Nematicidal activity of o-hydroxybenzaldehyde from common buckwheat methanol extract on Meloidogyne incognita.

The nematicidal activity of buckwheat (Fagopyrum esculentum Moench) on the root-knot nematode Meloidogyne incognita was tested. Dried plant methanol extract presented higher nematicidal activity than fresh plant extracts with an EC50 = 62.6 ± 26.0 and 40.8 ± 26.1 µg/ml after 48 and 72 hours of immersion, respectively. GC-MS analysis showed the presence of 17 aldehydes, with salicylaldehyde (o-hydroxybenzaldehyde) being the most abundant at 16%. Nematicidal activity of the latter refers to salicylaldehyde and other aldehydes with chemical similarities was then assessed. The most active aldehyde was o-hydroxybenzaldehyde followed by m-hydroxybenzaldehyde, p-hydroxybenzaldehyde and benzeneacetaldehyde with an EC50 of about 11.0 ± 1.0, 31.0 ± 22.0, 75.0 ± 23.0 and 168.1 ± 52.3 µg/ml after 1 day of immersion, respectively. Position 2 of the hydroxyl group in the benzene ring seems to be very important for the nematicidal activity, followed by positions 3 and 4. As a complementary experiment, synergistic activity was observed when we added o-hydroxybenzaldehyde to m-hydroxybenzaldehyde and to p-hydroxybenzaldehyde with an EC50 after 24 hours of immersion of 8.0 ± 2.5 and 6.1 ± 2.3 µg/ml, respectively. Antioxidant activity assessment showed that this latter is inversely proportional to nematicidal activity. Our results showed that F. esculentum and its major compound salicylaldehyde could be integrated into the pest management system.

Phytochemical profile and nematicidal activity of a hydroalcoholic extract from Cazahuate flowers (Ipomoea pauciflora M. Martens & Galeotti) against Haemonchus contortus infective larvae.

Haemonchus contortus (Hc) is a hematophagous parasite affecting the health and productivity of flocks. The administration of chemical anthelmintic drugs (AH) is the common method of deworming; however, generates resistance in the parasites to AH and it is a public health risk due to drug residues in milk, meat and sub-products. Natural compounds from plants are explored to diminish this parasitosis, improving their health and productivity, without the negative effects of AH. Ipomoea genus is a group of climbing plants belonging to the Convulvulaceae family possessing perennial leaves and tuberous roots. Medicinal properties has been attributed to this plant including nutritional agents, emetics, diuretics, diaphoretics, purgatives and pesticides. The objective of this study was assessing the in vitro nematocidal activity of a hydroalcoholic extract (HA-E) obtained from Ipomoea pauciflora (Cazahuate) flowers against Hc infective larvae (L3) and to identify its phytochemical profile (PhC-P). The assay was carried out using microtiter plates (MTP). Four HA-E concentrations were assessed and Ivermectin and distilled water were used as positive and negative control groups, respectively. Approximately 100 Hc L3 were deposited in each well (n=12) and incubated at 25-35°C for 7 days. Data were analyzed using ANOVA and a General Linear Model (GLM) followed by Tukey test (P<0.05). The treatments showing a concentration-dependent effect (CDE) were analyzed to identify their 50% and 90% lethal concentrations (CL50, 90) via a Probit Analysis. The highest mortality was observed at 50 mg/mL (82.64 ± 0.71%) and the lowest at 6.25 mg/mL (56.46 ± 2.49%), showing a CDE with increasing mortality from 6.25 to 50 mg/mL. The PhC-P revealed the presence of alkaloids, coumarins, flavonoids, tannins and triterpenes/ sterols. A HA-E from flowers of I. pauciflora will be considered to assess its potential use in the control of haemonchosis in small ruminants.

Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Nematicidal Activity of Cyclotides: Toxicity Against Caenorhabditis elegans.

Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.

Bio-directed Chemical Study of Pleurotus ostreatus Spent Substrate and Its Nematicidal Activity.

