Soy Isoflavones

The term soy isoflavones refers to three molecules that are present in food, although most famously contained in soy products; this includes:

• Genistein, and its glycoside Genistin at 30-60% of total soy isoflavones^[2]
• Daidzein and its glycoside Daidzin at 40-60% of total soy isoflavones^[2]
• Glycitein, and its glycoside Glycitin at around 1-13% of total isoflavones^[2]

Of these, genistein and daidzein are seen as the major components of soy due to their relative quantities and equol is also researched a fair bit (despite being a metabolite of daidzein not naturally occurring in soy) due to its potency.

Soy isoflavone is a term used to refer to three isoflavone molecules (genistein, daidzein, and glycitein) as well as their glycosides and metabolites. The content of genistein and daidzein are pretty equivalent in food, with glycitein being much lower

Despite being called 'soy' isoflavones (referring to the Glycine max plant) these above isoflavones can be found in a variety of common food sources including:

Daidzein Genistein Glycitein

• Glycine max (Soybean; main source)^[3] at 405-894µg/g (Genistein), 424-1,138µg/g (Daidzein), and 44-145µg/g Glycitein fresh weight^[2]
• Black soybean at 579-831µg/g genistein, 452-599µg/g daidzein, and 37-41µg/g glycitein fresh weight^[2]
• Green soybean at 335-701µg/g genistein, 630-650µg/g daidzein, and 39-44µg/g glycitein^[2]
• Boiled soybean at 354-428µg/g genistein, 270-369µg/g daidzein, and 9-24µg/g glycitein^[2]
• Roasted soybean at 892µg/g genistein, 970µg/g daidzein, and 144µg/g glycitein^[2] and Kinako (roasted soybean flour) at 830-1,280µg/g genistein, and 1,181-1844µg/g daidzein, 80-100µg/g glycitein fresh weight^[2]
• Okara at 48µg/g genistein, 45µg/g daidzein, and 12µg/g glycitein^[2]
• Soy milk (1.9-13.9μg/g genistein and genistin)^[4]
• Tofu (94.8-137.7μg/g combined genistein and genistin of which 80-112μg/g is genistein^[4][4]) with 75-106μg/g daidzein and 24-29μg/g glycitein fresh weight^[4]
• Fermented soy bean products (Miso and Natto) at 38.5-229.1μg/g genistein and 71.7-492.8μg/g genistin,^[4] with other sources suggesting higher level of genistein (296-440μg/g and 101-319μg/g in natto and miso, respectively), daidzein (323-342μg/g in natto and 108-363μg/g in miso), and glycitein (37-69μg/g in natto and 12-54μg/g in miso)^[4]
• Soy sauce at 1-4μg/mL genistein, 5-9μg/mL daidzein, and 1-2.5μg/mL glycitein^[4]
• Other legumes (more than 400mg/kg dry weight)^[5]
• Indian bread root^[5]
• Soy protein isolate (80-107mg per 40g) which can be reduced by alcohol extraction (4.4-6mg per 40g) with an isoflavone breakdown of 53-57% genistein, 20-35% daidzein, and 11-23%^[6][7]

When looking at food products, most soy based products seem to have practically comparable levels of isoflavones when looking at fresh weights (Kinako appears to have more due to a lower water content), although roasting and fermentation appear to increase the content of the aglycones (genistein, daidzein, glycitein) relative to the glycosides (genistin, daidzin, glycitin)

And other herbs that are sometimes used for supplemental or medicinal purposes including:

• Hydrocotyle sibthorpioides^[8]
• Kudzu root (pueraria lobata)^[9] and White Kwao Krua (Pueraria Mirifica) with the latter reaching 8.4-10.2% total estrogenic isoflavones by dry weight^[10]
• Psoralea species, including Psoralea corylifolia^[9]
• Butea Superba (Genistein and Daidzein)^[11]
• Hibiscus Sabdariffa (Daidzein)^[12]
• Oxytropis falcate (Genistin and Daidzein)^[13]

