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M.S. Padda and D.H. Picha

Sweetpotatoes may be potentially high in concentration of certain phytochemical compounds, including phenolics. Low temperature stress-induced phenolic compounds may enhance the nutraceutical value of sweetpotatoes. However, extended exposure to low temperature results in chilling injury. Cured and non-cured roots of `Beauregard' sweetpotatoes were exposed to low temperature storage (5 °C) for up to 4 weeks. The total phenolics and individual phenolic acid contents were determined at weekly intervals using Folin-Denis reagent and reversed-phase HPLC, respectively. Total phenolics and individual phenolic acids increased with length of low temperature exposure. Non-cured roots had a higher phenolic content than cured roots after 4 weeks. A 3-day exposure period to room temperature (22 °C) following removal from low temperature storage typically resulted in increased phenolics. In a comparison of different tissue locations, the highest phenolic content was found in peel tissue and the lowest in the pith tissue. The major individual phenolic acid in all root tissues was chlorogenic acid.

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Michael Cavalier, Armen Kachatryan, Evodokia Menelaou, Jack Losso, and Don LaBonte

Fresh leaves of six sweetpotato [Ipomoea batatas (L.) Lam.] genotypes, `Beauregard', `Bienville', L 99-35, L 00-8, L 01-145, and L 01-29 were characterized for lutein. Lutein is a carotenoid capable of delaying blindness-related macular degeneration. The content of lutein in sweetpotato ranged from 0.38 to 0.58 mg·g-1 fresh weight. Beta-carotene separated from lutein on HPLC chromatograms, and, when spiked in pure lutein extract, did not interfere with lutein separation. Stems were also characterized and found not to contain lutein. Our results showed that sweetpotato leaves are an excellent source of dietary lutein and surpass levels found in leafy crucifers. Leaves of sweetpotato and a related species are used as human food in some countries and could be a source of extracted lutein for commercial purposes.

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Roseann Leiner, Abraham Smyth, Rudy Candler, and Patricia S. Holloway*

Berries and vegetables can be sources of beneficial phytochemicals that may have antioxidant activity in the human diet. Information on type and quantity of phytochemicals may open new crop opportunities for berries and vegetables harvested in Alaska. A method was developed for detecting ascorbic acid and eight phenolic acids on an HPLC instrument using a reverse phase Merck Chromolith C18 column. The method used UV absorbance detection at 280nm to separate a standard solution of the following nine phytochemicals: ascorbic acid, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxyphenylacetic acid, caffeic acid, syringic acid, p-coumaric acid and ferulic acid. The mobile phase was a mixture (3.5% to 14% gradient) of organic solvent (5 parts acetonitrile: 2 parts methanol) and aqueous solvent (2 mmol aqueous trifluoroacetic acid - TFA) at a flow rate of 2 mL/min. In 2003, over 60 samples of berries and 60 samples of baby greens were extracted and analyzed. Plant samples were extracted by blending 10-20g of frozen plant tissue with 5 parts TFA. The extracts were centrifuged, diluted 4:1 and filtered (0.2 μm). Chromatograms from HPLC analysis for all samples were complex in peak size and number. Chromatograms for six extracts of high bush cranberries, Viburnum edule, exhibited intense peaks that indicate the presence of caffeic acid, based on retention times. Chromatograms for seven extracts of rose hips, Rosa acicularis, exhibited peaks that indicate the presence of ascorbic acid, based on retention times. Gallic acid and p-hydroxybenzoic acid are apparent minor components in the leaves of some baby greens. This research will continue in 2004 with more plant samples and further method development for detection of other phytochemicals.

