Summary:
The chemical composition of the olive fruit has been the focus of commercial and research interest in recent years due to its valuable bioactive components. Simple phenols, secoiridoids, flavonoids, terpenes, and lignans are some classes of components that have been detected in the olive fruit and have been attributed valuable biological activities, including antioxidant, antimetastatic, antiviral, anti-inflammatory, antimicrobial, and cardioprotective. Among them, secoiridoids are some of the most interesting bioactive components of the olive fruit, for which the European Food Safety Authority (EFSA), recognizing their beneficial effect on human health, has given an opinion in favor of a specific health claim, according to regulation 432 /2012 of the European Union.
Specifically, oleuropein is a dominant metabolite of the olive fruit with a range of biological activity attributions (anti-inflammatory, antioxidant, cardioprotective, neuroprotective, antidiabetic and antitumor effects). However, its content in the fruit is quite variable, as it is directly dependent on the ripening stage of the olive fruit, the variety, the geographical area, and the technical cultivation of the olive trees (organic or conventional), as well as on fruit infections by pathogens microorganism.
Another important component of the olive fruit, with equally proven biological activity, is oleacein, or otherwise the ester of the dialdehyde form of decarboxymethyl elenolic acid with hydroxytyrosol 3,4-DHPEA (3,4-DHPEA, 3, 4-DHPEA-EDA). However, its content in the fruit is usually at undetectable levels. It is a product of natural origin, with great biological, research and commercial interest for the pharmaceutical industry, the cosmetic industry and food technology. Extensive study of its pharmacological properties has shown that oleacein traps reactive oxygen free radicals, lowers blood pressure, limits the extent of inflammation, and at the same time appears to have a protective effect for many forms of cancer. To date, the only natural source of oleacein is olive oil, and especially extra virgin olive oil. But its content in the oil is quite variable, subject to many factors such as: the variety of the olive tree, the cultivation practices, the soil and climatic conditions, the ripening stage of the fruit, as well as the olive oil extraction process method and conditions. In addition, the limited half-life of oleacein in olive oil due to the presence of hydrolytic enzymes, as well as its oxidative instability during olive oil storage, are two additional factors that negatively affect its concentration and levels in the final product. At the same time, the lack of commercial synthetic oleacein, the small yields of its synthetic preparation, the large quantities of olive oil, the high cost of the raw material required for its isolation, and the great demand and commercial interest presented by specific secoiridoid from processing companies, suggest alternative natural sources of origin for the isolation of this very valuable and biologically active substance. It is indicated that oleacein is sold by the credible, well-known company Phytolab, for €28,000 per gram.
The purpose of this thesis is the study of the ripening stage of the olive fruit as expressed through phenolic content, the investigation of the optimal conditions for drying and extraction of the olive fruit with emphasis on secoiridoids, finding alternative sources to produce extracts rich in oleacein, as well as identifying methodologies for the isolation of oleacein from natural raw materials. Specifically, in the context of the implementation of this master’s thesis, the following were studied:
(a) the changes in the phenolic content of the olive fruit at two different stages of ripening. Specifically, by studying three different olive varieties, the levels of the main secondary metabolites were determined in ripe and unripe olives that came from the same olive tree. The results of these studies showed that the levels of oleuropein are higher in unripe fruits, in contrast to hydroxytyrosol and verbascoside where their content was higher in ripe olives, and this seems to happen regardless of olive variety.
(b) finding optimal conditions for olive fruit drying and processing based on the secoiridoid content. In particular, the content of the secoiridoids oleuropein and oleacein was studied in whole and pitted olives that had been dried in a natural way at room temperature, by heating to 140oC and by the freeze-drying technique. The aim of this study was to investigate the effect of the drying conditions and the contribution of the action of the enzymes located near the kernel of the olive fruit, on the content of secoiridoids. At this point it is worth noting that according to the literature, 98% of the peroxidase enzyme is located in the endocarp near the olive stone. The results of these studies showed that the levels of oleuropein in the flesh of dried-pitted olives were higher compared to its content in the pit-dried olive fruit, probably due to the removal of the peroxidase enzymes from the olive flesh during the ripening stage. In addition, the drying of the olive fruit at a temperature of 140oC, led to an increase in the levels of oleuropein in the olive fruit, likely due to the deactivation of the enzymes by high temperature. In other words, the drying of the olive fruit at high temperature likely inactivates the enzymes of the olive fruit (such as β-glucosidase, esterase, polyphenyloxidase etc.), resulting in the enzymatic reactions of transformation, oxidation, and degradation of oleuropein not being favored and therefore, presenting with increased oleuropein content. An important observation in this study was the results of drying the non-kerned olive fruit with the freeze-drying technique, where, for the first time in literature, the metabolite oleacein was detected and determined in the olive fruit, and at quite high levels. This observation can be explained as followsː During the thawing of the frozen olive fruit, the lysis of the fruit cells enables the interaction of the enzymes (β-glucosidase, esterase, polyphenyloxidase) and the polyphenols, which under normal conditions are in different cellular compartments. In this way, enzymatic reactions of transformation, oxidation, and degradation of oleuropein in secoiridoid by-products are favored. However, when the fruit is thawed in the absence of oxygen, as in the case of lyophilization, the oxidizing enzymes of the olive (polyphenyloxidase, peroxidase) are inactivated, while on the contrary, β-glucosidase and esterase continue to remain active, catalyzing the conversion of oleuropein in oleacein.
