Hydrophilic Radical Initiator Research Articles

Radical Scavenging Activity of Olive Oil Phenolic Antioxidants in Oil or Water Phase during the Oxidation of O/W Emulsions: An Oxidomics Approach.

Omics approaches are recently being applied also in food lipid oxidation, to increase knowledge of oxidation and antioxidation mechanisms. The so-called oxidomics throws a wider spot of light on the complex patterns of reactions taking place in food lipids, especially in dispersed systems. This research aimed to investigate the radical scavenging activity of olive oil phenolic antioxidants (OPAs) in O/W emulsions, as affected by the phase in which they were added. This allowed one to assess whether different behaviors could be expected from antioxidants originally present in phenolic-rich olive oils compared to natural antioxidants added in the water phase during emulsion production. Hydroperoxide decomposition kinetics and the analysis of volatile pattern provided an outline of antioxidation mechanisms. Though being effective in slowing down oxidation when added both in the oil and water phase, OPAs interfered in different ways with oxidation pathways, based on the phase in which they were added. OPAs added to the water phase were more effective in slowing down hydroperoxide decomposition due to the hydrophilic radical initiator. On the other hand, OPAs present in the oil were more effective in preventing radical propagation, with relevant consequences on the volatile pattern.

Mate extract is superior to green tea extract in the protection against chicken meat protein thiol oxidation

Water soluble caffeic acid derivatives in aqueous extracts of mate (Ilex paraguariensis) showed a direct dose-dependent response, protecting protein thiols in a chicken breast meat model system when oxidation was initiated by a lipophilic radical initiator (2,2′-azobis(2,4-dimethylvaleronitrile; AMVN). In contrast, an inverse dose-dependent response was obtained when initiated by a hydrophilic radical initiator (2,2′-azobis(2-amidinopropane)dihydrochloride; AAPH), pointing toward a mechanism, where a specific concentration of water-soluble polyphenol yields optimum protection. Optimal concentrations of mate extract for the protection of thiols against radicals initiated in the lipid phase (AMVN) or in the aqueous phase (AAPH) were 0.1–0.5% and 0.01–0.1%, respectively. Green tea (Camellia sinensis) extract showed a pro-oxidative effect involving quinones from oxidation of pyrogallol-type catechins (epigallocatechin-3-gallate (EGCG), gallocatechin-3-gallate (GCG), and epicatechin-3-gallate (ECG)) and not the catechol-type catechins (catechin and epicatechin).

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the Tm makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.

A fluorometric assay to determine antioxidant activity of both hydrophilic and lipophilic components in plant foods

This study aimed to develop a fluorometric method to determine total antioxidant activity of plant foods. The antioxidant activities in plant foods were determined after extracting (1) hydrophilic components with acidified methanol (methanol:glacial acetate acid:water=50:3.7:46.3), (2) lipophilic components with methanol followed by tetrahydrofuran (THF), or (3) both hydrophilic and lipophilic components using sequential extraction of acidified methanol and THF together. Both the hydrophilic assay [using the hydrophilic radical initiator 2,2′-azobis-(2-amidinopropane)dihydrochloride (10 mmol/L) and hydrophilic probe 2,7-dichlorodihydrofluorescein (DCFH)] and the lipophilic assay [using the lipophilic radical initiator [2,2′-azobis (4-methoxiy-2,4-dimethylvaleronitrile), 2 mmol/L], and the lipophilic probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-undecanoic acid (C11-BODIPY 581/591) (BODIPY: 2 μmol/L)] were used to measure antioxidant activity. The inhibition of BODIPY oxidation was significantly increased ( P<.01) when both the hydrophilic and lipophilic components were extracted using acidified methanol and organic solvent as compared to those extracted by organic solvent alone. In addition, the rate of DCFH oxidation was significantly delayed ( P<.05) when both components coexisted compared to DCFH oxidation of the hydrophilic component alone. The combination of lipophilic and hydrophilic components in these plant foods showed significantly greater antioxidant activity than that of either hydrophilic or lipophilic component alone. Thus, both hydrophilic and lipophilic components in plant foods and their interactions should be considered when determining their antioxidant activity.

