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.     

Citrus limonoids: analysis, bioactivity, and biomedical prospects

Limonoids are a prominent group of secondary metabolites in citrus fruit. The bitter character of some compounds in this group has historically compromised the quality of citrus fruit and juice. Detecting bitter limonoids in citrus, understanding their origins, and developing methods for their removal from citrus juices have provided the basis for citrus limonoid research. Evaluation of the biological activity of citrus limonoids has indicated the potential of these compounds to improve human health as anticancer, cholesterol-lowering, and antiviral agents. This review chronicles the evolution of citrus limonoid research from defining their participation in citrus bitterness to their potential utilization as important contributors to improving human health and well-being.     

Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization and electrospray ionization liquid chromatography-mass spectrometry

Improved analytical techniques for bitter limonoids in citrus and citrus juices can expedite the evaluation of freeze-induced citrus damage for citrus growers and juice quality for citrus juice producers. Microbore normal-phase and reverse-phase chromatography coupled to a mass spectrometer operating in a positive ion atmospheric pressure chemical ionization and electrospray ionization modes were found to be rapid, selective, and sensitive methods for the analysis of the bitter limonoids limonin and nomilin in citrus juices. Analysis was performed on a chloroform extract of citrus juice to which an internal standard was added. The methods are capable of detecting citrus limonoids in citrus juice in the 60-200 picogram range and quantifying citrus juice limonoids in concentrations as low as 120 picograms. An accurate "total limonoid bitterness" in citrus juice, as represented by the combined occurrence of limonin and nomilin, is easily determined by these methods.     

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.     

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.     

Calorimetric evidence of differentiated transport of limonin and nomilin through biomembranes

The effect exerted by two structurally similar limonoids possessing antifeedant and anticancer activity, limonin and nomilin, on the thermotropic behavior of model membranes constituted by dimyristoylphosphatidylcholine (DMPC) vesicles was studied by differential scanning calorimetry. Attention was directed to evaluate modifications in phytochemical-lipid interaction induced by compound structure and lipophilicity and to evidence their different membrane penetration. The two examined compounds, when dispersed in liposomes during their preparation, were found to exert a very different action on the L(beta)-L(alpha) gel-to-liquid crystal phase transition of DMPC multilamellar vesicles. Nomilin caused a detectable effect on the transition temperature (T(m)), shifting it toward lower values with a concomitant small decrease of the associated enthalpy (DeltaH) changes, while limonin was not able to modify the lipid vesicles thermotropic behavior. Modifications induced by nomilin were a function of phytochemical concentration, while the different behavior of limonin can be due to the different polarity induced by the presence of the single A ring in nomilin that possesses an acetyl group versus the A,A' ring system of limonin. Solid limonoids and aqueous dispersions of multilamellar (MLVs) or unilamellar vesicles (LUVs) (limonoids molar fraction 0. 045, 0.12, and 0.18) were left in touch for long incubation times at temperatures higher than T(m) to detect their spontaneous transfer through the medium. By following this procedure, no interaction was detected for limonin with lipid vesicles. The rate of transfer and interaction of nomilin was a function of the kind of vesicle species (faster for LUV, slower for MLV). The interaction, monitored by compound transfer from the solid phytochemical to the lipidic species after several periods of incubation, was on the same order as that detected by preparation carried out in organic solvent. The obtained results can be explained in terms of compound hydrophobicity, and a relation between compound structure and membrane interaction can be suggested. This allows the membrane interaction with nomilin, but the low water solubility of limonin hinders or totally blocks its transfer through the aqueous medium.     

