The involvement of P-glycoprotein in berberine absorption.

Abstract

Berberine is an important ingredient in a number of traditional Chinese medicines but has been shown to have poor bioavailability in the dog. The aim of this study was to use the P-glycoprotein (P-glycoprotein) inhibitors cyclosporin A, verapamil and the monoclonal antibody C219 in in vivo and in vitro models of intestinal absorption to determine the role of P-glycoprotein in berberine absorption. In the rat recirculating perfusion model, berberine absorption was improved 6-times by P-glycoprotein inhibitors. In the rat everted intestinal sac model, berberine serosal-to-mucosal transport was significantly decreased by cyclosporin A. In Ussing-type chambers, the rate of serosal-to-mucosal transport across rat ileum was 3-times greater than in the reverse direction and was significantly decreased by cyclosporin A. In Caco-2 cells, berberine uptake was significantly increased by P-glycoprotein inhibitors and by monoclonal antibody C219. P-glycoprotein appears to contribute to the poor intestinal absorption of berberine which suggests P-glycoprotein inhibitors could be of therapeutic value by improving its bioavailability.

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C Pharmacology & Toxicology 2002, 91, 193–197.
Printed in Denmark . All rights reserved
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ISSN 0901-9928
The Involvement of P-Glycoprotein in Berberine Absorption
Guo-yu Pan, Guang-Ji Wang, Xiao-Dong Liu, J. Paul Fawcett and Yuan-Yuan Xie
Center of Pharmacokinetics, China Pharmaceutical University, Nanjing, China and School of Pharmacy,
University of Otago, Dunedin, New Zealand
(Received January 9, 2002; Accepted May 23, 2002)
Abstract: Berberine is an important ingredient in a number of traditional Chinese medicines but has been shown to have
poor bioavailability in the dog. The aim of this study was to use the P-glycoprotein (P-glycoprotein) inhibitors cyclosporin
A, verapamil and the monoclonal antibody C219 in in vivo and in vitro models of intestinal absorption to determine the
role of P-glycoprotein in berberine absorption. In the rat recirculating perfusion model, berberine absorption was im-
proved 6-times by P-glycoprotein inhibitors. In the rat everted intestinal sac model, berberine serosal-to-mucosal transport
was significantly decreased by cyclosporin A. In Ussing-type chambers, the rate of serosal-to-mucosal transport across
rat ileum was 3-times greater than in the reverse direction and was significantly decreased by cyclosporin A. In Caco-2
cells, berberine uptake was significantly increased by P-glycoprotein inhibitors and by monoclonal antibody C219. P-
glycoprotein appears to contribute to the poor intestinal absorption of berberine which suggests P-glycoprotein inhibitors
could be of therapeutic value by improving its bioavailability.
Berberine is an isoquinoline alkaloid found in the plant gen-
era Berberis and Coptis. The plant has antibacterial and
antiinflammatory action and has been used in traditional
Eastern medicine for over two millennia to treat gastroen-
teritis and secretory diarrhoea (Taylor et al. 1999). In recent
years, a number of reports have described the use of berber-
ine and its derivatives in the treatment of tumours, inflam-
matory conditions and hypertension (Liu et al. 1999; Chung
et al. 2000; Zhou & Mineshita 2000). Despite the extensive
literature on the pharmacology of berberine, little infor-
mation is available relating to its pharmacokinetics. The
only report involved oral administration to Beagle dogs and
showed that berberine has poor bioavailability (Sheng et al.
1993).
P-Glycoprotein of epithelial cells plays an important role
in the intestinal barrier where its location in the apical
membrane is ideally suited to its action as a secretory xeno-
biotic efflux pump (Benet et al. 1999). Its broad substrate
specificity allows it to limit the oral bioavailability of many
drugs and can give rise to clinically relevant interactions
between drugs and certain foods (Hunter & Hirst 1997;
Asperen et al. 1998). Some reports suggest that berberine
alkaloids are members of a large group of cationic toxins
that can interact with P-glycoprotein (Stermitz et al. 2000).
