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Journal of Pharmacy And Pharmacology

Research Paper

Herbdrug pharmacokinetic interaction of articial calculus bovis with diclofenac sodium and chlorpheniramine maleate in rats
Can Penga,b, Mengying Lva,b, Jixin Tiana,b, Yin Huanga,b, Yuan Tiana,b,c and Zunjian Zhanga,b,c
a Key Laboratory of Drug Quality Control and Pharmacovigilance, bDepartment of Pharmaceutical Analysis, cState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China

Keywords articial calculus bovis; chlorpheniramine maleate; diclofenac sodium; herbdrug pharmacokinetic interaction; HPLC-MS/MS Correspondence Zunjian Zhang, Department of Pharmaceutical Analysis, China Pharmaceutical University, Tongjia Xiang 24, Nanjing 210009, China. E-mail: zunjianzhangcpu@hotmail.com Received December 15, 2012 Accepted March 18, 2013 doi: 10.1111/jphp.12069

Abstract
Objectives To investigate the herbdrug pharmacokinetic interaction of articial calculus bovis (ACB) with diclofenac sodium (DS) and chlorpheniramine maleate (CPM) in rats. Methods A sensitive high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous determination of DS and CPM in rat plasma. The proposed method was successfully applied to compare the herbdrug pharmacokinetic interaction of ACB with DS and CPM in rats following intragastric administration. Key ndings The proposed method had good linearity and no endogenous material interfered with the analytes and internal standard peaks. The lower limit of quantication of DS and CPM was 1 and 0.1 ng/ml, respectively. There was no apparent pharmacokinetic interaction between DS and CPM. Co-administration of ACB with DS noticeably increased the area under the concentrationtime curve (AUC0-) and peak plasma concentration (Cmax) of DS, while the parameters time of peak concentration (Tmax), clearance (ClZ/F) and apparent volume of distribution (VZ/F) of DS signicantly decreased. Meanwhile, co-administration of ACB with CPM noticeably increased the Tmax, ClZ/F and VZ/F of CPM. A marked decline in AUC0- and Cmax of CPM occurred in the presence of ACB. Conclusions This study indicated that co-administration of ACB with DS and CPM can result in an apparent herbdrug pharmacokinetic interaction in rats.

Introduction
As a representative natural medicine, Traditional Chinese Medicine (TCM) is becoming more widely used in many countries.[1,2] According to a World Health Organization report, nearly 80% of the global population relies on TCM as a part of disease treatment.[3] Even in the USA, where herbal remedies are only considered as dietary supplements, approximately one-fth of the population regularly consume TCM products.[4,5] The combination of TCM and conventional therapies necessitates a sufcient understanding of the use of different forms of treatment concurrently and especially herbdrug interactions. The common cold is a respiratory disease caused by a variety of viruses. Combination of TCM and Western medicine can improve clinical efcacy,[68] and this treatment
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model is accepted in many countries, such as China, Japan, India and South Korea.[911] Compound diclofenac sodium chlorphenamine maleate tablets, composed of articial calculus bovis (ACB), diclofenac sodium (DS) and chlorphenamine maleate (CPM), is an herbdrug preparation generally used to relieve the symptoms of headache, febricity, nasal obstruction and pharyngodynia.[12] DS, 2-((2, 6-dichlorophenyl) amino) benzene acetic acid, mainly used in the treatment of joint inammation, is a classic non-steroid anti-inammatory drug and a potent prostaglandin synthesis inhibitor.[1315] CPM, 1-p-chlorophenyl1-(2-pyridyl)-3-dimethylaminopropane maleate, is an alkylamine antihistaminic drug (H1RAS) commonly used in the prevention of symptoms of allergic conditions such as

