Cytotoxic effects of citrus aurantifolia crude leaf extracts on vero cells and C6/36 cells prior to test the anti-dengue viral activity of the extracts

Abstract

Citrus aurantifolia is a traditionally used medicinal plant, which can be tested against Dengue-virus (DENV) infections. The objective of this study was to identify in vitro cytotoxicity of C. aurantifolia leaf extracts against normal African green monkey kidney epithelial (Vero) cells and Aedes albopictus (C6/36) cells prior to antiviral studies against DENV infections. CytoTox-96®-Promega-USA is a colorimetric assay that quantitatively measures lactate dehydrogenase released upon cell lysis. C. aurantifolia leaves were ground and fresh, neat extract was diluted in normal saline to prepare the two-fold dilution series. Two 96-well assay plates were prepared separately with Vero and C6/36 cells. The assay was set up using CytoTox-96®- Promega-USA-kit manufacturer’s instructions, including 1. Negative control–without cells, 2.Vehicle control-untreated cells, 3.Positive control-lysis solution with four replicates. Leaf extract was added into the test wells at different concentrations and plates were incubated for 5 and 24 hours at 37 oC and at 28 oC. The absorbance data were measured using a standard 96-well plate reader (Labtech-LT-4500- Singapore) and the percentage cytotoxicity was calculated. The cytotoxicity-driven colour intensity, absorbance values and percentage cytotoxicity decreased with the decreasing concentrations of leaf extract. The percentage cytotoxicity against Vero cells for dilutions of 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512, 1/1024 were 104.99, 83.96, 75.33, 82.96, 22.23, 35.56, 25.97, 11.26, 5.82, respectively, for the 5-hour incubation and 104.55, 91.16, 53.26, 45.83, -17.61, -31.52, -34.47, -30.51, -25.73, respectively for the 24-hour incubation. The percentage cytotoxicity against C6/36 cells were 99.42, 80.34, 50.09, 36.46, 12.23, 7.12, 7.44, -7.19, -8.99, respectively for the 5 hours and 119.10, 96.59, 63.43, 42.04, 31.42, 20.14, 9.21, 5.19, 1.73, respectively for the 24 hours. High leaf extract concentrations were cytotoxic to both Vero and C6/36 cells. Cytotoxicity of C. aurantifolia leaf extracts helps select the minimum toxic concentrations for testing the anti-viral activity against DENV infections.