The Transport Mechanism Study Of Puerarin Based On MDCK

Caco-2and MDCK（Madin-Darby Canine Kidney）cell lines are monolayermodels used to predict permeability of intestinal barrier, in vitro. Like Caco-2, MDCKcells have differentiated into a columnal epithelium to form tight junctions, andshowed highly functionalized epithelial barrier with remarkable morphological andbiochemical similarity when cultured on semipermeable membrane. MDCK cellsgrows faster than Caco-2, which significantly shorten experimental period. Besides,compared with as Caco-2, MDCK cells express less subtypes. As a consequence, theresults showed higher reproducibility. P-gp is widely expressed in different histocytes.P-gp can pump absorbed drugs out of cells depend on energy, leading to amount ofpenetration and plasma concentration reduced. Verapamil is widely used as a inhibitor of P-gp.Puerarin, an isoflavones compound, is derived from the radix of puerarin lobata（Willd） Ohwi and considered as its main bioactive components. According to theancient Chinese literature and modern studies, puerarin possesses complex anddiverse pharmacology effects, such as hypotensive activity, anti-hyperlipidemia,anti-diabetes,anti-platelet-aggregratin,antioxidation,anti-inflammation,anti-arrhythmias, dilating the cardiac an cerebral vessels and neuroprotective effects, etc. The aim ismainly to raise oral bioavailability of puerarin in pharmaceutics research since its oralbioavailability is relatively lower.The objective of the present study is to develop MDCK monolayer model and asingle HPLC method for the identification and determination of puerarin and todiscuss effects on permeable efficiency of concentration, transport direction,temperature and verapamil（a inhibitor of P-gp）, respectively. The main contents weredivided into some sections as follows:1To establish an HPLC method for the determination of puerarinQuantitative determination of puerarin is performed on a Shimadzu HPLCinstrument（Japan）, which consists of a vacuum degasser, quaternary pump and UVdetector. Separation was achieved on a Shim-pack ODS column（4.6mm×250mm,5μm） at the temperature40℃. The elution was employed with methanol as solvent Aand an0.2%（v/v） aqueous solution of phosphate, PH3.5（adjusted with triethylamine）,as solvent B, at a flow rate of1.0ml·min-1,and the elution is30:70（A:B, v/v）. Effluentabsorbance was measured at250nm and20μL samples were injected into the column.In this condition, calibration curve of puerarin showed good relationship（R2﹥0.9999,n=7）,with the relative standard deviations（RSD） of intra-and inter-dayprecision≤2.13.2To develop MDCK monolayer modelMDCK cells were seeded50000per cm²on Millicell-CM Hanging CulturePlates Inserts. Confluence was reached within5-7days. Transepithelial electrical resistance values（TEER） were measured with Millicell ERS（Electirical ResistanceSystem） in order to indicate the integrity and compactness. Only monolayers withTEER above140·cm²were used.3Transport experimentsThe transport mechanism of puerarin across MDCK monolayer was investigatedby changing concentration, transport direction, temperature, and with or withoutverapamil. Pappof80μg mL^-1puerarin were （2.36±0.12）×10^-6and （6.10±0.36）×10^-7cm/s in the basolateral compartment,37℃and4℃, while （9.24±0.75）×10^-7and（2.14±0.25）×10^-7cm/s in the apical compartment, repectively. Added with verapamil,Pappof80μg mL^-1raised to （5.74±0.28）×10^-6cm/s in the basolateral compartment.The results showed that the rate and amount of penetration raised as the concentrationand temperature increasing. The process showed obvious directional. When verapamilwas added, absorption of puerarin was enhanced. The results indicated that, thetransport mechanism across MDCK includes not only simple diffusion but alsoactive transport, while puerarin is a substrate of P-gp. The efflux function of P-gpreduced absorption of puerarin.