Stabilization of azadirachtin A in neem formulations: effect of some solid carriers, neem oil, and stabilizers

Formulation of azadirachtin A on attapulgite, kaolinite, fuller's earth, hydrated calcium silicate, and fly ash revealed that it degraded to the tune of 70-95% on different solid carriers as compared to 56% in neem oil, during the 14 day heat storage studies at 54 +/- 1 degrees C in the laboratory. The degradation was reduced by 26-60% on different carriers by employing either anthraquinone or epichlorohydrin as stabilizer. Pyrogallol and hydroquinone enhanced the degradation. The cation exchange capacity and surface area of the carriers revealed a significant negative correlation with t(1/2) of azadirachtin A.     

Breakdown of azadirachtin A in a tropical soil amended with neem leaves and animal manures

A field investigation was conducted to assess the breakdown of azadirachtin A in a tropical coastal savanna soil amended with neem leaves （NL） combined with poultry manure （PM） or cow dung （CD） using gas chromatography. Samples in polythene bags 15 cm long and 4.8 cm in diameter were randomly placed to a depth of 14 cm in the soil, and azadirachtin A concentration was assessed on days 0, 14, 28, 42, 56, 70, and 84. Azadirachtin A degradation in the soil followed first-order reaction kinetics with different half-lives obtained for varying combinations of the amendments. Higher neem amendment levels of 100 g gave shorter half-lives of azadirachtin A than the lower levels of 50 g. Within the 50 g NL group the additions of the poultry manure and the cow dung gave significantly shorter （P 〈0.05） half-lives of azadirachtin A than the sole neem amendment, whereas in the 100 g NL group only additions of 10 g CD and 10 g PM were significantly less （P 〈 0.05） than the sole neem amendment. Different changes resulting from the kind and quantity of animal manure added were observed in the half-lives of azadirachtin A. The 100 g NL group had significantly higher （P 〈0.05） moisture content, which, coupled with the likely differeaces in microbial biomass, could be the major factor responsible for variations in the half-llfe of the compound. Therefore, the quantity of the neem leaves applied and the addition of animal manure affected the breakdown of azadirachtin A in the soil amended with neem leaves.     

Insecticidal activity of caffeine aqueous solutions and caffeine oleate emulsions against Drosophila melanogaster and Hypothenemus hampei

The bioactivity of caffeine aqueous solutions (0.20-2.00 wt %) and caffeine oleate emulsions (20 vol % oil, 2.00 wt % surfactant, 0.04 wt % caffeine, 0.05 wt % oleic acid) was assessed against two biological models: Drosophila melanogaster and Hypothenemus hampei. The caffeine aqueous solutions showed no insecticidal activity, whereas caffeine oleate emulsions had high bioactivity against both D. melanogaster and H. hampei. By preparing the caffeine oleate emulsions with anionic surfactants (i.e., sodium lauryl sulfate, sodium laureate, and sodium oleate), we obtained a lethal time 50 (LT50) of 23 min. In the case of caffeine oleate emulsions prepared with nonionic surfactants (i.e., Tween 20 and Tween 80), a LT50 of approximately 17 min was observed. The high bioactivity of the caffeine oleate emulsion against H. hampei opens the possibility of using this insecticide formulation as an effective way to control this pest that greatly affects coffee plantations around the world.     

Residues and persistence of neem formulations on strawberry after field treatment

