Propolis is a bee-produced substance derived from plant materials collected from buds, bark, and leaves. Its antiviral properties have been widely reported; however, the bioactive constituents responsible for this activity have not been identified through bioactivity-guided fractionation. The aim of this study is to purify and identify the antiviral constituents of propolis through bio-guided fractionation using TLC and LC-MS. Among the various extracts prepared from propolis, only the ethyl acetate fraction exhibited significant activity against Coxsackievirus B3 (CVB-3), a non-enveloped RNA virus, with a selectivity index of 50.32. In contrast, no activity was observed against herpes simplex virus type 2, differing from previous findings on enveloped viruses. Quantitative RT-qPCR analysis of the active fraction did not reveal a predominant mechanism underlying the antiviral effect. Four bioactive flavonoids (Isorhamnetin, Pinocembrin-5-methyl ether, Pinobanksin-5-methyl ether and Pinobanksin-5-methyl ether 3-O-acetate) were identified within the active fraction by HPLC-DAD-ESI-MS/MS following bio-guided fractionation. An exploratory molecular docking study on five putative cellular and viral targets suggested that the highest predicted binding affinity was associated with the CVB-3 capsid, especially for Isorhamnetin. However, as observed in the in vitro tests, the predicted binding energies across different targets were not sufficiently discriminative to indicate the predominance of one interaction over another, suggesting that complementary approaches and experimental validation remain necessary to fully elucidate the viral inhibition mechanisms of these bioactive constituents from propolis.

Acheta domesticus and Tenebrio molitor are two of the most consumed and studied edible insects worldwide. Anyway, several nutritional, chemical, and biological aspects must be further investigated. In this study, INFOGEST protocol was applied on A. domesticus and T. molitor to investigate the bioaccessibility of the released metabolites, using protein hydrolysis and bioavailability assay for amino acids, as well as NMR metabolomics for other compounds. Insect chitin content was also measured to avoid an overestimation of nitrogen determination. An optimized microwave-assisted procedure was used to quickly hydrolyse proteins, and the amino acids quantification was performed by a validated RP-HPLC-DAD method. Glutamate, alanine, cysteine, aspartate, leucine, proline, and valine were the most abundant amino acids in both insects. Differently, the amino acids composition in oral, gastric, and duodenal phases changed according to the digestion phase and insect species. In particular, alanine, serine, and phenylalanine were the main absorbed amino acids for both species, whereas threonine, glutamate, tyrosine, valine, isoleucine, and histidine were the lowest ones. Protein hydrolysis during digestion was also monitored by NMR spectroscopy, as well as other metabolites detected in the insects, such as trehalose and betaine, significantly higher in T. molitor, whereas taurine was only measured in A. domesticus.

Diospyros kaki Thunb. is a plant belonging to the Italian biodiversity and its leaves are included in the Belfrit list; therefore, they can be used as a source of food supplement ingredients. D. kaki polyphenolic extract was investigated for its capacity to act as anti-glycative/anti-aging agent for the first time, using different in vitro model systems to simulate the glycation reaction steps. The extract was able to completely trap dicarbonyl compounds such as glyoxal and methylglyoxal and had good inhibitory activity toward argpyrimidine-like and vesperlysine-like Advanced Glycation End products (AGEs) (about 61-70% and 70%, respectively) in the intermediate step of glycation; differently, a very high activity against vesperlysine-like AGEs was registered (> than 93%) in the final step. In the extract, thirteen compounds were characterized by RP-HPLC-DAD-ESI-MS/MS and tested to investigate their interaction with AGEs receptor (RAGE) by molecular modelling. All the compounds (mainly kaempferol-3-O-glucoside) were able to bind RAGE V domain, with the electrostatic term as the main driving force. Considering the possibility to use the extract as food ingredient, bioaccessibility and Caco-2 permeability were investigated by in vitro Infogest digestion protocol coupled with Caco-2 cell-based assay. Seven kaempferol and quercetin derivatives were selected as marker compounds and the bioaccessibility index (BI) was measured. It never exceeded 40% after oral phase, and only kaempferol-3-O-galactoside and kaempferol-3-O-glucoside had a BI value of 5% at intestinal level, but no metabolites were detected in Caco-2 permeated samples. In conclusion, these results are promising; however, the need of a suitable carrier to stabilize the extract and to improve the polyphenols bioaccessibility was equally evident.

