In vivo pharmacokinetic study on active constituents in Danhong Injection against cerebral ischemia of rats / 中草药

ABSTRACT

Objective: To develop an HPLC-DAD method for the simultaneous determination of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and hydroxysafflor yellow A (HYSA) in cerebral ischemic injury of rats, in order to provide an accurate and reliable analytical method to study the pharmacokinetics. Methods: The middle cerebral artery occlusion (MCAO) model was established, in which all the rats were iv injected with the active constituents in Danhong Injection simultaneously (danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA). The analysis of the measured components' plasma concentration was carried at 30℃ on the reversed-phase column of Agilent Eclipse XDB-C18 (150 mm×4.6 mm, 5 μm) and eluted with acetonitrile and water containing 0.4% phosphate acid as mobile phase in gradient mode. Detection wavelengths were 280 nm for danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and 403 nm for HYSA. The propyl p-hydroxybenzoate was used as the internal standard (IS). Results: Protocatechuic aldehyde rapidly metabolized in MCAO rats, but its metabolites, protocatechuic acid and vanillic acid, were easily detected. The linear ranges of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and HYSA were 0.35-140 (R2=0.9998), 0.15-60 (R2=0.9990), 0.05-20 (R2=0.9988), 0.25-100 (R2=0.9996), and 0.075-30 mg/L (R2=0.9985), respectively. The mean recoveries were between 80% and 120% and the RSD value of intra-day and inter-day was less than 10%. The results of stability meet the requirements for biopharmaceutical analysis. The main pharmacokinetic parameters of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and HYSA were as follows AUC0~∞ were (1143.862±230.840), (427.024±59.293), (135.785±47.631), (418.631±66.242), (288.788±87.809) mg·min/L and t1/2z were (71.496±29.067), (82.379±26.279), (40.331±6.006), (125.164±59.709), (177.577±112.836) min. Conclusion: The method is simple, rapid, precise and stable, which can be used to the pharmacokinetic studies on the compatibilities of danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA in MCAO rats.