0.3 mg/mL in liver micro-
somes or 15 nM in recombinant human supersomes, and a 30 min
reaction time were selected to ensure linear formation of metabo-
lites during the incubations. There was a 3 min preincubation at
37 ◦ C before the reaction was initiated by adding the NADPH-
generating system. The reaction was placed on ice and terminated
by adding 200 ␮L acetonitrile and an internal standard. The mixture
was centrifuged at 20,000g for 20 min and an aliquot of supernatant
was transferred for HPLC or LC–MS/MS analysis.
2.6. Pharmacokinetics study
Six male Wister rats (200–250 g) were obtained from the Lab-
oratory Animal Center of Liaoning Medical University. Approval
from the Institutional Animal Ethics Committee was sought and
the study protocols were approved before the commencement of
the studies. Psoralidin was administered intravenously into the tail
vein at a dose of 2 mg/kg. The blood samples were collected through
orbital venous plexus at 0.083, 0.3, 0.75, 1, 1.5, 2, 3.5, 4.5, 6 and
8 h after intravenous administration, and centrifuged at 2000g for
5 min at 4 ◦ C to obtain the plasma samples. The plasma (0.1 mL)
was mixed with 200 ␮L acetonitrile and phenacetin (100 ␮M) was
used as an internal standard. After centrifugation of the mixture
at 20,000g for 20 min at 4 ◦ C, the supernatant was collected and
analyzed by HPLC.
2.7. UGTs inhibition experiments
4-MU and recombinant UGT isoforms were used as the nonspe-
cific probed substrates and enzyme source to assess the inhibitory
effect of psoralidin against UGT isoforms. Incubation and ana-
lytical conditions were as described previously [25]. In brief,
the mixture (200 ␮L total volume) contained recombinant UGTs
(final concentrations: 0.125, 0.05, 0.025, 0.05, 0.025, 0.05, 0.05,
0.25, 0.05 and 0.2 mg/mL for UGT1A1, UGT1A3, UGT1A6, UGT1A7,
UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and 2B15, respec-
tively), 5 mM UDPGA, 5 mM MgCL2 , 50 mM Tris-HCl buffer (pH
7.4) and 4-MU in the absence or presence of different concen-
trations of psoralidin. The concentrations of 4-MU were 110 ␮M
for UGT1A1, 1200 ␮M for UGT1A3, 110 ␮M for UGT1A6, 15 ␮M
for UGT1A7, 750 ␮M for UGT1A8, 30 ␮M for UGT1A9, 80 ␮M for
UGT1A10, 1200 ␮M for UGT2B4, 350 ␮M for UGT2B7 and 250 ␮M
for UGT2B15. Psoralidin was dissolved in methanol and the final
concentration of methanol was 0.5% (v/v). After a 5 min prein-
cubation at 37 ◦ C, UDPGA was added to the mixture to initiate
the reaction. Incubation time was 120 min for UGT1A1, UGT1A3,
UGT1A10, UGT2B4, UGT2B7 and UGT2B15, 30 min for UGT1A6,
147
Inhibitors metabolites
furafylline O-deethylation phenacetin
sulfaphenazole 4 -hydroxylation diclofenac
quercetin 6␣-hydroxylation paclitaxel
xanthotoxin 7-hydroxylation coumarin
quinidine O-demethylation dextromethorphan
clomethiazole 6-hydroxylation chlorzoxazone
ketoconazole 6␤-hydroxylation testosterone
omeprazole 4 -hydroxylation S-mephenytoin
UGT1A7, UGT1A8 and UGT1A9, respectively. The reactions were
quenched by 200 ␮L acetonitrile and 7-hydroxycoumarin (100 ␮M)
which was used as an internal standard. The mixture was cen-
trifuged at 20,000g for 20 min and an aliquot of supernatant was
analyzed by HPLC with ultraviolet light (UV) detection.
2.8. CYP450inhibition experiments
Marker assays for CYP450s (CYP1A2, CYP3A4, CYP2D6, CYP2E1,
CYP2A6, CYP2C9, CYP2C19 and CYP2C8) were conducted using the
above incubation systems and conditions and consisted of liver
microsomes, the NADPH-generating system, probed substrates at
the concentration of their approximate Km value, and psoralidin
(or the control inhibitor). The positive control inhibitors of indi-
vidual CYP450 isoforms were as follows: furafylline (10 ␮M) for
CYP1A2, xanthotoxin (2.5 ␮M) for CYP2A6, quercetin (2 ␮M) for
CYP2C8, sulfaphenazole (10 ␮M) for CYP2C9, omeprazole (20 ␮M)
for CYP2C19, quinidine (10 ␮M) for CYP2D6, clomethiazole (50 ␮M)
for CYP2E1, ketoconazole for CYP3A4 and ABT (500 mM) for broad
CYP450s [26,27]. Furthermore, kinetic analysis was performed for
the isoforms which had been inhibited by more than 90% total
activity. IC50 values were obtained by incubating various psora-
lidin concentrations (0–15 ␮M for CYP1A2, 0–5 ␮M for CYP2C8,
0–100 ␮M for UGT1A1, and 0–5 ␮M for UGT1A7) with the isoforms.
Dixon and Lineweaver-Burk plots were generated to determine
the inhibition type. A second graph was created by plotting the
slopes from the Lineweaver-Burk plot versus the concentrations of
psoralidin to calculate the Ki values.
2.9. Identification of metabolites in human, rat, monkey, minipig,
rabbit and dog liver microsomes
In this study, in vitro metabolism of psoralidin among species
