Study On Synergistic Absorption Mechanism Of Gengen-Danshen Based On Intestinal Self-assembly TL/BSVs Formation

Objective:Intestinal tract is an important part of drug absorption to exert therapeutic effect.Studying the synergistic mechanism of drug pair from the dimension of intestinal endogenous environment is helpful to explain the connotation of modern science of drug pair compatibility.Many reports have shown that there is a phenomenon of oral absorption promotion between drug pairs,and explanations for this phenomenon mainly focus on the compatible ingredients may inhibit efflux proteins and affect the tight junctions between cells.However,there are still literature studies showing that in addition to the above mechanism,other drugs have the effect of promoting mutual absorption,such as Gegen-Danshen and Guizhi-Fuling,indicating that there are other factors that have not been clarified.Considering that self-assembled bile salt vesicles are an important phase state in the endogenous environment of the intestine,and that the formation of vesicle-like self-aggregates has been found to directly affect the solubility and permeability of encapsulated drugs,it is assumed that there is a definite link between the formation of bile salt vesicles and the reciprocal absorption of drug pairs.Based on the previous research,this project aims to investigate the contribution of bile salt vesicles to the mutual absorption of drug pairs,to improve the pharmacological evidence of synergistic effects of drug pairs,to enrich the scientific connotation of the rationality of the pairing of drug pairs,and to provide a scientific basis for the development of innovative formulations of Chinese medicine pairs,based on the in vitro and in vivo evaluations of Gegen-Danshen as a model drug pair.Methods:1.To verify the presence of TL/BSVs in intestinal environment:Bile was collected from male SD rats by common bile duct drainage,and simulated bile was prepared by membrane hydration method.The bile was characterized by Malvern laser scattering particle size analyzer and transmission electron microscope,and the particle size and appearance of their were analyzed and compared to confirm the presence of TL/BSVS in intestinal environment.2.Drug loading properties of TL/BSVs:Sodium Taurocholate and Egg Yolk Lecithin,were used as carriers,and puerarin,the main components of Gegen and the main fat（TSA and CTS）and water（DSS and SAB）soluble components of Danshen were used as model drugs.Blank TL/BSVs,4 co-loaded TL/BSVs and 5 single drug loaded TL/BSVs were prepared by thin film hydration method.The morphology of blank TL/BSVs and each drug loaded TL/BSVS was characterized by transmission electron microscopy and Malvern laser scattering particle size analyzer.The encapsulation rate and drug loading capacity were determined by HPLC to evaluate the solubilization effect of the co-loaded TL/BSVs system and the single-component TL/BSVs system.The drug precipitation,particle size changes and leakage rate of the co-loaded TL/BSVs system and the single-component TL/BSVs system were compared and analyzed to evaluate the stability of different drug loading systems.3.Simulation study on the formation of drug carrying TL/BSVs in vivo:All-atom molecular dynamics simulation technique（AA-MD）was used to simulate the behavior locus characteristics of the carrier and drug in vivo,so as to determine whether the carrier and drug will form drug carrying TL/BSVs in vivo.The stability of the systems was evaluated by the Root-mean-square deviation（RMSD）,the Radius of gyration（R_g）of the aggregates was calculated to reflect the compactness of systems I,II and III,and the degree of hydration of systems I,II and III was investigated by calculating Solvent-accessible Surface area（SASA）,and the three analyses were combined to explore the stability of drug carriers in different systems.4.Study on the effect of self-assembled TL/BSVs on drug absorption in the intestinal environment:The in vivo one-way intestinal perfusion test was combined with the common bile duct ligation technology.Firstly,the influence of different drug-carrying TL/BSVs systems prepared in vitro on drug absorption was investigated,the influence of TL/BSVs on drug absorption was analyzed,and other factors affecting drug absorption were excluded.On this basis,the effect of self-assembled TL/BSVs in intestinal environment on drug absorption without ligation was investigated.Results:1.Characterization results of rat bile and simulated bile:The size of rat bile was73.36±0.15nm,and the size of simulated bile was 92.61±0.14nm.The morphology of both of them was spheroid vesicular,indicating the existence of TL/BSVs in rat bile.Further studies showed that fat intake was beneficial to bile secretion.Therefore,the secretion of endogenous bile salts was increased,which was conducive to the formation of TL/BSVs.2.HPLC methods for the determination of PUE+TSA,PUE+CTS,PUE+DSS and PUE+SAB were successfully established.The preparation process was determined as follows:sodium taurocholate 320mg,egg yolk lecithin 80mg,methanol 20m L,spinning temperature 40℃,10m L ultrapure water hydration for 10min.The particle size results showed that the blank TL/BSVs was 76.14±0.07nm and the PDI was0.24,and the PDI was less than 0.3 for each drug loading system with a uniform distribution.Transmission electron microscopy results show that the spheres of the co-loaded system are darker than those of the single-loaded system,indicating that the co-loaded system is more compact and,as a side effect,the co-loaded TL/BSVs system is more stable.PT-TL/BSVs（PC-TL/BSVs）showed a significant increase in encapsulation rate and drug loading compared to TSA-TL/BSVs（CTS-TL/BSVs）.The change in drug precipitation,the change in particle size and the leakage rate of the drug in the different TL/BSVs showed that the stability of the co-loaded TL/BSV system was higher than that of the single drug TL/BSVs.3.The results of all-atom molecular dynamics simulations showed that sodium taurocholate and egg yolk lecithin carrier materials would self-aggregate with the active ingredients of“Gegen-Danshen”to form stable aggregates,while cholesterol did not participate in the aggregation behavior of the carrier and drug,and that the co-loaded TL/BSVs system would stabilize at 70 ns.The calculated results showed that the SASA and R_gof System I（co-loaded system）were smaller than those of System II and System III（single drug-loaded system）,further verifying that the stability of the co-loaded TL/BSVs system was better than that of the single drug-loaded TL/BSVs system.4.Intestinal absorption results showed that compared with P_appand K_aof PUE,DSS and SAB suspensions,TL/BSVs prepared in vitro was significantly improved,indicating that TL/BSVs could promote the absorption of drug pairs.The P_appand K_aresults of PUE,DSS and SAB suspensions in the bile model were also higher than those in the non-bile model,indicating that the intestinal absorption of PUE,DSS and SAB by TL/BSVs was also significantly improved.Conclusion:The bile salts and other endogenous components of the bile are stimulated by exogenous substances to self-assemble and form nano-vesicles in the intestine,in which the lipid-soluble components of the pair participate and are encapsulated,and the solubility and permeability of the components are improved.