Phytonutrients affecting hydrophilic and lipophilic antioxidant activities in fruits, vegetables and legumes

AbstractThe antioxidant activities of fruits (n = 21), vegetables (n = 67) and legumes (n = 7) commonly consumed in Korea were determined by both the lipophilic antioxidant performance assay (LAP) and the hydrophilic oxygen radical absorbance capacity assay (ORAC). The LAP assay used the lipophilic radical initiator MeO‐AMVN [2,2′‐azobis(4‐methoxy‐2,4‐dimethylvaleronitrile)] and the lipophilic probe BODIPY 581/591 [4,4‐difluoro‐5‐(4‐phenyl‐1,3‐butadienyl)‐4‐bora‐3a, 4a‐diaza‐s‐indacene‐3‐undecanoic acid]. The ORAC assay used the hydrophilic radical initiator AAPH [2,2′‐azobis(2‐amidinopropane) dihydrochloride] and the hydrophilic probe fluorescein. In addition, the lipid‐soluble phytonutrients, carotenoids and tocopherols were determined by a reverse‐phase HPLC system using a C30 column with a UV detector. The water‐soluble phytonutrient, ascorbic acid, was analyzed using an HPLC system with an electrochemical detector. Total phenols were determined by UV spectrophotometry. Tocopherols (r = 0.774, p &lt; 0.0001) and carotenoids (r = 0.569, p &lt; 0.0001) were significantly correlated with LAP in total samples (n = 95). ORAC was significantly correlated with total phenols (r = 0.893, p &lt; 0.0001), but not with ascorbic acid (r = 0.009, p = 0.929) in total samples (n = 95). These data indicate that carotenoids and tocopherols and total phenols are the major contributors to the lipophilic and hydrophilic antioxidant capacities, respectively. Therefore, the contribution of both the hydrophilic and lipophilic components of fruits and vegetables should be considered when determining the actual ‘total’ antioxidant activity of fruits and vegetables. Copyright © 2007 Society of Chemical Industry

Antioxidant Activity in Plasma and Tissues Distribution of (-)-Epigallocatechin Gallate after Oral Administration to Rats

The aim of the present work was to examine the antioxidative effect of EGCG after administering it perorally to rats at a dose of 150 mg/kg body weight. The antioxidative activity of rat plasma, and plasma levels and tissue distribution of EGCG were determined. The results showed that oral administration of EGCG increased the antioxidative activity of rat plasma, measured by the preservation of the inner lipophilic antioxidant α-tocopherol during ex vivo oxidation by hydrophilic radical initiator, at 2nd, 5th and 24th hours post dose. The levels of the total EGCG in rat plasma were 0.82 and 0.5 μ M at 2nd and 5th hours respectively as measured by developed high-performance liquid chromatographic method with electrochemical detection (HPLC - ED). The highest levels of EGCG were detected in rat small intestine and colon and were in the range 4.75 - 24.41 nmol/g. The EGCG levels in rat kidneys, liver, spleen, lung, brain, bladder and prostate were in the range 0.1 - 1 nmol/g. In conclusion, we showed that oral administration of EGCG at a dose of 150 mg/kg body weight increased the antioxidative strength of rat plasma, preserving α-tocopherol during ex vivo oxidation. In small intestine and colon EGCG reached values having anticancer effects in cell culture experiments.

