Multi-radical (ORAC_MR5) antioxidant capacity of selected berries and effects of food processing

2.1Antioxidant capacity methods

ORAC_MR5 methods described by Nemezer and Ou [13] were used for antioxidant capacity assay using peroxyl (ORAC), hydroxyl (HORAC), superoxide anion (SORAC), peroxynitrite (NORAC), and singlet oxygen (SOAC) radicals/oxidants. Peroxyl radical scavenging capacity (ORAC) was measured as previously reported [6, 14]. Fluorescein was used as the fluorescent probe. Hydroxyl radical scavenging capacity (HORAC) assay was performed according to a report by Ou et al. [15]. Peroxynitrite scavenging capacity (NORAC) was determined by monitoring the oxidation of 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (DHR-123) [16].

Superoxide anion scavenging assay (SORAC) was performed as described by Zhang et al. [17]. Hydroethidine (HE) was used to measure the O₂^– scavenging capacity. The mixture of xanthine and xanthine oxidase was used to generate O₂^– radicals. Nonfluorescent HE was oxidized by O₂^– to form a species of unknown structure that emits fluorescence signal at 586 nm. Addition of superoxide dismutase (SOD) inhibits the HE oxidation.

Singlet oxygen scavenging capacity (SOAC) assay was designed to accurately quantify singlet oxygen activity in a high throughput fashion (Ou et al., unpublished data, ICT, Milford, MA). Singlet oxygen is generated in ethanol by the molybdate-catalyzed disproportionation of hydrogen peroxide at 37 °C. Hydroethidine (HE), a non-fluorescent probe, was oxidized by singlet oxygen to form oxyethidium which exhibits a strong fluorescence signal at 590 nm. Therefore, the inhibition of HE’s fluorescence increase in the presence of antioxidant is an index of antioxidant capacity. Results are expressed as “μmole TE (micromole Trolox equivalent)/gram or liter”. Using the conversion factor of 1 μmole vitamin E eq/μmole TE, the final results were calculated.

Total ORAC_MR5 (Sum of peroxyl (ORAC), hydroxyl (HORAC), superoxide anion (SORAC), peroxynitrite (NORAC), and singlet oxygen (SOAC) radicals/oxidants) expressed as μmoles TE per g freeze dried berry or per serving for berry smoothies and purees.

2.2Pure phenolic/antioxidant compounds

Twenty three pure compounds were purchased from commercial vendors for the analysis of ORAC_MR5 as follows: β-carotene, protocatechuic acid, ferulic acid (Alfa Aesar/Thermo Fisher Scientific Inc., Waltham, MA); lycopene, astaxanthin, quercetin, rutin, baicalin, catechin, epicatechin, epicatechingallate, epigallocatechin (EGC), EGC gallate, naringenin, neohesperidin, gallic acid, caffeic acid, chlorogenic acid, Vitamin C (Sigma-Aldrich Corp., St. Louis, MO); Kaempferol, (INDOFINE Chemical Company, Inc., Hillsborough, NJ); hesperetin, resveratrol (Quality Phytochemicals LLC, New Jersey); procyanidin A2, (Extrasynthese,Genay,Cedex, France). Samples were analyzed in triplicate and data were expressed as μmoles Trolox Equivalents (TE) per mg of pure compound.

2.3Processed samples

Processed samples for analysis were obtained from commercial sources. Smoothies [Blueberry/pomegranate (BPS) and Mango/pineapple MPIS)] were obtained from a local McDonalds restaurant. BPS contained the following ingredients: in a low-fat Yogurt, Blueberry Puree, Water, Clarified Demineralized Pineapple Juice Concentrate, Raspberry Puree, Apple Juice Concentrate, Pomegranate Juice Concentrate, and contained 1% or less of Natural (Plant Source) and Artificial Flavors, Cellulose Powder, Peach Juice Concentrate, Pear Juice Concentrate, Citric Acid, Lemon Juice Concentrate, Xanthan Gum, Pectin, Fruit and Vegetable Juice For Color [18].

The Mango Pineapple Fruit smoothie (MPS) base contained the following ingredients in a low fat yogurt: Water, Clarified Demineralized Pineapple Juice Concentrate, Mango Puree Concentrate, Pineapple Juice Concentrate, Orange Juice Concentrate, Pineapple Puree (Pineapple and Ascorbic Acid), Apple Juice Concentrate, Contained less than 1% : Cellulose Powder, Natural (Plant Source) and Artificial Flavors, Xanthan Gum, Pectin, Citric Acid, Fruit and Vegetable Juice for Color, Turmeric Extract (Color) [19].

