Dynamics of the isoflavone metabolome of traditional preparations of Trifolium pratense L.

Ethnopharmacological relevance: The flowering tops of Trifolium pratense L., popularly known as red clover, are used in ethnic Western and Traditional Chinese medicine, in a variety of preparations, including infusions, decoctions and tinctures. Red clover has been reported to be helpful for treatment of menopausal symptoms, premenstrual syndrome, mastalgia, high cholesterol, and other conditions. Aims of the study: The aims were to compare the chemical dynamics between traditional preparations of infusions, decoctions, and tinctures, as well as to identify the chemical variability over time in a traditional red clover tincture. For this purpose, eight isoflavone aglycones as well as two glucosides, ononin and sissotrin, were used as marker compounds. Materials and methods: Quantitative NMR (qHNMR), LC-MS-MS, and UHPLC-UV methods were used to identify and quantitate the major phenolic compounds found within each extract. Results: Infusions, decoctions and tinctures were shown to produce different chemical profiles. Biochanin A and formononetin were identified and quantified in infusion, decoction, and tinctures of red clover. Both infusion and decoction showed higher concentrations of isoflavonoid glucosides, such as ononin and sissotrin, than 45% ethanolic tinctures. Dynamic chemical variability ("dynamic residual complexity") of the red clover tincture was observed over time (one-month), with biochanin A and formononetin reaching peak concentrations at around six days. Conclusions: Insight was gained into why different formulation methods (infusions, decoctions, and tinctures) are traditionally used to treat different health conditions. Moreover, the outcomes show that tinctures, taken over a period of time, are dynamic medicinal formulations that allow for time-controlled release of bioactive compounds.