Azadirachtin Research Articles

Azadirachtin Attenuates Carcinogen Benzo(a) Pyrene-Induced DNA Damage, Cell Cycle Arrest, Apoptosis, Inflammatory, Metabolic, and Oxidative Stress in HepG2 Cells.

Azadirachtin (AZD), a limonoid from the versatile, tropical neem tree (Azadirachta indica), is well known for its many medicinal, and pharmacological effects. Its effects as an anti-oxidant, anti-inflammatory, and anti-cancer agent are well known. However, not many studies have explored the effects of AZD on toxicities induced by benzo(a)pyrene (B(a)P), a toxic component of cigarette smoke known to cause DNA damage and cell cycle arrest, leading to different kinds of cancer. In the present study, using HepG2 cells, we investigated the protective effects of Azadirachtin (AZD) against B(a)P-induced oxidative/nitrosative and metabolic stress and mitochondrial dysfunction. Treatment with 25 µM B(a)P for 24 h demonstrated an increased production of reactive oxygen species (ROS), followed by increased lipid peroxidation and DNA damage presumably, due to the increased metabolic activation of B(a)P by CYP 450 1A1/1A2 enzymes. We also observed intrinsic and extrinsic apoptosis, alterations in glutathione-dependent redox homeostasis, cell cycle arrest, and inflammation after B(a)P treatment. Cells treated with 25 µM AZD for 24 h showed decreased oxidative stress and apoptosis, partial protection from DNA damage, and an improvement in mitochondrial functions and bioenergetics. The improvement in antioxidant status, anti-inflammatory potential, and alterations in cell cycle regulatory markers qualify AZD as a potential therapeutic in combination with anti-cancer drugs.

Azadirachtin exposure inhibit ovary development of Spodoptera litura (Lepidoptera: Noctuidae) by altering lipids metabolism event and inhibiting insulin signaling pathways

Lipids are main energy source for insects reproduction, which are becoming emerging target for pest management. Azadirachtin (AZA) is a multi-targeted and promising botanical insecticide, but its reproduction toxicity mechanism related to lipids metabolism is poorly understood. Here, we applied lipidomic and transcriptomic to provide a comprehensive resource for describing the effect of AZA on lipids remodeling in ovary of Spodoptera litura. The results showed that AZA exposure obviously altered the contents of 130 lipids subclasses (76 upregulated and 54 downregulated). In detail, AZA exposure changed the length and saturation degrees of fatty acyl chain of most glycerolipid, phospholipid and sphingolipid as well as the expression of genes related to biosynthesis of unsaturated fatty acids and fatty acids elongation. Besides, following the abnormal lipids metabolism, western blot analysis suggested that AZA induce insulin resistance-like phenotypes by inhibiting insulin receptor substrates (IRS) /PI3K/AKT pathway, which might be responsible for the ovary abnormalities of S. litura. Collectively, our study provided insights into the lipids metabolism event in S. litura underlying AZA exposure, these key metabolites and genes identified in this study would also provide important reference for pest control in future.

Salinity-induced modulation of growth and accumulation of phytochemicals composition in in vitro root cultures of Azadirachta indica

Salinity is a destructive abiotic factor that negatively impacts the growth and development of stress-intolerant plants. Although the effect of salinity on the growth of Azadirachta indica (neem) under in vitro conditions has been widely studied, little is known about the biochemical properties of in vitro root cultures exposed to salinity stress. Therefore, this study aimed to investigate the effects of varying concentrations of salinity on the biochemical properties of A. indica root cultures, specifically on total pigment and phenolic contents, antioxidant activities, and the presence of phytochemicals and azadirachtin compound, at different incubation periods. The results demonstrated that the root cultures coped with increasing NaCl concentrations by altering their phenotype. The highest antioxidant potential was observed in root cultures grown on MSN media (Murashige and Skoog media containing 3 mg L−1 NAA) supplemented with 0.5% w/v NaCl for 4 weeks. Additionally, this media composition and incubation period also resulted in the highest total phenolic and anthocyanin contents. The accumulation of photosynthetic pigments significantly decreased when exposed to NaCl treatments after 4 and 8 weeks. Interestingly, short-term exposure to NaCl had a positive impact on the azadirachtin (AZA) content, while longer treatment duration significantly decreased it. The highest amount of AZA was quantified from root cultures elicited with 1.5% w/v NaCl for 4 weeks. These findings contribute to a better understanding of the effects of salinity stress on the biochemical properties of in vitro root cultures of A. indica. The findings can aid in the development of strategies to enhance the production of bioactive compounds with potential applications in the food and pharmaceutical industries. Additionally, the study provides insights into the physiological mechanisms and manipulation of secondary metabolite production associated with salt tolerance in A. indica and other related species.