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W.Y.L. Poon and I.L. Goldman

Carotenoids have been shown to be important both nutritionally and medicinally. Carotenoid accumulation was compared during growth and storage of four carrot genotypes: YY y1y1y2y2RPRP, yyY1 Y1Y2Y2 RPRP, YY Y1 Y1Y2Y2 RPRP, and rprp. These genotypes exhibit orange, yellow, white, and pale-orange roots respectively. The orange and pale-orange genotypes are near-isogenic for rp, a gene that reduces total carotenoid content by 93%. Genotypes were grown in replicated field plots during 1996 and stored for 8 months at 4°C. Samples of root tissue were removed at 7-day intervals during vegetative growth and 4-week intervals during the postharvest period. Total carotenoid content were quantified using HPLC and spectrophotometric analyses. Increases in carotenoid content of 119% and 79% in rprp and YY y1y1y2y2RPRP and decreases of 6% and 64% in YYY1 Y1Y2Y2RPRP and yyY1 Y1Y2Y2RPRP, respectively, were measured between 62 and 100 days after planting. At 100 days after planting, YY y1y1y2y2RPRP exhibited 10-fold greater carotenoid content than rprp. Carotenoid content in yyY1 Y1Y2Y2RPRP and YY y1y1y2y2RPRP increased during the first 28 days of storage and decreased subsequently. Meanwhile, rprp began to decrease in carotenoid content at day 14 of storage. HPLC analysis at l = 445 nm revealed two large unique peaks in rprp with elution times of 27 and 28.7 minutes that were of lesser abundance in YY y1y1y2y2RPRP, suggesting that the rate of β- and α-carotene accumulation is not the only difference between YY y1y1y2y2RPRP and rprp.

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Todd C. Einhorn, Cecil Stushnoff, Ann E. McSay, Phil L. Forsline, Sam Cox, Joel R.L. Ehrenkranz, and Loretta Sandoval

Phlorizin is known for its role in reducing glucotoxicity and has a long history of use in diabetes research. In addition, its contribution to the pool of total phenolics adds to the overall health benefits attributed to fruit. Phlorizin is limited to Rosaceae family plants, of which apple comprises its current commercial source; however, limited information exists regarding its biodiversity among apple taxa. A subset of 22 taxa from a core collection of apple accessions representative of the global genetic diversity of apple was used to investigate the biodiversity of phlorizin present in apple shoots and in fruit relative to total phenolic content and free radical scavenging capacity. Fruit and shoots were harvested from the USDA Plant Genetic Resources Unit in Geneva, N.Y. Validation and quantification of phlorizin was conducted using a rigorous high-pressure liquid chromatography (HPLC) procedure. Total phenolics in fruit, assayed using a Folin-Ciocalteu method and expressed as gallic acid equivalents, ranged from 227 to 7181 mg·L-1

and were strongly related to 2,2' azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) antioxidant capacity for the core collection (r= 0.778). On a molar basis, phlorizin had lower antioxidant capacity than other major phenolic compounds present in apple fruit, but was more effective than ascorbic acid. Phlorizin yield in dormant apple shoots, expressed as percent weight, ranged from 0.9% to 5.5%. A rapid, 96 well micro-plate spectrophotometric assay was also developed to aid in the screening of multiple samples for selection of high phlorizin yielding apple taxa. Spectrophotometry overestimated phlorizin content as expected, but the calibration curve between HPLC and spectrophotometry was acceptable, r 2 = 0.88.

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Tyann Blessington, Douglas C. Scheuring, and J. Creighton Miller Jr.

