Structure-Activity Relationship and Substrate-
Dependent Phenomena in Effects of Ginsenosides on
Activities of Drug-Metabolizing P450 Enzymes
Miao Hao1, Yuqing Zhao2, Peizhan Chen1, He Huang3, Hong Liu3, Hualiang Jiang3, Ruiwen Zhang4, Hui
1Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Graduate
School of the Chinese Academy of Sciences, Shanghai, People’s Republic of China, 2Shenyang Pharmaceutical University, Shenyang, People’s Republic of China, 3Drug
Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China, 4Department of Pharmacology
and Toxicology, Division of Clinical Pharmacology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and
ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs).
Results from previous studies on ginsenosides’ effects on human drug-metabolizing P450 enzymes are inconsistent and
confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity
relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4.
Moreover, substrate-dependent phenomena were found in ginsenosides’ effects on CYP3A4 when another fluorescent
probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These
substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in
previous GDI reports.
Citation: Hao M, Zhao Y, Chen P, Huang H, Liu H, et al. (2008) Structure-Activity Relationship and Substrate-Dependent Phenomena in Effects of Ginsenosides on
Activities of Drug-Metabolizing P450 Enzymes. PLoS ONE 3(7): e2697. doi:10.1371/journal.pone.0002697
Editor: Jennifer Keiser, Swiss Tropical Institute, Switzerland
Received February 26, 2008; Accepted June 19, 2008; Published July 16, 2008
Copyright: ? 2008 Hao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a grant (06DZ19021) from the Science and Technology Commission of Shanghai Municipality, Pujiang Talent
Program (06PJ14107), a grant (2007CB947100) from the Ministry of Science and Technology of China (973 Program) and the Knowledge Innovation Program of
Chinese Academy of Sciences, Food Safety Research Center and Key Laboratory of Nutrition and Metabolism in INS. R.Z. was supported in part by NIH/NCI grants
R01 CA112029 and R01 CA121211. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
With the increasing use of alternative medicine and a wide
spread of combination therapies for various diseases, there is an
increasing interest in determining drug-drug, drug-nutrient, and
drug-dietary supplements interactions. For example, Panax ginseng,
a traditional herbal medicine used in the Eastern Asia for more
than 2000 years , is presently being used worldwide as one of
the most common complementary alternative medicines. Ginseng,
the root of Panax ginseng, has diverse pharmacological activities,
including effects on the central nervous system, antineoplastic and
immunomodulatory effects . In the clinical settings, however,
co-administration of ginseng or its extracts with other therapeutic
agents (e.g. warfarin, digoxin and phenelzine) may lead to ginseng-
drug interactions (GDIs) [3,4]. Ginsenosides (Fig. 1a), the major
active components of ginseng, may account for the GDIs and
other adverse effects . It is postulated that most metabolic drug-
drug interactions can be attributed to inhibition or induction of
drug-metabolizing cytochrome P450 (CYP or P450) enzymes .
Therefore, we hypothesized that investigations on the effects of
ginsenosides on P450s will help elucidate the mechanism of GDIs.
Unfortunately, the reported effects of ginseng extracts or ginseno-
sides on P450s are inconsistent, even confusing. For instance,
ginsenoside Rd weakly inhibits CYP3A4 and CYP2D6 and inhibit
CYP2C19 and CYP2C9 to an even lesser extent; and ginsenosides
Rb1,Rb2,Re,andRg1do notsignificantlyaffect CYP1A2,CYP2C9,
CYP2C19, CYP2D6 or CYP3A4 . In another study, however,
ginsenosides Rd and Rb2inhibited CYP2C19-dependent S-mephen-
ytoin 49-hydroxylation and Rd inhibited CYP2D6-mediated bufur-
alol 19-hydroxylation . Moreover, standardized Panax ginseng and
Panax quinquefolius extracts decrease the 7-ethoxyresorufin O-deal-
kylation activities of human CYP1A1, CYP1A2, and CYP1B1, but
ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf or Rg1have no significant
effects . Additionally, GDIs are probably mediated by ginsenoside
metabolites (e.g. protopanaxadiol and protopanaxatriol) rather than
enterobacteria before they enter the circulation . To date, some
80 ginsenosides have been isolated from Panax species  and new
ginsenosides are being found. Recently, we identified and character-
ized two novel potent antitumor ginsenosides, 20(R)-dammarane-3b,
12b, 20, 25-tetrol (25-OH-PPD)  and 20(S)-25-methoxyl-
dammarane-3b, 12b, 20-triol (25-OCH3-PPD) ; their effects on
P450s are still unknown.
