Study On The Absorption Mechanism Of The Main Components Of Compound Horseshoe Fragrance Anxiety Capsule Based On P - GP Transporter

The capsule Valeriana jatamansi Jones is used to treat anxiety-related diseases, and is composed of Valeriana jatamansi Rhizoma et Radix, Ziziphi Spinosae Semen, Albiziae Cortex and Junci Medulla, which is safe and effective to nourishing the nerves. However, the bioavailitity of active ingredient in the compound is poor. Due to the blood brain barrier, the drug concentrations in the brain are relatively low after the conventional administration of medicine, limiting the treatment of brain diseases. Therefore, it is necessary to study the absorption of the active ingredient compound preparations.P-glycoprotein (P-gp) is a transporter protein located on cell membrane.Because the efflux transporter P-glycoprotein(P-gp) is involved in the absorption, distribution, and excretion of many drugs and often participates in drug-drug interactions, the P-gp has became the research focus which has been widely expanded to include (drug-drug interaction DDI), drug screening and other aspects of the study drug interactions. We studied the effect of the capsule Valeriana jatamansi Jones in the P-gp-overexpressing cell line Caco-2 and MDCK-MDR1.This study aimed to the main active ingredient of the capsule Valeriana jatamansi Jones such as hesperidin, spinosin, (-)-Syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside(SAG) Based on P-gp. In vitro and in vivo model cell models can be used to evaluate the characteristics of drug permeability through mucous membrane.Specific studies are as follows:1 Cell toxicity of the main active ingredient in the capsule Valeriana jatamansi JonesCaco-2, MDCK often used in combination with MDCK-MDR1 cells, as in vitro cell model of drug transport research through in vivo. We have established the stable Caco-2, MDCK, MDCK-MDR1 cells culture and passage methods. Caco-2 cells was cultured in DMEM/F12 medium (containing 10% fetal bovine serum), cells were in good condition incubated in 37 ℃,5% CO2, showing stone-like, high transparency, refraction and strong MDCK, MDCK-MDR1 were cultured in DMEM medium (containing 10% fetal bovine serum), Cell growth in good condition, it can form tight junctions, and have the characteristics of high transparency, strong refraction, etc. which can be used to study the mechanism of drug action.Before the study of absorption mechanism from gastrointestinal to brain, we choose MTT method to evaluate the cell toxicity of drugs. Then we determine the dose in the following experiments, and preliminarily evaluate the safe dosage of the capsule Valeriana jatamansi Jones. Hesperidin, spinosin and SAG groups have no cell cytotoxicity in the concentration range of 0-200,0-150 and 0-140 μg·ml-1respectively. The survival rate of cells may be maintained at a relatively high level when the prescription as follow:hesperidin: spinosin (5:1), hesperidin:SAG (15:1), spinosin:SAG(2:1).2 Uptake of spinosin, SAG alone and combination with hesperidin in Caco-2 CellThe uptake and permeability of spinosin and SAG in Caco-2 cells were measured by HPLC. the two componds absorption in cells were explored by studying the time, PH, drug concentration and inhibitors on the uptake of spinosin and SAG in cells. The uptake of spinosin in Caco-2 cell was time-dependent and concentration-dependent, the uptake has saturation trend above 80μg·ml-1, suggesting be efflux by transporter proteins. The uptake increase significantly when Spinosin conjunction with P-gp inhibitors, which means the Spinosins is the Substrate of P-gp. SAG was readily absorbed in a concentration-related manner and easily permeated in Caco-2 cells (1.92*10-6cm·s-1), when combined with the P-gp inhibitor, the uptake increased significantly, indicating that SAG can be efflux by P-gp transporter proteins. Therefore, Spinosin and SAG both are the Substrate of P-gp.When the active ingredients combined with each other, It was observed that Hesperidin: spinosin (5:1), The uptake of spinosin is (0.69±0.013)μg·mg protein-1, which increase significantly compared with spinosin alone; Hesperidin:SAG (5:1),The uptake of spinosin is (3.01±0.26) μg·mg protein-1, which increase significantly compared with SAG alone; spinosin:SAG (2:1), The uptake of active ingredients increase significantly compared with alone. The results showed that the absorption of drugs increase than single drug.3 Transport characteristics of spinosin across Caco-2 and MDCK-MDR1 cell modelsWe choose Caco-2 and MDCK-MDR1 cells as in vitro models to study drug transport mechanism through gastrointestinal and BBB. The cells were seeded in transwell plate, and it can grow to 100% confluence 21-28 days,5-7 days respectively. Observed by