The Use of Merbromin Fluorescence Quenching for the Assay of Silymarin in its Natural Source and Pharmaceutical Preparations

Silymarin is a mixture of anti-hepatotoxic polyphenolic substances isolated from the milk thistle plant, Silybum marianum (L.) Gaertn. Silymarin is a static quencher, therefore, a new sensitive spectrofluorimetric method for quantitation of silymarin (as a total content) in its natural source and pharmaceutical formulations was developed. The method is based on the fluorescence quenching of merbromin (MER) upon the addition of silymarin. Calibration graphs correlating the fluorescence difference (Fo -F) or the fluorescence ratio (Fo /F) of MER before (Fo) and after (F) quencher (silymarin) addition versus the concentration of the latter are linear. First and second derivative treatment of the data was performed. Also, the study presents comparison between two statistical regression methods for handling data; least squares parametric regression method, which is considered the de facto standard method used for regression and Theil's non-parametric regression method. So, this work combines the advantages of the derivative techniques with their elimination of interferences, together with the reliability and efficacy of the non-parametric analysis of data. Theil's method was found to be superior to the method of least squares as it assumes that errors could occur in both x- and y-directions and they might not be normally distributed. In addition, it could effectively circumvent any outlier data points. The developed methods were successfully applied for the determination of total silymarin content in its natural source (Silybum marianum (L.) Gaertn.) and pharmaceutical formulations. The results were in a good agreement with the pharmacopoeial and label claims.