Isolation, Identificantion And Mechanism Of The Acaricidal Compound From Cinnamomum Camphora Against Tetranychus Cinnabarinus

The carmine spider mite,Tetranychus cinnabarinus?Boisduval??Acarina:Tetranychidae?,is highly polyphagous and is considered one of the most important crop pests,infesting over 100 crop species grown in field and greenhouse crops worldwides.Numerous mite biocontrol studies have been made due to the adverse effects of chemical pesticides,such as environmental pollution,resistance,toxicity,and pesticide residues.And pesticides based on plant extracts or phytochemicals are more frequently being considered for use as an alternative strategy to synthetic insecticide or acaricide treatments for controlling mite populations.In this study,the acaricidal properties of thirteen plant extracts from China were evaluated,and the leaf extract from Cinnamomum camphora?L.?Presl showed most significantly acaricidal activity against T.cinnabarinus.The active components of C.camphora were isolated and identified to be 2,4-di-tert-butylphenol and ethyl oleate.The mode of actions and stabilities of the two active compounds were evaluated.The biochemical mechanisms of the two active compounds in controlling T.cinnabarinus were also investigated at different time points by assessing the associated changes in toxic symptoms,potential defense enzyme,metabolic enzyme and target-related enzyme activities,seven neurotransmitters belonging to the biogenic amines?BAs?and free amino acid.2,4-di-tert-butylphenol and ethyl oleate were later formulated to develop as phytochemical acaricides and their effects were confirmed in both growth chamber and field in three provinces.The results were as following:1)In bioassay screening assays,the leaf extract from C.camphora showed significantly greater acaricidal activity against T.cinnabarinus than the other twelve plant extracts tested.The acaricidal activities were confirmed in slide-dip assays,leaf-dip assays and the greenhouse experiment,and the corrected mortality was still78.3%at 10000 mg kg^-1 after 7 d of treatment during the seedling stage.2)Five compounds were isolated and identified from the leaf extract of C.camphora via repeated column chromatography and GC-MS analysis.All the compounds presented acaricidal activity,with 2,4-di-tert-butylphenol and ethyl oleate exhibiting the greatest activity.The acaricidal activities of the two active compounds were confirmed in slide-dip assays,leaf-dip assays and the greenhouse experiment.The LC₅₀ values of 2,4-di-tert-butylphenol and ethyl oleate were found to be 1850.94and 2481.65 mg kg^-1,respectively,at 7 d after treatment in a potted seedling experiment.Orthogonal experimental design were used for optimization of2,4-di-tert-butylphenol and ethyl oleate extraction conditions from C.camphora by GC-MS.The optimal extraction conditions were dilution rate of 1:75,extraction time of 30 min and extraction 3 times.Moreover,the leaves of C.camphora was found to contain relatively high 2,4-di-tert-butylphenol and ethyl oleate,with the leaves collected in septemeber presented the highest content.Furthermore,the leaves of C.camphora from Guangxi province contain the highest 2,4-di-tert-butylphenol amount of 458.8 ng g^-1?w/w?of dry weight,and the content of ethyl oleate in leaves of C.camphora were most in Shanghai city with 61.38 ng g^-1?w/w?of dry weight.3)Both 2,4-di-tert-butylphenol and ethyl oleate exhibited significantly adulticidal,larvicidal and ovicidal activities against T.cinnabarinus in a concentration-dependent manner after treatment,with LC₅₀ values of 1256.51,625.39and 743.64 mg kg^-1 for 2,4-di-tert-butylphenol and 1621.57,859.25 and 987.26 mg kg^-1 for ethyl oleate,respectively.In addition to acaricidal activity,the two active compounds also showed significantly repellent,oviposition-deterrent and residual toxicity properties against adult females of T.cinnabarinus,but the stomach toxicity and systemic activity are limited.Stability assays revealed that 2,4-di-tert-butylphenol and ethyl oleate were relatively stable to environmental factors,and2,4-di-tert-butylphenol was more