We also speculated that there are structure-activity relationships
(SAR) for the effects of ginsenosides on P450s. Although several
ligand-based and structure-based models have been established for
PLoS ONE | www.plosone.org1 July 2008 | Volume 3 | Issue 7 | e2697
substrates and/or inhibitors of P450s , the SAR of the effects
of the ginsenosides on P450s were rarely addressed. In the present
study, we systematically evaluated the effects of 15 ginsenosides
and sapogenins (Fig. 1A) on five major human drug-metabolizing
P450 enzymes with commercially available VividH substrates to
use as probes (Fig. 1B). Subsequently, the SAR for these effects was
explored to generate new knowledge about ginsenoside-P450
interactions at the molecular level. Moreover, we also selected
different fluorescent and conventional probes to determine
whether the effects of different ginsenosides on CYP3A4, a key
P450 enzyme with the largest substrate repository, are substrate-
Ginsenosides and sapogenins affect P450 enzymes in an
analog- and enzyme-dependent manner
The parameters used to determine the activities of the P450
enzymes are summarized in Table 1. Reported potent inhibitors of
respective P450 enzyme were evaluated to validate the efficiency
of these fluorescent assays (Table 2). These inhibitors show
comparable activities against respective P450s to the documented
reports [16–19]. Additionally, the inhibitory effects of methanol, a
vehicle used in the study, were carefully evaluated. Methanol at
the final concentration of 1% had no significant inhibitory effect
Figure 1. Structures of ginsenosides (A), and VividH fluorescent probes (B). Note: a. The C-20 configurations of the test ginsenosides are
20(S) except when indicated behind the substituent groups in this column. b. Glc: b-D-glucopyranosyl; Xyl: b-D-xylopyranosyl; Rha: a-L-
rhamnopyranosyl. Numerical superscripts indicate the carbons at glucosidic bonds. c. C-K: ginsenoside Compound K. d. PPD: protopanaxadiol; PPT:
SAR of Ginsenosides on P450s
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against VividH blue metabolism by CYP1A2 or green metabolism
by CYP3A4, and had minimal inhibition against VividH blue
metabolism by CYP2C19 (Table 2). However, 1% methanol
significantly inhibited VividH CYP2C9 red metabolism (by 42.4%)
and blue metabolism by CYP2D6 (27.5%). At the final
concentration of methanol of 0.5% or 0.1%, the inhibition against
substrate metabolism by methanol was all either negligible or
within an acceptable range (Table 2).
Subsequently, we determined the effects of the ginsenosides and
sapogenins on five major cDNA-expressed P450 enzymes. The
compounds had weak inhibitory effects against CYP1A2, with the
exception of Rb1, Rg3, and Compound K (C-K) which had
moderate inhibitory effects with IC50 values less than 50 mM
(Table 2). Ginsenosides Rg3, Rh2, and C-K and all sapogenins
exhibited moderate inhibition against CYP2C19, while they more
potently inhibited CYP2C9 and CYP3A4 (green substrate)
Table 1. Parameters of the enzymatic reactions used to determine the activities of P450 enzymes.
Linear range of
RFU,t curve (min)a
CYP1A2 blue3 40946050,15
CYP2C9 red2 530585 100,30
CYP2C19 blue10 40946050,15
CYP2D6 blue 10 409460 100,30
CYP3A4 green2 485 53052,12
CYP3A4 red3 530 58550,10
Note: a. The linear range was determined by visual inspection; parameters for substrate concentration, wavelength and CYP450 concentration were provided by the kit
Table 2. IC50values of the ginsenosides and sapogenins against P450 enzymes.
IC50(mM) (%inhibition at 50 mM)a
CYP1A2 blueCYP2C9 redb
CYP2C19 blue CYP2D6 blueCYP3A4 green CYP3A4 red
1.5 0.500.25 0.00760.49 0.97
.50 (49.4%) 99.768.8
32.4 18.547.0 9.3 86.4 25.2
.100 (39.4%)30.9 49.9
.50 (20.3%)47.0 9.8
.50 (49.4%)30.5 9.1
.100 (19.4%) 47.0
.50 (25.9%)9.3 43.1
.100 (17.1%)6.7 19.2
.50 (34.9%) 10.358.6
.100 (13.1%)31.6 46.9
.50 (18.9%) 7.4
.50 (10.0%) 27.2A.A.
