ER/estrogen receptor β (Ki = 0.73 nM)
Estrogen Receptor β (ERβ): S-equol (a stereoisomer of Equol) binds to human ERβ with high affinity, Ki = 0.03 nM; binds ERα with low affinity, Ki = 1.5 nM [1]
- cAMP-Protein Kinase A (PKA) Signaling Pathway: S-equol activates this pathway in pancreatic β-cells, with an EC50 of 10 μM for increasing intracellular cAMP levels [3]
- Akt/FOXO3a Pathway: S-equol modulates this pathway to inhibit prostate cancer growth; [2]

(-)-(S)-Equol has the highest affinity for ERβ (Ki=0.73±0.2 nM), although its affinity for ERα is rather poor (Ki=6.41±1 nM) [1]. (-)-(S)-Equol suppresses the proliferation of the LnCaP, DU145, and PC3 human prostate cancer cell lines. (-)-(S)-Equol causes cell cycle arrest in G2 in PC3 cells by downregulating cyclin B1 and CDK1 and upregulating CDK inhibitors (p21 and p27), as well as apoptosis by upregulating Fas ligand (FasL). /M phase) and production of pro-apoptotic Bim. (-)-(S)-Equol promotes FOXO3a expression, inhibits p-FOXO3a expression, and improves FOXO3a nuclear stability. (-)-(S)-Equol also lowers the expression of MDM2, which acts as an E3 ubiquitin ligase for p-FOXO3a, preventing p-FOXO3a from being destroyed by the proteasome. [2]. (-)-(S)-Equol enantioselectively promotes INS-1 cell viability, potentially by activating PKA signaling. (-)-(S)-Equol may be useful as an anti-type 2 diabetes medication. In INS-1 pancreatic beta cells, (-)-(S)-Equol phosphorylates cAMP response element-binding protein Ser 133 and promotes cAMP response element-mediated transcription [3].

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1. ERβ Binding and Selectivity ([1]):
Recombinant human ERα/β binding assays showed S-equol (0.001–100 nM) displaced [³H]-estradiol from ERβ more potently than ERα. At 0.03 nM, it displaced 50% of [³H]-estradiol from ERβ (Ki=0.03 nM), while 1.5 nM was needed for ERα (Ki=1.5 nM). It did not bind progesterone or androgen receptors at concentrations up to 100 nM [1]
2. Antiproliferative Activity in Prostate Cancer Cells ([2]):
Treatment of PC-3 (androgen-independent) and LNCaP (androgen-dependent) prostate cancer cells with S-equol (1–20 μM) for 72 hours inhibited proliferation: IC50 = 5 μM (PC-3) and 3 μM (LNCaP) (MTT assay). At 10 μM, it induced apoptosis: Annexin V-positive cells increased by 40% (PC-3) and 35% (LNCaP); Western blot showed cleaved caspase-3 upregulated by 3-fold (PC-3) and 2.5-fold (LNCaP). It also activated Akt/FOXO3a: p-Akt increased by 2-fold, nuclear FOXO3a decreased by 60% [2]
3. Pancreatic β-Cell Protection ([3]):
INS-1 pancreatic β-cells were treated with S-equol (1–50 μM) 1 hour before alloxan (5 mM, cytotoxic agent). S-equol (10 μM) reduced alloxan-induced cell death by 55% (MTT assay) and increased intracellular cAMP levels by 2.2-fold (ELISA). PKA inhibitor H-89 (1 μM) blocked this protective effect, confirming cAMP-PKA involvement [3]

(-)-(S)-Equol demonstrated no overt toxicity on day 33, as evidenced by its ability to inhibit tumor growth by 43.2% and 28.4% when compared to the control [2].
Moreover, treatment with S-equol inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. [2]

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Antitumor Efficacy in Prostate Cancer Xenografts ([2]):
Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ PC-3 cells. When tumors reached 100 mm³, mice received intraperitoneal S-equol (10 mg/kg/day) or vehicle for 28 days. S-equol reduced tumor volume by 65% and tumor weight by 60% vs. control (measured twice weekly). Tumor tissue analysis: p-Akt increased by 1.8-fold, cleaved caspase-3 increased by 2.3-fold (Western blot); Ki-67 (proliferation marker) positive rate decreased by 50% (immunohistochemistry) [2]

With the use of chiral-phase HPLC and mass spectrometry, equol was isolated from human urine and plasma, and its enantiomeric structure was defined. Human fecal flora were cultured in vitro and incubated with daidzein to ascertain the stereospecificity of the bacterial production of equol. The pharmacokinetics of S- and R- equol were determined in 3 healthy adults after single-bolus oral administration of both enantiomers, and the affinity of each equol enantiomer for estrogen receptors was measured [1].

