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Nimbolide, a major limonoid constituent of Azadirachta indica , commonly known as neem, has attracted increasing research attention owing to its wide spectrum of pharmacological properties, predominantly anticancer activity. Nimbolide is reported to exert potent antiproliferative effects on a myriad cancer cell lines and chemotherapeutic efficacy in preclinical animal tumor models. The potentiality of nimbolide to circumvent multidrug resistance and aid in targeted protein degradation broaden its utility in enhancing therapeutic modalities and outcome. Accumulating evidence indicates that nimbolide prevents the acquisition of cancer hallmarks such as sustained proliferation, apoptosis evasion, invasion, angiogenesis, metastasis, and inflammation by modulating kinase-driven oncogenic signaling networks. Nimbolide has been demonstrated to abrogate aberrant activation of cellular signaling by influencing the subcellular localization of transcription factors and phosphorylation of kinases in addition to influencing the epigenome. Nimbolide, with its ever-expanding repertoire of molecular targets, is a valuable addition to the anticancer drug arsenal.

This study aimed to develop a highly selective, sensitive and fast liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the determination of obacunone in rat plasma. Sample preparation was accomplished by a simple solid-phase extraction procedure. Chromatographic separation was carried out on an ACQUITY BEH C 18 column using acetonitrile/methanol (1:1, v/v) and 0.1% formic acid in water as mobile phase at a flow rate of 0.4 mL/min. Quantification was performed with multiple reactions monitoring in positive ion mode with the precursor-to-product ion transitions at m/z 455.2 > 161.1 for obacunone and m/z 515.2 > 161.1 for nomilin (internal standard). The assay was demonstrated to be linear over the concentration range of 0.1-1,000 ng/mL with correlation coefficient >0.999 (r > 0.999). The intra- and inter-day accuracy ranged from -8.33 to 10.40%, while the precision was <10.41%. The mean extraction recovery was >75.32%, and the assay was free of matrix effect. The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of obacunone in rats after oral and intravenous administrations. The oral bioavailability of obacunone was 13.59%., (© 2020 John Wiley & Sons, Ltd.)

Virtual screening of phytochemicals was performed through molecular docking, simulations, in silico ADMET and drug-likeness prediction to identify the potential hits that can inhibit the effects of SARS-CoV-2. Considering the published literature on medicinal importance, 154 phytochemicals with analogous structure from limonoids and triterpenoids were selected to search potential inhibitors for the five therapeutic protein targets of SARS-CoV-2, i.e., 3CLpro (main protease), PLpro (papain-like protease), SGp-RBD (spike glycoprotein-receptor binding domain), RdRp (RNA dependent RNA polymerase) and ACE2 (angiotensin-converting enzyme 2). The in silico computational results revealed that the phytochemicals such as glycyrrhizic acid, limonin, 7-deacetyl-7-benzoylgedunin, maslinic acid, corosolic acid, obacunone and ursolic acid were found to be effective against the target proteins of SARS-CoV-2. The protein-ligand interaction study revealed that these phytochemicals bind with the amino acid residues at the active site of the target proteins. Therefore, the core structure of these potential hits can be used for further lead optimization to design drugs for SARS-CoV-2. Also, the medicinal plants containing these phytochemicals like licorice, neem, tulsi, citrus and olives can be used to formulate suitable therapeutic approaches in traditional medicines., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Limonin is a natural tetracyclic triterpenoid compound, which widely exists in Euodia rutaecarpa (Juss.) Benth., Phellodendron chinense Schneid., and Coptis chinensis Franch. Its extensive pharmacological effects have attracted considerable attention in recent years. However, there is no systematic review focusing on the pharmacology, toxicity, and pharmacokinetics of limonin. Therefore, this review aimed to provide the latest information on the pharmacology, toxicity, and pharmacokinetics of limonin, exploring the therapeutic potential of this compound and looking for ways to improve efficacy and bioavailability. Limonin has a wide spectrum of pharmacological effects, including anti-cancer, anti-inflammatory and analgesic, anti-bacterial and anti-virus, anti-oxidation, liver protection properties. However, limonin has also been shown to lead to hepatotoxicity, renal toxicity, and genetic damage. Moreover, limonin also has complex impacts on hepatic metabolic enzyme. Pharmacokinetic studies have demonstrated that limonin has poor bioavailability, and the reduction, hydrolysis, and methylation are the main metabolic pathways of limonin. We also found that the position and group of the substituents of limonin are key in affecting pharmacological activity and bioavailability. However, some issues still exist, such as the mechanism of antioxidant activity of limonin not being clear. In addition, there are few studies on the toxicity mechanism of limonin, and the effects of limonin concentration on pharmacological effects and toxicity are not clear, and no researchers have reported any ways in which to reduce the toxicity of limonin. Therefore, future research directions include the mechanism of antioxidant activity of limonin, how the concentration of limonin affects pharmacological effects and toxicity, finding ways to reduce the toxicity of limonin, and structural modification of limonin-one of the key methods necessary to enhance pharmacological activity and bioavailability., Competing Interests: The authors declare no conflict of interest.

