Obesity remains a major global health challenge, and pancreatic lipase (PL) inhibition represents a key strategy to reduce dietary fat absorption. This study evaluated the individual and combined inhibitory effects of three important olive phenolic compounds—hydroxytyrosol (HT), 3,4-dihydroxyphenylglycol (DHPG), and comselogoside (COM)—on PL activity in vitro. All three compounds showed concentration-dependent inhibition, with HT being the most potent (IC₅₀ = 0.294 mM), followed by DHPG (IC₅₀ = 0.464 mM) and COM (IC₅₀ = 0.858 mM). Kinetic analyses revealed competitive inhibition for HT and non-competitive inhibition for DHPG.Binary mixtures of HT:DHPG and HT:COM were tested at seven molar ratios (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10). Both combinations showed clear dose-dependent inhibition and marked synergistic effects across most molar ratios and inhibition levels (50–97%), as demonstrated by combination index (CI < 1) and isobologram analysis. The strongest synergism occurred at the 1:1 ratio, with CI values between 0.09 and 0.30 and high dose-reduction index (DRI) values (5–83-fold), indicating enhanced inhibitory potency at lower concentrations. Kinetic analyses confirmed that the HT:DHPG mixture followed a non-competitive pattern, whereas HT:COM maintained a competitive mechanism.These findings demonstrate that olive phenolics can act cooperatively to enhance PL inhibition, highlighting their potential as natural and sustainable agents for the development of functional foods and nutraceuticals aimed at lipid digestion and obesity management.

The olive industry is experiencing significant growth, held back only by the environmental and management issues that its by-products can cause. The two most polluting effluents produced are a new aqueous phase or ‘alpechín’ in the olive oil industry, and brine in the table olive ones. The high organic and phytotoxic load of both, together with the conductivity of the brines, makes their reuse difficult and poses a real technological challenge due to the environmental problems they are causing. This study demonstrates that the application of thermo-malaxation followed by three-phase centrifugation allows the removal of most of the most active phenolics, including hydroxytyrosol, 3,4-dihydroxyphenylglycol and tyrosol—from the alperujo in a 30%, yielding a partially detoxified solid phase and a liquid phase rich in the most active phenolics. Furthermore, the application of an industrial chromatographic process enables the removal of between 50% and 75% of the phenolic load from the liquid fraction of the aforementioned system, as well as from the brines of the table olive industry. The high antioxidant activity of the extracted phenolics has also been verified through in vitro assays.

Olive leaves (Olea europaea L.), an abundant by-product of olive cultivation, are a rich source of secoiridoids and other phenolic compounds with recognised bioactivity. In this study, aqueous infusions prepared from leaves of five Spanish cultivars at 70 °C and 90 °C were characterised in terms of phenolic profile and in vitro activities related to metabolic health. HPLC-DAD-TOF-MS analysis revealed a predominance of secoiridoids—particularly oleuropein and elenolic acid–derived glucosides—together with simple phenols, flavonoid glycosides and highly polar constituents. Extraction efficiency varied with cultivar and temperature, with 90 °C generally enhancing total phenolic diffusion, although individual compounds exhibited distinct temperature-dependent behaviours.All infusions exhibited antioxidant activity in DPPH, ABTS and FRAP assays, together with inhibitory effects against pancreatic lipase, α-glucosidase and angiotensin-converting enzyme (ACE) under the experimental conditions tested. Strong correlations between phenolic composition and antioxidant activity were observed for infusions prepared at 70 °C, whereas enzyme inhibitory responses showed a more complex behaviour not directly explained by total phenolic content alone. Infusions from Hojiblanca, Picual and Manzanilla prepared at 70 °C exhibited the strongest pancreatic lipase inhibitory activity among the samples evaluated.Overall, the results demonstrate that olive leaf infusions retain a chemically diverse mixture of secoiridoids and related phenolic compounds with measurable in vitro bioactivities. These findings contribute to the characterisation of olive leaf infusions as plant-based beverages and provide a basis for further studies addressing bioaccessibility, bioavailability and physiological relevance under in vivo conditions.

