azadirachtin-b and azadirachtin

This study identified the threshold concentration of limonoids for the complete inhibition of oviposition of Ceratitis capitata (Wiedemann) in grapes 'Itália.' Choice and no-choice experiments with the insect were performed. The three no-choice bioassays were conducted following a completely randomized design with 18 treatments (three densities of insects [one, two, or three females]×five concentrations of limonoids and control) and 20 replicates. In a free choice bioassay, two fruits per cage (a treatment grape and a control) were provided for ovipositing. Three densities of insects (one, two, or three females) were used, with 15 replicates. Bioassays were conducted at 25±2°C, 60±10% relative humidity, and a photoperiod of 14:10 (L:D) h. The inhibition of oviposition of C. capitata was concentration dependent, with infestation occurring at lower concentrations of azadirachtin (+3-tigloylazadirachtol) and complete inhibition occurring at concentrations at or exceeding 100 ppm azadirachtin (+28.5 ppm of 3-tigloylazadirachtol), maintaining protective effects even at the most densely populated treatment (three females per fruit). When the pest had a free choice of host grapes (treatment vs. control), severe inhibition was observed at concentrations≥50 ppm azadirachtin (+14.3 ppm of 3-tigloylazadirachtol). We conclude that a threshold concentration of 100 ppm azadirachtin (+28.5 ppm of 3-tigloylazadirachtol) is capable of preventing grape infestation. This concentration is likely to provide a reliable level of protection, as the experimental population density of three females per fruit usually does not occur in the field and wild flies usually have more host options.

Topics: Animals; Female; Insecticides; Limonins; Oviposition; Random Allocation; Tephritidae

A rapid and sensitive LC-ESI-MS method has been developed and validated for the quantitation of azadirachtin and 3-tigloylazadirachtol in deciduous tree matrices. The method involves automated extraction and simultaneous cleanup using an accelerated solvent technique with the matrix dispersed in solid phase over a layer of primary-secondary amine silica. The limits of quantification were 0.02 mg/kg for all matrices with the exception of Norway maple foliage (0.05 mg/kg). Validation at three levels (0.02, 0.1, and 1 mg/kg), demonstrated satisfactory recoveries (71-103%) with relative standard deviation <20%. Two in-source fragment ions were used for confirmation at levels above 0.1 mg/kg. Over a period of several months, quality control analyses showed the technique to be robust and effective in tracking the fate of these natural botanical insecticides following systemic injection into various tree species for control of invasive insect pest species such as the emerald ash borer and Asian longhorned beetle.

Topics: Chromatography, High Pressure Liquid; Limonins; Pesticide Residues; Phloem; Plant Leaves; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trees

Thirty-five limonoids, including 15 of the azadiradione type (1-15), five of the gedunin type (16-20), four of the azadirachtin type (21-24), nine of the nimbin type (25-33), and two degraded limonoids (34, 35), isolated from Azadirachta indica seed extracts, were evaluated for their cytotoxic activities against five human cancer cell lines. Seven compounds (3, 6, 7, 16, 18, 28, and 29) exhibited cytotoxic activity against one or more cell lines. Among these compounds, 7-deacetyl-7-benzoylepoxyazadiradione (7), 7-deacetyl-7-benzoylgeduin (18), and 28-deoxonimbolide (28) exhibited potent cytotoxic activity against HL60 leukemia cells with IC(50) values in the range 2.7-3.1 μM. Compounds 7, 18, and 28 induced early apoptosis in HL60 cells, observed by flow cytometry. Western blot analysis showed that compounds 7, 18, and 28 activated caspases-3, -8, and -9 in HL60 cells. This suggested that compounds 7, 18, and 28 induced apoptotic cell death in HL60 cells via both the mitochondrial- and the death receptor-mediated pathways. Futhermore, compound 7 was shown to possess high selective cytotoxicity for leukemia cells since it exhibited only weak cytotoxicity against a normal lymphocyte cell line (RPMI 1788).

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Azadirachta; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Limonins; Lymphocytes; Mitochondria; Molecular Structure; Receptors, Death Domain; Seeds

Azadirachtins are natural insecticides derived from the neem tree. The emerald ash borer (EAB) is an exotic invasive insect pest that infests various ash tree species and has the potential for significant economic, aesthetic and ecological impacts throughout North America. The initial translocation and foliar residue dynamics of azadirachtins were examined following direct injection into white and green ash trees growing in urban scenarios as a potential control for EAB.. Substantial concentrations of azadirachtins A and B [mean maxima > 0.98 mg kg(-1) fresh weight (f.w.)] were observed within 2 days of injecting a specifically designed formulation of azadirachtins. Foliar residues declined exponentially through time, with half-life estimates ranging from 5.1 to 12.3 days. At the time of leaf senescence, foliar residue levels approximated 0.01 mg kg(-1) f.w., strongly mitigating the potential effects of non-target biota in soil or aquatic compartments.. The magnitude and duration of exposures observed in this field study were considered to be above the thresholds required for biological effectiveness against both larval and adult life stages of EAB. Results support the use of azadirachtins as an environmentally acceptable systemic insecticide for control of EAB and protection of high-value ash trees in urban environments.

Topics: Animals; Coleoptera; Fraxinus; Insecticides; Limonins; Pesticide Residues; Plant Leaves; Time Factors

The degradation of the main azadirachtoids on tomatoes was studied after greenhouse treatment. These experiments were carried out at 1 and 5x the concentration recommended by the manufacturer. In all experiments the deposition of azadirachtin A (AZA-A) was below the maximum residue level (MRL). Even if at the highest dose, AZA-A half-life time calculated as pseudo first order kinetic was 1.2 days in agreement with the recommended preharvest interval (PHI) of 3 days. Experiments with a model system showed that sunlight photodegradation is the main factor influencing the rate of disappearance of AZA-A after greenhouse treatment while tomato epicuticular waxes doubled the photodegradation rate of AZA-A in a commercial formulation.

