Enhanced azadirachtin production in neem (Azadirachta indica) callus through NaCl elicitation: Insights into differential protein regulation via shotgun proteomics.

With their remarkable bioactivity and evolving commercial importance, plant secondary metabolites (PSMs) have gained significant research interest in recent years. Plant tissue culture serves as a credible tool to examine how abiotic stresses modulate the production of PSMs, enabling clear insights into plant stress responses and the prospects for controlled synthesis of bioactive compounds. Azadirachta indica, or neem has been recognized as a repository of secondary metabolites for centuries, particularly for the compound named azadirachtin, due to its bio-pesticidal and high antioxidant properties. Introducing salt stress as an elicitor makes it possible to enhance the synthesis of secondary metabolites, specifically azadirachtin. Thus, in this research, in vitro callus cultures of neem were micro-propagated and induced with salinity stress to explore their effects on the production of azadirachtin and identify potential proteins associated with salinity stress through comparative shotgun proteomics (LCMS/MS). To induce salinity stress, 2-month-old calli were subjected to various concentrations of NaCl (0.05-1.5%) for 4 weeks. The results showed that the callus cultures were able to adapt and survive in the salinity treatments, but displayed a reduction in fresh weight as the NaCl concentration increased. Notably, azadirachtin production was significantly enhanced in the salinity treatment compared to control, where 1.5% NaCl-treated calli produced the highest azadirachtin amount (10.847 ± 0.037 mg/g DW). The proteomics analysis showed that key proteins related to primary metabolism, such as defence, energy, cell structure, redox, transcriptional and photosynthesis, were predominantly differentially regulated (36 upregulated and 93 downregulated). While a few proteins were identified as being regulated in secondary metabolism, they were not directly involved in the synthesis of azadirachtin. In conjunction with azadirachtin elicitation, salinity stress treatment could therefore be successfully applied in commercial settings for the controlled synthesis of azadirachtin and other plant-based compounds. Further complementary omics approaches can be employed to enhance molecular-level modifications, to facilitate large-scale production of bioactive compounds in the future.

Using Azadirachta indica protein hydrolysate as a plant protein in Nile tilapia (Oreochromis niloticus) diet: Effects on the growth, economic efficiency, antioxidant-immune response and resistance to Streptococcus agalactiae.

A feeding trial for 90 days was conducted on Nile tilapia (Oreochromis niloticus) (average weight: 25.50 ± 0.05 g) to evaluate the effect of dietary inclusion of Azadirachta indica seed protein hydrolysate (AIPH). The evaluation included the impact on the growth metrics, economic efficiency, antioxidant potential, hemato-biochemical indices, immune response, and histological architectures. A total of 250 fish were randomly distributed in five treatments (n = 50) and received diets included with five levels of AIPH (%): 0 (control diet, AIPH0), 2 (AIPH2), 4 (AIPH4), 6 (AIPH6) or 8 (AIPH8), where AIPH partially replace fish meal by 0, 8.7%, 17.4%, 26.1%, and 34.8%, respectively. After the feeding trial, a pathogenic bacterium (Streptococcus agalactiae, 1.5 × 108 CFU/mL) was intraperitoneally injected into the fish and the survival rate was recorded. The results elucidated that AIPH-included diets significantly (p < 0.05) enhanced the growth indices (final body weight, total feed intake, total body weight gain, and specific growth rate) and intestinal morpho-metrics (villous width, length, muscular coat thickness, and goblet cells count) in comparison to the control diet, with the AIPH8 diet recording the highest values. Dietary AIPH inclusion significantly improved (p < 0.05) the economic efficacy indicated by reduced feed cost/kg gain and increased performance index. The fish fed on the AIPH diets had noticeably significantly higher (p < 0.05) protein profile variables (total proteins and globulin) and antioxidant capabilities (superoxide dismutase and total antioxidant capacity) than the AIPH0 group. The dietary inclusion of AIPH significantly (p < 0.05) boosted the haematological parameters (haemoglobin, packed cell volume %, and counts of red blood cells and white blood cells) and immune indices (serum bactericidal activity %, antiprotease activity, and immunoglobulin M level) in a concentration-dependent manner. The blood glucose and malondialdehyde levels were significantly (p < 0.05) lowered by dietary AIPH (2%-8%). The albumin level and hepatorenal functioning parameters (aspartate aminotransferase, alanine aminotransferase, and creatinine) were not significantly (p > 0.05) altered by AIPH diets. Additionally, AIPH diets did not adversely alter the histology of the hepatic, renal or splenic tissues with moderately activated melano-macrophage centres. The mortality rate among S. agalactiae-infected fish declined as dietary AIPH levels rose, where the highest survival rate (86.67%) was found in the AIPH8 group (p < 0.05). Based on the broken line regression model, our study suggests using dietary AIPH at the optimal level of 6%. Overall, dietary AIPH inclusion enhanced the growth rate, economic efficiency, health status, and resistance of Nile tilapia to the S. agalactiae challenge. These beneficial impacts can help the aquaculture sector to be more sustainable.

