Distribution, Quantification, and Identification of Tannins in Acorns from Red and White Oak Trees:
Tannins, as preservers of animal skins, were in commercial use long before there was any clue about their natural function. Their astringent properties, how they affect taste, may have an important role in their natural function. Their role in the taste of wines and other spirits is of major concern to the alcohol producing industry. In recent times, the operational definition of a vegetable tannin has been broadened to include “. . . water soluble phenolic compounds having molecular weights between 500 and 3,000 and besides giving the usual phenolic reactions have special properties such as the ability to precipitate alkaloids, gelatin, and other proteins.” The role of plant tannins at the ecological level is almost certainly a mixed function, involving a defense mechanism against living-plant enemies and a delay in decomposition when plant tissue becomes litter, either in the soil proper, as in roots, or on the soil surface, as in leaves. There is no compelling evidence that tannins have any role in plant physiological processes.
SAMPLE PREPARATION FOR MASS SPECTROSCOPY:
Cotyledons and embryos of Quercus palustris (Pin Oak), Quercus rubra (Red Oak), Quercus macrocarpa (Bur Oak), and Quercus muehlenbergii (Chinkapin Oak) were treated with the following method. The cotyledons and embryos were removed from the seed coat, allowed to dry at 37ºC and then pulverized with a mortar and pestle. 500mg of the dry material was added to 5mL of a solution of methanol/water (80:20 v/v) containing 0.8mM NaF to prevent sample oxidation. The solution was shaken on a Glas-Col bench top shaker for one hour and allowed to settle. The supernatant was removed and filtered with a 0.2 µm hydrophilic nylon membrane filter. The filtered extract was analyzed using LC/ESI/MS and LC/EI/MS.
INSTRUMENTATION – HPLC/DAD/ESI-MS/MS Analyses:
LC/ESI/MS/MS experiments were performed on an Agilent MSD XCT ion trap mass spectrometer (Palo Alto, CA) equipped with an electrospray ionization (ESI) interface, 1100 HPLC, a DAD detector, and Chemstation software. The column used was a 150 x .5 mm i.d., Zorbax XDB – C18 3.5 µm (Agilent, Palo Alto, CA). Flow rate was 5.00 µL/min, injection volume was 0.5 µL, and column temperature was 25ºC. The ESI parameters were as follows: nebulizer, 15 psi; dry gas (N2), 5.00 L/min; dry temperature, 325ºC; trap drive, 78.0; skim 1, -40V; lens 1, 5.00V;octopole RF amplitude, 200.0 Vpp; capillary exit, -200V. The ion trap mass spectrometer was operated in negative ion mode scanning from m/z 50 to m/z 2200 at a scan resolution of 13000 amu/s. Trap ICC was 70000 units and maximal accumulation time was 2000000 µs. MS-MS was operated at a fragmentation amplitude of 1.0V, and threshold ABS was 20000 units.
LIQUID CHROMATOGRAPHIC SEPARATION:
The constituents were separated using a water (A) and methanol (B) gradient (each containing 0.1% formic acid). Initial conditions were 3% methanol increasing to 25% methanol at 6 minutes increasing to 35% at 25 minutes increasing to 90 % at 35 minutes holding at 90% to 40 minutes and returning to starting conditions at 45 minutes. The detection wavelength was 254nm.
Our quantification of the tannins throughout the acorns did support the literature with regards to the increase of tannin levels from basal to apical segments of the acorn. Our observations were that while the amounts of tannins increased, the tannins varied in identity. As reported in the literature we were able to confirm that red oak acorns contain more total tannins than white oak acorns. However, these reports only considered the total polyphenolic compounds. We examined the amounts of ellagitannins and gallotannins and discovered that within the red oak acorns the concentration of gallotannins is much greater than ellagitannins, whereas the white oak acorns were much higher in ellagitannins.
We plan to look at the individual tannins to examine the possibility that certain tannins may be responsible for predator aversion towards acorn species and aversion to portions of individual acorns.
Tyler J. Marquart, Chad M. Scholes and James M. Chapman
Rockhurst University, Kansas City, MO
1. Phenolic Compounds and Fatty Acids From Acorns (Quercus Spp.), The Main Dietary Constituent of Free-Ranged Iberian Pigs. Emma Cantos, Juan Carlos Espin, Clemente Lopez-Bote, Lorenzo De La Hoz, Juan A. Ordonez, and Francisco A Tomas- Barberan. J. Agric. Food Chem. 2003, 51, 6248-6255.
2. Tannins: Does Structure Determine Function? An Ecological Perspective. William V. Zucker. The American Naturalist, 1983, Vol. 121, Issue 3, 335-365.