was compared with respect to metabolites, metabolic profiles and
catalytic efficiency using liver microsomes from humans (HLMs),
rabbits (RAMs), rats (RLMs), dogs (DLMs), minipigs (PLMs), and
monkeys (MLMs). Incubating conditions were conducted using the
reaction system described above. Metabolites were determined by
HPLC.
2.10. Assay with recombinant P450s
Six cDNA-expressed human CYP isoforms (CYP1A2, CYP2C9,
CYP2C19, CYP2D6, CYP2E1 and CYP3A4) were used to screen the
involved isoform(s) for metabolites of psoralidin. The incubations
were carried out with each of the CYP450 isoforms using the proto-
col described above. Psoralidin (10 ␮M) was incubated with each of
the recombinant CYP450s (15 nM) at 37 ◦ C for 30 min and possible
metabolites were monitored by HPLC.
2.11. Chemical inhibition assays
Initially, we used ABT (a broad-specificity P450 inactivator)
to screen the P450 isoform(s) responsible for the formation of
148 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Fig. 2. Representative HPLC profiles of psoralidin and its metabolites in cDNA-expressed human P450 isoforms liver microsomes from HLMs, DLMs, MLMs, PLMs, and RLMs.
Psoralidin (10 ␮M) was incubated with liver microsomes (0.3 mg/mL) from different species and an NADPH-generating system at 37 ◦ C for 30 min.
metabolites in HLMs. If ABT inhibited the formation of psora-
lidin metabolites, it suggested that CYP450s were responsible for
psoralidin metabolism in HLMs. Additionally, the metabolism of
psoralidin in HLMs in the absence or presence of selective inhibitors
for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1
and CYP3A4 were measured to explore the enzyme(s) involved in
this biotransformation of psoralidin.
2.12. Enzymatic kinetic study
The enzyme kinetics of psoralidin metabolism among six species
and the recombinant human CYP2C19 enzyme were investigated.
As the dominant metabolite of psoralidin, P3 was selected to con-
duct the kinetic parameter evaluation in each species and CYP2C19.
Preliminary experiments were carried out to ensure that the for-
mation of the metabolites was linear with the reaction time and the
concentration of the protein in liver microsomes. Concentrations of
the psoralidin dilutions were between 0.1 to 250 ␮M. Protein con-
tents of liver microsomes and recombinant human CYP2C19 were
0.3 mg/mL and 15 nM, respectively. Incubation time was 30 min.
All incubations were carried out in three independent experi-
ments. The apparent Km and Vmax values were calculated from
nonlinear regression analysis of experimental data according to
the Michaelis-Menten equation, and the results were graphically
represented by Eadie-Currently Hofstee plots. The intrinsic clear-
ance (CLint ) for formation of psoralidin metabolite was calculated
by Vmax /Km . Kinetic constants were estimated using origin 7.5 and
their estimates are presented as mean ± standard deviation (SD).
2.13. Analysis of metabolites by LC–MS/MS
Structure characterization of metabolites in HLMs, DLMs, PLMs,
RAMs, RLMs and MLMs were performed on UPLC–MS/MS. Mass
detection was performed in negative ion mode from m/z 100 to
1000. The injections volume was 0.5 ␮L using the partial-loop
mode, the column temperature was 55 ◦ C and the flow rate was
3 ␮L/min. The mobile phases consisted of LC grade water with 0.1%
formic acid (A) and LC grade acetonitrile (B) with the following
gradient profile: 0.0–10.0 min from 30% to 50% B, 10–11 min from
50% to 90% B, 11–12 min 90% B, 12–13 min from 90% to 30% B, and
13–15 min at 30% B.
2.14. Determination of P3 structure
As the main metabolite, P3 in HLMs was determined by AB SCIEX
4000 Q-TrapTM (Applied Biosystems, Foster City, CA) interfaced
online with an ekspertultra LC 100 system (Applied Biosystems)
in negative mode. The separation of samples was achieved on a
HYPERSIL BDS C18 column (4.6 mm × 150 mm, 5 ␮m, Dalian Elite
Analytical Instruments Co., Dalian, China). The mobile phases and
chromatographic condition were described as above. Multiple-
reaction monitoring (MRM) scans were used to detect and
identifyP3 . MS analyses were conducted with ion transitions m/z
351 → 351. The optimized MS instrument parameters were set as
follows: ion spray voltage, 4500 V; ion source temperature, 650 ◦ C;
curtain gas, 10 psi; gas 1, 50 psi; and gas 2, 50 psi.
2.15. Docking study of psoralidin into the reported structures of
CYP1A2, CYP2C8 and CYP2C19
Sybyl 6.9.1 software package was used for protein and ligand
preparation. AutoDock 4.02 was used for molecular docking. Dis-
covery Studio Visualizer was used for graphic display.
