Dissertation committee:
Αλέξιος Λέανδρος Σκαλτσούνης, Καθηγητής, Φαρμακευτική, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Σοφία Μητάκου, Καθηγήτρια, Φαρμακευτική, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Νεκτάριος Αλιγιάννης, Αναπληρωτής Καθηγητής, Φαρμακευτική, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Μαρία Χαλαμπαλάκη, Επίκουρη Καθηγήτρια, Φαρμακευτική, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Jean-Hugues Renault, Professor, Plant Science Department, Université de Reims Champagne Ardenne-Institute of Molecular Chemistry of Reims.
Hermann Stuppner, Professor, Institute of Pharmacy, University of Innsbruck.
Wirginia Kukula-Koch, Associate Professor, Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin.
Summary:
Vegetable oils (VOs) are regarded as an integral section of human diet and many early manuscripts refer to their multiple uses in daily life; from food to cosmetic purposes. They represent the main pool of fatty acids in human nutrition and besides their use is associated with food flavoring due to their aroma and their special organoleptic and sensory properties. According to studies, the average consumption of fat reaches the amount of 25 kg per person per year, with the 80% being covered from VOs. Moreover, they play important role in human well-being and enhancement of life quality, due to their dermo-cosmetic privileges. In parallel, the market needs for natural and edible based products in pharmaceutical and dermo-cosmetic sector is emerging year by the year. Taking into consideration all the above, the reasons that VOs are under the scientific focus is clearly evident. Chemical composition of VOs is characterized mainly by glycerides, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). The plethora of health beneficial effects provided from the different fatty acids of VO are wildly known. The most famous properties of MUFA are the cardiovascular diseases prevention through different mechanisms. In parallel, VOs are source of many PUFAs such as linoleic that among other properties is considered as an important anti-atherogenic and anti-thrombotic agent and it cannot be synthetized by the human organism. Furthermore, a great majority of VOs contain an essential fraction of mainly phenol-based chemical structures with interesting biological activities, colloquially called bio-phenols. These compounds represent a very small part of the total oil weight being around 2-5%. Many studies have proved that VOs bio-phenols are responsible for a series of human health beneficial activities. As a consequence, scientific and industrial interest were centered on the investigation of VO chemical composition and biological properties for the development of pharmaceutical and cosmetic products.
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Following the demand of the market for natural-based products, it is very important to note that the conception is not only to use starting materials coming from natural sources, but additionally to handle and treat them with green methodologies. The last years, plenty of eco-friendly techniques have been developed and have been optimized for applications on field of natural products. Non-toxic solvents, low energy and less time consuming extraction and separation procedures and use of recycle/reusable by-products are few of the criteria that were taking into account in order to transfer procedures from laboratory to pilot and industrial level. These characteristics can be fulfilled only from state of the art techniques with green environmental character, like liquid-liquid solid support free chromatography and extraction, supercritical fluids and food grade processes in combination with the use of edible or non-toxic reagents. After extraction and separation, another vital step is the biological evaluation of natural product extracts and pure compounds. Due to the increasing ecological awareness of the last years, a huge endeavor is given to supersede the in vivo models by in vitro experiments. The research axis of the current thesis, was based on the exploration of nine different cold pressed VOs. The thesis is divided into two different parts according to the significance and subsequent production and/or consumption of the studied VO. Two of them, sesame seed oil and olive oil, are considered as worldwide commercial oils and were discussed in the first part of the thesis. At the seconds part, seven oils of limited production were studied. These seed oils come from plants: (Foeniculum vulgare) fennel, grape (Vitis vinifera), pumpkin Cucurbita pepo), sunflower (Helianthus annuus), almond (Prunus dulcis), pomegranate (Punica granatum) and cannabis-hemp (Cannabis sativa) and are globally produced in low quantities. Based on the most recent data of Food Agriculture Organization of the United states (FAO), sesame and olive oil worldwide production reached 937,000 tonnes and 3,039,000 tonnes, respectively. Both of them, are straightly correlated with the food habits of plenty African,
