Hydroxytyrosol permeability comparisons and strategies to improve hydroxytyrosol stability in formulations.

There has been a growing interest in hydroxytyrosol (HT) due to its powerful antioxidant and free-radical scavenging properties when added to formulations such as pharmaceuticals and cosmetics. To study the stability and transdermal properties of hydrogels and creams (HT-based formulations), a high-performance liquid chromatography method was developed for determining HT. In the Franz diffusion cell system, both hydrogel and cream show a rapid and similar penetration profile through the Bama miniature pig skin. However, the Strat-M® membrane exhibits slightly lower permeability and is selective to different formulations; that is, the cream has a permeability value of 10.69%, while the hydrogel has a value of 5.27%. The dynamics parameters from the permeation assays indicate that the model using the Strat-M® membrane can be used as a screening tool to evaluate the skin uptake and permeation efficacy of different formulations. Adding 3-O-ethyl-L-ascorbic acid to HT-based formulations can effectively prevent discoloration under prolonged high-temperature storage, while combining multiple antioxidants delays degradation most effectively. This study provides novel ideas for functional formulation optimization to enhance the realism and reproducibility of cosmetic products containing HT and provides scientific evidence for the production, packaging, shelf life, storage, and transportation of products.

How the "Olive Oil Polyphenols" Health Claim Depends on Anthracnose and Olive Fly on Fruits.

Olive anthracnose, caused by Colletotrichum fungi, and the olive fruit fly Bactrocera olea are, respectively, the most important fungal disease and pest affecting olive fruits worldwide, leading to detrimental effects on the yield and quality of fruits and olive oil. This study focuses on the content of hydroxytyrosol (HYT) and its derivatives (the "olive oil polyphenols" health claim) in olive oils extracted from fruits of 'Galega Vulgar' and 'Cobrançosa' cultivars, naturally affected by olive anthracnose and olive fly. The olives, with different damage levels, were harvested from organic rainfed orchards, located in the center of Portugal, at four harvest times over three years. Galega oils extracted from olives with a higher anthracnose and olive fly incidence showed no conformity for the extra virgin olive oil (EVOO) and virgin olive oil (VOO) categories, presenting high acidity and negative sensory notes accompanied by the disappearance of oleacein. Conversely, no sensory defects were observed in Cobrançosa oils, regardless of disease and pest incidence levels, and quality criteria were still in accordance with the EVOO category. The total HYT and tyrosol (TYR) content (>5 mg/20 g) allows for the use of the "olive oil polyphenols" health claim on the label of all the analyzed Cobrançosa olive oils.

Hydroxytyrosol Alleviates Intestinal Oxidative Stress by Regulating Bile Acid Metabolism in a Piglet Model.

Infants and young animals often suffer from intestinal damage caused by oxidative stress, which may adversely affect their overall health. Hydroxytyrosol, a plant polyphenol, has shown potential in decreasing intestinal oxidative stress, but its application and mechanism of action in infants and young animals are still inadequately documented. This study selected piglets as a model to investigate the alleviating effects of hydroxytyrosol on intestinal oxidative stress induced by diquat and its potential mechanism. Hydroxytyrosol improved intestinal morphology, characterized by higher villus height and villus height/crypt depth. Meanwhile, hydroxytyrosol led to higher expression of Occludin, MUC2, Nrf2, and its downstream genes, and lower expression of cytokines IL-1ß, IL-6, and TNF-α. Both oxidative stress and hydroxytyrosol resulted in a higher abundance of Clostridium_sensu_stricto_1, and a lower abundance of Lactobacillus and Streptococcus, without a significant effect on short-chain fatty acids levels. Oxidative stress also led to disorders in bile acid (BA) metabolism, such as the lower levels of primary BAs, hyocholic acid, hyodeoxycholic acid, and tauroursodeoxycholic acid, which were partially restored by hydroxytyrosol. Correlation analysis revealed a positive correlation between these BA levels and the expression of Nrf2 and its downstream genes. Collectively, hydroxytyrosol may reduce oxidative stress-induced intestinal damage by regulating BA metabolism.