The X-ray structures of CYP 1A2 (PDB code 2HI4), 2C8 (PDB code
2VN0) and 2C19 (PDB code 4GQS) were obtained from RCSB Pro-
tein Databank (http://rcsb.org/). Heme moiety was considered as a
part of the protein and Tripos atom types were defined accordingly.
Polar hydrogens and partial charges were added for CYP proteins
 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Fig. 3. The plasma concentration profiles after intravenous administration of pso-
ralidin at a single dose of 2 mg/kg. Data represent mean ± SD of six rats.
using the Kollman United Atom charges with Sybyl 6.9.1, and
energy minimization was made employing both steepest descent
and conjugate gradients protocols.
The psoralidin structure was built by the Sybyl 6.9.1 soft-
ware package (Tripos Associates: St. Louis, MO, 2003), assigned
charges using the Gasteiger–Hückel method and minimized with
the Powell method (Tripos force field) with an energy gradient of
0.05 kcal/(mol Å). Flexible torsions in the ligands were assigned and
all dihedral angles were allowed to rotate freely.
Automated docking studies were performed with AutoDock 4.0.
A 80 × 80 × 80 Å grid box with a grid spacing of 0.375 Å was gener-
ated for the receptor. Affinity grid fields were generated using the
auxiliary program AutoGrid 4.0.
The Lamarckian genetic algorithm (LGA) was used to find the
appropriate binding positions, orientations, and conformations of
the ligands. The optimized AutoDocking parameters increased the
maximum number of energy evaluations to 25,000,000 per run, set
the iterations of Solis & Wets local search to 3000; set the number
of individuals in population to 300 and the number of generations
to 100. All other parameters were maintained as the default. Clus-
ter analysis with AutoDock results was performed to determine if
different binding sites had been produced from multiple runs.
2.16. Statistical analysis
Statistical analysis was performed with SPSS 13.0 software for
Windows operation system (SPSS Inc, Chicago, IL, USA). All grouped
data were expressed as means ± SD. The identification of signifi-
cance between different groups was carried out with student t-test.
Two-sided P < 0.05 was considered to be statistically significant.
3. Results
3.1. Analysis of psoralidin metabolites
After psoralidin was incubated with liver microsomes from
humans, monkeys, dogs, minipigs, rats or rabbits, five new
peaks (P1 –P5 ) were observed in RLMs and two new peaks
(P1 , P3 ) were found in HLMs, MLMs, DLMs, PLMs and RAMs
by HPLC as compared to the control group (CTRL) (Fig. 2).
The formation of these metabolites was time-, NADPH-, and
microsome-dependent. P3 , the dominant metabolite in the human
liver microsome, was also generated by all of the animal
liver microsomes. Therefore, P3 was selected to evaluate the
metabolism differences among different species. Peak areas of
P3 in the animal incubation were compared with its counter-
part from human incubation, which was normalized to 100%. As
a result, the peak areas of P3 generated by rats, dogs, minip-
igs, rabbits and monkeys were 214.38% ± 16.36%, 50.37% ± 3.82%,
149
Table 2
The pharmacokinetic parameters of psoralidin in rats after intravenous administra-
tion (n = 6).
Pharmacokinetic parameters Units Values (mean ± SD)
AUC (0-∞) mg/L/h 1.95 ± 0.50
t1/2 h 4.45 ± 0.95
V L/kg 6.22 ± 1.82
CL L/h/kg 1.12 ± 0.45
Cmax mg/L 2.19 ± 0.48
54.42% ± 4.29%, 140.74% ± 10.28% and 77.79% ± 5.23% of the human
levels. Metabolism of psoralidin exhibited significant differences
between species (P < 0.05).
3.2. Pharmacokinetics
After injecting psoralidin intravenously, the blood samples were
collected at different time points. These samples were processed
as described earlier and analyzed by the presently developed HPLC
method. The plasma concentration profile of psoralidin was shown
in Fig. 3. The pharmacokinetic (PK) parameters such as AUC(0-∞) ,
t1/2 , V, CL and Cmax were calculated by anoncompartmental method
using DAS 3.1 software package (Jiangxi University of Traditional
Chinese Medicine, Nanchang, China) and the values were showed
in Table 2.
3.3. Inhibition against UGTs and CYP450activities by psoralidin
Many herbs have inhibitory activity towards CYP450s or
UGTs, which may affect the concentration of co-administered
drugs and result in adverse effects of the drugs. Thus, we
determined the inhibitory potential of psoralidin against eight
CYP450s and ten UGTs. 100 ␮M of psoralidin inhibited the
activities of UGT1A1, UGT1A7, CYP1A2 and CYP2C8 by more
than 90%. The strong inhibitory effects against the activities of
UGT1A1, GT1A7, CYP1A2 and CYP2C8 were in a concentration-