For example, Lin et al. (1999) found that berberine treat-
ment could up-regulate P-glycoprotein expression in several
cancer cell lines and decrease retention of the P-glyco-
protein substrate rhodamine 123. On this basis, we reasoned
P-glycoprotein in normal intestinal epithelia could play an
important role in limiting berberine absorption. In this
study, we investigated this hypothesis using the classical P-
Author for correspondence: Guang-Ji Wang, Center of Pharmaco-
kinetics, China Pharmaceutical University, Nanjing, 210009, China
(fax 86–25–5303260, e-mail panguoyu/hotmail.com).
glycoprotein inhibitors cyclosporin A and verapamil in in
vivo and in vitro models of intestinal absorption.
Materials and Methods
Compounds and animal. Berberine, cyclosporin A and verapamil
were purchased from the China National Institute for the Control
of Pharmaceutical and Biological Products. The compounds were
dissolved in DMSO and diluted to desired concentration. Mono-
clonal antibody C219 was kindly supplied by the Division of Phar-
macology of China Pharmaceutical University. All other reagents
were of analytical grade. The final concentration of DMSO in solu-
tions (including control group) is 0.1%. Sprague-Dawley rats (male,
240–260 g, Grade II) were purchased from Shanghai Experimental
Animal Center, China. The protocols for the animal experiments
were reviewed and approved by the China Pharmaceutical Univer-
sity Animal Ethics Committee.
Cell culture materials. Dulbecco’s modified Eagle’s medium and
non-essential amino acids were obtained from GIBCOL Co. (USA).
Foetal calf serum, L-glutamine and penicillin/streptomycin (10,000
U/ml, 10,000 mg/ml) were obtained from TBD Co. (Tianjin, PR
China). Caco-2 cells were obtained from the ATCC (Rockville, MD,
USA). All other cell culture materials were obtained from Costar
(Cambridge, MA, USA).
In vivo absorption in the recirculating perfusion model. Experiments
were carried out as described previously with modification (Ikumi
et al. 1997; Stewart et al. 1997). Rats were fasted overnight,
anaesthetized with urethane (1 g/kg, intrapertoneally) and affixed
supine on a surface under suitable lighting to maintain body tem-
perature. The ileum was exposed by a midline abdominal incision,
and two polyethylene cannulae (outer diameter, 5 mm; inner diam-
eter, 3 mm) were inserted through small slits at the proximal and
distal ends (about 20 cm). To clear the gut, saline solution at 37æ
was passed slowly through it until the effluent was clear. The re-
maining perfusate was carefully expelled from the intestine by
means of air pumped from a syringe. A solution (50 ml) of 50 mM
berberine in D-PBS alone or with P-glycoprotein inhibitors (cyclo-
sporin A, 5 mM; verapamil, 200 mM) was circulated through the gut
at 2.0 ml/min. After 0, 40, 70, 100, 130 and 160 min., aliquots of

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GUO-YU PAN ET AL.
194
perfusion solution (0.2 ml) were removed from the reservoir for
analysis of berberine and replaced with equal volumes of D-PBS.
The D-PBS was maintained at 37æ under bubbling with an O2/CO2
(95/5) mixture throughout the experiment. A correction was made
for the change in volume of water in the intestinal lumen on the
basis of the change in concentration of the poorly absorbed marker,
phenolsulfophthalein (100 mM) present in the circulating fluid.
In vitro transport across everted intestinal sacs. Everted sacs of rat
jejunum and ileum were prepared using a previously described
method (Lo & Huang 2000). Rats were sacrificed by femoral artery
exsanguination under anaesthesia with urethane (1 g/kg, intraper-
itoneally). The whole small intestine was removed and washed with
50 ml D-PBS prewarmed to 37æ. The isolated jejunum and ileum (5
cm proximal to the ileocecal junction) was divided into five 10 cm
segments in ice-cold water. Each segment was everted, washed with
ice-cold D-PBS and allowed to equilibrate in D-PBS at 37æ for 20
min. before the start of experiments. D-PBS (1 ml) containing ber-
berine (50 mM) was placed in the everted sac (serosal side) and both
ends were ligated tightly. The sacs were then placed in 40 ml D-PBS
with or without P-glycoprotein inhibitors (cyclosporin A, 5 mM;
verapamil, 200 mM). Samples were taken after 10, 20, 30, 40, 50, 60
and 90 min., and replaced with an equal volume of buffer. The
medium was maintained at 37æ and stirred by bubbling with O2/
CO2(95/5%) throughout the process.
