Experimental Study On Acute Toxicity And Antipyretic Effect Of Aqueous Extract Of Ageratum Conyzoides.L. From Guangxi

Objective:To study the acute toxicity of the aqueous extract of Ageratum Conyzoides.L.from Guangxi on mice and to evaluate its safety.At the same time,it explores the antipyretic activity and possible mechanism of action of the aqueous extract of Ageratum Conyzoides.L.from Guangxi,and providing experimental and theoretical basis for further research on antipyretic activity.Methods:?1?Acute toxicity tests of aqueous extract of Ageratum Conyzoides.L.from Guangxi:The mice were tested with aqueous extract of different concentrations of Ageratum Conyzoides.L.from Guangxi,and their median lethal dose(LD₅₀) was measured to observe the survival of the mice.The mice were intragastrically administered with the maximum volume and the maximum concentration of aqueous extract of Ageratum Conyzoides.L.from Guangxi.The mice were continuously administered twice within 24 hours.Mice in the control group were given the same volume of double distilled water to observe the drug administration,and to observe the general toxicity symptoms,diet,and body weight changes after administration in mice in the administration group and the control group.After 14 days,blood was taken from the eyeball to detect the biochemical indexes such as ALT,AST,AKP,TP,ALB,CRE and BUN;the mice were sacrificed after cervical dislocation;the organs were observed by eyes;the heart,liver,spleen,lung,left and right kidney were weighed;the heart,liver,spleen,lung,left and right kidneys of the mice were dissected to calculate the visceral index,and Hematoxylin-eosin Staining was made to observe the pathological morphology of the viscera;the maximum dose?MTD?was calculated.?2?Antipyretic effect and its mechanism experiment of the aqueous extract of Ageratum Conyzoides.L.from Guangxi:Healthy male Sprague-Dawley rats were adapted for feeding for 3 days.The average body temperature was measured twice before the experiment as the basal body temperature of the rats.Except for the normal group,the rest of rats were subcutaneously injected with 20% yeast suspension 10 m L/kg to induce fever.After successful modeling of the building,according to the body temperature,the dosage of the model group,the aqueous extract of Ageratum Conyzoides.L.from Guangxi high-does group?50.14 g crude/kg?,the middle-does group?25.07 g crude/kg?,the low-does group?12.535 g crude/kg?and the aspirin group?100mg/kg?,mice were given intragastric administration,and the rectal temperature was measured once every 30 minutes for 3 hours,to observing changes in body temperature in rats to draw a cooling curve.The content of IL-1?,TNF-?and c AMP in serum and the content of PGE2,5-HT,and c AMP in the hypothalamus were detected by Enzyme Linked Immunosorbent Assay.The quantitative expression of IL-1?,TNF-?,COX-1 and COX-2 mRNA in the hypothalamus of rats was detected by Reverse Transcription PCR.Immunohistochemical staining sections of hypothalamus were made to observe and quantify the expression of COX-1 and COX-2 protein in the hypothalamus of rats in each group.Results:?1?Acute toxicity tests of aqueousextracts of Ageratum Conyzoides.L.from Guangxi:There was no death in the experimental mice,and the LD50 of aqueous extract of Ageratum Conyzoides.L.from Guangxi.could not be measured,the MTD of acute toxicity was 200.56 gcrude /kg.After 14 days of observation,the feeding amount and body weight of the mice in the administration group increased,but there was no significant difference between the blank group and the control group?P>0.05?.There was no significant difference in viscera index between each administration group and the blank group?P>0.05?;The results of Hematoxylin-eosin Staining showed no obvious pathological changes in heart,liver,spleen,lung,left kidney and right kidney of the blank group and the administration group;The serum levels of AST,ALT,AKP,TP,ALB,BUN and CRE were not significantly different from those in the control group?P>0.05?.?2?Antipyretic effect and its mechanism experiment of Ageratum Conyzoides.L.from Guangxi:After modeling,compared with the blank group,the body temperature of each group significantly increased?P<0.01?;After 3 hours of administration,compared with the rectal temperature of the model group,body temperature of each dose group of the Ageratum Conyzoides.L.from Guangxi and aspirin group were significantly reduced?P<0.01?.Compared with the model group,the content of IL-1? in the serum of each dose group of Ageratum Conyzoides.L.from Guangxi and aspirin group showed a significant decrease?P<0.01 or P<0.05?,Ageratum Conyzoides.L.from Guangxi high-dose,middle-dose group and aspirin group can significantly reduce serum TNF-? and c AMP levels?P<0.01 or P<0.05?,the content of low-dose group has a decreasing trend,but there is no statistical difference compared with the model group?P>0.05?;Compared with the model group,the concentration of PGE2 and c AMP in the hypothalamus of rats in the Ageratum Conyzoides.L.from Guangxi high and middle dose groups and aspirin groups was significantly lower?P<0.01 or P<0.05?,while the low-dose group had a decreasing trend,but with compared with the model group,the difference was not statistically significant?P>0.05?;There was no significant difference in the content of 5-HT between the hypothalamus and the model group?P>0.05?.Compared with the model group,there was no significant difference in the expression of TNF-? and COX-1 m RNA in the hypothalamus between the aspirin group and groupsof the Ageratum Conyzoides.L.from Guangxi?P>0.05?,while IL-1 ? and COX-2 mRNA expression was significantly decreased compared with the model group,and the difference was statistically significant?P<0.01 or P<0.05?.The results of Hematoxylin-eosin Staining showed that compared with the model group,the loose edema and demyelination of hypothalamus in the Ageratum Conyzoides.L.from Guangxi high and middle dose group and aspirin group were obviously improved,but no obvious change was found in the low dose group.The results of Immunohistochemical Staining showed that COX-1 showed positive reaction in hypothalamus cytoplasm of each administration group,model group and normal group.The COX-2 in hypothalamus of normal group was negative,but compared with model group,the brown granules of hypothalamus in Ageratum Conyzoides.L.from Guangxi high,medium and low dose groups and aspirin group were significantly decreased.Compared with the model group,there was no significant difference in average optical density of COX-1 protein between the aspirin group and drug administration group?P>0.05?.the COX-2 protein was significantly decreased in the aspirin and Ageratum Conyzoides.L.from Guangxi groups with high and middle doses?P<0.05?,while was no statistical significance in the low dose group?P >0.05?.Conclusions: 1.Aqueous extracts of Ageratum Conyzoides.L.from Guangxi had no obvious toxicity for mice and is safe for use.The maximum dose was 200.56 gcrude/kg.2.The aqueous extracts of Ageratum Conyzoides.L.from Guangxi had obvious antipyretic effect on dry yeast heat rats,the mechanism of the antipyretic effect may be through reducing the serum endogenous pyrogenic IL-1?,TNF-?and c AMP levels in fever rats,and reducing the content of PGE₂ and c AMP in the hypothalamus,related to the down-regulation of IL-1?gene,the inhibition of COX-2 gene and protein expression,and no effect on the 5-HT,COX-1 in the hypothalamus.