Estrogen receptor ß and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate.

Knockout of ERß in the mouse leads to nuclear expression of epidermal growth factor receptor (EGFR) in the prostate. To examine whether ERß plays a similar role in the human prostate, we used four cohorts of men: 1) a Swedish cohort of normal prostates and PCa (prostate cancer) of different Gleason grades; 2) men with benign prostatic hyperplasia (BPH) treated with the 5α-reductase inhibitor, finasteride, and finasteride together with the ERß agonists, soy isoflavones; 3) men with PCa above Gleason grade 4 (GG4), treated with ADT (androgen deprivation therapy) and abiraterone (AA), the blocker of androgen synthesis for different durations; and 4) men with GG4 PCa on ADT or ADT with the AR (androgen receptor) blocker, enzalutamide, for 4 mo to 6 mo. In men with BPH, finasteride treatment induced EGFR nuclear expression, but, when finasteride was combined with isoflavones, EGFR remained on the cell membrane. In GG4 patients, blocking of AR for 4 mo to 6 mo resulted in loss of ERß and PTEN expression and increase in patients with nuclear EGFR from 10 to 40%. In the men with GG4 PCa, blocking of adrenal synthesis of testosterone for 2 mo to 7 mo had the beneficial effect of increasing ERß expression, but, on treatment longer than 8 mo, ERß was lost and EGFR moved to the nucleus. Since nuclear EGFR is a predictor of poor outcome in PCa, addition of ERß agonists together with abiraterone should be considered as a treatment that might sustain expression of ERß and offer some benefit to patients.

Structure-Activity Relationship of Phytoestrogen Analogs as ERα/ß Agonists with Neuroprotective Activities.

A set of isoflavononid and flavonoid analogs was prepared and evaluated for estrogen receptor α (ERα) and ERß transactivation and anti-neuroinflammatory activities. Structure-activity relationship (SAR) study of naturally occurring phytoestrogens, their metabolites, and related isoflavone analogs revealed the importance of the C-ring of isoflavonoids for ER activity and selectivity. Docking study suggested putative binding modes of daidzein 2 and dehydroequol 8 in the active site of ERα and ERß, and provided an understanding of the promising activity and selectivity of dehydroequol 8. Among the tested compounds, equol 7 and dehydroequol 8 were the most potent ERα/ß agonists with ERß selectivity and neuroprotective activity. This study provides knowledge on the SAR of isoflavonoids for further development of potent and selective ER agonists with neuroprotective potential.

Pharmacological activation of ERß by arctigenin maintains the integrity of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal epithelial barrier dysfunction is deeply involved in the pathogenesis of inflammatory bowel diseases (IBD). Arctigenin, the main active constituent in Fructus Arctii (a traditional Chinese medicine), has previously been found to attenuate colitis induced by dextran sulfate sodium (DSS) in mice. The present study investigated whether and how arctigenin protects against the disruption of the intestinal epithelial barrier in IBD. Arctigenin maintained the intestinal epithelial barrier function of mice with DSS- and TNBS-induced colitis. In Caco-2 and HT-29 cells, arctigenin lowered the monolayer permeability, increased TEER, reversed the abnormal expression of tight junction proteins, and restored the altered localization of F-actin induced by TNF-α and IL-1ß. The specific antagonist PHTPP or shRNA of ERß largely weakened the protective effect of arctigenin on the epithelial barrier function of Caco-2 and HT-29 cells. Molecular docking demonstrated that arctigenin had high affinity for ERß mainly through hydrogen bonds as well as hydrophobic effects, and the protective effect of arctigenin on the intestinal barrier function was largely diminished in ERß-mutated (ARG346 and/or GLU305) Caco-2 cells. Moreover, arctigenin-blocked TNF-α induced increase of the monolayer permeability in Caco-2 and HT-29 cells and the activation of myosin light chain kinase (MLCK)/myosin light chain (MLC) pathway in an ERß-dependent manner. ERß deletion in colons of mice with DSS-induced colitis resulted in a significant attenuation of the protective effect of arctigenin on the barrier integrity and colon inflammation. Arctigenin maintained the integrity of the intestinal epithelial barrier under IBD by upregulating the expression of tight junction proteins through the ERß-MLCK/MLC pathway.

Selective Estrogen Receptor ß Agonists: a Therapeutic Approach for HIV-1 Associated Neurocognitive Disorders.

