Isolation Of Active Substances From Perilla Frutescens Against Stored Pests Of Traditional Chinese Medicine And Its Underlying Mechanism

Traditional Chinese medicine materials is the basis of traditional Chinese veterinary medicine,and its quality is directly related to the clinical curative effect.However,the control of pests in storage of traditional Chinese medicinal materials is a weak link in the modern industrial chain of traditional Chinese veterinary medicinal.At present,insect pests in stored products are controlled primarily with synthetic insecticides.However,excessive and inappropriate use of synthetic insecticides has led to human health risks,environmental pollution,pest resistance and pesticide residues.Hence,it is necessary to find safe and effective methods to control storage pests.In this study,Perilla frutescens was used as the research object,and Tribolium castaneum were used as the target insects.Chemical composition,biological activity and action mechanism of Perilla frutescens against Tribolium castaneum were investigated.It provided valuable compounds for the development of plant insecticide and basis for comprehensive utilization of Perilla frutescens resources.Main results and conclusions of this work are as follows:1.The volatile components of four essential oils of Perilla frutescens from three different habitats were analyzed by gas chromatography-mass spectrometry,and 63components were identified.The main components of essential oil from Perilla frutescens-1in Beijing were carvone（39.04%）,2-furyl methyl ketone（23.56%）and perilla aldehyde（6.72%）.The main components of essential oil from Perilla frutescens-2 and Perilla frutescens（Baisu）in Liaoning wereβ-caryophyllene（58.31%）、2-furyl methyl ketone（85.77%）andβ-asarone（11.70%）.The main components of essential oil from Perilla frutescens-3 in Guangxi were perilla aldehyde（59.65%）,limonene（11.86%）andβ-caryophyllene（11.45%）.It can be seen that the chemical composition and content of Perilla frutescens essential oil from different habitats are different,which may be caused by different collection sites,collection time and geographical environment.In addition,the contact activity of the four essential oils was evaluated.Among them,Perilla frutescens-1 essential oil showed the strongest contact toxicity(LD₅₀=9.30μg/adult),followed by Perilla frutescens-3 essential oil(LD₅₀=13.74μg/adult),Perilla frutescens（Baisu）essential oil(LD₅₀=32.32μg/adult)and Perilla frutescens-2 essential oil(LD₅₀=37.24μg/adult),indicating that the differences in the chemical composition and content of the essential oil may cause different insecticidal activities.2.The optimized essential oil of Perilla frutescens-1 with the most prominent contact toxicity was used for chemical composition study.Four compounds were isolated and identified from the essential oil by silica gel column chromatography and NMR spectral structure analysis,including carvone,2-furyl methyl ketone,perilla aldehyde,β-caryophyllen.The contact toxicity and repellent activity of Perilla frutescens monomer compounds were tested.The Perilla frutescens monomer compounds showed contact and repellent activities against Tribolium castaneum.Perilla aldehyde has good contact activity(LD₅₀=7.71μg/adult).At the same time,perilla aldehyde showed significant repellent activity.At the concentrations of 78.64 and 15.72 n L/cm~2,perilla aldehyde showed strong repellent activity against Tribolium castaneum at 2 h and 4 h,and the percent repellency were more than 90%.At the lowest concentration of 0.12 n L/cm~2,perilla aldehyde still showed good repellent activity against Tribolium castaneum at 2 h and 4 h,and the percent repellency were more than 50%.3.Perilla aldehyde and carvone with the best contact activity were selected for synergistic study.The contact activity of 10 formulations（carvone:perilla aldehyde=9:1,4:1,7:3,3:2,1:1,2:3,3:7,1:4,1:9,5.81:1）were tested,SR values were greater than 1.5,and all showed synergistic effect.When the ratio of carvone to perilla aldehyde was 4:1,the synergistic effect was the most obvious.At this time,the contact toxicity(LD₅₀=2.73μg/adult)of formula 2 was 2.8 and 4.0 times that of perilla aldehyde(LD₅₀=7.71μg/adult)and carvone(LD₅₀=10.93μg/adult),respectively.4.Transcriptome sequencing was performed for perilla aldehyde,carvone and mixed compound combination of Tribolium castaneum.Compared with the control group,1425,415and 164 genes were differentially expressed after 24 h treatment with carvone,perilla aldehyde and mixture,respectively.Among which 462,99,56 genes were up-regulated and963,316,108 genes were down-regulated.The differentially expressed genes related to detoxification metabolism were further screened.8 OBPs,26 CYPs,7 Car Es,9 GSTs,3UGTs and 10 MPRs were detected in the transcriptome of carvone,and 1 OBPs,4 CYPs and6 Car Es were detected in the transcriptome of perilla aldehyde,while only 1 OBPs,2 CYPs,2Car Es and 1 GSTs were detected in the transcriptome of formula.By comparing the common differential genes in the three groups of samples,it was found that CYP9E2-like and CYP6BQ11 genes were up-regulated by 2.17 and 3.62 folds in the carvone group,and 1.52and 1.43 folds in the compound combination,while no difference was found in the perilla aldehyde group.Thus,the insecticidal activity of different components may be related to the number and expression level of differentially expressed genes in Tribolium castaneum under stress.