Senecio Liver Toxicity Study

Senecio are distributed widely and have 1500 species in the world.In other country it be reported having toxicity.There are more than 160 species which 38 medicinal species be included in our country,but it has no reported in clinic.Though it be recognise differently in toxicity in history,it is be used as yet.Previous studies showed that Senecio commonly contained PAs.Unsaturated PAs have hepatic toxicity obviously.Foreign Senecio are different in species and toxic intensity from ours'.Senecio existed in our coutry widely,it has no research in the diferent of PAs content and toxic in the different area.In addition,it is used mostly in complex preparation in our country,but not pure PAs.because complex preparation have complex components which affect and act each other,the toxic relationship between complex preparation and PAs or single herb can't be over simplified into a sign of equivalence.It have no studies about chemistry and toxicology of S.scandens Bach-Ham at home and abroad at present,so this species is unclear in character and toxic tensity,wether it had any toxic and what about unsaturated PAs the function in detoxic about complex preparation.It is not be confirmed completely in the toxic mechanism,especially in the molecular level,so it should be reseach deeply.In order to make these clear.We take the methods of parallel comparing climbing water extract of groundsel herb with climbing groundsel herb total alkloids or Qianbaibiyanpian from the aspect of acute toxic accumulation toxic and genetic toxic combining chemistry experiment and contents of PAs. Studying the difference of these three,revealing climbing groundsel herb toxic character,the relationeship between toxic and quantity,the relationship between toxic and efficiency.All these studies can provide scientific discipline for climbing groundsel herb safty and reasonable application.To investgate DNA damages of livers of rats exposed to Climbling Groudsel Herb.We select four mono-compounds IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine to studies their toxicity to Hep G2 cell.Researching effection of GSH on pretecting the hepatic cell, using the methods of immunofluorescence and WESTERN-BLOT,studies the relationship micromolecule protein with the toxic of PAs,all these studies not only play some effect on getting the message of climbing groundsel herb,but also provide some information to prevent and treat the PA's toxicity of other genera botanicThis thesis take the following studies:Acute toxicity studies1.1 Comparative study on total alkloids and acute toxicity of climbing groundsel herb from different producing areaIn order make clear PAs content of climbing groundsel herb of different producing area and compare the difference of their toxic to offer science basis for select safty medical herb for clinic and production.We compare the toxic and PAs content of climbing groundsel herb from representative area of our country.The results show that the toxicity of climbing groundsel herb varies with its producing area and has relationship with its conent of PAs.The climbing groundsel herb from Henan has the the most PAs and the highest toxic one among these herbs.There is some degree of toxicity in climbing groundsel herb from Jiangsu,Zhejiang,but their toxicity are weaker and the PAs contents is lower than Henan.The toxicity of climbing groundsel herb varies with its producing area.No marked toxicity was found in climbing groundsel herb from Guangxi and Hubei,content of PAs in there are the lowest than others.There is some degree of toxicity in climbing groundsel herb from Jiangsu, Zhejiang..Pathological examination of died and survival mice has obviously hepatic toxic,A microscopic examination revealed more binuclear,macronuclear,and irregular nuclear cells in the liver,inflammatory cellular infiltration,multiple calcific foci,and dilatation and steas in hepatic central veins and hepatic sinusoid.Result show that when it is be used in clinic and Chinese formulated products produing,it should be select the specis which has lower contents of PAs and toxic to ensure its safty.1.2 Studies on acute toxicity of climbing groundsel herb and its compound preparation.Climbing groundsel herb from Henan which the toxic is highest are seclected to detect LD50 of mice rats and young mice,analysis the relationship of quantity and toxicity of acute medication. It can ensure its safty and provide information of clinic medication.The results show that mice,rats and young mice are perfused to the stomach with water extracts of climbing groundsel herb of Henan.It show obviously toxic and fetal when the highest dosage be used.It also has relationship with quantity and toxicity obviously.Compared on the relationship of quantity and toxicity in mice and rats.The tolerance in rates is greater than in mice.so climbing groundsel herb's toxicity is related to species difference.Water extacts of Qianbaibiyanpian were perfused to adult mice,it show obviously toxic and fetal when the highest dosage be used and also has the relationship with quantity and toxicity.The toxicity of Qianbaibiyanpian contained Henan climbing groundsel herb is lower than Water extacts of climbing groundsel herb.2.Studies of hepatic toxicity of long-term use climbing groundsel herb and its compound preparation.Water extracts,compound preparation and total alkloids of Henan climbing groundsel herb which the toxicity is highest are seclected four dosage of 1.5,3.0,6.0,20.0g/kg and perfused to rats 4 weeks tobserved the hepatic toxicity.Result show that 20.0g/kg