The Study On The Promoting Mechanism Of Menthol Involving The Absorption Of Tanshinol Through The P-glycoprotein

Objective: This study was to explore the effect of menthol on the intestinal absorption of tanshinol respectively from the cellular and behavioral levels by means of investigating the relationship between the effect of menthol on tanshinol and the translation function of P-glycoprotein(P-gp) via the Caco-2 cell monolayer model. Discovered the primary promoting mechanism of menthol affecting the absorption of tanshinol and provided a theoretical foundation for promoting enhancers improving the bioavailability of tanshinol and its development and application.Methods:1. Intestinal absorption experiment of everted intestinal sac method Used the everted intestinal sac method and the experimental groups was: blank group and verapamil group(positive control group), 20, 40, 80μg/ml Tanshinol group; absorption concentration added menthol group with volume ratio: 0.1%, 0.3%, 0.9%. Used HPLC to determinate the different concentration of tanshinol intestinal absorption, and the transformation of menthol intervention of tanshinol intestinal absorption for further research of cellular study.2. Establishment of Caco-2 cell monolayer model Using Transwell board to let the cells which was in logarithmic growth phase laid on the upper layer of the membrane, change the culture medium every 24 hours.The detection of the resistance on EVOM resistance tester substituted for a success basis on the cell model establishment.3. MTT drug concentration toxicity test Used the alternative production of CCK-8 on the cell model to test the toxicity by MTT assay, then used an enzyme mark instrument and measured OD values to calculate the cellular survival rate.4. Caco-2 monolayer model of cell transport experiment The Caco-2 cell monolayer model was cultured in medium supplemented with experimental groups was: blank group and verapamil group(positive control group), 10, 20, 40, 60 μm of tanshinol group; selective the better of the two groups to add the concentration: 2, 8, 16 μm(low, middle, high) of menthol group. We used HPLC to study the changes under the influence of menthol of the concentration of tanshinol and the transportation of different drug concentrations of tanshinol were determined.5. cell substrate accumulation and efflux experiments The Caco-2 monolayer model were cultured in medium and supplemented with experimental groups was : blank group and verapamil group(positive control group), 40, 60μm tanshinol group, 2, 16μ m(low dose group, mid and high dose group) of menthol group, tanshinol- menthol mixed group and the tanshinol-verapamil mixed group. The total protein content and fluorescence intensity were measured with enzyme labeling instrument, and the results expressed the amount of Rh123 by fluorescence intensity /mgpro, which can reflect the amount of Rh123 in Caco-2 cells.Results:1. The everted gut sac model of intestinal absorption test The experimental results show that tanshinol in 40 g/m L was the best concentration under the uptake and accumulation. After the addition of 0.1% menthol, uptake rate and uptake increased significantly; after adding 0.9% menthol, absorption rate decreased, uptake and accumulation had no significant change.2. The validation of Caco-2 cell model The integration of Caco-2 becomes higher during cell cultured, and the resistance value is gradually increased, the monolayer film is compact until about 21 days, the resistance value is greater than 400 Ω/cm2.3. The determination of the concentration of verapamil, menthol and tanshinol The toxicity test on cell model showed that tanshinol in drug concentration is not higher than 50 μm, Caco-2 cellular survival rate was bigger than 90%; menthol to drug concentration is not higher than 16 μm, Caco-2 cellular survival rate was more than 90%; the drug concentration of verapamil was under 30μm for 90% cellular survival rate. The mixed solution in tanshinol to concentration is below 50 μm, menthol concentration lower than 16 μm, Caco-2 cell survival rate was more than 90%; verapamil in tanshinol mixed drug concentration is not higher than 30 μm, Caco-2 cell survival rate of 90%, with tanshinol under the concentration of 40 μm.4. Caco-2 cell model for transport experiments Experimental results showed that the transport of tanshinol in 35 μm and 40 μm concentration under good permeability coefficient. Menthol with drug concentration of 2 μm, the permeability coefficient of tanshinol is improved obviously, transfer rate increased significantly; when the drug concentration was 16μm, tanshinol of permeability coefficient did not change significantly, transport rate had a downward trend; the absorption rate and transportation rate of tanshinol was increased by the increased concentration of verapamil.5. menthol on intestinal absorption of tanshinol and P-gp Cell accumulation and the outer row experimental results show, the accumulation of Rh123 in Caco-2 cells not with menthol concentration change, at low concentration was significantly higher than that of control group(only tanshinol)(P < 0.01), at high concentrations, but lower than the blank group; also of rhodamine 123 accumulation with the positive control in verapamil group to the increase of drug concentration increases. The amount of Rh123 on Menthol group in Caco-2 cells displaced in low concentrations with increased significantly, but the high concentration reduced compared with the blank group. In addition, the amount of Rh123 on the positive control group of verapamil in Caco-2 cells displaced with the increasing of concentration significantly.Conclusions:1. Low concentration of menthol can promote the intestinal absorption of tanshinol, and high concentration of menthol of tanshinol intestinal absorption have inhibitory effect;2. Menthol with low concentration can enhance the accumulation of Caco-2 cells on the P-gp substrate and reduce the efflux. High concentration has little significant influence.3. The effects of menthol tanshinol intestinal absorption mechanism and P-gp expression is related to low concentration of menthol could inhibit the expression of P-gp in Caco-2 cells, and high concentrations of menthol may promote the expression of P-gp in Caco-2 cells.