Estimation of Azadirachtin Content in Neem Extracts and Formulations

Abstract

A high performance liquid chromatographic reversed-phase procedure has been developed whereby azadirachtin content can be estimated in crude extracts of neem and in dust formulations of neem. An estimation of the azadirachtin content is achieved through the use of an external azadirachtin standard and valley-to-valley integration. Since azadirachtin seems to be the most potent insect feeding deterrent in these extracts and formulations, its content is a measurement of potency and represents an attempt at standardization.     

An HPLC method for determination of azadirachtin residues in bovine muscle

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.     

印楝素的合成、结构修饰及生物活性研究进展

自印楝素被分离鉴定以来, 其广谱而独特的生物活性受到人们的广泛关注, 已成为一种近乎完美的植物源杀虫剂; 且在医药、保健、日化等领域都有广泛的应用．其复杂而独特的分子结构使得科研工作者进行了大量的全合成尝试、结构修饰及构效关系等研究. 综述了印楝素A的片段合成、全合成和印楝素有关的结构修饰以及生物活性等方面的研究进展.     

Development and evaluation of thin-layer chromatography-digital image-based analysis for the quantitation of the botanical pesticide azadirachtin in agricultural matrixes and commercial formulations: comparison with ELISA

A simple thin-layer chromatography-digital image-based analytical method has been developed for the quantitation of the botanical pesticide, azadirachtin. The method was validated by analyzing azadirachtin in the spiked food matrixes and processed commercial pesticide formulations, using acidified vanillin reagent as a postchromatographic derivatizing agent. The separated azadirachtin was clearly identified as a green spot. The Rf value was found to be 0.55, which was similar to that of a reference standard. A standard calibration plot was established using a reference standard, based on the linear regression analysis [r2 = 0.996; y = 371.43 + (634.82)x]. The sensitivity of the method was found to be 0.875 microg azadirachtin. Spiking studies conducted at the 1 ppm (microg/g) level in various agricultural matrixes, such as brinjal, tomato, coffee, and cotton seeds, revealed the recoveries of azadirachtin in the range of 67-92%. Azadirachtin content of commercial neem formulations analyzed by the method was in the range of 190-1825 ppm (microg/mL). Further, the present method was compared with an immunoanalytical method enzyme-linked immonosorbent assay developed earlier in our laboratory. Statistical comparison of the 2 methods, using Fischer's F-test, indicated no significant difference in variance, suggesting that both methods are comparable.     

Purification of the seven tetranortriterpenoids in neem (Azadirachta indica) seed by counter-current chromatography sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography

Counter-current chromatography (CCC) sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography was used to isolate the seven bio-actives (azadirachtin A, azadirachtin B, azadirachtin H, desacetylnimbin, desacetylsalannin, nimbin and salannin) from the seed concentrate (NSC) of the neem tree (Azadirachta indica A. Juss). Reproducible, narrow polarity range, high purity fractions were obtained from repeated injections of the NSC (700 mg loadings/injection), on to a relatively small volume CCC coil (116 mL). The CCC biphasic solvent system chosen was hexane:butanol:methanol:water (1:0.9:1:0.9, v/v). A mass balance of injected material showed that 95+% were recovered.     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Biological studies of azadirachtin and its derivatives against polyphagous pest, Spodoptera litura

Azadirachtin-A (Aza-A, figure 1) was subjected to a variety of synthetic transformations and the antifeedant activity and toxicity of the azadirachtin derivatives were assessed against second instar larvae Spodoptera litura on castor leaves in comparison with the crude material containing 9.14% Aza-A, as well as relatively pure 91% Aza-A. A probable mechanism for the base initiated fragmentation of the diketocarbonate into a decalin fragment and a spiroketal moiety has been proposed. The present study indicates that the combination of insect toxicity and antifeedancy of azadirachtin provides good crop protection.     

Effect of Elicitors and Precursors on Azadirachtin Production in Hairy Root Culture of Azadirachta indica

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag⁺, Hg⁺², Co⁺², Cu⁺²) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l^?1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g^?1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 %?v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g^?1) with respect to the control (3.3 mg g^?1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l^?1 in 25 days) was found to be the highest with cholesterol (50 mg l^?1) addition as an indirect precursor in the medium.     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

Studies aimed at the total synthesis of azadirachtin. A modeled connection of C-8 and C-14 in azadirachtin

[reaction: see text] Studies on the connection between the right and left segments of azadirachtin are described. The Ireland-Claisen rearrangement of Li-enolate of the modeled ester with dichlorodimethylsilane in toluene afforded the desired limonoid framework stereoselectively in good yield.     