Synergistic protective effects of 3,4-dihydroxyphenylglycol and hydroxytyrosol in male rats against induced heat stress-induced reproduction damage.

Testicular heat stress disrupts spermiogenesis and damages testicular tissue. The study aims to assess 3,4-dihydroxyphenylglycol (DHPG) and hydroxytyrosol (HT) from olive oil as antioxidants to reduce heat-induced testicular damage. Seven groups of 35 male rats were used. Group I got normal saline. Group 2 had HS (43 °C for 20 min/day) and normal saline for 60 days. Groups 3-7 had HS and DHPG/HT doses (0.5 mg/kg DHPG, 1 mg/kg DHPG, 5 mg/kg HT, 0.5 mg/kg DHPG + 5 mg/kg HT, and 1 mg/kg DHPG + 5 mg/kg HT). The evaluation included tests on testicular tissue, sperm quality, oxidative status, gene activity, and fertility after 60 days. After DHPG and HT treatment, sperm motility, viability, and plasma membrane functionality, as well as levels of total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), and Bcl-2 gene expression, and in vivo fertility indexes increased. Meanwhile, abnormal morphology and DNA damage decreased, along with levels of glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA), and Bax, caspase-3, and caspase-9 gene expression, compared to the HS group. The study found that DHPG and HT have a more substantial synergistic effect when used together, improving reproductive health.

Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation.

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNetTM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein-peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet.

Proton-Coupled Electron Transfer and Hydrogen Tunneling in Olive Oil Phenol Reactions.

Olive oil phenols are recognized as molecules with numerous positive health effects, many of which rely on their antioxidative activity, i.e., the ability to transfer hydrogen to radicals. Proton-coupled electron transfer reactions and hydrogen tunneling are ubiquitous in biological systems. Reactions of olive oil phenols, hydroxytyrosol, tyrosol, oleuropein, oleacein, oleocanthal, homovanillyl alcohol, vanillin, and a few phenolic acids with a DPPH• (2,2-diphenyl-1-picrylhydrazyl) radical in a 1,4-dioxane:water = 95:5 or 99:1 v/v solvent mixture were studied through an experimental kinetic analysis and computational chemistry calculations. The highest rate constants corresponding to the highest antioxidative activity are obtained for the ortho-diphenols hydroxytyrosol, oleuropein, and oleacein. The experimentally determined kinetic isotope effects (KIEs) for hydroxytyrosol, homovanillyl alcohol, and caffeic acid reactions are 16.0, 15.4, and 16.7, respectively. Based on these KIEs, thermodynamic activation parameters, and an intrinsic bond orbital (IBO) analysis along the IRC path calculations, we propose a proton-coupled electron transfer mechanism. The average local ionization energy and electron donor Fukui function obtained for the phenolic compounds show that the most reactive electron-donating sites are associated with π electrons above and below the aromatic ring, in support of the IBO analysis and proposed PCET reaction mechanism. Large KIEs and isotopic values of Arrhenius pre-exponential factor AH/AD determined for the hydroxytyrosol, homovanillyl alcohol, and caffeic acid reactions of 0.6, 1.3, and 0.3, respectively, reveal the involvement of hydrogen tunneling in the process.

Valorization of Olive Pomace Using Ultrasound-Assisted Extraction for Application in Active Packaging Films.

