| Objective Ferulic acid(FA)has a good pharmacological effect and may be the main active ingredient of Danggui-Shaoyao-San(DSS)in the treatment of neurodegenerative diseases.FA is rapidly absorbed and rapidly eliminated in animal models,and its bioavailability is low.Studies have shown that the bioavailability of FA can be improved by compatibility.In this study,the Caco-2 monolayer cell model was used to verify the osmotic absorption of DSS,Angelica sinensis,Rhizoma Chuanxiong and FA in intestinal epithelial cells,and further explore the possible protein targets affecting this process and other major components in the study.The effects of DSS,Angelica sinensis,Rhizoma Chuanxiong and Fangzhong on the activity of CYP3A4 were investigated by rat liver microsome model.The effects of DSS on FA bioavailability and its mechanism were explored to provide experimental evidence for the compatibility of traditional Chinese medicine formula.Methods HPLC was used to quantify the FA contained in DSS,Angelica and Chuanxiong,and then the comparison of the permeation amount of FA in each group was examined on a Caco-2 cell model.After the P-gp and MRP2 specific inhibitors and other monomer components in the formula were administered,the osmotic change of FA was examined.The specific metabolite 6β-hydroxytestosterone of different groups of CYP3A4 was tested to compare the inhibitory effects of different groups on the liver drug CYP3A4,and the half-inhibition rate(IC50)of each group was calculated to compare the inhibitory effects of each group.The other monomer components in the addition side were added to the incubation system to screen for monomers which inhibited CYP3A4.HepG2 cells were used to investigate the effects of DSS,Angelica sinensis,Rhizoma Chuanxiong and FA on the expression of P-gp,MRP2 and CYP3A4 proteins.The effects of DSS,Angelica sinensis,Rhizoma Chuanxiong and FA on the activity of CYP3A4 were detected by ELISA kit.Results Compared with the FA group alone,the FA absorption increased significantly in the DSS(P<0.001).Angelica and Chuanxiong also increased FA absorption(P<0.001,P<0.01).Compared with the FA monomer group,FA absorption was significantly increased in the probenecid group and the verapamil group(P<0.001,P<0.01).The components of paeoniflorin,ligustilide and ligustrazine can significantly increase the osmotic absorption of FA(P<0.05,P<0.05,P<0.05).The IC50 of ketoconazole was 7.645μM·L-1,the IC50 of DSS group was 0.1017mg·mL-1,and the IC50 of Angelica group was 0.2948mg·mL-1.The results showed that DSS had better CYP3A4 inhibition than Angelica.P<0.001).FA also significantly inhibited CYP3A4(P<0.05).Paeoniflorin had no significant effect on CYP3A4 activity.Ligustilide and ligustrazine had certain inhibitory effects on CYP3A4,but there was no significant significance.DSS and FA inhibited the expression of CYP3A4 protein,but there was no significant significance.Angelica and Chuanxiong promoted the expression of CYP3A4;DSS and Angelica inhibited the expression of P-gp protein(P<0.05,P<0.05),but Chuanxiong showed It has the effect of promoting expression.The results of ELISA showed that DSS and FA significantly inhibited the activity of CYP3A4 enzyme(P<0.05,P<0.05).Angelica and Chuanxiong promoted the activity of CYP3A4,and the results were similar to those of WB protein.Conelusion The above results indicate that DSS,Angelica and Chuanxiong,as well as the main components of paeoniflorin,ligustilide and ligustrazine,can increase the intestinal epithelial absorption of FA;transport experiments and Western-blot detection of P-gp results indicate that DSS and FA are external Repressor P-gp has an inhibitory effect;liver microsome incubation test,CYP3A4 protein assay and ELISA assay show that DSS inhibits the activity of CYP3A4 enzyme.Taken together,the mechanism by which DSS increases FA bioavailability may be achieved by inhibiting the efflux transporter P-gp and the metabolic enzyme CYP3A4. |
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