Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Anthelmintic evaluation and essential oils composition of Hyptis dilatata Benth. and Mesosphaerum suaveolens Kuntze from the Brazilian Amazon.

Hyptis dilatata and Mesosphaerum suaveolens belong to Lamiaceae, are aromatic and medicinal subshrubs with antiparasitic potential and occurrence in the Amazon Region. The essential oils of both species were extracted, analyzed, and identified by GC and GC-MS and then evaluated their anthelmintic activities against the nematode Caenorhabditis elegans. Primary constituents of the samples of H. dilatata oils were limonene (72.6%), myrcene (11.5%), and p-cymene (10.3%) to PAMA19 sample, and camphor (25.5%), α-pinene (25.4%), 1,8-cineole (18.8%), ß-pinene (12.0%), and limonene (5.9%) to PAMA108 sample, while in the oil of M. suaveolens, PAMA131 sample, predominated bicyclogermacrene (23.5%, 1,8-cineole (23.0%), germacrene D (17.2%), and (E)-caryophyllene (10.4%). The sample oil of H. dilatata (PAMA108) exhibited the lower anthelmintic inhibitory concentration, with an IC50 value of 2.09 mg/mL for C. elegans Bristol N2 strain, while the oils of H. dilatata (PAMA19) and M. suaveolens (PAMA131) showed an IC50 up of 4 mg/mL for C. elegans IVR15 and Bristol N2 strains. These results suggest that the H. dilatata and M. suaveolens oils constituents' combination can be helpful as a nematicidal agent due to their synergistic action.

Parkia platycephala lectin enhances the antibiotic activity against multi-resistant bacterial strains and inhibits the development of Haemonchus contortus.

Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2 mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31 mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MIC = 50 mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and 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.

Myracrodruon urundeuva seed exudates proteome and anthelmintic activity against Haemonchus contortus.

Seed exudates are plant-derived natural bioactive compounds consisting of a complex mixture of organic and inorganic molecules. Plant seed exudates have been poorly studied against parasite nematodes. This study was undertaken to identify proteins in the Myracrodruon urundeuva seed exudates and to assess the anthelmintic activity against Haemonchus contortus, an important parasite of small ruminants. M. urundeuva seed exudates (SEX) was obtained after immersion of seeds in sodium acetate buffer. SEX was fractionated with ammonium sulfate at 0-90% concentration to generate the ressuspended pellet (SEXF1) and the supernatant (SEXF2). SEX, SEXF1, and SEXF2 were exhaustively dialyzed against distilled water (cut-off: 12 kDa) and the protein contents determined. Mass spectrometry analyses of SEX, SEXF1, and SEXF2 were done to identify proteins and secondary metabolites. The seed exudates contained protease, protease inhibitor, peptidase, chitinase, and lipases as well as the low molecular weight secondary compounds ellagic acid and quercetin rhamnoside. SEX inhibited H. contortus larval development (LDA) (IC50 = 0.29 mg mL-1), but did not affect larval exsheathment (LEIA). On the other hand, although SEXF1 and SEXF2 inhibited H. contortus LEIA (IC50 = 1.04 and 0.93 mg mL-1, respectively), they showed even greater inhibition efficiency of H. contortus larval development (IC50 = 0.29 and 0.42 mg mL-1, respectively). To the best of our knowledge, this study is the first to show the anthelmintic activity of plant exudates against a gastrointestinal nematode. Moreover, it suggests the potential of exuded proteins as candidates to negatively interfere with H. contortus life cycle.

Anthelmintic activity of plant extracts from Brazilian savanna.

Helminth infections represent a serious problem for the production of small ruminants that is currently aggravated by resistance to anthelmintic products and has induced a search for control alternatives, such as natural products. In this study, extracts of Turnera ulmifolia L. (leaves and roots), Parkia platycephala Benth. (leaves and seeds) and Dimorphandra gardneriana Tul. (leaves and bark), which have been cited in ethnoveterinary studies and selected naturally by goats in the cerrado (Brazilian savanna), were tested in vitro against Haemonchus contortus. Hydroacetonic (ACT) and hydroalcoholic (ETH) extracts were evaluated using an Egg Hatching Assay (EHA), a Larval Exsheathment Inhibition Assay (LEIA) and a Larval Development Assay (LDA). A second set of incubations was performed using polyvinylpolypyrrolidone (PVPP) to determine the influence of polyphenols on the anthelmintic effects of EHA and LEIA. Data from each extract were used to calculate inhibition concentrations (IC50). All tested extracts showed activity against at least one life stage of H. contortus. The use of PVPP revealed that the tannins are not the only extracts of secondary metabolites responsible for the anthelmintic effects. The results showed clear in vitro anthelmintic activities against H. contortus at different stages and indicated the potential use of these species as a promising alternative approach to control helminthic infections of small ruminants.

Detection of genotoxic, cytotoxic, and protective activities of Eugenia dysenterica DC. (Myrtaceae) in mice.

Eugenia dysenterica DC. (Myrtaceae), popularly known in Brazil as cagaiteira, is a widespread plant species in the Brazilian Cerrado. In folk medicine, the leaves of this plant are used to treat diarrhea and dysentery. The fruits are used for fresh consumption and industrial purposes. Because of the use of this plant as a therapeutic resource and food, the present study evaluated the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic effects of the lyophilized ethanolic leaf extract of E. dysenterica using the mouse bone marrow micronucleus test. The genotoxicity and antigenotoxicity of this extract were evaluated using the frequency of micronucleated polychromatic erythrocytes, and the cytotoxicity and anticytotoxicity were assessed by the polychromatic and normochromatic erythrocyte ratio. According to our results, the lyophilized ethanolic leaf extract of E. dysenterica exhibited genotoxic and cytotoxic effects at the higher doses and protection against cyclophosphamide-induced genotoxic and cytotoxic actions at all doses tested.

Assessment of Duguetia furfuracea genotoxic and cytotoxic activity in bacteria and mice.

Duguetia furfuracea (St. Hil.) Benth & Hook f. (1862), popularly known as "sofre-do-rim-quem-quer" and "araticum-seco", is a shrub of the Annonaceae family that occurs in several regions of Brazil. In folk medicine the infusion of its leaves and twigs is used to treat rheumatism and renal colic, whereas the seed powder is mixed with water to treat pediculosis. Previous studies have described biological activities of this plant with cytotoxic, antitumoral, trypanocidal, leishmanicidal, antiplasmodial and antiprotozoal effects. In the present work, genotoxicity and cytotoxicity of Duguetia furfuracea lyophilized leaf extract were evaluated using the prophage λ induction test (SOS-Inductest) and mouse bone marrow micronucleus test. Our results showed that Duguetia furfuracea lyophilized leaf extract did not present an increase either in the induction of prophage λ (P>0.05) using the SOS-inductest or in the micronucleated polychromatic erythrocytes (P>0.05) using the micronucleus test, suggesting absence of genotoxicity in both tests. On the other hand, a significant decrease in the number of bacteria, (P<0.05), as well as a significant decrease in the polychromatic erythrocytes and normochromatic erythrocytes ratio, (P<0.05), were observed, showing the cytotoxic action of Duguetia furfuracea lyophilized leaf extract. Thus, Duguetia furfuracea did not present the genotoxic action, but showed a cytotoxic effect in both assays utilized in the present work.