The soy isoflavones are found in a wide variety of vegetables and legumes

Food sources are typically as Genistin, which is a Genistein molecule bound to a sugar. It is biologically inactive, and during high-temperature heating is reduced to a smaller sized simple glycoside^[14] that can be hydrolyzed in the intestine to form the bioavailable genistein aglycone which is then absorbed.^[3] The former processing is traditional Eastern Asia processing, and soy foods made from soy flour (from hexane treatment of soybean flakes) standard in North America can either have their isoflavones lost in the hexane extract or decarboxylate their complex glycoside (6′′-O-malonyl-7-O-β-D-glucoside) into another structure (6′′-O-acetyl-7-O-β-D-glucoside) with altered pharmacokinetics.^[15] If not broken down into the simple glycoside by heat, genistein cannot be absorbed in the small intestine by the enzyme lactase phlorizin hydrolase.^[16]

Fermenting of food sources tends to break the glycoside, and release the free aglcyone (Genistein). Additionally, the free aglycone can be hydroxylated with further fermantation, which increases the anti-oxidative potential of isoflavones.^[17][15] Fermented soy sauces contain the compounds 6-hydroxygenistein, 8-hydroxygenistein, and genistein-7-tataric acid. These compounds are not in unfermented soy.^[15]

Food products tend to contain the glycosides (isoflavone attached to a sugar) which is readily broken into the free isoflavone in some intestinal bacteria and during the fermentation process

The total intake of isoflavonoids in the japanese diet have been reported to be 27.80 mg per day (daidzein 12.02 mg, glycitein 2.30 mg, and genistein 13.48 mg).^[2]

Daily intake of genistein has been suggested to be around 1.5-4.1mg per Japanese person.^[4]

Daily intake of genistin has been suggested to be around 6.3–8.3mg per Japanese person.^[4]

Genistein is an isoflavonoid compound, and is defined by having a hydroxy group on the 4' position of the outermost benzene ring.^[18]

The food bound glycosides Daidzin (Daidzein glycoside) and Genistin (Genistein glycoside) are enzymatically hydrolyzed in the small intestine by B-galactosidases, mostly in the jejunem.^[19][20] Some of these glycosides are not hydrolyzed in the jujenum and may reach the colon.^[21][22]

Daidzein can be metabolized by gut microflora (bacteria) into an estrogenic metabolite called Equol.^[23][22] In this process Daidzein is first hydrated to Dihydrodaidzein, which can then either turn into Equol or into O-Desmethylangolensin. This conversion is dependent on gut microflora and varies between individuals^[24] which is a reason for reported individual differences in estrogen status among people who consume soy.^[25] It has been noted that not all individuals may produce Equol from Daidzein^[26][23] and that only 33-50% of humans may possess this bacterial strain.^[25][21]

Persons who possess intestinal bacteria to metabolize Daidzein into Equol are classified as 'Equol Producers' and are more likely to experience estrogenic effects from soy.

Many actions on steroid metabolism come from Genistein's interactions with the aromatase enzyme. This is the rate-limiting enzyme that converts androsterone and testosterone to estrone and estradiol, respectively.^[27][28] Many isoflavones and flavones can interact with aromatase, in which the binding site for the androgen's D and C rings are occupied by the flavonoid's A and C rings, respectively.^[29][28] Aromatase is encoded and created by a single gene, the CYP19A1^[30] and has several promoters that are divided based on where they exist in the body.^[31][32] Currently known are I.1 through I.7, and PII promoters^[28] which are placenta (I.1) and placental minor (2a) specific, adipose specific (I.3), skin fibroblasts and preadipocytes (I.4), fetal (I.5), bone (I.6), brain (I.f) and endothelial cells (I.7).^{[33][34][35][36]} Although all these promoters vary by region, the encoded mRNA and final protein (aromatase enzyme) are structurally the same.^[37]

By aiming for the protein (aromatase), one can induce systemic wide effects. If aiming for protein transcription, one can hopefully induce more controlled effects based on promoter localization.

