Coexisted components of Salvia miltiorrhiza enhance intestinal absorption of cryptotanshinone via inhibition of the intestinal P-gp.
Everted rat gut sacs
Ciyptotanshinone, derived from the roots of Salvia miltiorrhiza Bge and Salvia przewalskii Maxim, is the major active component and possesses significant antibacterial, antidermatophytic, antioxidant, anti-inflammatory and anticancer activities. The objective of this study was to investigate the intestinal absorptive characteristics of cryptotanshinone as well as the absorptive behavior influenced by co-administration of the diterpenoid tanshinones and danxingfang using an in vitro everted rat gut sac model. The results showed a good linear correlation between cryptotanshinone of absorption and the incubation time from 10 to 70 min. The concentration dependence showed that a non-linear correlation existed between the cryptotanshinone absorption and the concentration at 100 [micro]g/m1. Coexisting diterpenoid tanshinones and danxingfang could significantly enhance the absorption of cryptotanshinone. Coexisting diterpenoid tanshinones and danxingfang, which influenced ciyptotanshinone's absorption, manifested as similar to that of the P-glycoprotein inhibitor. The underlying mechanism of the improvement of oral bioavailability was proposed that coexisting diterpenoid tanshinones and danxingfang could decrease the efflux transport of cryptotanshinone by P-glycoprotein.
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Cryptotanshinone is a major and characteristic abietane-type diterpene tanshinone derived from the roots of Salvia plants such as S. miltiorrhiza Bunge (SMB, Danshen) and S. przewalskii Maxim (SPM, Gansudanshen), which both are well-known traditional Chinese herbal medicine and have been widely used for treating coronary heart diseases, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure (Gao et al. 1979; An et al. 2004, 2005; Xu and Fu 2006; Su and Liu 2009). Chinese doctors have been using these two herbs in combination with other medicine such as Fufangdanshen tablet, Guanxinning tablet, Danx-iong Tongmai pellet and Danxiongfang (Kong 1989; Xu 1990; Chen et al. 2005; Li et al. 2007a, 2008a, 2008b; Chen et al., 2008; Wu et at., 2009), to treat the various diseases for many years. Cryptotanshi-none has attracted particular attention of chemists and clinicians all over the world due to its powerful and various kinds of pharmacological activities including antibacterial and anti-dermatophytic, antioxidant, anti-inflammatory and anticancer activities (Xue et al., 2000; Wang et al. 2005, 2008; Zhang et at. 2008). However, the pharmacokinetic studies demonstrated that cryptotanshi-none has a poor absorption and a very low bioavailability when administrated orally (Song et at. 2007). This was deduced to be due to the limited transportation of cryptotanshinone across gastrointestinal mucosa into the blood (Xue et at. 1999; Cui et at. 2005).
The efficacy of drugs depends critically on their ability to cross cellular barriers to reach the target tissue and cells. The small intestine is the principal site of drug absorption when the drug is administrated via the oral route. To study intestinal absorption of the oral drugs, several in vivo and in vitro models have been developed (Stewart et al. 1995; Barthe et al. 1998). The everted rat gut sac technique was first described by Wilson and Wiseman (1954). The reproducibility of the in vitro model suggests that the everted rat gut sac is a very useful screening tool for studying transport of P-glycoprotein (P-gp) substrates and potential P-gp modifiers, which is an ATP-dependant active transporter belonging to the ABC transporter superfamily (Bouer et al. 1999; Carreno-Gomez and Duncan 2000).
Recently, it was found that oral administration of cryptotanshi-none in rats had very low plasma drug concentration and poor oral bioavailability, while co-administration of coexisting diterpenoid tanshinones could markedly elevate the level of plasma drug concentration and improve the oral bioavailability of cryptotanshinone in rats, but the underlying transmembrane mechanisms of the drug disposition remain still unknown. Thus, our present study was to investigate the intestinal absorptive behavior of cryptotanshinone using the in vitro everted rat gut sac model in the absence and the presence of coexisting diterpenoid tanshinones, danxingfang and the P-gp inhibitor verapamil, in order to reveal the possible mechanisms of improving the bioavailability of cryptotanshinone in vivo. Danxiongfang is a useful preparation consisted of the Salvia extract mainly including the lipophilic diterpenoid tanshinones, the water soluble phenolcarboxylic acid (Salvianolic acids) and ferulic acid; and it could be used to treat the coronary heart diseases and cerebrovascular disease (Li et al. 2007a, 2007b, 2007c, 2008a, 2008b, 2010; Wu et al. 2009).