dosage of this three agent can reduce APTT.This three agent can effect the biochemical indicator AST.Histopathology show that 20.0g/kg dosage of those agent had the mild heptic toxicity.3.Studies on the mechanism of toxicity of climbing groundsel herb3.1 Studies on the damages of livers of rats exposed to Climbling Groudsel Herb.Liver of Rats which was perfused to water extracts of climbing groundsel herb.DNA damages were detected by single cell gel electrophoresis(comet assay).The results show that The incidence rates of comet cells in the experimental groups were significantly higher than those of the controls.Furthermore the incidence rates of comet has the relationship with the dosage.It demonstration that water extrats of climbling Groudsel Herb could induce DNA break and damage of rats hepatic cell cells.The frequency of damage is increase with dosage.So climbing groundsel herb can induce the damage DNA of hepatic cell and change of hepatic functions.3.2 Studies on the mutagenic action of climbing groundsel herb.Rats which was perfused to water extracts of climbing groundsel herb,then femoral bone was be maken to bone marrow slides,taking the bone marrow cell micronucleus test to ensure the mutagenic ation of climbing groundsel herb,total alkloids and Qianbaibiyanpian.Results show that 20.0g/kg dosage of this three objects can increase bone marrow cell micronuclear rates obviously and the data were significantly different compared with the control group(P＜0.05orP＜0.01).It demonstrate that climbing groundsel herb,total alkloids and Qianbaibiyanpian has the function of improving the one marrow cell micronuclear rates,but the function is weak3.3 Studies of the hepatic celluar toxicity of PAs on Hep G2.Using the model of Hep G2,We research the four chemical compounds of IntegerrimineA,IntegerrimineB,Monocrataline and Retrorsine cell toxicity,studies the changance of biochemical indicator and the effect to hepatic cell DNA in the progress of induing toxicity.All this can anticipate the toxicity of botanic herb which contents PAs.The results show that this four chemical compounds IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine has the higher inhibition ratio when it be used in the highest dosage.It can inhibit cell activity.Cell toxicity of Monocrataline,Retrorsine is higher than two other.Biochemical indicator was be detected and results show that biological enzyme in the cell has be released to the cell supematant,IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine can effect CK,LDH of hepatic cell,increase the release rate of this two biological enzyme. Monocrataline and Retrorsine has higher cell toxity than the other two objects,the suitable dosage of this two objects can increase the release ratio of AST,ALT.All this demonstrate Monocrataline,Retrorsine can induce serious hepatic cellular damage,especially to the Hep G2.Single cell gel electrophoresis(comet assay)show that this four chemical compounds can cause the DNA immigration,The incidence rates of comet cells in the experimental groups were significantly higher than those of the controls.It induce breaking of DNA and cellular necrosis and apoptosis.3.4 The effect of GSH on the toxicity of PAsContents of GSH was be detected when PAs cause the Hep G2 cellular toxicity.Studies the relationship between the toxicity of PAs and GSH.Cytoactive inhibition ratio and ALT which is the obvious indicator for the heptic cellular toxicity of PAs.BSO which is the catastaltic of GSH and NAC which is precurosor of GSH effect on the Hep G2,then contents of GSH in cell be inhibited and increase respectively.Approach the function of GSH in the process of PAs effect the heptic cellular toxicity.The results show that PAs have heptic cellular toxicity when we studies the effect of GSH on the PAs' hepatic cellular toxicity in Hep G2.BSO can increase the PAs' heptic cellular toxicity,NAC can reduce the PAs' heptic cellular toxicity,GSH can protect the cell indirectly. When NAC be added in the heptic cell culture fluid,cell can intake the NAC to syntheise GSH. Four hours later,concentration of GSH will increase.While,BSO be added in the heptic cell culture fluid,cell can' t continue to syntheise GSH.Intrinsic GSH has been exhausted, concentration of GSH in cell lower.So BSO and NAC have adverse effect on concentration of GSH in cell,and PAs' hepatic toxicity.Therefore,It can be consumed that GSH in cell has the relationship with PAs' heptic celluar toxicity.3.5 The effect of PAs to the micromolecule protein and cell cytokine in Hep G2Utilization the method of cytobiology,molecula biological,immunochemistry to studies the cellular toxicity of IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine,studies what's effect on the VEGF,GM130,Cav-1 and research its mechanism ofheptic toxicity.The results show that VEGF in Hep G2 has higher expression when PAs caused the cell toxicity.After IntegerrimineA,Monocrataline,Retrorsine effect cell 24 hours,VEGF in Hep G2 higher express.IntegerrimineA,Monocrataline,Retrorsine induce VEGF which have time dependence.When PALS effect Hep G2,cell be labeled with immunofluorence,this four PAs could reduce concentration of GM130 in intracytoplasm,IntegerrimineB,Monocrataline,Retrorsine could increase the concentration of Cav-1 in cytomembrance and intracytoplasm in Hep G2.The mechanism of PAs cellular toxicity may be that increasing Cav-1 can change the material transport and permeability,GM130 reducence can effect the function of endocytoplasmic reticulum,aggravate the damage of hepatic,promot the process of hepatic fibrosis.