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 1998. 

   
I. Introduction:  
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.

 

What characterizes a "Free Radical"?
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Unpaired electron in orbit around nucleus of atom

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Very unstable

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Caused by:

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Metabolic processes

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Cigarette smoke

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Smog

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Pesticides, drugs, etc

What radicals are made in biological systems?
bullet Peroxyl radical (ROO.), which is the most common radical in biological systems.
bullet Hydroxyl radical (.OH), which is always harmful.
bullet Superoxide radical (O2.-), which is produced by phagocytic cells and can be beneficial in that it inactivates viruses and bacteria.
bullet 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.
bullet Hydrogen peroxide(H2O2), 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 (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 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 J. 11:A649.


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.


VII. Acknowledgements:
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

The Author: 

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