|Antioxidant Capacity and
Health Benefits of Fruits and Vegetables
Blueberries, the Leader of the Pack
Ronald L. Prior, Ph.D.
USDA Human Nutrition Research Center on Aging
The following presentation
was given at the NABC meeting in Portland, Oregon in February
|For many years the free radical hypothesis
of aging has been utilized to suggest that age-related changes occur
as a result of an increasing inability to cope with oxidative stress
(OS) that occurs throughout the life-span and is associated with
increasing sensitivity to the effects of oxidative stressors. Oxidative
stress is defined as an imbalance between oxidants and antioxidants
in favor of the former, resulting in oxidative damage to molecules
such as DNA, lipids and proteins. After life-long free-radical insult
on an organ which already shows increased vulnerability to OS, functional
deficits are observed.
characterizes a "Free Radical"?
electron in orbit around nucleus of atom
are made in biological systems?
||Peroxyl radical (ROO.),
which is the most common radical in biological systems.
||Hydroxyl radical (.OH), which is always harmful.
||Superoxide radical (O2.-), which is produced by phagocytic cells
and can be beneficial in that it inactivates viruses and bacteria.
||Nitric oxide (NO.), which has beneficial effects in
that it is a vasodilator agent, can function as a neurotransmitter
and can be produced by macrophages and act to kill parasites.
Nitric oxide may also be harmful when it reacts with superoxide
to for the peroxynitrite anion.
which is not a free radical, but can cause damaging oxidative
events in cells.
|Free radicals are produced as products of normal
cellular oxidative metabolism through the following metabolic processes:
|O2 (Oxygen) è O2.- è H2O2 è .OH è H2O
The normal 70 kg human would utilize about 3.5 mL oxygen/kg/min
and if one assumed that about 1% of the oxygen is converted
to the superoxide radical, about 1.72 kg or 3.8 pounds of superoxide
radical would be produced per year.
An antioxidant, is any substance that, when present at low
concentrations compared to those of an oxidizable substrate,
significantly delays or prevents the oxidation of substrates.
The normal antioxidant defense systems in biological systems
consists of both enzymatic and nonenzymatic systems. Although
both are important in biological systems, as we consider the
role of food antioxidants, we will be considering the nonenzymatic
antioxidant systems which include substances such as: a-tocopherol (Vitamin E), ascorbic acid (Vitamin C), glutathione,
flavonoids, b-carotene (Vitamin A precursor),
uric acid and plasma proteins such as albumin, ceruloplasmin,
transferrin, metalothionein, etc.
We have developed a method called the oxygen radical absorbance
capacity (ORAC) assay which depends on the unique properties
of the protein, phycoerythrin (PE). The ORAC assay is, to date,
the only method that takes reaction of the free radical reactive
species to completion and uses an "area under the curve" (AUC)
technique for quantitation, and thus combines both inhibition
time and inhibition percentage of the reactive species action
by antioxidants into a single quantity. The ORAC assay depends
on the detection of chemical damage to R- or B-PE through the
decrease in its fluorescence emission. The fluorescence of PE
is highly sensitive to the conformation and chemical integrity
of the protein. Under appropriate conditions, the loss of PE
fluorescence in the presence of reactive species is an index
of oxidative damage of the protein. The inhibition of the reaction
by an antioxidant, which is reflected in the protection against
the loss of PE fluorescence in the ORAC assay, is a measure
of its antioxidant capacity against the reactive species.
