Joseph C. Scheerens

Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary phenolic antioxidants in black raspberry.

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.

Effect of Black Raspberry (Rubus occidentalis L.) Extract Variation Conditioned by Cultivar, Production Site, and Fruit Maturity Stage on Colon Cancer Cell Proliferation

Black raspberries have been shown to inhibit multiple stages of oral, esophageal, and colon cancer. The objective of this study was to evaluate how black raspberry extract variability conditioned by horticultural factors affected the antiproliferative activity of 75 black raspberry extracts using an in vitro colon cancer cell model. HT-29 cells grown in 96-well plates were treated with freeze-dried extracts at 0.6 and 1.2 mg of extract/mL of medium. Percent cell growth inhibition for each concentration of the extracts was determined using the sulforhodamine B assay. All extracts significantly inhibited the growth of HT-29 colon cancer cells in a dose-dependent manner. Cell proliferation was significantly influenced by cultivar, production site, and stage of maturity. The lack of correlation between growth inhibition and extract total phenolic and total monomeric anthocyanin assays suggested horticultural parameters influence bioactivity in a complex manner.

Total phenolic, anthocyanin contents and antioxidant capacity of selected elderberry (Sambucus canadensis L.) accessions.

Fourteen purple-black American elderberry accessions (Sambucus canadensis L.) obtained from various sites in midwestern USA and then grown at a single Ohio production site in USA were analyzed for their total phenolic (TP) and total monomeric anthocyanin (TMA) contents and for their antioxidant capacity by the ferric reducing antioxidant power (FRAP) and DPPH radical scavenging assays. Total phenolic and anthocyanin contents were measured using the Folin-Ciocalteu reagent and the pH differential methods, respectively. Overall, the phytonutrient contents and antioxidant capacity of our elderberry accessions were similar to those typically reported for black raspberries, blackberries and other dark-fleshed small fruits. Variability among accessions was greatest for TMA content (CV 37.5%); individuals ranged nearly threefold from 1308 to 4004 μg cy3-GE/g on a fresh weight basis. Variation among accessions was also evident for TP, FRAP and DPPH values (CV 14.4, 21.7 and 26.8%, respectively). TP and TMA values were very highly correlated (r = 0.93), although individuals differed in the estimated proportion of total phenolics attributable to anthocyanins. Both TP and TMA also highly correlated to antioxidant capacity values (r = 0.70–0.85). Within this limited study of 14 accessions, variability for phytonutrient content and antioxidant capacity suggested the employment of wild germplasm within an elderberry improvement program to incorporate an array of superior horticultural, post-harvest or processing traits into new or existing cultivars with superior phytonutrient profiles.

Nonanthocyanin secondary metabolites of black raspberry (Rubus occidentalis L.) fruits: identification by HPLC-DAD, NMR, HPLC-ESI-MS, and ESI-MS/MS analyses.

Nonanthocyanin secondary metabolites potentially contributing to the antiproliferative bioactivity of black raspberry ( Rubus occidentalis L.) fruits were extracted in ethyl acetate and isolated by semipreparative and analytical HPLC and analyzed by NMR, HPLC-ESI-MS, and ESI-MS/MS techniques. Here we present complete and partial structures of a variety of the chemical entities such as quercetin 3-glucoside, quercetin 3-rutinoside, myricetin glucoside, dihydrokaempferol glucoside, benzoic acid β-d-glucopyranosyl ester, 3,4-dihydroxybenzoic acid, epicatechin, caffeic acid, p-coumaric acid, p-coumaryl glucoside, p-coumaryl sugar ester, ellagic acid, methyl ellagic acid acetylpentose, methyl ellagic acid valerylpentose, trans-piceid, phloretin glucoside (phloridzin), dihydrosinapic acid, salicylic acid β-d-glucopyranosyl ester, a salicylic acid derivative without attached sugar, p-alkylphenyl glucoside, and a citric acid derivative. To our knowledge, 15 of these compounds were not previously reported in black raspberry fruits.

Root-zone temperature and nitrogen affect the yield and secondary metabolite concentration of fall- and spring-grown, high-density leaf lettuce.

