Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L-1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.

Two New Unspecific Peroxygenases from Heterologous Expression of Fungal Genes in Escherichia coli.

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.

Correction: A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

[This corrects the article DOI: 10.1371/journal.pone.0166561.].

Intramolecular cyclization of alkoxyaminosugars: access to novel glycosidase inhibitor families.

We report the synthesis of two novel families of iminosugars as glycosidase inhibitors involving an intramolecular cyclization between an N-alkoxyamino group and a latent aldehyde of a reducing sugar as the key step. Using this methodology we have prepared the hitherto unknown bicyclic polyhydroxylated N-(methoxy, benzyloxy)anhydroazepanes and N-benzyloxy-d-xylonojirimycin; all these novel compounds turned out to be moderate ß-glucosidase inhibitors in a pH-dependent manner.

Engineering Collariella virescens Peroxygenase for Epoxides Production from Vegetable Oil.

Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α−linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate.

Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements.

Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 Å, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two α-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides-reactive compounds with valuable cross-linking capabilities-due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.

Advances in enzymatic oxyfunctionalization of aliphatic compounds.

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as "dream biocatalysts" of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases.

Regioselective and Stereoselective Epoxidation of n-3 and n-6 Fatty Acids by Fungal Peroxygenases.

Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by 1H, 13C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

Dietary Oleocanthal Supplementation Prevents Inflammation and Oxidative Stress in Collagen-Induced Arthritis in Mice.

Oleocanthal (OLE), a characteristic and exclusive secoiridoid of Oleoaceae family, is mainly found in extra virgin olive oil (EVOO). Previous studies have reported its antioxidant, anti-inflammatory, antimicrobial, anticancer and neuroprotective effects. Since the pathogenesis of rheumatoid arthritis (RA) involves inflammatory and oxidative components, this study was designed to evaluate the preventive role of dietary OLE-supplemented effects in collagen-induced arthritis (CIA) murine model. Animals were fed with a preventive OLE-enriched dietary during 6 weeks previous to CIA induction and until the end of experiment time. At day 43 after first immunization, mice were sacrificed: blood was recollected and paws were histological and biochemically processed. Dietary OLE prevented bone, joint and cartilage rheumatic affections induced by collagen. Levels of circulatory matrix metalloproteinase (MMP)-3 and pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α, IL-17, IFN-γ) were significantly decreased in secoiridoid fed animals. Besides, dietary OLE was able to diminish COX-2, mPGES-1 and iNOS protein expressions and, also, PGE2 levels. The mechanisms underlying these protective effects could be related to Nrf-2/HO-1 axis activation and the inhibition of relevant signaling pathways including JAK-STAT, MAPKs and NF-κB, thus controlling the production of inflammatory and oxidative mediators. Overall, our results exhibit preliminary evidences about OLE, as a novel dietary tool for the prevention of autoimmune and inflammatory disorders, such as RA.

A New Peracetylated Oleuropein Derivative Ameliorates Joint Inflammation and Destruction in a Murine Collagen-Induced Arthritis Model via Activation of the Nrf-2/Ho-1 Antioxidant Pathway and Suppression of MAPKs and NF-κB Activation.

: Oleuropein (OL), an olive tree secoiridoid and its peracetylated derivate (Per-OL) have exhibited several beneficial effects on LPS-stimulated macrophages and murine experimental systemic lupus erythematosus (SLE). This study was designed to evaluate dietary Per-OL in comparison with OL supplementation effects on collagen-induced arthritis (CIA) murine model. Three-weeks-old DBA-1/J male mice were fed from weaning with a standard commercial diet or experimental enriched-diets in 0.05 % (w/w) OL, 0.05% and 0.025% Per-OL. After six weeks of pre-treatment, arthritis was induced by bovine collagen type II by tail base injection (day 0) and on day 21, mice received a booster injection. Mice were sacrificed 42 days after the first immunization. Both Per-OL and OL diets significantly prevented histological damage and arthritic score development, although no statistically significant differences were observed between both compounds. Also, serum collagen oligomeric matrix protein (COMP), metalloprotease (MMP)-3 and pro-inflammatory cytokines levels were ameliorated in paws from secoiridoids fed animals. Mitogen-activated protein kinases (MAPK)s and nuclear transcription factor-kappa-B (NF-κB) activations were drastically down-regulated whereas nuclear factor E2-related factor 2 (Nrf2) and heme-oxygenase-1 (HO-1) protein expressions were up-regulated in those mice fed with OL and Per-OL diets. We conclude that both Per-OL and its parent compound, OL, supplements might provide a basis for developing a new dietary strategy for the prevention of rheumatoid arthritis.

