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.     

Insect antifeedants from tropical plants: structures of dumnin and dumsenin

Two novel A-seco limonoids, dumnin and dumsenin, were isolated from the methanolic extract of Croton jatrophoides by bioassay-guided fractionation, and the structures were determined by nuclear magnetic resonance, circular dichroism, and mass spectrometry experiments. These compounds showed potent antifeedant activity (PC(50) 相似文献   

Insecticidal activity of Maytenus species (Celastraceae) nortriterpene quinone methides against codling moth, Cydia pomonella (L.) (Lepidoptera: tortricidae)

The insecticidal effects of nortriterpene quinone methides (pristimerin, tingenonee, and 20-alpha-hydroxytingenone) are reported for the first time. The natural products were isolated from Maytenus sp. (Celastraceae) and their effects tested on larvae of codling moth (Cydia pomonella, Lepidoptera: Tortricidae). The three metabolites produce the same effects on codling moth larvae that azadirachtin does, although at higher concentrations. 20-alpha-Hydroxytingenone was the most active compound, showing lethal, antifeedant, and insect growth regulation activities. Pristimerin showed also a high antifeedant activity together with its molt effect suppression. Tingenone showed the lowest activity. The differences in the activity of the three products are related to the structure of the E ring.     

Evaluation of calcium-alginate gel as an artificial diet medium for bioassays on common cutworms

A calcium-alginate gel diet was developed for Spodoptera litura larvae, and its reliability as a carrier for incorporating antifeedants as well as insecticides was investigated. The alginate gel diet was prepared with a simple protocol, which does not involve any heating process. When tested using this diet, acephate, a Bacillus thuringiensis endotoxin formulation and rotenone reproducibly showed insecticidal activity against the larvae, while neem oil and scabequinone deterred the larval feeding effectively. However, not only the insecticidal activity of acephate but also the antifeedant activity of neem oil was reduced by replacing the alginate component by agar in the diet, suggesting the usefulness of the alginate gel diet as an assay tool for testing a broad range of samples against the larvae.     

New mosquito larvicidal tetranortriterpenoids from Turraea wakefieldii and Turraea floribunda

The crude methanol extracts of the root barks of Turraea wakefieldii and Turraea floribunda were found to show mosquito larvicidal activity against third-instar larvae of Anopheles gambiae sensu stricto. Four new limonoids comprising a vilasininoid 1 and three havanensinoids 2-4 were isolated from the chloroform fractions of the methanol extracts of T. wakefieldii and T. floribunda, respectively. The structures of the compounds were elucidated by NMR spectroscopy. Compounds 1, 2, and 4 had LD50 values of 7.1, 4.0, and 3.6 ppm, respectively, and were more potent than azadirachtin, which had an LD50 value of 57.1 ppm when tested against larvae of A. gambiae.     

Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae)

Monoterpenoids (terpenes and biogenically related phenols) commonly found in plant essential oils were tested for acute toxicity via topical application to tobacco cutworms (Spodoptera litura Fab.). The most toxic among 10 such compounds was thymol (LD(50) = 25.4 microg/larva) from garden thyme, Thymus vulgaris. The compounds were then tested for sublethal effects, specifically inhibition of larval growth after topical application of low doses. Among 6 compounds tested, an LD(10) dose reduced growth by 20% on average 3 days after administration. Feeding deterrence was determined using a cabbage leaf disk choice test. The most deterrent compound was thymol, with a DC(50) of 85.6 microg/cm(2) leaf disk area. Because minor constituents in complex essential oils have been suggested to act as synergists, binary mixtures of the compounds were tested for synergy vis à vis acute toxicity and feeding deterrence. trans-Anethole acted synergistically with thymol, citronellal, and alpha-terpineol, in terms of both acute toxicity and feeding deterrence. On the basis of these findings, several complex mixtures were developed and tested as leads for effective control agents. Candidate mixtures demonstrated good synergistic effects. The observed LD(50) of mixture 3 was 40.6 microg/larvae compared to an expected value of 74.6 microg/larvae. The result of this research is a proprietary product suitable for commercial production.     

Growth inhibitory effects on fall armyworm Spodoptera frugiperda of some limonoids isolated from Cedrela spp. (Meliaceae)

Dichloromethane extracts of Cedrela salvadorensis and Cedrela dugessi afforded a photogedunin epimeric mixture, gedunin and cedrelanolide. These compounds and the photogedunin epimeric acetates 3 and 4 at the 23-OH position were evaluated against Spodoptera frugiperda. Toosendanin, isolated from Melia azedarach, was used as a positive control. When tested for activity on neonate larvae into the no-choice bioassays, gedunin, photogedunin epimeric mixture, and photogedunin acetates mixture caused significant larval mortality with LC(50) of 39.0, 10.0, and 8.0 ppm at 7 days, respectively, as well as growth reduction. All the compounds tested inhibited larval growth, compared to the control, in a concentration-dependent manner. In addition, it was possible to observe significant reduced pupal weights and adult emergence. All the tested compounds except cedrelanolide showed comparable activity to that of toosendanin.     

Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management

In the course of screening for novel naturally occurring insecticides from plants, the activity of the fruit extract of the Argentinian Melia azedarach L. (Meliaceae) and its recently described limonoid meliartenin were investigated. The antifeedant activity of the fruit extract was tested on a variety of herbivore and granivorous insects through choice tests. Sixteen of 17 species belonging to three orders consume significantly less food when treated with the extract. The bioactivity of the isolated active compound meliartenin and its interchangeable isomer 12-hydroxiamoorastatin (1) was further studied. In choice tests, compound 1 inhibited feeding of Epilachna paenulata Germ. (Coleoptera, Coccinellidae) larvae, with an ED(50) value of 0.80 microg/cm(2), comparable to that of azadirachtin (2) and lower than that of toosendanin (3) (0.72 and 3.69 microg/cm(2), respectively), both compounds used for comparison purposes. In no-choice tests, E. paenulata larvae reared on food treated with 1 or 2 ate less, gained less weight, and suffered greater mortality rates than control larvae. The activity of compound 1 was comparable to that of 2, with LD(50) values of 0.76 and 1.24 microg/cm(2), respectively, at 96 h. Shorter LT(50) values were recorded for 1 at 4 and 1 microg/cm(2) in comparison with 2. Thus, M. azedarach fruit extract and its active principle have interesting potential for use in pest control programs.     

Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study

The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from seeds of Citrus limon. These conversions focused on functional groups considered to be important for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7 afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared. Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows that insect species vary in their behavioral responses to these structural modifications. Highly significant antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and 10) was observed against S. frugiperda.     

Feeding deterrents from Dictamnus dasycarpus Turcz against two stored-product insects

The screening for insecticidal principles from several Chinese medicinal herbs showed that the root bark of Dictamnus dasycarpus possessed significant feeding deterrence against two stored-product insects (Tribolium castaneum and Sitophilus zeamais). From the methanol extract, two feeding deterrents were isolated by bioassay-guided fractionation. The compounds were identified as fraxinellone and dictamnine from their spectroscopic data. Fraxinellone was demonstrated to possess feeding deterrent activity against adults and larvae of T. castaneum as well as S. zeamais adults with EC50 values of 36.4, 29.1, and 71.2 ppm, respectively. Dictamnine was shown to have feeding deterrent activity against adults and larvae of T. castaneum as well as S. zeamais adults with EC50 values of 57.6, 47.9, and 91.7 ppm, respectively.     

Insecticidal activity of huperzine A from the New Zealand clubmoss, Lycopodium varium.

A bioactivity-directed investigation of an extract of the New Zealand clubmoss, Lycopodium varium, collected on subantarctic Campbell Island, has led to the isolation of the alkaloid huperzine A (1) as the major antifeedant and insecticidal component. Huperzine A showed insecticidal activity against the Australian carpet beetle, Anthrenocerus australis (LD(50) = 110 ppm), the Australian sheep blowfly, Lucilia cuprina (LD(50) = 2380 ppm), and the webbing clothes moth, Tineola bisselliella (LD(50) = 630 ppm). Feeding by A. australis was reduced by 97% at 63 ppm.     

Larvicidal activity of isobutylamides identified in Piper nigrum fruits against three mosquito species

The insecticidal activity of materials derived from the fruits of Piper nigrum against third instar larvae of Culex pipiens pallens, Aedes aegypti, and A. togoi was examined and compared with that of commercially available piperine, a known insecticidal compound from Piper species. The biologically active constituents of P. nigrum fruits were characterized as the isobutylamide alkaloids pellitorine, guineensine, pipercide, and retrofractamide A by spectroscopic analysis. Retrofractamide A was isolated from P. nigrum fruits as a new insecticidal principle. On the basis of 48-h LC(50) values, the compound most toxic to C. pipiens pallens larvae was pipercide (0.004 ppm) followed by retrofractamide A (0.028 ppm), guineensine (0.17 ppm), and pellitorine (0.86 ppm). Piperine (3.21 ppm) was least toxic. Against A. aegypti larvae, larvicidal activity was more pronounced in retrofractamide A (0.039 ppm) than in pipercide (0.1 ppm), guineensine (0.89 ppm), and pellitorine (0.92 ppm). Piperine (5.1 ppm) was relatively ineffective. Against A. togoi larvae, retrofractamide A (0.01 ppm) was much more effective, compared with pipercide (0.26 ppm), pellitorine (0.71 ppm), and guineensine (0.75 ppm). Again, very low activity was observed with piperine (4.6 ppm). Structure-activity relationships indicate that the N-isobutylamine moiety might play a crucial role in the larvicidal activity, but the methylenedioxyphenyl moiety does not appear essential for toxicity. Naturally occurring Piper fruit-derived compounds merit further study as potential mosquito larval control agents or as lead compounds.     