II. Antioxidant capacities of fruits and vegetables:
|Studies from our laboratory represent the first
attempt to measure total antioxidant capacity of fruits and vegetables
using the ORAC procedure (Wang et al 1996; Cao et al 1996). The
antioxidant capacity of common fruits and vegetables, and drinks
including green and black teas, commercial fruit juices and wines
were measured with the automated ORAC assay using a peroxyl radical
generator (ORACROO.) (Figure 1). Based upon
the weight of edible portion of the fruit, prunes, raisins, blueberry
and blackberry had an ORAC activity of over 20 expressed as mmole
Trolox eq./g followed by strawberry, plum, orange, red grape, kiwifruit,
pink grapefruit, white grape, banana, apple, tomato, pear, and honeydew
melon (Figure 1)(Wang et al 1996). Based upon the fresh weight of
vegetables, garlic had the highest antioxidant capacity against
peroxyl radicals (ORACROO.), followed by kale,
spinach, brussel sprouts, alfalfa sprouts and others (Table1). It
was calculated that the contribution of vitamin C to the total ORACROO. activity of these fruits was usually less than 15%, except for kiwifruit
and honeydew melon. This suggests that the major source of antioxidant
capacity of most fruits, and commercial fruit juices may not be
from vitamin C, but from other "unknown" antioxidants contained
in fruits. The nature of these phytochemicals will be discussed
|The measured ORAC values of common fruits and vegetables
are summarized in Figure 1. From these values, the amount of ORAC
activity in common serving sizes of fruit and vegetables was calculated
. Amounts in common servings range from a low of 28 to a high of
2289 mmol Trolox equivalents (TE). Data
from the 1989-91 Continuing Survey of Food Intake by Individuals
by the USDA was used to calculate a mean ORAC intake from fruits
and vegetables (Table 2) which was estimated to be 1232 (Table 2).
Although a number of assumptions went into these calculations, the
calculated ORAC intake seems to be a reasonable estimate. In a group
of 32 subjects (males, females, young and old) average ORAC intake,
based upon results from a food frequency questionnaire, was 1670±200.
The average servings of fruits and vegetables in this population
group was 5.0±0.4 which is higher than the number of servings observed
in much larger population surveys. Although the National Research
Council has recommended consumption of five servings of fruits and
vegetables daily, the NHANES II survey indicates that only 10% of
the US population consumed that number. When subjects from the above
study (Cao et al, unpublished data) were fed a 3-day rotating diet
of 10 servings of fruits and vegetables per day, average ORAC intake
was 3246±10. No special attempt was made in this study to include
fruits or vegetables that were particularly high in antioxidant
|ORAC intake which was calculated assuming an individual
consumed 3 servings of fruit and 4 servings of vegetables containing
low (melon, pear, apple, cucumber, lettuce, carrot, bean), medium
(plum, banana, white grape, potato, corn, beets) or high (blueberry,
strawberry, orange, spinach, kale, corn, beets) ORAC. Under these
conditions, total ORAC intake from fruits and vegetables was 1294,
2947 and 6875 respectively. These computations point out that the
estimated usual ORAC intake in the U.S. (1200-1700 mmol
Trolox eq.) is likely to be on the low end of the spectrum (1200-6000),
and could be increased 2- to 4-fold by the selection of a slightly
different mix of fruits and vegetables. In fact, in the study of
Cao et al (1997), ORAC intakes as high as 6000 were observed in
some individuals, which were explained by the subjects" inclusion
of prunes in their diets. Based upon data in Table 1, a dramatic
increase in ORAC intake can most easily be accomplished by increasing
consumption of fruits such as oranges, plums, strawberries, blackberries,
blueberries, raisins and prunes. Increasing the intake of any of
these by one serving per day could double the average ORAC intake.
Increased consumption of drinks such as grape, grapefruit or orange
juices or red wine would also markedly increase ORAC intake. Increases
in consumption of vegetables such as kale, spinach, brussel sprouts,
broccoli and beets will increase ORAC intake, but will require increases
in the number of servings from several of these vegetables to have
a similar impact as a single fruit might have.
|A reasonable question resulting from these observations
is whether increased consumption of foods high in ORAC would have
any health implications. In order to determine if there are health
implications, one needs to understand 1) what phytochemicals in
fruits and vegetables are responsible for the ORAC activities measured,
2) whether these substances can be absorbed and 3) what physiological
responses might be altered following their absorption.
Benefits of Consumption of Fruits and Vegetables:
|Cancer. The consumption of fruits
and vegetables has been associated with lower incidence and lower
mortality rates of cancer in several human cohort and case-control
studies for all common cancer sites (Ames et al 1993; Doll 1990;
Dragsted et al 1993; Willett 1994a).