Potatoes are stored to ensure a continuous supply; however, losses due to shrinkage and sprouting can be large. It is believed that ionizing irradiation will become more prominent for sprout inhibition due to the increasingly higher operating costs of low-temperature storage and possible phase-out of chemical sprout inhibitors. The effects of storage and ionizing irradiation (gamma and electron beam) on antioxidant activity (AOA), phenolic content, and carotenoid content were analyzed using the potato cultivar Atlantic. Tubers were subjected to 0, 75, and 200 Gy γ-irradiation doses, stored at 20 °C, and analyzed after 0, 10, 20, 75, and 110 days. Tubers from another harvest were subjected to a surface dose of 0 or 200 Gy e-beam irradiation, stored at 20 °C, and analyzed after 0, 10, 20, 75, and 110 days. AOA was measured via the DPPH method; phenolic content via the Folin-Ciocalteau method and individual phenolics via HPLC; and carotenoid content via absorbance at 445 nm and individual carotenoids via HPLC. During early storage, higher doses resulted in higher AOA, while, during longer storage, lower doses produced greater AOA. Phenolic content increased in storage during the γ-irradiation study, but decreased in the e-beam study, partly due to increases in chlorogenic acid in the former and decreases in caffeic acid in the latter. The e-beam dose of 200 Gy resulted in significantly greater total phenolics than 0 Gy. Total carotenoids and lutein decreased with storage, but were not affected by irradiation. Storage exerted a much greater influence on AOA, phenolic content, and carotenoid content than either irradiation treatment.

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David A. Starrett and Kenneth C. Gross

Antisense technology has shown that neither polygalacturonase nor pectin methylesterase alone are responsible for tomato fruit softening, leading to the likelihood that other enzymes or factors are important. Our laboratory recently found that α and β-galactosidase from avocado fruit solubilized tomato fruit pectin in vitro. Previously, Pressey (Plant Physiol. 1983,71:132) found that the activity of one of three α-galactosidase isozymes from tomato fruit increased during ripening and was capable of degrading cell wall galactan, suggesting a role for the enzyme in fruit softening. Increased β-galactosidase activity was observed in a number of other fruit during ripening. In the present study, NaCl extraction of tomato pericarp yielded relatively high levels of cc- and β-galactosidase activity. At least two isozymes of each were resolved during Mono-Q HPLC α-Galactosidase was further purified by additional Mono Q and Superose 12 gel filtration HPLC. Gel filtration and SDS-PAGE yielded an apparent molecular weight of 44 kD. The partially pure α-galactosidase had a specific activity of 294 μmol product/min per mg protein, a Km of 317 μm, a pl of 5.0, and a pH optimum of 5.5. Activity was inhibited 67% by α-d-galactose. Preliminary results show that β-galactosidase can also be purified by the same techniques. Following further purification, the isozymes will be sequenced and cloned. A second approach being used in an attempt to identify cDNA clones for the α- and β-galactosidase genes from tomato fruit involves using heterologous cDNA clones from guar (Overbeeke et al., 1989; Plant Molecular Biology 13:541-550) and carnation (Raghothama et al., 1991; Plant Molecular Biology 17:61-71), respectively, to screen a ripening tomato fruit cDNA library. Basic molecularbiological techniques will be used to elucidate the role of these enzymes in tomato fruit ripening.

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John R. Stommel and Bruce D. Whitaker

Eggplant (Solanum melongena L.) is ranked among the top ten vegetables in terms of oxygen radical absorbance capacity due to its fruit's phenolic constituents. Several potential health promoting effects have been ascribed to plant phenolic phytochemicals. We report here a first evaluation of phenolic acid constituents in eggplant fruit from accessions in the USDA eggplant core subset. The core subset includes 101 accessions of the cultivated eggplant, S. melongena, and 14 accessions representing four related eggplant species, S. aethiopicum L., S. anguivi Lam., S. incanum L., and S. macrocarpon L. Significant differences in phenolic acid content and composition were evident among the five eggplant species and among genotypes within species. Fourteen compounds separated by HPLC, that were present in many but not all accessions, were identified or tentatively identified as hydroxycinnamic acid (HCA) derivatives based on HPLC elution times, UV absorbance spectra, ES-—MS mass spectra, and in some cases proton NMR data. These phenolics were grouped into five classes: chlorogenic acid isomers, isochlorogenic acid isomers, hydroxycinnamic acid amide conjugates, unidentified caffeic acid conjugates, and acetylated chlorogenic acid isomers. Among S. melongena accessions, there was a nearly 20-fold range in total HCA content. Total HCA content in S. aethiopicum and S. macrocarpon was low relative to S. melongena. A S. anguivi accession had the highest HCA content among core subset accessions. Chlorogenic acid isomers ranged from 63.4% to 96% of total HCAs in most core accessions. Two atypical accessions, S. anguivi PI 319855 and S. incanum PI500922, exhibited strikingly different HCA conjugate profiles, which differed from those of all other core subset accessions by the presence of several unique phenolic compounds. Our findings on eggplant fruit phenolic content provide opportunities to improve eggplant fruit quality and nutritive value.

