Constituents of Geranium thunbergii SIEB. et ZUCC. VI. Differences of Tannin Activity caused by Structural Differences. (2). Colorimetry with Methylene Blue
A new colorimetric determination of tannin, measuring absorbances of the solutions after precipitating Methylene Blue with the sample, was investigated to develop an analytic method of higher sensitivity and reproducibility. Comparison of the calibration curves of tannic acid in Japanese Pharmacopoeia (J.P.), geraniin, chebulinic acid and chebulagic acid at various pH showed that the best result in sensitivity, linearity and reproducibility common to these 4 tannins is obtained at pH 7.0. The determined value (RMB) of geraniin by this method at pH 7.0, based on the calibration curve of tannin acid J.P. (RMBpH7) was 1, but appreciable differences between these 2 tannins were observed at other pH ranges. The RMB values of samples at pH ranges other than 7.0 were based on the calibration curve of tannic acid J.P. at pH 7.0. Marked similarity between RMBpH7 and RApH6 was observed for variation of the activity induced by hydrolysis of geraniin, and extracts of G. thunbergii. The RMBpH7 values for seasonal variation of tannin content in G. thunbergii, for tannin content of Geranium spp., and of several species of plants were obtained before and after treating the extracts with methanol. The RMB values of these specimens, based on the calibration curve of geraniin (RMBG), and RMB of geraniin and hydrolyzed geraniin based on the calibration curve of tannic acid J.P. at the same pH, were also obtained. The amount of the samples required for RMBph7 determinations was approximately 1/50 of those for RApH 6 determinations.