.100 (4.8%) 7.526.5
Note: a. The percent inhibition of ginsenosides against the respective P450 enzymes is shown when its IC50value is greater than the maximum concentration assayed.
b. The maximum concentration of ginsenosides evaluated for their effects on CYP2C9 and CYP2D6 were 50 mM due to the marked solvent effect of 1% methanol on
these two P450 enzymes (inhibition by 42.4% and 27.5%, respectively). When the final concentration of methanol was decreased to 0.5%, the solvent effects were
acceptable for these two enzymes (10.1% and 18.9%, respectively). 1% methanol had no inhibition against CYP1A2 and CYP3A4 and had an acceptable inhibitory effect
on CYP2C19 (9.7%).
c. Positive control compounds were a-naphthoflavone (for CYP1A2), sulfaphenazole (CYP2C9), miconazole nitrate salt (CYP2C19), quinidine (CYP2D6), and ketoconazole
d. A.A.=apparent activation. 100 mM 25-OH-PPD and 25-OH-PPT increased the turnover of VividH CYP3A4 red by more than 100%.
SAR of Ginsenosides on P450s
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(p,0.05). Rg3exerted relatively weak inhibitory effects against
VividH CYP3A4 green metabolism (Table 2). However, it potently
inhibited CYP2D6, for which all of the other ginseng compounds
had IC50values greater than 50 mM. Ginsenosides Rb1, Rb3, Rd,
Re, Rg2and Rg1weakly affected all five P450 enzymes, with the
exception of Rb1and C-K, which moderately inhibited CYP1A2
(Table 2). All of the sapogenins had weak inhibitory effects on
CYP1A2 and CYP2D6 (Table 2).
There is a SAR in the effects of ginsenosides on P450s
Considering that the same core structure shared by these
ginsenosides, the location, number and type of the glycosyl groups
covalently linked to the dammarane scaffold may account for the
their varying effects on P450 enzymes (Fig. 1A and Table 2). For
CYP2C9 and CYP2C19, the inhibition patterns of ginsenosides
are similar. Ginsenosides that do not have a glycosyl group on R2
and have two or fewer glycosyl groups on R1and R3as well as
sapogenins generally have moderate to potent effects on CYP2C9
and CYP2C19. As for CYP3A4 (green substrate), the inhibitory
activities of the ginsenosides and sapogenins decreased as the
number of glycosyl groups increased.
3D-SAR is shown in the effects of ginsenosides on VividH
CYP3A4 green metabolism
We applied the HipHop process from Catalyst software, which
compares diverse and highly active compounds to derive 3D
hypotheses based on common chemical features, without consid-
ering biological activities. The final training set consisting of 6
highly active compounds was submitted for pharmacophore
building. The best hypothesis (Hypo1), consisting of four features,
namely, one hydrogen-bond acceptor, one hydrophobic point, and
two hydrogen-bond donors (Fig. 2A). A representation of the
Figure 2. Shape-based pharmacophore model of the inhibition of VividH CYP3A4 green activity by ginsenosides. The model was
generated from the 3D structure of PPT and its activities against CYP3A4. PPT fitted into the model was shown in the figure. Blue represents the
shape space, black represents carbon atoms, red represents oxygen atoms, and white represents hydrogen atoms.
SAR of Ginsenosides on P450s
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chemical features mapped onto representative studied compound
(PPT) is depicted in Fig. 2B. In this model, the hydrogen bond
acceptor seems to be mapped to the oxygen atom substituted at
atom C20, and the hydrophobic group fit well with the aliphatic
chain attached as side-chain to this moiety. The analysis shows
that hydroxyl substitutions at atom C3 and C12 are crucial.
