Study On Compatibility Rule Of Shen Fu Formula In The Aspect Of Intestinal Absorption Using Caco-2Cells

Objective:Shen Fu Formula (SFF) is a typical traditional Chinese medicine formula constituted by Panax ginseng (root of Panax gens ing C. A. Mey; Araliaceae) and Aconitum carmichaeli (processed daughter root of Aconitum carmichaeli Debx.; Ranunculaceae). We aimed to establish the Caco-2cell monolayer model, so as to evaluate the compatibility rule of SFF in the aspect of intestinal absorption, and to finally provide technical support for further investigation and data support for component compatibility optimization.Methods:Panax ginseng extract and Aconitum carmichaeli extract were prepared and determined by UPLC/MS. The marker compounds of them were selected and quantified. Caco-2cells were inoculated in the inserts at the density of1×105cells/cm2and cultured21days to establish the monolayer model, of which the integrity, tightness and permeability were estimated with the transepithelial electrical resistance and the apparent permeability coefficient (Papp) of Lucifer Yellow and propranolol, respectively. Panax ginseng extract, Aconitum carmichaeli extract, their combination and the diester type alkaloid monomers were administered to the model. The marker compounds in their transport samples were quantified by UPLC/MS, and the Papp and efflux ratio values were calculated. Rhodamine123efflux experiment, RT-qPCR and western blot method were employed to analyze P-glycoprotein (P-gp) activities, multidrug resistance1(MDR1) mRNA and protein levels, respectively.Results:Six key ginsenosides in Panax ginseng extract, the ppt-type ginsenoside Rg1and ginsenoside Rf, the ppd-type ginsenoside Rb2, ginsenoside Rb1and ginsenoside Rc, and oleanane type ginsenoside Ro were selected as marker compounds due to their high contents and pharmacological effects. Eight key aconitum alkaloids in Aconitum carmichaeli extract, the diester type aconitine (AC), mesaconitine (MA) and hypaconitine (HA), the monoester type benzoylmesaconine and benzoylhypaconine, and the non-esterified type fuziline, neoline and talatisamine were selected as marker compounds due to their high contents, pharmacological effects or toxicities. After Caco-2cell monolayer model was established and validated, the transport of Panax ginseng extract was preliminarily analyzed. Only ginsenoside Ro probably had active efflux effect, so we transferred our focus on Aconitum carmichaeli. When Aconitum carmichaeli extract were transported through the Caco-2cells model, the highly toxic diester type alkaloids in it exhibited active efflux. When Panax ginseng extract was co-administered, the efflux of these diester type alkaloids increased, and the absorption of them decreased. When P-gp inhibitor was co-administered, the efflux of these diester type alkaloids was inhibited, indicating that P-gp possibly involved in their efflux. Then the transport experiments of diester type alkaloid monomers were performed and some transporter inhibitors were administered. Multiple transporters were found to be involved in their transport system, including efflux transpoters P-gp and breast cancer resistance protein, meanwhile not excluding apsorption organic cation transporter. Finally, P-gp activities, MDR1mRNA and protein levels were determined by rhodaminel23efflux experiment, RT-qPCR and western blot method, respectively. Panax ginseng was found to induce P-gp activity in Caco-2cells via increased MDR1/P-gp expression.Conclusion:Panax ginseng could facilitate the P-gp mediated efflux of highly toxic diester type alkaloids in Aconitum carmichaeli without having much influence on other active components. It would be beneficial to explain the toxicity reduction compatibility rule of SFF, and to finally provide technical support for further investigation and data support for component compatibility optimization.