| Objective:Based on the theory of traditional Chinese medicine,combined with traditional Chinese medicine literature and modern pharmacological toxicology research methods,two common compatibilityes of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen were used to study the compatibility of nutmeg and Juglandis Semen.using the appropriate techniques in vitro and in vivo,the effects of compatibility of nutmeg and Juglandis Semen on the hepatotoxicity of psoralea fructus were studied from the whole animal,isolated target organ and cells.Preliminary study on the effects of compatibility of nutmeg and Juglandis Semen on hepatotoxicity of psoralea fructus,"dose-time-toxicity",isolated liver toxicity and hepatotoxicity in vitro,In order to provide experimental basis for guiding the rational use of psoralea fructus.Methods:The classical acute toxicity test method was used to study the effects of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen on the acute toxicity of normal mice.The mice were treated with a single intragastric administration method.Study on the effect of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen compatibility on hepatic toxicity"quantity-time-toxicity"in normal mice,observe the death and toxicity of mice,detect serum ALT and AST levels,and calculate liver coefficient And the gross pathology and histopathological examination of the liver were performed.The effects of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen on the perfusion of isolated rat liver were studied by conventional rat perfusion method.After detecting the ALT and AST levels of the perfusate,the interference of other tissues and organs was excluded,the concentration of the drug was controlled,and the toxicity of the mixed nutmeg and Juglandis Semen on the isolated liver of rats was quantitatively compared.The skeletal fat-musk nutrient was treated by CCK8 method.And the effect of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen on L02 cells,observe the effect on the growth of L02 cells,and detect the levels of ALT,AST and LDH in the supernatant of the drug.The difference in the damage of L02 cells compared with the combination of nutmeg and Juglandis Semen.Results:Acute toxicity studies in vivo showed that psoralea fructus,psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen had acute toxic effects on normal mice,and LD50 value of psoralea fructus decoction was 95%.The confidence limit is56.028 g·kg-1·d-1(54.074?58.067 g·kg-1·d-1),which is equivalent to 392.2 times of daily dose of 70 kg adult per kg body weight,The MTD values of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction were 59.04 g·kg-1·d-1 and 72.0 g·kg-1·d-1,respectively,which were equivalent to clinical 70 kg.The daily dosage of adults per adult body weight was 413.28 and 504.0 times.The acute toxicity intensity is:psoralea fructus decoction>psoralea fructus+nutmeg decoction>psoralea fructus+Juglandis Semen decoction,the acute toxicity symptoms of mice mainly have mental retardation,agitation,prone and lethargy,convulsions,walking is not stable.The"time-toxic"relationship study showed that serum ALT and AST levels began to increase 0.5 h after 11.21 g·kg-1 psoralea fructus decoction in a single oral administration,reaching a peak at 6 h after drug administration.Compared with the group,serum ALT and AST levels were significantly increased 2 h to 72 h after drug administration?P<0.001?,and the liver coefficient was increased 2 to 24 h after drug administration?P<0.05?,and 6 h after drug administration?P<0.05?.reached the peak.The pathological examination of liver tissue showed that the liver tissue could be obviously damaged 8 hours after the drug;the mice were given a single oral administration of 44.28 g·kg-1 psoralea fructus+nutmeg decoction.Serum ALT and AST levels began to increase at 0.5 h,and peaked at 8 h after drug administration.Compared with the normal group,serum ALT and AST levels were significantly increased2 h to 72 h after drug administration?P<0.001?.The liver coefficient of the 2?24 h group was increased?P<0.05?,and reached the peak at 8 h?P<0.001?.There was no abnormality in the pathological morphology of the liver.The mice were given a single oral administration of 58.50 g·kg-1 The serum ALT and AST levels began to increase 1 h after the psoralea fructus+Juglandis Semen decoction,and reached the peak at 8 h and 12 h after the drug,respectively.Serum ALT and AST levels were significantly increased 2 h to72 h after drug administration?P<0.001?,and liver coefficient was increased 2 to 24 h after drug administration?P<0.05?,and 8 h after drug administration?P<0.001?reached the peak,and no abnormalities were found in the pathological morphology of liver tissue.The"quantity-toxicity"relationship study showed that compared with the normal group,the ALT level of the psoralea fructus decoction 6.29?11.21 g·kg-1 group and the AST level of the 9.25?11.21 g·kg-1 group were significantly increased.?P<0.001?,serum ALT and AST levels were dose-effect relationship,liver coefficient was increased in 9.25?11.21 g·kg-1group?P<0.05?,and pathological morphology of liver tissue in 11.21 