Research Of Xiongbing Microemulsion And The Evaluation System

1 ObjectiveThis research is the sub-item of the project "The Construction and Evaluation of local PK/PD Model's on chinese medicine by Microdialysis Technique" (No.30873443). To study the preparation of Xiongbing microemulsion, the formula is made up of Chuanxiong, ligustrazine, borneol and to establish the quality standards. To observe the body distribution in the mice and the protective effects on cerebral ischemia-reperfusion injury. In order to reflect the advantage of the Chinese herbal compound and development of new dosage form for reference,the research will be done by comparing with Ligustrazine microemulsion.2 Methods2.1 Preparation of Chuanxiong IntermediateTo establish the determination method of FA by HPLC. Comparing the ethanol extraction to steam distillation and using orthogonal experiments, a stable Chuanxiong extraction is prepared.2.2 Preparation of Xiongbing microemulsion(XB ME)The excipients was decided by the concentration of TMP. And the XB ME was prepared by using the methods of titration to prepare the Pseudo-ternary phase diagram. The stability, size, viscosity, distribution of XB ME is tested.2.3 Quality Assessment of Xiongbing Microemulsion Study the size distribution of Xiongbing microemulsion, pH value, viscosity and stability. To establish the TLC and the determination method of the main components of Xiongbing Microemulsion.2.4 Biodistribution of Xiongbing MicroemulsionTo study the distribution of Xiongbing microemulsion in the tissue of mice against the Ligustrazine microemulsion by testing the concentration of TMP in plasma, brain, liver, heart, lung and kidney in one hour.2.5 Pharmacodynamic of Xiongbing microemulsionCerebral ischemia-reperfusion models were established by inserting nylon thread in rats and clamping both common carotid arteries in mice.The cerebral water content and cerebral infarction area after infarction were observed. The level of MDA,SOD,NO in cerebral tissue were tested.3 Results3.1 Preparation of Xiongbing microemulsion(XB ME)Comparing the concentration of FA, the extraction method of ethanol extraction is 2 times the steam distillation. Orthogonal experimental of alcohol extraction shows that 80% ethanol 10 times extracted twice,1.5h for once is the best extraction method. Testing the extraction for three times, the concentration are 866.1,839.5,858.2μg·g-1, average 854.6μg·g-1, RSD=1.60%. The result shows the plan is feasible and reliable. Technology of eliminating tannin and extracting are studied.The extraction of Chuanxiong is made of adjusting pH value of 8, extracting with ethyl acetate in pH 2.3.2 Preparation of Xiongbing microemulsionThe result indicated that the TMP has a strong solubility in Ethyl Oleate, emulsifier and assistant emulsifiers. Microemulsion ternary phase diagram shows that the Ethyl Oleate /EL-35, OP (1:1)/glycerol, anhydrous ethanol (6:1) combination (Km= 1.5:1) has a large dairy region. To research the particle size, viscosity, stability based on the ternary phase diagram. The preparation method of Xiongbing microemulsion:Chuanxiong 500g, TMP 2.5g, borneol 0.5g, Ethyl Oleate 10g, EL-35 18g, OP 18g, glycerol 21g, alcohol 3g, adding distilled water to 1000mL. 3.3 Quality Assessment of Xiongbing MicroemulsionTesting three batches of Xiongbing microemulsion, the particle size are 17.2nm, 18.1nm,17.7nm, RSD 1.76%, the microemulsion made in this process have a uniform size. The microemulsion average pH value is 4.27, RSD= 1.59%,average viscosity is 2.15mpa·s, RSD= 1.87%, and has a good stability. The study of TLC and the content concentration required that the containing of TMP is not less than 2.50mg, ferulic acid is not less than 0.30mg, borneol is not less than 0.45mg in 1mL of Xiongbing microemulsion. The stability of the preparation was studied,and the results showed that the product was stable. However, the content of ferulic acid has a reduction in three months.3.4 Biodistribution of Xiongbing MicroemulsionIn mice, after oral administration of Xiongbing microemulsion and Ligustrazine Microemulsion, the TMP of Xiongbing Microemulsion has a better distribution than the Ligustrazine Microemulsion in blood and other tissues, especially in brain. The concentration TMP of Xiongbing Microemulsion is higher than the Ligustrazine Microemulsion in each tissue in mice, and has the feature of fast absorption,short Tmax.3.5 Pharmacodynamic of Xiongbing microemulsionCompared with the model group, it revealed that in Xiongbing microemulsion-treated,the cerebral water content and the cerebral infarction area after infarction were remarkably reduced, level of MDA and NO in cerebral tissue was markedly reduced, the activities of SOD in cerebral tissue was significantly elevated. It's similiar to control group and better than the Ligustrazine microemulsion.4 ConclusionThe Xiongbing microemulsion is feasible to prepare,convenient in determination and table. It has the benefits of fast absorption, high bioavailability, especially in brain. Pharmacodynamic of Xiongbing microemulsion shows that the action mechanism might be: inhibiting the production of NO, increasing the SOD activity and inhibiting lipoperoxidation.