Effects of natural phenolic compounds on the antioxidant activity of lactoferrin in liposomes and oil-in-water emulsions

The effect of natural phenolic compounds on the antioxidant and prooxidant activity of lactoferrin was studied in liposomes and oil-in-water emulsions containing iron. The antioxidants tested with lactoferrin were alpha-tocopherol, ferulic acid, coumaric acid, tyrosol, and natural phenolic extracts obtained from three different extra-virgin olive oils and olive mill wastewater. The natural extracts of olive oils and mill wastewaters were composed mainly of polyphenols and simple phenolics, respectively. Lipid oxidation at 30 degrees C was determined by the formation of hydroperoxides and fluorescent compounds resulting from oxidized lipid interactions. All phenolic compounds showed synergistic properties in reinforcing the antioxidant activity of lactoferrin in lipid systems containing iron. The highest synergistic effects were observed for the phenolic extracts rich in polyphenols of extra-virgin olive oils and lactoferrin. This synergistic effect was higher in liposomes than in emulsions.     

Evolution of antioxidant capacity during storage of selected fruits and vegetables

Interest in the consumption of fresh fruits and vegetables is, to a large extent, due to its content of bioactive nutrients and their importance as dietary antioxidants. Among all of the selected fruits and vegetables, strawberries and black grapes have relatively high antioxidant capacities associated with high contents of total phenolic compounds, ascorbic acid, and flavonols. More interesting, the results of this study indicated that in most fruits and vegetables storage did not affect negatively the antioxidant capacity. Better, in some cases, an increase of the antioxidant capacity was observed in the days following their purchase, accompanied by an increase in phenolic compounds. In general, fruits and vegetables visually spoil before any significant antioxidant capacity loss occurs except in banana and broccoli. When ascorbic acid or flavonoids (aglycons of flavonols and anthocyanins) were concerned, the conclusions were similar. Their content was generally stable during storage.     

Changes in antioxidant effects and their relationship to phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.) during maturation

Different fractions of sea buckthorn fruits were investigated for antioxidant activity and its relationship to different phytonutrients. Capacity to scavenge radicals of the crude extract, like the phenolic and ascorbate extracts, decreased significantly with increased maturation. The changes were strongly correlated with the content of total phenolics and ascorbic acid. Antioxidant capacity of the lipophilic extract increased significantly and corresponded to the increase in total carotenoids. The phenolic fractions made a major contribution to the total antioxidant capacity due to the high content of total phenolics. The lipophilic fractions were most effective if the comparison was based on the ratio between antioxidant capacity and content of antioxidants. The crude extract of fruits showed the highest inhibitory effect in both 2,2-azobis(2,4-dimethylvaleronitrile) (AMVN) and ascorbate-iron induced lipid peroxidations. The aqueous and ascorbate-free extracts showed higher inhibition in the AMVN assay, but lower inhibition in ascorbate-iron induced peroxidation, than the lipophilic extract.     

The relationship between the physicochemical properties of antioxidants and their ability to inhibit lipid oxidation in bulk oil and oil-in-water emulsions

Chain-breaking antioxidants differ in their effectiveness at inhibiting lipid oxidation because of their chemical properties and physical location within a food. Our objective was how the physicochemical properties of four structurally related lipid-soluble antioxidants were related to their antioxidant activity. Antioxidants differed in the number of methyl (alpha-tocopherol and delta-tocopherol) or hydroxyl (butylated hydroxytoluene (BHT) and 4-hydroxymethyl-2,6-ditertiarybutylphenol) groups. Surface activity of the antioxidants was in the order of delta-tocopherol > alpha-tocopherol approximately 4-hydroxymethyl-2,6-ditertiarybutylphenol > BHT. Free-radical scavenging activity was similar between alpha-tocopherol and delta-tocopherol as well as BHT and 4-hydroxymethyl-2,6-ditertiarybutylphenol. In bulk menhaden oil, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while alpha-tocopherol was more effective than delta-tocopherol. In menhaden oil-in-water emulsions, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while delta-tocopherol was more effective than alpha-tocopherol. These results indicate that the surface activity and polarity of lipid-soluble antioxidants were not the only determinants of their antioxidant effectiveness in food lipids.     

