Acacia farnesiana (L.) Willd is a shrub legume used as condiment, medicinal plant and bioactive herbage. This species is used in traditional medicine of several countries to relieve the symptoms of ...gastrointestinal diseases, diarrhoea, stomach pain and typhoid as well as astringent, antidysenteric and anthelmintic. Some studies have shown that this plant displayed anthelmintic activity against several gastrointestinal nematode parasites of livestock, and also against parasites of human beings, such as malaria.
This work describes the isolation and chemical identification of the anthelmintic compounds of Acacia farnesiana pods against eggs and infective larvae of the sheep parasitic nematode Haemonchus contortus. The bio-guided chemical fractioning of A. farnesiana pods using ethyl acetate against H. contortus eggs and infective larvae allowed for the identification of naringenin 7-O-(6″-galloylglucoside) (flavonol group) as the compound responsible for the anthelmintic activity against this important parasitic nematode.
Anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and mortality tests. A complete hydroalcoholic extract (HA-E) at 12.5–50 mg/mL, an aqueous fraction (Aq-F) at 3.12–25 mg/mL and an ethyl acetate fraction (EtOAc-F) at 3.12–25 mg/mL were analysed in the first selection phase. The purification of compounds through the chromatographic separation of the organic fraction resulted in nine less complex mixtures (C1F1, C1F2, C1F3, C1F4, C2F1, C2F2, C2F3, C2F4 and C2F5) that were assessed at 0.62–5 mg/mL concentrations. In addition, thiabendazole (0.6 mg/mL) and ivermectin (5 mg/mL) were used as positive controls. Likewise, distilled water and 4% methanol were used as negative controls. The bioactive compounds of EtOAc-F were obtained and characterised through chromatographic processes like open column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and gass chromatography-mass detection (GC-MS). Bioactive compounds were identified by spectroscopy (1H and 13C NMR) and mass spectrometric analysis. Additionally, the H. contortus eggs and infective larvae exposed to the bioactive compounds were observed through environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). Data were analysed based on a completely randomised design using ANOVA through a general linear model.
The EtOAc-F fraction showed the highest ovicidal and larvicidal activities, at close to 100% at 3.12 and 6.25 mg/mL, respectively. The treatments C1F2, C1F3 and C2F3 displayed the main ovicidal activity (80–100%) at 2.5 mg/mL. The major compounds found in these sub-fractions were identified as galloyl derivatives and flavanones, including gallic acid (1), methyl gallate (2), ethyl gallate (3), naringin (4), naringenin 7-O-(4″, 6″-digalloylglucoside) (5), naringenin 7-O-(6″-galloylglucoside) (6) and naringenin (7). Likewise, the ESEM and CLSM images showed that the assessed compounds adhered to the eggshell and the external cuticle of the larvae.
These results indicate that A. farnesiana pods contain nematocidal compounds and might be promising natural anthelmintic agents against H. contortus. This leguminous plant could be used as a nutraceutical food source for the control of gastrointestinal nematodes in small ruminants.
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Several plants of the Fabaceae family have been assessed regarding their high nutritional value and anthelmintic properties. The ovicidal effect of the hydroalcoholic extract (Bm-HAE) and ...subfractions from the aerial parts of Brongniartia montalvoana (Fabaceae) against a mixed strain of gastrointestinal nematodes (GIN) (Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp.) resistant to albendazole sulfoxide, ivermectin and levamisole was evaluated by the egg hatch test (EHT). The Bm-HAE was subjected to liquid-liquid chemical separation with ethyl acetate giving two fractions, an aqueous (Bm-Aq) and an organic (Bm-EtOAct). The purification of the bioactive fraction (Bm-EtOAct) through chromatographic separation resulted in four bioactive subfractions (BmR6, BmR7, BmR8 and BmR10). The treatments were designed as follows: Bm-HAE at 800, 1,500, 3,000 and 6,000 μg/mL, and Bm-Aq, Bm-EtOAct and subfractions (BmR6, BmR7, BmR8 and BmR10) at 100, 200, 400 and 800 μg/mL. Two properly negative controls (distilled water and 2% methanol) and thiabendazole (100 μg/mL) as a positive control were used for each bioassay. The chemical identification of the extract, fractions and subfractions was performed through chromatographic processes like open column chromatography, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC-PDA). Additionally, the GIN eggs exposed to the bioactive compounds were observed through confocal laser scanning microscopy (CLSM). The Bm-HAE showed 99.5% egg hatching inhibition (EHI) at 6,000 μg/mL with a lethal concentration (LC50) of 1110 μg/mL. The Bm-EtOAc fraction displayed 99.1% EHI at 800 μg/mL with LC50 = 180 μg/mL. The ovicidal activity of the four subfractions was similar at 800 μg/mL: BmR6 (92% EHI); BmR7 (100% EHI); BmR8 (97.8%); and BmR10 (99.1%). The HPLC-PDA analysis of the bioactive subfractions allowed identification of p-coumaric acid, ferulic acid and coumarin derivatives as major compounds. The CLSM analysis allowed observation of morphological alterations in unhatched larvae caused by bioactive compounds present in the Bm-EtOAc and BmR10. In addition, the flavonoids eriodyctiol, luteolin and cynaroside were described for the first time for B. montalvoana.