g·kg-1 group.Hepatocyte necrosis and inflammatory cell infiltration were observed,the hepatocyte cord arrangement was disordered and a small amount of inflammatory cell infiltration;compared with the normal group,psoralea fructus+nutmeg decoction 15.04?44.28 g·kg-1group ALT The level of AST in the level and 25.81?44.28 g·kg-1 group was significantly increased?P<0.001?.The serum ALT and AST levels were dose-effect relationship,and the liver coefficient was increased in 44.28 g·kg-1 group?P<0.05?.There was no abnormality in the pathological examination of liver tissue of each group of mice.Compared with the normal group,the ALT level of psoralea fructus+Juglandis Semen decoction 31.0?58.50g·kg-1 was significantly increased?P<0.001?,AST level was significantly increased?P<0.01?,serum ALT,AST levels were dose-effect relationship,58.50 g·kg-1 group Dirty coefficient increased?P<0.05?,groups of mice liver tissue revealed no pathological abnormalities.The dose range of hepatotoxicity induced by psoralea fructus decoction in mice is 6.29?11.21 g·kg-1,which is equivalent to 44.03?78.47 times of daily dose of 70 kg.The dose range of cerebral hepatotoxicity induced by psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction in mice was 10.03?29.52 g·kg-1 and19.08?36 g·kg-1,respectively.equivalent to 70.21?206.64 times and 133.56?252.0 times the daily dose of 70 kg.In vitro ex vivo liver perfusion studies showed that ALT levels in isolated liver perfusate of rats in the psoralea fructus decoction of 0.9 and 3.6 g·kg-1 were significantly increased from 75 to 120 min compared with 0 min?P<0.01?,ALT level in7.2 g·kg-1 group increased significantly at 45?120 min?P<0.01?.The AST level of isolated liver perfusate in rats with 0.9 g·kg-1 group was increased from 75 to 120 min?P<0.05?,and the AST level of 3.6 g·kg-1 was significant at 75?120 min.The increase was?P<0.01?,and the AST level of 7.2g·kg-1 group increased significantly at 60?120 min?P<0.01?.Compared with 0 min,the ALT level in isolated liver perfusate of rats in the 1.35 g·kg-1group was significantly increased at 120 min?P<0.01?,The ALT level in 5.40 g·kg-1 group was significantly increased from 90 to 120 min?P<0.01?,and the ALT level in group 10.80g·kg-1 was significantly increased from 75 to 120 min?P<0.01?.The AST level of isolated liver perfusate in rats of 1.35 g·kg-1 group was increased in 120 min?P<0.05?,and the AST level in 5.40 g·kg-1 group was significantly increase in 90?120 min?P<0.01?,and the AST level of 10.80 g·kg-1 group was significantly increased at 60?120 min?P<0.01?.Compared with 0 min,the levels of ALT and AST in isolated liver perfusate of rats in the5.48 g·kg-1 group were increased?P<0.05?,The levels of ALT and AST in the 11.68 g·kg-1group were significantly increased at 90?120 min?P<0.01?.The activity of ALT and AST in perfusate has obvious dose-effect and time-effect relationship.The effect of L02 cells in vitro showed that the half-inhibitory concentration?IC50?of psoralea fructusn,psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction in L02 cells for 24 h was 11.07,18.35 and 27.23 mg·L-1,respectively.With the increase of concentration,the damage of L02 cells increased with psoralea fructusn,psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction.At the same concentration,The inhibitory effect of psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction on L02 cell proliferation was lower than that of psoralea fructus decoction.The drug group had significant difference compared with the normal group?P<0.001?.The effect on biochemical indicators showed that compared with the normal group,ALT levels in the psoralea fructus decoction 2.5?40 mg·L-1 group were significantly increased?P<0.001?,The levels of AST and LDH in the5?40 mg·L?-11 cells supernatant were significantly increased?P<0.001?.Compared with the normal group,the ALT level of the supernatant of the psoralea fructus+nutmeg decoction 5?40 mg·L-1 was extremely significant.The levels of AST and LDH in the supernatant of 20?40 mg·L-1 group were significantly increased?P<0.01?.Compared with the normal group,psoralea fructus+Juglandis Semen decoction.The level of ALT in the supernatant of the 20?40mg·L-1 group was significantly increased?P<0.001?,and the levels of AST and LDH were significantly increased?P<0.01?.psoralea fructusn,psoralea fructus+nutmeg and psoralea fructus+Juglandis Semen decoction can inhibit the growth and proliferation of L02 cells,have toxic effects on L02cells,and the cytotoxicity is positively correlated with the dose,the cytotoxicity intensity is:psoralea fructus decoction>psoralea fructus+nutmeg decoction>psoralea fructus+Juglandis Semen decoction.Conclusion:Comprehensive internal and external experimental studies suggest that psoralea fructus combined with nutmeg and Juglandis Semen significantly reduce the hepatotoxicity of psoralea fructus,improve liver damage caused by psoralea fructus,the effect of Juglandis Semen on the hepatotoxicity of psoralea fructus is more obvious.provide experimental basis for guiding the rational use of psoralea fructus. |
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