Adulteration and contamination represent an ever-growing global problem, due to the emergence of increasingly sophisticated illicit commercial practices involving high-value and widely consumed products. Traditional chromatographic methods, such as liquid chromatography coupled with mass spectrometry, offer high sensitivity but their application is often limited by long analysis time, complex sample preparation, and the use of non-eco-friendly organic solvents. In contrast, enzymatic and immunoassay-based methods are rapid and require minimal sample preparation, but their applicability may be restricted by antibody specificity and potential cross-reactivity. In this context, near-infrared (NIR) spectroscopy is a rapid, non-destructive technique that does not require the use of solvents and is becoming increasingly relevant in food analysis. This review provides an overview of the potential of this spectroscopic technique, coupled with chemometrics, for the detection of adulterants and contaminants in various matrices. After a brief summary of the principles on which it is based and the instruments that can be used, the chemometric processes useful for data interpretation have been discussed, as well as the main applications in liquid and solid food, including oils, milk, juices, spices, cereals, coffee, and dietary supplements. The analyzed case studies indicated that even very low levels (ppm) of adulterants and contaminants can be detected with high accuracy and sensitivity using models such as PLSR, SVM, RF, and CNNs and new and emerging devices such as portable ones.

Life expectancy in high-income countries is increasing, leading to a higher incidence of age-related neurodegenerative diseases. To address this urgent medical need, several molecular targets have been identified, including advanced glycation end products (AGEs) and tyrosinase. Given the well-established role of diet in counteracting degenerative processes, this study aimed to identify a potential food ingredient with combined anti-tyrosinase and anti-glycative properties. Acanthus mollis L. was selected based on its inclusion in the BelFrIt list and its known content of tyrosinase inhibitors, such as benzoxazinones and verbascoside. Extraction of A. mollis leaves was optimized using a design of experiments approach, comparing microwave- and ultrasound-assisted techniques. Optimal conditions were achieved using microwave-assisted extraction with ethanol 80%, 80 °C, one cycle, drug-to-solvent ratio of 10 mL/g. The optimized extract (at 5 mg/mL) inhibited tyrosinase activity by approximately 47%, increasing to 58% after chlorophyll removal. Moreover, the extract reduces AGEs formation in presence of methylglyoxal, with an activity at 1 mg/mL comparable with that of a well-known anti-glycative agent. A similar trend was observed in the reduction in methylglyoxal and glyoxal levels. Overall, these results support the potential of the optimized A. mollis extract as a functional food ingredient to counteract aging-related neurodegeneration.

The present work introduces a sustainable strategy for biowaste valorization through the extraction and functionalization of biopolymers from Citrus limetta (bergamot) peel using natural deep eutectic solvents (NADES). A green NADES system composed of citric acid, glucose, and water (1:1:2 molar ratio) enabled an efficient extraction of polysaccharides, achieving a maximum yield of 28.11

Plant secondary metabolites can counteract glycation process responsible for the formation of Advanced Glycation End products (AGEs), involved in chronic degenerative diseases mediated by AGEs receptor (RAGE). The research aim was to evaluate the anti-glycative properties of Succisa pratensis extract rich in secoiridoids, mono- and di-caffeoylquinic acids, flavonoids, and saponins identified by RP-HPLC-DAD-ESI-MSn analysis. The extract reduced AGEs formation, mainly at the final step of glycation (100%), and showed a high capacity to trap methylglyoxal and glyoxal (almost 100%). In vitro simulated digestion indicated a low bioaccessibility and bioavailability of the extract compounds, even if a partial anti-glycative activity was retained, mainly attributed to akebia saponin D which had high affinity for RAGE, forming stable complexes, as evident by molecular docking simulations. Therefore, S. pratensis extract could represent a promising source of anti-glycative agents, although a suitable carrier will be necessary to stabilize the extract and improve its bioaccessibility and bioavailability.

