Compatibility of ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) and their protective effects on cerebral ischemia‑reperfusion injury in rats

Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) are two drugs commonly prescribed together, which are often used in the treatment of cerebrovascular diseases in China. Due to the complexity of the ingredients of Danhong, the present study focused on performing the orthogonal compatibility method on the primary effective molecules of this drug: Tanshinol, salvianolic acid A, salvianolic acid B and hydroxysafflor yellow A. These four molecules were studied to determine their protective effects and to screen for the most compatible ingredients to improve cerebral ischemia-reperfusion injury (IR) in rats. Focal middle cerebral artery occlusion was performed to establish the cerebral IR model in rats. Male Sprague-Dawley rats were randomly divided into sham operation group, IR group and nine orthogonal administration groups with different ratios of Danhong effective ingredients and Danhong injection group. Neurological deficit score and cerebral infarction volume were measured postoperatively. Morphological pathological alterations were observed via H&E staining. Bcl-2 and Bax were quantified using ELISA. Immunohistochemistry was conducted to analyze the expression of caspase-3 in the hippocampus. The expression levels of cytochrome c, apoptotic peptidase activating factor 1 (apaf-1), caspase-9, caspase-3 and p53 mRNA in the hippocampus were assessed via reverse transcription-quantitative PCR. The results demonstrated that different compatibility groups significantly reduced the neurological function score and decreased the volume of cerebral infarct compared with the IR group. These groups were also indicated to improve the pathological damage to the brain tissue. In addition, certain compatibility groups significantly decreased the number of caspase-3 positive cells in the hippocampus and the expression levels of cytochrome c, apaf-1, caspase-9, caspase-3 and p53 mRNA in the brain tissue. Orthogonal group 4 (30 mg/kg tanshinol; 2.5 mg/kg salvianolic acid A; 16 mg/kg salvianolic acid B; 8 mg/kg hydroxysafflor yellow A) was indicated to be the most effective. The four effective ingredients of Danhong exhibited a protective effect on rats with cerebral IR injury, potentially through the inhibition of apoptosis via the downregulation of key targets upstream of the caspase-3 pathway. In addition, the present study provided novel insights for the continued study of the drug compatibility rules of TCM.