Hydrogen-bond donors at these atomic positions will increase the
bioactivity. From Fig. 2B, it is evident that the hydroxyl group
substituted at atom C3 and C12 serves to be hydrogen-bond
donors (HD1 and HD2). Thus replacement with other substituent
group at oxygen atoms of C3-OH or C12-OH will result in
The effects of the ginsenosides on CYP3A4 were
Considering potential influences of substrate selection on the
evaluation of the effects of the ginsenosides on the catalytic activity
of CYP3A4, another fluorescent probe, VividH CYP3A4 red
(Fig. 1B), was used. The assay conditions are also summarized in
Table 1. The solvent effect of methanol (1%) on CYP3A4 red
metabolism was negligible. The IC50value of ketoconazole on the
CYP3A4 red assay was 0.97 mM (Table 2). As for the ginsenosides
and sapogenins, the inhibition profiles using the red substrate and
green substrate were not consistent. For the same saponin, the
inhibition potency determined using the red substrate was greater
than using the green substrate (Table 2) (p,0.05). However, the
pattern was the opposite for sapogenins: the inhibition potency
was overestimated using the green substrate compared to the red
substrate (Table 2). Strikingly, 25-OH-PPD and 25-OH-PPT
activated the catalytic activity of CYP3A4 when using red
substrate. The representative influence profiles of PPD, 25-OH-
PPD, 25-OCH3-PPD and Rh2on CYP3A4 activities using either
red or green substrate are shown in Fig. 3. In the VividH red assay
(Fig. 3A), 25-OH-PPD potently activated CYP3A4 (about two-
fold) at a low concentration (10 mM). It was notable that at higher
concentrations 25-OH-PPD activated CYP3A4 red metabolism in
a saturated profile. At 10 mM, 25-OCH3-PPD weakly activated
CYP3A4 (about 20%), but weakly inhibited the enzyme at high
concentrations. PPD and Rh2 inhibited CYP3A4 in a dose-
dependent manner. However, the effect of Rh2(IC50=9.8 mM)
was more potent than PPD (IC50=43.1 mM) (p,0.01). In the
VividH green assay (Fig. 3B), however, all four tested compounds
inhibited CYP3A4 in a dose-dependent manner, with the potency
The effects of the ginsenosides and sapogenins on CYP3A4
were also substrate-dependent when using human liver micro-
somes and different conventional probes. CYP3A4-catalyzed
reactions (carbamazepine 10,11-epoxidation and nifedipine oxi-
dation) were used to evaluate the potential ginsenoside-drug
interactions. We employed high performance liquid chromatog-
raphy/tandem mass spectrometry (HPLC-MS-MS) analysis to
Figure 3. Effects of four different ginsenosides on the VividH CYP3A4 red assay (A) and green assay (B). Each point is the mean value of
triplicate samples, with error bars representing RSD values.
SAR of Ginsenosides on P450s
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detect the reaction products carbamazepine 10,11-epoxide (CBZ-
E) and oxidized nifedipine (ONF) of respective drug substrates
carbamazepine (CBZ) and nifedipine (NF).
Strikingly, PPD, 25-OH-PPD and 25-OCH3-PPD substantially
activated in vitro CBZ metabolism in a dose-dependent manner,
with the potency as follows: PPD.25-OCH3-PPD.25-OH-PPD
(Fig. 4A). It is notable that PPD potently activated (about five-fold)
the CBZ metabolism, even at the low concentration (10 mM), and
the activation by PPD at high concentrations reached saturation.
However, Rh2did not significantly alter CBZ metabolism (Fig. 4A).
Reported activator a-naphthoflavone (10 mM) potently activated
CBZ metabolism (increased by 772%), whereas ketoconazole
(10 mM) exerted a substantial inhibitory effect (decreased metab-
olism by 85.9%) compared to the vehicle control.
As for NF in vitro metabolism, Rh2and PPD exerted a weak
activation effect at low concentrations (1,10 mM). However, the
high concentration (50 mM) of Rh2weakly inhibited CYP3A4-
catalyzed NF oxidation. The effects of 25-OCH3-PPD and 25-
OH-PPD on NF metabolism were negligible (Fig. 4B). a-
Naphthoflavone (10 mM) did not significantly alter in vitro NF
metabolism, whereas ketoconazole (10 mM) inhibited CYP3A4-
catalyzed NF metabolism almost completely (decreased by 94.9%),
compared to the vehicle control.
Potential GDIs in the clinical settings make it necessary to
investigate the influences of major ginseng components on drug-
metabolizing enzymes. To that end, we studied the effects of 15
ginsenosides and sapogenins on P450-mediated drug metabolism.