The persistence of HIV-1 associated neurocognitive disorders (HAND) in the post-cART era, afflicting between 40 and 70% of HIV-1 seropositive individuals, supports a critical need for the development of adjunctive therapeutic treatments. Selective estrogen receptor ß agonists, including S-Equol (SE), have been implicated as potential therapeutic targets for the treatment of neurocognitive disorders. In the present study, the therapeutic efficacy of 0.2 mg SE for the treatment of HAND was assessed to address two key questions in the HIV-1 transgenic (Tg) rat. First, does SE exhibit robust therapeutic efficacy when treatment is initiated relatively early (i.e., between 2 and 3 months of age) in the course of viral protein exposure? Second, does the therapeutic utility of SE generalize across multiple neurocognitive domains? Treatment with SE enhanced preattentive processes and stimulus-response learning to the level of controls in all (i.e., 100%) HIV-1 Tg animals. For sustained and selective attention, statistically significant effects were not observed in the overall analyses (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). However, given our a priori hypothesis, subsequent analyses were conducted, revealing enhanced sustained and selective attention, approximating controls, in a subset (i.e., 50%, n = 5 and 80%, n = 8, respectively) of HIV-1 Tg animals treated with SE. Thus, the therapeutic efficacy of SE is greater when treatment is initiated relatively early in the course of viral protein exposure and generalizes across neurocognitive domains, supporting an adjunctive therapeutic for HAND in the post-cART era. Graphical Abstract HIV-1 transgenic (Tg) and control animals were treated with either 0.2 mg S-Equol (SE) or placebo between 2 and 3 months of age (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). Neurocognitive assessments, tapping preattentive processes, stimulus response learning, sustained attention and selective attention, were conducted to evaluate the utility of SE as a therapeutic for HIV-1 associated neurocognitive disorders (HAND). Planned comparisons between HIV-1 Tg and control animals treated with placebo were utilized to establish a genotype effect, revealing prominent neurocognitive impairments (NCI) in the HIV-1 Tg rat across all domains. Furthermore, to establish the utility of SE, HIV-1 Tg animals treated with SE were compared to control animals treated with placebo. Treatment with 0.2 mg SE ameliorated NCI, to levels that were indistinguishable from controls, in at least a subset (i.e., 50-100%) of HIV-1 Tg animals. Thus, SE supports an efficacious, adjunctive therapeutic for HAND.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

The Natural Agonist of Estrogen Receptor ß Silibinin Plays an Immunosuppressive Role Representing a Potential Therapeutic Tool in Rheumatoid Arthritis.

Estrogens, in particular 17ß-estradiol (E2), have a strong influence on the immune system and also affect pathological conditions such as autoimmune diseases. The biological effects of E2 are mediated by two intracellular receptors, i.e., estrogen receptor (ER)α and ERß, which function as ligand-activated nuclear transcription factors producing genomic effects. Immune cells express both ERα and ERß that play a complex role in modulating inflammation. Phytoestrogens display estrogen-like effects. Among them, silibinin, the major active constituent of silymarin extracted by the milk thistle (Silybum marianum), has been suggested to have an ERß selective binding. Silibinin is known to have anti-inflammatory, hepatoprotective, and anticarcinogenic effects; however, the role of silibinin in modulating human immune responses and its impact on autoimmunity remains unclear. Aim of this study was to dissect the ability of the ERß natural ligand silibinin to modulate T cell immunity, taking into account possible differences between females and males, and to define its possible role as therapeutic tool in immune-mediated diseases. To this purpose, female and age-matched male healthy subjects and patients with active rheumatoid arthritis (RA) were recruited. We evaluated the ability of silibinin to modulate ERß expression in T lymphocytes and its effects on T cell functions (i.e., apoptosis, proliferation, and cytokine production). We also analyzed whether silibinin was able to modulate the expression of microRNA-155 (miR-155), which strongly contributes to the pathogenesis of RA driving aberrant activation of the immune system. We demonstrated that silibinin upregulated ERß expression, induced apoptosis, inhibited proliferation, and reduced expression of the pro-inflammatory cytokines IL-17 and TNF-α, through ERß binding, in T lymphocytes from female and male healthy donors. We obtained similar results in T lymphocytes from patients with active RA in term of apoptosis, proliferation, and cytokine production. In addition, we found that silibinin acted as an epigenetic modifier, down-modulating the expression of miR-155. In conclusion, our data demonstrated an immunosuppressive role of silibinin, supporting its application in the treatment of autoimmune diseases as drug, but also as dietary nutritional supplement, opening new perspective in the field of autoimmune disease management.

