Larval Exsheathment Inhibition Research Articles

Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis

Ethnopharmacological relevanceGastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. The aims of our studyFour medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. Materials and methodsTo evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). ResultsC. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42–41.80 μg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70–137.34 μg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20–67.00% of larvae exsheathment, (p < 0.05) and 40.51–24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. ConclusionsThe present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.

Impact of Seasonal and Organ-Related Fluctuations on the Anthelmintic Properties and Chemical Profile of Cladium mariscus (L.) Pohl Extracts.

The use of plants and their metabolites stands as a promising option to tackle parasitic infections by gastrointestinal nematodes (GIN) in integrated control strategies. Still, the influence of environmental and phenological factors, and their interactions, in the wild on the metabolomics and biological properties of target plant species, is often disregarded. In this work, we hypothesized that variations in the anthelmintic (AH) properties and chemical composition of extracts from the salt tolerant species Cladium mariscus L. Pohl (sawgrass) may be influenced by seasonal factors and organ-parts. To test this hypothesis, acetone/water extracts were prepared from dried biomass obtained from aerial organs collected from sawgrass in consecutive seasons and tested against Haemonchus contortus and Trichostrongylus colubriformis by the larval exsheathment inhibition assay (LEIA) and egg hatching inhibition assay (EHIA). To ascertain the role of plant organ, the activity of leaves and inflorescences extracts from summer samples was compared. The role of polyphenols in the anthelmintic activity depending on GINs and fluctuations across seasons and plant organs was assessed using polyvinylpolypyrrolidone (PVPP), coupled with an in-depth chemical profiling analysis using high-performance liquid chromatography completed with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). Main differences in anthelmintic activities were observed for summer and autumn samples, for both assays. Moreover, inflorescences’ extracts were significantly more active than those from leaves against both parasite species on EHIA and against H. contortus on LEIA. Application of PVPP totally inhibit the AH effects based on EHIA and only partly for LEIA. Non-treated PVPP extracts were predominantly composed of flavan-3-ols, proanthocyanidins, luteolin and glycosylated flavonoids, while two flavonoid glycosides were quantified in all PVPP-treated samples. Thus, the activity of such compounds should be further explored, although some unknown metabolites remain to be identified. This study reinforces the hypothesis of the AH potential of sawgrass and of its polyphenolic metabolites uses as nutraceutical and/or phytotherapeutic drugs.

Disclosing the bioactive metabolites involved in the in vitro anthelmintic effects of salt-tolerant plants through a combined approach using PVPP and HPLC-ESI-MSn

Strategies to reduce dependence on synthetic drugs for the treatment of gastrointestinal nematodes (GIN) infections in ruminants include the search for novel anthelmintic scaffolds on plants, yet salt-tolerant plants remain overlooked. This study aims to evaluate the in vitro anthelmintic properties of selected salt-tolerant plants against GIN, and identify the potential bioactive secondary metabolites involved. For that purpose, 80% acetone/water extracts were prepared from dried biomass of aerial organs of nine salt-tolerant plant species and tested against Haemonchus contortus and Trichostrongylus colubriformis by the Larval Exsheathment Inhibition Assay (LEIA) and Egg Hatching Inhibition Assay (EHIA). Pistacia lentiscus, Limoniatrum monopetalum, Cladium mariscus and Helychrisum italicum picardi were the most active in both GIN and life stages. To investigate the role of polyphenols in the anthelmintic activity, four selected extracts were treated with polyvinylpolypyrrolidone (PVPP), and non-treated and treated samples were further characterized by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MSn). While polyphenols seem responsible for the EHIA properties, they are partially accountable to LEIA results. Several phenolics involved in the anthelmintic effects were identified and discussed. In sum, these species are rich sources of anthelmintic compounds and, therefore, are of major interest for nutraceutical and/or phytotherapeutic applications against GIN in ruminants.

In vitro Evaluation of the Nutraceutical Potential of Theobroma cacao pod Husk and Leaf Extracts for Small Ruminants.

