6beta-hydroxygedunin from Azadirachta indica. Its potentiation effects with some non-azadirachtin limonoids in neem against lepidopteran larvae

The biological activity of 6beta-hydroxygedunin isolated from Azadirachta indica A. Juss. was assessed using the gram pod borer, Helicoverpa armigera (Hubner), and Asian armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), alone and in combination with other limonoids, gedunin, salannin, nimbinene, and azadirachtin. The compound exhibited growth inhibitory activity in artificial diet bioassays, with 24.2 and 21.5 ppm, respectively, inhibiting growth by 50%. This efficacy was higher in comparison to gedunin (EC(50) = 50.8 and 40.4 ppm), salannin (EC(50) = 74.5 and 72.0 ppm), and nimbinene (EC(50) = 391.4 and 404.5 ppm). Azadirachtin, however, remained the most active neem allelochemical against both insect species. Nutritional assays clearly demonstrated that, though relative consumption and growth rates of fourth instar larvae were reduced, gedunin-type compounds induced physiological toxicity, evident by reduced efficiency of conversion of ingested food (ECI) in feeding experiments. Salannin and nimbinene, on the contrary, induced concentration-dependent feeding deterrence only. In feeding experiments, combinations of the compounds revealed that when azadirachtin was present in a mixture, EC(50) values did not deviate from the individual efficacy of azadirachtin (0.26 and 0.21 ppm, respectively) against H. armigera and S. litura larvae. However, a combination without azadirachtin did show a potentiation effect with potent EC(50) values among structurally different molecules, i.e., when salannin or nimbinene was combined with 6beta-hydroxygedunin or gedunin rather than structurally similar salannin + nimbinene or 6beta-hydroxygedunin + gedunin. Obviously, azadirachtin being the most active compound in neem is not synergized or influenced by any other limonoid, but other non-azadirachtin limonoids were more potent in specific combinations vis-à-vis the structural chemistry of the compound. It is obvious from the present study that potentiation among non-azadirachtin limonoids having explicitly two different modes of action, such as feeding deterrence and physiological toxicity, may be playing a significant role in the potentiation effect.     

Comparative analysis of seed morphometric and allozyme data among four populations of neem (Azadirachta indica)

The objectives of this study were to determine the patterns of variability and to compare genetic distance between seed morphometric and allozyme data of neem (Azadirachta indica). Phenotypic variation from open pollinated seed materials at 12 isozyme loci was examined using starch gel electrophoresis for four populations from Thailand, Bangladesh and Kenya. All four populations exhibited high levels of variation for seed parameters and isozymes. UPGMA cluster analysis of Nei's genetic distance when based on isozymes revealed two genetic groups. The population from Kenya showed a close genetic relationship with the Bangladeshi populations. The population from Thailand exhibited a distant relationship with the other three populations. This result suggests that the Kenyan population was introduced from the Indian subcontinent. In contrast, cluster analysis of seed morphometric data indicated three different groups of populations. The population from Kenya showed a distant relationship with all other populations. The study demonstrates that allozyme data may not correspond with morphometric traits when measuring genetic distance. It is suggested that this controversy may depend on environmental influence on morphometric traits, possibly also on the enzyme patterns.     

Influence of physicochemical parameters of neem (Azadirachta indica A Juss) oils on nitrification inhibition in soil

The technology for the production of neem oil coated urea (NOCU) developed by the Indian Agricultural Research Institute is in the pipeline for adaption by several Indian fertilizer industries. Use of nitrification inhibitors is one of the methods of improving the nitrogen use efficiency (NUE) of nitrogenous fertilizers in agriculture. However, standard specifications for the neem oil as a raw material of NOCU are desired. Accordingly, the present study was undertaken to evaluate 25 samples of neem oils comprising 11 samples of expeller grade (EG) oils, 8 samples of cold-pressed (CP) oils, 3 samples of solvent-extracted oils, and 2 commercial formulations. NOCU was prepared using these oils (5000 ppm of urea-N). The soils fertilized with NOCUs (200 ppm of urea-N) were incubated at 27 degrees C and 50% water-holding capacity for a period of 15 days. Nitrapyrin (0.5% of N) coated urea served as the reference and prilled urea as control. Samples were analyzed for NH4+-N, NO2--N, and NO3--N using standard methods. The percent nitrification inhibition (NI) was calculated, and the results revealed that all of the neem oils caused NI ranging from 4.0 to 30.9%. Two samples of EG oils and two commercial formulations were found to be the best, causing 27.0-30.9% NI. Iodine, acid, and saponification values and meliacin content of all of the oils were analyzed and correlated with NI. The results revealed the direct influence of meliacin content of the neem oils on NI, which, however, was found to be negatively correlated with saponification and iodine values. There is, therefore, a need to introduce new Bureau of Indian Standards (BIS) specifications for neem oils as raw materials of NOCU.     