Whole berry/fruit Fruition purees [Mango Passion Fruition (MPF); Wild Blueberry Fruition (WBF); Cherry Fruition (CF) were obtained from Wyman’s of Maine (Milbridge, ME). The Fruition products are 100% of either mango, wild blueberry or cherry that have been “gently pressed and flash frozen”.

Aronia Juice concentrate was obtained from Mae’s Health and Wellness, LLC, Omaha, NE. This product is a highly concentrated version of the raw aronia berry in a convenient juice form. The product is stored frozen.

2.4Percent polymeric color analysis

Percent polymeric color (PC) of purees was determined using the spectrophotometric assay of Giusti and Wrolstad [20]. Sample extracts were diluted with water in order to have an absorbance reading between 0.5 and 1.0 at 512 nm. For analysis, 0.2 mL of 0.90 M potassium metabisulfite was added to 2.8 mL diluted sample (bisulfite bleached sample) and 0.2 mL of DI water was added to 2.8 mL diluted sample (non-bleached, control sample). After equilibrating for 15 min, but not more than 1 h, samples were evaluated at λ= 700 nm, 512 nm, and 420 nm. Color density was calculated using the control sample according to the following formula:

    Color Density = [(A_420 nm – A_700 nm) + (A_512 nm – A_700 nm)]×Dilution Factor

Polymeric color was determined using the bisulfite-bleached sample using the following formula:

    Polymeric Color = [(A_420 nm – A_700 nm) + (A_512 nm – A_700 nm)]×Dilution Factor

Percent polymeric color was calculated using the formula:

    % Polymeric color = (polymeric color/color density)×100

3.1Processing effects on ORACMR5

The effects of processing berries into whole fruit purees or smoothies on the antioxidant capacity were also investigated using ORAC_MR5 methods. Whole fruit Fruition purees of mango, wild blueberry and cherry contained 95, 85 and 67% respectively of the total ORAC_MR5 as singlet oxygen (SOAC) radical antioxidant protection (Table 2). However, freeze dried fresh wild blueberry from the same production season (2014) (Table 2) had only 28% as SOAC and 42 and 22% as ORAC and HORAC, respectively. Data from fresh wild blueberries (Wild BB 2014) are presented as a comparison to that of Wild Blueberry puree in the radial plot (Fig. 4). The remarkable observation is the relatively high singlet oxygen capacity in processed (pressed/pureed) blueberry samples had 50% lower amounts for ORAC (peroxyl radical) in the processed samples relative to the freeze dried wild blueberry sample. Comparing the unprocessed freeze dried wild blueberry to the wild blueberry Fruition, on a μmole basis, the increase in SOAC more than accounts for by the decrease in ORAC and HORAC such that the total ORAC_MR5 is 1721 compared to 1206 μmoles TE/g DM for the Fruition compared to the freeze dried wild blueberry. Although with processing there did not appear to be a decline in total ORAC_MR5 in the blueberry Fruition compared to whole blueberry, there was a definite shift in the amount of protection from peroxyl (ORAC) and perhaps from hydroxyl radicals (HORAC) into protection against singlet oxygen. The explanation for this shift is not clear. Processing did not cause a large increase in polymeric color (∼18%) (Table 2), as we have observed these levels in freeze dried wild blueberries. There does not appear to be any major shift in the anthocyanin profile in the Wild Blueberry Fruition (data not presented).

Mango and Cherry Fruition samples also had antioxidant capacity primarily against singlet oxygen (95 and 85% respectively) (Table 2). Unprocessed samples of mango and cherry were not available to compare with the processed samples. Sweet and tart cherries were shown to have 54% and 67%, respectively, of the ORAC_MR5 as SOAC. Blackberries were also been shown to have high SOAC (66% of total) and a quite high ORAC_MR5 (Table 1).

Commercially available Blueberry/Pomegranate and Mango/Pineapple smoothies had 70% and 77% of total ORAC_MR5 as SOAC, while antioxidant capacity against the peroxyl radical (ORAC) was 25% and 12%, respectively (Fig. 5, Table 3). The ORAC from a serving of the blueberry/pomegranate smoothie provided 2213 μmoles TE of ORAC (Table 3) which is equivalent to the estimated average of a serving of fruits and vegetables [9]. The Mango/Pineapple smoothie had a lower ORAC but higher SOAC and ORAC_MR5 than the blueberry/pomegranate smoothie (Table 3, Fig. 5). On a per serving basis (87 mL), the Fruition products had total ORAC_MR5 of 19,000 to ∼37,000 umoles TE and the smoothies had values of ∼9,000 ORAC_MR5 but mostly as SOAC. A serving of either of these smoothies would make a significant contribution to the daily antioxidant capacity intake in the diet. The Fruition products had higher ORAC_MR5 relative to calories in a serving compared to the Blueberry/Pomegranate and Mango/Pineapple smoothies. This can be important since the higher the caloric intake the more ROS will likely be generated. Additional studies are needed to determine the processing conditions responsible for the changes in antioxidant capacity within ORAC_MR5.