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rhinitis and urticaria.[16,17] Although numerous studies on the pharmacokinetics of DS and CPM individually have been carried out,[1822] the pharmacokinetic interaction of these two drugs has not yet been reported. As the primary substitute for calculus bovis (commonly known as Niuhuang in China), ACB is clinically useful for its various pharmacological actions such as sedation, immunoregulation, antihyperspasmia, fever-relieving and antiinammatory effects.[2325] Bile acids, bilirubin and taurine are viewed as the main effective components in ACB and there is a great variation in its internal quality because of diverse sources and species.[26,27] Several methods have been used to assay the effective components in calculus bovis and its substitutes. Kong et al. presented an ultra-performance liquid chromatography-evaporative light scattering detection method for the simultaneous determination of six bile acids and commented on the various inherent quality among different origins.[28] In our previous work, bilirubin, taurine and 11 bile acids were simultaneously quantied by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to control the quality of ACB.[29] In this prescribed preparation, ACB acts as the antipyretic analgesic. However, the effects of ACB on the pharmacokinetics and pharmacodynamics of DS and CPM are still elusive. HPLC-MS/MS has become increasingly common in the quantitative analysis of drugs owing to its high selectivity and sensitivity, particularly the selected reaction monitoring mode. Many methods have been developed to determine DS and CPM in plasma, among which HPLC-MS/MS is extensively adopted.[3033] However, to our knowledge, no study has reported the simultaneous determination of these two drugs in biological samples by HPLC-MS/MS. In this study, a simple HPLC-MS/MS method was successfully developed for the simultaneous determination of DS and CPM in plasma, and the herbdrug pharmacokinetic interaction of ACB with DS and CPM was investigated in rats to provide information on the combined use of ACB, DS and CPM in the clinical setting.

grade) was obtained from Merck (Darmstadt, Germany). Deionized water was prepared by a Milli-Q system (Millipore, MA, USA). All other chemicals were of analytical grade.

HPLC-MS/MS analysis of ACB

The HPLC-MS/MS system comprised a Finnigan Surveyor LC pump, a Finnigan Surveyor auto-sampler and a triple quadrupole TSQ quantum mass spectrometer via electrospray ionization (ESI) interface (Thermo Fisher, Palo Alto, CA, USA) for the identication and quantication of ACB. The column, ZORBAX SB-C18 (150 2.1 mm, i.d. 5 mm), was maintained at 30C. The mobile phase consisted of (A) methanol and (B) 10 mmol/l ammonium acetate in aqueous solution (adjusted to pH 3.0 with formic acid). Gradient elution program: 03 min, 1 : 99; 720 min, 70 : 30; 40 min, 80 : 20; 5070 min, 98 : 2 (A : B, v/v). The ow rate was 0.3 ml/min.

Animal studies
The study was approved by the Animal Ethics Committee of China Pharmaceutical University. A total of 36 SPF grade Wistar male rats (200 20 g) were provided by China Pharmaceutical University Laboratory Animal Center (Nanjing, China; certicate no. SCXK2009-0001). All rats were fed with standard rodent chow and water ad libitum, and then fasted for 12 h before the pharmacokinetic experiment. The rats were divided into six groups followed by oral administration. The dosage schedules were converted by means of body surface area to conform to the human clinical schedule as described in the commercial product.[34] The treatment groups were designated as: group 1 (DS, 10 mg/ kg), group 2 (CPM, 2 mg/kg), group 3 (DS, 10 mg/kg + CPM 2 mg/kg), group 4 (DS, 10 mg/kg + ACB 10 mg/kg), group 5 (CPM, 2 mg/kg + ACB 10 mg/kg) and group 6 (DS, 10 mg/kg + CPM 2 mg/kg + ACB 10 mg/kg). Blood samples (0.5 ml) were collected from each rat at predetermined time points: 0 (pre-dose), 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 480 min after administration. Plasma samples were obtained after centrifugation (12 000 rev/min, 14 000g) for 10 min and were frozen at -20C until analysis.

Materials and Methods

Chemicals and reagents
DS, CPM, ACB, midazolam, bilirubin, taurine, cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and hyodeoxycholic acid (HDCA) were purchased from National Institutes for Food and Drug Control (Beijing, China). Lithocholic acid (LCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA) and dehydrocholic acid (dhCA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Methanol (HPLC

Preparation of samples
Stock solutions of 1 mg/ml DS and CPM were prepared separately in methanol and stored at 5C. Working standard solutions were prepared by serially diluting the stock solution using methanol. The internal standard stock solution (midazolam, 50 mg/ml) was prepared in methanol. Calibration samples were prepared by mixing solutions of the standard mixture with blank rat plasma to form a concentration series of 50, 25, 12.5, 5, 0.5, 0.25 and 0.05 mg/ml for
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DS, and 5, 2.5, 1.25, 0.5, 0.05, 0.025 and 0.005 mg/ml for CPM. Quality control samples of DS and CPM in different concentrations were also prepared in a similar manner. All solutions were stored at -20C before use. All frozen standards and samples were allowed to thaw at room temperature and were then homogenized by vortex. A 100-ml plasma sample was transferred to a 1.5-ml centrifuge tube together with 10 ml of the internal standard. The sample mixture was mixed with 400 ml of methanol and vortexed for approximate 5 min, then allowed to stand for 5 min to deproteinize and the precipitate was removed by centrifugation at 16 000 rev/min (25 000g) for 10 min. The supernatant was then pipetted into an injected vial and a 10-ml aliquot was injected into the HPLC-ESI-MS/MS system for analysis.

