1a. Objectives (from AD-416)
Objective 1: Develop and evaluate procedures for quantitative extraction and/or fractionation of food materials by polarity. Sub-objective 1.A.: Develop an extraction procedure for sequential fractionation of the major groups of components from plant materials. Sub-objective 1.B.: Develop optimized extraction procedures for accurate quantification of individual phytochemicals in plant materials. Objective 2: Develop and evaluate spectral fingerprinting and chromatographic profiling methods to characterize components in lipid soluble, water soluble, and intermediate fractions of food materials. Sub-objective 2.A.: Develop spectral fingerprinting methods for identification of plant materials and individual components using direct analysis (no prior chromatographic separation) and pattern recognition algorithms. Sub-objective 2.B.: Develop chromatographic profiling methods for identification and quantification of individual components in plant materials. Objective 3: Develop methods to determine variability of biologically active components in food materials through profiles and/or fingerprints.
1b. Approach (from AD-416)
Objective 1: Methods will be developed for the quantitative extraction of macro and micro components from plant materials using commercial, high pressure/temperature extraction instrumentation. Soybeans will be tested initially as they contain both lipid soluble and highly polar molecules of health interest. Sequential and parallel extraction will be investigated. Extracted materials will be characterized using liquid chromatography with diode array and electrospray ionization/mass spectrometric detection (LC-DAD-ESI/MS). The new method will be applied to foods and botanical materials. In addition, optimized methods will be developed for specific families of compounds such as water-soluble vitamins, lipid-soluble vitamins, phenolic acids, and flavonoids. Objective 2: Spectral fingerprinting methods will be developed based on ultraviolet and visible molecular absorption (UV/Vis), infrared (IR), near-infrared (NIR), and mass spectrometric (MS) detection. The overlapping complex spectra will be interpreted using pattern recognition programs. The patterns will be used to determine the sensitivity of the different detection systems for discriminating between plant materials based on genera, species, variety, growing year, growing site, and processing conditions. These methods will be developed using 3 food materials and 3 botanical supplement materials. Repeat samples will be examined over a period of years to determine the stability of the spectra and the ability to compare spectra of new materials to archived spectra. The phenolic and vitamin content of the plant materials will also be determined using chromatographic profiling using LC-DAD-ESI/MS. This will make it possible to determine which compounds are contributing most to differences arising from the various growing factors. Objective 3: The spectral fingerprints can be used with nested analysis of variance to determine the relative variance contributed by each growing factor: species, variety, site, year, plant-to-plant variation, and analytical uncertainty. Samples will be obtained from collaborators across the country and representing a variety of foods and botanical supplements. UV/Vis, IR, and NIR spectra will provide variance data for the integrated chemical composition of the plant materials and MS will provide variance data for specific masses and, with the assistance of chromatographic profiling, specific compounds of health interest.
3. Progress Report
The influence of extraction solvents, solvent ratios, and specific conditions (e.g., temperature, pressure, sample volume, static time, and flush volume) on the assay of two classes of phenolic phytochemicals (flavonoids and bioflavonoids) from Ginkgo biloba was studied. It was observed that quantity of flavonoids and bioflavonoids in Ginkgo biloba decreased with time thus indicating that phenolic phytochemicals were not very stable. Spectral fingerprints for broccoli samples consisting of 2 different cultivars and 7 different growing conditions were compare using 6 different spectral methods: fourier transform infrared (FT-IR), fourier transform near-infrared (FT-NIR), molecular absorption in the ultraviolet region (UV), molecular absorption in the visible region (Vis), mass spectrometry with positive ionization (MS+), and mass spectrometry with negative ionization (MS-). All 6 methods provided statistical differentiation between the cultivars and the growing conditions. Based on analysis of variance, the UV and Vis gave the best discrimination, followed by the group of FT-NIR, MS+, and MS-, and finally FT-IR. Chinese (Panax ginseng) and American (Panax quinquefolius) Ginseng roots were purchased locally and a project to discriminate between the two species using UV, MS, and FT-NIR fingerprints is underway. This work was supported in part by an interagency agreement with the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH).
1. Chromatograpic comparison of skullcap (scutellaria lateriflora) and germanders (Teucrium canadense and Teucrium chamaedrys). Skullcap, a botanical commonly used in traditional herbal medicines, is frequently adulterated with germander which contains a hepatic toxin. Chromatographic analysis of the two plant materials using a standardized screening method developed by Food Composition and Methods Development scientists showed that 1% adulteration with germander could easily be distinguished based on its phenylethanoid content. This method can be further developed into a single-lab validated (SLV) method for validation by the Association of Official Analytical Chemists (AOAC International) for testing of all skullcap-containing herbal mixtures.
2. Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis. Methods for the rapid identification of plant and food materials are useful for discriminating between new and old materials and alleviating analytical work load. Direct injection (with no chromatographic separation) of sample extracts into the mass spectrometer (MS) (with either positive or negative ionization) was used to discriminate between two cultivars of broccoli (Majestic and Legacy), between conventionally and organically farmed broccoli, and between fully and partially (water-stressed)irrigated broccoli. In this study, 33% of the sample variance came from cultivar, 59% came from treatment, and 8% came from analytical uncertainty. Organic and amino acids and sugars were the most useful compounds for identifying cultivar and treatment. This research provides the proof of concept for further research to develop rapid identification of food materials and categorization into a repository reference spectral database.Brisibe, E.A., Umoren, U.E., Brisibe, F., Magalhaes, P.M., Ferreira, J.F., Luthria, D.L., Wu, X., Prior, R. 2009. Nutritional characterization and antioxidant capacity of different tissues of Artemisia Annua L. Food Chemistry. 115:1240-1246.