Stereoselective Regulations of P-Glycoprotein by Ginsenoside Rh2 Epimers and the Potential Mechanisms From the View of Pharmacokinetics
et al. (2012) Stereoselective Regulations of P-Glycoprotein by Ginsenoside Rh2 Epimers and the Potential
Mechanisms From the View of Pharmacokinetics. PLoS ONE 7(4): e35768. doi:10.1371/journal.pone.0035768
Stereoselective Regulations of P-Glycoprotein by Ginsenoside Rh2 Epimers and the Potential Mechanisms From the View of Pharmacokinetics
Jingwei Zhang. 0
Fang Zhou 0
Fang Niu 0
Meng Lu 0
Xiaolan Wu 0
Jianguo Sun 0
Guangji Wang 0
Shaida A. Andrabi, Johns Hopkins University, United States of America
0 Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University , Nanjing, Jiangsu , China
Chirality is an interesting topic and it is meaningful to explore the interactions between chiral small molecules and stereoselective biomacromolecules, with pre-clinical and clinical significances. We have previously demonstrated that 20(S)ginsenoside Rh2 is an effective P-glycoprotein (P-gp) inhibitor in vitro and in vivo. Considering the stereochemistry of ginsenoside Rh2, in our present study, the regulatory effects of 20(R)-Rh2 on P-gp were assayed in vivo, and the differential regulations of P-gp by ginsenoside Rh2 epimers in vivo were compared and studied. Results showed that 20(S)-Rh2 enhanced the oral absorption of digoxin in rats in a dose-dependent manner; 20(R)-Rh2 at low dosage increased the oral absorption of digoxin, but this effect diminished with elevated dosage of 20(R)-Rh2. Further studies indicated stereoselective pharmacokinetic profiles and intestinal biotransformations of Rh2 epimers. In vitro studies showed that Rh2 epimers and their corresponding deglycosylation metabolites protopanaxadiol (Ppd) epimers all exhibited stereoselective regulations of P-gp. In conclusion, in view of the in vitro and in vivo dispositions of Rh2 and the regulations of P-gp by Rh2 and Ppd, it is suggested that the P-gp regulatory effect of Rh2 in vivo actually is a double actions of both Rh2 and Ppd, and the net effect is determined by the relative balance between Rh2 and Ppd with the same configuration. Our study provides new evidence of the chiral characteristics of P-gp, and is helpful to elucidate the stereoselective P-gp regulation mechanisms of ginsenoside Rh2 epimers in vivo from a pharmacokinetic view.
Funding: This work was supported by China National Nature Science Foundation No. 30973583 (GW) and No. 30801411 (FZ); China Creation of New Drugs Key
Technology Projects No. 2009ZX09304-001 (HH) and 2009ZX09502-004 (GW); Jiangsu Province Nature Science Foundation No. BE2010723 (GW) and
No. BK2010437 (FZ). 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.
. These authors contributed equally to this work.
Chirality is a quite common feature for both biomacromolecules
and small-molecules in nature and in our daily life.
Biomacromolecules have the potential to stereoselectively recognize and dispose
the ligands. For example, it has been shown that S-verapamil is
significantly different from R-verapamil in plasma protein binding
and systemic clearance [1,2]. On the other hand, small-molecules
also stereoselectively take their biological actions. Taking
propoxyphene as an example, dextropropoxyphene is an analgesic,
whereas levopropoxyphene is an antitussive agent . Warfarin is
another example. At physiological concentrations, R-warfarin
interacts with pregnane X receptor (PXR) and significantly
induces CYP3A4 and CYP2C9 mRNAs, while S-warfarin does
not show such effects . As mentioned above, it is interesting and
important to explore the interactions between chiral small
molecules and stereoselective biomacromolecules, with pre-clinical
and clinical significances.
