Red blood cell hemolytic assay: an alternative to assess cytotoxicity of essential oils

Given the growing ethical questioning regarding the use of animal testing in the analysis of toxicity research and in vitro assay validation is a viable alternative to animal replacement. In this sense, this study evaluated the hemolytic activity of four essential oils extracted from Syzygiumjambolanum, Lippiagracilis, Myracrodruonurundeuva and Bixaorellanaplants, by applying the red blood cell hemolytic assay, since it is a simple technique, low cost, and effective to screening natural extracts cytotoxicity. With this purpose, a hemolytic assay was carried out in micro-centrifuge tubes containing 10% sheep erythrocytes suspension using samples in different concentrations previously established, at 37 °C/60 min. The hemolysis percentage was determined by spectrophotometer reading at 540 nm. Assays were performed in triplicate. The results showed values below 10% indicating that the essential oils did not show any hemolytic activity against the erythrocytes suspension, i.e. no toxicity was observed. These promising results are important for the use of the substances as well as to confirm the applicability and relevance of the test.