Journal of Ethnopharmacology 103 (2006) 425–432

The effects of sinomenine on intestinal absorption ofpaeoniflorin by the everted rat gut sac model

Kelvin Chana, Zhong Qiu Liua, Zhi Hong Jianga, Hua Zhoua,Yuen Fan Wonga, Hong-Xi Xub, Liang Liua,∗

a School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR Chinab Hong Kong Jockey Club Institute of Chinese Medicine Limited, 5/F, 2 Science Park East Avenue, Hong Kong Science Park,

Pak Shek Kok, Shatin, New Territories, Hong Kong, PR China

Received 26 May 2005; received in revised form 1 August 2005; accepted 16 August 2005Available online 19 September 2005

Abstract

Paeoniflorin and sinomenine, derived from the root ofPaeonia lactiflora Pall. (family Ranunculaceae) and the stem ofSinomenium acutumRehder & Wilson (family Menispermaceae), respectively, have been, and are currently, widely used for treatment of rheumatic and arthriticd aeoniflorini cteristics ofp odel. Ther . However,t s from 10 to1 cet il andq ern, whii enhancet vement ofp©

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iseases in China and Japan. Our previous studies demonstrated that sinomenine could significantly improve the bioavailability of pn rats, but the underlying mechanisms remain unknown. The present study aims to investigate the intestinal kinetic absorptive charaaeoniflorin as well as the absorptive behavior influenced by co-administration of sinomenine using an in vitro everted rat gut sac mesults showed a good linear correlation between the paeoniflorin absorption in sac contents and the incubation time from 0 to 90 minhe concentration dependence showed that a non-linear correlation exists between the paeoniflorin absorption and its concentration60�M, and the absorption was saturated at about 80�M of the drug. Sinomenine at 16 and 136�M concentrations could significantly enhan

he absorption of paeoniflorin (20�M) by 1.5- and 2.5-fold, respectively. Moreover, two well-known P-glycoprotein inhibitors, verapamuinidine, could significantly elevate the absorption of paeoniflorin by 2.1- and 1.5-fold, respectively. Furthermore, sinomenine in a pattch

nfluenced paeoniflorin’s absorption, manifested as similar to that of P-glycoprotein inhibitors. In conclusion, sinomenine significantlyhe intestinal absorption of paeoniflorin, subsequently improve the bioavailability of paeoniflorin. The mechanism underlying the improaeoniflorin’s bioavailability was proposed that sinomenine could decrease the efflux transport of paeoniflorin by P-glycoprotein.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Paeoniflorin; Sinomenine; Everted rat gut sacs; Absorption; Bioavailability; P-glycoprotein inhibitor

. Introduction

Paeoniflorin is a characteristic monoterpene glucosideFig. 1) derived from the root of a Chinese medicinal plant,aeonia lactiflora Pall. (family Ranunculaceae). Chinese andapanese doctors have been using this plant in combinationith other herbs to treat inflammatory and arthritic diseases

or hundreds of years. The therapeutic effects of this plant asell as its active component, paeoniflorin, have been confirmedy pharmacological results, including potent analgesic, anti-

nflammatory, sedative and anti-coagulative activities (Ishida

∗ Corresponding author. Tel.: +852 3411 2457; fax: +852 3411 2461.E-mail address: liuliang@hkbu.edu.hk (L. Liu).

et al., 1987; Liang et al., 1990; Ohta et al., 1994; Watan1997). However, the pharmacokinetic studies demonstratedpaeoniflorin has a poor absorption and thus a very low bioaability (3–4%) when administrated orally. This was deduto be due to the limited transportation of paeoniflorin acgastrointestinal mucosa into the blood (Takeda et al., 1991997).

Recently, we found that sinomenine (7,8-didehydrhydroxy-3,7-dimethoxy-17-methylmorphinan-6-one;Fig. 1),an alkaloid derived from the Chinese herbSinomenium acutumRehder & Wilson (family Menispermaceae), could markeimprove the oral bioavailability of paeoniflorin in rats. In jugulcatheterized male Sprague–Dawley rats, the co-administof sinomenine (90 mg/kg) markedly elevated the bioavailaity of paeoniflorin to more than 12 times in comparison w

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.08.020

426 K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432

Fig. 1. Chemical structures of paeoniflorin and sinomenine.