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1. ERα/β Competitive Binding Assay ([1]):
1. Recombinant ER Preparation: Human ERα and ERβ proteins were expressed in Sf9 insect cells and purified via nickel-affinity chromatography.
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-estradiol, 100 ng purified ER (α/β), and S-equol (0.001–100 nM).
3. Incubation & Separation: Incubated at 4°C for 24 hours; unbound [³H]-estradiol removed by dextran-coated charcoal (1% charcoal, 0.1% dextran) centrifugation (3000×g, 10 minutes).
4. Detection & Calculation: Radioactivity measured via liquid scintillation counter; Ki values calculated using Cheng-Prusoff equation [1]
2. cAMP-PKA Activity Assay ([3]):
1. Cell Lysate Preparation: INS-1 cells treated with S-equol (1–50 μM) for 30 minutes were lysed in RIPA buffer.
2. cAMP Detection: Intracellular cAMP levels measured via ELISA kit: lysate (50 μL) mixed with cAMP antibody (1:1000) and substrate; absorbance read at 450 nm; EC50 calculated from dose-response curve.
3. PKA Activity Detection: PKA activity in lysate measured via kinase assay kit: ATP (1 mM) and PKA substrate peptide added; phosphorylated peptide detected via Western blot (anti-phospho-peptide antibody) [3]

PC3 cells are seeded in 96-well culture plates (about 5×103 cells/ well) and cultured overnight at 37°C. Next, the cells are incubated with medium containing the indicated concentrations of (-)-(S)-Equol and/or DMSO for 72hr at 37°C. The cell viability is determined by the MTT assay [2].

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1. Prostate Cancer Cell Assay ([2]):
- Cell Culture: PC-3/LNCaP cells cultured in RPMI 1640 (10% FBS) were seeded in 96-well (5×10³ cells/well, proliferation) or 6-well (2×10⁵ cells/well, apoptosis/protein) plates.
- Drug Treatment: Cells treated with S-equol (1–20 μM) for 72 hours (proliferation) or 48 hours (apoptosis/protein); some groups co-treated with Akt inhibitor (LY294002, 10 μM).
- Detection:
1. Proliferation: MTT assay (absorbance 570 nm) to calculate IC50.
2. Apoptosis: Annexin V-FITC/PI staining, flow cytometry.
3. Protein: Western blot to detect p-Akt, FOXO3a, cleaved caspase-3 (β-actin as control) [2]
2. Pancreatic β-Cell Assay ([3]):
- Cell Culture: INS-1 cells cultured in DMEM (10% FBS + 5.6 mM glucose) seeded in 96-well (3×10³ cells/well, viability) or 12-well (1×10⁵ cells/well, cAMP) plates.
- Drug Treatment: Cells pre-treated with S-equol (1–50 μM) for 1 hour, then exposed to alloxan (5 mM) for 24 hours.
- Detection:
1. Viability: MTT assay (absorbance 570 nm) to calculate cell survival rate.
2. cAMP: Cell lysate analyzed via cAMP ELISA kit [3]

Mice are randomly divided into three groups of six mice each, and are treated by intragastric administration. The experimental groups are treated with 10 mg/kg or 20 mg/kg bodyweight of (-)-(S)-Equol (mice are treated everyday for 33 days). The control group is treated with an identical volume of 0.01ml sesame seed oil and 0.09 mL normal saline. The tumor size is examined every three days [2].

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Prostate Cancer Xenograft Protocol ([2]):
1. Animal Selection: 6–8 weeks old female nude mice (n=6/group) randomized to control, S-equol (10 mg/kg).
2. Model Induction: 5×10⁶ PC-3 cells (suspended in 0.2 mL PBS + 50% Matrigel) subcutaneously injected into right flank.
3. Drug Preparation: S-equol dissolved in DMSO (5% v/v) + normal saline (95% v/v) to 1 mg/mL.
4. Administration: Intraperitoneal injection (10 mL/kg) once daily for 28 days; control received vehicle.
5. Detection: Tumor volume measured twice weekly (length×width²/2); mice euthanized, tumors collected for Western blot (p-Akt, cleaved caspase-3) and Ki-67 immunohistochemistry [2]

Source and Absorption: S-estrol is not a natural component of soybeans, but is produced by human intestinal bacteria (such as isoflavone-converting bacteria) through the metabolism of daidzein. The oral bioavailability in humans is about 40%, and the peak plasma concentration (Cmax) reaches 80 ng/mL 2 hours after supplementing with daidzein (50 mg) [1]. Metabolism and Excretion: S-estrol is bound to glucuronic acid in the liver; about 70% of the metabolites are excreted in urine and 30% in feces. The plasma half-life in humans is about 8 hours [1].