Determination of azadirachtin in agricultural matrixes and commercial formulations by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza molecule. Rabbits were immunized with either bovine serum albumin (BSA)-azadirachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antisera by antibody capture assay showed that the antibody titer of antisera raised against OA-aza was 1:30,000. An indirect competitive ELISA was developed with BSA-azadirachtin as coating antigen and aza-specific antibodies raised against OA-aza immunogen. The immunoassay showed an inhibitory concentration (IC50) value of 75 ppb, with a range of detection from 0.5 to 1,000 ppb for azadirachtin [based on regression analysis, y= 85.87 (-18.89x); r2 = -0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23-dihydro-23beta-methoxy azadirachtin and 3-tigloylazadirachtol) was 33 and 29%, respectively. The indirect competitive ELISA was validated and evaluated by quantitating aza in spiked agricultural commodities and from neem formulations. Azadirachtin was spiked into 5 different agricultural commodities: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1,000 ppb and recovered at 62-100%. In samples drawn from 6 lots, the aza content in neem-seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2,000 ppm. The method developed may be applied to environmental monitoring of aza and quality assurance studies of aza-based commercial formulations.     

Production of the Biopesticide Azadirachtin by Hairy Root Cultivation of Azadirachta indica in Liquid-Phase Bioreactors

Batch cultivation of Azadirachta indica hairy roots was carried out in different liquid-phase bioreactor configurations (stirred-tank, bubble column, bubble column with polypropylene basket, and polyurethane foam disc as root supports) to investigate possible scale-up of the A. indica hairy root culture for in vitro production of the biopesticide azadirachtin. The hairy roots failed to grow in the conventional bioreactor designs (stirred tank and bubble column). However, modified bubble column reactor (with polyurethane foam as root support) configuration facilitated high-density culture of A. indica hairy roots with a biomass production of 9.2 g l^?1dry weight and azadirachtin yield of 3.2 mg g^?1 leading to a volumetric productivity of azadirachtin as 1.14 mg l^?1 day^?1. The antifeedant activity in the hairy roots was also evaluated by no choice feeding tests with known concentrations of the hairy root powder and its solvent extract separately on the desert locust Schistocerca gregaria. The hairy root powder and its solvent extract demonstrated a high level of antifeedant activity (with an antifeedant index of 97 % at a concentration of 2 % w/v and 83 % at a concentration of 0.05 % (w/v), respectively, in ethanol).     

Rapid and sensitive analysis of azadirachtin and related triterpenoids from Neem (Azadirachta indica) by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Based on reversed-phase high-performance liquid chromatography (RP-HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry, a HPLC-MS method was developed to permit the rapid qualitative and quantitative analysis of azadirachtin and related tetranortriterpenoids from seeds and tissue cultures of Neem (Azadirachta indica). APCI+ standard scanning mass spectra of the major Neem triterpenoids were recorded and utilized to select suitable ions for selected ion monitoring (SIM). Transitions for selective reaction monitoring (SRM) were based on MS-MS experiments. Using SIM, major Neem triterpenoids were detected in callus culture material and seed kernels of A. indica. The limit of detection for azadirachtin in extract samples (approximately 1 ng ml(-1) or 10 pg in SIM mode) was determined to be (with respect to injected absolute amounts) approximately 1000-times lower than values quoted in the literature for existing HPLC methods (approximately 200 ng ml(-1) or 10 ng). In addition to high sensitivity, the HPLC-MS method is able to tolerate minimal sample preparation and purification, dramatically reducing total analysis time.     

Synthesis of natural products from the Indian neem tree Azadirachta indica

The synthesis of five natural products (3, 6, 7, 10, and 14), isolated from the Indian neem tree Azadirachta indica, is reported from a common intermediate (2). The judicious choice of transacetalization conditions allows efficient access to both the azadirachtinin (9 and 10) and the azadirachtin (3, 6, 7, and 14) skeletons.     

