Red seaweed biorefinery: The influence of sequential extractions on the functional properties of extracted agars and porphyrans.

Red seaweeds are exploited for their hydrocolloids, but other fractions are usually overlooked. In a novel approach, this study aimed to evaluate cold-water (CWE), ethanolic (EE), and alkaline (SE) extractions, alone and in sequence, to simultaneously: i) decrease the hydrocolloid extraction waste (valorizing bioactive side-streams and/or increasing extraction yield); and ii) increase the hydrocolloids' texturizing properties. It is the first time these extractions' synergetic and/or antagonistic effects will be accessed. For Porphyra dioica, a combination of CWE and EE was optimal: a positive influence on the melting temperature (increasing 5 °C to 74 °C) and sulphate content (a 3-fold reduction to 5 %) was observed, compared to a direct porphyran extraction. The same was observed for Gracilaria vermiculophyla, recovering two additional bioactive fractions without impacting the hydrocolloid's extraction (agar with 220 g/cm2 gelling strength and 14 % yield was obtained). The sequential use of CWE, EE, and SE was the most beneficial in Gelidium corneum processing: it enhanced agar's texturizing capacity (reaching 1150 g/cm2, a 1.5-fold increase when compared to a direct extraction), without affecting its 22 % yield or over 88 % purity. Ultimately, these findings clarified the effects of cascading biorefinery approaches from red seaweeds and their pertinence.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Evolving biofilm inhibition and eradication in clinical settings through plant-based antibiofilm agents.

BACKGROUND: After almost 100 years since evidence of biofilm mode of growth and decades of intensive investigation about their formation, regulatory pathways and mechanisms of antimicrobial tolerance, nowadays there are still no therapeutic solutions to eradicate bacterial biofilms and their biomedical related issues. PURPOSE: This review intends to provide a comprehensive summary of the recent and most relevant published studies on plant-based products, or their isolated compounds with antibiofilm activity mechanisms of action or identified molecular targets against bacterial biofilms. The objective is to offer a new perspective of most recent data for clinical researchers aiming to prevent or eliminate biofilm-associated infections caused by bacterial pathogens. METHODS: The search was performed considering original research articles published on PubMed, Web of Science and Scopus from 2015 to April 2023, using keywords such as "antibiofilm", "antivirulence", "phytochemicals" and "plant extracts". RESULTS: Over 180 articles were considered for this review with a focus on the priority human pathogens listed by World Health Organization, including Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Inhibition and detachment or dismantling of biofilms formed by these pathogens were found using plant-based extract/products or derivative compounds. Although combination of plant-based products and antibiotics were recorded and discussed, this topic is currently poorly explored and only for a reduced number of bacterial species. CONCLUSIONS: This review clearly demonstrates that plant-based products or derivative compounds may be a promising therapeutic strategy to eliminate bacterial biofilms and their associated infections. After thoroughly reviewing the vast amount of research carried out over years, it was concluded that plant-based products are mostly able to prevent biofilm formation through inhibition of quorum sensing signals, but also to disrupt mature biofilms developed by multidrug resistant bacteria targeting the biofilm extracellular polymeric substance. Flavonoids and phenolic compounds seemed the most effective against bacterial biofilms.

Postharvest Quality Improvement of Tomato (Solanum lycopersicum L.) Fruit Using a Nanomultilayer Coating Containing Aloe vera.

The effectiveness of an alginate/chitosan nanomultilayer coating without (NM) and with Aloe vera liquid fraction (NM+Av) was evaluated on the postharvest quality of tomato fruit at 20 °C and 85% relative humidity (RH) to simulate direct consumption. Both nanomultilayer coatings had comparable effects on firmness and pH values. However, the NM+Av coating significantly reduced weight loss (4.5 ± 0.2%) and molds and yeasts (3.5-4.0 log CFU g-1) compared to uncoated fruit (16.2 ± 1.2% and 8.0 ± 0.0 log CFU g-1, respectively). It notably lowered O2 consumption by 70% and a 52% decrease in CO2 production, inhibiting ethylene synthesis. Visual evaluation confirmed NM+Av's efficacy in preserving the postharvest quality of tomato. The preservation of color, indicated by the Minolta color (a*/b*) values, demonstrated NM+Av's ability to keep the light red stage compared to uncoated fruit. The favorable effects of NM+Av coating on enhancing postharvest quality indicates it as a potential alternative for large-scale tomato fruit preservation.