Encased Gold Nanoparticle Synthesis as a Probe for Oleuropein Self-Assembled Structure Formation.

Stable oleuropein-coated gold nanoparticles in aqueous media were synthesized for the first time. Oleuropein (OLE) concentration in the reaction medium was found to greatly influence the outcome and stability of the resulting nanocolloid, with a marked decrease in particle size being found for the more concentrated oleuropein solutions. The protection mechanisms involved in the stabilized nanosystems were analyzed. Oleuropein self-assembled structures were found to be formed at a concentration threshold of [OLE] > 5 × 10-5 M, and observed through the use of CryoSEM imaging. Those structures were responsible for both the increased stability and the decrease in size observed at the more concentrated solutions.

High Epoxidation Yields of Vegetable Oil Hydrolyzates and Methyl Esters by Selected Fungal Peroxygenases.

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While CglUPO selectively produced "pure" epoxides (monoepoxides and/or diepoxides), MroUPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rHinUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of α-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.

Osteoarthritis treatment with a novel nutraceutical acetylated ligstroside aglycone, a chemically modified extra-virgin olive oil polyphenol.

Recent studies have shown that dietary patterns confer protection from certain chronic diseases related to oxidative stress, the immune system and chronic low-grade inflammatory diseases. The aim of this study was to evaluate the anti-inflammatory potential and the capacity to attenuate cartilage degradation using extra-virgin olive oil-derived polyphenols for the treatment of osteoarthritis. Results show that both nutraceuticals ligstroside aglycone and acetylated ligstroside aglycone showed an anti-inflammatory profile. Acetylated ligstroside aglycone significantly reduced the expression of pro-inflammatory genes including NOS2 and MMP13 at both RNA and protein levels; decreased nitric oxide release; and, importantly, reduced proteoglycan loss in human osteoarthritis cartilage explants. Our study demonstrated that a new synthetic acetylated ligstroside aglycone derivative offers enhanced anti-inflammatory profile than the natural nutraceutical compound in osteoarthritis. These results substantiate the role of nutraceuticals in osteoarthritis with implications for therapeutic intervention and our understanding of osteoarthritis pathophysiology.

Oleocanthal Modulates LPS-Induced Murine Peritoneal Macrophages Activation via Regulation of Inflammasome, Nrf-2/HO-1, and MAPKs Signaling Pathways.

The present study was designed to investigate the role of the canonical and noncanonical inflammasome, MAPKs and NRF-2/HO-1, signaling pathways involved in the antioxidant and anti-inflammatory activities of oleocanthal in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. Isolated cells were treated with oleocanthal in the presence or absence of LPS (5 µg mL-1) for 18 h. Oleocanthal showed a potent reduction of reactive oxygen species (ROS) (25 µM, 50. 612 ± 0.02; 50 µM, 53. 665 ± 0.09; 100 µM, 52. 839 ± 0.02), nitrites (25 µM, 0.631 ± 0.07; 50 µM, 0.652 ± 0.07; 100 µM, 0.711 ± 0.08), and pro-inflammatory cytokines levels when compared with LPS-DMSO-treated control cells. In terms of enzymes protein expression, oleocanthal was able to downregulate iNOS (25 µM, 0.173 ± 0.02; 50 µM, 0.149 ± 0.01; 100 µM, 0.150 ± 0.01; p < 0.001), COX-2 (25 µM, 0.482 ± 0.08; 50 µM, 0.469 ± 0.05; 100 µM, 0.418 ± 0.06; p < 0.001), and mPGES-1 (25 µM, 0.185 ± 0.11; 50 µM, 0.218 ± 0.13; 100 µM, 0.161 ± 0.15; p < 0.001) as well as p38 (25 µM, 0.366 ± 0.11; 50 µM, 0.403 ± 0.13; 100 µM, 0.362 ± 0.15; p < 0.001), JNK (25 µM, 0.443 ± 0.11; 50 µM, 0.514 ± 0.13; 100 µM, 0.372 ± 0.15; p < 0.001), and ERK (25 µM, 0.294 ± 0.01; 50 µM, 0.323 ± 0.01; 100 µM, 0.274 ± 0.01; p < 0.001) protein phosphorylation, which was accompanied by an upregulation of Nrf-2 (25 µM, 1.57 ± 0.01; 50 µM, 1.54 ± 0.01; 100 µM, 1.63 ± 0.05; p < 0.05) and HO-1(25 µM, 2.12 ± 0,03; 50 µM, 2.24 ± 0.01; 100 µM, 1.92 ± 0.05; p < 0.01) expression in comparison with LPS-DMSO cells. Moreover, oleocanthal inhibited canonical and noncanonical inflammasome signaling pathways. Thus, oleocanthal might be a promising natural agent for future treatment of immune-inflammatory diseases.