Nanobiotechnology is one of the emerging fields and its interventions in agriculture is been attracting the scientific community. Herein, the authors first to report on control of groundnut bruchid ( Caryedon serratus O.) using nanoscale zinc oxide (ZnONPs) particles and nanoscale chitosan (CNPs) particles-based Azadirachtin formulations. ZnONPs and CNPs were prepared using sol-gel and ion tropic gelation techniques, respectively. Neem seed kernel extract (NSKE) 5% and Neem oil (3000 and 1000 ppm) were encapsulated using the prepared nanoscale materials and characterised using the techniques such as dynamic light scattering, high-resolution transmission electron microscopy. Spherical-shaped nanoparticles were formed after encapsulation with the required bio-materials (ZnONPs 33.1 nm; CNPs 78.8 nm; neem oil encapsulated (3000 ppm) ZnONPs 182.9 nm; NSKE encapsulated ZnONPs 84.9 nm) and observed that the particles are stable (52.3 mV for ZnONPs, -36.2 mV for CNPs, -43.0 mV for neem oil encapsulated (3000 ppm) ZnONPs and -39.4 mV for NSKE encapsulated ZnONPs). NSKE encapsulated CNPs were able to contain groundnut bruchid up to 180 days with 54.61% weight loss compared to other formulations tested. Thus biomaterial encapsulated nanoscale material formulations are proved to be effective in controlling stored grain pests to reduce huge economic losses.

Nimbolide is a novel, natural compound with promising potential as a drug candidate for anticancer activity. It is isolated from the Indian traditional medicinal plant Azadirachta indica popularly known as neem. The present study was undertaken to explore the oral bioavailability and pharmacokinetic characteristics of nimbolide in rats using the LC/QTOF/MS method. A simple protein precipitation method using acetonitrile was employed for extracting nimbolide from rat plasma. The chromatographic separation of nimbolide and the internal standard (regorafenib) was attained on an Aquity BEH C18 column (100 × 2.1 mm, 2.7 μm), using ACN and 0.1% of formic acid in water as mobile phase components in a gradient elution mode at a flow rate of 0.45 mL/min over a short run time of 4 min. A mass detection was carried out using target ions of [M + H] + at m/z 467.2074 for nimbolide and m/z 483.0847 for the internal standard. The LC/MS method was validated and all the parameters were found well within the specified limits. The calibration curve was constructed in the range of 1-1000 ng/mL. The method shows good accuracy (91.66-97.12%) and precision (intra 2.21-6.92% CV and inter-day 2.56-4.62% CV). This developed LC/MS method was effectively applied to the pharmacokinetic study of nimbolide upon oral and intravenous administration in rats. In concordance with its physicochemical properties and high lipophilicity, we found that it shows poor oral absorption at different doses (10, 30 and 50 mg/kg). As expected, higher plasma levels were observed upon intravenous (10 mg/kg) administration. This method can be extended for evaluation of drug interaction and drug metabolism in rats as well as in humans. Moreover, our rapid and sensitive method may cater the need to accelerate the preclinical formulation development and lead optimization for future drug development of this potent anticancer agent. Further, our oral bioavailability studies demonstrated that nimbolide possesses poor oral absorption, which could be the probable reason for the delay in therapeutic translation of this promising agent for clinical use., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nimbolide is one of the main components in the leaf extract of Azadirachta indica (A. indica). Accumulating evidence from various in vitro and in vivo studies indicates that nimbolide possesses potent anticancer activity against several types of cancer and also shows potential chemopreventive activity in animal models. The main mechanisms of action of nimbolide include anti-proliferation, induction of apoptosis, inhibition of metastasis and angiogenesis, and modulation of carcinogen-metabolizing enzymes. Although multiple pharmacodynamic (PD) studies have been carried out, nimbolide is still at the infant stage in the drug development pipeline due to the lack of systematic pharmacokinetic (PK) studies and long-term toxicological studies. Preclinical PK and toxicological studies are vital in determining the dosage range to support the safety of nimbolide for first-in-human clinical trials. In this review, we will provide a comprehensive summary for the current status of nimbolide as an anticancer and chemopreventive lead compound, and highlight the importance of systematic preclinical PK and toxicological studies in accelerating the process of application of nimbolide as a therapeutic agent against various malignancies., Competing Interests: Conflicts of Interest: None declared.

