In vitro evaluation of methanol extracts of edible fungi Pleurotus ostreatus and Lentinula edodes against Rhyssomatus nigerrimus Fahraeus.

The study aimed to evaluate the insecticidal activity of extracts of edible mushrooms Pleurotus ostreatus and Lentinula edodes against Rhyssomatus nigerrimus. Methanol:water extracts (70:30) of P. ostreatus and L. edodes were made and evaluated in two in vitro tests (exposure and immersion toxic effect) against R. nigerrimus. Subsequently, the toxicity test of the extracts against Artemia salina was evaluated. These extracts were subjected to colorimetric tests and gas chromatography-mass spectrometry. The results showed a mortality effect against R. nigerrimus of 50% for the P. ostreatus 2 extracts at a concentration of 20% in the immersion test. Likewise, in the toxic effect test, 90% mortality was observed after five days of exposure to a concentration of 10%. On the other hand, for the toxicity test, the extract that showed the values with the highest mortality against A. salina was P. ostreatus, starting with 80% mortality at 100µg/mL. The functional groups present in the extracts were saponins, coumarins, and alkaloids. Furthermore, the presence of more than 7 compounds in the mushroom extracts evaluated is reported. This study demonstrates the insecticidal activity of P. ostreatus and L. edodes fungal extracts and indicates the importance of using different in vitro tests to elucidate the mechanism of action for future studies.

Insecticidal effect of metabolites identified in edible mushrooms against Rhyssomatus nigerrimus Fahraeus.

Excessive use of insecticides has led to resistance of some pathogenic organisms (nematodes, bacteria and fungi), environmental contamination, and the presence of hazardous residues. Therefore, the aim of the present study was to evaluate synthetic metabolites derived from previous studies with edible mushrooms against the soybean weevil Rhyssomatus nigerrimus Fahraeus (Curculonidae) because of the relevance of pest control in an economically important crop. Furthermore, this is one of the first studies where edible fungal molecules are evaluated for the control of these insects. Initially, two in vitro tests (toxic effect and immersion) were evaluated against R. nigerrimus. In these tests, sensitivity and viability were determined in the 2% Tween control in water. For these two tests, the synthetic metabolites pentadecanoic acid (PNA), palmitic acid (PMA), stearic acid (STA), linoleic acid (LNA), ß-sitosterol (ßT) were evaluated individually as well as in combinations, "the fraction of standards (E1)". Based on the results obtained, the dip test was selected to evaluate the mixtures of two standards (1. PMA + ßT, 2. PMA + PNA, 3. PMA + LNA, 4. PMA + STA, 5. STA + ßT, 6. STA + PNA, 7. STA + LNA, 8. PNA + ßT, 9. PNA + LNA, 10. LNA + ßT), three (1. PNA + ßT + LNA, 2. PNA + ßT + STA, 3. STA + LNA + PNA and 4. STA + LNA + ßT) and four (PNA, ßT, LNA and STA). The results showed that the mixture of three standards caused a higher percentage of mortality relative to the control group: l. PNA + ßT + LNA and 2. PNA + ßT + STA with 54.44 and 48% mortality of R. nigerrimus insects exposed for 15 days. These results show the importance of evaluating mixtures of molecules against R. nigerrimus.

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 nematicidal activity of two ferrocenyl chalcones against larvae of Haemonchus contortus (L3) and Nacobbus aberrans (J2).

The main goal of this work was to evaluate the in vitro biological activity of two ferrocenyl chalcones (FcC-1 and FcC-2) against Haemonchus contortus (third-stage larvae (L3)) and Nacobbus aberrans (second-stage juveniles (J2)). Both compounds were synthesized and characterized by usual spectroscopic methods and their molecular structures were confirmed by single-crystal X-ray diffractometry. Nematode strains were examined in terms of percentage mortality of H. contortus (L3) by the action of FcC-1, which showed an effectivity of 100% at a concentration of 342 µM in 24 h, with EC50 = 20.33 µM and EC90 = 162.76 µM, whereas FcC-2 had an effectivity of 72% at a concentration of 342 µM in 24 h, with EC50 = 167.39 µM and EC90 = 316.21 µM. The effect of FcC-1 against nematode phytoparasite N. aberrans showed a better percentage of 95% at a concentration of 342 µM, with EC50 = 7.18 µM and EC90 = 79.25 µM, whereas the effect of FcC-2 was 87% at 342 µM, with EC50 = 168 µM and EC90 = 319.56 µM at 36 h. After treatment, the scanning electron micrographs revealed deformities in the dorsal flank and posterior part close to the tail of H. contortus L3. They showed moderate in vitro nematicidal activity against H. contortus L3 and N. aberrans J2.

Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

An in vitro approach to evaluate the nutraceutical value of plant foliage against Haemonchus contortus.

