An improved butanol-HCl assay for quantification of water-soluble, acetone:methanol-soluble, and insoluble proanthocyanidins (condensed tannins)

BACKGROUND: Condensed tannins (CT) are the most abundant secondary metabolite of land plants and can vary in abundance and structure according to tissue type, species, genotype, age, and environmental conditions. Recent improvements to the butanol-HCl assay have separately helped quantification of soluble and insoluble CTs, but have not yet been applied jointly. Our objectives were to combine previous assay improvements to allow for quantitative comparisons of different condensed tannin forms and to test protocols for analyses of condensed tannins in vegetative plant tissues. We also tested if the improved butanol-HCl assay can be used to quantify water-soluble forms of condensed tannins. RESULTS: Including ~50% acetone in both extraction solvents and final assay reagents greatly improved the extraction and quantification of soluble, insoluble and total condensed tannins. The acetone-based method also extended the linear portion of standard integration curves allowing for more accurate quantification of samples with a broader range of condensed tannin concentrations. Estimates of tannin concentrations determined using the protocol without acetone were lower, but correlated with values from acetone-based methods. With the improved assay, quantification of condensed tannins in water-soluble forms was highly replicable. The relative abundance of condensed tannins in soluble and insoluble forms differed substantially between tissue types. CONCLUSIONS: The quantification of condensed tannins using the butanol-HCl assay was improved by adding acetone to both extraction and reagent solutions. These improvements will facilitate the quantification of total condensed tannin in tissues containing a range of concentrations, as well as to determine the amount in water-soluble, acetone:MeOH-soluble and insoluble forms. Accurate determination of these three condensed tannin forms is essential for careful investigations of their potentially different physiological and ecological functions.