Azadirachtoids were determined by liquid chromatography/mass spectrometry (LC/MS) in five methanolic seed extracts of the neem tree and in a commercial formulation. On average, seed extracts contain azadirachtin A (10.9%), azadirachtin B (3.5%), nimbin (10.4%), and large quantities of salannin (19.0%). The composition of the commercial formulations may present different azadirachtoids contents depending on the natural extracts used in the preparation. Because these compounds may also show insecticide activity, the efficacy on field of these formulations may be very different. Photodegradation of pure azadirachtoids was also studied. Azadirachtins and related compounds are very sensitive to sunlight, degrading rapidly, with half-lives of the order of 11.3 h for azadirachtin A and 5.5 h for azadirachtin B and few minutes for the other limonoids compounds studied. The residues of azadirachtins and the main constituents, e.g., salannin, nimbin, deacetylnimbin, and deacetylsalannin, of the neem seed extract were determined on strawberries after field treatment using two different formulations. This residue study on strawberry was carried out to assess not only the azadirachtin content but also the main azadirachtoids contents. Three days after field application at five times the dose recommended by the manufacturer, residues of azadirachtin A and B were 0.03 and 0.01 mg/kg, respectively, while residues of salannin (LOQ 0.01 mg/kg) and nimbin (LOQ 0.5 mg/kg) were not detectable.     

Analysis of fat-soluble vitamins. XXIX. Liquid chromatographic determination of vitamin D in AD concentrates: collaborative study

A simplified liquid chromatographic (LC) method for determining vitamin D in vitamin AD concentrates (greater than or equal to 5000 IU vitamin D/g) was collaboratively studied. In the simplified method, the 2 columns specified in AOAC LC method 43.101-43.109 are replaced by a single column, which separates the vitamin D isomers and the vitamin A esters. The procedure for oils includes dissolution and quantitation by normal phase LC. Dry multivitamin concentrates and aqueous dispersions are treated with an enzyme system and the vitamins are extracted with n-pentane. Six coded samples were distributed to 16 laboratories; 15 collaborators returned their results. Estimates of repeatability and reproducibility for the oil samples were 1.1 and 3.1%, respectively; for the high-level concentrated dry preparation 1.4 and 3.9% and for the low-level concentrated dry preparation 1.3 and 11.4%, respectively. These values are a considerable improvement over the results obtained in the 1979 multivitamin collaborative study. The method has been adopted official first action for determination of vitamin D in vitamin AD concentrates containing greater than or equal to 5000 IU vitamin D/g.     

Variability in Neem (Azadirachta indica) with respect to azadirachtin content

There is a controversy over variations in azadirachtin content in neem (Azadirachta indica) seeds among various provenances and countries. Also, variations in azadirachtins are usually attributed to climatic conditions such as temperature and humidity. The present study was undertaken to evaluate qualitative and quantitative variability in azadirachtins A and B among various neem provenances or individual neem trees. Forty-three provenances of India were examined for intraprovenance variability in azadirachtin A and B content and oil percentage. Twenty-eight individual neem trees from five provenances of different agroclimatic regions were also examined for interprovenance variability. The azadirachtins were quantified using reversed phase analytical HPLC. There were wide variations in oil and azadirachtin contents among different provenances. Azadirachtin A ranged from 556.9 to 3030.8 mg kg(-)(1) of kernels, whereas azadirachtin B was in the range 43.1-590.6 mg kg(-)(1) of kernel among the provenances investigated. Analysis of variance among various neem provenances showed significant differences in oil content, azadirachtin A, total azadirachtin (A + B), and A:B ratio. There were individuals with high and low azadirachtins within a single provenance, and this trend was observed in all of the provenances selected from five agroclimatic regions of the country. Variations among individual trees of a particular provenance indicated that climatic factors such as rainfall, humidity, or temperature did not influence azadirachtin content in the neem trees. The present study shows that there are individual genetic differences among neem trees. A systematic study for tree improvement with a population of mother trees with desired traits should be undertaken by performing half-sib progeny trials and further selections by clonal propagations. The role of genetic makeup needs further research.     

