Journal of Ethnopharmacology 155 (2014) 1473–1482

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Multifaceted interaction of the traditional Chinese medicinal herb

Schisandra chinensis with cytochrome P450-mediated drug metabolism
in rats
Baolian Wang, Shuang Yang, Jinping Hu, Yan Li n
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, No.1 Xiannongtan Street, Beijing 100050, China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Schisandra chinensis (SC), officially listed as a sedative and tonic in the
Received 20 December 2013 Chinese Pharmacopoeia, has been used as a common component in various prescriptions in Traditional
Received in revised form Chinese Medicine (TCM) and more recently in western medicine for its antihepatotoxic effect. To assess
14 April 2014
the possible herb–drug interaction, effects of SC extracts on hepatic cytochrome P450 (P450, CYP)
Accepted 15 July 2014
enzymes were studied.
Available online 1 August 2014
Material and methods: Effects of SC extracts on rat hepatic CYP450 enzymes in vitro and in vivo were
Keywords: investigated by probe substrates method, real-time RT-PCR assay and Western blotting analysis.
Schisandra chinensis Furthermore, the effects of SC alcoholic extract on the PK of four SC lignans and the drugs possibly co-
CYP450
administrated in vivo were studied in male Sprague-Dawley rat.
Inhibition
Results: SC aqueous extract and alcoholic extract showed significant inhibitory effect on the activities of
Induction
Herb–drug interaction rat liver microsomal CYP1A2, 2C6, 2C11, 2D2, 2E1 and 3A1/2 in vitro. Multiple administrations of SC
aqueous extract (1.5 g/kg, qd
7d) and alcoholic extract (1.5 g/kg, qd
7d) increased the activities, mRNA
Chemical compounds studied in this article:
and protein expressions of CYP2E1 and CYP3A1/2, and meanwhile, inhibited the activities and mRNA
Schisandrin (PubChem CID: 3001664)
expression of CYP2D2 in vivo. The in vivo metabolism of four SC lignans, such as schisandrin,
Schisantherin A (PubChem CID:151529)
Deoxyshisandrin (PubChem CID:155256) schisantherin A, deoxyshisandrin and γ-schisandrin, and chlorzoxazone was significantly accelerated,
γ-schisandrin (PubChem CID: 108130) exhibited by the reduced AUC and increased CLz/F, by 7-day pretreatment with SC alcoholic extract.
Chlorzoxazone (PubChem CID:2733) However, both single and multiple dosing treatments of SC alcoholic extract remarkably decreased the
Sertraline(PubChem CID:68617) in vivo metabolism of tacrolimus indicated by the enhanced AUC (7–12 fold) and elevated Cmax (10 fold).
Tacrolimus(PubChem CID: 5282315) Conclusion: These results revealed that the SC extracts exhibited multifaceted effects on rat hepatic
CYP450 enzymes. Herb–drug interaction should be paid intense attention between SC components and
drugs metabolized by different CYP450 enzymes.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cytochrome P450 (CYP) enzymes constitute a superfamily of

The use of herbs as the complementary and alternative med-
icine (CAM) is on the rise and gaining increased popularity (Kelly
et al., 2005). Meanwhile, it has been reported more and more
herb–drug interactions related to the use of herbs or natural
products, such as St. John's wort (Hypericum perforatum) (Zhou
et al., 2004), Garlic (Allium sativum) (Gallicano et al., 2003), Ginkgo
(Ginkgo biloba), Ginseng (Panax ginseng), Milk thistle (Silybum
marianum) and so on (Pal and Mitra, 2006). Based on these
evidences, the herb–drug interactions have drawn much attention
in the medical research community around the world.
n
Corresponding author. Tel.: þ 86 10 63165172.
E-mail addresses: wangbaolian@imm.ac.cn (B. Wang), yanli@imm.ac.cn (Y. Li).
hemoproteins that play an important role in the metabolism of
xenobiotics (Coon, 2005). As the expression of individual CYPs can
be regulated by both endogenous factors and foreign compounds
including drug and natural compounds (Mrozikiewicz et al., 2010),
herb–drug interactions may occur due to the induction or inhibi-
tion on specific CYPs when herb and a given chemical drug are co-
administered. Clearly, more studies are needed to assess the
potential impact of herbal components on hepatic CYPs so as to
predict herb–drug interaction advancing rational use of herbs in
clinic.
Schisandra chinensis (SC), officially listed as a sedative and tonic
in the Chinese Pharmacopoeia, has been used as a common
component in various prescriptions in Traditional Chinese Medi-
cine (TCM) and more recently in western medicine for its anti-
hepatotoxic effect (Pharmacopoeia of the PR China, 1988). It has

