Free radicals are produced as products of normal cellular oxidative
metabolism through the following metabolic processes:
O2 (Oxygen) č O2.- č
H2O2 č .OH č H2O
(water)
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 later.
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 capacity.
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.
III. Health
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.
IV. Evaluation
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 as bilberry.
V.
Conclusions:
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 ˝ 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.
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