Comprehensive Dissipation of Azadirachtin in Grapes and Tomatoes: The Effect of Bacillus thuringiensis and Tentative Identification of Unknown Metabolites.

Neem oil is a biopesticide normally applied together with Bacillus thuringiensis (Bt). However, neither its dissipation nor the influence of Bt has been previously evaluated. In this study, dissipation of neem oil was investigated when it was applied alone or together with Bt at 3 and 22 °C. A methodology involving solid-liquid extraction and liquid chromatography-high-resolution mass spectrometry was developed for that purpose. The method was validated obtaining recoveries from 87 to 103%, with relative standard deviations lower than 19% and limits of quantification from 5 to 10 μg/kg. Azadirachtin A (AzA) dissipation was fit to a single first order, being faster when neem oil was applied together with Bt and at 22 °C (RL50 = 12-21 days) than alone and at 3 °C (RL50 = 14-25 days). Eight related compounds were found in real samples with similar dissipation curves compared to AzA, and five unknown metabolites were identified in degraded samples, with increasing concentrations during parent compound degradation.

Phytochemical and Synergism of Plant and Microbial Insecticides to Eliminate Earias Vittella Gut Epithelial Cells

The spotted bollwom, Earias vittella is a very serious and polyphagous pest that attacks many plants of Malvaceae family. Such as Lady’s finger (Abelmoschus esculentus Moench), which attacks growing points of the plant. The intensive use of highly toxic and broad-spectrum pesticides threatens the sustainability of vegetable production, mainly due to the development of pest resistance. Consequently, it becomes more difficult to manage key pests and secondary pests, which assume major status due to the elimination of natural enemies from the eco system. In the present study, evaluation of the larvicidal, quantitative food utilization and histological activity of Manihot esculenta, Azadirachtin (AZA), Bacillus thuringiensis var. kurstaki (Btk), and synergistic effects, was carried out against Earias vittella. The GC-MS analysis of M. esculenta was estimated and seventeen different phytoactive compounds were identified. Larvicidal activity of M. esculenta, AZA, and Btk provided significant mortality against E. vittella. The lowest LC50 of individual treatment was from AZA, the LC50 being as follows: 0.122%, 0.131%, 0.150%, 0.157%, and 0.168%, and the LC50 of synergistic treatment was 1.434ppm, 1.469ppm, 1.531ppm, 1.701ppm, and 1.908ppm for I, II, III, IV, and V instars, respectively. The food utilization result showed that, the food intake of E. vittella was significantly reduced after the treatment. Here, synergistic effects controls the food intake, which is highly reduced, and fecal pallet egestion considerably reduces the amount of food consumed. Histological studies state that, after the treatment, the gut of E. vittella is drastically damaged by the biopesticides. M. esculenta, AZA and Btk and their synergism are very effective against the immature stage of E. vittella. Gut region of the insect finely eradicated by the biopesticide. Overall, this study concludes that the taken botanical insecticides are ecofriendly, harmless to mammals and other non-target animals.

Effects of sublethal azadirachtin on the immune response and midgut microbiome of Apis cerana cerana (Hymenoptera: Apidae)