http://dx.doi.org/10.1016/j.jep.2014.07.026
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
1474 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482
been shown that the extracts or major constituents (Schisandra
lignans) of SC have various biological activities, such as hepato-
protective effect (The State Pharmacopoeia of the Union of Soviet
Socialist Republics, 1966), antioxidative property (Lu and Liu,
1992), anticarcinogenic activity through the activation of key liver
enzymes (Liu et al., 1982) and strong inhibitory effect on human
immunodeficiency virus (HIV) (Fujihashi et al., 1995). Several
studies have reported the effects of SC extracts on CYPs. Tang
et al. indicated that aqueous extract of SC Baill induced the content
of rat hepatic microsomal cytochrome P450 and accelerated the
metabolism rate of imipramine (Tang et al., 2005). However,
Pei et al. found that deoxyshisandrin, lignans from SC, could
inhibit the activity of rat hepatic CYP3A and decrease the Vmax of
6-hydrotestosterone production (Pei et al., 2006). Iwata et al.
identified gomisins B, C (schisantherin A), G, and N and γ-
shizandrin, lignans from Schisandra fruit extract, having inhibitory
effects on CYP3A4 in human liver microsomes system (Iwata et al.,
2004). More recently, Su et al. reported that the unprocessed or
vinegar-processed Schisandra chinensis extract induced CYP3A4
enzyme activity and inhibited CYP1A2 enzyme activity in rats after
single or multiple dosing treatments. In addition, CYP2E1 enzyme
activity was induced after treatment with a single dose but was
inhibited after multiple doses (Su et al., 2013). Based on these
discrepant results, more studies are required to clarify the effects
of SC components on specific hepatic CYPs and give more insight
into the guideline of rational administration for using this herbal
medicine.
In the present study, we investigated the inhibitory potential of
SC extracts and four active SC lignans on six liver P450s (CYP1A2,
2C6, 2C11, 2D2, 2E1, 3A1/2), which are the isoforms involved in the
majority of clinical drug-metabolized reactions, in rat liver micro-
some incubation using a probe method in vitro. Moreover, the
effects of multiple administrations of SC extracts on rat liver P450
activities, mRNA and protein expressions were also characterized.
Finally, based on the results of the inhibition or induction experi-
ments, further studies were performed to investigate the effect of
alcoholic extract on pharmacokinetics (PK) of the drugs possibly
co-administrated in vivo. The results obtained from the study will
provide reliable experimental data for safe and effective use of SC
preparation in clinical practice.
2. Materials and methods
2.1. Materials
Schisandrin, schisantherin A, deoxyshisandrin, γ-schisandrin
(4 99% pure) and midazolam were purchased from the National
Institute for the Control of Pharmaceutical and Biological Products
(Beijing, China). Schisandra chinensis (Wuweizi) for preparation of
aqueous extract was obtained from Beijing Tongrentang Medicine
Corporation (Beijing, China). Dried alcoholic extract of SC (Batch
no.: SC070428), containing schisandrin (1.84%), schisantherin A
(1.54%), deoxyshisandrin (2.43%) and γ-schisandrin (1.23%),
was purchased from Nantong Sihai Plant Extracts Co., Ltd.
(Nantong, China). Phenacetin, acetaminophen, dextromethorphan,
dextrophan, diclofenac, 4-hydroxydiclofenac, mephenytoin,
4-hydroxymephenytoin, chlorzoxazone, 6-hydroxychlorzoxazone,
1-hydroxymidazolam, sertraline and tacrolimus (FK506) ( 499%
pure) were obtained from Shanghai Sigma-Aldrich Corporation.
High Capacity cDNA Reverse Transcription Kit, TaqMans Gene
Expression Assays, and TaqMans Real-Time PCR Master Mix were
products of Applied Biosystems (USA). Polyclonal antibodies
against rat CYP2E1 and CYP3A were products from Abcam Cor-
poration and Santa Cruz Corporation (Santa Cruz, USA),
respectively. Beta-actin antibody, HRP-labeled goat anti-rabbit
IgG were obtained from Beijing Zhongshan Goldenbridge Biotech-
nology Co., Ltd. Sertraline hydrochloride tablets and tacrolimus
hydrate were purchased from Pfizer Pharmaceutical Inc. and
Astellas Pharmaceutical Inc. (China), respectively.
Methanol was of HPLC grade (Fisher, USA). All other chemicals
were of analytical reagent grade. Ultrapure water, prepared using a
Milli-Q Reagent water system (Millipore, MA, USA), was used
throughout the study.
2.2. Preparation of aqueous SC extract
SC powder was mixed 1:3 with water (w/v) using a Soxhlet
apparatus for 3 h. The mixture was filtered and the filtrate was
dried under reduced pressure using a rotary evaporator at low
temperature(0–4 1C) as described previously (Pekthong et al.,
2008). The contents of the four lignans in the prepared aqueous
extract of SC, determined according to an established HPLC–MS
method (Wang et al., 2008), included 0.04% (w/v) schisantherin A
and 0.029% (w/v) deoxyshisandrin with undetectable schisandrin
and γ-schisandrin.
2.3. Inhibition of CYPs in vitro
The inhibitory effects of the SC aqueous extract, alcoholic extract
and the four pharmacologically active SC lignans on the activities of
CYP1A2, 2C6, 2C11, 2D2, 2E1 and 3A1/2 were investigated in normal
rat liver microsomes incubation. The tested concentrations of SC
extracts and lignans were as follows: 120 and 28 μg/mL for alcoholic
extract; 500 and 100 μg/mL for aqueous extract; 50, 10 and 5 μM for
the lignans. The activities of CYPs were determined by using probe
substrates, as described below. Control incubations without inhibi-
tor (but containing the solvent used to dissolve the inhibitor),
enzyme or substrate were routinely included.
2.4. Effect on P450s in vivo
All animal protocols were approved by Institutional Animal
Care and Welfare Committee of the Chinese Academy of Medical
Sciences. Sprague-Dawley rats (adult male), weighing 180–220 g,
were obtained from Beijing Vital River Experimental Animal Co.,
Ltd. The animals were quarantined for 1 week prior to study, and
were maintained on a 12-h light/12-h dark cycle at 227 1 1C and
60% relative humidity, with free access to solid pellet diet and
water throughout the study. No animal was replaced following the
beginning of the study. Rats, divided into 4 groups of 6 animals
each, were treated orally with aqueous extract (1.5 g/kg/day) or
alcoholic extract (1.5 g/kg/day) suspended in 0.5% sodium carbox-
ymethyl cellulose (CMC) once a day for 7 consecutive days. The
dosages were set according to the amounts of the four active
lignans used previously for pharmacological studies (Zhang et al.,
2001; Guo et al., 2006) and their contents in the extracts. Rats
from the control group were treated with the vehicle, under
similar conditions, while positive control rats were intraperitone-
ally injected with dexamethasone (DEX, 100 mg/kg/day) for 4 con-
secutive days. The animals were sacrificed by decapitation at 24 h
after the last administration and liver tissues were removed
rapidly for microsome preparation.
2.5. Preparation of liver microsomes
Liver microsomes were prepared by differential centrifugation as
described previously (Eriksson, 1978). The pellet was resuspended in
homogenization medium and stored at 80 1C until use. Protein
concentrations and contents of P450 enzymes were determined
 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482 1475

