| The long-term,large-scale and unreasonable use of chemical pesticides has caused some increasing problems such as safety of agricultural products,environmental damage,and resistance.The development of plant-derived pesticides has a great significance.Chinese chive?Allium tuberosum Rottl.ex Spreng?belongs to the genus Allium of the genus Liliaceae,which is a medicinal and edible plant in China,and its extract has good insecticidal properties.However,its mechanism of action and the main biological active substances are not clear.In this study,Plutella xylostella?Linnaeus?was used as test insects to study the insecticidal activity,and the secondary metabolites were isolated and identified by phytochemical methods and the main biological active ingredients and mechanism of the Chinese chive were discussed.The main findings are as follows:1.The agar-moisturizing leaf-leaching method and spot-drop method were used as the guide to evaluate the insecticidal activity of the essential oil and ethanol extracts of Chinese chive.At the same treatment concentration,the essential oil of Chinese chive?EOC?had a better repellent activity against to P.xylostella larvae than the ethanol extracts and thioether compounds.Especially,when the concentration was 5?L/mL,the repellent rate of EOC was96.67ą0.82%?mąSE?;the treatment concentration of ethanol extracts is 10000 mg/L,the avoidance rate can reach 83.33ą0.82%.And the LC500 of the contact toxicity of the ethanol extracts to Plutella xylostella was 3001.88 mg/L,but EOC was 0.56?L/mL.2.The composition and content of volatile components of Chinese chive were analyzed by hydro-distillation?HD?and Gas Chromatography-Mass Spectrometer?GC-MS?.The results showed that the content of volatile compounds in fresh Chinese chive was much higher than in steamed.There were 27 volatile compounds in fresh Chinese chive and 19 compounds in steamed,in addition,the pungent odor of steamed Chinese was significantly reduced.The rat kidney normal cells and liver cells were used as targets to evaluate the cytotoxicity of fresh and steamed Chinese chive.The results showed that the volatile components of fresh Chinese chive showed some toxicity to the normal rat liver cells and kidney cells.When the treatment concentration was 100?g/mL,the viability of these two cells decreased significantly,were25%and 30%respectively.3.Extraction from Chinese chive with 75%ethanol was extracted at room temperature three times and purified by HP-20 macroporous absorption resin.Then 10%,30%,50%,70%and 100%ethanol eluted respectively.The elutions were isolated by Sephadex LH-20 columnchromatography,and preparative HPLC system.46 compounds were identified on the basis of physic-chemical characters and spectroscopic analysis such as UV IR,NMR and MS,comparied with standard products and references,46 compounds were identified.These compounds are deoxyuridine?1?,thymine?2?,deoxythymidine?3?,thymidine?4?,guanosine?5?,deoxyguanosine?6?,adenine?7?,adenosine?8?,deoxyadenosine?9?,1-methyl-l,2,3,4-tetrahydro-carboline-3-carboxylic acid?10?,D-tryptophan?11?,D-phenylalanine?12?,?R?-2-?1-ethoxyl?-5-?2-ethoxyl?-pyrazine?13?,2-methyl-3-methylol-5-ethyl-pyrazine?14?,?R?-2-?1-ethoxyl?-6-ethyl-pyridazine?15?,?R?-2,3-di-methyl-6-?1-ethoxyl?-pyridazine?16?,2-[?1R,2R?-2-hydroxy-2-?4-hydroxy-3-methoxyphenyl?-1-?hydroxymethyl?-ethoxy]-5-?3-hydro xypropyl?-phenyl-?-D-glucopyranoside?17?,hydroxybenzoic acid?18?,?-D-glucopyranoside,4-hydroxy-3-methoxyphenyl?19?,4-[?2S,3R?-2,3-dihydro-3-?hydroxymethyl?-5-?3-hydroxypropyl?-7-methoxy-2-benzofuranyl]-2-methoxyphenyl-?-D-glucopyranoside?20?,?7R,8S?-dihydrodehydrodiconiferyl alcohol-di-9,9'-O-?-D-glucopyranoside?21?,[?2S,3R?-2,3-dihydro-2-?4-hydroxy-3-methoxyphenyl?-5-?3-hydroxypropyl?-7-methoxy-3-benzofuranyl]methyl-?-D-glucopyranoside?22?,2,3-dihydro-2-?4-hydroxy-3-methoxyphenyl?-3-?hydroxymethyl?-7-methoxy-,?2S,3R?-5-benzof uranpropanol?23?,3-?3,4-dihydroxyphenyl?-?2E?-2-propenoic acid?24?,1-[3-?4-hydroxy-3-methoxyphenyl?-2-propenoate]-D-glucopyranose?25?,quercetin-3-O-?6-trans-feruloyl?-?-D-glucopyranosyl-?1?2?-?-D-glucopyranoside-7-O-?