animals treated with paeoniflorin (150 mg/kg) alone (Liu et al.,2005). Pharmacologically, sinomenine has been demonstratedto significantly inhibit inflammatory reactions caused by var-ious phlogistic agents (Irino, 1958; Cheng et al., 1964). Ourprevious studies showed that sinomenine significantly amelio-rated arthritic pathologies in adjuvant arthritic rats and inhibitcell proliferation of lymphocytes and synovial fibroblasts (Liuet al., 1994, 1996). Clinically, in treating arthritis, Chinese andJapanese doctors usually prescribe both the root ofPaeonia lac-tiflora and the stem ofSinomenium acutum together (Wong andWu, 1936). This clinical practice likely suggested synergisticeffects between the two herbs in which the herb–herb interac-tion in intestinal drug absorption would be greatly implicated.To study intestinal absorption kinetics of the oral drugs, severalin vivo and in vitro models have been developed (Anderberget al., 1992; Park and Miltra, 1992; Hovgaard and Brondsted,1995; Lindmark et al., 1995; Stewart et al., 1995; Barthe et al.,1998). The everted rat gut sac technique was first described byWilson and Wisemen (Rubinstein, 1990). Researchers furtherdeveloped an improved everted rat gut sac system (Barthe etal., 1998) by using tissue medium, TC199. The reproducibilityof this in vitro model suggests that the everted rat gut sac is auseful screening tool for studying transport of P-glycoprotein(P-gp) substrates and potential P-gp modifiers (Carreno-Gomezand Duncan, 2000).

Using the in vitro everted rat gut sac model, Takeda et al.i avioo izedi oms rmea ility( ndt lowp n ofs atioa rini ainu testn rtedr P-gi ismso rini

2. Materials and methods

2.1. Reagents

Tissue culture medium, TC 199 (10× concentrated withEarle’s salts), glutamine, verapamil quinidine, digoxin, sodiumbicarbonate, pentoxifylline (internal reference standard forpaeoniflorin) were obtained from Sigma Chemical Co. (St.Louis, MO, USA). The reference standards of paeoniflorin andsinomenine hydrochloride (purity≥ 98%) were purchased fromthe Institute for the Control of Pharmaceutical and BiologicalProducts of China, Beijing, China.O-Dianisidine hydrochlo-ride, peroxidase, glucose oxidase, Triton-X 100 were obtainedfrom DBH Laboratory Supplies (USA), Folin-Ciocalteu’s phe-nol reagent was purchased from Sigma Chemical Co. (St. Louis,MO, USA). Acetonitrile and perchloric acid were obtained fromMerck (Darmstadt, Germany). All other reagents were of ana-lytical grade or HPLC grade.

2.2. Preparation of the everted rat gut sacs

This was carried out as a described previously (Bouer etal., 1999). Briefly, adult male Sprague–Dawley rats (250–300 g;11–12 weeks) were starved for 24 h, sacrificed by cervical dislo-cation and the small intestine removed and washed through threet ure.T d( rtedo e fill-i inge.T l sacs( res.E ining9 -i ine,w . Thefl , ther rb werer andb (sacc ighedb ately

nvestigated the intestinal absorptive and metabolic behf paeoniflorin, showing that paeoniflorin was not metabol

n the gut wall and only has a very low permeability frerosal side to mucosal side of the intestine. This low pebility of paeoniflorin may cause its poor oral bioavailabTakeda et al., 1995). In our previous studies, it was fouhat oral administration of paeoniflorin in rats had verylasma concentration of the drug, while co-administratioinomenine could markedly elevate the plasma concentrnd significantly improve the oral bioavailability of paeoniflo

n rats (Liu et al., 2005). The underlying mechanisms remnknown. Thus, the present study was to investigate the inal absorptive behavior of paeoniflorin using the in vitro eveat gut sac model in the presence of sinomenine, and somenhibitors, in order to reveal the possible underlying mechanf sinomenine improving the bioavailability of paeoniflo

n vivo.

r

-

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i-

p

imes with saline (0.9% NaCl solution) at room temperathe intestine was immediately places in 37◦C and oxygenateO2/CO2, 95%:5%) medium TC 199. The intestine was even a glass rod (2.5 cm in diameter) and one end clamp befor

ng with fresh, oxygenated medium using a 50 ml glass syrhe intestine was then sealed with a second clamp. Smal2–2.5 cm in length) were then tied using silk braided sutuach sac was placed in a 50 ml Erenemyer flask conta.0 ml of oxygenated media at 37◦C. 1 ml of medium, contain

ng paeoniflorin, sinomenine, digoxin, verapamil, or quinidas then added to the required concentration, accordinglyasks were stoppered with gas-tight silicon bungs. Finallyesulting gut sacs were incubated at 37◦C in an oscillating wateath (60 cycles/min). At the appropriate time points, sacsemoved, washed four times in saline (0.9% NaCl solution)lotted dry. The sacs were cut open and the serosal fluidontents) was drained into small tubes. Each sac was weefore and after serosal fluid collection to calculate accur