Genistein, directly on aromatase, can increase its activity as measured in ovarian cells and carcinoma cells.^[38][39] It can also stimulate the growth or aromatase in breast cells, and most notable estrogen-responsive breast cancer cells.^[40] It can negate the action of pharmaceutical anti-aromatases in these cells.^[41][40] In some scenarios, it has been shown to inhibit aromatase directly although it is weak in doing so; isoflavonoids in general are weaker than flavones at aromatase inhibition.^[29][42] When acting as an aromatase inhibitor, Genistein has an apparent Ki of 123+/-8uM.^[43]

Genistein, working through promoters, can suppress the adipose specific aromatase via I.3^[44] and in estrogen-non responsive breast cancers via the same promoter.^[28] At times, however, it has been shown to increase activity of this promoter in HepG2 cells and subsequently induce aromatase.^[45] It has been noted to reduce transcription in granulosa-luteal cells,^[46] and is synergistic with both Daidzein and Biochanin A in this regard.

One study investigating phytonutrients able to act as SSRIs noted that both genistein and daidzin were able to inhibit some degree of serotonin reuptake (11.5+/-11.6% and 5.7+/-6.7% respectively) at 50uM, but were unreliable and both much weaker than the active control of Imipramine (74.5+/-11.3% at 5uM).^[47]

It is unlikely that soy acts as an SSRI due to the high concentration required and low potency thereof even at these impractical doses

Consuming 60mg of Soy Protein isoflavones reduces the levels of various markers of cardiovascular disease in normal post-menopausal women^[48] although after cost-benefit analysis the recommendation to include soy protein foods is still controversial.^[49]

Numerous in vivo animal studies have linked Genistein consumption with a decreased fasting blood glucose in already diabetic animal models.^[50][51][52] The mechanism of action is hypothesized to be via acting on PI3K, an intermediate in insulin signalling cascades, and does so at concentrations of 10-50uM under conditions of normal glucose and 30uM under conditions of high glucose and was inhibited by introduction of an O-GlcNAcase inhibitor, suggesting another possible mechanism of action via decreasing O-GlcNAcylation.^[53]

Genistein appears to inhibit adipogenesis as well as induce fat cell apoptosis via AMP-Actiated protein Kinase (AMPK)^[54][53] and appears to be mediated through Reactive-Oxygen Species (ROS) release and was inhibited with treatment of an anti-oxidant.^[54][55]

Genistein can also inhibit GLUT4-mediated glucose uptake in adipocytes.^[56] Although genistein has the properties of a tyrosine-kinase protein inhibitor^[57] (of which the insulin receptor constitutes) the effects from Genistein are independent of direct receptor inhibition.^[56][58] These effects were seen best at a concentration of 20uM.

The anti-osteoporotic effects of Genistein are highly mediated via the ERa receptor (proliferative subset) and are drastically augmented with forms of mechanical resistance (exercise).^[59]

The high affinity immunoglobulin E (IgE) receptor FcεRI (usually a tetrameric receptor with an α and β subunit with two γ subunits^[60]) is involved in mast cell degranulation, since the ligand (IgE) binds to an α subunit which is a mandatory step^[61] (γ carries the signal into the cell and β, which is not mandatory, amplifies it^[62]) which causes allergic reactions in mast cells. The soy isoflavones can reduce FcεRIα expression after 24 hours of incubation at 5µM or higher, secondary to reducing mRNA transcription (genistein slightly reduced FcεRIβ expression, while daidzein and equol suppressed FcεRIγ) in a manner that is not related to ERK1/2 phosphorylation (the reduction thereof is associated with how green tea catechins are anti-allergic^[63]) nor estrogen.^[64]

