Biologically active components against Drosophila melanogaster from Podophyllum hexandrum

In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, a dichloromethane extract of Podophyllum hexandrum was found to give an insecticidal activity against larvae of Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation. The compound was identified as podophyllotoxin (1) by comparison of its spectroscopic characteristics with literature data. In bioassays for insecticidal activity, 1 showed a LC(50) value of 0.24 micromol/mL diet against larvae of D. melanogaster and a LD(50) value of 22 microg/adult against adults. Acetylpodophyllotoxin (1A), however showed slight insecticidal activity in both assays, indicating that the 4-hydroxyl group was an important function for enhanced activity of 1.     

Insecticidal sesquiterpene from Alpinia oxyphylla against Drosophila melanogaster

In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, an MeOH extract of Alpinia oxyphylla was found to possess insecticidal activity against larvae of Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation and identified as nootkatone (1) by GC, GC-MS, and (1)H and (13)C NMR spectroscopy. In bioassays for insecticidal activity, 1 showed an LC(50) value of 11.5 micromol/mL of diet against larvae of D. melanogaster and an LD(50) value of 96 microg/adult against adults. Epinootkatol (1A), however, showed slight insecticidal activity in both assays, indicating that the carbonyl group at the 2-position in 1 was the important function for enhanced activity of 1.     

Avocadofurans and their tetrahydrofuran analogues: comparison of growth inhibitory and insecticidal activity

The importance of the double bonds in the furan ring of avocadofurans with relation to their insecticidal activity was examined. The insecticidal activity of two naturally occurring avocadofurans, 2-(pentadecyl)furan and 2-(heptadecyl)furan, was compared to the toxicity of five tetrahydrofurans with alkyl chains at position 2 and varying side chains from 14 to 18 carbons. We found that eliminating the sites of unsaturation in the furan ring of avocadofurans significantly reduced the detrimental effects on the mortality and growth of the generalist insect herbivore Spodoptera exigua. In 7-day bioassays, S. exigua larvae were significantly more affected when fed a diet containing avocadofurans as compared to a larvae fed diet treated with the analogous tetrahydrofurans. Although larvae fed with the tetrahydrofurans showed reduced growth as compared to controls, larval mortality was not significantly increased. We conclude that the double bonds in the furan ring of avocadofurans play an important role in their insecticidal effects.     

Binding of PF2 lectin from Olneya tesota to gut proteins of Zabrotes subfasciatus larvae associated with the insecticidal mechanism

Zabrotes subfasciatus (Boheman) is the main pest of common beans ( Phaselous vulgaris ). Wild legume seeds from Olneya tesota contain a lectin, PF2, that shows insecticidal activities against this insect. The binding of PF2 to midgut glycoproteins of 20-day-old larvae was evaluated using PF2 affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the proteins retained on the gel revealed several putative glycoproteins, ranging in mass from 17 to 97 kDa. Subsequent protein digestion and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) provided amino acid fragments that identified an α-tubulin, cytochrome c oxidase subunit I, an odorant receptor, and a lysozyme from available insect sequence databases. The potential of these proteins to serve as part of the mechanisms involved in the insecticidal activity of PF2 to Z. subfasciatus is discussed.     

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 insecticidal activity of some novel fraxinellone-based esters

In continuation of a program aimed at the discovery and development of natural products-based insecticidal agents, two series of novel fraxinellone-based esters were synthesized by modification at the C-4 or C-10 position of fraxinellone and evaluated for their insecticidal activity against the pre-third-instar larvae of Mythimna separata in vivo. An efficient method for the stereoselective synthesis of 4α-hydroxyfraxinellone from fraxinellonone was developed, and the steric configuration of 6h was unambiguously confirmed by X-ray crystallography. Among 37 compounds, some derivatives displayed potent insecticidal activity; especially compounds 6h, 6q, 6t, and 7q showed more promising insecticidal activity than toosendanin, a commercial botanical insecticide derived from Melia azedarach . This suggested that introduction of the fluorine atom on the phenyl ring could lead to a more potent compound than one possessing chlorine or bromine. Meanwhile, introduction of the heterocyclic fragments at the C-4 or C-10 position of fraxinellone was essential for their insecticidal activity. This will pave the way for further design, structural modification, and development of fraxinellone as an insecticidal agent.     

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.     