Materials and methods
Chemicals and reagents
Cryptotanshinone was obtained from our laboratory, which was isolated and purified from the root of S. przewalskii Maxim and identified as pure compound from the melting point, IR, UV, MS, NMR and compared with the standard compound, the purity of cryptotanshinone was above 99% (Xue et at. 2000). Diterpenoid tanshinone extract was obtained with ethanol extraction of S. przewalskii Maxim (Origin ofGa nsu, China), in which the cryptotanshinone content was determined by a validated RP-HPLC method to be more than 50%. The determination of Danxiongfang and its main active ingredients was also performed by RP-HPLC according to a method previously used in our laboratory (Li et al. 2007a, 2008a, 20081). The mobile phase was a mixture of methanol-water containing 0.5% (v/v) glacial acetic acid employing gradient elution (from 40:60 to 75:25, v/v) at a flow rate of 1 ml/min. Danshensu and ferulic acid were determined at 281 nm and cryptotanshi-none and tanshinone IIA were detected at 254nm. The run time was 35 min. The HPLC chromatograms of the main constituents in danxiongfang preparation are shown in Fig. 1. Cryptotanshinone and tanshinone IIA were detected at 254 nm. The reference standards of cryptotanshinone, verapamil and nimodipine (the internal standard for cryptotanshinone) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Tissue culture medium and glucose were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methanol used was of HPLC grade and purchased from Fisher Scientific Products (Fair Lawn, NJ, USA). Water was triply distilled. Ethyl acetate and other reagents and solvents used were all of analytical grade. All other reagents were of analytical grade or HPLC grade.
Preparation of the everted rat gut sacs
Preparation of the everted rat gut sacs was carried out as a described previously (Bouer et al. 1999). Healthy Sprague-Dawley male rats (250-300g) were obtained from Animals Center of Capital Medical University (CCMU). The rats were starved for 24 and sacrificed by cervical dislocation. The abdominal cavity was quickly cut open and the small intestine removed and washed through three times with normal saline (0.9% NaCl solution) at room temperature. The intestine was immediately placed in 37 C and oxygenated ([O.sub.2]/[CO.sub.2], 95%:5%) in Tyrode buffer. The intestine was everted on a glass rod (2.5 cm in diameter) and one end was clamped before filling with fresh and oxygenated medium using a 50 ml glass syringe. Then the intestine was sealed with a second clamp. Small sacs (about 6cm in length) were tied using silk sutures. Each sac was placed in a 20 ml tube containing 9.0ml of oxygenated media at 37 [degrees]C. 1 ml of medium, containing cryptotanshinone, verapamil, diterpenoid tanshinone or danxiongfang, was then added to the required concentration, accordingly. The resulting gut sacs were incubated at 37 [degrees]C in an oscillating water bath. At the appropriate time points, the sacs were removed, washed four times in normal saline and blotted dry. The sacs were cut open and the serosal fluids (sac contents) were drained into small tubes. Each sac was weighed before and after serosal fluid collection to calculate accurately the volume inside the sac and to correct the serosal fluid for the actual volume. The area of each sac was measured. Samples of the medium and the serosal fluid of each sac were kept for HPLC analysis. From the analysis data, the sac content volumes and the absorption amounts of cryptotanshinone present in each sac were calculated. Each experiment was carried out using the small intestine from one animal. All studies on animals were in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of China.
Determination of cryptotanshinone in rat intestinal juice
All analyses were performed on an Agilent high performance liquid chromatography system (Series 1100, Agilent technology, Palo Alto, CA, USA) which consisted of a G1310A quaternary pump, G1322A vacuum clegasser, G1316A column thermostat, G1314A VWD and an auto-sampler injector. The chromatography data were recorded and processed with HP chemstation software. The analytical column was an Agilent HC C8 column (150 mm x 2.1 mm, i.d., 5 [micro]m; Thermo Electron, CA, USA). All chromatography was performed at 25 [degrees]C. The mobile phase was a mixture of methanol-water (75:25, v/v) at a flow rate of 0.8 ml/min. The solvent was filtered through a 0.45 [micro]m filter and degassed before using for the HPLC analysis. Cryptotans hinone was detected at 254 nm.
