| ObjectiveThe obtained new type tripterygium glycosides maintained therapy effect on nephritic syndrome by changing the chemical composition content of tripterygium glycosides.This study will provide toxicity evaluation in vitro and in vivo.The optimization of preparation process and research for main material basis could provide more safe and wide usegae of tripterygium glycosides in clinic.MethodsThe new type tripterygium glycosides maintained therapy effect on nephritic syndrome by changing the chemical composition content,according to the previous research of our group.As to the treatment of nephrotic syndrome,the WLL03 have similar anti-inflammatory effect compared with triptolide.Therefore,the new type tripterygium glycosides studies about toxicity and material basis of activity were carried out as following;1.The preparation of WLL03WLL03 was prepared through the hydrolysis of triptolide in phosphate buffer by using the chemical process and isolated by preparative liquid chromatography.2.The preparation of the new type tripterygium glycosideThe triptolide was turned into WLL03 by the chemical process of tripterygium glycosides.The content of triptolide and WLL03 were checked by ultra-high performance liquid chromatography(UPLC)in new type tripterygium glycosides after the addition of cetain amount of WLL03.3.The cellular toxicity of new type tripterygium glycoside in vitroMTT and CCK8 colorimetric methods were used to determine the cell livability in 24h and 48h to study cell toxicity of the WLL03,triptolide and tripterygium glycosides.4.The toxicity of the new type tripterygium glycoside in vivoThe tripterygium glycosides,the new type tripterygium glycosides were administered to mice in one day and compared with each other in accordance with acute toxicity test methods(LD50 methods).The symptom of toxicity was observed,the change of body weight and the cumulative death were recorded.It would provide the basis for the research and guide for clinical application on safety evaluation of Tripterygium wilfordii.5.Pharmacokinetics of WLL03An ultra-high performance liquid chromatography-mass/mass(UPLC-MS/MS)with multiple reaction monitoring(MRM)mode was adapted to determine the pharmacokinetics of triptolide in rats and its bioavailability.Results1.The main product after chemical process was WLL03 and the extraction rate was about 20%.2.The new type tripterygium glycosides Ⅱ,Ⅳ and Ⅴ were prepared according to the previous research.The contents of triptolide in these combinations were 232.34±2.01 μg/g、439,97±2.24 μg/g、232.82±1.93 μg/g respectively and the contents of WLL03 were 2380.29 μg/g、2322.13 μg/g、20589.03μg/g,respectively.3.WLL03 showed no inhibitory effect on cells.The 24h IC50 were HK-2(21919.73±1024.03 ug/mL)、HL-7702(9427.67±334.77 ug/mL)、GC-1(303862.92±9502.51 ug/mL)、GC-2(76578.35± 1627.39 ug/mL)、TM4(5562.77± 194.13 ug/mL)、Mouse podocyte(142.72±5.32 ug/mL),respectively.The 48 h IC50 were HK-2(10581.12±906.26 ug/mL)、HL-7702(1561.46±99.24 ug/mL)、GC-1(353.79±16.60 ug/mL)、GC-2(4300.40±264.90 ug/mL)、TM4(4224.97±133.39 ug/mL)、Mouse podocyte(220.06±0.13 ug/mL),respectively.The toxic level on cells were as follows:new type tripterygium glycosides Ⅳ,Ⅱ,tripterygium glycoside(in descending order).4.The LD50(95%confidence limit)of tripterygium glycoside was 571.52 mg/kg(486.78-682.51 mg/kg).The maximum dose of new type tripterygium glycosides Ⅱ and Ⅳ were 2400 mg/kg,620 mg/kg,which were equal to 1600,413 times of a 70 kg adult’s daily dried medicinal herb consumption,while the mortality rate were 1.67%and 16.67%respectively.5.The optimized fragmentation transitions of WLL03 was m/z 377.2→217.1 with the linear range from 0.975 ng/mL to 1000 ng/mL.There may be some interference in blank plasma from the extraction process at the same retention times of WLL03(377.2→217.1),such as solutions,dryness under a stream of nitrogen,and so on.ConclusionAccording to the preliminary experiments of our group,the contents of triptolide and other effective ingredients in Hunan Qianjin Xieli Pharmaceutical Company could meet the experimental requirements.The new type tripterygium glycosides Ⅱ,Ⅳ and Ⅴ were prepared by adding cetain amount of WLL03.In vitro and in vivo experiments have proved that inhibitory effect on cell are weaker and nontoxicity.The new type tripterygium glycosides Ⅱwhich was prepared by using chemical process method had less toxicity than tripterygium glycoside.It showed significant difference between these two glycosides in toxicity profile. |