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Kil Sun Yoo*, Julio Loaiza, Kevin Crosby, Leonard Pike, and Steve King

About 40 watermelon samples with various flesh colors (red, pink, orange, and yellow) were tested for their carotene, sugar, and ascorbic acid contents. Carotenoids were separated and purified by using a preparative HPLC system and identified by comparing the spectra with standard compounds by using a diode array detector. Sugar and ascorbic acid contents were measured by HPLC methods. Red and pink colored watermelon contained lycopene as the major carotenoid, with a wide range of variation (5 to 51 μg·g-1). Beta-carotene was the second major carotenoid and was less than 6 μg·g-1. There were also lutein and violazanthin in less than 1.5 μg·g-1 range. Yellow and orange flesh watermelons contained a complex mixture of carotenes. Prolycopene, lycopene, or beta-carotene was the major component, depending on the variety, and the contents were less than 24, 3, and 9 μg·g-1, respectively. There were also minor carotenoids, such as violaxanthin, lutein, neurosporene, zea-carotene with a 0 to 3.5 μg·g-1 range. Neurosporene, zea-carotene, and prolycopene were not found in the red watermelons. There was great variation in total sugar content, range being from 22 to 102 mg-1, while the °Brix was from 4.0 to 15.5. Sucrose, glucose, and fructose were the main sugars in the watermelon and their composition were grouped as sucrose-dominant or fructose-dominant groups. Some varieties with very low levels of sucrose were generally low in the total sugar content. Watermelon contained fairly low levels of ascorbic acid, less than 58 μg·g-1 and some varieties had nearly no ascorbic acid. Estimation of total carotenoid in the yellow watermelons by measuring absorbency at 435, 485, or 503 nm was tested and 435 nm showed the highest correlation coefficient (r 2 =0.845).

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Penelope Perkins-Veazie*, J.K. Collins, and Warren Roberts

Watermelons contain the carotenoids b-carotene, phytofluene, lycopene, and lutein. These carotenoids play an important role in plant oxidative protection and may serve to protect humans against oxidative assaults. Of the carotenoids, lycopene is the predominant pigment in red-fleshed melons (30-130 μg·g-1), b-carotene is present in small amounts (1-14 μg·g-1), and other carotenoids are present in minute amounts (1-3 μg·g-1). Seventy varieties were screened for lycopene content using scanning colorimetry, spectrophotometry, and HPLC techniques, and grouped as low, medium, high, or very high in lycopene. Pink-fleshed heirloom varieties such as Sweet Princess and Black Diamond contained low amounts of lycopene (<40 μg·g-1). A number of seeded and seedless varieties had medium amounts of lycopene (40-60 μg·g-1). Varieties in the high category (60-80 μg·g-1) were primarily seedless types, although `Dixie Lee', an open-pollinated, seeded variety had 69 μg·g-1, indicating that high lycopene content is not restricted to hybrid or seedless melon germplasm. Six selections were found to be very high in lycopene (>80 μg·g-1), including the minimelon Hazera 6008 (Extazy). Total carotenoids and carotenoid profiles were determined by HPLC for 23 varieties in 2003. Both seeded and seedless type melons had varieties high in bcarotene, lycopene, and total carotenoids. These results indicate that commercial watermelon varieties have a wide range in lycopene and b-carotene content, and that most commercially important varieties are high in lycopene and total carotenoids, providing important sources of phytonutrients to the human diet.