The rat intestine juice samples were collected in heparinzed tubes and centrifuged at 3500 rpm for 15 min immediately. The samples (0.4 ml) were spiked with 100 [micro]1 each of working solution containing cryptotanshinone (10-0.01 [micro]g/ml and 100 [micro]l of the IS stock solution (2.4 [micro]g/m1). Then 100 [micro]1 of 1 mol/1 HCI and 1 ml of ethyl acetate were added. The combined samples were adjusted to pH 3, vortex-mixed for 1 min and centrifuged at 3000 rpm for 10 min. Each sample was extracted two times and the upper organic potions were combined and evaporated to dryness under a stream of nitrogen flow at 40 [degrees]C. The residue was reconstituted in 100 [micro]l of the HPLC mobile phase before HPLC analysis and then an aliquot (20 [micro]l) was injected into the HPLC system.
The uptake of cryptotanshinone in everted rat gut sac
To evaluate the analytical method for the precision and accuracy of cryptotanshinone absorption in the everted rat gut sac model, the uptake amount (accumulation) of cryptotanshinone in gut tissue and the recovery of cryptotanshinone at the concentration of 10, 50 and 100 [micro]g/m1 from the incubation medium in the serosal side, the medium in the mucosal side and gut sac tissue after 10-70 min incubation were examined. Each sac tissue sample was accurately weighed, added with 1 ml iced normal saline, and homogenized for 10 min in iced water-bath. The mixture was centrifuged at 3000 rpm. 0.5 ml of the supernatant of each sample was then mixed with 100 [micro]l of 1 mol/1 HCI solution by vortex for 5 min. The denatured protein precipitate was further separated by centrifugation at 13,000 rpm for 10 min. Finally, 20 [micro]l of the supernatant of each sample was analyzed by HPLC and the uptake amount of cryptotanshinone in the gut sac tissue was calculated in sacs. The amount of cryptotanshinone both in the serosal side and in the mucosal side of medium were measured following HPLC method described as in section "Determination of cryptotanshinone in rat intestinal juice". The amount of accumulative transportation (Q) of cryptotanshinone within the time points was calculated from the following equation:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
where, n is the number of times for sampling, V is the volume of the solution in the intestinal sac, [C.sub.i] is the concentration of the sample, [V.sub.i] is the volume of the sample.
The apparent permeability coefficient, [P.sub.app] was calculated as follows:
[P.sub.app] = [increment of Q]/[increment of t]/A[C.sub.0]
where, [increment of Q]/[increment of t] is the linear appearance rate of the compound on the receiver side (intestinal serosal side), A is the membrane surface area ([cm.sup.2]), and [C.sub.0] is the initial drug concentration in the intestinal mucosal side.
Statistical analysis was performed using SPSS Software (SPSS Inc., version 13, Chicago, IL USA) on an IBM microcomputer. An independent samples t-test and one-way analysis of variance were used for the data. Data were presented as the means [+ or -] S.D. and the values of p < 0.05 were considered statistically significant.
Validation of HPLC detection method
The results for the selectivity of the determination method are shown in Fig. 2. The RP-HPLC method was selective and specific. The analysis from the samples showed that there were no endogenous substance peaks and drug metabolite peaks interfered with the analytes and I.S. at the retention ti mes. The retention times were 7.52 and 4.10 min for cryptotanshinone and the I.S. Fig. 1 showed the representative chromatograms of a blank intestinal juice, an intestinal juice samples spiked with both the analytes and the I.S. and an intestinal juice sample obtained from a rat intestinal sac after administration.
The linear regression of cryptotanshinone in rat intestinal juice displayed good linear relationships between the ratios of the peak areas of the analytes to the I.S. over the range of concentrations studied. For a standard curve the ratio of the chromatographic peak area (the analytes/I.S.) as ordinate variables was plotted versus the concentration of the drug as abscissa. The standard calibration for cryptotanshinone was linear over the range 0.01-10 [micro]g/m1 in intestinal juice. The regression equation of the analytes was: y = 1.9127x +0.0332. The [gamma] values were more than 0.9996.
The precision and accuracy of the method were assessed in intestinal juice by performing replicate analyses of spiked samples against calibration standards. The procedure was repeated on the same day and between five different days on the same spiked standard series. The within-day and between-clay precision and accuracy (R.S.D.%) were all less than 10%. The data indicated that the precision and accuracy of the method are valid and acceptable. The extract recoveries of cryptotanshinone were determined for five replicates in rat intestinal juice spiked with low, medium and high concentrations of the analytes. The mean recoveries were all more than 85%.