dependent manner. The IC50 values of UGT1A1, GT1A7, CYP1A2 and
CYP2C8 were 6.12 ± 0.43 ␮M, 0.38 ± 0.03 ␮M, 1.818 ± 0.22 ␮M and
0.28 ± 0.03 ␮M, respectively. The Ki values were 5.638 ± 0.46 ␮M,
0.248 ± 0.02 ␮M, 1.158 ± 0.13 ␮M and 0.25 ± 0.03 ␮M, respectively.
Dixon and Lineweaver-Burk plots showed that psoralidin non-
competitively inhibited UGT1A1, GT1A7, CYP1A2 and CYP2C8
(Supplemental Figs. S2–S5).
3.4. Identifying CYP450 involved in the metabolism of psoralidin
The enzymes involved in the formation of P3 were investigated
using cDNA-expressed human P450 isoforms. Quantities of P3 in
the CYP3A4, CYP2D6, CYP2E1, CYP2C9 and CYP1A2 incubation were
compared with its counterpart from CYP2C19 incubation which
was normalized to 100%. As shown in Fig. 4, the quantities of P3
generated by CYP3A4, CYP2D6, CYP2E1, CYP2C9 and CYP1A2 were
0.42%8 ± 0.15%, 13.58%8 ± 2.2%, 0.47%8 ± 0.11%, 6.94% ± 1.63% and
8.5%8 ± 1.32% of the CYP2C19 levels. CYP2C19 demonstrated a vast
ability to catalyze the formation of P3 , while other enzymes dis-
played limited ability to metabolize psoralidin. The key role of
CYP2C19 in metabolizing psoralidin was also confirmed by the
chemical inhibition assays. The formation of P3 could be potently
inhibited by ABT (a broad specificity CYP450 inactivator) and
omeprazole (a potent inhibitor of CYP2C19) to less than 10% activ-
ity, while selective inhibitors of other CYP450 isoforms had minor
effects on the formation of P3 (Fig. 5). According to the results of
the assays with recombinant CYP450 isoforms and chemical inhi-
bition experiments, CYP2C19 was the main enzyme involved in the
metabolism of psoralidin.
150 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Fig. 4. Representative HPLC profiles of psoralidin and its metabolites in recombinant CYP450 supersomes. Psoralidin (10 ␮M) was incubated with recombinant CYP450
supersomes (15 nM) and an NADPH-generating system at 37 ◦ C for 30 min.
3.5. Kinetic characteristics of psoralidin metabolism
To further characterize the kinetic differences among different
species and identify the optimal animal species to serve as a model
of metabolism in humans, psoralidin (0.1–200 ␮M) was incubated
in HLMs, MLMs, DLMs, PLMs, RLM, RAMs and CYP2C19. Under the
experimental conditions used, the metabolism of psoralidin obeyed
the typical monophasic Michaelis-Menten kinetics, as evidenced by
the Eadie-Hofstee plot (Fig. 6). The kinetic parameters (apparent Km
and Vmax ) and calculated intrinsic clearance (CLint ) are summarized
in Table 3.
Fig. 5. Inhibition assays of psoralidin metabolism by selective CYP450 inhibitors in
HLM. Results were mean ± SD at least 3 separate assays.
3.6. Structure characterization of metabolites by LC–MS/MS
In this study, psoralidin and eleven metabolites were identified
from HLMs, DLMs, RLMs, PLMs, MLMs and RAMs based on MS and
MS/MS data by using UPLC/Q-TOF-MS/MS in negative mode of an
ESI mass spectra.Eight (M1 –M5 , M7 –M9 ), seven (M1 –M5 , M10 and
M11 ), five (M1 , M2 , M3 , M5 and M9 ), nine (M1 –M5 , M8 –M11 ), ten
(M1 –M4 , M6 –M11 ) and three (M1 , M3 and M8 ) metabolites of pso-
ralidin were detected in HLMs, MLMs, PLMs, RAMs, RLMs and DLM,
respectively. The data of psoralidin and its metabolites are listed
in Table 4, and the structural skeleton of psoralidin and its poten-
tial metabolic pathways are shown in Fig. 7. TOF/MS/MS spectra of
psoralidin and its metabolites are shown in Fig. 8. Psoralidin (m/z
335.0919) exhibited six characteristic fragment ions: m/z 319.0608
(loss of methyl), m/z 317.0447, m/z 280.0374 (loss of butenyl), m/z
260.8776, m/z 251.0349 (loss of butenyl and carbonyl) and m/z
146.9665 (Fig. 8A). Fragment ion at m/z 146.9665 was also found in
all mass spectra of metabolites and was inferred to be the parent
nucleus of psoralidin.
M1 . The molecular ion at m/z 353.1035 (M1 , [M+H2 O]) (Fig. 8B)
was detected at a retention time of 8.17 min, with the product ions
at m/z 293.0459, 280.0381, 251.0356 and 146.9666. The molecular
ion of M1 was 18 Da more than the m/z of the parent psoralidin;
therefore, it was identified as the hydration product of psoralidin.
According to the fragment ions of M1 and psoralidin at m/z 280,
251 and 146, and analyzing the fragment ion of M1 at m/z 293, a
hydration reaction occurred at the double bond between C-2 and
C-3 , and the hydroxyl group was bonded to C-3 .
M2 and M3 . M2 and M3 showed an m/z of 16 Da more than
the parent compound indicating that the two metabolites were
hydroxylated psoralidin. The cleaved pathways and fragment ions
of M2 and M3 are shown in Fig. 8C and D.
 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156 151