3.2ORAC_MR5 of pure phytochemicals

Purified polyphenolic compounds found in many berries were also analyzed for ORAC_MR5 antioxidant capacity and the data is expressed as μmoles TE per mg of pure compound (Table 4) and SOAC and SORAC are presented in Figs. 6 and 7 and expressed as μmoles TE per μmole pure compound. There were large differences in antioxidant protection against the 5 oxidant sources. More than 95% of the ORAC_MR5 in vitamin C was as SORAC, (Tables 4). Using the peroxyl radical in the ORAC method, vitamin C was shown to have a relative value of 0.95 compared to Trolox [7], but levels in this study were non-detectable (Table 4).

The flavanols as a group had major SORAC protection as well as gallic acid (Table 4, Fig. 7). Green tea polyphenols were studied by Sato et al. [21] to determine structural characteristics for superoxide anion radical scavenging. Some structures among the green tea polyphenols were shown to actually produce superoxide anion as well as other structural characteristics had superoxide scavenging activity. In some cases the scavenging activity was stronger than the production activity. A net scavenging activity was observed for epigallocatechin-gallate, methyl-gallate, gallocatechin-epigallocatechin and epigallocatechin-epicatechin [21]. However, using our conditions of analysis, epigallocatechin and gallic acid were shown to have the highest superoxide anion quenching of the polyphenols (Table 4, Fig. 7) but epigallocatechin acted as a superoxide anion generator under the conditions used by Sato et al. [21]. Indoxyl sulfate has been shown to be an endogenous antioxidant with superoxide anion scavenging activity generated from activated neutrophils and also it has been shown to eliminate hydroxyl radicals [22]. Catecholamines have also been shown to have a direct effect on scavenging superoxide anion radicals [23]. Superoxide anion has been shown to be detrimental in some health related conditions. Physical frailty in older people is associated with superoxide anion overproduction by NADPH oxidase and low-grade chronic inflammation [24]. Diabetes-induced breakdown of the blood-retinal barrier (BRB) has been linked to hyperglycemia-induced expression of vascular endothelial growth factor (VEGF) and is likely mediated by an increase in oxidative stress mediated by superoxide anion [25]. Findings by Folbergrova and coworkers [26] suggested that increased superoxide generation precedes neuronal degeneration and may thus play a causative role in neuronal injury in the occurrence of oxidative stress in brain of immature rats during seizures. This may have clinical relevance for an approach for the treatment of epilepsy in children, suggesting that substances with antioxidant properties combined with the conventional therapies might provide a beneficial effect [26].

Astazanthin, beta-carotene and kaempferol had more than 90% of the ORAC_MR5 was as SOAC (Table 4). Astaxanthin is classified as axanthophyll. Singlet oxygen is produced by leukocytes during inflammatory and biochemical reactions and deactivated by producing reactive oxygen species and peroxides. It is generally accepted that free radical quenching is a key requirement for an efficient antioxidant. However, the antioxidant capacity does not depend solely on the efficiency of quenching/removal of the oxidizing free radical but also on the reactivity and the lifetime of the products of the quenching reaction [27]. While carotenoids are generally accepted as ‘antioxidants’, the reactivity/lifetime of such carotenoid radicals may lead to a switch from anti- to pro-oxidant behavior and such redox-controlled reactions may lead to deleterious as well as beneficial health effects [27]. Bohm et al. [27] have shown that in a mixture of carotenoids all carotenoid radical cations will be converted by lycopene back to the parent carotenoid and the lycopene may be sacrificed. It was noted that the radical cations arising from the three carotenoids present in the human macular (lutein, zeaxanthin and mesozeaxanthin) are all repaired efficiently by lycopene but not by β-carotene. Astaxanthin is being marketed as one of the most powerful antioxidants found in nature primarily because, unlike some other types of antioxidants, astaxanthin never becomes a pro-oxidant in the body so it can never cause harmful oxidation. Based upon the limited number of pure compounds that we analyzed, astaxanthin is definitely one that has the highest capacities for quenching singlet oxygen (Table 5), but it has low capacity for quenching the other radicals. Lycopene and beta-carotene have much lower SOAC and ORAC_MR5 than astaxanthin (Table 4, Fig. 6). The interaction of carotenoids with singlet oxygen species is important for the understanding of many important aspects of life such as photosynthesis, vision, various medical treatments from dermatology to cancer, as well as understanding any possible deleterious reactions affecting man.