In this study, a multimethodological approach was proposed as model to better define chemical changes and cell compatibility properties of digested Locusta migratoria, thus enhancing the definition of this less studied edible insect nutritional value. INFOGEST protocol was applied on a commercial sample of L. migratoria, observing the chemical changes during simulated digestion, as well as amino acids bioaccessibility and bioavailability, cell safety and biocompatibility. A chitin content of 13.19±1.25 g/kg was measured in raw sample. For targeted RP-HPLC-DAD amino acids analysis, optimization of microwave-assisted protein hydrolysis was carried out. A total amino acids content of 49.57±0.80 g/100 g was measured in raw sample. During in vitro digestion, amino acids release was observed in gastric and duodenal phases. Cysteine, serine, lysine, leucine, and phenylalanine had good absorption and bioavailability. Differently, histidine, threonine, valine, proline, alanine, and glutamate showed a limited bioavailability. NMR metabolomics also observed protein hydrolysis phenomena. Moreover, the behavior of organic acids, nitrogen-bases and derivatives, and other metabolites was observed. For instance, acetate, succinate and phosphorylcholine increased in duodenal phase.Cell viability on Caco-2 and Raw 264.7 murine macrophages was evaluated before and after digestion phases. Good cytocompatibility with Caco-2 and Raw 264.7 was observed for both undigested and digested phase, mainly in Caco-2 cells for duodenal phase. In addition, barrier integrity was evaluated for duodenal phase, reporting no significant changes in TEER values. Moreover, cell morphology was assessed by CLSM and SEM, confirming that no alterations of the epithelial barrier or monolayer integrity occurred.

Background: Nutraceuticals represent a strategy for maintaining health and constitute a brilliant market in Italy and across Europe. However, the absence of strict regulations regarding formulation requirements highlights a critical issue: their poor bioavailability. An example is coenzyme Q10 (CoQ10), a quinone known for its potential as a mitochondrial protective agent but characterized by low intestinal absorption. CoQ10 is a hydrophobic molecule with high molecular weight and poor water solubility, factors that significantly limit its intestinal bioaccessibility and, consequently, its oral bioavailability. Objectives: In this context, the present study describes a novel formulation designed to enhance CoQ10 bioaccessibility through in situ emulsification upon contact with gastroenteric fluids. This technology, termed Lipid-Based Auto-Emulsifying Drug Delivery System (LiBADDS), is unique because it combines a medium-chain triglyceride (MCT), a long-chain fatty acid, conjugated linoleic acid (CLA) with a high HLB solubilizer, Polysorbate 80 (PS80), and a sodium octenyl succinate starch derivative (SOS), which can create a nanometric emulsion simply by aqueous dispersion and upon contact with gastrointestinal fluids. This phenomenon promotes the prompt dispersion of CoQ10 and its rapid translocation into the serosal compartment of the intestinal epithelium. Methods: Its efficacy was evaluated in vitro through the Caco-2 cellular model and in vivo through a crossover study on healthy volunteers, measuring pharmacokinetic parameters such as AUC, Cmax, Tmax, ΔAUC, and ΔCmax. Results: Overall, LiBADDS demonstrated a significant improvement in both the bioaccessibility and bioavailability of CoQ10 compared to the unformulated substance. Conclusions: LiBADDS showed to be a promising tool to improve CoQ10 bioavailability by enhancing its bioaccessibility.

