Research On Insecticidal Active Substance From The Seeds Of Amorpha Fruticosa And Mechanism Of Action

Amorpha fruticosa is a perennial deciduous shrub native to North America, belongs to the family of Leguminosae. The plant was introduced into China around the 1920s and widely planted throughout China for windbreak and sand fixation. Current, there are abundant resources of this plant. Previous research showed that the seeds of A. fruticosa possess contact, stomach poison and antifeedant activity against various insect species. To systematically study the insecticidal compound, bioactivity and mechanism of action, different methods are adopted to extract and generate the essential oils and total flavonoids active ingredient of A. fruticosa seeds, then the isolation and identification of those active ingredients were carried out by employing modern separation technology such as GC-MS, column chromatograpy and preparative high performance liquid chromatography, and spectroscopic technique including UV, IR,1H-NMR and MS. The bioactivity of essential oils and total flavonoids active ingredient was individually assayed against different insect pests. Optimum extracting technology of the total flavonoids was studied by response surface methodology, and a 8% A. fruticosa total flavonoids microemulsion had been successfully developed. Meanwhile, a simple and rapid method using high performance liquid chromatography was developed to determine adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and energy charge levels of insect, and effect of total flavonoids of A. fruticosa and 8’-hydroxyrotenone on adenine nucleotides and energy charge were determined. Furthermore, inhibition effect and mechanism of action of 8’-hydroxyrotenone and rotenone on mitochondrial complex I of Culex pipiens pallens had been explored. The main results and conclusions were outlined as follows:The essential oils from the fruits of A. fruticosa which obtained by hydro distillation, petroleum ether, dichloromethane and n-hexane extraction, have been investigated by GC and GC-MS, and 84,112,75 and 93 compounds were identified by NIST mass spectral library search, respectively. The yield and quantity of essential oil extracted with Petroleum ether are more than the others. The highest percentage component of the oils extract with organic regent is y-muurolene, β-cubebene and y-cubebene. The bioassay revealed that all four essential oils exerted a promising insecticidal efficacy against Aphis citricola and the larvae of C. pipiens pallens, the highest potential was established from the essential oils extracted with petroleum ether. The larvicidal activity of the crude petroleum ether, ethyl acetate, acetone, chloroform and ethanol extracts of A. fruticosa seeds was individually assayed for toxicity against the early fourth-instar larva of the mosquito, C. pipiens pallens after 24h exposure. Of the tested extracts, the ethanol one exhibited the highest larvicidal activity (LCso=22.69mg/L) was isolated from the ethanol extract by column chromatograpy. The compound was isolated from the seeds of A. fruticosa by applying mixed solvents, the chemical structure was elucidated by UV, IR, 1H-NMR and MS spectral data, and It’s Amorphigenin (8’-hydroxyrotenone). The bioassay showed the compound had toxic activity against A. citricola, Pieris rapae, Plutella xylostella, C. pipiens pallens and Chironomus riparius after 24h treatment. Insecticidal effect of 8’-hydroxyrotenone for A. Citricola, P. rapae and C. pipiens pallens is better than that of rotenone. The LC50 values of 8’-hydroxyrotenone againt A. Citricola, P. rapae and C. pipiens pallens were 0.213,131.531 and 4.290μg/mL, the LC50 values of rotenone were 0.346,142.117 and 4.692ug/mL.The optimum extracting technology of the total flavonoids from A. fruticosa by using single factors test and response surface optimization was carried out. Among the variables screened of solid-liquid ratio, ultrasonic power, ethanol concentration and time, ethanol concentration and time were identified as the most significant variables. Through response surface methodology design experiments, the processing conditions were optimized as follows:ethanol concentration 77.43%, time 31.29min, solid-liquid ratio 1:21.22 (quality volume ration) and ultrasonic power 357.55W.A formulation of 8% A. fruticosa flavonoids microemulsion was successfully developed by screening of the solvent, auxiliary solvent, emulsifier and combination, and HPLC analytical method for 8’-hydroxyrotenone in the ME has been developed. Furthermore, according to the related universal standard of pesticide, the quality indexes for 8% A. fruticosa flavonoids ME were established. The bioassay of 8% A. fruticosa flavonoids ME and 8% A. fruticosa flavonoids EC al against A. citricola and the larvae of C. pipiens pallens was carried out, and the results showed that insecticidal efficacy of 8% A. fruticosa ME are better than 8% A. fruticosa EC.A simple and rapid method using high performance liquid chromatography (HPLC) was developed to determine ATP, ADP and AMP. The determination result indicated that of total flavonoids of A. fruticosa and 8’-hydroxyrotenone can reduce the contents of adenine nucleotides and energy charge of C. pipiens pallens with an inverse proportion relation, the larger the concentration of total flavonoids and 8’-hydroretenone, the lower the contents of adenine nucleotides and energy charge.8’-hydroxyrotenone and rotenone can significantly inhibit mitochondrial complex I enzyme activity of C. pipiens pallens, the IC50 of 8’-hydroxyrotenone and rotenone was 2.8592μM and 3.1375μM. The inhibition curve indicated that both of them are reversible and mixed-I type inhibitors.