Ginsenosides, the main effective constituents of ginseng, have a
broad range of therapeutic applications. The basic structure of
ginsenoside is tetracyclic triterpenoid, with many chiral carbones
in the molecule. Particularly, the chirality of carbon-20 contributes
to the two stereoisomers of each ginsenoside. They are called
epimers. It is very likely that the two epimers of ginsenoside have
different biological characteristics. 20(S)-ginsenoside Rg3 but not
20(R)-ginsenoside Rg3 inhibited the Ca2+, K+ and Na+ channel
currents in a dose- and voltage-dependent manner [5,6]. In
human fecal microflora, the amount of 20(S)-ginsenoside Rg3
transforming to 20(S)-ginsenoside Rh2 was 19-fold higher than
that of 20(R)-ginsenoside Rg3 transforming to 20(R)-ginsenoside
Rh2 . On the other hand, as the deglycosylation metabolite of
Rg3, ginsenoside Rh2 also exhibited stereoselective activities.
20(S)-ginsenoside Rh2 but not 20(R)-ginsenoside Rh2 inhibited
the proliferation of both androgen-dependent and independent
prostate cancer cells . Interestingly, 20(R)-ginsenoside Rh2 is a
selective osteoclastgenesis inhibitor without any cytotoxicity, while
20(S)-ginsenoside Rh2 showed weak osteoclastgenesis inhibition
but had strong cytotoxicity in osteoclasts . We have previously
examined the pharmacokinetic profile of ginsenoside Rh2 and
observed its poor bioavailability (absolute bioavailabilities were
about 4.06.4% when 19 mg/kg Rh2 were i.g. administered to
rats) . We found that stereochemistry was one of the causes to
poor oral absorption, because 20(S)-ginsenoside Rh2 and
20(R)ginsenoside Rh2 exhibited different membrane permeabilities
. Hence, the stereochemistry of the hydroxyl group at
carbon20 plays an important role in the activities of ginsenoside epimers.
P-glycoprotein (P-gp), a member of drug transporters, mediates
not only the transport of endogenous substances but also of the
exogenous therapeutic drugs. As biomacromoleucles, P-gp owns
the ability to distinguish the ligands stereoselectively, and
contributes to different dispositions of the chiral ligands .
For example, P-gp ATPase hydrolysis and P-gp substrate
recognition was stimulated by cis-flupentixol while inhibited by
trans-flupentixol . Recently, the structure of mouse P-gp, with
87% sequence identity to human P-gp, has been reported . It
was found that P-gp could distinguish between QZ59-RRR and
QZ59-SSS, two stereoisomers of cyclic peptides, through different
binding locations, orientation and stoichiometry with P-gp.
It is very interesting to discuss the interactions between P-gp and
chiral small molecules. However, the related reports are limited.
Recently, we have demonstrated that 20(S)-ginsenoside Rh2 is an
effective P-gp inhibitor both in vitro and in vivo . Considering
the stereochemistry of ginsenoside Rh2, in our present study, the
regulatory effects of 20(R)-Rh2 (Fig. 1) on P-gp were assayed in
vivo. For a comparative understanding of the differential regulation
of P-gp by ginsenoside Rh2 epimers in vivo, the pharmacokinetics
of Rh2 epimers in vivo, the possible metabolism, and evaluation of
P-gp regulatory effects in vitro were all included. Moreover, the
differential P-gp regulations of Rh2 epimers were further
confirmed by applying Rh2 epimers as P-gp regulators in reversal
of P-gp mediated multi-drug resistance. Our study provides a new
case describing the chiral characteristics of P-gp. It is also a
meaningful trial to elucidate the stereoselective P-gp regulation
mechanisms of ginsenoside Rh2 epimers in vivo from a
Effects of 20(S)-Rh2 and 20(R)-Rh2 on oral
pharmacokinetics of digoxin in rats
Digoxin has been proved as a classic P-gp substrate, and its
intestinal absorption is mainly restricted by P-gp [16,17]. When
20(S)-Rh2 was i.g. administered to rats prior to i.g. administration
of digoxin, the oral absorption of digoxin was enhanced with
increasing concentrations of 20(S)-Rh2 (Fig. 2A). The AUC and
Cmax of digoxin were elevated by 1.8-fold and 1.6-fold respectively
by 50 mg/kg 20(S)-Rh2 (Table 1). However, it was different in the
case of 20(R)-Rh2. When 20(R)-Rh2 was i.g. administered to rats
at 5 mg/kg prior to i.g. administration of digoxin, the AUC and
Cmax of digoxin were significantly enhanced (Table 1). But, when
the dosage of 20(R)-Rh2 was elevated to 50 mg/kg, the absorption
of digoxin was not changed significantly compared with control
group (Fig. 2B). The dose-effect trends of 20(S)-Rh2 and
20(R)Rh2 on the oral pharmacokinetics of digoxin were just opposite.