In silico estrogen-like activity and in vivo osteoclastogenesis inhibitory effect of Cicer arietinum extract.

Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

Estrogen Receptor (ER) Subtype Selectivity Identifies 8-Prenylapigenin as an ERß Agonist from Glycyrrhiza inflata and Highlights the Importance of Chemical and Biological Authentication.

Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERß activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/ß41 breast cancer cells (ERß). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERß agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.

Pharmacokinetics and safety profile of single-dose administration of an estrogen receptor ß-selective phytoestrogenic (phytoSERM) formulation in perimenopausal and postmenopausal women.

OBJECTIVE: Selected estrogen receptor ß-selective phytoestrogen (phytoSERM), a preparation of genistein, daidzein, and S-equol, has an 83-fold selective affinity for estrogen receptor (ER) ß, and may promote neuronal survival and estrogenic mechanisms in the brain without exerting feminizing activity in the periphery. The aim of this study was to assess the safety, tolerability, and single-dose pharmacokinetics of the phytoSERM formulation in perimenopausal and postmenopausal women. METHODS: Eighteen women aged 45 to 60 years from a 12-week clinical trial evaluating cognitive performance and vasomotor symptoms were randomly assigned to placebo, 50 mg, or 100 mg phytoSERM treatment groups. Plasma levels of the three parent phytoestrogens and their metabolites were measured before and at 2, 4, 6, 8, and 24 hours after ingestion by isotope dilution high-performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry. RESULTS: Plasma concentrations of genistein, daidzein, and S-equol peaked at 9, 6, and 4 hours, respectively, for the 50-mg dose, and at 6, 6, and 5 hours, respectively, for the 100-mg dose. The maximum concentration (Cmax) and area under the curve (AUC) for the three parent compounds were greater in the 100-mg dose group, indicating a dose-dependent change in concentration with the phytoSERM treatment. No adverse events were elicited. CONCLUSIONS: A single-dose oral administration of the phytoSERM formulation was well-tolerated and did not elicit any adverse events. It was rapidly absorbed, reached high plasma concentrations, and showed a linear dose-concentration response in its pharmacokinetics. These findings are consistent with previously reported parameters for each parent compound (Clinicaltrials.gov NCT01723917).

Synthesis of prenylated flavonols and their potents as estrogen receptor modulator.

Prenylated flavonols are known as phytoestrogen and have good bioactivties. However, their abundances in nature are pretty low. It is required to find an efficient synthesis technique. Icariin is a prenylated flavonol glycoside with low cost. It can be used to synthesize different prenylated flavonols. A combination of cellulase and trifluoacetic acid hydrolysis could effectively remove rhamnose and glucose from icariin. Icaritin, anhydroicaritin and wushanicaritin were the leading prenylated flavonol products. Their affinities to estrogen receptors α and ß were predicted by docking study. The weak affinity of wushanicaritin indicated that prenyl hydroxylation impaired its affinity to estrogen receptor ß. The prenyl cyclization led to a loss of affinity to both receptors. The interactions between icaritin and ligand binding cavity of estrogen receptor ß were simulated. π-π stacking and hydrophobic forces were predicted to be the dominant interactions positioning icaritin, which induced the helix (H12) forming an activated conformation.

Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor ß and increases skeletal muscle mass in young female mice.

Ubiquitin-specific protease 19 (USP19) is a key player in the negative regulation of muscle mass during muscle atrophy. Loss-of-function approaches demonstrate that 17ß-estradiol (E2) increases USP19 expression through estrogen receptor (ER) α and consequently decreases soleus muscle mass in young female mice under physiological conditions. Daidzein is one of the main isoflavones in soy, and activates ERß-dependent transcription. Here, we investigated the effects of daidzein on E2-increased USP19 expression and E2-decreased soleus muscle mass in young female mice. Daidzein stimulated the transcriptional activity of ERß in murine C2C12 cells and down-regulated USP19 expression. Consistently, daidzein inhibited E2-induced USP19 expression in a reporter activity using a functional half-estrogen response element (hERE) from Usp19. Daidzein inhibited E2-induced recruitment of ERα and promoted recruitment of ERß to the Usp19 hERE. Dietary daidzein down-regulated the expression of USP19 at the mRNA and protein levels and increased soleus muscle mass in female mice, but not in males. In soleus muscle from ovariectomized (OVX) female mice, dietary daidzein inhibited E2-increased USP19 mRNA expression and E2-decreased muscle mass. Furthermore, E2 induced the recruitment of ERα and ERß to the hERE, whereas daidzein inhibited E2-induced recruitment of ERα, and enhanced E2-increased recruitment of ERß, to the Usp19 hERE. These results demonstrate that dietary daidzein decreases USP19 mRNA expression through ERß and increases soleus muscle mass in young female mice, but not in male mice, under physiological conditions.