In vitro transport experiments in the Ussing-type chamber. Transport
experiments were performed as previously described (Hiroshi &
Aungst 1995; Ikumi et al. 1997). Rats were anaesthetized with ur-
ethane (1 g/kg) and an abdominal incision was made. Ileal segments
were pulled onto a glass rod and the fat adhering to the mesentery
was removed. The segments were cut open and washed with ice-cold
D-PBS before the tissue sheets were mounted vertically in the Uss-
ing-type chamber providing an exposed area of 0.72 cm2. Any sec-
tions containing Peyer’s patches were discarded. D-PBS containing
P-glycoprotein inhibitors (cyclosporin A, 5 mM; verapamil, 200 mM)
was put into both donor and receiving chambers and berberine (50
mM) was put into the donor chamber. The volume of bathing solu-
tion on each side was 10 ml. The solution was maintained at 37æ
and gassed with 95/5% O2/CO2.Samples (200 ml) were removed
from the receiving chamber after 0, 15, 30, 60, 90 and 120 min. and
replaced with drug-free buffer.
The cumulative amount of drug transport the membrane was cal-
culated based on the chamber volume and the volumes replaced
with drug-free buffer. It was calculated according to the equation:
TRcumΩAnπvsn
VR?
nª1
iΩ0
Ai
Anis berberine content in sample n, vs is the volume of sample n,
VRis the volume of buffer in reservoir. The apparent permeability
coefficient (Papp, cm/s) was calculated from the linear portion of
the amount permeating v. time plot according to the following
equation:
PappΩ(dQ/dt)/(C0¿A)
where dQ/dt is the rate at which the compound appears in the re-
ceiving compartment (mmol/s), C0is the initial concentration in the
donor compartment (mmol/ml) and A is the surface area of the ex-
posed intestinal membrane (cm2) (Palm et al. 1996).
Uptake by Caco-2 cells. Uptake of berberine by cultured mono-
layers of Caco-2 cells was examined according to methods described
previously (Batrakova et al. 1998;). Caco-2 cells were used between
passages 40–65. Experiments were performed with confluent epi-
thelial monolayers grown on 6-well plates. The culture medium was
renewed 3 hr before the start of the experiment. Cultured cells were
washed three times with 1 ml aliquots of Hank’s balanced salt solu-
tion (HBSS) at 37æ and then preincubated in HBSS for 20 min.
Uptake was initiated by adding 2 ml HBSS containing berberine
(50 mM) alone or with P-glycoprotein inhibitors (cyclosporin A, 5
mM; verapamil, 200 mM) or C219 (100 ng/ml) at 37æ. After 30 min.,
solutions were removed by suction and discarded. The monolayers
were washed three times with 1 ml aliquots of ice-cold HBSS and
then scraped into 1 ml HBSS solution. Cells were destroyed by three
freeze-thaw cycles. Protein concentration was determined by the
Coomassie Brilliant Blue method. Berberine concentrations were
assayed by HPLC and expressed per milligram of protein.
Assay methods. Berberine was assayed by reversed-phase HPLC on
an ODS column (150¿4.6 mm) with fluorescence detection (exci-
tation 348 nm, emission 526 nm) (Misaki et al. 1982; Shen & Xie
1993). The mobile phase consisted of acetonitrile: water (60:40, v/
v) plus KH2PO43.4 g/l and sodium dodecyl sulfate 1.7 g/l at a flow
rate of 1.0 ml/min. The retention time of berberine under these
conditions was 3.4 min.
Statistical methods. All data are given as mean∫S.D. Comparisons
between the means of various treatment groups were analyzed using
ANOVA. Differences were considered to be statistically significant
when P?0.05.
Results
In vivo absorption in the recirculating perfusion model.