Physiologically relevant concentrations of the soy isoflavones are known to suppress the receptor levels of FcεRI (all three subunit types), which are thought to reduce the amount of signalling into mast cells and thus reduce allergic reactions; this is not related to estrogenic signalling

Atopic disease tends to have FcεRIβ involved in its pathology^[65] and due to genistein suppressing FcεRIβ mRNA levels in vitro^[64] it may explain the benefits to NC/Nga mice (a model for atopic dermatitis) given 4-20mg/kg genistein daily for eight weeks since IgE and IL-4 concentrations are not affected (although the higher dose attenuated IFN-γ somewhat).^[66]

Animal research suggests that genistein may be of use to chronic dermatitis symptoms with daily ingestion with reasonable oral dosages

In regards to the alpha subset of the receptor (ERα), the isoflavones can bind to and activate signalling. Genistein has an affinity of 360nM^[67] (about 1% that of estrogen itself^[45]) and an EC₅₀ of 15μM^[68] whereas daidzein has an affinity of 3μM^[67] and an EC₅₀ of over 300μM.^[68] Equol seems to have an affinity similar to genistein^[69] and an EC₅₀ of 3.5μM,^[68] which is the most potent of the isoflavones but still less than estrogen itself (as 17β-estradiol at 30nM).

While they are inhernetly agonistic yet weaker than estrogen, it seems that they have the ability to active this receptor when concentrations of estrogen are low yet antagonize this receptor's activation at higher concentrations of estrogen via outcompeting it.^[70][71]

The soy isoflavones can signal through the alpha subset of the estrogen recepor (ERα) which is associated with the classical effects of estrogen. They are all weaker than estrogen itself (equol being the most potent) and while they activate this receptor in the absence of estrogen they can competitively inhibit it at higher estrogen concentrations

The three main isoflavones are known to bind to the beta-subunit of the estrogen receptor (ERβ) with genistein having the greatest affinity at 9nM^[67] and binding with a potency comparable or greater than estrogen,^[69][72] daidzein has the second highest affinity at 552nM^[67] and glycitein is the poorest binder.^[69] The glycosides of these isoflavones are poor binders^[69] and the ability of the aglycones to activate the receptors occurs at relatively low concentrations with an EC₅₀ of 30nM for genistein (17β-estradiol at 10nM^[68] and dihydrogenistein equally potent as genistein^[69]), daidzein (350nM),^[68] and equol (400nM).^[68]

The isoflavones are thought to be selective for this subset, since the affinity for this receptor is significantly higher than that of the alpha subset with 40-fold increased affintiy (genistein) and 5-fold (daidzein)^[73] and the signalling potency is significantly greater as well at 8.8-fold selectivity (equol), 500-fold (genistein) and over 800-fold (daidzein).^[68] Glycitein has been reported to also activate this receptor.^[69]

1μM of these isoflavones, sufficient to activate the receptor, seem to work in an additive manner with estrogen.^[74]

When looking at the beta subunit of the receptor (ERβ), the soy isoflavones appear to potently activate the receptor at concentrations comparable to estrogen and it does not appear that the antagonism seen with the alpha subunit extends to ERβ. Soy isoflavones are direct and effective agonists of ERβ in the nanomolar range

Oral intake of genistein at 10mg/kg in male rats has been noted to possess antiandrogenic activity in the testis, prostate, and brain (50-80% reductions in reporter activity) which failed to be antiandrogenic in skeletal muscle or lung tissue.^[1] It has been noted to reduce protein content of the androgen receptor itself at 1-50µM in prostate cells (increasing receptor ubiquination)^[75] but 10mg/kg for five days in rats failed to replicate this.^[1]

In castrated rats, 10mg/kg is able to activate androgen reporter activity in the testes and brain (2.6 and 2.7-fold higher than baseline) and without influence on skeletal muscle or prostate.^[1]