Antioxidant activity of a combinatorial library of emulsifier-antioxidant bioconjugates

A combinatorial chemistry approach was employed for the design and systematic synthesis of antioxidant-emulsifier bioconjugates to improve antioxidant activity in the interface between oil and water. A combinatorial library of 12 bioconjugates was synthesized from the phenolic antioxidants Trolox (a water-soluble alpha-tocopherol analogue), dihydroferulic acid, dihydrocaffeic acid, and gallic acid in combination with serine ethyl ester, serine lauryl ester, and lauroyl serine by esterification of the serine side chain or amidation, respectively. The bioconjugates were characterized by NMR and mass spectrometry, and each was tested for antioxidant activity by measuring the radical scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH (*)) in methanol, the radical scavenging rate of DPPH (*) in a heterogeneous solvent system, the rate of oxygen consumption in an oil-in-water emulsion with metmyoglobin initiated oxidation, and the lag phase for diene formation in unilamellar liposomes with free radical initiation in the aqueous phase; each case was compared to the antioxidant activity of the parent antioxidant. In general, the conjugates with longer chain derivatives exhibited improved antioxidative activity in heterogeneous systems, whereas no improvement was observed in homogeneous solution. The rate of oxygen consumption in oil-in-water emulsion was found to decrease with increasing octanol/water partition coefficient, which is suggested to correspond to a saturation of the water/oil interface with antioxidant bioconjugate to approach maximal protection.     

Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract

Postmenopausal women have an increased risk of coronary heart disease. Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis, and the presence of modified LDL (LDL(-)) in plasma appears to represent LDL oxidation in vivo. Because previous studies have demonstrated a strong antiatherogenic effect of estrogen due to its antioxidant activity and similar antioxidant activity was found for specific isoflavones derived from soy extract, the antioxidant activity of a phytoestrogen extract derived from soy and alfalfa was studied. Copper-mediated LDL oxidation was inhibited in the presence of soy and alfalfa extracts, and this effect was further enhanced in the presence of acerola cherry extract, which is rich in ascorbic acid. Male rabbit aortic endothelial cells pretreated with soy extract were resistant to the toxic effects of high levels of LDL and LDL(-), and a lesser, but significant protection, was also afforded by alfalfa extract. Cell-mediated oxidation of LDL, measured by LDL(-) formation, was inhibited in the presence of soy extract but not alfalfa extract. However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL(-). These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.     

Nutritional value of cherry tomatoes (Lycopersicon esculentum Cv. Naomi F1) harvested at different ripening stages

The average content of some classes of antioxidants is generally higher in cherry tomatoes than in normal-sized berries. The aim of this work was to assess the nutritional value of cherry tomato (cv. Naomi F1) by investigating the compositional pattern of berries harvested at different ripening stages and evaluating, in particular, all of the main antioxidants (carotenoids, ascorbic acid, phenolic compounds, and alpha-tocopherol) and the antioxidant activity of the water-soluble and water-insoluble fractions. Results confirmed the relatively high level of carotenoids in cherry tomato but showed that not all biologically active compounds necessarily increase in tomatoes picked at later stages of ripeness. Cherry tomatoes harvested at full ripeness exhibited the highest level of carotenoids and antioxidant activity in the water-insoluble fraction. On the other hand, no significant differences in ascorbic acid content were observed at different ripening stages, whereas the main phenolics content and the antioxidant activity of water-soluble fraction showed slight, but significant, decreases at later stages of ripeness.     

Antioxidant properties of pearled barley fractions

Two barley varieties (Falcon and AC Metcalfe) were separated by pearling into seven fractions and subsequently extracted with 80% methanol. The extracts, after solvent removal, were evaluated for their radical scavenging efficacy using Trolox equivalent antioxidant capacity (TEAC). The radical scavenging capacity of the extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC(FL)), and superoxide radical assays and a photoinduced chemiluminescence technique. In both barley varieties the outermost fraction (F1) yielded the highest phenolic content. In general, Falcon had a significantly higher total phenolic content than AC Metcalfe. A similar trend was observed for TEAC, DPPH, and superoxide radical scavenging capacities of the extracts. The contents of water-soluble antioxidants of Falcon and AC Metcalfe were 1.15-12.98 and 2.20-12.25 micromol of Trolox equiv/(g of defatted material), while the corresponding lipid-soluble counterparts varied from 1.44 to 4.70 micromol of alpha-tocopherol equiv/(g of defatted material). Phenolic acids, namely, vanillic, caffeic, p-coumaric, ferulic, and sinapic acids, were identified by HPLC in barley fractions.     