Nomilin is a potential anticancer agent. In this study, a rapid, sensitive, and simple ultra-performance liquid chromatography with tandem mass spectrometry methodology was established and validated to quantify nomilin in rat plasma. Plasma samples were prepared through liquid-liquid extraction using ethyl acetate. Chromatographic separation was performed using an Acquity HSS T3 column. Acetonitrile and water containing 0.1% (v/v) formic acid were used as mobile phases at a flow rate of 0.3 mL/min. Nomilin and quercetin (internal standard) were detected and quantified via a triple quadrupole tandem mass spectrometer in the positive ion mode with multiple reaction monitoring. Tandem mass spectrometry detection was performed by monitoring the fragmentations of m/z 515.3 → m/z 161.0 and m/z 303.2 → m/z 153.1 of nomilin and quercetin, respectively. Good linearity (R(2) > 0.996) was observed in the concentration range of 1 ng/mL to 500 ng/mL with a lower limit of quantification of 1 ng/mL for nomilin. The average extraction recoveries of nomilin and quercetin were > 82.3% and 82.0%, respectively. Intra- and interday precisions were less than 15% and accuracy ranged from 85.0% to 90.1%. Indeed, the proposed method was successfully applied to analyze the pharmacokinetics of nomilin after 3 and 50 mg/kg nomilin were administered to rats via intravenous and oral routes, respectively., (Georg Thieme Verlag KG Stuttgart · New York.)

Limonoids are heavily oxygenated, modified triterpenes dominant in Meliaceae and Rutaceae plant families. The term 'limonoid' is derived from limonin, which was first identified as the bitter constituent of Citrus seeds in 1841. This group of secondary metabolites exhibits a wide range of biological properties, including anticancer, antibacterial, antifungal, antimalarial, and antiviral activities. Significant progress on the role of limonoids as promising candidates for cancer chemoprevention and/or therapy has been achieved in particular in recent years. The aim of this review article is to discuss the recent developments on limonoids chemical aspects and biological activities with the relationship between structure and activity, supporting the new possibilities for the medicinal and/or nutraceutical use of these compounds.

Purpose: Extensive research over the past decade has revealed that the proinflammatory microenvironment plays a critical role in the development of colorectal cancer. Whether nimbolide, a limonoid triterpene, can inhibit the growth of colorectal cancer was investigated in the present study., Experimental Design: The effect of nimbolide on proliferation of colorectal cancer cell lines was examined by MTT assay, apoptosis by caspase activation and poly-ADP ribose polymerase cleavage, NF-κB activation by DNA-binding assay, and protein expression by Western blotting. The effect of nimbolide on the tumor growth in vivo was examined in colorectal cancer xenografts in a nude mouse model., Results: Nimbolide inhibited proliferation, induced apoptosis, and suppressed NF-κB activation and NF-κB-regulated tumorigenic proteins in colorectal cancer cells. The suppression of NF-κB activation by nimbolide was caused by sequential inhibition of IκB kinase (IKK) activation, IκBα phosphorylation, and p65 nuclear translocation. Furthermore, the effect of nimbolide on IKK activity was found to be direct. In vivo, nimbolide (at 5 and 20 mg/kg body weight), injected intraperitoneally after tumor inoculation, significantly decreased the volume of colorectal cancer xenografts. The limonoid-treated xenografts exhibited significant downregulation in the expression of proteins involved in tumor cell survival (Bcl-2, Bcl-xL, c-IAP-1, survivin, and Mcl-1), proliferation (c-Myc and cyclin D1), invasion (MMP-9, ICAM-1), metastasis (CXCR4), and angiogenesis (VEGF). The limonoid was found to be bioavailable in the blood plasma and tumor tissues of treated mice., Conclusions: Our studies provide evidence that nimbolide can suppress the growth of human colorectal cancer through modulation of the proinflammatory microenvironment., (©2013 AACR.)