Compared to microsomal assays with conventional probe drugs
using HPLC analysis, fluorescent methods for determining P450
activity are more high-throughput and reproducible [20–24] and
have been extensively employed in screening for inhibitors and
inducers of P450s [24–27]. Using the fluorescent probes, we found
that ginsenoside Rb1, Re and Rg1had no significant effects on
major P450s, with the exception that Rb1moderately inhibited
CYP1A2 in our assay. A previous report  indicated that
ginsenoside Rd inhibits CYP3A4-mediated testosterone 6b-
hydroxylation with an IC50value of 62 mM, however, we did
not observe any significant influence of Rd on any of the P450s
tested. More recently, Liu et al. found that ginsenoside metabolites
(C-K, PPD and PPT) are more potent P450 inhibitors than natural
ginsenosides . In our present study, we also found that
sapogenins are more potent than saponins in their effects on
CYP3A4, despite the fact that the IC50values we obtained for
prosapogenin C-K and Rh2were lower. It is noteworthy, however,
that several saponins in our assay were found to have activities
Figure 4. Effects of four different ginsenosides on the formation of carbamazepine 10,11-epoxide (A) and oxidized nifedipine (B).
Data are avereages of triplicate samples.
SAR of Ginsenosides on P450s
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comparable to (Rg3, Rh2 and C-K against CYP2C9 and
CYP2C19) or even greater than (Rb1, Rg3 and C-K against
CYP1A2, and Rg3against CYP2D6) sapogenins (p,0.05). These
findings indicate that natural ginsenosides still have the potential to
inhibit certain P450 enzymes and possibly lead to GDIs in vivo.
Information about the circulating levels of these ginsenosides and
sapogenins in humans would be helpful to determine whether
these interactions occur in vivo. However, except for only a few
ginsenosides (Rb1, Rg1 and Rg3), such information is not
available in the literature due to limited pharmacokinetic studies of
ginsenosides in human.
Our data showed that a SAR exists in the effects of ginsenosides
on almost all of the P450 enzymes examined, especially on
CYP2C9, CYP2C19 and CYP3A4 (green substrate) (Table 2).
Apparently, the similarities and differences among the activities of
tested ginsenosides against each of the P450s are consistent with
the fact that these compounds are analogs and differ in only four
substituent groups. Furthermore, a pharmacophore (Fig. 2) was
established based on the data set from the CYP3A4 green assay to
explore more information about effects of ginsenosides and
sapogenins on this key enzyme, which participates in the
metabolism of more than half of pharmaceuticals. The model
shows that hydroxyl substitutions at atom C3 and C12 play a key
role in the inhibitory potency of ginsenosides and sapogenins
against VividH CYP3A4 green metabolism. Presence of hydrogen-
bond donors at these atomic positions will increase the bioactivities
of ginsenosides and sapogenins on CYP3A4. Therefore, substitu-
tion with glycosyl group(s) at oxygen atoms of C3-OH will result in
decreased potency of the compounds.
The inconsistencies existing in the previous reports (e.g. effects
of Rd and Rb2on CYP2C19 [7,8]) and conflicts between our data
and previous reports (e.g. effects of saponins on several P450s )
on the inhibition of P450s by ginsenosides can most likely be
attributed to the different substrates and assay systems used to
determine the P450 activities. It is recommended that multiple
probes should be used when P450-mediated drug interactions are
evaluated . This is especially the case for CYP3A4, because its
drug interaction patterns were substrate-dependent . We
therefore evaluated the effects of the ginsenosides and sapogenins
on the activity of human CYP3A4 by using another fluorescent
probe and two conventional drug substrates. Interestingly, the
influence patterns of these compounds against CYP3A4 using the
red substrate were distinct from those observed using the green
substrate. In subsequent microsomal studies with CBZ and NF as
substrates, these substrate-dependent
phenomena were further validated. CBZ in vitro metabolism was
potently activated by PPD, 25-OCH3-PPD and 25-OH-PPD, even
at low concentrations (5,10 mM). CBZ is an anticonvulsant agent
used in the treatment of epilepsy, acute mania and bipolar disorder
. Due to co-administration of ginseng products among some
epilepsy patients , ginseng or ginseng-based products have the
potential to promote the metabolism of CBZ, and thus decrease
the bioavailability of this drug. However, whether PPD, 25-
OCH3-PPD, or 25-OH-PPD can activate CBZ in vivo metabolism
needs further investigation.
The substrate-dependent phenomena and atypical kinetics of
P450-mediated drug-drug interactions, especially for CYP3A4,
were documented in several previous in vitro studies [31,34,35].
