Red Clover Extract

Red Clover Extract (RCE) tends to refer to the plant known as trifolium pratense (of the fabaceae family) which is most well known for its content of Biochanin A, an estrogenic bioflavonoid. It tends to be sold as a herbal tea (the dried floral blossoms being steeped as tea) or as a supplement for menopausal symptoms^[7] since dietary isoflavones in general (usually from soy) are correlated with reduced hot flash symptoms^[8] and biochanin A can convert into the soy isoflavones.

This plant is not known to have usage as a traditional medicine, although tea made from the aerial parts of this plant appears to be a remedy for cough and bronchitis.^[9]

Trifolium pratense is known as the red clover, and it appears to be used as a dietary supplement due to having a high isoflavone content. This clover does not appear to have any traditional medicinal usage, although it is a recent 'folk remedy' for the treatment of cough

Red Clover Extract tends to contain:

• Biochanin A (4′-O- methylated genistein)^[7] and two biochanin A glycosides^[10] with total biochanin A aglycones totalling 0.009-0.116% (flower), 0.022-0.095% (stem), 0.067-0.339% (root), and 0.077-0.133% (leaf)^[10]
• Formononetin (4′-O- methylated daidzein)^[7] and its glycoside (Ononin) as well as diglycosides^[10] with total formononetin aglycones totalling 0.018-0.038% (flower), 0.027-0.056% (stem), 0.023-0.151% (leaf), and 0.019-0.096% (root)^[10]
• All three soy isoflavones (daidzein, glycitein, and genistein) and their glycosides (daidzin, glycitin, genistin; respectively).^[10] Genistein (in total aglycones) at highest levels in the roots (0.1-0.58%) while not being very detectable elsewhere, daidzein being in relatively low concentrations (less than 0.1%) in all plant part, and glycitein also being high in the roots (0.319-0.863%) but also the leaves (0.430-0.820%) and stems (0.219-0.729%)^[10]
• Calycosin (also found in Astragalus membranaceus) and its glycoside^[10] with the aglycone of calycosin totalling 0.066-0.126% leaf dry weight, 0.054-0.084% of the stem, 0.06-0.184% of the root, and 0.021-0.039% flower^[10]
• Pratensein glycosides^[10] with total pratensein aglycones reaching 0.043-0.074% (leaf), 0.009-0.029% (stem), 0.034-0.062% (root), and 0.006-0.01% (flower)^[10]
• Prunetin and two glycosides^[10] with total prunetin aglcyones being detectable in the leaves (0.149-0.282%), stem (0.133-0.235%), roots (0.133-0.288%), and flowers (0.036-0.052%)^[10]
• Pseudobaptigenin and two glycosides^[10] with total pseudobaptigenin aglycones totaling 0.029-0.372% (leaves), 0.069-0.585% (roots), 0.056-0.126% (stems), and 0.009-0.018% (flowers)^[10]
• Irilone (isoflavone with a catechol group on the C ring) and at least four glucosides^[10] with total aglycones totalling 0.121-0.167% (flowers), 0.038-0.169% (stems), 0.017-0.021% with an outlier at 0.907% (roots), and 0.532-0.737% with the same outlier at 0.02% (leaves)^[10]

Red clover appears to be a pretty robust source of total isoflavones, which seem to be at highest levels in the leaves and roots (surprisingly, not the flowers) and there appears to be more of an even distrubution of isoflavones rather than a large dose of Biochanin A

Total isoflavones tend to range from 0.307-0.633% of the flower, 0.740-1.850% of the stem, 1.36-2.853% of the root, and 1.75-2.272% of the leaf.^[10] These numbers are much higher than other clover (trifolium) species such as white clover (trifolium repens) which has 0.0213-0.0354%, alsike clover (trifolium hybridum) which has 0.0070-0.0431%, and hop trefoils (trifolium campestre) at 0.00028-0.00061%.^[10]

Red clover appears to have more total isoflavone content than other species of clover

Promensil® is a brand name for red clover extract which contains Biochanin A, Formononetin, Genistein, and Daidzein. This supplement is produced by Novogen Ltd. (Australia) which has had a sourcing and funding role in a majority of studies using Promensil^{[11][12][13][14]} and it appears that various sources have listed the concents of the 40mg capsules as:

• 26mg Biochanin A, 16mg Formononetin, 1mg Genistein and 500µg Daidzein (43.5mg isoflavones in a 40mg capsule)^[11]
• 25mg Biochanin A, 8mg Formononetin, 4mg Genistein, and 5mg Daidzein (42mg isoflavones in a 40mg capsule)^[15][16][17]

