Ricardo Lucas

Surface-active properties of lipophilic antioxidants tyrosol and hydroxytyrosol fatty acid esters: a potential explanation for the nonlinear hypothesis of the antioxidant activity in oil-in-water emulsions.

Our group has recently observed a nonlinear tendency in antioxidant capacity of different hydroxytyrosol fatty acid esters in fish oil-in-water emulsions, where a maximum of antioxidant efficiency appeared for hydroxytyrosol octanoate. These results appear to disagree with the antioxidant polar paradox. Because the physical location of the antioxidants in an oil-water interface has been postulated as an important factor in explaining this behavior, we have prepared a series of tyrosol and hydroxytyrosol fatty acid esters with different chain length and studied their surface-active properties in water, because these physicochemical parameters could be directly related to the preferential placement at the interface. We have found that tyrosol and hydroxytyrosol fatty acid esters are relevant surfactants when the right hydrophilic-lipophilic balance (HLB) is attained and, in some cases, as efficient as emulsifiers commonly used in industry, such as Brij 30 or Tween 20. Moreover, a nonlinear dependency of surfactant effectiveness is observed with the increase in chain length of the lipophilic antioxidants. This tendency seems to fit quite well with the reported antioxidant activity in emulsions, and the best antioxidant of the series (hydroxytyrosol octanoate) is also a very effective surfactant. This potential explanation of the nonlinear hypothesis will help in the rational design of antioxidants used in oil-in-water emulsions.

Effect of Lipophilization of Hydroxytyrosol on Its Antioxidant Activity in Fish Oils and Fish Oil-in-Water Emulsions

The effect of lipophilization of the antioxidant efficiency of hydroxytyrosol on fish oil enriched systems was studied. Hydroxytyrosol fatty acid esters with increasing size of the alkyl chain and different lipophilicity were tested in bulk fish oils and fish oil-in-water emulsions. Results showed a significant antioxidant activity of hydroxytyrosol esters in both systems especially in emulsions. The introduction of a lipophilic chain decreased the antioxidant effectiveness of hydroxytyrosol in homogeneous systems as fish oils. In emulsion systems, the presence of a short-medium lipophilic chain (acetate, butyrate or octanoate) improved the antioxidant efficiency of hydroxytyrosol favoring the physical location of the antioxidant in the interface, but longer alkyl chain (laurate) maintained or even decreased their antioxidant activity. A maximum of antioxidant efficiency seems to appear when the chain length of the hydroxytyrosol derivative is that of eight carbons which is probably associated with a preferential location of the diorthophenolic moiety in the right geometry. These results are of high importance for the optimum design of effective antioxidants for omega 3 enriched foods, which are very susceptible to suffer oxidation and, then, rancidity.

Metabolites and tissue distribution of resveratrol in the pig

SCOPE trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. METHODS AND RESULTS Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. CONCLUSION This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.

Highly Polar Carbohydrates Stack onto DNA Duplexes via CH/π Interactions

Carbohydrate-nucleic acid contacts are known to be a fundamental part of some drug-DNA recognition processes. Most of these interactions occur through the minor groove of DNA, such as in the calicheamicin or anthracycline families, or through both minor and major groove binders such as in the pluramycins. Here, we demonstrate that carbohydrate-DNA interactions are also possible through sugar capping of a DNA double helix. Highly polar mono- and disaccharides are capable of CH/π stacking onto the terminal DNA base pair of a duplex as shown by NMR spectroscopy. The energetics of the carbohydrate-DNA interactions vary depending on the stereochemistry, polarity, and contact surface of the sugar involved and also on the terminal base pair. These results reveal carbohydrate-DNA base stacking as a potential recognition motif to be used in drug design, supramolecular chemistry, or biobased nanomaterials.

A concise synthesis of glucuronide metabolites of urolithin-B, resveratrol, and hydroxytyrosol.

A simple and direct strategy to chemically synthesize O-beta-D-glucuronides of urolithin-B 4, resveratrol 5, and the corresponding hydroxytyrosol derivatives 6, 7 (as a regioisomeric mixture), and 8 is described. The critical glycosylation step has been optimized using a structurally simple phenol, urolithin-B, by modification of several reaction parameters (solvent, promoter, and glucuronide donor). Very high yields have been obtained in the first synthesis of the O-beta-D-glucuronide of urolithin-B 4. Extension of these reaction conditions was used for the synthesis of resveratrol-3-O-glucuronide 5 where a higher yield than previously reported was obtained by using the much more common trichloroacetimidate glucuronide donor. Finally, three O-beta-D-glucuronides of hydroxytyrosol 6, 7, and 8 have been synthesized for the first time using chemical synthesis.

