Ana A. Matias

Dunaliella salina has been recognized as an excellent biomass source of carotenoid, which can be used as a natural orange coloring agent for food products. The most eco-friendly approach for extracting carotenoid is through supercritical carbon dioxide extraction, as it yields highly concentrated extracts while preventing pigment thermal degradation. However, there are limitations when a lipophilic extract is considered a food ingredient, in particular very difficult handling and low solubility in water-based products. The aim of this study was to develop a hydrosoluble form of a natural carotenoid-rich extract recovered from algae biomass within a biorefinery concept to be incorporated in aqueous-based food products. A two-step process was developed, starting with the emulsification of the supercritical extract into a mixture of maltodextrin and gum arabic, using soy lecithin as an emulsifier. The emulsification was followed by a spray-drying step. The impact of process variables on the encapsulation yield, efficiency, emulsion properties, and particle characteristics was studied. The resulting particles exhibited an intense orange color and good water dispersibility, facilitating uniform yellow coloring when incorporated into an aqueous-based product. Overall, spray-drying emulsions containing carotenoids derived from Dunaliella salina prove to be a promising strategy for the global market demand for natural colorants.

Natural deep eutectic solvents (NaDES) were used to extract bioactive compounds from marine by-products: codfish bones, mussel meat, and tuna vitreous humor. NaDES were prepared using natural compounds, including lactic acid (Lac), fructose (Fru), and urea (Ur), and were characterized to define their physicochemical properties, including the viscosity, density, surface tension, and refractive index. FTIR and NMR analysis confirmed the presence of intermolecular hydrogen bonding in NaDES. The extracts obtained using these NaDES were characterized to define their composition. Results demonstrated that the extract’s composition differed highly, depending not only on the DES used, but also on the structure and composition of the raw material. Proteins and lipids were mainly present in extracts obtained from mussels, while ash content was highest in the extracts obtained from codfish bones. The biocompatibility of NaDES and the soluble fractions (SF) of the raw materials in NaDES was evaluated, and it was possible to conclude that the soluble ingredients obtained from the raw materials improved the biocompatibility of NaDES.

A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds (lactic acid, fructose, and urea). The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management.

Pressing strawberries for juice generates large amounts of pomace, containing valuable nutrients and therefore requiring more systematic studies for their valorization. This study compared conventional solid-liquid (SLE) and pressurized liquid (PLE) extractions with ethanol (EtOH) and H₂O for the recovery of bioactive compounds from strawberry pomace. The composition and bioactivities of the products obtained were evaluated. Among 15 identified compounds, quercetin-3-glucuronide, kaempferol-3-glucuronide, tiliroside, ellagic, malic, succinic, citric and p-coumaric acids were the most abundant constituents in strawberry pomace extracts. SLE-EtOH and PLE-H₂O extracts possessed strong antioxidant capacity in DPPH^• and ABTS^•+ scavenging and oxygen radical absorbance capacity (ORAC) assays. Cytotoxicity, antiproliferative and cellular antioxidant activities in human cells of PLE-EtOH and PLE-H₂O extracts were also evaluated. PLE-EtOH and PLE-H₂O extracts possessed strong antioxidant activity, protecting Caco-2 cells upon stress stimuli, while PLE-EtOH extract showed higher antiproliferative activity with no cytotoxicity associated. In general, the results obtained revealed that properly selected biorefining schemes enable obtaining from strawberry pomace high nutritional value functional ingredients for foods and nutraceuticals.

The beneficial health effects of Chlorella vulgaris have been associated with the presence of several nutrients and antioxidants, including carotenoids. However, the in vivo bioavailability of Chlorella is still poorly evaluated. In this work, a human intervention study was conducted in 11 healthy men to evaluate the bioavailability of carotenoids within 3 days after the intake of a single dose (6 g) of dried marine Chlorella vulgaris containing lutein (7.08 mg), β-carotene (1.88 mg) and zeaxanthin (1.47 mg). Subjects were instructed to follow a low carotenoid diet during the experimental phase, starting 1 week earlier. On the day of the experiment, dried microalgae formulated in vegetarian hard capsules were ingested, and blood samples were collected up to 72 h for the analysis of plasma carotenoids concentration by high-performance liquid chromatography with diode-array detection. For all carotenoids, the estimated AUC and C_max values were significantly different from zero (p < 0.05), indicating that a single dose of marine Chlorella vulgaris increased plasma concentrations of lutein (C_min-corrected AUC = 1002 µg·h/L, C_max = 20.4 µg/L), β-carotene (AUC = 1302 µg·h/L, C_max = 34.9 µg/L) and zeaxanthin (AUC = 122.2 µg·h/L, C_max = 3.4 µg/L). The bioavailability of other compounds, namely, polyunsaturated fatty acids and trace elements, was also assessed post-prandial for the first time, showing that linoleic acid, docosahexaenoic acid and iodine were absorbed after microalgae intake. These findings support the use of Chlorella vulgaris as a source of carotenoids, PUFA and essential trace elements with associated health benefits.