So overall it seems that more than two-thirds of the supplement by weight are the two isoflavones that dominate most red clover extracts (Biochanin A and Formononetin) while the soy isoflavones make a less relevant appearance. Another product by this company, Rimostil® (28.6mg isoflavones), is predominately Formononetin (25mg; 87%) with some Biochanin A (2mg; 7%) and trace levels of the soy isoflavones.^[17] The isoflavones are in the aglycone forms (without sugar)^[18] and each 40mg of isoflavones (in a 500mg capsule) seems to be extracted from the plant equivalent of 5 grams of red clover.^[19]

There are two formulations produced by one company which are just standardizations of the isoflavones in red clover, and these particular formulations appear to be used in the majority of the trials studying Red Clover Extract

There is another Red Clover isoflavone formulation known as MF11RCE (also known as Menoflavon®^[20]) which has been noted to decrease vaginal dryness in postmenopausal women and exerted estrogenicity as assessed by karyopyknotic, cornification, and basal cell maturity index.^[20] Most studies that note an estrogenic (or androgenic) effect associated with red clover isoflavnes tend to use this formulation, and the exact reason for this and how it differs from Promensil/Rimostil are not well known.

MF11RCE (Menoflavon®) is another formulation used in some studies which, at least in humans, are supported by the industry of which produces the formulation. Oddly, this formulation only (and not the aforementioned ones) appears to be estrogenic for unknown reasons

In humans given the active dose of total isoflavones (40-80mg daily) over the course of five weeks, it has been noted that the overall absorption exceeded 25%.^[18]

The methylated isoflavones appears to be absorbed, and (in accordance with methylation) may be slightly better absorbed than their nonmethylated variants of genistein and daidzein; the absorption is still not perfect though

Biochanin A appears to be able to be metabolized (demethylation) into genistein, one of the soy isoflavones, in vitro^[21][22] in a manner that can be mediated by a variety of P450 enzymes including CYP1A2, CYP2E1, CYP2C91, CYP2C19, and CYP2D6.^[22] Biochanin A can also be hydroxylated to form other metabolites including 5,7,3'-trihydroxy-4'-methoxyisoflavone, 5,7,8-trihydroxy-4'-methoxyisoflavone, and 5,6,7-trihydroxy-4'-methoxyisoflavone.^[23] Metabolism of Biochanin A into genistein following oral ingestion in humans occurs, but is not complete.^[24]

Formononetin (7-hydroxy-4'-methoxyisoflavone) appears to be able to be metabolized (via hydroxylation) into 6,7-dihydroxy-4'-methoxyisoflavone, 7,8-dihydroxy-4'-methoxyisoflavone, and 7,3'-dihydroxy-4'-methoxyisoflavone depending on where the hydroxylation occurs;^[22] formononetin can also be demethoxylated via CYP1A2, CYP2C91, CYP2A6, CYP2D61, or CYP2C19 to form free daidzein^[22] and similar to Biochanin A metabolism into daidzein from formononetin is not 100%.^[24]

The two main components of red clover extract can be metabolized by CYP enzymes in the liver and intestines to give the body soy isoflavones, or they can be hydroxylated to form another pool of potential metabolites

Oral intake of 80mg of total isoflavones from red clover extract (3.5:1 ratio of Biochanin A to Formononetin) has been noted to be excreted in the urine of participants at a level in the range of 18.9+/-23.6mg daily, suggesting very high variability between persons.^[24]

Urinary elimination of the methylated isoflavones appears to be variable from one person to the next, which is perhaps related to variable serum levels due to differences in absorption

Biochanin A inhibits aromatase with an IC₅₀ of 25µM in fibroblasts, a potency comparable to quercetin (30µM) but lesser than genistein or chrysin (3.6µM and 1.5µM; respectively)^[25] and elsewhere being less potent than hesperitin and myricetin when tested in placental cells where Biochanin A had an IC₅₀ of 10.2µg/mL (relative to 1.0µg/mL and 5.6µg/mL for the aforementioned two, respectively).^[26] This range of efficacy in comparable to other studies using biochanin A in vitro.^[27]

In MCF-7aro and SK-BR-3 breast cancer cells, biochanin A suppresses promoter 1.3 and II activity (25-50µM) which resulted in less aromatase mRNA and protein levels; this may be related to metabolism into genistein.^[21]

Aromatase inhibition has been confirmed to occur in extrahepatic tissue with biochanin A and formononetin, as these two bioflavonoids are peripherally metabolized by CYP1B1 or CYP1A1 into the soy isoflavones which then inhibit activity of both enzymes.^[23]

Biochanin A has been noted to suppress aromatase protein levels and transcription, as well as to directly inhibit its activity; relative to other bioflavonoid compounds however, it appears to be less potent and it has not been shown to be practically relevant following oral ingestion

