The anti-snake activity of Nectandra angustifolia flavonoids on phospholipase A2: In vitro and in silico evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Lauraceae family includes Nectandra angustifolia a species widely used in the folk medicine of South America against various maladies. It is commonly used to treat different types of processes like inflammation, pain, and snakebites. Snakes of the Bothrops genus are responsible for about 97% of the ophidic accidents in northeastern Argentina. AIM OF THE STUDY: To evaluate the anti-snake activity of the phytochemicals present in N. angustifolia extracts, identify the compounds, and evaluate their inhibitory effect on phospholipase A2 (PLA2) with in vitro and in silico assays. METHODS: Seasonal variations in the alexiteric potential of aqueous, ethanolic and hexanic extracts were evaluated by inhibition of coagulant, haemolytic, and cytotoxic effects of B. diporus venom. The chemical identity of an enriched fraction obtained by bio-guided fractioning was established by UPLC-MS/MS analysis. Molecular docking studies were carried out to investigate the binding mechanisms of the identified compounds to PLA2 enzyme from snake venom. RESULTS: All the extracts inhibited venom coagulant activity. However, spring ethanolic extract achieved 100% inhibition of haemolytic activity. Bio-guide fractioning led to an enriched fraction (F4) with the highest haemolytic inhibition. Five flavonoids were identified in this fraction; molecular docking and Molecular Dynamics (MD) simulations indicated the binding mechanisms of the identified compounds. The carbohydrates present in some of the compounds had a critical effect on the interaction with PLA2. CONCLUSION: This study shows, for the first time, which compounds are responsible for the anti-snake activity in Nectandra angustifolia based on in vitro and in silico assays. The results obtained in this work support the traditional use of this species as anti-snake in folk medicine.

Anti-inflammatory activity of Nectandra angustifolia (Laurel Amarillo) ethanolic extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Nectandra angustifolia belongs to the Lauraceae family and it is widely known in phytomedicine by local inhabitants of South America against various maladies. It is popularly used for the treatment of different types of inflammatory processes, like rheumatism, arthritis and its associated pain. AIM OF THE STUDY: To characterize the phytochemicals in an ethanolic extract of Nectandra angustifolia and to evaluate the total antioxidant content and its anti-inflammatory effect with multiparametric analyses through in vitro assays and an in vivo model. METHODS: Leaves and stems of Nectandra angustifolia were air-dried and an ethanolic extract (NaE) was further obtained. Total phenolic, flavonoid and tannin content were determined and the antioxidant activity was addressed by DPPH and FRAP assays. NaE was first analyzed by HPLC and then two tests were carried out as screening assays for anti-inflammatory activities: red blood cell membrane stabilization and protein denaturation. The non-cytotoxic concentration of NaE was determined for in vitro biological assays using RAW 264.7 (murine macrophages) cell cultures through cell counting with Trypan-blue and XTT assay. Subsequently, the cell cycle of RAW 264.7 cells exposed for 24 h to NaE was analyzed. Additionally, the anti-inflammatory capacity of NaE was evaluated by RT-qPCR of pro-inflammatory cytokines. Furthermore, NF-κB translocation was observed by confocal microscopy at different times. Finally, formalin-induced mice paw inflammation was used as an in vivo model. RESULTS: The chromatographic profile of NaE showed peaks compatible with flavonoids content. NaE exhibited better membrane stabilization effect on HRBC and protection of BSA denaturation than the standard drug (diclofenac). NaE diminished mRNA levels of pro-inflammatory cytokines when added 1-h prior LPS stimulation. Moreover, NaE prevented the translocation of NF-κB to the nucleus and in formalin-induced mice paw inflammation, reduced the edema and the stimulus of inflammatory phase. CONCLUSION: This study shows for the first time, that Nectandra angustifolia ethanolic extract has a high content of flavonoids and that possess antioxidant and anti-inflammatory biological properties as demonstrated by multiparametric analyses from in vitro assays and an in vivo model of inflammation.

Cytotoxic activity induced by the alkaloid extract from Ipomoea carnea on primary murine mixed glial cultures.

The prolonged consumption of Ipomoea carnea produces neurologic symptoms in animals and a typical histological lesion, cytoplasmic vacuolization, especially in neurons. The toxic principles of I. carnea are the alkaloids swainsonine and calystegines B1, B2, B3 and C1. In this study, primary brain cultures from newborn mouse containing mixed glial cells were utilized. These cells were exposed to Ipomoea extracts containing between 0 and 250 µM swainsonine for 48 h. Morphological changes were investigated through Phase Contrast microscopy and Rosenfeld's staining. The extract induced cytoplasmic vacuolization in astrocytes and microglia in a dose dependent manner, being more evident when cultures were exposed to 250 µM of swainsonine. In addition, acridine orange staining evidenced an increase in the number of lysosomes in both microglia and astrocytes cells. Consistent with this, scanning electron microscopy also showed that both types of cells presented morphological characteristics of cell activation. Ultrastructurally, cells showed vacuoles filled with amorphous material and surrounded by a single membrane and also multilayer membranes. Taken together, these findings suggest that swainsonine along with calystegines, are probably responsible for the activation of glial cells due to a possible lysosomal dysfunction and therefore intracellular storage. Our results demonstrate that this in vitro glial cell model is a very good alternative to in vivo studies that require several weeks of animal intoxication to observe similar neurotoxic effects.

Leptin regulates neuropeptides associated with food intake and GnRH secretion.