K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432 427

the volume inside the sac and to correct the serosal fluid forthe actual volume. The area of each sac was measured. Samplesof the medium and the serosal fluid of each sac were kept forHPLC analysis. From the analysis, the sac content volumes andthe absorption amount of paeoniflorin and digoxin (nmol/mm2)present in each sac were calculated. Each experiment was carriedout using the small intestine from one animal. All proceduresinvolving animal care were carried out according to the regula-tions of the Committee on the Use of Human & Animal Subjectsin Teaching and Research, Hong Kong Baptist University andDepartment of Health, Hong Kong.

2.3. Determination of paeoniflorin

An analytical HPLC system consisting of an Agilent qua-ternary HPLC model HP 1100 series (Hewlett-Packard, PaloAlto, CA), fitted with an Altima C18 column (250 mm× 4.5 mm,5�m), was employed. The mobile phase is a mixture of acetoni-trile (with 0.2% acetic acid and 0.5% triethylamine) and water(15:85), filtered through a 0.45�m Millipore filter (Millipore,Hong Kong). The flow-rate was maintained at 1.0 ml/min, andthe detection was performed at a wavelength of 231 nm underconstant temperature (25± 0.1◦C). Constant amount of pentox-ifylline (internal standard) was added to the samples collectedfrom the medium and serosal fluid of the everted rat gut sacs, andthen 80�l of each sample was injected into the HPLC system forac car-r f thec interf PLCr oxi-m

2

l HP1 usef ace-tfi at1 ngthom ands sys-t 015t erec e ot fromo timf

2t

ccur gut

sac model, the uptake amount (accumulation) of paeoniflorinin gut tissue and the recovery of paeoniflorin at the concentra-tion of 200�M from the incubation medium in the serosal side,the medium in the mucosal side and gut sac tissue after 45 minincubation were examined. Each sac tissue sample was accu-rately weighed, added with 1 ml iced-saline, and homogenizedfor 10 min in iced water-bath. The mixture was centrifuged at13,000 rpm. 0.5 ml of the supernatant of each sample was thenmixed with 50�l of 4.5% aqueous perchloric acid solution byvortex for 5 min. The denatured protein precipitate was furtherseparated by centrifugation at 13,000 rpm for 10 min at roomtemperature. Finally, 80�l of the supernatant of each samplewas analyzed by HPLC and the uptake amount of paeoniflorin inthe gut sac tissue was calculated in sacs. The amount of paeoni-florin both in the serosal side and in the mucosal side of mediumwere measured following HPLC method described as in Section2.3. The total recovery rate and the uptake rate for paeoniflorinwere calculated as follows:

Recovery (%) = [paeoniflorin amount (�g) in theserosal medium + paeoniflorin amount (�g) in the mucosalmedium + paeoniflorin amount (�g) in the sac tissue/actuallyadded amount of paeoniflorin (�g) in medium (10 ml)]× 100%.

Uptake rate (%) = [paeoniflorin amount (�g) in the sac tis-sue/actually added amount of paeoniflorin (�g) in medium(10 ml)]× 100%.

2

uts incu-b ifiedmp )w gentwO -d y thea ureda

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nalysis. The calibration curve was linear from 0.025 to 25�Moncentrations of paeoniflorin. Appropriate dilutions wereied out to obtain various concentrations within the range oalibration curve. The chromatogram system showed noerence with other components in the samples, and the Hetention times for paeoniflorin and pentofylline were apprately 16.1 and 33.2 min, respectively.

.4. Determination of digoxin

The HPLC system composed of the same HPLC mode100 series and the column in paeoniflorin analysis was

or digoxin analysis. The mobile phase was a mixture ofonitrile and water (28:78), filtered through a 0.45�m Milliporelter (Millipore Hong Kong). The flow-rate was maintainedml/min, and the detection was performed at a wavelef 218 nm under constant temperature (25± 0.1◦C). Eightyicrolitres of each sample collected from the medium

erosal fluid of the gut sac was injected into the HPLCem for analysis. The calibration curve was linear from 0.o 15�M concentrations of digoxin. Appropriate dilutions warried out to obtain various concentrations within the ranghe calibration curve. The system showed no interferencether components in the samples, and the HPLC retention

or digoxin was approximately 15.3 min.

