Studies On Acaricidal Substance And Mechanism Of Asarum

In this paper, systematic studies were done for the extraction, separation, chemical identification, mechanism and dosage formulation of medial plant asarum acaridal substances using techniques of HPLC-MS, GC-MS,1H-NMR, Electron microscopy et al. The acaricidal activity to Tetranychus urticae was confirmed, and the acaricidal mechanisms were primarily elucidated, also, an acaricidal monomer was isolated, of which chemical structure was identified, meanwhile, a practical dosage formulation was prepared. The research results laid a solid foundation for further development of asarum acaricidal substances.The toxicity of methanol extracts of 20 medicinal plants to female adults and eggs of T. urticae were evaluated. Bioassay showed that Areca catechu L. Asarum heterotropoides var. mandshuricum, Coptis chinensis Franch, Melia toosendan Sieb. et Zucc, and Sophora flavescens Ait possessed better biological activity toward the female adults and eggs of T. urticae, with 100% modified mortality for female adults at concentration of 10 mg/mL after 72 h treatment, and≤40% egg hatching rate at concentration of 10 mg/mL after 5 days treatment.Asarum demonstrated excellent and was chosen for further acaricidal assay. After 24h treatment of 1mg/mL asarum extract for female adults (24 h) and eggs (5 d), the modified mortality was 66.5% for female adults and 45.7% for eggs, and the LD50S were 0.57 mg/mL and 0.88 mg/mL for female adults and eggs respectively. The asarum extract showed oviposition inhibition, at concentration 1 mg/mL, the inhibition rate was 60.25% and 61.45% at 24 h and 48 h respectively, while at 4 mg/mL, the inhibition rate was 92.89% and 94.7% at 24 h and 48 h respectively. The asarum extract also showed deterrent activity, at 0.5 mg/ml, the deterrent rate was 57.14% after 24 h, it declined to 50% after 72 h; at concentrations more than 2 mg/ml, the deterrent rates from 24 to 72h were nearly to 100%. The asarum extract also showed stomach toxicity, which was indicated by the mortality of female adults and oviposition inhibition, but this toxicity was weaker than the direct touch toxicity. At concentration of 1 mg/mL, the mortality of female adults was 28.2%, and oviposition inhibition was 53.1% after 48 h treatment. At 8mg/mL, the modified mortality was 82.05% for female adults, and oviposition inhibition rate reached to 92.66%.The acaricidal mechanisms of asarum extract were elucidated. The mite ultrastructure after asarum treatment showed various changes of the epidermis and cell structure, such as water loss in surface, mite body shrinkage, the integumentary lobes gap becoming bigger, and the granules becoming small. The asarum extract was found to inhibit acetylcholinesterase, Na+, K+-ATPase and mixed function oxidases; increase activities of superoxide dismutase and catalase; increase early and decrease latter the activity of glutathiones-transferase, there were no significant changes found for monoamine oxidase and esterase. The results hint acetylcholinesterase, Na+, K+-ATPase and mixed function oxidases are the targets of asarum extract, and oxidative stress is involved in the mechanism of toxicity to T. urticae.The main acaricidal monomer of asarum was isolated and identified. Tracking acaricidal activity, separation and purification of acaridal substances of asarum were done. Applying column chromatography and crystallizatin/recrytallization techniques, a monomeric compound was isolated. HPLC-MS and GC-MS and 1H-NMR analysis revealed the monomer was (-) asarinin. Bioassay showed (-) asarinin possessed good activity against T. urticae with LC50 of 0.1237 mg/mL, indicating the toxicity of (-) asarinin was more than that of asarum extract.Emulsifiable concentrate of asarum extract was prepared. The final resulted formulation was 10% asarum extract+80% solvent (xylene)+10% emulsifier mixture (3% pesticide emulsifier 500+2% pesticide emulsifier 600+5% tween-80). Emulsification, dispersion, the stability, low temperature stability, heat storage stability and volatility of the prepared EC met the requirements of EC quality. Bioassay results for determining indoor toxicity against T. urticae showed that 400 X diluted asarum extracts EC possessed comparable toxicity with 3000 X diluted 15% pyridaben EC.The chemical components and effect on T. urticae of asarum essential oils were primarily investigated. Chemical analysis by GC-MS showed that 9 major constituents composed 86.15% of the total peak areas, methyleugenol (22.9%) was the most abundant, followed by safrole (19.8%). Bioassay showed that the asarum essential oils possessed fumigant toxicity and deterrent activity, at 8μg/mL, the mortality was 72.6%,100% after 24 and 48 respectively, the deterrent rate 86.1%. Asarum essential oils affected some enzymes of T. urticae, they decreased the mite activity of Na+, K+-ATPase, and increased at first and decreased latter the activity of glutathione S-transferase, suggesting the two enzymes are involved in the toxicity mechanism of asarum essential oils.