Compressed fluid-based technology for downstream isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus.

A dynamic compressed fluid-based separation process combining carbon dioxide and ethanol was explored to isolate portisins previously hemi-synthesized from blueberry surplus anthocyanins. The influence of process parameters such as pressure (100-500 bar), temperature (40-60 °C), and ethanol content in the compressed fluid mixture (20-50 wt%) on extraction yield, portisins yield, and portisins content in the extract was investigated. The two-step isolation process includes (1) a first step at 100 bar, 60 °C, and 20 wt% ethanol content in the compressed fluid mixture to remove the low polarity compounds; and (2) a second step at 500 bar, 40 °C, and 100 wt% ethanol to recover portisins, resulting in a 1.5-fold increase in portisins content. The performance of the two-step separation process was compared to centrifugal partitional chromatography and conventional reverse phase liquid chromatography already reported in terms of portisins content in the extract, process throughput, process efficiency, and total solvent used. The two-step separation process decreased the total solvent used, although with a decrease in the throughput and efficiency. Nevertheless, the choice of the best separation technology depends on the application, as these techniques result in different portisins purities. Overall, this study contributed to a scalable and more sustainable process for natural colorant production, specifically focusing on blue pigments, with several industrial applications.

Isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus using centrifugal partition chromatography.

Replacement of synthetic colorants with natural ones is a current marketing trend. Nevertheless, the naturally occurring blue color is rare compared to other colours. In this work, centrifugal partition chromatography (CPC) process was developed as a more efficient and sustainable alternative to reversed phase column chromatography (RP-CC) for the preparative-scale purification of portisins. The strategy began with the extraction of anthocyanins from blueberry surplus and hemi-synthesis of respective portisins. Then, the CPC method development started with the biphasic solvent system selection followed by the optimization of the operating parameters and ended up with a comparison with RP-CC. Aiming at maximizing the portisin content, process throughput, efficiency, and minimizing the environmental risk factor, the effect of sample load (100-500 mg/100 mL of column volume), mobile phase flow rate (10-20 mL/min), and rotation speed (1000-1600 rpm) was evaluated. The two-phase solvent system consisted of tert­butyl­methyl ether, n-butanol, acetonitrile, and water (volume ratio 2:2:1:5) acidified with 0.1 vol.% of HCl was selected. The best conditions were 464 mg of sample/100 mL of column volume, 20 mL/min of mobile phase flow rate, and 1600 rpm of rotation speed at reversed phase mode, allowing the purification of portisins by 5-fold. Compared to the RP-CC, the CPC process efficiency was 2.4 times higher, while the CPC process environmental risk factor was 5.5 times lower. Overall, this study suggests that CPC can be considered an effective, and sustainable alternative process for the preparative isolation of portisins. With this purification approach, the blueberry surplus has been valorized and a naturally derived product has been prepared, allowing its subsequent use as a natural blue colorant.

Lactic Acid-Based Natural Deep Eutectic Solvents to Extract Bioactives from Marine By-Products.

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.

Extraction of Bioactive Compounds From Cannabis sativa L. Flowers and/or Leaves Using Deep Eutectic Solvents.

The increasing demand for medical cannabis urges the development of new and effective methods for the extraction of phytocannabinoids. Deep eutectic solvents (DESs) are an alternative to the use of hazardous organic solvents typically used in the industry. In this study, hydrophilic and hydrophobic DESs were developed based on terpenes, sugars, and natural organic acids as green extraction media for the extraction of cannabis bioactive compounds. The factors influencing the extraction of bioactive components, such as the type of DESs and extraction time, were investigated. Initial screening in hemp showed that the DES composed of Men: Lau (a 2:1-M ratio) had a greater extraction efficiency of cannabidiol (CBD) and cannabidiolic acid (CBDA) (11.07 ± 0.37 mg/g) of all the tested DESs and higher than ethanol. Besides having a higher or equivalent extraction yield as the organic solvents tested, DESs showed to be more selective, extracting fewer impurities, such as chlorophyll and waxes. These results, coupled with the non-toxic, biodegradable, low-cost, and environmentally friendly characteristics of DESs, provide strong evidence that DESs represent a better alternative to organic solvents.

