|Grusak, Michael - Mike|
|Erdman Jr., John|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/2004
Publication Date: 3/1/2004
Citation: Yousef, G.G., Seigler, D.S., Grusak, M.A., Rogers, R.B., Knight, C.T., Kraft, T.F., Erdman, J.W., Lila, M.A. 2004. Biosynthesis and characterization of 14C-enriched flavonoid fractions from plant cell suspension cultures. Journal of Agricultural and Food Chemistry. 52(5):1138-1145. Interpretive Summary: Plants manufacture different kinds of bioactive phytochemicals that can benefit the health of humans. One such class of phytochemicals, the flavonoids, are found in foods such as grapes and berries, and their consumption has been associated with lowered incidences of human chronic diseases such as cardiovascular disease and certain cancers. Although we have identified various foods that contain flavonoids, we do not have a clear understanding of how dietary flavonoids are absorbed in the gut, or how they are distributed throughout the body to various target organs after they are absorbed. This lack of knowledge is primarily due to the fact that flavonoids are complex molecules that are difficult to synthesize in the laboratory, in forms that can be tracked throughout the body. Thus, new tools are needed to provide ways to study flavonoid absorption. In this study, we have developed a method for labeling cultured cells of berry and grape with radioactive carbon, in order to allow these cells to manufacture radioactively labeled flavonoids. We also have reported a technique for separating various flavonoid fractions from these cells, and have calculated how much radioactivity is incorporated into each fraction. This method will enable us to generate sufficient quantities of labeled (i.e., traceable) flavonoids for use in animal studies, thereby allowing us to investigate the absorption and subsequent whole body distribution of these important phytochemicals.
Technical Abstract: A range of radiolabeled proanthocyanidins, anthocyanins, and other flavonoids were accumulated in cell suspension cultures of two plant species, ohelo (Vaccinium pahalae) and grape (Vitis vinifera) by providing U-14C-sucrose to the medium. A semi-closed labeling chamber was used to safely and effectively incorporate 14C into the growing cells. The cells incorporated 68.7% to 74.6% of the initial 14C radioactivity, and about 50% was recovered in flavonoid-rich fractions. Monomers to oligomers of intact proanthocyanidins and anthocyanins were effectively labeled and isolated from flavonoid-rich fractions using a relatively gentle vacuum chromatography technique. While proanthocyanidins were extensively synthesized by ohelo cells, the predominant polyphenolics in grape were anthocyanins. Specific activity of flavonoid-rich fractions varied both between and within ohelo and grape species. Flavonoid-rich fractions reached specific activities of 150 muCi/g-1 and 444 muCi/g-1 in ohelo and grape, respectively. Our labeling system can be used repetitively to produce the same unique classes of natural phytochemical radiolabeled compounds. The biosynthesized phytochemicals in this study will be used for in vivo animal feeding studies to determine the bioavailability, metabolic fate, and accumulation in target organs such as brain, liver, and kidney.