Study On The Attenuation Effect And Mechanism Of Hepatotoxicity Of Toosendan In Processing Technology

Objective:This experiment were used to study the changes of chemical composition and toxicity before and after the processing of toosendan,and explored its mechanism of attenuating toxicity,and it was provide experimental basis for the rational clinical application of toosendan processed products.Methods:1.A study on the composition of toosendanin caused by different processed products of toosendan:according to the 2015 edition of Chinese pharmacopoeia,HPLC was used to detect the content of water and toosendanin indifferent processed products of toosendan,and compare the differences in the content of toosendanin between different processed products.2. Comparative study on acute toxicity of toosendan with different processed products in mouse:Kunming mice were randomly divided into the normal group and the different processed products of toosendan.The normal group was administrated with normal saline,and the processed product group was treated with 40m L.kg^-1of different processed products of toosendan,and the general behavioral observation and weight changes of the mice were recorded during continuous observation for 14 days.At the end of the 14-day observation period,serum were taken for the detection of ALT and AST activity.The liver,spleen,heart,kidney,and lungs were weighed and the organ coefficients were calculated.3.“Dose-time-toxicity-effect”relationship study on hepatotoxicity caused by different processed products of toosendan to mouse:?1?“Time-toxicity”relationship:Kunming mice were randomly divided into normal group and toosendan different processed products groups to receive the maximum tolerated dose?MTD?.Blood samples were collected at 1,2,6,12,24,48,and 72 hours after administration to determine the activity of ALT and AST in the serum.The liver,spleen,and thymus were weighed and the organ coefficient were calculated.The morphological changes of liver tissue were observed under optical microscope.?2?“Dose-toxicity”relationship:Kunming mice were randomly divided into the normal group and toosendan different processed products groups.According to the results of the pre-test,the raw products group was provided with 7 dose groups of MTD,0.85 MTD,0.85²MTD,0.85³MTD,0.85⁴MTD,0.85⁵MTD,and 0.85⁶MTD.Three dose groups were set for vinegar sunburn products group,fried coke products group,and the salt sunburn products group,which were MTD,0.85 MTD,and 0.85²MTD respectively.The wine sunburn products group was provided with 6 dose groups of MTD,0.85 MTD,0.85²MTD,0.85³MTD,0.85⁴MTD,and 0.85⁵MTD.The maximum non-toxic dose of each processed products groups were found respectively.The blood was taken for the detection of serum ALT and AST activity in 2 hours after administration.The liver,spleen,and thymus were weighed to calculate the organ coefficient.Morphological changes of liver tissue were observed under optical microscope.?3?“Dose-effect”relationship:Kunming mice were randomly divided into the normal group and the different processed products of toosendan.According to the results of the pre-test,7 groups were set in the raw product group,which were1/3*MTD,1/3*0.85MTD,1/3*0.85²MTD,1/3*0.85³MTD,1/3*0.85⁴MTD,1/3*0.85⁵MTD,1/3*0.85⁶MTD;vinegar sunburn products,wine sunburn products,fried coke products,salt sunburn products,three dose groups were set up,namely 1/3*MTD,1/3*0.85MTD,and 1/3*0.85²MTD;and after the mice were treated with different processed products of toosendan for 1 hour,the hot water tails of mice were measured time.4.Study on the mechanism of hepatotoxicity oxidative damage caused by different processed products in toosendan:Kunming mice were randomly divided into the normal group and the different processed products of toosendan.The maximum toxic dose,subtoxic dose and the maximum non-toxic dose were used for hepatotoxicity oxidative damage test in each processing group.Two hours after administration,the activity of SOD,GSH-Px,CAT,and XOD,and the contents of MDA and GSH in the liver tissue were measured according to the kit method,and the degree of peroxidative damage between the processed products was compared.5.Effect of different processing products of toosendan on the function of normal human liver cells L02 mitochondria:The effect of different processed products on L02 cell activity was detected by MTT assay.The mitochondrial complex enzymes I-IV activity were determined after treatment with500?g/m L,125?g/m L,31.25?g/m L different processed products of toosendan for 48 hours.Results:1.A study on the composition of toosendanin caused by different processed products of toosendan:The water content in the different processed products of toosendan does not exceed 15%.The results of the 5 kinds of different processed products of toosendan showed:fried coke products?0.113?