Neem leaf powder (Azadirachta indica) mitigates oxidative stress and pathological alterations triggered by lead toxicity in Nile tilapia (Oreochromis niloticus).

This study investigated the clinical and pathological symptoms of waterborne lead toxicity in wild Nile tilapia collected from a lead-contaminated area (the Mariotteya Canal: Pb = 0.6 ± 0.21 mg L-1) and a farmed fish after 2 weeks of experimental exposure to lead acetate (5-10 mg L-1) in addition to evaluating the efficacy of neem leaf powder (NLP) treatment in mitigating symptoms of lead toxicity. A total of 150 fish (20 ± 2 g) were alienated into five groups (30 fish/group with three replicates). G1 was assigned as a negative control without any treatments. Groups (2-5) were exposed to lead acetate for 2 weeks at a concentration of 5 mg L-1 (G2 and G3) or 10 mg L-1 (G4 and G5). During the lead exposure period, all groups were reared under the same conditions, while G3 and G5 were treated with 1 g L-1 NLP. Lead toxicity induced DNA fragmentation and lipid peroxidation and decreased the level of glutathione and expression of heme synthesis enzyme delta aminolaevulinic acid dehydratase (ALA-D) in wild tilapia, G2, and G4. NLP could alleviate the oxidative stress stimulated by lead in G3 and showed an insignificant effect in G5. The pathological findings, including epithelial hyperplasia in the gills, edema in the gills and muscles, degeneration and necrosis in the liver and muscle, and leukocytic infiltration in all organs, were directly correlated with lead concentration. Thus, the aqueous application of NLP at 1 g L-1 reduced oxidative stress and lowered the pathological alterations induced by lead toxicity.

Computational Evaluation of Azadirachta indica-Derived Bioactive Compounds as Potential Inhibitors of NLRP3 in the Treatment of Alzheimer's Disease.

BACKGROUND: The development of therapeutic agents against Alzheimer's disease (AD) has stalled recently. Drug candidates targeting amyloid-ß (Aß) deposition have often failed clinical trials at different stages, prompting the search for novel targets for AD therapy. The NLRP3 inflammasome is an integral part of innate immunity, contributing to neuroinflammation and AD pathophysiology. Thus, it has become a promising new target for AD therapy. OBJECTIVE: The study sought to investigate the potential of bioactive compounds derived from Azadirachta-indica to inhibit the NLRP3 protein implicated in the pathophysiology of AD. METHODS: Structural bioinformatics via molecular docking and density functional theory (DFT) analysis was utilized for the identification of novel NLRP3 inhibitors from A. indica bioactive compounds. The compounds were further subjected to pharmacokinetic and drug-likeness analysis. Results obtained from the compounds were compared against that of oridonin, a known NLRP3 inhibitor. RESULTS: The studied compounds optimally saturated the binding site of the NLRP3 NACHT domain, forming principal interactions with the different amino acids at its binding site. The studied compounds also demonstrated better bioactivity and chemical reactivity as ascertained by DFT analysis and all the compounds except 7-desacetyl-7-benzoylazadiradione, which had two violations, conformed to Lipinski's rule of five. CONCLUSION: In silico studies show that A. indica derived compounds have better inhibitory potential against NLRP3 and better pharmacokinetic profiles when compared with the reference ligand (oridonin). These compounds are thus proposed as novel NLRP3 inhibitors for the treatment of AD. Further wet-lab studies are needed to confirm the potency of the studied compounds.

