| Artemisia belongs to the Compositae family and has more than 350 species worldwide.Artemisia plants are abundant in China,with 187 species and 46 varieties distributed throughout the country.The vast majority of Artemisia plants are herbaceous,and only some are small shrubs or subshrubs.These plants are extremely adaptive and can grow at high altitudes and in extremely dry areas in Tibet,Artemisia plant finds its use in as medicine and animal feed in agriculture,food,grassland ecology and livestock.Metabolomics is a new bioomics technology,Plant Metabolomics,as an important branch of metabolomics,takes plant metabolites as the research object and uses qualitative and quantitative analysis methods to reveal the mechanism of plant metabolism.At present,the metabolomics analysis of Artemisia sieversiana,Artemisia wellbyi and Artemisia annua in Tibet and the biological activities of their extracts are remains unclear..In order to identify the metabolites of three Artemisia plants in Tibet,analyze their different metabolites and their metabolic pathways,and further study the biological activities and potential medicinal value of different extracts of three Artemisia plants.In this study,three Artemisia plants from Tibet were selected as research objects,and the metabolomics of three Artemisia plants and the biological activities of their different extracts were studied.The experimental results were as follows.1.Non-targeted metabolomics analysis of three Artemisia species in TibetIn order to understand the metabolites of Artemisia sieversiana,Artemisia wellbyi and Artemisia annua in Tibet and the differences of metabolites among different plants were investigated.For the first time,Non-targeted metabolomics was used to analyze three Artemisia species in Tibet.A total of 535 metabolites were detected from the three Artemisia species in the positive ion mode.A total of 220 metabolites were detected in the negative ion mode.The metabolites were mainly organic acids and their derivatives,ketones,phenols,terpenoids and coumarin.The differential metabolites of three Artemisia species were screened by principal component analysis,partial least square analysis and hierarchical clustering.In the positive-ion mode,262 different metabolites were screened from Artemisia sieversiana and Artemisia annua,312 different metabolites were screened from Artemisia wellbyi and Artemisia sieversiana,and 306 different metabolites were screened from Artemisia wellbyi and Artemisia annua.In the negative-ion mode,106 different metabolites were screened from Artemisia sieversiana and Artemisia annua,131 different metabolites were screened from Artemisia wellbyi and Artemisia sieversiana,and133 different metabolites were screened from Artemisia wellbyi and Artemisia annua.Artemisinin was detected for the first time in Artemisia wellbyi and Artemisia sieversiana.Artemisinin,as the representative differential metabolite,had the highest relative content in Artemisia annua.2.Widely targeted metabolomics analysis of three Artemisia species in TibetDue to the limited coverage and low sensitivity of non-targeted metabolomics technology,some metabolites had not been detected using this technology.Based on the previous non-targeted metabolomics analysis,in this study we applied a widely targeted metabolomics technique to comprehensively and accurately study the metabolites of three Artemisia plants in Tibet.A total of 1109 metabolites were comprehensively detected from three Artemisia species,there were 79 kinds of amino acids,101 kinds of nucleotides,155 kinds of lipids,168 kinds of phenolic acids,227 kinds of flavonoids,40 kinds of coumarin,86 kinds of alkaloids,56 kinds of terpenoids and 197 kinds of other classes.The differential metabolites of the three Artemisia species were further comprehensively analyzed by principal component analysis,OPLS-DA analysis and hierarchical clustering and 732 different metabolites were screened between Artemisia sieversiana and Artemisia annua,751 different metabolites were screened between Artemisia wellbyi and Artemisia sieversiana,768 different metabolites were screened between Artemisia wellbyi and Artemisia annua.Through the enrichment analysis of metabolic pathways,the differential metabolites of Artemisia sieversiana and Artemisia annua were mainly annotated and enriched in Purine metabolism pathway,2-Oxocarboxylic acid metabolism pathway,Tryptophan metabolism pathway;the differential metabolites of Artemisia sieversiana and Artemisia wellbyi were mainly annotated and enriched in Purine metabolism pathway,Tryptophan metabolism pathway;the differential metabolites in Artemisia wellbyi and Artemisia annua were mainly annotated and enriched in 2-Oxocarboxylic acid metabolism pathway,Purine metabolism pathway,Phenylpropanoid biosynthesis pathway.227 flavonoids and 56 terpenoid metabolites were identified from the three Artemisia species,including luteolin,quercetin,kaempferol,apigenin,artemisinin,artemisinin A,artemisinin B and dihydroartemisinin.These metabolites and their associated biological activities were determined.3.Bioactivity of different extracts of three Artemisia species in TibetIn the early stage,metabolomics analysis was carried out on Artemisia sieversiana,Artemisia wellbyi and Artemisia annua collected from Tibet.It was found that flavonoids and terpenoids were important metabolites,which provided theoretical basis for further study on their biological activities and medicinal value.The antibacterial activity of five extracts from three Artemisia species was tested,the results showed that it had different antibacterial activities against common pathogenic bacteria in veterinary clinic,including Staphylococcus aureus,Escherichia coli,Streptococcus,Salmonella,Bacillus subtilis,Proteus mirabilis and Pseudomonas aeruginosa.The petroleum ether extracts of three Artemisia plants showed better antibacterial activity.The total antioxidant capacity,scavenging capacity of DPPH free radical,ABTS free radical and hydroxyl free radical of five extracts from three Artemisia plants were determined.The results showed that the five extracts from three Artemisia plants had different antioxidant activities,the total antioxidant capacity of dichloromethane extract from Artemisia annua and Artemisia wellbyi was stronger,the total antioxidant capacity of the ethyl acetate extract from Artemisia sieversiana was stronger.Human hepatoma cells(Hep G2)were selected as the cell model in vitro,the petroleum ether extracts of three Artemisia plants were selected as the research objects in the preliminary test,the anticancer activity of petroleum ether extracts from three Artemisia plants was studied by MTT,Hoechst33258 nuclear staining and Western-blot.The results showed that the petroleum ether extracts of three Artemisia plants had significant inhibitory effects on the growth and proliferation of Hep G2 cells(P<0.05);Hep G2 cells were induced to undergo apoptosis,and the typical apoptotic characteristics such as dense chromatin,shrinking and strong bright white blue light were observed under fluorescence inverted microscope;after treatment on Hep G2 cells,the expression levels of mitochondria and death receptor-mediated apoptosis signaling pathway related proteins were changed,the expression levels of Bcl-2 protein were decreased(P<0.05),the expression levels of Bax,Fas and Fas L protein were increased(P<0.05),and it has a certain dose effect.This study was the first to systematically reveal the species of metabolite,differential metabolites and metabolic pathways of three Artemisia species in Tibet,and the biological activities of different extracts from three Artemisia plants were preliminarily discussed.The results showed that the metabolites of the three Artemisia plants in Tibet were rich in species and had obvious antibacterial,antioxidant and anticancer activities.The results of this study provide a new theoretical basis for the medicinal and comprehensive development and utilization of Artemisia in Tibet. |
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