Fig. 1. Inhibitory effects of Schisandra chinensis (SC) extracts and four schisandra lignans on rat liver microsomal P450 activities in vitro. The probe substrates tested and
metabolites detected are shown in Table 1. The incubation conditions and methods for metabolites analysis are shown in Section 2. A: SC alcoholic extract (120 μg/mL),
SC aqueous extract (500 μg/mL), individual lignans (50 μM); B: SC alcoholic extract (28 μg/mL), SC aqueous extract (100 μg/mL), individual lignans (10 μM); and C: individual
lignans (5 μM). Data represent means 7 SD (n ¼5). *Po 0.05; **Po 0.01; compared to the control group (no inhibitor).

using the Bradford method and Omura method (Omura and Sato,
1964), respectively.
2.6. Measurement of CYP activities with probe substrates
The activities of CYP450 isozymes were determined according
to the probe substrate methods reported previously (Zhang et al.,
2002; Li et al., 2007).The rates of metabolite formation for six
probe substrates, such as phenacetin for CYP1A2, diclofenac for
CYP2C6, S-mephenytoin for CYP2C11, dextromethorphan for
CYP2D2, chlorzoxazone for CYP2E1, and midazolam for CYP3A1/
2, were determined, as indicators of the activities of respective CYP
isoforms in rat liver microsomes. The incubation mixtures (final
volume¼0.5 mL) contained 50 mM Tris buffer (pH 7.4), probe
substrates, 4 mM MgCl2, and 1 mg/mL microsomal protein. The
reaction was initiated by the addition of an NADPH-generating
system (0.011 M β-NADP, 10 U glucose-6-phosphate dehydrogen-
ase, 0.1 M glucose-6-phosphate), and terminated by the addition
of an equal volume of ice-chilled acetonitrile. All metabolites were
determined using validated LC–MS/MS methods.
2.7. RNA extraction and real-time RT-PCR assay
Total RNA from liver tissue was isolated with TRIzols reagent
according to the manufacturer's instructions. Gel electrophoresis
was carried out with 1% (w/v) agarose gel to confirm the integrity
of the RNA sample. Two sharp bands of 18S and 28S rRNA
indicated intact RNA. cDNAs were synthesized using High Capacity
1476 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482

cDNA Reverse Transcription Kit (Applied Biosystems, USA). The
specific TaqMan Gene Expression Assays (Inventoried) for Cyp1A2
(assay ID, Rn 00561082m1), Cyp2C11 (Rn 01502203m1), Cyp2D2
(Rn 00562419m1), Cyp2E1 (Rn 00580624m1), Cyp3A1 (Rn
03062228m1), Cyp3A2 (Rn 00756461m1) and GAPDH (Rn
01775763g1) were commercially available and purchased from
Applied Biosystems. The real-time RT-PCR was performed on an
ABI Step One Plus System (Applied Biosystems) with Data Assist
software. The PCR conditions were as follows: activation of
AmpliTaq Gold DNA Polymerase at 50 1C for 2 min and 95 1C for
10 min, and then amplification with denaturation at 95 1C for 15 s,
and annealing and extension at 60 1C for 1 min. The amplified
products of CYP1A2, CYP2C11, CYP2D2, CYP2E1, CYP3A1, CYP3A2,
and GAPDH were detected directly by monitoring the fluorescence
of the reporter dye (FAM), for which an increase in fluorescence
signal was detected only if the target sequence was complemen-
tary to the probe and amplified by the PCR.
2.8. Western blotting analysis
The protein levels of CYP3A, CYP2E1, and β-actin were deter-
mined by Western blotting. Rat liver microsomal proteins were
separated on 12% (w/v) SDS-polyacrylamide gel and then trans-
ferred to PolyScreen PVDF membranes at 80 V for 40 min in
transfer buffer (20 mM Tris-base, 154 mM glycine, 20% methanol).
The membranes were blocked with 5% non-fat milk powder in
buffer (200 mM NaCl, 0.05% Tween 20 and 50 mM Tris–HCl,
pH 7.4) at room temperature for 2 h, incubated with primary
antibodies at 4 1C for 14 h, washed, and then incubated with
secondary antibodies at room temperature for 1 h. The blots with
HRP-conjugated antibodies were developed by the addition of ECL
reagents (Applygen Technologies Inc., Beijing). The relative abun-
dance of the detected proteins was determined by densitometry,
with use of the Quantity One analysis software (Bio-Rad company).
2.9. Effect of multiple administrations of SC alcoholic extract on PK of
the four SC lignans
Male Sprague-Dawley rats were divided randomly into treated
groups and vehicle-control group. Alcoholic extract (1.5 g/kg/day)
suspended in 0.5% CMC was given to rats via oral gavage once a
day for 7 consecutive days, while the control group received an
equal volume of the vehicle. On day 8, all rats were administrated
alcoholic extract of SC at a dosage of 1.5 g/kg. Blood samples
( 250 μL each) were collected from retro bulbar plexus at 0, 0.25,
0.5, 1, 2, 3, 4, 6, 8, 12, 24 and 36 h after dosing. Plasma was
separated by centrifugation and stored at  20 1C until analysis.

