| Azadirachtin, the most potent insect antifeedant and growth-regulating agent,is extracted from the seed of the neem tree, azadirachta indica A.Juss. At present, extensive researches have focused on the extraction of effective ingredients, structural determination and favorable application of azadirachtin. The method to extract azadirachtin is mostly traditional solvent extraction (TSE). However, there are few reports on microwave assistant extraction (MAE), on CO2 supercritical fluid extraction (SFE), on biological activeness of the main ingredients after extraction and on the research into antimicrobial and antifungal activity as well. At the same time, there are few reports on revising the character of azadirachtin to improve biological activity. In this thesis, we have systemly studied on the analyses, the method of the extraction, the revised character, biological activity and the factors, which could affect the stability for azadirachtin. The concrete work is listed below.1. The analytical method has been improved. The reversed-phase HPLC method is used to the measure the quantitative analysis of azadirachtin A in crude extracts. And the colorimetric method is applied to determine the quantity of the total azadirachtin related limonoids (AZRL) in neem extracts. Using the azadirachtin whose purification is 86.68% as a criterion, the interferences that come from other absorbing species are eliminated effectively. 2. The results of HPLC-MS indicate that azadirachtins mainly include azadirachtin A, 3-tigloylazadirachtol, salannolactame-21, 21-oxo-ochinolide, 6-deacetylnimbin, nimbin, 6-deacetylsalannin and salannin. Azadirachtin A whose content is 93.79% has been obtained by the method of a thin-layer chromatography and characterized by 1H NMR and 13C NMR spectra. At the same time, the author has obtained two mainly compounds, which are called nimbin and salannin . 3. The use of two new techniques for extracting azadirachtin A. The first method is that the neem seed power is extracted in the first place, and the neem oil is defatted with petroleum at last, meanwhile, 5%NH4Ac is added in liquid-liquid extraction. The second method is that the neem oil is squeezed by machine before extraction. The order of extraction rates for azadirachtin A, which are measured by using HPLC method are: microwave assistant extraction (MAE) > tradition solvent extraction (TSE) > CO2 supercritical fluid extraction (SFE). The results indicate that the yield of TSE is the highest under a suitable condition of temperature 23oC, methanol as solvent, the ratios of 1:3 (g/mL) and extraction time 5 h×3. The yield of oil seed and defatted seed are 0.5453 and 0.4951%, respectively. Compared with the TSE, MAE not only simplifies the craftwork but also improves the yield and reduces the extracted time from 5 h to 3 min. The results indicate that the yield is highest by using a suitable condition of microwave power 210 W, radiation time 3 min×3, methanol as solvent, ratios of solvent 1:3 (g/mL). And the yields of oil seed and defatted seed are 0.5612 and 0.5190%, respectively. The results indicate that the yield of SFE is highest by using a suitable condition of temperature 32oC, power 32 MPa, CO2 flux velocity 10 kg/h, methanol as modified solvent , ratios of solvent 1:3 (g/mL) and extraction time 120 min, the yields of oil seed and defatted seed are 0.2503 and 0.2084%, respectively .The mechanisms of the three extraction methods have been studied through using scan electron microscope (SEM).4．Azadirachtins bioassay against the third instars larva of spooptera exigua hübner are investigated, the results indicate that the bioassay of crude extracts are different with different extract methods. The order is SFE＞MAE＞TRE. The results show a wide spectrum of activity and species affected, LC50 (azadirachtin ) of spooptera exigua hübner and Plutella xylostella after treaded 7 days are 573.68 μg/mL,441.88 μg/mL,151.04 ug/mL,and 65.93 μg/mL ,41.37 ug/mL, 25.36 ug/mL, respectively. The separated fractions have less bioassay com...
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