Extraction Optimization Of DTD And Preparation And Activity Evaluation Of Its Nanoemulsions

As an invasive plant,the sustainable control and reuse of Ageratina adenophora is an important scientific issue.In recent years,secondary living substances have been reported in the plant,and the assessment of the corresponding bioactive potential is an important element,such as the insecticidal potential of 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8atetrahydronaphthalene-2,6(1H,7H)-dione(DTD),which is the basis for the construction of plant-derived insecticides.In this paper,a green,efficient and targeted extraction,separation and purification method was developed using Box-Behnken Design(BBD)to optimise the ultrasound-assisted extraction parameters of DTD,laying the foundation of raw material production technology for the application of DTD.In order to better exploit the insecticidal activity of DTD,DTD nanoemulsions were prepared by low-energy emulsification and evaluated for activity against important Lepidopteran pests and safety of non-target organisms,with a view to supporting the development of safe,green and effective alternative insecticides in the technological innovation of green control of maize pests.The main findings of the study are as follows:(1)The results of the condition screening test showed that the three factors that significantly influenced the extraction rate of DTD were extraction temperature,solid-liquid ratio and extraction time.The best extraction reagent for DTD was hexane,the best extraction time was 40 min,the best solid-liquid ratio was 1:30 and the best extraction temperature was 70℃.The best ultrasound-assisted extraction conditions for DTD were optimised using BBD based on the condition screening test:extraction temperature(60℃,70℃ and 80℃),solidliquid ratio(1:25,1:30 and 1:35)and extraction time(30 min,40 min and 50 min)were used as independent factors,and DTD extraction rate was used as the response value.The optimal extraction parameters obtained by BBD were:extraction time 39 min,solid-liquid ratio 1:31 and extraction temperature 68℃.The results showed that the ultrasonic-assisted extraction of DTD by BBD optimization could significantly improve the extraction rate.(2)DTD nanoemulsions were prepared using a low-energy emulsification method.The effects of different surfactant types,dosages and emulsification processes on the stability of DTD nanoemulsions were compared,and the properties of the DTD nanoemulsions prepared from the optimal formulation were evaluated:wetting properties(contact angle and tension),insecticidal activity and safety evaluation against non-target organisms.The results show that,among the surfactants studied(TW series),the best surfactant for the preparation of DTD nanoemulsions was found to be TW-80 with long term stability.the stability of the nanoemulsions increased with increasing surfactant dosage(0.5%～2%(w)).In terms of emulsification method,the water-in-oil preparation method can be used to prepare more stable nanoemulsions of DTD.Considering the production cost and stability,the optimal formulation of DTD is shown below:1%DTD,1.5%S-200,1%TW-80 and 96.5%deionised water.The results of the evaluation of the wettability of the nanoemulsions prepared with the optimum formulation are as follows:the contact angle and tension are low,indicating good wettability.The activity of the nanoemulsions against the test insects was evaluated as follows:The LC50(96h)of DTD nanoemulsions was 26.64 mg/L for Spodoptera frugiperda,1.57 mg/L for DTD nanoemulsions for Spodoptera litura,and 3.06 mg/L for DTD nanoemulsions for Ostrinia furnacali,the larvicidal toxicity of DTD nanoemulsions were similar to that of azadirachtin emulsion.The results of the biosafety evaluation on non-target organisms were as follows:the L-02 cell apoptosis rate was lower in the DTD nanoemulsions treated group,the toxicity to earthworms was lower and no adverse effects on maize growth were detected.These results indicate that the low energy emulsification method can produce DTD nanoemulsions with excellent performance and is green,effective and safe.