Time and concentration dependence of the cryptotanshinone absorptive profile
The intestinal kinetic absorptive behavior of cryptotanshinone was studied in the gut sac model at different incubation time periods from 10 to 70 min. The result, seen in Fig. 3, showed that the absorption amount of cryptotanshinone at three tested concentrations (10,50 and 100 [micro]/ml) was gradually increased within increase of incubation time. The correlation between the drug absorption (ng/[cm.sup.2]) and the incubation time (min) was nearly linear (y=0.1251x+3.4719, [R.sup.2]=0.9229 and y =0.1913x + 1.4045, [R.sup.2]=0.9844) when cryptotanshinone was added into the medium at the low and middle concentrations (10 and 50 [micro]g/m1). These data indicate that cryptotanshinone can be transported via the negative form from the medium into the serosal fluid of the gut sacs across intestinal epithelium. Thus, the culture system for the in vitro everted rat gut sacs used in this study was verified to be significantly functional. The correlation between the drug absorption (ng/[cm.sup.2]) and the incubation time (min) was a curve when the concentration of cryptotanshinone reached 100[micro]g/ml, suggesting that there was the active transport of the drug existed.
Tissue incubation from 10 to 70 min was conducted for studying the concentration dependence of cryptotanshinone absorptive profiles with the different concentrations (10,50 and 100 [micro]g/m1) of the drug. The results, seen in Fig. 3, demonstrated that there was a non-linear increase in cryptotanshinone absorption into the sac contents corresponding to the increase of cryptotanshinone concentration in the medium. These results indicated that the transport and absorption of cryptotanshinone in vitro gut sac system would be saturative effect with 100 [micro]g/ml.
Influence of tanshinone compounds on the absorptive profile of cryptotanshinone
In order to determine the possible effect of diterpenoid tan-shinone compounds on the enhancement of cryptotanshinone absorption in the small intestine, co-incubation tissue culture tests with cryptotanshinone (50 [micro]g/m1) and other tanshinone compounds were conducted. When treated with cryptotanshinone alone, there was good linear correlation between cryptotanshinone absorption and incubation time (shown in Fig. 4 with [R.sup.2]= 0.9844, y = 0.1913x + 1.4045). However, when tanshinone compounds were added to the intestine juice, this correlation became non-linear profile. After the 50 min co-incubation with other tanshinone compounds at the concentration of 50[micro]g/ml the absorption of cryptotanshinone significantly increased, especially within the 70 min. The absorption of cryptotanshinone was increased approximately by 33.9-40.7% from 50 to 70 min.The results suggested that tanshinone compounds could markedly enhance cryptotanshinone absorption in intestine.
Influence of danxiongfang on the absorptive profile of cryptotanshinone
In order to determine the potential effect of danxiongfang on the enhancement of cryptotanshinone absorption in the small intestine, co-incubation tissue culture tests with cryptotanshi-none (50 [micro]g/ml) and danxiongfang were performed. When treated with cryptotanshinone alone, there was good linear correlation between cryptotanshinone absorption and incubation time (shown in Fig. 5). However, when danxiongfang were added to the intestine juice, this correlation also became some non-linear profile. After 50 min co-incubation with other danxiongfang at the concentrations of 50 [micro]g/ml, the absorption of cryptotanshinone was significantly increased. The absorption of cryptotanshinone was increased approximately by 30.1-35.8% from 50 to 70 min. The results indicated that danxiongfang could also markedly enhance cryptotanshinone transportation and absorption in the intestine.
Influence of P-glycoprotein inhibitor on the absorptive profile of ctyptotanshinone
To determine the effect of intestinal P-gp on the absorption of cryptotanshinone, The P-gp inhibitor, verapamil was added to the medium containing 50 [micro]g/m1 of cryptotanshinone. Fig. 6 showed that there was the linear correlation between the amount of ayp-totanshinone absorbed and the incubation time ([R.sup.2]=0.9844) in the absence of verapamil. However, this correlation became also some non-linear profile when verapamil was added at a concentration of 50 [micro]g/ml. After the 50 min co-incubation with cryptotanshinone and verapamil together, cryptotanshinone absorption increased by 28.7% in comparison with the non-verapamil treated gut sacs. Within the 60 min co-incubation, cryptotanshinone absorption increased by 34.69%. Within the 70 min co-incubation, cryptotan-shinone absorption increased by 35.54%. These results indicated that the inhibition of the intestinal P-gp by verapamil could significantly enhance intestinal transportation as well as the absorption of cryptotanshinone in rat intestine.
