Studies on the properties of an extract from Opuntia ficus indica

Abstract

The heat shock response plays an important role in the protection of organisms against various aggressions, where they help the body respond to a number of chemical, physical, biological or mechanical traumas. Previous studies have shown that a natural extract from the prickly pear fruit Opuntia ficus indica (TEX-OE®) can accelerate the release of heat shock proteins during stress and decrease the symptoms and complications associated with exposure to such stressors. The current research primarily sought to determine the protective effect of TEX-OE® against chemical stressors such as alcohol both in vitro and in viva. It was shown that ethanolic stress of WRL-68 and HepG2 human hepatocyte cultures induced a significant increase in apoptotic cell death, whereas cultures previously treated with TEX-OE® were more resistant to cell death induced by ethanol. These observations were related to heat shock protein levels, where a significant difference in the expression of Hsp72 in response to stress appeared between treated and non-treated cultures when tested using both a sandwich-enzyme-immunoassay principle and quantitative indirect immunofluorescence microscopy techniques. Results from the in viva animal studies also demonstrated the protective effects of TEX-OE® against chemical stressors such as alcohol. Ethanolic stress of TEX-OE® pre-treated Angelfish (Pterophyllum scalare) induced a lower decrease in percentage survival when compared to non-treated fish. Histological differences in the muscular tissue of these fish were also identified, and can be possibly correlated with the considerable differences observed in the expression of Hsp72 between TEX-OE® treated and non-treated groups, where TEX-OE® treated samples gave a faster and greater increase in HSP levels. Studies to compare ethanolic stress with other stressors such as anoxia revealed similar results, where anoxic stress of non-treated Angelfish and Gilthead seabream (Sparus aurata) induced a significantly higher decrease in percentage survival when compared to TEX-OE® pre-treated fish. However, the effect of anoxic stress in Angelfish appeared to be less detrimental than that of ethanolic stress. The occurrence of a dose response to TEX-OE® supplemented feed was also demonstrated in these studies. Considering such positive results, these studies were extended to human clinical trials in order to assess the efficacy of the extract in the prevention of intoxication in humans by excessive alcohol consumption (binge drinking). Multiple physiological alterations are observed in individuals suffering from excessive alcohol intake, with these biological variations constituting a stress to the organism. In the first trial, a reduction in symptom severity scores of 55.8% (p<0.01) after 3 hours and 59.8% (p<0.01) after 12 hours (following morning) was observed with TEX-OE® when compared to placebo. Of the ten symptoms monitored, headache, dry mouth, and weakness were significantly reduced after 12 hours whilst blurred vision and the inability to speak coherently were significantly lower after 3 hours. The hypothesis that an acceleration in the production of heat shock proteins by TEX-OE® could possibly reduce the inflammatory mediators that cause the detrimental symptoms associated with excessive alcohol consumption and alcohol hangover was confirmed when a significant increase in the expression of Hsp72 was observed when subjects ingested TEX-OE®. Mild cognitive impairments are usually observed in healthy young individuals who are fatigued or intoxicated with alcohol, and hence the second trial aimed to compare the magnitude of cognitive impairment arising from increasing blood alcohol concentration using a computerised neuropsychological cognitive testing system (CogHealth™). Excessive alcohol consumption appeared to have an effect on cognitive performance, yet significant group effects were found between the placebo and TEX-OE® sessions for speed and accuracy of responses after 1 hour and 3 hours, where the magnitude of cognitive decline under the placebo condition was significantly greater than under the TEX-OE® condition. The analytical biochemical studies carried out involved the efficient extraction and purification of the active components present in Opuntia extract (TEX-OE®). For preparative separation, medium pressure liquid chromatography on a silica gel column was most suitable, whilst further purification was achieved by application of revered-phase high-performance liquid chromatography on C8 noctyl and C18 n-octyldecyl siloxane columns. High resolution gas chromatography-mass spectrometry in positive chemical ionization PCI mode revealed that the active molecule is a C29 hetero-compound, the molecular formula of which cannot be matched with any existing compound listed in the fragmentogram databases searched. This suggests that the molecule responsible for the characteristic biological effects of TEX-OE® is still unknown. Further analysis and characterisation of the molecule is currently underway. Finally, the biomolecular studies developed revealed important information on the mode of action of TEX-OE® in accelerating the increase in Hsp72 levels following stress. Results obtained indicate that HSFl is essential for Hsp72 expression under normal conditions, since after the addition of Anti-HSFl to cells, these were unable to increase Hsp72 levels. This inability to induce Hsp72 protein expression was also observed in cultures treated with TEX-OE® after the addition of Anti-HSFl, however cultures treated with TEX-OE® before the addition of Anti-HSFl were still capable of increasing the expression of Hsp72 following heat stress. This suggests that the mode of action of TEX-OE® could possibly involve an increase in heat shock promoter activation through HSF binding upon treatment of cells with TEX-OE® or an increase in Hsp70 message but no increase in protein accumulation until exposure of cells to heat stress. Further studies to test this hypothesis are currently being developed.