Influence of Azadirachta indica and Cnidoscolus angustidens Dietary Extracts on Equine Fecal Greenhouse Gas Emissions.

The present study was conducted to investigate the aqueous extracts of Azadirachta indica (AZN), Cnidoscolus angustidens (CNA), and their combination (MIX) at dosages of 0-, 0.6-, 1.2-, and 1.8- mL for their ability to reduce greenhouse gases and fermentation profiles in an in vitro study using horse feces and a nutrient-dense diet (as substrate). The quantity of greenhouse gas and fermentation profiles were determined in in vitro incubation for 48 h. Extracts of AZN, CNA, and MIX reduced total gas production of the incubated and degraded substrates in a dose-dependent and time-dependent manner. Production of CH4 was reduced (P < .05) by 4.41% to 54.54% with the incubated substrates and by 1.16% to 61.82% with the degraded substrates. However, AZN and MIX reduced (P < .05) CO by 4.43% to 12.85% with the incubated substrates and by 0.70% to 16.78% with the degraded substrates. In like manner, the plant extracts and combination reduced (P < .05) H2S production in a dose-dependent and time-dependent manner by 18.37% to 67.35% with the incubated substrates and by 8.51% to 67.23% with the degraded substrates. Extracts maintained pH within the normal range, reduced dry matter digestibility and metabolizable energy, and improved (P < .05) concentration of short chain fatty acids. Overall, aqueous extracts of AZN and CNA and their combinations had a positive effect on reducing the greenhouse gas production with no deleterious effect on fecal horses' fermentation activities.

Effect of increasing doses of neem (Azadirachta indica) seed oil on feed intake, nutrients digestibility, ruminal fermentation and nitrogen utilization of Omani sheep.

This study was conducted to examine the effects of neem (Azadirachta indica) seed oil (NSO) on feed intake, ruminal fermentability, feed digestibility, nitrogen balance, and blood parameters in Omani sheep. Nine Omani male sheep (BW; 54.44 ± 8 kg/sheep) were used in a replicated 3 × 3 Latin Square design with three dietary treatments and three periods, with each period lasting 21 days. The animals were randomly assigned into three treatment groups: basal diet without NSO (control); 20 mL/sheep per day NSO (low dose) or 40 mL/sheep per day NSO (high dose). Sheep fed with high dose presented lower (p < 0.05) intake, fiber digestibility, and excreted N in urine. However, diet supplemented with high NSO dose had a lower (p < 0.05) ruminal acetate proportion, while increased (p < 0.05) ruminal propionate compared to the control group. Moreover, sheep offered with NSO showed a linear decrease (p = 0.052) in ruminal ammonia nitrogen (NH3-N) concentration. The high dose increased (p < 0.05) blood glucose and cholesterol, while decreased urea nitrogen concentrations. Emission of predicted methane (CH4) reduced (p = 0.007) by 13% for high dose compared to the control group. In conclusion, these results suggested that NSO is a potential feed additive with up to 20 ml/kg of DM alters rumen fermentation profile, decreased CH4 production, feed digestibility, and nitrogen metabolism for sheep.

Antioxidant and Chemopreventive Effects of Azadirachta indica on Lead Acetate-induced Hepatotoxicity in Male Wistar Rats.