Antioxidant properties of Teaw (Cratoxylum formosum Dyer) extract in soybean oil and emulsions

The antioxidant activity of an extract from Teaw (Cratoxylum formosum Dyer) leaves was studied in soybean oil and soybean oil-in-water emulsions. Samples containing the extract or reference antioxidants including chlorogenic acid, which comprises 60% of the Teaw extract, were stored at 60 degrees C and analyzed periodically for peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) to allow both hydroperoxides and hydroperoxide degradation products to be monitored. Chlorogenic acid and the Teaw extract were more effective than alpha-tocopherol in inhibiting lipid oxidation in bulk oil but were less effective in an oil-in-water emulsion in accordance with the polar paradox. The PV/TBARS ratio for oil samples containing chlorogenic acid was higher than for alpha-tocopherol and BHT because chlorogenic acid inhibits both hydroperoxide formation by radical scavenging and hydroperoxide decomposition by metal chelation. The importance of the metal-chelating activity in retarding hydroperoxide decomposition was confirmed by studying the decomposition of oil samples containing added ferric ions. The PV/TBARS ratio was higher for citric acid than for alpha-tocopherol in the presence of added ferric chloride, but the order was reversed in samples lacking ferric chloride. Samples containing added chlorogenic acid gave the highest PV/TBARS ratios both in the presence and absence of ferric ions. The PV/TBARS ratios for the samples containing antioxidants fell rapidly to lower values in a soybean oil-in-water emulsion than in the soybean oil. This was due to increased hydroperoxide decomposition in the emulsion at the same PV. The Teaw extract contained 12% oil-soluble components, which contributed to a slightly higher oil-water partition coefficient than that of chlorogenic acid. The antioxidant activity of the aqueous phase of the Teaw extract was reduced more than that of chlorogenic acid by partitioning of the oil-soluble components into oil, which showed that the less-polar components contributed to the antioxidant activity of the Teaw extract in aqueous media.     

Antioxidant characterization of some Sicilian edible wild greens

Epidemiological studies have demonstrated that many antioxidants and the total antioxidant capacity (TAC) of the diet may protect against cancers and cardiovascular disease. Common fruits and vegetables are good sources of antioxidants, although in some Mediterranean areas traditional wild greens are responsible for a significant percentage of total dietary antioxidant intake. In the European Prospective Investigation into Cancer and Nutrition cohort of Ragusa (Sicily), a high number of subjects were found to frequently eat wild greens, including Sinapis incana and Sinapis nigra, Diplotaxis erucoides, Cichorium intybus, Asparagus acutifolius, and Borrago officinalis. On the basis of these observations, detailed characterization of single antioxidant components (i.e., polyphenols, carotenoids, chlorophylls, and ascorbic acid) and the TAC of these edible wild traditional plants was performed. The wild plants examined were found to be very rich in antioxidants, such as flavonoids and carotenoids, with high TAC values, suggesting that the importance of these vegetables, not only in the traditional but even in the contemporary diet, needs to be emphasized.     

Characterization of antioxidants and change of antioxidant levels during storage of Manilkara zapota L

Antioxidants found in fruits and vegetables play an important role via their protective effects against the onset of aging-related chronic diseases. Our previous research has indicated that unripe ciku fruits (Manilkara zapota L.) are an excellent source of antioxidants, with over 3000 mg of L-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. In this study, 24 antioxidants in an extract of ciku king were characterized through a free radical spiking test. Their chemical structures were proposed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and tandem MS (HPLC/MSn). The antioxidant capacity of ciku king fruits was mainly attributed to polyphenolics with basic blocks of gallocatechin or catechin or both. The changes of total antioxidant capacity (TAC) and total phenolics content (TPC) of ciku king fruits with storage time were also investigated. It was found that the TAC and TPC decreased significantly as the fruits gradually changed from the unripe to the overripe stage. The best time for one to consume ciku king fruits at a flavorful stage with high amounts of antioxidants with AEAC values ranging from 600 to 1200 mg per 100 g fresh sample is suggested. The change of the content of major antioxidant peaks was also consistent with changes of antioxidant levels during storage.     

Phenol antioxidant quantity and quality in foods: fruits.

The free and bound phenols have been measured in 20 fruits commonly consumed in the American diet. Phenols were measured colorimetrically using the Folin-Ciocalteu reagent with catechin as the standard after correction for ascorbic acid contribution. On a fresh weight basis, cranberry had the highest total phenols, and was distantly followed by red grape. Free and total phenol quality in the fruits was analyzed by using the inhibition of lower density lipoprotein oxidation promoted by cupric ion. Ascorbate had only a minor contribution to the antioxidants in fruits with the exception of melon, nectarine, orange, white grape, and strawberry. The fruit extracts' antioxidant quality was better than the vitamin antioxidants and most pure phenols, suggesting synergism among the antioxidants in the mixture. Using our assay, fruits had significantly better quantity and quality of phenol antioxidants than vegetables. Fruits, specifically apples and cranberries, have phenol antioxidants that can enrich lower density lipoproteins and protect them from oxidation. The average per capita consumption of fruit phenols in the U.S. is estimated to be 255 mg/day of catechin equivalents.     

