Green Extraction and Preliminary Biological Activity of Hydrolyzed Collagen Peptides (HCPs) Obtained from Whole Undersized Unwanted Catches (Mugil cephalus L.).

Considering the global increase in fish consumption, the growing side-streams coming from the fish supply chain (e.g., skin, fins, tail, heads…), also including undersized or "unwanted catches", have been recently proposed as source of high-value bioactive compounds (e.g., peptides and fatty acids). In this case study, hydrolyzed collagen peptides (HCPs) were extracted from different parts of Mugil cephalus L. using environmentally friendly techniques such as ultrasounds and enzymatic treatments. Both a mixed biomass derived from the skin, fins, and tail, and a whole fish, were considered as starting biomass, simulating the unsorted processing side-streams and an undersized/unwanted catch, respectively. The extracted HCPs were purified in fractions (<3 KDa and >3 KDa) whose yields (about 5% and 0.04-0.3%, respectively) demonstrated the efficiency of the hydrolysis process. The extraction protocol proposed allowed us to also isolate the intermediate products, namely the lipids (about 8-10%) and the non-collagenous proteins (NCs, 16-23%), whose exploitation could be considered. Each sample was characterized using Sircol, UltraViolet-Spectra, and hydroxyproline assay, and the viability of their collagen fractions was tested on human endothelial cells. Significant effects were obtained at a fraction of <3 KDa, in particular at a concentration of 0.13 µg/mL. The T-scratch test was also performed, with positive results in all fractions tested.

Determination of polycyclic aromatic hydrocarbons in bud-derived supplements by magnetic molecular imprinted microparticles and GC-MS: D-optimal design for a fast method optimization.

Within the world of natural food supplements, organic extracts deriving from young plant meristematic tissue (bud-derivatives) are becoming attractive, thanks to their richness in bioactive molecules. This natural source is scarce, but every year, tons of plant material, including buds, come from city pruning. If this sustainable source is rather promising from a circular economy point of view, the safety of the obtained supplements must be assessed. In fact, anthropic microcontaminants, such as polycyclic aromatic hydrocarbons (PAHs), could adsorb onto the urban buds, leading to a possible contamination of the bud-derivatives. In this study, we developed a magnetic dispersive solid phase extraction (m-dSPE) based on molecularly imprinted microparticles, combined with GC-MS, to quantify the 16 priority PAHs in such extracts. The D-optimal experimental design was implemented to maximize analytes' recovery with the smallest set of experiments. The optimized method was characterized by great selectivity thanks to the molecular imprinted polymer and ease of use provided by m-dSPE. Moreover, it complies with green principles, thanks to the minimum consumption of organic solvent (1.5 mL of acetone per sample). The recoveries ranged from 76 to 100% and procedural precision was below 10% for most PAHs. Despite the matrix complexity, low quantification limits (0.7-12.6 µg kg-1) were reached. This guaranteed the PAHs' quantitation at levels below those indicated as safe by a European Community regulation on food supplements. None of the analyzed samples, coming from different anthropically impacted areas, showed concerning PAHs levels.

In Silico and In Vitro Study of Antioxidant Potential of Urolithins.

In this work, quantum chemical calculations based on density functional theory (DFT) were performed to predict the antioxidant potential of four bioactive gut microbiota metabolites of the natural polyphenols ellagitannins (ETs) and ellagic acid (EA), also known as urolithins (UROs). In order to evaluate their ability to counter the effect of oxidative stress caused by reactive oxygen species (ROS), such as the hydroperoxyl radical (•OOH), different reaction mechanisms were investigated, considering water and lipid-like environments. Through our in silico results, it emerged that at physiological pH, the scavenging activity of all urolithins, except urolithin B, are higher than that of trolox and other potent antioxidants existing in nature, such as EA, α-mangostin, allicin, caffeine and melatonin. These findings were confirmed by experimental assays.

Vitis vinifera L. Pruning Waste for Bud-Preparations as Source of Phenolic Compounds-Traditional and Innovative Extraction Techniques to Produce New Natural Products.