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•Brongniartia montalvoana showed ovicidal activity against multi-drug resistant GIN.•The morphology of unhatched larvae was strongly affected by the organic fraction.•Coumarins, ferulic acid and p-coumaric acid were detected in active subfractions.•Three flavonoids were described for the first time for B. montalvoana.•Colocalization between compounds and larvae fluorescence was observed.
The indiscriminate use of chemical drugs to deworm livestock tends to trigger an anthelmintic resistance problem. In this context, the use of plant extracts rich in secondary metabolites could be an ...alternative method for the control of gastrointestinal nematodes. Baccharis conferta Kunth is a native plant species from Mexico that is widely used by several ethnic groups as forage for farm animals and medicinally to treat gastrointestinal diseases such as acute stomach ache, dysentery, diarrhoea, vomiting, indigestion, colic, intestinal spasms, urinary problems, and cramps. The aim of the present study was to isolate and characterise the ovicidal constituents of B. conferta and to determine a possible mode of action against Haemonchus contortus. The ovicidal activity was determined using the egg hatching inhibition test (EHI) to assess the methanol extract obtained from B. conferta foliage. The dry extract was partitioned (water/ethyl acetate) to obtain an ethyl acetate (BcEtOAc-F) and aqueous fraction. BcEtOAc-F showed an ovicidal activity of 72.32% EHI at 1 mg/mL. The chromatographic fractionation of BcEtOAc-F resulted in three active sub-fractions with higher ovicidal activity: BcC1R4 (99.15% EHI at 1.0 mg/mL); BcC1R5 (92.51% EHI at 0.75 mg/mL); and BcC1R8 (96.8% EHI at 3.0 mg/mL). Chemical analysis of the BcC1R4 fraction allowed the identification of the major active compound, isokaempferide (1, 98.06% EHI at 1 mg/mL). While, 4,5-di-O-acid caffeoylquinic (3; 96.8% EHI at 3 mg/mL) and an inactive flavone (vicenin-2, 2) were identified as the main compounds in BcC1R8. Chemical characterisation of the isolated compounds was performed via spectroscopic (NMR) and spectrometric (UPLC-MS) analyses. Additionally, the environmental and confocal scanning microscopy analyses revealed that isokaempferide was able to cross the eggshell layer without breaking it and attach itself to the embryo, causing its death. The flavonol, isokaempferide, and the hydroxycinamic acid, 4,5-di-O-caffeoylquinic, displayed powerful ovicidal effects, proving to be a potential alternative for the development of a phytodrug for the control of haemonchosis.
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•Baccharis conferta interrupt the egg hatching process of Haemonchus contortus.•Isokaempferide, vicenin-2 and 4,5-di-O-caffeoylquinic acid were the identified compounds.•Isokaempferide has a promising ovicidal activity against the H. contortus nematode.•Colocalisation between compounds and eggs fluorescence was observed.•Mode of action involves crossing the tri-layer complex of H. contortus eggs.