Synthesis and insect antifeedant activity of precocene derivatives with lactone moiety

Starting from precocenes I and II, four of their derivatives with a lactone moiety were obtained. The compounds have been assessed as antifeedants against several diverse insect species including the storage pests the confused flour beetle (Tribolium confusum Duv., larvae and adults), the granary weevil beetle (Sitophilus granarius L., adults), and the khapra beetle (Trogoderma granarium Ev., larvae) and against the herbivorous pest insects Colorado potato beetle (Leptinotarsa decemlineata Say, adults and larvae) and aphids (Myzus persicae Sulz.). Precocenes, especially precocene II, showed a very strong antifeedant effect against all storage pests and aphids. The introduction of a lactone moiety caused a decrease in antifeedant activity against these species. Both precocenes were moderately active against L. decemlineata adults. The best antifeedants to this species were precocene derivatives, especially iodolactones. The introduction of iodine into a molecule had a great effect on the antifeedant activity of those compounds.     

Synthesis and antifeedant activity of new oxadiazolyl 3(2H)-pyridazinones

A total of 20 new compounds containing the oxadiazolyl 3(2H)-pyridazinone moiety were synthesized. The structures of all the compounds were confirmed by (1)H NMR, IR, MS, and elemental analysis. Their insect antifeedant activities against Asiatic corn borer Ostrinia furnacalis (Guenee) were examined and compared with commercial azadirachtin. The compounds exhibited significant levels of activity. The feeding deterrency values of IIIa,j were 57% and 51% at 500 mg/kg concentration, respectively.     

Further studies on the anticancer activity of citrus limonoids

Research in this laboratory has shown that some citrus limonoids can inhibit the development of 7,12-dimethylbenz[a]anthracene-induced oral tumors. The data from these studies have suggested that certain rings in the limonoid nucleus may be critical to antineoplastic activity. Using the hamster cheek pouch model, three new limonoids (ichangensin, deoxylimonin, and obacunone) have now been tested for cancer chemopreventive activity. In the first experiment, it was found that the treatments with ichangensin had no effect on tumor number or burden. In the second experiment, obacunone reduced tumor number and burden by 25 and 40%, respectively, whereas deoxylimonin reduced tumor number and burden by 30 and 50%, respectively. The results with deoxylimonin were significant, p < 0.05. Overall, the data indicated that changes in the A ring of the limonoid nucleus can lead to a loss of anticancer activity, whereas changes in the D ring can be tolerated without any apparent loss of biological activity.     

Insect antifeedant activity of flavones and chromones against Spodoptera litura

The antifeedant polymethylated flavones 5-hydroxy-3,6,7,8,4'-heptamethoxyflavone, 5-hydroxy-3,6,7,8-tetramethoxyflavone, and 5,6-dihydroxy-3,7-dimethoxyflavone have been isolated from the cudweed, Gnaphalium affine D. Don (Compositae). These flavonoids and authentic analogues showed insect antifeedant activity against the common cutworm (Spodoptera litura F.). In a previous paper, it was suggested that there was no substituent on the B-ring of the flavonoid for the beneficial antifeedant activity against the common cutworm. These flavonoids having a phenyl group as the B-ring and the chromone as elimination of the B-ring from the flavonoids were used to test the hypothesis of the previously described B-ring effect. The known fact is that Sculletaria baicarensis (Rutaceae) produced the 2-phenyl flavone. Test compounds and their methylated derivatives were prepared from this material for the structure-activity relationship (SAR) study of insect antifeedant activity. In spite of the 2-phenyl flavonoids, some tested compounds did not show any insect antifeedant activity against the common cutworm, although these inactive flavonoids were deficient in the 6-substituent group on the A-ring of the flavonoid. This 6-position-substituted derivative almost showed strong insect antifeedant activity against common cutworm. Moreover, the tested flavonoids having a hydroxyl group as a substituent on any of the positions tended to increase the activity. These results suggested the importance of the 6-position substitution on the flavonoid; however, hydrophilic substituents decreased the activity. Baicalein (5,6,7-trihydroxyflavone) derivatives did not show any activity despite having the 6-substituent derivative. Although the activity of some chromones increased the activity of the flavone, the bulky B-ring was a disadvantage for the antifeedant activity. It was suggested that the charge on C(3) and C(5) of the flavonoid was important for the biological activity. Additionally, an adequate hydrogen bonding property, which is different from lipophilicity, was an advantage for the activity on the basis of a QSAR analysis.     

Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities

Thirty-nine fungal metabolites 1-39, including two new alkaloids, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6) and 3-hydroxyfumiquinazoline A (16), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach. Their structures were elucidated on the basis of detailed spectroscopic analysis (mass spectrometry and one- and two-dimensional NMR experiments) and by comparison of their NMR data with those reported in the literature. These isolated compounds were evaluated for in vitro antifungal activities against some phytopathogenic fungi, toxicity against brine shrimps, and antifeedant activities against armyworm larvae (Mythimna separata Walker). Among them, sixteen compounds showed potent antifungal activities against phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii), and four of them, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6), fumitremorgin B (7), verruculogen (8), and helvolic acid (39), exhibited antifungal activities with MIC values of 6.25-50 μg/mL, which were comparable to the two positive controls carbendazim and hymexazol. In addition, of eighteen that exerted moderate lethality toward brine shrimps, compounds 7 and 8 both showed significant toxicities with median lethal concentration (LC(50)) values of 13.6 and 15.8 μg/mL, respectively. Furthermore, among nine metabolites that were found to possess antifeedant activity against armyworm larvae, compounds 7 and 8 gave the best activity with antifeedant indexes (AFI) of 50.0% and 55.0%, respectively. Structure-activity relationships of the metabolites were also discussed.     

The effects of three phthalate esters on the larval development of the grass shrimp Palaemonetes pugio (Holthuis)

The effects of three phthalic acid esters, dimethyl phthalate (DMP), di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) on survival and development rate of larvae of the grass shrimp Palaemonetes pugio were investigated. Only 100 ppm DMP and 10 to 50 ppm DBP were acutely toxic to the larvae. DMP at a concentration of 100 ppm significantly increased the duration of larval development to the first postlarval stage. DEHP concentrations up to 1 ppm (the solubility limit of this ester) were without effect on either survival or development rate of the larvae. Phthalate esters were not detectable at or above the 2 ppm level in the tissues of larvae chronically exposed to phthalates for the duration of larval development, suggesting active metabolism of phthalates by this species.     

The limonoids and their antitobacco mosaic virus (TMV) activities from Munronia unifoliolata Oliv

Five new limonoids, named munronoids K-O (1-5), together with three known limonoids were isolated from Munronia unifoliola Oliv. These limonoids were involved in the skeletons of evodulone, gedunin, and peieurianin types of limonoids, and their structures were established on the basis of spectroscopic data. Compound 5 featuring a γ-lactone ring instead of the β-substituted furan ring was found in the peieurianin type for the first time. The antitobacco mosaic virus (anti-TMV) activities of compounds 1-8 were also evaluated with half-leaf, enzyme-linked immunosorbent assay, and Western blot methods, and limonoids 1, 5, and 8 showed stronger anti-TMV treatment activities than the positive control ningnanmycin. Six compounds (1-5 and 8) exhibited infection inhibition activities against TMV.     

Synthesis and insect antifeedant activity of aurones against Spodoptera litura larvae

A series of aurones were prepared from various phenols via phenoxy acetic acids and coumaranones and evaluated for insect antifeedant activity against the common cutworm (Spodoptera litura). The naturally occurring aurone was most active at an ED50 of 0.12 micromol/cm2. The synthetic precursor, coumaranones, showed that the introduction of methoxyl and methyl groups to the benzene ring increased insect antifeedant activity. Similarly, the tested aurones showed that the introduction of methoxyl group to the A and/or B rings increased the insect antifeedant activity, but 4,5,6- and 3',4',5'-trisubstituted compounds did not show this activity in this test. The hydroxylation of aurones in the B ring should be disadvantageous for insect antifeedant activity against S. litura. Although the melting points did not correlate well with the insect antifeedant activity, compounds that were nearly inactive had high melting points. A significant correlation was noted between biological activity (pED50) and a hydrogen-bonding parameter calculated from the Rf value obtained from SiOH thin-layer chromatography and a lipophilicity parameter (log k) calculated from the retention time in ODS high-performance liquid chromatography. The respective correlation coefficients (r) were -0.83 and -0.70. The introduction of alkoxy and alkyl groups along with adequate hydrogen bonding seems to contribute to the antifeedant activity of the compounds tested.