Study On The Effective Substance And Quality Standard Of Daphne Koreane Nakai Anti-toxoplasma Gondii

Objective:To study the effective substance and quality standard of Daphne koreane Nakai anti-Toxoplasma gondii.Method:The acute toxicity of the total extract of D.koreane in mice was determined by the maximum tolerance method.and the anti Toxoplasma effect of the total extract of D.koreane and the other four extracts in vitro was determined by MTT method.Separation and purification of dichloromethane extract from D.koreane by various chromatographic separation techniques(normal and reverse phase silica gel column chromatography and performance liquid chromatography et al).The qualitative and quantitative study of D.koreane was carried out by GC-MS analysis and content determination,and the research method of quality standard of D.koreane was established.Study on the anti Toxoplasma effect of anti-Toxoplasma drugs in vitro,using glide software in Schrodinger,combined with PubChem and PDB crystal database,high-throughput molecular docking is realized to optimize the active ingredients.Result:During the 14d observation period,no mice died,no changes in body weight and main organs were observed in the high(10 g/kg)and low(5 g/kg)dose groups.The median lethal dose of the total extract of D.koreane is higher than 10 g/(kg.body weight),which is actually non-toxic.Ethyl acetate and dichloromethane extract of D.koreane extracts,exhibit more obvious anti-Toxoplasma effect in vitro,with IC50 of 298.7 μg/mL,208.2 μg/mL and 298.6 μg/mL,206.1 μg/mL for HeLa cells before and after the infected with Toxoplasma gondii,and both selectivity indexs(SI)were 1.4.Five compounds were isolated from dichloromethane extract and identified as Naringenin(1)、Daphnetin(2)、Umbelliferone(3)、Syringin(4)and Daphnin(5)by spectral analysis.The contents of these five chemical components in different medicinal parts of D.koreane are obviously different.The extraction rate of volatile constituents was 0.56%,and 51 components were identified from 76 chromatographic pesks,accounting for 95.22%of the total of volatile components.The compound’s anti-toxoplasma gondii in vitro combined with molecular docking results showed that the effect of syringin against Toxoplasma gondii was more obvious in the dichloromethane extract of D.koreane.Conclusion:This experiment confirmed that the safety of D.koreane extract was high.The optimal medicinal substance for the preliminary evaluation of the anti-Toxoplasma gondii is syringin,which can provide a theoretical basis for further research on the material basis of the anti-Toxoplasma gondii.At the same time,the research method of D.koreane quality standard was established,which can provide reference for D.koreane quality standard.