Stereoselective LC-MS quantification of ginsenoside Rh2
epimers and the deglycosylation metabolites
ginsenoside Ppd epimers
The chromatograms shown in Fig. 3 demonstrated that the
present LC-MS conditions applied for analysis of Rh2 and Ppd
epimers provided appropriate separation with the retention time of
6.9, 7.9, 14.2, 14.7 and 6.7 min for 20(S)-Rh2, 20(R)-Rh2,
20(R)Ppd, 20(S)-Ppd and digitoxin respectively. The specificity of the
method was evaluated by screening blank biological matrix in
selected ion monitoring (SIM) mode, and no interference had been
observed. The method showed good linearity in a range of 1
1000 nM with a correlation coefficient R2 exceeding 0.995 for the
Stereoselective oral pharmacokinetics of ginsenoside Rh2
epimers in rats
As seen in Fig. 4, there was significant difference in oral
pharmacokinetics of ginsenoside Rh2 epimers in rats. With the
same dosage for oral administration, the Cmax and AUC of
20(S)Rh2 were 15-fold and 10-fold higher than those of 20(R)-Rh2
respectively: the Cmax of 20(S)-Rh2 was nearly 1000 nM while the
Cmax of 20(R)-Rh2 was no higher than 50 nM, which suggested
better oral absorption of 20(S)-Rh2 than 20(R)-Rh2 (Table 2).
Furthermore, chiral inversions between ginsenoside Rh2 epimers
were observed. When 20(S)-Rh2 was orally administered,
20(R)Rh2 was also detected in plasma, with Cmax only one eighth of
20(S)-Rh2 and AUC only one tenth of 20(S)-Rh2. Similarly, when
20(R)-Rh2 was orally administered, 20(S)-Rh2 was also detected in
plasma, and the concentrations of 20(S)-Rh2 were much lower
than those of 20(R)-Rh2. Otherwise, the deglycosylation
metabolite of 20(S)-Rh2 was also monitored in plasma when 20(S)-Rh2
was orally administered, and the configuration of Ppd was
confirmed by the standard substance of 20(S)-Ppd. But, no Ppd
was found in plasma after oral administration of 20(R)-Rh2.
Digoxin (0.25 mg/kg i.g.)
5 mg/kg 50 mg/kg 5 mg/kg 50 mg/kg
9.962.6 9.862.7 16.065.5* 1 16.962.9* 10.363.3
1.260.4 1.760.4 1.860.3
1.760.6 2.760.8 2.260.4
AUC 012 (nmol/L6h) 15.462.9 18.964.3 27.262.2* 11 34.966.0** 21.463.6 1
Effects of 20(S)-Rh2, 20(R)-Rh2, 20(S)-Ppd and 20(R)-Ppd
on P-gp functions in Caco-2 cells
Caco-2 cell model is a classic approach in the research of P-gp
. As shown in Fig. 6A, 20(S)-Rh2 decreased the efflux ratio of
digoxin crossing Caco-2 cell monolayers in a
concentrationdependent manner. However, low concentration of 20(R)-Rh2
(1 mM) significantly lowered the efflux ratio of digoxin. But, with
elevated concentrations of 20(R)-Rh2, the efflux ratio of digoxin
As shown in Fig. 6B, both 20(S)-Ppd and 20(R)-Ppd lowered the
efflux ratio of digoxin across Caco-2 cell monolayers
concentration-dependently. But the P-gp inhibitory effect of 20(S)-Ppd was
more pronounced than that of 20(R)-Ppd.