Influence of operating parameters on the use of the microwave-assisted process (MAP) for the extraction of azadirachtin-related limonoids from neem (Azadirachta indica) under atmospheric pressure conditions

The use of the microwave-assisted process (MAP) for the extraction of azadirachtin-related limonoids (AZRL) from various parts of the neem tree was investigated under different operating conditions. The influence of microwave power, solvent, and irradiation time on the recovery of AZRL was studied. The efficiency of the microwave-assisted extraction (MAE) of the seed kernel, the seed shell, the leaf, and the leaf stem was compared to that of conventional extraction methods. The content of AZRL in the extracts was estimated with a vanillin-based colorimetric assay and a multivariate calibration technique. The results showed that the MAE technique can enhance the extraction of AZRL from different parts of neem possessing microstructures. Investigation of the influence of the solvent also indicted that the solvent used not only influences the efficiency but also affects the selectivity of the MAE.     

Effect of emulsion size and shelf life of azadirachtin A on the bioefficacy of neem (Azadirachta indica A. Juss) emulsifiable concentrates

In a study of 33 recipes of neem oil based emulsifiable concentrates, the specific surface area of the emulsions and cream plus oil layer separation in emulsions at 24 h revealed a correlation of -0.6874 between them and correlations of -0.8940 and 0.6972, respectively, with bioefficacy (LC(50)) against the 3-day-old second-instar larvae of the Bihar hairy caterpillar, Spilosoma obliqua Walker. Nearly 96-99% of azadirachtin A in emulsifiable concentrates (aza-A content = 617.93-1149.65 ppm) degraded during the heat stability test at 54 +/- 1 degrees C for 14 days with half-lives ranging between 1.84 and 4.53 days. The LC(50) values against S. obliqua were, however, statistically at par in both the pre- and the post-heat-treated samples, suggesting a similar effect of azadirachtin A and its degradation products on the bioactivity. The half-life of azadirachtin A could be enhanced by storing the concentrates at lower temperatures. A low pH of the formulation solvent did not check the degradation of azadirachtin A, as reported with aqueous solutions in the literature.     

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.     

Response of neem (Azadirachta indica A. Juss) to indigenous arbuscular mycorrhizal fungi, phosphate-solubilizing and asymbiotic nitrogen-fixing bacteria under tropical nursery conditions

Neem (Azadirachta indica A. Juss) seedlings were inoculated with arbuscular mycorrhizal (AM) fungi, Glomus intraradices Schenck and Smith and G. geosporum (Nicol. and Gerd.) Walker, Azospirillum brasilense, and phosphate-solubilizing bacteria (PSB) individually or in various combinations in unsterile soil under nursery conditions. Seedlings were harvested at 60 and 120 days after transplantation. Microbial inoculation resulted in increased mycorrhizal colonization, greater plant height, leaf area and number, root collar diameter, biomass, phosphorus, nitrogen and potassium content, and seedling quality. Inoculated seedlings also had low root/shoot ratios and low nutrient utilization efficiencies. Populations of PSB declined with seedling growth; contrarily populations of A. brasilense increased. A. brasilense and PSB populations were related to each other and influenced root colonization by AM fungi. Microbial inoculation effects were greatest when seedlings were inoculated with a combination of microbes rather than individually. This clearly indicates that these microorganisms act synergistically when inoculated simultaneously, with maximum response being when both AM fungi were coinoculated with A. brasilense and PSB. The results emphasize the importance of microbial inoculations for the production of robust, rapidly growing seedlings in nurseries and illustrate the advantage of inoculating soils of a low microbial population with indigenous microbes.     

