Pure Azadirachtin Research Articles

Nano-Encapsulation Of Neem Based Pesticide Formulations

Neem based pesticide formulations are reported to control plant parasitic fungi, bacteria, virus, nematodes, insects and mites. Despite its many promising properties, low stability under field conditions, mainly due to a high rate of photo-degradation, as well as a short residence time and slow killing rates, compared to conventional pesticides hinder effective large-scale use of Neem. Although when two lines of Myzus persicae Were exposed to pure azadirachtin and refined Neem seed extract at the same concentration, it was found that after forty generations, the line treated with azadirachtin had developed ninefold greater resistance compared to a control line, whereas the line treated with the extract did not show resistance. To overcome these limitations, nanotechnology has emerged as a novel tool for agricultural sustainability. Many studies have shown that the encapsulation of agrochemicals in nanoparticulate systems can enhance the efficacy of the active ingredient, decrease toxicity toward the environment and humans, and reduce losses due to volatilization, leaching, and photobleaching. Thus, the use of nanoparticles provides an effective means of protecting neem oil against premature degradation, resulting in prolongation of its effect on the target pest. Due to nano encapsulation sustained release of the active component is achieved. Furthermore, the number of applications of neem oil can be reduced, bringing substantial economic benefits. Therefore, nano-encapsulation of neem oil may enable the production of more stable formulations for control of diseases and pests. Further, the use of nanotechnology is an excellent way to combat development of resistance in insects and pathogens due to indiscriminate use of neem oil.

Effects of Melia azedarach L. leaf and fruit on mineralization of carbon in soil

Carbon mineralization in soil increased significantly due to additions of pure azadirachtin and powdered leaves and fruits of Melia azedarach L. under in vitro incubation for 30 days at 28°C. Cumulative respired C(CO2) clearly increased with incubation time in all treatments except in soil mixed with pure azadirachtin (p < 0.001). Carbon mineralization ratio in soils mixed with single doses of powdered leaf and fruit were significantly higher than the other doses tested.

Comparison of the Effects of Tannin and Azadirachtin on Carbon Mineralization in Soils of Quercus coccifera from Eastern Mediterranean Region

In this study, organic matter mineralization was studied in the Quercus coccifera (Kermes oak, Fagaceae) soils mixed with Quercus leaves having high tannin content, Melia azedarach (Neem tree, Meliaceae) leaves and fruits having high azadirachtin content, pure tannin and pure azadirachtin. Carbon mineralization of all samples was determined by the CO2 respiration method over 30 days (28oC and 80% of field capacity). Addition of equal (1x) and half (1/2x) amounts of soil carbon containing leaves of both plants to soils separately increased microbial activity. The increase in microbial activity was higher in 1x leaves added soil compared with 1/2x leaves added soil and was only significant in 1x Melia leaves added soil compared with control (P<0,001). There was no significant difference in carbon mineralization between the 1/2x and 1x Quercus leaves added soils (P≥0.05) whereas the difference between the Melia leaves added (1/2x; 1x) soils was significant (P<0,001). It was determined that the carbon mineralization rates (%) of Quercus soil mixed with pure tannin and pure azadirachtin showed similar behaviours. When amounts of pure tannin and pure azadirachtin were increased, this rates reduced. Based on the results obtained from the findings of this study, it was determined that microorganisms used tannin and azadirachtin as carbon sources and preferred to use azadirachtin rather than tannin.

Are traditional neem extract preparations as efficient as a commercial formulation of azadirachtin A?