Effects of 20(S)-Rh2 and 20(R)-Rh2 on the sensitivity of
MCF-7/Adr cells to adriamycin
MCF-7/Adr cell line is an adriamycin resistant human breast
cancer cell line. It is derived from the parental human breast
cancer cell line MCF-7 by gradual adriamycin selection. Our
previous study showed that it is more resistant to adriamycin
compared with MCF-7. When series concentrations of adriamycin
were added to MCF-7/Adr cells in the presence of 20(S)-Rh2 or
20(R)-Rh2, these cells exhibited differential sensitivities towards
adriamycin. As seen in Table 4, 20(S)-Rh2 decreased the IC50 of
adriamycin in MCF-7/Adr cells in a concentration-dependent
manner. Although 20(R)-Rh2 (1 mM) lowered the IC50 of
adriamycin in MCF-7/Adr cells, with increasing concentrations
of 20(R)-Rh2, the sensitivity of MCF-7/Adr cells towards
adriamycin was restored to the initial level
Chirality is a basic characteristic of biological system.
Investigating the stereochemistry of either biomacromolecules or
exogenous small molecules plays an important role in exploring
the nature of life and promoting the health of people. Especially,
since the thalidomide tragedy in 1960s , people have realized
that the racemic mixtures and individual stereoisomers could
Stereoselective metabolic kinetics of ginsenoside Rh2
epimers by rat fecal microflora
Deglycosylation contributed greatly to the biotransformation of
ginsenoside Rh2 with fecal microflora [18,19]. As seen in Fig. 5A
and 5C, when 20(S)-Rh2 (1 mM and 10 mM) was incubated with
rat fecal microflora in anaerobic condition, the level of 20(S)-Rh2
decreased rapidly and the deglycosylation product 20(S)-Ppd
appeared as soon as one hour. In addition, a very small amount of
20(R)-Rh2 was also detected throughout the incubation. However,
when 20(R)-Rh2 (1 mM) was incubated with rat fecal microflora,
there was a marked decrease in the level of 20(R)-Rh2, and not
only a large amount of 20(R)-Ppd was found but also a small
amount of 20(S)-Rh2 and 20(S)-Ppd were detected (Fig. 5B).
Furthermore, when the concentrations of 20(R)-Rh2 were raised
to 10 mM, the level of 20(R)-Rh2 was decreased rather slowly. In
the incubation system, only 20(R)-Ppd could be detected, but not
Figure 3. Representative SIM chromatograms of ginsenosides in biological matrix. (A) blank rat intestinal microflora suspension;(B) blank
rat intestinal microflora suspension spiked with 20(S)-Rh2 (100 nM), 20(R)-Rh2 (100 nM), 20(S)-Ppd (1 mM), 20(R)-Ppd (1 mM) and digitoxin
(500 nM);(C) rat intestinal microflora suspension after incubation with 1 mM 20(S)-Rh2 for 1 h.
exhibit totally different physiochemical and biochemical properties
including carcinogenicity and teratogenicity . Developing
homochiral drugs has become a demanding tendency of the
pharmaceutical industry .
Ginsenoside Rh2 is a potential drug obtained from herbal
medicines, and its stereoselective properties have also gained much
attention [7,8,9]. In our previous studies, 20(S)-Rh2 was
demonstrated as a potent P-gp inhibitor . This leads us to
determine whether 20(R)-Rh2 could also inhibit P-gp. We
examined the effects of Rh2 epimers on the oral absorption of
P-gp substrate digoxin in rats. In contrast to 20(S)-Rh2 which
could promote the oral absorption of digoxin in a dose-dependent
manner, 20(R)-Rh2 showed the opposite P-gp inhibitory effect.
Then, pharmacokinetic profiles of Rh2 epimers were obtained
to elucidate this interesting phenomenon, assuming that different
concentrations of Rh2 epimers in vivo might lead to differential
Pgp regulations. Actually, our previous studies had shown that the
stereoselectivity of Rh2 epimers was one of the factors contributing
to the poor oral absorption of Rh2 . However, the
stereoselective absorptions of Rh2 epimers were only analyzed
on models in vitro, without further confirmation in vivo. Moreover,
our previous LC-MS method could not distinguish the
configurations of Rh2, and therefore the potential inversions between two
configurations of Rh2 were not revealed.
