RESUMO
This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.
RESUMO
The nanoencapsulation of biocomposites with anthelmintic action has been proposed as an alternative for improving their efficiency. Thus, the current study aimed to evaluate the efficacy of carvacryl acetate nanoencapsulated with biopolymers (nCVA) in the control of sheep gastrointestinal nematodes. CVA was nanoencapsulated with chitosan/chichá gum and characterized in terms of its efficacy of encapsulation (EE), yield and zeta potential. The acute toxicity of nCVA was evaluated in mice. For the fecal egg count reduction test, 40 animals were divided into four groups (n = 10) and orally administered the following treatments: G1, 250 mg kg−1 CVA; G2, 250 mg kg−1 nCVA; G3, chitosan/chichá gum (negative control) and G4, 2.5 mg kg−1 monepantel (positive control). Feces were collected on days 0 and 16 posttreatment to determine the eggs per gram of feces (epg). The EE and yield of nCVA were 72.8 and 57.5%, respectively. The nanoparticles showed a size of 764.5 ± 302.5 nm, and the zeta potential at pH 3.2 was +22.0 mV. nCVA presented a 50% lethal dose (LD50) of 2609 mg kg−1. By 16 days posttreatment, CVA, nCVA and monepantel reduced the epg by 52.9.7, 71.5 and 98.7%, respectively, and the epg of sheep treated with nCVA differed from that of the negative control (P > 0.05) but did not differ from that of sheep treated with CVA. In conclusion, the nanoencapsulation of CVA reduced its toxicity, and nCVA showed anthelmintic activity.
Assuntos
Quitosana , Nematoides , Infecções por Nematoides , Doenças dos Ovinos , Animais , Fezes , Bebidas Fermentadas , Camundongos , Monoterpenos , Infecções por Nematoides/tratamento farmacológico , Infecções por Nematoides/veterinária , Ovinos , Doenças dos Ovinos/tratamento farmacológicoRESUMO
Anacardic acids are the main constituents of natural cashew nut shell liquid (CNSL), obtained via the extraction of cashew shells with hexane at room temperature. This raw material presents high technological potential due to its various biological properties. The main components of CNSL are the anacardic acids, salicylic acid derivatives presenting a side chain of fifteen carbon atoms with different degrees of unsaturation (monoene-15:1, diene-15:2, and triene-15:3). Each constituent was isolated by column chromatography using silica gel impregnated with silver nitrate. The structures of the compounds were characterized by nuclear magnetic resonance through complete and unequivocal proton and carbon assignments. The effect of the side chain unsaturation was also evaluated in relation to antioxidant, antifungal and anticholinesterase activities, and toxicity against Artemia salina. The triene anacardic acid provided better results in antioxidant activity assessed by the inhibition of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), higher cytotoxicity against A. salina, and acetylcholinesterase (AChE) inhibition. Thus, increasing the unsaturation of the side chain of anacardic acid increases its action against free radicals, AChE enzyme, and A. salina nauplii. In relation to antifungal activity, an inverse result was obtained, and the linearity of the molecule plays an important role, with monoene being the most active. In conclusion, the changes in structure of anacardic acids, which cause differences in polarity, contribute to the increase or decrease in the biological activity assessed.
RESUMO
Calotropis procera is among the species of medicinal plants that have traditionally been used for the treatment of parasites in small ruminants, stimulating the scientific validation of anthelmintic effects. This study aimed to investigate the chemical composition of ethyl acetate extract of Calotropis procera latex (EAECPL), assess the in vitro effect against Haemonchus contortus and the structural changes caused in the adult worm. The latex was collected, lyophilized and subjected to washing with the ethyl acetate solvent to obtain EAECPL. The constituents of the extract were isolated by column chromatography and identified by (13)C and (1)H nuclear magnetic resonance spectroscopy. The egg hatching test (EHT), larval development test (LDT) and adult worms motility test (WMT) were conducted to evaluate the effectiveness of EAECPL on eggs, larvae and adult of H. contortus, respectively. The worms obtained from the WMT, after 24h exposure to EAECPL or controls were observed on a scanning electron microscope (SEM). The results were analysed by variance analysis and compared with Tukey's test (P<0.05). Three compounds were isolated from EAECPL and identified as urs-19(29)-en-3-yl acetate, (3ß)-Urs-19(29)-en-3-ol, and 1-(2',5'-dimethoxyphenyl)-glycerol. In the EHT, EAECPL inhibited larval hatching by 91.8% at dose of 4mg/ml. In the LDT 1mg/ml inhibited 99.8% larval development. In the WMT, EAECPL in the concentration of 100µg/ml inhibited 100% motility of worms, 12h post-exposition. In the SEM, obvious differences were not detected between the negative control worms and the worms treated with EAECPL. In this study, EAECPL showed an effect on inhibition egg hatching, larval development and motility of the adult worms of H. contortus. This should be related both to the identified compounds, as well as the other compounds present in the EAECPL, acting alone or synergistically.