Topics: Chromatography, High Pressure Liquid; Food Technology; Half-Life; Insecticides; Kinetics; Limonene; Limonins; Photolysis; Solanum lycopersicum; Spectrometry, Mass, Electrospray Ionization; Sunlight; Triterpenes

Persistence of azadirachtins (A+B) and of the other limonoids (nimbin, salannin, deacetylnimbin, and deacetylsalannin) on peach leaves and fruits was studied using a commercial formulation (form. C) compared with an experimental formulation (form. E) prepared with coformulations allowed in organic culture. Field experiments were carried out using three concentrations: 1x, 5x, and 10x the dose recommended by the manufacturer. The EU maximum residue level (MRL) in fruits and vegetables for azadirachtin A is 1 mg/kg with a preharvest interval (PHI) of 3 days. At the recommended dose, azadirachtin A residue on fruits was not detectable (LOQ < 0.8 microg/kg). After field treatment at the 5x concentration, azadirachtoids were found with 22% in the epicuticular waxes and the remaining 78% on the fruit surface. No residues were found in the fruit pulp. The experimental formulation (E) produced lower residues on leaves and fruit compared with the commercial formulation (C), although formulation E showed greater stability. This is probably due to the amount of the active ingredients that diffuse into the epicuticular wax layer thus enhancing photostability of azadirachtoids.

Topics: Azadirachta; Fruit; Half-Life; Insecticides; Limonins; Pesticide Residues; Plant Leaves; Prunus

To study the systemic effects of active neem ingredients, the substrate of bean plants was treated with a 170 g kg(-1) azadirachtin (NeemAzal-U; Trifolio-M GmbH, Lahnau, Germany, registration pending). This product was used at a dose rate of 10 mg AZA (azadirachtin a) and 1.2 mg 3-tigloyl-azadirachtol (azadirachtin b) per treated bean plant. Afterwards, the translocation and persistence of AZA and 3-tigloyl-azadirachtol and the effects on western flower thrips, Frankliniella occidentalis (Pergande), were studied. Residues of AZA and 3-tigloyl-azadirachtol from substrates with different contents of organic matter [pure culture substrate (CS), CS-sand mixture] and from various plant parts were quantified by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The dissipation trends of AZA and 3-tigloyl-azadirachtol were similar within the same substrates. A slower decline of both active ingredients was measured with CS than with CS-sand mixture. Residue analysis of the bean plants showed that only small proportions of the initial amounts of AZA and 3-tigloyl-azadirachtol applied to the substrate were present in the plant (0.3-8.1%). Variable amounts of residues of the active components in relation to plant parts and time of analysis indicated a different translocation pattern for the two active ingredients. Higher residues of the active ingredients were found in roots and stems after neem application using CS, whereas higher residues were found in leaves after CS-sand mixture treatments. Mortality of F. occidentalis after NeemAzal-U soil applications reached up to 95% on CS-sand mixture, compared with 86% in CS.

Topics: Animals; Insecta; Limonins; Pesticide Residues; Phaseolus; Soil

Azadirachtoids were determined by liquid chromatography/mass spectrometry (LC/MS) in five methanolic seed extracts of the neem tree and in a commercial formulation. On average, seed extracts contain azadirachtin A (10.9%), azadirachtin B (3.5%), nimbin (10.4%), and large quantities of salannin (19.0%). The composition of the commercial formulations may present different azadirachtoids contents depending on the natural extracts used in the preparation. Because these compounds may also show insecticide activity, the efficacy on field of these formulations may be very different. Photodegradation of pure azadirachtoids was also studied. Azadirachtins and related compounds are very sensitive to sunlight, degrading rapidly, with half-lives of the order of 11.3 h for azadirachtin A and 5.5 h for azadirachtin B and few minutes for the other limonoids compounds studied. The residues of azadirachtins and the main constituents, e.g., salannin, nimbin, deacetylnimbin, and deacetylsalannin, of the neem seed extract were determined on strawberries after field treatment using two different formulations. This residue study on strawberry was carried out to assess not only the azadirachtin content but also the main azadirachtoids contents. Three days after field application at five times the dose recommended by the manufacturer, residues of azadirachtin A and B were 0.03 and 0.01 mg/kg, respectively, while residues of salannin (LOQ 0.01 mg/kg) and nimbin (LOQ 0.5 mg/kg) were not detectable.

Topics: Azadirachta; Fragaria; Fruit; Half-Life; Limonene; Limonins; Plant Extracts; Sunlight; Triterpenes

Three new azadirachtin derivatives, named azadirachtins O-Q (1-3), along with the known azadirachtin B (4), azadirachtin L (5), azadirachtin M (6) 11alpha-azadirachtin H (7), 11beta-azadirachtin H (8), and azadirachtol (9) were isolated from seed kernels of Azadirachta excelsa. Their structures were established by spectroscopic techniques, and the structure of 3 was confirmed by X-ray analysis. Compounds 1-7 and 9 exhibited toxicity to the diamondback moth (Plutella xylostella) with an LD50 of 0.75-1.92 microg/g body weight, in 92 h.

Topics: Animals; Azadirachta; Crystallography, X-Ray; Limonins; Moths; Plants, Medicinal; Seeds; Thailand

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.

Topics: Antifungal Agents; Antinematodal Agents; Azadirachta; Chromatography, High Pressure Liquid; Limonins; Spectrometry, Mass, Electrospray Ionization