Hydroxytyrosol (HTyr) and tyrosol (Tyr) are the most well studied phenolic alcohols of olive oil and olive products demonstrating numerous and significant beneficial health effects. However, their activity in the human organism as food bioactives is strongly associated with their bioavailability and metabolism, while manifested through their metabolites. Nevertheless, there are limited studies investigating their biotransformation and mainly catabolism by gut microflora under a holistic interpretation close to the human organism. Thus, in the present study, the GastroIntestinal Dialysis (GIDM)-colon model, a continuous flow in vitro dialysis system mimicking physiological conditions during human gastrointestinal digestion, was used to explore the metabolism of HTyr and Tyr as pure compounds. The GIDM–colon model simulates absorption from the lumen to the mucosa, followed by the colon phase using pooled human fecal suspensions. Samples were collected at different time points and analyzed via LC–Orbitrap MS. An integrated approach combining Multivariate Data Analysis (MVA) and thorough dereplication procedures led to the identification of HTyr and Tyr metabolites in different phases (gastric, small intestine, and colon), yielding also valuable information about metabolites kinetics. To our knowledge, this is the first study reporting full spectrometric data of HTyr and Tyr metabolites along with possible transformation mechanisms in the GI tract.

Background: Olive leaves are a rich source of polyphenols, predominantly secoiridoids, flavonoids, and simple phenols, which exhibit various biological properties. Extracts prepared from olive leaves are associated with hypoglycemic, hypotensive, diuretic, and antiseptic properties. Upon ingestion, a substantial fraction of these polyphenols reaches the colon where they undergo extensive metabolism by the gut microbiota. Host characteristics, like age, can influence the composition of the gut microbiome, potentially affecting the biotransformation of these compounds. Therefore, it can be hypothesised that differences in the gut microbiome between young and elderly individuals may impact the biotransformation rate and the type and amount of metabolites formed. Methods: An in vitro biotransformation model was used to mimic the conditions in the stomach, small intestine and colon of two age groups of healthy participants (20–30 years old, ≥65 years old), using oleuropein as a single compound and an olive leaf extract as test compounds. The bacterial composition and metabolite content were investigated. Results: The study revealed that, while the same metabolites were formed in both age groups, in the young age group, less metabolite formation was observed, likely due to a reduced viable cell count. Most biotransformation reactions took place within the first 24 h of colon incubation, and mainly, deglycosylation, hydrolysis, flavonoid ring cleavage, and demethylation reactions were observed. A bacterial composition analysis showed a steep drop in α-diversity after 24 h of colon incubation, likely due to favourable experimental conditions for certain bacterial species. Conclusions: Both age groups produced the same metabolites, suggesting that the potential for polyphenols to exert their health-promoting benefits persists in healthy older individuals.

Glutathione (GSH), a tripeptide that consists of cysteine, glutamate and glycine, is present in all mammalian tissues in the millimolar range. Besides having numerous cellular functions, GSH is an important antioxidant and is considered a valuable biomarker in evaluating oxidative stress. This paper provides a sensitive analytical method using HPLC‐ECD to quantify GSH in erythrocytes, validated using the ICH guidelines for Bioanalytical Method Validation. The sample preparation was optimised using centrifugal filtration and a hypotonic phosphate buffer for extracting GSH from erythrocytes. HPLC‐ECD parameters were adjusted to allow a fast, reversed phase, isocratic separation in 10 min. The detector response was linear between 0.3 and 9.5 μg/mL with a satisfactory regression coefficient and a LOQ of 0.11 μg/mL. Intra‐ and inter‐day repeatability ranged between 1.10% and 8.57% with recoveries ranging from 94.3% to 106.0%. Dilution integrity, benchtop, freeze–thaw and long‐term stability were investigated. Samples were stable for up to 6 months at −80°C. This method has a good linear response and is repeatable, precise and accurate. It minimises GSH auto‐oxidation using a centrifugal filter during sample preparation, instead of acidification. Therefore, this analytical method is suitable for quantifying GSH in erythrocytes as a marker of oxidative stress.

