Evaluation of Four Assays to Determine Cytotoxicity of Selected Crude Medicinal Plant Extracts In vitro

Aims: Cytotoxic assessment of plant extracts is crucial during the pre-clinical screening of medicinal preparations selected for further development. The aim of this study was to compare four assays used to test the cytotoxicity of eight African plant extracts, as well as to determine whether the antioxidant properties of the extracts potentially diminished the reliability of the resazurin conversion assay.
Methodology: HepG2 cells were exposed to hot water or methanol extracts of Acokanthera oppositifolia, Boophane disticha, Solanum aculeastrum and Tabernaemontana elegans for 72 h. Cell viability was determined using neutral red uptake, sulphorhodamine B staining, MTT and resazurin conversion assays. Phytochemical interference in the resazurin conversion assay was assessed in a cell-free environment and antioxidant activity of the crude extracts was determined using the Trolox Equivalence Antioxidant Capacity assay.
Results: Compared to the other three assays, the resazurin conversion assay failed to detect cytotoxicity, even at the highest concentration tested. The sulphorhodamine B staining assay showed the highest reproducibility, and compared well to the neutral red uptake and MTT conversion assays. Although extracts possessed moderate antioxidant activity, this did not contribute to the spontaneous conversion of resazurin in the cell-free environment, inferring that cellular conversion of resazurin was up-regulated by the crude extracts, leading to the false negative result of cytotoxicity.
Conclusion: Due to the potential interference between samples and substrates used in cytotoxicity assays, assessment of the assay’s suitability should always be conducted. It is possible that the crude extracts increased resazurin-specific enzyme activity through up-regulation, and as such led to higher conversion. Due to the interference, the resazurin conversion assay should not be used when assessing cytotoxicity of plant extracts. It is recommended that in vitro toxicological evaluation be performed using multiple cytotoxicity assay, preferably those based on different mechanistic principles to ensure higher accuracy.