Progress 02/01/04 to 07/31/08
Outputs
OUTPUTS: Consumption of dark and colorful fruits and vegetables has been associated with reduced incidence of chronic disease. Animal, clinical and in vitro studies provide compelling evidence that commodities rich in anthocyanins are powerful antioxidants and may be chemoprotective to gasintestinal tract. With close to 600 anthocyanin structure present in nature, there is a need to increase understanding structure/function relationships. Our efforts have been focused on increasing the understanding of the role that anthocyanins from fruits and vegetables play on health promotion. For that reason we have selected 7 different anthocyanin sources, with distinctive but different anthocyanin profiles and tested them in an in vitro system, to evaluate the effect of anthocyanin chemical structure and chemoprevention on the colon cancer model system. A fractionation procedure hereby has been developed to prepare pure sample suitable for evaluating structure/function relationship of anthocyanins in cell culture. Moreover, we explored how horticultural and processing conditions affected their incidence in foods and other food components also played an effect on anthocyanin biological activity in a colonic cancer cell model. We have studied the possible interactions between anthocyanins (ACN) and other phenolics (OP) present in the extracts, trying to determine possible additive, synergistic or antagonistic effects. Other food ingredients, such as protein and tannins, which contents were affected by processing conditions, were evaluated their impact on anthocyanin biological activities. I have developed a course: "Food colors and Pigments" targeted to training students on the importance and potential health benefits of natural pigments mainly on anthocyanins. We have participated in national and international meetings to enhance our collaborations and exchange information with other researchers working in this field of research. In August 2007 I presented our research at the American Chemical Society national meeting in Boston. Our research was chosen by the media for an associated press release, and featured in more than 10 different countries, and in different languages. We also participated on a collaborative effort sponsored by the USDA and the PR China Ministry of Agriculture. We visited China, including Beijing, Shanghai, Nanjing and other cities in the Jiangsu province. In all different cities we visited about 7 local universities and/or research institutions where we disseminated results from our research and exchanged ideas for future collaborations. Collaboration with Jiangsu University has been established to investigate Asian radish cultivars as sources of natural colorants and nutraceuticals for food applications. Dissemination of this project also was done through presentations by the students at the annual IFT meeting, as well as by presentations by the PI at research institutions in Peru (International Potato Center, Universidad Nacional Agraria), at the 4th international congress on pigments in food in Germany, as well as to the food industry at companies in USA, Peru and Bolivia. PARTICIPANTS: M Monica Giusti, Principal investigator, coordinated the work, supervised personnel, and supervised experimental design, data collection, interpretation, publications. The PI was also encharged of dissemination of the informatio in other venues such as press releases and presentations at professional meetings. Collaborators: Steven Schwartz - suppported the chemical characterization of poliphenolics, mainly at the initial stages of the project. Joshua Bomser provided the facilities for the cell work, and assisted on the data interpretation of biological tests. Bernadene Magnuson assisted on experimental design of cell work as well as with data interpretation. Pu Jing conducted a large portion of the experiments including characterization of plant materials, and cell proliferation tests both as student and as a post-doctoral researcher. Students: Pu Jing, Jian He, Taylor Wallace. This project allowed training and supported the studies of the students listed above. As a result of the project and the press coverage of the wrk, the PI has been invited to present the work at scientific meetings as well as to highschool science teachers, as part of a continued education program in Ohio. This project allowed the investigators to establish contacts with a number of research Institutions around the world, including: - Jiangsu University, China - Universidad Nacional Agraria, Lima, Peru - International Potato Center, Lima, Peru TARGET AUDIENCES: Scientific community - information has been shared and disseminated in the form of presentation at scientific meetings, abstracts, proceedings, manuscripts, book chapters. Food Industry - information has been shared as presentations to the scientific communitiy as well as to specific companies with interest in the use of anthocyanin-rich commodities and/or natural anthocyanin-based colorants. Consumers - information regardig the potential health benefits of increased consumption of anthocyanin-rich fruits and vegetables has been shared with the media. Also, information will be shared with high school teachers, which will help disseminate the infomration to a wider audience. PROJECT MODIFICATIONS: The scope of the project was expanded as needed to include puple corn, a material that showed special potential. The work was focused on the HT-29 cell lines and little work was done with the normal cells as the data showed at the begining that anthocyanins had little effects on normal cells. Also, the fractionation procedures for anthocyanins and other phenolics were optimized. The work in this area was expanded to improve the separation of the compounds and to obtain more valid results on the contribution of anthocyanins to the biological effects being observed.
