Selective cytotoxicity of Crotalaria agatiflora subspecies agatiflora Schweinf

Abstract

Crotalaria species have been widely used in Chinese traditional medicine to treat several types of internal cancers. Crotalaria agatiflora is used as a medicinal plant in several African countries for the treatment of bacterial and viral infections as well as cancer. Researchers have found this species to relieve spasms and reduce blood pressure. The aims of the study were to investigate the potential anti-cancer as well as cancer preventative activity of C. agatiflora subspp. agatiflora and ultimately to evaluate the possible mechanism of action.

Water and ethanolic extracts were prepared after which the cytotoxcity of the samples were determined on four cancerous and one noncancerous cell lines, using XTT (Sodium 3’ –[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro) benzene sulfonic acid hydrate) colorimetric assay. Antioxidant activity was determined using DPPH (1,1-Diphenyl-2-picryl hydrazyl). Light microscopy (eosin and hematozylin staining), flow cytometry (Annexin-V and propidium iodide) and micro-Raman spectroscopy were used to evaluate the mechanism of action of the ethanol leaves extract of C. agatiflora and one of the isolated compounds. Both water extracts were relatively non-toxic against all cell lines tested. The ethanol extract, was found to be the most active extract against the proliferation of human leukemic U-937 cells (Inhibitory concentration of 50% of the cell population, IC50 = 73.9 µg/mL) and human oesophageal carcinoma (SNO) cells (IC50 = 111.5µg/mL). The ethanol extract exhibited an IC50 value of 352.4 µg/mL against non-cancerous African green monkey kidney (Vero) cells. The best selectivity index (SI = 4.8) of the ethanol extract was seen against U-937 cells and thus further investigations were focused on these cancerous cells. It was evident that the ethanol extract showed the highest antioxidant activity, with an IC50 (Inhibitory concentration of 50% of free radicals) value of 18.89 µg/mL, while the water extracts had similar IC50 values between 27 and 29 µg/mL respectively. A bioassay-guided fractionation led to the isolation of two bioactive compounds, namely madurensine and doronenine, from the alkaloidal fraction of the ethanolic extract of the leaves. It was found that doronenine was the most active having an IC50 of 87.7 µg/mL, while madurensine had an IC50 of 136.5 µg/mL against U-937 cells. Both compounds were relatively non-toxic having IC50 values higher than 2mg/mL on Vero cells. The SI values were higher than 100 for madurensine and higher than 30 for doronenine. Although doronenine was more active than madurensine, it was not further investigated due to madurensine having higher SI value and the small quantity of the sample. The crude extract treated U-937 cells showed definite signs of cell death during light microscopic investigation, while little apoptosis (10-20%) and necrosis (<2%) were detected with flow cytometry in cells treated cells. Raman spectroscopy confirmed the decrease in cell size and thus the apparent decrease in the concentration of proteins and lipids within several treated single analyzed U-937 cells. The ethanolic extract was the most active sample tested both for cytotoxicity and antioxidant activity. The mechanism of action was hypothesized as autophagy, but should be confirmed with further analysis. Crotalaria agatiflora subspp. agatiflora could be further investigated as a chemo-preventative drug in the future.