Antioxidant composition in cherry and high-pigment tomato cultivars

Fourteen cultivars of cherry tomatoes and four cultivars of high-pigment tomato hybrids were cultivated in southern Italy, and the red-ripe fruits were analyzed for their content in different classes of antioxidants and for their antioxidant activity. Among the different cultivars, significant differences were found between lycopene, beta-carotene, alpha-tocopherol, vitamin C (ascorbic acid and dehydroascorbic acid), and total phenolic and flavonoid contents. LS203 and Corbus appear to be the cultivars with the highest content of lipophilic and hydrophilic antioxidants among cherry tomatoes, respectively. All cultivars of high-pigment tomato hybrids showed an expected exceptionally high lycopene content. Among them, the highest content of lipophilic and hydrophilic antioxidants was found in cv. HLY 13. Hydrophilic and lipophilic antioxidant activities were both significantly influenced by genotype. Such results highlight an existing unexploited variability in tomato germplasm and stress the need to evaluate the biodiversity and to support conventional breeding programs to improve tomato nutritional value.     

Antioxidant potential of hydroxycinnamic acid glycoside esters

Hydroxycinnamic acids are natural antioxidants found in fruits, vegetables, and cereals. In this study, the antioxidant activity of various types of hydroxycinnamoyl glycoside esters that mimic the structure of polymeric carbohydrates was studied in different model systems prone to oxidation, namely, liposomes and emulsions. In addition, radical scavenging activity against the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was tested. It was found that the esterification in the primary hydroxyl group of the glycoside resulted in the improved radical scavenging activity of both sinapoyl and feruloyl glycosides compared to conjugation to the secondary hydroxyl group. Increased activity was also observed, particularly in the case of feruloyl glucosides in inhibiting the oxidation of liposomes emulsions. The results showed that sinapic and ferulic acid glycoside esters were as effective or more efficient antioxidants than their free forms. In conclusion, the strength of their antioxidant effect depends on the nature of conjugation.     

Antioxidant activity of 3-dehydroshikimic acid in liposomes,emulsions, and bulk oil

The antioxidant activity of 3-dehydroshikimic acid (DHS), an intermediate in the biosynthesis of aromatic amino acids, was evaluated in three assay systems: bulk oil (lard), liposomes, and a 10% corn oil-in-water emulsion. Upon initiation of peroxidation in the liposome or emulsion systems, DHS exhibited weak antioxidant activity. In contrast, DHS displayed strong antioxidant activity in lard, suppressing peroxidation with activity comparable to that of tert-butylhydroquinone, propyl gallate, and gallic acid and superior to that of alpha-tocopherol. Two major DHS oxidation products, gallic acid and protocatechuic acid, were identified by gas chromatography/mass spectral analysis of lard extracts; both compounds are effective antioxidants in the bulk oil system. In the liposome system, DHS remained intact throughout the assay period. A small amount of gallic acid was observed in extracts of the emulsion; however, protocatechuic acid was not detected. A mechanism to explain the different activities of DHS in the three lipid systems is proposed.     

Antioxidants, low molecular weight carbohydrates, and total antioxidant capacity in strawberries (Fragaria x ananassa): effects of cultivar, ripening, and storage

Four cultivars of strawberries (Senga Sengana, BFr77111, Elsanta, and Honeoye) were studied for their content of antioxidants, total antioxidant capacity, and low molecular weight carbohydrates in relation to harvest year, ripening stage, and cold storage. For ascorbic acid, chlorogenic acid, ellagic acid, and total antioxidative capacity, measured in both water-soluble and water-insoluble extracts, there was a 2-5-fold variation among cultivars. Unripe berries contained lower concentrations of chlorogenic acid and p-coumaric acid and also quercetin and kaempferol compared with riper berries. During cold storage for up to 3 days, relatively few changes in the concentration of the different antioxidants occurred. The concentrations of several investigated parameters were interrelated, for example, for ascorbic acid and water-soluble antioxidant capacity and for ellagic acid and water-insoluble antioxidant capacity. The dominating sugars in strawberries were fructose and glucose, but considerable amounts of sucrose were also present, and their contents varied among cultivars, giving a predicted glycemic index of approximately 81. Verbascose, raffinose, and stachyose were found in only minor amounts. The study shows that the concentration of a number of bioactive compounds in strawberries varied according to cultivar, ripening stage, and storage. This information should make it possible to select strawberries with an optimal content of bioactive compounds.     