When looking at the androgen receptor, it appears that genistein may negatively regulate it in the presence of androgens yet positively regulate it in the absence thereof; it also shows Selective Androgen Receptor Modulator (SARM) properties due to not being active in lungs or skeletal muscle

Genistein is an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD) via competing with pregnenolone for binding (competitive) and via inhibiting the NAD+ activation of the enzyme.^[76] It also can inhibit 17β-HSD with an IC₅₀ of 85+/-15nM^[77] and the inhibition on 3β-HSD has been replicated^[78][79][80] and does not extend to suppressing the actual expression of 3β-HSD mRNA or its protein content.^[81]

When looking at the 5α-reductase enzyme, Genistein and Equol can inhibit the enzyme with an IC₅₀ of 710μM and 370μM respectively while daidzein is ineffective; both underperforming relative to the active control of riboflavin (17μM).^[82]

When looking at the enzymes of testosterone synthesis, genistein may inhibit the hydroxysteroid dehydrogenases and secondary to that very high doses may be anti-fertility and anti-androgenic. The inhibitory effects on 5α-reductase are likely not relevant due to the high concentration needed

The aforementioned impairment of enzymatic activity may cause developmental impairment in rat pups (10uM in vitro)^[83][84] which is equivalent to a human dose of 100mg/kg.

In rats given 40mg/kg genistein, serum testosterone trended to be reduced (nonsignificant) while 10mg/kg was without affect.^[77]

40mg/kg genistein (but not 10mg/kg) to rats is able to increase circulating LH by 29%.^[77]

10-40mg/kg genistein is unable to influence circulating FSH in rats over the course of 21 days.^[77]

Genistein is able to inhibit cortisol synthesis (induced by ACTH) in adrenal cells with an IC₅₀ in the range of 1-4µM with near complete suppression at 40µM, which is due to inhibiting the P450c21 enzyme and preventing the conversion of 17α-hydroxyprogesterone to 11-deoxycortisol.^[81]

While 10mg/kg was ineffective, 40mg/kg of genistein for 21 days in rats is able to half serum corticosterone while ACTH is unaffected.^[77]

It appears that genistein and daidzein can stimulate DHEA and DHEA-S production in isolate adrenal cells with an EC₅₀ value in the range of 1-4µM^[81] probably related to its estrogenic properties as estrogen itself can stimulate DHEA production in this tissue.^[85]

The inhibition of cortisol from genistein in adrenal tissue is due to inhibition of P450c21,^[81] and it is thought that the backlog of the immediate metabolite (17α-hydroxyprogesterone) causes a relative increase in how much substrate is available for DHEA synthesis.^[81]

In isolated adrenaocorticol cells genistein and daidzein appear to be able to suppress ACTH-induced cortisol synthesis with an EC₅₀ in the range of 1-4µM and 40µM suppressing cortisol to near basal levels, and this is mimicked by estrogen^[81] which is known to suppress cortisol production.^[85] DHEA production in isolated adrenal cells appears to be stimulated with genistein or daidzein with an EC₅₀ in the range of 1-4µM^[81] which is also seen with estrogen,^[85] and since both cortisol and DHEA are synthesized from the same parent molecule (pregnenolone) which is thought to be due to inhibition of P450c21 (converts 17α-hydroxyprogesterone to 11-deoxycortisol^[86]) causing a partitioning effect of 17α-hydroxyprogesterone from cortisol towards DHEA.^[81] This property (P450c21 inhibition) is shared with Apigenin^[87] and due to the inhibition of 3β-HSD from soy isoflavones there is also a decrease in androstenedione synthesis in the adrenals.^[87]

Genistein appears to inhibit P450c21 in the adrenal glands, which causes a reduction in cortisol and relative increase in DHEA synthesis. Androstenedione also appears to be reduced

Concentrations of up to 40µM are not toxic to adrenal cells.^[81]

When looking at the weight and histology of the testicles in genistein fed rats, they appear to be normal suggesting no toxic properties.^[77]

Genistein is most notably in its effects against Breast Cancer. It (Soy intake) has been correlated with a reduced risk of breast cancer in numerous epidemiological studies.

