Titolo della tesi: CHARACTERIZATION OF BIOACTIVE COMPOUNDS IN AGRIFOOD PRODUCTS
In the last two decades, agri-food matrices have assumed a fundamental role as a source of sustenance and a reservoir of beneficial chemical compounds. For this purpose, research increasingly aims to make food the focal point of our health in preventing and improving immune responses. The identification of bioactive compounds and the subsequent enhancement of agri- food products have thus become the forefront of research. Functional foods have taken on an increasingly important role in recent years, thanks to their ability to maintain good health by preventing various pathologies due to their content of bioactive molecules. When appropriately isolated, characterized, and quantitatively determined, these substances can be used in the formulation of so-called nutraceutical compounds. The importance of characterizing bioactive compounds is not only linked to discovering molecules with beneficial effects on health. Still, it can also help detect possible food adulterations or determine composition and genuineness. To comprehensively characterize food and isolate individual bioactive compounds or phyto- complexes, it is necessary to use and develop advanced analytical techniques for extraction, purification, separation, and characterization. Liquid chromatography coupled with high-resolution mass spectrometry is currently the most effective tool for obtaining a "fingerprint" of the examined food matrix.
In this doctoral project, ample attention will be given to some classes of bioactive molecules with a specific focus on the polar lipidome of hemp. Although phytocannabinoids are the most recognized hemp bioactives due to their therapeutic potential, other lesser-studied classes of chemicals can exert synergistic effects to enhance the bioactivities of phytocannabinoids and other health-promoting activities. As such, a detailed characterization of the composition of this plant could help future research further exploit the beneficial effects of hemp compounds on human health. In this context, this doctoral project contributes to a detailed characterization of minor components of industrial hemp in terms of fatty acids, policosanols, carotenoids, and fat-soluble vitamins. The analytical approaches commonly used for determining fatty acids and policosanols involve gas chromatographic techniques with prior derivatization of the compounds under examination. The development of more straightforward methods based on liquid chromatography techniques, which do not require chemical derivatization, is one of the goals that this doctoral thesis achieved. The localization of double bonds is a crucial step for the structural characterization of fatty acids.
For this reason, another aim of this thesis was the development of a procedure that allows the identification of carbon-carbon double bond locations. In particular, although the consumption of hempseeds and its derivatives is rapidly increasing for the high nutritional value of its bioactive compounds, including a rich lipidome with elevated concentrations in PUFAs with optimum ω- 6/ω-3 ratio, comprehensive characterization of the polar lipidome was missing, especially regarding the regioisomer composition of fatty acids bound to glycerophospholipids. To achieve this goal, an analytical platform, including derivatization by a photochemical [2+2] cycloaddition with 6- azauracil was developed for pinpointing the regiochemistry of carbon-carbon double bonds in polar lipids extracted from hempseeds. This approach allowed the determination of double bond locations of free and conjugated fatty acids, that is essential to further understand their biological roles and biophysical properties.
In the wide range of studied phytochemical compounds in industrial hemp, carotenoids and fat- soluble vitamins have scarcely been considered, even though their various bioactivities. For the first time, in this doctoral project, the carotenoid and fat-soluble vitamin profile of Cannabis inflorescences has been extensively characterized in both qualitative and quantitative fashion after an extraction approach aimed at avoiding the generation of artifacts.
Another aim of this thesis was the development of proper mass-spectrometric and data-analysis methods for the comprehensive characterization of phenolic compounds. Thanks to their demonstrated potential health benefits, phenolic compounds are perhaps the most explored natural compounds. Hence, exploring and identifying new phenolic compounds from natural sources have become a primary research focus in developing natural products. In particular, since Andean berries are a rich source of a wide variety of phenolic compounds, untargeted MS analyses coupled with a dedicated data processing workflow allowed us to expand the current knowledge on these berries grown in the unique Andean ecosystem, also improving our understanding of the fate of phenolic compounds after fermentation. Moreover, an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity was developed.
Therefore, this doctoral project aimed to develop advanced analytical methodologies for determining these classes of bioactive compounds and overcoming obstacles still present in their extraction, identification, and complete characterization.