.5. Recovery of the assay and the uptake of paeoniflorin inhe gut sac tissue

To evaluate the analytical method for the precision and aacy of paeoniflorin absorption in the in vitro everted rat

-

d

f

e

-

.6. Validation of the viability of the everted rat gut sacs

In order to verify the integrity and viability of the gacs, glucose measurement was conducted both in theation medium and in the gut sac contents using a modethod described byDahlqvist (1968). Briefly, 20�l of sam-le of medium (made up to 100�l with double distilled wateras incubated for 45 min with 1 ml of glucose oxidase reahich consists of: 0.2% Triton-X 100 (w/v in ethanol), 10�g/ml-dianisidine-HCl, 1�g/ml peroxidase, 200�g/ml glucose oxiase in 0.5 M Tris–HCl, pH 7.2. The reaction was stopped bddition of 2 ml of 5 M HCl, and the absorbance was meast 525 nm with a Beckman Coulter (DU530 UV–vis, USA).

.7. Statistical analysis

All values are expressed as mean± S.D. Analysis of variatioNOVA followed by post hoc test with LSD test was used fvaluation of data andP values lower than 0.05 were acceps statistically significant.

. Results

.1. Tissue uptake and recovery of paeoniflorin in theverted rat gut sacs

The data for uptake and recovery of paeoniflorin at 200�Mfter 45 min incubation in TC 199 medium are showed inTable 1.he total recovery rate included paeoniflorin amount inedium of the serosal side, of the mucosal side and in the g

issue was higher than 97%; whereas the paeoniflorin upta

428 K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432

Table 1Tissue uptake and recovery of paeoniflorin at 200�M concentration over 45 min incubation in the in vitro everted rat gut sacs system (n = 5)

Testedsac no

Addedpaeoniflorin(�g)

Paeoniflorin measuredin mucosal sidemedium (�g)

Paeoniflorin measuredin serosal side medium(�g)

Paeoniflorin measuredin sac tissue (�g)

Paeoniflorin uptakerate in sac tissue (%)

Total recovery ofpaeoniflorin (%)

X X̄ ± S.D.a X X̄ ± S.D.b

1 100 78.1 4.3 9.1 9.1 91.52 100 79.9 5.2 8.4 8.4 10.0± 1.7 93.5 97.2± 5.83 100 88.4 3.6 11.8 11.8 R.S.D.% = 17.0 103.9 R.S.D.% = 6.04 100 84.9 6.1 12.0 12.0 102.95 100 80.5 4.7 8.9 8.9 94.1

a The tissue uptake amount denotes the ratio of the measured value in sac tissue over the added amount of paeoniflorin.b The total recovery rate means the ratios of the measured amount of paeoniflorin contents in the medium of the mucosal and serosal sides, and the amount inthe

sac tissue over the added amount of paeoniflorin.

sac tissue was about 10%. The content of paeoniflorin detectedin the sac tissue inferred that paeoniflorin could be transportedacross the small intestinal epithelium by the transcellular routeinto the sac fluid of the serosal side.

3.2. Viability of the gut sacs

In the presence of paeoniflorin at 200�M, or sinomenine at136�M, or verapamil at 100�M, or quinidine at 1.3 mM, theratios of the glucose contents in the serosal fluid to the mucosalside were increased with the incubation time up to 120 min(Table 2), which indicated that the tissue of the gut sacs wereviable, i.e., those compounds at the tested concentrations did notinduce toxicity to the gut sac tissue.

3.3. Time dependence of the paeoniflorin absorptive profile

The intestinal kinetic absorptive behavior of paeoniflorin wasstudied in the gut sac model with TC 199 medium culture sys-tem at different incubation time periods from 0 to 90 min. Theresult showed that the absorption of paeoniflorin was graduallyincreased along with increase of incubation time, up to 90 min.The correlation between the drug absorption (Y, nmol/mm2)and the incubation time (X, min) was linear (Y = 0.0004X,R2 = 0.9931) when paeoniflorin was added into the medium at200�M concentration. These indicate that paeoniflorin can bet guts m fort ed tob

Fig. 2. Concentration dependence of the paeoniflorin absorptive profile at therange of concentrations from 10 to 160�M in TC199 medium of the evertedrat gut sac system. The data represent the means± S.D. (n = 5 sacs; from oneanimal).