Bioactive compounds that can be recovered by the solid wastes of the olive oil sector, such as polyphenols, are known for their significant antioxidant and antimicrobial activities with potential application in nutraceutical, cosmetic, and food industries. Given that industrial demands are growing, and the polyphenol market value is ever increasing, a systematic study on the recovery of natural antioxidant compounds from olive pomace using ultrasound-assisted extraction (UAE) was conducted. Single-factor parameters, i.e., the extraction solvent, time, and solid-to-liquid ratio, were investigated evaluating the total phenolic content (TPC) recovery and the antioxidant activity of the final extract. The acetone-water system (50% v/v, 20 min, 1:20 g mL-1) exhibited the highest total phenolic content recovery (168.8 ± 5.5 mg GAE per g of dry extract). The olive pomace extract (OPE) was further assessed for its antioxidant and antibacterial activities. In DPPH, ABTS, and CUPRAC, OPE exhibited an antioxidant capacity of 413.6 ± 1.9, 162.72 ± 3.36 and 384.9 ± 7.86 mg TE per g of dry extract, respectively. The antibacterial study showed that OPE attained a minimum inhibitory activity (MIC) of 2.5 mg mL-1 against E. coli and 10 mg mL-1 against B. subtilis. Hydroxytyrosol and tyrosol were identified as the major phenolic compounds of OPE. Furthermore, active chitosan-polyvinyl alcohol (CHT/PVA) films were prepared using different OPE loadings (0.01-0.1%, w/v). OPE-enriched films showed a dose-dependent antiradical scavenging activity reaching 85.7 ± 4.6% (ABTS) and inhibition growth up to 81% against B. subtilis compared to the control film. Increased UV light barrier ability was also observed for the films containing OPE. These results indicate that OPE is a valuable source of phenolic compounds with promising biological activities that can be exploited for developing multifunctional food packaging materials.

Commercial wine yeast nitrogen requirement influences the production of secondary metabolites (aroma, hydroxytyrosol, melatonin and other bioactives) during alcoholic fermentation.

During alcoholic fermentation, Saccharomyces cerevisiae synthesizes different compounds, which are crucial for product quality: volatile compounds with sensory impact, and bioactive compounds such as melatonin (MEL) and hydroxytyrosol (HT), linked to health benefits. As many of these compounds are related with yeast's nitrogen metabolism, their production have been studied in four different commercial strains with different nitrogen requirement (Red Fruit, Uvaferm VRB, Lalvin Rhone 2323 and Lalvin QA23) being, Uvaferm UVR the higher nitrogen demander strain. All strains produced the secondary metabolites, notably Uvaferm UVR produced the highest HT concentration, despite its low growth. Uvaferm UVR emerged also as a significant producer of MEL, indicating a potential role in fermentation related stress. Moreover, Uvaferm UVR shows the highest total concentrations of volatile compounds. Multivariate analysis revealed distinct clustering based on nitrogen requirements of the strains, highlighting the strain-dependent metabolic responses.

Polyphenol-Rich Extracts from Agroindustrial Waste and Byproducts: Results and Perspectives According to the Green Chemistry and Circular Economy.

Polyphenols are natural secondary metabolites found in plants endowed with multiple biological activities (antioxidant, anti-inflammatory, antimicrobial, cardioprotective, and anticancer). In view of these properties, they find many applications and are used as active ingredients in nutraceutical, food, pharmaceutical, and cosmetic formulations. In accordance with green chemistry and circular economy strategies, they can also be recovered from agroindustrial waste and reused in various sectors, promoting sustainable processes. This review described structural characteristics, methods for extraction, biological properties, and applications of polyphenolic extracts obtained from two selected plant materials of the Mediterranean area as olive (Olea europaea L.) and pomegranate (Punica granatum L.) based on recent literature, highlighting future research perspectives.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis.

Hydroxytyrosol, a naturally occurring compound with antioxidant and antiviral activity, is widely applied in the cosmetic, food, and nutraceutical industries. The development of a biocatalytic approach for producing hydroxytyrosol from simple and readily accessible substrates remains a challenge. Here, we designed and implemented an effective biocatalytic cascade to obtain hydroxytyrosol from 3,4-dihydroxybenzaldehyde and l-threonine via a four-step enzymatic cascade composed of seven enzymes. To prevent cross-reactions and protein expression burden caused by multiple enzymes expressed in a single cell, the designed enzymatic cascade was divided into two modules and catalyzed in a stepwise manner. The first module (FM) assisted the assembly of 3,4-dihydroxybenzaldehyde and l-threonine into (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and the second module (SM) entailed converting (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid into hydroxytyrosol. Each module was cloned into Escherichia coli BL21 (DE3) and engineered in parallel by fine-tuning enzyme expression, resulting in two engineered whole-cell catalyst modules, BL21(FM01) and BL21(SM13), capable of converting 30 mM 3,4-dihydroxybenzaldehyde to 28.7 mM hydroxytyrosol with a high space-time yield (0.88 g/L/h). To summarize, the current study proposes a simple and effective approach for biosynthesizing hydroxytyrosol from low-cost substrates and thus has great potential for industrial applications.

