Azadirachtin Content Research Articles

Treating green pea aphids, Myzus persicae, with azadirachtin affects the predatory ability and protective enzyme activity of harlequin ladybirds, Harmonia axyridis

As a natural enemy of green peach aphids, harlequin ladybirds, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), are also indirectly affected by azadirachtin. In this study, we evaluated the effects of ladybird exposure to azadirachtin through azadirachtin-treated aphids. About 2 mg/L azadirachtin treated aphid can deliver the azadirachtin to ladybird larvae in 12 and 24 h. And azadirachtin treatment affected the rate at which fourth instar larvae and adult ladybirds preyed on aphids. Furthermore, the antifeedant effect increased with increasing azadirachtin concentrations. Twelve hours after exposing fourth instar ladybird larvae to aphids treated with 10 mg/L azadirachtin, the antifeedant effect was 47.70%. Twelve hours after exposing adult ladybirds to aphids treated with 2 mg/L azadirachtin, the antifeedant effect was 67.49%. Forty-eight hours after exposing ladybird larvae to azadirachtin-treated aphids, their bodyweights were 8.37 ± 0.044 mg (2 mg/L azadirachtin), 3.70 ± 0.491 mg (10 mg/L azadirachtin), and 2.39 ± 0.129 mg (50 mg/L azadirachtin). Treatment with azadirachtin affected the ability of ladybirds to prey on aphids. The results indicated that the instant attack rate of ladybird larvae and adults and the daily maximum predation rate were reduced by azadirachtin treatment. Superoxide dismutase (SOD), peroxidase (POD), and peroxide (CAT) enzyme activities of ladybirds were affected after feeding on aphids treated with azadirachtin. Azadirachtin has certain antifeedant effects on ladybirds and affects the ability of ladybirds to prey on aphids and the activities of SOD, POD, and CAT enzymes, which results in inhibition of normal body development.

Acute toxicity of organophosphate pesticide profenofos, pyrethroid pesticide λ cyhalothrin and biopesticide azadirachtin and their sublethal effects on growth and oxidative stress enzymes in benthic oligochaete worm, Tubifex tubifex

The present study was aimed to assess the acute toxicity of organophosphate pesticide, profenofos; synthetic pyrethroid pesticide, λ cyhalothrin and biopesticide, azadirachtin and their sublethal effects on growth rate and oxidative stress biomarkers in Tubifex tubifex in vivo. The results showed that 96 h LC50 value of profenofos, λ cyhalothrin and azadirachtin to Tubifex tubifex are 0.59, 0.13 and 82.15 mg L−1 respectively. Pesticide treated worms showed several behavioral abnormalities including increased mucus secretion, erratic movements, wrinkling activity and decreased clumping tendency during acute exposure. The percentage of autotomy increased significantly (p < 0.05) with the increasing concentration of the pesticides at 96 h of exposure. Sublethal concentrations of profenofos (0.059 and 0.118 mg L−1), λ cyhalothrin (0.013 and 0.026 mg L−1) and azadirachtin (8.2 and 16.4 mg L−1) caused significant alterations in growth rate and oxidative stress enzymes in T. tubifex during 14 days exposure period. The growth rate of the pesticide exposed worms decreased significantly (P < 0.05) in a concentration and duration-dependent manner. Superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-s-transferase (GST) and glutathione peroxidase (GPx) demonstrated a noteworthy (p < 0.05) initial induction followed by a subsequent reduction, while catalase (CAT) and malondialdehyde (MDA) exhibited noteworthy induction (p < 0.05) all through the exposure time. Through principal component analysis, correlation matrix, and integrated biomarker response, the effects of profenofos, λ cyhalothrin and azadirachtin on T. tubifex were distinguished. These results indicate that exposure to profenofos, λ cyhalothrin and azadirachtin affect survivability, change the behavioral responses, reduce the growth rate and induce oxidative stress enzymes in T. tubifex.

Deleterious action of azadirachtin against the mutualistic fungus of leaf-cutting ants.