Hence, in our present study, a stereoselective LC-MS method
for quantification of ginsenoside Rh2 epimers and the
deglycosylaFigure 4. Plasma concentration-time curves of ginsenoside Rh2 epimers and their deglycosylation metabolites Ppd epimers after
i.g. administration of 20(S)-Rh2 or 20(R)-Rh2 in male SD rats. (A) 25 mg/kg 20(S)-Rh2, (B) 25 mg/kg 20(R)-Rh2. Data are expressed as mean 6
S.E., n = 5 per group.
Table 2. Pharmacokinetic parameters of ginsenoside Rh2 epimers and their deglycosylation metabolites Ppd epimers after i.g.
administration of 25 mg/kg 20(S)-Rh2 or 20(R)-Rh2 in rats.
ND, Not detected.
Table 3. The AUCs of ginsenoside Rh2 epimers and their deglycosylation metabolites Ppd epimers after incubation of ginsenoside
20(S)-Rh2 or 20(R)-Rh2 in rat fecal microflora.
ND, Not detected.
tion metabolites ginsenoside Ppd epimers were developed firstly.
Then, this method was successfully applied to the stereoselective
oral pharmacokinetic studies of Rh2 epimers. Although there were
inversions between 20(S)-Rh2 and 20(R)-Rh2 after oral
administration of a single configuration of Rh2, the inverted proportion
was limited to ,10%. This indicated that the initial configuration
of Rh2 was predominant after oral absorption in vivo. Furthermore,
20(S)-Rh2 was absorbed into plasma more and better than
20(R)Rh2 with higher concentrations and larger values of AUC, which
was in accordance with our previous in vitro results .
Otherwise, with the fall of plasma concentrations of 20(S)-Rh2,
the concentrations of the deglycosylation metabolite ginsenoside
Ppd were raised, and the configuration of Ppd was approved to be
20(S)-Ppd by comparing with the authentic standard. However,
20(R)-Ppd was not detected in plasma after oral administration of
20(R)-Rh2. There are two possible reasons: one is that 20(R)-Rh2
was not deglycosylated into 20(R)-Ppd; the other is that 20(R)-Ppd
was not absorbed into plasma. In order to elucidate the specific
reason, metabolisms of 20(S)-Rh2 and 20(R)-Rh2 in rat fecal
microflora were studied. The results showed that both 20(S)-Rh2
and 20(R)-Rh2 could be largely deglycosylated into Ppd which was
the main metabolite of Rh2 in intestine . The configuration of
Ppd was primarily in accordance with the initial configuration of
Rh2 that was added into the incubation system, which
demonstrated some steps of the proposed metabolic pathway of
ginsenoside Rg3 epimers by intestinal bacteria . Thus, it could
be concluded that 20(R)-Rh2 was deglycosylated into 20(R)-Ppd in
intestine, but 20(R)-Ppd was hardly absorbed into plasma.
Through these experiments, the differential absorption of
ginsenoside Rh2 epimers were confirmed in vivo, which indicated
that the ginsenoside with R-configuration possessed lower
membrane permeability and poorer absorption than the one with
S-configuration. This might be attributed to the geometrical
arrangement of hydroxyl groups at the chiral centers,
inaccessibility to water, and compact structure of 20(S)-ginsenoside. As we
all known, compounds with high hydrophobicity, weak metabolic
activity and little efflux by transporters have better membrane
permeability and oral absorption. For 20(S)-ginsenosides, it is
speculated that hydroxyl group at carbon-20 is geometrically close
to the one at carbon-12; alkene chain at carbon-20 has a stably
fixed orientation and is packed tightly near the terpenoid. These
characteristics prevent 20(S)-ginsenosides from accessing to water
molecules. However, alkene chain at carbon-20 in
20(R)-ginsenosides protrudes further outside with flexibility, which makes
20(R)ginsenosides more accessible to water molecule and more polar
than 20(S)-ginsenosides. Therefore, more hydrophobic
20(S)ginsenosides have better membrane permeability than
Furthermore, Caco-2 cells were chosen as an ideal model
analyzing P-gp-mediated drug-drug interactions. The
pharmacokinetic studies of Rh2 epimers in vivo showed that Rh2 was largely
metabolized into Ppd in intestine, which suggested the
unneglectable role of Ppd in regulation of P-gp. So the P-gp inhibitory
effects of Rh2 and Ppd were all evaluated on Caco-2 cell
monolayers using digoxin as P-gp substrate. It was found that the
P-gp inhibitory effect of Rh2 epimers in vitro was in accordance
with the studies in vivo from the concentration-effect viewpoint. As
only a little amount of Rh2 was transformed into Ppd in Caco-2
cell incubation buffer , the observed differential P-gp
regulation effect of Rh2 epimers could be attributed to Rh2 itself.
