Artepilina C: isolamento, desenvolvimento e validação de metodologia de quantificação por CLUE-EM-Q-TOF e avaliação das atividades biológicas em Caenorhabditis elegans e Zebrafish

Abstract

Artepillin C is the main chemical marker used in quality control of green propolis, which is produced especially in Minas Gerais and marketed mainly to Japan, being Baccharis dracunculifolia DC. (Asteraceae) its main botanical source. Among the techniques that can be used for the analysis of artepillin C, ultra high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) has many advantages. Regarding its biological activities, artepillin C shows antioxidant and antitumor action, and its embryotoxicity and antiaging activity have not been evaluated yet. The aim of this work was to isolate artepillin C, to develop and validate the analytical method of quantification of this substance by UHPLC-MS-Q-TOF, as well as to determine its antiaging activity in Caenorhabditis elegans nematodes and its toxicity in Zebrafish embryos. To isolate the artepillin C from green propolis sample, basic extraction was performed followed by acidification, with subsequent fractionation by vacuum liquid chromatography (VLC) and semipreparative high performance liquid chromatography (HPLC). HPLC and nuclear magnetic resonance (NMR) 1H and 13C analysis were performed for the substance identification. After isolation, the substance was used as standard for validation of quantification methodology of artepillin C in green propolis by UHPLC-MS-Q-TOF. The methodology was specific, precise, accurate and robust for the evaluated conditions. About the biological assays in C. elegans nematode, artepillin C was not toxic for the animal at any of evaluated concentrations. However, artepillin C (125 μM) was not able to prolong its life span. In Zebrafish embryos, the substance was toxic at 250, 125 e 50 μM concentrations, showing the need for further studies about its embryotoxicity.