The results of this study of absorption across intestinal
loops are shown in fig. 1. Berberine absorption in the first
100 min. was negligible and after 160 min. only 2.5% of the
berberine was absorbed. In the presence of P-glycoprotein
inhibitors, berberine absorption was increased. Absorption
at 160 min. increased to 14.8% for cyclosporin A group
and 17.2% for verapamil group. The difference in amount
absorbed was significant at almost all time points in the
cyclosporin A group and at 70 min. and 160 min. in the
verapamil group.
In vitro transport across everted intestinal sacs.
The results of experiments with everted sacs are shown in
fig. 2. Serosal-to-mucosal flux produced a final concen-
tration of 62.6∫7.5 nM after 90 min. The effect of verapam-
Fig. 1. Intestinal absorption across the rat small intestine in the
recirculating perfusion model. Berberine left in rat smalll intestine
was measured in the presence or absence of 5 mM cysclosporin A
(g) and 200 mM verapamil (S). Control group (M). (* means
cysclosporin A versus control group; ! means verapamil versus con-
trol group. Statistically significant difference, P?0.05, nΩ5).

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195
P-GLYCOPROTEIN INHIBITION AND BERBERINE ABSORPTION
Fig. 2. Berberine (50 mM) transport in everted sacs of rat intestine
model. Berberine concentration on mucosal side was measured in
the presence or absence of 5 mM cysclosporin A (g) and 200 mM
verapamil (S). Control group (M). (* means cysclosporin A versus
control group; ! means verapamil versus control group. Statistically
significant difference, P?0.05, nΩ4 or 5).
il on berberine transport was not significant although after
60 min. berberine secretion was reduced. By contrast, cyclo-
sporin A significantly decreased berberine transport after
40 min. (P?0.01).
In vitro transport experiments in the Ussing-type chamber.
Serosal-to- mucosal flux was 3 times greater than in the
reverse direction (fig. 3). The relationship between cumula-
tive berberine transport and time is shown in fig. 4. With
P-glycoprotein inhibitors on the mucosal side, berberine
transport was significantly slower after 60 min. in the cyclo-
sporin A group. With verapamil, berberine transport was
also slower but the decrease was not significant. This is re-
flected in the Papp values which decreased from 9.41∫2.24
(cm/s¿10ª6) to 5.11∫1.79 (cm/s¿10ª6) in the cyclosporin
A group and to 6.01∫2.76 (cm/s¿10ª6) in the verapamil
group.
Fig. 3. Berberine transport across rat ileum in Ussing-type
chambers. Data are berberine flux (nmol/cm2) after administration
of 50 mmol to the donor side over 120 min. (nΩ6). Serosal-to-mu-
cosal flux (M); mucosal-to-serosal flux (k). (* Statistically signifi-
cant difference P∞0.05).
Fig. 4. Cumulative berberine (50 mM) secretion across rat ileum in
modified Ussing chamber. Berberine concentration was measured
in the presence or absence of 5 mM cysclosporin A (g) and 200 mM
verapamil (S). Control group (M). (* means cytosporin A versus
control group; ! means verapamil versus control group. Statistically
significant difference, P?0.05, nΩ6).
Uptake by Caco-2 cells.
The uptake of berberine was time-dependent reaching
steady-state after 20 min. incubation (data not shown).
Using berberine uptake at 30 min. as a measure of berberine
transport, increasing concentrations of berberine (5–200
mM), were accompanied by an increase in steady-state up-
take, although the extent of the increase was smaller in the
presence of 200 mM berberine than the extent of increase at
50 mM berberine. There was an increase in Caco-2 cell up-
take of berberine from 5 to 50 mM, followed by a decrease
at 200 mM. The inhibitory effects of P-glycoprotein inhibi-
tors and monoclonal antibody on the uptake of berberine
were also studied. As shown in table 1, the increase in up-
take caused by C219, verapamil and cyclosporin A was sig-
nificant at almost every concentration except 5 mM. The
significant increase in berberine uptake by monoclonal anti-
body C219 provides further evidence that P-p has a signifi-
cant impact on berberine transport.