Whereas Ppd epimers all showed inhibitory effects on P-gp
function in a positive concentration-dependent manner but with
different inhibitory abilities.
In view of the in vitro and in vivo dispositions of Rh2 and the
regulations of P-gp by Rh2, it is indicated that the strong P-gp
inhibitory effect of 20(S)-Rh2 in vivo actually is a double actions of
both 20(S)-Rh2 and 20(S)-Ppd. However, it is complex for
20(R)Rh2. Previously, Berginc et al reported that aged garlic extract
increased darunavir efflux while decreased saquinavir efflux in
both HepG2 cells and rat liver slices, which were attributed to
different binding sites in P-gp [28,29]. Accordingly, we put
forward the following speculations. At low dosage, 20(R)-Rh2 was
rapidly transformed into 20(R)-Ppd (Fig. 5B). Then, the small
amount of 20(R)-Rh2 might compete with digoxin for the same
binding site, and inhibited the efflux of digoxin. Otherwise,
20(R)Ppd was largely resided in the intestine and exhibited its P-gp
inhibitory effect (Fig. 6B). The net effects of 20(R)-Rh2 and
20(R)Ppd showed inhibition. When the dosage of 20(R)-Rh2 was
elevated, the transformation rate of 20(R)-Rh2 into 20(R)-Ppd was
significantly lowered (Fig. 5D). Large amount of both 20(R)-Rh2
and 20(R)-Ppd were coexistent in the intestine. And 20(R)-Rh2
might not only compete for digoxin binding site, but also has
affinity for other regulatory site in P-gp. This probably caused the
transition of digoxin binding site from low to high affinity
conformation, and resulted in higher extrusion . Thus, the
final net effects of 20(R)-Rh2 and 20(R)-Ppd did not exhibit
remarkable P-gp regulation.
Since Rh2 epimers could differentially regulate P-gp functions in
vitro and in vivo, their MDR reversal effects based on P-gp
inhibition were also detected. Cell growth inhibition assay was
performed on multi-drug resistant cancer cells with high P-gp
expression. It turned out that 20(R)-Rh2 at low concentrations
could synergistically enhance the cytotoxic effect of adriamycin.
However, unlike 20(S)-Rh2, when the concentrations of
20(R)Rh2 were increased, the synergistic effect of 20(R)-Rh2 were
decreased and disappeared (Table 4), which again demonstrated
the stereoselective regulation of P-gp by Rh2 epimers.
In conclusion, the differential regulations of P-gp by ginsenoside
Rh2 epimers in vivo were observed in our present study.
Considering the dispositions of Rh2 epimers themselves in vivo
and the regulations of P-gp by Rh2 and Ppd in vitro, the P-gp
regulatory effects in vivo should be a net effect of Rh2 and its
deglycosylation metabolite Ppd. Upon those, the Rh2 epimers
were also applied in reversal of MDR and the differential reversal
effects were again observed. Our study revealed the stereoselective
P-gp regulation effects of ginsenoside Rh2 epimers in vivo and the
possible mechanisms from a view of pharmacokinetics.
Materials and Methods
Chemicals and reagents
20(S)-ginsenoside Rh2, 20(R)-ginsenoside Rh2,
20(S)-protopanaxadiol and 20(R)-protopanaxadiol were all purchased from Jilin
University (Changchun, China). Digoxin, digitoxin and verapamil
were purchased from Sigma Chemical Co. (St. Louis, MO).