Table 1
Effect of in vivo treatments of rats with Schisandra chinensis extracts on the activities of six liver microsomal P450sa.

Rates (pmol/min/mg protein)

Enzyme Probe (concentration)
Control Alcoholic extract Aqueous extract Dexamethasone

CYP1A2 Phenacetin (50 μM) 110 724 1347 12 1377 26 125 717
CYP2C6 Diclofenac (20 μM) 544 7106 565 7 103 593 7 178 527 794
CYP2C11 S-mephenytoin (100 μM) 52 710 337 7n 397 12 75 713n
CYP2D2 Dextromethorphan (15 μM) 262 725 967 11nnn 1247 21nnn 234 736
CYP2E1 Chlorzoxazone (100 μM) 354 79 13707 180nnn 653 7 132nn 369 737
CYP3A1/2 Midazolam (20 μM) 119 78 1717 17nnn 1537 17nn 268 729nnn

a
Data are expressed as means 7SD, n ¼5. Statistical analysis is performed using analysis of variance (ANOVA) and the Student–Newman–Keuls post-hoc test.
n
Po 0.05 compared to vehicle control.
nn
Po 0.01 compared to vehicle control.
nnn
Po 0.001 compared to vehicle control.

Fig. 2. Effect of in vivo treatments of rats with Schisandra chinensis (SC) extracts on the mRNA expression of six liver CYP450s. Adult male rats were treated orally with
aqueous or alcoholic SC extracts (1.5 g/kg), or with vehicle alone, once a day for 7 days, while positive control rats were intraperitoneally injected with dexamethasone (DEX,
100 mg/kg/day) for 4 consecutive days. The mRNA levels of these P450s were normalized to that of GAPDH. The normalized mRNA level of CYP450s is shown relative to the
level in control group. Each column represents the mean7 SD (n¼ 6). Significance is examined using the Student's t-test. **Po 0.01 vs control.
 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482
Fig. 3. Effect of in vivo treatments of rats with Schisandra chinensis (SC) extracts on
the protein expression of liver CYP2E1 and CYP3A. Adult male rats were treated
orally with aqueous or alcoholic SC extracts (1.5 g/kg), or with vehicle alone, once a
day for 7 days, while positive control rats were intraperitoneally injected with
dexamethasone (DEX, 100 mg/kg/day) for 4 consecutive days. Hepatic microsomes
were analyzed on immunoblots with an anti-CYP2E1 or anti-CYP3A antibody, as
described in Section 2. The same amount of protein was loaded in each lane (5 mg).
Relative levels of β-actin protein were also determined, as a loading control.
Densitometry results (in arbitrary units) represent means 7SD (n¼6). Representa-
tive blots are shown for one set of 6 microsomal samples, analyzed in duplicate.
*Po 0.05; **Po 0.01; vs vehicle control.
Plasma concentrations of the four lignans were determined by the
established LC–MS method (Wang et al., 2008).
2.10. Effect of SC alcoholic extract on the PK of sertraline,
chlorzoxazone and tacrolimus
Male Sprague-Dawley rats were divided randomly into 7-day
SC treatment groups, 1-h SC treatment groups, and corresponding
vehicle control groups, for each co-administered drug. For the
7-day SC treatment groups, rats were treated orally with alcoholic
extract (1.5 g/kg/day) suspended in 0.5% CMC once a day for
7 consecutive days, while the 1-h SC treatment groups and control
groups received an equal volume of the vehicle. On day 8, all rats
were orally administrated sertraline (20 mg/kg), chlorzoxazone
(100 mg/kg) or tacrolimus (1 mg/kg) except the 1-h SC treatment
groups which were pretreated orally with SC alcoholic extract
(1.5 g/kg) ahead of 1 h. Blood samples (  250 μL) were collected
from retro bulbar plexus at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and
24 h after oral dosing. Plasma was separated by centrifugation and
stored at  20 1C for analysis.
Plasma concentration of sertraline, chlorzoxazone or tacroli-
mus was determined by established LC–MS/MS methods (Jia et al.,
2007; Ansermot et al., 2008; Wang et al., 2010).
2.11. Data analysis
All plasma concentration data fitting and pharmacokinetic
parameter calculations were carried out using analysis software
1477
Phoenix WinNonlin (Pharsight, USA). Non-compartmental analysis
was chosen for the calculation of pharmacokinetic parameters.
Data are expressed as means 7SD. Statistical analysis was per-
formed using analysis of variance (ANOVA) and the Student–
Newman–Keuls post-hoc test. Differences between groups were
considered significant at P o0.05.
3. Results
3.1. Inhibition of SC extracts and four lignans on the activity of rat
liver microsomal CYPs in vitro
The inhibitory effects of SC components on the activities of six
CYPs were determined in rat liver microsomal incubations in vitro.
The activities of CYP enzymes were characterized by the rates of
metabolism of probe drugs. As shown in Fig. 1A, the activities
representing the six CYPs are all significantly inhibited by the
alcoholic extract at 120 μg/mL or the aqueous extract at 500 μg/
mL; these concentrations are confirmed in the range attainable in
gastrointestinal tract in vivo after oral administration of SC extracts
to rat in a preliminary experiment. Moreover, when the concen-
trations of alcoholic extract and aqueous extract in the incubation
were reduced by 80%, the six CYPs were still obviously inhibited,
by 39–73% (alcoholic) and 18–59% (aqueous) (Fig. 1B). In addition,
the four active SC lignans, such as Schisandrin, schisantherin A,
deoxyshisandrin and γ-schisandrin, also caused significant, but
CYP- and lignin-specific, inhibitions at the concentrations of
50 μM (Fig. 1A) and 10 μM (Fig. 1B), while no inhibition was
observed at 5 μM (Fig. 1C).
3.2. Effect of SC extracts on liver microsomal CYPs in rats in vivo
Significant increases in liver microsomal P450 content were
observed after seven daily oral administrations of either alcoholic
(0.90 70.07 nmol/mg protein; mean 7SD; P o0.01, n ¼5) or aqu-
eous (0.737 0.08 nmol/mg protein; P o0.05, n ¼5) SC extracts (at
1.5 g/kg), compared to values for vehicle-treated animals
(0.59 70.09 nmol/mg protein). As a positive control, treatment of
rats with DEX at 100 mg/kg/day (i.p.) for 4 consecutive days
increased hepatic microsomal P450 content to 0.92 70.33 nmol/
mg protein (Po0.01, n¼ 5, compared to vehicle control). Besides,
in the alcoholic extract group, the activities of CYP2E1 and CYP3A,
as indicated by microsomal activities toward their respective
probe substrates metabolism (6-hydroxychlorzoxazone and
1-hydroxymidazolam), were markedly increased by 3.9-fold and
1.5-fold, respectively, compared with those of the vehicle-control
group (Table 1). On the other hand, the activities of CYP2C11 (64%
of control) and CYP2D2 (37% of control) appeared to be inhibited
by the alcoholic extract. The aqueous extract of SC caused an 1.8-
fold and 1.3-fold induction of CYP2E1 and CYP3A1/2, respectively,
but a 53% suppression of CYP2D2 (Table 1). Consistent with the
previous results, the CYP2C11 and CYP3A1/2 were strongly
induced by intraperitoneal injection of dexamethasone (DEX,
100 mg/kg/day) for 4 days. These results showed that multiple
administrations of SC extracts cause divergent effects on different
CYPs, inducing certain isoforms while suppressing others, even
though the total P450 content was increased.
3.3. Effect of SC extracts on the expression of rat liver CYPs mRNA
Real time RT-PCR assays were performed to characterize the
gene expression of specific liver CYP450s in rats (Fig. 2). After
multiple doses of SC alcoholic extract or aqueous extract, the
hepatic mRNA expressions of CYP2E1, CYP3A1 and CYP3A2 were
significantly increased, while the levels of CYP2C11 and CYP2D2
1478 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482

