Fractionation of biologically active components by supercritical carbon dioxide

Obtainment and further fractionation of bioactive components from Vitis vinifera (grape) has been evaluated by using supercritical fluid extraction and conventional solvents (hexane, ethanol, acetone, ethyl-acetate). Supercritical fluids have been widely used to extract target components from varied matrices, at analytical and commercial scales. This is an environmentally friendly alternative to conventional organic solvent extraction2. Organic solvent clean products can be obtained, and their natural properties are preserved due to the moderate temperatures used. The most widely used supercritical solvent for separation of natural products is carbon dioxide, typically in the ranges of 10-50 MPa pressures and 35-80°C temperatures. The focus of this work is to extract and fractionate bioactive natural products as polyphenols (proanthocyanidins) and fatty acids from the seeds of Vitis vinifera1, 3. Controllable ratios of the major biologically active components offer the possibility of tailor made product development as food additive or supplement or for cosmetic purposes. Conventional Soxhlet extraction with 50:50 % ethanol-water mixture was performed to obtain the raw extract. Cellulose was used as a carrier to turn the texture of the alcoholic extract into a solid material (mass ratio alcoholic extract-cellulose: 1-1) capable to form a porous bed. Further investigations aims to fractionate this solidified raw extract and compare the possibilities of selective extractions directly from grape seeds, from cold pressed oil and grape seed flour. Effects of cosolvent concentrations (0-15 % ethanol in carbon dioxide) were studied in detail at constant pressure of 30 MPa. The pressure of extraction was varied between 100 and 450bar in case of pure carbon dioxide. The temperature was kept constant at 45ºC and the solvent flow was 5-6kg/h. Studies on Vitis vinifera solidified raw extract revealed slightly increasing extraction yields with increasing ethanol concentrations: 2.53, 4.12 and 7.29% yields at 5, 10 and 15% ethanol concentrations, respectively. Supercritical fluid extraction with pure CO2 at 450bar led to 4.38% of extraction yield. The antioxidant activity was evaluated by using the DPPH* (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging assay4, based on to the concentration where 50% of inhibition of the DPPH* was achieved (IC50). It resulted in 7.04±0.76 μg/ml for the initial grape seed extract. In reference to the supercritical extracts, this factor was achieved at 28.54±2.80, 76.31±2.77 and 197.82±27.61μg/ml for the extracts obtained with 15, 10 and 5% of ethanol, respectively. There was no relevant antioxidant activity in the supercritical extract with pure CO2. In the light of these results it is observed that there is a correlation between the polarity of the extractant solvent and the antioxidant activity of the sample, therefore the interesting substance for this purposes could be the supercritical raffinate. The research work was supported by Marie Curie DoHiP European Project (Training Programme for the Design of Resource and Energy Efficient Products by High Pressure Processes). E. Székely thanks the János Bolyai Research Fellowships of the Hungarian Academy of Sciences