Conventional extraction of fucoidan from Irish brown seaweed Fucus vesiculosus followed by ultrasound-assisted depolymerization.

Fucoidan has attracted considerable attention from scientists and pharmaceutical companies due to its antioxidant, anticoagulant, anti-inflammatory, anti-tumor, and health-enhancing properties. However, the extraction of fucoidan from seaweeds often involves the use of harsh chemicals, which necessitates the search for alternative solvents. Additionally, the high viscosity and low cell permeability of high molecular weight (Mw) fucoidan can limit its effectiveness in drug action, while lower Mw fractions exhibit increased biological activity and are also utilized as dietary supplements. The study aimed to (1) extract fucoidan from the seaweed Fucus vesiculosus (FV) using an environmentally friendly solvent and compare it with the most commonly used extraction solvent, hydrochloric acid, and (2) assess the impact of ultrasound-assisted depolymerization on reducing the molecular weight of the fucoidan extracts and examine the cytotoxic effect of different molecular weight fractions. The findings indicated that the green depolymerization solvent, in conjunction with a brief ultrasound treatment, effectively reduced the molecular weight. Moreover, a significant decrease in cell viability was observed in selected samples, indicating potential anticancer properties. As a result, ultrasound was determined to be an effective method for depolymerizing crude fucoidan from Fucus Vesiculosus seaweed.

Sequential extraction of fucoidan, laminarin, mannitol, alginate and protein from brown macroalgae Ascophyllum nodosum and Fucus vesiculosus.

The study involves development of a green biorefinery process for obtaining fucoidan, laminarin, mannitol, alginate and protein from dry and fresh Fucus vesiculosus and Ascophyllum nodosum using hydrochloric acid and a green extraction solvent. After the extraction of fucoidan which was the targeted biomolecule, an extract and by-product (residual biomass) were obtained. The extract was passed through an ultrafiltration membrane, where fucoidan was obtained in the ultrafiltration retentate while ultrafiltration permeate was analysed for laminarin and mannitol. The residual biomass was used for obtaining alginate using ultrasound (20 kHz, 64 % amplitude and 32 min, optimum parameters for alginate extraction based on our previous study). All the samples, showed good results for alginate, laminarin and mannitol, indicating that the by-products can be utilised using this green extraction process. The comparison of both dry and fresh seaweed is relevant from an industry perspective, as fresh seaweed can directly be used for extraction, avoiding drying which adds significantly to the cost of the process. Life cycle impact assessment of the complete seaweed value chain has been carried out to identify the energy demand and key environmental hotspots. This biorefinery process can be used by industry to improve their processes and utilise the by-products generated efficiently.

Polyphenols in Agricultural Grassland Crops and Their Health-Promoting Activities-A Review.

Grassland crops are emerging reservoirs of undisturbed, natural antioxidants and phytochemicals, such as phenolic acids and flavonoids. The present review will focus on the most commonly cultivated crops, namely Lolium perenne L, Cichorium intybus L, Plantago lanceolata L. and Trifolium pratense L, which have been recognized for their polyphenolic composition. However, these crops are often undervalued and underutilized, yet have the means of potentially creating novel, value-added food and nutraceutical products. Previous studies relating to these crops have identified them as rich sources of caffeic acid, chlorogenic acid, daidzein, kaempferol, luteolin, and quercetin. The key to harnessing the hidden potential of these species is the recovery, identification, and characterization of the phytochemicals they contain. Considering the upsurge of research studies on alternative plant-based diets for the health of humans and the planet earth, there is a necessity to understand the phytochemical composition and the bioactivity that they possess. This review summarizes recovery methods of phytochemicals from the aforementioned grassland crops and their compositional and functional (antioxidant, anti-cancer, and anti-diabetic) characterization and discusses the potential for grassland crops as an abundant reservoir of health-promoting ingredients which can increase the nutritional composition within novel food innovations or within nutraceuticals.

The intracellular C-terminal domain of mGluR6 contains ER retention motifs.

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Purification and Molecular Characterization of Fucoidan Isolated from Ascophyllum nodosum Brown Seaweed Grown in Ireland.