Olive leaves (Olea europaea L.) are an abundant by-product of olive cultivation and a rich source of phenolic compounds such as secoiridoids, flavonoids, and simple phenolics, which are associated with antioxidant, anti-inflammatory, and cardioprotective properties. This study analyzed the phenolic composition of leaves from 21 olive cultivars using two methods: a conventional ethanol–water extraction and autoclave-assisted extraction of dry leaves. The extraction method significantly affected both the total phenolic yield and the profile of individual compounds. Ethanol–water extraction was more effective for recovering flavonoid glycosides such as luteolin glucoside and apigenin derivatives, while autoclave-assisted extraction promoted the release oleuropein glucoside, hydroxytyrosol derivatives, and 3,4-dihydroxyphenylglycol. In some cultivars, including Picual, Hojiblanca, and Arbequina, dry leaves yielded higher phenolic content than fresh leaves. Antioxidant activity followed similar trends, highlighting the importance of cultivar selection and extraction conditions to optimize phenolic recovery from olive leaves for food, nutraceutical, and pharmaceutical applications.

Olive leaves, a significant byproduct of Olea europaea cultivation, are a rich source of phenolic compounds. Among these, 3,4-dihydroxyphenylglycol (DHPG) stands out for its potent antioxidant capacity, reportedly surpassing hydroxytyrosol. Despite its potential, DHPG has been consistently elusive in conventional olive leaf extracts, often going undetected or appearing in negligible amounts in literature, which predominantly relies on organic solvent methods. This study investigates the efficient recovery of DHPG from 21 diverse olive cultivars, comparing a thermal extraction method against traditional ethanolic extraction. Our results unequivocally demonstrate that thermal treatment (120 °C, 30 min in autoclave) vastly superiorly enhances DHPG yields across all cultivars, confirming its crucial role in DHPG release. Mass spectrometry analysis indicates that this process mediates the hydrolysis and conversion of DHPG precursors. This robust and sustainable thermal strategy unlocks the full potential of olive leaf biomass, providing a high-value source of DHPG for nutraceutical, pharmaceutical, and functional food applications, thereby addressing a critical gap in olive leaf valorization.

This study introduces an innovative biorefinery process aimed at maximizing the utilization of olive leaves. The proposed approach seeks to extract valuable phenolic compounds, lignocellulosic material, and biomethane from olive leaves. To achieve this, a combined technique involving mechanochemical extraction and sustainable solvents, specifically natural deep eutectic solvents (NADES), was employed. Unlike conventional methods, NADES were simultaneously formed and utilized for extraction within a ball mill. The optimal conditions for maximum extraction of high-value compounds were 0.5 g of OL using 10 mL of ChCl:Gl (3:1) and 1.1 h in a ball mill. The recovered yields were 14.5 f 2.9 g/kg OL of lignocellulosic fraction and 1.3 f 0.3 g gallic acid eq./kg OL of phenolic compounds. OL waste generated after the optimized extraction process was evaluated for methane production, obtaining 142 f 53 mL CH4/g VS OL waste compared to 126 f 30 mL CH4/g VS untreated OL. Therefore, NADES represent promising solvents for the biorefinery process and reduce dependence on organic solvents.