Some agroindustrial by-products could be used as nutraceutical materials for small ruminants helping with their nutrition while controlling their gastrointestinal nematodes. This study evaluated the potential invitro nutraceutical value of pod husks and leaves of three varieties of Theobroma cacao using two Haemonchus contortus isolates with different polyphenol susceptibility. Leaves and husks from three T. cacao varieties (AZT, CAL and CEY) were evaluated for their bromatological composition, invitro dry matter digestibility and polyphenol content. Acetone:water extracts (70:30) of each plant variety were evaluated using the egg hatch and larval exsheathment inhibition tests, using two isolates (FESC and PARAISO) of H. contortus. Effective concentrations 50% (EC50) were determined for both tests. The role of polyphenols was confirmed using polyvinylpolypyrrolidone. L3 exposed to CAL leaf extract were submitted to transmission electron microscopy. Both plant materials showed a good nutritional value to complement protein-rich diets for small ruminants. Extracts inhibited exsheathment of H. contortus L3 more effectively than the egg hatching, and the leaf extracts were more active than husk extracts in the L3 exsheathment inhibition. The FESC isolate was more sensitive to extracts. Polyphenols blocked exsheathment inhibition of leaf extracts. Structural damage was observed in the sheath and muscles of L3 exposed to CAL leaf extracts. The two T. cacao materials tested showed their potential to be used as ruminant feeds. Extracts affected H. contortus by blocking L3 exsheathment, particularly with the leaf extracts. The in vivo nutraceutical value should be confirmed in small ruminants.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL−1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

Anthelmintic effect of four extracts obtained from Caesalpinia coriaria foliage against the eggs and larvae of Haemonchus contortus.

To investigate the in vitro anthelmintic efficacy of dividivi (Caesalpinia coriaria), a traditional medicinal plant used in Central America and the northern part of South America, extracts from the foliage of this plant were subjected to the egg hatching test (EHT) and larval exsheathment inhibition test (LEIT), against Haemonchus contortus. Four different extracts were evaluated: acetone-water (AW), methanol-water (MW), acetone-water-dichloromethane (AWD) and methanol-water-dichloromethane (MWD). The concentrations used for the EHT and LEIT tests ranged from 500 to 4000 µg mL-1 and six repetitions per concentration. The effective concentrations (EC50) were calculated using Probit analysis. The EC50 for EHT were 2947.0, 3347.0, 3959.6 and 4538.7 µg mL-1 for MWD, MW, AW and AWD, respectively. The EC50 for LEIT were 2883.4, 5927.4, 9876.3 and 9955.4 µg mL-1 for AWD, AW, MWD and MW, respectively. The methanol extracts were the most effective in inhibiting the hatching of eggs, while the acetone extracts showed efficacy in inhibiting larval exsheathment. This study explains the importance that C. coriaria has as a medicinal plant in Central and South American countries.

In Vitro Anthelmintic Evaluation of Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce: Fingerprint Analysis of Extracts by UHPLC-Orbitrap Mass Spectrometry.

In the present work, the anthelmintic activity (AA) of ethanolic extracts obtained from Gliricidia sepium, Leucaena leucocephala, and Pithecellobium dulce was evaluated using the third-stage-larval (L3) exsheathment inhibition test (LEIT) and egg hatch test (EHT) on Haemonchus contortus. Extracts were tested at concentrations of 0.3, 0.6, 1.2, 2.5, 5.0, 10, 20, and 40 mg/mL. The larval exsheathment inhibition (LEI) results showed that G. sepium achieved the highest average inhibition of 91.2%, compared with 44.6% for P. dulce and 41.0% for L. leucocephala at a concentration of 40 mg/mL; the corresponding IC50 values were 22.4, 41.7, and 43.3 mg/mL, respectively. The rates of egg hatching inhibition (EHI) at a concentration of 5 mg/mL were 99.5% for G. sepium, 64.2% for P. dulce, and 54% for L. leucocephala; the corresponding IC50 values were 1.9 mg/mL for G. sepium, 3.9 mg/mL for P. dulce, and 4.3 mg/mL for L. leucocephala. The species extracts studied here were also analyzed by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry (UHPLC-Q/Orbitrap/MS/MS), resulting in the compounds’ identification associated with AA. Glycosylated flavonoids and methoxyphenols were observed in all three species: fatty acids in G. sepium and P. dulce; phenylpropanoids, anthraquinone glycosides, amino acids and glycosylated phenolic acids in G. sepium; and flavonoids in L. leucocephala. Comparatively, G. sepium presented a greater diversity of compounds potentially active against the control of gastrointestinal nematodes, which was associated with the results obtained in the applied tests.

Ellagitannins Inhibit the Exsheathment of Haemonchus contortus and Trichostrongylus colubriformis Larvae: The Efficiency Increases Together with the Molecular Size.