As a wildly used plant-derived insecticide, azadirachtin (AZA) is commonly reported as harmless to a range of beneficial insects. However, with the research on the effect of AZA against pollinators in recent years, various negative physiological effects on other Apidae species have been demonstrated. Thus to explore the safety of azadirachtin to Apis cerana cerana, the different physiological effects of sublethal concentration of azadirachtin on worker bees A.c.cerana has been studied. With the exposure of 5 mg·L-1 and 10 mg·L-1 azadirachtin for 5 d, the relative expression of Apidaecin, Abaecin and Lysosome genes in workers has decreased significantly at 1, 2,3 and 5 d, and the mRNA levels of Defensin 2 and Hymenoptaecin were also significantly inhibited by 10 mg·L-1 azadirachtin at each check point. Besides, the activity of midgut antioxidant enzymes Superoxide Dismutase (SOD) and Catalase (CAT) which are the first line of defence in antioxidant systems was not affected by AZA, the activity of Peroxidase (POD) showed a fluctuating pattern at 24 h and 48 h, while the activity of polyphenol oxidase (PPO) has significantly inhibited by AZA. However, through 16sRNA analysis it was observed that 5 mg·L-1 AZA did not affect the midgut microbiome colony composition and relative abundance, as well as its main function. Therefore, to a certain extent, azadirachtin is safe for workers, but we should pay more attention to the sublethal effect of AZA that also detrimental to the healthy development of the honeybee colony.

Azadirachtin Attenuates Lipopolysaccharide-Induced ROS Production, DNA Damage, and Apoptosis by Regulating JNK/Akt and AMPK/mTOR-Dependent Pathways in Rin-5F Pancreatic Beta Cells.

Pancreatic inflammation and the resulting cellular responses have been implicated in pancreatitis, diabetes, and pancreatic cancer. Inflammatory responses due to the bacterial endotoxin, lipopolysaccharide (LPS), have been demonstrated to alter cellular metabolism, autophagy, apoptosis, and cell proliferation in different cell populations, and hence increases the risks for organ toxicity including cancer. The exact molecular mechanism is however not clear. In the present study, we investigated the role and mechanism of an antioxidant, azadirachtin (AZD), a limonoid extracted from the neem tree (Azadirachta indica), against LPS-induced oxidative stress in the pancreatic β-cell line, Rin-5F. We demonstrated that cells treated with LPS (1 µg/mL for 24 h) showed increased reactive oxygen species (ROS) production, DNA damage, cell cycle arrest, and apoptosis. Our results also showed that LPS induced alterations in the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathways, suppressing autophagy and augmenting apoptosis. Treatment with Azadirachtin (25 µM for 24 h), on the other hand, rendered some degree of protection to the pancreatic cells from apoptosis by inducing the autophagy signals required for cell survival. These results may have significance in elucidating the mechanisms of pancreatic β-cell survival and death by balancing the molecular communication between autophagy and apoptosis under inflammatory and pathological conditions.

Physicochemical, Druggable, ADMET Pharmacoinformatics and Therapeutic Potentials of Azadirachtin - a Prenol Lipid (Triterpenoid) from Seed Oil Extracts of Azadirachta indica A. Juss.

Azadirachtin (AZA) is the most abundant bioactive secondary metabolite (BASM) in neem seed oil extract (NSOE) of Azadirachta indica A. Juss. AZA is localised in different parts of the plant (seeds, fruits, flowers, leaves, stem, bark and root) however, with varying degree of concentration. It has been documented that maximum concentration of AZA is present to the tune of 48000 μg g-1 in the seeds. It has been established that the environmental conditions determines the overall content and composition of BASM in different parts of the plant. Neem plant parts are most commonly used as therapeutic agents in remote villages in India for its ethnomedicinal therapeutic potentials; however, its physicochemical, druggable and pharmacological properties inadequately described. In the present study an attempt has been made to evaluate the physicochemical, druggable and pharmacological properties of Azadirachtin in NSOE of A. indica from ADMET perspectives.
 Keywords: NEEM; Azadirachta indica; Azadirachtin; Pharmacoinformatics; ADMET; Drug-Likeness; Toxicology

Effects of pesticide NeemAzal-T/S on thyroid, stress hormone and some cytokines levels in freshwater common carp, Cyprinus carpio L

In this study, the effects of azadirachtin (AZA) on thyroid axis hormones [thyroid stimulating hormone (TSH), free/total triiodothyronine (FT3/TT3), free/total thyroxine (FT4/TT4)], stress axis [cortisol, adrenocorticotropic hormones (ACTH)] and cytokines [tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)] levels in carp fish exposed to 1.20 ppm and 2.40 ppm AZA for 4 days and 30 days were investigated. As a result of our study, the increase in serum TSH, FT3, FT4, ACTH, cortisol, TNF-α, IL-1β and IL-6 levels and decreases in serum TT3 and TT4 levels were found to be statistically significant depending on the dose and time (p < 0.05).