3.4. Concentration dependence of the paeoniflorinabsorptive profile

Tissue incubation in TC 199 medium for 60 min was con-ducted for studying the concentration dependence of paeoni-florin absorptive profiles with different concentrations (0,10,20, 40, 80, 160�M) of the drug. The results, shown inFig. 2,demonstrate that there was a non-linear increase in paeoniflorinabsorption into the sac contents corresponding to the increaseof paeoniflorin concentration in the TC 199 medium. Whenpaeoniflorin concentration was increased three-fold, i.e., from10 to 40�M, the paeoniflorin absorption was increased by a

TI bility of the in vitro everted gut sacs over the time periods of incubation indicated by ther the mucosal side

I

Sinomenine at 136�M Verapamil at 100�M Quinidine at 1.3 mM

1.0± 0.0 1.0± 0.0 1.1± 0.01.3± 0.1 1.0± 0.0 1.2± 0.01.4± 0.1 1.4± 0.0 1.6± 0.32.0± 0.2 1.8± 0.1 2.2± 0.4

1 2.3± 0.1 2.5± 0.4 2.6± 0.5

T

ransported from the medium into the serosal fluid of theacs across intestinal epithelium. Thus, the culture systehe in vitro everted rat gut sacs used in this study was verifie significantly functional.

able 2nfluences of paeoniflorin, sinomenine, verapmil and quinidine on the viaatios of glucose contents in the serosal fluid of the sacs: the medium of

ncubation time (min) Ratio of glucose content (%)

Blank Paeoniflorin at 200�M

0 1.1± 0.0 1.0± 0.030 1.2± 0.0 1.2± 0.060 1.5± 0.1 1.5± 0.190 1.8± 0.2 2.1± 0.320 2.3± 0.2 2.5± 0.4

he data represent mean± S.D. (n = 3).

K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432 429

Fig. 3. The absorptive characteristics of paeoniflorin in the everted rat gut sac system influence by sinomenine, verapamil and quinidine. The data represent themeans± S.D. (n = 5 sacs; from one animal).* P < 0.05. (A) Sinomenine at 16�M; (B) sinomenine at 136�M; (C) verapamil at 100�M; and (D) quinidine at 1.3 mM.

factor of about 10. However, this factor was not continuouslyincreased when paeoniflorin concentration in the medium wasfurther increased to 160�M. These results indicate that the trans-port and absorption of paeoniflorin in this in vitro gut sac systemwould be saturated at the concentration of about 80�M.

3.5. Influence of sinomenine on the absorptive profile ofpaeoniflorin

In order to determine the possible effect of sinomenine onthe enhancement of paeoniflorin absorption in intestine, co-incubative tissue culture tests with paeoniflorin (20�M) andsinomenine (16 and 136�M) were conducted. When treatedwith paeoniflorin alone, there was good linear correlationbetween paeoniflorin absorption and incubation time (shownin Fig. 3A and B with R2 = 0.9766 and 0.9689, respectively).However, when sinomenine was added to the TC 199 medium,this correlation became non-linear. After 45 min incubation, theabsorption of paeoniflorin significantly increased. That is, whenpaeoniflorin was co-incubated with sinomenine at concentra-tions of 16�M (Fig. 3A) and 136�M (Fig. 3B), the absorptionof paeoniflorin was increased approximately to 1.5- and 2.5-fold,respectively. These results suggest that sinomenine can enhancepaeoniflorin absorption in small intestine.

3.6. Influences of P-glycoprotein inhibitors on thea

-gpi rs,

verapamil or quinidine were added to the medium containing20�M paeoniflorin.Fig. 3C and D show that there is good lin-ear correlation between the amount of paeoniflorin absorbedand the incubation time (R2 = 0.9970 and 0.9980, respectively)in the absence of verapamil or quinidine. However, when ver-apamil is added at a concentration of 20�M, this correlationbecome non-linear. After 45 min incubation with paeoniflorinand verapamil together, paeoniflorin absorption increased up toabout 2.1-fold in comparison with the non-verapamil treated gutsacs. When quinidine was added at a concentration of 1.3 mM,this correlation become non-linear too. When paeoniflorin andquinidine were added together, there was about 1.5-fold increasein paeoniflorin absorption from the medium (Fig. 3C and D).These results indicate that the inhibition of the intestinal P-gp byverapamil and quinidine could significantly enhance intestinaltransportation as well as the kinetic absorption of paeoniflorinin rat intestine.