A novel, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of eight main active ingredients (evodiamine, rutaecarpine, dehydroevodiamine, limonin, ginsenoside Rb1, Rd, Re and Rg1) in rat plasma after oral administration of Wu-Zhu-Yu (WZY) decoction, which is a celebrated and widely used Traditional Chinese Medicine formula for the treatment of headache. The analytes and internal standard (IS) were separated on a SHIM-PACK XR-ODS II column, and the detection was performed on a UFLC-MS/MS system with turbo ion spray source. The lower limits of quantification were 1.5, 0.5, 1.0, 2.0, 2.0, 1.0, 0.5 and 0.2 ng ml(-1) for evodiamine, rutaecarpine, dehydroevodiamine, limonin, gensenoside Rb1, Rd, Re and Rg1, respectively. Linearity, accuracy, precision and absolute recoveries of the eight analytes were all within satisfaction. The IS-normalized matrix factor was adopted for assessing the matrix effect and accompanied with a satisfactory result. The validated method has been successfully applied to compare pharmacokinetic profiles of the eight active ingredients in rat plasma between normal and headache rats after administration. Exact pharmaceutical effect of WZY decoction on headache was demonstrated by the ethological response of headache rats induced by nitric oxide donor after administration. The results indicated that the absorption of evodiamine, rutaecarpine, gensenoside Rb1, Re and Rg1 in headache group were significantly higher than those in normal group with similar concentration-time curves while no significant differences existed in limonin and ginsenoside Rd between the two groups., (Copyright © 2013 John Wiley & Sons, Ltd.)

A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of limonin in human urine using podophyllotoxin as internal standard. The analyte and IS were extracted with solid-phase extraction and separated by a rapid isocratic elution with 1% formic acid/methanol (v:v, 40:60) on an C(18) column (150 mm × 2.1 mm I.D.). The detection was performed by mass spectrometry in the multi-reaction-monitoring mode. The precursor to product ion transitions of m/z 471.3→161.2 and m/z 397.2→313.1 were used to measure the analyte and the IS. The assay was linear over the concentration range of 0.0783-10 ng/mL for limonin in human urine. The lower limit of quantification was 0.0783 ng/mL and the extraction recovery was larger than 76.7% for limonin. The inter- and intra-day precision of the method at three concentrations was less than 7.4%. The method was successfully applied to pharmacokinetic study of limonin in humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A sensitive, rapid and specific LC-MS-MS method was established and validated for determination of methyl kulonate, a major bioactive constituent isolated from Meliae Cortex, in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed using a Capcell Pak C₁₈ column (100 x 4.6 mm, 5 μm) with the mobile phase comprising a mixture of methanol, 10 mM ammonium formate and formic acid (95:5:0.1, v/v/v). Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive atmospheric ionization at m/z 467 --> 311 for methyl kulonate, and m/z 469 --> 451 for dubione B (internal standard), respectively. A good linear response was observed over the concentration range 1.00-500 ng/mL with the lower limit of quantification 1.00 ng/mL in rat plasma. The method also afforded satisfactory results base on sensitivity, specificity, precision, accuracy, recovery, freeze-thaw and long-time stability. The validated method was successfully applied to determine the pharmacokinetic properties of methyl kulonate in rats after oral administration at dose of 100 mg/kg. This pharmacokinetic study of methyl kulonate is reported here for the first time.

