Variable Isoflavone Content of Red Clover Products Affects Intestinal Disposition of Biochanin A, Formononetin, Genistein, and Daidzein

Abstract

Marketed red clover (Trifoleum pratense) products use a wide variety of labels, and the isoflavone content from the label is ambiguous. In the present study, we analyzed the content of various isoflavone products, and determined (1) the content and (2) how the sample matrix of red clover products affects the intestinal disposition of main isoflavones within it, using the human intestinal Caco-2 cell model. Analysis using high- and ultraperformance liquid chromatography indicates that the isoflavone content varied significantly (p < 0.05) between the chosen products. Consequently, rates of isoflavone absorption across the Caco-2 cell monolayers varied (p < 0.05) greatly. Unexpectedly, permeabilities of biochanin A and formononetin (two key biomarkers) were found to be significantly affected (p < 0.05) by the product matrix. As expected, biochanin A was the only isoflavone with noticeable metabolite peaks in both the apical and basolateral sides. Interestingly, rates of metabolism and the polarity of the glucuronidated biochanin A excretion were also significantly altered (p < 0.05) by the product matrix. Studies using the breast cancer resistance protein (BCRP) inhibitor, dipyridamole, showed that both the apical and basolateral excretion of biochanin A glucuronides were significantly (p < 0.05) reduced (7.5- and 9.4-fold, respectively) when dipyridamole is present. This provides evidence that BCRP is the main transporter responsible for the apical efflux of isoflavone glucuronides. The isoflavone content of the marketed red clover products is highly variable, and the product matrix significantly affected the intestinal disposition of red clover isoflavones by altering their absorption rates, permeabilities, biochanin A glucuronide excretion rates, and the polarity of biochanin A glucuronide excretion. This research provides scientific evidence to support the standardization effort, so that consumers can make intelligent product choices.