Raquel Mateos

Protection of Human HepG2 Cells against Oxidative Stress by Cocoa Phenolic Extract

Cocoa is a rich source of flavanols and procyanidin oligomers with antioxidative properties, providing protection against oxidation and nitration. The present study investigated the potential protective effect of a polyphenolic extract from cocoa on cell viability and antioxidant defenses of cultured human HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Pretreatment of cells with 0.05-50 microg/mL of cocoa polyphenolic extract (CPE) for 2 or 20 h completely prevented cell damage and enhanced activity of antioxidant enzymes induced by a treatment with t-BOOH. Moreover, lower levels of GSH caused by t-BOOH in HepG2 cells were partly recovered by a pretreatment with CPE. Increased reactive oxygen species (ROS) induced by t-BOOH was dose-dependently prevented when cells were pretreated for 2 or 20 h with CPE. These results show that treatment of HepG2 in culture with CPE (within the physiological range of concentrations) confers a significant protection against oxidation to the cells.

New Lipophilic Tyrosyl Esters. Comparative Antioxidant Evaluation with Hydroxytyrosyl Esters

New lipophilic esters of tyrosol, a naturally occurring phenol with interesting biological properties, have been synthesized in good yields by a chemoselective procedure, using lipase from Candida antarctica or p-toluenesulfonic acid as catalysts. Their antioxidant activities have been evaluated by the Rancimat test in lipophilic food matrices, as well as by FRAP and ABTS assays in methanolic solutions, and compared with those of previously synthesized hydroxytyrosyl esters. Free tyrosol, hydroxytyrosol, butylhydroxytoluene, and alpha-tocopherol were used as standards. All methods used for the antioxidant activity evaluation emphasized the high influence of the ortho-diphenolic structure on the antioxidant capacity, tyrosol and its derivatives being less active than hydroxytyrosol and its analogues and even less than BHT and alpha-tocopherol. In addition, the Rancimat test revealed a lower activity for ester derivatives than for their respective reference compounds (HTy or Ty), in agreement with the polar paradox. On the other hand, FRAP and ABTS methods reported an opposite behavior between the synthetic esters and their respective references. Thus, hydroxytyrosyl esters were more active than HTy, whereas tyrosyl esters were less active than Ty. The length and nature of the acyl side chain did not seem to play an important role in the antioxidant activity of either the hydroxytyrosyl or tyrosyl ester series, since no significant differences were observed among them.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples

Abstract Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F2-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Regular consumption of a cocoa product improves the cardiometabolic profile in healthy and moderately hypercholesterolaemic adults

Cocoa products present great health potential due to their high content of polyphenols, mainly of flavanols. However, the antioxidant, anti-inflammatory and other health effects of regularly consuming cocoa products seem to depend on the intake and health status of the consumer, etc. and need to be further clarified. A randomised, controlled, cross-over, free-living study was carried out in healthy (n 24) and moderately hypercholesterolaemic (>2000 mg/l, n 20) subjects to assess the influence of regularly consuming (4 weeks) two servings (15 g each) of a cocoa product rich in fibre (containing 33·9 % of total dietary fibre (TDF) and 13·9 mg/g of soluble polyphenols) in milk v. consuming only milk (control) on (1) serum lipid and lipoprotein profile, (2) serum malondialdehyde levels, carbonyl groups, ferric reducing/antioxidant power, oxygen radical absorbance capacity and free radical-scavenging capacity, (3) IL-1β, IL-6, TNF-α, IL-10, IL-8, monocyte chemoattractant protein-1, and vascular and intracellular cell adhesion molecule levels, and (4) systolic and diastolic blood pressure and heart rate. Throughout the study, the diet and physical activity of the volunteers, as well as any possible changes in weight or other anthropometric parameters, were also evaluated. The intake of TDF increased (P< 0·001) to the recommended levels. Serum HDL-cholesterol (HDL-C) levels were increased (P< 0·001), whereas glucose (P= 0·029), IL-1β (P= 0·001) and IL-10 (P= 0·001) levels were decreased. The rest of the studied cardiovascular parameters, as well as the anthropometric ones, remained similar. In conclusion, regularly consuming a cocoa product with milk improves cardiovascular health by increasing HDL-C levels and inducing hypoglycaemic and anti-inflammatory effects in healthy and hypercholesterolaemic individuals without causing weight gain.

Synthesis of Hydroxytyrosyl Alkyl Ethers from Olive Oil Waste Waters

The preparation of a new type of derivatives of the naturally occurring antioxidant hydroxytyrosol is reported. Hydroxytyrosyl alkyl ethers were obtained in high yield by a three-step procedure starting from hydroxytyrosol isolated from olive oil waste waters. Preliminary results obtained by the Rancimat method have shown that these derivatives retain the high protective capacity of free hydroxytyrosol.