-D-gl ucopyranoside?26?,kaempferol-3-O-?6-trans-feruloyl?-?-D-glucopyranosyl-?1?2?-?-D-glucopyranoside-7-O-?-Dglucopyranoside?27?,quercetin-3-O-?6-trans-p-coumaroyl?-?-D-glucopyranosyl-?1?2?-?-D-glucopyranoside-7-O-?-D-glucopyranoside?28?,3-[[2-O-[6-O-[?2E?-3-?3,4-dihydroxyphenyl?-1-oxo-2-propen-1-yl]-?-D-glucopyranosyl]-?-Dglucopyranosyl]oxy]-7-??-D-glucopyranosyloxy?-5-hydroxy-2-?4-hydroxyphenyl?-4H-1-benzo pyran-4-one?29?,7-??-D-glucopyranosyloxy?-5-hydroxy-2-?4-hydroxyphenyl?-3-[[2-O-[6-O-[?2E?-3-?4-hydroxy phenyl?-1-oxo-2-propen-1-yl]-?-D-glucopyranosyl]-?-D-glucopyranosyl]-oxy]-4H-1-Benzopyr an-4-one?30?,3-phenylpropyl-?-D-glucopyranoside?31?,3,7-bis??-D-glucopyranosyl-oxy?-5-hydroxy-2-?4-hydroxyphenyl?-4H-1-benzopyran-4-one?32?,7-[?6-deoxy-?-L-mannopyranosyl?-oxy]-3-[?2-O-?-D-glucopyranosyl-?-D-glucopyranosyl?-oxy]-5-hydroxy-2-?4-hydroxyphenyl?-4H-1-benzopyran-4-one?33?,3-[?2-O-?-D-glucopyranosyl-?-D-glucopyranosyl?-oxy]-5,7-dihydroxy-2-?4-hydroxyphenyl?-4H-1-benzopyran-4-one?34?,3-[?2-O-?-D-glucopyranosyl-?-D-glucopyranosyl?-oxy]-7-??-D-glucopyranosyloxy?-5-hydroxy-2-?4-hydroxyphenyl?-4H-1-benzopyran-4-one?35?,2-?3,4-dihydroxyphenyl?-3-[?2-O-?-D-glucopyranosyl-?-D-glucopyranosyl?-oxy]-5,7-dihydrox y-4H-1-benzopyran-4-one?36?,ethyl-?-D-xylopyranoside?37?,butyl-?-D-glucopyranoside?38?,p-methylphenol?39?,lignan-8-O-4'-glucoside?40?,3-[?2S,3R?-2,3-dihydro-2-?4-hydroxy-3-methoxyphenyl?-3-?hydroxymethyl?-7-methoxy-5-benzofuranyl]propyl-?-D-Glucopyranoside?41?,3-[2-[4-??-D-glucopyranosyloxy?-3-methoxyphenyl]-2,3-dihydro-3-?hydroxymethyl?-7-methox y-5-benzofuranyl]propyl-?2-trans?-?-D-Glucopyranoside?42?,kaempferol?43?,7-[?4-O-?-D-Glucopyranosyl-?-D-glucopyranosyl?oxy]-3,5-dihydroxy-2-?4-hydroxyphenyl?-4H-1-benzopyran-4-one?44?,quercetin?45?and ferulic acid?46?.Compounds 13,14,15,16,21,26,27,and 28 were the new compounds,wherein compounds 13,14,15 and 16 were pyrazine compounds with a particular roasted aroma of Chinese chive,compounds 26,27 and 28 are new flavonoids which isolated from Chinese chive.4.The Plutella xylostella larvae was used as test insects,and the agar agar-soaking leaf method was used as a guide to study the repellent activity of the new pyrazine compounds.The action mechanism of EOC,ethanol extracts of Chinese chive?CE?and pyrazine compounds were discussed by using the antennal potential method and enzyme assays.At the same concentration,the activities of?R?-2-ethoxy-6-ethyl-pyridazine and?R?-2-hydroxy-5-hydroxyethyl-pyrazine were significantly better than the others.When the concentration ? 1 mg/m L,?R?-2-Ethoxy-6-ethyl-pyridazine has the best avoidance activity.With a treatment concentration of 5 mg/m L,the repellence rate of?R?-2-ethoxy-6-ethyl-pyridazine against Plutella xylostella can reach 100%.The activity of pyrazine compounds was better than the EOC and CE.In EAG response measurement experiments,it can be found that there was a good dose-response relationship between EAG response and treatment concentration,and the EAG response result was consistent with the performance of the repellent activity.When the treatment concentration of?R?-2-ethoxy-6-ethyl-pyridazine was 100 ?g/m L,the EAG response to Plutella xylostella could reach 5.68 m V,at the same concentration,the EOC was 4.33 m V.This result was consistent with the experimental results of repellent activity,which can be specaluted that EOC andpyrazines showed repellence activity through the antennae of Plutella xylostella.In the enzyme assays,EOC and CE can inhibit the activities of two important detoxification enzymes GSTs and CarE,and the results were consistent with the bio-assay results.This article clarified the repellent and contact activity of the Chinese chive extract against to Plutella xylostella larvae,determined the main insecticidal active ingredients in Chinese chive,and revealed the repellent activity and mechanism of pyrazine compounds on the tested insects.The results provided a theoretical basis for the development of insecticides based on Chinese chive as a resource and established a foundation for the discovery of new insecticidal lead compounds. |
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