Therapeutic utility of natural estrogen receptor beta agonists on ovarian cancer.

Ovarian cancer is the deadliest of all gynecologic cancers. Despite success with initial chemotherapy, the majority of patients relapse with an incurable disease. Development of chemotherapy resistance is a major factor for poor long-term survival in ovarian cancer. The biological effects of estrogens are mediated by estrogen receptor alpha (ERα) and estrogen receptor beta (ERß). Emerging evidence suggests that ovarian cancer cells express ERß that functions as a tumor suppressor; however, the clinical utility of ERß agonists in ovarian cancer remains elusive. We tested the utility of two natural ERß agonists liquiritigenin (Liq), which is isolated from Glycyrrhiza uralensis and S-equol, which is isolated from soy isoflavone daidzein, for treating ovarian cancer. Both natural ERß ligands had significant growth inhibition in cell viability and survival assays, reduced migration and invasion, and promoted apoptosis. Further, ERß agonists showed tumor suppressive functions in therapy-resistant ovarian cancer model cells and sensitized ovarian cancer cells to cisplatin and paclitaxel treatment. Global RNA-Seq analysis revealed that ERß agonists modulate several tumor suppressive pathways, including downregulation of the NF-κB pathway. Immunoprecipitation assays revealed that ERß interacts with p65 subunit of NF-κB and ERß overexpression reduced the expression of NF-κB target genes. In xenograft assays, ERß agonists reduced tumor growth and promoted apoptosis. Collectively, our findings demonstrated that natural ERß agonists have the potential to significantly inhibit ovarian cancer cell growth by anti-inflammatory and pro-apoptotic actions, and natural ERß agonists represent novel therapeutic agents for the management of ovarian cancer.

A Selective Estrogen Receptor Beta Agonist for the Treatment of Hot Flushes: Phase 2 Clinical Trial.

OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, and efficacy of a selective estrogen receptor beta (ERß) agonist, Dr. Tagliaferri's Menopause Formula (MF102), to treat the symptoms of menopause. METHODS: An open-label trial of MF102 taken for 12 weeks by 30 postmenopausal women aged 40-65 years, who experienced a minimum of five moderate to severe hot flushes per day. The primary efficacy outcome was a change in the frequency of moderate to severe hot flushes from baseline to week 12. A change in the frequency of hot flushes that woke participants from their sleep from baseline to 12 weeks was a secondary endpoint. Lipid profile and endometrial thickness were also evaluated. RESULTS: Thirty postmenopausal women with an average of nine moderate to severe hot flushes per day were treated with MF102 4 g/day; 27 participants completed the study. The median percent reduction in moderate to severe hot flushes was 71% (p < 0.001). The median percent reduction in hot flushes that woke participants from their sleep was 54% (p < 0.001). Low-density lipoprotein (LDL-C) and total cholesterol both declined significantly from baseline. There were no serious adverse events, reports of abnormal uterine bleeding, or significant changes in double-wall endometrial thickness. CONCLUSIONS: Treatment with MF102 resulted in a marked decrease in the frequency of moderate to severe hot flushes, was well-tolerated, and demonstrated no safety concerns.

Estrogenic effects of flavonoid components in Xiaoyao powder.

The objective of this study was to evaluate the estrogenic effects and mechanisms of three flavonoid components in Xiaoyao powder: quercetin, kaempferol, and isorhamnetin. The drugs were used to treat estrogen receptor (ER)-positive human breast cancer MCF-7 cells, and proliferation was measured using the MTT method. The expression of proteins and mRNA of the ER subtype were measured using western blotting and real time polymerase chain reaction. The quercetin (10(-2) µM, 10(-3) µM), kaempferol (100 µM, 10(-2) µM), and isorhamnetin (10(-3) µM) promoted the proliferation of MCF-7 cells, and the expression of ERα and ERß proteins and mRNA were all increased significantly (P < 0.05). These effects were reversed by treatment with 0.1 µM estrogen antagonist ICI182780. Three flavonoid components in Xiaoyao powder increased the expression of proteins and mRNA of ERα and ERß and promoted the proliferation of MCF-7 cells. These estrogenic effects were mediated by the ER.