PURPOSE: In the present study, the nematicidal activity of the chemical fractionation of the spent substrate of the edible mushroom Pleurotus ostreatus against eggs and L3 larvae of Haemonchus contortus was evaluated. METHODS: The hydroalcoholic extract of the spent substrate was subjected to a bipartition with ethyl acetate giving two fractions: one aqueous (F. Ac) and one organic (F. AcOET). Both fractions were evaluated against eggs and L3 larvae at different concentrations (5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and 2% methanol, PBS and thiabendazole (5 mg/mL) as controls. Chemical fractionation of F. AcOET was performed in open column chromatography where 76 fractions were obtained and when analyzed by thin layer chromatography (TLC) were grouped into 11 mixtures (R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11). These mixtures were evaluated at 10 mg/mL against eggs and L3 larvae. The data obtained were analyzed by ANOVA and a Tukey test in the SAS V9 program. RESULTS: The results showed that, in the evaluation of F. AcOET and F. Ac, for the inhibition of egg hatching, the highest percentages were 78.80 and 76.89% at 5 mg/mL, respectively. As for the percentage of larval mortality, F. AcOET obtained 60.91 and F. Ac 29.77% at 5 mg/mL. The results of the evaluations of the mixtures showed that mixtures R4, R5 obtained 100 and 95.41% larval mortality and mixtures R6 and R7 presented 100% inhibitory activity of egg hatching, so these were analyzed by gas chromatography mass spectrometry finding compounds such as vanillin, ß-sitosterol, ρ-methyl ρ-hydroxycinnamate and ρ-hydroxybenzaldehyde. CONCLUSION: The results of the present study demonstrate that the spent substrate of P. ostreatus has potential anthelmintic activity against H. contortus. Moreover, by reusing and taking advantage of this substrate, its environmental pollution effects can be reduced.

Ultrasound assisted cyanotoxin extraction for nematode inhibition in soil.

Root-knot nematodes are one of the plant damaging nematodes in agriculture causing a projected annual yield loss of ∼12 % (∼$160 billion) worldwide. Conventional solutions to control these plant-parasitic nematodes involve chemical nematicides. To reduce the use of harmful chemicals, microalgal extracts can be used as greener alternatives for nematode management. Microalgae produce valuable metabolites, including cyanotoxins which can aid in nematode suppression. In this study, two microalgae species, Trichormus variabilis and Nostoc punctiforme, were treated with ultrasound for intensified recovery of secondary metabolites. Ultrasound results in cell wall disruption of the microalgal species, thus resulting in enhanced release of secondary metabolites. Microalgal biomass was treated with an ultrasound probe at 50 % amplitude, 20 kHz frequency, using water as the extraction medium, for 5-30 min. The extraction efficiency was determined in terms of the total chlorophyll (Chl) content of the extract. Microscopic images of the treated cells were also investigated to gain insight into the effect of the ultrasonication time on the cell morphology. Our results suggest that ultrasonication resulted in the intensified release of secondary metabolites, as established through the total chlorophyll content of the ultrasonicated microalgal samples as well as the microscopic images of the ruptured cells. The best extraction for Trichormus variabilis was achieved with 15 min extraction time where the Total Chl content increased by 29 times (compared to the non-ultrasonicated sample), and for the Nostoc punctiforme, 30 min extraction time gave the highest metabolite recovery of 6.4 times higher than the non-ultrasonicated sample. Ultrasonicated algal extracts were then tested for their nematicidal potential against root-knot nematode, Meloidogyne hapla, in infested field soil samples. Experimental study was conducted using different concentrations of each microalga, Trichormus sp. and Nostoc sp., individually, as well as in combination. The nematode count for the treated soil was compared with that of the control (untreated soil). Ultrasonicated microalgal extracts showed 66% to 100% inhibition on root-knot nematodes in the soil samples tested.

Bioassay-Guided Isolation of Nematicidal Artemisinic Acid and Dihydroartemisinic Acid from Artemisia annua L. and Evaluation of Their Activity against Meloidogyne incognita.