inverted microscope, the cells form tight junctions, grow to become single cell membranes. By measuring TEER values, rapid evaluate integrity of single cell membranes, Approximate MDCK-MDR1, Caco-2 TEER values were 200-300,400-500 Ω·cm2 respectively. Standard drug absorption was consistent with literature reports. The cell monolayer model function normally, and it can be used for drug transport. After P-gp inhibitor verapamil pretreatment, The fluorescence intensity of Rho-123 in Caco-2 and MDCK-MDR1 cell was significantly increased, compared with the control, which showed the P-gp function is normal.Different concentration spinosin (2、10、40、80 ug·ml-1) transport change significantly in A→B directions in Caco-2 monolayer. The apparent permeability coefficients (A→B) is(1.36±0.41), (2.57±0.43), (12.4±1.98), (13.5±3.21) 10-6cm·s-1 respectively,The (B→A)/(A→B) permeability Re of spinosin is 0.005,0.04,0.135,2.84, respectively, which showed that spinosin transport in both directions was concentration-dependent and saturable, when the Re was more than 2 in the cell model, indicating the intestinal absorption of spinosin is passive diffusion as the dominating process and active transportation were mediated, was affected by P-gp. the Papp (A→B) of spinosin between (1～10)×10-6 cm·s-1in Caco-2 models, which showed that it is medium absorbed. Meanwhile, Papp (A→B) is more than 3×10-6 cm·s-1in MDCK-MDR1 models, which showed that it is good absorbed through the BBB. Hesperidin and SAG significantly increased the absorbtion of spinosin in both directions.4 Mechanism research of spinosin efflux when combined with hesperidin and SAGThe levels of the TJ proteins localized in the cell-cell junctions were same when cells exposed to spinosin or SAG. In particular, the intensities of actin were slowly reduced after cells exposing to spinosin or SAG when the concentration more than 150μg·ml-1.Flow cytometry results showed that in Caco-2 cells, fluorescence enhancement values of spinosin and SAG were (119.94±3.54)%, (193.22±7.65)%, the fluorescence intensity of the cells increase significantly compared with the control group (P<0.05), which means SAG shows strong inhibition of P-gp function. At the same time, hesperidin had no significant differences compared with the control group. While, fluorescence intensity increased value of spinosin combined with hesperidin is (125.19±2.34), compared with a blank value is also significantly increased (P<0.05), when combined with hesperidin and SAG is (167.55 ± 5.67), which increased significantly compared with control (P<0.05), Molecular docking were employed to explore the interaction modes of hesperidin, spinosin and SAG. the molecular docking results showed that PHE332,SER725 are the binding sites of hesperidin and spinosin, TYR306,SER725 are the binding sites of hesperidin and SAG, SER725,TYR303 are the binding sites of spinosin and SAG. SER725 is the same binding sites of the three.Observed the results of P-gpATP activity, we found that spinosin and SAG can inhibit the P-gp ATP which is concentrations-dependent. At the same time, the P-gp ATP activity when combined with the drug at different ratios had increase significant difference (P<0.05) compared to the control group.5 in-situ single-pass intestinal perfusion method to detect the absorption of the drug alone and combinedThis experiment uses the classic shake-flask method for the determination of the oil-water partition coefficient of spinosin and SAG. In the octanol-water system, spinosin and SAG apparent oil-water partition coefficient logP<1, wihin the scope of optimal absorption.The oil-water partition coefficient different with the pH.The absorption rate constant (Ka) of spinosin and SAG in duodenum, jejunum, ileum and colon in the single-pass intestinal perfusion (SPIP) model were saturation. The Ka increase significantly combined with P- gp inhibitors compared to single-use group. At the same time, it was found Ka increase significantly combination with hesperidin.Summary:Based on the P-gp transporter, we study the absorption mechanisms of the active ingredient in the capsule Valeriana jatamansi Jones used the in vivo and invitro model. Used the cell monolayer model, we found that spinosin and SAG are P-gp substrate, the absorption of the active ingredient increase significantly associated with P-gp inhibitors or hesperidin. By observing the actin protein, the fluorescence enhancement values of P-gp、 molecular docking and P-gpATP activity, we found that the Mechanism of enhance absorption is locked amino acid positions of P-gp, inhibit P-gpATP activity, decrease P-gp drug efflux, increasing its absorption, in the single-pass intestinal perfusion method, we verify hypothesis that drug combinations can reduce P-gp drug efflux.