Molecular interactions between 3,4-dihydroxyphenylglycol and pectin and antioxidant capacity of this complex in vitro.

This study explored the interaction of pectin with 3,4-dihydroxyphenylglycol (DHPG), a potent phenolic antioxidant naturally found in olive fruit, via encapsulation into pectinate beads. MALDI TOF-TOF analysis supported the formation of complexes between DHPG and pectin. A combination of covalent bonds (ester bonds) and non-covalent interactions, mostly hydrogen bonding, were suggested as the cause of DHPG-pectin complex formation. Free radical scavenging assays confirmed that DHPG maintained its antioxidant activity after complexation and after a digestion simulated in vitro with gastric and intestinal fluids. Therefore, DHPG-pectin beads could reach the large intestine and contribute to a healthy antioxidant environment.

Oleuropein down-regulated IL-1ß-induced inflammation and oxidative stress in human synovial fibroblast cell line SW982.

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory autoimmune disease mainly characterized by aggressive hyperproliferation of synovial fibroblasts (SFs). It is accompained by a massive infiltration of inflammatory immune cells inducing progressive matrix degradation, destruction of cartilage and bone erosion through the production of inflammatory mediators. Oleuropein is the most prevalent phenolic component in olive leaves, seed, pulp and peel of unripe olives and is responsible for the characteristic bitter taste of unprocessed olives. This secoiridoid possesses well-documented pharmacological properties, including antioxidant and anti-inflammatory properties, and is available as a food supplement in Mediterranean countries. However, to date, anti-arthritic effects of oleuropein on SFs have not been yet elucidated. Thus, the aim of the present study was to investigate the potential effects of oleuropein, on IL-1ß-induced production of inflammatory mediators and oxidative stress in the human synovial sarcoma cell line (SW982). In order to gain a better insight into mechanisms of action, signaling pathways were also explored. Cell viability was determined using the sulforhodamine B (SRB) assay. The expression of inflammatory cytokines IL-6, TNF-α, MMP-1 and MMP-3 was evaluated by ELISA. Moreover, changes in the protein expression of cyclooxygenase (COX)-2, microsomal prostaglandin E synthase-1 (mPGES-1) as well as mitogen-activated protein kinase (MAPKs), nuclear factor kappa B (NF-κB), and nuclear factor-erythroid 2-related and heme oxygenase-1 (HO-1) signalling pathways were analysed by western blot. Oleuropein exerted anti-inflammatory and anti-oxidant effects via down-regulation of MAPK and NF-κB signaling pathways and induction of Nrf2-linked HO-1 controlling the production of inflammatory mediators decreasing IL-6 and TNF-α cytokines, MMP-1 and MMP-3 levels and mPGES-1 and COX-2 overexpression. Thus, oleuropein might provide a basis for developing a new dietary strategy for the prevention and management of RA.

Correction: Oleuropein down-regulated IL-1ß-induced inflammation and oxidative stress in human synovial fibroblast cell line SW982.

Correction for 'Oleuropein down-regulated IL-1ß-induced inflammation and oxidative stress in human synovial fibroblast cell line SW982' by Maria Luisa Castejón, et al., Food Funct., 2017, DOI: 10.1039/c7fo00210f.