BACKGROUND:The inhibition of carbohydrate-digesting enzymes, α-glucosidase and α-amylase, is a key strategy in managing postprandial hyperglycemia in type 2 diabetes. Natural phenolic compounds isolated from olive mill by-product (alperujo), such as hydroxytyrosol (HT) and trans-p-coumaroyl secologanoside (comselogoside, COM), have shown potential in this regard. However, the individual effect of the 3,4-dihydroxyphenylglycol (DHPG), another potent phenolic antioxidant naturally found in olive fruit with the same ortho-diphenolic structure as HT, but with an additional hydroxyl group in the β position, and their combination with HT and COM on these enzymes remain underexplored. RESULTS:The present study evaluated the inhibitory effects of HT, DHPG and COM on α-glucosidase and α-amylase. DHPG exhibited the strongest inhibition of α-glucosidase (IC50 = 288.2 μm), comparable to the anti-diabetic drug acarbose (IC50 = 281.9 μm), whereas HT and COM showed moderate activity. Binary combinations of HT and DHPG (1:1) displayed synergistic inhibition of both enzymes, significantly enhancing their efficacy. Conversely, DHPG and COM combinations resulted in antagonistic effects on α-amylase inhibition, although they showed a significant inhibitory effect on α-glucosidase (IC50 = 100.7 μm). CONCLUSION:These findings highlight the potential of phenolic compounds derived from olive oil by-products as natural alternatives for postprandial glucose control. The synergistic interactions between specific compounds suggest an enhanced therapeutic effect that could be used for diabetes management. Further studies are needed to elucidate the mechanisms underlying these interactions and assess their in vivo efficacy. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Olive mill wastewater (OMW), a byproduct of the olive oil industry, is a potential source of natural bioactive phenolic polymers. In this work, a column chromatography technique was used for the isolation of a new complex polymer (named OMW-2000XAD) from OMW via fractionation on Amberlite® XAD16 resin. The developed procedure was simple and proved to be reproducible using OMW from two different sources. OMW-2000XAD was further characterized by elemental, glycosidic, and amino acid composition analysis, as well as spectroscopic techniques. The polymer’s molecular size, which was estimated via gel filtration chromatography, was 1960 kDa, which is significantly larger than other high-molecular weight fractions previously isolated from OMW or other agro-industrial wastes. OMW-2000XAD was mainly composed of phenolic compounds (89.8%). It also contained polysaccharides (16.1%) and proteins (10.3%), with glucose (12.25%) and cysteine (1.71%) being the most abundant sugar and amino acid, respectively, as well as metals (1.29%, primarily potassium). However, due to its low solubility, complexity, and heterogeneous composition, it was not possible to identify all phenolic compounds or elucidate a definitive structure via MS, FTIR, and NMR. OMW-2000XAD exhibited strong radical scavenging antioxidant capacity (ABTS•+, DPPH• and peroxyl radicals), with results up to 7415 µmol Trolox equivalent/mol (ORAC method), but showed no antiproliferative effects, highlighting the need for further research.

BACKGROUND:Cardiovascular diseases, largely driven by hypertension, remain the leading cause of mortality worldwide. Angiotensin-I-converting enzyme (ACE) inhibition is a well-established therapeutic approach; however, synthetic inhibitors are frequently associated with adverse effects. Natural bioactive compounds, particularly those derived from olives, have been proposed as potential alternatives or complementary agents. RESULTS:This study evaluated the ACE-inhibitory potential of olive-derived triterpenic acids (oleanolic acid (OA) and maslinic acid (MA)) and phenolic compounds (hydroxytyrosol (HT), 3,4-dihydroxyphenylglycol (DHPG), verbascoside (VER), luteolin (LUT), and comselogoside (COM)). Among the individual compounds, OA (half-maximal inhibitory concentration (IC50) = 18.43 μmol L-1) and MA (IC50 = 25.66 μmol L-1) exhibited the strongest ACE inhibition, acting through different mechanisms: OA as a competitive, MA as an uncompetitive, and COM as a non-competitive inhibitor. Notably, certain binary combinations revealed notable interactions, particularly HT:COM, which displayed strong synergistic inhibition at concentrations where individual components were inactive. In contrast, combinations such as COM:MA and COM:LUT demonstrated additive or antagonistic effects depending on molar ratios and concentration. CONCLUSIONS:These findings highlight the potential of individual olive-derived compounds, especially OA and MA, as natural ACE inhibitors with distinct mechanisms of action. Moreover, specific synergistic interactions, such as HT:COM, represent promising candidates for the development of safer and more effective dietary interventions in hypertension management. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Limited data exist on the difference in polysaccharide-polyphenols conjugation using two methods: adsorption technique and free radical induction. This study investigates the structural and antioxidant properties of complexes formed between hydroxytyrosol (HT) and two commercial pectins (apple and citrus) using both approaches. The adsorption method generates non-covalent bonds, while the free radical method creates covalent bonds with the aid of ascorbic acid and hydrogen peroxide. Findings indicate that HT-pectin complexes exhibit distinct structural and antioxidant properties depending on the conjugation method. Proton nuclear magnetic resonance (1H NMR) analysis of HT-pectin conjugates showed that the free radical method induced more significant changes in the chemical environment of HT and pectin protons compared to the adsorption method, suggesting stronger interactions.The retention of HT was higher in complexes formed via adsorption, with citrus pectin showing the highest HT retention (94.7 mg/g) compared to the free radical method (60.2 mg/g). Additionally, the antioxidant activity of each complex was directly related to the proportion of HT conjugated. In vitro antioxidant assays (DPPH and reducing power), revealed that adsorption method enhanced the antioxidant capacity of free HT more effectively than the free radical approach. Simulated gastrointestinal digestion showed substantial HT release in the gastric phase and partial degradation in the intestinal phase, with 27%-35% of HT retained in the free radical method and 34.6%-48.1% in the adsorption method. These results suggest that HT bound to pectin could serve as an effective antioxidant in the colon. Conjugating HT with pectin may provide specific benefits for gut health by facilitating the development of functional foods that support a balanced intestinal microbiota.

