| Baicalein is one of the effective ingredients of Chinese herbs Scutellaria baicalensis Gerogi,it can antibiosis,antiviral,anticancer and other pharmacological actions.Because of slight solubility in water and easy metalolism,its orally bioavailability is very low,and so there is no pharmaceutical product in the market.Objective:To establish UV and HPLC methods for the determination of baicalein and its related substances.To inspect the properties of baicalein and the restrict key factors to develop it for injection.A new phytosome technique was studied to improve the solubility of baicalein and overcome the restriction.To prepare a safe,stable and efficacious submicron-emulsion of baicalein.Method:①Preformulation study A UV spectrophotometric method was utilized to study the solubility of drug.An HPLC method was used to determine the stability of baicalein in different pHs solution.②Phytosome study A reasonable criterion of accurate evaluation was established by which such factors as solvent,time,temperature, the ratio of baicalein to lecithin and the concentration of reactant was examined. Investigate the phase state and spectral characteristics of phytosome with UV,IR,DSC, XRD,31P-NMR.The solubility of baicalein in phytosome was determined.③Baicalein submicron-emulsion study Baicalein with phytosome form was dissolved in oil,then shape into a submicron-emulsion by high pressure homogenizer.The properties of emulsion of emulsion of mean particle size,zeta potential,the stability of centrifugation and autoclaving,were evaluated to investigate the influence of the emulsifier,stabilizer, preparation parameters and so on on the stabilization of emulsion.The optimized formulation and preparation process were studied by orthogonal experimental design.④Pharmacokinetics study A simple HPLC method was developed for the simultaneous determination of baicalein and its major metabolite(baicalin) in rat plasma.Comparison of the pharmacokinetics between submicron-emulsion and solution was performed in rats.⑤Pharmacodynamics study Viral pneumonia model established by influenza A1 virus FM1(H1N1)in mice.The mice were randomized into five groups:controlled model group,baicalein emulsion high dose group,baicalein emulsion middle dose group,baicalein emulsion low dose group,ribavirin group.Mortality and pulmonary-index were observed after injection of baicalein emulsion.Results:①Preformulation study The solubility of baicalein in water was 17.5μg/mL,in soybean oil less than 0.3 mg/mL.Along with the rise of pH values,the solubility increased.But it's easy to degrade at pH＞7.0.②Phytosome study The optimized preparations for baicalein phytosome were obtained as follows:solvent was Tetrahydrofuran, the ratio of baicalein to lecithin was 1:3.5(w/w),reaction time was 0.5h,temperature was ambient,the concentration of reactant was 60mg/mL.The results showed that the binding ratio of baicalein and lecithin was 101.1%.The solubility of baicalein phytosome in water was 142.3μg/mL,in soybean oil more than 30mg/mL.The results of UV,IR,DSC, XRD,31P-NMR suggest the formation of phytosome with non-covalent.③Baicalein submicron-emulsion study After the single factor screening experiment,we determined lecithin/F68(3:5) as the emulsifier.The pressure and cycle of homogenization had a noticeable influence on the emulsion diameter.The optimization preparation conditions obtained as follow:the homogenization pressure was 800Bar,the cycle of homogenization was 5,the amount of emulsifier was 3.2%,and homogenized at ambient temperature.The emulsion was saturated with nitrogen gas and sterilized by autoclaving(121℃,15min).The particle size of emulsion was 139 nm.The zeta potential was -22.40mv.the pH was 5.83. The emulsion is stable after storage for six month at the room temperature and 4℃.④Pharmacokinetics study Under the assay condition,no interference from the endogenous plasma was observed at the retention time of both the analytes and the internal standard.Baicalein and baicalin showed a good linear relationship at the range of 0.06 to 48.0μg/mL,with accuracy and precision conformed with the requirement of biological specimen analysis.The LOQ was 0.02μg/mL for both baicalein and baicalin.The method recoveries of these drugs varied from 95.47%to 105.27%and the extraction recoveries were all higher than 69%.The pharmacokinetics results demonstrated that both baicalein in emulsion and its free form were quickly metabolized to baicalin in vivo.The disposition of baicalein in rats was conformed to a two-compartment model.The main pharmacokinetic parameters of emulsion group and control group were t1/2β 16.30min and 10.88min,AUC 90.51(μg/mL)·min and 62.57(μg/mL)·min,respectively.Compared to baicalein,the elimination of its major motabolite(baicalin) was relative slow,which could still be detected 10h post-dosing.⑤Pharmacodynamics study Compare with the controlled group,pulmonary-index and mortality decreased significantly by baicalein emulsion at the dose of 10mg/kg(P＜0.05),20mg/mL(P＜0.01) and 30mg/mL(P＜0.01).Conclusion:By studing on phytosome,the solubility of baicalein was improved markedly in soybean oil.Based on this,submicron-emulsion was prepared.The baicalein submicron-emulsion made by optimum formulation and preparative technique was fitted to be used for intravenous administration.Comparison of metabolic pharmacokinetics of baicalein emulsion and its solution in rats indicated that baicalein emulsion made the blood drug concentration for longer time and had higher bioavailability.Baicalein emulsion administratation by iv.has antiviral activity against viral pneumonia in mice and have a possibility used for clinic. |
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