M A Zarza-Albarrán1, A Olmedo-Juárez2, R Rojo-Rubio3, P Mendoza-de Gives4, M González-Cortazar5, D Tapia-Maruri6, J Mondragón-Ancelmo3, C García-Hernández3, Ever A Blé-González7, A Zamilpa8. 1. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Carr. Fed. Cuernavaca-Cuautla No. 8534, Jiutepec, Morelos, Mexico; Centro Universitario UAEM Temascaltepec, Universidad Autónoma del Estado de México, Km 67.5 Carr. Fed, Toluca-Tejupilco, Temascaltepec, Mexico, Mexico. 2. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Carr. Fed. Cuernavaca-Cuautla No. 8534, Jiutepec, Morelos, Mexico. Electronic address: olmedo.agustin@inifap.gob.mx. 3. Centro Universitario UAEM Temascaltepec, Universidad Autónoma del Estado de México, Km 67.5 Carr. Fed, Toluca-Tejupilco, Temascaltepec, Mexico, Mexico. 4. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Carr. Fed. Cuernavaca-Cuautla No. 8534, Jiutepec, Morelos, Mexico. 5. Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica del Sur, Argentina No. 1, Xochitepec, Morelos, Mexico. 6. Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Carretera Yautepec-Jojutla Km 6, calle CEPROBI N°8, Col. San Isidro, Yautepec, Morelos, C.P. 62731, Mexico. 7. Universidad Juárez Autónoma de Tabasco, Carretera Cunduacán-Jalpa Km. 0.5, 86690, Cunduacán, Tabasco, Mexico. 8. Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica del Sur, Argentina No. 1, Xochitepec, Morelos, Mexico. Electronic address: azamilpa_2000@yahoo.com.mx.
Abstract
ETHNOPHARMACOLOGY RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
ETHNOPHARMACOLOGY RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Authors: Gloria Sarahi Castañeda-Ramírez; Juan Felipe de Jesús Torres-Acosta; José Ernesto Sánchez; Pedro Mendoza-de-Gives; Manases González-Cortázar; Alejandro Zamilpa; Laith Khalil Tawfeeq Al-Ani; Carlos Sandoval-Castro; Filippe Elias de Freitas Soares; Liliana Aguilar-Marcelino Journal: Biomed Res Int Date: 2020-06-24 Impact factor: 3.411
Authors: Agustín Olmedo-Juárez; Ana Laura Jimenez-Chino; Alejandro Bugarin; Alejandro Zamilpa; Pedro Mendoza-de Gives; Abel Villa-Mancera; María Eugenia López-Arellano; Jaime Olivares-Pérez; Edgar Jesús Delgado-Núñez; Manases González-Cortazar Journal: Plants (Basel) Date: 2022-09-28