BACKGROUND Understanding the effects of temperature and nitrogen levels on key variables, particularly under field conditions during cool seasons of temperate climates, is important. Here, we document the impact of root-zone heating and nitrogen (N) fertility on the accumulation and composition of fall- and spring-grown lettuce biomass. A novel, scalable field system was employed. RESULTS Direct-seeded plots containing a uniform, semi-solid, and nearly stable rooting medium were established outdoors in 2009 and 2010; each contained one of eight combinations of root-zone heating (-/+) and N fertility (0, 72, 144, and 576 mg day(-1)). Root-zone heating increased but withholding N decreased biomass accumulation in both years. Low N supplies were also associated with greater anthocyanin and total antioxidant power but lower N and phosphorus levels. Tissue chlorophyll a and vitamin C levels tracked root-zone temperature and N fertility more closely in 2009 and 2010, respectively. CONCLUSIONS Experimentally imposed root-zone temperature and N levels influenced the amount and properties of fall- and spring-grown lettuce tissue. Ambient conditions, however, dictated which of these factors exerted the greatest effect on the variables measured. Collectively, the results point to the potential for gains in system sustainability and productivity, including with respect to supplying human nutritional units.

NMR-based metabolomic investigation of bioactivity of chemical constituents in black raspberry (Rubus occidentalis L.) fruit extracts.

Black raspberry (Rubus occidentalis L.) (BR) fruit extracts with differing compound profiles have shown variable antiproliferative activities against HT-29 colon cancer cell lines. This study used partial least-squares (PLS) regression analysis to develop a high-resolution (1)H NMR-based multivariate statistical model for discerning the biological activity of BR constituents. This model identified specific bioactive compounds and ascertained their relative contribution against cancer cell proliferation. Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were the predominant contributors to the extract bioactivity, but salicylic acid derivatives (e.g., salicylic acid glucosyl ester), quercetin 3-glucoside, quercetin 3-rutinoside, p-coumaric acid, epicatechin, methyl ellagic acid derivatives (e.g., methyl ellagic acetyl pentose), and citric acid derivatives also contributed significantly to the antiproliferative activity of the berry extracts. This approach enabled the identification of new bioactive components in BR fruits and demonstrates the utility of the method for assessing chemopreventive compounds in foods and food products.

Nutritional Yield: A Proposed Index for Fresh Food Improvement Illustrated with Leafy Vegetable Data

Consumer interest in food products, including fresh vegetables, with health promoting properties is rising. In fresh vegetables, these properties include vitamins, minerals, dietary fiber, and secondary compounds, which collectively impart a large portion of the dietary, nutritional or health value associated with vegetable intake. Many, including farmers, aim to increase the health-promoting properties of fresh vegetables on the whole but they face at least three obstacles. First, describing crop composition in terms of its nutrition-based impact on human health is complex and there are few, if any, accepted processes and associated metrics for assessing and managing vegetable composition on-farm, at the origin of supply. Second, data suggest that primary and secondary metabolism can be ‘in conflict’ when establishing the abundance versus composition of a crop. Third, fresh vegetable farmers are rarely compensated for the phytochemical composition of their product. The development and implementation of a fresh vegetable ‘nutritional yield’ index could be instrumental in overcoming these obstacles. Nutritional yield is a function of crop biomass and tissue levels of health-related metabolites, including bioavailable antioxidant potential. Data from a multi-factor study of leaf lettuce primary and secondary metabolism and the literature suggest that antioxidant yield is sensitive to genetic and environmental production factors, and that changes in crop production and valuation will be required for fresh vegetable production systems to become more focused and purposeful instruments of public health.

Phenotypic variation of agronomic traits among coyote gourd accessions and their progeny