Impacts
Anthocyanin-rich extracts (AREs) from chokeberry (Aronia meloncarpa E.), bilberry (Vaccinium myrtillus L.), purple carrot (Daucus carota L.), grape (Vitis vinifera), radish (Raphanus sativus), purple corn (Zea mays L.), and elderberry (Sambucus nigra L.) were selected and tested to determine the concentration needed to inhibit growth of a human colorectal adenocarcinoma (HT29) cell line by 50% (GI50). Our research found that all anthocyanin rich extracts (ARE) suppressed HT29 cell growth to varying degrees. Purple corn ARE showed the highest growth inhibition (GI50~14 ug/mL as cyanidiin-3-glucoside equivalent), followed by chokeberry and bilberry. Elderberry and radish AREs showed the lowest growth inhibition (GI50:~130 ug/mL; ~108 ug/mL). AREs at these concentration ranges and under similar conditions had little effect on NCM460 normal colon cells. We developed and optimized a methodology for fractionation of anthocyanins from other phenolics present in ARE. Anthocyanins played a major role in AREs' chemoprotection and exerted an additive interaction with other non-anthocyanin phenolics. Statistical analyses demonstrated that type of anthocyanin aglycone, glycosylation, and/or acylations affected growth inhibition activity. Pelargonidin 3-glucoside to have lower biological effect than cyanidin 3-glucoside. Nevertheless they both showed higher inhibitory activity than their respective 3-sophoroside-5-glucoside forms. Deacylation significantly increased the growth-inhibitory activity for cyaniding-3-sophoroside-5-glucoside acylated with cinnamic acids but not for pelargonidin in a similar structure pattern. Purple corn anthocyanin mixture exerted higher inhibitory effect after the removal of aliphatic acids on anthocyanins via saponification. We also found that growth conditions affected purple corn the degree of non-acylated anthocyanins, which may contribute to more inhibitory effect than acylated ones. Formation of anthocyanin complexes with macromolecules during purple corn colorant processing conditions reduced but did not eliminate the chemoprotective effects of these anthocyanins. These findings can be used for the development of new anthocyanin applications in functional foods, as well as for crop and cultivar selection and promotion of processing condition of anthocyanin-type colorants or nutraceuticals. Identification of health promoting components in anthocyanin extracts would increase the demand of these commodities by consumers and the food industry expanding the market of fresh and processed anthocyanin-containing foods. Consequently, our results may expand the market of anthocyanin-rich commodities as value added ingredients for functional foods. Anthocyanin colorants represent an excellent source of polyphenols, and can improve the visual appeal of foods as an alternative to the use of synthetic dyes. An increased consumption of polyphenols can be then obtained while improving the visual appeal of food products. Incorporation of these compounds into the regular diet may help improve health. The added value of these commodities would open a new window of opportunities for use of these extracts in a variety of food applications.
Publications
- GIUSTI, MM. 2007. Effect of glycosylation and acylation on the chemoprotective effects of anthocyanins. American Chemical Society, Division of Agricultural & Food Chemistry. 234th National Meeting & Exposition, Boston, MA August 19-23
- Wallace, T., and Giusti MM. 2008. Berberis boliviana pigments as source of natural colorants. J. Food Science. In press.
- Jing, P., Noriega, V.; Schwartz, S.J. and Giusti, M.M. 2007. Effects of growing conditions on Purple Corncob (Zea mays L) anthocyanins. J. Agr Food Chem 55, 8625-8629.
