Morfoanatomia foliar, efeitos toxicológicos e anti-inflamatório em embriões/larvas de zebrafish (Danio rerio) após a exposição à diferentes extratos e ao óleo essencial de Siparuna guianensis Aublet (Siparunaceae)

Abstract

Siparuna guianenis Aublet is an aromatic plant, popularly known as negramina. Its leaves are traditionally used in the treatment of several diseases, as an anti-inflammatory and antipyretic.

This work aims to verify the anatomical characteristics of the leaf blade, the toxicity, the anti- inflammatory potential, the capacity of caudal fin regeneration and activity of antioxidant

enzymes of the aqueous (AE), hydroethanolic (HE), methanolic (ME) and essential oil (EO) extracts of S. guianensis leaves in zebrafish embryos and larvae. To verify the anatomical characteristics, an equation was determined to estimate the leaf area, the quantification and characterization of stomata and different histochemical methods were used. From the leaves, three types of extracts were prepared using different solvents (methanol, alcohol 70o and distilled water) and the EO was obtained by hydrodistillation. The main phenolic compounds present in the extracts were determined using HPLC analysis and the identification and quantification of the EO by chromatographic analysis. Toxicity and teratogenicity were investigated using the zebrafish model. Zebrafish embryos were treated with different concentrations of the extracts (10 to 0.08mg/ml) and OE (200 to 0.39μg/ml) for a period of 120 hours, and mortality, hatching and malformations were evaluated. The non-lethal and teratogenic concentrations were used for the analyses of neutrophil migration; caudal fin regeneration; apoptosis and activity of the antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). It was identified in the anatomical analysis of the leaves the presence of stomas in the epidermis to classify the leaves as anfihypoestomatics. The existence of stellate tector trichomes in the abaxial epidermis and the presence of secondary metabolites such as phenolic compounds, alkaloids, tannin and lignin were identified and the mathematical equation obtained was through linear regression by means of the length and width relationship, using the model Y=3.5833+0.6715x, with a determination coefficient of 0.97. It was identified in the extracts the presence of phenolic compounds with trans-cinnamic acid. The extracts showed toxicity, being for EA at concentrations from 2.5 to 10mg/ml and for EH and EM between 0.62 to 10mg/ml. The larvae treated with EA didn’t exhibit malformations. All tested extracts demonstrate anti-inflammatory activity, however they do not show good performance in caudal fin regeneration. The extracts did not influence SOD and CAT activity. EA didn’t induce apoptotic cells unlike EH and EM. In OE was identified the presence of the monoterpene β-Myrcene (24.72%). In the inflammation test, the different concentrations showed ability to reduce the migration of neutrophils to the site of the larvae, in the regeneration of the caudal fin the EA showed a lower regeneration rate than the controls (DMSO 1% and Medium E3). No influence of EO on antioxidant enzymes was verified. Finally, apoptotic cells were identified in the region of the heart at the concentration of 12.5μg/ml. Conclude that the stomata of this species are classified as paracytic and the EA, EH, EM and the OE of the leaves of S. guianensis present anti-inflammatory activity by reducing the migration of neutrophils.