Herbal products are now considered among the most important sources of phenolic compounds: the FINNOVER project aimed at the creation and development of sustainable supply chains to extract and use natural biologically active agents. Vitis vinifera is one of the most utilised herbal products derived from buds and sprouts as polyphenolic food supplements for its homeostatic and astringent properties. This research was aimed to describe the antioxidant capacity and the phytochemical composition of V. vinifera herbal products by the application of spectroscopic and chromatographic fingerprints considering phenolics as potential markers to significantly differentiate traditional preparations (macerates) from innovative extracts obtained by an ultrasound extraction from V. vinifera buds. Two different commercial products were also considered. Flavonols were the most abundant class in ultrasound extracts (45%), while phenolic acids were the most important class in traditional macerates (49%) and commercial bud-preparations (about 50%). This study may support the potential use of V. vinifera bud-products (starting from pruning byproducts) as food supplements to integrate human diet with good amounts of phenolics. Finally, the use of different extraction methods on the same plant material could be an important development to produce innovative herbal products with a phytochemical composition similar to traditional preparations.

Presynaptic Release-Regulating Alpha2 Autoreceptors: Potential Molecular Target for Ellagic Acid Nutraceutical Properties.

Polyphenol ellagic acid (EA) possesses antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic and cardio protection activities, making it an interesting multi-targeting profile. EA also controls the central nervous system (CNS), since it was proven to reduce the immobility time of mice in both the forced swimming and the tail-suspension tests, with an efficiency comparable to that of classic antidepressants. Interestingly, the anti-depressant-like effect was almost nulled by the concomitant administration of selective antagonists of the noradrenergic receptors, suggesting the involvement of these cellular targets in the central effects elicited by EA and its derivatives. By in silico and in vitro studies, we discuss how EA engages with human α2A-ARs and α2C-AR catalytic pockets, comparing EA behaviour with that of known agonists and antagonists. Structurally, the hydrophobic residues surrounding the α2A-AR pocket confer specificity on the intermolecular interactions and hence lead to favourable binding of EA in the α2A-AR, with respect to α2C-AR. Moreover, EA seems to better accommodate within α2A-ARs into the TM5 area, close to S200 and S204, which play a crucial role for activation of aminergic GPCRs such as the α2-AR, highlighting its promising role as a partial agonist. Consistently, EA mimics clonidine in inhibiting noradrenaline exocytosis from hippocampal nerve endings in a yohimbine-sensitive fashion that confirms the engagement of naïve α2-ARs in the EA-mediated effect.

Increased Water-Solubility and Maintained Antioxidant Power of Resveratrol by Its Encapsulation in Vitamin E TPGS Micelles: A Potential Nutritional Supplement for Chronic Liver Disease.

Children affected by chronic liver disease exhibit impaired neurocognitive development and growth due to the low absorption and digestion of nutrients. Furthermore, malnutrition is an adverse prognostic factor in liver transplantation as it is associated with an increase in morbidity and mortality. D-α-tocopheryl-polyethylene-glycol-succinate (TPGS) is currently administered per os as a vitamin E source to improve children's survival and well-being; however, TPGS alone does not reverse spinocerebellar degeneration and lipid peroxidation. To potentiate the effects of TPGS, we loaded micelles with resveratrol (RES), a natural polyphenol, with antioxidant and antiinflammatory activities, which has demonstrated protective action in the liver. Firstly, we investigated the suitability of TPGS to encapsulate RES in micelles by means of a phase-solubility study, then RES-TPGS formulations were prepared via solvent casting and solvent diffusion evaporation methods. RES-TPGS colloidal dispersions showed small mean diameters (12 nm), low polydispersity, and quite neutral Zeta potentials. The formulations showed a sustained drug release and a good drug loading capacity, further confirmed by infrared spectroscopy and differential scanning calorimetry. RES-TPGSs exhibited unaltered antioxidant activity compared to pristine RES via the DPPH assay and a significant reduction in toxicity compared to empty TPGS on HaCaT cells. Thus, RES-TPGS micelles may overcome the challenges of current liver disease therapy by providing more protective effects thanks to the antioxidant activity of RES and by reducing the surfactant toxicity on normal cells.

Use of an Animal Model to Evaluate Anxiolytic Effects of Dietary Supplementation with Tilia tomentosa Moench Bud Extracts.