Cardiovascular diseases (CVDs), including coronary heart disease, cerebrovascular disease, and atherosclerosis, remain the leading cause of global mortality, accounting for nearly one-third of all deaths. Despite advancements in prevention strategies, the prevalence of CVDs has risen over the past decade, highlighting the urgent need for novel interventions. Among modifiable risk factors, diet plays a pivotal role in cardiovascular health. The Mediterranean diet, rich in polyphenol-containing foods such as olives, has been extensively associated with reduced CVD risk. In this thesis, the potential cardiovascular benefits of olive polyphenols are explored through a multidisciplinary approach. The polyphenolic profiles of olive leaf and fruit extracts were characterised using an optimised UPLC-ESI-QToF-MS method. Olive leaf extract exhibited a broader spectrum of polyphenols, including oleuropein, hydroxytyrosol, verbascoside, flavonoids, and phenolic acids, compared to the fruit extract. The reduced diversity in the fruit extract is likely due to polyphenol degradation during maturation. Biotransformation of oleuropein and olive leaf extract was studied using an in vitro gastrointestinal model simulating the stomach, small intestine, and colon phases, incorporating faecal samples from healthy young (20-30 years old) and elderly (≥ 65 years old) individuals. Despite age-related differences in gut microbiota composition, similar biotransformation reactions, primarily deglycosylation, demethylation, and hydrolysis, were observed, with hydroxytyrosol and 3-(4-hydroxyphenyl) propionic acid as key metabolites. Hydroxytyrosol was further evaluated for its vascular effects. In vitro and ex vivo studies demonstrated its ability to enhance antioxidant enzyme expression and autophagic activity in endothelial cells, and to reduce aortic stiffness in the Fbn1C1039G+/– mouse model. A double-blind, placebo-controlled, randomised clinical trial assessed the effects of a commercial olive extract on blood pressure and cardiovascular biomarkers in 56 participants with elevated systolic blood pressure (≥130 mmHg). A significant decrease in systolic blood pressure was observed between the start and end of the trial in the intervention and placebo groups. However, the reported change was comparable with blood pressure reductions in placebo arms of hypertension trials, resulting in a non-significant difference between the groups. Both groups showed reductions in cholesterol markers, though no changes in oxidative stress biomarkers were detected. In conclusion, olive polyphenols hold the potential to aid in CVD disease prevention. Further research into the bioavailability of olive polyphenols after biotransformation by the gut microbiome and investigations into liver biotransformation are necessary to clarify which metabolites are responsible for the reported beneficial effects and to expand the mechanistic insights into the cardiovascular health benefits of olive polyphenols.

Despite advances in reducing cardiovascular disease (CVD) incidence, CVD mortality has increased, emphasising the need for new preventive strategies. Polyphenol-rich olive extracts have been proposed to lower blood pressure by reducing oxidative stress and enhancing nitric oxide production. This double-blind, placebo-controlled, randomised study aims to examine whether a standardised olive extract can lead to a reduction in blood pressure, lipid levels, and biological markers of oxidative stress after supplementation in the context of primary CVD prevention. In this trial, 56 participants with a systolic blood pressure ≥ 130 mmHg received capsules containing 440 mg olive dry extract (123.5 ± 9.4 mg oleuropein, 25.0 ± 3.8 mg hydroxytyrosol) or placebo on a daily basis over eight weeks. Both groups showed significant reductions in systolic blood pressure with a larger decrease in the intervention group as compared to the control group (intervention: −8.3 ± 2.2 mmHg, control: −7.3 ± 2.2 mmHg). However, this decrease was not found significantly different between the two groups. Moreover, total cholesterol, LDL, and apo B levels decreased in both groups, but with no significant difference between them. These results indicate that the olive extract was well-tolerated, although no significant hypotensive benefit was observed, with effects paralleling typical placebo responses in hypertension trials. Literature reports conflicting results on the hypotensive effect of olive polyphenols, indicating the need for further research with well-designed clinical trials. This trial was registered on Clinicaltrials.gov in April 2021, ID NCT04874961.