Red clover has been noted in a case study to cause symptoms of methotrexate toxicity in a person on high dose methotrexate, suggesting a drug drug interaction increasing circulating levels of the drug;^[28] possible explanations for this include inhibiting drug efflux transporters (mostly OAT3,^[29] MRP3,^[30] BCRP^[31]) or hepatic aldose reductase which metabolizes methotrexate into 7-hydroxymethotrexate (7-33% of orally ingested methotrexate).^[32]

Potential interaction with methotrexate, which has a low therapeutic index and is more susceptable to adverse drug-drug interactions, with oral supplementation of red clover in one case study; needs to be evaluated further

One study using a red clover extract at 398mg (120mg isoflavones) daily for one year in postmenopausal women noted that supplementation was associated with reductions in anxiety after one year, but not after three months and not alongside improvements in any other menopausal symptom.^[33] This was later expanded on with supplementation of 80mg isoflavones (MF11RCE) but over 90 days in a study funded by the aforementioned supplement's producers, but supplementation was associated with a significant reduction in anxiety assessed by the Hospital Anxiety and Depression Scale (HADS) reaching 76.9% alongside reductions in depressive symptoms (former scale plus Zung's Self Rating Depression Scale; SDS) by 78.3-80.6%.^[34]

Potential anxiety and depression reducing effects in menopause, although the one study that had a potential conflict of interest noted a much larger effect size (near 80% reductions) than the independent study (which did not report anti-depressive effects, and the improvement in anxiety was minor); requires more research to pinpoint the exact magnitude of effect

These effects are somewhat in line with the effects on quality of life in postmenopausal women, as one study using 80mg of the isoflavones daily for a period of 90 days noted a significant improvement in self-rated mood (on a 0-100 VAS rating scale, the improvement reached 66-68 points whereas placebo increased 8-15)^[35] although a later study assessing quality of life via MENQOL noted that the reduction was lesser in magnitude and not significantly different than placebo.^[36]

Similar to anxiety symptoms, quality of life appears to improved with red clover extract although the magnitude is equally variable and requires more research into it

In postmenopausal women over 60 given Rimostil (formononetin rich isoflavone extract) daily for six months, there appeared to be an improvement in the block design test relative to placebo (visuospatial reasoning) with impairments in digit span and verbal memory testing, and most other cognitive parameters being unaffected.^[37]

A formononetin rich extract of red clover has shown both beneficial (visuospatial reasoning) and negative (short term memory) effects on the cognition of postmenopausal women

Supplementation of 40-80mg of red clover isoflavones for five weeks in otherwise healthy postmenopausal women appears to increase arterial compliance by up to 23% as assessed by ultrasound, and this occurred without any changes in blood pressure.^[18] It was later expanded upon and arterial stiffness measured again, and red clover isoflavones were again able to reduce stiffness (assessed by arterial compliance and pulse wave velocity)^[19] without any effect on blood flow (assessed by flow mediated vasodilation) nor blood pressure.^[19] One study where an increase in blood flow noted, however, used 50mg isoflavones from red clover (predominately formononetin) in postmenopausal women with type II diabetes which coincided with a decrease in blood pressure.^[38]

There may be an increase in arterial compliance (reactivity) that could reduce the risk of cardiovascular complications, but this does not appear to be related to any alteration in blood flow nor pressure and instead seems to be more related to reducing arterial stiffness; mechanisms underlying this are not yet known

It was noted that formononetin, by itself, was able to reduce circulating VCAM-1 concentrations by 11% relative to placebo in postmenopausal women and older men.^[19]

It has been noted that dietary isoflavone intake is negatively correlated with serum homocysteine concentrations in premenopausal women^[39] but when testing 86mg of red clover extract daily supplementation has failed to significantly influence serum homocysteine or folate concentrations over the course of four menstrual cycles.^[40]

There may be a minor antiinflammatory effect following oral ingestion, which theoretically would reduce the risk of atherosclerosis over the long term. The effect seems to be less than many other dietary supplements

Supplementation of 80mg red clover isoflavones daily for 90 days in menopausal women, despite improving symptoms of menopause, failed to influence blood pressure^[41] and this failure has been noted elsewhere^[42][19][43] but may not apply to type II diabetics, as one study in postmenopausal women with type II diabetics given 50mg of the isoflavones daily for a period of four weeks was able to reduce systolic (8mmHg) and diastolic (3.4mmHg) blood pressure relative to control.^[38]

A decrease in blood pressure has once been noted in diabetic persons, but twice failed to occur in persons without diabetes even when given to women in menopause. The increase in arterial compliance (mentioned in the blood flow section) can occur without any changes in blood pressure

