| Chebulae Fructus,the dried and ripe fruit of Terminalia chebula Retz.or Terminalia chebula Retz.var.tomentella Kurt,which is a commonly medicine of ethnic medicines such as Mongolian medicine and Tibetan medicine.Chebulae Fructus tastes bitter,sour,astringent,and calm in nature.It has the effects of relieving diarrhea with astringents,constricting the lung to stop cough,and eliminating pathogenic fire to benefit pharynx.Because of its extensive pharmacological function,Chebulae Fructus has a broad application prospect in medical treatment and food development.Based on the preliminary investigation of the market,it is found that the current construction of Tibetan medicine standard system is relatively backward,and the medicine source of Chebulae Fructus is complex.The basic plant T.chebula Retz.var.tomentella Kurt.occupies a relatively large share in the current market.However,there are more studies on the pharmacology of Chebulae Fructus and less reports on toxicology.For the safety and rationality of clinical medication,the toxicity and target organs of T.chebula Retz.var.tomentella Kurt.decoction were determined by the acute toxicity experiment and accumulated toxicity experiment of mice in the first part of study.And the repeat dosing toxicity experiment of different rodents was used to determine toxicity to rats and mice with different dosage.Then the totoxicity of Chebulae Fructus was evaluated by systematic animal toxicology experiments.High content screening has an automatic quantitative cell imaging system,labeled with fluorescent reagents for specific targets,which could optimize imaging schemes and perform complex data analysis.It is a simple,high-sensitivity,and high-throughput screening technology,which is widely used in the screening of active ingredients and drug toxicity of traditional Chinese medicine.In the second part of this project,fluorescent dyes were used to specifically stain the corresponding targets.Then the high-content cell imaging analysis platform and DILI Assay TemPlate were used to jointly investigate the toxic effects of 10 compounds in Chebulae Fructus on HepG2 and L02 cells.By detecting 5 indicators such as cell number,DNA content,ROS content,MMP change,and GSH level,a high-content method of hepatotoxicity screening was established,which can screen the test compounds for cell morphology,replication,cycle,transcription,translation and apoptosis,to determine the risk of hepatotoxicity caused by the compound.This paper mainly discusses the hepatotoxicity and its material basis of T.chebula Retz.var.tomentella Kurt from two parts.In the first part,the toxicity and target organs of T.chebula Retz.var.tomentella Kurt were determined by toxicological experiments with different dosages and administration time to diverse rodents.In the second part,the toxic substance basis of T.chebula Retz.var.tomentella Kurt.was studied by high-content screening and high-performance liquid chromatography.Part 1 To determine the hepatotoxicity of T.chebula Retz.var.tomentella Kurt.1.Acute toxicity of T.chebula Retz.var.tomentella Kurt.in miceAcute toxicity experiments were conducted to determine the toxicity and target organs of T.chebula Retz.var.tomentella Kurt..The classical method of 14 days acute toxicity was used,after single administration,the changes of body weight,physiological state and death of mice were observed and recorded during the experiment.,the biochemical indicators,organ coefficients of liver and kidney were measured,and the LD50 was calculated.The lethal dose LD50 of its decoction is 18.93 g/kg,which is 147.8 times the maximum daily dosage of 70 kg people.The mice in the high-dose group showed abnormal clinical manifestations,diarrhea,and watery diarrhea.The mice began to die after 12 hours of administration,and the time of concentrated death was between 24 h and 72 h.Combined with the results of pathological sections and serum biochemical tests,alanine aminotransferase and aspartate transaminase in the experimental group increased abnormally,hepatocellular swelling,enlarged cell nucleus,loose cytoplasm,and obvious liver cell damage which indicated that the liver was damaged.It was speculated that the target organ of acute toxicity experiment of decoction of T.chebula Retz.var.tomentella Kurt.was liver.2.Accumulation toxicity test of T.chebula Retz.var.tomentella Kurt.in miceThe classic 20-day accumulation toxicity test method was used to determine the toxicity target organs and toxicity characteristics of T.chebula Retz.var.tomentella Kurt.,which provided a reference for chronic toxicity experiments and other toxicity experiments.Accumulation toxicity experiment showed that the accumulation coefficient of decoction of T.chebula Retz.var.tamentella Kurt.was more than five,which was mild accumulation.After the second stage of administration,the hair on the back of mice was disordered,diarrhea,lying and moving less.Compared with the control group,the growth of body weight of male mice was slow,with significant difference.In the fourth stage after administration,the mice died,the surviving mice had reduced food intake,decreased activity,and some abdominal abdomen bulging.Combining the results of serum biochemistry and pathological section,it is speculated that the target organ for accumulating toxicity in the decoction of T.chebula Retz.var.tomentella Kurt.is the liver,which is consistent with the results of the acute toxicity experiment.3.28-day repeated dosing experiment in mice of T.chebula Retz.var.tomentella Kurt.Through the continuous intragastric administration of the decoction of T.chebula Retz.var.tomentella Kurt.in mice for 28-days,the clinical adverse reactions and judgments of drug vaccination,the safety and the toxic organ of the drug were predicted during the repeated administration of T.chebula Retz.var.tomentella Kurt.The mice in the experimental group were gavage water extract,15 g/kg in the high-dose group,7.5 g/kg in the middle-dose group,and 1.5 g/kg in