Potential Ophthalmological Application of Extracts Obtained from Tuna Vitreous Humor Using Lactic Acid-Based Deep Eutectic Systems.

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.

Alternative Extraction and Downstream Purification Processes for Anthocyanins.

Anthocyanins are natural pigments displaying different attractive colors ranging from red, violet, to blue. These pigments present health benefits that increased their use in food, nutraceuticals, and the cosmetic industry. However, anthocyanins are mainly extracted through conventional methods that are time-consuming and involve the use of organic solvents. Moreover, the chemical diversity of the obtained complex extracts make the downstream purification step challenging. Therefore, the growing demand of these high-value pigments has stimulated the interest in designing new, safe, cost-effective, and tunable strategies for their extraction and purification. The current review focuses on the potential application of compressed fluid-based (such as subcritical and supercritical fluid extraction and pressurized liquid extraction) and deep eutectic solvents-based extraction methods for the recovery of anthocyanins. In addition, an updated review of the application of counter-current chromatography for anthocyanins purification is provided as a faster and cost-effective alternative to preparative-scale HPLC.

Low-Phytotoxic Deep Eutectic Systems as Alternative Extraction Media for the Recovery of Chitin from Brown Crab Shells.

The versatility of chitin and its derivatives has allowed their utilization in a wide range of applications, from wastewater treatment to pharmaceutical or biomedical industries. However, even though the extraction method used industrially is extremely efficient, it involves the use of strong acids and bases and results in the disposal of large quantities of toxic effluents. Deep eutectic systems (DESs) have emerged as a promising new class of alternative solvents, including for chitin recovery. Yet, the assessment of their toxicity has often been neglected. Therefore, in this work, the phytotoxicity of choline chloride (ChCl)/organic acid-based DESs toward wheat seeds was evaluated by measuring different growth parameters and stress biomarkers. DESs were then explored for the efficient recovery of chitin contained in brown crab shell residues at varying conditions of temperature and processing time as well as with and without water addition. The obtained chitin was then characterized through different analytical techniques and compared to a standard as well as to chitin obtained by a conventional acid/alkaline hydrolysis. Results have shown that by applying a ChCl/lactic acid-based DES (which was the system that showed the least phytotoxic effects on wheat; EC50 ≥ 1.6 mg/mL) at 130 °C, it was possible to obtain pure chitin (up to 98%) with characteristics similar to those presented by commercial chitin or chitin recovered by conventional hydrolysis in a shorter time (more than 8-fold faster), thus suggesting that ChCl/organic acid-based DESs can truly represent a low-phytotoxic alternative extraction media for the recovery of chitin from the crab shell biomass.

A Single Dose of Marine Chlorella vulgaris Increases Plasma Concentrations of Lutein, ß-Carotene and Zeaxanthin in Healthy Male Volunteers.

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), ß-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 Cmax values were significantly different from zero (p < 0.05), indicating that a single dose of marine Chlorella vulgaris increased plasma concentrations of lutein (Cmin-corrected AUC = 1002 µg·h/L, Cmax = 20.4 µg/L), ß-carotene (AUC = 1302 µg·h/L, Cmax = 34.9 µg/L) and zeaxanthin (AUC = 122.2 µg·h/L, Cmax = 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.

Recovery of Bioactive Compounds from Strawberry (Fragaria × ananassa) Pomace by Conventional and Pressurized Liquid Extraction and Assessment Their Bioactivity in Human Cell Cultures.

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.

Lipid-based nanostructures as a strategy to enhance curcumin bioaccessibility: Behavior under digestion and cytotoxicity assessment.

The aim of this study was to evaluate the behavior of different lipid-based nanostructures during in vitro digestion, in particular on curcumin's bioaccessibility, and to access their potential toxicity. Solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE) were submitted to harmonized static in vitro digestion and their cytotoxicity and cellular transport were evaluated using Caco-2 cell line. NE presented the highest curcumin's bioaccessibility followed by NLC and SLN, 71.1%, 63.7% and 53.3%, respectively. Free fatty acids percentage increased in the following order: NLC ≤ NE < SLN. Non-digested nanostructures and excipients presented no cytotoxicity; however, digested NE and NLC presented cytotoxicity due to MCT oil, which presented cytotoxicity after digestion. The apparent permeability coefficient of NLC was higher than SLN and NE. These results showed that lipid-based nanostructures' physical state and composition have a high influence on particles' behavior during digestion, and on their cytotoxicity/intestinal permeability, and highlights the importance of conducting cytotoxicity assessments after in vitro digestion. This work contributes to a better understanding of the behavior of lipid-based nanostructures under digestion/adsorption, and this knowledge will be useful in design of nanostructures that afford both safety and an increased bioactive compounds' bioavailability.