Cryptotanshinone and wogonin up-regulate eNOS in vascular endothelial cells via ERα and down-regulate iNOS in LPS stimulated vascular smooth muscle cells via ERß.

Phytoestrogens were widely used as natural alternatives to estrogen for treating cardiovascular diseases. They have been reported to have cardioprotective and anti-inflammatory response, but the mechanisms remain unclear. In this study, we found cryptotanshinone and wogonin exhibited phytoestrogenic property in an estrogen-responsive reporter assay. In EA.hy926 cells, treatment of cryptotanshinone and wogonin led to significant increase in NO production levels, which were inhibited by co-incubation of estrogen receptor (ER)α antagonist methyl-piperidino-pyrazole (MPP). The expression of endothelial NO synthase (eNOS) and ERα were up-regulated with the same treatment, indicating they stimulate NO and eNOS expression via ERα-dependent pathway in endothelial cells. While in lipopolysaccharide activated vascular smooth muscle cell line A7r5, cryptotanshinone and wogonin exerted anti-inflammatory effects by inhibiting NO and inducible NO synthase expression via ERß-dependent pathway. The reduction of NO synthesis was not affected by MPP, and was abrogated by ERß antagonist R,R-tetrahydrochrysene. Our findings provide the potential molecular mechanism of cryptotanshinone and wogonin as phytoestrogens for their cardioprotective effects, which exerted regulatory effects on NO synthesis through differential regulation of estrogen receptors. It can be employed as a basis for evaluating the beneficial effects of phytoestrogens in the treatment of patients at risk of cardiovascular disease.

Estrogen receptor-ß in mitochondria: implications for mitochondrial bioenergetics and tumorigenesis.

Estrogen enhances mitochondrial function by enhancing mitochondrial biogenesis and sustaining mitochondrial energy-transducing capacity. Shifts in mitochondrial bioenergetic pathways from oxidative phosphorylation to glycolysis have been hypothesized to be involved in estrogen-induced tumorigenesis. Studies have shown that mitochondria are an important target of estrogen. Estrogen receptor-ß (ERß) has been shown to localize to mitochondria in a ligand-dependent or -independent manner and can affect mitochondrial bioenergetics and anti-apoptotic signaling. However, the functional role of mitochondrial ERß in tumorigenesis remains unclear. Clinical studies of ERß-related tumorigenesis have shown that ERß stimulates mitochondrial metabolism to meet the high energy demands of processes such as cell proliferation, cell survival, and transformation. Thus, in elucidating the precise role of mitochondrial ERß in cell transformation and tumorigenesis, it will be particularly valuable to explore new approaches for the development of medical treatments targeting mitochondrial ERß-mediated mitochondrial function and preventing apoptosis.

Divide and Conquer May Not Be the Optimal Approach to Retain the Desirable Estrogenic Attributes of the Cyclopia Nutraceutical Extract, SM6Met.

The genus Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, contains phytoestrogenic compounds. An extract from C. subternata, SM6Met, displays three desirable estrogenic attributes for future development of a phytoestrogenic nutraceutical, namely, ERα antagonism, ERß agonism, and also antagonism of E2-induced breast cancer cell proliferation. Activity-guided fractionation of SM6Met was used in an attempt to isolate and identify compounds conferring the desirable estrogenic profile to SM6Met. Initial liquid-liquid fractionation of SM6Met yielded a polar fraction (PF) and a non-polar fraction (NPF), with the desirable estrogenic attributes retained in the NPF. Subsequent high performance counter-current chromatography (HPCCC) fractionation of the NPF yielded three fractions (F1-F3). Interestingly, the fractions revealed separation of the previously demonstrated positive estrogenic attributes of the NPF into separate fractions, with F1 and F2 acting as ERα antagonists, only F2 inducing antagonism of E2-induced breast cancer cell proliferation and only F3 retaining robust ERß agonist activity. In terms of major polyphenols, quantitative HPLC and liquid chromatography tandem mass spectrometry (LC-MS/MS) indicated that HPCCC fractionation resulted in a divergence of polyphenolic classes, with F1 emerging as the dihydrochalcone-rich fraction and F2 as the flavanone- and benzophenone-rich fraction, while the xanthones, flavones and phenolic acids were retained in F3. F3 was re-engineered into F3R by reassembling the major polyphenols identified in the fraction. F3R could, however, not replicate the effect of F3. In conclusion, although activity-guided fractionation results suggest that retention of all the desirable estrogenic attributes of the original SM6Met in one fraction is not an attainable goal, fractionation is a useful tool to enhance specific desirable estrogenic attributes.