Worldwide, parasitic gastrointestinal nematodes continue to threaten animal health, welfare, and production in outdoor breeding systems of small ruminants. For more than 50 years, the control of these parasitic worms has relied on the use of commercial synthetic anthelmintics. However, anthelmintic resistance in worm populations is nowadays widespread and requires novel solutions. The use of tannin-rich plants has been suggested as an alternative to synthetic anthelmintics to control gastrointestinal nematodes. The majority of previous studies have focused on the activity of proanthocyanidins (syn condensed tannins), and less is known about ellagitannins. In this study, the effects of 30 structurally unique ellagitannins on the exsheathment of third-stage infective larvae were examined on Haemonchus contortus and Trichostrongylus colubriformis by the in vitro larval exsheathment inhibition assay. Ellagitannins were found to be promising natural anthelmintics as they showed direct inhibition on larval exsheathment for both nematode species. In general, ellagitannins were more efficient at inhibiting the exsheathment of H. contortus larvae than those of T. colubriformis. The efficiency of inhibition increased as the degree of oligomerization or the molecular weight of the ellagitannin increased. Otherwise, we found no other structural features of ellagitannins that significantly affected the anthelmintic activity on the third-stage infective larvae. The effective concentrations were physiologically relevant and should be achievable in the gastrointestinal tract also in in vivo conditions.

Optimal age of Trichostrongylus colubriformis larvae (L3) for the in vitro larval exsheathment inhibition test under tropical conditions

This study identified the optimal age of Trichostrongylus colubriformis larvae (L3) under tropical conditions for the in vitro evaluation of plant extracts using the larval exsheathment inhibition test (LEIT). Two T. colubriformis isolates with different anthelmintic (AH) susceptibility status were used for this study. The L3 of both isolates were maintained on refrigeration (4−5 °C) until use. For the LEIT, the isolates were tested every week during 16 weeks, using a stock solution of Acacia pennatula acetone:water extract at different dilutions (80–1200 μg/mL). Respective positive controls (levamisole 12.5 mM) and negative controls (PBS) were included. Effective concentrations 50 % (EC50), 90 % (EC90) and respective 95 % confidence intervals (95 %CI) for exsheathment inhibition were calculated at 60 min after exposure to chlorine solution. Motility of L3 (migration percentages (M%)) was recorded weekly using the larval migration test (LMT) as an indicator of L3 fitness over time. No correlation was found between L3 age or M% and the extract’s EC50 or EC90 values. However, the EC50 values for the A. pennatula extract ranged from 80 to 200 μg/mL from weeks 2–10. Beyond week 12, larval exsheathment was irregular, with higher EC50 and EC90 values and wider 95 %CI. The M% decreased below 85 % on week 7 for Paraiso isolate, and on week 10 for FMVZ isolate. A linear negative relationship was observed between the age of L3 and M% for both T. colubriformis isolates. The relationship (slope) for both isolates was similar therefore a single linear equation was estimated describing all M% data (r2 = 0.771, df = 164, P < 0.05). Thus, when using these T. colubriformis isolates under our tropical conditions for the evaluation of AH activity of plant extracts with LEIT, the optimal age of L3 is between weeks 2–7, when M% remained above 85 %. The latter may ensure consistent and reproducible exsheathment results for T. colubriformis. Each laboratory must identify optimal conditions to perform the LEIT.

Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia punctata

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC50) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6–9.8 mg mL−1 and 0.15–2.4 mg mL−1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC50 estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FICindex). Combinations had a highly synergistic interaction against larval exsheathment (FICindex < 0.5) except for coumarin:rutin against egg hatching (FICindex> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC50 of bioactive molecules in a range of 43%–64% and 68%–83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC50 for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.

Evaluation of cinnamic acid and six analogues against eggs and larvae of Haemonchus contortus

This study evaluated the in vitro anthelmintic (AH) activity of cinnamic acid and six analogues against eggs and larvae of Haemonchus contortus. Stock solutions of each compound (trans-cinnamic acid, p-coumaric acid, caffeic acid, trans-ferulic acid, trans-sinapic acid, 3,4-dimethoxycinnamic acid, and chlorogenic acid) were prepared in PBS:Tween-20 (1%) for use in the egg hatch test (EHT) and larval exsheathment inhibition test (LEIT) at different concentrations (25–400 μg/mL). The respective effective concentration 50% (EC50) values with 95% confidence intervals were estimated. Mixtures made of all cinnamic acid and its analogues as well as some selected individual compounds were also tested in the EHT. Only ferulic and chlorogenic acids showed AH activity in the EHT (EC50: 245.2 μg/mL (1.26 mM) and 520.8 μg/mL (1.47 mM), respectively) (P < 0.05). A higher EC50 (1628.10 μg/mL) of the mixture of cinnamic acid and its analogues was required to observe activity against eggs mostly blocking the larvae hatching. The analogues’ mixtures tested were less active than ferulic or chlorogenic acid alone. The activity of ferulic and chlorogenic acids against eggs was associated with larvae failing to hatch, and the two compounds exhibited antagonistic effects when evaluated together. All standards had an EC50 lower than 0.42 mM in the LEIT. Caffeic acid had the best activity in the LEIT (EC50 0.04 mM), followed by ferulic acid (EC50 0.11 mM) (P < 0.05). There was no clear, definitive structure-activity relationship for these non-flavonoid polyphenols against eggs or larvae of H. contortus in vitro. This study is the first to directly evaluate cinnamic acid and its derivatives as active compounds against eggs and larvae of H. contortus.

