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S.M. Javad Feghahati and R. Neil Reese

Echinacea angustifolia DC., the common coneflower of the western Great Plains, is difficult to propagate by achenes due to inherent seed dormancy. The effects of light and prechilling on seed germination were examined, alone and combined with scarification (mechanical, acid) and ethylene (ethephon) treatments. The results showed that a 2-week prechill treatment combined with ethephon and continuous light, followed by a 2-week germination period in light (16 hours per day) at 25C, could induce >95% seed germination in E. angustifolia. This was a significantly higher percentage of germination over a shorter period of time than any other method examined or previously described. This treatment also synchronized germination, with most viable seeds germinating in <1 week after being placed at 25C in the light. Chemical name used: 2-chloroethylphosphonic acid (ethephon).

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Mustafa Ozgen, Artemio Z. Tulio Jr., A. Raymond Miller, R. Neil Reese, and Joseph C. Scheerens

In preliminary studies, we found that relative and absolute antioxidant (AO) levels varied within and among small fruit types. AO levels were affected by assay method used, time of reaction, volume of sample, and the ratio of reactants to total AO activity. To identify the physicochemical parameters that affect accuracy and reproducibility, a series of experiments were conducted to test the roles of AO assay, different AOs, and AO concentration on measured AO content and reaction kinetics. Three assays (DPPH, FRAP, ABTS) were used to evaluate AO capacity of seven fruit types (black and red raspberry, blackberry, strawberry, grape, elderberry, and cranberry) and nine purified AOs (ascorbic, caffeic, chlorogenic, gallic, and ellagic acids, α-tocopherol, trolox, cyanidin-3-glucoside, and quercetin). Ascorbic acid, trolox, caffeic acid, chlorogenic acid, and α-tocopherol exhibited simple reaction kinetics and reached endpoints quickly, regardless of assay. Gallic and ellagic acids, quercetin, cyanidin-3-glucoside, and all fruit extracts exhibited more complex kinetics and long reaction times (>70 min) to reach an endpoint. Moreover, the latter four AOs had the highest AO capacity among the compounds tested. We observed differences in reactivity between assays, compounds and fruit extracts, but relative AO activity was comparable, although the absolute values differed. Since AO capacity of fruit extracts is a composite of the individual AOs present, it is important that reactions progress to near steady state, assay reactants are in excess of (30–50×) the AO capacity being measured, more than one assay is used to describe the total AO activity of fruit samples. Thus, there may not be a single AO assay method that completely defines the AO activity of a given fruit.

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Mustafa Ozgen, Artemio Z. Tulio Jr., Ann M. Chanon, Nithya Janakiraman, R. Neil Reese, A. Raymond Miller, and Joseph C. Scheerens

To investigate phytonutrient accumulation in black raspberries, fruits of `Jewel' and `MacBlack' were harvested at stages from the onset of color development (S1) to ripe fruit (S7). S1–S7 samples were characterized for color reflectance and then frozen at –28 °C within an hour of harvest. Additional ripe fruit were maintained at 20 °C for 3 days to overripen (S8) before freezing. After storage, samples were analyzed for dry weight (DW), total soluble solids (TSS), fructose (FRU), glucose (GLU), and organic acid (ORG) contents; total phenolic (PHE) and anthocyanin (ACY) contents; individual cyanidin glycoside levels (ICG); and antioxidant capacity (FRAP and ABTS) by standard methodology. `Jewel' and `MacBlack' ripened similarly. Chroma values and DW percentage decreased while TSS levels, sugar contents (FRU+GLU), PHE, ACY, the ACY: PHE ratio, and ICG increased with progressive ripening stages (S1–S7). Values of PHE, ACY, and ICG were highly correlated (r < +0.95) with FRAP and ABTS values. ACY levels in S6 fruit were 18% to 23% less than those of S7; lower S6 ACY levels were associated with reduced antioxidant capacity in `MacBlack', but not `Jewel'. Overripened fruit (S8) exhibited increased DW (11% to 25%) and decreased sugar contents (16% to 17%), consistent with moisture and respiratory losses after harvest. After correction for these losses, S7 and S8 levels of PHE, ACY, FRAP, and ABTS were similar in `MacBlack'. However, as `Jewel' overripened, ACY levels and antioxidant activity increased 44% and 22% to 26%, respectively. Our data suggests that significant changes in the antioxidant behavior of black raspberries can occur during the periods surrounding peak ripeness.