Nutraceutical plants provide nutrients for the animal as well as secondary compounds that can affect the biology and survival of gastrointestinal nematodes (GIN). Current screening of nutraceutical plants is based on in vitro evidence of anthelmintic (AH) activity against different life stages of GIN, but nutritional information is omitted or scarce. This study proposes an integral in vitro screening protocol to identify the nutraceutical value of the foliage from plant species consumed by small ruminants, using Haemonchus contortus as a biological model. The leaves from Acacia collinsii, A. pennatula, Bunchosia swartziana, Gymnopodium floribundum, Havardia albicans, Leucaena leucocephala, Lysiloma latisiliquum, Mimosa bahamensis, Piscidia piscipula, and Senegalia gaumeri were evaluated for their chemical composition and in vitro digestibility. Acetone:water extracts (70:30) from leaves of each plant were evaluated using the egg hatch assay and larval exsheathment inhibition assay. Respective effective concentrations 50% (EC50) were determined for each assay. The ten plant species showed good nutritional value for ruminants, including crude protein (> 10%), metabolizable energy (> 2.9 MJ/kg DM), and varied CT content (from 1.0 to 37.6%). The best AH activity against H. contortus eggs (EC50 = 401.8 µg/mL) and L3 (EC50 = 83.1 µg/mL) was observed for S. gaumeri extract. Although all the plant species showed in vitro nutraceutical potential, the leaves of S. gaumeri had the best values. The proposed in vitro protocol showed to be useful for the integral assessment of the nutraceutical potential of different plant species as it included the nutritional value and the AH activity against eggs and L3 in the selected plant species.

Is there a negative association between the content of condensed tannins, total phenols, and total tannins of tropical plant extracts and in vitro anthelmintic activity against Haemonchus contortus eggs?

In vitro studies using plant extracts suggest a relationship between their polyphenol contents and their anthelmintic (AH) activity against Haemonchus contortus. High polyphenol content appears to increase the efficacy of plant extracts against H. contortus as assessed by the larval exsheathment inhibition assay (LEIA) while appearing to reduce the AH efficacy measured using the egg hatch assay (EHA). In addition, some plants lack AH activity. Therefore, the present study investigated the relationship between the contents of condensed tannins (CT), total phenols (TP), and total tannins (TT) in methanol:water extracts (70:30) obtained from ten tropical plant species consumed by small ruminants as well as their AH activity against H. contortus evaluated by LEIA and EHA. Extracts of Acacia collinsii, Lysiloma latisiliquum, Havardia albicans, Senegalia gaumeri, Mimosa bahamensis, Piscidia piscipula, Acacia pennatula, Gymnopodium floribundum, Leucaena leucocephala, and Bunchosia swartziana were examined. Positive correlations were found between the effective concentration 50% (EC50) (EHA) of extracts and their CT (r = 0.6809, P < 0.05, n = 10) and TP (r = 0.9152, P < 0.05, n = 10) content, suggesting that their concentration negatively affected AH activity against eggs. Based on the LEIA, there was no significant association between the EC50 and the CT, TP, or TT of all extracts evaluated. Thus, if sheep and goats consume a complex feed mixture with high amounts of CT, TP, and TT, it might be difficult to observe an AH effect against H. contortus egg hatching. However, the AH effect upon L3 establishment might be feasible.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.

Susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenol-rich plants. Part 2: Infective L3 larvae.

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200µg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200µg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P<0.05). The EC50 values for A. pennatula extract ranged from 36 to 501µg/mL (RR: 2.11-13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003µg/mL (RR: 1.25-7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.

In vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of two tannin rich plants.

The aim of the study was to examine the variation in the in vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins using respective egg hatch assays (EHA) with acetone:water extracts of two tannin containing plants, chimay (Acacia pennatula) and sainfoin (Onobrychis viciifolia). Fresh eggs were incubated in PBS with different concentrations of each extract (0, 600, 1200, 2400, 3600, 5000 and 8000 µg/ml PBS). Additional concentrations were tested for O. viciifolia (75, 100, 200 and 400 µg/ml PBS). Effective concentrations 50% (EC50), with the corresponding 95% confidence interval (95% CI), were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated to compare the isolates, using the most susceptible isolate for each extract as the respective reference. A second set of incubations were made using polyvinylpolypyrrolidone (PVPP) (0, 5000 µg/ml, 5000 µg/ml+PVPP) to determine the influence of polyphenols on the AH effect. The proportion of morulated eggs, eggs with L1 larvae failing eclosion (%LFE), and emerged larvae were estimated at different extract concentrations. Data of each isolate was used to calculate the effective concentration 50% (EC50) for each extract. The EC50 of each isolate was used to determine resistance ratio (RR) for the different isolates. For the 2 extracts, a susceptibility variation in egg hatching was observed for the different H. contortus isolates. The EC50 values for A. pennatula ranged from 2203 to 14106 µg (RR from 2.01 to 6.40). The O. viciifolia extract showed higher variability with EC50 values ranging from 104 to 4783 µg (RR from 3.66 to 45.74). The main AH effects of the two extracts tested on the ten isolates consisted in blocking the emergence of L1 larvae (higher% LFE). Additional observations on emerged larvae showed that extract exposure caused alterations in the internal structure, separating the cuticle from the pharynx, bulb and intestinal cells. The use of PVPP revealed that (a) condensed tannins were not the sole plant secondary metabolites responsible for the AH effects, and (b) different H. contortus isolates showed variability in the role of tannins either on the ovicidal effect or the %LFE.