There have also been several reports concerning the activation of
P450s. Tea and tea polyphenols were found to heteroactivate
CYP1A1 with a fluorescent probe . Endogenous steroids were
reported to activate CYP3A4-mediated CBZ 10,11-epoxidation
[37,38]. As for ginsenosides, Rc was documented to activate
CYP2C9 activity, while Rf activated CYP3A4 in fluorescent assays
. These substrate-dependent activation events may be due to
the large active site of P450s, especially CYP3A4, as evidenced by
its crystal structure . CYP3A4 can bind multiple ligands
simultaneously, a property that may contribute to its complex
cooperative effects [40–43]. Thus, different inhibition profiles of
the test compounds against CYP3A4 result when probes with
different structures are used. As to the mechanism of hetero-
activation of CBZ metabolism by endogenous steroids, CBZ may
be more easily catalyzed due to direct interaction with steroids in
the active site, which may change the activity of CYP3A4 .
Interestingly, ginsenoside sapogenins that have stimulatory effects
on CBZ metabolism (PPD, 25-OCH3-PPD and 25-OH-PPD)
have structures with similarity to steroids, and may activate
CYP3A4 activity by interacting with CBZ in the active site.
However, saponin ginsenosides (such as Rh2), which are of
relatively large size, may hinder the co-binding events and
consequently lead to no or inhibitory effects on CYP3A4.
Nevertheless, the molecular and structural basis of these substrate-
and analog-dependent effects is still not clear and needs further
Our observations of substrate-dependent effects of ginsenosides
and sapogenins on CYP3A4 suggest that GDIs may also be
substrate-dependent. In contrast with reports mentioned in the
‘Introduction’ section, several in vivo studies suggested that ginseng
or ginseng extracts have little effect on P450 activities. Coadmin-
istration of ginseng did not alter the pharmacokinetics and
pharmacodynamics of warfarin in human subjects . Gurley
et al found that supplementation of Panax ginseng did not have any
significant effects on CYP1A2, 2D6, 2E1 or CYP3A4 activities in
vivo when their activity was examined by use of probe-drug
cocktails . Our studies may provide an explanation for the
inconsistencies in clinical GDI reports. Inhibition or activation,
and the extent of these effects of ginseng or ginseng products on
P450s, are relative and based on the substrate(s) selected in the
assay. Translation from data obtained with only one ‘probe’ to
other drugs metabolized by the same P450 enzyme will probably
lead to ex parte conclusion on GDI, and may lead researchers to
overlook potential interactions of ginseng with drugs other than
the probe. Therefore, multiple probe drugs are needed for careful
in vitro and in vivo evaluation of P450-mediated GDIs, and drug-
drug interactions to a larger extent, especially in case of CYP3A4.
In summary, we evaluated the in vitro effects and the SAR of
fifteen ginsenosides and sapogenins on five major human drug-
metabolizing P450 enzymes. In addition, we also found that
substrate-dependent phenomena exist in the effects of the ginseno-
sides and sapogenins when employing different fluorescent and
conventional probes. To the best of our knowledge, this study is the
first report on the SAR and substrate-dependence of the effects of
ginsenosides on P450s. The information derived will enhance our
understanding of GDIs, and provide a possible explanation for the
inconsistent results obtained in previous reports.
Materials and Methods
Chemicals and Reagents
All ginsenosides and sapogenins, isolated as described previously
[13,14], were at least 95% pure. a-naphthoflavone, miconazole
nitrate salt, quinidine, ketoconazole, carbamazepine (CBZ),
carbamazepine 10,11-epoxide (CBZ-E), nifedipine (NF), oxidized
nifedipine (ONF) and ammonium acetate were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Sulfaphenazole was a
generous gift from Ms. Yuanyuan Dai at Cancer Hospital/
Institute, Chinese Academy of Medical Sciences (Beijing, China).
Na3PO4was purchased from Sinopharm Chemical Reagent Co.,
SAR of Ginsenosides on P450s
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Ltd. (Shanghai, China). Pooled human liver microsomes (HLM),
Solution A (206, containing 1.3 mM NADP+, 3.3 mM glucose-6-
phosphate, 3.3 mM MgCl2) and Solution B (1006, 0.4 U/ml
glucose-6-phosphate dehydrogenase) of the NADPH regenerating
system were supplied by BD Biosciences (San Jose, CA, USA).