Neem seeds contain many substances with insecticidal properties, the main insecticidal ingredient being azadirachtin A. In developing countries such as Mali, a neem seed water extract is prepared by soaking ground seeds in water for three or seven days. The aim of this study was to check the effectiveness of this extract in terms of azadirachtin A extraction yield and insecticidal activity. The yield of extraction was 0.19 g azadirachtin A/100 g seeds. The concentration of azadirachtin A in the seed extract was approximately 200 mg l−1, eight times higher than the recommended concentration of commercial products (25 mg l−1). A comparison of the extractive capacity of different solvents indicated that the best solvents were water and methanol. The azadirachtin A concentration declined in extracts stored for more than 3 days at a temperatures higher than 30 °C. Bioassays were performed on target insects (the leafhopper Macrosteles quadripunctulatus, the moth Spodoptera littoralis and the tobacco whitefly Bemisia tabaci) in order to compare the insecticidal activity of the neem extract with that of the commercial product Neemazal T/S and of a solution of pure azadirachtin A. The bioassays conducted on the leafhopper and the moth demonstrated that the neem extract at the recommended concentration (25 mg l−1 active ingredient) was as effective as the azadirachtin-based commercial product at the same concentration, while for the control of the whitefly B. tabaci a higher concentration of the water extract was needed.

Laboratory evaluation of the side effects of insecticides on Aphidius colemani (Hymenoptera: Aphidiidae), Aphidoletes aphidimyza (Diptera: Cecidomyiidae), and Neoseiulus cucumeris (Acari: Phytoseidae)

The side effects of methoxyfenozide, indoxacarb, pyridaben, acetamiprid, azadirachtin A, spinosad, and propargite on Aphidius colemani, Aphidoletes aphidimyza, and Neoseiulus cucumeris were tested under laboratory conditions. Methoxyfenozide had low toxic effect on all three species, causing mortality after 24 h in 4.4, 11.4, and 29.3% of N. cucumeris, A. colemani, and A. aphidimyza, respectively. Similarly, indoxacarb caused mortality after 24 h in 11.9, 20.0, and 24.9% of A. aphidimyza, N. cucumeris, and A. colemani, respectively. In general, N. cucumeris exhibited the lowest sensitivity to all the insecticides. In contrast, A. colemani was highly sensitive to most of the tested insecticides. Methoxyfenozide was shown to significantly reduce fecundity of A. aphidimyza. In contrast, there was no effect of pure azadirachtin A on A. colemani fecundity. Results showed that both methoxyfenozide and indoxacarb would be suitable for use in the integrated pest management (IPM) because of their low toxic effect against all of the tested model species of natural enemies.

Toxic effects of neem cake extracts on Aedes albopictus (Skuse) larvae

In order to investigate its insecticide potential, the neem cake methanol extract was first analyzed and then separated by different solvents. The high-performance liquid chromatography analysis showed that the neem cake methanol extract still contained relevant quantities of nortriterpenes. Fractions of increasing polarity were obtained from the separation process: hexane fraction (Hp), EtOAc fraction (Ep), n-BuOH fraction (Bp), and aqueous fraction (Wp). The activity of the fractions on Aedes albopictus (Skuse) eggs and larvae was tested, and the Ep fraction exhibits the most relevant larvicide effect. The nuclear magnetic resonance fingerprint analysis of this phytocomplex isolated on EtOAc fraction was performed. The larvicidal effectiveness of the phytocomplex isolated on EtOAc, compared to that of pure and commercial azadirachtin solutions of different concentrations, was checked. The results showed that the activity of the phytocomplex, as a whole, was significantly higher than those of isolated compound solutions. As a consequence, the neem cake is a promising low-cost, easily available on the market, and natural resource to develop a new bioinsecticide, mainly in developing countries.

Ecological Risk Assessment of Neem-Based Pesticides

A tiered process was used to evaluate the risks of pure azadirachtin (AZA) and two neem-based insecticides (Neemix™ and Bioneem™) on six aquatic animals [crayfish (Procambarus clarkii), white shrimp (Penaeus setiferus), grass shrimp (Palaemonetes pugio), blue crabs (Callinectes sapidus), water fleas (Daphnia pulex), and mosquito larvae (Culex quinquefasciatus)] through short term acute toxicity tests. The risk was calculated using the level of concern endpoints (Q values) and relative hazard index (RHI) for acute and chronic exposure scenarios. The Q values of Neemix™, Bioneem™, and pure AZA derived from acute exposure tests indicated that D. pulex is the only sensitive species to the test pesticides. Furthermore, the RHI values of Neemix™ and Bioneem™ for D. pulex were above the critical limit of 10 indicating that these pesticides may pose a moderate hazard to this species and related crustaceans in acute exposure scenarios. The RHI values of the two pesticides and pure AZA were all below the critical limit of 10 for P. clarkii, P. setiferus, P. pugio, C. sapidus, and C. quinquefasciatus. The aquatic risk assessment process showed that the risk values of tested pesticides did not exceed the criteria, and therefore, no ecological hazard is likely to result from their use.