Of the pure compounds analyzed (Table 4), procyanidin dimer-A2 had extremely high ORAC levels and asta xanthin had higher ORAC levels than most of the pure compounds analyzed (Table 4). Of the berries, blueberries, elderberries and Açaí fruit pulp had relatively high ORAC levels (Table 1) which has been documented previously [9]. ORAC levels in numerous fruits and vegetables have been published previously [9].

Nitric oxide is an important intracellular messenger molecule that activates guanylyl cyclase to convert guanosine triphosphate to cyclic GMP which causes smooth muscle relaxation [28]. Nitric oxide converts rapidly to NO₂ under aerobic conditions (Fig. 1). Nitric oxide is known to have beneficial effects on human health [29]. Its removal by antioxidants may lead to detrimental effects on health. In contrast, NO₂ is a strong radical oxidant and known only to cause damage to biomolecules and human health [30]. NO₂ can be converted to peroxynitrite or nitric oxide can react with superoxide anion to form peroxynitrite (Fig. 1). Peroxynitrite oxidizes protein thiols forming disulfide and can nitrate tyrosyl groups in proteins to form nitrated proteins leading to either a loss or gain of enzymatic activity or interfere with signal transduction mechanisms. Peroxynitrite (NORAC) protection was relatively low in all berry samples tested (<1–5% of ORAC_MR5 (Table 1). Most of the pure compounds tested also had relatively low antioxidant protection against peroxynitrite except baicalin and procyanidin dimer-A2 which had NORAC values which were 31.5 and 8.4% of ORAC_MR5, respectively (Table 4). Baicalin is a polyphenol flavonoid (flavone) naturally extracted from Scutellaria sp. (skullcap) including Scutellariabaicalensis and Scutellarialateriflora. It has numerous health promoting properties, including anti-inflammatory, calming, and sleep promoting benefits, and is contained in plants which have been used for centuries in traditional medicine.

Baicalin and procyanidin dimer-A2 were also compounds with relatively higher HORAC levels (Table 4). Berries that had relatively higher levels of HORAC included Aronia juice concentrate, Açaí and wild blueberry particularly from the 2013 production year. There is no data available suggesting that any of these berries have biacalin or procyanidin dimer-A2. Procyanidin dimer-A2 had much higher levels of ORAC and NORAC than HORAC (Table 4).

3.3Dietary antioxidant intake and health effects

Data available from the published literature [2] indicates that daily intakes in the range of 10–14,000 umoles TE antioxidant capacity against the peroxyl radical (ORAC) is sufficient to have beneficial health effects. A recent study [13] using a supplement mixture of berries and other ingredients that provided 44,000 units of ORAC_MR5 and 10,700 units of ORAC, was effective in deceasing oxidative stress following a meal. This study is the first reported that combined a mixture of antioxidant sources to arrive at a reasonable balance between the antioxidants and also to demonstrate an in vivo effect on lowering free radicals in the plasma following a meal.

In addition to studies referenced in a recent review [2], more recent studies have also demonstrated that increased intake of dietary antioxidants/phytochemicals can improve health outcomes. Increased dietary antioxidant intake was associated with less oxidative stress among men diagnosed with prostate cancer [31] as indicated by lower urinary 8-isoprostane concentrations. Ingestion of anthocyanin-rich beverages improved antioxidant enzyme activities and plasma antioxidant capacity, thus protecting the body against oxidative stress, a hallmark of ongoing atherosclerosis [32]. Diets naturally rich in polyphenols positively influence fasting and postprandial triglyceride-rich lipoprotein (TRL) concentrations and reduced oxidative stress. Marine long chain n-3 polyunsaturated fatty acids reduced TRLs of exogenous origin. Through their effects on postprandial lipemia and oxidative stress, polyphenols may favorably affect cardiovascular disease risk [33].

Ingestion of a phytonutrient-poor food and its individual fat/protein or sugar components increased plasma oxidative activity, which was not observed after ingestion of a kilojoule-equivalent phytonutrient-rich food [34]. A decrease in resting luminol enhanced whole blood chemiluminescence was observed following regular strawberry consumption which may suppress baseline formation of oxidants by circulating phagocytes and decrease the risk of systemic imbalance between oxidants and anti-oxidants and be one of mechanisms of the health-promoting effects of strawberry consumption [35]. The major antioxidant protection in strawberries is from peroxyl, hydroxyl and superoxide-anion radicals (Table 1).

Attenuated oxidative and inflammatory responses were observed with Montmorency tart cherry concentrate consumption which may be efficacious in combating post-exercise oxidative and inflammatory cascades that can contribute to cellular disruption. Additionally, consumption of Montmorency tart cherry concentrate may be beneficial in repeated days of cycling and conceivably in other sporting scenario’s where back-to-back performances are required [36].