Triglycerides appear to be slightly attenuated over 90 days in postmenopausal women given 80mg of the isoflavones (9%, although there was an increase in placebo)^[41] which was replicated elsewhere to a similar degree in postmenopausal women (9.7%)^[17] but has failed to affect a sample of older men and women given the same dose^{[24][42][43][44]} or in premenopausal women given 80mg for three menstrual cycles.^[45]

One study noting a minor decrease in triglycerides barely reaching significance (9.7% reduction^[17]) noted that the results were significantly larger when controlled for women with baseline triglycerides over 178mg/dL; the reduction being seen with 40mg Promensil twice daily (33%).^[17]

Most evidence at this point in time does not support a significant reduction in triglyceride concentrations with oral supplementation of red clover extract in menopausal women, although in women with very high triglycerides at the beginning of the study (baseline) there may be a more relevant reductions that needs to be investigated more

Biochanin A appears to signal via one of the estrogen receptor subsets (ERα) to induce apolipoprotein A1 mRNA synthesis (reaching 600% of control), with activity at concentrations as low as 500nM and exerts maximal effects at 10µM, with 500-1,000nM biochanin A being comparable in potency to 1nM 17β-estradiol (300% above control).^[46] This signalling was dependent on the estrogen receptor alpha (ERα), since it was replicated in HepG2 cells made to express this receptor (which they normally do not^[47]) while expressing ERβ only failed to respond to biochanin A.^[46] This mechanism also occurs with genistein, a metabolite of biochanin A.^[48]

Estrogenic signalling, which biochanin A is capable of activating, is known to increase the transcription of Apolipoprotein A1 which is thought to be a cardioprotective mechanism

Oral ingestion of 40mg isoflavones (Biochanin A and Formononetin in a 3.5:1 ratio) twice daily over the course of six weeks has been noted to reduce LDL cholesterol by 9.5% in men but not women, although this study noted that the intervention group trended (nonsignificantly) to have a higher baseline LDL concentration with men.^[24] This same dose has elsewhere failed to influence LDL, HDL, and total cholesterol in postmenopausal women over 90 days^[41] to a year^[43] and a failure has been noted in premenopausal women given the supplement over three menstrual cycles where all subsets of HDL (HDL, HDL₂, and HDL₃) as well as Apolipoprotein A were unaffected.^[45] Apolipoprotein A has been once noted to be reduced in overweight, but nor normal weight, postmenopausal women with supplementation of red clover extract (MF11RCE) at 80mg over 90 days.^[44]

There have been some positive studies with Red Clover supplementation,^[49][50][51] but are usually low powered pilot studies^[49] or lacking a placebo control despite blinding^[50] with only one study with a blinded control noting an increase in HDL-C (18.1%).^[14] When looking at well controlled positive studies, one conducted over the course of one year in otherwise healthy postmenopausal women given 40mg of isoflavones once daily noted a 21% increase in HDL-C within three months which persisted at the same magnitude until a year of supplementation^[51] and another study (failing to note an increase in HDL-C) noted an improvement in LDL and total cholesterol only in overweight postmenopausal women.^[44]

In women diagnosed with high cholesterol (mild to moderate; 5-9mM), supplementation of red clover isoflavones at 40-80mg for 12 weeks after a four week run-in period noted that supplementation failed to reduce any cholesterol biomarker relative to control.^[42]

There are highly variable results when looking at supplementation of red clover on cholesterol, and at this moment in time there may be an increase in HDL-C (admittedly unreliable both in whether it occurs or not, and in its magnitude) while LDL may only be reduced, and weakly so, in men or overweight/obese postmenopausal women. While a benefit cannot be ruled out right now, red clover does not seem to be the best supplement to use for improving cholesterol

While in otherwise healthy premenopausal women there is no influence of red clover supplementation in insulin resistance at 80mg over the course of three menstrual cycles^[45] and isoflavones in general (usually referring to the soy isoflavones tend to be associated with less insulin resistance over three months,^[52] 40-80mg of red clover extract (Promensil) daily in postmenopausal women was associated with a decrease in insulin sensitivity as assessed by QUICKI after three months (despite the estrogen patch control being insulin sensitizing and no influences of isoflavones on hormones).^[53]

Fasting blood glucose, insulin, and HbA1c have been noted to be unaffected by supplementation (50mg isoflavones, mostly formononetin, daily over four weeks) in type II postmenopausal diabetics.^[38]

Isoflavones (as a structural class) seem to have varying effects on insulin sensitivity, but the ones from red clover have either been noted to have no significant influence or there may be a reduction in insulin sensitivity (increase in insulin resistance) seen with supplementation in postmenopausal women

