2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Phenolics are a class of secondary metabolites synthesized by plants and are ubiquitous throughout the plant kingdom. Over 8,000 different phenolic compounds belonging to four major groups namely flavonoids, phenolic acids, coumarins and tannins have been isolated from different natural products. Each group is further subdivided into further subgroups, based on their structures. Phenolic compounds are found in substantial levels in commonly consumed fruits, vegetables, herbal products, and beverages. There are numerous epidemiological studies that suggest that consumption of fruits, vegetables and herbal products correlates with reduced risk of chronic diseases associated with oxidative stress. The precise role that phenolics have in human health is not well understood because these compounds may either be present in the free aglycon form or in conjugate form with acids, sugars. The accurate database values of phenolic compounds in commonly consumed foods are also not known. In addition, levels of phenolic compounds in plants, fruits, vegetables and herbal products are influenced by genotype (cultivar or variety), agronomic practices (irrigation, fertilizations, pest management), climatic and regional conditions (temperature, light, moisture, Ultra Violet (UV) radiation), maturity at harvest, post harvest and storage conditions. These variations cause significant challenges for development of accurate and reliable analytical methods for quantitation of phenolic compounds in food and herbal products. To understand more about the association between dietary intake and protective health effects it is important to identify and quantify them in natural form in foods that we eat every day. Robust analytical measurements system for accurate determination of these components in free and conjugated form must be developed. We are developing analytical procedures that will be used to fill these knowledge gaps. This project has three specific objectives:. 1)to develop extraction, fractionation, and separation methods and measurement systems for the quantitative determination of glycosylated flavonoids in foods;. 2)develop robust extraction method(s) and measurement system(s) for the quantitative determination of prominent phenolic acids in foods;. 3)develop 'fingerprinting' method using instrumental and multivariate statistical analysis to provide differentiation of foods, herbals and botanicals based on total phenolics. The research to be undertaken falls under National Program 107 - Human Nutrition and addresses performance goal 3.1.2.B as described in the National Program Action Plan. Goal 3.1.2 is "Food Composition and Consumption: Develop techniques for determining food composition, maintain national food composition databases, monitor the food and nutrient consumption of the U.S. population, and develop and transfer effective nutrition intervention strategies." This project directly addresses Priority Objective B. Develop Analytical Methodology for Nutrients. Phenolic compounds, which are prevalent in most foods, have biological activities that could significantly improve the health of individuals there by reducing health care costs. In addition, identification of foods that are enriched in phenolics may result in development of new value-added foods products. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004): Glycosylated Flavonoids *Develop library of standards. *Prepare multiflavonoid standard for use in evaluating methodology. *Develop standardized method/s for extraction, separation and detection. Phenolic Acids *Procure phenolic acids standards. *Develop method for extraction and saponification of phenolic acids. *Develop HPLC-UV/Vis method for analysis of phenolic acids. Fingerprinting *Examine preparation/extraction methods for food materials. *Acquire preliminary data. *Acquire training on chemometrics. Year 2 (FY 2005): Glycosylated Flavonoids *Continue purchasing standards for library. *Continue detection of flavonoids and initiate development of methods for quantitation. *Initiate development of food reference materials. Phenolic Acids *Initiate investigation of hydrolysis of phenolic acids conjugate by acid, base and enzymatic digestion. *Continue work on development of extraction methodology of phenolic acids from different food matrices. Fingerprinting *Compare preparation/extraction methods for food materials using different solvents with varying polarity. *Compare different detection methods. *Establish collaborations to expand chemometrics capacity and data interpretation. Year 3 (FY 2006): Glycosylated Flavonoids *Continue purchasing standards for library. *Continue development of methods for quantitation. *Continue development of food reference materials. Phenolic Acids *Finish investigation of hydrolysis of phenolic acids conjugate by acid, base and enzymatic digestion. *Initiate synthesis/purchase of 13C labeled phenolic acids for stable isotope methods for phenolic acids. *Continue to evaluate the impact of sample preparation on assay of different classes of phenolic compounds from different