Effects of bioactive constituents in functional cocoa products on cardiovascular health in humans

Cocoa manufacturers are producing novel products increasing polyphenols, methylxanthines or dietary fibre to improve purported health benefits. We attempt to explain the contribution of cocoa bioactive compounds to cardiovascular effects observed in previous studies, placing particular emphasis on methylxanthines. We focused on a soluble cocoa product rich in dietary fibre (DFCP) and a product rich in polyphenols (PPCP). Effects of regularly consuming DFCP (providing daily 10.17 g, 43.8 mg and 168.6 mg of total-dietary-fibre, flavanols and methylxanthines, respectively) as well as PPCP (providing daily 3.74 g, 45.3 mg and 109.8 mg of total-dietary-fibre, flavanols and methylxanthines, respectively) on cardiovascular health were assessed in two controlled, cross-over studies in free-living normocholesterolemic and moderately hypercholesterolemic subjects. Both products increased HDL-cholesterol concentrations, whereas only DFCP decreased glucose and IL-1β levels in all subjects. Flavanols appeared to be responsible for the increase in HDL-cholesterol, whereas insoluble-dietary-fibre and theobromine in DFCP were associated with the hypoglycemic and anti-inflammatory effects observed.

Protection of human HepG2 cells against oxidative stress by the flavonoid epicatechin.

Flavanols, such as epicatechin (EC), constitute an important part of the human diet; they can be found in green tea, grapes and cocoa and possess different biological activities such as antioxidant, anti‐inflammatory and anticarcinogenic. This study investigated the potential chemo‐protective effect of EC against oxidative stress induced by tert‐butylhydroperoxide (t‐BOOH) on human HepG2 cells. Cell viability by lactate dehydrogenase assay and markers of oxidative status: reduced glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), glutathione peroxidase (GPx) and glutathione reductase (GR) were evaluated. Pretreatment of cells with EC for 20 h prevented the enhanced cell damage and GPx and GR activities as well as the decrease in GSH induced by t‐BOOH. The increased ROS generation induced by t‐BOOH was also partly prevented by a pretreatment for 20 h with EC. In addition, pretreatment of cells with EC for 20 h recovered the t‐BOOH‐induced MDA concentration to control values. A pretreatment for 2 h with EC did not reduce cell damage but partly recovered GSH, reduced ROS levels and muffled the increase of GPx and GR after exposure to t‐BOOH. Treatment of HepG2 cells with concentrations of EC in the micromolar range confers a significant protection against oxidative stress. Copyright

Biscuit melanoidins of different molecular masses protect human HepG2 cells against oxidative stress.

Soluble melanoidins from biscuits were enzymatically solubilized and isolated by sequential ultrafiltration and separated by molecular mass in three different fractions, below 3 kDa, between 3 and 10 kDa, and over 10 kDa; the latter was subsequently digested by simulating gastric plus pancreatic digestive conditions. The four fractions were investigated for their protective effect against an oxidative challenge in HepG2 cells. Pretreatment of cells for 20 h with 0.5-10 microg/mL of any of the four fractions prevented the increased cell damage evoked by the challenge but, except for the intermediate size fraction, did not suppress the increased reactive oxygen species. Antioxidant defenses were rapidly restored after the challenge, and the increase of the oxidative stress biomarker malondialdehyde was prevented by the pretreatment with all but the undigested high molecular mass fraction. The results show that treatment of HepG2 cells with concentrations of biscuit melanoidins within the expected physiological range confers on the cells a significant protection against an oxidative challenge.

Determination of esters of fatty acids with low molecular weight alcohols in olive oils.

A simple and precise analytical method was developed for the simultaneous determination of squalene and methyl, ethyl, propyl, and butyl esters of fatty acids present in olive and olive pomace oils. A fraction containing squalene and fatty acid alkyl esters was isolated from the oil by solid phase extraction on silica gel cartridges and quantitatively analyzed by gas chromatography. A modification of the procedure allowed the isolation of squalene and esters separately. Repeatability and recovery of the method were good. The method was applied to extra and lampant virgin olive oil categories and also to oils obtained from olive pomace by second centrifugation and solvent extraction. Extra virgin olive oils contained low amounts of fatty acid methyl and ethyl esters, while oils obtained from altered olive or olive pomace showed high concentrations of fatty acid alkyl esters, mainly ethyl esters. Correlation between oil acidity and ethyl esters concentration was poor.

Cocoa-rich diet prevents azoxymethane-induced colonic preneoplastic lesions in rats by restraining oxidative stress and cell proliferation and inducing apoptosis

Cocoa is a rich source of bioactive compounds with potential chemopreventive ability but up to date its effectiveness in animal models of colon carcinogenesis has not been addressed. Herein, we investigated the in vivo effect of a cocoa-rich diet in the prevention of azoxymethane (AOM)-induced colon cancer and the mechanisms involved. Our results showed that cocoa feeding significantly reduced AOM-induced colonic aberrant crypt foci formation and crypt multiplicity. Oxidative imbalance in colon tissues seems to be prevented by cocoa as indicated by reduced oxidation markers levels and increased enzymatic and non-enzymatic endogenous defences. Cocoa-rich diet also exhibited antiproliferative effects by decreasing the levels of extracellular regulated kinases, protein kinase B and cyclin D1 together with pro-apoptotic effects evidenced by reduced Bcl-x(L) levels and increased Bax levels and caspase-3 activity. Our findings provide the first in vivo evidence that a cocoa-rich diet may inhibit the early stage of colon carcinogenesis probably by preventing oxidative stress and cell proliferation and by inducing apoptosis.