Antiparasitic activities of hydroethanolic extracts of Ipomoea imperati (Vahl) Griseb. (Convolvulaceae).

Ipomoea imperati is widely used in tropical areas to treat several pathological conditions. The effect of this plant against parasitic species has not been investigated even being used for this purpose in the Brazilian northeastern. This study aimed to evaluate the anthelmintic and acaricide potential of a hydroethanolic extract of I. imperati leaves and stolons. I. imperati leaves and stolons were crushed and subjected to maceration in ethanol 70% (v/v), after which the solvent was removed using a rotary evaporator. The chromatographic profile of the extract was obtained by UV Spectrum high-performance liquid chromatography and compounds were identified by liquid chromatography/electrospray ionization tandem mass spectrometry. Identification of the compounds present in the extract was achieved by comparing their retention times and UV spectra with data in the literature. Anthelmintic activity was evaluated by larval exsheathment inhibition assays using Haemonchus contortus larvae and five concentrations of each extract ranging from 0.07 to 1.2 mg/mL. Acaricide activity was evaluated via larval immersion of Rhipicephalus microplus in eight concentrations of each extract ranging from 5.0 to 25.0 mg/mL. Live and dead larvae were counted after 24 hours. The median inhibitory concentration (IC50) for H. contortus larvae and the median lethal concentration (LC50) for R. microplus larvae were calculated. Twelve compounds were observed in the hydroethanolic extract of leaves, with a predominance of the aglycone form of flavonoids and tannins. This extract was effective against H. contortus larvae, presenting an average inhibitory concentration of 0.22 mg/mL, but showed no activity toward R. microplus larvae. The stolon hydroethanolic extract presented 11 compounds, with phenolic acids and glycosylated flavonoids prevailing. This extract showed low activity on R. microplus and no effect on inhibiting H. contortus larval exsheathment at the concentrations tested. This study is the first to assess the anthelmintic and acaricidal activities of I. imperati. Data reported confirm promising potential of I. imperati leaves hydroethanolic extract against H. contortus. This effect could be due to its secondary compounds presents in this extract, such as procyanidin, kaempferol, isoquercitrin and rutin.

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 (> 2.9MJ/kg DM), and varied CT content (from 1.0 to 37.6%). The best AH activity against H. contortus eggs (EC50 = 401.8μg/mL) and L3 (EC50 = 83.1μg/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.

In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0–90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.

Effects of selected Palestinian plants on the in vitro exsheathment of the third stage larvae of gastrointestinal nematodes

BackgroundGastrointestinal parasites are one of the main restrictions to small ruminant production. Their pathological importance is primarily related to the major production losses, in quantity or quality, induced by the direct action of worms. Control of these parasites is based exclusively on the frequent use of anthelmintic drugs. However, the resistance to anthelmintics in worm populations after commercialisation of chemical drugs is now widespread. Therefore, there is a need to find new natural resources to ensure sustainable and effective treatment and control of these parasites. The aim of this study was to evaluate the anthelmintic activity, as minimum inhibitory concentration (IC50 mg/mL), of different plant extracts using larval exsheathment inhibition assay using a two-species but steady population of parasitic nematodes (ca. 20% Teladorsagia circumcinta and 80% Trichostrongylus colubriformis).ResultsThe study showed that the ethanolic extracts of 22 out of the 48 plant extracts, obtained from 46 plant species, have an inhibitory effect >50% (at concentrations of 100 mg/mL) on the third stage larvae (L3) of the nematodes exhibited the strongest inhibition activity (94%) with IC50 of 0.02 mg/mL, where other members of the Rhamnaceae family have shown to possess strong anthelmintic activity (70–89%).ConclusionsPlant extracts are potential rich resources of anthelmintics to combat helminthic diseases. Our results suggest that extracts from Rhamnus elaternus, Epilobium hirsutum, Leucaena leucocephala and Rhamnus palaestinus have promising anthelmintic activity, with potential applications in animal therapeutics and feed.

