Department of Pharmacy
Library of the School of Science

2025-04-15

2025

Kalampokis Evangelos

Μαρία Χαλαμπαλάκη, Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Λέανδρος Αλέξιος Σκαλτσούνης, Καθηγητής, Τμήμα Φαρμακευτικής, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Εμμανουήλ Μικρός, Καθηγητής, Τμήμα Φαρμακευτικης, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Σοφία Μιτάκου, Καθηγήτρια, Τμήμα Φαρμακευτικής, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Ιωάννης Κωστάκης, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικης, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Ιωάννης Παπουτσης, Καθηγητής, Τμήμα Ιατρικής, Σχολής Επιστημών Υγείας, ΕΚΠΑ
Thomas Brück, Καθηγητής, Τμήμα Χημείας, Technical University of Munich, Garching, Germany

Investigation of metabolization pathways of bioactive molecules of edible, medicinal plants and their products in various in vitro and in vivo systems. Isolation and identification of metabolites thereof.

English

Investigation of metabolization pathways of bioactive molecules of edible, medicinal plants and their products in various in vitro and in vivo systems. Isolation and identification of metabolites thereof.

This thesis explores the metabolic pathways of bioactive molecules from edible, medicinal plants and their products in various in vitro and in vivo systems, with a particular focus on olive biophenols and turmeric sesquiterpenes. Understanding of the mechanisms of action of food bioactives (FBs) and their role in promoting health requires detailed elucidation of their metabolic pathways, beginning with the precise identification of their metabolites. To achieve this, advanced analytical platforms such as high-resolution LC/GC-MS and NMR are typically employed. The critical step of identifying metabolites or biomarkers is largely dependent on databases and existing literature. However, the substantial structural diversity of FBs and their metabolites, coupled with the scarcity of reference standards, as well as the fact that these compounds are often present in human biological fluids at very low concentrations makes their precise identification and quantification a significant analytical challenge.
To address these challenges, the preparative isolation of metabolites of interest in pure form from human urine or other substrates, presents a promising alternative for achieving unambiguous identification. Purification on a milligram scale would not only provide comprehensive spectral data from LC-MS as well as critical information such as chromatographic behavior and polarity but also allows 1 and 2D NMR experiments. The present thesis is structured into four chapters, each addressing a specific aspect of the sample preparation, preparative isolation of FBs metabolites and biological evaluation of these compounds, as well as biochemical pathways analysis. The overarching goal is to advance our understanding of how the bioactive compounds from medicinal plants or foods are metabolized in the human body and their potential applications in personalized nutrition and disease prevention.
Chapter 1 focuses on the development of a novel multiscale workflow for the preparative isolation of FBs-related metabolites from human urine by embedding established chromatographic tools from the field of natural products chemistry and the unambiguous structural characterization thereof. Chapter 1 is divided into two parts. Ιn part I, the primary aim was to develop a unified urine pretreatment workflow that is compatible with typical profiling techniques while also allowing for the isolation of biotransformation products and metabolites, contributing to their unambiguous identification. Towards this purpose, hydroxytyrosol (ΗΤ) was supplemented as a soft capsule (15 mg/day) to a healthy volunteer for one month and 24-h samples were collected. Typical analytical protocols used for biological samples pre-treatment were applied together with enrichment methodologies which are employed in natural products research. All derived extracts were analyzed via HPLC-DAD/ELSD, UPLC-HRMS and NMR to investigate their chemical profile and evaluated in terms of recovery yield, metabolite richness, and specificity based on the chemical classes of identified metabolites. Ultimately, the proposed methodology was successfully applied for large-volume urine pretreatment, offering the potential for metabolite isolation. The novelty of the current work lies in the development of a comprehensive workflow that facilitates both analytical profiling and the isolation of urine metabolites. To the best of our knowledge, such an integrated concept has not been reported in literature before. Another innovative aspect of this approach is the use of multiple analytical platforms in combination, providing complementary results for highly accurate metabolite identification. It is important to highlight that both qualitative and quantitative assessments were performed, focusing on the number of identified metabolites, their chemical classes, and their related biochemical pathways. To our knowledge, no similar study has investigated the efficiency of a urine pretreatment method while considering these aspects, which are crucial for accurate and complete interpretation of results towards biochemical pathways analysis and biological role interpretation.
