A micro-Raman spectroscopic investigation of leukemic U-937 cells treated with Crotalaria agatiflora Schweinf and the isolated compound madurensine

In South Africa traditional medicine plays an important role in primary health care and therefore it is very important that the medicinal use of plants is scientifically tested for toxicity and effectiveness. It was established that the ethanolic extract of the leaves of Crotalaria agatiflora, as well as the isolated compound madurensine, is moderately toxic against leukemic U-937 cells. Light microscopic investigations indicated that symptoms of cell death are induced during treatments, but flow cytometry analysis of treated cells, using annexin-V and propidium iodide, showed that apoptosis and necrosis are insignificantly induced. The Raman results suggested that protein extraction and DNA melting occur in the cells during treatment with the ethanolic extracts (IC50 value 73.9 μg/mL), drastically changing the molecular content of the cells. In contrast, treatment with madurensine (IC50 value 136.5 μg/mL), an isolated pyrrolizidine alkaloid from the ethanolic extract of the leaves, did not have the same effect. The results are also compared to that of cells treated with actinomycin D, a compound known to induce apoptosis.The investigation showed that micro-Raman spectroscopy has great promise to be used for initial screening of samples to determine the effects of different treatments on cancerous cell lines together with conventional methods. The results highlight the fact that for many natural products used for medicinal purposes, the therapeutic effect of the crude plant extract tends to be significantly more effective than the particular action of its individual constituents.

Graphical abstractLeukemic U-937 cells were treated with different concentrations of the ethanolic extract of Crotalaria agatiflora subspp. agatiflora and the isolated pyrrolizidine alkaloid, madurensine. Different biochemical responses were observed with micro-Raman spectroscopy after treatments.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Crotalaria agatiflora Schweinf has an ethnobotanic use against cancer. ► An ethanolic extract of the plant was tested against leukemic U-937 cells. ► Raman spectra showed changes in cell morphology and biochemical constituents. ► Crude plant extracts might be more bioactive than isolated compounds. ► Raman spectroscopy has potential as an initial screening tool in phytomedicine.