Microencapsulating properties of sodium caseinate

Emulsions were prepared with 5% (w/v) solutions of sodium caseinate (Na Cas) and soy oil at oil/protein ratios of 0.25-3.0 by homogenization at 10--50 MPa. Emulsions were spray-dried to yield powders with 20--75% oil (w/w). Emulsion oil droplet size and interfacial protein load were determined. Microencapsulation efficiency (ME), redispersion properties, and structure of the powders were analyzed. The size of emulsion oil droplets decreased with increasing homogenization pressure but was not influenced by oil/protein ratio. Emulsion protein load values were highest at low oil/protein ratios. ME of the dried emulsions was not affected by homogenization pressure but decreased from 89.2 to 18.8% when the oil/protein ratio was increased from 0.25 to 3.0, respectively. Mean particle sizes of reconstituted dried emulsions were greater than those of the original emulsions, particularly at high oil/protein ratios (>1.0), suggesting destabilization of high-oil emulsions during the spray-drying process.     

Gas chromatographic method for determination of fenitrothion in fenitrothion technical and in formulated products: collaborative study

A gas chromatographic (GC) method for determination of fenitrothion in fenitrothion technical and formulated products has been subjected to a collaborative study with 7 participating laboratories. Formulations are extracted with chloroform containing dibutyl sebacate as an internal standard and are analyzed by gas chromatography using an OV-210 column. Collaborators were furnished matched pairs of technical product and water-dispersible powder and emulsifiable concentrate formulations. Relative standard deviations for reproducibility (RSDR) for the paired samples were 0.54, 1.00, and 1.56%, respectively, for technical fenitrothion, water-dispersible powders, and emulsifiable concentrates. The method has been approved interim official first action as an alternative to the present official first action AOAC method 6.A19-6.A24, which uses a polyphenyl ether, 6 ring (PPE-6R) column packing and fluoranthene as internal standard.     

Hydroxytyrosol prevents oxidative deterioration in foodstuffs rich in fish lipids

Hydroxytyrosol, a natural phenolic compound obtained from olive oil byproduct, was characterized as an antioxidant in three different foodstuffs rich in fish lipids: (a) bulk cod liver oil (40% of omega-3 PUFAs), (b) cod liver oil-in-water emulsions (4% of omega-3 PUFAs), and (c) frozen minced horse mackerel ( Trachurus trachurus) muscle. Hydroxytyrosol was evaluated at different concentration levels (10, 50, and 100 ppm), and its antioxidant capacity was compared against that of a synthetic phenolic, propyl gallate. Results proved the efficiency of hydroxytyrosol to inhibit the formation of lipid oxidation products in all tested food systems, although two different optimal antioxidant concentrations were observed. In bulk oil and oil-in-water emulsions, a higher oxidative stability was achieved by increasing the concentration of hydroxytyrosol, whereas an intermediate concentration (50 ppm) showed more efficiency, delaying lipid oxidation in frozen minced fish muscle. The endogenous depletion of alpha-tocopherol and omega-3 polyunsaturated fatty acids (omega-3 PUFAs) was also inhibited by supplementing hydroxytyrosol in minced muscle; however, the consumption of the endogenous total glutathione was not efficiently reduced by either hydroxytyrosol or propyl gallate. A concentration of 50 ppm of hydroxytyrosol was best to maintain a longer initial level of alpha-tocopherol (approximately 300 microg/g of fat), whereas both 50 and 100 ppm of hydroxytyrosol were able to preserve completely omega-3 PUFAs. Hydroxytyrosol and propyl gallate showed comparable antioxidant activities in emulsions and frozen fish muscle, and propyl gallate exhibited better antioxidant efficiency in bulk fish oil.     