Production of Biopesticides in an In Situ Cell Retention Bioreactor

The seeds of Azadirachta indica contain azadirachtin and other limonoids, which can be used as a biopesticide for crop protection. Significant variability and availability of seed only in arid zones has triggered biotechnological production of biopesticides to cope up with its huge requirement. Batch cultivation of A. indica suspension culture was carried out in statistically optimized media (25.0 g/l glucose, 5.7 g/l nitrate, 0.094 g/l phosphate and 5 g/l inoculum) in 3 l stirred tank bioreactor. This resulted in 15.5 g/l biomass and 0.05 g/l azadirachtin production in 10 days leading to productivity of 5 mg l(-1) day(-1). Possible inhibition by the limiting substrates (C, N, P) were also studied and maximum inhibitory concentrations identified. The batch kinetic/inhibitory data were then used to develop and identify an unstructured mathematical model. The batch model was extrapolated to simulate continuous cultivation with and without cell retention in the bioreactor. Several offline computer simulations were done to identify right nutrient feeding strategies (with respect to key limiting substrates; carbon, nitrate and phosphate) to maintain non-limiting and non-inhibitory substrate concentrations in bioreactor. One such continuous culture (with cell retention) simulation was experimentally implemented. In this cultivation, the cells were propagated batch-wise for 8 days. It was then converted to continuous cultivation by feeding MS salts with glucose (75 g/l), nitrate (10 g/l), and phosphate (0.5 g/l) at a feed rate of 500 ml/day and withdrawing the spent medium at the same rate. The above continuous cultivation (with cell retention) demonstrated an improvement in cell growth to 95.8 g/l and intracellular accumulation of 0.38 g/l azadirachtin in 40 days leading to an overall productivity of 9.5 mg l(-1) day(-1).     

Synthetic studies on azadirachtin: stereoselective construction of the ABCE ring system

A new method for constructing the ABCE ring system of azadirachtin in a stereoselective manner was developed. The synthesis of the model compound features (1) stereoselective construction of the highly hindered C8 quaternary center by an intermolecular addition reaction of an allylborane with an aldehyde and (2) construction of the E ring moiety by the Pd-catalyzed Nazarov cyclization.     

Azadirachtol,a tetranortriterpenoid from neem kernels

The title compound, di­methyl (?)‐(2aR,3R,4R,4aS,5R,7aS,8R,10S,10aR)‐3,8,10‐tri­hydroxy‐4‐[(2R,6R)‐2‐hydroxy‐11‐methyl‐5,7,10‐trioxatetra­cyclo­[6.3.1.0^2,60^9,11]­dodec‐3‐en‐9‐yl]‐4‐methyl­per­hydro­isobenzo­furano­[5,4,3a‐cd]­isobenzofuran‐5,10a‐di­acetate, C₂₈H₃₆O₁₃, which exhibits higher antifeedant activity than azadirachtin‐A, a known potent antifeedant, was isolated from neem kernels. The asymmetric unit of the structure contains two independent mol­ecules, which differ in the conformations of their functional groups and also in the conformations of some of the rings. The relative orientation between the decalin and furan­yl moieties is similar to that observed in the majority of azadirachtin structures, but is different from that in azadirachtin‐A. The two symmetry‐independent mol­ecules are linked into dimeric units by intermolecular O—H?O hydrogen bonds.     

Synthesis of the tricyclic dihydrofuran moiety of azadirachtin: efficient transformation of the Claisen rearrangement intermediate into a functionalized tricyclic dihydrofuran core

Azadirachtin is a C-seco limonoid, derived from the neem tree. Previously, we reported the synthesis of the right and left segments of azadirachtin, in addition to the coupling reaction of the modeled ester utilizing Claisen rearrangement. Herein we report the transformation of the modeled rearrangement product into a tricyclic dihydrofuran, which would be expected to have bioactivity against insects.     

Asymmetric Formal Synthesis of Azadirachtin

An asymmetric formal synthesis of azadirachtin, a potent insect antifeedant, was accomplished in 30 steps to Ley’s synthetic intermediate (longest linear sequence). The synthesis features: 1) rapid access to the optically active right‐hand segment starting from the known 5‐hydroxymethyl‐2‐cyclopentenone scaffold; 2) construction of the B and E rings by a key intramolecular tandem radical cyclization; 3) formation of the hemiacetal moiety in the C ring through the α‐oxidation of the six‐membered lactone followed by methanolysis.