A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

The chemical synthesis of new lipophilic polyphenols with improved properties presents technical difficulties. Here we describe the selection, isolation and identification of lipolytic bacteria from food-processing industrial wastes, and their use for tailoring a new set of compounds with great interest in the food industry. These bacteria were employed to produce lipolytic supernatants, which were applied without further purification as biocatalysts in the chemoselective and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols. The chemoselectivity of polyphenols acylation/deacylation was analyzed, revealing the preference of the lipases for phenolic hydroxyl groups and phenolic esters. In addition, the alcoholysis of peracetylated 3,4-dihydroxyphenylglycol resulted in a series of lipophilic 2-alkoxy-2-(3,4-dihydroxyphenyl)ethyl acetate through an unexpected lipase-mediated etherification at the benzylic position. These new compounds are more lipophilic and retained their antioxidant properties. This approach can provide access to unprecedented derivatives of 3,4-dihydroxyphenylglycol with improved properties.

Preventive effects of dietary hydroxytyrosol acetate, an extra virgin olive oil polyphenol in murine collagen-induced arthritis.

SCOPE: Hydroxytyrosol acetate (HTy-Ac), an extra virgin olive oil (EVOO) polyphenol, has recently been reported to exhibit antioxidant and anti-inflammatory effects on LPS-stimulated macrophagesand ulcerative colitis. This study was designed to evaluate dietary HTy-Ac supplementation effects on collagen-induced arthritis (CIA) in mice. METHODS AND RESULTS: DBA-1/J mice were fed from weaning with 0.05% HTy-Ac. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrificed 42 days after first immunization. Blood was recollected and paws were histological and biochemically processed. HTy-Ac diet significantly prevent edarthritis development and decreased serum IgG1 and IgG2a, cartilage olimeric matrix protein (COMP) and metalloproteinase-3 (MMP-3) levels, as well as, pro-inflammatory cytokines levels (TNF-α, IFN-γ, IL-1ß, IL-6 and IL-17A). The activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-κB) pathways were drastically ameliorated whereas nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions were significantly up-regulated in those mice fed with HTy-Ac. CONCLUSION: HTy-Ac improved the oxidative events and returned pro-inflammatory proteins expression to basal levels probably through JAK/STAT, MAPKs and NF-κB pathways. HTy-Ac supplement might provide a basis for developing a new dietary strategy for the prevention of rheumatoid arthritis.

Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary supplements.

This work evaluated the effects of extra virgin olive oil (EVOO) phenols, hydroxytyrosyl acetate (2) and 3,4-dihydroxyphenylglycol (3), as well as two new acyl derivatives of 3, 4-(1,2-di(butanoyloxy)ethyl)benzene-1,2-diol (7) and 4-(1,2-di(lauroyloxy)ethyl)benzene-1,2-diol (8), on LPS-stimulated murine peritoneal macrophages in comparison with hydroxytyrosol (HTy, 1). Compounds 2, 3, 7, and 8 showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease on iNOS expression [2 (50 µM, 0.44 ± 0.03; 100 µM, 0.44 ± 0.01; p < 0.01); 3 (50 µM, 0.37 ± 0.03; 100 µM, 0.37 ± 0.01; p < 0.001); 7 (50 µM, 0.45 ± 0.06; p < 0.01)] . However, only 2 and 3 down-regulated COX-2 expression [2 (50 µM, 0.72 ± 0.04, p < 0.05; 100 µM, 0.54 ± 0.06, p < 0.01); 3 (50 µM, 0.56 ± 0.05, p < 0.05; 100 µM, 0.37 ± 0.04; p < 0.001)] and prevented IKBα degradation [2 (100 µM, 1.63 ± 0.14, p < 0.01); 3 (100 µM, 1.82 ± 0.09; p < 0.01)] ; the diacylated compounds 7 and 8 showed worse anti-inflammatory activity than the parent 3. In conclusion, 2 and 3 phenolic derivatives could play an important role in the anti-inflammatory effect of EVOO. The implication of this study for the nutrition and general health of the population rests in the possible use of natural HTy derivatives with better hydrophilic/lipophilic balance, thus improving its pharmacodynamic and pharmacokinetic profiles, as new dietary supplements in foods.