An Eco-Friendly Supercritical CO2 Recovery of Value-Added Extracts from Olea europaea Leaves.

An eco-friendly approach towards the recovery of value-added extracts from olive tree leaves with the aid of supercritical CO2 at 30 MPa was carried out. The impact of extraction temperature (35-90 °C) and presence of co-solvents (ethanol, water, and aqueous ethanol) on the total phenolic, flavonoid, and pigment content, as well as oleuropein, hydroxytyrosol, tyrosol, and α-tocopherol content was determined. In addition, the antioxidant activity of extracts from tree leaves using DPPH, ABTS, and CUPRAC assays was investigated. The results of the study showed that the most effective supercritical CO2 extraction was at 90 °C with an addition of ethanol, which enabled the separation of extract with the highest content of tested compounds. Some of the highest recorded values were for oleuropein 1.9 mg/g, for carotenoids 5.3 mg/g, and for α-tocopherol 2.0 mg/g. Our results are expected to contribute to the efforts towards the valorization of olive leaves as a sustainable source of valuable compounds, and boost local economies as well as the interest of pharmaceutical, food, and cosmetic industries for novel food by-product applications.

Hydroxytyrosol, a Promising Supplement in the Management of Human Stroke: An Exploratory Study.

Hydroxytyrosol (HT) is a bioactive olive oil phenol with beneficial effects in a number of pathological situations. We have previously demonstrated that an HT-enriched diet could serve as a beneficial therapeutic approach to attenuate ischemic-stroke-associated damage in mice. Our exploratory pilot study examined this effect in humans. Particularly, a nutritional supplement containing 15 mg of HT/day was administered to patients 24 h after the onset of stroke, for 45 days. Biochemical and oxidative-stress-related parameters, blood pressure levels, serum proteome, and neurological and functional outcomes were evaluated at 45 and 90 days and compared to a control group. The main findings were that the daily administration of HT after stroke could: (i) favor the decrease in the percentage of glycated hemoglobin and diastolic blood pressure, (ii) control the increase in nitric oxide and exert a plausible protective effect in oxidative stress, (iii) modulate the evolution of the serum proteome and, particularly, the expression of apolipoproteins, and (iv) be beneficial for certain neurological and functional outcomes. Although a larger trial is necessary, this study suggests that HT could be a beneficial nutritional complement in the management of human stroke.

Anti-Cancer, Anti-Angiogenic, and Anti-Atherogenic Potential of Key Phenolic Compounds from Virgin Olive Oil.

The Mediterranean diet, renowned for its health benefits, especially in reducing cardiovascular risks and protecting against diseases like diabetes and cancer, emphasizes virgin olive oil as a key contributor to these advantages. Despite being a minor fraction, the phenolic compounds in olive oil significantly contribute to its bioactive effects. This review examines the bioactive properties of hydroxytyrosol and related molecules, including naturally occurring compounds (-)-oleocanthal and (-)-oleacein, as well as semisynthetic derivatives like hydroxytyrosyl esters and alkyl ethers. (-)-Oleocanthal and (-)-oleacein show promising anti-tumor and anti-inflammatory properties, which are particularly underexplored in the case of (-)-oleacein. Additionally, hydroxytyrosyl esters exhibit similar effectiveness to hydroxytyrosol, while certain alkyl ethers surpass their precursor's properties. Remarkably, the emerging research field of the effects of phenolic molecules related to virgin olive oil on cell autophagy presents significant opportunities for underscoring the anti-cancer and neuroprotective properties of these molecules. Furthermore, promising clinical data from studies on hydroxytyrosol, (-)-oleacein, and (-)-oleocanthal urge further investigation and support the initiation of clinical trials with semisynthetic hydroxytyrosol derivatives. This review provides valuable insights into the potential applications of olive oil-derived phenolics in preventing and managing diseases associated with cancer, angiogenesis, and atherosclerosis.

