Despite the changes Bate-Smith (1977) made to the original protocol, the time- and temperature sensitivity of the iodate assay, as well as the cross-reactivity with ellagitannins negatively impact the reproducibility of this method. Inoue and Hagerman (1988) developed the rhodanine assay. Rhodanine (2-thio-4-ketothiazolidine; 4.1) reacts with the vicinal hydroxyl groups of gallic acid to produce a red complex that can be detected spectrophotometrically at 520 nm. This reaction is specific for gallic acid, and can thus be used for the detection of gallotannins. This requires acid hydrolysis of the gallotannins before the reaction with rhodanine. Tannins are extracted from the plant tissue in 1 mL 70% (w/v) acetone in water per 100 mg dry sample in a sonicator at 4°C. The extract is then filtered through a sintered glass filter. The filtrate containing the tannins is collected into a glass ampule. The residue in the filter is then washed with 5 mL 2N sulfuric acid, which is added to the ampule. The ampule is frozen and vacuum-sealed, and then heated at 100°C for 26 h. to hydrolyze the gallotannins.
The contents of the ampule are diluted in water to a final volume of 50 mL. A 1-mL sample is then taken for the assay. To this sample 1.5 mL 0.667% (w/v) rhodanine in methanol is added. After exactly 5 min. 1 mL 0.5N KOH is added. After 2.5 min. water is added to a final volume of 25 mL. The absorbance is read at 520 nm after a 5-10 min. incubation. A standard curve is made by reaction of gallic acid in 0.2N sulfuric acid with the rhodanine solution. Hagerman and Butler (1989) argued that this assay is more suitable than the potassium iodate assay for the determination of hydrolysable tannins, although it has to be kept in mind that the rhodanine assay is sensitive to any gallic acid ester, including those in non-tannin compounds.
A helpful review of the different methods for the analysis of tannins and the rationale for choosing one over the other was presented by Hagerman and Butler (1989). They also pointed out that it was critical to choose a suitable standard to compare the experimental data with. Given the
difficulty of isolating chemically pure tannins, they recommended using either non-tannin standards (such as gallic acid or cathechin), in which case the data are expressed in 'equivalents of the standard', or using commercially available tannic acid preparations. In the latter case it is critical to indicate the source of the standard, since the commercial preparations vary from each other considerably.
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