Safflower Injection Bioassay Method

Objective:Choose the methods to measure the biological activity of safflower injection, then optimize experimental conditions, identify its main active components and toxic substances, establish the dosage limits and quality criteria, and explore the mechanism of the main active ingredients.Methods:(1) Choose 1 or 2 appropriate methods from 5 relevant pharmacodynamic methods to test the biological activity of safflower injections which were from 4 different manufactuers. (2) Optimize the experimental conditions by using orthogonal test, then establish the dosage limits and quality criteria of the safflower injection under the optimized condition, which were validated through testing the bioactivities of safflower injections. (3) The active components of safflower injections were identified by measuring the bioactivities of the qulified safflower injections which contained different contents of HSYA A (HSYA) and total flavonoids. (4) Mice were treated with safflower injections (0.5ml, i.v.) which contained different contents of .HSYA and K~+ . Record the death and survival of mice within 48h after intravenous injection and identify the toxic substances. (5) Measure the changes of SOD, MDA in left ventricular myocardial in mice acute myocardial ischemia induced by pituitary (Pit, i.p.). The TTC staining of myocardial tissue was also observed.Result:(1) Mice thrombosis test in vivo and rabbit platelet aggregation test in vitro were consistent with the requirements of safflower injection biological activity measurement. (2) The optimal experimental conditions of thrombosis test was that safflower injection was injected continuously for 3 days with the concentration of safflower injection 3.34g crude drug / kg as the limit of its biological activity test dosage, and thrombosis inducer (concentration ratio of collagen is 0.6mg/ml to adrenaline 0.02mg/ml) was injected after 30 minutes of the last injection of safflower injection. The protective rate of anti-thrombus was≥50%, as well there were significant differences compared with the negative control group, which were defined as the quality criteria of mice thrombosis test. The optimal experimental conditions of rabbit platelet aggregation test is that 200μl rabbit platelet-rich plasma (PRP) was taken with the concentration of safflower injection 5.01g crude drug / kg as the limit of its biological activity test dosage, 50μl ADP with 50μg/ml was added. When the rabbit platelet aggregation inhibition was≥25%, and there were significant differences compared with the negative control group, which was defined as quality criteria of rabbit platelet aggregation test . According to the research on 6 batches from 6 manufacturers of safflower injections on the biological activity assay, the pass rate was 83.3%. (3) With the same content of total flavonoid, the protective effects were decreased in thrombus formation in vivo and platelet aggregation rate in vitro with the content of HSYA reduced; while with the same content of HSYA, the protection effects were declined in thrombus formation in vivo and the rabbit platelet aggregation rate in vitro with the content of total flavonoid reduced. When the contents of HSYA and total flavonoid in safflower injection were both lower than the other one, then the protective effects were also lower. Therefore, the results demonstrated that HSYA and total flavonoid were both the cardinal active ingredients of safflower injection, and its bioactivity was positive relative with the contents of active ingredients, which had synergistic effect. (4) The mice injected with the safflower injections were observed for 48 hours, and the number of death was recorded. It found that when K~+ was lower than 1600 mg/ml, there was no death in mice. However, when K~+ is greater than 1600 mg/ml, the contant of HSYA in safflower injection was between 0.03mg/ml and 0.82mg/ml, there were deaths in each group. With the procedure of removing K~+, there were no deaths in mice. (5) HSYA increased the activity of SOD and decreased content of MDA in Pit treated rats, results showed a dosage-relationship between the dose of HSYA and the effect of antioxidation, there were significant differences between HSYA treatment group and the model group. By staining of TTC, infarct area in HSYA group was less than that of model group, with also a dosage relationship.Conclusion: Mice thrombosis test and rabbits anti-platelet aggregation test.were chosen as biological activity measurement methods The limit dosage of two bioassay methods were determined: 3.34g crude drug / kg in thrombosis test in vivo and 5.01g crude drug / kg in anti-platelet aggregation in vitro. The bioassay criteria of safflower injection was determined. HSYA and flavonoids were proven as the main active substances of safflower injection. The primary toxic substance which caused the deaths in mice was K~+. The preliminarily research suggested that the mechanism of the main active substances of safflower injection was related to the effect of antioxidation.