Evaluation of methods for measuring polyphenols in conifer foliage

There is a resurgence of interest in the quantification of polyphenols in plant tissues because of their presumed ecological importance in plant-litter-soil and plant-animal interactions. The influence of sample preparation, extracting solvent, foliage quality, and assay method was investigated for the quantification of total phenols and condensed tannins in conifer foliage. Our results suggest that it is not possible to recommend a single optimal protocol for quantification of total phenol and condensed tannin fractions from plant materials. In general, the use of aqueous acetone (50-70% v/v) with freeze-dried materials gave the highest recovery. The Folin-Ciocalteau method for total phenols and the butanol-HCl hydrolysis method for condensed tannins appear superior to other common assays tested. There were large differences (1.4-2.2 times) in the reactivity of purified condensed tannins among species, indicating the importance of an appropriate standard for polyphenol quantification. A solid-state C-13 NMR method with an improved "interrupted decoupling" pulse sequence yielded the highest concentrations for condensed tannins. Assuming that C-13 NMR provides an accurate measure of total condensed tannin, the other extraction/assay methods used in this study recovered 50-86% of the condensed tannin fraction. The recovery rate is correlated with the nitrogen content of the foliage, which suggests that the formation of protein-tannin complexes may limit the extractability of condensed tannins. While C-13 NMR condensed tannin values may give the best value for total condensed tannin concentrations, the water-soluble fraction may have the greatest physiological and/or ecological significance.