Introduction: Ayahuasca is a traditional psychoactive tea of Amazonian indigenous, used medicinal and spiritual purposes. Wide variation in the concentration of N,N-dimethyltryptamine (DMT), Harmaline (HRL), Harmine (HRM) and Tetrahydroharmine (THH) alkaloids in Ayahuasca has been reported worldwide. Objective: To evaluate the causes of variations in alkaloids concentrations of Ayahuasca prepared with fresh and dehydrated plants from different environments and determine the best drying method to plants according to alkaloids content and cytotoxicity of Ayahuasca tea. Material and methods: The environment interference on the alkaloids of the two species was evaluated in samples of Ayahuasca tea prepared with fresh plants. The most suitable drying process to the two species was evaluated in sample Ayahuasca tea prepared with plants submitted to drying under the sun conditions and five different temperatures in forced circulation oven. The concentration of the alkaloids determined by high performance liquid chromatography with UV-vis detector with diode array detection (HPLC-DAD). The in vitro cytotoxicity of Ayahuasca was evaluated in human keratinocytes cells (HaCaT) by colorimetric assay. Results: Environmental characteristics, preparation process and temperature of plants drying interfered on DMT, HRL, HRM and THH concentrations of Ayahuasca. No effect cytotoxicity was detected with relationship to psychoactive alkaloids in samples of Ayahuasca tea prepared with fresh or dried plants. Conclusion: Concentration of DMT, HRL, HRM and THH alkaloids in Ayahuasca are influenced by plants environmental. The most suitable drying process was obtained in forced circulation oven at 43 and 45°C to P. viridis leaves and B. caapi stems respectively. The Ayahuasca prepared with fresh or dry plants no showed cytotoxicity in human keratinocytes cells.

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