Leaf-cutting ants have a beneficial and obligatory relationship with the fungus that they grow. This mutualism allowed the evolutionary success of these ants. The great defoliation capacity of these insects, which often exceeds the level of tolerable economic damage, includes them as severe pests in many cultures. However, given the close relationship between these two agents of mutualism, it is expected that an impact on the fungus will reflect on the performance of the colony as a whole. Therefore, the effect of azadirachtin on the development, and the macronutrient composition of Leucoagaricus gongylophorus was evaluated. Azadirachtin reduced the final fungal mass at the end of treatment at all concentrations tested, but did not reduce the final growth area. A reduction in the amount of hyphae produced with increasing azadirachtin concentration was also observed. Regarding macronutrients, the compound did not affect their total amount in the fungus. Thus, it is observed that azadirachtin did not alter the composition of L. gongylophorus macronutrients, but inhibited its growth by reducing the number of hyphae produced. This reduction reflects directly on the amount of nutrients offered to the workers and the queen and may improve the management of these insects.

Microencapsulated extracts from Azadirachta indica seeds: Acquisition, characterization, and use in controlling Helicoverpa armigera

Biodegradable insecticides of Azadirachta indica A. Juss (Meliaceae) can be sustainable alternatives for agriculture. The goal of this study was to develop spray-dried microencapsulated extracts from A. indica seeds and to evaluate their effects in vitro on Helicoverpa armigera Hübner. Five polymeric matrices based on different proportions of gum arabic and maltodextrin were produced and characterized by evaluating photostability profile, drying yield, encapsulation efficiency, water activity, and azadirachtin content. In the photostability study, sample 3 showed the lowest loss of azadirachtin at the end of 15 days (62.90%). In the bioassays, the deterrence percentage was 100%. It was observed that in a 15-day period there was 100% mortality among the caterpillars that had a diet containing about 288 to 576 μg.mL−1 of microencapsulated extract. The microencapsulated extract from A. indica seeds presented increased azadirachtin photostability and showed potential as a bioinsecticide in the control of H. armigera.

Bioindicator potential of Spathosternum prasiniferum prasiniferum (Orthoptera; Acridoidea) in pesticide (azadirachtin)-induced radical toxicity in gonadal/nymphal tissues; correlation with eco-sustainability

Abstract Acridids are highly abundant living-organism/major-component in agricultural field, globally. It may act as a dependable bio-indicator species in response to environmental-stress. Antioxidants protect insects by scavenging free radicals. Here, we investigated the dose dependant azadirachtin (C35H44O16 AZT) toxicity on oxidative-biomarker; Alkaline-phosphatase (ALP), malondialdehyde (MDA), non-protein-soluble-thiol (NPSH) and antioxidants like superoxide-dismutase (SOD), catalase (CAT), glutathione-peroxidase (GPx) and amylase of gonads from both sexes and juvenile tissues of common grasshopper, Spathosternum prasiniferum prasiniferum (Walker, 1871). The newly hatched adults (female: male = 1:1) are exposed to 1 to 20 ppm AZT for 48 h and have compared to control and 6 h incubation with same concentration of azadirachtin for in vitro experiments. Both in vivo and in vitro experiment demonstrated significant influences on oxidative biomarkers with increasing antioxidant enzymatic activities in either sex. The male gonads represents decreasing antioxidant enzyme activities compared to female gonads. Lesser protection by CAT and SOD are noticed in male than female in response to AZT exposure. This experiment suggests that azadirachtin increased the major biomarkers with decreasing antioxidant enzyme activities resulting in more free-radicals related threat in adult male gonads. Variable dose responses were noticed on ALP, AchE and MDA in either gender suggesting multiphasic action of the pesticides. Higher mortality rate is noticed in male with lower nymphal life span. Moreover, nymph IInd is more susceptible than nymph IVth in vitro intoxication of azadirachtin. Possible life threat of vast representatives of agricultural ecosystem by pesticide should be avoided to maintain different bio-geo cycle and eco-sustainability.

Antifeeding effects of azadirachtin on the fifth instar Spodoptera litura larvae and the analysis of azadirachtin on target sensilla around mouthparts.