Sambucus nigra flower and berry extracts for food and therapeutic applications: effect of gastrointestinal digestion on in vitro and in vivo bioactivity and toxicity.

The bioavailability of natural compounds should be assessed through different perspectives. Studying the behaviour of the extracts after digestion is often overlooked but is crucial for success in the development of active food ingredients. Thus, the bioaccessibility of S. nigra (flower and berry) extracts after in vitro gastrointestinal digestion and their effect on toxicity and bioactive potential were studied. The flower extract had a higher content of phenolic compounds, like rutin, chlorogenic acid and rosmarinic acid, while in the berry extract, rutin, resveratrol, ferulic acid and chlorogenic acid were the main phenolic compounds. The effect of the non-digested and digested extracts was significantly different on different cell lines. The IC50 of the normal cell line (L929) was the highest, indicating low toxicity. The IC50 of the cancerous cell lines (HeLa and HT29) was lower, particularly the extract obtained from the flower upon digestion. In the presence of an oxidant agent - tbHP, only the berry extract was able to significantly reduce the formation of ROS in the L929 cell line, while in the HeLa cells, all the extracts were able to reduce ROS formation. The in vivo Artemia salina lethality bioassay demonstrated a dose-dependent effect of extracts, and the berry digested extract induced the lowest mortality rate. The promising results obtained on the chemical and biological evaluation of the extracts indicate that the natural compounds isolated from S. nigra by-products can be used as potential ingredients for functional food formulations and/or as bio-therapeutic agents.

Unveiling the Antioxidant Therapeutic Functionality of Sustainable Olive Pomace Active Ingredients.

Olive pomace (OP) is the main residue that results from olive oil production. OP is rich in bioactive compounds, including polyphenols, so its use in the treatments of diseases related to oxidative stress, such as cancer, could be considered. The present work aimed to study the biological properties of different OP extracts, obtained by ohmic heating-assisted extraction and conventional heating, using water and 50% ethanol, in the treatment and prevention of colorectal cancer through Caco-2 cell models. Additionally, an in-silico analysis was performed to identify the phenolic intestinal absorption and Caco-2 permeability. The extracts were chemically characterized, and it was found that the Ohmic-hydroethanolic (OH-EtOH) extract had the highest antiproliferative effect, probably due to its higher content of phenolic compounds. The OH-EtOH induced potential modifications in the mitochondrial membrane and led to apoptosis by cell cycle arrest in the G1/S phases with activation of p53 and caspase 3 proteins. In addition, this extract protected the intestine against oxidative stress (ROS) caused by H2O2. Therefore, the bioactive compounds present in OP and recovered by applying a green technology such as ohmic-heating, show promising potential to be used in food, nutraceutical, and biomedical applications, reducing this waste and facilitating the circular economy.

Green Extraction Techniques as Advanced Sample Preparation Approaches in Biological, Food, and Environmental Matrices: A Review.

Green extraction techniques (GreETs) emerged in the last decade as greener and sustainable alternatives to classical sample preparation procedures aiming to improve the selectivity and sensitivity of analytical methods, simultaneously reducing the deleterious side effects of classical extraction techniques (CETs) for both the operator and the environment. The implementation of improved processes that overcome the main constraints of classical methods in terms of efficiency and ability to minimize or eliminate the use and generation of harmful substances will promote more efficient use of energy and resources in close association with the principles supporting the concept of green chemistry. The current review aims to update the state of the art of some cutting-edge GreETs developed and implemented in recent years focusing on the improvement of the main analytical features, practical aspects, and relevant applications in the biological, food, and environmental fields. Approaches to improve and accelerate the extraction efficiency and to lower solvent consumption, including sorbent-based techniques, such as solid-phase microextraction (SPME) and fabric-phase sorbent extraction (FPSE), and solvent-based techniques (µQuEChERS; micro quick, easy, cheap, effective, rugged, and safe), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), in addition to supercritical fluid extraction (SFE) and pressurized solvent extraction (PSE), are highlighted.

2'-O-(N-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'-O-(N-(Aminoethyl)carbamoyl)methyl (2'-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2'-O-methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages. It is therefore particularly interesting and unprecedented that the 2'-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2'-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2'-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

Chemical Characterization of Sambucus nigra L. Flowers Aqueous Extract and Its Biological Implications.

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extract's antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50 = 1250 µg mL-1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes.

An oral antisense oligonucleotide for PCSK9 inhibition.