Olive oil extraction by two-phase systems generates a by-product called “alperujo” which presents several difficulties for its valorization. The present work evaluated an industrial approach, based on the application of thermal treatments to alperujo followed by solid/liquid separation using standard two-phase olive oil mill equipment. Treatments consisted of the thermo-malaxation of alperujo at 70 °C for 45 or 90 min, with or without acid addition, followed by solid/liquid separation in an industrial decanter. The solid was characterized concerning subsequent use for composting, while total and hydrophilic phenolics were analyzed in liquid for their recovery. Additionally, a laboratory-scale trial to compare phenolic purification by ethylic acetate extraction with chromatographic procedures was also included. The static respiration index showed that solid fractions presented higher susceptibility to biodegradation processes than raw alperujo. The phenolic content of treated liquid fractions was higher than in raw alperujo. Total phenolics were maximum at the longest exposure time without acid addition, while hydrophilic phenolics were highest at the shortest exposure time in acidified samples. The use of non-ionic resins seemed attractive for obtaining highly concentrated phenolic fractions. The proposed thermal treatments can be applied in olive oil industries, allowing in situ pomace valorization and the recovery of phenolic-enriched liquid fractions.

Unlike olive orchards intended for olive oil, the production of table olives in narrow hedgerows is very recent and the information available about this cropping system is yet very scarce. In this work, we studied for the first time in table olive hedgerows (3.75m x 1.35m, N-S oriented) of Manzanilla de Sevilla and Manzanilla Caceren similar to a cultivars, the distribution within the canopy of yield components and physical-chemical fruit quality traits in response to the incident irradiance. Hedgerow canopies were divided into four positions on each side (East and West) according to height aboveground: 0.5-1.0m, 1.0-1.5m, 1.5-2.0m, 2.0-2.5m, and an additional top canopy position (> 2.5m) integrated for both sides. Total production, number of fruits and size distribution were recorded for each position and the following fruit traits were assessed: fresh weight, length, equatorial diameter, pulp-to-pit ratio, color index, bruising percentage, oil content, water content, total sugars and total phenols. Individual phenolic compounds were also quantified. The incident irradiance at each position was calculated over the fruit growth period using a model based on hedgerow parameters, resulting in a positive gradient from base to top canopy positions. For both cultivars, yield components and most quality traits were positively affected by incident irradiance as they followed a similar trend showing larger mean values with increasing canopy heights. Conversely, the total contents in sugar and phenols and individual phenolic compounds were not affected by the canopy positions. No remarkable differences between East and West sides of the hedgerow were observed.