|Steinmetz and Potter (1991a, 1991b, 1996) reviewed
the scientific literature from 206 human epidemiological studies
and 22 animal studies on the relationship between vegetable and
fruit consumption and the risk of cancer. Evidence for a protective
effect of greater vegetable and fruit consumption is consistent
for cancers of the stomach, esophagus, lung, oral cavity and pharynx,
endometrium, pancreas and colon. The types of vegetables or fruits
that most often appeared to be protective against cancer were raw
vegetables (85% of studies), followed by allium vegetables (onions,
garlic, scallions, leeks and chives), carrots, green vegetables,
cruciferous vegetables (i.e. broccoli, cauliflower, brussel sprouts
and cabbage) and tomatoes; for each of these latter groups, 70%
or more of the studies showed a protective association. In an earlier
review, Block et al (1992) reviewed approximately 200 studies for
the relationship between fruit and vegetable intake and cancers
of the lung, colon, breast, cervix, esophagus, oral cavity, stomach,
bladder, pancreas and ovary. For most cancer sites, individuals
with low fruit and vegetable intake experienced about twice the
risk of cancer compared with those with high intake. A statistically
significant protective effect of fruit and vegetable consumption
was found in 128 of 156 dietary studies in which results were expressed
in terms of relative risk. Thus, the scientific evidence regarding
a role for vegetable and fruit consumption in cancer prevention
is generally consistent and is supportive of current dietary recommendations.
However, what is not clear from the available literature is what
phytochemicals are responsible for the anticarcinogenicity and if
specific fruits or vegetables might be more effective than others
in preventing age-related diseases.
|Cardiovascular Disease: A highly significant negative
association between intake of total fresh fruits and vegetables
and ischemic heart disease mortality was reported by Armstrong and
coworkers (1975) in Britain and by Verlangieri and coworkers (1985)
in the United States. A significant negative association was also
reported between fruit and vegetable consumption and cerebrovascular
disease mortality (Acheson and Williams 1983). Other epidemiological
data, human clinical trials, and animal studies suggest that dietary
antioxidants and diets rich in vegetables and fruits increase longevity,
and decrease cardiovascular disease.
|Health Promoting Effects of Bilberry: Several studies
have been undertaken with a highly purified extract of V. myrtillus L., designated Myrtocyan®, which contains 36% anthocyanosides
(Morazzoni and Bombardelli, 1996). Cyanidin 3-glucoside, a major
component of Myrtocyan®,was shown to be the most active
compound tested against carbon tetrachloride induced lipoperoxidation
(Morazzoni and Bombardelli, 1996) and was the anthocyanin with the
highest ORAC that we tested (Wang et al., 1997). In addition to
the antioxidant activity, Myrtocyan® has been shown to
1) prevent or control interstitial fluid formation and contribute
to controlling the blood flow redistribution in the microvascular
network, 2) modulate capillary resistance and permeability, improving
visual function by promoting dark adaptation after dazzling, 3)
promote wound-healing and 4) have anti-ulcer and anti-atherosclerotic
activity (Morazzoni and Bombardelli, 1996). However, studies have
not been done to determine whether consumption of anthocyanins from
other Vaccinium species might have similar health benefits.
of Antioxidant Properties of Blueberries:
|ORAC, total anthocyanins, total phenolics,
and vitamin C: Blueberries are of particular interest because
of their high antioxidant capacity. However, we anticipated that
the antioxidant capacity might vary considerably because of the
wide range of reported anthocyanin concentrations.
|Total antioxidant capacity, measured
as ORAC, ranged from a low of 13.9 to 44.6 mmol
TE/g fresh berries in the acetonitrile extracts of the different
cultivars of blueberries (Tables 1 and 2). The overall mean of all
commercially available cultivars was 24.0±2.0. The highbush varieties
of "Rancocas", "Rubel", "Bladen", and the late harvest on the rabbiteye
cultivars "Tifblue" and "Brightwell" had ORAC values (i.e. 32.4,
37.1, 42.3, 37.8, and 34.3 respectively) that approached that observed
for the Bilberry (44.6). There appears to be two clusters of ORAC
values in the lowbush blueberries. The first included lowbush from
Prince Edward Island and Nova Scotia, and Fundy lowbush blueberries
which were relatively high in ORAC (mean: 41.8), anthocyanins, and
total phenolics. The second cluster included lowbush from Maine,
"Cumberland", and "Blomidin"lowbush blueberries which were lower
in ORAC (mean: 27.5). At this point, it is not clear as to the source
(genetics, location, maturity, etc.) of this variation. Anthocyanins
in the lowbush blueberries were not as high as the bilberry relative
to ORAC values as reflected in the ratio of anthocyanins to ORAC
(0.37 vs. 0.57).
|The relationship between ORAC and total
anthocyanin or total phenolic content in all these different blueberry
samples is presented in Figures 1 and 2. A significant linear relationship
was observed between ORAC and total anthocyanin or total phenolic
content. The correlation coefficient was much higher between ORAC
and total phenolics (rxy = 0.85) compared to ORAC and
anthocyanins (rxy = 0.77) (Figures 1 and 2).
|Ascorbate concentrations (1.3-16.4 mg/100
g) showed a significant variability between cultivars and species.
