A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2′-azobis(2-amidinopropane)
dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 μmol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple ) red grape > green grape. The CAA
assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its positively charged amino group. This interaction allows stable covered vesicles (chitosomes) to be developed as a suitable targeted carrier and controlled release system. This study investigated the effect of chitosomes on the activation of cranberry proanthocyanidins (PAC) in Raw 264.7 macrophages. Chitosomes were characterized according to size, zeta potential, PAC-loading, and release properties. Results showed an increase in the net positive charge and size of the liposomes as the concentration of chitosan was increased, suggesting an effective covering of the vesicles by means of electrostatic interactions, as shown by transmission electron microscopy and fluorescence microscopy. About 85% of the PAC that was loaded remained in the chitosomes after release studies for 4 hours in phosphate-buffered saline. Cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are associated with inflammation. Activated RAW 264.7 macrophages increase the expression of COX-2 and iNOS in response to bacterial infection and inflammation; we, therefore, tested the ability of the PAC-loaded chitosomes to attenuate COX-2 and iNOS expression in LPS (lipopolysaccharide)-stimulated macrophages. Increasing the amount of PAC loaded into the chitosomes caused a dose-dependent attenuation of iNOS and COX-2 expression in LPS-stimulated macrophages. A 2% v/v PAC-loaded chitosomes formulation almost completely attenuated the LPS-induced expression of iNOS and COX-2. PAC-loaded chitosomes were more active than PAC alone, suggesting that the macrophage response to LPS occurs after endocytosis of the PAC-loaded chitosomes.

Flavonoids can bind the divalent cations frequently used to evaluate LDL antioxidant capacity in vitro. Flavonoids in cranberry juice (CBJ) may serve as antioxidants and promote cardiovascular health. This in vitro study characterizes CBJ effects on metal and non-metal based oxidation of human LDL. For cupric ion-initiated oxidation of LDL, thiobarbituric acid reactive substances (TBARS) formation and relative electrophoretic mobility (REM) were significantly inhibited by CBJ at a dilution of 1:10,000. Diene formation during LDL oxidation was evaluated by continuous measurement of absorbance at 234 nm. The time required for cupric ion-initiated LDL oxidations to reach maximum reaction velocity was significantly delayed by 1:10,000 dilutions of CBJ. Non-metal initiated LDL oxidation by 2,2'-azobis-amidinopropane was significantly inhibited by CBJ at dilutions of 1:10,000 and 1:5,000 for REM and TBARS tests, respectively. Protection of LDL from both metal and non-metal based oxidative injury confirms that the effects of CBJ are not due to flavonoid chelation of oxidants but due to a true and potent antioxidant capacity.

Abstract: Antioxidant activity of six fractions of cranberry phenolic compounds was determined by
inhibition of Cu2+-induced low-density lipoprotein (LDL) oxidation. The phenolic composition of each fraction was determined by high-performance liquid chromatography. The phenolic fractions were mixed with aliquots of modified human serum prior to LDL isolation. The serum was modified to remove very-low-density lipoprotein and chylomicrons that may bind phenolic compounds. Only fractions 5 and 6 that contained proanthocyanidins (PAs) significantly increased the lag time of LDL oxidation, and the lag time for fraction 6 was significantly higher than for fraction 5. The mass distribution of PAs in these fractions was obtained by matrix-assisted laser desorption/ionisation time- of-flight mass spectrometry, a technique that allows rapid characterisation of the molecular weight distribution in mixtures of oligomeric compounds. Fraction 5 contained trimers through heptamers, whereas fraction 6 contained pentamers through nonamers. In addition, fraction 6 contained PA oligomers with more doubly linked, A-type interflavan bonds. Results indicate that PAs specifically associate with LDL in modified serum and increase the lag time of Cu2+-induced oxidation. Differences between fractions 5 and 6 in PA structure and effects on LDL oxidation suggest that the degree of polymerisation and the nature of the interflavan bond influence antioxidant properties

Abstract:Proanthocyanidin-rich extracts were prepared by fractionation of the fruit of theNorthAmerican cranberry (Vaccinium macrocarpon). In vitro growth inhibition assays in eight tumor cell lines showed that selected fractions inhibited the growth of H460 lung tumors, HT-29 colon and K562 leukemia cells at GI50 values ranging from 20 to 80 μgml−1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of one of these fractions found it to be composed of polyflavan-3-ols, which are primarily tetramers through heptamers of epicatechin containing one or two A-type linkages. Whole cranberry extract and the proanthocyanidin fractions were screened for effect on the expression of matrix metalloproteinases in DU 145 prostate carcinoma cells. The expression of MMP-2 and MMP-9 was inhibited in response to whole cranberry extract and to a lesser degree by the proanthocyanidin fractions

