Comparison of HPLC-UV and NMR methodologies for the quantification of Silymarin complex in Silybum marianum fruit extracts

Silybum marianum (L.) Gaertn. (Asteraceae) is one of the most investigated plant extracts with known mechanism of action. Milk thistle preparations have been used to treat a variety of ailments, particularly against liver damage. The beneficial properties of S. marianum are ascribed to silymarin, a mixture of (at least) six flavonolignans and one flavonoid, used in clinical research as well as in dietary supplements. The separation of individual compounds from the complex mixture of regioisomers remains a challenging task. This study presents two validated HPLC-DAD and NMR methods, according to the ICH guidelines, for the simultaneous determination and quantification of six bioactive compounds in S. marianum extracts.

A HPLC method was developed for determination of flavonolignans incorporating rapid separation with highly sensitive UV detection. The method, beside the analysis, was used for their quantification in the fruit extracts. As Silybum flavonolignans are difficult to obtain as fully characterized pure compounds, the development of a non-targeted approach by quantitative ¹H NMR represented an attractive alternative to conventional chromatographic analysis. Besides quantification, qNMR provides valuable structural information, requires simple sample preparation and reasonably short measuring times, especially with contemporary NMR instrumentation. Silybins and isosilybins exhibit near-identical ¹H NMR spectra, which led us to quantify them in pairs. Measurement of two different types of extracts showed comparable results in composition between the two different techniques.

For more than five decades, silybins and isosilybins have presented a wealth of challenging interdisciplinary research problems. The successful implementation of the current work could comprise an established approach for future investigations, giving new insight into the quantification of bioactive compounds of other plant species containing complex mixtures of isomers.