- Jing, P. and Giusti, M.M. 2007. Effects of extraction conditions on improving the yield and quality of an anthocyanin-rich purple (Zea Mays L.) corn color extract. J. Food Sci. 72, 1517-1522.
- He, J.; Rodriguez-Saona, L.E.; Giusti, M.M. 2007. Mid-Infrared Spectroscopy for Juice Authentication - Rapid Differentiation of Commercial Juices. J Agr Food Chem, 55, 4443-4452.
- Giusti, M.M. and Wrolstad, R.E. 2008. Color Quality of Maraschino Cherries. Chapter in Color quality of fresh and processed fruits and vegetables. Eds. Wrolstad and Culbert. ACS publications. In Press.
- Rodriguez-Saona, L.E., Giusti, M.M. and Wrolstad, R.E. 2008. Expanding the Potato Industry: Exotic Colored-Fleshed Tubers. Chapter in Color quality of fresh and processed fruits and vegetables. Eds. Wrolstad and Culbert. ACS publications. In Press.
- Schwartz, S.J., Von Elbe, J.H., and Giusti, M.M. 2007. Colorants. Ch. 9 In Fennema's Food Chemistry. S. Damodaran, K.L. Parkin, O.R. Fennema (Eds.), 4th edition. CRC Press. Pp 571-638.
- GIUSTI, M.M. Jing, P. 2007. Analysis of Pigments and Colorants: Analysis of Anthocyanins. Ch 6.3 In Food Colorants: Chemical and Functional Properties, C. Socaciu (Ed). CRC Press. Pp. 479-506.
- GIUSTI, M.M. Jing, P. 2007. Natural pigments of berries: functionality and applications. Chapter 4 in Berry fruit: Value-added products for health promotion, Yanyun Zhao (Ed.). CRC Press. Pp 105-146.
- Jing, P., Joshua Bomser, M. Monica Giusti, 2007. Intrinsic and extrinsic factors affecting anthocyanin chemoprotection. Presentation at the Institute of Food Technologists Annual Meeting and Food EXPO. Chicago, IL, July.
- Anonymous. 2007. Darker fruits could fight cancer. BBC News. Monday august 20, 2007. www.news.bbc.co.uk.
- Wallace, TC, M. Monica Giusti. 2007. Berberis boliviana anthocyanins as natural colorants for yogurt. Presentation at the Institute of Food Technologists Annual Meeting and Food EXPO. Chicago, IL, July.
- He, J and MM Giusti. 2007. High-purity Fractionation of Anthocyanins from Fruit and Vegetables - Advantages of Ion-exchange Techniques over Conventional Methods. Presentation at the Institute of Food Technologists Annual Meeting and Food EXPO. Chicago, IL, July.
- Keatley, KE, J He, R Klein, MM Giusti, LA Kresty, ML Failla. 2007. Inhibition of proliferation of human bladder epithelial cancer cell lines by anthocyanin-rich fraction from black raspberry. Presentation at the Institute of Food Technologists Annual Meeting and Food EXPO. Chicago, IL, July.
- Anonymous. 2007. Color Esperanza: Antocianinas y cancer al colon. Suplemento Somos, diario El Comercio, August 26, 2007. Article highlight study in Dr. Giusti's lab on chemoprotective effects of anthocyanin rich commodities.
- Anonymous. 2007. Study: dark colored fruits and veggies fight cancer. Fox News, Tuesday August 21, 2007. www.foxnews.com.