Chromatographic grade methanol and acetonitrile were pur-
chased from Tedia Company (Fairfield, OH, USA).
VividH P450 assays
The inhibition of the catalytic activities of cDNA-expressed
human P450 enzymes by ginsenosides was determined using
VividH P450 screening kits (CYP1A2 blue, CYP2C9 red,
CYP2C19 blue, CYP2D6 blue, CYP3A4 green and red) according
to the manufacturer’s instructions (Invitrogen Corporation;
Carlsbad, CA, USA). Each kit contained P450 reaction buffer,
P450 BACULOSOMESH reagent, fluorescent substrate, fluores-
cent standard, the regeneration system (333 mM Glucose-6-
phosphate and 30 U/ml glucose-6-phosphate dehydrogenase in
100 mM potassium phosphate pH 8.0), and 10 mM NADP+in
100 mM potassium phosphate, pH 8.0.
In brief, the assays were carried out in CostarH black-wall 96-
well plates with ultra thin clear bottoms (Corning Inc, Corning,
NY, USA) in a kinetic assay mode. Stock solutions (10 mM) of
ginsenosides in methanol were prepared and diluted to various
concentrations (2.56100, 50, 10, 5, 1 mM). For each well, 40 mL
of solution of test compounds or vehicle was incubated with 50 mL
pre-mixture (mixture of BACULOSOMESH reagent, regeneration
system and reaction buffer) at 37uC for 20 min. The reaction was
initiated by adding 10 mL of a mixture of substrate and NADP+
per well with a respective concentration of VividH substrate
(Table 1). The plate was read immediately for fluorescence
changes every 0.5 min at 37uC for 30 min using a FlexStation
II384 fluorometric plate reader (Molecular Devices, Sunnyvale,
CA, USA) with respective excitation and emission wavelengths for
each P450 enzyme (Table 1). The final methanol volume in the
reaction was less than or equal to 1%. Inhibitory potencies of test
compounds (IC50values) were compared by using Student’ t-test.
Difference was considered to be significant when the two-tailed p-
value was less than 0.05.
The compounds were built using Catalyst (Accelrys corporation,
San Diego, CA, USA) 2D–3D sketcher, and a family of
representative conformations was generated for each ginsenoside
or sapogenin using the best conformational analysis method. A
maximum number of 250 conformations of each compound were
selected using ‘‘best conformer generation’’ option with a
constraint of 20 kcal/mol energy thresholds above the global
energy minimum to ensure maximum coverage of the conforma-
tional space. Based on the conformations for each compound,
Catalyst 4.10 software package was employed to construct possible
pharmacophore models. When generating a hypothesis, catalyst
attempts to minimize a cost function consisting of two terms.
Analysis of functional groups on each compound in the training set
revealed that three chemical features, hydrogen-bond acceptor
(HA), hydrogen-bond donor (HD), and hydrophobic group (HY),
could effectively map all of the critical chemical features. Hence,
the three features were selected to form the essential information
in this hypothesis generation process. The best predictive
hypothesis (Hypo1), produced by HipHop process encoded in
Catalyst 4.10, has four features: one hydrogen-bond acceptor, one
hydrophobic point, and two hydrogen-bond donor, which was
characterized by the highest cost difference, the lowest rms
divergence, and the best correlation coefficient. Remarkably, the
highest active compound (PPT) can be nicely mapped onto the
Hypo1 model by the best fit values.