Anxiety disorders are common and complex psychiatric syndromes affecting a broad spectrum of patients. On top of that, we know that aging produces an increase in anxiety vulnerability and sedative consumption. Moreover, stress disorders frequently show a clear gender susceptibility. Currently, the approved pharmacological strategies have severe side effects such as hallucinations, addiction, suicide, insomnia, and loss of motor coordination. Dietary integration with supplements represents an intriguing strategy for improving the efficacy and the safety of synthetic anxiolytics. Accordingly, a recent article demonstrated that glyceric bud extracts from Tilia tomentosa Moench (TTBEs) exert effects that are consistent with anxiolytic activity. However, the effects of these compounds in vivo are unknown. To examine this question, we conducted behavioral analysis in mice. A total of 21 days of oral supplements (vehicle and TTBEs) were assessed by Light Dark and Hole Board tests in male and female mice (young, 3 months; old, 24 months). Interestingly, the principal component analysis revealed gender and age-specific behavioral modulations. Moreover, the diet integration with the botanicals did not modify the body weight gain and the daily intake of water. Our results support the use of TTBEs as dietary supplements for anxiolytic purposes and unveil age and gender-dependent responses.

Bud-Derivatives, a Novel Source of Polyphenols and How Different Extraction Processes Affect Their Composition.

The use of herbal food supplements, as a concentrate form of vegetable extracts, increased so much over the past years to count them among the relevant sources of dietetic polyphenols. Bud-derivatives are a category of botanicals perceived as a "new entry" in this sector since they are still poorly studied. Due to the lack of a manufacturing process specification, very different products can be found on the market in terms of their polyphenolic profile depending on the experimental conditions of manufacturing. In this research two different manufacturing processes, using two different protocols, and eight species (Carpinus betulus L., Cornus mas L., Ficus carica L., Fraxinus excelsior L., Larix decidua Mill., Pinus montana Mill., Quercus petraea (Matt.) Liebl., Tilia tomentosa Moench), commonly used to produce bud-derivatives, have been considered as a case study. An untargeted spectroscopic fingerprint of the extracts, coupled to chemometrics, provide to be a useful tool to identify these botanicals. The targeted phytochemical fingerprint by HPLC provided a screening of the main bud-derivatives polyphenolic classes highlighting a high variability depending on both method and protocol used. Nevertheless, ultrasonic extraction proved to be less sensitive to the different extraction protocols than conventional maceration regarding the extract polyphenolic profile.

Dendrimer Nanodevices and Gallic Acid as Novel Strategies to Fight Chemoresistance in Neuroblastoma Cells.

Human neuroblastoma (NB), a pediatric tumor inclined to relapse, after an initial response to therapy, usually develops resistance. Since several chemotherapeutics exert anticancer effect by increasing reactive oxygen species (ROS), NB cells overproduce antioxidant compounds becoming drugs-resistant. A strategy to sensitize NB cells to chemotherapy involves reducing their antioxidant defenses and inducing ROS overproduction. Concerning this, although affected by several issues that limit their clinical application, antioxidant/pro-oxidant polyphenols, such as gallic acid (GA), showed pro-oxidant anti-cancer effects and low toxicity for healthy cells, in several kind of tumors, not including NB. Herein, for the first time, free GA, two GA-dendrimers, and the dendrimer adopted as GA reservoir were tested on both sensitive and chemoresistant NB cells. The dendrimer device, administered at the dose previously found active versus sensitive NB cells, induced ROS-mediated death also in chemoresistant cells. Free GA proved a dose-dependent ROS-mediated cytotoxicity on both cell populations. Intriguingly, when administered in dendrimer formulations at a dose not cytotoxic for NB cells, GA nullified any pro-oxidant activity of dendrimer. Unfortunately, due to GA, nanoformulations were inactive on NB cells, but GA resized in nanoparticles showed considerable ability in counteracting, at low dose, ROS production and oxidative stress, herein induced by the dendrimer.

Biodegradable and biocompatible spherical dendrimer nanoparticles with a gallic acid shell and a double-acting strong antioxidant activity as potential device to fight diseases from "oxidative stress".