The present study investigates the molecular characteristics of fucoidan obtained from the brown Irish seaweed Ascophyllum nodosum, employing hydrothermal-assisted extraction (HAE) followed by a three-step purification protocol. The dried seaweed biomass contained 100.9 mg/g of fucoidan, whereas optimised HAE conditions (solvent, 0.1N HCl; time, 62 min; temperature, 120 °C; and solid to liquid ratio, 1:30 (w/v)) yielded 417.6 mg/g of fucoidan in the crude extract. A three-step purification of the crude extract, involving solvents (ethanol, water, and calcium chloride), molecular weight cut-off filter (MWCO; 10 kDa), and solid-phase extraction (SPE), resulted in 517.1 mg/g, 562.3 mg/g, and 633.2 mg/g of fucoidan (p < 0.05), respectively. In vitro antioxidant activity, as determined by 1,1-diphenyl-2-picryl-hydrazyl radical scavenging and ferric reducing antioxidant power assays, revealed that the crude extract exhibited the highest antioxidant activity compared to the purified fractions, commercial fucoidan, and ascorbic acid standard (p < 0.05). The molecular attributes of biologically active fucoidan-rich MWCO fraction was characterised by quadruple time of flight mass spectrometry and Fourier-transform infrared (FTIR) spectroscopy. The electrospray ionisation mass spectra of purified fucoidan revealed quadruply ([M+4H]4+) and triply ([M+3H]3+) charged fucoidan moieties at m/z 1376 and m/z 1824, respectively, and confirmed the molecular mass 5444 Da (~5.4 kDa) from multiply charged species. The FTIR analysis of both purified fucoidan and commercial fucoidan standard exhibited O-H, C-H, and S=O stretching which are represented by bands at 3400 cm-1, 2920 cm-1, and 1220-1230 cm-1, respectively. In conclusion, the fucoidan recovered from HAE followed by a three-step purification process was highly purified; however, purification reduced the antioxidant activity compared to the crude extract.

Antioxidant Guided Fractionation of Blackberry Polyphenols Show Synergistic Role of Catechins and Ellagitannins.

In the present study, blackberry extract was prepared using a previously optimized solid-liquid extraction method in 70% aqueous acetone aimed at the recovery of its principal phenolics. Subsequently, 0.5 g of freeze-dried extract was subjected to flash chromatography fractionation, which was conducted on a C18 column using a binary solvent system of water and methanol at 10 mL/min. The total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) activities of the obtained 42 flash fractions were determined, and a strong positive correlation (r ≥ 0.986) was exhibited among them. Furthermore, the graph of the antioxidant indices of the flash fractions resembled the flash chromatogram, suggesting a good correlation among the compounds within the chromatographic peaks and the antioxidant indices. LC-MS/MS identified as many 28 phenolics, including cinnamtannin A2 reported for the first time in blackberries. This study further established the role of dominant anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside), but uniquely those of ellagitannins and catechins on the antioxidant capacity of blackberries.

Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug.

BACKGROUND: The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells. METHODS: Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression. RESULTS: Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3. CONCLUSIONS: Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

Development of Antioxidant and Nutritious Lentil (Lens culinaris) Flour Using Controlled Optimized Germination as a Bioprocess.

Germination is an efficient and natural strategy that allows the modification of the nutritional value and the nutraceutical properties of seeds, enabling one to tailor the process according to its final use. This study aimed at optimization of germination conditions to produce novel lentil flours with improved nutritional and functional features. Response Surface Methodology (RSM) was applied to model the effect of temperature (15-27 °C) and time (1-5 days) on different nutritional and quality parameters of lentil flours including proximate composition, content and profile of fatty acids, content of phytic acid, ascorbic acid and γ-aminobutyric acid (GABA), content and profile of phenolic compounds, antioxidant activity, expected glycemic index (GI) and color during germination. As shown by RSM polynomial models, sprouting promoted the reduction of phytic acid content and enhanced the levels of ascorbic acid, GABA, insoluble phenolic compounds, antioxidant activity and expected GI, and modified the color of the resultant lentil flours. RSM optimization of germination temperature and time using desirability function revealed that the optimal process conditions to maximize the nutritional, bioactive and quality properties of sprouted lentil flours were 21 °C for 3.5 days.