Hydroxytyrosol acetate from olive leaves (Olea Europaea L.) induces apoptosis via mitochondrial pathway in BEL7402 cell line.

Hydroxytyrosol acetate is one of the polyphenolic compounds in olive leaves. Hydroxytyrosol acetate has a variety of biological activities, such as antibacterial, antioxidant, anti-inflammatory, cognitive improvement and neuroprotective effects. However, there is no report on the antitumor activity and the antitumor mechanism of hydroxytyrosol acetate. In our study, we studied the antitumor activity of hydroxytyrosol acetate by MTT assay and determined the antitumor mechanism by DNA ladder assay, mitochondrial membrane potential assay and western blot assay. We found that hydroxytyrosol acetate could inhibit cell proliferation, and the inhibition rate was 78.08%. The further researches showed that hydroxytyrosol acetate could downregulate Bcl-2 protein while upregulate Bax protein. It also could induce mitochondrial depolarisation and release of cytochrome C. These results indicated that hydroxytyrosol acetate might induce BEL7402 cells apoptosis via mitochondrial pathway.

Stability of monovarietal Sicilian olive oils under different storage condition: Chemical composition, sensory characteristics, and consumer preference.

Monovarietal olive oils, known for their distinct aromatic profiles, face challenges in preserving their initial quality due to variations in stability and susceptibility to oxidative deterioration. This study focused on the storage stability of two Italian cultivars, 'Biancolilla' and 'Cerasuola', from Sicily, chosen for their aromatic complexity and divergent storage stability. Oils, whether filtered or unfiltered, underwent storage at two thermal regimes (18 °C and - 20 °C) over a year. The combination of filtration and low-temperature storage was employed to mitigate oxidative deterioration and hydrolytic processes, as filtration removes suspended particles and emulsified water, while low temperatures slow down enzymatic activities and oxidative reactions, thereby enhancing the overall stability and shelf life of the olive oils. Unfiltered samples at room temperature showed a significant increase in secoiridoid aglycone derivatives (Hydroxytyrosol and Tyrosol) due to hydrolytic processes, along with a decrease in secoiridoid aglycone. Filtration delayed these processes, with a more pronounced effect observed when combined with -20 °C storage. Sensory analysis identified the emergence of the "fusty" defect in the less resilient 'Biancolilla' cultivar after six months, a phenomenon mitigated by filtration and freezing. Consumer tests validated these findings. In summary, the synergistic approach of combining filtration with low-temperature storage emerges as a promising strategy for maintaining high-quality standards, especially for less stable monovarietal extra virgin olive oils. This strategy ensures compliance with EU regulations beyond the conventional 12-month shelf life, offering a practical solution for preserving the nutritional and sensory quality of olive oil.

Proteomics in Patients with Fibromyalgia Syndrome: A Systematic Review of Observational Studies.

PURPOSE OF REVIEW: Fibromyalgia syndrome (FMS) is a disease of unknown pathophysiology, with the diagnosis being based on a set of clinical criteria. Proteomic analysis can provide significant biological information for the pathophysiology of the disease but may also reveal biomarkers for diagnosis or therapeutic targets. The present systematic review aims to synthesize the evidence regarding the proteome of adult patients with FMS using data from observational studies. RECENT FINDINGS: An extensive literature search was conducted in MEDLINE/PubMed, CENTRAL, and clinicaltrials.gov from inception until November 2022. The study protocol was published in OSF. Two independent reviewers evaluated the studies and extracted data. The quality of studies was assessed using the modified Newcastle-Ottawa scale adjusted for proteomic research. Ten studies fulfilled the protocol criteria, identifying 3328 proteins, 145 of which were differentially expressed among patients with FMS against controls. The proteins were identified in plasma, serum, cerebrospinal fluid, and saliva samples. The control groups included healthy individuals and patients with pain (inflammatory and non-inflammatory). The most important proteins identified involved transferrin, α-, ß-, and γ-fibrinogen chains, profilin-1, transaldolase, PGAM1, apolipoprotein-C3, complement C4A and C1QC, immunoglobin parts, and acute phase reactants. Weak correlations were observed between proteins and pain sensation, or quality of life scales, apart from the association of transferrin and a2-macroglobulin with moderate-to-severe pain sensation. The quality of included studies was moderate-to-good. FMS appears to be related to protein dysregulation in the complement and coagulation cascades and the metabolism of iron. Several proteins may be dysregulated due to the excessive oxidative stress response.