Statistical analysis
Statistical analysis was performed using Microsoft Excel 2010 and BAPP software. The pharmacokinetic software DAS 2.0 package based on the non-compartment model was used to calculate the pharmacokinetic parameters (Mathematical Pharmacology Professional Committee of China, Shanghai, China). Statistical signicance was assessed by one-way analysis of variance followed by the Tukey post-hoc test (SPSS version 20.0, SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically signicant. All results were expressed as mean SD.

Results
Identication and quantitative determination of ACB
The contents of the effective components in ACB were quantied by a HPLC-MS/MS method described in our previous work.[29] The ACB used in this study contained 4.387 mg/mg LCA, 1.274 mg/mg UDCA, 27.931 mg/mg HDCA, 8.975 mg/mg CDCA, 24.173 mg/mg DCA, 75.923 mg/mg CA, 12.037 mg/mg GCDCA, 68.277 mg/mg GCA, 8.214 mg/mg TLCA, 4.597 mg/mg TDCA, 29.229 mg/mg TCA, 36.747 mg/mg taurine and 9.470 mg/mg bilirubin (Figure 1).

HPLC-MS/MS assay
Liquid chromatographic separation and mass spectrometric detection were performed using the Finnigan TSQ Quantum Discovery MAXTM LC-MS/MS system comprising a Finnigan Surveyor LC pump, a Finnigan Surveyor auto-sampler and a triple quadrupole TSQ Quantum mass spectrometer via ESI interface (Thermo Fisher). The chromatographic separation was on a ZORBAX SB-C18 (150 2.1 mm, i.d. 5 mm) analytical column. An isocratic elution lasting for 3.5 min was obtained with a mobile phase consisting of methanol and 10 mmol/l ammonium acetate in aqueous solution (adjusted to pH 3.0 with formic acid) (85 : 15, v/v) at a ow rate of 0.25 ml/min. The HPLC efuent was introduced into the mass spectrometer without splitting. The column temperature was set at 30C and the sample tray temperature was maintained at 16C. The mass spectrometer was operated in the positive mode and ran with Xcalibur 2.0 software (Thermo Fisher). The capillary voltage was 3900 V and the temperature of the capillary was set at 300C. Nitrogen was used as the sheath (49 arb) and auxiliary gas (20 arb). Argon was used as the collision gas at a pressure of 0.2 Pa. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The precursor product ion pairs used for MRM of DS, CPM and internal standard were m/z 296.0213.8 (collision energy: 33 V), m/z 275.0229.8 (18 V) and m/z 326.0291.0 (24 V), respectively, with a scan time of 0.5 s per ion pair. The scan width for MRM was 0.1 m/z and both Q1 and Q3 were set at 0.7 unit mass resolution.

Quantitative basis and method validation

The HPLC-MS/MS method described was selective and specic. Analysis of the plasma samples conrmed that no endogenous material or drug metabolite peaks interfered with the analytes and the internal standard at the retention times. The retention times of DS, CPM and internal standard were 2.35, 1.58 and 2.01 min, respectively. As shown in Figure 2, the proposed HPLC-MS/MS method showed satisfactory results for the simultaneous determination of DS and CPM in rat plasma and was successfully used for the investigation of the herbdrug pharmacokinetic interaction of ACB with DS and CPM in rats after intragastric administration. The method exhibited an excellent linear response over the range of the selected concentration by weighted (1/c2) least-squared linear regression analysis. The standard calibration for DS and CPM was linear over the range 0.0550 and 0.0055 mg/ml, respectively. The mean values of the regression equation of the analytes in rat plasma were: y = 0.3435x + 0.0138 (r = 0.9981, DS) and y = 0.0444x 0.0003 (r = 0.9991, CPM), where y corresponds to the peak area ratios of the analytes to the internal standard and x refers to the concentrations of DS and CPM added to plasma. The lower limit of quantication proved to be 1 ng/ml for DS and 0.1 ng/ml for CPM.