Limonin existed in citrus fruits has been shown to have anti-bacterial, anti-viral, anti-feedant, anti-nociceptive, anti-inflammatory activities and anti-carcinogenic activities. But the clinical use is limited by its low bioavailability. The aim of this study is to observe the absorption and secretion transport mechanisms of limonin in intestine which can pave the way for the further study and clinical use. The transport characteristics and mechanisms of limonin in rat were studied by in situ intestine perfusion and in vitro Caco-2 cells method. The intestinal absorption of limonin was probably via a facilitated diffusion pathway which was poor and without segment-selection. Verapamil and ketoconazole improved the absorption remarkably according to the result of in vitro Caco-2 cells study; however, probenecid had no significant effect on the absorption. The P-gp efflux and CYP3A4 metabolism were involved in the poor intestinal absorption and low bioavailability of limonin. The exploration of the intestinal absorption mechanism is crucial to the design of dosage form and clinical use of limonin.

Background: Azadirachtins are natural insecticides derived from the neem tree. The emerald ash borer (EAB) is an exotic invasive insect pest that infests various ash tree species and has the potential for significant economic, aesthetic and ecological impacts throughout North America. The initial translocation and foliar residue dynamics of azadirachtins were examined following direct injection into white and green ash trees growing in urban scenarios as a potential control for EAB., Results: Substantial concentrations of azadirachtins A and B [mean maxima > 0.98 mg kg(-1) fresh weight (f.w.)] were observed within 2 days of injecting a specifically designed formulation of azadirachtins. Foliar residues declined exponentially through time, with half-life estimates ranging from 5.1 to 12.3 days. At the time of leaf senescence, foliar residue levels approximated 0.01 mg kg(-1) f.w., strongly mitigating the potential effects of non-target biota in soil or aquatic compartments., Conclusion: The magnitude and duration of exposures observed in this field study were considered to be above the thresholds required for biological effectiveness against both larval and adult life stages of EAB. Results support the use of azadirachtins as an environmentally acceptable systemic insecticide for control of EAB and protection of high-value ash trees in urban environments., (Copyright © 2011 Society of Chemical Industry.)

The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive pest discovered in North America in 2002, is now well established and threatens ash (Fraxinus spp.) trees throughout the continent. Experiments were conducted to 1) examine the efficacy of an alternative natural pesticide, azadirachtin, to control emerald ash borer, and 2) determine foliar uptake and dissipation patterns after systemic injections of azadirachtin into trunks of small (2.2 cm diameter at breast height [dbh]), uninfested green ash trees. We found no evidence of mortality of adult beetles. In contrast, fewer larvae completed their development at dose levels > or = 1.7 mg (AI)/cm dbh and development ceased beyond the second instar at dose levels > or = 13.6 mg (AI)/cm dbh. Substantial concentrations (11.2 microg/g dry mass [SD = 7.55]) of azadirachtin were present in leaves within 7 d of treatment. After rapid initial uptake, concentrations in leaves declined logarithmically during the 55 d after injection. A similar pattern was observed in a separate experiment that examined the uptake and translocation of azadirachtin in larger green ash trees (22 cm dbh) treated with 250 mg (AI) /cm dbh with the EcoJect injection system. In another experiment, recently infested plantation green ash trees treated with doses > or = 40 mg (AI)/cm dbh had significant reductions in adult emergence approximately 1 yr postinjection. Given the inhibition of larval development, reduction of adult emergence, and the occurrence of foliar residues at biologically active concentrations, we conclude that azadirachtin is effective in protecting ash trees from emerald ash borer.

Previous studies have indicated the chemopreventive potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonoids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), and deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of phase II enzymes in female A/J mice. Our results indicated the highest induction of glutathione S-transferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroquinoline 1-oxide (4NQO), while the same compound in the stomach induced GST by 51% compared to the control. The DNAG treated group induced 55% in stomach homogenates. Another phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 32% in liver and lung homogenates, respectively. Defuran limonin induced QR in lung homogenates by 45%. Our results indicated that modification of limonin has differential induction of phase II enzymes. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in the detoxification of xenobiotics.