Effects of aqueous extract of neem seed kernel and azadirachtin on the fecundity, fertility and post‐embryonic development of the melonfly, Bactrocera cucurbitae and the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae)

Abstract:Aqueous extract of neem seed kernel (NSKE) was tested for its effect on the fecundity and post‐embryonic development of Bactrocera cucurbitae and Bactrocera dorsalis, when fed as water source. Comparisons were made with pure azadirachtin to ascertain its role as a compound responsible for these effects. Three oviposition dates (10, 15 and 20) after adult emergence were chosen. In the 10 day experiment no egg laying was recorded at a concentration of 0.25% NSKE for both the species. By the 20th day, mean number of eggs laid were 93 for B. cucurbitae and 100 for B. dorsalis at the same concentration as compared with 241 and 243 eggs in respective controls. This pattern of recovery was observed for the first time using neem extracts on B. cucurbitae and B. dorsalis. Effect on fertility were evident as the per cent egg‐hatch in the 10‐day experiment ranged from 56 to 72 and 61 to 75% for B. cucurbitae and B. dorsalis respectively, compared with 99 and 99% in controls. The overall egg hatch ranged between 56 and 85% for both the species, covering the reproductive age of the flies. There was no significant effect on pupation. Per cent pupation of the hatched larvae was &gt;90%, while the adult emergence calculated from the number of larvae that pupated was &gt;62%, for both the species, however, when calculated from the initial number of eggs laid the post‐embryonic effects were evident. The effects on fecundity, fertility and adult emergence were confirmed with pure azadirachtin. As with NSKE, pupation was not much affected, however, significant effects on adult emergence and behaviour were recorded. The post‐embryonic effects of neem seed kernel extracts on the two species of fruit flies was confirmed for the first time. Being safe, cheap and renewable, neem extract can be effectively used as an excellent alternative to synthetic insecticides.

An efficient method for the purification and characterization of nematicidal azadirachtins A, B, and H, using MPLC and ESIMS.

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.

Acute Toxicity Assessment of Azadirachtin‐Based Pesticides Using Murine Hybridoma and Oyster Cells

In vitro acute toxicities of azadirachtin‐containing pesticides (Neemix™ and Bioneem™), formulated with neem tree extracts, and pure azadirachtin (AZA), the believed active ingredient, were studied using hybridoma and oyster cells and were compared to results obtained using the standard in vivo Daphnia pulex toxicity assay. Neem‐based pesticides showed relatively high toxicity to both hybridoma and oyster cells at concentrations of 1 μg AZA/mL and higher. The IC50 values for hybridoma cells were 2.15 μg AZA/mL for Neemix™ and 1.67 μg AZA/mL for Bioneem™. Oyster cells had IC50 values of 2.18 μg AZA/mL for Neemix™ and 9.46 μg AZA/mL for Bioneem™. Purified AZA, however, did not appear to be as toxic as the formulations. D. pulex was also more sensitive to neem‐based pesticide exposure than that of pure AZA. The applications and limits of these two in vitro models for testing the acute toxicity of AZA‐based pesticides are discussed in comparison with the in vivo D. pulex test.