Neem-based oil-in-water (O/W) emulsion as a biopesticide

Neem tree (Azadirachta indica) is well-known to contain active components, such as azadirachtin (AZA), nimbin, and nimbidin that are potent to eliminate pesky insects. These active components can be found in large numbers in the neem oil. The favourable properties of neem oil to kill pests with minimum impacts to the ecosystem makes it veritably attractive to replace synthetic chemical pesticide. In this research, the neem oil is mixed with water and emulsifiers in the homogenizer to generate oil-in-water (O/W) emulsions. The droplet size, the size distribution, the stability, and the effectiveness of emulsion are analysed to determine the best formulation of neem-based biopesticide. The results showed that the formulation of 10%- w neem oil and 5%-w non-ionic emulsifier Lutensol® TO 6 produces the most stable emulsion with d32 of 0.50 μm. The fastest mortality rate for Musca domestica larvae is achieved at 20 ppm and 8 minutes with emulsion formulae of 10%-w neem oil and 5%-w non-ionic emulsifier Lutensol® TO 8.

Field assessment on management of South American pin worm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in tomato

Field trials were conducted for three years during kharif season of 2014, 2015 and 2016 to evaluate the integrated pest management (IPM) module against pinworm (Tuta Absoluta) in tomato in comparison with farmer’s practice. Seasonal incidence studies revealed that population increased gradually from September and touched its peak during December (28.00 adults/trap). The insect was found to be higher at the fruit maturity stage and infestation found decreased with increase in temperature. IPM module reduced the cost of cultivation by Rs. 9615.66 ha-1 and the net returns were increased by Rs. 53282.00 per hectare. The average benefit cost ratio was improved with 2.39 in treatment compared to farmers practice 1.92. The results based on pooled data showed that reduction in the fruit damage by Tuta absoluta and good yields is due to the integrated management strategy taken up by installing pheromone traps 2 weeks after transplanting by which awareness on timely spraying of Azadirachtin 1500ppm @ 5ml/lt or combination of Azadirachtin with Chlorantraniliprole 18.5% SC @ 0.3 ml or Flubendiamide 480 SC @0.3ml or Indoxacarb 14.5% SC @ 1.75ml carried in practicing farmers field based on ETL level of adult catches and burning of infested plants and remnants of the crop immediately after the fruits have been harvested also found effective in managing the pest.

Effects of chemical and biological plant protection products on R&D colonies of the buff-tailed bumblebee Bombus terrestris: (2.5 Part 1)

Bumblebees (Bombus terrestris) are exposed daily to Plant Protection Products through their foraging and feeding activities. Through all possible means of contact with pesticides, consumption through sugarwater is the most severe. In the present study, lethal and sublethal effects of the consumption of sugarwater solutions with the pesticides Sivanto WG (flupyradifuron), Exalt SC (spinetoram) and Oikos EC (azadirachtin) were studied using a sequential dilution testing scheme of 1/1 and 1/10 of the maximum field recommended concentration (MFRC). For the weekly assessment, parameters such as the survival of the mother queen, of workers and drones, the formation of gynes, and the weight and volume of the colonies were recorded. Moreover, by the end of the colony’s life, the total number of formed workers/drones, the number of newborn gynes and queen brood were also recorded. The IOBC side-effect classes for laboratory trials were applied in order for the results to be categorized and conclusions made. Both tested concentrations of Sivanto WG (flupyradifuron) were slightly harmful for queen, worker and drone populations, Exalt SC (spinetoram) was harmful at 1/1 dilution but only slightly harmful at the 1/10 dilution, and both concentrations of Oikos EC (azadirachtin) were slightly harmful for workers and drones but toxic for queens at both dilutions.

Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica.

