Studies Of Biological Activity And Kinetic Chemical Reaction Of Active Compounds From Neem Seeds

This paper aimed at searching the extraction technology, quantitative methods, degradation kinetics and biological characteristics, etc. Basic data were obtained to supposed neem industrialization. The study was under taken on the techniques of the analysis, isolation and purification of standard azadirachtin-A(contenting 95% azadirachtin-A). Uniform design method was adopted to optimize the two techniques, including traditional solvent extraction(TSE) and CO₂ supercritical fluid extraction(SFE). Then a practical technology was established by integrating the advantages of two techniques. Water, temperature, pH, time etc. impact the degradation of azadirachtin. The degradation kinetic model was constructed. The biological activity of neem extracts against Diaphania indica and Sitophilus zeamais Motschulsky had been researched. The main conclusions are as follows:1. Establishing a quantitative analysis of aza-A. The detection conditions were: chromatographic column C18, 3.9mm×150mm; mobile phase: acetonitrile : water = 30:70 ; current velocity: 1mL/min; detection wave length: 215nm. Minimum detection limit was 0.5ug/mL. Retention time Aza-A was 3.15min. UV-Spectrophotometry was used in the analysis method of azadirachtins in the neem biological pesticide. Active compounds were extracted from neem biological pesticide by using dichloromethane under a microwave condition. Blue-green compounds could be got when alcohol solution was added, which was containing sulfuric acid and vanillin. At 572nm wavelength maximum absorption was observed.2. Dichloromethaner crude extract was higer concentration of azadirachtin-A. The crude extract was separated by Flash Column Chromatography and TLC. Thus, the pure azadirachtin-A(>99%) and other homologous compounds(>95%) were obtained. Then, their chemical constructions were identified by HPLC, UV and LC-MS: azadirachtin-A, retention time 3.15min, molecular weight720; 6-deacetylnimbin, retention time 10.37 min, molecular weight 498; 6-deacetylsalannin, retention time 12.61 min, molecular weight 554; Nimbin, retention time 16.72 min, molecular weight 540; Salannin, retention time 22.10 min, molecular weight 596。3. After the optimal technological data were compared with practical production conditions, the optimal data were: the extracting volume 230mL and the time 20 min of light petroleum, the extracting volume 310mL and the time 345 min of methanol, the extracting volume 120mL and the time 20 min of ethyl acetate. Hence, the most of azadirachtin-A could be extracted by solvent method from neem seeds. The optimal conditions of SFE CO₂ were the pressure 37MPa, the temperature 43 ℃, the time 165min, and the volume 13mL of methanol. A new industrialization technology was recommended with combining the TSE and SFE.4. The neem oil was qualitatively and quantitatively analyzed by GC-MS and GC-FID. The fatty acids extracted with TSE had same components with SFE. The main components were determined as 8-octadecenoic acid 51.15%, octadecanoic acid 19.13%, hexadecanoic acid 16.78%, 7,10-octadecadienoic acid 9.86%, eicosanoic acid 1.24%. And other under 1% fatty acids were cis-3-octyl-oxirane octanoic acid, docosanoic acid, 9-octadecenoic acid, 11-eicosenoic acid, 9-hexadecenoic acid and heptadecanoic acid etc.5. The pH value, temperature, water, UV light, solvents, antioxidant and light stabilizers were systematically investigated to explain their influence to chemical reaction grade, reaction rate coefficient, Arrhenius coefficient, temperature effect quotient(Q), activation energy(Ea), activation enthalpy(â–³H) and activation entropy(â–³S) of azadirachtin-A and other active compounds. The test showed that weak acid condition was good for azadirachtin-A, otherweise, accelerated its degradation. When the reaction temperature was within the range of 4℃ and 25℃, azadirachtin-A was stable. When temperature higher than 35 ℃, the degradation was accelerated. The degradation rate is biggest at 65℃. After 14 days the testing concentration of azadirachtin-A was almost zero. With the increasing of water amout in reaction system, the degradationrate become bigger. UV light was an important factor azadirachtin-A degradation. After 6h UV irradiation the azadirachtin-A in pure sample and crude extract decomposed respectively 60.86% and 55.57%. 2 days later the percentages became 92.95% and 89.11%.Va and Vc both could restrain the degradation of azadirachtin -A, Va was better than Vc. When amout of Va was 0.5, 1.0, 1.5 times that of azadirachtin-A, the degradations were 50.97%, 48.00% and 40.29% at 55℃. There was no more difference between Neem oil and alkali lignin as light stabilizer.6. Tested with 3 different neem extracts(neem SFE extracts , neem ethyl acetate extracts and oil) against Diaphania indica. The results showed that those extracts strongly deterred anti-feeding, growth inhibition and oviposition deterrent, and the toxicity against the low larva were better than the old ones. The bioactivities of those extracts increased with the growing of their dosage;.Azadirachtin had some contact toxicity against to the low instar larva, but few to the the old ones.The biological activity of 4 different neem extracts against adults of Sitophilus zeamais Motschulsky had been researched. The repellence, fumigant, contact toxicity and population inhibition activity were assayed. Repellence activity: all of 4 different neem extracts showed good repellencies against adults of Sitophilus zeamais Motschulsky, and the repellence grades reached grade V at the treatment of high concentration. The 4 different neem extracts showed great difference of fumigant against adults of Sitophilus zeamais Motschulsky. The fumigant activity of the 4 different neem extracts was neem oil>neem SFE extracts>neem ethyl acetate extracts>azadirachtin A. All of 4 different neem extracts showed weak contact toxicity against adults of Sitophilus zeamais Motschulsky. But 4 extracts showed good population inhibition activity against Sitophilus zeamais Motschulsky.