Gastrointestinal nematodes (GIN) are responsible for enormous economic losses worldwide. The use of anthelmintic drugs reduces the parasitic burden in ruminants. However, the excessive use of these ...drugs triggers anthelmintic resistance in these parasites, which leads to a worrisome inefficacy of most of the commercially available antiparasitic drugs. Caesalpinia coriaria is an arboreal legume possessing medical properties, although the antiparasitic potential of this plant against animal parasitic nematodes has not yet been studied. The aim of this study was to assess the in vitro ovicidal activity of a hydro-alcoholic extract (HA-E) from C. coriaria fruits against GIN and to identify the compounds responsible for this activity through an egg hatch inhibition (EHI) assay. GIN eggs obtained from cattle faeces were used in bio-guided assays. The HA-E was subjected to a liquid-liquid extraction using water and ethyl acetate to obtain two fractions, an organic fraction (EtOAc-F, 27% yield) and an aqueous (Aq-F, 73% yield) fraction. The chromatographic fractionation of the EtOAc-F (2 gr) was performed on a glass column packed with silica gel and eluted with dichloromethane/methanol with 10% ascending polarity. The bioactive compounds were analysed using high-performance liquid chromatography (HPLC) with UV detection, nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS). The HA-E extract and the EtOAc-F showed ovicidal activity at a LC50 of 0.92 and 0.16 mg/mL, respectively. A concentration-dependant effect was observed in both treatments. Chromatographic fractionation of the EtOAc-F, allowed for the isolation and characterisation of three important compounds: methyl gallate (1), gallic acid (2) and an unidentified compound (UC). The bioactive molecules (2 and UC) displayed an ovicidal activity close to 100% at 1 mg/mL concentration. The results of this work show that gallic acid (2) isolated from C. coriaria fruits is responsible for its ovicidal activity. The use of Caesalpinia coriaria could be explored in future studies as an environmentally-friendly alternative for the control of GIN in ruminants.
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•A bioguided study with a Caesalpinia coriaria extract showed its nematocidal activity.•The C. coriaria ethyl acetate fraction was lethal to ruminant parasitic nematodes.•Bioactive compounds were separated through TLC and HPLC chromatographic procedures.•Molecule identification was performed using nuclear magnetic resonance.•Gallic acid was the main compound responsible for the observed ovicidal activity.
Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study ...showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one ‘Katahdin’ crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3–68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.
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•The anthelmintic effect of an organic fraction from Acacia farnesiana was assayed.•Haemonchus contortus artificially infected sheep were used.•EtOAc-F was orally administered at 100 mg/kg BW.•A. farnesiana fraction reduced (almost 70%) the EPG and the faecal larvae means.•Galloyl derivatives and flavonoids were found as the major phytochemical compounds.
Byrsonima crassifolia (Malpighiaceae) has been used in traditional medicine for the treatment of some mental-related diseases; however, its specific neuropharmacological activities remain to be ...defined. The present study evaluates the anxiolytic, anticonvulsant, antidepressant, sedative effects produced by the extracts of
Byrsonima crassifolia, and their influence on motor activity in ICR mice. Additionally, we determine the acute toxicity profiles of the
Byrsonima crassifolia extracts and the presence of neuroactive constituents. Our results show that the methanolic extract of
Byrsonima crassifolia produces a significant (
P
<
0.05) antidepressant effect in the forced swimming test in mice at 500
mg/kg dose. However, it does not possess anxiolytic, sedative, or anticonvulsant properties, and does not cause a reduction of mice locomotion (
P
>
0.05). Although the main compound of the methanolic extract was identified as quercetin 3-
O-xyloside (12
mg/kg), our findings suggest that flavonoids, such as rutin (4.4
mg/kg), quercetin (1.4
mg/kg) and hesperidin (0.7
mg/kg), may be involved in the antidepressant effects. To the best of our knowledge, the present study constitutes the first report on the presence of the flavonoids with neuropharmacological activity rutin and hesperidin in
Byrsonima crassifolia. In conclusion, the present results showed that the methanolic extract standardized on flavonoids content of
Byrsonima crassifolia possesses potential antidepressant-like effects in the FST in mice, and could be considered as relatively safe toxicologically with no deaths of mice when orally administered at 2000
mg/kg.
