Withania somnifera as a topical anti-melanoma agent

PhD (Pharmaceutics), North-West University, Potchefstroom CampusIn recent years people have become more attuned to the use of natural products for medicinal purposes as the belief is that natural products have fewer side effects. While it is true that natural products have medicinal value it is wise to have scientific evidence backing the suggested medicinal uses of natural products in order to ensure safe and effective use of these products. In some cases the natural product is not used in its natural form but it becomes the source of a lead compound for drug synthesis (Rishton, 2008:43D; Cragg & Newman, 2013:3671). There are numerous anti-cancer compounds of natural origin that are currently on the market or being investigated for use in different cancers. In this study the anti-melanoma activity of Withania somnifera, which has been reported to have anti-cancer activity was investigated. W. somnifera is a medicinal plant commonly used in Ayurveda to treat different ailments within the home. The plant extract and compounds originating from the plant have been reported to be active against breast cancer, colon cancer, pancreatic cancer, melanoma, arthritis, hypertension and diabetes (Malik et al., 2009:1508; Nagella & Murthy, 2010:6735; Samadi et al., 2012; Vel Szic et al., 2014:1179). It was decided in this study to investigate and compare the anti-cancer activity of crude plant extracts and that of two active metabolites present in the plant. The metabolites that were chosen were withaferin A and withanolide A. Withaferin A has been reported to be a very potent bioactive constituent of W. somnifera hence its use in this study (Kulkarni & Dhir, 2008:1095). The influence of solid lipid nanoparticles (SLNs) and niosomes on delivery and anti-cancer efficacy of the W. somnifera extracts was then investigated. Nanoformulations have been said to have the ability to increase the intracellular concentrations of active pharmaceutical ingredients (APIs) in cancer cells thus in turn enhancing the efficacy of anti-cancer compounds (Sanna et al., 2013a:144). Soxhlet extraction of W. somnifera leaves was done using water, ethanol and 50% ethanol as solvents to come up with three different crude extracts. Each extract contained both withaferin A and withanolide A at different percentages. The extracts were encapsulated in niosomes and SLNs then the formulations were used to determine release and skin diffusion properties. A three month stability study was conducted on the formulations at room temperature in order to determine any potential stability issues. With respect to the in vitro efficacy studies, both the pure compounds and crude extracts were utilised for the treatments so as to see any differences between the use of pure compounds and a blend of compounds. Cytotoxicity and apoptosis assays were conducted on human cutaneous melanoma cells (A375 cells) and keratinocytes (HaCaT cells) and the selectivity of W. somnifera for cancerous as opposed to normal cells was determined. Selected in vitro efficacy studies were also done using SLN formulations of the plant extracts in conventional two-dimensional (2D) cell culture and in three-dimensional (3D) cell culture. For the 3D cell culture A375 cells were seeded in Matrigel®and treated with particular treatments then assessed for cytotoxicity and apoptosis. Therefore in this study the cytotoxic effects of W. somnifera crude extracts were compared with that of two pure metabolites in 2D and 3D cell culture. Additionally the influence of SLNs on the cytotoxicity was also investigated. All the extracts that were prepared contained withaferin A and withanolide A with different percentage compositions and this probably influenced the differences that were seen with the Franz cell diffusion studies and efficacy results. Withaferin A and withanolide A were both released from the ethanol extract niosomes, 50% ethanol extract niosomes and all the SLN formulations. This meant that the two compounds were available for diffusing into or through the skin from the formulations in question. None of the compounds however were detected as having diffused through the skin from the formulations. Tape-stripping results revealed that withaferin A permeated into the stratum corneum-epidermis from all the formulations except the water extract niosomes. However, only the 50% ethanol extract SLNs managed to achieve deeper penetration of withaferin A to the epidermis-dermis. On the other hand all the SLN formulations resulted in withanolide A reaching both stratum corneum-epidermis and the epidermis-dermis but only the 50% ethanol extract niosomes succeeded in bringing about the permeation of withanolide A into the stratum corneum-epidermis. Stability testing of these formulations revealed that the formulations were not very stable possibly due to the presence of unidentified compounds in the crude extracts and the effects of lyophilisation without the use of a lyoprotectant (Hua et al., 2010:8). In vitro efficacy studies showed that withaferin A was toxic to melanoma cells and the presence of withanolide A enhanced the anti-melanoma effects of withaferin A. Withaferin A and withanolide A were generally more active than the crude extracts with respect to inducing apoptosis in melanoma cells. Selectivity for inducing apoptotic and necrotic cell death in melanoma cells versus keratinocytes was observed for all the treatments. The SLNs however did not have a notable influence on the apoptosis inducing effects of the plant extracts. Deoxyribonucleic acid (DNA) fragmentation, caspase 3/7 activation, increase in membrane permeability and a decrease in mitochondrial membrane potential were taken to indicate the occurrence of apoptosis in the 2D assays. For the 3D assays with the plant extracts and SLN formulations the externalisation of phosphatidylserine and reduced uptake of CellTracker™ Red dye were indicators of the occurrence of apoptosis or cell death. In this study it was revealed that although W. somnifera crude extracts have activity against melanoma, withaferin A and a withaferin A/withanolide A combination had the greatest activity and maintained selectivity for melanoma cells over keratinocytes. The SLNs displayed superior ability to carry the marker compounds into the skin layers but not through the skin. This was favourable as dermal delivery was desired and not systemic delivery. The results of this study support the further study of W. somnifera and its constituent compounds for use in the topical treatment of melanoma.Doctora