Gallic acid (GA) is a natural polyphenol with remarkable antioxidant power present in several vegetables and fruits. A normal feeding regime leads to a daily intake of GA which is reasonably regarded as "natural" and "safe" for humans. It owns strong potentials as alternative to traditional drugs to treat several diseases triggered by oxidative stress (OS), but poor gastrointestinal absorbability, pharmacokinetic drawbacks, and fast metabolism limit its clinical application. In this work, a fifth-generation polyester-based dendrimer was firstly prepared as a better absorbable carrier to protect and deliver GA. Then, by its peripheral esterification with GA units, a GA-enriched delivering system (GAD) with remarkable antioxidant power and high potential against diseases from OS was achieved. Scanning electron microscopy results and dynamic light scattering analysis revealed particles with an average size around 387 and 375 nm, respectively, and an extraordinarily spherical morphology. These properties, by determining a large particles surface area, typically favour higher systemic residence time and bio-efficiency. Z-potential of - 25 mV suggests satisfactory stability in solution with tendency to form megamers and low polydispersity index. GAD showed intrinsic antioxidant power, higher than GA by 4 times and like prodrugs, and it can carry contemporary several bioactive GA units versus cells. In physiological condition, the action of pig liver esterase (PLE), selected as a model of cells esterase, hydrolyses GAD to non-cytotoxic small molecules, thus setting free the bioactive GA units, for further antioxidant effects. Cytotoxicity studies performed on two cell lines demonstrated a high cell viability. Graphical Abstract Graphical Abstract.

Pulsed Ultrasound-Assisted Extraction as an Alternative Method to Conventional Maceration for the Extraction of the Polyphenolic Fraction of Ribes nigrum Buds: A New Category of Food Supplements Proposed by The FINNOVER Project.

In this research, for the first time, an alternative method to produce Ribes nigrum bud derivatives is presented. Pulsed ultrasound-assisted extraction (PUAE), using a food-grade solvent according to green chemistry principles, has been employed and compared to the conventional extraction method. Traditionally, bud derivatives, a category of botanicals marketed as plant food supplements in the European Community, are produced by macerating meristematic tissues of trees and plants mainly spontaneously collected. Buds are a challenging raw material for the UAE, since meristematic tissues are much softer and fragile than their corresponding adult phenological stage. It is therefore important to assess whether the polyphenolic fraction, very susceptible to degradation, is conserved after UAE. Untargeted polyphenolic fingerprints (UV-Visible and fluorescence) coupled with chemometrics are employed to quickly screen the best extraction conditions, evaluated by the design of experiment (DoE) method. The polyphenolic fraction of the optimized PUAE extract was quantified by targeted HPLC fingerprint and its antiradical activity was determined. PUAE on a lab pilot reactor was proven to be the most practical approach for a rapid (20 min vs. 21 days maceration) and efficient extraction of bioactive polyphenolics from Ribes nigrum buds, encouraging the scale up to an industrial plan.

Ellagic acid a multi-target bioactive compound for drug discovery in CNS? A narrative review.

It is well-known that the health properties attributed to several fruits, herbs, seeds and their processed foods/beverages are due to an important group of natural polyphenols classified as hydrolysable tannins (HT) named ellagitannins (ETs), that encompass both one or more gallic acid (GA) units and one or more hexahydroxydiphenoic acid (HHDP) units, ester-connected with a sugar residue. In vivo, ETs are rather not absorbed and in gastrointestinal tract (GIT), they are hydrolysed providing mainly ellagic acid (EA). Due to its trivial water-solubility, first pass effect, metabolism in GIT, or irreversible binding to cellular DNA and proteins, EA has a very low bioavailability. Some authors are studying methods to increase EA water-solubility and thus to improve its bioavailability. At the same, EA metabolism to urolithins (UROs), whose concentration and activity is inter-individual and intra-individual dependent, is still under study and not completely elucidate. Numerous in vitro and in vivo studies have been carried out to define the molecular and cellular events underlying the beneficial effects that this compound and its metabolites exert in pathological conditions. The anti-inflammatory and the antioxidant properties of EA attracted the interest of researchers for its potential health benefits in humans, including anti-cancer, anti-diabetes activities and cardio-protection. Nevertheless, lately the attention paid to EA is focusing on its potential protective action towards several neurodegenerative disorders. Thus, EA is investigated as a potential "lead compound" endowed with multi-target pharmacological properties on CNS. Since the identification of the pharmacophore(s) responsible for both health benefits and collateral effects of this compound is crucial in drug discovery, this review aims to provide an all-round updated analysis of the literature concerning EA involvement in several CNS disorders, hoping that such information will be useful to researchers involved in multi-target drug design for CNS.

