~ 19 ~ American Journal of Essential Oils and Natural Products 2014; 2(1): 19-23 ISSN: 2321 9114 AJEONP 2014; 2(1): 19-23 © 2014 AkiNik Publications Received: 03.07.2014 Accepted: 18.09.2014 Yahima Frión-Herrera Food and Pharmacy Institute, Havana University, Cuba Alexis Díaz-García Laboratories of Biopharmaceuticals and Chemistry Productions (LABIOFAM) Ave. Independencia, Km 16 ½, Stgo de las Vegas, Boyeros. La Habana, Cuba Hermis Rodríguez-Sánchez Microbiology Department, Tropical Medicine Institute “Pedro Kouri” Havana, Cuba Jenny L. Ruiz-Fuentes Microbiology Department, Tropical Medicine Institute “Pedro Kouri” Havana, Cuba Lianet Monzote Fidalgo Microbiology Department, Tropical Medicine Institute “Pedro Kouri” Havana, Cuba William N. Setzer Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA Correspondence: Alexis Díaz-García Laboratories of Biopharmaceuticals and Chemistry Productions (LABIOFAM) Ave. Independencia, Km 16 ½, Stgo de las Vegas, Boyeros. La Habana, Cuba Cytotoxic effect of Cuban propolis extracts on normal cells and in-vitro basal toxicity assay to estimate acute oral toxicity Yahima Frión-Herrera, Alexis Díaz-García, Hermis Rodríguez-Sánchez, Jenny L. Ruiz-Fuentes, Lianet Monzote Fidalgo and William N. Setzer Abstract In-vitro toxicity tests are recognized as alternative methods to animal acute toxicity testing. The aim of this study was to assess toxicity of 16 Cuban propolis extracts of different chemical types: brown (1, 4, 5, 16, and 17 BCP), red (9, 29, 35, 37, 45 and 72 RCP) and yellow (18, 39, 41, 50 and 60 YCP) against Balb/c 3T3 and Vero cells. Cells were treated at different concentrations (12.5; 25; 50 and 100 μg/mL) of propolis for 72 h and the IC50 value was determined. Furthermore we employed in-vitro cytotoxicity test described by Spielmann et al., red uptake (NRU) assay to estimate acute oral toxicity. Propolis showed differential cytotoxicity toward normal cells in a dose and tissue-dependence. RCP was the most interesting group because they did not affect normal cells evaluated. In Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, IC50 values to RCP-45 and RCP-37 were 86.8 ± 1.14 and 96.1 ± 1.02 µg/mL, respectively. The LD50 values of these extracts were 558 and 579 mg/kg, respectively. For both samples the start dose for acute oral toxicity studies is 550 mg/kg in the case of using the Up-and-Down Procedure (UDP) and 300 mg/kg when Fixed Dose Procedure (FDP) and Acute Toxic Class Methods (ATC) were used. Results presented in this work can contribute to understand the toxicological profile of Cuban propolis and reduce the number of animals required in subsequent pharmacological / toxicological studies. Keywords: Cuban propolis, cytotoxicity, normal Cells, NRU assay. 1. Introduction Natural products constitute one of the most promising sources to obtain biologically active compounds. During the last decade, propolis has been found to exhibit a broad spectrum of therapeutic effects, including antibacterial, antifungal, antitumor, antioxidant, immunomodulatory and other beneficial properties [1, 2] . This bee product is derived from various plant resins and widely employed in traditional medicine. Cuban propolis has been classified according to its composition: brown Cuban propolis type (BCP type), rich in polyisoprenylated benzophenones [3] , red type (RCP type), containing isoflavonoids as the main constituents, and yellow type (YCP type) with a variety of triterpenoids as the major chemical components [4] . Recently, extracts of BCP, RCP and YCP have shown potential activity against tumor cells, using murine breast carcinoma, human breast adenocarcinoma and human lung adenocarcinoma [5] , and antiprotozoal properties against Leishmania amazonensis, Trichomonas vaginalis and Plasmodium falciparum [6,7] . In this sense, extensive cytotoxic studies should be carried out with Cuban propolis. Therefore, is reasonable to use, at the primary screening stage, in-vitro toxicity assays on normal cells to select the less toxic compounds among the most active ones. The toxicological potential of a compound is analyzed through the affectation of vital functions of normal and tumor cell lines from different tissues and different species [8] . Moreover, the fulfilling of this criterion and also the combination with the estimation of the initial dose for acute toxicity tests in rodents, using in-vitro data, facilitate the selection of the most promising compounds while the reduction of laboratory animals is accomplished [9] . Basal cytotoxicity is considered the starting point for an integrated assessment of potential in- vivo acute systemic toxicity [10, 11] .