NMR analytical approach to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of antioxidants in tea using 1,1-diphenyl-2-picrylhydrazyl

(+)-catechin, ethyl gallate, ascorbic acid, and alpha-tocopherol were reacted with 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the reaction mixtures were subjected to 13C-nuclear magnetic resonance (NMR) analyses to clarify the molecular mechanisms of the antioxidative and radical-scavenging activities of each antioxidant. When ascorbic acid was reacted with DPPH, it was oxidized to dehydroascorbic acid by DPPH. When a mixture of ascorbic acid and (+)-catechin was reacted with DPPH, ascorbic acid scavenged DPPH radical faster than (+)-catechin. Ascorbic acid also scavenged DPPH radical faster than ethyl gallate and alpha-tocopherol. When (+)-catechin was reacted with DPPH, the B-ring of (+)-catechin changed to an o-quinone structure. However, it was reduced to (+)-catechin by ethyl gallate or alpha-tocopherol. alpha-Tocopherol and ethyl gallate had almost identical antioxidative activities. Therefore, the order of radical-scavenging ability (speed) suggested by our 13C NMR study was as follows: ascorbic acid > alpha-tocopherol = ethyl gallate > (+)-catechin.     

Novel low-density lipoprotein (LDL) oxidation model: antioxidant capacity for the inhibition of LDL oxidation

A novel model of peroxyl radical initiated low-density lipoprotein (LDL) oxidation (LDL oxidation model for antioxidant capacity, or LOMAC) was developed to assess the free radical scavenging capacity of antioxidants and the extracts of natural products. A water-soluble free radical initiator, 2,2'-azobis(amidinopropane) dihydrochloride, was used at physiological temperature (37 degrees C) to generate peroxyl radicals to catalyze lipid oxidation of LDL isolated from human plasma samples. Headspace hexanal, a major decomposition product of LDL oxidation, was measured by a headspace gas chromatograph as an indicator of antioxidant capacity of different concentrations of pure antioxidants (vitamins C and E) and the extracts of natural products (fresh apple phytochemical extracts). All vitamin C and E and apple extract concentrations tested resulted in increasing partial suppression and delay of LDL oxidation. On the basis of the median effective dose (EC(50)) calculated for each compound or extract tested, the LOMAC value of 100 g of apple against LDL oxidation was equivalent to 1470 mg of vitamin E or to 402 mg of vitamin C. This study shows that the LOMAC assay can be routinely used to analyze or screen antioxidants or phytochemical extracts against LDL oxidation to prevent cardiovascular disease. The food-specific LOMAC values will be very useful as a new alternative biomarker for future epidemiological studies of cardiovascular disease.     

Effect of apple cell walls and their extracts on the activity of dietary antioxidants

The effect of dietary fiber in the form of apple cell walls and pectin extracts on natural antioxidants was examined. Cell walls (CW), isolated from apples ( Malus domestica Borkh. cv. "Pacific Rose"), were incubated with ascorbic acid (AA) or quercetin in N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) buffer (pH 6.5) at 37 degrees C for 2 h. The resulting supernatants were characterized by a ferric reducing antioxidant power (FRAP) assay and cyclic voltammetry (CV). The experiments were repeated with pectin isolated from the apple cell walls and commercial pectins and showed that polysaccharide preparations stabilized AA effectively but offered little protection against quercetin oxidation. The water-soluble components from cell walls appeared to be responsible for the observed effects of cell-wall polysaccharide preparations on antioxidant activity.     

Modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (abts) method to measure antioxidant capacity of Selected small fruits and comparison to ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods

The measurement of antioxidant capacity in fruits differs from that of other biological samples due to their low pH and very low lipophilic antioxidant capacity. In this report, we present a modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method for fruits and compare its performance with the other commonly used antioxidant methods of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The antioxidant capacity and reaction kinetics of four phenolic compounds, two antioxidant standards, and five fruits were also investigated. The modified ABTS method prepared at a pH of 4.5 with sodium acetate buffer is highly stable and easily applied to fruit samples as compared to the standard (pH 7.4) version. The measured antioxidant capacity of samples varied with the assay method used, pH, and time of reaction. Traditional antioxidant standards (trolox, ascorbic acid) displayed stable, simple reaction kinetics, which allowed end point analysis with all of assays. Of the phenolic compounds examined, chlorogenic and caffeic acids exhibited the most complex reaction kinetics and reaction rates that precluded end point analysis while gallic acid and quercetin reached stable end points. All fruit extracts exhibited complex and varied kinetics and required long reaction times to approach an end point. Because the antioxidant capacity of fruit extracts is a function of the array of individual antioxidants present, accurate comparisons among fruit samples require that reaction times be standardized and of sufficient length to reach steady state conditions and that more than one assay be used to describe the total antioxidant activity of fruit samples.