Antioxidant activity of citrus limonoids, flavonoids, and coumarins

A variety of in vitro models such as beta-carotene-linoleic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH), superoxide, and hamster low-density lipoprotein (LDL) were used to measure the antioxidant activity of 11 citrus bioactive compounds. The compounds tested included two limonoids, limonin (Lim) and limonin 17-beta-D-glucopyranoside (LG); eight flavonoids, apigenin (Api), scutellarein (Scu), kaempferol (Kae), rutin trihydrate (Rut), neohesperidin (Neh), neoeriocitrin (Nee), naringenin (Ngn), and naringin(Ng); and a coumarin (bergapten). The above compounds were tested at concentration of 10 microM in all four methods. It was found that Lim, LG, and Ber inhibited <7%, whereas Scu, Kae, and Rut inhibited 51.3%, 47.0%, and 44.4%, respectively, using the beta-carotene-linoleate model system. Lim, LG, Rut, Scu, Nee, and Kae showed 0.5% 0.25%, 32.2%, 18.3%, 17.2%, and 12.2%, respectively, free radical scavenging activity using the DPPH method. In the superoxide model, Lim, LG, and Ber inhibited the production of superoxide radicals by 2.5-10%, while the flavonoids such as Rut, Scu, Nee, and Neh inhibited superoxide formation by 64.1%, 52.1%, 48.3%, and 37.7%, respectively. However, LG did not inhibit LDL oxidation in the hamster LDL model. But, Lim and Ber offered some protection against LDL oxidation, increasing lag time to 345 min (3-fold) and 160 min (33% increase), respectively, while both Rut and Nee increased lag time to 2800 min (23-fold). Scu and Kae increased lag time to 2140 min (18-fold) and 1879 min (15.7-fold), respectively. In general, it seems that flavonoids, which contain a chromanol ring system, had stronger antioxidant activity as compared to limonoids and bergapten, which lack the hydroxy groups. The present study confirmed that several structural features were linked to the strong antioxidant activity of flavonoids. This is the first report on the antioxidant activity of limonin, limonin glucoside, and neoeriocitrin.     

Antifeedant effects of the limonoids from Entandrophragma candolei (Meliaceae) on the gram pod borer, Helicoverpa armigera (Lepidoptera: Noctuidae)

The biological activity of the limonoids prieurianin and epoxyprieurianin isolated from Entandrophragma candolei (Harms) (Meliaceae) and their respective acetates was assessed using the gram pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). The compounds exhibited strong antifeedant activity in a diet choice bioassay with epoxyprieurianin acetate being most effective with 48.3 ppm deterring feeding by 50% (DI50) and prieurianin the least effective (DI50 = 91.4 ppm). The effect on growth of larvae was concomitant with the reduced feeding by neonate and third instar larvae. In nutritional assays, all the compounds reduced growth and consumption when fed to larvae without any effect on efficiency of conversion of ingested food (ECI), suggesting antifeedant activity alone. No toxicity was observed nor was there any significant affect on nutritional indices following topical application, further suggesting that prieurianin-type limonoids act specifically as feeding deterrents.     

水土保持措施降低河网平原区果园地表氮磷铜流失

河网平原因地下水位高、内排水较差,进行水果生产需要深沟排水以便适合果树根系的生长,但这些直接与水体连接的排水沟也大大促进了果园氮、磷等养分的流失。为了解不同水土保持措施对水网平原果园地表养分流失的影响,本文采用天然降雨条件下的小区试验研究了生物措施(种植黑麦草)、化学措施(表施沸石+石灰石粉)和工程措施(在排水沟内设置沉砂坑)对水网平原果园地表径流氮、磷、铜流失的影响。结果表明,生物措施能减少18.6%的径流水量流失;生物措施、化学措施和工程措施能分别减少52.6%、16.3%和38.3%的泥沙流失。地表泥沙携带侵蚀是果园氮、磷、铜流失的主要途径,生物措施能使总氮流失减少23.6%;生物措施、化学措施和工程措施能使总磷流失分别减少38.66%、18.53%和22.68%,总铜流失分别减少41.4%、18.4%和29.9%。三种水土保持措施对降低磷、铜流失的效果好于降低氮流失的效果。同时采用生物措施、化学措施和工程措施可使氮、磷、铜流失量明显降低,有助于果园周围水体环境的保护。     

Analysis of limonoid glucosides from citrus by electrospray ionization liquid chromatography-mass spectrometry

An electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) method for the detection and quantitation of limonoid glucosides has been developed. Negative ions [M - H(+)](-) characteristic of six limonoid glucosides can be detected and quantified from selected ion monitoring chromatograms using carminic acid as an internal standard. The described method has been applied to the analysis of limonoid glucoside content in various liquid and solid Citrus spp. samples as well as complex mixtures of partially purified limonoid glucosides. Rapid and sensitive qualitative screening of samples for limonoid glucosides can also be accomplished with slight modifications of the method.     

Insect antifeedants from tropical plants II: structure of zumsin

A novel A-seco limonoid was isolated from methanolic extract of Croton jatrophoides and designated as zumsin. This compound showed potent antifeedant activity against two lepidopteran larvae, pink bollworm, Pectinophora gossypiella (PC(50) = 1 microg/cm(2), PC(95) = 8 microg/cm(2)), and fall armyworm, Spodoptera frugiperda (PC(50) = 2 microg/cm(2), PC(95) = 16 microg/cm(2)). The structure of zumsin was determined as 1 using a variety of spectroscopic methods including nuclear magnetic resonance, mass spectrometry, and circular dichroism. The structure consists of an A'-B trans-fused ring while dumsin (2), a constituent of the same source, maintains an A'-B cis-fused ring, and suggests two unique biosynthetic processes after A ring oxidative expansion.     