>raw products?0.097?>wine sunburn products?0.096?>salt sunburn products?0.094?>vinegar sunburn products?0.089?.Compared with raw products,the content of toosendanin in vinegar sunburn products was significantly decreased?P<0.01?.The content of toosendanin in fried coke products was significantly increased?P<0.01?.There was no significant change in the content of toosendanin of wine sunburn products and salt sunburn products.2.Comparative study on acute toxicity of toosendan with different processed products in mouse:The general behavioral observation showed that after administration of raw products,vinegar sunburn products,wine sunburn products,fried coke products and salt sunburn products,all mice showed varying degrees of body licking and scratching,and after administration of large doses of fried coke products and salt sunburn products,male mouse had hair loss.Weight change found that compared with the normal group,there was no significant difference in raw products group,salt sunburn products group,vinegar sunburn products group,and wine sunburn products group of female mice,while the weight of fried coke products group was decreased significantly on the 1^stday?P<0.05?.Compared with the normal group,there was no significant difference in salt sunburn products group,vinegar sunburn products group,and fried coke products group of male mice,while the weight of the raw products group was decreased significantly on the 2^ndday?P<0.01?,and the weight of salt sunburn products group on the 1st day decreased significantly?P<0.05?.The organ coefficient results showed that compared with the liver coefficient of the normal group,a large dose of raw,vinegar,wine,and salt extracts of toosendan can cause the liver enlargement of male and female mice after 14 days and the coefficient was increased significantly?P<0.05 or P<0.01?.Compared with the normal group kidney coefficient,the salt sunburn products group can cause kidney enlargement,and the kidney coefficient was increased significantly?P<0.01?.Compared with the spleen coefficient of the normal group,the vinegar sunburn products group can cause spleen enlargement,and the spleen coefficient was increased significantly?P<0.01?,while there was no significant difference in lung coefficient and heart coefficient results.The ALT and AST vitality results showed that after administration of the cumulative maximum doses of raw products,vinegar sunburn products,wine sunburn products,fried coke products,and salt sunburn products at one time,the serum ALT level had a tendency to increase compared with the normal group,but there was no significant difference,while the AST value showed a distinct increase in different degrees.3.“Dose-time-toxicity-effect”relationship study on hepatotoxicity caused by different processed products of toosendan to mouse:The results of the“time-toxicity”relationship study show that compared with the normal group,the serum ALT and AST levels were increased in varying degrees from 1 to 12 hours after administration of the raw product group,and showing a certain“time-toxicity”relationship.ALT and AST values were both at the peak of 2 hours after administration,and the duration of the toxicity of ALT was about 12h,and AST was about 6h.Liver tissue showed diffuse moderate water degeneration of hepatocytes,showing some punctate necrosis and hepatic cells scattered in small focal steatosis.Compared with the raw product group,the duration of toxicity of ALT and AST in the vinegar sunburn products group,wine sunburn products group,fried coke products group,and salt sunburn products group was shortened,and the pathological damage degree of the liver tissue was reduced,and the serum ALT levels were decreased significantly?P<0.05?at 12 h after administration of wine sunburn products,and the serum ALT levels were decreased significantly at 2h and 12h?P<0.01?after administration of fried coke products.Serum ALT and AST values significantly were decreased at 2h after administration of salt sunburn products.The results of the“Dose-toxicity”relationship show that the maximum toxic dose of the raw products group was MTD,the minimum toxic dose was0.85³MTD,and the maximum nontoxic dose was 0.85⁴MTD.The maximum toxic dose of the vinegar sunburn products group,the fried coke products group,and the salt sunburn products group was MTD,the minimum toxic dose was 0.85MTD,and the maximum nontoxic dose was 0.85²MTD,while compared with the raw products group,the toxic dose