Phytochemicals of the Month
Phytoestrogens
Special expanded section on Phytoestrogens and Human Health
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For basic information on isoflavones, see the Phenolics Intermediate page.

Genistein and daidzein, two phytoestrogens from legumes

Notice that the only difference between these two molecules is the additional hydroxyl (-OH)  group on genistein. Both are typical isoflavones. Genistein, however, is considerably more estrogenic than daidzein; chemists attribute this to the influence of the additional hydroxyl group (highlighted). The hydroxyl groups are important for binding to estrogen receptors.

Biochanin A and formononetin, two isoflavones from red clover

These two isoflavones are just like genistein and daidzein, except that they have methyl groups (-CH₃) replacing the hydroxyl groups (highlighted in blue). Biochanin A and formononetin are considerably less estrogenic in their original forms; because of the shape of the methyl groups, they are not able to bind to estrogen receptors very well. However, once these molecules are ingested, colon bacteria clip off the methyl groups; biochanin A becomes genistein and formononetin becomes daidzein. Daidzein can be further metabolized to equol (see below). So in the long run, biochanin A and formononetin can be the source of considerable estrogenic activity.

Glycitein

This is glycitein, another an isoflavone found in the soybean. It bears a methoxy group (-O-CH₃, highlighted) which is more difficult for colon bacteria to remove than a methyl group. Glycitein accounts for some 5 to 10% of the total isoflavones in soy foods; in the soy germ, it makes up ~ 40% of total isoflavones.

The major lignans from flax: secoisolariciresinol and matairesinol

These are the most common lignans in the human diet. Once ingested, bacteria transform them into the mammalian lignans enterodiol (from secoisolariciresinol) and enterolactone (from matairesinol). Notice that the basic structures are similar, but the methoxy groups of the plant lignans have been replaced by hydroxyl groups in the mammalian lignans, and there are fewer functional groups overall:

Coumestrol, a highly estrogenic compound

Coumestrol is a coumestan (isoflavones, lignans, and coumestans are all polyphenols); although the structure is quite different from an isoflavone such as genistein, this molecule also has hydroxyl groups at both ends which allow it to bind to estrogen receptors. Coumestrol is found mainly in red clover sprouts, mature alfalfa (lucerne) and alfalfa sprouts, soybean sprouts, and kudzu leaf. It is highly estrogenic, but only small amounts are present in the human diet. Low-coumestrol varieties of alfalfa and red clover are bred for forage and for commercial extraction of other phytoestrogens.

Equol is an estrogenic metabolite of daidzein

In some 30 to 40 percent of the population, equol is produced as a major metabolite of daidzin (the glucoside of daidzein). Equol is known as an 'isoflavan' rather than an 'isoflavone' - it lacks the carbonyl group (=O) that makes its precursor an 'one.' In estrogen-receptor assays, equol exhibits considerably more binding affinity than its precursor; it is roughly equal to genistein in this respect. The differences between the two molecules (blue highlight) seem slight, but equol is more able to fit into and be retained by the estrogen receptor.

Pharmacokinetics of isoflavones and lignans

In plants isoflavones occur mainly as water-soluble glucosides, acetylglucosides, and/or malonylglucosides. Inside the human digestive system, enzymes known as beta-glycosidases snip off the sugar units of these molecules and free the aglycones for absorption. Some of these enzymes are endogenous to the small intestine,¹ but bacterial glycosidases from gut microflora are also involved in the process.² Researchers have noted that there can be a wide range of plasma isoflavone levels when different individuals ingest equal amounts of the compounds. This may be partially attributable to the fact that different people can have very different microfloral populations: some populations may be much more efficient at hydrolyzing the glycosides than others.

After deglycosylation, the free aglycones are either absorbed directly (for genistein and daidzein) or demethylated by colon bacteria (biochanin A --> genistein; formononetin --> daidzein) prior to absorption.³ In the liver, the majority of the aglycones (~ 97%) are conjugated with glucuronic acid and (to a lesser degree) with sulfate; they then enter the entero-hepatic circulation. This conjugation allows the isoflavones to remain in circulation for a considerable time; endogenous estrogens undergo a similar process.

Setchell et al. demonstrated that while both glucosides and aglycones were efficiently absorbed, it took an average of 5.2 hours after ingestion to reach peak plasma concentrations for free genistein and 6.6 hours for free daidzein. However, when subjects ingested the glucoside forms (genistin and daidzin), peak levels were attained after 9.3 hours and 9.0 hours, respectively. Peak levels of glycitein occurred ~ 4 hours after administration of glycitin. Overall, the bioavailability of the glucosides exceeded that of the free aglycones. The researchers speculated that this was because the sugar molecule was acting as a 'protector group' to stabilize the isoflavone against degradation in the digestive system. It was also noted that the isoflavones biochanin A and formononetin (predominant in red clover) were quickly demethylated after ingestion, and were subsequently detectable as genistein and daidzein in the plasma.⁴

Interestingly, one new study⁵ found no significant difference in absorption between native glucosides and pre-hydrolyzed aglycones ingested in a soy beverage. These findings would seem to be in opposition to the study discussed above; however, this may be due to some as yet unknown influence in the food matrix.