To clarify the types, number, and distribution of sensilla on the head of the fifth instar Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae and identify the main sensilla of azadirachtin acting on larvae, scanning electron microscopy was used to study the morphology of the head and sensilla on the mouthparts. The four sensilla-sensillum basiconicum, sensillum chaeticum, sensillum styloconicum, and sensillum trichodeum-on the head of the fifth instar larvae were treated with 0, 0.1, 0.5, 1, 2, and 4 mg/kg azadirachtin by a microdrop method. The larvae showed an obvious antifeeding effect with azadirachtin. And higher the concentration of azadirachtin, the more obvious the phenomenon of antifeeding activity. The sensillum styloconicum and the sensillum trichodeum were the main sensilla for azadirachtin. When 1 mg/kg azadirachtin was used to treat sensillum styloconicum and sensillum basiconicum, the fifth instar larvae of S. litura showed obvious antifeedant activity and the cumulative feed intake for 24 hr was no more than 30% of the leaf area. Quantitative reverse-transcription polymerase chain reaction verified the expression patterns of some Grs, indicating that Grst43a was upregulated by 1.3- and 3.9-fold, Gor24 was upregulated by 2.5- and 3.3-fold, Gr5a was downregulated by 0.6-fold and upregulated by 2.0-fold, and Gr28a was downregulated by 0.8-fold and upregulated by 3.6-fold upon treatment with 0.5 mg/kg and 1 mg/kg azadirachtin in 24 hr. Gr genes participated in the identification of bitterness and we speculated that Gr genes may indirectly lead to the occurrence of antifeeding behavior.

Development and stability enhancement of neem oil based microemulsion formulation using botanical synergist

The present study investigated the stability of neem oil based microemulsion (a water based formulation) by switching synthetic synergist with botanicals. Aqueous extract of Prosopis Juliflora was used as a botanical synergist. Microemulsion with different concentrations of neem oil i.e. 2%, 4%, 6%, 8%, 10% and 12% (w/w) were developed using a blend of ionic and anionic surfactant and 2-Butanol as a co-surfactant. Stability study has been done at first day and after 14 days of preparation at 54 °C by using HPLC, ATR-FTIR and DLS. The phase diagram study was also done to observe the feasibility of the prepared formulation. The concentration of azadirachtin in microemulsion samples with and without botanical synergist range 95% and 40% of the initial concentration respectively. Particle size of all the microemulsion samples were range 14.47 nm–32.36 nm with PI (polydisprsity index) value 0.19 to 0.74. This signifies that the formulated microemulsions are in one phase with homogeneous distribution of the molecules. Bioassay study with Spodoptera Litura also showed decrease in LD50 value from 40% to 10%. Thus the botanical synergist used in this study stabilize the azadirachtin content of neem oil in water based formulation.

The Synergy Effect of Azadirachtin and Minor Components of Neem Seed Oil on Antifeedant Activity of Spodoptera litura

Azadirachtin in neem oil ( Azadirachta indica A.Juss ) might have potential synergism effect with a minor compound in the neem oil as an antifeedant. This study aimed to determine the content of minor components that have the most significant contribution to the effect of synergy with the main component (azadirachtin) in antifeedant activity against Spodoptera litura . Neem oil was fractionated with n-hexane, ethyl acetate, and water. The antifeedant activity of three neem oil fractions was evaluated using the 4 th instar of S. litura larvae. The bioassay was conducted using the choice leaf disc test with 2.5; 5; 10; and 20 % (v/v) concentrations of each fraction. The concentration of the fractions showed equal azadirachtin content, hence the antifeedant activity represented the effect of the minor component in the fraction. The best activity was shown by water fraction (84-100 %). Probit analysis of the antifeedant activity of all fractions showed that water fraction has the best effective inhibitory (EI 50 ) at 1.0 %. The minor component in the water fraction was dominated by phenol compounds (48.5 %) which were suspected to synergize with the main component (azadirachtin) in increasing the antifeedant activity on S. litura . These results indicated the phenol compound in this fraction has the role in increasing azadirachtin antifeedant activity against S. litura .

Influence of fungal elicitor on bio production of Azadirachtin in cell cultures of Azadirachta indica

The present investigation involves the influence of elicitor feeding on bioproduction of important secondary metabolites in cell suspension cultures of Azadirachta indica. The content of azadirachtin in MS media supplemented with NAA (1 mg/l) + Kinetin (0.5 mg/l) and Dicamba (2 mg/l) + Kinetin (1 mg/l) hormonal combinations was found to be (0.42±0.04 mg/l) and (0.30 mg/l±0.07) respectively. Out of these two combinations, NAA (1 mg/l) + Kinetin (0.5 mg/l) combination was found optimum for bioproduction of azadirachtin. The addition of Fusarium solani significantly enhanced the amount of azadirachtin over control cultures. The cell extracts (CE) and culture filtrates (CF) of Fusarium solani were added in different concentrations (5% v/v and 10% v/v) to the cell cultures of A. indica separately, while maintaining suitable control. There is no significant change in the amount of azadirachtin between control and cell extract (CE) treated cultures. The addition of culture filtrate (5% v/v) of Fusarium solani showed significant improvement in bioproduction of azadirachtin (0.87±0.12 mg/l) over to control cultures (0.38±0.07 mg/l). It showed approximately 2-fold improvement in the bioproduction of azadirachtin. Present study constitute an important milestone in realizing commercial production of these metabolites.

