Study On The Effect And Mechanism Of Fangfeng Extract In Promoting Intestinal Absorption Of Paeoniflorin

ObjectiveBased on the formulation principle of Tongxie Yaofang decoction,this paper applied network pharmacology techniques to predict the potential active compounds,targets and mechanism of Tongxie Yaofang decoction in treating ulcerative colitis.The rat inverted intestinal sac model and in situ single pass perfusion model were further used to explore the effect of the Fangfeng extract on the intestinal absorption of paeoniflorin,which proving a scientific evidence for the mechanism of Fangfeng as“envoy drug”in Tongxie Yaofang decoction.Methods（1）Study on the potential components,targets and mechanism prediction of Tongxie Yaofang decoction in the treatment of ulcerative colitis.The chemical components of the four herbs in Tongxie Yaofang decoction were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）,whereas the other relevant information was obtained from the literature.Targets of the active compounds of Tongxie Yaofang decoction were obtained from the TCMSP and Swiss Target Prediction database.Meanwhile,the relevant disease targets of ulcerative colitis were collected from Dis Ge NET and Gene Cards database.Cytoscape 3.7.2 software was used to construct the network diagram of single herb-active compounds-targets of Tongxie Yaofang decoction,and the STRING database was used to build the target protein-protein interaction（PPI）network.The top 3 target proteins with moderate value in the network diagram were selected for molecular docking with the top 6 active ingredients.The potential targets received the GO analysis and KEGG pathway enrichment analysis by DAVID database.（2）The preparation of Fangfeng extract and the determination of its total chromone content.According to the identification items of radix saposhnikovia stipulated in the first part of the Chinese Pharmacopoeia of2020 edition,the Fangfeng extract was prepared by heating reflux method with 70%ethanol.To establish a method for the determination of total chromone content in Fangfeng extract.（3）The effect of Fangfeng extract on intestinal absorption of paeoniflorin.The rat model of inverted intestinal sac was established and the apparent absorption coefficient(P_app)was used as the evaluation index to investigate the absorption of paeoniflorin in the duodenum,jejunum,ileum and colon under the same concentration conditions,and compare the effects of Fangfeng extract on the absorption of paeoniflorin in different intestinal segments.（4）Study on the mechanism of Fangfeng extract promoting intestinal absorption of paeoniflorin.Taking the absorption rate parameters（K_a）and effective permeability coefficient(P_eff)as evaluation indicators,in vivo singal intestinal perfusion model was established to the optimal absorption intestinal segment of paeoniflorin after Fangfeng extract,and the effects of mass concentration,Fangfeng extract,P-glycoprotein（P-gp）inhibitor（verapamil hydrochloride）,multidrug resistance related protein 2（MRP2）inhibitor（pronecid）and antibiotic（norfloxacin）on paeoniflorin intestinal absorption were investigated.Results（1）In Tongxie Yaofang decoction,53 ulcerative colitis related potential active compounds and 168 potential targets were obtained,among which paeoniflorin in radix paeoniae alba and wogonin in radix saposhnikovia were the most moderate active ingredients.The molecular docking results showed that the active ingredients and target proteins had certain binding activity,and the target cyclooxygenase-2（PTGS2）was the most association with the active ingredient,suggesting that it might be an important therapeutic target.The results of GO and KEGG enrichment analysis showed that the efficacy of Tongxie Yaofang decoction involved mulitiple molecular functions and biological processes,and was associated with cancer pathways,tumor necrosis factor signaling pathway,Toll-like receptor signaling pathway,and PI3K/Akt signaling pathway and other signaling pathways to synergistically treat ulcerative colitis.（2）This product is the dried root of Saposhnikovia divaricate（Turcz.）Schischk.,which is the genuine of the Chinese Pharmacopoeia.Each 1 g of Fangfeng extract is equivalent to 2 g of original medicinal materials,of which the contents of total chromones in the extract from Hebei and Neimenggu was（20.36±0.06）%and（8.59±0.20）%,respectively.（3）Paeoniflorin was absorbed in the whole intestinal segment of rats,but there was no ignificant difference（P>0.05）in permeability coefficient P_appof the same concentration of paeoniflorin in each intestinal segments.Compared with the paeoniflorin group,Fangfeng extract has a certain trend of promoting the absorption of paeoniflorin in each intestinal segments,especially in ileum（P<0.01）.（4）Within the experimental concentration range(20～80μg·m L^-1),the absorption of K_aand P_effof paeoniflorin showed no significant difference with the increasing of paeoniflorin concentration,indicating that the main transport mode of paeoniflorin into the systemic circulation may be passive transport.Compared with the paeoniflorin group(40μg·m L^-1),Fangfeng extract（P<0.05）,verapamil hydrochloride,probenecid and norfloxacin had significant effects on the K_aand P_effof paeoniflorin（P<0.01）,suggesting that paeoniflorin may be the substrate of P-gp and MRP2,and regulating intestinal flora can increase its intestinal absorption.ConclusionThis paper predicted the main pharmacodynamic material basis and key targets of Tongxie Yaofang decoction in the treatment of ulcerative colitis,and demonstrated that paeoniflorin（Baishao）and wogonin（Fangfeng）are the main active ingredients in the prescription and the pharmacological effects of Tongxie Yaofang decoction in the treatment of ulcerative colitis may be closely related to it.In order to further confirm the effect of radix saposhnikovia,paeoniflorin was used as a model drug to study the effect of Fangfeng extract on the intestinal absorption of paeoniflorin by rat inverted intestinal sac model and in situ single pass perfusion model.Paeoniflorin can be absorbed in the whole section without specific absorption window.The Fangfeng extract has a certain trend of promoting the intestinal absorption of paeoniflorin,of which the ileum is the most significant.Paeoniflorin may be a substrate of P-gp and MRP2,and its absorption mechanism in the intestinal tract may be passive transport.The mechanism of paeoniflorin intestinal absorption promoted by Fangfeng extract may be related to the inhibition of drug efflux transporter（P-gp and MRP2）activities and the regulation of intestinal flora.