Magnetic molecularly imprinted polymers using ternary deep eutectic solvent as novel functional monomer for hydroxytyrosol separation.

In this work, magnetic molecularly imprinted polymers (MIPs) for specific recognition of Hydroxytyrosol (HT) were designed by vinyl-modified magnetic particles (Fe3O4@SiO2@VTEOs) as carrier, ternary deep eutectic solvent (DES) as functional monomer, while ethylene glycol dimethacrylate (EGDMA) as crosslinker. The optimum amount of DES was obtained by adsorption experiments (molar ratio, caffeic acid: choline chloride: formic acid = 1:6:3) which were 140 µL in total. Under the optimized amount of DES, the maximum adsorption capacity of the MIPs particles was 42.43 mg g-1, which was superior to non-imprinted polymer (4.64 mg g-1) and the imprinting factor (IF) is 9.10. Syringin and Oleuropicrin were used as two reference molecules to test the selectivity of the DES-MIPs particles. The adsorption capacity of HT was 40.11 mg g-1. Three repeated experiments show that the polymer has high stability and repeatability (RSD = 5.50).

Stability study and validation of a liquid chromatography tandem mass spectrometry method for the quantitative analysis of polyphenols in fish feed ingredients.

The perception of polyphenols as a safe, healthy, and sustainable solution for replacing synthetic antioxidants has been an important factor for their rapid growing in the global food market. Therefore, it is essential to use reliable methods for their quantification in commercial products intended for animal or human consumption. The purpose of this study is to evaluate the performance of some solvents used for the extraction of selected polyphenols, explore their stability under different experimental conditions, and validate a liquid chromatography tandem mass-spectrometry method for their quantification in commercial fish feed ingredients by using the standard addition method. The regression models for gallic acid, hydroxytyrosol, catechin, oleuropein, carnosol and carnosic acid were linear in the range 0-30 µg/mL, limit of detection and quantification around 0.03 and 0.1 µg/mL, respectively, and accuracy within ± 15 % of the nominal concentrations. The method was successfully applied to the determination of specific polyphenols in commercial fish feed ingredients supplemented with polyphenols from olive and rosemary extracts.

Functional differentiation of olive PLP_deC genes: insights into metabolite biosynthesis and genetic improvement at the whole-genome level.

KEY MESSAGE: This study identified 16 pyridoxal phosphate-dependent decarboxylases in olive at the whole-genome level, conducted analyses on their physicochemical properties, evolutionary relationships and characterized their activity. Group II pyridoxal phosphate-dependent decarboxylases (PLP_deC II) mediate the biosynthesis of characteristic olive metabolites, such as oleuropein and hydroxytyrosol. However, there have been no report on the functional differentiation of this gene family at the whole-genome level. This study conducted an exploration of the family members of PLP_deC II at the whole-genome level, identified 16 PLP_deC II genes, and analyzed their gene structure, physicochemical properties, cis-acting elements, phylogenetic evolution, and gene expression patterns. Prokaryotic expression and enzyme activity assays revealed that OeAAD2 and OeAAD4 could catalyze the decarboxylation reaction of tyrosine and dopa, resulting in the formation of their respective amine compounds, but it did not catalyze phenylalanine and tryptophan. Which is an important step in the synthetic pathway of hydroxytyrosol and oleuropein. This finding established the foundational data at the molecular level for studying the functional aspects of the olive PLP_deC II gene family and provided essential gene information for genetic improvement of olive.