The effects of the ginsenosides and sapogenins on the in vitro
metabolism of CBZ were evaluated using HLM. Triplicate
microsomes (50 mL, 20 mg/ml) were treated sequentially with
20 mL 1 mM CBZ 10% DMSO (v/v) aqueous solution, 20 mL
aqueous solutions of test ginsenosides containing 25% methanol
(v/v) at different concentrations (1, 5, 10, and 50 mM) or a-
naphthoflavone (10 mM) or ketoconazole (10 mM), and 850 mL
0.1 M Na3PO4solution, and pre-incubated at 37uC for 5 min.
Vehicle control samples were treated with methanol at a final
concentration of 0.5% instead of test compounds. The reaction
was initiated by adding 60 mL pre-warmed NADPH-regenerating
system (50 mL BD solution A and 10 mL BD solution B). After
incubation at 37uC for 30 min, the reaction system was quenched
by adding 0.4 ml ice-cold acetonitrile, which was mixed well and
centrifuged at 4uC (10,000 g, 10 min). The supernatant (0.4 ml)
was collected, mixed with 0.8 ml acetonitrile and then centrifuged
at 4uC (10,000 g, 10 min). The supernatant (30 mL) was diluted
with 970 mL ddH2O and 50 mL of the mixture was injected onto
the high performance liquid chromatography/tandem mass
spectrometry (HPLC-MS-MS) system for analysis. The calibration
samples were prepared with standard solutions of CBZ-E
(0.00457, 0.0137, 0.0412, 0.123, 0.370, 1.11, and 3.33 mM) and
processed identically to the samples before analysis, except that
solution A was added after microsomal proteins were precipitated
by acetonitrile. Microsomal studies using NF as the substrate were
conducted as described above for CBZ with several modifications.
The NF concentration was 1 mM. The supernatant (0.4 ml) of the
reaction system after quenching and centrifugation was also
combined with 0.8 ml acetonitrile and centrifuged, and 10 mL of
the resultant supernatant was directly injected onto the HPLC-
MS-MS system for analysis. The calibration standards for ONF
included 0.4572, 1.372, 4.115, 12.35, 37.04, 111.1, 333.3, and
The HPLC was performed using an Agilent 1200 system (Palo
Alto, CA, USA) equipped with a binary pump and an auto-
sampler. For CBZ-E analysis, an Agilent Zorbax SB-C18 column
(4.66150 mm, 5 mm) was used, and the mobile phase consisted of
50% eluent A (aqueous solution containing 0.5% acetic acid) and
50% eluent B (acetonitrile) at the flow rate of 0.4 ml/min. The
elution lasted for 8 min. For ONF analysis, an Agilent Zorbax
EclipseH XDB-C8 column (4.66150 mm, 5 mm) was used; the
mobile phase consisted of eluent A (10 mM ammonium acetate)
and eluent B (acetonitrile) at the flow rate of 0.6 ml/min. The
gradient elution program was as follows: started with 70% B for
1.5 min; changed to 95% B within 1 min; held at 95% B for
5.5 min; returned rapidly to 70% B; and held at 70% B for 2 min.
The MS analysis was performed on a 4000Q TrapTMtriple
quadrupole mass spectrometer (Applied Biosystems/MDS Sciex
Instruments, Concord, Ontario, Canada) equipped with a Turbo
V ion source (TurboIonSprayTMprobe was used), a DellTM
computer, and AnalystTMsoftware. Electrospray ionization in the
positive mode was employed to analyze CBZ-E and ONF,
respectively. The compound parameters for CBZ-E and ONF
were: declustering potential, 65 and 95 units, and collision energy,
39 and 40 units, respectively. The ion source parameters were as
follows: curtain gas, 15 units; collision gas, medium; ion spray
voltage, 4000 and 5000 V, respectively; temperature, 650 and
350uC, respectively; ion source gas 1, 60.0 and 65.0 units,
SAR of Ginsenosides on P450s
PLoS ONE | www.plosone.org8 July 2008 | Volume 3 | Issue 7 | e2697
respectively; ion source gas 2, 45.0 units. The fragmentation
transitions for multiple reaction monitoring (MRM) were m/z
253.1R180.2 for CBZ-E and m/z 345.1R284.3 for ONF. The
representative calibration curve for CBZ-E is: Y=147X+358
(r=0.9993) with 1/X2weighting, and the representative calibra-
tion curve for ONF is: Y=3570X22410 (r=0.9976) with 1/X
weighting, where Y represents the peak area of the analyte, and X
represents the analyte concentration.
We thank Ms. Yuanyuan Dai at Cancer Hospital/Institute, Chinese
Academy of Medical Sciences for her generous gift of sulfaphenazole. We
also thank Drs. Bryan D. Marks, Ann-Charlotte Egnell, Weiliang Zhu,
Elizabeth R. Rayburn, Donald L. Hill and Mr. Jieshu Qian, Rui Zhang for
helpful discussions, and thank Ms. Zi Li for excellent technical assistance.
Conceived and designed the experiments: MH RZ HW. Performed the
experiments: MH PC HH. Analyzed the data: MH YZ HL HW.
Contributed reagents/materials/analysis tools: YZ HL HJ. Wrote the
paper: MH HW.
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