Antifeedant activity of fruit and seed extracts ofMelia azedarach andazadirachta indica on larvae ofSesamia nonagrioides

Methanolic extracts of seeds and fruits of the chinaberry tree,Melia azedarach L. (Meliaceae), showed strong antifeedant activity against 2nd instar larvae ofSesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae), a very serious pest of maize(Zea mays L.) in Mediterranean countries. Extracts were applied in an artificial diet at concentrations of 1000 and 2000 ppm. The parameters used to evaluate the activity were larval growth rates; quantity of food ingested; phagodepression/phagostimulation index; quantity of frass produced; quantity of material ingested; duration of larval development; and cumulative mortality. Seed extract showed high bioactivity at both doses, while fruit extract proved to be less active, and only at the higher dose used (2000 ppm) did it display a slight antifeedant activity. The activity of theM. azedarach seed extract at the higher dose (2000 ppm) was comparable to that of pure azadirachtin applied at a dose of 1.25 ppm, or to ‘Mubel’, a commercial extract ofAzadirachta indica A. Juss. (Meliaceae), applied at a dose of 75 ppm.

Neem (Azadirachta indica) seed kernel extracts and azadirachtin as oviposition deterrents against the melon fly (Bactrocera cucurbitae) and the oriental fruit fly (Bactrocera dorsalis)

Neem(Azadirachta indica A. Juss.) seed kernel (NSK) extracts,viz., NSK aqueous suspension (NSKS), ethanolic extract of NSK (EtOH. NSK), hexane extract of NSK (neem oil), ethanolic extract of the hexane extract (EtOH. oil) and acetone extract of deoiled NSK powder (Acet. DNSKP) at 1.25-20% concentrations, and pure azadirachtin at 1.25-10 ppm, were evaluated as oviposition deterrents toBactrocera cucurbitae (Coq.) andB. dorsalis Hendel. NSKS at ≥5% under choice test conditions, and at all concentrations (≥1.25%) in no-choice tests significantly deterred oviposition in both species. Similarly, EtOH. NSK was significantly active at all the concentrations tested for both species in choice and no-choice tests. However, with neem oil and EtOH. oil sensitivities of the two species differed considerably. Both extracts deterred oviposition byB. cucurbitae at all the concentrations tested under both choice and no-choice test conditions. On the other hand, withB. dorsalis, neem oil was significantly deterrent only at 20% in both test regimes and at 5% and 20% for EtOH. oil under choice and no-choice test conditions, respectively. Acet. DNSKP significantly deterred oviposition by both species at all concentrations tested. Azadirachtin failed to deter oviposition in either species.

Molluscicidal activity of neem ( Azadirachta indica A.Juss)

Molluscicidal property of Azadirachta indica A. Juss (neem) against the snails Lymnaea acuminata and Indoplanorbis exustus was studied. It was observed that the molluscicidal activity of the leaf, bark, cake, neem oil and the neem-based pesticides, achook and nimbecidine, was both time- and dose-dependent. The toxic effect of pure azadirachtin against both the snails was greater than the synthetic molluscicides.

Repellency and Toxicity of Azadirachtin and Neem Concentrates to Three Stored-Product Beetles

Repellency and toxicity of azadirachtin (98% AZA, which contains 98% azadirachtin) and 3 neem extracts (48, 23, and 7% AZA) to 3 stored-product insects, the rusty grain beetle, Cryptolestes ferrugineus (Stephens), the rice weevil, Sitophilus oryzae (L.), and the red Aour beetle, Tribvlium castaneum (Herbst), were investigated in the laboratory. Each test material repelled all 3 species in a standard repellency test using a food preference apparatus. Significant negative correlations were found between insect settling response and extract concentrations. T. castaneum was more sensitive to the repellent action of neem than the other 2 species. The test materials were also toxic to the 3 pest species, with C. ferrugineus being the most susceptible. Six-week LC50 values for 48, 23, and 7% AZA for C. ferrugineus were 18.8, 37.0, and 127.3 ppm, respectively. The F1 adults of all 3 insect species in almost all treatments were significantly reduced compared with controls. This reduction was significantly dose dependent. The relationship between bioactivity of neem materials and their azadirachtin content was established and is discussed. We confirmed that azadirachtin was largely responsible for both repellent (behavioral) and toxic (physiological) actions of neem on stored-product insects. However, the neem extracts are slightly more active than pure azadirachtin when applied at equivalent azadirachtin concentrations, indicating that azadirachtin is not the only active compound in neem.

