Study On The Insecticidal Activities Of Neem Leaves And Active Components Against Oxya Chinensis

Azadirachta indica A. Juss (Syn Melia azadirachta L.) commonly known as neem is a traditional medicine plant possessing-wide biological and pharmacological activities, including antiparasitic, antioxidant, anticancer, antibacterial, antiviral, fungicidal, antiulcer, spermicidal, antidiabetic, anti-implantation, immunomodulating, molluscidal, insecticidal effects, etc. It has prompted the screening of neem extracts for the biological effects on pests in recent years. However, there is no information about neem leaves against Oxya chinensis. In order to develop a new botanical insecticide, this thesis focus on the insecticidal activities of neem leaves and active components against 5rd instar Oxya chinensis. The extraction, isolation, components analysis and mode and mechanism of action of neem leaves were studied by a bioassay-directed fractionation. The main results as follows:1. Study on the insecticidal activities of different extracts by different solvents from neem leaves against 5rd instar Oxya chinensis:Neem leaves were extracted by petroleum ether,95% ethanol and water, and the insecticidal activities of the three extracts were tested by leaf-soaking and larva-soaking method. The three extractions, which showed high insecticidal activity, were further purified. The result showed that the rates of petroleum ether,95% ethanol and water extracts were 2.20%,25.95% and 46.80%, respectively. The water extract was found to have no significant insecticidal activity, and the petroleum ether extract was found to have the highest insecticidal activity against 5rd instar Oxya chinensis, followed by the 95% ethanol extract. The 95% ethanol extract had a little lower insecticidal activity (The mortalities of the 95% ethanol extract and the petroleum ether extract were 33.33% and 26.67% at 72h post-treatment.) but much higher extraction rate than the petroleum ether extract. So the 95% ethanol extract was chosen for a further study.2. Study on the tracing of active fractions of the 95% ethanol extract of neem leaves:The 95% ethanol extract of neem leaves was isolated by system solvents, and the insecticidal activities of the extractions were tested by leaf-soaking and larva-soaking method. The extractions which showed the highest insecticidal activity were chosen for a further study. The result showed that the rates of petroleum ether, chloroform, ethyl acetate, n-butyl alcohol and water extracts were 13.88%,7.33%,4.22%,20.80% and 48.34%, respectively. At a concentration of 25mg/mL, the ethyl acetate extract, n-butyl alcohol extract and water extract were found to have no significant insecticidal activity, and the petroleum ether extract and chloroform extract displayed different levels of insecticidal activities. And the mortalities of 24h,48h and 72h were 23.33% and 23.33%, 73.33% and 30.00%,80.00% and 43.33%, respectively. The results indecated that the petroleum ether extract showed the highest insecticidal activity to 5rd instar Oxya chinensis. And the median lethal concentration (LC50) of 48h and 72h post-treatment were 20.0667mg/mL and 14.9262mg/mL.3. Study on the isolation of active components of the petroleum ether extract of the 95% ethanol extract of neem leaves:A bioassay-directed fractionation of the petroleum ether extract was conducted by the separation with silica gel column chromatography, and the components of active fraction were identified by GC-MS. Three fractions obtained from the petroleum ether extract by column chromatography were investigated for insecticidal activities against 5rd instar Oxya chinensis by leaf-soaking and larva-soaking method. The result showed that the first fraction (Fractionâ… ) displayed the highest insecticidal activity, followed by the second fraction (Fractionâ…¡) and the third fraction (Fractionâ…¢). At 24h post-treatment, the LC50 of the three fractions were 14.3563mg/mL,24.5616mg/mL and 23.7878mg/mL, respectively. The main components of Fraction I were studied by GC-MS. The result indicated that there were 6 alkanes (pentacosane, hexacosane, heptacosane, nonacosane, triacontane and hentriacontane),1 olefin (squalene),2 esters (hexadecanoic acid, ethyl ester and 9,12.15-octadecatrienoic acid, ethyl ester, (Z,Z,Z)-) and 1 amide (erucyl amide).4. Study on the mode and mechanism of action of the petroleum ether extract of the 95% ethanol extract of neem leaves against Oxya chinensis:The antifeedant activity, contact toxicity, stomach poisoning and growth restraining effect of the petroleum ether extract against 5rd instar Oxya chinensis were tested. The larvae were treated with 25mg/mL petroleum ether extract by leaf-soaking and larva-soaking method. Midguts were cut off after 24h,48h and 72h respectively for paraffin section and electron microscopic. The result showed that the mode of action was antifeedant. Obvious pathological changes in the midgut of the grasshoppers, Oxya chinensis were observed. Paraffin section observation showed that midgut cells swelled, degenerated, followed by partial loss, then fell off completely, leaving only the circular muscle layer. Electron microscopic observation showed that the structure of the midgut cells were damaged seriously, the microvilli fractured and fell off; mitochondria swelled and their cristae were disorganized and myelin figures emerged; rough endoplasmic reticulums swelled obviously, fractured and vacuolated, and the ribosomes denuded. All the above observations showed that damaging the normal structure of midgut and endomembrane organization may be one of the primary mechanisms.