An analytical approachfrom natural sources of bioactive compounds to nutraceuticals and functional foods. Development of extraction, characterization and bioactivity evaluation strategies

Resumen

The current report encompasses all the results found during work carried out for the PhD Thesis entitled: “An analytical approach: from natural sources of bioactive compounds to nutraceuticals and functional foods. Development of extraction, characterization, and bioactivity evaluation strategies”. This Thesis provides data on the extraction and characterization of different plant matrices such as Moringa oleifera and Pistacia lentiscus leaves, and various melon varieties. In addition, the in vitro bioactivity of different nutraceuticals made from cranberry (Vaccinium macrocarpon) is evaluated, and the in vivo bioactivity of functional foods such as docosahexaenoic-acid-enriched canola oil is assessed. This work is divided into two main sections: Introduction and Experimental part, results and discussion. The first part of the Introduction section presents the state of the art of nutraceuticals and functional foods and the workflow for their development, from the search for new sources of bioactive compounds to their marketing. Firstly, bioactive compounds are classified and their health implications are reviewed, focusing mainly on the three target families of bioactive compounds studied in this Thesis: phenolic compounds, plant sterols, and unsaturated fatty acids. Next, conventional (LLE, SLE, SPE) and alternative extraction techniques (UAE, SFE, PLE, and MAE) used for plant matrices and extraction techniques for biological matrices are described. Afterwards, the chromatographic analytical techniques for the separation (HPLC and GC) and detection (UV-Vis, QTOF-MS for HPLC and FID as well as Q-MS for GC) of bioactive compounds and the spectrophotometric methods (TPC, TFC, DMAC, and ELISA assays) for the quantification of bioactive compounds are detailed. After a section dedicated to the fractionation and purification of bioactive compounds, a section is included to describe the methods used to evaluate bioactivity. At the same time, this section is divided into methods for determining antioxidant activity and methods for evaluating antimicrobial activity. Finally, the plant matrices, nutraceuticals, and functional foods under study are briefly presented. The Experimental part, results, and discussion section is split into two subsections. The first focuses on searching for new sources of bioactive compounds while the second evaluates the in vitro and in vivo bioactivity of nutraceuticals and functional foods. Specifically, Subsection I examines the optimization of extraction systems for producing extracts rich in bioactive compounds from different matrices such as Moringa oleifera, Pistacia lentiscus, and three Cucumis melo varieties (melon) for a subsequent characterization and evaluation of their antioxidant activity. This subsection is divided in 5 chapters. In Chapter 1, two extraction systems, namely ultrasound-assisted extraction and conventional solid-liquid extraction, are optimized using different solvents and water-solvent mixtures for producing an extract with the highest total phenolic content from Moringa oleifera leaves. The optimal extract was characterized by HPLC–ESI–QTOF–MS, a powerful analytical tool that enabled the tentative characterization of 59 compounds, 30 of which, mainly flavonoids, were tentatively characterized for the first time in this matrix. Chapter 2 and Chapter 3 were prepared in collaboration with the Institute of Food Science Research (CIAL-CSIC), Madrid, after a 3-month-long predoctoral mobility fellowship called “Estancias breves FPU”. Due to the great potential of moringa leaves, in Chapter 2, a comparative analysis was made between two green extraction techniques, i.e. microwave-assisted extraction and pressurized liquid extraction. This time, both extraction methods were optimized through multi-response surface methodology. The parameters studied were temperature, solvent percentage, and extraction time while the extraction yield, total phenolic content, total flavonoid content, and the antioxidant activity measured by TEAC and DPPH assays were considered as response variables. Both extraction systems proved efficient for the extraction of phenolic compounds. However, pressurized liquid extraction allowed the recovery of more polar and temperature-sensitive compounds compared to the microwave-assisted extraction, which was better for the extraction of flavonoids and their