100-1,000nM of Biochanin A in isolated stem cells derived from adipose tissue (ADSCs) in a growth medium over the course of 12 days has been noted to reduce the formation of adipocytes (33.6%) in a concentration dependent manner to 20.7-27.3%, which was due to promoting differentiation of ADSCs into osteoblasts with greatest potency at 300nM.^[54]

One in vitro study noted that biochanin A could influence the conversion of stem cells away from fat cells and towards bone cells, suggesting a potential long term benefit to bone mass and fat mass. This mechanism is seen with numerous flavonoid and phenolic compounds, and practical significance to oral supplementation is not known

While fat mass per se is not frequently measured, overall weight has been measured in numerous trials on supplementation of red clover extract in postmenopausal women; there appears to be a unanimous failure for red clover extract to reduce weight in these subjects even when supplementated at the higher end (80mg) for up to a year.^{[41][13][42][38][55][51][37]}

Red Clover Extract supplementation does not appear to significantly reduce weight in any study in which weight was actually measured, even when looking at the highest doses taken over a period of one year

When looking at red clover extract overall, 6mg/kg of red clover extract in the food supply (MF11RCE; equivalent to 240mg in humans) noted a preservation of estrogen and was able to preserve bone mass and mineral density relative to ovarectomized control (effectively normalized)^[56] and preserved bone strength;^[56][57] this preservation was additive with a bicarbonate mixture (part sodium bicarbonate and also potassium carbonate) at levels higher than normal in the rat diet^[56] and isoflavones from red clover have been noted elsewhere to be more beneficial for bone mass when paired with the bicarbonate mixture (to reduce acidity).^[56]

Oral ingestion of 10mg/kg formononetin to ovarectomized rats over the course of four weeks was noted to attenuate the loss in tibia (7.4%) and femur (5.3%) bone diameter with no influence on bone length, and was able to improve mechanical properties by improving maximal and fracture loads (force required to break bones) although with a potency lesser than the reference drug of 17β-estradiol (200µg/kg).^[58]

When looking at rodent studies, there appears to be an increase in bone strength relative to the menopausal (ovarectomized) control group with a potency lesser than estrogen as a reference drug

Isoflavone supplements are known to promote bone growth in postmenopausal women, and when tested 40mg Promensil twice daily over the course of 12 weeks in otherwise healthy postmenopausal women failed to significantly influence circulating osteocalcin or urinary N-Tx suggesting no influence on bone turnover.^[17] While the other branded product of mostly formononetin (Rimostil) has been noted to increase bone mineral density over the course of six months by 3-4.1% (57-85.5mg daily) in an blinded preliminary study^[50] it was also noted to fail in significantly influencing osteocalcin and N-Tx later in a controlled trial.^[17]

One study aiming to measure bone loss during menopause noted that supplementation of 40mg red clover isoflavones (Promensil) attenuated the loss of lumbar spine bone mineral density over a year (attenuating a 1.86% loss into a 1.08% loss) although hip density and mineral content were unaffected.^[55] This was not associated with any changes in serum biomarkers such as PINP or ALP, which were not significantly different between groups.^[55]

Human studies have found very minor beneficial effects at the level of the lumbar spine, but not hip; most moderate length studies have failed to find changes in the biomarkers that would indicate bone growth and no long term studies exist. At the moment the anti-osteoporetic effect seems very minor, and perhaps mostly just due to Formononetin (found in other plants as well such as astragalus membranaceus, licorice, or pueraria lobata)

There are three receptors known as 'estrogen related receptors' (ERRs) consisting of the isoforms of ERRα,^[59] ERRβ,^[59] and ERRγ;^[60][61] these steroid hormone receptors have a large structural homology to estrogen receptors, but are not activated by endogenous estrogens (they appear to be constituitively active without a ligand^[62]). These receptors have a smaller binding pocket for ligands than do the estrogen receptors^[63] and it appears that they may respond to some flavonoids such as biochanin A which activated all three subsets (10µM) with nonsignificantly greater potency than daidzein (one of the soy isoflavones) by enhancing the ERR affinity to the coactivator PNRC.^[63]

It is thought that ERRs are involved in estrogen signalling (uncertain how) since ERRα and ERRγ are negatively and positively associated with good biomarkers in breast cancer, respectively.^[64]

Although the role of estrogen-related receptors in the human body is not completely certain, biochanin A appears to be an agonist of all three subsets with comparable potency as daidzein

When looking at the level of estrogen signalling, Promensil in isolated BT-474 cells was able to signal through the estrogen receptors when used at a 10-fold diluation (from a basic stock solution) weakly at 100-fold and nothing at 1,000-fold.^[65] Isolated irilone, a minor catchol isoflavone, appears to exert estrogenic properties in vitro.^[66]