Evidence suggests that the food matrix in which isoflavones are ingested has an influence on the relative plasma levels of genistein and daidzein. Soy germ produces higher levels of daidzein and lower levels of genistein. Soy protein, on the other hand, produces more genistein and less daidzein in the plasma. Studies show the specific cholesterol-lowering properties of isoflavones may depend on the food matrix: isolated compounds did not lower cholesterol, while those in the soy protein matrix did.⁴ Several studies reported better absorption of isoflavones from fermented food sources (such as miso) vs. non-fermented sources (such as soymilk).^7,8,9 Some strains of bacteria used in the manufacture of the fermented soy product tempeh have been shown to create various polyhydroxylated metabolites from the isoflavones in soybeans.¹⁰

In some of the subjects in the Setchell study, equol (a metabolite of daidzin, with an estrogen binding affinity similar to that of genistein) appeared in the plasma some 6 - 8 hours after ingestion of its precursor. Previous studies have noted that about 1/3 of the population can produce equol and the remaining 2/3 do not.¹¹  Equol tends to remain in circulation longer than daidzein or genistein, which might result in a stronger cumulative estrogenic effect in persons who produce it.

Isoflavones are excreted in the urine and feces; (3) reported that 50% of ingested daidzein, 38% of glycitein, and 20% of genistein were excreted in urine, mostly within 24 hours after ingestion; these numbers can vary considerably between individuals. Another study found 17.4 - 87.7% excretion for daidzein, 19.7 - 91.3% for glycitein, and 8.5 - 69.6% for genistein.¹² Metabolites of the isoflavones are also found in urine, including dihydrodaidzein, O-desmethylangolensin (ODMA) and equol (in ~ 30 - 40% of the population) from daidzein, and p-ethylphenol and dihydrogenistein from genistein. Urinary glycitein metabolites include 6,7,4'-trihydroxyisoflavone, 5'-hydroxy-O-desmethylangolensin, and 5'-methoxy-O-desmethylangolensin.

Normal concentrations of phytoestrogens in plasma (nm/L)¹³

Average everyday serum concentrations given for W = women (n = 125) and M = men (n = 102). Participants were > 40 y old. For soy-formula fed infants, average plasma concentrations of total isoflavones can reach ~ 7,000 nm/L.¹⁴ An excellent short review of isoflavone pharmacokinetics (as well as information on their role in cancer prevention) can be found in this paper.

Lignan pharmacokinetics

The main dietary lignans are matairesinol and secoisolariciresinol; the latter is present in plants as secoisolariciresinol diglucoside. These are transformed by colon bacteria into the 'mammalian lignans' enterolactone and enterodiol. Studies with flaxseed show that the plasma and urinary levels of enterolactone and enterodiol increase in a dose-dependent manner. One report observed that after 8 days of supplementation with 25 g raw flaxseed/day, the average plasma concentrations were ~26 nm/L for enterolactone and ~58 nm/L for enterodiol in premenopausal women. No plateau was observed with intake up to 25 mg; increased intake may lead to higher levels of the lignans in the body. There was no difference in plasma levels of the two compounds with raw vs. cooked flax seed.¹⁵

The same study found that total plasma lignan levels significantly increased by 9 hours after initial dosing and remained high for at least 24 hours afterwards; this is in contrast to levels of soy isoflavones, which revert to baseline considerably sooner. After several days of dosing, plasma concentrations of lignans were maintained at an elevated level even when the flax was ingested only once daily.

Back to Phytoestrogens and Human Health

References

1 Day, A. J. et al. 1998. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glycosidase activity. FEBS Lett. 436: 71 - 75.
2 Xu, X. et al. 1995. Bioavailability of soybean isoflavones depends upon gut microflora in women. J. Nutr. 125: 2307 - 2315.
3 Hur, H. and F. Rafii. 2000. Biotransformation of the isoflavonoids biochanin A, formononetin, and glycitein by Eubacterium limosum. FEMS Microbiol. Lett. 192: 21 - 25.
4 Setchell, K. D. R. et al. 2001. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J. Nutr. 131: 1362S - 1375S.
5 Richelle, M. et al. 2002. Hydrolysis of isoflavone glycosides to aglycones by beta-glycosidase does not alter plasma and urine pharmacokinetics in post-menopausal women. J. Nutr. 132: 2587 - 2592.
6  Birt, D. F., S. Hendrich, and W. Wang. 2001. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacology and Therapeutics. 90: 157 - 177. Abstract.
7 Hutchins, A. M. et al. 1995. Urinary isoflavonoid phytoestrogen and lignan excretion after consumption of fermented and unfermented soy products. J. Am. Diet. Assoc. 95: 545 - 551.
8 Slavin, J. L. et al. 1998. Influence of soybean processing, habitual diet, and soy dose on urinary isoflavonoid excretion. Am. J. Clin. Nutr. 68: 1492S - 1495S.
9 Izumi, T. 2000. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr. 130: 1695 - 1699.
10 Klus, K. and W. Barz. 1995. Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempeh. Arch. Microbiol. 164: 428 - 434.
11 Kelly, G. E. et al. 1993. Metabolites of dietary (soya) isoflavones in human urine. Clin. Chim. Acta 223: 9 - 22.
12 Zhang, Y. et al. 1999. Urinary disposition of the soybean isoflavones daidzein, genistein, and glycitein differs among humans with moderate fecal isoflavone degradation activity. J. Nutr. 129: 957 - 962.
13 Morton, M. S. et al. 2002. Phytoestrogen concentrations in serum from Japanese men and women over forty years of age. J. Nutr. 132: 3168 - 3171.
14 Badger, T. M. et al. 2002. The health consequences of early soy consumption.  J. Nutr. 132: 559S - 565S.
15 Nesbitt, P. D., L. Yi and L. U. Thompson. 1999. Human metabolism of mammalian lignan precursors in raw and processed flaxseed.  Am. J. Clin. Nutr. 69: 549 - 555.

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