3.7. Influence of sinomenine on the absorptive profile ofdigoxin

Digoxin is a well-known substrate for P-glycoprotein, whoseabsorption can be significantly enhanced by administration ofP-gp inhibitors, such as verapamil and quinidine (Bouer etal., 1999). In order to investigate the effect of sinomenine onenhancement of the intestinal absorption of substrate-like drugso me-n cs s

bsorptive profile of paeoniflorin

To determine the possible potential role of intestinal Pn the kinetic absorption of paeoniflorin, two P-gp inhibito

f the P-glycoprotein, an experiment of co-incubation of sinoine (136�M) with digoxin (13�M) in the everted rat gut saystem was conducted. The results inFig. 4show that there wa

430 K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432

Fig. 4. Influence of sinomenine at 136�M concentration on the absorption ofP-gp substrate digoxin in the everted rat gut sac system. The data represent themeans± S.D. (n = 5 sacs; from one animal).* P < 0.05.

good linear correlation between digoxin absorption and incuba-tion time (R2 = 0.9924). However, when sinomenine was addedto the medium at a concentration of 136�M, this correlationbecame non-linear. After 45 min incubation, the digoxin absorp-tion was increased up to about 2.5-fold in comparison with thatin the non-sinomenine treated gut sacs. These results indicatethat sinomenine can influence digoxin absorption as verapamiland quinidine do.

4. Discussion

There are many factors that can influence the bioavailabil-ity, for example, drug solubility, the gastrointestinal pH, gas-tric emptying, gastrointestinal transit and different interactions(Barthe et al., 1998). In addition, there are some other possi-bilities, where the presystemic elimination of the product willreduce the quantity reaching the circulation by degradation inthe intestinal lumen and/or the metabolism in the enterocyteduring passage leading to low bioavailability. The previous stud-ies of paeoniflorin showed that oral administration of the drughad a very low bioavailability due to the limited transportationof the drug across intestinal epithelium into the blood circu-lation (Takeda et al., 1995, 1997). However, other investigationdemonstrated that the intestinal bacteria could quickly transformpaeoniflorin into a major metabolite, paeonimetabolin I, whichc gen-e l-i porta sm ot harm singa wedt theb onw indi-c eonfl ainu

t sat sti-

nal absorption behavior of paeoniflorin. This model was firstlydescribed byWilson and Wiseman (1954)and further improvedby Barthe et al. (1998), using a complex tissue culture medium(TC 199) as a replacement of the salt medium. However, the mainconcern of this model is the tissue viability like the in vitro modelof two chambers of intestine. Previous studies showed that wheneach sac was incubated with gentle shaking in the oxygenatedTC 199 medium, the tissue viability could be maintained for upto 120 min, as demonstrated by oxygen consumption, glucoseand amino acid uptake (Barthe et al., 1998). To check the sacviability and integrity, glucose concentration can be monitoredinside and outside the everted sacs during the experiments. Theresults (Table 2) showed that along with increasing of the incu-bation time of the everted gut sacs from 0 to 120 min, the ratio ofthe glucose contents in the serosal side to the mucosal side wasincreased in the presence of paeoniflorin, or sinomenine, or ver-apamil, or quinidine. This implies that the gut sacs were viableand functional during the experiments. As glucose is activelytransported, there is an increase on the internal concentrationcompared with external medium, and hence a concentrationgradient. Moreover, this model is a two-compartment systempermitting calculation of the kinetics of uptake into the epithe-lial tissue by assaying materials in the cells, and transfer acrossthe entire epithelial layer, by measuring materials that is foundinside the sacs at the end of the incubation period. The presentstudy showed that the paeoniflorin contents both inside the sacsa d thetc s arev

thatt sorp-t thes m 0t r. Tos 0, 20,4 nts,a tationa in int en-t oni ,w orinm thatn nre-s rationo con-c wasd theCp me-n s ofp esultss tedw C1 t ofp up to

ould be rapidly absorbed from the intestinal tract into theral circulation (Heikal et al., 1997). Thus, the low bioavailabi

ty of paeoniflorin may result from the poor intestinal transnd absorption, as well as the degradation and metaboli

he drug in the intestinal lumen. Our previous studies on pacokinetic interaction of paeoniflorin and sinomenine un in vivo model in unrestrained conscious male rats sho

hat co-administration of them could significantly elevateioavailability of paeoniflorin up to 12 times in comparisith the animals treated with paeoniflorin alone. Theseate that sinomenine can function as an enhancer of paorin bioavailability, while the underlying mechanisms remnknown.