Generally, there are two main ways by which drugs cross cell membrane, i.e. by diffusing directly through the lipid membrane, and by combination with a transmembrane transporter and consumption of ATP that binds a molecule on one side of the membrane, then changes conformation and releases it on the other side. Meanwhile, many factors influence the drug transport and bioavailability, such as drug lipophilicity or solubility, the gastrointestinal pH value, gastric emptying, gastrointestinal first pass effect and drug interactions (Barthe et al. 1998). The previous studies showed that oral administration of cryptotanshinone had a very low bioavailability (3-4%) due to the limited transportation of the drug across intestinal epithelium into the blood circulation (Song et al. 2006). Our previous investigation demonstrated that the in vivo drug enzymes could quickly transform cryptotanshinone at least into two major metabolites, tanshinone IIA and hydoxytanshinone (Xue et al. 2002; Dai et al. 2008), which could also be rapidly absorbed from the intestinal tract into the general circulation. Thus, the low bioavailability of cryptotanshinone may result from the poor intestinal transport and absorption, as well as the metabolism of the drug in vivo. Further, our studies on pharmacokinetic interaction of cryptotanshinone and danxiongfang in rats showed that co-administration could significantly elevate the bioavailability of cryptotanshinone in comparison with the animals treated with cryptotanshinone alone, which danxiongfang was the compound preparation contained the main lipophilic tanshinones or hydrophilic phenolic acid components (Li et al. 2007a, 2008a, 2008b, 2010; Liu et at. 2010). The results indicated that these compounds in danxiongfang could be function as an enhancer of cryptotanshinone bioavailability. The co-administration of coexisting diterpenoid tanshinones in rats markedly elevated the bioavailability of cryptotanshinone to more than 3 folds in comparison with animals treated with cryptotanshinone alone (Song et al. 2007), while the underlying transmembrane transport mechanisms remain still unknown.
In the present study, we employed the everted rat gut sac technique as an in vitro model for the study of the intestinal absorption behavior and transmembrane mechanism of cryptotanshinone, especially with the lipophilic tanshinone compounds, danxiong-fang compounds, as the lipophilic and hydrophilic components (Li et at. 2007b, 2007c, 2008a, 2008b, 2010), and the P-glycoprotein inhibitor verapamil. The everted rat gut sac model was firstly described by Wilson and Wiseman and further improved by Barthe (Wilson and Wiseman 1954; Barthe et at. 1998). Our present study showed that the cryptotanshinone contents both inside the sacs and the intestinal tissue are significantly measurable, and the total recovery rate of cryptotanshinone is 98.28 [+ or -] 6.21%, which is considered as the evidence confirming that the gut sacs are viable.
Cryptotanshinone at the tested concentration (10 and 50 [micro]g/ml) in rat gut sacs showed that there was a good linear correlation between the drug absorption from the medium across the intestinal epithelium into the sac contents and the test time. The incubation time from 10 to 70 min manifested a significant time-dependent manner. To study concentration dependence, three concentrations of 10, 50 and 100 [micro]g/ml cryptotanshinone were designed in the experiments. The results showed a non-linear increase in transportation and absorption with increasing 100 [micro]g/m1 concentration of cryptotanshinone. Along with the increase of cryptotanshinone concentration from 10 to 100 [micro]g/ml, a saturation of the drug absorption into the sac contents appeared at about 100 [micro]g/ml concentration, indicating that the intestinal absorption of cryptotanshinone might be a transport way with energy-dependent carrier at the higher drug concentration. Our previous in vivo studies with the unrestrained conscious rats demonstrated that with co-administration of danxiongfang and cryptotanshinone, the peak plasma concentration of cryptotanshinone in rats was elevated, the peak time was delayed, the [AUC.sub.o-t] was increased and the mean remain time was prolonged (Liu et at. 2010). The present in vitro studies examined the effects of some compounds on the intestinal transport kinetics of cryptotanshinone using the everted rat gut sac system, and the results indicate that in the gut sacs treated with these compounds in the medium for incubation, the absorption amount of cryptotanshinone in the sac contents was markedly elevated up to about 30-40%, respectively, in comparison with the amount in the non treated sacs.