Toxic metals such as lead (Pb) cause severe liver damage in humans and animals, with oxidative stress prominently implicated in the pathogenesis of lead acetate­induced liver injury. Azadirachta indica is hepatoprotective due to its antioxidative effect. This study investigated the antioxidative role of A. indica (AI) and its chemopreventive effect on lead acetate (LA)-induced hepatocellular dysfunction with seventy adult male rats classified into group A- Control (distilled water), group B 0.1% LA only, group C and D- 0.1% LA + 100 mg/kg and 0.1% LA + 200 mg/kg AI respectively, group E- 0.2% LA, group F and G- 0.2% LA + 100 mg/kg and 0.2% LA + 200 mg/kg AI. Oxidative stress markers (MDA and H2O2), antioxidant parameters (GSH, SOD, CAT, GPx, GST), inflammatory markers (MPO and NO), alanine aminotransferase (ALT) and histopathological studies of the liver were evaluated. The results showed that LA administration caused a decrease in GSH, GPx, and GST while AI co-administration increased the activities of the antioxidants. Moreover, LA administration increased MPO, NO, MDA, and H2O2 levels whereas AI significantly reduced (P<0.05) these parameters. Histopathological examination revealed necrosis and mild infiltration by inflammatory cells in LA administered rats, whereas these lesions were absent in AI administered rats. In conclusion, A. indica demonstrates a protective role in lead acetate-induced hepatotoxicity, mainly via oxidative stress inhibition.

Efficacy of the Aqueous Extract of Azadirachta indica Against the Marine Parasitic Leech and Its Phytochemical Profiling.

Zeylanicobdella arugamensis (Hirudinea), a marine parasitic leech, not only resulted in the mortality of the host fish (Groupers) but also caused economic losses. The current study aimed to elucidate the antiparasitic efficacy of the aqueous extract of the Azadirachta indica leaves against Z. arugamensis and to profile the composition via LC-Q Exactive HF Orbitrap mass spectrometry. Different concentrations (25, 50 and 100 mg/mL) of A. indica extract were prepared and tested on the parasitic leeches. The total mortality of leeches was noticed with an exposure to the A. indica aqueous extract. The average times required for the aqueous extract at concentrations of 25, 50 and 100 mg/mL to kill the leeches were 42.65 ± 9.20, 11.69 ± 1.11 and 6.45 ± 0.45 min, respectively, in a dose-dependent manner. The Orbitrap mass spectrometry analysis indicated the presence of five flavonoids (myricetin 3-O-galactoside, trifolin, isorhamnetin, quercetin and kaempferol), four aromatics (4-methoxy benzaldehyde, scopoletin, indole-3-acrylic acid and 2,4-quinolinediol), three phenolics (p-coumaric acid, ferulic acid and phloretin) and two terpenoids (pulegone and caryophyllene oxide). Thus, our study indicates that A. indica aqueous extract is a good source of metabolites with the potential to act as a biocontrol agent against the marine parasitic leech in aquaculture.

Azadirachta indica MicroRNAs: Genome-Wide Identification, Target Transcript Prediction, and Expression Analyses.

MicroRNAs are short, endogenous, non-coding RNAs, liable for essential regulatory function. Numerous miRNAs have been identified and studied in plants with known genomic or small RNA resources. Despite the availability of genomic and transcriptomic resources, the miRNAs have not been reported in the medicinal tree Azadirachta indica (Neem) till date. Here for the first time, we report extensive identification of miRNAs and their possible targets in A. indica which might help to unravel their therapeutic potential. A comprehensive search of miRNAs in the A. indica genome by C-mii tool was performed. Overall, 123 miRNAs classified into 63 families and their stem-loop hairpin structures were predicted. The size of the A. indica (ain)-miRNAs ranged between 19 and 23 nt in length, and their corresponding ain-miRNA precursor sequence MFEI value averaged as -1.147 kcal/mol. The targets of ain-miRNAs were predicted in A. indica as well as Arabidopsis thaliana plant. The gene ontology (GO) annotation revealed the involvement of ain-miRNA targets in developmental processes, transport, stress, and metabolic processes including secondary metabolism. Stem-loop qRT-PCR was carried out for 25 randomly selected ain-miRNAs and differential expression patterns were observed in different A. indica tissues. Expression of miRNAs and its targets shows negative correlation in a dependent manner.

