Research Article pubs.acs.org/journal/ascecg
Naphthoquinones from Walnut Husk Residues Show Strong Nematicidal Activities against the Root-knot Nematode Meloidogyne hispanica Carla Maleita,*,†,‡ Ivânia Esteves,‡ Rita Chim,† Luís Fonseca,‡ Mara E. M. Braga,† Isabel Abrantes,*,‡ and Hermínio C. de Sousa*,†
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CIEPQPF−Chemical Process Engineering and Forest Products Research Centre, Chemical Engineering Department, University of Coimbra, Rua Sílvio Lima, Pólo II, Pinhal de Marrocos, 3030-790 Coimbra, Portugal ‡ CFE−Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal ABSTRACT: Naphthoquinones exhibit important biological activities and are present in walnut husk residues in significant amounts. However, their potential as alternatives to synthetic nematicides has not been fully explored. This work aimed to assess the effects of pure naphthoquinones (juglone; 1,4-naphthoquinone; plumbagin) on the mortality of the root-knot nematode Meloidogyne hispanica second-stage juveniles (J2). Extracts from Juglans spp. walnut husks were characterized, and the effects of J. nigra extracts on attraction and life cycle of M. hispanica were evaluated. 1,4-Naphthoquinone was the most effective compound causing 42% J2 mortality at 50 ppm. The extract from in natura J. nigra walnut husks presented similar effects on J2 mortality to those observed for pure 1,4-naphthoquinone. The extract from dried husks was repellent and reduced nematode root penetration but did not affect reproduction. Therefore, walnut residues can be valorized as renewable sources of naphthoquinone-based products and potentially employed as bionematicides against Meloidogyne spp. KEYWORDS: 1,4-Naphthoquinone, Extracts, Juglans spp., Juglone, Naphthoquinone-based bionematicides, Plumbagin
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INTRODUCTION The worldwide crop losses caused by plant-parasitic nematodes (PPN) were estimated to be higher than US$ 80 billion/y, and root-knot nematodes (RKN, Meloidogyne spp.) are in the top 10 PPN responsible for the major ecological and economic impacts caused worldwide. 1 Meloidogyne hispanica is a polyphagous RKN for several economically relevant crops, such as bean, corn, potato, and tomato and a species of emerging importance. Since is suited to soil temperatures around 25 °C, with the predicted future climate changes, there is a risk of this species spreading all over Southern Europe and northwards.2,3 So far, it has been recorded worldwide, in countries such as Australia, Brazil, Burkina Faso, Cape Verde Islands, the Fiji Islands, France, Korea, Malawi, Martinique, Puerto Rico, Spain, South Africa, The Netherlands, and the United States (as mentioned in the work of Maleita et al.2) and is able to infect and reproduce in a wide range of crops.2 Once RKN are established in soil their eradication becomes difficult and current management strategies are mostly focused on reducing nematode population densities to limit damage to an economically acceptable level.4 In the past century, synthetic-origin nematicides were widely used to minimize crop losses caused by PPN. However, their adverse impact on the environment and human/animal health has urged the © 2017 American Chemical Society
development of safer and sustainable alternatives such as the use of natural-origin nematicides, obtained/derived from plant extracts.5 A large number of plant secondary metabolites have been extracted, identified, and demonstrated to present moderate-to-strong nematicidal activity.5−8 According to the Food and Agriculture Organization of the United Nations (FAO),9 the world production of shelled walnuts was estimated to be around 3 420 000 tonnes with China being the largest producer (1 700 000 tonnes). Europe accounted for 175 000 tonnes, and France was the largest European producer (37 000 tonnes). Walnut, Juglans spp., is a highly nutritious food, rich in bioactive natural products and is an important component of the Mediterranean diet.10 Walnut husks and leaves are abundant agro-industrial residues obtained after walnut farming and processing activities.11 Several bioactive substances, including phenolic compounds and naphthoquinones (NTQ), such as chlorogenic, p-coumaric, ellagic, ferulic, gallic, protocatechuic, sinapic, syringic and vanillic acids, (+)-catechin, myricetin, juglone (5hydroxy-1,4-NTQ), and 1,4-naphthoquinone (1,4-NTQ), have Received: January 5, 2017 Revised: February 27, 2017 Published: March 10, 2017 3390
DOI: 10.1021/acssuschemeng.7b00039 ACS Sustainable Chem. Eng. 2017, 5, 3390−3398
Research Article