In the present study, we employed the everted rat guechnique as an in vitro model for the study of the inte

s

f-

i-

c

nd the intestinal tissue are significantly measurable, anotal recovery rate of paeoniflorin is 97.19± 5.78%, which isonsidered as the evidence confirming that the gut saciable.

Paeoniflorin tested by this in vitro gut sac system showedhere was a good linear correlation between the drug abion from the medium across the intestinal epithelium intoac contents and the incubation time of the gut sacs froo 90 min, manifesting a significant time-dependent mannetudy concentration dependence, five concentrations of 10, 80, 160�M paeoniflorin were designed in the experimend the results showed a non-linear increase in transpornd absorption with increasing concentration of paeoniflor

he medium. Along with the increase of paeoniflorin concration from 10 to 160�M, a saturation of the drug absorptinto the sac contents appeared at about 80�M concentrationhich indicates that the intestinal absorption of paeoniflight be a transport way with energy-dependent carriereeds to be expounded. Our previous in vivo studies with utrained conscious rats demonstrated that with co-administf sinomenine (90 mg/kg) and paeoniflorin, the peak plasmaentration of paeoniflorin in rats was elevated, the peak timeelayed, the AUC0−t was increased, the MRT was prolonged,L was decreased, and the Vd was reduced (Liu et al., 2005). Theresent in vitro studies firstly examined the effects of sinoine on the intestinal transport and the absorptive kineticaeoniflorin using the everted rat gut sac system, and the rhown inFig. 3A and B indicate that in the gut sacs treaith sinomenine at 16 and 136�M concentrations in the T99 medium for 45 min incubation, the absorption amounaeoniflorin in the sac contents was markedly elevated

K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432 431

about 1.5- and 2.5-fold, respectively, in comparison with theamount in the non-sinomenine treated sacs.

In order to further investigate the possible mechanisms of theinteractions on paeoniflorin absorption influenced by sinome-nine, two inhibitors and one substrate of P-gp were employedin the experiments. Given the presence of P-gp activity alongthe rat intestine, the in vitro everted gut sac model can alsobe useful to study the action of intestinal P-gp on intestinaldrug absorption (Barthe et al., 1998). By comparing the trans-port kinetics of a substrate in the absence or the presence ofpotential P-gp inhibitors, the method has potential as a sim-ple and efficient tool to evaluate the role of P-gp inhibitorsthat may improve the bioavailability of many drugs suscepti-ble to transport by P-gp (Barthe et al., 1998). The results ofthe present study showed that when the P-gp inhibitors, vera-pamil and quinidine, were administered, paeoniflorin absorptioninto the sac contents were markedly elevated, i.e., using 20�Mconcentration of paeoniflorin in the medium for 45 min incu-bation, the addition of verapamil at 100�M concentration orquinidine at 1.3 mM concentration led to significant increaseof paeoniflorin absorption in the sac content up to 2.1- and1.5-fold, respectively (Fig. 3C and D). This indicates that inhibi-tion of P-gp induced by verapamil and quinidine can markedlyenhance the intestinal transportation and absorption of paeoni-florin, and subsequently increase its bioavailability, while theinfluencing pattern induced by sinomenine is almost similar tot lesst e ot es-t ues(

asp ndm on-t Thep iflorim amia orpt er,o forp A,v eonfl A.M s fop rugr r rest rt ofp

t fact )r orini littleb tabo ine.I rinw .,1 tab-

olized by gut wall, liver and lung; while its poor absorptionfrom the intestine resulted in extremely low bioavailability andthe unabsorbed fraction of paeoniflorin was degraded by theintestinal flora (Takeda et al., 1997). Hsiu et al. found the agly-cone of paeoniflorin, paeoniflorgenin, in rats’ plasma after oraladministration ofPaeonia lactiflora decoction, which suggeststhat hydrolysis to the aglycone is the major metabolic pathwayof paeoniflorin. Moreover, no further conjugated metabolitesof the aglycone of paeoniflorin could be found (Hsiun et al.,2003).

