Bioavailability Of Levistolide A And Its Influence On Aβ Clearance

BackgroundAlzheimer’s disease (AD) is a common progressively Aggravated neurodegenerative disease, and can result in be death due to complications. The incidence increases with age, the prevalence rate increases with the coming of the aging society gradually. AD will become a major health problem in the medical community and the publicconcern, and there is no ideal drug treatment, and traditional Chinese medicine correlational researches are becoming aresearch hotspots because of the therapeutic mechanism of their multi way and multi targets.ObjectiveExplored the effects of LA on the expression of p-amyloid protein (Ap) from M146L cell line, providing the reference for the research of anti AD. Otherwise, through discussing the determination of Danggui-Shaoyao-San alcohol extraction, Chuanxiong alcohol extraction and an effective component of Ligusticum chuanxiong levistolide A(LA) and its pharmacokinetic process the determination of content in plasma and brain of SD rat, to analize how to improve the bioavailability of LA a method for finding more effective means to the prolong absorption.Methods1. Pharmacodynamics experiment(1) Determination of different doses of LA on cell viability of M146L cell line (Cell Counting Kit-8, CCK-8):Detected cell survival rate, made clear that the best medicine non-toxic concentration and action time.(2) Detected the effect of LA on Aβ1-42 generation on M146L cell line by ELISA.(3) Detected the effect of LA with different doses on expression of total Aβ1-42 of M146L cell line.2. Pharmacokinetics experiment(1) The determination of Danggui-Shaoyao-San aocohol extraction and Chuanxiong alcohol extractionPreparaed Danggui-Shaoyao-San alcohol extraction and Chuanxiong alcohol extraction, respectively, established and validated a HPLC-UV method for determination of LA content, including verification of linearity, accuracy, repeatability, detection of limit, quantification and stability.(2) A comparative pharmacokinetics study of LA and the alcohol extract of chuanxiong and Danggui-Shaoyao-Sanâ‘ Establish a method of determination of drug concentration in rats plasma of LA by LC-MS/MS.About 32 300g male SD rats were randomly divided into 4 groups:single gavage group, herbs gavage group, compound gavage group (dose of 20mg/kg) and a single static injection group(dosage of 2mg/kg), serial blood samples (500μL)were collected via the postorbital vein plex from each rat at the corresponding time point, detected the serum content by LC-MS/MS, drawing time-drug concentration curve, pharmacokinetic parameters were calculated by using NONMEM Program version 1.1 and were compared between each group.â‘¡Establish a method of determination of drug concentration in rats brain of LA by LC-MS/MS.The 24 male SD rats weight 300g were randomly divided into into 4 groups, 6 rats in each group with intravenous injection of LA 2mg/kg, after administration of 0.03,0.17,0.5,4h rats. Determinated the content of brain by LC-MS/MS, rendered time-drug concentration curve, dynamic parameters using NONMEM Program version 1.1 software were calculated by using NONMEM Program version 1. land then compared.Results1. Pharmacodynamics experiment(1) The concentration of LA solution was at 20 mol/L and had effects on cell activity, the concentration of 15,10,5,1,0 mmol/L(final concentration of DMSO was 0.08%) had no effect on cell viability and had no cell toxicity.(2) ELISA experimental results showed that after using different doses of LA to meddle on M146L cells 24 hours, the dose of 15 mM, lOmM can reduce the content of extracellular Ap1-42(P<0.05).(3) WB results showed that concentration of 15 mM,10 mM can down regulate the expression of intracellular Api-42(P<0.05).2. Pharmacokinetics experiment(1) After verification methodology, the HPLOUV method for determination of of LA in Danggui-Shaoyao-San alcohol extraction and Chuanxiong alcohol extraction content in accord with the methodology requirements for the determination of drug content in vitro. After the test, content in LA extraction of Danggui-Shaoyao-San alcohol and Chuanxiong alcohol were respectively 0.119% and 3.2%.(2) After verification methodology, the HPLC-UV method for determination of of LA in Danggui-Shaoyao-San alcohol extraction and Chuanxiong alcohol extraction in rat plasma content in accord with the methodology requirements for the determination of drug content in vitro. Plasma concentrations of LA were measured and analyzed that LA in rat pharmacokinetic process was two compartment model. The AUC0â†'∞ and t1/2 values of DSS group were larger than the other group (P<0.05), the bioavailability of LA was 7.6%.(3) After verification methodology, the HPLC-UV method for determination of of LA in Danggui-Shaoyao-San alcohol extraction and Chuanxiong alcohol extraction in rat brain content in accord with the methodology requirements for the determination of drug content in vitro. Plasma concentrations of LA were measured and analyzed that LA can penetrate the blood brain barrier, at 30minutes after administration came to a maximum concentration (about 43ng/mL).Conelusion1. LA can penetrate the blood brain barrier, which played the scavenging effect on Aβ1-42, which may be related to the mechanism for up regulation of P-gp and the efflux of Aβ1-42, thereby reducing its accumulation in the brain, that might have certain value and significance on related research of treatment for AD.2. This study developed and validated a rapid, simple, repeatable, and stablly viable method of LA determination by LC-MS/MS, which was applied in pharmacokinetic study in rats.3. Comparing between LA monomer and the alcohol extract of chuanxiong, Danggui-Shaoyao-San can significantly increase the bioavailability of LA, which has ease of administration and low risk, high degree of absorption and longer duration of action.