It is thought that there are direct estrogenic effects in humans since oral supplementation of 80mg isoflavones from red clover over 90 days in menopausal women appears to improve vaginal cytology including karyopyknotic, cornification and basal cell maturation indices;^[41] indicative of estrogenic effects. This failure has been replicated elsewhere^{[13][14][65][67]} and when measuring breast density (another biomarker of in vivo estrogenic effects) there are no whole-group effects with 40mg Promensil over the course of one to three years,^[68][11] although there may be a minor antiestrogenic effect assessed by reduced breast density in women overexpressing the estrogen receptors (ESR1 genotype)^[11] but not necessarily in women with a family history of breast cancer.^[68]

Despite the above (which used the Promensil extracts of Red Clover Extract), one extract known as Menoflavon has been noted to have estrogenic effects in vaginal tissue at an equivalent dose (80mg daily) although said study was funded by the producers of Menoflavon^[20] and another study (independent) using this extract actually noted a decrease in endometrium thickness associated with no changes in estrogen and a 22% increase in testosterone.^[69]

It appears that red clover isoflavones can be directly estrogenic after oral ingestion in humans

In postmenopausal women given 80mg of red clover isoflavones daily for 90 days, there appears to be no significant influence on circulating 17β-estradiol concentrations relative to placebo^[41] and this failure noted elsewhere^[13] and with 40mg over the course of three months^[53] to a year.^[11]

Despite the aforementioned estrogenicity, there are no significant alterations in the circulating estrogen concentrations of menopausal women given red clover extract

When tested in vitro, Promensil has been noted to have an androgen antagonistic action (63% when incubated with a 10-fold dilution of stock solution) with no appreciable agonistic activity;^[65] Promensil failed to interact with progesterone receptors.^[65]

In postmenopausal women given 80mg of red clover isoflavones daily for 90 days, no significant influences on testosterone are noted relative to placebo^[41] which has been noted with 40-80mg over the same time period elsewhere.^[53] One lone study with MF11RCE at 40mg twice daily has noted an increase in the testosterone concentrations of menopausal women by 22% associated with less of a decline in LH seen in placebo,^[69] but this occurred without changes in any other hormonal parameters.

Testosterone does not appear to be influenced for the most part, although one study using the MF11RCE (which seems to be hormonal for some unknown reason) has noted an increase in testosterone in the women taking the supplement; there does not appear to be enough evidence to support usage of this supplement for either reductions in testosterone or improving it secondary to aromatase inhibition

In postmenopausal women given 80mg of red clover isoflavones daily for 90 days, FSH appears to be unaffected.^[41]

In postmenopausal women given 80mg of red clover isoflavones daily for 90 days, there doesn't appear to be any influence on LH concentrations in serum.^[41]

IGF proteins are involved in the pathology of colon cancer somewhat, as they generally promote normal and cancerous cell growth^[70][71] and higher circulating IGF levels are seen in persons with colon cancer relative to normal controls^[72] and while the major binding protein (IGFBP-3; sequesters up to 90% of bound IGF) is not^[72][73] the less active IGFBP-1 and IGFBP-2 appear to be.^[74][75]

One pilot study in otherwise healthy women noted that red clover (Promensil, 40mg capsules twice daily for a month) there was a nonsignificant trend to reduce IGF-1 concentrations in premenopausal women which was not present in the whole group (including postmenopausal women) and at no time was there an influence on binding proteins (IGFBP-1 and IGFBP-3).^[49]

A study using Promensil capsules (40mg) with breakfast and dinner over the course eight weeks in men with a family history of colorectal cancer, supplementation failed to alter circulating levels of insulin like growth factors (free IGF-I, total IGF-II, IGF binding proteins 1-3) when assessing the whole group but in men designated as equol producters (23% of the sample) there was a 15.1% decrease in total IGF-1 (4.1-27.2% range).^[15] Serum IGF levels negatively correlated with equol, but not with genistein^[15] and elsewhere in a sample of women with a family history for colorectal cancer there was still a failure of 40mg Promensil twice daily to influence circulating IGFs or their binding proteins, with confirmation that the mRNA levels for IGF in colonic tissue were similarly unaffected^[16] but there was a large variation seen in IGFBP-2 levels^[16] and while that study did not measure equol production it has elsewhere been noted to positively correlate with other binding factors such as IGFBP-3.^[49]

Lowering total IGF concentrations in serum or increasing two of the binding proteins (IGFBP-1 and IGFBP-2) seem to be protective against colon cancer, but red clover extract has failed to influence any of these biomarkers in either sex. There may be some interactions with equol production status that need to be evaluated further

