Study On The Effects Of Rhein On The Absorption Of The Main Active Ingredients From Rehmannia Based On Absorption Models In Vivo And In Vitro

Objective:To investigate the effects of rhein on the absorption of catalpol and rehmannioside D,the main active ingredients in Rehmannia,to establish the foundation for further exploring the compatibility mechanism of Sheng Dihuang Decoction.Methods:1.Pharmacokinetic study in vivo: Methods for the determination of catalpol and rehmannioside D in the plasma of SD rats were established using LC-MS/MS.After the rats were administered orally with catalpol 400 mg/kg,200 mg/kg,100 mg/kg and rehmannioside D 76 mg/kg,38mg/kg,19 mg/kg,as well as catalpol and rehmannioside D combined with rhein,respectively,the contents of catalpol and rehmannioside D in rat plasma were measured,and their pharmacokinetic parameters were obtained.2.In situ single-pass intestinal perfusion: Methods for the determination of catalpol,rehmannioside D,and rhein in the intestinal perfusate of SD rats were established using HPLC.The effect of rhein on intestinal absorption of catalpol and rehmannioside D was studied by in situ single-pass intestinal perfusion,and the effects of different concentrations,p H,intestinal segments,efflux inhibitors and tight junction regulator on drugs' absorption were also investigated.3.Transmembrane transport study in vitro Caco-2 cell monolayer model: LC-MS/MS was used to establish a method for the determination of catalpol,rehmannioside D,and rhein in cell incubation solutions.The appropriate drug concentration was screened by MTT and a cell monolayer model was established to study the effect of rhein on transmembrane transport of catalpol and rehmannioside D,and to explore the effects of different concentrations,p H,efflux inhibitors and tight junction regulator on drug absorption.Results:1.Pharmacokinetic study in vivo: The linear ranges for the determination of catalpol and rehmannioside D in plasma by LC-MS/MS were 1 to 40,000 ng/m L and 1 to 10,000 ng/m L,respectively.And their precision,stability,carry-over effect,extraction recovery and matrix effects all meet the detection requirements of biological samples.After oral administration of catalpol and rehmannioside D,both the Cmax and AUC increased with the concentration of the drugs.After the combination of catalpol and rhein at low doses,its Cmax and AUC increased significantly by 1.71 times and 1.29 times(p < 0.05),and at the middle doses,its Cmax and AUC increased by 1.41 and 1.67 times,respectively(p < 0.05).After the combination of rehmannioside D and rhein at low doses,its Cmax and AUC increased by 3.96 times and 2.98 times,respectively(p < 0.05),and after medium-dose combination administration,Cmax and AUC increased by 1.56 and 1.50 times,respectively(p < 0.05),Cmax and AUC significantly increased by 2.60 and 2.01 times after high-dose combination administration(p < 0.05).2.In situ single-pass intestinal perfusion: The determination methods of catalpol,rehmannioside D and rhein in the intestinal perfusate were established by HPLC that all met the requirements of biological samples.After single-pass intestinal perfusion in rats,it was found that rhein had an effect on promoting intestinal absorption of catalpol and rehmannioside D.The addition of indomethacin,an MRP2 inhibitor,significantly increased the Papp values of catalpol in the ileum and colon by 1.81 and 2.79 times(p < 0.05),and the Papp values of rehmannioside D in the ileum increased by 2.33 times(p < 0.05),as well as the Papp values of rhein significantly increased by 1.46 times and 2.48 times in the ileum and colon,respectively(p < 0.05).After the addition of the BCRP inhibitor reserpine,the Papp values of catalpol increased significantly in the jejunum,ileum,and colon by 2.80 times,4.11 times,and 3.46 times,and the Papp values of rehmannioside D in the ileum increased significantly by 2.10 times,and the Papp values of rhein in ileum increased significantly by 1.49 times,respectively(p < 0.05).There were no significant changes were observed in the Papp values of the P-gp inhibitor and the tight junction regulator EDTA to the four intestinal segments of catalpol and rehmannioside D.Compared with p H6.8 and p H5.5,catalpol at p H7.4 significantly increased the Papp values in the duodenum and jejunum(p < 0.05).3.Caco-2 cell monolayer model: The determination methods of catalpol,rehmannioside D,and rhein in cell incubation solutions were established by LC-MS/MS that all met the requirements for the detection of biological samples.According to the MTT method,the ranges of the safe administration concentrations of catalpol,rehmannioside D,and rhein were 110.5~663.0 ?M,4.37~65.60 ?M,and 1.76~26.41 ?M,respectively.After the Caco-2 cell monolayer model was cultured for 21 days,the transmembrane resistance value(TEER)of the monolayer cells was greater than 400?·cm2,and the tight junction has been formed,which met the requirements of transmembrane transport.Through transmembrane transport studies,it was found that Papp(AP-BL)of catalpol increased significantly and Papp(BL-AP)of rehmannioside D significantly decreased after they were combined with rhein(p < 0.05).The MRP2 inhibitor indomethacin significantly increased the Papp(AP-BL)value of catalpol by 4.29 times,the Papp(BL-AP)value decreased by 1.89 times,and the Papp(BL-AP)value of rhein significantly decreased by 1.59 times(p < 0.05).Reserpine,a BCRP inhibitor,significantly increased the Papp(AP-BL)value of rehmannioside D by 1.34 times,and significantly reduced the Papp(BL-AP)value of rhein by 1.70 times(p < 0.05).Different p H had no significant effect on the rate of rehmannioside D transport.At p H8,the Papp(AP-BL)of catalpol increased significantly by 2.76 times and 3.87 times compared to p H5 and p H7,respectively(p < 0.05).EDTA had no significant effects on the Papp values of catalpol and rehmannioside D.Conclusion:This article comprehensively explored the effects of rhein on the promotion of catalpol and rehmannioside D from the overall animal level,tissue level,and cell level.It was found that rhein can promote the membrane permeability of catalpol and rehmannioside D in the intestinal epithelial cells.And rhein can reduce the binding amount of catalpol and rehmannioside D to the efflux transporter by competitively binding catalpol with MRP2 and competitively bindind rehmannioside D with BCRP,further promoting the amount of transmembrane transport of catalpol in intestinal epithelial cells,reducing the efflux of rehmannioside D in intestinal epithelial cells.Furthermore,it promotes intestinal absorption of catalpol and rehmannioside D,and finally promotes absorption of catalpol and rehmannioside D in vivo.