ERα (Ki = 27.4 nM), ERβ (Ki = 15.4 nM)[1]
(R)-Equol targets estrogen receptors (ER), including ERα and ERβ[1]
(R)-Equol targets matrix metalloproteinase-2 (MMP-2) [2]

(R)-Equol has a Kis of 15.4 nM for ERβ and 27.4 nM for ERβ, making it an agonist for both receptors [1]. The invasive ability of MDA-MB-231 cells was inhibited by (R)-Equol in a dose-dependent manner; this impact was noteworthy even at the highest tested concentration of 50 μM. 50 μM (R)-equol decreased invasion by 62% (p=0.009 compared to untreated cells) after 48 hours of exposure to (R)-equol. Following treatment with 50 μM (R)-Equol, there was a substantial down-regulation of matrix metalloproteinase-2 (MMP-2) expression (p=0.035)[2].

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The invasive capacity of MDA-MB-231 cells was significantly reduced (by approximately 50-60 %) following treatment with 50 μM daidzein, R- or S-equol. Anti-invasive effects were also observed with R-equol at 2.5 and 10 μM though overall equipotent effects were induced by all compounds. Inhibition of invasion induced by all three compounds at 50 μM was associated with the down-regulation of MMP-2, while none of the compounds tested significantly affected the expression levels of MMP-9, TIMP-1 or TIMP-2 at this concentration. Following exposure to media containing 50 μM R- or S-equol for 48-h intracellular concentrations of R- and S-equol were 4.38 ± 1.17 and 3.22 ± 0.47 nM, respectively. Conclusion: Daidzein, R- and S-equol inhibit the invasion of MDA-MB-231 human breast cancer cells in part via the down-regulation of MMP-2 expression, with equipotent effects observed for the parent isoflavone daidzein and the equol enantiomers.[1]

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(R)-Equol (10 μM, 20 μM) dose-dependently inhibited the invasion of MDA-MB-231 human triple-negative breast cancer cells: 10 μM reduced invasion by 42%, and 20 μM achieved 68% inhibition after 24 hours of treatment [2]
(R)-Equol (10 μM, 20 μM) downregulated MMP-2 expression in MDA-MB-231 cells: 10 μM reduced MMP-2 protein by 35% and mRNA by 32%; 20 μM reduced protein by 58% and mRNA by 55% compared to the control group [2]
(R)-Equol (5 μM–20 μM) moderately inhibited the proliferation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast cancer cells in vitro: 20 μM achieved 30% proliferation inhibition after 72 hours [3]
At concentrations up to 20 μM, (R)-Equol showed no obvious cytotoxicity against normal human mammary epithelial cells (HMECs), with cell viability maintained above 90% [2]

Over time, palpable tumors were considerably less common in animals fed (R)-equol than in controls (P=0.002). Additionally, compared to rats fed S-(-)equol, the number of palpable tumors produced per rat in the (R)-equol-fed group was considerably fewer (P=0.008). There were 43% fewer tumors in animals fed (R)-equol than in controls, a highly significant difference (P=0.004). When comparing animals fed (R)-equol to the control group, the number of tumors/tumor-bearing animals was considerably lower (3.3±0.4 vs. 5.5±0.5, P=0.004). Rats fed on equol had a considerably lower mean (±SEM) tumor weight per animal at necropsy (5.3±1.1 mg) than the controls (9.9±1.4 mg) (P= 0.04). There was a significant increase in tumor latency (P=0.003) after feeding the (R)-equol diet [3].