stable.High temperature,long exposure to light and ultraviolet radiation,and low pH decreased the efficacy of ethyl oleate,and it will work best under neutral or alkaline conditions and no exposed to high temperatures or strong light for long periods during storage.In contrast,the efficacy of2,4-di-tert-butylphenol was not as dramatically sensitive as that of ethyl oleate,except to pH.The application and storage of acaricides based on 2,4-di-tert-butylphenol are better under neutral or acidic conditions.4)The biochemical mechanisms of 2,4-di-tert-butylphenol and ethyl oleate in controlling T.cinnabarinus were also investigated.Using dynamic symptomology observations,typical neurotoxic symptoms including excitation,convulsion and paralysis were observed in the mites treated with 2,4-di-tert-butylphenol and ethyl oleate.Furthermore,the two active compounds exerted significant inhibitory activity on monoamine oxidase?MAO?in adult T.cinnabarinus females in vitro and in vivo and had little effect on acetylcholinesterase?AChE?activity.The content levels of the seven biogenic amines?BAs?analyzed by UPLC-3QMS were higher in the mites treated with 2,4-di-tert-butylphenol and ethyl oleate than in the controls,except for phenethylamine?PEA??for 2,4-di-tert-butylphenol and ethyl oleate?and octopamine?OA??for ethyl oleate?.These results demonstrate that both 2,4-di-tert-butylphenol and ethyl oleate exert effects on T.cinnabarinus that are possibly consequences of their preventive effects on the deamination of BAs in the nervous system,most likely through inhibitory effects on MAO or MAO-like enzymes and/or interactions with certain special biogenic amine G protein-coupled receptors.In addition,both2,4-di-tert-butylphenol and ethyl oleate exhibited inhibitory activity on ATPase of adult T.cinnabarinus females in some degree,with 2,4-di-tert-butylphenol exhibiting the greatest activity on Ca²⁺-Mg²⁺-ATPase and ethyl oleate presenting most remarkable inhibition to Na⁺-K⁺-ATPase.Thus,it is possibility that Ca²⁺-Mg²⁺-ATPase and Na⁺-K⁺-ATPase was one of the targets of action for 2,4-di-tert-butylphenol and ethyl oleate,respectively.5)2,4-di-tert-butylphenol and ethyl oleate were formulated to develop as phytochemical acaricides?20%2,4-di-tert-butylphenol microemulsion and 30%ethyl oleate emulsifiable concentration?and their effects were confirmed in both growth chamber and field.The determination and control the content of active components were conducted with GC-MS.The 2,4-di-tert-butylphenol and ethyl oleate formulations showed excellent acaricidal activities against T.cinnabarinus,and the formulations clearly increased the control effect compared to the active compounds not only in vitro but also in the seedling stage.The LC₅₀ values of the two active compound formulations were 497.22 and 770.30 mg kg^-1,respectively,after 7 d of treatment during the seedling stage.Furthermore,the acaricidal activities of2,4-di-tert-butylphenol and ethyl oleate formulations have been further verified under field conditions in three provinces of China.The results indicated that the two active compound formulations at a high level displayed the best remarkable efficacy in controlling the mites,the control effect were 83.28-97.76%and 81.63-98.86%after14 d of treatment,and they were equally or more effective than commercial acaricides?bifenazate,pyridaben and chlorfenapyr?that have been widely used in China at a recommended concentration.The results of acute toxicology showed that the actue oral,dermal and inhalation toxicity of 2,4-di-tert-butylphenol and ethyl oleate formulations were micro toxic and they were weak sensitizers owing to no skin and eye irritability in rabbits and guinea pigs.In conclusion,the present study demonstrated that the C.camphora and C.camphora-derived materials represent potentially promising alternatives to synthetic acaricides for use in the agricultural industry,and they could be developed as novel natural acaricides for the management of T.cinnabarinus.Moreover,this study provides the basis for further development of phytochemical acaricides using C.camphora as the active material.