The extract of Artemisia annua L. has been shown to possess the nematicidal activity but the potent constituents were unclear. Herein, two nematicidal sesquiterpenoid acids artemisinic acid (ArA) and dihydroartemisinic acid (DHArA) were isolated from the methanol extract of the aerial parts of A. annua L. by the bioassay-guided isolation method using Meloidogyne incognita second stage juveniles (J2s) as the screening target. The in vitro activity, control efficacy in the pot experiment, and toxic effects of these two natural compounds against M. incognita were evaluated. The in vitro results showed that the EC50/48h values of ArA and DHArA were 0.37 mM and 0.76 mM against J2s, respectively. In the pot experiment, ArA and DHArA at the dose of 5 mg (a.i.)/pot could achieve the same level of control efficacy compared with avermectins at 2 mg (a.i.)/pot. Microscopic observations indicated the obvious toxic symptoms of J2s after ArA- and DHArA-treatment, including the shrinking body, imperfect body wall, and undiscerning organs. The physiological and biochemical studies, together with the toxic symptoms, revealed that ArA and DHArA had great impacts on the membrane system of J2s. Additionally, ArA occurring the α, ß-unsaturated carbonyl was demonstrated to be reactive with glutathione (GSH) and cause the decrease of GSH content in J2s. Taken together, the present study suggests that ArA and DHArA or ArA- and DHArA-based extracts of A. annua L. have a substantial potential to be used as botanical agents for integrated disease management programs against root-knot nematodes in crop protection.

Nematicidal activity of seaweed-synthesized silver nanoparticles and extracts against Meloidogyne incognita on tomato plants.

The purpose of this study was to test the nematicidal activity of extracts of two marine algae (Colpomenia sinuosa and Corallina mediterranea) and their synthesized silver nanoparticles against root-knot nematodes (Meloidogyne incognita) that infest tomato plants. Scanning electron microscopy (SEM) revealed that nanoparticles had aggregated into anisotropic Ag particles, and transmission electron microscopy (TEM) revealed that the particle sizes were less than 40 nm. Fourier Transform Infrared Spectroscopy (FT-IR) analysis revealed that the obtained nanoparticles had a sharp absorbance between 440 and 4000 cm-1, with 13 distinct peaks ranging from 474 to 3915 cm-1. Methylene chloride extracts and nanoparticles synthesized from both algae species were used to treat M. incognita. C. sinuosa nanoparticles had the highest nematicidal activity of any treatment. Furthermore, and in contrast to other treatments, C. sinuosa nanoparticles reduced the number of nematode galls, egg-masses per root, and eggs/egg mass, while also improving plant growth parameters. C. sinuosa's methylene chloride extract was more active than C. mediterranea's, and the most effective eluent of this solvent was hexane: methylene chloride: ethyl acetate (1: 0.5: 0.5, v/v/v). When applied to M. incognita, the third fraction of this eluent was the most effective, resulting in 87.5% mortality after 12 h and 100% mortality after 24 and 72 h of exposure. The presence of seven bioactive constituents was discovered during the analysis of this fraction. In conclusion, the silver nanoparticles synthesized from C. sinuosa could be used as alternative chemical nematicides.

Nematicidal activity of leaf extract of Moringa oleifera Lam. against Haemonchus contortus and Nacobbus aberrans.

In the present study, the nematicidal activity of a Moringa oleifera ethyl acetate leaf extract against the eggs and larvae of Haemonchus contortus and Nacobbus aberrans, nematodes of agricultural importance, was evaluated. The experimental design for the evaluation of the effects against both nematodes consisted of eight treatments (n = 4). Distilled water, Tween (4%) and a commercial anthelmintic agent (ivermectin, 5 mg/mL) were used as controls, and for treatments 4-8, the concentrations of the extract were 20, 10, 5, 2.5 and 1.25 mg/mL, respectively. Readings were taken at 12 h and 24 h for N. aberrans and 48 h and 72 h for H. contortus post-treatment under an optical microscope (10× and 40×). The data obtained were analysed by analysis of variance through a completely randomized factorial design using the SAS V9 program. The results show that, for H. contortus egg hatching, 85.88% inhibition was obtained at a concentration of 20 mg/mL at 48 h, while for third-stage larva (L3) mortality, the highest percentage was 68.19% at 1.25 mg/mL at 72 h. In the case of N. aberrans, the greatest inhibition of egg hatching was 90.69% at 5 mg/mL at 12 h post-treatment, and for larval mortality, it was 100% at 10 mg/mL at 24 h post-treatment. The main major compounds identified by qualitative analysis and by gas chromatography coupled to mass spectrometry were 9,12,15-octadecatrienoic acid, (Z,Z,Z)-, n-hexadecanoic acid and 2,4-di-tert-butylphenol, and the minor compounds included phytol, γ-sitosterol and α-tocopheryl acetate. It was demonstrated that the ethyl acetate leaf extract of M. oleifera Lam. shows great potential for combating agricultural nematodes.

