Dissertation committee:
Maria Halabalaki, Assistant Professor, School of Pharmacy, National and Kapodistrian University of Athens
Leandros A. Sklatsounis, Professor, School of Pharmacy, National and Kapodistrian University of Athens
Emmanouil Mikros, Professor, School of Pharmacy, National and Kapodistrian University of Athens
Emerson Ferreira Queiroz, Senior Lecturer, School of Pharmaceutical Sciences, University of Geneva
Nikolaos Tentolouris, Professor, School of Medicine, National and Kapodistrian University of Athens
Sofia Mitakou, Professor, School of Pharmacy, National and Kapodistrian University of Athens
Elissavet Dotsika, Researcher A’ , Institute of Nanoscience and nanotechnology, National Center for Scientific Research Demokritos
Summary:
Nature still remains an untapped source of bioactive molecules and an important driving force in drug discovery process. Natural products (NPs) are characterized by high degree of structural diversity and uniqueness which is incomparable to chemically synthesized molecules. Only in the area of cancer, from 1940s to 2007, 73% of the 155 small molecules are other than synthetic, with 42% actually being either NP or directly derived from NP [1]. The main sources of NPs are medicinal plants but also marine organisms and microorganism as well as edible plants and foods.
In our days much attention has been given to dietary and nutritional habits for the prevention as well as the treatment of human health complications and diseases. Dietary supplements have been introduced in the daily routine of the general population especially in western countries aiming to the improvement of life quality and health [2]. Nevertheless, the definition of dietary supplements differs from country to country and the general terminology includes natural health products, complementary medicines, food supplements or functional foods [3]. No global consensus exists for the classification of these products and their assortment into specific groups is difficult due to the existence of large number of products, their varying content and the large number of properties and claims.
The existing regulations concerning such products can be characterized as a grey zone, and in European Union (EU) dietary supplements are regarded as foods [2]. Many botanicals are also classified as dietary supplements and typically labelled as natural foods while health claims are often accompanying them. The lack of legislation allows for the marketing of many preparations without sufficient or solid scientific data, mainly related to quality, efficacy and safety. Even in cases that active compounds or entities are contained for which their activity and safety are scientifically substantiated, it is not the case for the final product in the market. Moreover, despite efficacy and safety which are commonly underestimated, one of the most common limitation in supplements is the lack of knowledge for the bioavailability and generally ADMET properties, which are directly connected to efficacy. Finally, the absence of quality assessment of the dietary and/or food supplements is commonly observed.
Amongst foods, olive oil (OO) has attracted the scientific interest after the publication of two heath claims concerning its chemical composition and the respective positive impacts to human health [4,5]. Nowadays, OO has been established in the consciousness of people as one of the most superior nutritional and health promoting edible oils world-wide, with exceptional organoleptic and sensory properties. Responsible for health and nutrition beneficial effects as well as its recognition as superior edible oil is its unique chemical composition. OO composition is extremely variable and complex and depends on several exogenous factors like olive variety, microclimate conditions, cultivation practice, production procedure, storage etc. Such factors influence its content in the contained compounds [6]. Interestingly, till today there is not a satisfactory and generally accepted method for quality control of OO. Considering the economic value of OO for EU and especially for Mediterranean countries, the investigation of factors infusing the composition of OO, the identification of certain chemical markers as well as the establishment of a proper methodology for quality control purposes is of outmost importance.
Towards the elucidation of OO composition, numerous studies have been carried out aiming to the identification of its constituents but also the exploration of biological properties thereof. Recently, OO polyphenols, the small fraction of polar compounds of OO are in the center of research. It is worth noting that till today there are still compounds in OO which haven’t been structurally characterized and new ones are reported [7]. Especially, OO polyphenols exert important biological properties and constantly new studies are published investigating their role. However, a critical drawback which complicates and delays significantly this process is the lack of reference compounds in the market for the investigation of their activity. For instance, oleocanthal (OLEA) and oleacein (OLEO) are two of the major secoiridoids in OO categorized in polyphenols group which according to recent studies exert a plethora of promising biological properties but they are not commercially available. Thus, the establishment of methods for the isolation in pure form as well as in satisfactory yield of such compounds is the basic step enabling their further investigation. This will allow the performance of in vitro but more importantly in vivo experiments which will facilitate significantly the elucidation of their biological role and consequently will assist to decipher the properties of OO itself.
Another field which is also overlooked in the area of NPs and it is affected from the lack of reference compounds in required yield and purity is the exploration of ADMET properties. Studies related to such properties are rare [8] also in well-established active natural entities despite the fact that Absorption, Distribution, Metabolism, Excretion and Toxicity of a compound influence the possible drug levels and kinetics and hence influence the performance and pharmacological activity of a compound as a drug. This fact, resulting to incomplete information for an active entity and it is decisive step for the development of a compound as a drug. Similar pattern is observed also in OO polyphenols and limited are the studied concerning the investigation of ADMET characteristics thereof. Despite, the numerous studies related to the activity of OO secoiridoids, the lack of such information is vast even though their unstable and highly reactive chemical nature is well-known. Therefore, the introduction of ADMET in the general workflow of drug discovery process in the area of NPs is a critical factor. ADMET information together with complete isolation and identification data as well as biological profile can optimize significantly the entire process.
