Study On The Absorption Mechanism And Transport Properties Of Total Glycosides Of Pterocephali Herba Based On Monolayer Model Of Caco-2 Cells

Objective:The purpose of this project is to establish a Caco-2 cell monolayer for the study of the absorption mechanism of the main components iridoid glycosides,curcumin,stiblenin,stutrin,and the active monomeric curcumin aglycones in the total glycosides of PterocepHalus model.Simultaneously based on molecular docking techniques to explore the binding,To investigate the infiltration and transmembrane transport mechanism of curcumin,evodiside,stutrin and active monomeric logiin aglycone in the monolayer model of Caco-2 cells in the extract of total glycosides of P.frondosa and action of P-gp and MRP2 transporter inhibitors to the curcumin aglycones.To provide further experimental evidence for further understanding of the absorption and bioavailability of total glycosides of PterocepHalus in vivo,It also provides scientific basis for the development of new formulations of curcumin,evodialdehyde,scochioside and Chinese herbal medicines containing these three components.Methods:1.Caco-2 cells were cultured to establish and evaluate Caco-2 cell monolayer models:Logarithmic growth phase cells were used to suspend cells in complete medium and inoculated into 12-well Transwell plates at a density of 1x10⁵cells/ml（3.6x10⁴cells/cm²）.0.5 ml of DMEM containing cell suspension was added to the AP side,and 1.5 ml of medium was added to the BL side.In the first week,the fluid was changed every other day.After one week,the fluid was changed once a day.The transmembrane resistance（TEER）was measured within 21 days after inoculation.The tightness of the model was examined.Permeability of the Caco-2 cell monolayer model was evaluated by examining the permeability of propranolol and fluorescent yellow on a Caco-2 cell model.2.Molecular docking techniques were used to predict and investigate the binding modes and forces of transporter inhibitors P-gp and MRP2 and curcumin aglycones.3.The ultra-high performance liquid pHase method was used to measure the concentration of the sample through the model,and the apparent permeability coefficient was calculated to study the bidirectional transport of the loganin aglycone in the Caco-2cell monolayer model.The study time,drug concentration,pH,P-gp and MRP2transporter inhibitors on their uptake.4.Ultra-high performance liquid phase method was used to measure the concentration of the sample through the model,and the apparent permeability coefficient was calculated to study the transport mode and characteristics of curcumin,evodiamine,and tartarin in the extract of total glycosides of PterocepHalus.Results:1.Establishment and Evaluation of Caco-2 Cell Monolayer Model:The morpHology of Caco-2 cell monolayer model was normal.After 21 days of modeling,the cell monolayer resistance value was above 500Ω·cm².The integrity and tightness of the cell model met the requirements.The rate of propranolol in the Caco-2 cell monolayer model was<10×10^-6cm·s^-1.Both met the requirements.The Caco-2 cell monolayer model was established to meet the experimental requirements.2.Linkage between Loganetin and p-gp and MRP1 molecules:Molecular docking results showed that when the docking protein is 4F4C,the free energy values of verapamil and the loganin aglycone are quite different,and the arginine and P-gp form unstable through hydrogen bonding and hydropHobic interactions.When the docking protein was 2CBZ,there was no significant difference in the free energy value of the MRP2 inhibitor cyclosporine and the loganin aglycone,and there was no significant difference in the free energy value of the selected docking conformation.3.A two-way translocation result of loganetin the Caco-2 cell monolayer model:In the two-way transmembrane transport of Caco-2 cell monolayer model,the loganin showed a decreasing trend of transmissivity,with the same absorption characteristics and time and concentration dependence.There was no significant difference between Papp（BL-AP）and Papp（AP-BL）（P>0.05）,and there was no obvious directionality.Papp decreased with time;the change of pH of solvent to the translucency of arium aglycone.No significant effect.P-gp inhibitors had no significant effect on the transport of arnicaside（P>0.05）.Cyclosporine inhibitors significantly increased the transmissibility of B-side glycosides in A→B transport,with significant differences（P<0.05）.There was no significant change in the transmissivity of B-A side primulin.The transdermal rate of argylen is in the range of 0.1-3×10^-66 cm/s.4.Bidirectional transport results of total glycosides of PterocepHalus in Caco-2 cell monolayer model:In Caco-2 cell monolayer bi-directional transmembrane transport process,the concentrations of loganin,stutrin,and evodiamine at high,medium,and low concentrations and at different time points all exhibited concentration-and time-dependence.In the two-way transmembrane transport process of Caco-2 cell monolayer model,loganin,arbutin,and evodiside showed a decreasing trend of transmissivity and had the same absorption characteristics.In addition,Papp（BL-AP）and Papp（AP-BL）were close,both less than 2,and no obvious directionality,Papp（BL-AP）and Papp（AP-BL）no significant difference（P>0.05）.Conclusions:1.Establishment and Evaluation of Caco-2 Cell Monolayer Model:The Caco-2 cell monolayer model was established in accordance with the experimental requirements and can be used for subsequent transport experiments.2.The conclusion of molecular docking and bidirectional transport of Loganetin:The results showed that the transmembrane transport mode of arginin was passive transport,and the results of molecular docking and Caco-2 model revealed that argomigenin is not a substrate of P-gp,and there is no efflux of P-gp transporter.Since cyclosporine is an inhibitor of both P-gp and MRP2 transporters and transport of curium aglycone in the Caco-2 cell monolayer does not have the efflux function of the P-gp transporter,it can be deduced that MRP2 The transporter may affect the transmembrane transport of the loganin aglycone in the Caco-2 cell monolayer.There may be an MRP2transporter-mediated efflux function.The docking results revealed that the loganin aglycone may be MRP2.The results of the docking with the molecular revealed that the arionin is likely to be the substrate for MRP2.The marquini aglycone is an MRP2substrate and is effluxed by MRP2.When used clinically,the MRP2 inhibitor can be used in combination to increase the bioavailability of the formulation with the curcumin as an active ingredient.According to the papp value of the Loganetin,it is known that the arionine is a substance having an absorption rate of between 1%and 100%,and the degree of absorption in the body is moderate.Therefore,the transportation mode of Loganetin was passive transport.3.Bidirectional transport of total glycosides of PterocepHalus in Caco-2 cell monolayer model:The results of bidirectional transport of the above total glycosides of PteropHyllus in the Caco-2 cell monolayer model show that:（1）It was suggested that the main components of total glycosides of curcuma pilosula,loganin,evodiamine,and tartarin,were transported and absorbed by passive transport.（2）The changes in the contents of the three components in the total glycosides of Bilateral P.edulis were the same and the characteristics of the three components were the same over time,indicating that the three components in the total glycosides of P.multiflora may undergo extensive conversion during transmembrane transport and are transformed The speed of the drug accelerates with time,and may be related to the up-regulation of the expression of related metabolic enzymes by total glycosides of P.aeruginosa.（3）The three components of total glycosides of PteropHyllus can be detected on the permeate side,From this,it can be judged that the total glycosides of PterocepHalus has good absorption and high bioavailability.In summary,the transport studies of argentinoids and total glycosides of P.aeruginosa in a Caco-2 cell monolayer model showed that their transport in vivo was passive transport,with good absorption performance,medium absorption,and Caco’s The quantitative correlation between-2 cell monolayer model and in vivo absorption provides an experimental basis for the related clinical application of total glycoside extract of P.frutescens.