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

Abstract

Marketed red clover products use a wide variety of labels and the isoflavone contents from the lable is ambiguous. In the present study, we analyzed the content of various isoflavone products, and determined a) the content and b) how sample matrix of red clover products affects intestinal disposition of main isoflavones within it using the human intestinal Caco-2 cell model. Analysis using high and ultra-performance 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 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 product matrix. Studies using breast cancer resistance protein 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. In conclusion, the isoflavone contents of the marketed red clover products are highly variable, and product matrix significantly affected 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.

Characterization of Chrysin Glucuronidation in UGT1A1-Overexpressing HeLa Cells: Elucidating the Transporters Responsible for Efflux of Glucuronide

“…5C). DIPY has been also used as a BCRP inhibitor in the literature (Zhang et al, 2005;Wang et al, 2008;Liu et al, 2012). Although DIPY at 10 mM did not alter the rate of CG excretion, DIPY at 20 mM resulted in a significant increase (110%, P , 0.05) in excretion of CG (Fig.…”

“…Compared with Ko143, use of DIPY as a BCRP inhibitor is relatively limited (Zhang et al, 2005;Wang et al, 2008;Liu et al, 2012). This may be because little is known about its inhibitory selectivity toward other transporters.…”

Role of Metabolism in the Effects of Genistein and Its Phase II Conjugates on the Growth of Human Breast Cell Lines

“…Genistein glucuronides and sulfates have poor lipid solubility and carry net negative charges at physiological pH levels, so they must transverse the cell membrane (into the cells) by a carrier-mediated transport before they can react with the hydrolytic enzymes inside the cells. Efflux transporters existed in breast cells, such as MRP2 and BCRP (37,38), limit their presence in cells by excreting them rapidly (39)(40)(41), all of which could lead to weak hydrolytic metabolism. Hence, transporters mediating the influx or efflux of genistein conjugates probably play an important role in the metabolism of genistein conjugates in breast cells, and further studies are underway to define these transporters.…”

Soybean isoflavone exposure does not have feminizing effects on men: a critical examination of the clinical evidence

“…Isoflavones have a limited distribution in nature, and among commonly consumed foods they are found in physiologically relevant amounts only in soybeans and foods derived from this legume (4), although a variety of plants such as red clover (48) are also rich sources. One serving of a traditional soy food contains approximately 25 mg of isoflavones (approximately 3.5 mg per gram of protein; isoflavone amounts in this text refer to the aglycone weight) (46).…”