Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology

An experimental setup based on a 2(3) full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R-2=0.91, p=0.0139), but not for the antiradical activity of the extracts (R-2=0.67, p=0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 +/- 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography-electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3',7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3'-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R-2=0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.