ACS Sustainable Chemistry & Engineering already been identified in these residues.12,13 Moreover, moderate-to-strong nematistatic and/or nematicidal effects have also been reported for some of these compounds.6−8 NTQ, an important class of quinones and a group of highly reactive phenolic compounds, are widespread in nature as products of micro-organisms, fungal, and plant secondary metabolism.14 Among NTQ, 1,4-NTQ and two of its derivatives, juglone and plumbagin, are arousing great research and application interests due to their broad-range of potential biological activities. In plants, juglone is stored in vacuoles as hydrojuglone-β-D-glucopyranoside and can be enzymatically degraded by hydrojuglone-β-D-glucopyranoside-β-glucosidase. The release of juglone into the soil occurs by exudation from roots, leaching from leaves, and through decomposition of plant materials.15 The persistence of juglone in the soil varies from moderate to low, and it is particularly short-lived in soils supporting microbial activity.15,16 Juglone may accumulate in subsurface soils, due to its reduced microbial degradation and be available for uptake by deep-rooting plants.16 Plumbagin is usually obtained from Plumbago species roots,17 but it has also been reported, in smaller amounts than juglone and 1,4-NTQ, in J. nigra (black walnut), J. regia, and J. cinerea roots, bark, xylem, and leaves.18 The objectives of this work were (i) to determine the in vitro toxicity of pure juglone, 1,4-NTQ (alone or combined), and plumbagin, against M. hispanica infective second-stage juveniles (J2); (ii) to obtain, characterize, and evaluate the effects of J. nigra and J. regia walnut husks extracts on M. hispanica J2 mortality, attraction/repellence, penetration, and reproduction; and (iii) to evaluate the potential of these NTQ-based natural products/extracts toward the development of novel, safer, and environmentally friendly RKN management strategies.
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i.d., 5 mm). Employed chromatographic assays were performed according to Jakopič et al.12 with some modifications in elution profile [0−24 min, 5−20% (v/v) B; 24−25 min, 30% B; 25−65 min, 35% B; 35−75 min, 80% B; 75−80 min, 85% B; and an equilibration time of 10 min]. The diode array detector was set to 254 nm. Extract samples were filtered (0.2 μm) before injection. The concentrations of 1,4NTQ (purity ≥97% w/w), juglone (purity ≥95%, w/w), and of ellagic (HPLC grade, purity ≥95%, w/w), p-coumaric (HPLC grade, purity ≥98%, w/w), and syringic (purity ≥95%, w/w) acids were calculated based on previously obtained calibration curves. The standards from Sigma-Aldrich were used without purification. In vitro Mortality Bioassays Using Pure NTQ. Pure bioactive compounds, juglone, 1,4-NTQ, and plumbagin (purity ≥95% w/w) were solubilized in Triton X-100 (laboratory grade) aqueous solutions (5000 ppm) to obtain final NTQ concentrations of 500, 250, 175, 100, and 50 ppm. Water and Triton X-100 were used as controls. Solutions were stirred for 3 days, at 37 °C, away from light. Each treatment consisted of five replicates, and each mortality experiment was repeated three times. Twenty four hours, RKN J2 hatched from egg masses were collected, and 20 nematodes were placed on a glass-staining block containing 1 mL of each preprepared NTQ solution/water/Triton X100. Glass-staining blocks were maintained in a moist chamber, in the dark, at room temperature (20−22 °C), and nematode mortality was monitored at 3, 6, 12, 24, 48, and 72 h after exposure (HAE). NTQ solutions were not replaced as it was assumed that NTQ activity was preserved during the tested period. Nematodes not showing movements when touched with a bristle were transferred to water and considered dead if they still failed to react. Lower NTQ concentrations (100 and 50 ppm) were only tested when the mortality at 150 ppm reached at least 70%. Additional experiments were performed using mixtures of 1,4-NTQ and juglone (1:2 w/w), since these substances were found at this relative composition in J. regia walnut husks extracts.12 These mixtures were prepared in order to achieve final solution concentrations of 500, 250, and 150 ppm (1,4-NTQ + juglone), and the effects on M. hispanica mortality were studied. Bioassays Using J. nigra Extracts. J. nigra extracts were solubilized in Triton X-100 5000 ppm and stirred for 3 days at 37 °C in the dark. The amounts of extracts to be added to the test solutions were calculated taking into consideration the relative composition of the extracts in 1,4-NTQ (Table 2). The J. nigra extract