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Mustafa Ozgen, Artemio Z. Tulio Jr., Ann M. Chanon, Nithya Janakiraman, R. Neil Reese, A. Raymond Miller, and Joseph C. Scheerens

To investigate the variation in the phytonutrients of Cornelian cherry (Cornus mas L.), fruit was harvested at the blush (S1), red (S2), and ripe (S3) stages from five genotypes maintained at the Secrest Arboretum, Wooster, Ohio. The S1-S3 samples were characterized for color reflectance and then frozen at –28 °C. After storage, samples were analyzed for dry weight (DW), total soluble solids (TSS), sugars (FRU + GLU), organic acids (ORG), total phenols (PHE), total anthocyanins (ACY), individual anthocyanins (IA), hydroyzable tannins (HT), and antioxidant capacity (FRAP and ABTS). From S1 to S3, DW and TSS increased by 24% and 21%, respectively, and L, hue angle, and chroma values decreased. On a DW basis, all analytical parameters were significantly influenced by genotype and stage. The ACY levels rose 7-fold during ripening, but PHE contents declined by 10%. In ripe fruit, HT comprised the bulk of the PHE constituents, whereas ACY accounted for only 7.6% of PHE levels. Variability among genotypes was moderate for all ripe fruit parameters but ACY. Ripe fruit varied little in color parameters and ACY (fwb) and IA (fwb) were not significantly different among cultivars. The Cy 3-gal and pel 3-gal levels were negatively correlated. Antioxidant capacity declined 16% to 18% during ripening. Ripe fruit FRAP and ABTS values were higher than those reported for most fruits, averaging 596 ± 85 and 629 ± 85 μmol TE eq./gDW, respectively. ABTS and FRAP values were highly correlated with each other and with PHE and HT contents, but were loosely and negatively related to ACY levels. Considering our limited sample size, we concluded that the phytonutrient capacity of cornelian cherry is substantial, predominantly associated with tannins and moderately variable among genotypes.

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Mustafa Ozgen, Faith J. Wyzgoski, Artemio Z. Tulio Jr, Aparna Gazula, A. Raymond Miller, Joseph C. Scheerens, R. Neil Reese, and Shawn R. Wright

Black raspberries (Rubus occidentalis L.) are rich in phytonutrients that have demonstrated chemoprotective properties against certain degenerative diseases. To estimate variability in phytonutritional quality among sources of black raspberry, 19 samples representing four common midwestern cultivars obtained from eight production sites were assayed for their antioxidant capacity [2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing ability of plasma (FRAP) methods], total phenolic content (TP), total monomeric anthocyanin levels (TMA), and levels of cyanidin 3-rutinoside. The antioxidant potential among samples averaged 2.92 ± 0.29 and 4.62 ± 0.88 mmol TE·100 g−1 fresh weight by the DPPH and FRAP methods, respectively; TP, TMA, and cyanidin 3-rutinoside means averaged 449 ± 62, 336 ± 109, and 244 ± 84 mg·100 g−1 fresh weight, respectively. Levels of FRAP, TP, TMA, and cyanidin 3-rutinoside were strongly correlated (r = +0.85 to +0.96). Mean antioxidant capacities and phenolic constituent levels were similar among ‘Bristol’, ‘Jewel’, and ‘MacBlack’ samples; values for a single sample of ‘Haut’ were lower but comparable to levels found in individual samples of the other three cultivars. Black raspberry production site differences were statistically significant for FRAP, TMA, cyanidin 3-rutinoside, and titratable acidity (TA) levels. Inverse relationships (r = –0.65 to –0.74) among black raspberry samples for FRAP, TMA, or cyanidin 3-rutinoside levels versus levels of TA suggested that site differences may be partially attributable to fruit ripeness at harvest. Relationships among these parameters versus regional differences in soil temperatures were also significant but weak. Regardless of its environmental or physiological drivers, point-source variation in fruit phytonutrient contents may be a relevant concern in health-related studies or clinical applications. Moreover, it may impact the nutritional benefits to the consumer and affect the quality advantages associated with direct-marketed black raspberries.