茶多酚对淀粉酯纳米颗粒及其稳定的Pickering乳液性质的影响

为探究茶多酚（Tea Polyphenols, TPs）对辛烯基琥珀酸酐（Octenyl Succinic Anhydride, OSA）酯化淀粉纳米颗粒（Starch Nanoparticles,SNPs）及其稳定的Pickering乳液性质的影响,该研究在制备OSA-SNPs的过程中添加TPs,研究TPs对OSA-SNPs的理化性质和乳化性能的影响。结果发现,添加TPs使OSA-SNPs的平均粒径增加、表面Zeta电位绝对值下降、接触角减小（P<0.05）。通过傅立叶红外光谱扫描发现,TPs与OSA-SNPs之间存在氢键和疏水相互作用。在TP-OSA-SNPs稳定的乳液中,增加TP-OSA-SNPs的质量浓度（从0.5 g/mL至2.0 g/mL）,乳滴平均直径明显减小（P<0.05）;当TP-OSA-SNPs的质量浓度增加至2 g/mL时,乳液形成了油滴紧密堆积的界面结构,能够抑制油滴迁移。通过加速氧化试验发现,与OSA-SNPs相比,TP-OSA-SNPs稳定的乳液中氢过氧化物值（Peroxide Value, POV）相对较低（P<0.05）,说明TP-OSA-SNPs具有延缓乳液中油脂氧化的作用。结果表明,这种新型具有抗氧化功能的食品级颗粒乳化剂,对构筑淀粉基Pickering乳液载体具有潜在价值。     

Effect of partial replacement of gum arabic with carbohydrates on its microencapsulation properties.

Gum arabic solutions (10% w/v) were emulsified with soy oil at oil/gum ratios of 0.25-5.0. At oil/gum ratios <1.0, it was established that gum arabic could be partially replaced with a nonsurfactant carbohydrate. To assess different carbohydrates as replacers for gum arabic, emulsions and spray-dried emulsions of soy oil and mixed solutions (10% w/v) of gum arabic and a range of carbohydrate wall materials (oil/gum = 0.5) were prepared and analyzed. Maize starch and glucose were ineffective as partial replacers of gum arabic, but maltodextrins of various dextrose equivalence values (5.5-38) successfully replaced 50% of the gum arabic. The microencapsulation efficiency of the gum arabic/maltodextrin stabilized powders was further increased by increasing total solids of the feed to the dryer and by increasing the atomizer nozzle diameter.     

Bovine serum albumin produces a synergistic increase in the antioxidant activity of virgin olive oil phenolic compounds in oil-in-water emulsions

Virgin olive oil is valued for its flavor, but unacceptable off-flavors may develop on storage in food products containing this oil due to oxidation. The oxidative stability of oil-in-water emulsions containing bovine serum albumin (BSA) and virgin olive oil phenolic compounds was studied. Four oil-in-water emulsions with and without BSA and phenols isolated from virgin olive oil were prepared. These model systems were stored at 60 degrees C to speed up lipid oxidation. Primary and secondary oxidation products were monitored every three days. Peroxide values and conjugated diene contents were determined as measures of the primary oxidation products. p-Anisidine values and volatile compounds were determined as measures of the secondary oxidation products. This latter determination was carried out by headspace solid-phase microextraction coupled with gas chromatography. Although olive oil phenolic compounds and BSA contributed some antioxidant activity when present as individual additives, the combination of BSA with phenols in an emulsion showed 58-127% synergy, depending on which analytical method was used in the calculation. The emulsion containing phenolic compounds and BSA showed a low level of deterioration after 45 days of storage at 60 degrees C.     

Ability of surfactant micelles to alter the physical location and reactivity of iron in oil-in-water emulsion

The purpose of this research was to determine how surfactant micelles influence iron partitioning and iron-promoted lipid oxidation in oil-in-water emulsions. Lipids containing ferric ions were used to produce oil-in-water emulsions, and continuous-phase iron concentrations in emulsions were measured as a function of varying continuous-phase polyoxyethylene 10-lauryl ether (Brij) concentrations. Continuous-phase iron concentrations increased with increasing surfactant micelle concentrations (0.1-2.0%) and storage time (1-7 days). At pH 3.0, the concentration of continuous-phase iron was higher than at pH 7.0. Similar trends in iron solubilization by Brij micelles were observed when either hexadecane or corn oil was used as the lipid phase. Lipid oxidation rates, as determined by the formation of lipid hydroperoxides and headspace hexanal, in corn oil-in-water emulsions containing iron decreased with increasing surfactant concentrations (0.5-2.0%). These results indicate that surfactant micelles could alter the physical location and prooxidant activity of iron in oil-in-water emulsions.     