Method validation
The method was validated in terms of selectivity, linearity, accuracy, precision, stability, extraction recovery and matrix effect according to FDA guidelines for the bioanalytical method validation.[35]
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4 5 6 7 8 9 10 12 11

100 90 80 70 60 50 40 30 20 10 0

13 1 2 3

30 40 50 60 70 Time (min) 1. UDCA 2. HDCA 3. CDCA 4. DCA 5. LCA 6. CA 7. TLCA 8. TCA 9. Bilirubin 10. TDCA 11. GCDCA 12. GCA 13. Taurine
Figure 1 Selected reaction monitoring chromatogram of a representative articial calculus bovis sample.

10

20

The precision and accuracy of the proposed method were determined in rat plasma by conducting replicate analyses of spiked samples based on the calibration standards. The procedure was repeated on the same day and on 7 different days by the same spiked standard series. The precision (RSD%) was less than 9.8%, which conrmed that the precision and accuracy of the method were acceptable according to FDA guidelines. The recovery was determined for six replicates of rat plasma with low, medium and high concentrations of the two analytes (0.10, 2.00, 40.00 mg/ml for DS, and 0.01, 0.20, 4.00 mg/ml for CPM). The mean absolute extraction recovery of DS was 85.1 4.3%, 89.7 3.5% and 90.7 4.9%, and the mean recovery of CPM was 97.6 6.2%, 101.3 4.5% and 96.8 3.8%. The RSD% of DS was 0.874.65% and of CPM was 2.685.02%. The data indicated that the recovery of the analytes was acceptable. All the ratios of the peak area of analytes resolved in the blank sample (the nal solution of blank plasma after extraction) to that of standard solutions at the same concentration were between 90 and 107%, which means that there were no signicant matrix effects in the proposed method.

Pharmacokinetic interaction study

The plasma concentrationtime proles for DS alone and DS co-administrated with ACB and CPM are shown in Figure 3. The plasma concentrationtime proles for CPM alone and CPM co-administrated with ACB and DS are shown in Figure 4. The main pharmacokinetic parameters of DS and CPM in different groups are presented and further statistically analysed in Tables 1 and 2, respectively. From the comparison of the main pharmacokinetic parameters among groups 1, 2 and 3, there was no signicant difference in each parameter (P > 0.05), which indicated no drug pharmacokinetic interaction between DS and CPM. Compared with group 1, the pharmacokinetic parameters of area under the concentrationtime curve (AUC0-) and peak plasma concentration (Cmax) in groups 4 and 6 were much greater (P < 0.01), and the time of peak concentration (Tmax), clearance (ClZ/F) and volume of distribution (VZ/F) of DS were lower than that of group 1 (P < 0.05). There were statistically signicant differences in these parameters. There was no statistically signicant difference in the t12 among these groups. Additionally, compared with group 2, the pharmacokinetic parameters of AUC0- and Cmax in groups 5 and 6 showed a greater degree
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100 80 60 40 20 0 100 Relative abundance 80 60 40 20 0 100 80 60 40 20

m/z 275.0229.8 (18V)

CPM RT=1.58

m/z 296.0213.8 (33V)

DS RT=2.35

m/z 326.0291.0 (24V)

IS RT=2.01

0 0.0

0.5

1.0

1.5 Time (min)

2.0

2.5

3.0

3.5

Figure 2 Typical multiple reaction monitoring chromatograms of diclofenac sodium (DS), chlorpheniramine maleate (CPM) and the internal standard (IS) in a rat plasma sample.

50
Concentration (g/ml)

group 1 (DS alone) Concentration (g/ml) group 3 (DS+CPM) group 4 (DS+ACB)

group 2 (CPM alone) group 3 (DS+CPM)

40 30 20 10 0
0 2 4

group 6 (DS+ACB+CPM)

group 5 (CPM+ACB) group 6 (DS+ACB+CPM)

0 6 Time (h) 8 10 0 1 2 3 4 5 6 7 8 9 Time (h)

Figure 4 Plasma concentrationtime proles of chlorpheniramine maleate (CPM). ACB, articial calculus bovis; DS diclofenac sodium. Values are the mean SD, n = 6.

Figure 3 Plasma concentrationtime proles of diclofenac sodium (DS). ACB, articial calculus bovis; CPM, chlorpheniramine maleate. Values are the mean SD, n = 6.

