| The herb-pair, the fixed combination of two herbs, is a basic form of compatibilityapplication of traditional Chinese Medicine (TCM). Compatibility would enhance thefunction of drugs, change the dissolution rate of active components and affect theabsorption and metabolism of drugs. Oral administration is the preferred route for TCM.The intestinal absorption would be the key step for the action of TCM formulas.Intestinal transport proteins form a barrier in the absorption of compounds. The herb-pairof Rhizoma Corydalis (Yuanhu) and Radix Angelicae Dahuricae (Baizhi) is a classicanalgesia formulas included in the Chinese Pharmacopoeia. The main object of our studywas to investigate the absorption characteristic of compounds in the Yuanhu-Baizhiherb-pair, find out the potential substrate of intestinal transporter and explore theinteraction between the intestinal transporters and the compounds. Ultimately, we couldelucidate the rationality on the compatibility of the herb-pair in the view of intestinalabsorption. Considering the complexity and integrity of TCM, our study would explorethe absorption mechanism of herb-pair and the effect of compatibility from three aspectsmonomer, single herb, and herb-pair using rat single-pass intestinal perfusion in situ andCaco-2cell monolayers transport model in vitro. The detailed results were presented asfollows.1. The LC-MS/MS assay of the compounds from the Yuanhu-Baizhi herb-pair in ratperfusion fluidsAgilent1260liquid chromatography and a6460triple quadrupole mass spectrometerwas used for simultaneous quantification of11major compounds in the herb-pair fromrat intestinal perfusion experiment, including eight alkaloids and three coumarins. Thechromatographic separation was performed on an Agilent Poroshell120EC-C18columnusing a mobile phase consisting of a mixture of acetonitrile and water containing0.0015%formic acid and2mM ammonium formate, respectively, gradient elutionpumped at0.3mL/min.All compounds were detected by tandem mass spectrometry inpositive ionization mode and quantified using multiple reaction monitoring mode.Theblank perfusion fluids were found to be free of interferences with the compounds ofinterest and IS.Good linearity was presented in related range of each compound.Intra-day and inter-day precisions were less than12.84%. The matrix effect was found tobe within the acceptable range (81.02%～113.65%).The recoveries of compounds weregreater than79.12%. In conclusion, the developed LC-MS/MS method meets the criteria set in FDA guidelines for bioanalytical methods and could be applied to thedetermination of the compounds from the herb-pair in rat perfusion fluids.2. Absorption characteristics of Yuanhu-Baizhi herb-pair and the effect ofcompatible application by rat single-pass intestinal perfusion model(SPIP)To study the absorption characteristics of the herb-pair, the LC-MS/MS method wasused to determine the compounds, and the absorption rate constant (Ka) and apparentpermeability coefficient permeability (Papp) of compounds were calculated by thegravimetric correction method. The Pappvalue of11compounds were all greater than0.2×l0-4cm/s. The Pappvalue of eight alkaloids appeared by the following order:THB>CYD>THP>GUC>BBR, PTP, ACP, JAR and that of three coumarins:IPT>IIPT>BGP. Alkaloids could pass through duodenum easily. PTP, THB, GUC, CYDwere best absorbed in ileum, significantly, by one-way ANOVAwith Dunnet’s test. Therewere no significant differences of permeability from three coumarins among threeintestinal segments. Compared with that of single herb group, the Pappvalue ofBBR,PTP,ACP,JAR increased significantly in three intestinal segments (>50%),THP,CYD,BGP,IIPT in duodenum and GUC,THP,CYD,BGP,IIPT in jejunum(P<0.05) inYuanhu–Baizhi herb-pair group. Compared with that of control group,the Pappvalue ofeight alkaloids increased significantly in jejunum after perfusing Baizhi herbextract.These data demonstrated that the enhanced absorption of herb-pair and therationality of compatible application.3.The effect of intestinal transporters on the absorption of compounds in Yuanhuand BaizhiThe inhibitors of transporter P-gp and MRP2were used for probe drug to investigatethe effect of two transporters on compounds from Yuanhu and Baizhi in this study.Compared with the absence of verapamil, the Pappvalues of BBR in three intestinalsegments, PTP,JAR in rat duodenum, and BGP in jejunum were raised significantly byabout50%, which indicated that BBR,PTP,JAR,BGP would be the substrates of P-gp.The Pappvalue of alkaloids significantly increased and there were no significantdifferences in the values of coumarins which suggested that the transport of alkaloidswould be mediated by MRP2.4. Absorption characteristics of Yuanhu Baizhi herb-pair and the effect ofcompatible application using Caco-2cell mononlayer transport modelThe LC-MS/MS method for the determination of compounds in rat perfusion fluids was slightly modified. The mobile phase was adjusted to pH4.5using4mM ammoniumformate. All compounds were quantified by dynamic multiple reaction monitoring mode.The blank matrix in Caco-2cell didn’t interfere with the compounds.Good linearity ofeach compound was observed in the corresponding range. The intra-day and inter-dayprecisions were within0.69～12.15%and2.70～14.41%,respectively.The matrix effectwas over80.97%.The modified LC-MS/MS method was thus applied to thedetermination of the compounds from the herb-pair in Caco-2cell mononlayer transportexperiment.The Caco-2cell mononlayer transport experiment was performed to study theabsorption characteristics of compounds of interest in the Yuanhu-Baizhi herb-pair inthree states of monomer, single herb, and herb-pair. The efflux ratio of THP monomerwas less than2, and IPT,IIPT less than1. The Pappvalue of quaternary-amine typealkaloids, such as BBR and JAR, were2.76×10-6cm/s, and1.88×10-6cm/s,respectively.The Pappvalue for tertiary-amine type alkaloids, such as ACP,GUC,THP,PTP,CYD, weremore than10×10-6cm/s and they could easily permeate through membranes. BBR andJAR were less permeable among the alkaloids whose efflux ratios were8.43and9.70inYuanhu single herb, respectively. The efflux ratio of other alkaloids were within2～3,which indicated that their main transport modes might involve active transport. Theefflux ratio of BBR and JAR were decreased to4.55and3.86in Yuanhu-Baizhi herb-pairwhich suggested that the compatibility could enhance the absorption of BBR and JAR. |
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