Insecticidal and fumigant activities of Cinnamomum cassia bark-derived materials against Mechoris ursulus (Coleoptera: attelabidae)

The insecticidal and fumigant activities of Cinnamomum cassia (Blume) bark-derived materials against the oak nut weevil (Mechoris ursulus Roelofs) were examined using filter paper diffusion and fumigation methods and compared to those of the commercially available Cinnamomum bark-derived compounds (eugenol, salicylaldehyde, trans-cinnamic acid, and cinnamyl alcohol). The biologically active constituent of the Cinnamomum bark was characterized as trans-cinnamaldehyde by spectroscopic analysis. In a test with the filter paper diffusion method, trans-cinnamaldehyde showed 100 and 83.3% mortality at rates of 2.5 and 1.0 mg/filter paper, respectively. At 2.5 mg/paper, strong insecticidal activity was produced from eugenol (90.0% mortality) and salicylaldehyde (88. 9%), whereas trans-cinnamic acid revealed moderate activity (73.3%). At 5 mg/paper, weak insecticidal activity (50.0%) was produced from cinnamyl alcohol. In a fumigation test, the Cinnamomum bark-derived compounds were much more effective against M. ursulus larvae in closed cups than in open ones. These results indicate that the insecticidal activity of test compounds was attributable to fumigant action, although there is also significant contact toxicity. As a naturally occurring insect-control agent, the Cinnamomum bark-derived materials described could be useful as a new preventive agent against damage caused by M. ursulus.     

苏云金芽孢杆菌WB9菌株cry2Ac4基因的克隆及表达

WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)菌株,经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267,被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-,得到的工程菌能正常表达70 kD蛋白,形成方形晶体。生物测定结果表明,cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。     

Influence of edaphic factors on the mineralization of neem oil coated urea in four Indian soils

The utility of neem (Azadirachta indica A Juss) oil coated urea as a value-added nitrogenous fertilizer has been now widely accepted by Indian farmers and the fertilizer industry. In the present study, the expeller grade (EG) and hexane-extracted (HE) neem oils, the two most common commercial grades, were used to prepare neem oil coated urea (NOCU) of various oil doses, for which mineralization rates were assessed in four soils at three incubation temperatures (20, 27, and 35 degrees C). Neem oil dose-dependent conservation of ammonium N was observed in NOCU treatments in all of the soils. However, a longer incubation period and a higher soil temperature caused depletion of ammonium N. Overall, the nitrification in NOCU treatment averaged 56.6% against 77.3% for prilled urea in four soils. NOCU prepared from EG neem oil was consistently superior to that derived from hexane-extracted oil. The performance of NOCUs was best in coarse-textured soil and poorest in sodic soil. The nitrification rate (NR) of the NOCUs in the soils followed the order sodic > fine-textured > medium-textured > coarse-textured. The influence of edaphic factors on NR of NOCUs has been highlighted. The utility of the present study in predicting the performance of NOCU in diverse Indian soils was highlighted through the use of algorithms for computation of the optimum neem oil dose that would cause maximum inhibition of nitrification in any soil.     

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.     

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.     

Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis

The insecticidal activity of hexane extracts from the roots and leaves of Aristolochia malmeana was evaluated against Anticarsia gemmatalis larvae by topical application. Extract from the roots was the most active and caused 50% mortality in larvae at 308.4 microg/microL. From this extract, a clerodane diterpene, (-)-kolavenic acid, and three lignans, (-)-kusunokinin, (-)-hinokinin, and (8 S,8' R,9 S)-cubebin, were isolated by chromatography and partition procedures and then evaluated for their insecticidal activities either individually or in pairs. (-)-Kusunokinin showed higher activity against A. gemmatalis (LD10=9.3, LD50=230.1 microg/microL) than the crude extract, and its activity was dose-dependent, whereas the other constituents did not exhibit any significant activity. Together with (-)-kusunokinin and (-)-hinokinin, (-)-copalic acid, (-)-2-oxokolavenic acid, (-)- ent-6-beta-hydroxy-copalic acid, (8 R,8' R,9 R)- and (8 R,8' R,9 S)-cubebins, (-)-fargesin, and (-)-phillygenin were isolated from the hexane extract of the leaves. The compounds were identified on the basis of spectroscopic analysis.     

Expression of the Truncated vip3A Gene at the 3'-terminus from Bacillus thuringiensis in Escherichia coli

将苏云金芽孢杆菌(Bacillus thuringiensis)缺失C端154个氨基酸编码区的vip3A基因(vip3T)插入原核表达载体pQE30，构建了重组表达载体pQEvip3T，并转化大肠杆菌(Escherichia coli ) M15进行IPTG诱导表达，比较了完整的Vip3A蛋白和C端缺失的蛋白Vip3T的可溶性和杀虫活性。与Vip3A不同，融合蛋白Vip3T以不可溶的包含体形式存在，诱导表达的菌液中没有检测到可溶性Vip3T蛋白。生物测定结果表明，M15(pOTP)诱导表达的Vip3A蛋白对初孵斜纹夜蛾(Spodoptera litua)和甜菜夜蛾(S. exigua)幼虫具有较高的杀虫活性，其提纯的包含体无毒，但包含体的碱性裂解液却又恢复了对夜蛾科害虫的活性；M15(pQEvip3T)菌液、包含体及其碱性裂解液对这两种昆虫幼虫则完全无毒，说明在大肠杆菌中，Vip3A蛋白C端氨基酸可能对Vip3A蛋白的可溶性和杀虫活性具有重要的影响。     

Larvicidal and adulticidal activity of alkylphthalide derivatives from rhizome of Cnidium officinale against Drosophila melanogaster