Preparation of Protein Isolate from Neem Seed: Biochemical Evaluation, Antinutrients and In Vitro Digestibility Study

Neem protein isolate (NPI) was prepared from neem seed cake (NSC) resulting in five fold increase of crude protein content, amounting to 82%. The recovery of protein was highest (49%) when the NSC was solubilised at pH 12 for 2h and precipitated at pH 4. Crude fibre, nitrogen free extract and total ash contents were significantly (P<0.05) reduced substantially in the isolate compared to the NSC. The NPI has a balanced amino acid composition with high levels of methionine and arginine. A significant decrease of anti-nutritional factors such as oxalate (87.50%), phytic acid (70.50%), tannin (72.73%), cyanide (50.98%) and azadirachtin content (79.06%) was found in the isolate, implying that the isolation process was effective in the removal of toxic components. A significant (P<0.05) increase in the in vitro protein digestibility (90.4) was also obtained for the protein isolate, suggesting that the NPI as a promising feed ingredient.

IMMUNOMODULATION OF HOUSE FLIES EXPOSED TO AZADIRACHTIN

In this study the lethal and sub-lethal effects of azadirachtin on adult house flies, and the main variations in theirimmune-related gene expression levels were detected. Flies acquiring azadirachtin by ingestion showed differentdegrees of susceptibility depending on concentration and time of exposure to this compound, with LC50valuecorresponding to 101.14 µg/ml, after five days of exposure to a dose of 7.5 µl/fly/day. Flies surviving ingestion of sub-lethal concentrations showed significant decrease in their lifespan and reproductive performance, including fecundityand percentage of egg hatching.A significant immune-stimulation effect of lower azadirachtin concentrations (25 µg/ml), and a generalimmunosuppression of most AMPs (i.e. attacin, cecropin, defensin,diptericin and muscin) at higher concentrationlevels (100 µg/ml) were observed. This study highlights the immunodeficiency potential of azadirachtin, providing new insights into understandingthe physiological response of Musca domesticato this botanical compound at the molecular level.

Toxicity Potential Of Different Azadirachtin Against Plutella Xylostella (Lepidoptera; Plutellidae ) And Its Natural Enemy, Diadegma Species

The present study was conducted to assess different concentrations of azadirachtin against 3rd and 4thinstar larvae of P. xylostella and to determine their effects on natural enemy, Diadegma sp. Four different concentrations (0.31, 0.50, 0.60 and 1%) of azadirachtin were evaluated in the choice and non-choice tests methods

Seed yield and azadirachtin content of Azadirachta indica in four ecosystems of southwest China

Azadirachta indica (neem) is a globally recognized plant resource suitable for the development of botanical insecticides, and its seeds have been promoted as an industrial source for the manufacture of biopesticides. The purpose of this study was to evaluate the yield potential of neem seeds and azadirachtin from four ecosystems of southwest China during the periods of 2008 (Year 1) and 2009 (Year 2). The results showed that each ecosystem in Year 1 had higher neem seed production, while each ecosystem in Year 2 had markedly higher contents of azadirachtin A (azA) and total azadirachtin A and B [az(A + B)]. There were significant differences (P < 0.05) between the four ecosystems in the mean tree height (H), mean tree basal diameter (BD), and mean tree canopy diameter (CD) during the two consecutive years, but no significant differences were found in the weight of per seed (SW), the length/width ratio of seeds (RLW), or the kernel percentage (KP). The seed yield per hectare was significantly (P < 0.05) higher for the trees in the tropical sub-humid region (Yuanyang), with a maximum of 1763.4 kg ha−1 in Year 1 and an average of 1650.3 kg ha−1 for the two years, than those in the subtropical arid (Yuanmou) and semi-arid (Yuanjiang) regions. The content and yield of az(A + B) for the seeds produced in the tropical semi-arid region (Gejiu) were the highest, with respective average values of 8.2 g kg−1 and 5.84 kg ha−1 throughout the two evaluation years. Compared with the other three locales, the trees from the subtropical arid region (Yuanmou) produced significantly (P < 0.05) lower seed yield and az(A + B) yield in the drought year of 2009.