Our present studies have confirmed that paeoniflorin couldnot be metabolized in the everted gut sac. Several other pilotexperiments in vitro for metabolism of paeoniflorin were doneby us, and the results showed that paeoniflorin could not bemetabolized by Caco-2 cells lysate, intestinal microsomesand liver microsomes (data not shown). This suggests that thelow bioavailability of paeoniflorin seems not be due to thephase I metabolic enzymes involved in P450 nor the phaseII metabolic enzymes including glucuronosyltransferasesand sulfotransferases. Instead we hypothesize that it mayresult from one or more of the following three processes: (1)degradation by intestinal flora (Takeda et al., 1997); (2) lowintestinal permeability (Takeda et al., 1997); and (3) activity ofintestinal P-glycoprotein, which might be responsible for thelow bioavailability.

To investigate the property of sinomenine on the substrateo osei uini-d ithi ,t dm p toa ninet as ani s as

5

sys-t as af andk pro-fi cedb sacc n thei f them abil-i mila rt andt ilarp thats iflorinv or,a oni-fl nalP

he pattern induced by those two P-gp inhibitors. Neverthehe actual effects as well as the mechanism of sinomeninhe impairment of intestinal P-gp activity still need to be invigated with other in vitro cell culture systems and techniqWatkins, 1997).

The above-mentioned results showed paeoniflorin hoor bioavailability not only due to its poor absorption aetabolism by intestinal bacterial; but also owing to the c

ribution of efflux transporters such P-gp in the intestine.resent studies have provided data showing that paeonay be likely a P-gp substrate since P-gp inhibitors (verapnd quinidine) could significantly enhance the intestinal abs

ion of paeoniflorin from apical (A) to basolateral (B). Moreovur studies on Caco-2 cell culture model (to be submittedublication) also showed that P-gp inhibitors (cyclosporinerapamil and quinidine) could enhance the transport of paorin from A to B, and decrease the transport from B tooreover, P-gp seems to be the main efflux transporteraeoniflorin in intestine since the inhibitors of MRPs (multidesistance-associated proteins) and BCRP (breast canceance protein) could not significantly influence the transpoaeoniflorin in Caco-2 cell culture model.

Drug metabolism has been considered as an importanor of the pharmacokinetics of drugs.Takeda et al. (1995eported that, after intravenous administration of paeonifln rats, about 50% of the dose was excreted in urine, witheing excreted in bile, suggesting that paeoniflorin is melized in the body and the rest is excreted mainly in ur

n contrast, they found that the bioavailability of paeonifloas 3–4%, with extremely low fecal excretion (Takeda et al995). They further reported that paeoniflorin was not me

,n

a

nl-

i-

r

is-

-

-

f P-gp, digoxin, a well-known P-gp substrate-like drug, whntestinal absorption can be enhanced by verapamil and qine (Emi et al., 1998), was employed in the experiment. W

ncubation of digoxin at 13�M concentration in the mediumhe addition of sinomenine at 136�M concentration coularkedly enhance the intestinal absorption of digoxin ubout 2.5-fold, in comparison with that of the non-sinome

reated gut sacs, which indicates that sinomenine actsnhibitor of intestinal P-gp activity while paeoniflorin acts aubstrate of intestinal P-gp.

. Conclusion

In the present study, an in vitro everted rat gut sacem cultured with TC 199 medium has been developedunctional technique to investigate the intestinal transportinetic absorption of paeoniflorin. The overall absorptivele of paeoniflorin by the rat small intestine can be enhany co-incubation of sinomenine in the medium of the gutulture system. This enhancement effect of sinomenine ontestinal absorption of paeoniflorin was proved to be one o

echanisms on the improvement of paeoniflorin’s bioavailty in vivo. We further verified that the P-gp inhibitors, verapand quinidine, can markedly enhance the intestinal transpo

hereafter the kinetic absorption of paeoniflorin, with a simattern as sinomenine did. Therefore, it could be concludedinomenine can enhance the intestinal absorption of paeonia inhibition of the intestinal P-gp activity like a P-gp inhibitnd subsequently improve the oral bioavailability of paeorin, while paeoniflorin is likely a substrate of the intesti-gp like digoxin.

432 K. Chan et al. / Journal of Ethnopharmacology 103 (2006) 425–432

Acknowledgements

The Hong Kong Jockey Club Charities Trust funded thisresearch (Project code: JCICM-6-02).

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