Liquid chromatographic determination of carbadox, desoxycarbadox, and nitrofurazones in pork tissues

A liquid chromatographic (LC) method has been developed for the determination of carbadox, desoxycarbadox, and nitrofurazones in the 10-40 ppb range in pork muscle, liver, and kidney tissues. Tissues were homogenized in absolute ethanol, and the homogenates were treated with metaphosphoric acid and reduced in volume by rotovaporization. Hexane was added to the concentrates, which were then centrifuged to remove fat. After addition of KH2PO4 to the aqueous phase and extraction with ethyl acetate, the extracts were passed through alumina columns before analysis by reverse phase LC. Overall average recoveries (10-40 ppb range) for carbadox and desoxycarbadox from spiked tissues were 53% +/- 13.6 and 61% +/- 7.2, respectively; overall average recoveries for nitrofurazone and furazolidone were 43% +/- 7.3 and 77% +/- 10.9, respectively. Before these optimum determinations, degradation by even minimal incandescent light was found to reduce recovery especially of desoxycarbadox. The results of this photochemical degradation are reported and briefly discussed.     

甲氰胺混配乳油对柑桔全爪螨的药效试验及联合毒力测定

由甲氰菊酯（灭扫利）和水胺硫磷复配而成的22％甲氰胺混配乳油，其稀释3000～4000倍液，对柑桔全爪螨（Panonychuscitri）有良好的防治效果，室内药效达99．38％～100％。田间药效为94．70％～100％。室内毒力测定结果，甲氰胺混配乳油的LC50为21．02mg／L，灭扫利的为28.75mg／L，水胺硫磷的为153.62mg／L，甲氰胺的毒力显著高于两种单体；共毒系数达205.79，表现出显著的增效作用。     

Extraction and colorimetric determination of azadirachtin-related limonoids in neem seed kernel.

A colorimetric method was developed for the determination of total azadirachtin-related limonoids (AZRL) in neem seed kernel extracts. The method employed acidified vanillin solution in methanol for the colorization of the standard azadirachtin or neem seed kernel extracts in dichloromethane. Through the investigation of various factors influencing the sensitivity of detection, such as the concentration of vanillin, acid, and the time required for the formation of color, optimum conditions were selected to perform the assay. Under the optimum conditions, a good linearity was found between the absorbance at 577 nm and the concentration of standard azadirachtin solution in the range of 0.01-0.10 mg/mL. In addition, different extraction procedures were evaluated using the vanillin assay. The HPLC analysis of the extracts indicated that if the extractions were performed in methanol followed by partitioning in dichloromethane, approximately 50% of the value determined by the vanillin assay represents azadirachtin content.     

Characterization of the antioxidant activity of sugars and polyhydric alcohols in fish oil emulsions