were obviously inhibited compared to those of the control group.
The mRNA expression of CYP1A2 remained unchanged in all the
treated groups. Agreeing with the previous report, the mRNA
expression of CYP2C11, CYP3A1 and CYP3A2 was strongly induced
by the injections of dexamethasone (DEX) as a positive control.
extract enhanced CYP2E1 and 3A enzyme activities through up-
regulation of their protein expression.
3.5. Effect of multiple administrations of SC alcoholic extract on PK of
the four SC lignans

As several P450s that are inhibited or induced by SC components

3.4. Effect of SC extracts on the expression of rat liver CYP2E1 and
CYP3A proteins
Consistent with the results of CYP activity determination, both
alcoholic and aqueous extracts of SC significantly elevated the
protein levels of hepatic CYP2E1 and CYP3A in rats (Fig. 3).
Compared with the aqueous extract, the alcoholic extract was
more potent in the induction of CYP2E1, but the two preparations
caused similar induction of CYP3A. Consistently, dexamethasone
(DEX) strongly increased the expression of CYP3A, but not CYP2E1.
These results suggested that SC alcoholic extract and aqueous
after multiple administrations to rats may be involved in the
metabolism of the SC lignans, we further examined the effects of
the multiple dosing of SC alcoholic extract (for seven consecutive
days) on the PK profiles of the four lignans contained in the SC
alcoholic extract that was given on the 8th day (Fig. 4). We found
that most of the PK parameters of the four lignans including Cmax,
AUC, MRT and CLz/F were significantly reduced in rats pretreated
with the alcoholic extract for seven days, compared with rats that
received vehicle alone during the pretreatment period (Table 2).
These results suggest that overall the in vivo metabolism of the
lignans was accelerated by pretreatment of rats with the SC extract.

Fig. 4. Effects of the 7-day pretreatment with Schisandra chinensis (SC) alcoholic extract on systemic clearance of the four SC lignans. Adult male rats were treated orally with
the SC alcoholic extract (1.5 g/kg), or with vehicle alone, once a day for 7 days. At 24 h after the 7th dose, rats in both groups were treated once with SC alcoholic extract
(at 1.5 g/kg), and blood samples were obtained, at various times thereafter, for determination of SC lignan levels, as described in Section 2. A: schisandrin; B: schisantherin A;
C: deoxyshisandrin; and D: γ-schisandrin. Data represent means 7 SD (n¼5).

Table 2
Effects of the 7-day pretreatment with Schisandra chinensis (SC) alcoholic extract on pharmacokinetic parameters of the four SC lignansa.

