Solvent-free Microwave Extraction Of Essential Oil From Elsholtiza Bodinieri Vaniot And Research On Its Nematodes Activity

Objectives:There is a great loss to global agriculture up to $100 billion every year due to plant nematodes.At present,the prevention and control of plant nematodes is still dominated by chemical control.Although chemical control has quick and effective,it poses a serious threat to human health and ecological security because of its characteristics of high toxicity,much residue and easy occurrence of drug resistance.At present,plant essential oil is a new source to prevention and cure nematode.And it is one of the research hotspots in the field of plant nematode control.This thesis makes a study of Elsholtiza bodinieri Vaniot which is a kind of rich natural plant resource in Yunnan province.The solvent-free microwave extraction method is adopted to extract essential oil from the plant and then GC-MS is used to analyze chemical constituents of the essential oil.Further,C.elegans is considered as the model organism to investigate the nematocidal activity of Elsholtiza bodinieri Vaniot.Finally,nematicides mechanism of the essential oil will be explored on the level of gene.The result will provide the scientific basis for the development and utilization of Elsholtiza bodinieri Vaniot and also development of green and new nematicides.Methods:The essential oil was extracted from Elsholtiza bodinieri Vaniot by solvent-free microwave extraction.The effects of material liquid ratio?soaking time?extraction time and microwave power on the yield of essential oil were tested and studied.Based the single factor experiment data and orthogonal test method,the solvent-free microwave extraction process for the was optimized.Gas chromatography-mass spectrometry(GC-MS)was used to analyze the chemical constituents of the essential oil.The 24h half-lethal concentration of essential oil on C.elegans was determined by acute toxicity test,and the toxicity of essential oil on reproduction and locomotion of C.elegans was evaluated at physiological level.After 24h exposure to essential oil,the gene library of up-regulated and down-regulated of C.elegans were obtained by RNA-seq sequencing method.Then,the main pathways of molecular functions and the biological processes of C.elegan were analyzed through the GO annotation and the KEGG annotation.All statistical analysis was performed using SPSS 21.0 software.P<0.05 was statistically significant.Results:The optimum extraction condition of solvent-free microwave extraction was that:material liquid ratio 1:6?soaking time 2h?extraction time 60min and microwave power 600 W,the yield of Elsholtiza bodinieri Vaniot essential oil was 0.096mL/50g.The chemical components were analyzed by GC-MS,and 43 peaks were separated and identified.The test results showed that the main component of the essential oil of Elsholtiza bodinieri Vaniot was terpinyl acetate,and its relative content was 56.25%,followed by geranyl propionate(6.8%)?paracetamol(4.83%)?caryophyllene oxide(4.41%),ccetic acid(Z)-5-lanolin alcohol(3.28%)?a-terpineol(2.91%)?thymol thyme(1.6%)?(S)-(+)-5-(1-hydroxy-1-methylethyl)-2-methyl-cyclohexene-1 ketone(1.18%)?terpineol(1.14%)and trimethyl(1.1%).The 24h half-lethal concentration(LC50)values of essential oil against C.elegans was 271mg·mL-1.The offspring number?head thrashes and body bends of C.elegans.in control group and treatment group were as follows:(183.1±3.88)pcs and(117.4±3.71)pcs?(87.2±2.92)times per minute and(55.3±3.30)times per minute?(29.4±3.33)times per 20s and(17.9±2.83)times per 20s.RNA-Seq analysis was carried out to explore the gene expression profiles of C.elegans in response to essential oil treatment using Illumina sequencing techno logy.We found 830 differentially expressed genes.Among them,381 genes were up-regulated and 449 genes were down-regulated.After GO annotation,the differentially expressed genes of C.elegans treated with essential oil were involved in three fields:molecular function,biological process and cell components.After GO annotation,some genes related to energy metabolism?transmembrane transport and oxidation-reduction process were highly expressed during nematode-trapping process;KEGG annotation revealed the metabolic pathways which were involved in differentially expressed genes include:fatty acid degradation?drug metabolism-cytochrome P450?metabolism of xenobiotics by cytochrome P450?lysosome and ABC transporters.Conclusions:Solvent-free microwave extraction has a high extraction rate of essential oil from Elsholtiza bodinieri Vaniot.Toxic effects of essential oils on C.elegans were studied,and the results showed that essential oil could inhibit the offspring number?head thrashes and body bends of C.elegans.After 24h exposure to essential oil,it showed that the essential oil had reproductive toxicity and neurotoxicity.After GO annotation,some genes related to energy metabolism?transmembrane transport and oxidation-reduction process were highly expressed during nematode-trapping process.In the KEGG enrichment analysis,two major detoxifying metabolic enzymes were identified:the detected P450 genes of drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450 could be up-regulated and the detected genes of ABC transporter could be down-regulated by essential oil.It was speculated that cytochrome P450 and ABC transporter played an important role in the metabolism of essential oils.