Temperature-dependence of rate of oxidation of rapeseed oil encapsulated in a glassy food matrix

Oxidation of stripped rapeseed oil, encapsulated in a carbohydrate/protein glassy matrix, initiated by a lipophilic free radical initiator (or not initiated) and monitored as peroxide value and conjugated dienes for 45 days, developed differently depending on storage temperature. At low temperature (5 °C), the encapsulated oil and bulk oil, as reference, showed little oxidation which, moreover, could be accounted for by the oxygen dissolved in the oil. At intermediate temperatures (25 and 45 °C), oxidation exceeded the level corresponding to dissolved oxygen and became dependent on oxygen transport through the matrix. At high temperature (60 °C), a rapid, linear increase in peroxide concentration was followed by an autocatalytic phase with a rapid increase in peroxides, subsequently reaching a steady-state concentration. The oxidation of the encapsulated oil was found to have a lower energy of activation (around 60 kJ/mol) than the bulk oil (around 80 kJ/mol), resulting in a protection of the encapsulated oil at higher temperatures. However, the temperature-dependence of the zeroth order rate constants for initial peroxide formation in the encapsulated oil showed a shift from a rate determining reaction at low temperature with a high energy of activation to a reaction at higher temperature with a smaller energy of activation, especially for the encapsulated oil without initiator added. At low temperature, lipid oxidation seems rate-determining while, at higher temperature, oxygen permeation through the matrix with a lower energy of activation becomes rate-determining. The glassy matrix yields only partly protection against lipid oxidation as it allows permeation of oxygen and other small molecules, as further confirmed by the effect of a hydrophilic radical initiator, incorporated in the matrix, on peroxide value of the encapsulated oil.

(−)-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (−)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation ( IC 50=0.72 μM ) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, α-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared α-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment ( IC 50=4.37 μM ). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled α-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.

Interaction between .ALPHA.-Tocopherol, Tocotrienols and Astaxanthin in Liposomes, Subjected to Lipid Peroxidation

We studied the cooperative interaction between α-tocopherol, tocotrienols and astaxanthin, incorporated in soybean phosphatidylcholine (PC) multilamellar vesicles (MLV) against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), 2,2′-azobis (2,4-dimethyl-valeronitrile) (AMVN), or 1,3-morpholinosydnonimine hydrochloride (SIN-1) induced lipid peroxidation. Tocotrienols spared α-tocopherol consumption in hydrophilic or lipophylic radicals initiated oxidation. The combination of tocotrienols and astaxanthin had the greatest protective effect on α-tocopherol consumption in MLV, oxidized by the hydrophilic radical initiator. α-Tocopherol and tocotrienols protected astaxanthin against AAPH-, AMVN-, and SIN-1-induced oxidations. In the three oxidations α-tocotrienol was consumed first, followed by α-tocopherol and the tocotrienols in the sequence: β > γ > δ. Astaxanthin was consumed as fast as γ-tocotrienol in AAPH oxidation, as fast as α-tocopherol in AMVN oxidation, and before δ-tocotrienol in SIN-1 oxidation. In conclusion, α-tocopherol, tocotrienols, and astaxanthin cooperated in protecting against MLV lipid peroxidation. α-Tocotrienol was the most effective antioxidant among them. Astaxanthin was as effective as the chain-breaking antioxidants against lipid peroxidation.

Scavenging Effect of Aged Garlic Extract and its Constituents on Active Oxygen Species

The effect of aged garlic extract (AGE) and its major organosulphur constituents, S-allylcysteine (SAC), S-allylmercaptocysteine (SAMC) and alliin on active oxygen species were examined in an in vitro system. AGE and three compounds have demonstrated a scavenging effect on hydrogen peroxide, and also inhibited the chain oxidation induced by a hydrophilic radical initiator.