Fig. 6. Michaelis-Menten plots for psoralidin in HLMs (A), RLMs (B), PLMs (C), MLMs (D), RAMs (E), DLMs (F) and CYP2C19 (G). The corresponding Eadie-Hofstee plots were
shown as insets. Each data point represents the mean of triplicate determinations.

Table 3
Kinetic parameters of P3 production from psoralidin in pooled human liver microsomes (HLMs), rat liver microsomes (RLMs), minipig liver microsomes (PLMs), monkey liver
microsomes (MLMs), rabbit liver microsomes (RAMs), dog liver microsomes (DLM) and recombinant CYP2C19.

Enzyme sources HLMa RLMa PLMa MLMa RAMa DLMa CYP2C19b

Vmax 16.27 ± 1.21 57.21 ± 3.18 40.42 ± 3.58 27.04 ± 1.56 34.76 ± 2.40 28.61 ± 2.1 3.01 ± 0.23
Km 5.171 ± 0.43 21.04 ± 1.53c 9.07 ± 0.65c 4.81 ± 0.43 4.41 ± 0.35 11.25 ± 0.93c 2.42 ± 0.15
CL(Vmax /Km ) 3.15 ± 0.22 2.72 ± 0.21 4.45 ± 0.31 5.61 ± 0.41 7.87 ± 0.59 2.54 ± 0.19 1.24 ± 0.14
a
Km , Vmax , and CLint were expressed in units of ␮M, nmol min−1 mg−1 protein, and mL min−1 mg−1 protein, respectively.
b
Km , Vmax , and CLint were expressed in units of ␮M, nmo min−1 pmol−1 CYP, and mL min−1 pmol−1 CYP, respectively.
c
Indicates Km value is statistical significant at P < 0.05 as compared to the Km value in HLM. All grouped data were expressed as means ± SD.

Fig. 7. Proposed chemical structures and major metabolic pathway of psoralidin in six species (including rat, monkey, dog, rabbit, minipig and human).
152 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156

Fig. 8. Mass spectrum (MS/MS) of psoralidin and its metabolites.

 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156 153

M4 , M5 , M6 and M7 . M4 (tR = 6.82 min), M5 (tR = 7.39 min), M6

52.79
1.59

5.06

0.02
N.D.

N.D.
N.D.
N.D.
N.D.