Polyols have been incorporated into fish oil emulsions as a means for the inhibition of lipid oxidation and suppression of fishy flavor. However, the role of sugars and polyhydric alcohols as antioxidants has not been clearly established. Selected polyols were evaluated for their performance as antioxidants and modifiers of oxidation pathways in a model system. Oil/water (O/W) emulsions were prepared with freshly steam-deodorized menhaden oil. A layer of emulsion in aluminum pans held at 5 degrees C was exposed to 2550 lx fluorescent lights for 24 h before peroxide values and volatile flavor compounds were analyzed by GC headspace entrainment procedure. Antioxidant activity was confirmed for fructose, sucrose, raffinose, sorbitol, or mannitol when incorporated at 16% of the aqueous phase into model fish oil-in-water emulsions. Peroxide values were suppressed 10-18% in treated samples compared to control samples. Viscosity data did not exclude possible contributions from a restricted oxygen diffusion mechanism in the antioxidant activity, but revealed that emulsion viscosity did not govern fish oil oxidation rates. Combining polyols with phenolic antioxidants (alpha-tocopherol, BHT, or TBHQ) frequently diminished the antioxidant activity compared to that for individual phenolic antioxidants, which was interpreted as indicating that the H-donating activity of phenolic antioxidants was hindered by the H-bonding activity of polyols. A viscosity-based inhibition of the retroaldol conversion of (E,Z)-2,6-nonadienal to (Z)-4-heptenal with a high fructose concentration (67%) was attributed to a restriction of molecular mobility of reactants, but the conversion was only slightly inhibited by the concentration of fructose (16%) used in experimental emulsions. The data supported a hypothesis that either or both free radical scavenging and transition state metal chelation activities were provided by polyols in fish oil emulsions. Also, polyols retarded the water-requiring retroaldol decomposition of (E,Z)-2,6-nonadienal to (Z)-4-heptenal in the model systems and the reaction may be involved in some suppression of fishy flavors in emulsions.     

Protection against oxidation of fish-oil-enriched milk emulsions through addition of rapeseed oil or antioxidants

The ability of rapeseed oil and/or different antioxidants (alpha- and gamma-tocopherol mixture, ascorbyl palmitate, and EDTA) to protect fish-oil-enriched milk emulsions against oxidation was investigated. Tocopherol isomers in concentrations similar to those found in natural rapeseed oil were added to rapeseed oil stripped of natural tocopherols. The rapeseed oil with added tocopherols significantly inhibited oxidation in the fish-oil-enriched milk emulsions. In contrast, the emulsions with only fish oil and added alpha- and gamma-tocopherol were less stable than the emulsions with fish oil alone. When added individually, the gamma-tocopherol seemed to inhibit oxidation more efficiently than alpha-tocopherol. Ascorbyl palmitate (AP) almost completely retarded oxidation in the fish-oil-enriched milk emulsions, as determined by PV, volatile oxidation products, and sensory evaluation. AP also prevented the otherwise prooxidant effect of tocopherols added to fish oil before emulsification. No interactions between AP, tocopherols, and EDTA were observed, and EDTA added alone to fish oil did not show antioxidant properties in the milk emulsions. Overall, the results showed that addition of AP or rapeseed oil containing natural tocopherols to fish oil was equally efficient in inhibiting oxidation in the fish-oil-enriched milk emulsions.     

An efficient method for the purification and characterization of nematicidal azadirachtins A,B, and H,using MPLC and ESIMS

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.     

Experimental and modeling studies showing the effect of lipid type and level on flavor release from milk-based liquid emulsions

The purpose of this work was to study two key parameters of the lipid phase that influence flavor release-lipid level and lipid type-and to relate the results to a mass balance partition coefficient-based mathematical model. Release of 10 volatile compounds from milk-based emulsions at 10, 25, and 50 degrees C was monitored by 1-min headspace sampling with a solid-phase microextraction fiber, followed by GC-MS analysis. As compared to the observations for milk fat, changing to a lipophilic lipid (medium-chain triglycerides, MCT) and adding a monoglyceride-based surfactant did not influence the volatiles release. However, increasing the solid fat content was found to increase the release. At 25 degrees C, and even more so at 10 degrees C, concurrent with an increase in their solid fat content, hydrogenated palm fat emulsions showed increased flavor release over that observed for emulsions made with coconut oil, coconut oil with surfactant, milk fat, and MCT. However, at 50 degrees C, when hydrogenated palm fat emulsions had zero solid fat content, there was no difference in flavor release from that observed for milk fat emulsions. Varying milk fat at nine levels between 0 and 4.5% showed a systematic dependence of the release on the lipid level, dependent on compound lipophilicity. Close correlations were found between the experimental and model predictions with lipid level and percent liquid lipid as variables.