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Asian and European civilizations. Their high nutritional properties and the wide medical uses were the stimulus for their further speculation. Numerous studies emphasize the importance of these two oils bioactive constituents on human health. These findings trigger the need to develop methodologies for the recovery of bioactive compounds, known as bio-phenolic fraction, from sesame and olive oil in sufficient amounts, in order to get feasible their biological investigation in deep. To the best of our knowledge, until now there is no scientific reference in pilot scale extraction of bio-phenolic fraction neither for olive oil, nor for sesame oil. Aim of this thesis first part, is the development of extraction methodologies for the recovery of phenolic fraction from the two oils, employing pioneering liquid-liquid centrifugal techniques and the further fractionation and purification of bioactive solutes, mainly with liquid-liquid countercurrent solid support free chromatography, in quantities that reach up to gram scale. Precisely, regarding sesame oil, two synchronous extraction liquid-liquid centrifugal methods, Annular Centrifugal Extraction (ACE) and Centrifugal Partition Extraction (CPE), were compared in lab scale, with the intention to obtain its bio-phenolic fraction that is rich in two lignans, sesamin and sesamolin. For the fractionation of extract and the isolation of sesamin and sesamolin in pure form, over 95%, preparative Centrifugal Partition Chromatography (CPC) was applied. In parallel four minor lignans, samin, sesamol, sesaminol and episesaminol were isolated in high purity. All the minor and major lignans were finally evaluated in relation to their dermo-cosmetic properties against tyrosinase, elastase, collagenase and hyaluronidase enzymes. Moreover, this study was amplified with the development of a ground-breaking methodology for the recovery of sesame oil extract in pilot scale, in order to meet the in vivo experiment needs. Combining liquid-liquid centrifugal extraction of sesame oil with DMSO and food
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grade adsorption resins, discussed in the current manuscript for the first time, the recovery of major lignans in high quantities was succeed. For olive oil, several samples were analyzed and the richest in high added value molecules like oleocanthal (OLEO), oleacein (OLEA), monoaldehydic form of oleuropein aglycone (MFOA), monoaldehydic form of ligstroside aglycone (MFLA) and hydroxytyrosol (HT) were selected for extraction. For the first time a pilot scale extraction process of EVOO was took part on liquid-liquid centrifugal extractor. The starting experiments were employed on lab scale instrumentation and scaled up on pilot level providing an enriched bio-phenolic extract of hundreds grams. Prep-CPC analysis of EVOO extract led to the production of enriched fractions with the compounds of interest. Moreover, OLEO, OLEA, MFOA, MFLA and HT were purified on gram scale from CPC fractions with prep-HPLC-DAD. During the purification process of the target compounds, four minor solutes were isolated, three of which are new natural products (EDA lactone, (1R, 8E)-1-ethoxy-ligstroside aglycon and (1S, 8E)-1-ethoxy-ligstroside aglycon) and one more which is isolated for the first time from EVOO ((9E, 11E)-13-oxotrideca-9,11-diencoic acid). The second part of the thesis discusses the seven limited production cold pressed seed oils and their corresponding by-products. A totally, green pipeline was selected for all the steps of the employed treatment. With the purpose of decreasing at minimum the ecological fingerprint, seed oils were eluted straightly through food grade resins and their extracts were obtained, while for their by-products, experiments with Ultrasound Accelerated Extraction (UAE) and Supercritical Fluid Extraction (SFE) were conducted. Only non-toxic extraction solvents, ethanol and water, were used for the UAE, while SFE extraction was accomplished with CO2 and ethanol as co-solvent. Crude seed oils’ fatty acids were identified after GC-MS analysis, while all the extracts were evaluated for their antioxidant activity and their total phenolic
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content (TPC). Finally, dermo-cosmetic activities against collagenase, elastase, and tyrosinase enzymes were determined. The two most interesting seed oils and their by-products extracts (grape and fennel), in terms of enzymatic inhibition and their chemical profile, were further investigated. Grape seed oil resin extract and grape seed paste ultrasound assisted extract were fractionated with CPC. Concerning grape seed paste extract CPC fractions, were also evaluated against tyrosinase, collagenase and elastase enzymes. Fennel seed oil extract was found rich in anethole, a bioactive compound of high importance for the food industry. After one step CPC fractionation of extract, pure anethole was isolated in high amount, while in parallel a fraction enriched in isomeric forms of 1-(4-methoxyphenyl) propane-1,2-diol was eluted. Supercritical Fluid Chromatography (SFC) was chosen for the complete separation of isomeric forms. Moreover, other minor compounds were identified after prep-TLC treatment of CPC fractions. High interest had also the by-product extract of fennel seeds. After CPC analysis a fraction enriched in syringin was obtained that with the support of prep-HPLC syringin was purified.
To finalize, the current thesis was based on an effort to contribute in the existing knowledge for the ecological extraction and chromatographic treatment of edible vegetable oils as pool of bioactive compounds with health beneficial effects. Novel extraction and separation methodologies were developed for the recovery of high added value compounds, from extensive use oils, sesame and olive oil, with the implementation of state of the art green techniques. Compounds of high scientific interest were isolated in grams, while minor solutes were also isolated and reported for the first time. Additionally, a holistic green strategy for the screening of extracts from seven limited production seed oils (fennel, grape, pumpkin, sunflower, almond, pomegranate and cannabis-hemp seed oils) their respective by-products was designed. Innovative mindsets were proposed for the treatment of the starting materials
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with minimum environmental fingerprint. The final objective was the dermo-cosmetic evaluation with enzymatic assays of the produced extracts and the further chemical investigation of the most promising of them.