Enhanced Biosynthesis Synthesis of Copper Oxide Nanoparticles (CuO-NPs) for their Antifungal Activity Toxicity against Major Phyto-Pathogens of Apple Orchards.

PURPOSE: The present study made an attempt to develop copper nanoparticles (Cu-NP) with antifungal property using green synthesis method. Copper oxide nanoparticles (CuO-NPs) botanically synthesized using Neem leaf extract (Azadirachta indica A. Juss) were characterized by using different techniques like; UV-visible spectrophotometry, FTIR, XRD, SEM and TEM. METHODS: Materials were chosen the disease free and fresh Azadirachta indica A. Juss were collected and identified at Center of Biodiversity and Taxonomy. The plant samples were vigorously washed with distilled water then shade dried followed by sterilization with 0.1% mercuric chloride for 20 s and again it was washed with distilled water. 15 g powder form of plant material was added to 200 ml double distilled, CO2 free and deionized water and kept in shaker at 80°C and 1500 rpm for six hours. After agitation, the extract was separated by regular centrifugation at 10,000 rpm followed by filtration by using whatmann filter paper. The final volume of 100 ml of supernatant was collected as pure extract and stored in cool place for further use. RESULTS: The final results confirm a significant inhibition of CuO-NPs for the test fungi. Additionally, CuO-NPs demonstrated an enhanced effect when combined with Neem leaf extract. A total of 20-30% improvement in activity was noticed after combination, which correlates with commonly used synthetic fungicides. The toxicity results reveal that A. indica extract and their combined fractions with CuO-NP were less toxic to the test seeds of experimental plant while as bulk Cu followed by biosynthesized CuO-NPs influenced the germination rate as compared to control pots. CONCLUSIONS: The study drops a concern of research and offers a promising route of developing Copper based green fungicides that can help to combat with modern issues of synthetic fungicides. An average size of 80 ± 15 nm monoclinic cupric oxide (CuO) and cubic cuprous oxides (Cu2O) nanocrystals that existed in mixed form were successfully developed.

Melanogenesis-inhibitory activities of limonoids and tricyclic diterpenoids from Azadirachta indica.

The chemical constituents of the roots and bark of Azadirachta indica were investigated, leading to the isolation of six tricyclic diterpenoids and four limonoids including a new compound, azadirachtin J (4). The structures were elucidated on the basis of NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Furthermore, melanogenesis-inhibitory activities of the isolated compounds were evaluated. As a result, compounds 1-3 and 10 exhibited superior inhibitory activities against melanogenesis with no, or almost no, toxicity to the cells (86.5-105.1% cell viability). Western blot analysis showed that compounds 1 and 3 exhibited melanogenesis inhibitory activities in α-MSH-stimulated B16 melanoma cells due to, at least in part, inhibition of the expression of MITF, followed by a decrease in the expression of tyrosinase, TRP-1, and TRP-2. Compounds 1 and 3 exhibited tyrosinase inhibitory activities (IC50 values of 44.86 µM and 69.85 µM respectively). Docking results confirm that the active inhibitors strongly interact with tyrosinase residues.

Neem, pawpaw and bamboo leaf meal dietary supplementation in broiler chickens: Effect on performance and health status.