Assuntos
Calotropis/química , Hemoncose/veterinária , Haemonchus/efeitos dos fármacos , Látex/química , Látex/farmacologia , Abomaso/parasitologia , Acetatos , Animais , Fezes/parasitologia , Liofilização/veterinária , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Haemonchus/crescimento & desenvolvimento , Haemonchus/fisiologia , Haemonchus/ultraestrutura , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura/veterinária , Contagem de Ovos de Parasitas/veterinária , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/parasitologiaRESUMO
Carvacrol is a compound isolated from some essential oils. It has been reported to possess anthelmintic activity. Acetylation of this monoterpene has been proposed as a potential way to reduce the toxicity and enhance the pharmacological effects of carvacrol. This study aimed to evaluate the effect of carvacryl acetate (CA) using in vitro and in vivo assays with gastrointestinal nematodes of small ruminants. The egg hatching test (EHT), larval development test (LDT) and adult worm motility (AWM) assessment were conducted to evaluate the effect of the acetylated product and pure carvacrol on Haemonchus contortus eggs, larvae and adults. The structural changes induced in adult H. contortus were assessed using scanning electron microscopy (SEM). CA and carvacrol acute toxicity was evaluated in mice. Finally, the efficacy of 250 mg/kg CA and 2.5mg/kg monepantel (positive control) were evaluated in 30 sheep naturally infected with gastrointestinal nematodes by the fecal egg count reduction test (FECRT). In vitro tests were analyzed by analysis of variance (ANOVA) followed by comparison with Tukey's test. The efficacy was calculated by the Boot Street program using the arithmetic average. The number of eggs in feces (epg) of the groups were transformed to log (x+1) and subjected to ANOVA to compare differences among the groups by Tukey's test. The level of significance was P<0.05. CA and carvacrol inhibited larval hatching by 89.3 and 97.7% at doses of 8.0 and 1.0mg/ml, respectively. At the concentration of 2mg/ml, CA and carvacrol inhibited 100% of larval development. At a concentration of 200 µg/ml, CA and carvacrol inhibited the motility of adult worms by 100% and 58.3% at 24h post-exposure, respectively. CA caused cuticle and vulvar flap wrinkling and bubbles to emerge from the tegument. Carvacrol caused more discreet effects on the cuticle and vulvar flap. The LD10 and LD50 of CA were 566.7 mg/kg and 1544.5mg/kg, respectively. The LD10 and LD50 of carvacrol were 546.8 mg/kg and 919 mg/kg, respectively. CA and monepantel reduced the epg of sheep by 65.9 and 96.4%, respectively, at 16 days post-treatment. CA showed in vitro and in vivo anthelmintic activity and was less toxic than carvacrol.
Assuntos
Comportamento Animal/efeitos dos fármacos , Monoterpenos/farmacologia , Monoterpenos/toxicidade , Nematoides/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Anti-Helmínticos/toxicidade , Cimenos , Fezes/parasitologia , Feminino , Larva/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Monoterpenos/uso terapêutico , Atividade Motora/efeitos dos fármacos , Nematoides/ultraestrutura , Infecções por Nematoides/tratamento farmacológico , Infecções por Nematoides/veterinária , Óvulo/efeitos dos fármacos , Contagem de Ovos de Parasitas , Distribuição Aleatória , Ovinos , Doenças dos Ovinos/tratamento farmacológicoRESUMO
Aiming to find a phytotherapeutic compounds to treat animal fungal infections, plants commonly found in Northeastern Brazil were evaluated in vitro against Microsporum canis and Candida spp. strains isolated from dogs and cats. The leaf ethanol extracts of Momordica charantia, Calotropis procera, Peschiera affinis and Piper tuberculatum and decoction of Mangifera indica were initially evaluated by the agar-well diffusion method. Four extracts induced growth inhibition zones against M. canis: P. tuberculatum (20 mm), M. indica (14 mm), M. charantia (13 mm) and P. affinis (11 mm). None of them were active against Candida spp. Broth microdilution tests were performed for M. canis strains (n=5), to find the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). The geometric means for the MIC values were 590, 370, 350, 170 ug/mL, and for the MFC values were 1190, 750, 700, 340 ug/mL for M. charantia, P. affinis, P. tuberculatum and M. indica, respectively. Therefore, extracts from M. charantia, P. affinis, P. tuberculatum and M. indica are good candidates to produce antifungal phytotherapics since these extracts demonstrated good activity against M. canis.