This study explores the remarkable dual antiviral potential of Pelargonium graveolens, an edible aromatic plant widely valued in traditional medicine and culinary applications. The ethanol extract from P. graveolens leaves and its ethyl acetate fraction demonstrated significant inhibitory efficacy against both Herpes Simplex Virus Type 2 (HSV-2) and Coxsackievirus B3 (CVB-3), with notable selectivity index of 134 and 83, respectively. Mechanistic investigations revealed distinct modes of action: the ethanol extract primarily targets viral entry processes and exhibits direct virucidal effects against HSV-2, whereas the ethyl acetate fraction specifically inhibits CVB-3 post-infection. Bioassay-guided fractionation and purification using TLC and HPLC-DAD-ESI-MSn led to the identification of two bioactive compounds: quinic acid from the ethanol extract and kaempferol dimethyl ether from the ethyl acetate fraction. Molecular docking analyses yielded results not markedly different from our experimental findings, demonstrating the most favorable interactions between quinic acid and the HSV-2 fusion regulator complex gH-gL, which is crucial for viral entry, as well as between kaempferol dimethyl ether and CVB-3 RNA polymerase. Drug-likeness assessment confirmed both compounds comply with Lipinski’s Rule of Five, suggesting favorable pharmacokinetic profiles. The presence of two bioactive compounds within Pelargonium graveolens, each exhibiting distinct antiviral mechanisms against different viruses, highlights the plant as a promising source for the development of novel broad-spectrum antiviral therapeutics.

In this report, Castanea sativa shell extracts have been investigated for their activity against Herpes Virus type 2 (HSV-2). Among four organic extracts only 75% ethanol extract demonstrated activity against HSV-2 with a 50% inhibitory concentration of 6.45 μg/mL. The study of the mechanism underlying the antiviral activity demonstrated this extract strongly inhibits HSV-2 by direct contact and moderately protects the cell from virus recognition and binding. The active compound has been identified as oleuropein by HPLC-DAD-ESI-MSn. This compound which is widely known in the Oleaceae family has been identified in the chestnut shells for the first time. The in vitro results of the mechanism underlying anti HSV-2 action were confirmed by an in silico study which revealed a strong binding free energy of -9.08 kcal/mol between oleuropein and the glycoprotein D of HSV-2. In summary, our findings suggest that C. sativa shells may constitute a promising natural source of anti-HSV-2 agents.

The generally very low bioaccessibility of polyphenols can be enhanced through several different strategies, especially when these metabolites are components of extracts used as food ingredients. This work explores the efficacy of pectin-zein beads as carriers for delivering p-coumaric acid), the main component of rice husk extract. Ten formulations were prepared using the ionic gelation technique, employing a Taghuci Design of Experiments to optimize zein, pectin, and CaCl2 concentrations. Zein content was found as the main parameter affecting the encapsulation efficiency. The highest value (51.77 ± 1.13%) was achieved using 10% zein, 3% pectin, and 4% CaCl2. p-coumaric acid bioaccessibility in the raw and encapsulated extracts was evaluated by adopting the Infogest digestion protocol and simulating a colon phase with Pectinex® Ultra SPL enzymes, evidencing that pectin-zein beads effectively improved p-coumaric acid stability in the extract. The encapsulation highly preserves p-coumaric acid during the gastric phase (bioaccessibility index 34%); conversely, an increased release was registered at the intestinal level, reaching approximately 80% and 100% during the duodenal and colon steps, respectively. Therefore, pectin-zein beads were demonstrated to be a promising tool for the development of active ingredients suitable for functional foods/food supplements aimed at enhancing health benefits through controlled intestinal delivery of bioactives.