High-Pressure Processing on Whole and Peeled Potatoes: Influence on Polyphenol Oxidase, Antioxidants, and Glycaemic Indices.

Polyphenol oxidase (PPO) inactivation in five whole and peeled Irish potato cultivars was investigated using high-pressure processing (HPP) at 400 MPa and 600 MPa for 3 min. PPO activity was significantly lower in most of the HPP-treated samples, while the highest PPO inactivation was observed after HPP at 600 MPa. No significant (p > 0.05) changes were observed on the total phenolic content and antioxidant activity of all the HPP-treated potatoes. Regarding individual phenolic acids, chlorogenic acid was decreased significantly (p < 0.05) in all studied varieties with a concomitant increase (p < 0.05) in caffeic and quinic acid. Similarly, ferulic acid was also increased (p < 0.05) in all studied varieties after the HPP treatment, while there was a variation in rutin and 4-coumaric acid levels depending on the cultivar and the sample type. Anthocyanins in the coloured whole potato varieties (i.e., Kerr's Pink and Rooster), tentatively identified as pelargonidin-O-ferulorylrutinoside-O-hexoside and pelargonidin-O-rutinoside-O-hexoside, also exhibited significantly (p < 0.05) higher levels in the HPP-treated samples as opposed to those untreated. Glycaemic indices of the potatoes treated with HPP did not differ with the corresponding untreated cultivars.

A Review on Chromatography-Mass Spectrometry Applications on Anthocyanin and Ellagitannin Metabolites of Blackberries and Raspberries.

Berries have been widely assessed for their beneficial health effects, predominately due to their high (poly)phenol content of anthocyanins and ellagitannins. After ellagitannins and ellagic acid are metabolized by the gut microbiome, a class of compounds known as urolithins are produced, which exert potential advantageous health effects. Anthocyanins, on the other hand, undergo a complex metabolic pathway after their interaction with microbial and endogenous enzymes, forming a broad range of metabolites and catabolic products. In most cases, in vitro models and cell lines are used to generate metabolites, whereas their assessment in vivo is currently limited. Thus far, several analytical methods have been developed for the qualitative and quantitative analysis of phenolic metabolites in berries, including liquid chromatography, mass spectrometry, and other hyphenated techniques, and have been undoubtedly valuable tools for the detailed metabolite characterization and profiling. In this review, a compilation of studies providing information on the qualitative and quantitative analysis of (poly)phenol metabolites in blackberries and raspberries after the utilization of in vitro and in vivo methods is presented. The different analytical techniques employed are assessed, focusing on the fate of the produced metabolic compounds in order to provide evidence on their characteristics, formation, and beneficial effects.

Influence of molecular weight fractionation on the antimicrobial and anticancer properties of a fucoidan rich-extract from the macroalgae Fucus vesiculosus.

The objective of this study was to investigate the antimicrobial and anticancer properties of a fucoidan extract and subsequent fractions isolated from the macroalgae Fucus vesiculosus. The fractions obtained (>300 kDa, <300 kDa, <100 kDa, <50 kDa and <10 kDa) could inhibit the growth of B. subtilis, E. coli, L. innocua and P. fluorescens when assayed at concentrations between 12,500 and 25,000 ppm. The bacterial growth was monitored by optical density (OD) measurements (600 nm, 24 h) at 30 °C or 37 °C, depending upon on the strain used. The extracted fractions were also tested for cytotoxicity against brain glioblastoma cancer cells using the Alamar Blue assay for 24 h, 48 h and 6 days. The >300 kDa fraction presented the lowest IC50 values (0.052% - 24 h; 0.032% - 6 days). The potential bioactivity of fucoidan as an antimicrobial and anticancer agent was demonstrated in this study. Hence, the related mechanisms of action should be explored in a near future.

The Effect of Carnosol, Carnosic Acid and Rosmarinic Acid on the Oxidative Stability of Fat-Filled Milk Powders throughout Accelerated Oxidation Storage.