glycosylated derivatives. Continuing with the same matrix, Chapter 3 approaches the composition of bioactive compounds from different fractions of moringa leaves after developing a green platform based on a three-step downstream processing that includes the use of supercritical fluid extraction, following by gas-expanded liquid (GXL), and ending with pressurized hot-water extraction (PHWE), all previously optimized. The extract by SFE was analyzed by GC–MS while the extract gained by PHWE was analyzed by HPLC–ESI–QTOF–MS. Meanwhile, the extract by GXLs was characterized employing both analytical techniques. The chromatographic analysis revealed that the volatile fraction from moringa leaves was a good source of alcohols, ketones, fatty acids, esters, and alkanes while the mid-polarity fraction and the most polar fraction were rich in glycosylated flavonoids and phenolic acids, respectively. Moreover, the content of total phenolics and total flavonoids was calculated and the antioxidant activity was also evaluated by TEAC assay. In Chapter 4, an analytical method was optimized in order to characterize the leaves of Pistacia lentiscus, commonly known as mastic tree, by means of a metabolite-profiling strategy. This chapter was prepared in collaboration with the Mira University, Bejaia, Algeria. The main parameters optimized were the mobile-phase composition, elution gradient, flow rate, and volume of injection to achieve the best resolution and sensitivity by HPLC–ESI–QTOF–MS. The optimized method enabled the tentative identification of 46 compounds, 20 of which were identified for the first time in this matrix. In addition, the quantification revealed flavonoids and phenolic acids as the most abundant in mastic leaves. Chapter 5 is dedicated to comparing the polar fraction of 14 extracts from 3 varieties of melon (Galia, Cantaloupe, and Piel de Sapo) most consumed in Spain. The HPLC–ESI–QTOF–MS analysis allowed the tentative characterization of amino acids and derivatives, nucleosides, organic acids, phenolic acids, esters, lignans, flavonoids, and other polar compounds. Furthermore, a principal component analysis (PCA) was applied to explore the distribution of the 3 varieties according to their composition, revealing a good differentiation between them. The Subsection II evaluates the stability as well as the in vitro and in vivo bioactivity of nutraceuticals and functional foods. This subsection is made up of 3 chapters. Chapters 6 and 7 were prepared in collaboration with the Pediatric Unit of San Cecilio Hospital from Granada and Chapter 7 was also included collaboration with the Microbiology Department from the aforementioned hospital. Chapter 6 was dedicated to the study of the stability of commercial cranberry syrup irradiated with gamma radiation stored for 6 months at 25°C and 60% relative humidity and under accelerated stability conditions. Compounds were characterized and quantified through HPLC–ESI–QTOF–MS and the proanthocyanidin quantification was also made by DMAC assay, both at the beginning and after 1, 3, and 6 months of storage. The majority of the quantified compounds were stable until 3 months of storage at 25ºC. In Chapter 7, 25 fractions from commercial cranberry capsules were isolated by semi-preparative chromatography. All the fractions and the complete extract were characterized by HPLC–ESI–MS and 13 fractions where chosen (the purest one) to assess their in vitro effect against a mix of 14 uropathogenic strains of Escherichia coli adherence and biofilm formation. The results suggest that apart from proanthocyanidins, other compounds, mainly flavonoids, could have antibacterial effects against uropathogenic E. coli by altering the surface hydrophobicity and inhibiting the biofilm formation in vitro. Lastly, Chapter 8 was prepared in collaboration with the “Richardson Centre for Functional Foods and Nutraceuticals” from the University of Manitoba (Canada) with a long-term predoctoral CEI-Biotic mobility fellowship of 3 months. In this chapter, the impact of different canola oils —conventional canola, high oleic canola oil, and high oleic canola oil enriched with docosahexaenoic acid (DHA)— was studied with respect to the sterol metabolism and PCSK9 concentration (a novel proprotein that regulates the cholesterol metabolism discovered in 2003) in plasma from 54 volunteers with at least one symptom of metabolic syndrome. The results demonstrated that the intake of omega-3 could reduce the risk factors associated to cardiovascular disease by diminishing the plasma PCSK9 concentration.