| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00691 | |
| Title | *Plant Antioxidants as Free Radical Scavengers:Implications for Human Health and Plant Survival |
| Investigator(s) | Stushnoff, C; Holm, DG; |
| Department | Horticulture and Landscape Architecture |
| Objectives | 1. To develop analytical protocols for identifying and quantifying antioxidant constituents in plant tissues, food, and beverages using microplate reader spectrophotometry, GC, GC/MS, and HPLC analytical techniques. 2. To examine important commodities grown in Colorado for antioxidant properties, and to explore opportunities that might be developed to add value, and to possibly create new products. The first priority will be to examine potato and apple. 3. To investigate opportunities to collaborate with scientists who conduct research to improve Colorado grown crops. 4. To investigate opportunities to collaborate with medical researchers with an interest in using food and antioxidant derivatives from agricultural plants to improve human health and nutrition. 5. To integrate and apply assays based upon free radical chemistry and antioxidant biochemistry to plant injury caused by reactive oxygen species, from desiccation, salinity and temperature stresses in plants. |
| Approach | We will analyze genetic core subsets that represent the biodiversity of potato and apple germplasm available to breeders. Environmental factors, including temperature and water stress have been reported to impact phenolic content of plant tissues. We will examine environmental effects, whenever they can be quantified, on antioxidant content and activity . Total phenolics (phase one) will be analyzed using Folin-Ciocalteu reagent for potato tuber tissue, apple tissue, and apple juice. Freeze dried samples, and tuber samples that have been subjected to heat to determine temperature stability will be analyzed. Our preliminary results show a strong relationship between phenolic content and radical scavenging capacity. We thus propose to select 10 percent of the high and low phenolics determinations for further, more complex and time consuming analyses. In the second phase, selected entries will be analyzed for radical scavenging potential based upon a modified Trolox vitamin E antioxidant equivalent assay using the ABTS assay and, or the TRAP assay. A lipid peroxidation assay, modified to measure plant tissues will also be used for selected entries. While the ABTS and TRAP assays provide data on water and lipid soluble antioxidant activity that can impact most biological systems, the FOX assay specifically detects lipid peroxidation activity, believed to be important in oxidation of LDL lipids leading to cardiac diseases. These assays provide a measure of activity to supplement the total phenolic content analyses conducted initially. Experience with these assays was obtained while on a sabbatical leave at Crop and Food Research, and at Lincoln University, Lincoln, New Zealand. The equipment necessary to implement these assays is available in our laboratory. In the third phase we will select the most interesting taxa with very high and low levels for HPLC or GC/MS screening to search for specific unique phenolic compounds and flavonols. Data from phase two and three should determine unique specific compounds and estimate antioxidant activity to supplement the composition data in phase one. This step-wise approach should permit efficient initial screening of the maximum number of accessions, followed by more intensive investigation of specific components and their mode of action. |
| Keywords | apple, potato, antioxidants, free radical scavengers, Temperature stress, Moisture stress, Salt stress, phytochmicals, phenolics |
| Progress Reports | |
| 2002 | Post-harvest storage properties of new potato cultivars can determine successful adaptation and special market niches. While sprout inhibitors have dramatically increased storage life, continuance of their registration has been questioned, and their use for organic markets is prohibited. Thus to test inherent tuber dormancy in the absence of sprout inhibitors, we initiated a three-year storage trial at five storage temperatures with newly introduced cultivars grown in Colorado. Reducing-sugar content was also tested. Storage at 1.1 and 2.2C vs 3.3, 4.4 or 6.7C extended dormancy by 30-60 days, but the reducing sugars were also highest at 1.1 and 2.2C. Natural tuber dormancy (days to 10% bud break) was: R. Nugget (112), R. Norkotah #3 (109), R. Norkotah #8 (95), Cherry Red (89), Durango Red (87), Chipeta (87) and Keystone R. (<30). Reducing sugars (fructose & glucose) ranked highest to lowest in Keystone R ., R. Nugget, Durango Red, R. Norkotah #8, R. Norkotah #3, Cherry Red and Chipeta. No symptoms of chilling injury were detected at any temperature. These data suggest that while 1.1C provides the greatest storage longevity without sprout inhibitors, warm temperature conditioning would be necessary to remove sweetness and to enhance high temperature processing properties imparted by high reducing sugar content. Several