The ovicidal and larvicidal effect of a full aqueous extract (FE) and two phases: an aqueous (Aq-Ph) and an ethyl acetate (EtOAc-Ph) from
(
) stems and leaves against
(
) were assessed. The egg ...hatching inhibition (EHI) assay and larval mortality (LM) test were performed by triplicate in 96-well micro-titration plates (n=4 wells). The FE against
eggs and larvae was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL; and 30, 60, 90, 120,150 and 200 mg/ mL, respectively. The ovicidal effect of Aq-F and EtOAc-F was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL. Plates were incubated at 28 °C for 48 (ovicidal assay) and 72 h (larvicidal assay). The EHI results were considered based on the mean number of eggs hatching failure after 48 h exposure. The LM was recorded after 72 h exposure to the
phases and expressed as mortality percentage. The
FE caused 96 and 100% EHI at 10 and 20 mg/mL, respectively; meanwhile, 74% LM was recorded at 200 mg/mL (p<0.05). The Aq-Ph showed 78.5% EHI at 2.5 mg/mL. Likewise, the EtOAc-Ph caused 100% EHI in almost all concentrations. Eighteen compounds including alkaloids, cumarins, triterpens, flavonoids, tannins, saponins and sterols were identified by GC-MS analysis. The results indicate that the aqueous extract from
possesses bioactive compounds with
nematocidal activity against
; mainly in the EtOAc-Ph. Further studies should be performed to elucidate those compounds searching for alternative methods of control of the sheep haemonchosis.
Acacia cochliacantha is a small tree whose foliage is traditionally used in Mexico for treatment of kidney pain, gastrointestinal illnesses and to kill intestinal parasites. In recent decades, the ...study of vegetal extracts has offered other possible alternatives for the control of Haemonchus contortus. Considering that this nematode affects dramatically the health and productivity of small ruminants, the aim of this study was to identify the anthelmintic compounds from A. cochliacantha hydro-alcoholic extract (HA-E) through an ovicidal test.
In vitro egg hatch assay was conducted to determinate the anthelmintic effects of a HA-E (60g). Liquid-liquid ethyl acetate/water extraction gave two fractions (EtOAc-F, 1.92g; Aq-F; 58.1g). The less polar compounds from ethyl acetate fraction were extracted by addition of dichloromethane offering a precipitate phase (Mt-F, 1.25g) and a soluble mixture (DCMt-F 1.15g). All fractions were evaluated for ovicidal activity obtaining the egg hatching inhibition (EHI, 0.07–25mg/mL). Ivermectin (0.5mg/mL) was used as a reference drug (positive control), and distilled water, 2.5% DMSO and 2% methanol were used as negative controls. The isolated compounds from the most active fractions were subjected to spectroscopic (1H NMR) Spectrometric (MS) and UV HPLC analysis in order to identify the bioactive compounds.
The less polar treatments (AcOEt-F, DCMt-F, DCMt-P) showed the highest ovicidal activities (98–100% EHI; at 0.62–1.56mg/mL) and the major compounds found in these fractions were identified as caffeoyl and coumaroyl derivatives, including caffeic acid (1), p-coumaric acid (2), ferulic acid (3), methyl caffeate (4), methyl-p-coumarate (5), methyl ferulate (6) and quercetin. In case of the less active fractions (Aq-F, Mt-F) were constituted principally by glycosylated flavonoids.
These results show that caffeoyl and coumaroyl derivatives from Acacia cochliacantha leaves had promising anthelmintic activity against Haemonchus contortus. This leguminous may offer an alternative source for the control of gastrointestinal nematodes of small ruminants.
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An infusion prepared with aerial parts from
Galphimia glauca has been widely used in Mexican traditional medicine as a remedy for nervous excitement. The sedative activity of a methanolic extract ...from this plant has been demonstrated by neuropharmacological tests. This effect was attributed to the nor-secotriterpene named galphimine B (GB). In the present work, the anxiolytic and antidepressant-like effects of
G. glauca methanolic extract (standardized on GB content, 8.3
mg/g) were assayed by using the elevated plus-maze, light–dark test and the forced swimming paradigm, on ICR albino mice. This extract, administered orally, three times (24, 18 and 1
h before the test), and in different doses (125, 250, 500, 1000 and 2000
mg/kg) was able to increase significantly (
p
<
0.05
) the number of entries, as well as the time spent in the open arms of the elevated plus-maze, indicating an anxiolytic-like effect. A similar effect was observed in the light–dark paradigm test, the time spent in the light box was increased in treated mice. Nevertheless, this treatment was unable to change any parameter in the forced swimming test. Altogether, these results suggest an anxiolytic-like effect to the methanolic standardized extract of
G. glauca on ICR inbred mice.
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