Simultaneous Extraction and Separation of Oil and Azadirachtin from Seeds and Leaves of Azadirachta indica using Binary Solvent Extraction

Conventional extraction of oil and azadirachtin, a botanical insecticide, from Azadirachta indica involves defatting the seeds and leaves using hexane followed by azadirachtin extraction with a polar solvent. In order to simplify the process while maintaining the yield we explored a binary extraction approach using Soxhlet extraction device and hexane and ethanol as non-polar and polar solvents at various ratios and extraction times. The highest oil and azadirachtin yields were obtained at 6 h extraction time using a 50:50 solvent mixture for both neem leaves (44.7 wt%, 720 mg(Aza)/kg(leaves)) and seeds (53.5 wt%, 1045 mg(Aza)/kg(seeds)), respectively.

Potential of Azadirachtin and Neem (Azadirachta indica) Based Saponins as Biopesticides for In vitro Insect Pests Cellulase (Beta-1,4-Endoglucanase) Enzyme Inhibition and In vivo Repellency on Tribolium castaneum.

Aims: The work was undertaken to identify the role of Neem derived compounds (saponins and azadirachtin) on the digestive cellulose hydrolyzing enzyme activity of red flour beetle (T. castaneum), rice grasshopper (Oxya chinensis) and red pumpkin beetle (Aulacophora foveicolis). Place and Duration of Study: The work was carried out at the Institute of Biochemistry and Biotechnology University of the Punjab Lahore Pakistan. Methodology: Total cellular proteins were isolated from the insect’s gut and salivary glands and were tested for cellulose hydrolyzing activity on substrate agar plates. Saponins and Azadirachtin were isolated from Azadirachta indica tissues and used for enzyme inhibition studies. Repellancy test was performed for T. Castaneum, using saponins and Azadirachtin. For computational studies sequence of endoglucanase gene was identified from T. castaneum genome and protein structure was deduced. Results: Saponins were able to inhibit beta-1,4-endoglucanase enzyme activity, present in all the three insect pests. A computational dissection of T. castaneum endoglucanase enzyme, deduced from T. castaneum genome, showed that there were five cys involved in the formation of disulphide bridges in the molecule. The disulfide bridges did not provide any protection to endoglucanase active site. Azadirachtin had no effect on cellulase activity of Oxya chinensis and Aulacophora foveicolis, while beta-1,4- endoglucanase activity of T. castaneum was inhibited. Repellency test for T. castaneum revealed that each group of compounds (Saponins and Azadirachtin) was able to repel the insect. Conclusion: Neem derived compounds had a considerable inhibitory effect on the digestive cellulose hydrolyzing enzyme of T. castaneum.

Acute toxicity of azadirachtin to a teleost, Heteropneustes fossilis / Toxicidade aguda da azadiractin para um teleósteo, Heteropneustes fossilis

The acute static renewal test of a botanical pesticide - azadirachtin for the freshwater catfish, Heteropneustes fossilis has been performed to determine the LC50 values at different exposure period. The LC50 values at various exposure periods are 173.06 mg L-1 for 24h; 80.69 mg L -1 for 48h; 58.57 mg L-1 for 72h and 52.35 mg L -1 for 96h. The upper confidence limits were 196.87, 86.91, 79.20 and 70.04 mg L-1 for 24, 48, 72 and 96 h and lower confidence limits were 154.01, 74.24, 37.33 and 33.83 mg L-1, respectively. These results indicate that azadirachtin exposure to the fish caused toxic effects.

A renovação do ensaio estático agudo de um pesticida botânico - azadiractin para o peixe de água doce, Heteropneustes fossilis foi realizada para determinar os valores de LC50 em diferentes períodos de exposição. Os valores de LC50 em diferentes períodos de exposição são 173,06 mg L-1 por 24h; 80,69 mg L-1 por 48h; 58,57 mg L-1 por 72 h e 52,35 mg L-1 por 96h. Os limites de confiança superiores foram 196,87; 86,91; 79,20 e 70,04 mg L-1 para 24, 48, 72 e 96h os limites inferior e confiança foram 154,01; 74,24; 37,33 e 33,83 mg L-1, respectivamente. Estes resultados indicam que a exposição do peixe à azadiractin causou efeitos tóxicos.