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Table 1

Pharmacokinetic parameters of diclofenac sodium in plasma Group 1 Group 3 DS + CPM 23.313 2.337 0.750 0.224 10.874 0.958 2.150 0.452 0.433 0.046 1.342 0.301 Group 4 DS + ACB 65.824 7.926 0.236 0.034* 33.769 6.548** 2.088 0.715 0.154 0.018* 0.467 0.174*
**

Group 6 DS + ACB + CPM 61.958 8.764** 0.236 0.034** 31.311 3.197** 1.975 0.393 0.164 0.025* 0.461 0.074**

Parameter AUC0-(mg/ml) Tmax (h) Cmax (mg/ml) t12 (h) ClZ/F (l/h/kg) VZ/F (l/kg)

DS alone 23.047 3.35 0.750 0.224 10.742 1.184 1.933 0.595 0.442 0.069 1.222 0.345

AUC0-, area under the concentrationtime curve to innity; Tmax, time of peak concentration; Cmax, peak plasma concentration; t12, biological halflife; ClZ/F, clearance; VZ/F, apparent volume of distribution. ACB, articial calculus bovis; CPM, chlorpheniramine maleate; DS, diclofenac sodium. Values are mean SD, n = 6. *P < 0.05, signicantly different compared with the DS alone group. ** P < 0.01, signicantly different compared with the DS alone group. Table 2 Pharmacokinetic parameters of chlorpheniramine maleate in plasma Group 2 Parameter AUC0- (mg/ml) Tmax (h) Cmax (mg/ml) t12 (h) ClZ/F (l/h/kg) Vz/F (l/kg) CPM alone 5.775 0.472 0.667 0.129 2.475 0.095 1.800 0.344 0.348 0.029 0.915 0.242 Group 3 CPM + DS 5.651 0.123 0.667 0.129 2.499 0.105 1.367 0.167* 0.354 0.008 0.698 0.081 Group 5 CPM + ACB 2.483 0.071 0.917 0.129* 0.932 0.014** 1.972 0.571 0.810 0.020** 2.296 0.669*
**

Group 6 DS + ACB + CPM 2.575 0.102** 0.958 0.102** 0.932 0.013** 2.313 0.413 0.778 0.030** 2.598 0.501**

AUC0-, area under the concentrationtime curve to innity; Tmax, time of peak concentration; Cmax, peak plasma concentration; t12, biological halflife; ClZ/F, clearance; VZ/F, apparent volume of distribution. ACB, articial calculus bovis; CPM, chlorpheniramine maleate; DS, diclofenac sodium. Values are mean SD, n = 6. *P < 0.05, signicantly different compared with the CPM alone group. **P < 0.01, signicantly different compared with the CPM alone group.

of decline, the Tmax, ClZ/F and VZ/F of CPM were markedly higher than that of group 2, and there were statistically signicant differences in these parameters (P < 0.05). Although the t12 of CPM was found to be slightly different among these groups, there was no signicant difference because of large variations in the data. From the results above, we concluded that no apparent herbdrug pharmacokinetic interaction existed between DS and CPM, and the combination of ACB, DS and CPM noticeably altered the absorption, distribution and disposition of the drug.

Discussion
The pharmaceutical action and therapeutic value of traditional Chinese medicines are expressed by multiple chemical components. In our previous work, we quantitatively determined the content of bile acids, bilirubin and taurine in ACB.[29] The batch of herb with higher levels of these effective components was used in this study to ensure the reliability of results. TCM is considered as an alternative and complementary medicine system in many Asian and Western countries. An integration of TCM and Western medicine has begun in several international medical centers. The combination of TCM and Western medicine necessitates a comprehensive