The in vitro antioxidant effects of the most potent antioxidants of rosemary, namely carnosol, carnosic acid and rosmarinic acid (c: ca: ra) were assessed in fat-filled milk powders (FFMPs) under accelerated conditions (40 °C and relative humidity (RH) 23%) over 90 days. Lipid oxidation was assessed in FFMPs by measuring peroxide values (PVs), thiobarbituric acid reactive substances (TBARS) and aroma volatiles using headspace (HS) solid-phase microextraction (SPME) coupled to gas-chromatography-mass spectrometry (GC-MS). The antioxidant potency of c: ca: ra exhibited a concentration-related effect (308 ppm > 200 ppm > 77 ppm), with the highest concentration being the most effective at controlling the formation of TBARS and PVs. At a concentration of 308 ppm c: ca: ra were particularly effective (p < 0.05) in inhibiting all the evaluated oxidation indices (primary and secondary) compared to the control samples, but in some cases less effectively (p < 0.05) than butylated hydroxyanisole: butylated hydroxytoluene (BHA: BHT) (200 ppm).

Ultrasound processing of coffee silver skin, brewer's spent grain and potato peel wastes for phenolic compounds and amino acids: a comparative study.

Awareness towards utilizing food-processing by-products are increasing in health as well as environmental purview. Coffee silver skin (CSS), potato peel (PP) and brewer's spent grain (BSG) are voluminous by-products in their respective processing industries. The present study compared these three by-products for their prospective utilization in producing polyphenols-rich aqueous extracts by using ultrasound-assisted extractions (UAE). A probe-type sonicator was used for ultrasound treatments. The total phenolic contents in the extracts were assessed by Folin-Ciocalteu assay, while the phenolic profiles of the extract was characterized by LC-Q-TOF mass spectrometry. The microstructure of the samples after UAE was evaluated by scanning electron microscopy (SEM). Ultrasound treatment enhanced the rate of extraction and recovered 2.79, 2.12 and 0.66 mg gallic acid equivalents/g of TPC from CSS, PP and BSG, respectively in 30 min, which correspond to recoveries of 97.6%, 84.5% and 84.6%, respectively, compared to conventional solid-liquid extractions carried out for 24 h. The extraction yield was dependent on the particle size of the raw materials and the highest yield was obtained from the materials with 100-250 µm particle size. The SEM imaging revealed that ultrasound treatment caused prominent tissue damage. Extracts contained mainly hydroxycinnamic acid derivatives of phenolic acids. PP and CSS had the highest amounts of umami free amino acids (0.13 mg/g in each), while BSG contained the highest amount of essential amino acids (92 mg/g). The present work shows that CSS, PP and BSG are good sources of polyphenols and UAE can be employed to enhance the extraction efficiency as means of a green approach.

High-Pressure Processing and Ultrasonication of Minimally Processed Potatoes: Effect on the Colour, Microbial Counts, and Bioactive Compounds.

HPP at 600 MPa alone, and in combination with US at 20 kHz (200 W), was applied to minimally processed potatoes of two commonly grown cultivars in Ireland. Changes in colour and microbial load (Enterobacteriaceae, total aerobic count, Salmonella, yeasts, and moulds) were monitored in vacuum-packaged potatoes during 14 days of storage at 4 °C. HPP and HPP/US significantly (p < 0.05) affected the colour parameters a*, b*, L*, and ΔE of minimally processed potatoes compared to the controls. Microbial growth was delayed in most of the treated samples with respect to those untreated (controls), while HPP completely inactivated Enterobacteriaceae in both cultivars. Total phenolic content and antioxidant activities were not altered in the treated samples of both varieties when compared to the controls. The levels of chlorogenic acid, ferulic acid, and caffeic acid were decreased after both treatments, with a significant (p < 0.05) increase in quinic acid in the treated samples as opposed to those untreated. A significant (p < 0.05) decrease in the levels of glycoalkaloids, namely α-chaconine and α-solanine, in HPP- and HPP/US-treated potatoes was also observed. These findings suggest that HPP and US can extend the shelf-life of minimally processed potatoes with a negligible impact on their antioxidant activity and phenolic content.

Data sharing in PredRet for accurate prediction of retention time: Application to plant food bioactive compounds.