Acute toxicity of azadirachtin to a teleost, Heteropneustes fossilis - doi: 10.4025/actascibiolsci.v34i2.9455

The acute static renewal test of a botanical pesticide - azadirachtin for the freshwater catfish, Heteropneustes fossilis has been performed to determine the LC50 values at different exposure period. The LC50 values at various exposure periods are 173.06 mg L-1 for 24h; 80.69 mg L-1 for 48h; 58.57 mg L-1 for 72h and 52.35 mg L-1 for 96h. The upper confidence limits were 196.87, 86.91, 79.20 and 70.04 mg L-1 for 24, 48, 72 and 96 h and lower confidence limits were 154.01, 74.24, 37.33 and 33.83 mg L-1, respectively. These results indicate that azadirachtin exposure to the fish caused toxic effects.

Efficacy evaluation of a commercial neem cake for control of Haematobia irritans on Nelore cattle / Avaliação da eficácia de uma torta comercial de nim para o controle de Haematobia irritans em bovinos Nelore

Much attention has been given to the development of botanical insecticides to provide effective natural control of cattle ectoparasites without harming animals, consumers, and environment. This study evaluated the efficacy of a commercial neem cake in controlling Haematobia irritans infestation on cattle. The study was conducted at the Embrapa Southeast Cattle Research Center (CPPSE), in São Carlos, SP, Brazil, from April to July 2008. The neem cake mixed in mineral salt in a 2 percent concentration was provided to 20 Nelore cows during nine weeks and had its efficacy evaluated by comparison of the infestation level against a control group. Fly infestations were recorded weekly by digital photographs of each animal from both groups and the number of flies was later counted in a computer-assisted image analyzer. Quantification of neem cake components by high-performance liquid chromatography revealed the presence of azadirachtin (421 mg.kg-1) and 3-tigloyl-azadirachtol (151 mg.kg-1) in the tested neem cake. Addition of the 2 percent neem cake reduced mineral salt intake in about 22 percent. The 2 percent neem cake treatment failed to reduce horn fly infestations on cattle during the 9-week study period.

Muita atenção tem sido dada ao desenvolvimento de inseticidas vegetais buscando-se um efetivo controle de ectoparasitas de bovinos, sem prejudicar animais, consumidores e meio ambiente. Este estudo, realizado de abril a julho de 2008, na Embrapa Pecuária Sudeste, em São Carlos, SP, Brasil, avaliou a eficácia de uma torta comercial de nim (Azadirachta indica) no controle da mosca-dos-chifres (Haematobia irritans) em bovinos. A torta de nim, misturada ao sal mineral na concentração de 2 por cento, foi fornecida a 20 vacas Nelore, durante nove semanas, e sua eficácia foi monitorada através de contagens semanais nos grupos tratado e controle. Infestações individuais foram registradas por meio de fotos digitais em todos os animais de ambos os grupos, e o número de moscas foi, posteriormente, quantificado com o auxílio de um sistema de análise de imagem computadorizado. A quantificação dos componentes da torta de nim, por cromatografia líquida, revelou a presença de azadiractina (421 mg.kg-1) e 3-tigloyl-azadirachtol (151 mg.kg-1). A adição da torta de nim a 2 por cento reduziu o consumo de sal mineral em cerca de 22 por cento. O tratamento com torta de nim a 2 por cento não reduziu as infestações por mosca-dos-chifres em bovinos durante as nove semanas do estudo.