understanding of the interaction between drugs, particularly herbdrug interactions.[2,5,36,37] As a treatment for the common cold, compound diclofenac sodium chlorphenamine maleate tablets are a herbdrug preparation composed of ACB, DS and CPM. Although the concomitant administration of these three drugs is often used in clinical treatment, there have been no studies systematically and comprehensively determining their pharmacokinetic interactions. The aim of this study was to elucidate the herb drug pharmacokinetic interaction of ACB with DS and CPM in rats. Previous investigations revealed that DS had rapid absorption, high plasma protein binding (>99.7%) and minimal tissue binding, which is consistent with the present results.[38,39] Compared with the DS alone group, there were signicant differences in the AUC0-, Cmax, Tmax, ClZ/F and VZ/F for DS after co-administration of ACB with DS. The DS concentration increased to 33.769 6.548 mg/ml (Cmax) and 31.311 3.197 mg/ml (Cmax), with a Tmax range of 1015 min in groups 4 and 6, respectively, which increased 3.1-fold and 2.9-fold compared with the DS alone group. There was a signicant difference in the AUC0- among the three groups (P < 0.01). The DS from groups 4 and 6 presented a relative bioavailability 2.9-fold and 2.7-fold greater than that of the DS alone group. The increase in
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Cmax and AUC0- indicated that ACB was effective in promoting the drug absorption of DS. Meanwhile, the clearance of DS from groups 4 and 6 was 2.7-fold lower than that of the DS alone group, and the apparent volume of distribution for DS was decreased by 2.6-fold owing to the co-administration of ACB. These results suggest that ACB signicantly inhibited the rate of elimination of DS. Interestingly, the effects of ACB on the pharmacokinetic behaviour of CPM were virtually the opposite of its effects on DS. ACB apparently decreased the relative bioavailability and promoted the rate of elimination for CPM (as shown in Table 2). In contrast with DS, CPM distributed rapidly and extensively in body tissues such as kidney, brain and lung, and the concentration in these tissues was more than 31-fold higher than the plasma level.[40] Although the mechanism of the herbdrug pharmacokinetic interaction is still unclear, the different pharmacokinetic and distribution behaviours between DS and CPM may contribute to the observed opposite effects, and further investigations are required to clarify this. Cytochromes P450 (CYP) are the primary enzymes related to drug metabolism. Therefore, slight changes in CYP activity may alter the pharmacokinetic prole of a therapeutic agent and lead to many important drugdrug interactions. Previous ndings indicated that the metabolism of DS mainly involved CYP2C9 and CYP3A4, and CPM was a substrate of CYP2D6.[41,42] Since CYP participates in the metabolic process, substrates that can alter CYP activity may account for the variability in the pharmacokinetic behaviour of DS and CPM. In the present study, the pharmacokinetic prole of DS and CPM were both altered in the presence of ACB, which indicated that ACB was a possible effective inducer and/or inhibitor of CYP and had the potential to alter CYP activity. The exact mechanism would require further investigation. The stereoselective pharmacokinetics of CPM merit attention. Although CPM is administered as a racemate in the products market, its pharmacological activity primarily resides in the (S)-(+)-enantiomer.[43] Koch et al. investigated the potential effect of ranitidine on the stereoselective pharmacokinetics of CPM and the results demonstrated that ranitidine had no effect on the pharmacokinetics of either the (S)-(+)- or (R)-()-enantiomer, indicating no pharmacokinetic drugdrug interaction.[44] The present study has conrmed that ACB had an inuence on the pharmacokinetic prole of CPM. Whether such stereoselective pharmacokinetics exist between ACB and CPM would need to be assessed in a future study.

Concurrent use of TCM and Western medicine can improve the clinical efcacy, expand the scope of treatment and have positive effects on some diseases.[1,45] However, traditional Chinese medicines comprise multiple chemical components and so the interaction mechanisms that cause the therapeutic effects are complicated, and this may in turn cause more difculty in investigating the interaction between TCM and Western medicine. The combination of TCM and Western medicine has an impact on drug absorption, distribution, metabolism, excretion and other related body process, but may sometimes have a negative impact on the pharmacodynamics, decreasing the effects of the medicine or even producing adverse reactions. The apparent impact of ACB on the pharmacokinetic behaviour of DS and CPM has been conrmed in this study, and future research will focus on the effects of ACB on the drug metabolism of both, as well as the adverse reactions arising from concomitant administration of TCM and Western medicine.

Conclusions
A sensitive HPLC-MS/MS method was successfully applied to characterize the herbdrug pharmacokinetic interaction of ACB with DS and CPM in rats. Co-administration of ACB with DS noticeably increased the AUC0- and Cmax of DS, and the parameters of Tmax, ClZ/F and VZ/F of DS were signicantly decreased. Co-administration of ACB with CPM increased the Tmax, ClZ/F and VZ/F of CPM. A marked decline in AUC0- and Cmax of CPM occurred in the presence of ACB. However, there was no apparent pharmacokinetic interaction between DS and CPM. This study indicates that co-administration of ACB with DS and CPM can result in an apparent herbdrug pharmacokinetic interaction in rats.

Declarations
Conict of interest
The Author(s) declare(s) that they have no conicts of interest to disclose.

Funding
This work was supported by the Xiansheng Innovation Fund (CX11B-003XS) and the Fundamental Research Funds for the Central Universities (JKY2011037).
napur (west) district of Bengal. EthnoMed 2007; 1: 3745. 3. Foster BC et al. Natural health products and drug disposition.

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