Antioxidative and antimutagenic activities of 4-vinyl-2,6-dimethoxyphenol (canolol) isolated from canola oil

A potent antioxidative compound in crude canola oil, canolol, was recently identified, and reported herein are studies of its scavenging capacity against the endogenous mutagen peroxynitrite (ONOO(-)). ONOO(-) is generated by the reaction between superoxide anion radical and nitric oxide, both of which are produced by inflammatory leukocytes. Among various antioxidative substances of natural or synthetic origin, canolol was one of the most potent antimutagenic compounds when Salmonella typhimurium TA102 was used in the modified Ames test. Its potency was higher than that of flavonoids (e.g., rutin) and alpha-tocopherol and was equivalent to that of ebselen. Canolol suppressed ONOO(-)-induced bactericidal action. It also reduced intracellular oxidative stress and apoptosis in human cancer SW480 cells when used at a concentration below 20 microM under H(2)O(2)-induced oxidative stress. In addition, canolol suppressed plasmid DNA (pUC19) strand breakage induced by ONOO(-), as revealed by agarose gel electrophoresis.     

Effect of pH on binding of mutagenic heterocyclic amines by the natural biopolymer poly(gamma-glutamic acid)

Poly(gamma-glutamic acid) (gamma-PGA), a nontoxic and biodegradable macropolymer, was evaluated for its efficiency in binding three mutagenic heterocyclic amines (HAs), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-p-2), as affected by pH in a batch mode. The maximum HA sorption was attained for pH 3-7 and decreased sharply for pH less than 3. Binding isotherms obtained at pH 2.5 and 5.5 showed different isotherm shapes that belong to S and L types, respectively. The isotherm data at pH 2.5 were well described by a linear form of the Langmuir equation, while at pH 5.5 it showed two distinct curves, which were precisely fitted as multiple Langmuir curves. The deviation of linearity in Scatchard plot proved the multisite HA sorption. The Brunauer-Emmett-Teller equation also fitted better to isotherm data at pH 5.5, suggesting a multisite sorption caused by multimolecular HA layers on gamma-PGA. High HA sorption levels of 1250, 667, and 1429 mg/g at pH 2.5 and 1429, 909, and 1667 mg/g at pH 5.5 were observed for MeIQ, 4,8-DiMeIQx, and Trp-p-2, respectively. Among the HAs studied, the sorption capacity correlated directly with hydrophobicity of HAs and inversely with the number of methyl groups in HA molecules. The plausible binding mechanism of HAs on gamma-PGA may include a combination of hydrophobic, hydrogen-bonding, ionic, and dipole-dipole interactions.     

Changes in the levels of polymethoxyflavones and flavanones as part of the defense mechanism of Citrus sinensis (cv. Valencia Late) fruits against Phytophthora citrophthora

Phytophthora citrophthora causes serious losses in Citrus fruits through brown rot lesion. The effect of infection with P. citrophthora on Citrus sinensis (cv. Valencia Late) fruits was studied, with particular reference to the levels of the flavanones hesperidin and isonaringin and the polymethoxyflavones sinensetin, nobiletin, tangeretin, and heptamethoxyflavone, because flavonoids are most probably involved as natural defense or resistance mechanisms in this genus. Changes in the levels of these flavonoids were detected after infection. The hesperidin and isonaringin contents fell by 13 and 67%, respectively, whereas the contents of their corresponding aglycons, hesperetin and naringenin, increased, suggesting the hydrolyzing effect of this fungus on the glycosylated flavanones. The heptamethoxyflavone, nobiletin, sinensetin, and tangeretin levels increased by 48, 28, 26, and 24%, respectively. The in vitro study revealed that these compounds acted as antifungal agents, the most active being the aglycons (naringenin and hesperetin), followed by the polymethoxyflavones and flavanone glycosides. The participation of these flavonoids in the defense mechanism of this Citrus species is discussed.     

Anthraquinones isolated from Cassia tora (Leguminosae) seed show an antifungal property against phytopathogenic fungi

The fungicidal activities of Cassia tora extracts and their active principles were determined against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani using a whole plant method in vivo and were compared with synthetic fungicides and three commercially available anthraquinones. The responses varied with the plant pathogen tested. At 1 g/L, the chloroform fraction of C. tora showed a strong fungicidal activity against B. cinerea, E. graminis, P. infestans, and R. solani. Emodin, physcion, and rhein were isolated from the chloroform fraction using chromatographic techniques and showed strong and moderate fungicidal activities against B. cinerea, E. graminis, P. infestans, and R. solani. Furthermore, aloe-emodin showed strong and moderate fungicidal activities against B. cinerea and R. solani, respectively, but did not inhibit the growth of E. graminis, P. infestans, P. recondita, and Py. grisea. Little or no activity was observed for anthraquinone and anthraquinone-2-carboxylic acid when tested at 1 g/L. Chlorothalonil and dichlofluanid as synthetic fungicides were active against P. infestans and B. cinerea at 0.05 g/L, respectively. Our results demonstrate the fungicidal actions of emodin, physcion, and rhein from C. tora.     