UV radiation is known to increase the kinase activity of Mixed-lineage kinase 3 (MLK3) which then acts via both the MKK4/JNK/c-Jun pathway as well as the MKK3/6/p38/MSK1 pathways (via phosphorylating MKK4 and MKK3/6 independently) to promote inflammation and apoptosis;^[76][77] it seems that biochanin A is a direct inhibitor of MLK3 and can prevent its increased activity in response to UV radiation by blocking its ATP binding site.^[78]

Biochanin A appears to directly dock onto MLK3 and prevent it from mediating an inflammatory response from UV radiation towards skin inflammation

Biochanin A has been noted to have melanin inhibiting properties in vitro by inhibiting melanogenesis in B16 cells in the range of 11-22µM, able to normalize melanogenesis to control levels and comparable to 400µM arbutin.^[79] This potency did not correlated with tyrosinase inhibition (which was partial at 22µM only^[79]) and was replicated with formononetin.^[79] Both Biochanin A and formononetin both also demonstrated depigmenting properties in vitro with 88-176µM being comparable to 400µM arbutin, and when tested in mice given a topical application of a cream containing 2% biochanin A twice daily over the course of one week showed skin whitening properties.^[79]

Appears to have skin whitening properties when tested in vitro and in mice, and it appears to have respectable potency at this based on the limited evidence for it

Oral ingestion of 80mg red clover isoflavones daily for 90 days appears to be associated with improved subjective ratings of skin quality (moisture content, texture, and 'overall condition' as assessed by a VAS scale of 0-100).^[35] There was high variability, but the overall improvement was at 17.7-18.6 points (on the scale of 0-100) whereas placebo say a 5.0-6.2 point increase.^[35]

At least one study has noted improvements in skin quality over the course of ninety days of supplementation in postmenopausal women

Oral supplementation of 80mg red clover extract daily for 90 days in postmenopausal women appears to increase the self-reported quality of scalp hair (thickness, fragility, and overall quality) as assessed by a 0-100 rating scale, where improvements seen where between 6.3-7.3 points (placebo at 0.2-4.2) but with high variability.^[35]

Oral supplementation of 80mg red clover extract daily for 90 days in postmenopausal women appeared to be associated with increased subjective ratings of nail quality in the latter half of the study (said study being double blind crossover) reaching a 10.8+/-19.3 point increase (on a 0-100 rating scale) although the first group given supplementation only noted a trend to increase subjective ratings.^[35]

Supplementation of 80mg of the red clover isoflavones appears to reduce postmenapausal symptoms after 90 days as assessed by the Kupperman index by 78%, whereas placebo experienced a 23% reduction over the same time period^[41] with a similarly structured study using 80mg isoflavones noted a reduction in menopausal symptoms as assessed by the Kupperman Index (75.4%) as well as both hot flashes and night sweats (72.2-73.5%) relative to placebo, and this benefit was not observed three months after supplement cessation.^[80]

40mg of red clover isoflavones daily for twelve weeks has been noted to reduce hot flashes when measured at four weeks, but over the full course of the study there was a failure to reduce overall menopausal symptoms or hot flashes relative to placebo^[13] and this failure was then replicated when using 40-160mg Promensil and assessing hot flashes (29-34% reduction; placebo noted a 35%) or overall symptoms (26-44% reduction whereas placebo reached 46%).^[14] Using Promensil at this dose (40mg) over the course of a year does not appear to confer protective effects either, even if controlled for urinary isoflavones or genotypes (CYP19, CYP17, ESR1)^[11] and the 40-120mg dosage range of red clover isoflavones has failed elsewhere over a year where the reduction in symptoms seen (Kupperman Index) were similar to placebo.^[33][81]

At times the brand Promensil is used (red clover isoflavones paired with soy isoflavones), and when using this supplement it appears that supplementation of 80mg is able to significantly reduce hot flashes by 33% (placebo unchanged) within three weeks which increases to a 44% reduction after 12 weeks.^[12] Overall, menopausal symptoms as assessed by Greene Climacteric Scale (12.8% reduction) were improved relative to placebo^[12] although this has not been noted elsewhere with the same dose and time frame.^[82]

One study has noted that, despite no overall benefits at the end of the 12 week intervention seen with supplementation of 80mg Promensil, that there was a quick decline in hot flashes in the early parts of the trial in supplementation only;^[82] another study with a 4-week placebo run in before Promensil supplementation noted that the expected drop occurred within 1-3 weeks (33% from baseline, greater than placebo)^[12] suggesting an acute effect that fades with time. Elsewhere, while many studies note that supplementation can increase urinary isoflavones and said increase in variabile from one subject to another^{[13][14][12][82]} that urinary isoflavones may not be correlated with individual benefits;^[82] instead, subjects who are overweight may be more likely to see benefits.^[82]