The effect of the diet supplemented with leaf meals (LM) of neem (NLM), pawpaw (PLM), bamboo (BLM) and their composite leaf mix (CLM) on broiler chickens was assessed. Three hundred 1-day old broiler chickens were distributed to five diets: (control/no LM), (5 g/kg NLM), (5 g/kg PLM), (5 g/kg BLM) and (5 g/kg NLM+PLM+BLM 1:1:1). Body weight gain of birds fed BLM and CLM supplemented diets were similar but higher (p < 0.05) than those fed the control, NLM and PLM supplemented diets at day 42. The glucose, triglycerides, cholesterol, alanine aminotransferase and creatinine levels in LM supplemented diets were (p < 0.05) lower compared to the control. The superoxide dismutase, glutathione peroxidase and catalase were (p < 0.05) higher in LM supplemented diets compared to the control. The growth promoting potentials of bamboo leaf meal and the composite leaf mix can be harnessed for broiler chicken production. PRACTICAL APPLICATIONS: The use of herbs as the growth promoter is currently gaining research attention in most countries. In particular, herbal supplementation in poultry diets had been proposed as a means of enhancing the performance, stabilizing the physiological state, boosting immunity and antioxidative status in the birds. This study showed that improved body weight gain of broiler chickens at 42 days of age with attendant low serum glucose, triglycerides and cholesterol can be achieved when leaf meals from neem, pawpaw, bamboo and their composite mix were used as phyto-additives. The bamboo leaf meal and the composite leaf meal can be used to reduce the occurrence of arteriosclerosis which predisposes sudden death syndrome in well-grown and healthy broiler chickens. The procedures of raising broiler chickens to reach about 3 kg in less than 2 months impose a heavy burden on the heart, particularly overloading on the ventricular arteries leading to arteriosclerosis with age.

Natural plant extracts as an economical and ecofriendly alternative for harvesting microalgae.

The study investigated the ability of plant based natural coagulants from Azadirachta indica; Ficus indica; Moringa oleifera; Citrus sinensis; Punica granatum and Musa acuminata to harvest the microalgal biomass. Influence of eluent type (water and NaCl) and concentration (1-5 N) on coagulant extraction; coagulant dosage (1-5 g) and volume (20-100 ml); pH (6-12) and algal concentration (0.1-1 g l-1) on harvesting were analyzed. The results obtained were compared with alum and chitosan. FTIR and biochemical analysis confirmed the presence of bioactive compounds to aid coagulation. Biomass removal efficiency of 75.50% was obtained with M. oleifera extracts (8 mg ml-1) at pH 7.5-7.8, within 100 min. The harvesting efficiency increased to 95.76% when 4 mg ml-1M. oleifera extracts was combined with 0.75 mg ml-1 chitosan. The life cycle and cost analysis acknowledged the eco-friendly coagulants as strong alternative for conventional coagulants used in microalgal harvesting, thereby improvising the overall bioprocess.

Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension.

BACKGROUND: Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree. RESULTS: Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways. CONCLUSIONS: Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through 13C limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors.

Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids.

Azadirachta indica A. Juss, commonly known as Neem, is the reservoir of triterpenoids of economic importance. Metabolite analysis of different developmental stages of leaf and fruit suggests tissue-specific accumulation of the major triterpenoids in this important tree. Though biosynthesis of these complex molecules requires substrate flux from the isoprenoid pathway, enzymes involved in late biosynthetic steps remain uncharacterized. We established and analyzed transcriptome datasets from leaf and fruit and identified members of gene families involved in intermediate steps of terpenoid backbone biosynthesis and those related to secondary transformation leading to the tissue-specific triterpenoid biosynthesis. Expression analysis suggests differential expression of number of genes between leaf and fruit and probable participation in the biosynthesis of fruit-specific triterpenoids. Genome-wide analysis also identified members of gene families putatively involved in secondary modifications in late biosynthetic steps leading to the synthesis of highly oxygenated triterpenoids. Expression and molecular docking analyses suggest involvement of specific members of CYP450 family in secondary modifications for the biosynthesis of bioactive triterpenoids. This study generated rich genomic resource and identified genes involved in biosynthesis of important molecules, which will aid in the advancement of tools for functional genomics and elucidation of the biosynthesis of triterpenoid from this important tree.

Evaluation of anthelmintic activity of biologically synthesized silver nanoparticles against the gastrointestinal nematode, Haemonchus contortus.