Lignans Treatment t1/2z (h) Cmax (ng/mL) Tmax (h) AUClast (ng/mL h) MRTlast (h) CLz/F (mL/h/kg)

Schisandrin Vehicle 5.5 71.2 1847.2 7 409.6 6 23048.4 7 6707.3 9.4 7 0.4 1242.57 277.9
SC 13.7 76.2n 421.0 7 111.1nnn 1 1727.3 7 576.0nnn 4.9 7 0.6nnn 14304.3 7 3415.8nnn
Schisantherin A Vehicle 9.1 72.0 654.0 7 126.0 6 10743.5 7 3043.5 12.17 0.7 2086.1 7 425.2
SC 7.6 71.6 362.17 59.1nn 1 3833.07 339.4nnn 7.9 7 0.6nnn 5323.0 7 587.5nnn
Deoxyshisandrin Vehicle 5.9 70.8 732.17 123.3 6 9042.17 2941.5 9.17 0.6 4242.87 1180.7
SC 2.3 70.5nnn 628.0 7 86.3 1 2302.3 7 1184nn 2.9 7 0.5nnn 17887.1 7 6165.7nn
γ-schisandrin Vehicle 11.2 75.9 313.8 7 102.8 8 4746.17 1088.8 13.17 1.1 3454.5 7 442.3
SC 6.2 73.1 808.17 108.3nnn 1 3830.8 7 1860 4.5 7 0.8nnn 5242.87 1702.8

a
Data shown in Fig. 4 are used for calculation of pharmacokinetic parameters. The results are shown as means 7SD (n¼ 5). Statistical analysis is performed using
analysis of variance (ANOVA) followed by the Student–Newman–Keuls test – comparison between vehicle and SC groups.
n
Po 0.05.
nn
Po 0.01.
nnn
Po 0.001.
 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482
Fig. 5. Plasma concentration–time profiles of sertraline (A), chlorzoxazone (B), and
tacrolimus (C) for rats pretreated with the alcoholic extract of Schisandra chinensis
(SC) for one hour or seven days. For the 7-day SC treatment groups, rats were
treated orally with alcoholic extract (1.5 g/kg/day) once a day for 7 days, while the
control groups received vehicle alone. On day 8, all rats were orally administrated
sertraline (20 mg/kg), chlorzoxazone (100 mg/kg) or tacrolimus (1 mg/kg). For the
1-h SC treatment and control groups, rats were pretreated orally with alcoholic
extract (1.5 g/kg), or vehicle, at 1 h before administration of one of the three test
drugs. Plasma concentration of sertraline, chlorzoxazone or tacrolimus was
determined as described in Section 2. Data represent means 7 SD (n¼ 5).
3.6. Effect of SC alcoholic extract on the PK of sertraline,
chlorzoxazone and tacrolimus
Further in vivo interaction studies were performed to investi-
gate the impact of SC alcoholic extract on PK of three drugs
selectively metabolized by CYP2D (sertraline, an antidepressant),
CYP2E1 (chlorzoxazone, a probe drug), and CYP3A (tacrolimus, an
immunosuppressant). The mean plasma concentration–time pro-
files for the three drugs in rats pretreated with a single oral dosing
(1-h SC treatment group) or multiple oral administrations (7-day
SC treatment group) of SC are shown in Fig. 5(A–C), and the
relevant pharmacokinetic parameters are listed in Table 3.
For sertraline, the Cmax value in 7-day SC treatment group was
relatively higher than that in control group. Except that, no
significant differences in other calculated PK parameters were
observed among the three treatment groups.
1479
For chlorzoxazone, a moderate, but significant increase in
clearance was observed in the multiple-dose SC treatment group,
as indicated by a 35% decrease in AUC value and a 20% decrease in
Cmax value, compared to the control group; however, the single-
dose SC pretreatment group was not significantly different from
the control group.
For tacrolimus, large and significant decreases in clearance
were found for both single-dose and 7-day SC treatment groups,
compared to controls, as indicated by substantial increases in AUC
(6–13 fold) and Cmax (  10 fold) values (Table 3). These results
demonstrate the multifaceted nature of interactions between SC
and drugs metabolized by different P450 enzymes.
4. Discussion
Natural products, as used by the general population, are usually
complex mixtures of many compounds. Both the putative active
ingredient(s) and other constituents present in the mixture have
the potential to cause interactions with various classes of drugs (Pal
and Mitra, 2006). It has been proved that hepatic P450 enzymes
were involved in the metabolism of most clinical drugs. The
induction and inhibition of CYPs were considered as the major
mechanism of metabolic drug–drug interactions (Flockhart and
Oesterheld, 2000). Therefore, it is important and meaningful to
study the modulation of CYPs by herbal components for clarifying
mechanisms of herb–drug interactions.
Previous studies have indicated that the components from SC
have an induction or inhibition on CYPs, exemplified by the
declaration that aqueous extract of SC Baill was shown to induce
the content of rat hepatic microsomal P450. The lignans from SC
like deoxyshisandrin, gomisin B, C (schisantherin A), G, N and
γ-shizandrin, could significantly inhibit the activity of rat hepatic
CYP3A. Lately, it was been found that unprocessed or vinegar-
processed SC extract could induce CYP3A4 enzyme activity and
inhibit CYP1A2 enzyme activity in rats after single or multiple
dosing treatments. Meanwhile, CYP2E1 activity was induced after
a single dose treatment but was inhibited after multiple doses. All
these discrepant studies most focused on the modulation of total
P450 content or CYP3A activity. More studies were necessary to
clarify the effects of SC components on other important CYP450
isozymes, and furthermore, the possible herb–drug interactions.
Considering most of the results reported before were observed in
rat, we chose rat as the target animal for the studies in vivo and
in vitro. In the present work, we systematically investigated the
effects of SC components on hepatic P450s in rats in vivo and
in vitro. Moreover, the metabolic herb–drug interaction was
studied between SC components and possible coadministrative
drugs. These results may provide more reliable experimental data
and scientific explanations for its rational clinical application or
related herb–drug interactions.
It was reported that CYP1A2, CYP2C9, CYP2C19, CYP2D6,
CYP2E1 and CYP3A4 were the major CYPs involved in the
metabolism of more than 95% of available clinical pharmaceuticals.
Therefore, the relevant isozymes in rat, CYP1A2, CYP2C6, CYP2C11,
CYP2D2, CYP2E1 and CYP3A1/2, were selected as the main CYPs
observed in the study. The activities of six major hepatic CYPs
were determined by a sensitive, precise and reliable probe
method. The related mRNA and protein expression of CYPs were
also investigated using real-time PCR and western blot methods.
The results indicated that six CYPs could be differentially either
induced or inhibited, even though total P450 was enhanced by
multiple administrations of SC extracts, which was interesting,
novel and different from the results reported before.
Among the six enzymes, CYP3A, the largest single portion of
P450 protein in human, acts as a substantial role in the metabolism
1480 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482

Table 3
Pharmacokinetic parameters of sertraline, chlorzoxazone, and tacrolimus in rats pretreated with the alcoholic extract of Schisandra chinensis (SC) for one hour or seven daysa.