For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2–0.6 mg L−1) and 2,4-D (0.2–0.6 mg L−1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L−1 of TDZ resulted in the highest fresh weight (3.38 ± 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus (214.53 ± 33.63 mg g−1 dry weight (DW)) induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 µg mL−1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic β-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with β-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic β-cells. Furthermore, AZD supplementation in pancreatic β-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

Assessment of 8-hydroxy-2-deoxyguanosine activity, gene expression and antioxidant enzyme activity on rainbow trout (Oncorhynchus mykiss) tissues exposed to biopesticide

The goal of this study was to determinate toxicity mechanism of biopesticide with antioxidant enzymes parameters such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and malondialdehyde (MDA) levels, oxidative DNA damage (8-hydroxy-2-deoxyguanosine (8-OHdG)), transcriptional changes of heat shock protein 70 (HSP70), and cytochromes P4501A (CYP1A), sod, cat, and gpx in liver and gill tissues of Oncorhynchus mykiss. For this aim, plant-based (natural pesticides, azadirachtin (AZA)) and synthetic pesticides (deltamethrin (DLM)) were exposed on the fish at different concentrations (0.0005 and 0.00025ppm of DLM; 0.24 and 0.12ppm of AZA) for 21 days.According to the results of the study, the activity of SOD, CAT and GPx decreased, but malondialdehyde (MDA) level and activity of 8-OHdG increased in the gill and liver of rainbow trout (p<0.05). Additionally sod, cat and gpx were down regulated; HSP70 and CYP1A were up regulated for transcriptional observation. The downwards regulation of antioxidant (sod, cat and gpx) and the upregulation of HSP70 and CYP1A was obvious with doses of AZA or DLM (p<0.05). The findings of this study suggest that biopesticide can cause biochemical and physiological effects in the fish gill and liver by causing enzyme inhibition, an increase in 8-OHdG levels and changes in both transcriptional parameters (sod, cat, gpx, HSP70 and CYP1A). We found that excessive doses of plant-based pesticide are nearly as toxic as chemical ones for aquatic organisms. Moreover, 8-OHdG, HSP70 and CYP1A used as a biomarker to determinate toxicity mechanism of biopesticide in aquatic environment.

Rationale for Water Extraction of Lipophilic Azadirachtin ( Tetranor Triterpenoid) from Neem Seeds - A Conundrum

Objectives: To extract lipophilic Azadirachtin from seed kernels of Azadirecta indica (Neem) with water and the rationale behind it as its solubility in water is very negligible (0.26 gm/lit of water).Methods: Neem seeds obtained from local market were dehulled to obtain kernels which were cold pressed to remove oil and extracted with four volumes of water at room temperature. Azadirachtin content in cold pressed neem kernel was determined using High Performance Liquid Chromatography (HPLC) with reverse phase silica column (RP18).Proteincontentinaqueousextractwasdeterminedby micro-Kjeldahl’s method and sugars by acid hydrolysis, permethylation and Gas Chromatography (GC). Findings: Aqueous extract of neem seed kernel was found to contain about 670 ppm of azadirachtin (AZA). A small portion of this aqueous extract when lyophilized yielded a hygroscopic material showing the presence of 4% AZA (HPLC method), 25% proteins (micro-Kjeldahl’s method) and about 50% of carbohydrates and other aliphatic compounds. The aqueous solution when re-extracted into ethyl acetate followed by precipitation using n-hexane yielded azadirachtin of 40% purity.Application: Lipophilic azadirachtins get extracted into water may be due to micelle formation of polysaccharides and proteins that cooccur in neem seed kernel.This hypothesis has been substantiated by addition of external azadirachtin to aqueous extract of neem seed kernel thereby enhancing its solubility from initial 670 ppm by more than two fold to a level of 1800 ppm. To obtain azadirachtin of about 40% purity directly from neem seed kernels would be possible only when first extracted with water.

Field evaluation of three biopesticides for control of the raspberry cane midge, Resseliella thobaldi (Barnes) in Bulgaria

The raspberry cane midge, Resseliella theobaldi is a key pest on red raspberry, Rubus idaeus. The larvae of the insect severely attack the raspberry canes, resulting in premature death of the plant canes. In the last decade, organic production of raspberry fruits has significantly increased in Bulgaria. At the same time there are few products of botanical or microbiological origin that might be used for control of this pest. In present study the effect of NeemAzal® T/C (azadirachtin A), Sineis 480 SC® (spinosad), and Bacillus subtilis on R. theobaldi was evaluated. The experiments were conducted in two raspberry fields at different altitude. In the field at lower altitude (196 m), the raspberry cane midge has developed four generation per year, while in the field at higher altitude (960 m) three generations of the pest have been completed. Lowest number of larvae in raspberry canes was observed after application of NeemAzal® T/C, and B. subtilis in both raspberry fields. Both products demonstrated highest efficacy at 7th day after treatment, when the number of larvae per splits was 67.1-82.5% for NeemAzal® T/C, and 75.1-81.2% for B. subtilis lower compared with the control at the two experimental sites.