from in natura walnut husks was only tested on M. hispanica mortality, due to the insufficient amount of the extract. The J. regia dried extract was not employed on any bioassay, due to their low contents of 1,4-NTQ and juglone (Table 2). Mortality Bioassays. These bioassays were carried out as previously described, for 1,4-NTQ concentrations of 175 and 50 ppm (235 and 67 ppm of juglone, respectively) for the in natura black walnut husks extract, and of 175, 100, and 50 ppm (166, 95, and 47 ppm of juglone, respectively) for the dried black walnut husks extract. Chemotaxis Assays. Plates (5 cm ⌀) were filled with 1% water-agar (5 mL/dish). Two wells (0.5 cm ⌀) made at opposite sides were filled with 50 μL of the dried black walnut husks extract solutions (175:166, 100:95 and 50:47 ppm, 1,4-NTQ:juglone) 4 h before the inoculation of 20 M. hispanica J2 at the center. Water, Triton X-100 5000 ppm, 1% v/v gacial acid acetic (repellent) and 50 μg/mL salicylic acid (purity ≥95%, w/w, attractant) solutions were used as controls.7 Each treatment was replicated three times and assayed twice. Plates were kept in the dark at room temperature (20−22 °C) and after 2 h, nematode positions were recorded using a counting template divided into 16 segments. The number of nematodes at the attractive and repellent zones of plates was registered. Results were presented as the number of nematodes on attractive zones divided by the number of nematodes on the repellent ones (chemotaxis factor, Cf). Extracts are classified as attractive (Cf > 2), repellent (Cf < 0.5), and neutral (0.5 ≤ Cf ≤ 2) (adapted from the work of Wuyts et al.7). Infectivity and Reproduction. Twenty-four tomato plants cv. Coraçaõ de Boi (two-weeks old) were transferred to pots (50 cm3) containing autoclaved sandy loam soil and sand (1:2, v/v). Eight
EXPERIMENTAL SECTION
Nematode Isolate. The M. hispanica isolate was maintained on tomato, Solanum lycopersicum, cv. Coraçaõ de Boi, in pots containing sterilized sandy loam soil and sand (1:1 v/v), at 25 ± 2 °C, and its identification was confirmed by esterase phenotype analysis.19 Juglans regia and J. nigra Extracts. Walnuts husks of J. nigra and J. regia, cv. Franquette, collected in two localities of Portugal, Alcobaça (September 2014) and Arraiolos (October 2014), respectively, were transported and surface-sterilized with a 1% (v/v) NaOCl (reagent grade, 10−15% chlorine) solution for 10 min, and rinsed 3 times with distilled water. J. nigra raw material was used in the in natura form (comminuted after surface sterilization/rinsing in a knife-mill, for 4 min) or dried, while the J. regia raw material was used only in a dried form. Due to the quick oxidation after harvest and hulling, it was not possible to obtain sufficient in natura form J. regia material for extraction. Drying procedures started after sterilization in an air-circulated oven, at ∼35 °C, and at atmospheric pressure, for 6 days. Dried materials were comminuted and sieved. Only the solid fractions, collected at the mesh 60 sieve (Tyler series), were selected. All raw materials were stored in sealed recipients and protected from light. A solid−liquid low pressure solvent extraction was used to obtain three walnut husks extracts: in natura and dried J. nigra and dried J. regia walnut husks. Extraction of the in natura J. nigra raw material was performed 1 day after the comminution and from dried raw materials 1−2 days after drying, the extractions being conducted at 70 °C, for 3 h, using a mixture of ethanol (p.a., purity ≥99.9% w/w) and ethyl acetate (p.a., purity >99.9%) (1:1 v/v) and a solid-to-solvent ratio of 1:50 (m/v). Extracts were vacuum-dried (BÜ CHI Rotavapor R-114) at 50 °C, stored at 4 °C, and kept away from light. Chromatographic analyses were carried out in an HPLC system (Shimadzu, UFLC, pump LC-20AD coupled to Diode array detector SPDM20A) and using a Eurospher column (100-C18 RP, 250 × 4 mm 3391
DOI: 10.1021/acssuschemeng.7b00039 ACS Sustainable Chem. Eng. 2017, 5, 3390−3398
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Corrected cumulative mortality (%) of Meloidogyne hispanica J2 exposed to concentrations of (a) juglone, (b) 1,4-NTQ, (c) 1,4NTQ:juglone (1:2 w/w), and (d) plumbagin: (I) 500; (II) 250; (III) 150; (IV) 100; and (V) 50 ppm. Water and a 5000 ppm Triton X-100 aqueous solution were used as controls. Data are average of five replicates, and bars represent standard errors. Presented average followed by the same lower case, at the same exposure time, do not differ significantly (p > 0.05) according to the Fisher LSD test or to the Kruskal−Wallis test.