While isolated studies have noted benefit to menopausal symptoms (usually either assessing overall symptoms via a rating scale, or frequency/magnitude of hot flashes assessed by personal journal) overall these benefits seem to be unreliable in both the magnitude of benefits and when they occur. Many studies are also partially funded by producers of the brand name products whereas the frequency of failure tends to increase in independent studies, so at this moment in time it seems prudent to assume either a minor or nonexistent benefit to general menopausal symptoms

Red clover (as tea or tincture) appears to have traditional usage for the treatment of cough and bronchitis^[9] and when tested in isolated guinea pig trachea, 10-30µM of Biochanin A appears to reduce ovalbumin induced contractions with an IC₅₀ of 8.1+/-0.8μM^[9] and this may be related to its inhibitory actions on PDE4 (IC₅₀ of 8.5μM) that are more potent than its inhibitory actions on PDE1 (29.1μM) and PDE2 (27.9μM; no influence on PDE3 nor PDE5), differing from genistein (more selective towards PDE2) and daidzein (PDE3 selective).^[83] Interestingly, while PDE4 has its activity inhibited with an IC₅₀ of 8.5μM yet failed to significantly displace rolipram from PDE4 up to 300μM,^[9] resulting in a PDE4_H/PDE4_L of over 35 (which is thought to be a desirable ratio for treating asthma^[84]).

Red clover, particularly biochanin A, is thought to have some anti-asthmatic properties in the lungs by acting as a bronchodilator (widening the bronchus and enhancing breathing)

Oral ingestion of 100µM/kg Biochanin A (but not 30µM/kg) to mice is able to significantly suppress methacholine induced airway hyperresponsiveness associated with reductions in inflammatory cell infiltration and most inflammatory cytokines with exception to IFN-γ.^[9]

Oral ingestion has been noted to, in rodents, reduce airway hyperresponsiveness

Biochanin A is reported to be an aromatase inhibitor (thought to be therapeutic for breast cancer that is responsive to estrogen^[85]) and in MCF-7aro breast cancer cells incubated with biochanin A a day before androgens, proliferation (due to conversion of androgens to estrogen) was suppressed with 12.5µM biochanin A and abolished with 25µM.^[21]

Biochanin A was noted to proliferate MCF-7 breast cancer cells with an EC₅₀ of 9nM, lesser than 17β-estradiol (4pM) but greater than genistein (32nM).^[25]

Biochanin A has shown both estrogenic and antiestrogenic properties in isolated MCF-7 cells, but the estrogenic properties seem to occur at a lower concentration and may be more practically relevant to oral ingestion

One previous study has noted, in noncancerous females taking isoflavone supplements for over one year, that there are no changes in breast density indicative of estrogenic effects^[86] and when this was replicated with red clover extract it was noted that 40mg Promensil daily for one year failed to have either estrogenic or antiestrogenic properties in breast tissue overall;^[11] controlling for urinary isoflavones (absorption marker) failed to show any significance, but looking only at women expressing an ESR1 genotype (estrogen receptor, where an increase in receptor content may be slightly associated with increased risk^[87] and isoflavone supplemented exerted minor antiestrogenic effects relative to placebo) noted an interaction while CYP17 and CYP19 genotypes were not associated.^[11]

In general there does not appear to be any estrogenic nor antiestrogenic properties on the breast tissue of women in menopause without a history of breast cancer, although there may be a slight antiestrogenic effect particularly in those women with a higher level of estrogen receptors (genetics)

Isoflavones from red clover appear to protect dopaminergic neurons from inflammatory (LPS induced) neurological damage, with greatest potency coming from pratensein which preserved 80.2% of dopamine uptake (reduced to 37.2% with LPS alone) at a concentration of 2.5μM;^[88] this exceeded formononetin (60.7%) and daidzein (70.1%) in potency^[88] and elsewhere Biochanin A was able to preserve 88.7% of dopamine uptake relative to control.^[89] More practically, 250nM of Biochanin A was able to increase dopamine uptake (36.7% in LPS control) to 55.9%, minor but statistically significant protective effects^[89] and this protective effect has been noted to occur in microglia at 2.5μM for all isoflavones by attenuating inflammatory cytokine release.^[88][89]

In rats subject to 6-OHDA (dopaminergic) lesions, supplementation of 200mg/kg red clover extract (26% formononetin and 12% Biochanin A) for four days prior to lesions partially attenuated the lesion size and dopamine less.^[90]

Preliminary evidence both in vitro and in rats fed higher than normal doses of red clover extract (around 2g in a normal weight human) have noted minor protective effects against damage to dopaminergic (dopamine releasing) neurons, thought to be due to general antioxidative and antiinflammatory properties