Con el objetivo de encontrar compuestos fitoterapéuticos para tratar las infecciones por hongos de los animales, plantas que se encuentran comúnmente en el noreste de Brasil se evaluaron in vitro frente a cepas de Microsporum canis y Candida spp. aisladas de perros y gatos. Los extractos etanólicos de hojas de Momordica charantia, Calotropis procera, Peschiera affinis y Piper tuberculatum y la decocción de Mangifera índica fueron evaluados inicialmente por el método de difusión en pocillos de agar. Cuatro extractos indujeron zonas de inhibición del crecimiento contra M. canis: P. tuberculatum (20 mm), M. índica (14 mm), M. charantia (13 mm) y P. affinis (11 mm). Ninguno de ellos fue activo contra Candida spp. Se realizaron pruebas de microdilución en caldo para las cepas de M. canis (n = 5), para encontrar la concentración mínima inhibitoria (CIM) y la concentración fungicida mínima (CFM). Las medias geométricas de los valores de CIM fueron 590, 370, 350, 170 mg/ml, y para los valores de CFM fueron 1.190, 750, 700, 340 mg/ml de M. charantia, P. affinis, P. tuberculatum y M. indica, respectivamente. Por lo tanto, los extractos de M. charantia, P. affinis, P. tuberculatum y M. indica son buenos candidatos para la producción de fitoterápicos antifúngicos ya que estos extractos demostraron una buena actividad contra M. canis.
Assuntos
Antifúngicos/farmacologia , Candida , Microsporum , Extratos Vegetais/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The high incidence of fungal infections has led to the continuous search for new drugs. Extracts of Luehea paniculata, a tree of multiple medicinal uses, were evaluated for anti-Candida activity, as well as its modulator potential of the Fluconazole antibiotic. Chemical prospecting of ethanol extracts of leaf and bark was carried out, the quantification of total phenols and flavonoids, characterized by the HPLC-DAD technique. The rosmarinic acid and the vitexin flavonoid were observed as major constituents in ELELP and ESWELP, respectively. Antioxidant activity was also evaluated by the method of scavenging the free radical DPPH, and quercetin was used as standard, obtaining IC50 values: 0.341 (mg/mL) for ELELP and 0.235 (mg/mL) for ESWELP. The microdilution assay was performed for antifungal activity against strains of Candida albicans, C. krusei, and C. tropicalis and showed minimum inhibitory concentrations values ≥1024 µg/mL. In the modulator action of extracts on Fluconazole against multiresistant clinical isolates of Candida (subinhibitory concentration minimum of 128 µg/mL), a significant synergism was observed, indicating that the extracts potentiated the antifungal effect against C. tropicalis, where antioxidant flavonoids could be responsible. This is the first report about modifying activity of the antibiotic action of a species of the genus Luehea.