The present study focuses on the in vitro anthelmintic activity of silver nanoparticles (AgNPs) synthesized using the aqueous extract of Azadirachta indica against Haemonchus contortus. The synthesized AgNPs were characterized by ultraviolet-visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies. The UV-Vis spectrum exhibited a sharp peak at 420 nm, which was validated by electron microscopy, indicating the preparation of spherical nanoparticles measuring 15-25 nm in size. The in vitro study was based on an egg hatch assay (EHA) and adult motility inhibition assays. Six concentrations of AgNPs were used for EHA, ranging from 0.00001 to 1.0 µg/ml, and a range of 1-25 µg/ml was used for adult worms. The highest concentration induced 85 ± 2.89% egg hatch inhibition. The IC50 value for EHA was 0.001 µg/ml, whereas in vitro adult H. contortus motility inhibition was produced at 7.89 µg/ml (LC50). The effectiveness of A. indica leaf extract (aqueous) was also evaluated, which showed an IC50 value for EHA of 115.67 µg/ml, while the LC50 against adult H. contortus was 588.54 µg/ml. The overall findings of the present study show that the experimental plant extract contains reducing properties for the synthesis of AgNPs which, in turn, showed potent anthelmintic properties. This is the first report where AgNPs have been tested for their anthelmintic properties in an in vitro model.

An Improved Genome Assembly of Azadirachta indica A. Juss.

Neem (Azadirachta indica A. Juss.), an evergreen tree of the Meliaceae family, is known for its medicinal, cosmetic, pesticidal and insecticidal properties. We had previously sequenced and published the draft genome of a neem plant, using mainly short read sequencing data. In this report, we present an improved genome assembly generated using additional short reads from Illumina and long reads from Pacific Biosciences SMRT sequencer. We assembled short reads and error-corrected long reads using Platanus, an assembler designed to perform well for heterozygous genomes. The updated genome assembly (v2.0) yielded 3- and 3.5-fold increase in N50 and N75, respectively; 2.6-fold decrease in the total number of scaffolds; 1.25-fold increase in the number of valid transcriptome alignments; 13.4-fold less misassembly and 1.85-fold increase in the percentage repeat, over the earlier assembly (v1.0). The current assembly also maps better to the genes known to be involved in the terpenoid biosynthesis pathway. Together, the data represent an improved assembly of the A. indica genome.

In vivo and in vitro effectiveness of Azadirachta indica-synthesized silver nanocrystals against Plasmodium berghei and Plasmodium falciparum, and their potential against malaria mosquitoes.

Malaria transmission is a serious emergence in urban and semiurban areas worldwide, becoming a major international public health concern. Malaria is transmitted through the bites of Anopheles mosquitoes. The extensive employ of synthetic pesticides leads to negative effects on human health and the environment. Recently, plant-synthesized nanoparticles have been proposed as highly effective mosquitocides. In this research, we synthesized silver nanoparticles (AgNP) using the Azadirachta indica seed kernel extract as reducing and stabilizing agent. AgNP were characterized by UV-vis spectrophotometry, SEM, EDX, XRD and FTIR spectroscopy. The A. indica seed kernel extract was toxic against Anopheles stephensi larvae and pupae, LC50 were 232.8ppm (larva I), 260.6ppm (II), 290.3ppm (III), 323.4ppm (IV), and 348.4ppm (pupa). AgNP LC50 were 3.9ppm (I), 4.9ppm (II), 5.6ppm (III), 6.5ppm (IV), and 8.2ppm (pupa). The antiplasmodial activity of A. indica seed kernel extract and AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of A. indica seed kernel extract were 63.18µg/ml (CQ-s) and 69.24µg/ml (CQ-r). A. indica seed kernel-synthesized AgNP achieved IC50, of 82.41µg/ml (CQ-s) and 86.12µg/ml (CQ-r). However, in vivo anti-plasmodial experiments conducted on Plasmodium berghei infecting albino mice showed moderate activity of the A. indica extract and AgNP. Overall, this study showed that the A. indica-mediated fabrication of AgNP is of interest for a wide array of purposes, ranging from IPM of mosquito vectors to the development of novel and cheap antimalarial drugs.

Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica).

BACKGROUND: Neem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI(+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized. RESULTS: Quantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA ΙΙ platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively. CONCLUSIONS: Triterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.