稻秸还田对油菜季径流氮磷及COD流失的影响

通过田间小区试验,设置CK(无施肥+无秸秆)、F(常规施肥)、SF(秸秆还田+常规施肥)和SFR(秸秆还田+常规施肥减15%)4个处理,研究在巢湖地区稻油轮作模式下,稻秸还田对油菜季农田径流氮磷及化学需氧量(COD)流失影响。结果表明,秸秆还田处理显著增加土壤微生物细菌、真菌和放线菌数量,显著降低径流总氮浓度,但增加总磷和COD含量,土壤微生物数量与径流氮磷及COD含量呈显著正相关关系。与F处理相比,SF、SFR处理总氮流失量分别减少16.9%~19.8%,27.1%~29.3%,总磷流失量分别降低2.4%~4.0%,4.0%~5.6%,COD流失量分别增加6.1%~10.0%,2.8%~6.1%。研究结果为揭示土壤微生物数量与径流氮磷及COD流失的作用机理提供了参考,为合理利用秸秆还田技术提供了理论依据。     

高温酸性木聚糖酶的储存稳定性研究

随着生物技术和酶工程技术在工业上的普遍应用,木聚糖酶应用越来越广泛。在应用过程中,由于稳定性低而导致应用效果大幅下降,从而增加木聚糖酶的应用成本。本文通过研究木聚糖酶ACPC的酶学性质,确定其最佳反应温度和最佳反应pH值分别为75℃、5.5,属于高温酸性木聚糖酶。通过单因素实验研究,该酶在pH 5.5条件下储存稳定性最好,NaCl、甘油、蔗糖和EDTA对其储存稳定性有很大的提高。通过正交实验,获得一个液体酶稳定剂：30%甘油、15%NaCl、10%蔗糖和1%EDTA,木聚糖酶在该稳定剂的保护下,25℃储存木聚糖酶的T0.9（表示酶的残余活力保持90%以上的时间）〉90d,稳定剂将T0.9延长了75d以上,50℃高温储存15 d残余酶活还保留约80%以上的活力。     

Use of glutaraldehyde and benzalkonium chloride for minimizing post-harvest physio-chemical and microbial changes responsible for sucrose losses in sugar cane

Sugar cane is sensitive to enormous sucrose losses induced by physio-chemical and microbial changes, the severity being increased during the time lag between harvest and crushing in the mills. Minimization of the sucrose losses in the field is essential for better sugar recovery and prevention of sucrose losses. An experiment was conducted to evaluate the efficacy of glutaraldehyde and benzalkonium chloride for their effects on the microbial counts and physio-chemical changes responsible for sucrose losses. Glutaraldehyde and benzalkonium chloride (1000 + 250 ppm) reduced the losses in sucrose content to 7.1% as compared to the 30.8% loss in the control, thus improving the performance by 76.9%. The application of chemicals reduced the acid invertase activity (by 60%), lowered weight loss, titrable acidity, reducing sugars content, dextran, ethanol, and ethylene production and respiration rates. The application led to the reduction in the total bacterial, fungal, Leuconostoc, and yeast counts by 67.92, 51.3%, 26.08, and 51.2%, respectively.     

添加木质素和生物炭对土壤氮、磷养分及水分损失的影响

研究添加木质素和生物炭对土壤氮、磷养分及水分损失的影响,降低土壤改良剂使用成本,增加造纸黑液中木质素的利用途径,促进木质素在土壤改良中的应用。试验选取生物炭改良剂与木质素作为对比,通过土柱淋溶和静态吸收法,研究不同添加量(质量分数为0,1%,2%,4%)的木质素和生物炭对土壤氮磷养分、水分、脲酶活性以及pH的影响。土壤中添加木质素和生物炭均能减缓pH变化程度;抑制土壤脲酶的活性,且在添加氮肥后的1～20天抑制效果明显,其抑制效果与添加量呈正相关。添加量为1%,2%,4%的木质素和生物炭与对照组相比,铵态氮挥发量分别显著减少8.29%,14.29%,14.86%和3.79%,11.65%,15.26%;全氮淋溶量分别显著减少32.37%,37.70%,42.49%和25.43%,30.70%,39.54%;全磷淋溶量分别显著减少23.68%,40.48%,48.12%和6.97%,22.88%,35.30%;水分淋溶量分别显著减少7.71%,15.82%,9.29%和9.91%,15.00%,16.06%。在本试验中,木质素、生物炭质量分数分别为2%和4%时对降低土壤氮、磷养分和水分损失的效果最佳。因此,可以说明木质素和生物炭在水肥保持上的效果相近,在一定程度上可以替代生物炭改良剂在土壤中的应用。     

Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth,reproduction and activity of the earthworm Lumbricus terrestris (L.)