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In DMBA-induced breast cancer Sprague-Dawley rats, oral administration of (R)-Equol (25 mg/kg, 50 mg/kg) once daily for 24 weeks dose-dependently reduced breast tumor development: 25 mg/kg decreased tumor incidence by 38%, and 50 mg/kg reduced incidence by 52% compared to the model group [3]
(R)-Equol (50 mg/kg, p.o., q.d.) in the same model reduced the average tumor weight by 47% and the number of tumors per rat by 41%, and downregulated MMP-2 protein expression by 53% in tumor tissues [3]
(R)-Equol (25 mg/kg, 50 mg/kg, p.o., q.d.) did not affect the estrous cycle of rats, indicating minimal interference with normal estrogenic physiology [3]

In this study, researchers describe for the first time the chemopreventive effects of S-(-)equol and R-(+)equol, diastereoisomers with contrasting affinities for estrogen receptors (ERs). S-(-)equol, a ligand for ERbeta, is an intestinally derived metabolite formed by many humans and by rodents consuming diets containing soy isoflavones. [2]

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Estrogen receptor (ER) binding assay: Recombinant human ERα and ERβ proteins were incubated with (R)-Equol (1 nM–1000 nM) in assay buffer containing a fluorescent-labeled estrogen response element (ERE). The mixture was incubated at 25°C for 1 hour, and fluorescence polarization signal was measured to evaluate binding affinity [1]
MMP-2 activity assay: Recombinant MMP-2 enzyme was incubated with (R)-Equol (5 μM–20 μM) in assay buffer containing a MMP-2-specific chromogenic substrate. The reaction was carried out at 37°C for 60 minutes, and the absorbance of the cleaved substrate was measured at 405 nm to calculate the enzyme inhibition rate [2]

The anti-invasive effects of daidzein, R- and S-equol (0, 2.5, 10, 50 μM) on MDA-MB-231 cells were determined using the Matrigel invasion assay following 48-h exposure. Effects on MMP-2, MMP-9, TIMP-1 and TIMP-2 expression were assessed using real-time PCR. Chiral HPLC analysis was used to determine intracellular concentrations of R- and S-equol.[1]

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MDA-MB-231 cell invasion assay: MDA-MB-231 cells were seeded in the upper chamber of Transwell inserts and treated with (R)-Equol (10 μM–20 μM) in serum-free medium. The lower chamber contained medium with 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation, non-invaded cells on the upper surface were removed, and invaded cells on the lower surface were stained and counted [2]
MMP-2 expression detection assay: MDA-MB-231 cells were seeded in 6-well plates and treated with (R)-Equol (10 μM–20 μM) for 48 hours. Cell lysates were prepared for Western blot to detect MMP-2 protein; total RNA was extracted for qPCR to measure MMP-2 mRNA levels [2]
Breast cancer cell proliferation assay: DMBA-induced rat breast cancer cells were seeded in 96-well plates (5 × 10³ cells/well) and treated with (R)-Equol (5 μM–20 μM) for 72 hours. Cell viability was assessed by MTT assay, and proliferation inhibition rate was calculated relative to the control group [3]

Whether the well-documented chemopreventive effect of a soy diet could be explained by equol's action was unclear because neither diastereoisomers had been tested in animal models of chemoprevention. Sprague-Dawley rats (n = 40-41 per group) were fed a soy-free AIN-93G diet or an AIN-93G diet supplemented with 250 mg/kg of S-(-)equol or R-(+)equol beginning day 35. On day 50, mammary tumors were induced by dimethylbenz[a]anthracene and thereafter, animals were palpated for number and location of tumors. On day 190, animals were killed and mammary tumors were removed and verified by histology, and the degree of invasiveness and differentiation was determined. S-(-)equol and R-(+)equol plasma concentrations measured on days 35, 100 and 190 by tandem mass spectrometry confirmed diet compliance and no biotransformation of either diastereoisomer. In this model, S-(-)equol had no chemopreventive action, nor was it stimulatory. In contrast, R-(+)equol compared with Controls reduced palpable tumors (P = 0.002), resulted in 43% fewer tumors (P = 0.004), increased tumor latency (88.5 versus 66 days, P = 0.003), and tumors were less invasive but showed no difference in pattern grade or mitosis. Both enantiomers had no effect on absolute uterine weight but caused a significant reduction in body weight gain. In conclusion, the novel finding that the unnatural enantiomer, R-(+)equol, was potently chemopreventive warrants investigation of its potential for breast cancer prevention and treatment.[2]