Drug Treatment t1/2z (h) Cmax (ng/mL) Tmax (h) AUClast (ng/mL h) MRTlast (h) CLz/F (mL/h/kg)

Sertraline Control 6.4 7 0.4 336.17 33.6 2 3651.3 7 589.4 7.7 7 0.2 5111.0 7879.0
1-h SC 6.6 7 0.3 403.17 67.1 3 3996.0 7 890.2 7.6 7 0.2 4727.8 71018.2
7-day SC 7.17 1.5 437.9 7 49.7nn 3 3954.9 7 305.1 7.4 7 0.8 4544.0 7381.7
Chlorzoxazone Control 2.17 0.1 27767.7 7 2117.3 2 160471.6 7 4708.1 4.3 7 0.2 623.2 718.1
1-h SC 2.2 7 0.2 26596.8 7 1550.3 2 153658.4 7 11817.5 4.17 0.3 653.5 752.3
7-day SC 2.17 0.3 21728.8 7 6000.1 1 101878.17 5135.5nnn 3.7 7 0.6 983.2 749.8nnn
Tacrolimus Control 10.5 7 1.4 2.3 7 0.9 0.5 12.5 7 0.7 8.2 7 0.5 64112.9 74600.4
1-h SC 5.8 7 0.2nnn 26.17 6.2nnn 0.5 171.4 7 38.0nnn 6.8 7 0.8n 5715.9 71268.0nnn
7-day SC 8.17 2.0 21.2 7 4.3nnn 0.5 79.17 22.8nnn 5.9 7 0.6nnn 11739.8 72403.8nnn

a
Data shown in Fig. 5 are used for calculation of pharmacokinetic parameters. The results are shown as means 7SD (n¼ 5). Statistical analysis is performed using
analysis of variance (ANOVA) followed by the Student–Newman–Keuls test – comparison among three treatment groups.
n
Po 0.05.
nn
Po 0.01.
nnn
Po 0.001.

