Degradation Kinetics And Pharmacokinetics Of Baicalin Magnesium Salt And Its Inhibitory Effect On HepG2 Cells

In the early stage of our team,the extraction process of Baicalin was investigated for the poor water solubility and low bioavailability of Baicalin.It was found that Baicalin was present in the form of its magnesium saltin the scutellaria.The water solubility,bacteriostatic effect and hepatoprotective effect of Baicalin magnesium salt were studied.The results showed that the solubility of Baicalin magnesium salt in water was 129.1 mg·ml^-1,which was2225 times higher than that of Baicalin,and its antibacterial effect and hepatoprotective effect were significantly better than Baicalin.However,there was no significant difference in pharmacokinetic parameters between Baicalin magnesium salt and Baicalin during oral administration,suggesting that Baicalin magnesium salt was converted to Baicalin under the action of gastric acid.At the same time,due to high solubility and efficacy results,It is more suitable for the preparation of injection for the magnesium salt of Baicalin.In view of the factors that may affect the stability of the injection,this experiment investigated the stability of Baicalin magnesium salt monomer under different p H,temperature,ionic strength and compatibility with common infusion solutions;In addition,the pharmacokinetic characteristics of Baicalin magnesium salt injection administration were studied and compared with Baicalin.On the basis of the known protective effect of Baicalin magnesium salt,the effect of Baicalin magnesium salt on apoptosis of human hepatoma Hep G2 cells was further investigated.Provide reference for the research and development of the preparation of Baicalin magnesium salt.Objectives:1. Establish an accurate and reliable detection method to study the degradation of Baicalin magnesium salt under different p H,temperature and ionic strength conditions,and to investigate the compatibility of Baicalin magnesium salt and clinical common infusion agent.2.To study the pharmacokinetic characteristics of Baicalin magnesium salt injected into the tail vein of rats,and to compare the pharmacokinetic parameters of Baicalin magnesium salt and Baicalin.3.To investigate the effect of Baicalin magnesium on the apoptosis of human hepatoma Hep G2 cells and the expression of related proteins,and provide reference for the further development of Baicalin magnesium salt.Methods:1. Investigate the change of the content of Baicalin magnesium salt under different p H,temperature and ionic strength with time by HPLC.Forecast its validity(t_0.9).At the same time,Study the compatibility of Baicalin magnesium with common infusion solutions?0.9%sodium chloride injection,5%glucose injection,10%glucose injection?.2. After the injection of Baicalin magnesium and Baicalin in the tail vein of rats,take the blood from the eyelids at different time points. Determine the drug concentration at each time point by HPLC,and Draw drug concentration versus time?C-t?curve.Calculate the pharmacokinetic parameters by DAS 3.0software.Perform statistical analysis using SPSS 19.0 software.3.In vitro cell culture,determine the inhibition of the growth of Hep G2cell by MTT assay.The effect of Baicalin magnesium on the expression of apoptosis-related proteins Bcl-2,Caspase-3 and Caspase-9 was detected by enzyme-linked immunosorbent assay kit.Results:1.The degradation reaction of Baicalin magnesium salt under different temperature and acid-base conditions follows the first-order kinetics.It is unstable in a peracid or overbased environment,and is most stable in a solution environment with a p H of 6 at this time t_0.9=103.33 h,The damage of Baicalin magnesium salt was obvious at high temperature,and the degradation rate of Baicalin magnesium salt was accelerated significantly in the environment above 70?.Na⁺and Cl^-had no significant effect on the degradation of Baicalin magnesium salt.The color of the Baicalin magnesium salt and the three infusion agents did not change,no bubbles were generated,no insoluble impurities were precipitated,and the p H value and the content were slightly decreased,but the general quality requirements of the preparation were met.2. When the dosage of Baicalin magnesium salt was 25,50 and 100mg·kg^-1,there was a good linear relationship?r=0. 95?between the AUC_0-tand the dose?r=0.95?,and there was no significant difference in other pharmacokinetic parameters among the dose groups.The mean residence time(MRT_0-t)of Baicalin magnesium salt group was significantly higher than that of Baicalin group?P<0.05?.AUC_0-t,AUC_0-?,elimination half-life(t_1/2z),apparent distribution volume?V_z?were all increased,but the difference parameters were not statistically significant.3. Baicalin magnesium salt can induce apoptosis of human hepatoma Hep G2 cells. When the doses are 50,100 and 200?g·m L^-1,he inhibitory effect of Baicalin magnesium salt on human hepatoma Hep G2 cells showed dose-effect,time-effect relationship.When the concentration of Baicalin magnesium salt was 50?g·m L^-1for 48 h,the inhibitory rate on human hepatoma Hep G2 cells was 50.78%,close to 50%inhibition concentration(IC₅₀).With the increase of drug concentration,the expression of Bcl-2 was decreased,and the expression of Caspase-9 and Caspase-3 protein was increased,and the difference was statistically significant?P<0.05 or P<0.01?.Conclusions:1.The stability of Baicalin magnesium salt is greatly affected by p H and temperature,and the p H and temperature should be paid attention to during preparation.The compatibility of Baicalin magnesium salt monomer with three clinical commonly used infusion agents is good,which provides a basis for the subsequent development of Baicalin magnesium salt injection.2.The pharmacokinetic characteristic of Baicalin magnesium salt and Baicalin was similar,but the average retention time of Baicalin magnesium salt in vivo was longer than that of Baicalin.The reason is further explored.3. Baicalin magnesium salt can induce apoptosis of human hepatoma hepg2 cells. Its mechanism may be related to down-regulation of bcl-2expression,improvement of mitochondrial membrane permeability,elevation of caspase-9 protein expression level,and promotion of caspase-3 protein expression and activation...