In vitro cytotoxic activity of medicinal plants from Nigeria ethnomedicine on Rhabdomyosarcoma cancer cell line and HPLC analysis of active extracts

Cytotoxic studies were determined in human Rhabdomyosarcoma (RD) cells (CDC, Atlanta, USA), African green monkey kidney (Vero) cell (WHO Reference Polio laboratory, UCH, Ibadan, Nigeria) and the normal human prostate (PNT2) cell line obtained from the (WHO Reference Polio laboratory, UCH, Ibadan, Nigeria). Cells were grown in Eagle’s MEM supplemented with 10% FBS, 100 units/mL of penicillin, 100 mg/mL of streptomycin, 2 mM L-glutamine, 0.07% NaHCO₃, and 1% non-essential amino acids and vitamin solution. Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C and passaged bi-weekly.

Cell viability was examined by the ability of the cells to cleave the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (Sigma, Chem, St. Louis, MO), by the mitochondrial enzyme succinate dehydrogenase following the procedure as described earlier [19]. Briefly, each extract was pre-solubilized in dimethylsulphoxide (DMSO) at 37 °C to give a stock solution (SS) of 1 mg/mL. Serial ten-fold dilutions were made from SS to give working concentrations of 1000–0.01 μg/mL, making sure that the final concentration of DMSO in the tested dilutions was not higher than 1%. Confluent monolayers of RD/Vero cells were grown in 96 well-microtitre plates for 24 h. Cells were incubated with various concentrations of the test extracts in triplicate at 37 °C in a CO₂ environment for 72 h. The negative control was performed using growth medium alone instead of plant extract, while cyclophosphamide (CTX) was used as the positive control. In addition, the cell viability was examined microscopically for the presence or otherwise of cytopathic effect (CPE). At the expiration of the 72 h treatment period, supernatants were removed from the wells and 25 μL of the MTT solution (2 mg/mL in PBS) was added to each well. The plates were incubated for 1.5 h at 37 °C, and 125 μL of DMSO was added to each well to dissolve the formazan crystals. The plates were placed on a shaker for 15 min and the optical density was determined at 492 nm on a multi-well spectrophotometer (Multiskan, Thermo Fisher Scientific, Waltham, MA). The 50% cytotoxic concentration (CC₅₀) was defined as the extract/compound concentration required for the reduction of cell viability by half. The CC₅₀ value and the standard error mean (SEM) were calculated using a non-linear regression curve contained in the Graph pad prism statistical software.

The normal African green monkey kidney epithelial cell line (Vero) and the normal human prostate (PNT2) cell line were used to measure the SI. The SI of the most active fraction was calculated as the ratio of cytotoxic effect on normal cell line to the cytotoxic effect on cancer (RD) cell line.

The active fractions were subjected to HPLC analysis using the Dionex HPLC System 2695 (Waters). Reversed-phase analytical HPLC experiments were performed on a thermoscientific NX 5 μM C18 column (250 × 4.6 mm). The column temperature was set at 25 °C. A variable wavelength ultraviolet–visible detector was set at 235 nm. 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in methanol (solvent B) was used as the eluent. For the analysis, the mobile phase composed of 70% of A and 30% B at 0 min, then linear gradient to 100% of B over 40 min and held at that composition for 10 min at a flowrate of 1 mL/min. To determine that nature of the compounds present in these active fractions, the peaks generated in the chromatogram of each active fraction were compared to the compounds collection contained in the HPLC library.