Although antioxidant systems help control the level of reactive oxygen species they may be overwhelmed during periods of oxidative stress. Evidence suggests that oxidative stress components as well as inflammatory mediators may be involved in the pathogenesis of vascular disorders, where localized markers of oxidative damage have been found. In this regard we investigated the putative antioxidant and anti-inflammatory effects of blueberry and cranberry anthocyanins and hydroxycinnamic acids against H2O2 and TNF induced damage to human microvascular endothelial cells. Polyphenols from both berries were able to localize into endothelial cells subsequently reducing
endothelial cells vulnerability to increased oxidative stress at both the membrane and cytosol level. Furthermore, berry polyphenols also reduced TNF induced up-regulation of various inflammatory mediators (IL-8, MCP-1 and ICAM-1) involved in the recruitment of leukocytes to sites of damage or inflammation along the endothelium. In conclusion, polyphenols isolated from both blueberry and cranberry were able to afford protection to endothelial cells against stressor induced up-regulation of oxidative and inflammatory insults. This may have beneficial actions against the initiation and development of vascular diseases and be a contributing factor in the reduction of age-related
deficits in neurological impairments previously reported by us

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response.
Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity
relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC50 values of 5.4, 1.6, 0.9, and 0.7 μM, respectively. Levels of ET-1 mRNA showed a similar
pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.

This study hypothesized that ginger (Zingiber officinale) and cranberry (Vaccinium macrocarpon) extracts would alter the physiological response to exercise as well as markers of muscle damage, and mRNA expression for the inflammatory cytokines tumour necrosis factor-a (TNF-a), interferon-g (IFN-g) and interleukin-6 (IL-6) after an exhaustive bout of exercise in horses. Nine unfit Standardbred mares (age 10 ^ 4 years, ,450 kg) completed three graded exercise tests (GXTs) in a crossover design, where they were assigned to the initial order of treatment in a randomized fashion. The GXTs were conducted between 07.00 and 12.00 hours, 7 days apart. Mares received either water (2 l), cranberry (,30 g in 2 l of water) or ginger (,30 g in 2 l of water) extract 1 h prior to testing. Blood samples were taken prior to dosing (pre-exercise), at the end of each step of the GXT, at the end of the exercise and at 2, 5 and 30 min, 1, 2, 4 and 24 h post-GXT. Plasma total protein (TP) concentration and haematocrit (HCT) were analysed immediately following the tests. Analysis of creatine kinase (CK) and aspartate aminotransferase (AST) was done commercially. There was no effect of treatment (P > 0.05) on VO2max, run-time to fatigue, core temperature, TP or HCT. CK was substantially elevated (P 0.05) in the ginger group at 4 h post-GXT. All CK levels returned to baseline 24 h post-GXT. No change (P > 0.05) was noted in AST. A slight increase (P 0.05) in CK was seen in all groups at 2 h post-GXT. The cranberry group had significantly lower TNF-a mRNA expression than the control and ginger groups. Ginger appeared to influence (P 0.05) the upregulation and expression of IFN-g mRNA at 30 min post-GXT, but, more strikingly, significantly decreased recovery time defined as the time for VO2 to recover from the peak observed at fatigue to a post-exercise plateau (ginger = 101 ± 3 s, water = 130 ± 14 s, cranberry = 131 ± 16 s). No effect of treatment or exercise (P > 0.05) was seen on IL-6 mRNA expression. Results suggest that cranberry extract blunts the upregulation and expression of TNF-a mRNA, while ginger extract reduces cardiovascular recovery time in horses completing a short, exhaustive bout of exercise.

It is well known that antioxidants present in various fruits, vegetables, and juices have the potential to protect the urinary bladder from free radical damage. What is not well understood, however, is how well antioxidant activities detected by chemical methods such as the CUPRAC assay for total antioxidant activity (TAA) predict the level of physiological protection available. It is hypothesized that the level of antioxidant reactivity found by the CUPRAC assay will positively correlate with increased protection in a model of in vitro ischemia/reperfusion. To test this hypothesis, the CUPRAC assay was utilized to determine the antioxidant reactivity of a series of fruits, vegetables, and juices, and the results were compared to the protective ability of selected juices in an established in vitro rabbit bladder model of ischemia/reperfusion. The results of the CUPRAC test showed that cranberry juice had the highest level of antioxidant reactivity, blueberry juice had an intermediate activity, and orange juice had the lowest. It was determined, however, that contrary to the hypothesis, the orange juice was significantly more potent in protecting the bladder against ischemia/reperfusion damage than either blueberry or cranberry juice. Thus, it is concluded that chemical tests for TAA do not necessarily correlate with their physiological activity.

Phenolics from the American cranberry (Vaccinium macrocarpon) were fractionated into a series of proanthocyanidins and other flavonoid compounds by vacuum chromatography on a hydrophilic, porous polyvinylic gel permeation polymer. Antioxidant activity was not restricted to a particular class of components in the extract but was found in a wide range of the fractions. Significant chemopreventive activity, as indicated by an ornithine decarboxylase assay, was localized in one particular proanthocyanidin-rich fraction from the initial fractionation procedure. Further fractionation of the active anticarcinogenic fraction revealed the following components: seven flavonoids, mainly quercetin, myricetin, the corresponding 3-O-glycosides, (-)-epicatechin, (+)-catechin, and dimers of both gallocatechin and epigallocatechin types, and a series of oligomeric proanthocyanidins.