Background: Italy’s plant biodiversity, characterized by many plant species, is an important source of bioactive secondary metabolites that help reduce the risk of the development of advanced glycation end product (AGE)-related diseases. AGEs are involved in various diseases, such as diabetes, cardiovascular, and neurodegenerative disorders. Therefore, the aim of the study was to investigate the antiglycative, hypoglycemic, and neuroprotective properties of nine edible plant extracts using different in vitro assays. Methods: The ability of the extracts to counteract AGE formation was evaluated at different stages of the glycation reaction using in vitro systems based on the determination of Amadori products and the co-incubation of a model protein with a dicarbonyl compound under different experimental conditions. In addition, the extracts’ methylglyoxal (MGO) and glyoxal (GO) trapping ability was investigated. Hypoglycemic activity was assessed by measuring α-amylase inhibition, while the neuroprotective effects were explored by testing amyloid β peptide 1-42 (Aβ1-42) fibrillogenesis inhibition. Results: All extracts generally had a dose-related capacity for the inhibition of AGE formation, mainly at the intermediate stage of the glycation reaction; high trapping capacity against MGO and GO; and promising hypoglycemic properties. In addition, they affected the fibrillogenesis process by reducing mature amyloid fibril formation and altering fibril morphology. Conclusions: All tested extracts had promising anti-fibrillogenic properties. Rosa canina extract was the most active among the tested plant species given its antiglycative activity (about 80% inhibition of AGE formation), trapping capacity against MGO and GO (almost 100%), hypoglycemic effects (66.20 ± 0.88%), and anti-fibrillogenic effects (69.00 ± 4.49% inhibition), indicating its suitability in the management of AGE-related diseases and for the potential development of a novel food ingredient.

Background: The in vivo accumulation of Advanced Glycation End products (AGEs) is associated with the development of several chronic aging-related and degenerative diseases, as they alter protein structures and activate oxidative and inflammatory processes through interactions with the receptor for AGEs (RAGE). Plant secondary metabolites play a key role in counteracting the glycation process through various mechanisms of action. Therefore, Aloysia citrodora leaf polyphenolic extract could represent a source of anti-glycative compounds. Methods: The methanolic extract was characterized by RP-HPLC-DAD-MSn, and its anti-glycative properties were investigated using several in vitro assays mimicking the different steps of the glycation reaction. In parallel, molecular docking studies were carried out to evaluate potential interactions between the identified metabolites and RAGE. Furthermore, A. citrodora metabolites' stability under simulated in vitro digestion was assessed, and the anti-glycative activity of the bioaccessible fraction was investigated. Results:A. citrodora extract, rich in iridoid glycosides, phenylethanoid glycosides, and flavones, strongly inhibited AGE formation (from 10% to 100%) in both the middle and end step of the reaction and had high methylglyoxal and glyoxal trapping capacity. However, the digestion process affected extract stability, particularly under intestinal conditions, yielding an overall bioaccessibility of about 40% and leading to a subsequent reduction in anti-glycative properties. Finally, molecular modeling analysis highlighted the ability of the studied metabolites to bind RAGE. Conclusions:A. citrodora represents a promising source of natural anti-glycative agents with potential applications as food ingredients. However, it is essential to improve the extract bioaccessibility and to preserve its anti-glycative properties by developing a suitable formulation.

In recent years, nutraceuticals have emerged as a promising strategy for maintaining health and represent a high-growth market in Italy and across Europe. However, the lack of strict regulations regarding formulation requirements and proof of efficacy raises serious concerns about their poor bioavailability and, consequently, their uncertain health benefits. An emblematic example is t-resveratrol (RES), a cardioprotective stilbene polyphenol that undergoes extensive metabolism in the intestine and liver, resulting in a bioavailability of <1 %. This manuscript describes a novel technological matrix developed with the primary goal of improving RES oral bioavailability. This technology can be classified as a lipid-based autoemulsifying drug delivery system (LIBADDS), in which RES is thoroughly solubilized in a hot liquid phase composed of lipids and surfactants, and the mixture is further adsorbed onto a powder composed of polysaccharides and sodium caseinate (NaC), along with inert excipients, and then compressed. In this study, NaC was used for the first time to trigger pancreatin-mediated hydrolysis of an enteric-coated tablet, allowing micellar delivery of RES to the small intestine. The RES-containing tablets were characterized via differential scanning calorimetry (DSC) and X-ray diffraction (PXRD). The digested formulation, with simulated gastric and enteric fluids, was dimensionally assessed via dynamic light scattering (DLS). Finally, calculations of the bioaccessible fraction, dissolution tests, and in vitro permeability experiments using Caco-2 cell monolayers were carried out to preliminarily define the overall efficiency and applicability of this new technology in improving RES intestinal permeability.

