Planta Med 2015; 81 - PM_160
DOI: 10.1055/s-0035-1565537

Application of Zebrafish embryos toxicity test to evaluate the alkaloid fraction of Psychotria deflexa

PR Bertelli 1, EP Rico 2, LD Grünspan 2, R Biegelmeyer 1, LC Klein-Júnior 1, 3, Y Vander Heyden 3, AL Gasper 4, SAL Bordignon 5, DL Oliveira 2, AT Henriques 1
  • 1Laboratory of Pharmacognosy and Quality Control of Phytomedicines, Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
  • 2Department of Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
  • 3Department of Analytical Chemistry and Pharmaceutical Technology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium
  • 4Laboratory of Botany, Universidade Regional de Blumenau, Blumenau, Brazil
  • 5Laboratory of Biology, Centro Universitário La Salle, Canoas, Brazil

The Psychotria L. genus is chemically characterized by the presence of indole alkaloids. Our research group has already demonstrated that these alkaloids are able to inhibit enzymes in vitro, such as cholinesterases and monoamine oxidases. The fish embryo acute toxicity (FET) test is an important dose-screening tool: it is a rapid, simple and effective method. This screening established by the OECD 236 (Organization for Economic Co-operation and Development) was used to define the best dose that can be use in the future studies to evaluate the biochemical parameters in the nervous system of adults Zebrafish. In addition, a preliminary chemical evaluation of enriched alkaloids fraction (EFA) obtained from P. deflexa leaves is also objective of this study. The leaves of P. deflexa were collected in the Rain Forest at Blumenau (SC-Brazil). The air-dried material was powdered and macerated with EtOH. After solvent evaporation, the crude extract was submitted to acid-base partition to obtain the EAF. This fraction was analyzed by HPLC-DAD and High-resolution Mass Spectrometry (HRMS) to determine their chemical profiles. In addition, the FET evaluation of the EAF (1; 5; 10; 20; 30; 40; 50; 75 e 100 µg/mL) was performed. The following parameters were evaluated: coagulation of fertilized eggs; lack of somite formation; lack of detachment of the tail-bud from the yolk sac; and lack of heartbeat. The HPLC-DAD analysis showed the presence of several monoterpene indole alkaloids in the EAF. HRMS analysis identified the major product having a molecular ion m/z= 517.2188 (C26H32N2O9) as strictosidinic acid. The FET test demonstrated that the EAF (1 – 50 µg/mL) did not produce morphological changes after 96 hpf. However, higher concentrations resulted in coagulation of embryos at the period of 48 hpf. From these results it was possible to establish an appropriate dose for the study of biochemical parameters in zebrafish central nervous system such as cholinergic activity.