Study On Toxicological Safety Of "Ruyanning" Water Extract And Its Effect On Antioxidation And Immune Function In Mice

Object:In this study,mice were used as the main experimental animals to carry out acute and subchronic toxicity tests by gavage to evaluate the safety of the water extract of"Ruyanning";The antibacterial effect of the water extract of"Ruyanning"was investigated through the in vitro and in vivo antibacterial test of the main pathogenic bacteria of cow mastitis;Through gavage of different concentrations of"Ruyanning"water extract to normal mice,the effects of different concentrations of"Ruyanning"water extract on the antioxidant and immune functions of mice were investigated,providing theoretical basis for the safe and reasonable application of"Ruyanning"in clinical practice.Methods:（1）Experiment 1:Mice were gavaged with the water extract of"Ruyanning"at 5,10,20 and 40g/kg body weight,and the control group was gavaged with normal saline.The toxicological reaction and death of mice were observed and recorded,and LD₅₀ was measured;The maximum concentration of"Ruyanning"water extract（equivalent to 2.0 g/m L of crude drug）and the maximum tolerance volume of mice（0.04 m L/g body weight）were administered by gavage once,and the control group was given physiological saline by gavage to determine the maximum tolerance of mice.（2）Experiment 2:The low,medium and high dose groups were gavaged with 4,8 and 16 g/kg body weight respectively,while the control group was gavaged with normal saline once a day for 30 consecutive days.The general condition of mice was observed during the experiment.After the experiment,the organ index,blood routine index,blood biochemical index and pathological section of mice were measured respectively.（3）Experiment 3:Staphylococcus aureus,Streptococcus lactis and Escherichia coli were tested for the diameter of the bacteriostatic ring by the method of perforation,and the MIC and MBC were determined by the broth dilution method;The mice were randomly divided into five groups,namely blank control group（normal saline group）,positive control group（ciprofloxacin group）,low（0.4 g/m L）,medium（0.8 g/m L）and high（1.6 g/m L）dose groups of"Ruyanning"water extract.The mice in each group were gavaged at a fixed time every day,twice a day,for 7 consecutive days,and the protective rate of the water extract of"Ruyanning"on mice infected with mixed pathogenic bacteria was determined.（4）Experiment 4:The blank control group（normal saline group）,the positive control group（vitamin E group）,the low（0.4 g/m L）,medium（0.8 g/m L）,and high（1.6g/m L）dose groups of"Ruyanning"water extract were gavaged once a day for 7 days.After the experiment,the organ index,serum biochemical index,serum and liver antioxidant index and immune index of mice were measured.（5）Experiment 5:The blank control group（normal saline group）,the positive control group（levamisole hydrochloride group）,the low（0.4 g/m L）,medium（0.8 g/m L）,and high（1.6 g/m L）dose groups of"Ruyanning"water extract were gavaged once a day for 7 days.After the test,the immune organ index,mononuclear-macrophage phagocytosis,erythrocyte immune function,NK cell activity,peripheral blood lymphocytes and their subsets were measured.Results:（1）The mice in each dose group of"Ruyanning"water extract did not die and were in good physical condition.The LD₅₀ was more than 40000 mg/kg and the maximum tolerance was 80 g/kg according to the pathological changes of tissues and organs.（2）The water extract of"Ruyanning"at different concentrations had no significant effect on body weight,blood routine index,blood biochemical index,organ index and organ structure of mice.（3）The MIC of the water extract of"Ruyanning"against Staphylococcus aureus,Streptococcus lactis and Escherichia coli were 0.0625,0.0625 and 0.125 g/m L respectively;MBC is 0.125,0.125 and 0.250 g/m L respectively.The diameter of bacteriostatic ring of"Ruyanning"water extract（1.6g/m L）against Staphylococcus aureus,Streptococcus lactis and Escherichia coli was 11.38,11.56 and 11.23mm respectively,which was moderately sensitive;the protective rate of mice infected with mixed pathogenic bacteria is 62.5%,and the bacteriostatic effect is good.（4）Compared with the blank control group,the lung index in the middle and high dose groups of"Ruyanning"water extract was significantly higher（P<0.05）;the contents of TG,CREA,BUN,ALT and ALP in serum of mice in each dose group of"Ruyanning"water extract were significantly decreased（P<0.05）,in serum of mice in the middle dose group,the contents of LDL,IL-1β,IL-4,IL-6,IL-2,IL-10,Ig G,IL-17 and Ig A increased significantly（P<0.05）,AST content decreased significantly（P<0.01）,the content of IL-6 in liver was significantly decreased（P<0.05）;the water extract of"Ruyanning"can significantly increase the activity of CAT,GSH-Px,SOD and T-AOC in serum and liver tissue of mice（P<0.05）,and reduce the content of MDA（P<0.05）.（5）Compared with the blank control group,the thymus index of mice in the middle-dose group of"Ruyanning"water extract was significantly higher（P<0.01）,the clearance index and phagocytic coefficient increased significantly（P<0.05）,the percentage of C_3bRR in red blood cells of mice increased significantly（P<0.05）,NK cell activity increased significantly（P<0.05）,the percentage of CD3⁺cells in peripheral blood of mice increased significantly（P<0.05）.Conclusion:（1）"Ruyanning"water extract LD₅₀>40000 mg/kg,maximum tolerance is 80 g/kg.（2）The concentration of the water extract of"Ruyanning"used in the test has no subchronic toxicity to mice.（3）The water extract of"Ruyanning"has antibacterial effect on Staphylococcus aureus,Streptococcus lactis and Escherichia coli,and the best effect is at the concentration of 1.6 g/m L.（4）Each dose group of"Ruyanning"water extract can improve the antioxidant function of mice,and 0.8 g/m L has the best effect.（5）The water extract of"Ruyanning"can improve the immune function of mice at the concentration of 0.8 g/m L and 1.6g/m L,and 0.8 g/m L has the best effect.