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seedlings were inoculated with 150 M. hispanica J2 (initial nematode population density, Pi) which were exposed for 3 days to a solution of dried J. nigra extract (50:47 ppm, 1,4-NTQ:juglone). Eight seedlings were also inoculated with 150 M. hispanica J2 exposed to water or 5000 ppm Triton X-100 aqueous solution (controls). Three days after inoculation (DAI), four roots/treatment were removed, washed, stained with fuchsin acid20 (purity ≥70%, w/w), and the number of nematodes inside the roots recorded. The remaining four tomato plants/treatment were transferred into new pots (150 cm3). At 30 DAI, the plants were harvested, and the root systems were washed and rated for galls.21 Eggs were extracted using a 0.52% NaOCl solution,22 the final nematode population (Pf) was determined and the reproduction factor (Rf = Pf/Pi) was calculated. Statistical Data Analyses. Data on mortality was converted to percentage cumulative mortality and corrected by Schneider Orelli’s formula (100 × [(% mortality in treatment − % mortality in control)/ (100 − % mortality in control)]),23 considering the Triton X-100 solution as the control. The data was checked for normality using the Kolmogorov−Smirnov test and for variance homogeneity using the Hartley, Cochran, and Bartlett’s tests. For each tested pure compound and for each J. nigra extract, the effects of the concentrations on nematode mortality were compared by ANOVA, followed by a posthoc Fisher LSD statistical test. When the assumptions of ANOVA were not fulfilled, even after data transformation (logarithmic and square-root), the nonparametric Kruskal−Wallis test was applied. Data for the effects of J. nigra extract on the nematode penetration and reproduction were also analyzed. Statistical analysis was performed using StatsoftStatistica 7.0 for Windows. Data on mortality, derived from the 24 and 72 h observations, were subjected to Probit analysis,24 using PriProbit1.63 software. The lethal concentrations causing 50% mortality were also calculated.
RESULTS In Vitro Mortality Bioassays Using Pure NTQ. The M. hispanica J2 mortality in the Triton X-100 solution control (cumulative mortality = 0−1%) was not significantly different from that observed in the water control (cumulative mortality = 0−3%). However, the J2 mortality was significantly affected by the presence of the three pure NTQ at all concentrations (p < 0.05, Figure 1). No significant differences (p > 0.05) in nematode mortality were found for 500 and 250 ppm solutions for all treatments. At these concentrations, all pure NTQ were equally effective, presenting >90% mortality at 72 HAE (Figure 1). However, juglone induced 100% mortality within 12−24 h at 500 and 250 ppm, respectively; 1,4-NTQ within 6 h at 500 ppm and 12 h at 250 ppm; and 1,4-NTQ:juglone and plumbagin induced 100% mortality within 48 h of exposure, at 500 ppm (Figure 1). Significant differences in M. hispanica mortality were found for juglone and 1,4-NTQ at 150, 100, and 50 ppm (p < 0.05). Plumbagin was the least effective at 150 ppm as it induced mortality in only 23% of M. hispanica J2. The combination of 1,4-NTQ and juglone led to similar results at 150 ppm. Therefore, 1,4-NTQ emerged as the most effective tested pure NTQ, inducing mortality in 42% M. hispanica J2, at 50 ppm and 72 HAE, with LC50 values of 119.34 (24 HAE) and 63.42 ppm (72 HAE) (Table 1). During the assays, nematodes remained immotile after the first hours of exposure and only recovered their movements when touched with a bristle or transferred into water. Dead nematodes showed a straight or a slightly bent shape and their bodies were strongly vacuolated. In juglone, it 3392
DOI: 10.1021/acssuschemeng.7b00039 ACS Sustainable Chem. Eng. 2017, 5, 3390−3398
