Analysis Of Chemical And Nutritional Components And Toxicological Safety Evaluation Of Ambrosia Trifida

Ambrosia trifida is an annual herb of Ambrosia genus,Compositae,which is widely distributed in 23 provinces and autonomous regions of central China,east China,south China,north China,northeast China,southwest China（Guizhou）and northwest（Xinjiang）.As an alien invasive species in China,it has the characteristics of vigorous vitality,strong regeneration,rapid diffusion and great destruction.In China,large scale plant population dominance has been formed,which poses a great threat to China’s biosecurity,ecological security,environmental security and human health.The severity of the harm has aroused great attention from environmental protection departments at all levels in China.At present,the research on A.trifida is mainly focused on biological and ecological characteristics,occurrence and spread rule,chemical composition and allelopathy,harm and prevention and control techniques,but basic toxicological data about A.trifida toxicity,main toxic substances and their mechanism of toxic action are still lacking.Therefore,this study took A.trifida as the experimental object.Through systematic chemical composition pretest,alkaloid component TLC analysis and GC-MS detection,the chemical composition of A.trifida was preliminarily determined,especially the species and content of alkaloids.The nutritional value of A.trifida was analyzed and evaluated by routine nutrient composition,mineral element content and amino acid type and content determination.Through the acute toxicity and subchronic toxicity tests of mice,the toxic effects of A.trifida on mice were clarified.The cell activity test was conducted to screen and evaluate the cytotoxicity of extract parts of A.trifida,which provided the basis for the confirmation of toxic substances.This study is not only insufficient to complete the basic toxicological data of A.trifida,but also can provide an important theoretical basis for further study on the toxic mechanism of A.trifida and resource utilization.The following results were obtained:1.Analysis of chemical constituents and alkaloids in A.trifida:Through systematic preliminary test of chemical constituents,it was detected that A.trifida contained alkaloids,terpenoids,steroids,volatile oils and lipids,amino acids,glycosides,polypeptides,sugars,tannins,organic acids,phenols,flavonoids and terpenoids.Traditional alkaloid extraction of A.trifida alkaloid extraction and separation,by TLC and GC-MS detection showed that alkaline methylene chloride and alkaline n-butanol extraction yield of 0.40%and 1.86%respectively,thin layer chromatography detection respectively in 5 and 1 orange flecks,alkaloids component part focused on alkaline methylene chloride.Three alkaloids,N,N-Dimethylethanolamine,1-Pyrrolidinecarboxaldehyde and 1-Acetylpyrrolidine were identified by GC-MS,and compared with the mass spectrometry database.The content of three alkaloids was 0.16%,1.34%and 0.19%,and the content of 1-Pyrrolidinecarboxaldehyde was the highest.2.Nutrient composition analysis of A.trifida:The results showed that the contents of crude protein,crude fiber,crude fat,crude ash and dry matter in conventional nutrients were9.55%,62.62%,1.20%,11.88%and 90.56%.The contents of Ca,P,Mg,Fe,Zn,Mn,Cu and Se were 23487.25 mg/kg,3178.75 mg/kg,3530.75 mg/kg,356.18 mg/kg,40.19 mg/kg,33.54mg/kg,10.65 mg/kg and 0.15 mg/kg;In addition,There are 17 kinds of amino acids,with a total content of 8.19%and 9 essential amino acids,accounting for 42.27%of the total amino acid content and 8 non-essential amino acids,accounting for 57.72%of the total amino acids.The above results showed that A.trifida has abundant nutrients,,reasonable ratio and the potential of forage development and utilization.3.Acute toxicity test of A.trifida:the acute toxicity test of A.trifida was detected by intragastric poisoning of mice according to toxicological test method.Accoriding to this study,the mice in the high-dose group had the clinical symptoms showing respiratory and neurological dysfunction,compared with the mice in the low-dose group which developed similar symptoms.The LD₅₀ of A.trifida extract was 45.90 g/kg with 95%confidence limit of40.01～53.23 g/kg.According to the classification standard of toxicity,clinical manifestation and mortality,A.trifida was judged to be weakly toxic.4.Subchronic toxicity test of A.trifida:Mice were fed with rat chow supplemented with different doses of grass meal（0%,20%and 40%）for 90 days.Clinical symptoms of the mice were recorded,body weight and physiological and biochemical indexes were analyzed,and tissue sections were made to observe pathological changes.The results showed that the low-dose group did not show clinical symptoms,and part of the high-dose group developed clinical symptoms.The body weight and organ coefficient of the mice did not change significantly compared with the control group,however the blood physiological indexes and serum biochemical indexes were changed in two experimental group.Physiological indexes of PLT,HCT,MCV,WBC,Lymph and HGB decreased,biochemical indexes of ALT,TP,GLO,CRE and T-chol decreased,and A/G and BUN increased.There was a statistical difference between the control mice and treatment mice.Histopathological results have shown minor damag to the liver,kidney and lung tissues in the low-dose group,and significant damage to the liver,kidney,lung,cerebellum and ovarian tissues in the high-dose group.5.Cytotoxicity test of A.trifida:CCK-8 test was used to explore the effect of activity of extracts from five extracts on TCMK-1 cells,and IC₅₀ of each extract was calculated.The results showed that The IC₅₀ was 956.4μg/m L for Alkaline dichloromethane site of A.trifida,with 95%confidence interval 771.8～1185.0μg/m L.The IC₅₀ of dichloromethane site was4322.0μg/m L,with 95%confidence interval 4198.0～4450.0μg/m L.The IC₅₀ of basic n-butanol site was 3389.0μg/m L,with 95%confidence interval of 2147.0～5349.0μg/m L.The IC₅₀ of n-butanol site was 2294.0μg/m L,with 95%confidence interval 1719.0～3063.0μg/m L,and the IC₅₀ of petroleum ether site was 3681.0μg/m L,with 95%confidence interval3479.0～3895.0μg/m L.The toxicity of different extraction sites to TCMK-1 cells was different,and the order was alkaline dichloromethane site>n-butanol site>basic n-butanol site>petroleum ether site>dichloromethane site.It could be seen that the alkaline dichloromethane site was the most toxic and had the most obvious inhibition effect.