Although most of the samples had an ascorbate concentration between
9-16 mg/100 g, no consistent pattern emerged relative to ORAC or
anthocyanins or to total phenolics. Using an ORAC value for ascorbate
of 5.6 mmol TE/g, it was calculated that the antioxidant capacity
contributed by ascorbate to the total antioxidant capacity, measured
as ORAC, was 2.3% for the highbush and rabbiteye berries. Ascorbate
in lowbush berries contributed only 1.5% while in the bilberry sample,
the contribution of ascorbate to ORAC was only 0.2%. Thus, it is
clear that ascorbate does not make a major contribution to the antioxidant
capacity of any of the blueberries sampled. In calculations with
other fruits, ascorbate has generally contributed less than 10%
of the total antioxidant capacity (Wang et al., 1996).
|Maturity effects: Maturity at
harvest had a marked effect on ORAC, total anthocyanins and total
phenolics of the berries, for the "Brightwell" and "Tifblue" cultivars
of rabbiteye blueberries which were the only two cultivars evaluated.
Berries harvested immediately after turning blue had lower ORAC
and total anthocyanins than berries well matured that were harvested
49 days later. ORAC and total anthocyanins increased 224% and 261%
respectively, in the "Brightwell" cultivar, while in "Tifblue" they
increased 164% and 176% respectively, with increasing maturity.
Total phenolics increased by 169% and 113% in the "Brightwell" and
"Tifblue" cultivars, respectively, with increased maturity.
|The results presented in this presentation
represent the first data on the total antioxidant capacity in blueberries.
On a fresh weight basis, blueberries have the highest antioxidant
capacity of all the fresh fruits and vegetables tested to date.
However, considerable variability seemed to exist among the initial
analyses that were performed on blueberry samples obtained from
the commercial supermarket, suggesting that variation exists in
the antioxidant capacity of different varieties of the Vaccinium species. We have previously analyzed the antioxidant capacity
of anthocyanins (Wang et al., 1997) and other flavonoids (Cao et
al., 1997) and found them to have 2 to 6 times the activity found
in common antioxidants such as ascorbate, glutathione, etc. Thus,
in our current studies, we also determined the anthocyanin and total
phenolic concentrations in the different blueberry samples. Previous
reports of anthocyanin content in blueberries have also indicated
a large variation (Mazza and Miniati, 1993). Highbush blueberries
have been reported to have an anthocyanin content of 25-495 mg/100
g (Mazza and Miniati, 1993). Highbush blueberry (V. cormbosum
L.) and lowbush blueberry (Vacccinium angustifolium Ait.)
are the primary species of blueberries used by the food industry
in the United States. Rabbiteye blueberries (Vacccinium ashei Reade) grown in the southern U.S., have been reported
to have an anthocyanin content of 210- (Tifblue) to 272- (Bluegem)
mg/100 g (Gao and Mazza, 1994). Gao and Mazza (1994) reported, using
HPLC techniques to measure anthocyanins, that most lowbush blueberry
cultivars contained 150-200 mg anthocyanins/100 g and highbush blueberry
samples contained about 100 mg anthocyanins/100 g. Bilberry (Vacccinium
myrtillus L.), native to parts of Europe and northern regions
of Asia, has been reported to have the highest anthocyanin content
(300-698 mg anthocyanin/100 g)(Mazza and Miniata, 1993). Lowbush
blueberries (V. angustifolium Ait.), which are grown in Maine
and Eastern Canada, are reported to have about 138 mg anthocyanins
per 100 g (Kalt and McDonald, 1996). We observed anthocyanin concentrations
in the range of 62 mg/100 g for "Reveille" blueberries to 300 mg/100
g for bilberries (V. myrtillus L.). Our results in general
seem to be a little lower than some of the other reports; however,
the particular anthocyanin compound used as a standard and its associated
molar absorption coefficient can influence the absolute amounts
calculated. The 3-glucoside(s) and 3-galactoside(s) of delphinidin,
malvidin, petunidin, cyanidin and peonidin are the primary anthocyanins
that have been identified in blueberries (Mazza and Miniati, 1993;
Gao and Mazza, 1994). Bilyk and Sapers (1986) found that 4 varieties
of highbush blueberry ("Earliblue", "Weymouth", "Coville" and "Bluetta")
had total anthocyanin concentrations that varied by about 15%. Anthocyanin
content of the different blueberry samples was linearly related
to the ORAC measurement (rxy = 0.77; p<0.01) (Fig.