In Part II we tried to develop a novel method for the separation, isolation and identification of FBs metabolites from human urine as well as to investigate their biochemical role through molecular pathways analysis. The opportunity of recognizing and quantifying the pathways in which FBs metabolites are involved will allow us to understand in depth their mechanisms of action and the interactions that may exert their beneficial effect. A major prerequisite in unraveling the underlying mechanisms is either organic synthesis or preparative isolation of these metabolites, which hampers their structural elucidation and biological assessment. Isolation of metabolites from human urine can be a useful alternative source for the recovery of bioactive compounds and an important driving force in drug discovery process since urine encompasses a wide range of chemical classes (including gut microbial metabolites and xenobiotics), it may be collected in large volumes non-invasively, making it an ideal starting material for purification, and is rich in small molecule metabolites and has a very low concentration in macromolecules. Using hydroxytyrosol (HT) as a case study, part II outlines a novel methodology for isolating and characterizing FB metabolites from human urine by embedding established chromatographic tools from the field of natural products chemistry to biological samples. Our approach could be used for the discovery of unknown metabolites that are not present in experimental databases, contributing to their full characterization. We believe that the proposed protocol will be valuable not only in traditional urine metabolomic studies but also in cases where unambiguous identification of metabolites is crucial, particularly in the analysis of biochemical pathways and the investigation of FB mechanisms of action within the framework of personalized nutrition.
Chapter 2 shifts focus on the biotechnological production of hydroxytyrosol using E. coli biofactories. Microbial production of bioactive molecules provides a promising alternative to traditional isolation or chemical synthesis approaches, enabling high-yield production of pure compounds without the need for heavy-metal catalysts, harsh conditions, or complex plant-based processes. E. coli has been widely used to synthesize diverse natural bioactive compounds. While microbial factories have been effective for HT production, optimization of yield and recovery process remain a challenge. Furthermore, isolation of HT and its metabolites continues to suffer from inefficiencies during extraction, separation, and purification even when biosynthetic pathways are successful. The chapter addresses the challenges associated with the biotechnological production, extraction and purification of HT from fermentation broths, which are often complex and contain impurities. To overcome these challenges, the chapter introduces a systematic multistep protocol that incorporates centrifugal extraction and partition chromatographic techniques, such as Annular Centrifugal Extraction (ACE) and Centrifugal Partition Chromatography (CPC). These methods enable the efficient isolation and purification of HT and its metabolites, facilitating their structural elucidation using HRMS/MS and NMR. This innovative approach not only optimizes the production of HT but also provides insights into the metabolic pathways of engineered microorganisms. This can significantly aid in pathway optimization by enabling efficient monitoring of the process and optimizing metabolic flux. This methodology aims to generate new insights into the mechanistic understanding of microbial metabolic biotransformations during fermentation but also to introduce centrifugal chromatography methodologies in biotechnological workflows.
In Chapter 3 the aim was to evaluate the activity of the olive oil biophenols (OBs) isolated in the preceding two chapters utilizing two model organisms: Caenorhabditis elegans and zebrafish. Part A investigates the modulatory role of OBs on increasing lifespan, stress resistance and the pathogenesis of Parkinson's disease in C. elegans experimental model. The study demonstrates that OBs, particularly HT and oleuropein, can modulate oxidative stress and reduce the aggregation of α-synuclein, a key factor in PD. Part B explores the anticonvulsant activity of OBs in a zebrafish (Danio rerio) model of epilepsy. The findings suggest that OBs, such as oleuropein and HT, exhibit protective effects against seizures, highlighting their potential as novel therapeutic agents for neurological disorders.
Chapter 4 examines the metabolic fate of bisabolane sesquiterpenes derived from turmeric essential oil (TEO) in the human body. Despite the well-documented health benefits of TEO, the bioavailability and metabolism of its sesquiterpenes remain poorly understood. Structural modifications in ΤEΟ bioactives are expected to result in different and potentially improved biological effects. This chapter proposes a multiplatform analytical approach combining Centrifugal Partition Chromatography (CPC), UHPLC-Orbitrap-MS, NMR, and GC/MS for the profiling, isolation and structural elucidation of TEO metabolites. The multiplatform approach provides a clear advantage to expanding the coverage of metabolome and improves our confidence in metabolite identification. The study aims to uncover novel metabolites with enhanced pharmacological activity, providing new insights into the metabolic pathways of turmeric sesquiterpenes and their potential health benefits.
In summary, this thesis contributes to the growing body of knowledge on the metabolism and bioactivity of natural bioactive compounds. By developing innovative methodologies for metabolite isolation and characterization, the research provides a deeper understanding of how these compounds exert their health benefits. The findings have significant implications for the investigation of metabolic pathways of medicinal plants in human organism and the development of novel therapeutic agents derived from natural sources.

Science

Metabolization pathways, Food bioactives, Urine, Metabolism, Metabolic profiling, Sample preparation, High-scale extraction, Metabolite identification, Hydroxytyrosol metabolites, NMR spectroscopy, LC-MS, Centrifugal Partition Chromatography, Escherichia coli, Annular Centrifugal Extraction, Caenorhabditis elegans, zebrafish, Curcuma longa, turmeric essential oil metabolism, bisabolene sesquiterpenoids biotransformation, ar-turmerone, urine metabolites isolation

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https://pergamos.lib.uoa.gr/uoa/dl/object/3479685