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

Advances in Extraction Methods to Recover Added-Value Compounds from Seaweeds: Sustainability and Functionality.

Seaweeds are a renewable natural source of valuable macro and micronutrients that have attracted the attention of the scientists in the last years. Their medicinal properties were already recognized in the ancient traditional Chinese medicine, but only recently there has been a considerable increase in the study of these organisms in attempts to demonstrate their health benefits. The extraction process and conditions to be used for the obtention of value-added compounds from seaweeds depends mainly on the desired final product. Thermochemical conversion of seaweeds, using high temperatures and solvents (including water), to obtain high-value products with more potential applications continues to be an industrial practice, frequently with adverse impact on the environment and products' functionality. However more recently, alternative methods and approaches have been suggested, searching not only to improve the process performance, but also to be less harmful for the environment. A biorefinery approach display a valuable idea of solving economic and environmental drawbacks, enabling less residues production close to the much recommended zero waste system. The aim of this work is to report about the new developed methods of seaweeds extractions and the potential application of the components extracted.

Algal proteins: Production strategies and nutritional and functional properties.

Animal-based proteins are the most consumed worldwide given their well-balanced nutritional composition. However, the growing demand for animal proteins will not be sustainable due to their low conversion efficiency and high environmental footprint. Specific consumers' dietary restrictions and modern trends emphasize the importance of finding alternative sustainable non-animal sources to meet future food (and, in particular, protein) global needs. Algal biomass is considered a relevant alternative, presenting advantages over terrestrial biomass such as higher growth rate, low water consumption, no competition for arable land, carbon-neutral emissions, and production of numerous bioactive compounds. This review provides an overview of recent research advances on algae as source of proteins, including production strategies from relevant protein-producing species. Particular emphasis will be given to algae protein current applications and forthcoming challenges of their use. Nutritional and functional aspects of algae biomass or its protein-enriched fractions will be overviewed.

Encapsulated Pine Bark Polyphenolic Extract during Gastrointestinal Digestion: Bioaccessibility, Bioactivity and Oxidative Stress Prevention.

Polyphenolic extracts from pine bark have reported different biological actions and promising beneficial effects on human health. However, its susceptibility to environmental stresses (temperature, storage, etc.) and physiological human conditions prequires the development of efficient protection mechanisms to allow effective delivering of functionality. The aim of this work was to encapsulate pine bark extract rich phenolic compounds by spray-drying using maltodextrin, and understand the influence of encapsulation on the antioxidant and antimicrobial activity and bioaccessibility of phenolic compounds during gastrointestinal digestion. The optimized process conditions allowed good encapsulation efficiency of antioxidant phenolic compounds. The microencapsulation was effective in protecting those compounds during gastrointestinal conditions, controlling their delivery and enhancing its health benefits, decreasing the production of reactive oxygen species implicated in the process of oxidative stress associated with some pathologies. Finally, this encapsulation system was able to protect these extracts against acidic matrices, making the system suitable for the nutritional enrichment of fermented foods or fruit-based beverages, providing them antimicrobial protection, because the encapsulated extract was effective against Listeria innocua. Overall, the designed system allowed protecting and appropriately delivering the active compounds, and may find potential application as a natural preservative and/or antioxidant in food formulations or as bioactive ingredient with controlled delivery in pharmaceuticals or nutraceuticals.

Green and Sustainable Valorization of Bioactive Phenolic Compounds from Pinus By-Products.

In Europe, pine forests are one of the most extended forests formations, making pine residues and by-products an important source of compounds with high industrial interest as well as for bioenergy production. Moreover, the valorization of lumber industry residues is desirable from a circular economy perspective. Different extraction methods and solvents have been used, resulting in extracts with different constituents and consequently with different bioactivities. Recently, emerging and green technologies as ultrasounds, microwaves, supercritical fluids, pressurized liquids, and electric fields have appeared as promising tools for bioactive compounds extraction in alignment with the Green Chemistry principles. Pine extracts have attracted the researchers' attention because of the positive bioproperties, such as anti-inflammatory, antimicrobial, anti-neurodegenerative, antitumoral, cardioprotective, etc., and potential industrial applications as functional foods, food additives as preservatives, nutraceuticals, pharmaceuticals, and cosmetics. Phenolic compounds are responsible for many of these bioactivities. However, there is not much information in the literature about the individual phenolic compounds of extracts from the pine species. The present review is about the reutilization of residues and by-products from the pine species, using ecofriendly technologies to obtain added-value bioactive compounds for industrial applications.