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ACS Sustainable Chemistry & Engineering Table 1. Estimated Values of Lethal Concentration (ppm) Necessary to Result in 50% Meloidogyne hispanica J2 Mortality (LC50), at 24 and 72 h after Exposure to Juglone, to 1,4-NTQ, to 1,4-NTQ:Juglone (1:2 w/w), and to Plumbagin lethal concentration (LC50, ppm) hours after exposure
juglone
1,4NTQ
1,4-NTQ:juglone (1:2 w/w)
plumbagin
24 72
137.42 113.27
119.34 63.42
124.70 115.40
249.46 177.70
was observed a change in the color to yellowish of the dead nematodes body contents, possibly due to the uptake of this substance (data not shown). No changes in the mobility and body shape were registered in J2 exposed to Triton X-100. Extraction Yields and Characterization of J. regia and J. nigra Extracts. Extraction yields were quite similar for both J. nigra extracts but were lower than the extraction yield obtained with dried J. regia walnut husks (Table 2). Employed HPLC analyses of the extracts from in natura or dried J. nigra and dried J. regia walnut husks identified and quantified 3−5 substances: juglone, 1,4-NTQ, and ellagic, p-coumaric, and syringic acids (Figure 2; Table 2). In the extracts from in natura J. nigra walnut husks, juglone and 1,4-NTQ were found at higher concentrations. These NTQ amounts were markedly smaller in the extracts from the dried J. nigra and even smaller for the dried J. regia extract (Table 2). Bioassays Using J. nigra Extracts. Mortality Bioassays. The extract from in natura J. nigra walnut husks (175 ppm of 1,4-NTQ) and the pure 1,4-NTQ solution at the same concentration were equally effective, both inducing ≈89% J2 mortality within 72 h (Figures 1 and 3). At 50 ppm, this extract was less effective than pure 1,4-NTQ as it only induced ≈20% J2 mortality while the pure 1,4-NTQ induced around 42% mortality (Figures 1 and 3). After the first day of exposure to in natura extracts, lack of movement was noticed in most nematodes and only recovered their movement when touched with a bristle or transferred into water (data not shown). The extract from dried J. nigra walnut husks, at 175, 100, and 50 ppm of 1,4-NTQ in the test solutions, was less effective on M. hispanica mortality if compared to the pure compound (Figures 1 and 3). Chemotaxis Assays. The acetic (1% v/v) and salicylic acids (50 μg/mL) solutions used as repellent and attractant controls, respectively, were not completely effective for these purposes. The acetic acid was found to be from neutral to repellent (Cf = 0.44 ± 0.34) and the salicylic acid as neutral to attractant (Cf = 2.11 ± 1.76). Nevertheless, the chemotactic behavior of M. hispanica J2, when using the extract from dried walnut husks of J. nigra clearly showed that this extract was repellent at all the
Figure 2. HPLC chromatograms of the extracts from in natura or dried Juglans nigra and J. regia, cv. Franquette, walnut husks. Extracts were obtained by solid−liquid ethanol:ethyl acetate (1:1 v/v) solvent extraction.
concentrations (175, 100, and 50 ppm): Cf = 0.07 ± 0.07, 0.17 ± 0.20, and 0.13 ± 0.02, respectively. Infectivity and Reproduction. M. hispanica J2 were found inside the tomato roots three DAI in all treatments but their relative numbers were significantly different (Figure 4a). A negative impact on M. hispanica J2 penetration was detected after the nematode exposure to the dried J. nigra extract solution (1,4-NTQ 50 ppm). The J2 numbers found inside tomato roots decreased approximately in 50% when compared to the controls (p > 0.05, Figure 4a). At 30 DAI, M. hispanica reproduction was also influenced by the exposure to the extract (Figure 4b). The number of galls (47 to 88 galls/root system) found in tomato roots was not significantly different between all treatments (data not shown), but a significant reduction of the Rf values was observed when J2 were exposed to the extract, and when compared to the treatment with 5000 ppm Triton X100 solution (p > 0.05, Figure 4b). No significant differences were found between the controls (p < 0.05, Figure 4b).
