Effect And Primary Basis Of MHE On The Vivo Behavior Of P-gp Substrate-Digoxin

Objective:The micromolecular hydrophilic extract from vinegar baked radix bupleuri(MHE), is the residue of vinegar Bupleurum water extract which the polysaccharides and n-butanol extraction were removed. The early experiment have proven that it was the effective part of soothing the liver and relieving depression of VBRB, and it could inhibit gene and protein expression of P-glycoprotein and increase the uptake of substrate on Pgp-HEK293 cell. Thus, in this paper, the effect and primary basis of MHE on the vivo behavior of P-glycoprotein substrate-digoxin were studied. Methods:1. Pgp resistance cell line MES-SA/MX2 was used to evaluate the reversal effect of MHE on doxorubicin. The toxicity effects of MHE, verapamil on MES-SA and MES-SA/MX2 cells were determined by MTT method to obtain the safe dose range. The anti-multiresistance effect of MHE were determined by comparing toxic effect of doxorubicin using along or combined with MHE on MES-SA/MX2 cell line. Cellular concenrtration of Rh123 were used to evaluate the effect of MHE on the uptake activity of Pgp.2. Effect of MHE on Pgp in vivo was studied by observing pharmacokinetics change of P-gp substrate digoxin. Firstly, an LC-MS method was established to determine the concentration of digoxin in rats’ plasma which was collected in determined time points after orally administered with digoxin or MHE combined with digoxin. Thereafter the difference of pharmacokinetic parameters of digoxin alone or combined with MHE was compared. Secondly, the concentration of digoxin in rats’ tissues which were collected after being administrated with drugs was determined, and the effect of MHE on thedigoxin of tissues was compared.3. The mechanism of MHE effect in vivo of digoxin was studied through investigation of the transporter expression on tissues. Real time fluorescence quantitative PCR was used to determine the relative expression of m RNA of the P-glycoprotein and orangic anion transporters peptides 2(Oatp2) in 5 min, 15 min, 3 h of the digoxin single-dose group and digoxin combined with MHE group of liver, kidney, small intestine, heart.4. Components of MHE were analyzed. The carbohydrate and the free amino acid of MHE were derivatized by three methyl silane and AQC respectively, and detected by the GC-MS and the HPLC-UV respectively. Result:1. The safe dose of MHE and verapamil were under 50 mg/m L and 10 μM respectively; the reversal fold of MHE at 1 mg/m L, 10 mg/m L concentration and 1μM Verapamil on MES-SA/MX2 cells were 0.23, 0.42, 9.49 respectively. Compared with the control group, the relative exposure rate of Rh123 of different doses of MHE showed no significant difference.2. The linear range of digoxin was 0.156 ~50 ng/m L, the extraction recovery rate was 70.19~98.99%, the matrix effect was among 96.08~114.2%, the accuracy was among 85.7~114.3%, the intra and inter day precision RSD was less than 15%, the stability of RSD was less than 15%. Compared with the monotherapy group, AUC(0-t), AUC(0-∞), MRT(0-t) of the MHE combined with low-dose of MHE were reduced by 31.54%,24.92 %,24.42%(P<0.05)respectively, meanwhile CLz / F increased by 29.60%, while at high-dose combination group decreased by 42.98%(P<0.01),36.31%(P<0.01),21.81%(P<0.05), CLz / F increased by 59.02%(P <0.05). MHE changed the distribution of digoxin in vivo, and increased digoxin concentraion in liver, while reducing it in the small intestine and plasma, but had marginal effect on heart. The relative uptake rate(RUE) of digoxin in liver increased by 14.1%, and the RUE of digoxin in small intestine and plasma reduced by 58.1% and 36.5%, and RUE of the heart and kidney kept almost unchang; the RTE of digoxin in liver, kidney, heart were 76.9%, 57.1%, 52.6% respectively, while the RTE in small intestine was-35.0%.3. Compared with the control group, the expression of liver Pgp m RNA of the combination group was decreased significantly in 5 min( P <0.05); and the expression of m RNA of Oatp2 in 3 h were rising(P <0.05). Pgp m RNA expression of kidney in 5min and 15 min decreased significantly(P<0.05); Oatp2 m RNA at 3h was descending significantly. Intestinal Pgp m RNA significantly increased at 3 h(P <0.05). The expression of Pgp and Oatp2 of remaining organs in other time points were not significant differences.4. The components of MHE: five kinds of oligosaccharide: sucrose, maltose, lactose, mannose disaccharide, gentiobiose; 10 kinds of monosaccharide(including sugar alcohols and aldehydes acid): xylitol, sorbitol, D-glucose, D-mannose, D-glucuronic acid, Dfructose, D-mannitol, sedoheptulose, D-arabinopyranose, D-ribose; 5 kinds of organic acids: succinic acid, acetic acid, malonic acid, succinic acid, fumaric acid; the rest: cetyl alcohol, inositol, cis-13-oleic acid, 11-cis-octadecenoic acid, trans-9-octadecenoic acid, stearic acid, and the 18 kinds of free amino acids which made of protein and γ-aminobutyric acid were found. Conclusion:Although MHE couldn’t reverse the MDR of doxorubicin in MES-SA/MX2 caused by the P-glycoprotein in virto, it did affect the pharmacokinetics and distribution of the digoxin which is the substrate of the P-gp significantly in vivo, and the reason partly related with the Pgp activity change induced by MHE, which indicated that we should take more attention on the efficacy and toxicity of the combination between the vinegar baked radix bupleuri or MHE and the substrate of the P-gp in clinic. MHE contained a variety of monosaccharides, oligosaccharides, amino acids, however, the effective component remains to be further explored.