Unravelling the Biological Potential of Pinus pinaster Bark Extracts.

Natural compounds from agro-food by-products have fostered interest in food industries. The aim of this study was to unravel potential uses for Pinus pinaster bark extracts (PBE). As functional features of this type of extracts are usually attributed to phenolic compounds, the extraction process was studied. Different PBEs were achieved, with high content in phenolic compounds, using different water/ethanol combinations as a solvent. These PBEs were chemically characterized, and their bioactivity and in vitro cell viability were evaluated. Extracts obtained with hydroethanolic solvents had higher content in phenolic and flavonoid compounds. All the PBEs presented high antioxidant, antibacterial and antihyperglycemic activities. Moreover, PBEs have low cytotoxicity and a selective activity against cancer cells as these were negatively affected. These features may allow the extracts to be used in food formulation and processing (as preservatives, antioxidants or bioactive ingredients), but they showed also potential for the pharmaceutical or nutraceutical sectors.

Recent trends on seaweed fractionation for liquid biofuels production.

Concerns about fossil fuels depletion has led to seek for new sources of energy. The use of marine biomass (seaweed) to produce biofuels presents widely recognized advantages over terrestrial biomasses such as higher production ratio, higher photosynthetic efficiency or carbon-neutral emissions. In here, interesting seaweed sources as a whole or as a residue from seaweed processing industries for biofuel production were identified and their diverse composition and availability compiled. In addition, the pretreatments used for seaweed fractionation were thoroughly revised as this step is pivotal in a seaweed biorefinery for integral biomass valorization and for enabling biomass-to-biofuel economic feasibility processes. Traditional and emerging technologies were revised, with particular emphasis on green technologies, relating pretreatment not only with the type of biomass but also with the final target product(s) and yields. Current hurdles of marine biomass-to-biofuel processes were pinpointed and discussed and future perspectives on the development of these processes given.

Antisense Oligonucleotides for Splice Modulation: Assessing Splice Switching Efficacy.

Today, there are emerging numbers of oligonucleotide therapies being approved by the governmental authorities. These types of therapies present a different mode of action when compared to the traditional small molecules, acting at the RNA level instead of the protein level. In drug development, drug potency is defined by the drug affinity to the target biomolecule (target engagement), together with the ability to initiate a response at the molecular, cellular, tissue, or system level (efficacy). In oligonucleotide therapies, affinity and efficacy can be both easily evaluated by gene expression analysis. Although more advanced techniques can be used, this chapter presents a protocol to evaluate splice switching oligonucleotide efficacy that can be easily applied in a molecular biology laboratory without the need of advanced equipment. It describes all steps from sample preparation to data analysis.

Enrofloxacin and copper plant uptake by Phragmites australis from a liquid digestate: Single versus combined application.

Application of anaerobically digested sludges and manure (digestates) to agricultural fields reduces the need for fertilizer application, but might also pose environmental risks due to the introduction of various pollutants -including metal trace elements and pharmaceuticals- to amended soils. The simultaneous presence of different contaminants can affect plant uptake, altering the performance of phytoremediation. This work aims to investigate and compare the single and combined effects of Enrofloxacin (Enro) and Cu on their plant uptake from digestate solutions. Plant uptake experiments were carried out by exposing, for 5 days, Phragmites australis to three different concentrations of a liquid digestate doped with Cu and/or Enro. Contaminant concentrations were afterward determined in both plant tissues and digestate solutions. Contaminant speciation in solution and Cu speciation in plant roots were also assessed. Cu plant uptake (mostly in plant roots, Cu concentration varied between 55 and 254 µg·g-1) was observed, especially at low concentration of digestate. At high digestate concentration, Cu uptake decreased due to the formation of Cu-DOC complexes rendering Cu less bioavailable. The presence of the antibiotic slightly further reduced Cu plant uptake due to the formation of Cu-Enro complexes. Plant roots also accumulated Enro, an accumulation also higher for the lowest concentration of digestate (an increase of 40%), and that slightly increased in the presence of Cu (up to 226 µg·g-1). However, proportion of free Enro in solution decreased (up to 70%) in the presence of Cu due to complexation. Cu speciation in plant roots confirmed the implication of the complexation in the uptake of Cu and Enro when they are simultaneously present in solution. Thus, the presence of amendments (such as digestates) increasing the DOC content, in addition to the interactions between contaminants, should be taken into account for field crop soils as well as for phytoremediation technologies.