| Tripterygium wifordii Hook.f.is known as duan chang cao or huang teng mu,which is a plant in Tripterygium of Celastraceae.lt is native to Fujian,Hunan and Zhejiang province in China.The property of Tripterygium wifordii Hook.f.is warm,slightly bitter and it has a long history of use in traditional Chinese Medicine(TCM)for treating swelling,inflammation,ulcer,fever and so on.Preparations of Tripterygium were firstly used to treat inflammation and rheumatoid arthritis and then for cancer,chronic nephritis,systemic lupus erythematosus,ankylosing spondylitis,and avariety of skin conditions.Although the efficacy of Tripterygium has been confirmed,the obvious side effects limited its widely use in clinic.It could make liver injury in the therapeutic dose range.Triptolide is believed to be the major hepatotoxicity component of Tripterygium extracts,and other compounds have seldom researched.What elements or components might induce hepatotoxicity is not clear so far.To ensure the safety of using Tripterygium and exploit the most advantage in the treatment of illness,the toxicity and efficacy components of Tripterygium extracts have to be reasearched,which might discover the components with low hepatotoxicity and high anti-inflammation and immunosuppression effects.The long term toxicity studies and the target organs(or cells)affinity analysis method are adopted to observe the distribution and accumulation of different extraction parts in animal’s liver after different administration time.Choose mice’s liver impacted by evident toxic parts through toxicity reaction analyse and gain the information of higher affinity and accumulative component,which can be considered as the hepatotoxicity component.It has been analysed that the interaction of the components of Tripterygium with liver cells as toxicity target organ,and spleen cells and macrophages as effect target organ.The corresponding affinity of composition were compared and try to determine those components with low liver toxicity,high immunosuppressive and anti-inflammatory effects.The study seeks to clarify the active material basis with low liver toxicity of Tripterygium,which provides new ideas and methods for reseraching the toxic and efficient components/constituents groups.This study includes 5 parts as following:1.The determination of the liver toxic extracts in Tripterygium.The method of system solvent was adopted to extract different parts from Tripterygium(ethanol extract parts,ethyl acetate extract parts,n-butanol extract parts and the water extract parts).ICR mice,male and female,were given these four parts of Tripterygium.they were divided into blank group,alcohol high and low dose group,ethyl acetate high and low dose group,butanol high and low dose group and water high and low dose group.The low dose group is the clinical equivalent amount,which is equivalent to 3.25g/kg of crude drug.The high dose group is 5 times of the clinical equivalent,which is equivalent to 16.25g/kg of crude drug.The blank group was given the same volume of saline.There have four time points of one,two,three and four weeks and each time point has nine experimental groups.Taking blood to measure AST、ALT、LDH、ALB and TP in serum after administrated for 12 hours in the end of one week,two weeks,three weeks and four weeks,respectively.Then make a tissue biopsy and observe the liver changes by light microscope.The experimental results showed that ethanol extract parts,ethyl acetate extract parts,n-butanol extract parts and the water extract parts of Tripterygium all had liver toxicity.The ethanol extract parts,ethyl acetate extract parts,n-butanol extract parts had significant liver toxicity while the water extract parts had less.2.To Compare toxicity between different compounds of Tripterygium.Ethyl acetate extract part and n-butanol extract part were separated by silicagel column chromatography.Toxicity screening by liver cells(L-02)might identify the composition of liver toxicity.The experimental results showed that procyanidin-B、catechin and epicatechin was separated from ethyl acetate extract part;the mixture of sucrose and cellobiose was separated from n-butanol extract part.But these components have no cytotoxicity.3.The analysis of stock composition of hepatotoxicity in Tripterygium.The mice administrated 95%alcohol extract of high-dose(16.25 g crude drug/kg)of Tripterygium 10 days and control mice were taken to obtain livers and homogenated the organs respectively.The blank group’s liver was taken,homogenated and added ethanol extract solution(16.25 g crude drug/kg)of Tripterygium.Three liver homogenates were divided into two and extracted with ethyl acetate and dichloromethane,and then take the organic solvent layer and evaporated solvent,respectively.Then 95%methanol solution were used to dissolve the samples and made a total of six samples.LC-MS was adopted to analyze organ affinity ingredients.Experimental results show that tridioltonide,triptoquinoneE,epigallocatechin,triptoquinoneH,triptobenzeneN,triptininB,(+)-dehydroabietane,triptolide,triptocallol,hypoglaucum lactone A,(+)-isolariciresinol,triptoquinoneC,13-epi-19-nor-manoyloxide-18-oicacid,and ray phenol terpene acid J were the potential liver toxic ingredients.4.The analysis of the immunosuppressive active ingredient of Tripterygium.Primary rat macrophages,mouse spleen cells and liver cells were made in suspension with RPMI-1640 medium and seeded in 24-well plates.The cell concentration is about 1-2 × 106 and cultured 1h in 5%CO2,37℃ saturated humidity conditions.Water extracts of Tripterygium were added in concentration of 2g/ml and the mixture were cultured for another 0.5h.The cell supernatant were collected and analyzed to compare the differences among the composition of liver cells,spleen cells and macrophages by liquid chromatography-mass spectrometry technology.Experimental results show that phenol terpene alcohol、triptonodiol、wilforolF、triptobenzeneL、triptoquinoneG、(-)-syringaresinol、triptoquinoneC、triptocallol、dibutylphthalate、triptobenzene A、triptoquinone D、triptobenzene M、12,14-Dihydroxy-3-oxo-abieta-8,11,13-triene、11-hydroxy-14-methoxyl-dehydrobaietane、methoxyl-dehydrobaietan,dibutylphthalate might be the active composition with low liver toxicity.5.To investigate the hepatotoxicity of Tripterygium with different species of animals.288 SD rats,half male and half female,were choosed and administrated with 75%ethanol extract of Tripterygium.The rats were randomly divided into low dose group(1 times of the clinical equivalent dose group:2.25 g(crude drug)/kg),medium dose group(2.5 times of the amount:5.625 g(crude drug)/kg),high dose group(5 times of the amount:11.25 g(crude drug)/kg)and control group(saline).Each time point of the experiment included four test groups and each group consisted of 12 rats,half male,half female.Animals were oral administrated once a day and the serum and livers were obtained in one,two,three,four,six,and eight weeks after the administration.The contents of ALT,AST,TP and ALB in serum were measured and the liver tissue biopsy were conducted to observe the liver changes under light microscope.Experimental results show that the extract of Tripterygium had the obviously hepatotoxicity in rats in the second week. |
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