2006 Annual Report
Phenolics are a class of 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, which have been isolated from different natural products. Each group is further subdivided into 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 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, fertilization, and pest management), climatic and regional conditions (temperature, light, moisture, and UV radiation), maturity at harvest, and post harvest, and storage conditions. These variations cause significant challenges for the 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 phenolic compounds in natural form in foods that are eaten every day. Robust analytical measurement systems for accurate determination of these components in free and conjugated form must be developed. The Lab is developing analytical procedures that will be used to fill these knowledge gaps.
This project has three specific objectives: A) to develop extraction, fractionation, and separation methods and measurement systems for the quantitative determination of glycosylated flavonoids in foods; B) to develop robust extraction method(s) and measurement system(s) for the quantitative determination of prominent phenolic acids in foods; and C) to develop a 'fingerprinting' method using instrumental and multivariate statistical analysis to provide a differentiation of foods, herbals, and botanicals based on total phenolics.
Phenolic compounds, which are prevalent in most foods, have biological activities that could significantly improve the health of individuals thereby reducing health care costs. In addition, identification of foods that are enriched in phenolics may result in development of new value-added foods or food products.
2. Phenolic Acids *Procure phenolic acids standards. *Develop method for extraction and saponification of phenolic acids. *Develop HPLC-Ultraviolet/Visible (UV/Vis) method for analysis of phenolic acids.
3. Fingerprinting *Examine preparation/extraction methods for food materials. *Acquire preliminary data. *Acquire training on chemometrics.
Year 2 (FY 2005):
1. Glycosylated Flavonoids *Continue purchasing standards for library. *Continue detection of flavonoids and initiate development of methods for quantitation. *Initiate development of food reference materials.
2. Phenolic Acids *Initiate investigation of hydrolysis of phenolic acid conjugates by acid, base, and enzymatic digestion. *Continue work on development of extraction methodology of phenolic acids from different food matrices.
3. 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):
1. Glycosylated Flavonoids *Continue purchasing standards for library. *Continue development of methods for quantitation. *Continue development of food reference materials.
2. Phenolic Acids *Finish investigation of hydrolysis of phenolic acids conjugates by acid, base, and enzymatic digestion. *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.
3. Fingerprinting *Evaluate chemometrics analysis results on reparation/extraction methods for food materials using different solvents with varying polarity. *Continue collaborations to expand chemometrics capacity and data interpretation.
Year 4 (FY 2007):
1. Glycosylated Flavonoids *Continue purchasing standards for library. *Analyze foods for nutrient database.
2. Phenolic Acids *Determine phenolic acid content in foods. *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.
3. Fingerprinting *Compare different detection methods (mass spectrometry (MS, infrared(IR), near infrared (NIR), ultraviolet/visible (UV/Vis)) for fingerprinting. *Continue collaborations to expand chemometrics capacity and data interpretation. *Continue study of correlation of fingerprints with component concentrations.
Year 5 (FY 2008):
1. Glycosylated Flavonoids *Continue purchasing standards for library. *Analyze foods for nutrient database.
2. 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.
3. Fingerprinting *Continue to compare different detection methods (MS, IR, NIR, UV/Vis) for fingerprinting. *Continue collaborations to expand chemometrics capacity and data interpretation. *Continue study of correlation of fingerprints with component concentrations.
Database for Flavonoid Content of Selected Foods updated: results from the Food Composition Laboratory were used to update the USDA Database for Flavonoid Content of Selected Foods (Release.
A high performance liquid chromatography-diode array detection (DAD) procedure developed in the Food Composition Laboratory was used for separating and quantifying 16 phenolic acids in 10 market classes and 15 varieties of dry beans (Phaseolus vulgaris L.) that are commonly consumed in the United States. Total phenolic acid content among all samples varied between 19.1-48.3 mg/100 g of bean samples. Ferulic acid was the most abundant phenolic acid present in all samples, whereas intermediate levels of p-coumaric acid and sinapic acid were extracted from all bean samples. This work was conducted in collaboration with scientists at the Vegetable Laboratory, ARS, USDA, Beltsville, MD.