A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control (Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar (S. tuberosum L. cv. Désirée). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity.     

Short-term and long-term effects of human trampling on above-ground vegetation, soil density, soil organic matter and soil microbial processes in suburban beech forests

Understanding the effects of disturbance by human trampling on ecosystem processes is essential for the management of recreational areas. Discussions on recreational impacts are based either on data from trampling experiments or on field survey data from sites subjected to long-term recreational use, but rarely on a combination of both. We examined whether results from a short-term trampling experiment reflect the impact of long-term trampling around frequently used fire places. We compared short- and long-term effects of human trampling on above-ground forest vegetation and soil physical, chemical and microbial characteristics. We found both similarities and differences in short- and long-term trampling effects. Both short- and long-term trampling reduced plant cover, plant height and species density, though long-term effects were more pronounced than short-term effects. In both approaches, leaf litter biomass decreased, whereas soil density increased with trampling intensity. Other soil characteristics including soil moisture, total soil organic matter content and total organic nitrogen content were not or only marginally affected by short- and long-term trampling. Furthermore, soil microbial biomass and the activity of dehydrogenase did not change in both approaches. In contrast, the activity of β-glucosidase was only reduced by short-term trampling, whereas activity of phosphomonoesterase was reduced only by long-term trampling. Soil compaction was one factor reducing microbial activities at low and medium trampling intensities in our experiment and in the highly compacted area around the fire rings. We conclude that it could be problematic to use the results of short-term trampling experiments to predict general long-term trampling effects. Our results imply also that the restoration of degraded sites might be hampered by the low nutrient turnover resulting from the reduced litter layer and changes in enzyme activities, mitigating a successful re-establishment and growth of plants.     

持续秸秆还田减施化肥对水稻产量和氮磷流失的影响

[目的] 探究持续性秸秆还田减施化肥对水稻产量和氮磷随径流流失的影响,为当地农业资源循环再利用和防控农业面源污染提供科学依据。[方法] 在四川省广汉市开展连续3 a (2018—2020年)的田间小区试验,设置常规施肥处理(T₁)和秸秆还田+常规施肥减氮28.57%,减磷25.11%(T₂)2种施肥方式,分别测定了地表径流中氮磷浓度、流失量,水稻秸秆、籽粒的产量和氮磷吸收量、水稻收获时土壤养分。[结果] 随着秸秆还田年限的增加,T₂可达到显著的增产效果,其中2020年T₂比T₁增产16.93%。与T₁相比,T₂的总氮和硝态氮流失量分别增加6.25%～14.97%,6.99%～15.03%,可溶性总氮、总磷和可溶性总磷流失量分别降低0.94%～6.03%,4.66%～10.32%和5.77%～21.15%。土壤中全磷、速效磷、硝态氮和铵态氮含量的年际变化显著(p<0.05)。与T₁相比,T₂处理显著降低了土壤8.79%的全磷和30.56%的速效磷。[结论] 持续秸秆还田与减施化肥在保证作物产量的同时,减少了化肥投入量,降低了磷素的径流流失量,但增加了氮素径流流失的风险,在实际农业生产中应进一步优化处理。     

Chemical characterization of Citrus sinensis grafted on C. limonia and the effect of some isolated compounds on the growth of Xylella fastidiosa

Citrus sinensis grafted on C. limonia produces a considerable number of compounds that are not common in both plants developed from germination of seeds. The chemical profile of scion and rootstock differ notably for absence in the form of flavonoids and coumarins containing C5 prenyl groups attached to the carbon atoms of aromatic and heterocyclic systems or to oxygen. Only linear pyranocoumarins xanthyletin and xanthoxyletin were found in scion. This observation indicates that the prenylated compounds once biosynthesized in the roots could have been translocated to other organs. Xylella fastidiosa colonizes the xylem of plants causing diseases on several economically important crops such as citrus variegated chlorosis (CVC). A number of flavonoids, coumarins, alkaloids, dihydrocinnamic acid derivative, anacardic acid, triterpenes, and limonoids were tested for in vitro activity on the growth of Xylella fastidiosa. Azadirachtin A was the most active. Hesperidin, which occurs in great amounts in cells of the mesophyll of the affected leaves with CVC, showed a moderate activity suggesting that it can act as a good barrier for small-size colonies from X. fastidiosa.