Discussion
The clinical use of berberine is limited to gastroenteritis and
secretory diarrhoea where it acts within the gut. However
in clinical trials, it was found that co-administration of ber-
berine caused a significant elevation (29.3%) in the steady-
state blood concentration of cyclosporin A in renal trans-
plant recipients (Li et al. 2001). Since cyclosporin A is a
classical P-glycoprotein inhibitor, this result suggests that
berberine may also interact with P-glycoprotein. This paper
describes the first investigation of the involvement of P-gly-
coprotein in berberine absorption.
The rat recirculating perfusion model was applied to test
berberine absorption across the small intestine. As pre-
viously reported (Sheng et al. 1993), it was found that ber-
berine itself is poorly absorbed (?5%). However, when co-
administered with cyclosporin A, berberine absorption was

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GUO-YU PAN ET AL.
196
Table 1.
Berberine uptake in Caco-2 cells. Berberine (Ber) concentration was measured in the presence or absence of 5 mM cysclosporin A, 200 mM
verapamil and 100 ng C219. (* means statistically significant difference, P?0.05, nΩ3. Unit is nmol/mg protein).
Ber conc. (mM) ControlC219 Ver CsA
5 79.8∫8.8
218.9∫12.1
600.9∫64.7
736.8∫71.1
92.9∫4.9
259.4∫15.8*
754.0∫51.1*
1214.4∫257.1*
40.6∫11.3
321.0∫18.9*
908.6∫77.7*
1376.7∫323.8*
95.0∫12.3
327.8∫19.7*
1262.3∫394.9*
1525.2∫199.3*
25
50
200
Abbreviations: Berberine (Ber), Verapamil (Ver), Cysclosporin A (CsA).
C219, C219 p-glycoprotein monoclonal antibody.
improved almost 6 times and the characteristics of the ab-
sorption were changed. The absorption process took place
mostly within 0–40 min. Verapamil, another classical P-gly-
coprotein inhibitor, also improved berberine absorption,
but its effect was not as obvious as that of cyclosporin A.
The ability of cyclosporin A and verapamil to reduce ber-
berine absorption indicates that intestinal P-glycoprotein is
involved (Ikumi et al. 1997). This hypothesis is further sup-
ported by the measurement of serosal-to-mucosal and mu-
cosal-to-serosal flux in the Ussing-type chamber. Berberine
serosal-to-mucosal flux was shown to be 3 times greater
than in the reverse direction indicating an active transport
system exists in intestinal epithelial cells which pumps ber-
berine into the intestinal lumen (Prueksaritanont et al.
1998). The secretory efflux was markedly reduced by cyclo-
sporin A and to a lesser extent by verapamil. This phenom-
enon was also observed in the everted intestinal sac model.
The results clearly indicate that P-glycoprotein reduces net
berberine absorption in the gut.
Cultured Caco-2 cells were used to investigate berberine
uptake in intestinal epithelial cells. P-Glycoprotein inhibi-
tors significantly increased berberine uptake providing
further evidence that P-glycoprotein acts to pump berberine
out of intestinal epithelial cells and thereby decrease its ab-
sorption (Cormet-Boyaka et al. 1998; Yumoto et al. 1999).
The P-glycoprotein monoclonal antibody C219 also in-
creased berberine uptake despite the fact that C219 has
been shown to recognize a cytosolic epitope of P-glyco-
protein (Elias et al. 1991). This suggests C219 can be taken
up by Caco-2 cells or that its ability to increase berberine
uptake is the result of a non-specific effect. Other workers
have shown that C219 can significantly increase rhodamine
123 accumulation in brain capillary endothelial cells sup-
porting the former hypothesis (He et al. 2002). Further
study is required to elucidate this finding.
In summary, the results in the present study show that
berberine is a xenobiotic with poor bioavailability (?5%)
which can be increased markedly by cyclosporin A and to
a lesser extent by verapamil. The results suggest berberine
is a P-glycoprotein substrate and that its poor bioavail-
ability is due to its secretion by P-glycoprotein into the in-
testinal lumen. Our paper supports the idea that powerful
P-glycoprotein inhibitors may be of use in extending the
clinical use of berberine.
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