Ethanol extracts of twenty Southern African plants were prepared and tested for their in vitro cytotoxic activity against various cancerous and non-cancerous cell lines. The cancerous cell lines included human melanoma (A375), epidermoid carcinoma (A431) and adenocarcinoma cervical cancer (HeLa) and the non-cancerous cell line was human embryonic kidney cells (HEK-293). The plants studied were selected based on their traditional usage for the treatment of cancer and various skin disorders which could lead to the development of skin and cervical cancer.
The twenty plant extracts were also tested for their chemo-preventive activity by using the DPPH (2,2-Diphenyl-2-picryl hydrazyl) assay and the nitric oxide (NO) scavenging assay. All the extracts showed dose-dependent curves which were used to calculate the fifty percent inhibitory concentrations (IC50). The Syzygium jambos extract showed the most promising radical scavenging capacity (DPPH) with activity even greater than that of the positive control, Vitamin C, with an IC50 value of 1.17±0.26μg/ml. Rapanea melanophloeos showed the greatest potential in the nitric oxide scavenging assay with activity also greater than that of Vitamin C with an IC50 value of 63.73±0.4μg/ml.
The cytotoxicity of the plant extracts was measured using the Sodium 3‟-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate (XTT) colorimetric assay at concentrations ranging between 3.125μg/ml to 400μg/ml. The Helichrysum odoratissimum extract showed the highest cytotoxic activity with pronounced effects against the A431 cell line. The dose-dependent studies revealed an IC50 value of 15.5±0.2μg/ml on the A431 cell line and relative toxicity on the non-cancerous cells with an IC50 value of 37.1±4.8μg/ml.
Synergistic studies on the active plant extracts were also performed to determine whether a greater toxicity could be obtained against the A431 cell line. The H. odoratissimum extract and the S. jambos extract were tested in combination at various concentrations and ratios. At the most active ratio (9:1) an IC50 value of 19.53±0.4μg/ml was obtained which was determined to be non-interactive. As a result of the extracts being non-interactive all morphological and cytokine studies were performed on H. odoratissimum only.
Light microscopy, where haematoxylin and eosin staining was used to evaluate morphological changes in the A431 cell line when exposed to various concentrations of H. odoratissimum extract. Morphological changes included signs of condensed and fragmented nucleus, the formation of apoptotic bodies, cell shrinkage and cellular debris. These observations suggested an increase in morphological changes associated with apoptosis.
H. odoratissimum was also evaluated for its cytokine production or suppression in phorbol-12-myristate 13-acetate (PMA) stimulated U937 cells. The U937 cells were exposed to various concentrations of H. odoratissimum extract to determine whether there was an increase in IL-12 production or IL-8 suppression. It was noted that the extract was able to increase the production of IL-12 and decrease the production of IL-8 in U937 cells.
The antioxidant activity of the twenty plant extracts based on NO scaveniging activity has been reported for the first time in this study, whereas based on the DPPH scavenging activity, a few plant extracts have been reported earlier by other researchers. Furthermore, many of the plant extracts have been reported for the first time in this study for cytotoxicity against A431, A375 and HeLa cells. H. odoratissimum has been reported for the first time as a possible anti-cancer agent on A431 cells and therefore, a provisional South African patent has been filed.
Future consideration would be to perform other mechanistic studies which would give more possible answers on whether H. odoratissimum is inhibiting specific enzymes which would inhibit cancer cell growth, such as COX-2 and SPHk1, as well as techniques such as raman and infrared spectroscopy. Furthermore would be to consider bio-assay guided fractionation of H. odoratissimum to determine the active compounds which are responsible for the anti-cancer activity on A431 cells.