In Vitro Nematocidal Properties from Two Extracts: Lippia graveolens Leaves and Delonix regia Flowers Against Eggs and Infective Larvae of Haemonchus contortus.

The objective of this study was to assay the ovicidal and larvicidal effects of two hydroalcoholic extracts (HA-E) and their aqueous fractions (Aq-F) and organic fractions (EtOAc-F) from Lippia graveolens (leaves) and Delonix regia (flowers) against the parasitic nematode Haemonchus contortus. The egg hatching inhibition test (% EHI) and mortality of infective larvae (% mortality) were used as biological models. The treatments of each plant (mg/mL, % EHI, and % mortality) were assigned as follows: HA-E (12.5-50, EHI; 25-200, mortality), Aq-F (6.25-25.00, EHI; 12.5-50, mortality), EtOAc-F (0.37-25, EHI; 3.12-50, mortality), negative controls (distilled water and methanol at 2% and 4%), and positive control (ivermectin at 5 mg/mL). Data were analyzed using a completely randomized design through an analysis of variance, considering the EHI and mortality percentages as the response variables. To identify the secondary metabolites, both plants were subjected to qualitative chemical analysis and HPLC. The results showed that both plants exhibited highly ovicidal and larvicidal effects. The bipartition in each HA extract allowed the increase of the anthelmintic effect in EtOAc-F, achieving a total ovicidal activity at 1.56 mg/mL and a larvicidal effect higher than 90% at 25 mg/mL. The qualitative chemical profile of both vegetal species identified alkaloids, flavonoids, condensed tannins, terpenes, and saponins. These results suggest that either L. graveolens leaves or D. regia flowers could be potential candidates for the control of parasitic gastrointestinal nematodes in small ruminants. However, in vivo studies with experimental animals to evaluate their possible toxicity and anthelmintic effect are necessary.

Nematocidal activity of hydroalcoholic extracts of spent substrate of Pleurotus djamor on L3 larvae of Haemonchus contortus.

The objective of this study was to evaluate and compare the in vitro lethal effect of the hydroalcoholic extract of the spent substrate of Pleurotus djamor ECS-123, obtained at 15 days of colonization (SPS) and at the first (SPS1) and second (SPS2) harvests, against infective larvae L3 of Haemonchus contortus. The in vitro lethal effect was evaluated by the L3 larval mortality test (LM) using six concentrations: 1.25, 2.5, 5, 10, 20, and 40 mg/mL, with ivermectin and thiabendazole (5 mg/mL) as controls. The first harvest extract (SPS1) of strain ECS-123 was subjected to liquid-liquid bipartition, which resulted in two fractions: aqueous (PdAcO) and ethyl acetate (PdAct). The chemical fractionation of PdAct with the highest mortality rate (80.11 %) was carried out with open-column chromatography, giving a total of 13 fractions, which were analyzed by thin-layer chromatography (TLC) and grouped into 5 mixtures (R1;1-3, R2;4-7, R3;8-9, R4;10-11 and R5;12-13). Subsequently, the mixtures were evaluated against H. contortus L3 larvae. Finally, the components of the mixtures with the highest nematocidal effects were evaluated by gas chromatography coupled to mass spectrometry (GC-MS). The data were analyzed with a completely randomized design through ANOVA using the generalized linear model (GLM) with the "R" program. The purification and characterization of R4 and R5 by GC-MS revealed the presence of the following compounds: veratryl alcohol, 4-hydroxy-3,5,5 trimethyl-4-[3-oxo-1-butenyl]-2- cyclohexen-1-one, caffeine and 5,6-dimethoxy-1(3 H) isobenzofuranone. This information allowed for the identification of nematocidal compounds in the degraded substrate of P. djamor, an activity that had not been reported previously.

Nematicidal and Molecular Docking Investigation of Essential Oils from Pogostemon cablin Ecotypes against Meloidogyne incognita.