reports suggest that tomato products in the diet may intervene in the development of prostate cancer. Because many diverse tomato cultivars are grown in home gardens and available in markets, we examined antioxidant properties in cultivars with different colored fruit and under different ripening conditions. Green-mature fruit exposed to 24 hour light contained more lycopene than those exposed to 8 hours light during ripening. Even though lycopene and total phenolics were higher in red, black, and orange than in yellow fruited cultivars, radical scavenging capacity based upon 2 ,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) assay did not differ among cultivars, suggesting that some other component such as vitamin C may also contribute to ABTS activity. |
| 2003 | A phytochemical antioxidant screening program was initiated to aid selection of healthier cultivars in the CO potato breeding program. Total phenolics (TP), radical scavenging capacity , 'Trolox' vit. E equivalents (TEAC) and vitamin C content were determined for 62 advanced selections and 9 standard cultivars. TP of uncooked samples ranged from 6.4 to 118.8 mg/gdw. The cultivar mean was 28.4 compared to 72.0 mg/gdw for the highest 10 selections. Radical scavenging ranged 0.07 to 6.0 mM vitamin E equivalents, with a mean of 3.89 mg/gdw for the 10 highest selections. Vitamin C ranged from 0.2 to 1.85 mg/gdw for the 62 selections. Cooking reduced antioxidant content and capacity 22 to 40%. Fruit from 7 freshly harvested apple cultivars, grown at 7 CO high elevation west slope locations was compared to antioxidant properties of the same cultivars obtained from local supermarkets, but grown in CA and WA. CO grown Fuji, Gala, and Pink Lady had higher antioxidant properties and soluble solids than fruit from CA and WA, but CO grown G. Delicious and Granny Smith were lower than CA and WA sources. Highly colored red clones had the highest TP and TEAC. |
| 2004 | While potato has long been recognized as an important source of vitamin C in the human diet, additional health attributes from phenolic- based antioxidants prevalent in potato are receiving increasing attention. We analyzed 72 advanced selections and 15 cultivars from the 2003 CO breeding program for total phenolics, ABTS radical scavenging capacity and vitamin C. Of the 15 entries falling into the highest classes, 13 had red or purple flesh, and only 2 had russet skins with white flesh. One of the latter has been introduced as Rio Grande Russet, and one of the purple- fleshed selections is slated for introduction in 2005. All of the lowest 15 entries had white or yellow flesh. Among the highest vitamin C selections, all but 2 were white or yellow fleshed, thus antioxidant contributions from vitamin C were genetically different from the phenolic antioxidants. Breeding for combined attributes should be possible, but will require choice of parents based upon analytical detection of both phenolic-based antioxidants and vitamin C. Phlorizin is a phenolic dihydrochalcone found only in the Roscaceae family and most abundantly in apple tissues. Phlorizin has received recent research attention for its potential role in diabetes research, attributed to reversal of glucose toxicity and to lowering of postprandial hyperglycemia. It also possesses antioxidant properties. While these putative effects are in the early stages of investigation, identification of the most potent source of this natural phytochemical appears prudent. Using HPLC, we analyzed biodiversity of phlorizin content in stems and fruit juice of 22 taxa, known to have high total phenolic content. The 22 taxa, (10% of the genetic core of apple germplasm) varied six-fold (0.89 to 5.5 % of stem dw) and from 0 to 112 mg/L in juice. There was no relationship between total phenolic and phlorizin content. We also developed a high-throughput spectrophotometric microplate assay that was highly correlated to HPLC data (R2 = 0.8764). This rapid assay could be applied to quickly screen large numbers of samples at minimal cost. |
| 2005 | Our previous research on biodiversity of antioxidant properties with Colorado potatoes identified a number of advanced selections developed by the breeding program that are substantially higher in antioxidant content and free radical scavenging capacity. In order to sharpen our focus on putative active antioxidant phytochemicals, six cultivars that had previously been identified with diverse antioxidant properties were selected for analyses by LC/MS and HPLC. Two of these cultivars were also discovered to inhibit growth of two human mammary cancer cell cultures. Major glycoalkaloids and phenolic components in extracts from oven baked tubers, identical to those used in the cancer cell culture experiments, were identified and concentrations estimated from known standards. Concentrations of several specific compounds varied greatly among cultivars, as did our previous analytical estimates of antioxidant properties. Two major (alpha-chaconine, alpha-solanine) and two minor glycoalkaloids (solasonine and solandine dehydrodimer) were identified. Alpha-chaconine was slightly more abundant in all but Russet Nugget. One cultivar, Rio Grande Russet had double the content of all others, but this was well below federal and industry standards for newly introduced cultivars. The purple and red flesh pigmented cultivars Purple Majesty and Mountain Rose contained 3 to 4 times the content of three chlorogenic acid isomers, compared to Russet Burbank, Russet Nugget and Yukon Gold. Eight different anthocyanin pigments were identified in the purple flesh of Purple Majesty and six different anthocyanin pigments were identified in the red flesh of Mountain Rose. Yukon Gold had the lowest phytochemical content of all cultivars |