As a prelude to domestication efforts, variation of agronomic traits was determined among accessions of the polytypic, xerophytic cucurbit, coyote gourd [Cucurbita digitata subsp. digitata (DIG), palmata (PAL), cylindrata (CYL), and cordata (COR)] and among and within their progeny. Oil content in 60 accession seed lots (x = 27.8%, CV 21.4%) was more variable than protein content (x = 33.1%, CV = 13.5%). Punicic acid (c,t,c-9,ll,13-octadecatrienoic acid) levels in seed oils were also variable (x = 12.0%, CV = 20.6%) among accession seed lots. Substantial differences among and within subspecies were noted in 40 progeny lines for fruit/plant (x = 55.2, CV = 47.5%), seed wt/plant (x = 0.89 kg, CV = 51.1%), seed wt/fruit (x = 17.4 g, CV = 39.8%), seed no./fruit (x = 356, CV= 30.8%), 100-seed wt (x = 4.8 g, CV = 21.6%) and fruit diameter (x = 77 mm, CV 4.8%). Correlations among parameters suggested selection for fruit/plant to be the most effective primary strategy for seed yield improvement, but among high fruit-yielders, selection for seed wt/fruit was also important. The two subspecies PAL and CYL exhibited superior seed wts/plant. CYL types matured high fruit-loads, but their fruits were smaller and contained a smaller number of lighter seed than PAL or PAL hybrids. In contrast, PAL and PAL hybrids displayed lower fruit-yields/ plant but their fruits were larger and contained higher seed wts/fruit than their CYL counterparts. Root wt/plant was also variable (x = 1.55 kg, CV = 63.2%). Roots of DIG were larger and less branched than those of other subspecies. Proximate and liquid Chromatographic analyses of selected accession seed lots and controlled crosses failed to reveal advantages for the inclusion of specific types in a breeding program for the development of high oil or high/low punicic acid lines.RésuméComo un preludio a los esfuerzos de domesticación, se determinó la variatión de caracteres agronómicos entre ejemplares delpolitipo de cucurbitas xerofitas, “calabacillas coyote ” [Cucurbita digitata subspp. digitata (DIG), palmata (PAL), cylindrata (CYL), y cordata (COR)] asi como en y entre su progenie. El contenido de aceite en 60 lotes de semillas de los ejemplares (x = 27.8%, CV = 21.4%) fué más variable que el contenido protéico (x = 33.1%, CV = 13.5%). Los nivelés de ácido punícico (c,t,c-9,11,13-ácido octadecatrienóico) en los aceites de las semillas fureon también muy diversos y variables (x = 12%, CV = 20.6%), entre los lotes de semillas de la colección. Se apreciaron diferencias substantiates entre y dentro de las subespecies en las 40 líneas de progenie para la relatión fruto/planta (x = 55.2, CV = 47.5%), peso de semilla/planta (x = 0.89 kg, CV= 51.1%), peso de semilla/fruto (x = 17.4 g, CV=21.6%), numúro de semillas/ fruto (x = 356, CV = 30.8%), peso de 100 semillas (x = 4.8 g, CV = 21.6%) y diámetro defruto (x = 77 mm, CV = 4.8%). Las correlaciones entre los parâmetros sugieren que seleccionar por la relatión fruto/planta, seíria la estrategia primaria más efectiva para fines de mejorameiento en el aspecto de rendimiento de semilla, pero entre los productores de fruta, seleccionar por la relatión peso de semilla/fruto, es también importante. Las dos subespecies, PAL y CYL mostraron una relatión peso de semilla/planta superior. Los tipos CYL maduraron altos lotes de fruto, pero losfrutos eran mas pequeños y contenían un menor número de semilla liviana que aquellos del tipo PAL o sus híbridos. En contraste, PAL y sus híbridos tuvieron menores rendimientos de fruta/planta pero los frutos fueron mas grandes y con una relatión peso de semilla/fruto mayor que en el caso de CYL. El peso radicular/planta también varió (x = 1.55 kg, CV = 63.2%). Las raices de DIG fueron mayores y menos ramificadas que aquellas de otras subespecies. Los análisis proximales y de cromatografía líquida de lotes específicos de semillas de la colección no revelaron ninguna ventaja para la inclusión de tipos específicos en un programa de mejoramiento para el desarrollo de líneas con alto contenido de aciete o alto/bajo ácido punícico.

Grafting, Scion and Rootstock Effects on Survival Rate, Vegetative Growth and Fruit Yield of High Tunnel-grown Grafted Pepper (Capsicum annuum L.) Plants

The objective of this study was to develop information useful to pepper (Capsicum annuum L.) rootstock breeders, grafted plant propagators and growers managing commercial plantings in semi-protected settings. To accomplish this, an experiment involving four phenotypically diverse scion cultivars and five rootstocks grafted in all combinations along with self-grafted and non-grafted cultivars as controls was completed twice. The effects of the grafting process, scion cultivars and rootstock genotypes on grafted plant performance were delineated by measures of post-grafting plant survival (%), plant vegetative vigor and by early and total season yield parameters. The grafting process significantly increased vegetative vigor parameters, most notably in 2014 whereas the yield parameters of self-grafted plants exceeded their non-grafted counterparts only in 2015. In both years, scion performance in graft combinations remained characteristic of the cultivar type; long-fruited cultivars ‘Eigman’ and ‘Kurtovszka Kapia’ had lower survival rates, generally exhibited greater vegetative vigor, and amassed greater yields per m2 than blocky-fruited ‘Toronto’ and ‘Zedinca’. In general, graft combinations using commercial pepper rootstocks 52-03 RZ and Tan Tan (No: 12G076) outperformed those composed of standard cultivars or breeding lines used as rootstocks. Scions on these rootstocks exhibited greater vegetative vigor in both years and higher yields per m2 than non-grafted controls in 2014. The superior performance of 52-03 RZ and Tan Tan (No: 12G076) in this study exemplified the advantage of using rootstocks specifically bred for optimum root system performance and compatibility with a variety of scions. However, significant scion × rootstock interactions and seasonal differences in performance suggested, that as new rootstocks are developed or as new graft combinations are introduced, it will be necessary to rigorously test them in multiple environments to insure commercial success.