P-glycoprotein exists in many cell plasma membranes and acts as an efflux transporter of xenobiotics and drugs. It was reported that P-gp functions as a gatekeeper against xenobiotics in the gut and other tissues (Doige and Ames 1993). To investigate the property of cryptotanshinone on the substrate of P-gp, verapamil, a well-known P-gp substrate-like drug, some drug intestinal absorption can be enhanced by verapamil (Emi et al. 1998), was employed in the experiment. In order to further investigate the possible mechanisms of cryptotanshinone absorption influenced by tanshinones and danshen compounds, an inhibitor of P-gp was employed. By comparing the transport kinetics of the substrate in the absence or the presence of potential P-gp inhibitors, this method has potential as an efficient tool to evaluate the role of P-gp inhibitors that may improve the bioavailability of some drugs susceptible to transport by P-gp (Barthe et al. 1998). Our results showed that when the P-gp inhibitors, verapamil, was administered, cryptotanshi-none absorption into the sac contents were markedly elevated, i.e., the addition of verapamil at 50 [micro]g/m1 concentration led to significant increase of cryptotanshinone absorption in the sac content up to 35.54%. This indicates that inhibition of P-gp induced by verapamil can markedly enhance the intestinal transportation and absorption of cryptotanshinone, and subsequently increase its bioavailability. Cryptotanshinone has a low bioavailability in vivo not only clue to its poor absorption and metabolism: but also owing to the contribution of efflux transporters such P-gp in the intestine. Our data indicated that cryptotanshinone could be one of the P-gp substrates since P-gp inhibitor could significantly enhance the intestinal absorption of cryptotanshinone.
Drug metabolism is an important influence factor for the pharmacokinetics of drugs. We have reported that less 8% of the parent drug dose was excreted only via urine and a little was excreted via bile after intravenous administration of cryptotanshinone in pigs, suggesting that cryptotanshinone is easily metabolized in vivo and the metabolites are excreted mainly in urine (Xue et at. 1999; Cui et at. 2005). We also found that cryptotanshinone existed in rat bile after oral administration, suggesting that hydrolysis to form the aglycone of the drug is one of the major metabolic pathways of cryptotanshinone. Moreover, the conjugated metabolites of cryp-totanshinone could also be found (Dai et at. 2008). Our studies have confirmed that cryptotanshinone could not be metabolized in the everted rat gut sac. The experiments in vitro for the biotransformation of cryptotanshinone were also performed by us, and the results showed that cryptotanshinone could be metabolized to form the hydroxylation, dehydrogenation, furan ring cleavage and oxidation metabolites by the liver microsomes (Xue et at. 2002; Dai et at. 2008). The data suggests that the low bioavailability of cryptotanshinone seems not only be due to the phase I metabolic enzymes involved in [P.sub.450] and the phase II metabolic enzymes including glucuronosyltransferases and sulfotransferases, but also be clue to the P-gp substrate action. We hypothesize that the low bioavailability of the drug may result from more of the following two processes: (1) metabolism in vivo, and (2) activity of intestinal P-glycoprotein.
In the present investigation, an in vitro everted rat gut sac system has been developed as a functional technique to study the intestinal transport and absorption of cryptotanshinone and drug interaction. The overall absorptive profile of cryptotanshinone by the rat small intestine can be enhanced by co-incubation of coexisting diterpenoid tanshinones and danxingfang in the gut sac system. This enhancement effect on the intestinal absorption of cryptotanshinone was proved to be one of the major mechanisms on the improvement of cryptotanshi-none's bioavailability in vivo. We have verified that the P-gp inhibitors, verapamil, can markedly enhance the intestinal absorption of cryptotanshinone, with a similar pattern as the coexisting diterpenoid tanshinone compounds and danxingfang did. Therefore, it could be concluded that coexisting diterpenoid tanshinones and danxingfang can enhance the intestinal absorption of cryptotanshinone via inhibition of the intestinal P-gp activity like a P-gp inhibitor, and subsequently improve the oral bioavailability of cryptotanshinone.
The authors thank the National Foundation of Natural Sciences of China (Nos. 30472057, 30772611 and 81173121), Beijing Natural Science Foundation Program (No. 7052007), the Key Program of Beijing Municipal Commission of Education (KM201110025024) and Funding Project for Academic Human Resources Development in Institutions of High Learning under the jurisdiction of Beijing Municipality (PHR201007111) for their financial supporting.
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* Corresponding author. Tel.: +86 1083911520: fax: +86 10 83911520.
E-mail address: firstname.lastname@example.org (M. Xue).
(1.) These two authors are co-first authors.
0944-7113/$ - see front matter [c] 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.08.007
Haixue Dai (1), Xiaorong Li (1), Xiaoli Li, Lu Bai, Yuhang Li, Ming Xue *
Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China