Infections caused by Staphylococcus aureus are particularly difficult to treat due to the high rate of antibiotic resistance. S. aureus also forms biofilms that reduce the effects of antibiotics and disinfectants. Therefore, new therapeutic approaches are increasingly required. In this scenario, plant waste products represent a source of bioactive molecules. In this study, we evaluated the antimicrobial and antibiofilm activity of the rice husk extract (RHE) on S. aureus clinical isolates. In a biofilm inhibition assay, high concentrations of RHE counteracted the formation of biofilm by S. aureus isolates, both methicillin-resistant (MRSA) and -sensitive (MSSA). The observation of the MRSA biofilm by confocal laser scanning microscopy using live/dead cell viability staining confirmed that the bacterial viability in the RHE-treated biofilm was reduced. However, the extract showed no or little biofilm disaggregation ability. An additive effect was observed when treating S. aureus with a combination of RHE and oxacillin/cefoxitin. In Galleria mellonella larvae treated with RHE, the extract showed no toxicity even at high concentrations. Our results support that the rice husk has antimicrobial and antibiofilm properties and could potentially be used in the future in topical solutions or on medical devices to prevent biofilm formation.

Background: Fruit and vegetable seeds represent an important source of bioactive (proteins, polysaccharides, flavonoids, carotenoids, lignans, and vitamins) useful ingredients for food supplements and functional foods, providing health benefits and having important implications mainly in the prevention of cardiovascular and neurological disorders. Bioactive protein hydrolysates and peptides recovered from food wastes have significant functional and nutraceutical value, acting mainly as antihypertensive, hypoglycemic, hypocholesterolemic, and antioxidant agents. Scope and Approach: This review article discusses the main bioactivities of peptides deriving from different fruit and vegetable seed wastes, focusing on their molecular mechanisms of action. The protein extraction, purification, fractionation, and analysis approaches to obtain the highest amount and the hugest typology of bioactive peptides are also commented. Findings and conclusions: As a matter of fact, an important issue that over time limited the use of seed wastes was the notable costs of extraction and purification of plant proteins: the here reported modern approaches could ensure a higher recovery of bioactive peptides with very promising applications at industrial level. In addition, the optimization of detoxification processes resolved another potential disadvantage, that is the presence of toxic derivatives in some plant seeds. At the present, digestion studies useful to better clarify the peptide bioavailability and to predict their effective role in vivo and potential re-use are still lacking. However, they are an important issue for the development and commercial perspectives of new protein-based products and therefore should be mandatory.

Recently, pectin, a versatile polysaccharide with different industrial applications, has gained significant attention as an eco-friendly and functional ingredient. This study investigates pumpkin peels (Cucurbita maxima L., Mantua variety) as a novel source of pectin, using a microwave-assisted extraction method with citric acid-acidified water as solvent. The extraction conditions were optimized using a Design of Experiments approach, considering the solvent-to-solid ratio (SSR), pH, temperature, and extraction time. The optimized conditions (94.8 °C, 5 min, pH 1.5, and 46 mL/g SSR) resulted in a pectin yield of 18.05%. A comprehensive characterization of the extracted pectin was performed, including FT-IR spectroscopy, DSC, TGA, rheological properties, and techno-functional assessments such as water holding capacity and fat binding capacity. The results indicated a high degree of esterification (56.19 ± 0.87%), classifying the pumpkin peels (PP) extract as a high methoxyl pectin. PP pectin demonstrated potential as a stabilizer and emulsifying agent, although its high methoxyl content limits its use as a carrier for targeted bioactive delivery. The findings support the viability of using agricultural by-products to obtain valuable polysaccharides, contributing to waste valorization and sustainable industrial practices.