2), however, the agreement as indicated by the correlation coefficient
was not as high as between total phenolics and ORAC (rxy = 0.85; p<0.01) (Fig. 3) although both were significant.
|The phytochemicals responsible for the
antioxidant capacity most likely can be accounted for by the phenolic
acids, anthocyanins and other flavonoid compounds (Cao et al., 1997).
We are in the process of identification of the compounds represented
in our HPLC chromatograms, the results of which will be published
at a later time.
|The polyphenolic components present
within blueberries may have multiple health benefits which at this
point are difficult to understand. The potential beneficial effects
of the high antioxidant capacity and protection of cells from free
radical attack seem clear, but other possible effects which might
be independent of antioxidant effects remain open to question. Anthocyanins
in blueberries may have potential health benefits that are independent
of or in addition to their antioxidant effects. Based upon our measurements
of antioxidant capacity, other Vaccinium species might be
equally good as a source of anthocyanins and other antioxidants
|Studies are continuing in our laboratory
of the implications of consuming foods containing increased quantities
of ORAC. Increased plasma ORAC has been observed in humans following
a single meal containing 3.4 mmole ORAC from either strawberries,
spinach or the phenolics in red wine (Cao et al., unpublished data).
Also, an increase in serum ORAC was observed following a diet change
from 5 servings of fruits and vegetables per day to 10 servings
per day (Cao et al., unpublished data). The antioxidant rich phytochemicals
in strawberries have been shown in rat models to reduce or retard
the central nervous system deficits seen in aging (Bickford et al.,
1997). Dietary supplementation to rats of an extract from blueberries
or strawberries has been shown to protect against the oxidative
stress caused by 100% oxygen exposure (Sofic et al., 1997; Cao et
al., unpublished data). Since the antioxidant capacity of blueberries
is higher than for strawberries, a benefit of consuming antioxidants
from blueberries would also be expected. Furthermore, consumption
of a more concentrated source of antioxidants will have the greatest
impact on in vivo antioxidant capacity. Consumption of ½ cup of
blueberries per day (72.5 g) would increase ORAC intake by 1-3.2
mmol, depending upon the blueberry variety and maturity. Blueberries
are one of the richest sources of antioxidant phytonutrients of
the fresh fruits and vegetables we have studied.
VI. Selected References:
|Acheson, R.M.; Williams, D.R.R. (1983)
Does consumption of fruit and vegetables protect against stroke? The Lancet 1(8335): 1191 - 1193.
|Armstrong, B.K.; Mann, J.I.; Adelstein,
A.M.; Eskin, F. (1975) Commodity consumption and ischemic heart
disease mortality, with special reference to dietary practices. J..Chron. Dis. 28 : 455 - 469
|Bickford, P.C., Chadman, K., Taglialatea,
G., Shukitt-Hale, B., Prior, R.L., Cao, G. and Joseph, J.A. (1997)
Dietary strawberry supplementation protects against the age-accelerated
CNS effects of oxidative stress. FASEB J. 11:A176.
|Block, G.; Patterson, B.; Subar, A.
(1992) Fruit, vegetables, and cancer prevention: A review of the
epidemiological evidence. Nutrition and Cancer 18:1-29.
|Cao, G., Alessio, H.M. and Culter, R.G.
(1993) Oxygen-radical absorbance capacity assay for antioxidants.
Free Radic. Biol. Med. 14:303-311.