the low-dose group.The control group was given an equal volume of saline,and the material was taken once every 14 days.After the fourth day of continuous dosing,the mice died,the number of mice in the middle dose group was the most,and that in the high-dose group was the second.According to the results of organ coefficient and serum biochemistry,the liver was presumed to be the target organ of toxicity,which was consistent with the results of the acute toxicity and accumulated toxicity experiments.4.Toxicity test of 28-day repeated administration in rats of T.chebula Retz.var.tomentella Kurt.In this study,rats were used to administer the decoction of T.chebula Retz.var.tomentella Kurt.for 28 consecutive days,and then.two weeks recovery period was set up to observe the nature,degree,dose-effect and time-effect relationship and reversibility of the toxic reaction caused by water decoction of T.chebula Retz.var.tomentella Kurt,and the toxicity of water decoction of chebula villosa to rats and its toxic target organs were determined.In the stage of the experiment,the high-dose group 18.9 g/kg,the medium-dose group 9.5 g/kg,the low-dose group 1.9 g/kg,etc.were given the corresponding concentration of water decoction and the control group was given the same volume of saline.In the subacute toxicity experiment in rats,males in the high-dose group died on the 16th day,and females in the middle-dose group died on the 18th day and there was no rat death after that.After 14 days of repeated administration,there was no liver injury in the experimental group.After 28 days of repeated administration,the ALT of rats in the three high-dose and low-dose groups was significantly different from the control group,indicating that there might be liver damage.In the recovery period of the experiment,the biochemical indexes of the rats in the three dose groups of high,middle and low were not abnormal,indicating that the liver injury of rats may be reversible.Part 2 Preliminary study on the substance basis of liver toxicity of T.chebula Retz.var.tomentella Kurt.by high content analysis technology1.Determine the proliferation inhibition rate of HepG2 and L02 cellsThe inhibition rates of gallic acid,benzoic acid,shikimic acid,1,2,3,4,6-O-pentagalic glucose,corilagin,procatechin acid,ellagic acid,chebulic acid,chebulinic acid and chebulagic acid were determined on the proliferation of HepG2 and L02 cells under different incubation times.The absorbance values of the compounds at different dosages and incubation times were measured,and the inhibition rates of the compounds on different cells were calculated.The experimental results showed that the growth inhibition rates of gallic acid,1,2,3,4,6-O-penta galloyl glucose at a concentration of 200 ?g/mL on HepG2 cells are 73.96%and 93.77%,the growth inhibition rate of L02 hepatocytes was 99.02%and 98.98%%.Comprehensive analysis of the proliferation inhibition rate of different compounds on HepG2 and L02 cells,the top four were gallic acid,1,2,3,4,6-O-penta galloyl glucose,chebulinic acid and chebulagic acid.All of them had significant toxic effects on L02 hepatocytes and HepG2 hepatocytes,but benzoic acid and shikimic acid had no significant cytotoxic effects.2.HCS technology was used to screen the toxic components of T.chebula Retz.var.tomentella Kurt.on HepG2 cellsUsing high content analysis technology to determine the effect of the compound on HepG2 cell number,DNA content,GSH reduction level,ROS content and MMP changes at different doses,whether it exceeds its safety threshold,and hepatotoxicity of the compound sort.In the order of DILI Assay TemPlate,the top four ranked as gallic acid,1,2,3,4,6-O-pentagalloyl glucose,chebulinic acid and chebulagic acid.Gallic acid has the greatest hepatotoxicity to HepG2 cells,which is higher than that of positive control ticlopidine,30 ?g/mL is the critical concentration of gallic acid-induced liver injury.It was concluded that gallic acid has the highest risk of hepatotoxicity,and then from high to low were 1,2,3,4,6-O-pentagalloyl glucose,chebulinic acid,chebulagic acid,corilagin,ellagic acid,protocatechin acid,benzoic acid,chebulic acid and shikimic acid.3.HCS technology was used to screen the toxic components of T.chebula Retz.var.tomentella Kurt.on L02 liver cellsThe high-content screening technology was used to analyze the toxic effects of the compounds in Chebulae Fructus on L02 hepatocytes,and the damage mechanism of the toxic compounds was preliminarily discussed.The experimental results showed that in the ranking of DILI Assay TemPlate,the top five were gallic acid,chebulagic acid,1,2,3,4,6-O-pentagalyl glucose,chebulinic acid and corilagin,which is consistent with the screening results of HepG2 cells.Gallic acid has the greatest hepatotoxicity to L02 cells.When the concentration more than 10 yg/mL,the cell number,DNA content,GSH reduction,ROS content and MMP change all exceed their safety thresholds.Gallic acid has the highest risk of hepatotoxicity,and from high to low were chebulagic acid,1,2,3,4,6-O-penta gallic acid glucose,chebulinic acid,corilagin,ellagic acid,benzoic acid,protocatechin acid,chebulic acid,shikimic acid.4.The content of toxic components in T.chebula Retz.var.tomentella Kurt.was detected by HPLCThe method for the determination of the content of chebulic acid,gallic acid,corilagin,chebulagic acid,chebulinic acid,protocatechuic acid,etc.in T.chebula Retz.var.tomentella Kurt.was established by high performance liquid phase dual wavelength.It was found that the content of chebulic acid was the highest,up to 38.21 mg/g,followed by gallic acid,which was 10.74 mg/g,and the content of corilagin was 9.76 mg/g.Compared with the measured content of the ingredients with the toxicity ranking of the compounds in the T.chebula Retz.var.tomentella Kurt.screened by the HCS technology,it is speculated that gallic acid and corilagin were the material basis for the liver toxicity. |
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