Evaluation location: Oregon, United States
Abstract:
Fruits from 107 genotypes of Vaccinium L., Rubus L., and Ribes L., were analyzed for total anthocyanins (ACY), total phenolics (TPH), and antioxidant capacities as determined by oxygen radical absorbing capacity (ORAC) and ferric reducing antioxidant power (FRAP). Fruit size was highly correlated (r ) 0.84) with ACY within Vaccinium corymbosum L., but was not correlated to ACY across eight other Vaccinium species, or within 27 blackberry hybrids. Certain Vaccinium and Ribes fruits with pigmented flesh were lower in ACY, TPH, ORAC, and FRAP compared to those values in berries with nonpigmented flesh. ORAC values ranged from 19 to 131 ímol Trolox equivalents/g in Vaccinium, from 13 to 146 in Rubus, and from 17 to 116 in Ribes. Though ACY may indicate TPH, the range observed in ACY/TPH ratios precludes prediction of ACY from TPH and vice versa for a single genotype. In general, TPH was more highly correlated to antioxidant capacity than ACY was. This study demonstrates the wide diversity of phytochemical levels and antioxidant capacities within and across three genera of small fruit.
Safety. There is an explosion hazard as liquid nitrogen becomes gaseous. For proper venting procedures during liquid nitrogen milling
of frozen materials, refer to Rodriguez-Saona and Wrolstad (15).
Sampling Procedures. Ripe fruit samples, as judged by flavor and color, were harvested during summer 2000 from two Willamette Valley sites: Oregon State University North Willamette Experiment Station (Aurora, OR) and the U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository (Corvallis, OR). Approximately 60 g of fruit was collected from 1 to 4 clones of each genotype. The highbush blueberry, V. corymbosum L. cv. Summit, was picked very early and very late in its season. Fruit was placed immediately on ice in the field and frozen at -10 C later that same day. Care was taken to avoid unripe, damaged, or overripe fruit. Samples were prepared according to Rodriguez-Saona and Wrolstad (15). About 40 g of berries were counted as each sample was weighed to determine average berry size. The frozen fruits were further cooled in liquid nitrogen; then they were cryogenically milled in a stainless Waring blender jar containing a lid modified with a chimney. Chilled tubes were filled with milled fruit powder and weighed, and then the powder was extracted with acetone, followed by two additional extractions with 70:30 acetone/water. The pooled supernatants were partitioned with two volumes of chloroform. The nonpolar phase was discarded, and the aqueous extracts were stored at -10 C or at -70 C if for antioxidant analysis.
Determination of Total Anthocyanins (ACY). Anthocyanin quantitation was performed by the pH differential method of Giusti and
Wrolstad (16). Samples were diluted 1:150 in pH 1.0 and pH 4.5 buffers, then measured at 520 and 700 nm in a Shimadzu 300 UVVisible spectrophotometer. ACY was based on a cyanidin 3-glucoside molar extinction coefficient of 26,900 and a molecular weight of 449.2. Resultant values were expressed in terms of mg of anthocyanin/100 g of fresh-frozen fruit.
Determination of Total Phenolics (TPH). The Folin-Ciocalteu method (17) was used to determine total soluble phenolics (TPH).
Extracts were diluted 1:500 or 1:1000 before incubation at 40 C. Absorption was measured at 755 nm. TPH was expressed as mg of gallic acid/100 g of fresh-frozen fruit.
Determination of Antioxidant Capacity. Antioxidant capacity was determined by ORAC and FRAP assays at the Linus Pauling Institute, Oregon State University. The ORAC assay was performed as described by Cao et al. (18) and adapted for use in a 96-well microplate fluorometer (model Cytofluor 4000, PerSeptive Biosystems, Framingham, MA). ORAC values, derived from triplicate analyses, are expressed as ímol Trolox equivalents (TE) per g of fresh-frozen fruit.
Trolox is a water-soluble tocopherol analogue used as a reference compound for antioxidant capacity. The FRAP assay (19) was adapted
for use in a 96-well microplate spectrophotometer (ThermoMax, Molecular Devices, Foster City, CA). FRAP values, derived from
triplicate analyses, are expressed as ímol of ferric iron reduced per g of fresh-frozen fruit.
Statistical Analysis. Correlation and regression analyses were performed using Microsoft Excel Data Analysis. Differences at p > 0.05 were considered significant.