Antioxidant role of cellular reduced coenzyme Q homologs and α-tocopherol in free radical-induced injury of hepatocytes isolated from rats fed diets with different vitamin E contents

Reduced coenzyme Q 9 (CoQ 9H 2) and reduced coenzyme Q 10 (CoQ 10H 2) as well as α-tocopherol (α-Toc) are known to be potent lipid-soluble antioxidants in mammalian tissues. Reduced coenzyme Q homolog (CoQ n H 2) appears to show antioxidant activity independent of that of α-Toc (Matsura, T., Yamada, K. and Kawasaki, T. (1992) Biochim. Biophys. Acta 1123, 309–315). To further confirm this, we have studied the antioxidant role of cellular CoQ n H 2 and α-Toc using hepatocytes isolated from rats fed diets containing deficient, sufficient, and excess amounts of vitamin E(VE). Cellular damage was induced with a hydrophilic radical initiator. 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The concentration of α-Toc in VE-deficient hepatocytes was approximately 1/12 that in VE-sufficient hepatocytes, whereas the concentration of α-Toc in VE-excess hepatocytes was approximately 7-fold that in VE-sufficient hepatocytes. The molar ratios of α-Toc to CoQ n H 2 (CoQ 9H 2 plus CoQ 10H 2) in VE-deficient, sufficient and excess cells were 0.03, 0.33 and 2, respectively. In the hepatocytes in these three dietary groups, α-Toc status had little effect on the concentration of CoQ homologs. These hepatocytes were incubated with 50 mM AAPH for 4 h. The cell viability in all groups of hepatocytes decreased rapidly after 3 h of AAPH treatment, and was associated with the increase of lipid peroxides. The loss of cell viability and the increase of lipid peroxidation in VE-deficient cells were more pronounced than those in the hepatocytes of the other two groups. The endogenous CoQ 9H 2 content of each group of hepatocytes decreased linearly with a reciprocal increase in oxidized CoQ 9 after addition of AAPH, whereas the decrease of endogenous CoQ 10H 2 in each group during AAPH treatment was much less than that of endogenous CoQ 9H 2. α-Toc in the three VE dietary groups of hepatocytes was also consumed without a time lag addition of AAPH, and it was not spared by CoQ n H 2, even VE-deficient cells where the CoQ n H 2 concentration was 38-fold that of α-Toc. These results indicate that CoQ n H 2, especially CoQ 9H 2, is a lipid-soluble antioxidant, which is as effective as α-Toc in rat hepatocytes under the conditions employed in this study, and acts independently of α-Toc to inhibit lipid peroxidation.

Difference in antioxidant activity between reduced coenzyme Q 9 and reduced coenzyme Q 10 in the cell: studies with isolated rat and guinea pig hepatocytes treated with a water-soluble radical initiator

A possible difference in antioxidant activity between reduced coenzyme Q 9 (CoQ 9H 2) and reduced coenzyme Q 10 (CoQ 10H 2) in animal cells was studied by incubation of hepatocytes with a hydrophilic radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). Two kinds of hepatocytes differing in their content of CoQ homologs were used: rat, total (oxidized plus reduced) CoQ 9: total CoQ 10 6:1, guinea pig, 1:5. The sum of total CoQ 9 and CoQ 10 in rat and guinea-pig hepatocytes was about 780 and 400 pmol/mg protein, respectively. The concentration of CoQ 9H 2 in rat hepatocytes decreased linearly after the addition of AAPH, whereas that of oxidized CoQ 9 showed a reciprocal increase. No loss of cell viability or increase of lipid peroxidation was observed until most of the CoQ 9H 2 had been consumed. Cellular CoQ 9H 2 was consumed probably through scavenging of lipid peroxyl radicals produced by incubation with AAPH. On the other hand, CoQ 10H 2 was not significantly consumed in the AAPH-treated rat hepatocytes during incubation compared with the control cells. In guinea-pig hepatocytes, cellular CoQ 10H 2 as well as CoQ 9H 2 was consumed by addition of AAPH. α-Tocopherol also showed linear consumption with incubation time regardless of the cell types used. It is concluded that CoQ 9H 2, together with α-tocopherol, constantly acts as a potential antioxidant in hepatocytes when incubated with AAPH, whereas CoQ 10H 2 mainly exhibits its antioxidant activity in cells containing CoQ 10 as the predominant CoQ homolog.