The present review focused on the most important effects of leptin on the hypothalamus and on how leptin regulates neuropeptides associated with food intake and GnRH secretion. This review of the literature suggests that a reduction in leptin serum concentrations results from lower body energy reserves or poor energy availability, leading to hypothalamic secretion of neuropeptides such as NPY/AgRP and QRFP to stimulate food intake. Under these negative metabolic conditions, GnRH secretion is reduced, impairing reproductive functions. In contrast, when metabolic status is inversed by an increase in food availability, energy reserves or both, leptin serum concentrations increase to an action threshold reversing the pattern of secretion: i.e., reducing NPY/AgRP and QRFP and increasing POMC and Kisspeptin, and thereby reducing food intake and stimulating GnRH secretion to promote reproductive function.

Supplementation with rumen-protected L-arginine-HCl increased fertility in sheep with synchronized estrus.

The aim of the present study was to evaluate the effects of L-arginine-HCl supplementation on ovulation rate, fertility, prolificacy, and serum VEGF concentrations in ewes with synchronized oestrus. Thirty Suffolk ewes with a mean body weight of 45 ± 3 kg and a mean body condition score (BCS) of 2.4 ± 0.28 were synchronized for estrus presentation with a progestin-containing sponge (20 mg Chronogest® CR) for 9 days plus PGF2-α (Lutalyse; Pfizer, USA) on day 7 after the insertion of the sponge. The ewes were divided into two groups; i.e., a control group (n = 15) that was fed on the native pasture (basal diet) and an L-arginine-HCl group (n = 15) that received 7.8 g of rumen-protected L-arginine-HCl from day 5 of the sponge insertion until day 25 after mating plus the basal diet. The L-arginine-HCl was administered daily via an esophageal probe between days 5 and 9 of the synchronization protocol and every third day subsequently. Blood samples were drawn from the jugular vein every 6 days throughout the entire experimental period. The results revealed that the L-arginine-HCl supplementation increased fertility during the synchronized estrus (P = 0.05). However, no effects were observed on the final BCS (P = 0.78), estrus presentation (P = 0.33), multiple ovulations (P = 0.24), prolificacy (P = 0.63), or serum VEGF concentration. In conclusion, L-arginine-HCl supplementation during the period used in this study increased fertility in sheep with synchronized estrus possibly due to improved embryo-fetal survival during early pregnancy.

SDS-PAGE: una herramienta útil en la evaluación preliminar de la actividad alexítera de extractos vegetales / SDS PAGE: a useful tool for preliminary screening of antisnake activity of plant extracts

Some results obtained during our research work in the search of anti-snake compounds from plant origin, allow us to propose sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a valuable method for a fast and reliable screening in order to evaluate plant extracts activity on snake proteins from Bothrops diporus (yarará chica). Such approach will allow to process a larger number of plant extracts and to select the active ones. Venoms used in this study came from B. diporus which was previously vacuum dried. Extracts (aqueous, alcoholic and hexanic) were from native plants: Aristolochia elegans, Aristolochia gibertii, Asclepia curassavica, Cissampelos pareira, Dorstenia brasiliensis, Eclipta prostrata, Iresine diffusa, Mikania micrantha, M. periplocifolia, M. coridifolia, Nectandra angustifolia, N. megapotamica, Sapium haematospermum and Trixis divaricata. The results obtained by SDS-PAGE were compared with those obtained from in vitro assays (coagulation and hemolysis inhibition). The correlation between results obtained from electroforetic and in vitro assays allowed to suggest SDS-PAGE as a suitable technique to assist in preliminary plant screenings for anti-snake activity by snake venom protein interaction with plant compounds.

El desarrollo de nuestro trabajo de investigación en la búsqueda de compuestos alexíteros de origen vegetal nos permite proponer la electroforesis en geles de poliacrilamida en condiciones desnaturalizantes, como método de screening rápido y confiable, para evaluar la actividad de extractos vegetales sobre proteínas del veneno de yarará, de manera de procesar mayor número de muestras vegetales y seleccionar aquellas que son activas. Para el desarrollo de la metodología, se utilizó un pool de veneno de Bothrops diporus desecado al vacío y extractos acuosos, alcohólicos y hexánicos de plantas autóctonas Aristolochia elegans, A. gibertii, Asclepia curassavica, Cissampelos pareira, Dorstenia brasiliensis, Eclipta prostrata, Iresine diffusa, Mikania micrantha, M. periplocifolia, M. coridifolia, Nectandra angustifolia, N. megapotamica, Sapium haematospermum y Trixis divaricata. Se realizaron pruebas in vitro (inhibición de la coagulación y hemólisis) para contrastar con los resultados obtenidos por SDS-PAGE. La correlación de los resultados obtenidos con técnicas in vitro validadas, permite sugerir el empleo de la técnica de SDS-PAGE como una herramienta útil en la evaluación preliminar de la actividad alexítera de extractos vegetales, propiedad evidenciada por la modificación en el perfil de bandas proteicas cuando se compara el veneno puro con el producto de la interacción extracto vegetal-veneno.

Environmental effect on essential oil composition of Aloysia citriodora from Corrientes (Argentina).

Lemon verbena (Aloysia citriodora Palau) is indigenous to South America and was introduced into Europe. It is cultivated mainly due to the lemon-like aroma emitted from its leaves, which are utilized for the preparation of herbal tea reputed to have antispasmodic, antipyretic, sedative and digestive properties. In this work we introduce the enantiomeric distribution of sabinene and limonene by bidimensional gas chromatography (chiral GC-GC) as a genuine quantitative parameter in order to improve the knowledge so far available on A. citriodora oil. Multivariate analysis afforded information on the similarities and differences of wild and cultivated A. citriodora populations during different seasons in the same environmental conditions. The results indicated that it was possible to discard the environmental and seasonal effect on the chemical composition of A. citriodora for wild and cultivated materials belonging to the same genetic origin.