Root-knot nematode, Meloidogyne incognita is one of the most destructive nematodes worldwide. Essential oils (EOs) are being extensively utilized as eco-benign bionematicides, although the precise mechanism of action remains unclear. Pogostemon cablin Benth. is well-known as "Patchouli". It is native to South East Asia and known for ethno-pharmacological properties. In this study, chemical composition and potential nematicidal effect of EOs hydrodistilled from the leaves of P. cablin grown at three different locations in India were comprehensively investigated to correlate their mechanism of action for target specific binding affinities toward nematode proteins. Aromatic volatile Pogostemon essential oils (PEO) from Northern India (PEO-NI), Southern India (PEO-SI) and North Eastern India (PEO-NEI) were analyzed by Gas Chromatography-Mass Spectrometry (GC/MS) to characterize forty volatile compounds. Maximum thirty-three components were identified in PEO-NEI. Sesquiterpenes were predominant with higher content of α-guaiene (2.3-24.4 %), patchoulol (6.1-32.7 %) and α-bulnesene (5.9-27.1 %). Patchoulol was the major component in PEO-SI (32.7±1.2 %) and PEO-NEI (29.2±1.1 %), while α-guaiene in PEO-NI (24.4±1.2 %). In vitro nematicidal assay revealed significant nematicidal action (LC50 44.6-87.0 µg mL-1 ) against juveniles of M. incognita within 24 h exposure. Mortality increases with increasing time to 48 h (LC50 33.6-71.6 µg mL-1 ) and 72 h (LC50 27.7-61.2 µg mL-1 ). Molecular modelling and in silico studies revealed multi-modal inhibitive action of α-bulnesene (-22 to -13 kJ mol-1 ) and α-guaiene (-22 to -12 kJ mol-1 ) against three target proteins namely, acetyl cholinesterase (AChE), odorant response gene-1 (ODR1), odorant response gene-3 (ODR3). Most preferable binding mechanism was observed against AChE due to pi-alkyl, pi-sigma, and hydrophobic interactions. Structure nematicidal activity relationship suggested the presence of hydroxy group for nematicidal activity is nonessential, rather highly depends on synergistic composition of sesquiterpene hydrocarbons.

Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans.

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the "HitFinder" library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

Capsicum frutescens and Citrus limon: a new take on therapy against experimental trichinellosis.

Trichinellosis is a zoonotic disease that endangers human health and can lead to death. Restricted absorption and poor results of conventional therapies demand new effective natural remedies to treat both enteral and parenteral trichinellosis. This study assessed the antiparasitic and anti-inflammatory effects of Citrus limon and Capsicum frutescens on murine trichinellosis and compared them with those of albendazole and prednisolone, which are conventionally used to treat trichinellosis. Overall, 50 Swiss albino male mice were divided into five groups, with ten mice in each group: negative control, positive control, albendazole combined with prednisolone, C. limon, and C. frutescens. Mice were sacrificed 7 and 35 days after infection, for intestinal and muscular phase analyses. Drug efficacies were parasitologically, biochemically, histopathologically and ultrastructurally assessed. Our results demonstrated the efficacy of C. frutescens and C. limon extracts as antiparasitic agents, showing a substantial decrease in adult and larval counts. Moreover, both extracts had the ability to decrease serum tumour necrosis factor-α levels during the intestinal and muscular phases. In addition to the improved histopathological changes in the small intestine and muscles, the destructive effects on adults and larvae were ultrastructurally evident on transmission electron microscopy. In conclusion, C. frutescens and C. limon extracts are promising remedies for the treatment of experimental trichinellosis, particularly, the C. frutescens extract.

Chemical Characterization of Plant Extracts and Evaluation of their Nematicidal and Phytotoxic Potential.

Nacobbus aberrans ranks among the "top ten" plant-parasitic nematodes of phytosanitary importance. It causes significant losses in commercial interest crops in America and is a potential risk in the European Union. The nematicidal and phytotoxic activities of seven plant extracts against N. aberrans and Solanum lycopersicum were evaluated in vitro, respectively. The chemical nature of three nematicidal extracts (EC50,48h ≤ 113 µg mL-1) was studied through NMR analysis. Plant extracts showed nematicidal activity on second-stage juveniles (J2): (≥87%) at 1000 µg mL-1 after 72 h, and their EC50 values were 71.4-468.1 and 31.5-299.8 µg mL-1 after 24 and 48 h, respectively. Extracts with the best nematicidal potential (EC50,48h < 113 µg mL-1) were those from Adenophyllum aurantium, Alloispermum integrifolium, and Tournefortia densiflora, which inhibited L. esculentum seed growth by 100% at 20 µg mL-1. Stigmasterol (1), ß-sitosterol (2), and α-terthienyl (3) were identified from A. aurantium, while 1, 2, lutein (4), centaurin (5), patuletin-7-ß-O-glucoside (6), pendulin (7), and penduletin (8) were identified from A. integrifolium. From T. densiflora extract, allantoin (9), 9-O-angeloyl-retronecine (10), and its N-oxide (11) were identified. The present research is the first to report the effect of T. densiflora, A. integrifolium, and A. aurantium against N. aberrans and chemically characterized nematicidal extracts that may provide alternative sources of botanical nematicides.

