ABSTRACT
Bufadienolides are the main active compounds in the Bufonidae family of frogs. Recent studies have demonstrated cytotoxic and/or antitumor activity in these molecules. A HPLC-DAD method was developed and validated to quantify three bufadienolides (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of the cane toad poison frogs and smooth-sided toad. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 × 4.6 mm, 5 µm), using gradient elution with acetonitrile and water, at a flow rate of 1.0 mL min(-1) and detection at 296 nm. The method showed linearity (r > 0.999) and adequate recovery values (86%-111%). The limits of quantification of bufadienolides were 7.4 µg mL(-1) for telocinobufagin, 4.2 µg mL(-1) for marinobufagin and 4.0 µg mL(-1) for bufalin. Intraday and interday values of the method were evaluated and presented standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of the cane toad, which showed a content of 60% of marinobufagin. The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise, requiring the development of a technique that presents a greater selectivity.
Subject(s)
Amphibian Venoms/chemistry , Bufanolides/analysis , Bufo marinus/metabolism , Bufonidae/metabolism , Chromatography, High Pressure Liquid/methods , Spectrophotometry, Ultraviolet/methods , Animals , Limit of Detection , Reproducibility of ResultsABSTRACT
This study examined the effect of glyphosate-based herbicide (Roundup Original), the major herbicide used in soybean crops in Mato Grosso state, at concentrations of 0, 2.25, 4.5, 7.5, and 15 mg L(-1) on metabolic and behavior parameters of the hybrid fish surubim in an acute exposure lasting 96 h. Glycogen content, glucose, lactate, and protein levels were measured in different tissues. Plasma levels of cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also determined. Ventilatory frequency (VF) and swimming activity (SA) were considered behavior parameters. Results showed that herbicide exposure decreased plasma glucose levels and increased it in surubim liver. Lactate increased in both plasma and liver but decreased in muscle. Protein levels decreased in plasma and muscle but increased in liver. After herbicide exposure, liver and muscle glycogen was decreased. Cholesterol levels decreased in plasma at all concentrations tested. Plasma ALT increased, and no alterations were recorded for AST levels. VF increased after glyphosate exposure (5 min) and decreased after 96 h. SA showed differences among all groups (5 min). At the end of 96 h, SA was altered by the 7.5 mg L(-1) concentration. Fish used anaerobic glycolysis as indicated by generally decreased glycogen levels and decreased lactate levels in muscle but increased ones in plasma and liver. We suggest that the studied parameters could be used as indicators of herbicide toxicity in surubim and may provide extremely important information for understanding the biology of the animal and its responsiveness to external stimuli (stressors).