Efeito do nim (Azadirachta indica) na mosca-das-frutas Ceratitis capitata (Diptera: Tephritidae) e seu parasitoide Diachasmimorpha longicaudata (Hymenoptera: Braconidae) / Effect of neem (Azadirachta indica) oil on the fruit-fly Ceratitis capitata (Diptera: Tephritidae) and its parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

Este trabalho teve como objetivo avaliar a ação do inseticida botânico nim em larvas de Ceratitis capitata, bem como no parasitismo destas larvas por Diachasmimorpha longicaudata. Para tanto, larvas de 3o ínstar de C. capitata foram mergulhadas por 30 segundos em soluções contendo as concentrações de 0,5%, 1% e 1,5% de um produto comercial à base de óleo de nim a 0,37% (3.686 ppm), além da testemunha mergulhada apenas em água destilada. Em seguida, as larvas foram expostas ao parasitismo de D. longicaudata por um período de 2 horas e 30 minutos. No experimento sem chance de escolha, para cada tratamento, uma única "unidade de parasitismo" foi pendurada no interior da gaiola. No experimento com livre escolha, as "unidades de parasitismo" foram penduradas no interior de uma mesma gaiola, de forma que os parasitoides tivessem livre acesso a todos os tratamentos. No experimento sem livre escolha, foi constatado que o aumento da concentração de nim diminuiu a atração das fêmeas, o número de fêmeas que efetuaram postura, o índice de parasitismo e a emergência dos parasitoides. No experimento com livre escolha, verificou-se que a emergência dos parasitoides e o índice de parasitismo também foram prejudicados nas maiores concentrações de nim. A ação isolada do nim, quando em contato com as larvas de 3o instar, não afetou a emergência de C. capitata.

The purpose of this work was to evaluate the action of the botanic insecticide neem oil on larvae of Ceratitis capitata, as well as in the parasitism of these larvae by Diachasmimorpha longicaudata. Third-instar larvae were immersed for 30 seconds in a neem solution, in concentrations of 0.5%, 1% and 1.5% of a commercial product composed of 0.37% (3.686 ppm) neem oil, while the controls were immersed only in distilled water. Soon afterwards the larvae were exposed to parasitism of D. longicaudata for 2 hours and 30 minutes. In the no-choice experiment, for every treatment, a single "parasitism unit" was hung inside the cage. In the experiment with free choice, "parasitism units" were hung inside another cage, so that the parasitoids had free access to all treatments. In the no-choice experiment, it was ascertained that the increase of the neem oil concentration decreased the attraction of the females, the number of females that lay, the parasitism rate and parasitoid emergency. In the experiment with free choice, it was ascertained that the emergence of parasitoids and parasitism rate were harmed at the larger neem oil concentrations. The isolated action of neem oil, when in contact with the third-instar larvae, did not affect the emergence of C. capitata.

Effects of azadirachtin in Rhodnius prolixus: data and hypotheses

The effects of azadirachtin A, a tetranortriterpenoid from the neem tree Azadirachta indica J., on both development and interaction between Trypanosoma cruzi, the causative agent of Chagas' disease, and its vector Rhodnius prolixus were studied. Given through a blood meal, a dose-rsponse relationship of azadirachtin was established using antifeedant effect and ecdysis inhibition as effective parameters. A singlo dose of azadirachtin A was able to block the onset of mitosis in the epidermis and ecdysteroid titers in the hemnolymph, determined by radioimmuneassay, were too low for an induction of ecadysis. The survival of T. cruzi was also studied in R. prolixus treated with the drug. If the trypomastigotes were fed in presence of azadirachtin A the number of parasites drastically decreased. If the drug was applied after infection of the bug with T. cruzi, the parasite was still abolished from the gut. If the insect was pretreated with azadirachtin A before infection the same observation was obtained. A single dose of azadirachtin A was enough for a permanent resistance of the insect host against its reinfection with T. cruzi and for blocking the ecdysis for a long time. The effects of azadirachtin A on the hormonal balance of the host and growth inhibition of the parasite will be discussed on the basis of the present results.