food matrices. *Extract and analyze phenolic acids content of different food materials. Fingerprinting *Evaluate chemometrics analysis results on preparation/extraction methods for food materials using different solvents with varying polarity. *Continue collaborations to expand chemometrics capacity and data interpretation. Year 4 (FY 2007): Glycosylated Flavonoids *Continue purchasing standards for library. *Analyze foods for nutrient database. Phenolic Acids *Continue development of method using 13C labeled phenolic acids. *Evaluate impact on phenolic acid content in food grown under different environmental conditions. *Continue to evaluate the impact of sample preparation on assay of different classes of phenolic compounds from different food matrices. *Continue to extract and analyze phenolic acids content of different food materials. *Evaluate the impact of environmental changes on total phenolic content of different foods. Fingerprinting *Finish comparison of different detection methods (Mass, Infra-Red, Near Infra-Red, and Ultraviolet/Visible Spectrometry) for fingerprinting *Continue collaborations to expand chemometrics capacity and data interpretation. *Continue study of correlation of fingerprints with component concentrations. Year 5 (FY 2008): Glycosylated Flavonoids *Continue purchasing standards for library. *Analyze foods for nutrient database. Phenolic Acids *Finish development of method using 13C labeled phenolic acids. *Determine phenolic acids in foods for nutrient database. *Continue to evaluate the impact of sample preparation on assay of different classes of phenolic compounds from different food matrices. *Evaluate the impact of environmental changes on total phenolic content of different foods. Fingerprinting *Finish comparison of different detection methods (Mass, Infra-Red, Near Infra-Red, and Ultraviolet/Visible Spectrometry) for finger printing. *Continue collaborations to expand chemometrics capacity and data interpretation. *Continue study of correlation of fingerprints with component concentrations. *Carry out systematic analyses of different foods. 4a.What was the single most significant accomplishment this past year? A screening method has been developed as the first step in identifying and quantifying the thousands of glycosylated flavonoids in food materials. Quantification of flavonoids in food materials is of considerable interest because of recent research connecting phytochemicals with reduced risk of chronic disease. The screening method provides a standardized extraction, separation, and mass spectrometric detection procedure for initial identification of flavonoid aglycones, glycosylated flavonoids, and phenolic acids. This methodology and the resulting database will allow clinical and epidemiological studies to clarify the effect of these phytochemicals on human health. 4b.List other significant accomplishments, if any. An approach to optimize sample preparation for extraction of bioactive phenolic phytonutrient was developed using black cohosh (a dietary supplement that is currently under investigation by National Institute of Health) as a model substrate. This research will be important for all research professionals carrying out any kind of analysis. This includes all the professionals in the entire value chain from producers to consumers. Sample preparation is often overlooked and is frequently considered as "means to an end". It is estimated that an average analyst spends 2/3 of the analysis time on sample preparation and over 30% of analytical error stems from sample preparation step. Optimizing sample preparation procedures for assay of structurally diverse phenolic compounds from food matrices will immensely improve the quality of analysis and enable researchers to accurately establish their roles as it relates to health and establish appropriate dietary intake guidelines and establish accurate nutrient database values. 4c.List any significant activities that support special target populations. None. 4d.Progress report. The quantitative high performance liquid chromatography-photodiode array detection method developed by this Laboratory was used for analysis of phenolic acids from broccoli samples grown under different conditions with varying selenium concentration. This work was done in collaboration with Grand Forks Human Nutrition Research Center, North Dakota. The impact of extraction methodology and polarity of extraction solvents on assay of phenolic compounds was investigated using parsley (Petroselinum crispum) flakes as a model substrate. This systematic study was undertaken to address substantial variations in the extraction procedures, solvents and conditions as described in the recent literature. Five different extraction procedures (shaking, vortexing, sonication, stirring and pressurized liquid extraction or PLE), and three different solvents (methanol, ethanol, and acetone), with five different solvent to water ratios per solvent were used for extraction. Extracts were analyzed for phenolic content by high performance liquid chromatography and Folin-Ciocalteu assays. The major objectives of this