Considering the above observation, the biotransformation of oleuropein to oleacein was further studied during thawing in ambient conditions of frozen non-pitted olive fruit. Olive fruit from two different olive varieties was frozen at -20oC for one day and then left to thaw at room temperature. Then, after the stone was removed, the flesh of the olive fruit was extracted with methanol following the proposed extraction methodology. The results of this study showed that in this case the biotransformation of oleuropein into oleacein took place similarly to the case of lyophilization, as extracts rich in oleacein were produced.
(c) finding optimal conditions for olive fruit extraction to produce extracts rich in oleacein. Specifically, 9 extraction protocols were applied to unripe and ripe olives from the same olive tree and from three different olive varieties (Kalamon, Megaritiki and Koroneiki) using as extraction solvent water, aqueous solution of Na2SO4, 0.1 M and aqueous solution of NaHCO3, 0.1M, in three different extraction temperatures of 25oC, 50oC, and 85oC. Aqueous extraction was chosen to investigate the effect of enzymes on the phenolic content of the olive fruit by varying the ionic strength of the extractant and the extraction temperature. At this point it is worth noting that the aqueous extracts underwent an additional process of liquid-liquid extraction with ethyl acetate to remove salts and sugars, and to enrich them. The results of these studies showed that the extraction of fresh olive fruits, with water and an aqueous solution of Na2SO4, 0.1 M in a temperature of 85oC, followed by an additional stage of liquid-liquid extraction with ethyl acetate is more effective to produce extracts rich in oleacein, and this seems to happen regardless of ripening stage and olive variety. However, a disadvantage of this process is that the low extraction yields ˂ 2% (dried extract / 100 grams of olive fruit). The increased levels of oleacein from the extraction of the olive fruit with aqueous solution of Na2SO4, 0.1 M is assumed to result from the increased ionic strength of the extraction solution, which likely favors the stabilization of the enzyme structure and the induced enzymatic biotransformation reactions of oleuropein to oleacein.
(d) the optimization of olive fruit processing and extraction conditions with the aim of finding rapid and economically viable production methodologies for extracts rich in oleacein. Specifically, in unripe olives of the Kalamon variety, the conditions of olive fruit processing and extraction were investigated. Considering the extraction yields and oleacein levels of the olive extracts, resulting from the application of the drying, processing and extraction methodologies, the optimization tests were chosen to be carried out in the protocol where initially the olive fruit is frozen at -20οC and then thawed at environment conditions. The parameters chosen to be studied wereː the time interval of thawing of the olive fruit (1 hour, 8 hours and 24 hours), the type of olive fruit (pitted-without the stone or whole-with the stone), the way of pulping the fruit (with solvent, in the absence of solvent or in the presence of dry ice), as well as the type of solvent and the extraction temperature. Specifically, the extraction of the olive fruit with methanol and isopropanol was tested, at a temperature of 0oC, 25oC and 60oC. The results of these studies showed that freezing the olive fruit at -20oC for 24 hours, then thawing at room temperature for 8 hours and then extracting at 25oC for 30 minutes by pulping the whole fruit in Methanol is an economical and productive method for receiving olive extracts rich in oleacein. In this way, olive extracts can be produced in a very good yield which can reach 15%, in which the oleacein content ranges from 1 to 10% depending on the variety and the stage of fruit ripening.
This thesis is completed with the development and application of oleacein isolation methodologies from enriched olive fruit extracts. For this purpose, a quantity of pitted olive fruit of the Kalamon olive variety, which was initially frozen at -20oC and then thawed at room temperature, was extracted with an aqueous solution of Na2SO4, 0.1M, under stirring, at 85oC, for 1 hour. This was followed by defatting with hexane to remove fatty components, before subjecting the aqueous extract to liquid-liquid extraction with ethyl acetate to remove salts and sugars, and enable its enrichment. Then, the enriched extract was fractionated by Centrifugal Partition Chromatography (CPC) and the most oleacein-enriched CPC fraction was used to isolate oleacein by High Performance Preparative Liquid Chromatography (HPLC-UV). With the above procedure, oleacein was isolated to a high degree of purity (≥98%). Purity control was confirmed by HPLC-DAD High Performance Liquid Chromatography, while structure identification was performed by High Resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance Spectroscopy (NMR).
In summary, this study managed to transform, in a completely natural way, oleuropein into oleacein and then to isolate this secoiridoid on a pilot scale from an alternative natural source such as the olive fruit.
