Interaction And Compatibility Mechanism Of The Main Active Components In Shengmai Formula Based On Sodium Taurocholate Co-transport Polypeptide(NTCP)

Backgroud:Shengmai Formula?SMF?,including Shengmai Yin,Shengmai San and Shengmai Injection and other traditional Chinese medicine preparations,is widely used to treat disease such as chronic heart disease,coronary atherosclerotic heart disease,viral myocarditis and others.Previous studies of our research group have shown that OATP1B1/1B3 mediates the interactions between ophiopogon D and ginsenoside Rb1/Rd,which are the major active components in SMF.Sodium taurocholate co-transport polypeptide?NTCP?,another important hepatic uptake transporter,is capable of mediating transmembrane transport of many endogenous and exogenous substances and plays vital roles in drug absorption,distribution and excretion.In recent years,the drug-drug interactions based on NTCP have also drawn much attention from many scholars.Objective:The purpose was to deeply study the effect of the main active components in SMF on the transport functions of NTCP and the interaction and compatibility mechanism among the complex components in SMF based on NTCP to provide further experimental basis for the clinical safety and rational use of SMF using HEK293T cell models stably expressing NTCP.Methods:1.Quantitative methods of LC-MS for TCNa,ginsenoside Rb1,Rd,Rg1,Re,ophiopogonin D,ophiopogon D?,methylophiopogonanone A,methylophio-pogonanone B,schizandrin A,schizandrin B,schizandrol A and schizandrol B in HEK293T cells were established,respectively.2.The effect of gradient time and drug concentrations on the uptake of TCNa in NTCP-HEK293T cells was investigated to explore the optimized conditions for the uptake of TCNa in NTCP-HEK293T cells.3.The effect of the main active fractions and active components in SMF on TCNa transport in NTCP-HEK293T cells was investigated,and their respective IC₅₀values were calculated by Graphpad Prisms and the influence mechanisms were futher studied.4.The uptake and pharmacokinetic properties of the main active components in SMF in NTCP-HEK293T cells were investigated and the corresponding V_max and K_m values were calculated.5.The interaction of complex components in SMF based on NTCP was investigated and the possible compatibility mechanism was further studied.Results:1.The LC-MS quantitative analysis methods for TCNa,ginsenoside Rb1,Rd,Rg1,Re,ophiopogonin D,ophiopogon D?,methylophiopogonanone A,methylophiopogonanone B,schizandrin A,schizandrin B,schizandrol A and schizandrol B show good specificity,precision and accuracy in this experiment,which accord with the relevant requirements.2.The uptake kinetic parameters K_m and V_max of TCNa in NTCP-HEK293T cells were determined as 9.41±1.22?M and 342.63±17.36 pmol/mg protein/min,respectively.3.The main active fractions ginseng total saponins?GTS?,ophiopogon total saponins?OTS?,ophiopogon total flavonoids?OTF?and fructus schisandrae total lignans?STL?in SMF all had an obvious inhibitory effect on NTCP-mediated uptake of TCNa,and they inhibited transport activity by 44%,29%,49%and 43%at 50?M,respectively.And only ginsenoside Rg1,ophiopogon D?and schizandrin A exhibited obvious inhibitory effect on the uptake of TCNa among the 12 active components in SMF,they inhibited TCNa uptake by 73%,58%and 69%at 100?M,respectively.Among them,ophiopogon D?was the strongest NTCP inhibitor with an IC₅₀0 value of6.71±0.70?M.Ginsenoside Rg1 and schizandrin A exhibited weaker inhibitory effects on NTCP with IC₅₀ values of 50.49±4.24?M and 45.80±3.10?M,respectively.Ginsenoside Rg1 and ophiopogon D?may inhibit the NTCP-mediated uptake of TCNa by noncompetitive inhibition,and the V_max values decreased from357.90±19.68 pmol/mg protein/min to 267.13±16.78 pmol/mg protein/min and from 357.90±19.68 pmol/mg protein/min to 212.13±13.77 pmol/mg protein/min,respectively,while the K_m values remained unchanged.Schizandrin A showed a mixed competitive inhibition,with a K_m value that increased from 10.09±1.46?M to15.52±2.31?M and a V_maxax value that decreased from 357.90±19.68 pmol/mg protein/min to 308.86±21.89 pmol/mg protein/min.4.Only ginsenoside Re and schizandrin B may be the substrates of NTCP and could be transported by NTCP among 12 active components observed in SMF,and their uptake kinetic parameters(K_m and V_max)for NTCP were determined to be 47.13±2.73?M and 18.61±1.49 pmol/mg protein/min and 82.43±11.36?M and 32.62±2.65 pmol/mg protein/min,respectively.Furthermore,their uptake in NTCP-HEK293T cells was significantly inhibited by the classical inhibitor cyclosporin A.5.OTS,OTF,STL,ginsenoside Rg1,ophiopogon D?and schizandrin A significantly inhibited the uptake of ginsenoside Re in NTCP-HEK293T cells,this uptake was decreased by 78%,95%,96%,36%,58%and 49%at 50?M,respectively.Similarly,GTS,OTS,OTF and ophiopogon D?significantly inhibited the uptake of schizandrin B in NTCP-HEK293T cells,and this uptake was decreased by 42%,41%,43%and 39%at 50?M,respectively.However,the uptake of schizandrin B in NTCP-HEK293T cells was not affected by ginsenoside Rg1 and schizandrin A.Conclusions:1.The main active fractions GTS,OTS,OTF,STL and main active components ginsenoside Rg1,ophiopogon D?and schizandrin A in SMF could inhibit the uptake of TCNa mediated by NTCP in different degrees.They are potential inhibitors of NTCP,which may interact with clinical drugs based on NTCP.2.Ginsenoside Re and schizandrin B are the potential substrates of NTCP.Their transport mediated by NTCP could be inhibited by the classical inhibitor cyclosporin A.3.OTS,OTF,STL,ginsenoside Rg1,ophiopogon D?and schizandrin A in SMF could inhibit the uptake of ginsenoside Re mediated by NTCP in different degrees;GTS,OTS,OTF and ophiopogon D?could inhibit the uptake of schizandrin B mediated by NTCP in different degrees.There are interaction and compatibility mechanism of the main complex components in SMF based on NTCP.