Prediction of retention times (RTs) is increasingly considered in untargeted metabolomics to complement MS/MS matching for annotation of unidentified peaks. We tested the performance of PredRet (http://predret.org/) to predict RTs for plant food bioactive metabolites in a data sharing initiative containing entry sets of 29-103 compounds (totalling 467 compounds, >30 families) across 24 chromatographic systems (CSs). Between 27 and 667 predictions were obtained with a median prediction error of 0.03-0.76 min and interval width of 0.33-8.78 min. An external validation test of eight CSs showed high prediction accuracy. RT prediction was dependent on shape and type of LC gradient, and number of commonly measured compounds. Our study highlights PredRet's accuracy and ability to transpose RT data acquired from one CS to another CS. We recommend extensive RT data sharing in PredRet by the community interested in plant food bioactive metabolites to achieve a powerful community-driven open-access tool for metabolomics annotation.

Phytochemicals in Food and Health.

Consumption of plant-based diets, rich in phytochemicals, has been associated with reduced risk of degenerative diseases, improved overall health and well-being [...].

Anticholinesterase Activities of Different Solvent Extracts of Brewer's Spent Grain.

Cholinesterases, involved in acetylcholine catabolism in the central and peripheral nervous system, have been strongly linked with neurodegenerative diseases. Current therapeutic approaches using synthetic drugs present several side effects. Hence, there is an increasing research interest in naturally-occurring dietary polyphenols, which are also considered efficacious. Food processing by-products such as brewer's spent grain (BSG) would be a potential bio-source of polyphenols. In this study, polyphenol-rich BSG extracts using 60% acetone and 0.75% NaOH solutions were generated, which were further subjected to liquid-liquid partitioning using various organic solvents. The water-partitioned fractions of the saponified extracts had the highest total polyphenol content (6.2 ± 2.8 mgGAE/g dw) as determined by Folin-Ciocalteu reagent, while the LC-MS/MS showed ethyl acetate fraction with the highest phenolics (2.9 ± 0.3 mg/g BSG dw). The best inhibitions of acetyl- (37.9 ± 2.9%) and butyryl- (53.6 ± 7.7%) cholinesterases were shown by the diethyl ether fraction of the saponified extract. This fraction contained the highest sum of quantified phenolics (99 ± 21.2 µg/mg of extract), and with significant (p < 0.01) inhibitory contribution of decarboxylated-diferulic acid. Amongst the standards, caffeic acid presented the highest inhibition for both cholinesterases, 25.5 ± 0.2% for acetyl- and 52.3 ± 0.8% for butyryl-cholinesterase, respectively, whilst the blends insignificantly inhibited both cholinesterases. The results showed that polyphenol-rich BSG fractions have potentials as natural anti-cholinesterase agents.

Shotgun proteomics for the preliminary identification of biomarkers of beef sensory tenderness, juiciness and chewiness from plasma and muscle of young Limousin-sired bulls.

Label free shotgun proteomics was used to analyse plasma and Longissimus muscle biopsies of Limousin-sired bulls, classified as 5 high-quality and 5 low-quality meat based on sensory texture traits (tenderness, juiciness and chewiness). A total of 31 putative protein biomarkers (16 in plasma and 15 in muscle) differed significantly in abundance between the two quality groups. The proteins were associated with muscle structure, energy metabolism, heat shock proteins, oxidative stress and proteolysis related pathways. Among them, B2M, AHSG, APOA4 and HP-20 (plasma), PFKM, MYH2, PTER, GSTM1 and MYPN (muscle) were good predictors of the three texture quality traits. Further, significant correlations were identified for FETUB, SERPINA7, ASL, TREH, HP, HP-25, AZGP1, APCS and SYT15, which are novel biomarkers from plasma that warrant further evaluation. This study is a significant step forward in elucidating proteomic profiles in bovine bio-fluids and muscle tissue, which may ultimately provide opportunities to processors for early assessment of beef sensory quality.

Involvement of the C-terminal domain in cell surface localization and G-protein coupling of mGluR6.

Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gßγ subunits via its intracellular C-terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840-871) was necessary for mGluR6 cell surface localization and G-protein coupling using mGluR6-CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C-terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate-induced G-protein-mediated responses, whereas a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface localization and glutamate-dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.