Emodin isolated from Cassia obtusifolia (Leguminosae) seed shows larvicidal activity against three mosquito species

Mosquito larvicidal activity of Cassia obtusifolia (Leguminosae) seed-derived materials against the fourth-instar larvae of Aedes aegypti, Aedes togoi, and Culex pipiens pallens was examined. The chloroform fraction of C. obtusifolia extract showed a strong larvicidal activity of 100% mortality at 25 mg/L. The biologically active component of C. obtusifolia seeds was characterized as emodin by spectroscopic analyses. The LC(50) values of emodin were 1.4, 1.9, and 2.2 mg/L against C. pipiens pallens, A. aegypti, and A. togoi, respectively. Pirimiphos-methyl acts as a positive control directly compared to emodin. Pirimiphos-methyl was a much more potent mosquito larvicide than emodin. Nonetheless, emodin may be useful as a lead compound and new agent for a naturally occurring mosquito larvicidal agent. In tests with hydroxyanthraquinones, no activity was observed with alizarin, danthron, and quinizarin, but purpurin has an apparent LC(50) value of approximately 19.6 mg/L against A. aegypti.     

Inhibitory effects of (-)-epigallocatechin gallate on the mutation,DNA strand cleavage,and DNA adduct formation by heterocyclic amines

Green tea is known to be a potential chemopreventive agent against cancer. In this study, we investigated the inhibitory activities of tea extracts, and in particular the polyphenolic component (-)-epigallocatechin gallate (EGCG), against heterocyclic amine-induced genotoxicity. The tea extracts displayed inhibition of 2-hydroxyamino-6-methyldipyrido[1,2-a,3',2'-d]imidazole (Glu-P-1(NHOH))-induced mutagenicity. This inhibition can be accounted for by the presence of EGCG in the extracts. The mutagenic effect of Glu-P-1(NHOH), which induces single-strand cleavage in supercoiled circular DNA under neutral conditions, was inhibited by EGCG. Using the Drosophila repair test, a test for gross DNA damage, and DNA adduct detection by (32)P-postlabeling, we showed that EGCG prevented 2-amino-3,8-dimethylimidazo[4,5-f]quinoline-induced DNA damage and adduct formation in insect DNA. EGCG was found to accelerate the degradation of Glu-P-1(NHOH) in vitro. This observation suggested that the inhibition by EGCG is associated with an accelerated degradation of metabolically activated heterocyclic amines.     

Varietal differences in the bioaccessibility of beta-carotene from mango (Mangifera indica) and papaya (Carica papaya) fruits

Mango and papaya, which are rich sources of beta-carotene, are widely consumed in India. In this study, beta-carotene content and its bioaccessibility were determined in six locally available varieties of mango, namely, Badami, Raspuri, Mallika, Malgoa, Totapuri, and Neelam, and two varieties of papaya, namely, Honey Dew and Surya. Varietal differences were evident in both beta-carotene content and its bioaccessibility in the case of mango. beta-Carotene content in ripe mango ranged from 0.55 +/- 0.03 mg/100 g in the Malgoa variety to 3.21 +/- 0.25 mg/100 g in the Badami variety. Similarly, in the Honey Dew and Surya varieties of papaya, beta-carotene contents were 0.70 +/- 0.10 and 0.74 +/- 0.12 mg/100 g, respectively. Bioaccessibility of beta-carotene ranged from 24.5% in Badami to 39.1% in Raspuri varieties of mango. Considering both the percent bioaccessibility and the inherent beta-carotene content, the amount of bioaccessible beta-carotene was highest in the Mallika variety (0.89 mg/100 g), followed by Badami (0.79 mg/100 g). Because mango and papaya are also consumed as a blend with milk, the influence of the presence of milk on the bioaccessibility of beta-carotene from these fruits was also examined. Addition of milk generally brought about a significant increase in the bioaccessibility of beta-carotene from mango, the increase ranging from 12 to 56%. Bioaccessibility of beta-carotene from the two varieties of papaya examined was similar (31.4-34.3%). Addition of milk increased this bioaccessibility by 19 and 38% in these two varieties. Considering the beta-carotene content of mango and papaya, the latter has to be consumed in amounts roughly 3 times that of mango to derive the same amount of beta-carotene. Thus, this study has indicated that varietal differences exist in the content and bioaccessibility of beta-carotene in mango and that the addition of milk is advantageous in deriving this provitamin A from the fruit pulp of mango and papaya.     

