Study On Constituents Of Polygoni Multiflori Radix Praeparata And The Effect On Bilirubin Transport Mediated By OATP1B1/MRP2

Objective1.A RP-HPLC method was established to simultaneously determine nine active components,Gallic acid?Gal?,5-Hydroxymethy furfural?5-HMF?,Catechin?Cat?,L-Epicatechin?Epi?,2,3,5,4'-Tetrahydroxystilbene-2-O-?-D-glucopyranoside?Tet?,Emodin-8-O-?-D-glucoisde?Emo-G?,Physcion-8-O-?-D-glucoside?Phy-G?,Emodin?Emo?and Physcion?Phy?in Polygoni Multiflori Radix Praeparata?PMP?.Fingerprint of PMP from different regions was established to evaluate their quality and provide a scientific basis for the use and development of PMP.2.MDCK cell models expressed OATP1B1 and OATP1B1/MRP2 were stably constructed.The cell models will provide technology platform to explore the effect of PMP on OATP1B1-medicated uptake and MRP2-mediated efflux of bilirubin.3.MDCK cell models were constructed to explore and disclose the mechanism of bilirubin related adverse reaction or diseases caused by PMP and its main active components at the molecular level.The bilirubin in cells and transport buffer was detected by RP-HPLC.Method1.9 active components in PMP were simultaneously determinated and established the fingerprint of PMP?1?PMP was processed by the braising method according to"Chinese pharma-copoeia".?2?To build a RP-HPLC method to detect the main components in PMP from differ-rent origins.?3?PMP extraction was prepared by reflux extraction.?4?To establish the fingkerprint of PMP from different regions.2.The MDCK models expressing OATP1B1 and OATP1B1/MRP2 were constructed.?1?The PEGFP-C1/OATP1B1 recombinant plasmid was transferred into the wild type MDCK?to build the high expression cell model.?2?mRNA and protein expression of OATP1B1 and MRP2 in the wild type MDCK?,MDCK/OATP1B1and MDCK/OATP1B1/MRP2 cells were verified by Q-PCR and Immunoblot analyses,respectively.Furthermore,the protein positions were located by confocal laser scanning microscope technology.?3?The gene expression of OATP1B1 and MRP2 in MDCK/OATP1B1 and MDCK/OATP1B1/MRP2 cells was induced by sodium butyrate and its appropriate concentration was confirmed.3.Effect of PMP on bilirubin transport based on the models of MDCK/OATP1B1/MRP2 was investigated.?1?A HPLC method for determination of bilirubin in cells samples was established.?2?The optimum condition for the transport was investigated by exploring the effect of bilirubin concentration and the transport time on uptake of bilirubin.?3?The pharmacokinetic parameters(e.g.,V_max,K_m)of bilirubin in MDCK/OATP1B1/MRP2 were calculated.The effect of PMP and its main active components on bilirubin transport was evaluted.Results1.A convenient,accurate and sensitive RP-HPLC method was established to simul-taneously determine 9 active components in PMP.Meanwhile,the fingerprint of PMP from different regions was sucessfully established.2.The MDCK cell models expressing OATP1B1 and OATP1B1/MRP2 were success-fully constructed.?1?The hybridoma cell lines were obtained after additional selection with G418.?2?No mRNA of OATP1B1 or MRP2 could be detected in wild cells.mRNA expression of OATP1B1 in the MDCK/OATP1B1 cells was 394.87±75.89 fold higher than that of the wild type.mRNA expression of OATP1B1 and MRP2 in MDCK/OATP1B1/MRP2 were 938.40±56.42 and 633.59±40.90 fold higher than that of the wild type,respectively.All the cell lines only expressed the objective proteins,and the objective proteins were not detected in the wild cells.OATP1B1 and MRP2 located in the cell membrane.?3?The result showed that the amount of proteins was increased with increasing concentration of sodium butyrate.The optimal concentration of sodium butyrate was 10mM.3.Effect of PMP and its components on bilirubin transport based on the MDCK/OATP1B1/MRP2 cell models was investigated.?1?The HPLC method showed good specifity,sensitivity and precision,and can be used to detect bilirubin samples.?2?The volume of bilirubin transported by MDCK/OATP1B1/MRP2 cells increased rapidly in 0-45 min and get saturation at 45 min.The bilirubin transport volume was increased with the increase of bilirubin concentration.When the concentration of bilirubin was up to 0.75??,the uptake velocity became slowly.The kinetic parameter K_m was0.13±0.02??,V_max was 53.56±1.94 nmol?min^-1?mg^-1protein.?3?PMP strongly inhibited biliruin transport?P<0.05?and the PMP-4?Shan Dong Province?displayed the most strong inhibitiory effect.Tet,Emo,Phy and Cat in PMP showed different capacity ininhibiting uptake and transport of bilirubin,Emo displayed stronger inhibitiory effect on OATP1B1 than the other components.Tet and Cat showed stronger inhibitiory effect on MRP2.Conclusion1.A fingerprint of PMP was established successfully.And a valuable advice on the revision of quality criterion of PMP in the"Chinese pharmacopoeia?2015?"was presented.2.The processed PMP met the criterion of the"Chinese pharmacopoeia".And the content of the active ingredients in PMP from different regions demonstrated obvious discrepancy.Unfortunately,PMP from several regions were even unqualified.3.Bilirubin can be transported by MDCK/OATP1B1/MRP2 cells.PMP and its com-ponents inhibited the OATP1B1-medicated uptake and MRP2-mediated efflux of bilirubin,which might result in bilirubin accumulation in the body,and then causing jaundice and the other bilirubin related adverse reactions or diseases.