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.

The effects of Cissampelos pareira extract on envenomation induced by Bothropsdiporus snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophidian accidents are a serious public health problem in Argentina; the Bothrops species is responsible for 97% of these accidents, and in particular, B. diporus is responsible for 80% of them. In the northeast of the country (Corrientes Provinces), Cissampelos pareira L. (Menispermaceae) is commonly used against the venom of B. diporus; its use is described in almost all ethnobotanical literature from countries where the plant grows. AIM OF THE STUDY: In this study, the in vitro and in vivo antivenom activities of C. pareira extracts were evaluated against B. diporus venom, with a particular focus on the local effects associated with envenoming. The seasonal influence on the chemical composition of the active extracts was also studied, in order determine the associated range of variability and its influence on the antivenom activity. MATERIALS AND METHODS: This research was conducted using aerial parts (leaves, flowers, tender stems) and roots of Cissampelos pareira collected from two different phytogeographic regions of Corrientes (Argentina); Paso de la Patria and Lomas de Vallejos. In addition, to perform a seasonal analysis and to evaluate the metabolic stability, material was collected at three different growth stages. In vivo and in vitro anti-snake venom activities were tested, and a bio-guided chromatographic separation was performed in order to determine the active chemicals involved. The fractions obtained were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the chemical profile of the most active constituent was analyzed by ultra high-performance liquid chromatography coupled to quadrupole/high-resolution mass spectrometry (Q-Orbitrap). (UHPLC-MS). RESULTS: The alcoholic extract was found to be the most active The bio-guided fractionation allowed selection one fraction to be analyzed by UHPLC-MS in order to identify the components responsible for the activities found; this identified five possible flavonoids. CONCLUSIONS: Our studies of the activity of C. pareira against the venom of B. diporus have confirmed that this species possesses inhibitory effects in both in vitro and in vivo models. Moreover, the present data demonstrate that certain flavonoids may mitigate some of the venom-induced local tissue damage.

Neutralizing effects of Nectandra angustifolia extracts against Bothrops neuwiedi snake venom.

Leaves extracts and essential oil of Nectandra angustifolia were explored for the first time for neutralization of Bothrops neuwiedi diporus snake venom. The ethanol extract was the most active and inhibited both venom activities (hemolytic and coagulant), while the oil was only active on the coagulant activity. These observations confirmed that certain medicinal plants from Corrientes and Chaco Provinces possess significant snake venom neutralizing capacity and need further examination for their active constituents. Analysis by GC and GC-MS of the essential oil and the enantiomeric excess found for alpha-pinene, beta-pinene and limonene allowed a better characterization of this species.

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.

Chemical variability of essential oils of Lippia alba (Miller) N. E. Brown growing in Costa Rica and Argentina.

Lippia alba (Verbenaceae) is a shrub whose essential oil has important biological, pharmacological, and aromatizing properties. The species has a natural range from Central America to Argentina, being cultivated for its commercial value in Brazil, Argentina and Guatemala, and has been introduced into India and Spain. To reach the economic potential of the plant, the present study was aimed at evaluating L. alba for different chemotypes. The composition of the essential oil from two native populations of L. alba, collected from Argentina and two accessions from Costa Rica, were screened by GC and GC-MS. The results obtained led us to adopt the concept of a biodistribution map, as was proposed previously for the species, representing an approach to the natural biological distribution of the species in America based on the chemotypes described and their geographical distribution. Moreover, the biodiversity reported for the species (seven of eight chemotypes described for L. alba are present in Argentina) suggests the southern region of South America as the centre of distribution for L. alba.