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Mustafa Ozgen, Artemio Z. Tulio Jr., Aparna Gazula, Joseph C. Scheerens, R. Neil Reese, A. Raymond Miller, Shawn R. Wright, Brent L. Black, and Ingrid M. Fordham

Autumnberry (Elaeagnus umbellata, “A”) and cornelian cherry (Cornusmas, “CC”) genotypes were examined for mineral composition, anthocyanin, phenolic and tannin contents, antioxidant characteristics and levels of individual phenolic compounds via GC-MS. Values were compared with those of 58 cultivars of blackberries (“B”), black raspberries (“BR”), cranberries (“C”), elderberries (“E”), grapes (“G”), red raspberries (“RR”) and strawberries (“S”). The phenolic content of “CC” (6955 μg·gfw-1) was greater than 2× that of “B”, “BR” and “E”. Phenolic contents of “A” samples (1058-1776 μg·gfw-1) were similar to those of “RR”, red “G” and “S”. Anthocyanin levels in “CC” (270 μg·gfw-1) resembled those in “C”. “A” did not contain anthocyanins. Fruit of “CC” and “A” possessed high tannin levels (9291 μg·gfw-1 and 1410–5403 μg·gfw-1, respectively) and exhibited high antioxidant potential (μmol·gfw-1 trolox equiv.). DPPH and FRAP values of “CC” (72.1 and 94.9, respectively) were greater than 2× those of “BR”. DPPH values of “A” (23.9–56.2) were ≥ values for “BR”, whereas “A” FRAP values (13.3–34.0) were similar to those of “B” and “RR”. However, the lipid-soluble antioxidant potential of lycopene-rich “A” was substantial. Levels of individual compounds varied among cultivars. Ca and Mg contents of “A” were less than those found in “CC” and “BR”. Other mineral levels were comparable.

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Artemio Z. Tulio Jr., Ann M. Chanon, Nithya Janakiraman, Mustafa Ozgen, Gary D. Stoner, R. Neil Reese, A. Raymond Miller, and Joseph C. Scheerens

This study was conducted to determine the effects of postharvest storage temperatures on the antioxidant capacity, anthocyanin compounds, phenolic constituents, and physico-chemical properties of black raspberries. Fresh `MacBlack' berries were stored at 4, 12, 20, and 28 °C for up to 11, 6, 4, and 3 days, respectively. Results showed that higher storage temperatures promoted tissue deterioration (cellular leakage), fungal growth, and moisture loss. The levels of the two major anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, increased by up to 2.7- and 1.9-fold, respectively, with increasing storage temperatures. The antioxidant capacity of berries, as measured by FRAP and ABTS assays, increased by up to 1.5- and 1.4-fold, respectively, which was accompanied by increases in soluble solids, total sugars, total phenolics, and total anthocyanin contents. Our findings indicate that postharvest storage at higher temperatures increases the level of bioactive compounds and antioxidant capacity in black raspberries, but this increase may be due in part to moisture loss and sugar metabolism. Storage at 4 °C maintained the level of bioactive compounds and antioxidant capacity present at harvest and prolonged the effective shelf life of the product. Further studies of black raspberry bioactive components as influenced by postharvest conditions and processing procedures (e.g., IQF, freeze-drying, air-drying) are warranted.