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Progress 01/01/06 to 12/31/06
Outputs
The long-term goal of this study is to expand the markets of anthocyanin-rich agricultural commodities by identifying anthocyanin-rich products with added value due to the presence of health-promoting components. This long-term goal is being fulfilled through investigation in two main specific areas, described by our supporting objectives: 1. Screen the health-promoting potential of anthocyanin-rich extracts using an in vitro biological model system. 2. Determine the structure/function relationship between anthocyanins and other phenolics and biological activity. AREs with different anthocyanin profiles from purple corn (Zea mays L.), chokeberry (Aronia meloncarpa E.), bilberry (Vaccinium myrtillus L.), purple carrot (Daucus carota L.), grape (Vitis vinifera L.), radish (Raphanus sativus L.), and elderberry (Sambucus nigra L.) were tested for their GI50 value which is the concentration needed to inhibit growth by 50%, tested on a human colorectal adenocarcinoma (HT29)
cell line. Extracts were fractionated into an anthocyanin fraction (ACN) and other phenolics fraction (OPF) by using a solid phase extraction procedure and tested for synergistic, additive or antagonistic effect. The effects of type of aglycone, glycosylation pattern and acylation with aliphatic or cinnamic acids on anthocyanin chemoprotection were evaluated. Pure cyanidin/pelargonidin 3-glucoside and their 3-sophoroside-5-glucoside and acylated forms were also evaluated for their antiproliferative activity on HT29. All AREs inhibited colon cancer cell proliferation to varying degrees. Purple corn ARE showed the highest growth inhibition (GI50~14ug/ml), followed by chokeberry and bilberry. Elderberry and radish showed the lowest inhibition (GI50:~130ug/ml; ~108ug/ml). These differences on bioactivity were attributed to the differences in chemical structure, including the type of aglycone, anthocyanin glycosylation, and /or acylations. ACN, rather than other OPF, played an important
role in the chemoprotective effects of AREs although generally they exerted additive interactions towards the total inhibitory effects. From our structure/function study, we found pelargonidin 3-glucoside to have lower biological effect than cyanidin 3-glucoside. Nevertheless they both showed higher inhibitory activity than their respective 3-sophoroside-5-glucoside forms. Deacylation significantly increased the growth-inhibitory activity for cyaniding-3-sophoroside-5-glucoside acylated with cinnamic acids but not for pelargonidin in a similar structure pattern. Purple corn anthocyanin mixture resulted in an increased inhibitory effect after the removal of aliphatic acids on anthocyanins via saponification and purification on a C18-cartridge. Anthocyanin-based colorants may be chemoprotective and therefore valuable ingredients for functional foods. Our results should provide light on what anthocyanin structures to choose for further application in functional foods and crop and
cultivar selection. In addition, the structure/function study should provide structural targets to study behind mechanisms that enable anthocyanins to exhibit their chemoprotection.
Impacts
Anthocyanin-based colorants may protect against chronic diseases and therefore they may become valuable ingredients for functional foods. Our results provide light on what anthocyanins may be selected for application in functional foods and crop and cultivar selection when looking for health benefits. In addition, the structure/function study should provide structural targets to study behind mechanisms that enable anthocyanins to exhibit their chemoprotection. This study provides important information that may increase the market value of anthocyanin-containing commodities. Identification of health promoting components in anthocyanin extracts would increase the demand of these commodities by consumers and the food industry expanding the market of fresh and processed anthocyanin-containing foods. Additionally, incorporation of these compounds in more foods as part of the regular diet may help improve health. The added value of these commodities would open a new window
of opportunities for use of these extracts in a variety of food applications.
Publications
- Jing, P, Bomser, JA, Magnuson, BA, Giusti, MM, 2006. Structure-function relationship between anthocyanins and their chemoprotective properties. In Pigments in Food: A Challenge to Life Sciences, Section 1, Analysis and chemistry of pigments. Carle, Schieber and Stintzing (Eds). The 4th International Congress on Pigments in Food, Stuttgart-Hohenheim, Germany, October, 2006, p 47-49. - Outstanding poster contribution.
- Jing, P., Bomser, J., Schwartz, S., Giusti, M.M. 2006. Chemical structure and biological activity of anthocyanins from different edible sources. Book of abstracts. Ann. Mtg., Inst. Food Technol. Orlando, FL. June 24-28.
- Jing, P., Bomser, J., Schwartz, S., Giusti, M.M. Effects of chemical composition of anthocyanin-rich commodities on their chemoprotective properties. 2006. Knowledge Bank. The Ohio Agricultural Research and Development Center (OARDC). Columbus, Ohio, April 21.