of a vast array of pharmaceutical products. CYP2E1 is considered to
be responsible for the metabolism of diverse kinds of small
molecular chemicals and drugs, accounting for 7% of total hepatic
CYPs in human. It was found that multiple dosages of both aqueous
and alcoholic extracts of SC significantly induced the activity, mRNA
and protein expressions of rat hepatic CYP3A and CYP2E1, which
suggested that the induction of CYP3A/2E1 by SC could be con-
ducted through the enhancement of mRNA transcription and post-
transcription.
CYP2D, characterized by its genetic polymorphism in human, is
the metabolic enzyme involved in the most clinical antidepres-
sants and diazepam-drugs. Different from the results for CYP3A
and CYP2E1, the mRNA expression and activity of CYP2D were
significantly inhibited by multiple dosages of SC extracts in the
study. Certainly, more studies are still needed to confirm the
interesting result by determination of CYP2D protein expression.
Besides, there is CYP2C subfamily composing 20% of total P450
which mainly participates in the metabolism of drugs for nervous
system, and CYP1A2 is believed to be very active in the detoxifica-
tion or activation of xenobiotics. For CYP1A2, SC extracts treatment
had no obvious effect on its activity or mRNA expression. However,
for CYP2C, further studies will be still needed considering the
mRNA expression of CYP2C11 was not agreed with the result of
activity.
Numerous studies demonstrated that the induction of meta-
bolic enzymes contributed to approximately 30% of drug interac-
tions, while the inhibition accounted for the rest 70%. It is valuable
to study the inhibitory effect of herbal components on CYPs in vitro
for the prediction of drug–drug interaction in vivo. Our results
revealed that both SC aqueous extract and alcoholic extract, at the
attainable concentration in vivo (28–120 μg/mL of alcoholic extract
and 100–500 μg/mL of aqueous extract), had significant inhibition
on six CYPs, while four active lignans at 10–50 μM also showed the
inhibitions on above enzymes. According to the plasma concen-
tration of four lignans (r5 μM) after oral administration of SC
extracts reported before (Wang et al., 2008), it was suggested that
four lignans might have possible inhibitory effect on CYPs in
gastrointestinal tract, whereas no impact on hepatic P450s. Except
the four active lignans, there might be other compounds which
contributed to the inhibition on CYPs in SC extracts. So the further
purification of extracts needs to be carried out for the clarification
of unknown potent inhibitors.
All results from above studies suggested that herb–drug inter-
action might happen between components of SC and coadminis-
trative drugs due to the inhibition or induction of CYPs. The
inhibitory effect is possibly leading to an increase in plasma drug
concentration during short time concomitant administration of SC
and prescribed drugs. In contrast, prolonged intake may result in
subtherapeutic drug concentrations due to the induction of CYPs.
However, xenobiotics metabolism and disposition in vivo were
affected by many factors considering the complicated biological
conditions. Further study at whole animal level is necessary to
confirm whether drug pharmacokinetic process will be changed
significantly by the interaction mediated by CYPs, which could
provide more reliable information and reference value for drug
application in clinic.
As multiple P450s involved in the metabolism of four effective
lignans were obtained from SC, we first observed their PK profile
difference between single and multiple dosing of SC alcoholic
extract to rat. The result revealed that multiple dosing of SC
alcoholic extract accelerated the metabolism of four lignans
possibly by induction of P450 isozymes in rat. The dosage might
need to be adjusted properly when SC preparations were used for
a long time in clinic. However, more studies are still needed to
confirm or explain the mechanism for the Cmax increase of γ-
schisandrin.
In addition, we selected tacrolimus, chlorzoxazone and sertra-
line, the respective substrate drugs of CYP3A, CYP2E1 and CYP2D
whose activities were inducted/inhibited significantly in our study,
to investigate the pharmacokinetics interaction with alcoholic
extract of SC.
Tacrolimus (FK506) is a new type of immunosuppressive drug
mostly metabolized by hepatic CYP3A. Because of its narrow
therapeutic window and unexpected toxic effect, plasma concen-
trations of patients have to be monitored in clinical application.
As liver transplant patients usually take tacrolimus with the
preparation of SC for hepatoprotection, CYP3A mediated interac-
tion between two of them may happen in all probabilities, which
may result in organ rejection or adverse effect. The present result
showed that the plasma concentration of tacrolimus was elevated
significantly (11-fold of Cmax, 13-fold of AUC) by single dose of
alcoholic extract of SC (1-h SC treatment), which was most
probably mediated by inhibition of CYP3A. However, the concen-
tration of tarcrolimus was also enhanced (9-fold of Cmax, 6-fold of
AUC) by multiple dosing of SC (7-day SC treatment) although the
CYP3A was induced. It was reported that tacrolimus was a CYP3A
and P-gp substrate. In this case, multiple dosing of SC might inhibit
the activity or expression of P-gp, which prevailed over the
induction of CYP3A and resulted in the elevation of tacrolimus
plasma concentration. Based on the data, the dosage of tacrolimus
should be adjusted to avoid the occurring of adverse effect when
this drug was used in combination with components from SC.
Chlorzoxazone, a probe drug of CYP2E1, was selected to study
the PK interaction after SC treatment. The result demonstrated
that single dose of alcoholic extract (1-h SC treatment) had no
obvious effect on chlorzoxazone PK profile, while multiple dosages
 B. Wang et al. / Journal of Ethnopharmacology 155 (2014) 1473–1482
(7-day SC treatment) accelerated the metabolism of chlorzoxa-
zone, indicated by the obvious changes of AUC and CLz/F, through
the induction of CYP2E1 activity, mRNA and protein expression.
Therefore, it would be suitable to modify the dosage of the drug
specifically metabolized by CYP2E1 when SC components were co-
administrated for a long time.
It was reported recently that antidepression agent sertraline,
substrate drug of CYP2D, induced adverse effect of severe liver
damage for which its plasma concentration should be controlled in
the safe range. In the present study, no significant change, except
the Cmax in 7-day SC group, on sertraline metabolism was observed
in both 1-h and 7-day SC treatment groups. The result might be
explained by the involvement of multiple P450 isozymes including
CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, in the biotrans-
formation of sertraline in vivo (Obach et al., 2005). The compen-
sative metabolism of sertraline could be dedicated by other
CYP450 isozymes when CYP2D was inhibited. Still, more studies
are necessary to determine the PK interaction between SC com-
ponents and the drugs exclusively metabolized by CYP2D.
The molecular mechanism for the modulation of SC compo-
nents on rat CYPs has not been explored in the present study.
A ligand-activated transcriptional factor, pregnane X receptor
(PXR), has been previously shown to mediate the effects of
xenobiotics on ligand-mediated expression of CYP3A gene. And
the aryl hydrocarbon receptor can mediate the effects of xenobio-
tics on ligand-mediated expression of the CYP1A2 gene in human
and other animals (Kliewer et al., 1998). Moreover, it was also
reported that the PXR was activated by extracts or constituents
from SC, CYP3A and CYP2C, which the affected enzymes included
(Mu et al., 2006). Therefore, the molecular mechanism of CYPs
induction/inhibition by SC components may be elucidated by the
activation of the PXR. Further studies are needed for the purifica-
tion and identification of the SC components as PXR agonists.
In summary, the present study has determined the inhibitory
and inductive effects of SC aqueous extract and alcoholic extract
on activity, mRNA and protein expression of six liver CYP450s in
rat in vitro and in vivo. Furthermore, the effects of SC alcoholic
extract on the PK of four SC lignans and the drugs possibly co-
administrated in vivo were investigated. The study provides useful
experimental data for rational assessment of the herb–drug
interaction between SC components and drugs metabolized by
CYP450 enzymes. Certainly, further investigation in clinic should
be considered to validate the conclusion because of the species
differences of CYP subfamilies between human and animals.
Acknowledgments
We are very grateful to Dr. Xinxin Ding (Laboratory of Molecular
Toxicology, Wadsworth Center, New York State Department of
Health, USA) to review the manuscript. This work was supported
by the National Natural Science Foundation of China [Grants
81072696 and 81202990] and the National Science and Technology
Major Project of China [Grant 2012ZX09301002-001-007].
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Glossary TCM: traditional Chinese medicine;

HIV: human immunodeficiency virus;
PK: pharmacokinetics;
SC: Schisandra chinensis; LC–MS: liquid chromatography–mass spectrometry;
CYP450: cytochrome P450; PXR: pregnane X receptor;
CYPs: cytochrome P450s; SD: standard deviation.
CAM: complementary and alternative medicine;