Selectivity assessment of two biorational insecticides, azadirachtin and pyriproxyfen, in comparison to a neonicotinoid, acetamiprid, on pupae and adults of a Neotropical strain Eretmocerus mundus Mercet

Assessment of the susceptibility of natural enemies of pests to selective pesticides is relevant for a sustainable agriculture with low impact on the environment. The aim of this study was to assess the toxicity of two biorational insecticides, azadirachtin and pyriproxyfen in comparison to a neonicotinoid insecticide, acetamiprid, on pupae and adults of a Neotropical strain of Eretmocerus mundus. Adult emergence and survival were evaluated as lethal effects whereas the sublethal effects were assessed through the reproductive capacity, sex ratio, and longevity of the surviving first progeny. Adult emergence from treated pupae was reduced by all three insecticides, but azadirachtin at its maximum field recommended concentration (MFRC) proved the most toxic insecticide. The survival probability of emerged adults was reduced by the three insecticides below than 50% from 2 to 5 days after the adult emergence. Malformations in nonemerged adults from treated pupal hosts were observed at the MFRC of all three insecticides. Sublethal effects on survivors from pupal treatment could be evaluated at only the lowest azadirachtin concentration. At that concentration, though azadirachtin did not affect the reproductive capacity of females, the sex ratio and the longevity of the first progeny were disrupted. The survival of parasitoid adults after adult exposure was reduced by all three insecticides, pyriproxyfen at the MFRC being the most toxic. All insecticides at their half of MFRCs induced sublethal effects in the survivors’ adults, with pyriproxyfen being the most harmful to the reproductive capacity of females. In conclusion, both biorational insecticides were toxic to E. mundus.

Development Rate and Ultrastructure Changes of Puparia of Megaselia scalaris (Loew) (Diptera: Phoridae) Induced by Azadirachtin.

The effect of azadirachtin on the development and mortality of the scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae) was investigated in the laboratory by using neem extract, Achook®, containing 0.15% azadirachtin. Different biological changes were recorded after treatment 1st larval instar with different concentrations of azadirachtin as larval mortality, pupation rate and adult emergence of. Azadirachtin-LC50 against the 1st instar larvae of M. scalaris was 13.79 mg/L. Exposing larvae to LC50 of azadirachtin caused many changes in the puparia such as having folded cuticle and swelling of the integument compared to control. Distortion of the anterior spiracle and papillae found on respiratory horns were also observed. The present study suggests that azadirachtin hampers the development rate of M. scalaris by reducing the insect population and causing several scanning electron microscopic changes in the puparia of the insect.

Lethal and sublethal effects of Embelia ribes and two commercial pesticides on a generalist predator

AbstractEven as new substances show promise as biopesticides for controlling pests due to their natural properties and high effectiveness in inhibiting pests, their side effects on non‐target organisms must nevertheless be evaluated before they can be included into integrated pest management systems. In this study, a crude extract from dried leaves of Embelia ribes was evaluated together with two commercial pesticides: azadirachtin (a natural product) and amitraz (a synthetic acaricide). We examined both lethal and sublethal effects on the predatory potential of the lynx spider Oxyopes lineatipes, which is among the most dominant predator in tropical agricultural agroecosystems. We found that the spider's mortality increased with rising concentration of both commercial products, azadirachtin and amitraz, but not with rising concentration of the extracts from E. ribes. The greatest mortality occurred when amitraz was used. That material caused almost 100% spider mortality in the doses recommended for field spraying. Azadirachtin significantly reduced the rate at which O. lineatipes captured prey, while there was no significant difference in capture rates among spiders exposed to a control treatment and the E. ribes treatment. Considering its absence of unfavourable impacts on O. lineatipes in terms of mortality and predatory activity, the plant extract from E. ribes shows promise as a new biopesticide material. In contrast, azadirachtin, which has been considered as safe for non‐target organisms, exhibited slight lethal effect only in higher concentrations and strong sublethal effect by reducing spiders’ predation rate.