| 2006 | Nutrient attributes of fresh potatoes generally drop during post-harvest handling and storage . Earlier we reported that purple and red flesh pigmented potato taxa contained substantially higher levels of phenolic based antioxidants, and higher radical scavenging capacity. Preliminary data also suggested that purple and red pigment content increased during storage. In this study we compared antioxidant properties, following six months storage, of four traditional white and yellow fleshed cultivars (Russet Norkotah, Russet Burbank, Russet Nugget, Yukon Gold) to three pigmented cultivars (All Blue, Mountain Rose, Purple Majesty) and six pigmented selections from the Colorado breeding program. After storing the four non-pigmented cultivars for six months, total phenolics dropped an average of 15%, ABTS radical scavenging capacity dropped 28% and vitamin C dropped 39 %. By comparison average values for the nine pigmented taxa increased 23% for total phenolics and 40% for ABTS radical scavenging capacity, while vitamin C content dropped 28%. Plant stress tolerance, regulated by an elaborate network of physiological and biochemical changes is frequently induced by exposure to environmental stresses. In an earlier cold hardiness study we generated both sense and anti sense alpha galactosidase transgenic plants of Mitchell diploid petunia. Eight different asexually propagated T5 and T6 lines were compared to their wild type counterparts for responses to drought and salt stresses. Even though the same gene was inserted, each antisense line differed in stress response, some more resistant, others less than the wild types. Sense lines were weak demonstrating that raffinose metabolism influences plant growth and tolerance to water and salt stress. |
| Impact | |
| 2002 | Storage profiles of new potato cultivars can be used by producers to optimize management in the event sprout inhibitors are delisted, and for organic marketing. Home gardeners seeking high lycopene content should grow red fruited cultivars that are ripened while exposed to light. |
| 2003 | Considerable diversity in antioxidant properties of potato breeding lines provides an opportunity to select and introduce new cultivars that can appeal to health conscious diets and aid marketing. Apple cultivars produced in high elevation CO orchards with significantly higher antioxidant properties provide growers with information to aid their marketing strategies. |
| 2004 | Antioxidant and other health attributes of potatoes can be significantly increased, primarily by breeding new cultivars that have (1) purple or red flesh, and (2) higher vitamin C content. Such attributes will likely boost consumer appeal in the present health conscious atmosphere. Progress through breeding for these attributes will require analytical chemical analyses for content and bioactivity of active antioxidants. Considerable biodiversity appears to exist in existing germplasm, but more extensive investigations of potato genetic resources may be prudent. Significant biodiversity for phlorizin content is present in stems and fruit juices of apple cultivars and species. The high throughput microplate assay developed from this research can be used as an initial screen to identify genetic lines with high phlorizin content. |
| 2005 | Germplasm diversity for antioxidant traits provides opportunity for developing new specialty potato cultivars with potentially improved health attributes. These data provide the first comprehensive phytochemical composition of purple and red flesh potatoes, and suggest key components that may be candidates for additional studies. This study also provides data to support marketing of new cultivars with potential health benefits. |
| 2006 | While potato nutrients such as vitamin C typically decline during storage in non-pigmented cultivars, purple and red fleshed taxa increased in phenolic anthocyanin antioxidants during storage. This may be a valuable attribute that maintains nutrient properties during storage. The biochemical basis for this intriguing low temperature metabolic process requires further investigation. Variation in raffinose related drought and salt stress tolerance detected among transformed lines suggests that well replicated and carefully regulated environmental stress imposition is essential to maximize successful selection of desired transformants. |