In order to better understand and optimize the sorption of limonin (the major navel orange juice bitter principle) by various plasticized polymeric films, a sorption and plasticizer migration study was carried out using high-performance liquid chromatography (HPLC) and gas chromatography (GC). Low molecular weight (LMW) poly (vinyl chloride) (PVC) plasticized with dioctyl adipate (DOA) gave the best results for both limonin sorption and low DOA migration. Thick films did not significantly sorb more limonin than thin films in the timeframe of our experiments, as the absorption involved rapid surface sorption followed by slow bulk diffusion. The debittering efficiency was a 1000-fold greater than that obtained with current polystyrene divinylbenzene resin beads, with potential for industrial scale debittering.

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

In the present study, the pharmacokinetics of limonin (LM) were investigated in male and female rats. LM concentrations in the plasma were determined after the oral administration of 36 mg/kg LM or after intravenous (i.v.) injection of LM 3.6 mg/kg respectively. Concentrations in the tissues, urine, feces and bile were also analyzed following the oral administration of 36 mg/kg of the test product. It was found that the plasma concentrations of LM in female rats were significantly higher (P < 0.01) than those in male rats. Assessment of the effects of limonin based on the C(max) and AUC in female rats showed that levels were about 50-fold higher than those in male rats after oral administration of 36 mg/kg LM. Furthermore, after i.v. administration of 3.6 mg/kg LM, the C(max) and AUC in female rats was found to be about 3-fold higher than those in male rats. The total excretion of LM in the urine and bile of female rats was also found to be significantly higher than in male rats, which displayed lower concentrations of LM in the tissues, amounting to around one-half to one-tenth of those in female rats, apart from levels in the rectum and duodenum. In conclusion, the present results demonstrate the existence of marked gender difference in LM pharmacokinetics in rats.

A neem-based insecticide, Neemix 4.5, was applied to forest pond enclosures at environmentally realistic concentrations (i.e., below the worst-case expected environmental concentration of 35 microg L(-1)). Crustacean zooplankton communities were examined by multivariate ordination (nonmetric multidimensional scaling) and time-course analyses of population trends among indicator taxa over two field seasons to determine application effects on community structure and recovery patterns. Significant effects on zooplankton community structure were detected at all main test concentrations (n = 5) of 28, 17, and 10 microg L(-1) azadirachtin. There was also evidence of adverse effects on zooplankton communities at an auxiliary test concentration (n = 2) of 5 microg L(-1) azadirachtin. Community-level effects resulted primarily from reductions in adult copepods with short-term, reciprocal increases in cladocerans. Copepod nauplii were not significantly affected. Response patterns suggested that the reductions in adult copepods resulted from growth-regulating effects of the active ingredient azadirachtin, or other neem compounds, and not from formulation ingredients. There was no evidence of recovery among adult copepods within the season of application. At the beginning of the second sampling season, there were apparent carryover effects similar to the community responses in the previous year. By the end of the second season, there was evidence indicating recovery of community structure at the two lower test concentrations of 10 and 17 microg L(-1), but not at 28 microg L(-1). The selective toxicity to adult copepods is problematic in that this group has a relatively long life cycle (1 year), contributes a major component of zooplankton biomass and respiration, and occupies critical functional guilds within zooplankton food web structures. Mitigation measures such as reductions in application rates and efforts to avoid deposition of sprayed materials on water bodies will be required to reduce the risk of harmful effects on zooplankton communities of forest ponds and other shallow, standing-water bodies.

This study utilizes liquid chromatography/mass spectrometry (LC-MS) to analyze the plasma of four groups of four healthy male and female subjects administered high doses of pure limonin glucoside (0.25-2.0 g in 200 mL of buffered water) for the presence of limonin to establish the absorption, metabolism, and bioavailability of citrus limonoids to humans. The plasma analysis revealed increasing amounts of limonin associated with increasing doses of limonin glucoside among the subject groups in mean maximum concentration amounts ranging from 1.74 to 5.27 nmol/L. A high degree of variability in the analyzed limonin concentration was observed within the subject groups. The mean time to maximum concentration was 6 h. A second compound with MS/MS characteristics identical to limonin was detected in amounts up to 5.13 nmol/L and is hypothesized to be a limonin epimer. Poststudy health evaluation established no ill effects among study subjects consuming high doses of limonin glucoside.