Assuntos
Candida/efeitos dos fármacos , Malvaceae/química , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Antifúngicos/administração & dosagem , Antifúngicos/química , Candida/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/fisiologia , Compostos Fitoquímicos/administração & dosagem , Compostos Fitoquímicos/químicaRESUMO
In recent years, the Brazilian Health Ministry and the World Health Organization have supported research into new technologies that may contribute to the surveillance, new treatments, and control of visceral leishmaniasis within the country. In light of this, the aim of this study was to isolate compounds from plants of the Caatinga biome, and to investigate their toxicity against promastigote and amastigote forms of Leishmania infantum chagasi, the main responsible parasite for South American visceral leishmaniasis, and evaluate their ability to inhibit acetylcholinesterase enzyme (AChE). A screen assay using luciferase-expressing promastigote form and an in situ ELISA assay were used to measure the viability of promastigote and amastigote forms, respectively, after exposure to these substances. The MTT colorimetric assay was performed to determine the toxicity of these compounds in murine monocytic RAW 264.7 cell line. All compounds were tested in vitro for their anti-cholinesterase properties. A coumarin, scoparone, was isolated from Platymiscium floribundum stems, and the flavonoids rutin and quercetin were isolated from Dimorphandra gardneriana beans. These compounds were purified using silica gel column chromatography, eluted with organic solvents in mixtures of increasing polarity, and identified by spectral analysis. In the leishmanicidal assays, the compounds showed dose-dependent efficacy against the extracellular promastigote forms, with an EC50 for scoporone of 21.4µg/mL, quercetin and rutin 26 and 30.3µg/mL, respectively. The flavonoids presented comparable results to the positive control drug, amphotericin B, against the amastigote forms with EC50 for quercetin and rutin of 10.6 and 43.3µg/mL, respectively. All compounds inhibited AChE with inhibition zones varying from 0.8 to 0.6, indicating a possible mechanism of action for leishmacicidal activity.
Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.
Assuntos
Inibidores da Colinesterase/isolamento & purificação , Leishmaniose Visceral/prevenção & controle , Plantas Medicinais/parasitologia , Cumarínicos/isolamento & purificação , Quercetina/isolamento & purificação , Rutina/isolamento & purificaçãoRESUMO
In recent years, the Brazilian Health Ministry and the World Health Organization have supported research into new technologies that may contribute to the surveillance, new treatments, and control of visceral leishmaniasis within the country. In light of this, the aim of this study was to isolate compounds from plants of the Caatinga biome, and to investigate their toxicity against promastigote and amastigote forms of Leishmania infantum chagasi, the main responsible parasite for South American visceral leishmaniasis, and evaluate their ability to inhibit acetylcholinesterase enzyme (AChE). A screen assay using luciferase-expressing promastigote form and an in situ ELISA assay were used to measure the viability of promastigote and amastigote forms, respectively, after exposure to these substances. The MTT colorimetric assay was performed to determine the toxicity of these compounds in murine monocytic RAW 264.7 cell line. All compounds were tested in vitro for their anti-cholinesterase properties. A coumarin, scoparone, was isolated from Platymiscium floribundum stems, and the flavonoids rutin and quercetin were isolated from Dimorphandra gardneriana beans. These compounds were purified using silica gel column chromatography, eluted with organic solvents in mixtures of increasing polarity, and identified by spectral analysis. In the leishmanicidal assays, the compounds showed dose-dependent efficacy against the extracellular promastigote forms, with an EC50 for scoporone of 21.4µg/mL, quercetin and rutin 26 and 30.3µg/mL, respectively. The flavonoids presented comparable results to the positive control drug, amphotericin B, against the amastigote forms with EC50 for quercetin and rutin of 10.6 and 43.3µg/mL, respectively. All compounds inhibited AChE with inhibition zones varying from 0.8 to 0.6, indicating a possible mechanism of action for leishmacicidal activity.(AU)
Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.(AU)
Assuntos
Leishmaniose Visceral/prevenção & controle , Plantas Medicinais/parasitologia , Inibidores da Colinesterase/isolamento & purificação , Cumarínicos/isolamento & purificação , Rutina/isolamento & purificação , Quercetina/isolamento & purificaçãoRESUMO
The aims of this study were to test the antifungal activity, toxicity and chemical composition of essential oil from C. sativum L. fruits. The essential oil, obtained by hydro-distillation, was analyzed by gas chromatography/mass spectroscopy. Linalool was the main constituent (58.22%). The oil was considered bioactive, showing an LC50 value of 23 µg/mL in the Artemia salina lethality test. The antifungal activity was evaluated against Microsporum canis and Candida spp. by the agar-well diffusion method and the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were established by the broth microdilution method. The essential oil induced growth inhibition zones of 28 ± 5.42 and 9.25 ± 0.5 for M. canis and Candida spp. respectively. The MICs and MFCs for M. canis strains ranged from 78 to 620 and 150 to 1,250 µg/mL, and the MICs and MFCs for Candida spp strains ranged from 310 to 620 and 620 to 1,250 µg/mL, respectively. C. sativum essential oil is active in vitro against M. canis and Candida spp. demonstrating good antifungal activity.