research is to develop a systematic approach for extraction of phenolic compounds from a food matrix and create awareness regarding the importance and influence of sample preparation. Parsley (Petroselinum crispum) was selected as a model food substrate as it one of the most common herbs consumed globally and is one of the richest source of a commonly occurring phenolic aglycon, apigenin. Broccoli, one of the most commonly consumed vegetables in North America, is a rich source of various nutrients such as folic acid, phylloquinone, lutein, zeaxanthin, vitamin C, dietary fibers, sulforophanes, phenolic acids, selenium, etc. When grown in the presence of available selenium (Se), broccoli will accumulate large concentrations; human studies suggests that selenium reduces the incidence of cancer when taken in higher doses than most diets supply, and the form of Se in broccoli may be especially efficacious. Preliminary results of fingerprinting approach utilizing simple and cost effective spectroscopy (Ulterviolet/Visible, Infra-Red Near Infra-Red, direct Mass Spectrometry) techniques in conjunction with chemometrics showed that it was possible to differentiate broccoli samples grown in different environment with varying concentration of sodium selenate. These approaches shows tremendous potential for differentiating newly developed functional foods. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is the second year of the approved project. In the first year a quantitative high performance liquid chromatography-photodiode array detection method separating 16 phenolic acids was developed. Since no reference standard materials were available, this method was validated by carrying out a collaborative study with Dr. Scott Bean from Grain Marketing and Production Research Center, USDA, Manhattan, Kansas. This accomplishment was important since this robust method can be used for analysis of 16 most prominent phenolic acids in a single run, thus increasing efficiency as well as reducing cost. This method can be easily adopted for accurate analysis of phenolic acids from different food matrices. The phenolic acids values generated by this method can be potentially used for generating accurate phenolic acid nutrient database. This procedure was used for analysis of phenolic acids from broccoli samples grown under different conditions with varying selenium concentration. This work was done in collaboration with Grand Forks Human Nutrition Research Center, North Dakota. The impact of extraction methodology and polarity of extraction solvents on assay of phenolic compounds was investigated using parsley (Petroselinum crispum) flakes as a model substrate. This systematic study was undertaken to address substantial variations in the extraction procedures, solvents and conditions as described in the recent literature. Five different extraction procedures (shaking, vortexing, sonication, stirring and pressurized liquid extraction), and three different solvents (methanol, ethanol, and acetone), with five different solvent to water ratios per solvent were used for extraction. Extracts were analyzed for phenolic content by high performance liquid chromatography and Folin-Ciocalteu assays. The major objectives of this research is to develop a systematic approach for extraction of phenolic compounds from a food matrix and create awareness regarding the importance and influence of sample preparation. Parsley (Petroselinum crispum) was selected as a model food substrate as it one of the most common herbs consumed globally and is one of the richest source of a commonly occurring phenolic aglycon, apigenin. Broccoli, one of the most commonly consumed vegetables in North America is a rich source of various nutrients such as folic acid, phylloquinone, lutein, zeaxanthin, vitamin C, dietary fibers, sulforophanes, phenolic acids, selenium, etc. When grown in the presence of available selenium (Se), broccoli will accumulate large concentrations; human studies suggests that selenium reduces the incidence of cancer when taken in higher doses than most diets supply, and the form of Se in broccoli may be especially efficacious. Preliminary results of fingerprinting approach utilizing simple and cost effective spectroscopy (UV, IR, NIR, direct MS) techniques in conjunction with chemometrics showed that it was possible to differentiate broccoli samples grown in different environment with varying concentration of sodium selenate. These approaches shows tremendous potential for differentiating newly developed functional foods. This project was carried out in collaboration with Dr. John Finley 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? None. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None. Review Publications Finley, J.W., Keck, A.S., Robbins, R.J., Hintze, K.J. 2005. Selenium enrichment of broccoli: interactions between selenium and secondary plant compounds. Journal of Nutrition. 135:1236-38. Robbins, R.J., Keck, A., Banuelos, G.S., Finley, J.W. 2005. Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate and sulforaphane content of broccoli. Journal of Medicinal Food. 8:204-214.