Among these lines, together with unknown or recently uncovered polyphenols, there are also well known compounds in OO with established biological and pharmacological properties. One of the most potent compound which is also generally considered as one of the most active NPs is hydroxytyrosol (HT) [9]. A lot effort has been made so far for the interpretation of its properties and its role mainly as a strong antioxidant agent has been proven. Special attention has been given after the EFSA health claim connecting HT and its derivatives in OO with protective effect of blood proteins from oxidation. HT but also its derivatives can been found in several dietary supplements in the market usually in OO or as an enrichment factor. However, very limited are the studies exploring the efficacy of such products, most of the cases the levels of individual constituents are not determined and even less are the interventions in humans. HT has been detected in urine, plasma and tissues (even in the brain) however it is easily biotransfomed [10]. Unfortunately, despite the commercialization of many products containing HT, there is limited information for the impact of HT to human metabolome. Most of the studies investigate the positive impact of HT supplementation for the prevention or treatment of a specific pathology or its concentration in human tissues and few studies have been published for the followed biosynthetic path in human organism. This situation which as mentioned before concerns the majority of supplements resulting to consumer or patient confusion and on the other hand doesn’t allow truly beneficial products to be disclosed and further investigated and developed. Therefore, there is a strong need for solid scientific data for dietary supplements substantiating the complete composition as well as their efficacy. Most importantly human studies are critical ensuring the potency against a clear complication or disease as well as safety; both critical factors for the general population.
In the current study, we tried to develop methods and approaches aiming to offer possible answers to certain complications, limitations and inadequacies in NPs drug discovery process and NPs applications and uses. As a model starting material olive oil was selected. Olive oil is a perfect candidate since it is characterized by insufficient methods for quality evaluation, is a rich source of highly potent, promising and unknown compounds while OO itself or its constituents in different forms and mixtures are circulated in the market. Moreover, limited are the studies relayed to the ADMET properties of OO constituents, in vivo evaluation towards specific targets and nearly absent controlled human studies. In the center of this effort was the development of chromatographic and analytical methods for the isolation of active compounds, quality control and quantitation of actives in different biological fluids. Especially, mass spectrometry was intensively used to accomplished these goals as well as metabolic profiling and metabolomic approaches.
Based on the above in the current study a complete work-flow was designed and established exploring OO and its constituents. Specifically, starting from a library of OO samples (approx. 300) from all over Greece, quality control aspects for OO were examined aiming to the determination of the effect of specific quality parameters to OO composition and identification of marker compounds. OO characteristic compounds, hydroxytyrosol (HT), tyrosol (T), oleacein (OLEA) and oleocanthal (OLEO) were quantified based on International Olive Council (IOC) HPLC-DAD method revealing important information. Moreover, taking advantage of the ultra high resolution and high accuracy of mass spectrometry platforms in combination with chemometric tools, statistical significant metabolites were determined and identified for their final correlation with specific quality factors. This is the first time that a complete metabolic profiling approach is developed and applied in Greek EVOOs with two different HRMS platforms and a list of identified marker compounds were determined.
Secoiridoids were found to be one of the major chemical categories in the generated list of markers. Thus, selected secoiridoid derivatives and recently reported iridoid derivatives were isolated for the performance of in vitro and in vivo tests as well as a pharmacokinetics study. OLEO and OLEA were two of them, characterized by strong anti-inflammatory and antioxidant properties. Both of them as well as three polyphenols extracts (TPFs) with high, medium and low levels of these metabolites were tested in vitro for evaluation of their cytotoxicity effects and proteasome activity in human fibroblasts cells. Moreover, TPFs were administered as dietary supplement to a Drosophilla in vivo model for investigation of their impact to healthy aging. Both molecules as well as TPF revealed positive impact to healthy aging promoting cytoprotective pathways and suppressed oxidative stress in both mammalian cells and flies. Consequently, an in vivo mouse model was designed for determination of OLEO pharmacokinetic characteristics. OLEO metabolism and biotransformation in vivo was investigated for the first time.
Another statistical significant compound was HT which is also a constituent of several products in the market. Thus, an intervention study was designed and performed to investigate the effect of an enriched in HT soft capsule supplemented in two different doses (5mg/day and 15mg/day) based on suggested dose from EFSA, to obese/overweight women in double-blinded study for a six-month period. Capsule was firstly evaluated for its final quality; total ingredients were identified and the final HT concentration was accurately determined. A targeted UPLC-triple-quadropole methodology was developed and validated for quantitation of HT in urine during the whole intervention period and excretion levels were established. Consequently, un untargeted high-resolution mass spectrometry method was developed for the exploration of blood and urine metabolome during intervention. Biotransformation reactions of HT were monitored and possible phase I and phase II metabolization products were identified. Statistical significant weight and fat loss was observed after six months of supplementation in high doss (15mg/day) and it is the first time that HT is related to obesity in a human cohort. Moreover, different pathways were seemed to be followed depending on the supplemented dose. Using metabolomics approaches novel data were provided concerning the association of certain endogenous metabolites with HT supplementation.