The insecticidal activity of the chloroform extract of Cnidium officinale rhizomes and its constituents was investigated against larvae and adults of Drosophila melanogaster and compared with that of rotenone. Bioassay-guided isolation of the chloroform extract of C. officinale resulted in the isolation and characterization of four alkylphthalides, cnidilide (1), (Z)-ligustilide (2), (3S)-butylphthalide (3), and neocnidilide (4). The structures of these compounds were established by spectroscopic analysis. The isolated compounds 2, 3, and 4 exhibited LC50 values of 2.54, 4.99, and 9.90 micromol/mL of diet concentration against larvae of D. melanogaster, respectively. Against both sexes (males/females, 1:1) of adults (5-7 days old), compound 3 showed the most potent activity of the compounds isolated with the LD50 value of 5.93 microg/adult, comparable to that of rotenone (LD50 = 3.68 microg/adult). Structure-activity relationships of phthalides isolated suggest that the presence of conjugation with the carbonyl group in the lactone ring appeared to play an important role in the larvicidal activity. Acetylcholinesterase (prepared from the adult heads of D. melanogaster) inhibitory activity was also investigated in vitro to determine the insecticide mode of action for the acute adulticidal activity.     

Insecticidal fatty acids and triglycerides from Dirca palustris.

Five compounds, 1-5, were isolated from the seed hexane extract of Dirca palustris. Compounds 1-3 were triglycerides, and 4 and 5 were linoleic and oleic acids, respectively. Compounds 1-3 were not biologically active; however, 4 (linoleic acid) and 5 (oleic acid) were insecticidal against fourth instar Aedes aegyptii larvae and exhibited potent feeding deterrent activity against neonate larvae of Helicoverpa zea, Lymantria dispar, Orgyia leucostigma, and Malacosoma disstria.     

Larvicidal activity of lignans identified in Phryma leptostachya Var. asiatica roots against three mosquito species

The insecticidal activity of phytochemicals isolated from the roots of Phryma leptostachya var. asiatica against third instar larvae of Culex pipiens pallens, Aedes aegypti, and Ocheratatos togoi was examined. The two constituents of P. leptostachya var. asiatica roots were identified as the leptostachyol acetate (I) and 8'-acetoxy-2,2',6-trimethoxy-3,4,4',5'-dimethylenedioxyphenyl-7,7'-dioxabicyclo[3.3.0]octane (II) by spectroscopic analysis. Compound I was lethal to C. pipiens pallens, A. aegypti, and O. togoi at 10 ppm. Compound II showed weak or no insecticidal activity against three mosquito species at 10 ppm. The LC(50) values of I against C. pipiens pallens, A. aegypti, and O. togoi were 0.41, 2.1, and 2.3 ppm, respectively. Naturally occurring P. leptostachya var. asiatica root-derived compounds merit further study as potential mosquito larval control agents or lead compounds.     

Design, synthesis, and biological activities of arylmethylamine substituted chlorotriazine and methylthiotriazine compounds

Heterocyclic rings were introduced into the core structure of s-triazine to design and synthesize a series of novel triazines containing arylmethylamino moieties. These compounds were characterized by using spectroscopic methods and elemental analysis. Their herbicidal, insecticidal, fungicidal, and antitumor activities were evaluated. Most of these compounds exhibited good herbicidal activity, especially against the dicotyledonous weeds, and compound F8 was almost at the same level as the control compound atrazine. Their structure-activity relationships were discussed. At the same time, some triazines had interesting fungicidal and insecticidal activities, of which F4 exhibited 100% efficacy against Puccinia triticina even at 20 ppm, and F5 showed Lepidopteran-specific activity in both leaf-piece and artificial diet assays. Moreover, these compounds showed antitumor activities against leukemia HL-60 cell line and lung adenocarcinoma A-549 cell line.     

Protective effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids against oxidative stress-induced DNA damage of rat liver in vivo

The present study was undertaken to know the effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat liver in vivo. Male Wistar rats were fed a diet containing fish oil or safflower oil with high n-6 PUFA at 50 g/kg of diet and an equal amount of vitamin E at 59 mg/kg of diet for 6 weeks. Livers of rats fed fish oil were rich in n-3 PUFA, whereas those of rats fed safflower oil were rich in n-6 PUFA. Ferric nitrilotriacetate was intraperitoneally injected to induce oxidative stress. The degree of lipid peroxidation of the liver was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS), and the degree of oxidative DNA damage was assessed by comet type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-2'-deoxyguanosine levels. The levels of TBARS of the livers of the fish oil diet group increased to a greater extent than those of the safflower oil diet group, whereas the levels of the hydroperoxides of the livers of both diet groups increased to a similar extent. The vitamin E level of livers of the fish oil diet group was remarkably decreased. The degree of DNA damage of both diet groups was increased, but the increased level of the fish oil diet group was remarkably lower than that of the safflower oil diet group. The above results indicate that fish oil supplementation does not enhance but appears to protect against oxidative stress-induced DNA damage and suggest that lipid peroxidation does not enhance but lowers the DNA damage.     

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.