The Gut Microbiota Ellagic Acid-Derived Metabolite Urolithin A and Its Sulfate Conjugate Are Substrates for the Drug Efflux Transporter Breast Cancer Resistance Protein (ABCG2/BCRP)

The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect the pharmacological and toxicological properties of many molecules. Urolithins, metabolites produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. This study evaluated whether urolithins (Uro-A, -B, -C, and -D) and their main phase II metabolites Uro-A sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport assays. Uro-A and, to a lesser extent, Uro-A sulfate showed a significant increase in apically directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of the tested compounds. Data were confirmed for murine, human, bovine, and ovine BCRP-transduced subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone transport in a dose-dependent manner. This study reports for the first time that Uro-A and its sulfate conjugate are ABCG2/BCRP substrates. The results suggest that physiologically relevant concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-mediated transport processes and mechanisms of cancer drug resistance. Further in vivo investigations are warranted.

Effects of long-term consumption of low doses of resveratrol on diet-induced mild hypercholesterolemia in pigs: a transcriptomic approach to disease prevention

Metabolic and cardiovascular diseases (CVDs) have risen to alarming proportions, and there is a need for therapeutic and preventive measures. The polyphenol resveratrol (RES) protects against CVDs, but in vivo molecular mechanisms responsible for protection are not yet understood. Peripheral blood mononuclear cells (PBMNCs) are involved in the development of atherosclerosis and metabolic disorders. The identification of PBMNCs genes responding to dietary compounds might help to understand the mechanisms underlying the effects of polyphenols. We determined gene expression differences between PBMNCs from pigs fed a high-fat diet manifesting a mild increase of cholesterol and pigs fed a high-fat diet containing low doses of RES. Although the consumption of RES did not modify the levels of cholesterol, microarray analyses indicated that some of the differentially expressed genes, collagens (COL1A, COL3A), lipoprotein lipase (LPL) and fatty-acid binding proteins (FABPs) involved in CVDs and lipid metabolism were up-regulated by the high-fat diet and down-regulated by RES. Reverse transcriptase polymerase chain reaction confirmed that RES and RES-containing grape extract prevented the induction of FABP4 in PBMNCs in female pigs fed a high-fat diet. Low micromolar concentrations of RES and its metabolite dihydroresveratrol exerted a minor but significant reducing effect on the induction of FABP4 expression in human macrophages treated with oxidized low-density lipoprotein. Our results show that the consumption of low doses of RES modulates the expression of genes related to lipid metabolism and metabolic disorders that are affected by a high-fat diet and suggest that some of the circulating RES metabolites may contribute to these effects.

Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations

Oligoribonucleotide conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two, or four glucose and galactose residues at the 5′-end have been prepared using phosphoramidite chemistry. Carbohydrate-modified siRNA duplexes have similar inhibitory properties than unmodified RNA duplexes in HeLa cells transfected with oligofectamine. When HeLa cells were treated with siRNA carrying one, two, or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues, although the efficacy of the process is not enough for efficient RNA interference experiments.

Effects of sugar functional groups, hydrophobicity, and fluorination on carbohydrate-DNA stacking interactions in water.

Carbohydrate-aromatic interactions are highly relevant for many biological processes. Nevertheless, experimental data in aqueous solution relating structure and energetics for sugar-arene stacking interactions are very scarce. Here, we evaluate how structural variations in a monosaccharide including carboxyl, N-acetyl, fluorine, and methyl groups affect stacking interactions with aromatic DNA bases. We find small differences on stacking interaction among the natural carbohydrates examined. The presence of fluorine atoms within the pyranose ring slightly increases the interaction with the C-G DNA base pair. Carbohydrate hydrophobicity is the most determinant factor. However, gradual increase in hydrophobicity of the carbohydrate does not translate directly into a steady growth in stacking interaction. The energetics correlates better with the amount of apolar surface buried upon sugar stacking on top of the aromatic DNA base pair.

Apolar carbohydrates as DNA capping agents

Mono- and disaccharides have been shown to stack on top of DNA duplexes stabilizing sequences with terminal C-G base pairs. Here we present an apolar version of glucose and cellobiose as new capping agents that stack on DNA increasing considerably its stability with respect to their natural polyhydroxylated mono- and disaccharide DNA conjugates.