Genistein appears to act as a pro-carcinogen in breast cancer lines that express Estrogen Receptor Alpha (ERa) predominately at normal doses (6-8uM), although in supra-physiological doses (more than 10uM) this effect is reversed.^{[88][89][57][90]}

In regards to prostate cancer, genistein (via soy intake) has been implicated in epidemiological studies to be associated with a decreased risk of prostate cancer.^[91][92] This is somewhat backed up by lower rates of prostate cancer in Asian countries relative to Western countries with lower soy intakes^[93][1] paired with asian immigrants matching western rates of prostate cancer upon immigration.^[94]

In vitro results suggest that genistein can act via inhibiting NF-kB in various cells^[95] and suppression of metalloproteins associated with cancer.^[96] Other possible mechanisms of action include preventing an upregulation in 5a-reductase activity in the prostate from a high-fat diet.^[97]

These results have been applied to animal models showing genistein intervention capable of reducing prostate cancer metastasis^[98] and acting as an anti-carcinogen.^[98]

Via AMPK activation^[54], Genistein can aid in regulating a cell cycle's lifespan.^[99][100]

Tamoxifen is a pharmaceutical anti-estrogen that directly competes with estrogen at the level of the receptor and is commonly used for the treamtent of breast cancer.^[101][102]

1,000ppm genistein in the diet (1%) has been noted to reduce the inhibitory effects of tamoxifen on MCF-7 tumor growth in mice^[103] and elsewhere studies using mixed isoflavones (0.22%)^[104] or genistein (0.14%)^[105] at low doses have found inhibitory effects that were not seen with 0.44% mixed isoflavones.^[104] Due to these studies, a study using scaling doses of genistein (0.25-1%; causes circulating levels of 1.4–3.3μM^[106]) in tumor bearing mice noted that while 0.25% reduced the effects of tamoxifen and 0.5% abolished it the highest tested dose (1%) failed to inhibit tamoxifen.^[41] When looking at daidzein, it appears to enhance the effects of tamoxifen.^[105]

The above information is in accordance with the role of genistein in signalling through the alpha subset of the estrogen receptor (ERα) which occurs at low concentrations but not so much at higher concentrations (and which daidzein is a poor activator of). Genistein has been noted to stimulate the growth of breast cancer cells that expresss ERα specifically at physiologically relevant concentrations,^{[107][90][106]} and it is thought that it is outcompeting tamoxifen at the level of the receptor due to its high affinity.

Genistein, but not daidzein, is known to interact with the anti-breast cancer drug tamoxifen (an estrogen receptor antagonist). It is thought that genistein is outcompeting tamoxifen at the level of the receptor, and thus it would be prudent to avoid soy based supplements during tamoxifen therapy

Letrozole is an aromatase inhibiting pharmaceutical that alone is effective in the treatment of breast cancer,^[108] and is sometimes used alongside estrogen receptor antagonists such as tamoxifen. Genistein is also an aromatase inhibitor at high concentrations (10μM)^[109] but not physiologically relevant concentrations (1μM)^[110]

Dietary genistein at 250-1,000ppm of the mouse diet is able to prevent the inhibitory effects of letrozole (aromatase inhibitor) on breast tumor growth in a dose-dependent manner.^[40]

May also adversely interact with the anti-breast cancer drug letrozole, an aromatase inhibitor

As the bacterial conversion of Daidzein to Equol (in Equol producers) appears to be a significant aspect of the estrogenicity of soy products, it was investigated as to whether consumption of probiotics could influence this conversion. Two studies investigating Lactobacillus Acidophillus and Bifidobacterium longum found that oral administration of these probiotics does not alter Equol producing status.^[111][112]