HPLC-grade acetonitrile and methanol were purchased from
Sigma Chemical Co. (St. Louis, MO). Deionized water was
prepared by Milli-Q system (Millipore, Milford, MA, USA) and
was used throughout. Ethylacetate and all of other reagents,
solvents were commercially available and of analytical grade.
Male healthy SpragueDawley rats (200250 g) were supplied
by the Experimental Animal Breeding Center, Nanjing General
Hospital of Nanjing Military Command (Nanjing, China). All the
rats were maintained in room temperature (2262uC),5060%
relative humidity and automatic day-night rhythm (12 h-cycle).
The animals were acclimatized to the facilities for one week, and
then fasted overnight (12 h) with free access to water prior to each
experiment. Rats were randomly assigned to different
experimental groups. The animal experiments in this investigation were
carried out in accordance with the Guidelines for Animal
Experimentation of China Pharmaceutical University (Nanjing,
China) and protocol was approved by the Animal Ethics
Committee of this institution.
Effects of 20(S)-Rh2 and 20(R)-Rh2 on oral
pharmacokinetics of P-gp substrate digoxin in rats
The rats were divided into five groups with five each. One group of
rats received i.g. single dose of the vehicle (0.5% CMC-Na) serving as
the control. Two groups were i.g. administered 20(S)-Rh2 suspended
in 0.5% CMC-Na at the doses of 5 mg/kg and 50 mg/kg
respectively, while another two groups i.g. administration of
20(R)Rh2 suspended in 0.5% CMC-Na at the doses of 5 mg/kg and
50 mg/kg respectively. Two hours later, P-gp substrate, digoxin
(0.25 mg/kg) was given to the rats by i.g. administration. Blood
samples were collected before the P-gp substrate dosing and at 0.08,
0.17, 0.25, 0.5, 1, 2, 3, 6, 8 h post-digoxin-dosing. Plasma was
obtained by centrifugation at 5000 g for 10 min and stored at
220uC before analysis. Plasma concentrations of digoxin were
determined as described previously .
Pharmacokinetic Studies of 20(S)-Rh2 and 20(R)-Rh2 in
To investigate the differences of pharmacokinetic characteristics
between 20(S)-Rh2 and 20(R)-Rh2, rats were divided into 2
groups with five rats for each group. One received a single dose of
20(S)-Rh2 intragastrically at 25 mg/kg suspended in 0.5%
CMCNa, while the other received 20(R)-Rh2 at the same dosage. Blood
samples were collected at 0, 30, 60, 180, 240, 300, 360, 480, 660
and 840 min after oral administration into heparinized tubes.
Plasma was obtained by centrifugation at 5000 g for 10 min and
stored at 220uC before analysis.
Metabolism of 20(S)-Rh2 and 20(R)-Rh2 in Rat Fecal
Fresh feces of healthy rats were collected and suspended in
anaerobic medium (1 g : 3 ml). After filtration, the rat intestinal
microflora suspension was ready for anaerobic incubation of
ginsenoside. An aliquot of 1 ml rat intestinal microflora suspension
was spiked with 20(S)-Rh2 or 20(R)-Rh2, and then was incubated
under anaerobic condition. At designated time, samples were taken
Caco-2 cells (HTB-37), obtained from American Type Culture
Collection (Rockville, MD, USA), were routinely cultured in
DMEM supplemented with 10% fetal bovine serum, 1%
nonessential amino acids, 1 mM sodium pyruvate, and 100 U/
ml penicillin and streptomycin (Gibco-Invitrogen, USA). MCF-7/
Adr cells were obtained from Institute of Hematology and Blood
Diseases Hospital (Tianjin, China), and cultured in RPMI 1640
supplemented with 10% fetal bovine serum, and 100 U/ml
penicillin and streptomycin (Gibco-Invitrogen, USA). The cells
were grown in an atmosphere of 5% CO2 at 37uC and cell
medium were changed every other day.
