Characterization and cytotoxic activity on glial cells of alkaloid-enriched extracts from pods of the plants Prosopis flexuosa and Prosopis nigra (Fabaceae) / Caracterización, y actividad citotóxica en células gliales, de extractos enriquecidos con alcaloides de vainas de las plantas Prosopis flexuosa y Prosopis nigra (Fabaceae)

Introduction: Prosopis spp. pods have shown to be a potential source of protein and energy in livestock. However, prolonged ingestion of some of these species produces neurological symptoms in ruminants. Objective: In the present study, the alkaloid content and the in vitro neurotoxic activity of alkaloid enriched-extracts from P. flexuosa and P. nigra pods were determined in order to elucidate the mechanism of animal poisoning caused by these species. Methods: The main alkaloids present in both extracts were analysed by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). The cytotoxic activity of Prosopis alkaloid enriched-extracts in primary mixed glial cell culture was assessed by phase contrast microscopy and using neutral red, and lactate dehydrogenase (LDH) activity assays. Results: Juliprosine and juliprosopine were identified in P. flexuosa pods, while the absence of these alkaloids in P. nigra was confirmed. Both extracts (5-30 μg/mL) induced in a dose dependent manner, morphological alterations, such as swelling, enlargement and detachment from the culture surface. Consistent with this, decrease in cell viability and release of LDH 48 hours after exposure, revealed that P. flexuosa pods was significantly more cytotoxic than P. nigra. Conclusions: In P. flexuosa pods, juliprosine and juliprosopine alkaloids were identified for the first time. Moreover, the present study suggests that the cytotoxic effect displayed by both extracts is due to its alkaloid content. However, the presence of piperidine alkaloids in P. flexuosa could explain the greater cytotoxicity on glial cells with respect to P. nigra that was not shown to contain these alkaloids.

Introducción: Las vainas de diversas especies de Prosopis muestran ser una potencial fuente de proteínas y energía para el ganado. Sin embargo, la ingestión prolongada de algunas de estas especies produce síntomas neurológicos en los rumiantes. Objetivo: En el presente estudio se determinó el contenido de alcaloides y la actividad neurotóxica in vitro de los extractos enriquecidos con alcaloides obtenidos en las vainas de P. flexuosa y P. nigra, con el fin de dilucidar el mecanismo de la intoxicación animal causada por estas especies. Métodos: Los principales alcaloides presentes en ambos extractos se analizaron mediante cromatografía líquida de alto rendimiento-espectrometría de masas de alta resolución (HPLC-HRMS). La actividad citotóxica de los extractos enriquecidos con alcaloides de Prosopis se determinó en cultivos primarios de células gliales mixtas y se evaluó mediante microscopía de contraste de fase y utilizando ensayos de actividad de rojo neutro y de deshidrogenasa láctica (LDH). Resultados: Se identificaron la juliprosina y la juliprosopina en las vainas de P. flexuosa, mientras que se confirmó la ausencia de estos alcaloides piperidínicos en P. nigra. Ambos extractos (5-30 μg/mL) indujeron, de manera dependiente a la dosis, alteraciones morfológicas, como hinchazón, agrandamiento y desprendimiento de la superficie de cultivo. En consecuencia, la disminución de la viabilidad celular y la liberación de la LDH después de 48 horas de exposición, reveló que las vainas de P. flexuosa eran significativamente más citotóxicas que las de P. nigra. Conclusiones: El presente estudio muestra la presencia de los alcaloides juliprosina y juliprosopina en vainas de P. flexuosa y sugiere que el efecto citotóxico mostrado por ambos extractos se debe al contenido de alcaloides. Sin embargo, la presencia de estos alcaloides piperidínicos en P. flexuosa podría explicar la mayor citotoxicidad en las células gliales con respecto a P. nigra que no mostró que tuviera estos alcaloides.

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)