Short and long term effects of some bio-insecticides on Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) and its coexisting generalist predators in tomato fields

Tomato leaf miner, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), is one of the most destructive pests of tomato in many parts of the world including Iran. Field studies were conducted to determine the short and long term effects of Bacillus thuringiensis var Kurstaki (Bt), azadirachtin (AZ), a mix of AZ + Bt, and indoxacarb, as a current chemical insecticide, on T. absoluta larvae. Also, effects of the insecticides were studied on the coexisting generalist predators, Coccinella septempunctata L., Chrysoperla carnea Stephens and Syritta sp. Sampling of T. absoluta and its coexisting generalist predators were performed 1 day before treatment (DBT) and one, 5, 8, 14 and 19 days after treatment (DAT). The results indicated significant short term effect of indoxacarb on the pest larvae. Indoxacarb reduced T. absoluta density and damages. Bt, AZ and mixture of them significantly suppressed the larval density at 19 DAT and caused significant reduction in leaf, stem and fruit damage. The highest long term effect on the pest abundance and damage were observed in Az + Bt caused 100% reduction in fruit and foliage damage compared to the control. The highest and lowest adverse effects on C. carnea, C. septempunctat and Serrita sp. were observed in indoxacarb and Bt treatments, respectively. Findings of this study imply that the mixture of Az + Bt has the highest selective toxicity on the pest and the lowest effect on its coexisting generalist predators.

Effect of neem (Azadirachta indica A. Juss) leaf extract on resistant Staphylococcus aureus biofilm formation and Schistosoma mansoni worms

Ethnopharmacological relevanceThere are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms. Materials and methodsQuantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy. ResultsIt was observed the presence of AZA in the extract (0.14±0.02mg/L). Testing Neem EE sub-inhibitory concentrations, a significant biofilm adherence inhibition from 62.5µg/mL for a sensitive S. aureus and 125µg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000µg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50µg/mL at 72h of incubation, while 300µg/mL at 24h of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96h of incubation with 100µg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300µg/mL (male) and 100µg/mL (female) of the extract showed severe morphological changes after 24h of treatment. ConclusionsNeem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.

INSECTICIDAL AND OXIDATIVE EFFECTS OF AZADIRACHTIN ON THE MODEL ORGANISM Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE).

The insecticidal effects, specifically, changes in hemolymph total protein and malondialdehyde (MDA) levels, and antioxidant enzyme activities of azadirachtin (AZA) given to the wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) larvae via force feeding were investigated. Bioassays showed that the LD50 and LD99 (lethal dose) values of AZA were 2.1 and 4.6 μg/larva, respectively. Experimental analyses were performed with five doses of AZA (0.5, 1, 1.5, 2, and 3 μg/larva). Total protein level in larval hemolymph increased at all AZA doses at 24 h whereas a considerable decrease was observed at 2 and 3 μg/larva doses, and only an increase displayed at 1.5 μg/larva at 72 h. The level of MDA increased at 2 and 3 μg/larva doses at 24 h compared with controls. This trend was also observed at 1.5, 2, and 3 μg/larva doses at 72 h and MDA levels were lower when compared with those of 24 h at all doses except for 1.5 μg/larva dose. Catalase activity decreased at 1, 1.5, and 2 μg/larva doses at 24 h whereas increased at all doses except for 0.5 μg/larva at 72 h compared with controls. AZA led to a decline in superoxide dismutase activity at all experimental doses at 24 and 72 h except for 3 μg/larva doses at 72 h. An increase in glutathione-S-transferase (GST) activity was evident at all AZA doses at 24 h. AZA displayed 68% decline in GST activity at 72 h post treatments when compared to 24 h. Consequently, We infer that the toxicity of AZA extends beyond its known actions in molting processes to redox homeostasis.