| Publications | |
| 2002 |
McSay, A.E., Stushnoff, C., Holm, D. and Davidson, R. 2002. Storage characteristics of new potato introductions. XXVIth International Horticultural Congress & Exhibition. Toronto, Canada. P133. (Abstr.). Stushnoff, C., McSay, A.E., Forsline, P.L., and Luby, J.L. 2002. Diversity of phenolic antioxidant content and radical scavenging capacity in the apple germplasm core collection. XXVIth International Horticultural Congress & Exhibition. Toronto, Canada, P358 (Abstr.). |
| 2003 |
Cox, S.E., Stushnoff, C. and Sampson, D. 2003. Relationship of fruit color and light exposure to lycopene content and antioxidant properties of tomato. Can. J. Plant Science 83:913-919. Javanmardi, J., Stushnoff, C., Locke, E., and Vivanco, J.M. 2003. Antioxidant activity and total phenolics content of Iranian Ocimum accessions. Food Chemistry 83:547-550. McSay, A. E., Stushnoff, C., Holm, D., Davidson, R., and Shabba, M. 2003. Storage characteristics of new potato introductions. XXVI International Horticultural Congress. Symposium 3(Potatoes-Healthy Food for Humanity: Interational Developments in Breeding, Production, Protection and Utilization). Acta Horticulturae. (ISHS) 619:463-469. Stushnoff, C., McSay, A.E., Luby, J.J. and Forsline, P.L. 2003. Diversity of phenolics antioxidant content and radical scavenging capacity in the apple germplasm core collection. XXVI International Horticultural Congress. Symposium 21 (Plant Genetic Resources: The Fabric of Horticulture's Future). Acta Horticulturae 623: 305-312. |
| 2004 |
Bunning, M., Kendall, P.A., Stone, M.B., Stushnoff, C., and Stonaker, F. 2005. Effects of seasonal variation on sensory properties of five lettuce cultivars. Abstract# 30526. Institute of Food Technologists Annual Meeting, July 2005. Esparza, J.R., Stone, M.B., Kendall, P.A., Stushnoff, C., and Pilon-Smits, E. 2005. Quality of fresh cut green leaf lettuce treated with ascorbic acid applied by two hydrocooling methods. Abstract # 30780. Institute of Food Technologists Annual Meeting, July 2005. Kulen, O., and Stushnoff, C. 2003. Release of potato minituber bud dormancy. Abstract. HortScience 38(5):778. Locke, E.L., Stushnoff, C., Pennycooke, J.C., and Jones. M.L. 2004. Effects of salinity and drought stresses on Petunia transformed for alpha-galactosidase expression. Abstract. HortScience 39(4): 897. Pennycooke, J.C., Vepachedu, R., Stushnoff, C., and Jones, M.L. 2004. Expression of an alpha-galactosidase gene in petunia is upregulated during low-temperature deacclimation. J. Amer. Soc. Hort. Sci. 129(4):491-496. |
| 2005 |
Chiang, Y.J., Stushnoff, C., McSay, A.E., Jones, M.L. and Bohnert, H.J. 2005. Overexpression of mannitol-1-phosphate dehydrogenase increases mannitol accumulation and adds protection against chilling injury in petunia. J. Amer. Soc. Hort. Sci. 130: 605-610. Einhorn, T.C., Stushnoff, C., McSay, A.E., Forsline, P.L., Cox, S., Ehrenkranz, J.R.L., and Sandoval, L. 2005. Biodiversity of the flavonoid phlorizin in a subset of the USDA apple germplasm core collection (Abstract). HortScience 49: 1067. Mathers, H.M. and Stushnoff, C. 2005. Screening Malus seedlings for cold hardiness. HortScience 40:318-322. Pennycooke, J.C., Cox, S. and Stushnoff, C. 2005. Relationship of cold acclimation, total phenolic content and antioxidant capacity with chilling tolerance in petunia (Petunia x hybrida). Environmental and Experimental Botany 53:225-232. |
| 2006 |
Alenazi, M. M., Hughes, H.G., Stushnoff, C. and Holm, D.G. 2006. Influence of storage temperature and time in storage on pigment content of potato (Solanum tuberosum L.) (Abstract) . HortScience, 41:1014. Bunning, M., Kendall, P., Thilmany, D., Stonaker, F., Kulen, O., Guenther, D., Stushnoff, C. 2006. Comparison of vitamin C, total phenolic content, and antioxidant activity of ten melon cultivars grown using conventional and organic methods. (Abstract) 66th Annual Meeting Institute of Food Technologists, June 24-28, Orlando, FL. No. 039G-03. Esparza Rivera, J.R., Stone, M.B., Stushnoff, C., Pilon-Smits, E. and Kendall, P. 2006. Effects of ascorbic acid applied by two hydrocooling methods on physical and chemical properties of green leaf lettuce stored at 5˚ C. J. Food Science 71:1-7. Locke, E.L. and Stushnoff C. 2006. Raffinose family oligosaccharides in protection from osmotic stresses and a review of plant responses to chilling, freezing, drought and salinity. Invited Chapter 13, In Floriculture, Ornamental and Plant Biotechnology, Volume III, Global Science Books, 123-131. Stushnoff, C., Kendall, P., Thilmany, D., and Stonaker, F., 2005. Differentiating small farm produce offerings through nutritionally superior cultivars, marketing, and extension programs. Abstract. USDA/CSREES Sustainable Agriculture, Ecosystem Management, and Small-Midsized Farms Project Directors Meeting, Oct. 31-Nov. 2, 2005, Washington DC. Troxell, H., Bunning, M., Kendall, P., Stonaker, F., Stushnoff, C. 2007. Impact of cultivar and growing method on sensory qualities of melon. (Abstract) 67th Annual Meeting of the Institute of Food Technologists, July 28, Chicago, IL. |