This study explored the impact of complexing comselogoside (COM) with n-cyclodextrin (n-CD) on antioxidant capacity and investigated its in vitro inhibitory effects against a-glucosidase and angiotensin I-converting enzyme (ACE). The COM: n-CD complex in three molar ratios (1:2, 1:1, and 2:1) showed significantly higher antioxidant activity compared to free COM, assessed by DPPH and ferric reducing power assays. COM exhibited weak to moderate a-glucosidase inhibition (IC50 1221 mu M) and notable ACE inhibition (IC50 119.4 mu M). Encapsulation improved ACE inhibition notably for the 1:2 and 2:1 M ratios. The cleavage of secoiridoid moiety of COM by n-glucosidase further enhanced ACE inhibition from IC50 of 63.91 to 41.75 mu g/mL in the hydrolysed mixture. In vitro gastrointestinal digestion revealed 34-40% bioaccessibility of COM and its n-CD complex. This study demonstrates the potential of encapsulated COM as a functional food or supplement for preventing and treating diabetes, hypertension, and oxidative stress-related diseases.

This research explores the health-promoting properties of the pectin–polyphenol complex extracted from alperujo, a by-product of olive oil production. This study investigates the chemical composition and antioxidant activity of the extracts, revealing their high antioxidant activity in vitro. Cell viability assays conducted on colon carcinoma cells (Caco-2) demonstrate the inhibitory effect of the extracts on cell proliferation. However, the extracts do not affect the viability of differentiated Caco-2 cells, suggesting a selective antiproliferative action. Additionally, the extracts reduce intracellular reactive oxygen species (ROS) and nitrite (NO) production in LPS-stimulated murine peritoneal macrophages. Furthermore, the extracts exhibit anti-inflammatory effects by downregulating the secretion of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in these macrophages. These findings highlight the potential of pectin–polyphenol complexes as functional ingredients with significant health benefits, demonstrating antioxidant, antiproliferative, and anti-inflammatory properties.

The extensive production of olive mill solid waste (OMSW) from olive oil industry in the Mediterranean basin claims effective treatments and valorization strategies. This study aims to elucidate the potential of anaerobic digestion (AD) and anaerobic fermentation (AF) to convert pre-treated OMSW into biogas (CH4) and volatile fatty acids (VFA), respectively. The two thermal treatment conditions (65 °C and 180 °C) that are being implemented in the industry that manages the OMSW were tested. Comparing the two treatments aims to demonstrate the influence on the AD process of the degree of solubilization and degradation of the metabolites produced from the same substrate. AD of OMSW treated at low-temperature (65 °C) exhibited similar methane yields (195 ± 8 mL CH4/g volatile solid (VS)) to raw OMSW. AD of the solid phase (SP) after high-temperature treatment with acid addition at 180 °C resulted in methane yields comparable to raw OMSW while the liquid phase (LP) exhibited low methane yields (85 ± 10 mL CH4/g VS). Nevertheless, LP/180 °C exhibited the highest VFA bioconversion at 27.6 %, compared to less than 10 % for SP/180 ºC, SP/65 °C, and raw OMSW. The VFA profile showed notable variations with thermal treatment temperatures. Propionic acid dominated at SP/65 °C, while acetic acid became the primary VFA at 180 °C. Furthermore, significant degradation rates of phenolic compounds and furans were observed during the final day of both anaerobic processes. Overall, these findings suggest that AD is more suitable for raw OMSW, treated at low temperature and SP at high temperature, while AF offers a promising alternative for high-temperature-treated LP.

p-Coumaroyl-6'-secologanoside (comselogoside) is a secoiridoid identified in large amounts in olive fruits, although no studies in vitro or in vivo of comselogoside have been reported. This work focuses on the recovery and purification of this compound from olive mill waste (alperujo). The successive isolation on Amberlite XAD-16 and Sephadex LH-20 resins, allowed a comselogoside extract with 80-85% of purity. A photoisomerization of the vinyl-double bond in the p-coumaroyl moiety occurred when the extract was exposed to ultraviolet radiation and a mixture of the trans and cis-isomers was obtained. Both isomers were characterized using NMR, mass spectroscopy, and UV spectrometry. The J (coupling constant) of the protons on the C7 and C8 on the unsaturated chain were found to be the difference between cis (12.8 Hz) and trans- (15.9 Hz) comselogoside. Cis-isomer exhibited lower radical-scavenging activity than trans, although a synergistic effect occurred when the cis-isomer was supplement by the trans-isomer.

