Nitrous acid oxidation

Ellagic acid concentration can be determined through an oxidation reaction with nitrous acid, and two methods have been described by BateSmith (1977) and by Wilson and Hagerman (1990), respectively.

Ellagitannins can be isolated from plant tissue by extraction of freeze-dried and ground tissue in 2N sulfuric acid (5-10 mg of sample/mL of acid), followed by freezing in a dry ice/2-propanol bath, vacuum sealing, and heating at 100°C for 10 h. in glass ampules. After hydrolysis, the ampules are cooled to room temperature, opened, cooled in an ice bath for 10 min. and filtered through a membrane filter. The residue on the filter (containing the ellagic acid) is then washed with several volumes of ice-cold wash solvent (acetone/H2O/concentrated HC1 (70:30:1 v/v/v) and air-dried. When the residue is dry, both sample and filter are transferred to a test tube and dissolved in 10 mL pyridine. Undissolved material is removed by a second filtration. Samples are filtered through a glass-fiber filter supported on a fine sintered glass filter to remove insolubles (undissolved membrane filter and plant material). The final filtrate is assayed for ellagic acid (Wilson and Hagerman, 1990).

Scalbert et al. (1989) summarized the method of Bate-Smith (1977): In a tube sealed with a Teflon-lined screw cap, 0.2 mL of the aqueous plant extract (1 mL if the extract is not very concentrated) is added to 1.8 mL of 1:1 methanol/water (1 mL of 9:1 methanol/water if the sample is not concentrated) and 0.16 mL of aqueous 6% (v/v) acetic acid. Nitrogen is bubbled for 5-10 min and 0.16 mL of an aqueous solution of 6% (w/v) sodium nitrite (NaNO2) is added. Nitrogen is bubbled through the solution for a few more seconds, after which the tube is sealed and incubated in a 25°C water bath for 100 min. A blue product is formed during the reaction, which is quantified by reading the absorbance with a spectrophotometer at 590 nm. The concentration of HHDP esters is expressed as 4, 6-hexahydroxydiphenoyl-glucose equivalents (emop 2169 L mol-1cm-1).

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