Study on Quality Control of Danhong Injection and Study on Pharmacodynamic of Brain Based Microdialysis
|School||Zhejiang University of Traditional Chinese Medicine|
|Keywords||Danhong injection HPLC-DAD Total flavonoids Chinese herbalcompound Microdialysis cerebra Isehemia amino acid transmitter|
Objective:To develop a HPLC-DAD method for the simultaneous quantitation of Salvianic acid A, Protocatechuic aldehyde, Caffeic acid, Rosmarinic acid, Salvianolic acid B and Salvianolic acid A in Danhong injection; and determine the content of total flavonoids in Danhong injections with ultraviolet-visible spectrophotometer(UV-VIS). To develop a HPLC-DAD method for the simultaneous quantitation of three effective constituents (Salvianic acid A, Hydroxysafflor yellow A and Rosmarinic acid) in Danhong extract; and taking Danhong as a model drug to explore the feasibility of microdialysis technique to be used in pharmacokinetic study of Chinese herbal compound.To develop a HPLC method for the simultaneous quantitation of three amino acid transmitter(Asp,Glu and GABA) in hippocampus. And determine the content of three amino acid in hippocampus.Methods:(1)Samples were analyzed on an Agilent Eclipse XDB-C18column (4.6x150mm,5μm), and eluted with acetonitrile and water containing0.1%phosphate acid as mobile phase. The flow rate was kept at1.0mL·min-1, and the column temperature was set to30℃. The DAD detector wavelengths were280nm and330nm. Using rutin as control product, the content of total flavonoids in Danhong injection was determined by UV-VIS.(2)The samples were obtained by microdialysis method, determined by HPLC-DAD gradient elution to calculate the in vitro recovery rate (RR) of specific components. To study the difference of RR of Salvianic acid A, Hydroxysafflor yellow A and Rosmarinic acid in dialysis mediums of Salvia extract, Safflower extract and Danhong extract, and the effect of flow rates and concentration on RR.(3)Male SD rats were subjected to60minoftransient middlecere bralartery occlusion(MCAO) followed by reperfusion, Danhong injection(2mL) was injection in tail vein10min before MCAO. Then determine the content of three amino acid in hippocampus by HPLC.Results:(1)The good separation of six constituents was achieved. It showed good linear relationship (R>0.999,n=6), and the average recoveries of the six constituents were98.98%,100.86%,100.44,98.73%,101.35%,100.17%. Per milliliter Danhong injection of each batch contains six constituents over11.5mg totally; Per milliliter Danhong injection of each batch contains over9.0mg total flavonoids.(2)The good separation of Salvianic acid A, Hydroxysafflor yellow A and Rosmarinic acid was achieved within30min, and the three constituents showed good linear relationship (R>0.999, n=6). When the flow rate was1.0μL·min-1, the RR of Salvianic acid A sodium, Hydroxysafflor yellow A and Rosmarinic acid in the mixed standard solution were11.05%,5.81%,6.73%, and along with the increase of number of substances in the dialysis medium, the RR of specific components showed a downward trend. The RR reduced with the increasing flow rate in the same dialysis medium, but the RR was independent of the concentration of the component in the dialysis medium.(3) MACO resulted in a rapid increase in the striatum dialysate levels of three amino acids(30min peak), and then gradually decline. Compared with the model group after tail vein injection, Asp, Glu increased amplitude decreased,GABA increased the rate of increase.Conclusion:(1)The method was proved to be accurate, sensitive, repeatable, and can be used as evidence for the quality control of Danhong injection.(2)The microdialysis technique can be used in pharmacokinetics study of Chinese herbal compound. The developing of HPLC-DAD method in the study can be used as the method for determination of the RR of Danhong in the study of microdialysis. The factors influencing the RR should be considered in the microdialysis experiment, and choosing the appropriate conditions to make a convenient detecting.(3)Treatmentwith Danhong injection significantly depressed ischemia induced efflux of amino acids.