Experimental Study On Toxicology And Efficacy Evaluation Of Detoxicated Aconitum Carmichaelii Debx. Dispensing Granules

Objective Detoxicated Aconitum carmichaelii Debx.Dispensing Granules was prepared by standardized process,and the quality control was completed by determining the content of main alkaloids by HPLC.The safety of detoxicated Aconitum carmichaelii Debx.Dispensing Granules was evaluated by acute toxicity test of mouse model,and whether detoxicated Aconitum carmichaelii Debx.Dispensing Granules had pharmacodynamic effect on cartilage injury of osteoarthritis was discussed and demonstrated from the whole animal model.Methods 1.The preparation and process evaluation of detoxicated Aconitum carmichaelii Debx.:Divide the decoction pieces into four groups,add the same amount of water to decoct twice in each group,and the decoction time is 1 h,2 h,4 h,6 h respectively,combine the two decoctions,and filter.The components of water decoction were detected at 1 h,2 h,4 h and 6 h,and the contents of six toxic alkaloids in B decoction at different decocting time were determined by HPLC.2.The preparation and the analysis of toxic components of detoxicated Aconitum carmichaelii Debx.Dispensing Granules:Take the decoction pieces,add water to decoct twice,the first time 2 hours,the second time 2 hours,combine the two decoctions,and filter,concentrate the filtrate to a clear paste with relative density of 1.06～1.14(65℃),add proper amount of auxiliary materials,mix evenly,dry and granulate to obtain the finished product.The content of six toxic alkaloids in the aqueous solution was determined by HPLC after the detoxicated Aconitum carmichaelii Debx.Dispensing Granules was dissolved in water.3.The acute toxicity evaluation of detoxicated Aconitum carmichaelii Debx.Dispensing Granules:Take 40 mice and divide them randomly into 5 groups(blank control group,detoxicated Aconitum carmichaelii Debx.Dispensing Granules group,group decocted with water for 0.5h,group decocted with water for 1h,group decocted with water for 2h),and then the mice in each group were given intragastric administration.The effects of Aconitum carmichaelii Debx.at different decocting time and preparation of granules on toxicity in mice were compared by observing daily,behavioral and various indexes.4.The efficacy evaluation of detoxicated Aconitum carmichaelii Debx.Dispensing Granules:In vivo experiment:30 rats were randomly divided into 3 groups(blank control group,kOA model control group and detoxicated Aconitum carmichaelii Debx.Dispensing Granules group).After the same time of intragastric administration,the curative effect of detoxicated Aconitum carmichaelii Debx.Dispensing Granules on rat knee joint was evaluated by analyzing the pain behavior index,histopathology and immunohistochemistry.In vitro test:Healthy rat serum and knee joint specimens were taken for primary culture of chondrocytes,and then subculture was grouped into models.The effect of drug-containing serum on chondrocyte proliferation was determined by CCK-8 method.Results 1.After Aconitum carmichaelii Debx.was decocted for more than 2 hours,the content of aconitine and mesaconitine was almost undetectable,and the content of benzoyl hypacoitine,benzoyl mesacoitine and benzoyl acoitine increased at first and then decreased,and reached the peak level at 2 and 4 hours.Compared with the group decocted for 2 hours,the content of benzoyl hypacoitine in detoxicated Aconitum carmichaelii D ebx.Dispensing Granules group was lower,while the benzoyl mesacoitine content was significantly higher than that in the group decocted for 2 hours.The content of benzoyl acoitine was similar to that in the group decocted for 2 hours,and hypacoitine and mesacoitine was lower than that group,but acoitine was undetectable.2.The results of acute toxicity test in mice showed that both detoxicated Aconitum carmichaelii Debx.Dispensing Granules group and groups decocted in water for more than 2 hours had no acute toxicity to mice.According to the results of body mass changes in mice,the toxicity of detoxicated Aconitum carmichaelii Debx.Dispensing Granules group and group decocted for 2h was the lowest;According to the serum biochemical results of mice,the toxicity of group decocted for 0.5h was the highest,and the serum biochemical indexes of mice in group detoxicated Aconitum carmichaelii Debx.Dispensing Granules group and group decocted for 2h were similar,which was closest to the mice in the normal control group.3.In vivo experiments showed that detoxicated Aconitum carmichaelii Debx.Dispensing Granules can obviously improve the pain condition of kOA model rats,and repair the defect and morphological destruction of knee chondrocytes in kOA model rats.In detoxicated Aconitum carmichaelii Debx.Dispensing Granules group,the defect of chondrocytes of rats was improved obviously,and a large number of normal chondrocytes could be seen.Compared with rats in kOA model group,the hypertrophic degeneration of chondrocytes of rats was improved,but there was still a tendency of hypertrophy,and the cartilage matrix gradually returned to normal.By comparing the results of MMP-13 immunohistochemical staining in the knee tissue sections of rats in each group,it was found that the positive expression rate of MMP13 was about 22.40%,suggesting that the synovitis was improved.The results of in vitro experiments suggested that after 24 hours of administration of detoxicated Aconitum carmichaelii Debx.Dispensing Granules-containing serum with different concentrations,the growth of rat chondrocytes can be obviously promoted,and with the increase of the concentration of drug-containing serum,the effect on cell proliferation was more obvious,and the effect of 20%drug-containing serum was the most significant.Conclusion Detoxicated Aconitum carmichaelii Debx.Dispensing Granules retained the effective components and achieved the effect of attenuation.There was no aconitine component in detoxicated Aconitum carmichaelii Debx.Dispensing Granules aqueous solution by HPLC,and there was no acute toxicity;In the aspect of drug effect,for osteoarthritis rats,in vivo and in vitro experiments proved that detoxicated Aconitum carmichaelii Debx.Dispensing Granules had obvious protective effect on cartilage.