Selection for resistance to azadirachtin in the green peach aphid,Myzus persicae

Two lines ofMyzus persicae of the same origin were treated repeatedlywith pure azadirachtin (aza), or a refined neem seed extract (NSE), at the equivalent concentration of aza. After 40 generations, the aza-selected line had developed 9-fold resistance to aza compared to a non-selected control line, whereas the NSE-selected line did not. These results suggest that a blend of active constitutents in a botanical insecticide such as neem might diffuse the selection process, mitigating the development of resistance compared to that expected with a single active ingredient.

Insect growth regulating effects of neem extract and azadirachtin on aphids

Neem, Azadirachta indica (A. Juss.), seed oil (NSO) applied to leaf discs at a concentration of 1.0% resulted in 94% to 100% mortality of second instar nymphs of currant-lettuce aphid, Nasonovia ribis-nigri (Mosley), and green peach aphid, Myzus persicae (Sulzer), after nine days. The equivalent amount of pure azadirachtin (AZA) (ã40 ppm), the principle active ingredient of neem, was as effective as NSO. The survival of adult aphids was unaffected by NSO or AZA, but the survival of offspring from treated adult M. persicae and N. ribis-nigri was reduced significantly. The lethal concentration of AZA resulting in 50% mortality of second instar nymphs of nine species of aphids ranged from 2.4 ppm for M. persicae on pepper to 635.0 ppm for the strawberry aphid, Chaetosiphon fragaefolii (Cockerell), on strawberry. For M. persicae, the growth regulating effect of AZA was influenced by the host plant and the nymphal instar treated.

Combined Florisil, Droplet Counter-Current, and High Performance Liquid Chromatographies for the Preparative Isolation and Purification of Azadirachtin from Neem (Azadirachta Indica) Seeds

Abstract An alternate prepurification procedure was developed for the isolation and purification of azadirachtin, a biologically active limonoid, from neem (Azadirachta indica) seeds by the use of Florisil and droplet counter-current chromatographies. The procedure, combined with the use of preparative high performance liquid chromatography, yielded 364.8 mg of >99% pure azadirachtin from 1.5 kg of Indian neem seeds.

Influence of azadirachtin on insect nutrition and reproduction

Neem extracts and pure azadirachtin influence the behaviour and physiology of large number of insects. These compounds affect the sensory receptors, thereby inhibit food intake. This anti-feedant property is exploited by using neem extracts and azadirachtin for plant protection. The gut motility and movement of food in the digestive tube are regulated by azadirachtin.Holotrichia serrata, the beetle of the ‘white grub’ feeds on neem leaves and no ill-effect of azadirachtin is reported. Azadirachtin and neem extracts regulate the reproductive functions of insects through their endocrine system. Morphogenesis, ovarian development, fecundity, egg viability and moulting are adversely affected by azadirachtin. Radioimmunoassays show that the ecdysteroid level falls drastically on azadirachtin treatment. Juvenile hormone synthesis also is inhibited inLocusta on administration of azadirachtin. InOncopeltus fasciatus, high doses of azadirachtin inhibit adult development resulting in permanent larvae which show oocyte differentiation with yolk incorporation equivalent to day 4 of adult development.

Preparative Reversed-Phase Liquid Chromatographic Isolation of Azadirachtin from Neem Kernels (1)

Abstract Azadirachtin is a tetranortriterpenoid insect feeding deterrent and growth regulator present in the kernels of the neem tree, Azadirachta indica A. Juss. A procedure was developed whereby 8.7 g of >90% pure azadirachtin was obtained from 48.2 kg of neem kernels by the use of open-column reversed-phase liquid chromatography on Phase-bonded C-18 Hi-Flosil and high-performance liquid chromatography on μ Bondapak C-18 for monitoring (217 nm) the purification.