Reshaped as polyester-based nanoparticles, gallic acid inhibits platelet aggregation, reactive oxygen species production and multi-resistant Gram-positive bacteria with an efficiency never obtained.

Natural polyphenols such as Gallic Acid (GA) form an important class of bioactive chemical entities that, having innumerable biological properties, could represent a safer alternative to common drugs against several disorders, including platelet aggregation, radical oxygen species (ROS) hyperproduction, oxidative stress (OS) and bacterial infections. Unfortunately, their clinical uses are limited by pharmacokinetics drawbacks and high sensitivity to environmental factors. In order to overcome these problems and to exploit the GA curative potentials, it has been linked to a biodegradable nanospherical dendrimer matrix, capable of protecting it, thus obtaining a GA-enriched nanosized dendrimer (GAD) endowed with a strong antioxidant capacity. GAD activity as an inhibitor of platelet aggregation and ROS accumulation and its antibacterial efficiency are evaluated here and compared to those of free GA, obtaining outcomes never achieved. Regarding platelet aggregation induced by thrombin and collagen, the GAD proved to be stronger by 7.1 and 7.3 times, respectively. Furthermore, the GAD showed a ROS inhibitory activity higher than that of GA by 8.1 (thrombin) and 6.9 (collagen) times. Concerning the antibacterial activities, evaluated on eleven multi-resistant Gram-positive strains of clinical relevance, the GAD is far more potent than GA, by exerting a growth inhibitory activity at MIC (µM) concentrations lower by factors in the range 12-50.

Olive fruits and vacuum impregnation, an interesting combination for dietetic iron enrichment.

In this study vacuum impregnation (VI) was employed for the iron enrichment of olive fruits, which are very interesting as food vehicle for VI mineral supplementation for the porosity of their pulp. NaFeEDTA was chosen for olives fortification since it prevents iron from binding with compounds that could hinder it from being efficiently absorbed and since it causes few organoleptic problems. In order to improve the efficiency of the VI process, several parameters of the whole process were studied by design of experiment techniques. First of all D-optimal design was employed for a preliminary screening of the most significant process variables and showed that the concentration of VI solution was by far the most significant process variable, though its time in contact with olives was also significant. A factorial design was then applied to the remaining variables and it showed that the speed of the addition of VI solution was also significant. Finally, the application of a face centered composite design to the three selected variables allowed to detect processing conditions leading to final iron contents of 1.5-3 mg/g, corresponding to an introduction of 10-15 mg Fe with four or five fortified olive fruits. No effect on olive taste was observed at these concentrations. The results showed that olive fruits were the most interesting vehicles for the supplementation of both iron and other minerals.

Green Extraction from Pomegranate Marcs for the Production of Functional Foods and Cosmetics.

The aim of this study was to investigate the potential of retrieving polyphenolic antioxidants directly from wet pomegranate marcs: the fresh by-products obtained after pomegranate juice processing. These by-products mainly consist of internal membranes (endocarp) and aril residues. Even if they are still edible, they are usually discharged during juice production and, thus, they represent a great challenge in an eco-sustainable industrial context. Green technologies, such as ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE), have been employed to convert these organic residues into recycled products with high added value. UAE and MAE were used both in parallel and in series in order to make a comparison and to ensure exhaustive extractions, respectively. Water, as an environmentally friendly extraction solvent, has been employed. The results were compared with those ones coming from a conventional extraction. The most promising extract, in terms of total polyphenol yield and radical scavenging activity, has been tested both as a potential natural additive and as a functional ingredient after its incorporation in a real food model and in a real cosmetic matrix, respectively. This study represents a proposal to the agro-alimentary sector given the general need of environmental "responsible care".