N.D.
N.D.
N.D.
Dog
(tR = 7.52 min) and M7 (tR = 7.68 min) were isomers with the same
molecular formula of C20 H16 O7 (m/z 367.0818) and 32 Da more
than the parent compound psoralidin. Exact mass determination

Human

30.09
and the corresponding chemical composition showed the addition

2.29
0.34
6.80
0.16

0.24
0.03

0.03
0.01
N.D.

N.D.
N.D.
of two hydroxyl groups to the parent molecule. Structural formula
Rabbit and fragmentation pathway of M4 –M7 are shown in Fig. 8E–G.

42.56
M8 . The molecular formula of M8 was determined to be
2.09
0.28
5.80

0.11

0.12

0.11
0.40

0.05

0.06
N.D.
N.D.
C20 H18 O6 (m/z 353.1025) based on its MS data. According to its
molecular formula and MS/MS spectra, M8 was a reduction prod-
39.66
1.36

3.28
0.25

0.45

0.21
N.D.

N.D.
N.D.
N.D.

N.D.
N.D.
uct of M3 (2 Da more than M3 ), and two hydrogen were bonded to
Pig

C-2 and C-3 (Fig. 8H).

M9 . The mass spectrum of M9 (Fig. 8I) with a molecular ion at
Monkey

28.09

m/z 349.0712 was produced by a dehydrogenation reaction from

9.33
4.10

0.42
1.30
0.04

0.07
0.06
N.D.
N.D.
N.D.
N.D.
Peak area (%)

M3 . Psoralidin and M9 had the same fragment ions at m/z 321.0769

and 305.0464. Thus, the dehydrogenation reaction of M3 occurred
23.79

13.61

at C-4 and produced the M9 metabolite.

8.83

2.23

0.94
0.96
0.13
0.08

0.07
0.11
0.09
N.D.
Rat

M10 and M11 . M10 and M11 were isomers with the same molec-
ular formula (C20 H18 O7 ) but different retention times (6.30 and
Hydroxylation + Dehydrogenation

6.56 min). According to the MS/MS spectra and information on the

2 × Hydroxylation + Reduction
2 × Hydroxylation + Reduction

fragment ions, M10 and M11 were identified as the reduction and
Hydroxylation + Reduction

two hydroxylation products of psoralidin (Fig. 8J).

Metabolic reaction

2 × Hydroxylation
2 × Hydroxylation
2 × Hydroxylation
2 × Hydroxylation

3.7. The inference about P3 and M3

Hydroxylation
Hydroxylation

The HPLC, total ion and extracted ion chromatograms were

Hydration

showed in Fig. S6. P3 and P4 were observed in HPLC chromatogram,

parent
Accurate mass measurement for the protonated molecules of metabolites in rat, monkey, pig, rabbit, human and dog liver microsomes.

and M2 and M3 were found in total ion chromatogram. P3 , P4 ,

M2 and M3 were not found in control group. The retention time
of P3 (10.75 min) and P4 (11.02 min) in HPLC chromatogram was
319.0608, 317.0447, 280.0374, 279.0298, 260.8776, 146.9665
353.1035, 351.0880, 293.0459, 280.0381, 251.0359, 146.9666
333.0777, 321.0773, 317.0416, 305.0461, 280.0381, 146.9667

321.0767, 309.1137, 305.0455, 280.0383, 260.8782, 146.9667

same with M3 and M2 in total ion chromatogram, respectively.

321.0767,309.1137,305.0455,280.0383,260.8782,146.9667

The corresponding extracted ion chromatograms showed M2 and

M3 had same molecular ion at m/z 351. According to retention time
333.0776, 321.0766, 305.0454, 266.0236, 146.9664
349.0725, 317.0466, 307.0617, 260.8782, 146.9666

and peak area described in Table 4, the frontal peak in total ion
chromatogram was M3 and the latter peak was M2 . Thus, M3 was
inferred to be the metabolite P3 and M2 was inferred to be the
349.0725, 337.0726, 296.0322, 146.9667

294.0134, 280.0381, 279.0306, 146.9666

321.0769, 305.0464, 280,0373, 146.9664
325.1082, 280.0383, 240.0427, 146.9667
325.1082, 280.0383, 240.0427, 146.9667

metabolite P4 .