Evaluation of bitter masking flavanones from Herba Santa (Eriodictyon californicum (H. and A.) Torr., Hydrophyllaceae)

Products made from Herba Santa (Eriodictyon californicum (H. & A.) Torr.) have been used as bitter remedies for some pharmaceutical applications for many years, but they are actually too aromatic to be useful for many food or pharmaceutical applications. In sensory studies flavanones homoeriodictyol (1), its sodium salt (1-Na), sterubin (2), and eriodictyol (4) could significantly decrease the bitter taste of caffeine without exhibiting intrinsic strong flavors or taste characteristics. Further investigations on 1-Na elicited a broad masking activity between 10 and 40% toward different chemical classes of bitter molecules (e.g. salicin, amarogentin, paracetamol, quinine) but not toward bitter linoleic acid emulsions. For caffeine and amarogentin, dose-response studies were performed; the masking activity toward bitter taste for both compounds reached a plateau at higher concentrations of 1-Na. Due to these facts, homoeriodictyol sodium salt (1-Na) seems to be a very interesting new taste modifier for food applications and pharmaceuticals.     

Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments

The analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by soaking and drying treatments were studied. The various carotenoids in the flowers of daylily were analyzed using a reversed-phase C(30) HPLC column and a mobile phase of methanol/methylene chloride/2-propanol (89:1:10, v/v/v) with methanol/methylene chloride (45:55, v/v) as sample solvent. Twenty-one pigments were resolved, of which 14 carotenoids were identified, including neoxanthin, violaxanthin, violeoxanthin, lutein-5,6-epoxide, lutein, zeaxanthin, beta-cryptoxanthin, all-trans-beta-carotene, and their cis isomers, based on spectral characteristics and Q ratios. Prior to hot-air-drying (50 degrees C) or freeze-drying, some of the daylily flowers were subjected to soaking in a sodium sulfite solution (1%) for 4 h. Under either the hot-air- or the freeze-drying treatment, the amounts of most carotenoids were higher in the soaked daylily flowers than in those that were not soaked. With hot-air-drying, the amount of cis carotenoids showed a higher yield in soaked samples than in nonsoaked samples. However, with freeze-drying, only a minor change of each carotenoid was observed for both soaked and nonsoaked samples. Also, air-drying resulted in a higher loss of carotenoids than freeze-drying.     

Insecticidal activity of rhamnolipid isolated from pseudomonas sp. EP-3 against green peach aphid (Myzus persicae)

Microorganisms capable of growth on oils are potential sources of biopesticides, as they produce complex molecules such as biosurfactants and lipopeptides. These molecules have antimicrobial activity against plant pathogens, but few data are available on their insecticidal activity. The present study describes the insecticidal activity of a rhamnolipid isolated from diesel oil-degrading Pseudomonas sp. EP-3 (EP-3). The treatment of cell-free supernatants of EP-3 grown on glucose-mineral medium for 96 h led to > 80% mortality of aphids (Myzus persicae) within 24 h. Bioassay-guided chromatography coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MADLDI-TOF MS) and (1H, 13C) nuclear magnetic resonance (NMR) analyses was employed to isolate and identify the EP-3 insecticidal metabolites. Dirhamnolipid, with molecular formulas of C??H??O?? and C??H??O??, was identified as a main metabolite exhibiting insecticidal activity against aphids. Dirhamnolipid showed a dose-dependent mortality against aphids, producing about 50% mortality at 40 μg/mL and 100% mortality at 100 μg/mL. Microscopy analyses of aphids treated with dirhamnolipid revealed that dirhamnolipid caused insect death by affecting cuticle membranes. This is the first report of rhamnolipid as an insecticidal metabolite against M. persicae. Rhamnolipid shows potential for use as a pesticide to control agricultural pests.