Cichorium intybus L. or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being harnessed. We developed and optimized a new strategy for SL extraction that can contribute to the conversion of chicory root waste into valuable products to be used in human health-promoting applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical CO₂. A response surface methodology was used to optimize the process parameters (pressure, temperature, flow rate, and co-solvent percentage) for the extraction performance. The best operating conditions were achieved at 350 bar, 40 °C, and 10% EtOH as a co-solvent in a 15 g/min flow rate for 120 min. The extraction with supercritical CO₂ revealed to be more selective for the SLs than the conventional solid–liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene lactones (8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin), which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at different concentrations for their toxic profile and anti-inflammatory potential towards a human calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin inhibited the activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. These results reveal a possible exploitable green technology to recover potential anti-inflammatory compounds from chicory roots waste after inulin extraction.

The characterization of terpene-based eutectic solvent systems is performed to describe their solid–liquid phase transitions. Physical properties are measured experimentally and compared to computed correlations for deep eutectic solvents (DES) and the percentage relative error e_r for the density, surface tension, and refractive index is obtained. The thermodynamic parameters, including the degradation, glass transition and crystallization temperatures, are measured using DSC and TGA. Based on these data, the solid–liquid equilibrium phase diagrams are calculated for the ideal case and predictions are made using the semi-predictive UNIFAC and the predictive COSMO RS models, the latter with two different parametrization levels. For each system, the ideal, experimental, and predicted eutectic points are obtained. The deviation from ideality is observed experimentally and using the thermodynamic models for Thymol:Borneol and Thymol:Camphor. In contrast, a negative deviation is observed only experimentally for Menthol:Borneol and Menthol:Camphor. Moreover, the chemical interactions are analyzed using FTIR and ¹H-NMR to study the intermolecular hydrogen bonding in the systems.

Therapeutic deep eutectic systems (THEDES) have dramatically expanded their popularity in the pharmaceutical field due to their ability to increase active pharmaceutical ingredients (APIs) bioavailability. However, their biological performance has not yet been carefully scrutinized. Herein, THEDES based on the binary mixture of perillyl alcohol (POH) and ibuprofen (IBU) were prepared using different molar ratios. Our comprehensive strategy includes the characterization of their thermal and structural behavior to identify the molar ratios that successfully form deep eutectic systems. The in vitro solubility of the different systems prepared has demonstrated that, unlike other reported examples, the presence of the terpene did not affect the solubility of the anti-inflammatory agent in a physiological simulated media. The biological performance of the systems was studied in terms of their antimicrobial activity against a wide panel of microorganisms. The examined THEDES showed relevant antimicrobial activity against all tested microbial strains, with the exception of P. aeruginosa. A synergistic effect from the combination of POH and IBU as a eutectic system was verified. Furthermore, the cytotoxic profile of these eutectic systems towards colorectal cancer (CRC) in vitro cell models was also evaluated. The results provide the indication that the cell viability varies in a dose-dependent manner, with a selective THEDES action towards CRC cells. With tunable bioactivities in a ratio-dependent manner, THEDES enhanced the antimicrobial and anticancer properties, representing a possible alternative to conventional therapies. Therefore, this study provides foreseeable indications about the utility of THEDES based on POH and IBU as strong candidates for novel active pharmaceutical systems.

Cichorium intybus L. has recently gained major attention due to large quantities of health-promoting compounds in its roots, such as inulin and sesquiterpene lactones (SLs). Chicory is the main dietary source of SLs, which have underexplored bioactive potential. In this study, we assessed the capacity of SLs to permeate the intestinal barrier to become physiologically available, using in silico predictions and in vitro studies with the well-established cell model of the human intestinal mucosa (differentiated Caco-2 cells). The potential of SLs to modulate inflammatory responses through modulation of the nuclear factor of activated T-cells (NFAT) pathway was also evaluated, using a yeast reporter system. Lactucopicrin was revealed as the most permeable chicory SL in the intestinal barrier model, but it had low anti-inflammatory potential. The SL with the highest anti-inflammatory potential was 11β,13-dihydrolactucin, which inhibited up to 54% of Calcineurin-responsive zinc finger (Crz1) activation, concomitantly with the impairment of the nuclear accumulation of Crz1, the yeast orthologue of human NFAT.

Defatted by supercritical CO₂, Viburnum opulus berry pomace (VOP) was subjected to consecutive extraction with pressurized ethanol (E) and water (W) and yielded 23% of VOP-E and 8% of VOP-W, respectively. The major phytochemical groups covering 42 identified and quantified constituents in VOP extracts were organic and phenolic acids, iridoids, quercetin and (epi)catechin derivatives, flavalignans, procyanidins, and anthocyanins. The on-line HPLC-DPPH^•-scavenging assay revealed the presence of numerous antioxidants. VOP-E had a higher total phenolic content, was a stronger antioxidant (equivalent to 0.77, 0.42, and 0.17 g trolox/g in oxygen radical absorbance capacity (ORAC), ABTS, and DPPH assays, respectively), and recovered the major part of phenolics from the pomace; however, both extracts demonstrated similar antioxidant activity in the cellular assay. VOP-E inhibited HT29 cancer cells at non-cytotoxic concentrations. The results of this study revealed that VOP contains valuable phytochemicals possessing antioxidant and antiproliferative activities. Consequently, extracts from VOP substances may be of interest in developing functional ingredients for healthy foods, nutraceuticals, and cosmeceuticals.