3.8. Molecular modelling

CYP2C19 is the enzyme involved in the metabolism of pso-

ralidin, and psoralidin showed strong inhibitory effects against
CYP1A2 and CYP2C8. Therefore, the interactions between pso-
ralidin and CYP1A2 or CYP2C8 at a molecular basis were
studied through molecular docking. Psoralidin bound within the
Fragment

hydrophobic pocket of CYP1A2 containing residues Val220, Phe226,

Phe260, Phe315, Gly316, Phe319, and Leu497 (Fig. 9A), within
the hydrogen-bonded and hydrophobic pocket of CYP2C8 con-
taining residues Ser103, Phe205, Ala297, Val366, Val477 and Ile
Error (ppm)

113 (Fig. 9B), and within the hydrogen-bonded and hydrophobic

pocket of CYP2C19 containing the residues Val113, Phe114, Ala297,
3.9
1.4
3.7

3.3
1.4

2.4
3.8
2.5
0.7
2.6
4.0

3.0

Thr301, Leu361, Ile362, Leu368, Phe476, and Ala477 (Fig. 9C).

335.0919
353.1039
351.0878
351.0871
367.0846

367.0846
367.0846
353.1035
349.0717
369.0841
369.0841
367.0808

4. Discussion
Mass

CYP450 and UGT are two imperative enzymes responsible for

the metabolism of most drugs. The inhibition of these two enzyme
C20 H16 O5
C20 H18 O6
C20 H16 O6
C20 H16 O6
C20 H16 O7
C20 H16 O7
C20 H16 O7
C20 H16 O7
C20 H18 O6
C20 H14 O6
C20 H18 O7
C20 H18 O7
Formula

systems could result in drug-drug interactions and severe adverse

side effects. As a conservative approach, the inhibitor Cmax at the
highest clinical dose expected should be used in the estimation of
AUC change. Both the Food and Drug Administration and European
tR (min)

Medicines Agency stated that an interaction would likely occur for

N.D., No detect.
8.76
8.17
7.74
7.24
6.82
7.39
7.52
7.68
7.74
7.82

6.56
6.30

reversible inhibition if the ratio of inhibitor Cmax /Ki was greater

than 1. Interactions are possible if the ratio is between 0.1 and 1 and
Table 4

M10
M11
NO.