range shorten.The maximum toxic dose of wine sunburn products was MTD,the minimum toxic dose was 0.85⁴MTD,and the maximum nontoxic dose was 0.85⁵MTD,while compared with the raw product group,the toxic dose range was increased.Under the microscope,the liver tissues of the raw products group showed diffuse and moderate water degeneration of hepatocytes,individual punctate necrosis and a small amount of inflammatory cells infiltrated into the portal area.Compared with the raw product group,the extent of liver tissue pathological damage was slightly reduced after administration of vinegar sunburn products,the fried coke products,and the salt sunburn products.The results of the“Dose-effect”relationship study showed that compared with the normal group,the time for stretching the tail was significantly prolonged in the raw products dose of 1/3*0.85²MTD,1/3*0.85⁵MTD,1/3*0.85MTD vinegar,1/3*0.85MTD The time of tailing of fried coke product and 1/3*0.85²MTD salt licking product was significantly prolonged?P<0.05?,but did not show a clear“quantity-effect”relationship.4. Study on hepatotoxicity oxidative damage in mouse caused by different processed products in toosendan:Compared with the normal group,The raw products group were increased the content of MDA in the liver tissue,decreased the activity of CAT,GSH-Px and the content of GSH,increased the XOD activity,and led to the occurrence of hepatotoxicity in mice.The vinegar sunburn products group were increased the content of MDA in liver tissue and decreased CAT activity,but had no significant difference in SOD,GSH-Px,XOD activity and GSH content.The wine sunburn products group were increased the content of MDA in liver tissue,decreased CAT,GSH-Px activity,increased the XOD activity,but had no significant difference in SOD and GSH content.The fried coke products group were decreased the activity of CAT and GSH-Px,increased the activity of XOD,and decreased the content of GSH,but had no significant difference in SOD activity and MDA content.The salt sunburn products group were decreased CAT activity,but had no significant difference in SOD,GSH-Px,XOD activity and GSH,MDA content.5. Effect of different processing products of toosendan on the function of normal human liver cells L02 mitochondria:The inhibitory effect of the raw products and wine sunburn products on L02 cells was obvious.Compared with the control group,the proliferation of L02 cells were significantly inhibited by the generation of the raw products in the range of 125 to 2000?g/ml?P<0.05 or P<0.01?.Compared with each dose group of the raw products,the proliferation of L02 cells were significantly increased by the generation of the vinegar sunburn products and salt sunburn products in the range of 62.5 to 2000?g/ml?P<0.05 or P<0.01?,while there was no significant change in the dosage groups of fried coke products.However,the proliferation of L02 cells were significantly inhibited by the generation of the wine sunburn products in the range of 62.5 to 500?g/ml?P<0.05 or P<0.01?.The order of toxicity of different processed products to L02 cells in vitro was as follows:wine sunburn products>raw products>fried coke products>salt sunburn products>vinegar sunburn products.The inhibitory effect of raw products and wine sunburn products on the mitochondrial respiratory chain complex was obvious.Compared with the normal group,raw products on mitochondrial respiration?-?polymerase chain compound enzyme activity were inhibited significantly?P<0.05 or P<0.01?.Compared with the corresponding dose of the normal group,the high dose of vinegar sunburn products were significantly reduced the inhibition of the mitochondrial respiratory chain complex I and III of the raw products?P<0.05?.The high dose of fried coke products were significantly reduced the inhibition of the mitochondrial respiratory chain complex I of the raw product?P<0.05?.The high dose of salt sunburn products were significantly reduced the inhibition of the mitochondrial respiratory chain complex I of the raw product?P<0.05?.Conclusion:The raw material can cause obvious hepatotoxicity,showing a certain“dose-time-toxicity”relationship.Vinegar sunburn products,fried coke products,and salt sunburn products can significantly reduce hepatotoxicity in mice,mainly as the duration of toxicity is shortened,the scope of toxic dose is reduced,and the pathological degree of liver tissue is reduced,while the hepatotoxicity of wine sunburn products is enhanced.The attenuated mechanism of vinegar sunburn products,fried coke products,and salt sunburn products may be related to the reduction of oxidative stress damage and the reduction of mitochondrial dysfunction.