Effects of 20(S)-Rh2, 20(R)-Rh2, 20(S)-Ppd and 20(R)-Ppd
on P-gp Mediated Bidirectional Transport of Digoxin
across Caco-2 cell Monolayers
The Caco-2 cell transport model was established as described
previously . Then, Hanks balanced salt solution (HBSS)
containing 20(S)-Rh2, 20(R)-Rh2, 20(S)-Ppd, 20(R)-Ppd or 0.1%
DMSO (control) was loaded into both apical and basolateral
chambers. After incubation at 37uC for 1 h, 5 mM digoxin was
added to either apical or basolateral side to evaluate the transport in
absorptive and secretory direction respectively. After incubation for
just another 2 h, samples were taken from the receiving chamber for
analysis. Verapamil (10 mM) was used as a positive control. Digoxin
was determined by LC-MS/MS. All experiments were conducted in
Effects of 20(S)-Rh2 and 20(R)-Rh2 on Adriamycin
Sensitivity in P-gp highly-expressed MCF-7/Adr Cells
MTT colorimetric assay was used to measure the cell growth
inhibition after incubation with various concentrations of
adriamycin in the absence or presence of 20(S)-Rh2 or 20(R)-Rh2 at 37uC
for 72 h. The concentrations required to inhibit growth by 50%
(IC50) were calculated from survival curves using the Bliss method.
LC-MS analysis of 20(S)-Rh2, 20(R)-Rh2 and the
deglycosylation metabolites 20(S) - Ppd and 20(R)-Ppd
The 20(S)-Rh2, 20(R)-Rh2 and the deglycosylation metabolites
20(S)-Ppd and 20(R)-Ppd were quantified simultaneously by
reversed-phase LC-MS. An aliquot of 100 ml sample spiked with
digitoxin as internal standard was extracted by 1 ml ethylacetate.
The analysis was performed on Finnigan LC-MS system (Thermo
Electron, San Jose, CA, USA) with a Lux Cellulose-1 Chiral
Column (25064.6 mm, 5 mm, Phenomenex, USA). The column
and autosampler tray temperatures were 40 and 4uC, respectively.
The mobile phase was consisted of methanol, acetonitrile and 0.1%
formic acid with gradient elution (Table 5). Mass spectrometer was
operated in positive ESI mode. MS parameters were as follows:
spray voltage, 5.0 kV; sheath gas/auxiliary gas, nitrogen; sheath gas
pressure, 356105 Pa; auxiliary gas pressure, 206105 Pa; ion
transfer capillary temperature, 300uC. Quantification was
performed using SIM mode with [M+Na]+ peak: m/z 645.4 for Rh2;
m/z 483.3 for Ppd; m/z 787.5 for digitoxin (internal standard).
The pharmacokinetic parameters of digoxin, 20(S)-Rh2 and
20(R)-Rh2 in rats were obtained by noncompartmental analysis
using DAS (Drug and Statistics, version 2.1, Chinese
Pharmacological Society). The area under the plasma concentration-time
curve (AUC) was calculated using the trapezoidal method.
For the transport assay, the apparent permeability coefficient (Papp)
and efflux ratio (ER) were calculated as reported previously .
Data are expressed as mean 6 S.E.. Comparisons for
betweengroups were performed using Students t test. For multiple
comparisons, one-way analysis of variance (ANOVA) followed
by Post-Hoc test was adopted. The difference was considered to be
statistically significant if the probability value was less than 0.05
The authors wish to sincerely thank Dr. Chaonan Zheng, Hua Ai and
Yuan Sun (Key Lab of Drug Metabolism and Pharmacokinetics, China
Pharmaceutical University, Nanjing, China), Dr. Yi Gu (DMPK/Tox,
Hutchison MediPharma Ltd., Shanghai, China) and Dr. Yu Lu
(PerkinElmer, Inc, Shanghai, China) for their kind assistance and hard
work in the performance of the experiments and the review of the paper.
Conceived and designed the experiments: JZ FZ GW. Performed the
experiments: JZ FN ML XW. Analyzed the data: JZ FZ ML. Contributed
reagents/materials/analysis tools: JS GW. Wrote the paper: JZ FZ.
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