In Vitro Nematocidal Effect and Anthelmintic Activity of Artemisia cina Against Haemonchus contortus in Gerbils and Relative Expression of Hc29 Gene in Transitional Larvae (L3-L4).

PURPOSE: (1) To assess the in vitro activity of Artemisia cina against Haemonchus contortus L3 (HcL3) and in transitional (L3-L4) larvae (HcTrL3-L4); (2) to quantify the relative expression of the Hc29 gene in HcTrL3-L4 exposed to the A. cina n-hexane extract; and (3) to assess the anthelmintic activity (AA) of the A. cina organic extracts in gerbils artificially infected with H. contortus (HcArt/inf/gerbs). METHODS: The in vitro assay was carried out in 96-well microtitration plates. The following A. cina extracts: ethyl acetate (Ac-EtOAcEx), n-hexane (Ac-n-HexEx), and methanol (Ac-MethEx) were assessed at 1 and 2 mg/mL against HcL3 and HcTrL3-L4 at 24 h exposure. Relative expression of the Hc29 gene in HcTrL3-L4 was obtained by RT-PCR. For assessing the AA, six groups of five HcArt/inf/gerbs were used. Groups were treated orally with 4 mg/kg BW of A. cina extracts. Five days after treatment, the gerbils were necropsied and nematodes counted. RESULTS: The highest in vitro activities (75 and 82.6%) were shown by Ac-n-HexEx at 1 and 2 mg/mL, respectively. For HcTrL3-L4 the highest in vitro activities (69 and 23%) were shown by Ac-n-HexEx and isoguaiacine at 0.625 mg/mL, respectively. Also, upregulation of H. contortus Hc29 gene by 13- and 80-fold (p < 0.01) was observed on the HcTrL3-L4 stage after exposure to Ac-n-HexEx extract and isoguaiacine at 0.078 mg/mL, respectively. Reduction percentage was 100% in HcArt/inf/gerbs treated with Ac-n-HexEx. CONCLUSIONS: We conclude that the Ac-n-HexEx and isoguaiacine compound had anthelmintic efficacy against H. contortus and L3 and HcTrL3-L4.

Acute Anisakiasis: Pharmacological Evaluation of Various Drugs in an Animal Model.

BACKGROUND: The accidental ingestion of the third larval stage of Anisakis can cause acute clinical symptoms, which are relieved via extraction of the larvae. Although this is a highly effective technique, it can only be practiced when the larvae are found in accessible areas of the gastrointestinal tract, and therefore instead the condition has often been treated using various different drugs. AIMS: This study evaluates the effectiveness of gastric acid secretion inhibitors (omeprazole and ranitidine), gastric mucosal protectants (sucralfate) and anthelmintics (mebendazole and flubendazole) in treating anisakiasis in Wistar rats. METHODS: Rats were infected with Anisakis-type I larvae and administered the drugs via a gastric probe. Data were recorded regarding the number of live and dead larvae, their location both within the animal and in its feces, and the presence of gastrointestinal lesions. Additionally, gastric pH was measured and histology performed. RESULTS: While rats in all experimental groups exhibited lesions; those treated with ranitidine and mebendazole showed significantly fewer lesions (50% and 35% of rats exhibited lesions, respectively). Histological examination of the gastric lesions revealed infection-induced changes, but no significant differences were observed between the treated and untreated rats. CONCLUSIONS: Mebendazole was found to be most efficacious in preventing gastrointestinal lesions, followed by ranitidine, which was the most effective antacid of those studied. Both these drugs could thus be considered as part of the conservative management of anisakiasis.

Recent advances on synthesis and biological activities of aurones.