Determination of the isoflavone composition and estrogenic activity of commercial dietary supplements based on soy or red clover.

Dietary supplements high in isolated isoflavones are commercially available for human consumption primarily to alleviate menopausal symptoms in women. The isoflavone composition, quantity and importantly their estrogenic potency are poorly standardised and can vary considerably between different products. The aim of this study was to analyse the isoflavone composition of 11 dietary supplements based on soy or red clover using the HPLC/MS/MS technique. Furthermore, we investigated the transactivational potential of the supplements on the estrogen receptors (ER), ERα and ERß, performing luciferase reporter gene assays. As expected, we found that the isoflavone composition varies between different products. The measured total isoflavone contents in various supplements were mostly comparable to those claimed by the manufacturers in their product information. However expressing the isoflavone content as isoflavone aglycone equivalents, soy-based supplements had a clearly lower quantity compared to the manufacturer information. All supplements transactivated more or less ERα and ERß with a preference for ERß. The transactivational efficiency exceeded partly the maximal 17ß-estradiol induced ER activation. While the different soy-based supplements revealed similar transactivation potential to both ERs, red clover-based supplements differed considerably. We conclude that different commercial dietary supplements based on soy or red clover vary in their isoflavone composition and quantity. They are estrogenically active, although especially the red clover-based supplements show considerable differences in their estrogenic potential to ERα and ERß. Thus, different isoflavone-rich products cannot be necessarily compared regarding possible biological effects.

Estrogen modulation properties of mangiferin and quercetin and the mangiferin metabolite norathyriol.

Mango fruit contain many bioactive compounds, some of which are transcription factor regulators. Estrogen receptor alpha (ERα) and beta (ERß) are two regulators of gene transcription that are important in a variety of physiological processes and also in diseases including breast cancer. We examined the ability of the mango constituents quercetin, mangiferin, and the aglycone form of mangiferin, norathyriol, to activate both isoforms of the estrogen receptor. Quercetin and norathyriol decreased the viability of MCF-7 breast cancer cells whereas mangiferin had no effect on MCF-7 cells. We also determined that quercetin and mangiferin selectively activated ERα whereas norathyriol activated both ERα and ERß. Despite quercetin, mangiferin and norathyriol having similar polyphenolic structural motifs, only norathyriol activated ERß, showing that bioactive agents in mangoes have very specific biological effects. Such specificity may be important given the often-opposing roles of ERα and ERß in breast cancer proliferation and other cellular processes.

Enhanced action of apigenin and naringenin combination on estrogen receptor activation in non-malignant colonocytes: implications on sorghum-derived phytoestrogens.

Activation of estrogen receptor-ß (ERß) is an important mechanism for colon cancer prevention. Specific sorghum varieties that contain flavones were shown to activate ER in non-malignant colonocytes at low concentrations. This study aimed to determine positive interactions among estrogenic flavonoids most relevant in sorghum. Apigenin and naringenin were tested separately and in combination for their ability to influence ER-mediated cell growth in non-malignant young adult mouse colonocytes (YAMC). Sorghum extracts high in specific flavanones and flavones were also tested. Apigenin reduced ER-mediated YAMC cell growth comparable to physiological levels of estradiol (E2, 1 nM) at 1 µM; naringenin had similar effect at 10 µM. However, when combined, 0.1 µM apigenin plus 0.05 µM naringenin produced similar effect as 1 nM E2; these concentrations represented 1/10th and 1/200th, respectively, of the active concentrations of apigenin and naringenin, demonstrating a strong enhanced action. A sorghum extract higher in flavones (apigenin and luteolin) (4.8 mg g(-1)) was more effective (5 µg mL(-1)) at activating ER in YAMC than a higher flavanone (naringenin and eriodictyol) (28.1 mg g(-1)) sorghum extract (10 µg mL(-1)). Enhanced actions observed for apigenin and naringenin were adequate to explain the level of effects produced by the high flavone and flavanone sorghum extracts. Strong positive interactions among sorghum flavonoids may enhance their ability to contribute to colon cancer prevention beyond what can be modeled using target compounds in isolation.