The protective effects of Opuntia extract against stress conditions in vitro : methodologies for the determination of biological activity and for isolation of the active molecule responsible for such effects

Abstract

The heat shock response is an immediate, transient response to a wide variety of physiological stresses. Heat shock proteins (HSPs) are synthesised when cells are exposed to heat as well as other types of cellular injury. They are evolutionarily highly conserved, and have an essential function in the survival of organisms, protecting them from harmful traumas. Unfortunately, the stress response in the human body is not so immediate, and it can only react after a period of time, when the stress damage is already done, by repairing the damaged tissue. The prickly pear cactus ( Opuntia) holds a secret deep in its genes that enables it to induce and amplify the rapid synthesis of heat shock proteins produced by the body under conditions of stress, thus minimising the harmful effects of the stress condition. This explains why such interest has been shown in this area of study. In the first part of the study, different techniques enabling the efficient and accurate biological analysis of Opuntia extracts (TEX-OE®) in vitro were evaluated. Using a dye exclusion method for the determination of cell viability, it was found that heat stress of non-treated HL60 cultures induced a significant decrease in cell viability (31 %), whereas cultures previously treated with TEX OE® showed to be somewhat resistant to cell death induced by hyperthermia (5.5%). Morphological evaluation of the HL60 cells confirmed these results, where cultures treated with TEX-OE® prior to heat stress showed a clear-cut resistance to apoptotic cell death. The same response was exhibited in HaCaT cultures subjected to heat stress and tested using the XTT viability assay, where heat stress of non-treated cells also induced a significant decrease in cell viability in comparison with the TEX-OE® treated cultures (53.6% and 10.7% respectively). Results obtained for the XTT assay were similar to those obtained from the dye exclusion method, thus indicating that the latter procedure represents a suitable model for monitoring biological activity of TEX-OE®. Studies carried out to show the effect of recurrent heat stress on HL60 cultures demonstrated a dramatic decrease in cell viability for non-treated cells over the three-day experimental period (78.4%), and a significant resistance to cell death in TEX OE® treated cultures (12.1 %). It was also shown that the duration of protection after heat shock achieved by TEX-OE® treatment is three days. Observations for cell viability were related to HSP72 levels, where a significant difference in the expression of HSP72 in response to each heat stress was observed between treated and non-treated cultures. A rapid increase in HSP72 levels (17-fold) which were maintained at high values throughout the experiment were observed for treated cultures, whereas non-treated cells showed a relative inability to respond immediately to the stress, with higher levels (<2-fold) only being achieved after the second day of experimentation. These results thus indicate that the protective role of Opuntia against stress induced lesions and cell death can be related to the heat shock response. The second part of the study involved the development and assessment of efficient methods for the extraction, separation, and purification of the active components for Opuntia. For preparative separation, medium pressure liquid chromatography on a silica gel column was most suitable. Using a hexane-acetone solvent system, the active components were efficiently separated into one main fraction, the activity of which was confirmed in vitro using the technique for the determination of HSP72 levels. Further purification of this fraction was achieved by the application of a normal-phase high-performance liquid chromatographic method utilising a Hypersil® 12µm silica column eluted with heptane-diethyl ether (95:5%). The active molecule responsible for the biological effects of Opuntia was isolated using revered-phase high-performance liquid chromatography on a Kromasil 5µm C1s n-octyldecyl siloxane column. Isolation of the active molecule was found to be most complete with the use of a 95:5% methanol-water solvent system. Identification of the active molecule remains to be determined. Various methods and techniques in order to achieve this are currently under investigation.