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DISCUSSION Only a few studies have been previously conducted to evaluate the in vitro toxicity of NTQ on RKN.6,25 In work conducted by
Table 2. Extraction Yields (% g/g) and Contents (mg/g) of Some Phenolic Compounds Identified at the Extracts from in Natura or Dried Juglans nigra and from J. regia, cv. Franquette, Walnut Husksa identified/quantified substances (mg/g)
a
raw material
extraction yield (% g/g)
J. nigra (in natura) J. nigra(dried) J. regia (dried)
2.0 ± 0.1 1.9 ± 0.2 9.5 ± 1.0
ellagic acid
juglone
1,4-NTQ
p-coumaric acid
syringic acid
― 0.3 ± 0.1
49.4 ± 0.3 3.7 ± 0.1 0.3 ± 0.1
36.8 ± 0.3 3.9 ± 0.1 0.7 ± 0.3
3.5 ± 0.1 0.5 ± 0.1 0.1 ± 0.1
― 5.7 ± 0.2
Extracts were obtained by solid−liquid ethanol:ethyl acetate (1:1 v/v) solvent extraction. 3393
DOI: 10.1021/acssuschemeng.7b00039 ACS Sustainable Chem. Eng. 2017, 5, 3390−3398
Research Article
ACS Sustainable Chemistry & Engineering
Figure 3. Corrected cumulative mortality (%) of Meloidogyne hispanica J2 exposed to solutions of extracts from (a) in natura and from (b) dried J. nigra walnut husks, at 175 (I), 100 (II), and 50 ppm (II) of 1,4-naphthoquinone in the test solutions. Water and a 5000 ppm Triton X-100 aqueous solution were used as controls. Data are averages of five replicates, and bars represent the corresponding standard errors. Presented averages followed by the same lower case, at the same exposure time, do not differ significantly (p > 0.05) according to the Fisher LSD test or to the Kruskal−Wallis test. Please note that the corrected cumulative mortality axes are different in parts a and b.
Figure 4. (a) Number of Meloidogyne hispanica J2 found inside tomato cv. Coraçaõ de Boi roots and (b) reproduction factor, three and 30 days after inoculation, respectively. Before inoculation, J2 were soaked for three days in water, in a 5000 ppm Triton X-100 solution, and in a solution containing the extract from dried J. nigra walnut husks at 50 ppm of 1,4-NTQ in the test solution. Each bar represents the average ± standard deviation of four replicates and bars denoted by different letters differ significantly at p > 0.05 (according to the Fisher’s LSD test).
Dama25 to assess the efficacy of plumbagin, juglone, and lawsone on M. javanica mortality, plumbagin promoted 100% of J2 mortality, juglone 97.94%, and lawsone 58.31%, at 20 ppm, 24 HAE. In a different study, a concentration of 1100 ppm juglone was efficient against M. javanica J2, 48 HAE.6 Our results demonstrated that juglone, 1,4-NTQ, plumbagin, and 1,4-NTQ:juglone strongly affected M. hispanica mobility and mortality, which supports the in vitro nematostatic and nematicidal properties of these compounds. In general, the pure 1,4-NTQ was found to be more active than juglone and, in contrast to Dama,25 plumbagin was less effective. Despite their nematostatic and nematicidal activities, 1,4NTQ exhibit a broad range of toxic effects and present relevant biological activities (antibacterial, antifungal, antiviral, insecticidal, anti-inflammatory, antiproliferative, antipyretic, and herbicidal).7,14,25−28 However, the biological mechanisms of the 1,4-NTQ effects and the structure−function/activity relationship are still not fully understood. 1,4-NTQ have a general biological tendency to be reduced to semiquinones and then to hydroquinones, which are highly reactive and unstable species. Semiquinones and hydroquinones can also be auto/ reoxidized donating an electron, leading to the formation of the original NTQ and of reactive oxygen species (ROS).29 ROS are strong oxidizing agents that are usually associated with several toxic and undesired biological effects (cell membranes damage, alkylation of thiol or amine groups of proteins and other
biomolecules, inhibition of mRNA synthesis and of electron transport, DNA breakage and oxidation, cell cycle disruption, and mitochondrial membrane depolarization leading to programmed cell death).29 Thus, the nematicidal activity of NTQ results, probably, from their instability in aqueous solutions and consequent formation of semiquinones and hydroquinones with release of ROS. The properties of 1,4-NTQ and of their derived intermediates will be strongly influenced by their relative stabilities/reactivities in aqueous media, which in turn are a result of their specific chemical structures, namely the presence/absence and position of their substituent groups.18,30−34 While plumbagin is characterized by the presence of R5-OH and R2-CH3 groups, juglone lacks the R2-CH3 group and 1,4-NTQ lacks both groups. These differences are sufficient to lead to distinct single/two-electron reduction potentials in aqueous media.18,30,35 Thus, the biological activities that can be promoted by redox cycling/ oxidative stress are likely to follow the general series juglone >1,4-NTQ > plumbagin. This trend was already found for other biological systems such as epidermal human keratinocytes, protozoa and tobacco BY-2 cells.18,36,37 In this study, the results obtained with M. hispanica followed the sequence: 1,4-NTQ ≥ juglone > plumbagin. At lower concentrations (