Aurones are naturally occurring structural isomerides of flavones that have diverse bioactivities including antiviral, antibacterial, antifungal, anti-inflammatory, antitumor, antimalarial, antioxidant, neuropharmacological activities and so on. They constitute an important class of pharmacologically active scaffolds that exhibit multiple biological activities via diverse mechanisms. This review article provides an update on the recent advances (2013-2020.4) in the synthesis and biological activities of these derivatives. In the cases where sufficient information is available, some important structure-activity relationships (SAR) of their biological activities were presented, and on the strength of our expertise in medicinal chemistry and careful analysis of the recent literature, for the potential of aurones as medicinal drugs is proposed.

Identification and characterization of Vietnamese coffee bacterial endophytes displaying in vitro antifungal and nematicidal activities.

The endophytic bacteria were isolated from coffee roots and seeds in Vietnam and identified with 16S rDNA sequencing as belonging to the Actinobacteria, Firmicutes and Proteobacteria phyla with the Nocardia, Bacillus and Burkholderia as dominant genera, respectively. Out of the thirty genera recovered from Coffea canephora and Coffea liberica, twelve were reported for the first time in endophytic association with coffee including members of the genera Brachybacterium, Caballeronia, Kitasatospora, Lechevalieria, Leifsonia, Luteibacter, Lysinibacillus, Mycolicibacterium, Nakamurella, Paracoccus, Sinomonas and Sphingobium. A total of eighty bacterial endophytes were characterized in vitro for several plant growth promoting and biocontrol traits including: the phosphate solubilization, the indolic compounds, siderophores, HCN, esterase, lipase, gelatinase and chitinase production. A subset of fifty selected bacteria were tested for their potential as biocontrol agents with in vitro confrontations with the fungal pathogen Fusarium oxysporum as well as the coffee parasitic nematodes Radopholus duriophilus and Pratylenchus coffeae. The three most efficient isolates on F. oxysporum belonging to the Bacillus, Burkholderia, and Streptomyces genera displayed a growth inhibition rate higher than 40%. Finally, five isolates from the Bacillus genus were able to lead to 100% of mortality in 24 h on both R. duriophilus and P. coffeae.

Utilization of Cladophora glomerata extract nanoparticles as eco-nematicide and enhancing the defense responses of tomato plants infected by Meloidogyne javanica.

Tomato (Solanum Lycopersicum L.) is an important vegetable crop that belongs to the family Solanaceae. Root-knot nematodes reflect the highly critical economically damaging genera of phytoparasitic nematodes on tomato plants. In this study, the eco-nematicide activity of freshwater green macroalga Cladophora glomerata aqueous extract and their synthesized silver nanoparticles (Ag-NPs) against root-knot nematodes Meloidogyne javanica was investigated on tomato plants. The formation and chemical structure of Ag-NPs was examined. The aqueous extract from C. glomerata was applied against the root-knot nematodes besides the biosynthesized green silver nanoparticles with 100, 75, 50, and 25% (S, S/2, S/3, S/4) concentrations. To investigate the plant response toward the Green Synthesized Silver Nanoparticles (GSNPs) treatment, expression profiling of Phenylalanine Ammonia-Lyase (PAL), Poly Phenol Oxidase (PPO), and Peroxidase (POX) in tomato were examined using Quantitative Real-Time PCR (Q-PCR). The results indicated that GSNPs from C. glomerata exhibited the highest eco-nematicide activity in the laboratory bioassay on egg hatchability and juveniles (J2S) mortality of M. javanica compared with the chemical commercial nematicide Rugby 60%. Also, results showed a significant reduction in galls number, egg masses, females per root system/plant, and mortality of juveniles. The results of PAL and PPO enzyme expression for the control plants remained relatively stable, while the plant inoculated with nematode M. javanica as well as the activity of genes in scope was increased from 14 to 28 Days after Nematode Inoculation (DANI). These activities were improved in inoculated plants and treated with C. glomerata extract and their green syntheses of Ag-NPs and the other plants treated with Rugby 60% (4 mL/L). The greatest activities of the three enzymes were evident after 14 days after the nematode inoculation. It can be concluded that the green synthesized nanoparticles using C. glomerata could be used as potent nematicides against M. javanica which induces the immune system to defend against nematode infection.