Improving the vanillin-sulphuric acid method for quantifying total saponins

The colorimetric assay used for saponin quantification in plant extracts is subject to interference by common solvents used to extract the saponins from plant materials. Therefore, the degree of interference of ten common solvents was investigated. It was found that the presence of acetone, methanol and n-butanol in the reaction solution caused an intense darkening of the reaction solution in the absence of saponins, which likely could lead to erroneous saponin content values. Using aescin to construct standard curves with different solvents—such as water, ethanol, and methanol— also showed significant differences in the standard curves obtained, which led to different values when they were applied to quantify the saponin content of an ethanol extract from dried and powdered Gac (Momordica cochinchinensis Spreng) seed kernels. To improve the method, a solvent evaporation step was added prior to the colorisation reaction to prevent undesired solvent interference during the reaction step. Using this modified protocol for the aescin standard curve and the Gac seed kernel extract eliminated any solvent interference. Thus, this improved protocol is recommended for the quantification of the saponin content of plant extracts irrespective of which extraction solvent is used.