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.

The use of 1 H-NMR Metabolomics to Optimise the Extraction and Preliminary Identification of Anthelmintic Products from the Leaves of Lysiloma latisiliquum.

Tannin-rich forages are recognised as an important alternative for the control of gastrointestinal nematodes in small ruminants. Lysiloma latisiliquum, a forage commonly consumed by goats and sheep, has shown anthelmintic activity when tested against Haemonchus contortus. However, to date, the metabolites responsible for the activity are not known. To use 1 H-NMR metabolomics in the extraction and identification of anthelmintic metabolites from L. latisiliquum. Eight different solvent systems were compared for the optimum extraction of anthelmintic metabolites from L. latisiliquum. 1 H-NMR spectra of the tannin-free extracts were measured in methanol-d4 using trimethylsilylpropanoic acid (TSP) as internal standard. Extracts were also evaluated for their anthelmintic activity using the larval exsheathment inhibition assay against H. contortus. These data were correlated by multivariate analysis [principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA)] and analysed. To validate the results obtained after the OPLS-DA, a bioassay-guided isolation of bioactive metabolites was conducted. The PCA of the 1 H-NMR data allowed the identification of hydrophilic solvents as those best suited for the extraction of anthelmintics from L. latisiliquum and indicated that the bioactive metabolites are high-polarity, glycosylated products. Similarly, OPLS-DA of the data enabled the detection of activity-related signals, assigned to the glycosylated metabolites quercitrin and arbutin obtained from the bioassay-guided purification of the extract. The results of this investigation confirm metabolomics as a useful tool in the detection of bioactive metabolites in plants without previous phytochemical studies. Copyright © 2017 John Wiley & Sons, Ltd.

In vitro action of Mimosa caesalpinifolia ketone extract on Haemonchus contortus and Trichostrongylus colubriformis

This study aimed to evaluate the in vitro anthelmintic activity of Mimosa caesalpinifolia (sabiá) ketone extract, which is rich in condensed tannins (CT), on Haemonchus contortus and Trichostrongylus colubriformis. The leaves and stems of M. caesalpinifolia were collected, dried in the shade, and ground to a dry powder from which ketone extracts were obtained for in vitro assays. The extracts were diluted in 5% acetone and PBS to concentrations in the range 0.25-2.80 mg mL-¹ and the inhibition of egg hatchability was assayed. Feces collected from goats naturally infected with H. contortus (55%) and T. colubriformis (45%) were macerated and the helminth eggs were collected to conduct in vitro egg hatchability tests. Nematode larvae collected from the feces were used for a larval exsheathment inhibition assay of M. caesalpinifolia leaf and M. caesalpinifolia leaf and stem ketone extracts at concentrations of 0.6 and 1.2 mg mL-¹; three replicates were performed for each concentration. PBS was used as a negative control. Polyvinylpolypyrrolidone (PVPP) was added to the extracts to assess the effect of phenolic compounds on anthelmintic activity. An inhibitory effect of 80.7% and 82.3% was observed on hatchability after treatment with 1.0 and 2.0 mg mL-¹ M. caesalpinifolia leaf ketone extract, respectively. An inhibition rate lower than 75% was observed for 1.0 mg mL-¹ M. caesalpinifolia leaf and stem ketone extract. The M. caesalpinifolia leaf ketone extract larval exsheathment assay resulted in inhibition of 97.3% and 99.8% at concentrations of 0.6 and 1.2 mg mL-¹, respectively. For the leaf and stem ketone extract, the larval exsheathment inhibition was 94.3% at 1.2 mg mL-¹. The leaf and leaf and stem extracts showed no inhibitory effects on larval exsheathment after the addition of PVPP. These results showed that cuticular loss was related to the action of the phenolic compounds in the M. caesalpinifolia extracts. M. caesalpinifolia leaf ketone extract inhibited egg hatchability, but the leaf and stem extract showed lower efficiency, which indicated a greater abundance of active compounds in the leaves. The M. caesalpinifolia ketone extracts were effective in the inhibition of larval exsheathment at the highest concentration tested. Based on these results, in vivo studies should be performed to demonstrate the anthelmintic activity of this plant in goats.

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.