remote if below 0.1 [28]. Using UGT1A1-overexpressing HeLa cells,

M0
M1
M2
M3
M4
M5
M6
M7
M8
M9

Sun et al. found that psoralidin showed strong inhibition against

154 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
Fig. 9. Binding modes of compound with CYP 1A2 (A), 2C8 (B) and 2C19 (C). The compounds were displayed in green sticks, residues were displayed in blue thin sticks,
hydrogen bonds were displayed in green dot lines, and hydrophobic interactions were displayed in pink dot line. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
UGT1A1 [29]. In this study, we found psoralidin strongly inhibited
the activities of UGT1A1, UGT1A7, CYP1A2 and CYP2C8 with the
calculated Cmax /Ki values of1.15, 27.12, 5.67, and 26.21, respec-
tively. The results of the in vitro-in vivo extrapolation were well
above the thresholds for clinical DDI evaluation as recommended
in the regulatory guideline. This indicated that the most likely in
vivo adverse effects are due to the large Cmax /Ki values of pso-
ralidin against UGT1A1, UGT1A7, CYP1A2 and CYP2C8. CYP1A2,
CYP2C8, UGT1A1 and UGT1A7 are particularly significant enzymes
because they are involved in the metabolism of many important
drugs and endogenous substances such as bilirubin, raltegravir,
caffeine and paclitaxel [30–33]. Thus, potential drug-drug inter-
actions should be monitored when psoralidin is co-administered
with clinical medications.
We identified the CYP450 isoforms involved in psoralidin
metabolism by screening assays with recombinant CYP450 super-
somes including CYP1A2, CYP3A4, CYP2E1, CYP2C9, CYP2D6 and
CYP2C19 and chemical inhibition studies. CYP2C19 played a vital
role in the metabolism of psoralidin, while all other CYP isoforms
displayed an insignificant effect compared to CYP2C19. CYP2C19 is
one of important CYP450 isoforms, which catalyzes the metabolic
reaction of many drugs such as R-warfarin [34]. In addition, the
Km value of psoralidin in CYP2C19 was similar to its Km value in
HLMs which further indicated that CYP2C19 was the major enzyme
involved in its metabolism. These results demonstrated that pso-
ralidin was selectively catalyzed by CYP2C19 in humans.
In preclinical studies to evaluate pharmacokinetics and toxic-
ity, animal models have been commonly used to replace humans.
However, it should be understood that suitable animal models for
in vivo drug metabolism and pharmacokinetics studies are impor-
tant and should have similar metabolic behaviors as compared to
humans. The models should include identical metabolic profiles,
equivalent or similar metabolic enzymes, and comparable catalytic
efficiency. Consequently, we distinguished the in vitro metabolism
behaviors of psoralidinin HLMs, RLMs, DLMs, RAM, MLMs and PLMs.
The kinetic parameters including Km , Vmax , and CLint for the forma-
tion of P3 were determined and varied among species.Our findings
also revealed significant differences in the metabolism of psora-
lidin between species. The Km values in DLMs, RLMs and PLMs
were much higher than those in HLMs and all the differences are
statistically significant (all P < 0.01). In comparison, Km values in
MLMs were comparableto the Km values of HLMs. The Km values in
HLMs and MLMs indicated comparable binding affinities towards
the metabolic site and reflected species similarities [35,36]. Simul-
taneously, the chromatogram and P3 peak area of monkeys shown
in Fig. 2 was also similar to humans. These results indicated that
monkeys might be a suitable choice to serve as a surrogate model
for drug metabolism and pharmacokinetics studies of psoralidin.
This conclusion agrees well with previous publications in which
monkeys are reported to be closely related to humans and share
a common ancestor from roughly 25 million years ago. Because
they are genetically and physiologically similar to humans, mon-
keys are the most widely used nonhuman primate in basic and
applied biomedical research [37].
Wang and Geng found that psoralidin produced 13 metabolites
by cyclization, dehydration, hydroxylation and glucuronidation
reaction [38,39], 9 of which metabolites were not identified in our
study. However, we identified 7 metabolites (M3, M5, M7–M11)
that were not reported by them. The inconsistent between our
and previous studies may be caused by differences in the selec-
tion of model.Compared with their studies using rats as model,
livermicrosomes from different species were used to simulate the
in vivo metabolism of psoralidin in our study which can identify
the metabolic differences between different species. Additionally,
Sun et al.resported that psoralidin generated one or two monoglu-
curonides in HLMs and UGT1A9 was responsible for 99.6% of
psoralidin-3-O-glucuronidation [40]. The monoglucuronides did
not be detected in our experiment. Due to the purpose of our study
that more and non-target metabolites of psoralidin were expected
to explore, the data acquiring and ananlysis method developing
would not be most suitable for monoglucuronides of psoralidin. Our
results were valuable and helpful for understanding and depicting
the metabolism profiles of psoralidin comprehensively. Further-
more, structural skeleton and potential metabolic pathways of
psoralidin and its metabolites among different species were deter-
mined and analyzed. Metabolites of psoralidin yielded in DLMs
were less than those in other species, which was consistent with
the results determined by HPLC-UV. M1 and M3 were found in
all species and their peak areas were more than 1%, which indi-
cated that hydration and hydroxylation were the major metabolic
transformations of psoralidin. In particular, M3 had biggest peak
area among the metabolites. Furthermore, we inferred M3 was
the metabolite P3 described in Fig. 2. In this study, all metabolites
were only measured by mass spectrometry, and more spectromet-
ric methods (i.e., NMR) are warranted in the future.
Molecular modeling studies were carried out to explore the
interactions between psoralidin and CYP1A2, CYP2C8 or CYP2C19
to further understand the key residues involved in binding
and recognition between the substrate and enzymes. Psora-
lidin interacted with CYP1A2 exclusively through hydrophobic
interactions, while it interacted with CYP2C8 through hydrogen
bonds and hydrophobic interactions. The hydrogen bonds formed
between CYP2C8 and psoralidin imply that psoralidin exhibited
stronger interactions with CYP2C8 than CYP1A2. These molecular
 X. Shi et al. / J. Chromatogr. B 1029 (2016) 145–156
modeling findings also agree with the above-mentioned experi-
mental results in which psoralidin had lower IC50 and Ki values
for CYP2C8 than CYP1A2. In addition, psoralidin also interacted
with CYP2C19 through hydrogen bonding and hydrophobic inter-
actions. These results support the high binding affinity between
CYP2C19 and psoralidin and that CYP2C19 was the major enzyme
participating in psoralidin metabolism.
5. Conclusion
Psoralidin exhibited potent inhibition against UGT1A1, UGT1A7,
CYP1A2 and CYP2C8, which may lead to drug-drug interactions.
Eleven metabolites were observed in the liver microsome incu-
bation system. CYP2C19 was the major isozyme involved in the
metabolism of psoralidin. Monkeys possessed the metabolic capac-
ity that is most similar to humans and could be used as the animal
selection in advanced toxicology or pharmacokinetic studies of pso-
ralidin in the future.
Conflict of interests
None.
Acknowledgment
We acknowledged that financially supported fromthe Natural
Science Foundation of Liaoning Province (Project No. 2014022005).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016.
06.031.
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