Study On The Metabolic Characteristics Of Geniposide Influenced By Genistein/Daidzein Regulating Intestinal Flora

Isoflavones from Semen Sojae Praeparatum are phytoestrogens with both estrogen and anti-estrogen effects,with genistein and daidzein as the main active components.Studies have shown that Semen Sojae Praeparatum can affect intestinal flora,affect the structure and composition of intestinal flora,and then affect human physiological activities,such as the absorption and digestion of diet,absorption,distribution,metabolism and excretion（ADME）of the drug.Geniposide is a cycloiridoid glycoside from Gardeniae Fructus of Rubiaceae,which has anti-inflammatory,analgesic,cholagogic and anti-inflammatory effects.Previous studies showed that the hepatotoxicity of Gardeniae Fructus was related to geniposide,an active metabolite of Gardeniae Fructus,and the compatibility of Semen Sojae Praeparatum with Gardeniae Fructus could up-regulate the abundance of Lactobacillus in intestinal flora and thus play the role of attenuating toxicity.Genistein and daidzein are the main effective substances of Semen Sojae Praeparatum for regulating intestinal flora.The present study verifies this fact,and the specific contents are as follows:1.To explore whether the combination of genistein and daidzein can play a role in regulating intestinal flora,and to screen the best administration conditions that can be compared with the effect of Semen Sojae Praeparatum.We used the composition of 3ratios of genistein-daidzein（1:1）,genistein-daidzein（2:1）and genistein-daidzein（3:1）to gavage rats,and used 16S r RNA high-throughput sequencing method to analyze and determine the fecal flora structure of each group,and compared theαdiversity,βdiversity and species composition at the phylum or genus level between the dosing group and the control group after 7 days,14 days,21 days and 28 days after administration.The results showed that genistein and daidzein could change the structure and composition of the intestinal flora of rats,mainly by inhibiting the growth of some bacteria in the phylum Bacteroides and promoting the reproduction of bacteria associated with the phylum Firmicutes.The administration of genistein-daidzein（1:1）was finally selected for 21days to regulate the abundance of Lactobacillus in the intestinal flora of rats.Compared with traditional Chinese medicine,the composition is clear and the quality is controllable,which lays the foundation for the development of new Chinese medicine based on traditional prescription medicine.2.To further verify whether the metabolism of geniposide in vivo is affected by it,under the premise of isoflavones regulating the abundance of Lactobacillus in the intestinal flora.In this chapter,the metabolic characteristics of geniposide and its five metabolites in intestinal tract（genipin,geniposidic acid,genipin sulfate,hydrolysis glucuronide conjugation of geniposide and geniposide glucoside conjugate and hydrolysis glucuronide conjugation and dehydrogenation of geniposide）in 24 hours were analyzed by LC-MS/MS.The pharmacokinetic parameters of C_max,T_max,t_1/2,MRT_0-t,AUC_0-t and AUC_0-∞were calculated by DAS2.0 software,and the average peak area ratio-time curves were drawn.The results showed that compared with the blank control group,the AUC_0-t,AUC_0-∞and C_max of geniposide and genipin,and the in vivo exposure of geniposide and genipin,were reduced in the isoflavone administration group and the Lactobacillus administration group.It was shown that after the intervention of isoflavones and Lactobacillus,the metabolism of geniposide to genipin in vivo changed,and the effect of Lactobacillus was more significant than that of isoflavones,which provided another evidence for the participation of intestinal flora in drug metabolism,but the specific participation method needs to be further studied.3.The absorption characteristics of geniposide in the duodenum,jejunum,ileum and colon segments of the blank control group and the Lactobacillus administration group in the absence of intrinsic factors such as blood circulation and nerve control were studied by the extracorporeal ectropic capsule model,and after verifying that Lactobacillus can affect the metabolism of geniposide in rats more than isoflavones.The relative cumulative absorption and apparent permeability coefficient of geniposide in different intestinal segments were calculated,and the P-glycoprotein（P-gp）inhibitor verapamil was used to explore whether P-glycoprotein was involved in the absorption and transport of geniposide.The results showed that the intervention of Lactobacillus reduced the absorption of geniposide in the isolated intestinal sac of rats;Verapamil can significantly increase the absorption and apparent permeability coefficient of horidoside,which indicates that Lactobacillus mediates the absorption of geniposide,and P-glycoprotein participates in the efflux transport process of geniposide,which provides another direction for the subsequent study of how isoflavones affect the metabolism of horicumside after regulating intestinal flora.It is of great significance for studying the drug-intestinal flora-drug interaction.4.The intestinal tissues of normal rats,isoflavones-administered rats and Lactobacillus-administered rats were used as experimental subjects to further study the effect of isoflavones on intestinal absorption.Real Time Quantitative PCR（RT-q PCR）and Western blot（WB）were used to detect the m RNA and protein expression levels of excretory proteins and tight ligand proteins in intestinal tissues,and to investigate the effect of i Lactobacillus on intestinal permeability.To explore whether Lactobacillus affect drug absorption by regulating paracyte uptake and efflux transport pathways.RT-q PCR results showed that isoflavones and Lactobacillus administration could significantly reduce the m RNA level of Claudin-5 and significantly increase the m RNA level of P-gp and Mrp2;WB results showed that Lactobacillus administration could significantly increase the protein expression levels of the above three proteins.For the decrease in the m RNA level of Claudin-5 and the increase in protein level,it is speculated that the drug acts on the translation stage of Claudin-5,reducing the degradation of the Claudin-5 protein,and may promote the reduction of the m RNA of Claudin-5 due to the"negative feedback"mechanism of action.In addition,GCMS technology was also used to detect the content of short-chain fatty acids in the cecal contents of rats in each group,and the results showed that both isoflavones and Lactobacillus could significantly increase the content of butyric acid in the cecum of rats,and the effect of Lactobacillus was significant,which was consistent with the results of the previous two chapters,suggesting that isoflavones may increase the content of butyric acid in the intestine by increasing the abundance of Lactobacillus in the intestinal flora,and finally act on related proteins to improve their protein expression levels.Through the above research,this project confirmed that genistein and daidzein can be combined to form a new compound preparation to replace Semen Sojae Praeparatum to regulate the intestinal flora,explore the relationship between the structure of isoflavones of Semen Sojae Praeparatum-intestinal flora-intestinal microbial metabolite-intestinal transporter-geniposide metabolism,The relationship between the exposure degree of geniposide and the intestinal flora-mediated transporter was clarified,and the mechanism of action of isoflavone components regulating the structure of intestinal flora after entering the body was revealed,promoting the production of intestinal microbial metabolites,and then upregulating the expression of efflux transporters,which ultimately led to the mechanism of action of reduced bioavailability of geniposide and genipin,which provided research ideas for the safe and effective application of clinical compound application of traditional Chinese medicine and combination of traditional Chinese medicine and chemical drugs.