Comselogoside (COM) is a secoiridoid, derivative of p-coumaric acid with a wide range of potential beneficial properties, although its poor aqueous solubility and low stability limit its study and application. The formation of an inclusion complex with β-cyclodextrin (β-CD) could improve the solubility and photostability of COM. The complexation of COM with β-CD in an aqueous solution has been studied for the first time, using the freeze-drying method with high encapsulation yield and efficiency. The formation of the inclusion complex was confirmed by electrospray ionization (ESI) mass and 1H NMR spectroscopy. Phase solubility studies provided an AN-type diagram with an increase in aqueous solubility by 100% and the formation of a high stoichiometric order complex COM: β-CD. The 1H NMR analysis with different COM/ β-CD molar ratio established the formation of the 2:1 stoichiometric complex by Job ҆ s plot. In addition, the complexation of COM with β-CD improved the stability of COM against irradiation with UV light at 254 nm. Finally, combined docking and DFT calculations have been performed to study the formation and interactions of the COM: β-CD complexes. The computational results show excellent agreement with experimental observations.

This research evaluates process instability together with microbial population dynamics of the startup of an anaerobic digestion of a mild pretreated solid olive oil waste. The pretreatment consisted of a mild thermal treatment called thermo-malaxation and a subsequent dephenolized process of the olive mill solid waste. The anaerobic digestion process of the mild pretreated and partially dephenolized biomass was studied for three Hydraulic Retention Times (HRTs), with 21 days each HRT, with an organic load rate of 1 g VS/L d, carried out at mesophilic temperature (35 ± 1 °C). The average value of methane yield decreased from 204 ± 9 mL CH4/g VS d on day 21, the last day of the first HRT, to 87 ± 24 mL CH4/g VS d on day 60, the last day of the third HRT. The alkalinity decreased drastically, indicating instability of the anaerobic digestion process. Although phenolic compounds were partially extracted in the pretreatment, the observed increase in phenolic compounds during reactor operation might be contributed to the methane production decay. Interestingly, volatile fatty acids decreased with time, indicating that not only the methanogenic stage but also the hydrolysis stage was affected. Indeed, the microbial analysis showed that the abundance of hydrolytic bacteria decreased over time. It is also worth noticing that hydrogenotrophic methanogens, while present during the first two HRTs, were not observed at the end of the last HRT. This observation, together with the increase in the relative abundance of acetoclastic methanogens, showed a shift in the methane production pathway from hydrogenotrophic methanogenesis to acetotrophic methanogenesis.

The main by-product from olive oil extraction (alperujo) was extracted with hot water, citric acid, natural deep eutectic solvent (choline chloride: citric acid), and only choline chloride. The purified extracts were composed of macromolecular complexes constituting polyphenols associated with pectin. The extracts were structurally characterized by FT-IR and solid-NMR spectroscopy and an in vitro test revealed distinct antioxidant and antiproliferative activity, depending on the extracting agents. The choline chloride-extracted complex contained the highest amount of polyphenols among the examined agents, which exhibited a strong antioxidant activity and significant antiproliferative capacity. However, the complex extracted by hot water showed the highest antiproliferative capacity in vitro against the colon carcinoma Caco-2 cell line. In this finding, choline chloride could be used as a novel, green and promising alternative to the conventional extracting agent for the production of complexes that combine the antioxidant activity of phenolic compounds and the physiological effects of pectic polysaccharides.