Study On The Toxicity And Antibacterial Activity Of Extracts Of Eupatorium Odoratum

Objective:To study the acute toxicity and cytotoxicity of Eupatorium odoratum extract in mice and to provide the basis of safe medication for further research on Eupatorium odoratum.At the same time,through the in vivo and in vitro antibacterial activity,the antibacterial effect of the extract of Eupatorium odoratum was detected,which provided the basis for the clinical research of new antibacterial drugs and provided the basis for the study of the antibacterial mechanism of Eupatorium odoratum.Methods:1.Acute toxicity study:Through the lethal dose method(LD₅₀)and the maximum dose method with intragastrical administration,the acute toxicity symptoms of the mice were observed within 7 days after the administration and the hematological changes were detected.At the same time,the mice were dissected and visually observed important organ changes,and the pathological changes of liver,lung and kidney were observed by HE staining.2.Cytotoxic activity:The extracts of Eupatorium odoratum were prepared with different concentration gradients.The cytotoxicity of each extract to Vero cells by adding CCK-8 reagent was detected,then calculate the IC₅₀and 95%confidence interval of each extract.3.In vitro antibacterial activity:The diameter of the zone of inhibition of each extract to the bacterias was measured by the paper diffusion method;the MIC of each Eupatorium odoratum extract was detected by the micro broth dilution method.4.In vivo antibacterial activity:According to the results of antibacterial activity in vitro,the suspension of the extract of leaves,the ethanol extract of the stems,the acetone extract of the stems and the ethanol extract of the aerial part were prepared,and Staphylococcus aureus ATCC25923 was selected as the experimental strain,and the bacterial solution was injected intraperitoneally.Intragastric adminstration after half an hour,observe and record the symptoms and mortality of mice in each group,and dissect mice to observe different organ changes and pathological changes in the livers,lungs,and kidneys by HE staining.Results:1.Acute toxicity study:The LD₅₀of the acetone extract of the stem of the experimental group was 25.29 g/kg,and 95%confidence interval was（22.54-28.38）the LD₅₀of the acetone extract of the aerial part was 23.96 g/kg,and 95%confidence interval was（21.35-26.88）.The MTD of the methanol extract of the leaves,the ethanol extract of the leaves,the acetone extract of the leaves,the water extract of the leaves,the methanol extract of stem,the ethanol extract of stem,the water extract of stem,the methanol extract of aerial part,the ethanol extract of aerial part and the water extract of aerial part could be measured,and the MTD values of which are 35.2g/kg,48.4 g/kg,25.44 g/kg,73.2 g/kg,34.8 g/kg,41.2 g/kg,58.8 g/kg,32.4 g/kg,44 g/kg,64 g/kg.Compared with the normal group,the WBC of the ethanol extracts of leaves,the acetone extracts of leaves,the acetone extracts of stems,and the acetone extracts of aerial part was significantly different from that of normal group,P<0.05;the NEUT%in the acetone of leaves extracts was significantly different from that normal group,P<0.05;ALT in the water extract of leaves,the acetone extract of stems,and the acetone extract of aerial partes was significantly different from the normal group,P<0.05;LYMPH%,RBC,HGB,PLT,AST,Scr and TBIL of the Eupatorium odoratum groups are not significantly different from the normal group.The pathological results show that different kind of extract causes different degrees of damage to liver,lung and kidney,and the pathological damage of water extract was relatively light.2.Cytotoxic activity:The extracts from different parts of the Eupatorium odoratum have different degrees of toxicity to Vero cells,and the higher the concentration of the extract of the Eupatorium odoratum,the greater the toxicity to cells,among which acetone extracts of the Eupatorium odoratum are the most toxic and water extracts of the Eupatorium odoratum are the least toxic.3.In vitro antibacterial activity:The diameter of the zone of inhibition in methanol extracts of leaves,ethanol extracts of leaves,acetone extracts of leaves,water extracts of leaves,ethanol extracts of stems,acetone extracts of stems,ethanol extracts of aerial part against S.aureus ATCC25923 were 11 mm,11 mm,11.5 mm,10 mm,12 mm,12.5 mm,11 mm;the diameter of the zone of inhibition in methanol extracts of leaves,ethanol extracts of leaves,acetone extracts of leaves,water extracts of leaves,ethanol extracts of stems,acetone extracts of stems,ethanol extracts of aerial part against S.aureus CMCC26001 were 12 mm,11 mm,12 mm,11 mm,8 mm,13 mm;the diameter of the zone of inhibition in methanol extract of the leaves,ethanol extract of the leaves,acetone extracts of leaves,methanol extract of stem,ethanol extract of stem,acetone extract of stem,methanol extract of aerial part,ethanol extract of aerial part,acetone extract of aerial part against pneumonia Streptococcus ATCC49619 were 11 mm,11.5 mm,11 mm,11 mm,12 mm,12 mm,12.5 mm,13 mm,12 mm.the diameter of the zone of inhibition of water extract of leaves against E.coli ATCC25922 and P.aeruginosa ATCC27853 were 7.5 mm and 9mm respectively.the minimum in hibitory concentration（MIC）of each extract was different for different experimental strains.4.In vivo antibacterial activity:After intragastric administration to the mice infected with S.aureus ATCC25923,the mortality rate of the mice in the methanol extract of the leaves,the ethanol extract of the leaves,the acetone extract of the leaves,the water extract of the leaves,the ethanol extract of stem,the acetone extract of stem and the ethanol extract of aerial part were 20%,50%,80%,0,50%,80%,and 70%.the results showed that the water extract of leaves and the methanol extract of leaves had the strongest protective effect on infected mice.The WBC and the NEUT%in the experimental group were significantly different from those in the normal group,P<0.001.the pathological results showed that the liver,lung and kidney of the mice in the each experimental group had different degrees of damage.The lung damage was the most serious,manifested by a large number of inflammatory cell infiltration,congestion and edema of lung tissue,and fibrosis in some groups.Conclusions:According to the results of LD₅₀and MTD of the extracts,we found that the extracts were actually non-toxic to the mice after oral administration,but they can cause hematological changes and pathological damage to liver,lung and kidney after high-dose administration.The results of cytotoxic activity show that differnt extract has different degrees of toxicity to cells,but the water extracts had the least toxicity,which can provide the basis for safe medication in clinic.In vivo and in vitro antibacterial activity showed that the extracts of Eupatorium odoratum had a certain antibacterial effect,especially on Gram-positive bacteria（S.aureus and Streptoccus pneumoniae ATCC49619）.Only the water extracts of leaves had a weak antibacterial effect on Gram-negative bacteria（E.coli ATCC25922 and P.aeruginosa ATCC27853）.Combined with the toxicological results of the extract,we can provide a scientific basis for the clinical development of new antibacterial drugs.