The impact of extraction methodology and polarity of extraction solvents on assay of phenolic acids was also investigated using eggplant samples. This systematic study was undertaken to address substantial variations in the extraction procedures, solvents and conditions as described in the recent literature.
The screening method for glycosylated flavonoids and other phenolic compounds was applied to more then 170 flavonoid standards and 200 food materials. The phenolic make-up of Mexican oregano, celery, cashew apples, and ledum tea (a Native American drink) were identified.
The Food Composition Lab determined catechins in 24 chocolate materials, ranging from raw cocoa and extract to finished milk and dark chocolates with partial support under a Trust Agreement between ARS and the American Cocoa Research Institute, now known as Chocolate Manufacturers Association (Vienna, VA).
The impact of extraction methodology and polarity of extraction solvents on assay of phenolic compounds was investigated using parsley (herb), Black Cohosh (dietary supplement), and eggplant (vegetable) as model substrates. This systematic study was undertaken to address substantial variations in the extraction procedures, solvents, and conditions as described in the recent literature. Extracts were analyzed for phenolic content by high performance liquid chromatography and Folin-Ciocalteu assays. The major objective of this research was to develop a systematic approach for extraction of a diverse group of phenolic compounds present in different food matrices and create awareness regarding the importance and influence of sample preparation. Sample preparation is often overlooked and is frequently considered as a "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 steps.
Broccoli, one of the most commonly consumed vegetables in North America, is a rich source of nutrients and bioactive compounds (e.g., folic acid; phylloquinone; lutein; zeaxanthin; vitamins A, C, and E; dietary fiber; sulforophanes; phenolic compounds; and organoselenium). When grown in the presence of available selenium (Se), broccoli will accumulate large concentrations; human studies suggest 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 the 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 environments with varying concentration of sodium selenate. These approaches show tremendous potential for differentiating newly developed functional foods. This project was carried out in collaboration with Grand Forks Human Nutrition Research Center.
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 effects of these phytochemicals on human health.
Reeves III, J.B., Delwiche, S.R., Daughtry, C.S., Luthria D.L. 2005. Near infrared (NIR) research at the Beltsville Agricultural Research Center. NIR News. 1618:12-13.
Luthria D.L. 2006. Optimization of pressurized liquid extraction technology for extraction of phenolic phytonutrients using green solvents, IUPAC sponsored 2nd International Symposium on Green/Sustainable Chemistry 2006, Jan. 10-14, 2006, New Delhi, India.
Luthria D.L. 2005. Assay of phenolic compounds from dry beans (Phaseolus vulgaris L.), Biannual Meeting of the Bean Improvement Cooperative - BIC 2005, held Oct. 29 to Nov. 2, 2005, Newark, Delaware.
Luthria D.L. 2005. Functional food analysis: Challenges and opportunities. United States and Japan: Cooperative Program in Natural Resources Meeting, Oct. 2005. Mt. Fuji, Japan, p. 276-279.Luthria, D.L., Pastor-Corrales, M.A. 2005. Phenolic acid content of fifteen dry edible beans (phaseolus vulgaris l.) varieties. Journal of Food Composition and Analysis. 19:205-211.
Mukhopadhyay, S., Robbins, R.J., Luthria, D.L. 2006. Optimization of extraction process for phenolic compounds from black cohosh (Cimicifuga racemosa) by pressurized liquid extractor. Journal of the Science of Food and Agricultural. 86:156-162.
Luthria, D.L., Mukhopadhyay, S., Kwansa, A. 2006. A systematic approach for extraction of phenolic compounds using parsley (petroselinum crispum) flakes as a model substrate. Journal of the Science of Food and Agriculture. 86:1350-1358