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Artemio Z. Tulio Jr., Mustafa Ozgen, R. Neil Reese, Steven J. Schwartz, Qingguo Tian, Gary D. Stoner, A. Raymond Miller, and Joseph C. Scheerens

Anthocyanins in black raspberry extracts may play a key role in the regulation of oncogene expression in cancer cell cultures. Variations in anthocyanin levels of `Jewel', `Mac Black', and `Bristol' black raspberries grown at seven commercial farms in Ohio were investigated using HPLC and uv-vis spectrometry. Cyanidin-3-rutinoside (cy-3-rut) and cyanidin-3-(2G-xylorutinoside) (cy-3-2-xyl), the two major compounds present in all cultivars (≈2:1), were highly correlated with total anthocyanin contents. Sample variation in total anthocyanin, cy-3-rut, and cy-3-2-xyl levels was greater among commercial farms than among cultivars grown at the same location. The antioxidant activities of cy-3-rut, cyanidin-3-glucoside, cyanidin-3,5-diglucoside (cy-3,5-diglc), and pelargonidin-3-glucoside from purified extracts were determined using the free radical scavenging assays DPPH and ABTS, and the ferric reducing power assay FRAP. All pure anthocyanins showed strong antioxidant potentials except for cy-3,5-diglc. Cy-3-rut was identified and quantified as the dominant anthocyanin in black raspberries and was also the most potent antioxidant. Results suggest that anthocyanins, cy-3-rut in particular, may function as the primary antioxidants in black raspberries. Genetic and environmental variation in the anthocyanin contents necessitate characterization of the antioxidant and anthocyanin levels in fruits from any given source prior to measuring biological and medicinal activities.

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Joseph C. Scheerens, Todd E. Steiner, Artemio Z. Tulio Jr., Mustafa Ozgen, Diane D. Miller, R. Neil Reese, A. Raymond Miller, and David C. Ferree

Eight unique varietal grape juices were examined for their antioxidant characteristics and commercial potential compared to that of commercial `Niagara' and `Concord'. Grape juices were cold-pressed from mature grapes, clarified, preserved, analyzed for pH, soluble solid and titratable acidity levels, pasteurized at 73 °C for 12 seconds, and sampled for microbial testing. A preliminary panel of 41 routine evaluators assessed all juices for 18 quality characteristics against known and blind controls. Based on these results, `Reliance', `Traminette', and New York 73 juices were presented to a 107-member panel of untrained judges. Panelists rated experimental juices against commercial controls for color, appearance, aroma and flavor intensity, sweetness, tartness, overall quality, and preference. Among juices tested, `Reliance' and NY 73 offer the greatest potential as specialty grape juices. `Traminette', `Chardonel', `Chambourcin', and NY 62 may also have potential as grape juice cultivars, if processed to improve their color and clarity. Small juice lots were hand-pressed from mature grapes and examined for total anthocyanin and phenolic content, antioxidant characteristics (DPPH and FRAP) and levels of individual phenolic compounds via GC-MS. Total anthocyanin and phenolic contents of experimental juices varied from 0–1460 μg·gfw-1 and 1001–2850 μg·gfw-1, respectively, and were highest in NY 73. Estimates of antioxidant activity differed slightly among tests, but activity appeared highest in `Chambourcin' and NY 73 and lowest in `Reliance'. Levels of individual compounds varied substantially among juices.

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Faith J. Wyzgoski, A. Raymond Miller, Joseph C. Scheerens, Peter L. Rinaldi, Bert L. Bishop, R. Neil Reese, Mustafa Ozgen, Artemio Z. Tulio Jr., M. Monica Giusti, and Joshua A. Bomser

We have developed a Nuclear Magnetic Resonance (NMR)-based approach to metabolomics research that enables the identification of bioactive compounds in crude plant extracts. For this work, we used black raspberries, which are known to contain compounds that exhibit chemopreventive activity toward oral, esophageal, and colon cancers. To ascertain bioactive components and their interrelationships, NMR results for black raspberry samples from four cultivars grown on commercial farms in Ohio were examined using principal component analysis. Multivariate analysis that included anthocyanin content (HPLC), antioxidant activity (DPPH, ABTS, FRAP), total phenolics (Folin-Ciocalteau assay), and bioactivity as measured by inhibition of colon cancer HT-29 cell line proliferation showed correlations with specific regions of NMR spectra at 400 MHz. Correlations were also observed for major and minor groupings of the black raspberry samples. Replicate black raspberry samples were examined with a 750 MHz NMR spectrometer equipped with a cryoprobe that provided a 4- to 5-fold improvement in sensitivity. In this manner, even minor bioactive components in black raspberries could be examined to determine additive and synergistic effects.