Insecticidal products from Azadirachta indica and Plectranthus glandulosus growing in Cameroon for the protection of stored cowpea and maize against their major insect pests

Botanical insecticides are more biodegradable than synthetic chemicals and could be a source of more environmental-friendly insecticides. Accordingly, the effectiveness of oils from Azadirachta indica seeds and pulverized seeds of this plant and leaf powder of Plectranthus glandulosus were tested against Callosobruchus maculatus on cowpea seeds and Sitophilus zeamais on maize grains at fixed laboratory conditions. The azadirachtin A contents of A. indica seed oils and powders from sun-dried kernels, shade-dried kernels, sun-dried seeds and shade-dried seeds, and the chemical composition of P. glandulosus powders from sun-dried leaves, were determined, before admixing each product with cowpea seeds or maize grains for the toxicity and progeny reduction bioassay studies. Mortality counts were determined after 6 d (C. maculatus) or 7 d (S. zeamais). After the mortality counts, the grains were kept until all the emerging F1 progeny were recorded. From the results, Azadirachtin A content was higher in A. indica seed oil than in powder. The average content of Azadirachtin A in the seed powder was 1.20g/kg and this irrespective of the drying regime. On the contrary, the oil from the shade-dried seeds (3.69g/kg) had a higher azadirachtin A content than that from the sun-dried seeds. About 45 volatile compounds were found in the sun-dried leaves of P. glandulosus. Generally, P. glandulosus powder caused greater mortality to C. maculatus and S. zeamais than A. indica seed powder, but the neem seed oil was more active towards both insects than the powders. The A. indica products were more effective in suppressing progeny emergence in both insects than P. glandulosus leaf powders. Insecticidal products from the present study could form a major component of the integrated storage protection package for cowpea and maize against beetle infestations.

Variation in seed morphometric characters, oil content and azadirachtin content of seeds, in vitro shoot cultures and callus cultures among different populations of Azadirachta indica

We studied seed morphometric characters, oil content and azadirachtin content in a medicinally important and insect repellent tree species Azadirachta indica A. Juss. Ten populations were selected from two central Indian states of Madhya Pradesh and Chhattisgarh and twenty candidate plus trees were selected from each population based on tree attributes. Azadirachtin content was estimated in seeds, in vitro shoots and callus cultures through HPLC. Significant variation (p < 0.05) was observed in seed morphometric characters, oil content and azadirachtin content among the ten populations. Maximum value for 100 seed weight was obtained in seeds of Raigarh (30.51 g) which was on par with seeds of Bargi (28.37 g) and Chhatarpur (26.36 g). The ratio of highest kernel to seed was also obtained in seeds of Raigarh (60.11%). Kernels from Chhatarpur population recorded the highest oil content (24.20%), which was equal to that in Sihore (23.75%) and Katni (21.75%). We recorded highly significant variation in azadirachtin content in seed samples, in vitro shoot samples and callus cultures. Maximum azadirachtin was present in the seeds of Gwalior (0.882%) which was on par with azadirachtin content in seeds of Chhatarpur, Katni, Sihore, Shahdol and Chhindwara. Maximum azadirachtin in the in vitro shoots was present in the cultures of Gwalior (0.218%) which was statistically on par with Sihore (0.176%). Similarly, in callus cultures of nodal segments maximum azadirachtin was recorded in the Gwalior population (0.033%) which was statistically on par with Sihore (0.028%). The trees of Gwalior and Sihore populations can be produced in large numbers through vegetative propagation to obtain seeds with higher azadirachtin content.

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.

Production of biopesticide azadirachtin using plant cell and hairy root cultures.

The extensive use of nondegradable chemical pesticides for pest management has developed serious environmental hazards. This has necessitated the urgent need to switch over to an alternative mode of biopesticide development for mass agriculture and field crop protection. Azadirachta indica A. Juss (commonly known as neem) houses a plethora of bioactive secondary metabolites with azadirachtin being the most active constituent explored in the sector of ecofriendly and biodegradable biopesticides characterized by low toxicity toward nontarget organisms. It has been reported that the highest content of azadirachtin and related limonoids is present in the seeds, available once in a year. Moreover, the inconsistent content and purity of the metabolites in whole plant makes it imperative to tap the potential of in vitro plant tissue culture applications, which would allow for several controlled manipulations for better yield and productivities. This review gives a summarized literature of the applied research and achievements in plant cell/hairy cultures of A. indica A. Juss mainly in context with the biopesticide azadirachtin and applications thereof.