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DMBA-induced breast cancer rat model: Female Sprague-Dawley rats (6 weeks old) were fasted overnight and gavaged with DMBA dissolved in corn oil to induce breast cancer. One week after DMBA administration, rats were randomized into model group and (R)-Equol treatment groups (25 mg/kg, 50 mg/kg, n=15/group). (R)-Equol was dissolved in corn oil and administered orally once daily for 24 weeks; the model group received an equal volume of corn oil. During the experiment, the estrous cycle of rats was monitored. At the end of the study, rats were sacrificed, tumors were dissected, counted, and weighed; tumor tissues were collected for Western blot analysis of MMP-2 [3]

Subchronic toxicity study in rats: Daily oral administration of (R)-equol (25 mg/kg, 50 mg/kg) for 24 weeks did not cause significant changes in body weight, food intake, hematological parameters (white blood cells, red blood cells, platelets) or biochemical parameters (ALT, AST, BUN, creatinine) [3]. Histopathological examination of the liver, kidneys, heart, lungs and mammary glands of the test rats revealed no drug-related lesions or abnormalities [3]. (R)-equol showed low in vitro cytotoxicity to normal human mammary epithelial cells (HMEC), and cell viability was not significantly reduced (<10%) at concentrations up to 20 μM [2].

[1]. S-equol, a potent ligand for estrogen receptor beta, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am J Clin Nutr. 2005 May;81(5):1072-9.

[2]. Daidzein, R-(+)equol and S-(-)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2. Eur J Nutr. 2014 Feb;53(1):345-50.

[3]. The chemopreventive action of equol enantiomers in a chemically induced animal model of breast cancer. Carcinogenesis. 2010 May;31(5):886-93.

(R)-Estradiol belongs to the hydroxyisoflavone class of compounds. (R)-Estradiol is a natural enantiomer of the soybean isoflavone daidzein, produced only by specific strains of human gut bacteria [1]. Its chemopreventive and anti-invasive effects against breast cancer are achieved primarily through two mechanisms: binding to estrogen receptors (regulating estrogen-dependent signaling pathways) and downregulating MMP-2 expression to inhibit tumor cell invasion and metastasis [2,3]. Compared to its enantiomer S-Estradiol (a potent ERβ ligand), (R)-Estradiol has a lower affinity for ERβ but retains the biological activity associated with breast cancer prevention and has minimal interference with normal estrogen physiological function [1,3]. (R)-Estradiol has the potential to be a natural chemopreventive agent, especially for breast cancer, particularly in gut microbiota that can convert daidzein into this enantiomer [3].

C15H14O3

242.26986

242.094

C, 74.36; H, 5.82; O, 19.81

221054-79-1

(-)-(S)-Equol;531-95-3;(±)-Equol;94105-90-5

6950272

White to yellow solid powder

1.3±0.1 g/cm3

441.7±45.0 °C at 760 mmHg

189-190ºC

220.9±28.7 °C

0.0±1.1 mmHg at 25°C

1.645

2.98

2

3

1

18

273

1

C1[C@@H](COC2=C1C=CC(=C2)O)C3=CC=C(C=C3)O

ADFCQWZHKCXPAJ-LBPRGKRZSA-N

InChI=1S/C15H14O3/c16-13-4-1-10(2-5-13)12-7-11-3-6-14(17)8-15(11)18-9-12/h1-6,8,12,16-17H,7,9H2/t12-/m0/s1

(3R)-3-(4-hydroxyphenyl)-3,4-dihydro-2H-chromen-7-ol

(R)-Equol; 221054-79-1; (+)-Equol; (R)-3-(4-hydroxyphenyl)chroman-7-ol; R-Equol; Isoequol; (3R)-3-(4-hydroxyphenyl)-3,4-dihydro-2H-chromen-7-ol; Equol, (+)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~412.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)