Physicochemical Characterization and Simulation of the Solid-Liquid Equilibrium Phase Diagram of Terpene-Based Eutectic Solvent Systems.

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 er 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.

Supercritical CO2 Extraction as a Tool to Isolate Anti-Inflammatory Sesquiterpene Lactones from Cichorium intybus L. Roots.

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 11ß,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 11ß,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.

Assessing the Intestinal Permeability and Anti-Inflammatory Potential of Sesquiterpene Lactones from Chicory.

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 11ß,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.

Valorization of European Cranberry Bush (Viburnum opulus L.) Berry Pomace Extracts Isolated with Pressurized Ethanol and Water by Assessing Their Phytochemical Composition, Antioxidant, and Antiproliferative Activities.

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.

Silver nanocomposites based on the bacterial fucose-rich polysaccharide secreted by Enterobacter A47 for wound dressing applications: Synthesis, characterization and in vitro bioactivity.

This study demonstrates the potential of a high molecular weight fucose-containing polysaccharide secreted by the bacterium Enterobacter A47, named FucoPol, and its silver nanocomposite as potential bioactive materials for wound dressings applications. A green, simple, light-assisted method was used for the synthesis of silver nanoparticles (AgNP) using FucoPol, as stabilizing and reducing agent. The synthesized nanoparticles were spherical, and the main population had a particle size in number ranging between 13 and 30 nm for percentiles 50 and 90, respectively. FucoPol, as well as the functionalized material, besides having no cytotoxicity towards human skin keratinocytes and mouse fibroblasts, also promoted in vitro keratinocytes migration. These observations not only show the safety of FucoPol and FucoPol/AgNP biocomposite, but also their wound healing ability. Moreover, the biocomposite had a strong antimicrobial activity against Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae CECT 8453, two representative strains of known skin commensal pathogens. These findings demonstrate for the first time the potential of FucoPol for the development of wound healing formulations. Additionally, the FucoPol/AgNP biocomposite might find use in antimicrobial biomaterials, including antibacterial wound healing formulations, which further strengthens the establishment of FucoPol as a bioactive biopolymer.

Hyaluronic acid and Chondroitin sulfate from marine and terrestrial sources: Extraction and purification methods.

Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products.

Phytochemical Composition, Antioxidant and Antiproliferative Activities of Defatted Sea Buckthorn (Hippophaë rhamnoides L.) Berry Pomace Fractions Consecutively Recovered by Pressurized Ethanol and Water.

This study aimed at valorisation of sea buckthorn pomace (SBP) for the production of extracts containing valuable bioactive compounds. For this purpose, SBP defatted by supercritical CO2 was subjected to consecutive fractionation with pressurized ethanol and water, which yielded 11.9% and 4.8% of extracts, respectively. The extracts were evaluated for their antioxidant potential, phytochemical composition and antiproliferative effects against cancer cells. Water extracts exhibited remarkably higher values in Folin-Ciocalteu assay of total phenolic content, oxygen radical absorbance capacity (ORAC), ABTS●+/DPPH scavenging and cellular antioxidant activity (CAA) assays and more efficiently inhibited proliferation of HT29 cells at non-cytotoxic concentrations measured in non-tumoral Caco2 cells. Among 28 detected and 21 quantified phytochemicals, flavonols with the structures of isorhamnetin (five compounds), quercetin (three compounds), kaempferol (three compounds) glycosides and catechin (six compounds) were the most abundant in the extracts. In conclusion, the applied method of fractionation of SBP produces promising natural antioxidant complexes with antiproliferative properties that could find potential applications in nutraceuticals, functional foods and cosmeceuticals.

Polyphenol-Rich Extracts Obtained from Winemaking Waste Streams as Natural Ingredients with Cosmeceutical Potential.