Cao, G., Verdon, C.P., Wu, A.H.B.,
Wang, H. and Prior, R.L. (1995) Automated oxygen radical absorbance
capacity assay using the COBAS FARA II. Clin. Chem. 41:1738-1744.
|Cao, G., Sofic, E. And Prior, R.L. (1996)
Antioxidant capacity of tea and common vegetables. J. Agric.
Food Chem. 44:3426-3431.
|Cao, G., Sofic, E. and Prior, L. R.
(1997) Antioxidant and prooxidant behavior of flavonoids: structure-activity
relationships. Free Radic. Biol. Med. 22:749-760.
Doll, R. (1990) An
overview of the epidemiological evidence linking diet and cancer. Proc. Nutr. Soc. 49:119-131.
Dragsted, L.O.; Strube, M.; Larsen, J.C. (1993)
Cancer-protective factors in fruits and vegetables: biochemical
and biological background. Pharmacology & Toxicology 72: (suppl 1) 116-135.
Gao, L. & Mazza, G. (1994) Quantitation and
distribution of simple and acylated anthocyanins and other phenolics
in blueberries. J. Food Sci. 59(5):1057-1059.
Kalt, W. and McDonald, J.E. (1996) Chemical composition of lowbush
blueberry cultivars. J. Amer. Soc. Hort. Sci. 121:142-146.
Mazza, G. and Miniati, E. (1993) Anthocyanins in Fruits, Vegetables,
and Grains. CRC Press, Boca Raton, 362 pp.
Morazzoni, P. and Bombardelli, E. (1996) Vaccinium myrtillus L. Fitoterapia LXVII:3-29.
Sofic, E., Cao, G., Shukitt-Hale, B., Joseph, J.A.,.
Prior, R.L and Bickford, P.C. (1997) Effects of 100% oxygen exposure
on the antioxidant status of rats fed a diet supplemented with a-tocopherol or strawberry extract. FASEB
Steinmetz, K.A.; Potter, J.D. (1991a) Vegetables, fruit, and cancer,
I: Epidemiology. Cancer Causes Control 2:325-357.
Steinmetz, K.A.; Potter, J.D. (1991b) Vegetables, fruit, and cancer,
II: Mechanisms. Cancer Causes Control 2:427-442.
Steinmetz, K.A.; Potter, J.D. (1996) Vegetables, fruit, and cancer
prevention: A review. J. Am. Dietetic Assoc. 96:1027-1039.
Verlangieri, A.J.; Kapeghian, J.C.; el-Dean, S.; Bush, M. (1985)
Fruit and vegetable consumption and cardiovascular mortality.
Medical Hypothesis 16:7 - 15.
Wang, H., Cao, G. and Prior, R.L. (1996)
Total antioxidant capacity of fruits. J. Agric. Food Chem. 44:701-705.
Willett, C.W. (1994) Diet and health: what should we eat? Science 264: 532 - 537.
|The collaboration of the following is
acknowledged: Dr. Guohua Cao, Dr. Antonio Martin, Dr. Emin Sofic,
John McEwen, Christine O"Brien, Neal Lischner, Dr. Mark Ehlenfeldt,
Dr. Willy Kalt, Dr. Gerard Krewer, and Dr. C. Mike Mainland. In addition to samples provided by the collaborators, appreciation
is expressed to the following for providing blueberry samples for
these studies: Chad Finn, USDA-ARS, Hort Crops Res. Lab., 3420 NW
Orchard Ave., Corvallis, OR 97330 (Jersey); Dave Trinka, MBG Marketing,
04726 CR 215, Grand Junction, MI 49056 (Jersey, Bluecrop, Rubel,
Little Giant); Denny Doyle, Tru Blu CoOp, 194 Magnolia-New Lisbon
Rd., New Lisbon, NJ 08064 (Lowbush); Sonja and Wilhelm Dierking,
Wilhelm Dierking Beernobst, OT Nienhagen, Gilton, Germany D-29690
(Bilberry); Bill Whaley, Berryhill Foods, 30360 Old Yale Road, Abbotsford,
British Columbia, Canada V4X 2N7 (Rancocas). Supported in part by
funds from the North American Blueberry Council, 4995 Golden Foothill
Parkway, Suite #2, El Dorado Hills, CA 95762
Ronald L. Prior, Ph.D.
USDA Human Nutrition Research Center on Aging
711 Washington Street
Boston, MA 02111
Copyright 2002 - U.S. Highbush Blueberry Council