- Jing, P., and Giusti, M.M. Effects of chemical composition of anthocyanin-rich commodities on their chemoprotective properties. 2006. Presented at the 20th Annual Edward F. Hayes Graduate Research Forum, The Ohio State University, April 22
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Progress 01/01/05 to 12/31/05
Outputs
Anthocyanins are potent antioxidant and may be chemoprotective. However, the relationship between anthocyanin chemical structure and its potential biological activity is not well understood. There are more than 600 anthocyanins already reported in nature, and they differ on the type of aglycones, the type and number of sugars substitutions and in the presence, type and numbers of organic acids attached to the molecule. For that reason we have selected 7 different anthocyanin sources, with distinctive but different anthocyanin profiles and tested them in an in-vitro system, to evaluate the effect of anthocyanin chemical structure and chemoprevention on the colon cancer model system. In addition, we are exploring the possible interactions between anthocyanins and other phenolics present in the extracts, trying to determine possible additive, synergistic or antagonistic effects. For this purpose, we used a fractionation procedure that was developed during the early stage
of this grant, and previously reported. Anthocyanin-rich extracts (AREs) from chokeberry (Aronia meloncarpa E.), bilberry (Vaccinium myrtillus L.), purple carrot (Daucus carota L.), grape (Vitis vinifera), radish (Raphanus sativus), purple corn (Zea mays L.), and elderberry (Sambucus nigra L.) were tested to determine the concentration needed to inhibit growth of colon cancer cells (HT-29) by 50% (GI50). An anthocyanin fraction (ACN) and other phenolics fraction (OPF) were separated with a C18-cartridge and tested for synergistic, additive or antagonistic effects. Anthocyanins and concomitant phenols were quantified as either cyanidin-3-glucoside equivalent or gallic acid equivalents, respectively, and cell proliferation was determined by using the sulforhodamine B assay. All AREs inhibited colon cancer cell proliferation at varying degrees. Purple corn ARE showed the highest growth inhibition (GI5014 ug/ml), followed by chokeberry and bilberry. Radish showed the lowest inhibition,
with a GI50 of 131 ug/ml. This may be attributed to the presence of anthocyanin diglycosides, type of aglycone (pelargonidin) and/or cinnamic acid acylations. More research is being carried out trying to determine the role of each of these factors on chemoprotection by anthocyanins. Both, cytostatic and cytotoxic (>100% cell growth inhibition) effects were observed with AREs on HT-29 colon cancer cells. Fractionation of ACN suggested that anthocyanins play an important role on the chemoprotective effects of AREs. Combining ACN with OPF in the media showed that their inhibitory effects were mostly additive. Saponification of purple corn ACN resulted on an increased inhibitory effect of HT-29 cell proliferation, suggesting that non-acylated anthocyanins are more effective chemoprotective agents than their corresponding acylated anthocyanins. We are currently trying to verify this hypothesis by testing saponified radish extracts.
Impacts
Anthocyanins are compounds widely distributed in nature, and that provide color to most orange, red, purple and blue fruits and vegetables. For this reason, anthocyanins have been used by the food industry as natural alternatives to the use of artificial dyes. However, using natural pigments is more challenging and expensive than using their artificial counterparts. Our research is showing that anthocyanins provide more than just color: they may protect some tissues (such as colon tissues) from cancer development. By understanding which chemical structure (or which anthocyanins) may be more biologically active; we also provide a valuable tool for future research. It would make it easier to determine what anthocyanin-rich fruits or vegetables should be target of further research as chemoprotective agents. It also provides a tool for plant breeders to determine what traits to look for, given that some varieties may tend to accumulate higher proportions of certain
pigments. Eventually, the impact from our research would be to expand the market of anthocyanin-rich commodities as value added ingredients for functional foods.
Publications
- GIUSTI, M.M. 2005. Absorption, excretion, and in vivo transformations of different individual anthocyanins as affected by their chemical structure. Book of abstracts. Ann. Mtg., Inst. Food Technol., New Orleans, LA, July 16-20.
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