Phenolics present in grapes have been explored as cosmeceutical principles, due to their antioxidant activity and ability to inhibit enzymes relevant for skin ageing. The winemaking process generates large amounts of waste, and the recovery of bioactive compounds from residues and their further incorporation in cosmetics represents a promising market opportunity for wine producers and may contribute to a sustainable development of the sector. The extracts obtained from grape marc and wine lees, using solid-liquid (SL) extraction with and without microwave (MW) pretreatment of the raw material, were characterized in terms of antioxidant activity through chemical (ORAC/HOSC/HORAC) and cell-based (keratinocytes-HaCaT; fibroblasts-HFF) assays. Furthermore, their inhibitory capacity towards specific enzymes involved in skin ageing (elastase; MMP-1; tyrosinase) was evaluated. The total phenolic and anthocyanin contents were determined by colorimetric assays, and HPLC-DAD-MS/MS was performed to identify the main compounds. The MW pretreatment prior to conventional SL extraction led to overall better outcomes. The red wine lees extracts presented the highest phenolic content (3 to 6-fold higher than grape marc extracts) and exhibited the highest antioxidant capacity, being also the most effective inhibitors of elastase, MMP-1 and tyrosinase. The results support that winemaking waste streams are valuable sources of natural ingredients with the potential for cosmeceutical applications.

Isolation of Strong Antioxidants from Paeonia Officinalis Roots and Leaves and Evaluation of Their Bioactivities.

Paeonia officinalis extracts from leaves and roots were tested for their antioxidant potential using in vitro chemical (Folin-Ciocalteu, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), oxygen radical absorbance capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), hydroxyl radical scavenging capacity HOSC)) and cellular antioxidant activity (CAA) assays. Leaf extracts were stronger antioxidants than root extracts, while methanol was a more effective solvent than water in chemical assays. However, the selected water extract of leaves was a stronger antioxidant in CAA than the methanol extract (0.106 vs. 0.046 µmol quercetin equivalents/mg). Twenty compounds were identified by ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF) mass spectrometer, while on-line screening of their antioxidant capacity by high performance liquid chromatography (HPLC) with a DPPH•-scavenging detector revealed that gallic acid derivatives are the major peony antioxidants. Root water and leaf methanol extracts inhibited α-amylase in a dose dependent manner. The IC50 value for the strongest inhibitor, the methanol extract of leaves, was 1.67 mg/mL. In addition, the cytotoxicity assessment of extracts using human Caco-2 cells demonstrated that none of them possessed cytotoxic effects.

Cytotoxicity and biological capacity of sulfur-free lignins obtained in novel biorefining process.

Lignin is one of the most promising and versatile products obtained in biorefineries due to its diverse therapeutic properties such as antimicrobial, antioxidant and anti-inflammatory activity. However, these properties depend on the source of lignin and the way it was isolated from the biomass. In this study, four different lignins are compared (extracted with Aquasolv (ASL1, ASL2), Organosolv (OSL) and Alkali (ALK) processes) for their cellular antioxidant capacity, anti-diabetic activity, free radical scavenging and cytotoxicity. Alkali and Organosolv lignins showed the highest antioxidant capacity 1159.815 µmol TE g-1 and 1463.415 µmol TE g-1, respectively, in agreement with their highest amount of free -OH groups. Additionally, OSL showed the highest inhibition in the antidiabetic assay followed by ASL1 with values for α-amylase of 3.6 mg/ml and 4.3 mg/ml respectively, and α-glucosidase 1.6 mg/ml and 2.5 mg/ml correspondingly. Nevertheless, cell-based assays revealed that ASL has the lowest cytotoxic effect in Caco-2 cells and, thus, is 10 times less cytotoxic than Alkali and OSL. This work suggests the applicability of ASL for high value applications such as cosmetics or pharmaceuticals. INDUSTRIAL RELEVANCE: Industrially, Liquid Hot Water (LHW) and Organosolv processes may appear as promising biorefining technologies in the following years. Lignin produced is free of sulfur, can be labelled as clean and environmentally-friendly and in this study, was proven that LHW lignin is non cytotoxic. The findings in this paper showed that different sources of lignin can be used in product formulation for life science purposes, thus opening a broad spectrum of possibilities for lignin valorisation in biorefineries.