Interactions between Plant Extracts and Cell Viability Indicators during Cytotoxicity Testing: Implications for Ethnopharmacological Studies

Purpose: To compare the cytotoxicity of six medicinal plants ( Acmella ciliata , Amaranthus tricolor , Coriandrum sativum , Glebionis coronaria , Kyllinga brevifolia and Tradescantia zebrina ) using 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) assays. Methods: Hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts were obtained for each plant by sequential solvent extraction. Cytotoxicity was evaluated in triplicate, from 640 to 5 μg/mL, two-fold, serially on monkey kidney epithelial (Vero) cells. Results: The hexane, chloroform and ethyl acetate extracts of the six plants were more toxic to the Vero cells compared to the ethanol, methanol and water extracts. Thirty one percent (11/36) and 75 % (27/36) of the extracts showed significant cytotoxicity (p < 0.05) in MTT and NRU assays, respectively. The 78, 52 and 7 % cytotoxicity levels detected in 27 extracts using the MTT assay were significantly (p < 0.05) underestimated at 640, 320 and 160 μg/mL, respectively, using NRU assay. Nine extracts from five plants exhibited significantly lower (p < 0.05) 50 % cytotoxic concentration (CC 50 ) when NRU assay was employed compared to MTT assay. At 640 μg/mL, 10 of the 21 extracts were also found to react chemically with MTT, causing a 2.0 – 29.1-fold increase in the absorbance value (550 nm) compared to control. Conclusion: The plant extracts of A. ciliata , A. tricolor , C. sativum , G. coronaria , K. brevifolia and T. zebrina show concentration- and extraction method-dependent cytotoxicity using MTT and NRU assays. NRU assay appears to be more sensitive and reliable than MTT assay for cell viability evaluation of the plant extracts. Keywords: Acmella ciliata , Amaranthus tricolor , Coriandrum sativum , Glebionis coronaria , Kyllinga brevifolia and Tradescantia zebrina , Extraction, Medicinal plant, Neutral red uptake assay, Vero cell