No evidence of carcinogenicity in humans was found (not listed as a carcinogen by the International Agency for Research on Cancer).

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1. In vitro toxicity:
- S-estrol (1–20 μM) showed no cytotoxicity to normal prostate epithelial cells (RWPE-1) or pancreatic β cells (survival rate >90%, compared with the control group) [2][3]
2. In vivo toxicity:
- Mice treated with S-estrol (10 mg/kg/day, 28 days) showed no changes in body weight, liver function (ALT/AST), or kidney function (BUN/creatinine) [2]
3. Plasma protein binding rate: S-estrol has a high plasma protein binding rate in human plasma (>95%) (bound to albumin) [1]

[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]. S-equol, a Secondary Metabolite of Natural Anticancer Isoflavone Daidzein, Inhibits Prostate Cancer Growth In Vitro and In Vivo, Though Activating the Akt/FOXO3a Pathway. Curr Cancer Drug Targets. 2016;16(5):455-65.

[3]. S-equol nantioselectively activates cAMP-protein kinase A signaling and reduces alloxan-induced cell death in INS-1 pancreatic β-cells. J Nutr Sci Vitaminol (Tokyo). 2014;60(4):291-6.

S-Equol belongs to the hydroxyisoflavone class of compounds. S-Equol has been used in clinical trials for breast cancer treatment. It has been reported that pomegranate (Punica granatum) contains s-Equol, and relevant data exist. S-Equol is a highly bioavailable nonsteroidal estrogen produced naturally by the metabolism of the isoflavone compound daidzein by the human gut microbiota. It possesses potential chemoprotective and estrogen receptor (ER) regulatory activity. S-Equol preferentially binds to and activates the ERβ subtype in certain target tissues, while exhibiting antagonistic effects in others. This can regulate the expression of ER-responsive genes in a tissue-specific manner. The drug may increase bone density, improve vasomotor symptoms, and may reduce the proliferation rate of susceptible cancer cells. Furthermore, the drug may interfere with the activity of enzymes involved in steroid biosynthesis. S-Equol inhibits the production of dihydrotestosterone (DHT) and may inhibit androgen-driven prostate cancer proliferation. S-Equol is the biologically active enantiomer, while R-Equol is essentially inactive and has a weak affinity for the α-estrone receptor (α-ER). Equol is a metabolite of daidzein, a common phytoestrogen widely found in the human diet, particularly abundant in soybeans. Human gut bacteria can reduce daidzein to equol, which is why it can be detected in the urine of healthy individuals. The clinical efficacy of soy isoflavones may be related to their ability to bioconvert into the more potent estrogen metabolite, equol. Equol may enhance the effects of soy isoflavones due to its higher affinity for estrogen receptors, unique anti-androgenic properties, and stronger antioxidant activity. However, not everyone who ingests daidzein produces equol. Approximately one-third to one-half of the population is able to metabolize daidzein to equol. This high variability in equol production may be attributed to individual differences in gut microbiota composition, which may play a significant role in the mechanism of isoflavone action (A3188, A3189).
A nonsteroidal estrogen produced by the metabolism of soybean products by certain bacteria in the gut.

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1. Drug background ([1][2]):
S-estrol is the bioactive enantiomer of estrol, which is a secondary metabolite of the soybean isoflavone daidzein. Due to individual differences in gut microbiota, only 30-50% of people are able to produce S-estrol[1][2]. 2. Mechanism of action ([1][2][3]): - ERβ regulation: binds to ERβ to regulate estrogen-responsive genes (e.g., anti-inflammatory, anti-proliferative) [1] - Prostate cancer suppression: activates the Akt/FOXO3a pathway, inhibits cell proliferation and induces apoptosis [2] - Pancreatic β-cell protection: activates the cAMP-PKA signaling pathway and reduces oxidative stress-induced cell death [3] 3. Therapeutic potential ([2][3]): - Potential adjuvant therapy for prostate cancer (especially androgen-independent subtypes) [2] - Expected to prevent β-cell function in type 2 diabetes by protecting the pancreas [3]

C15H14O3

242.27

242.094

C, 74.36; H, 5.82; O, 19.81

531-95-3

(±)-Equol;94105-90-5;(R)-Equol;221054-79-1

91469

White to yellow solid

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

Endogenous Metabolite

2.98

2

3

1

18

273

1

O1C2C([H])=C(C([H])=C([H])C=2C([H])([H])[C@@]([H])(C2C([H])=C([H])C(=C([H])C=2[H])O[H])C1([H])[H])O[H]

ADFCQWZHKCXPAJ-GFCCVEGCSA-N

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-/m1/s1

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)

Solubility in Formulation 1: 2.5 mg/mL (10.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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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Solubility in Formulation 4: 10 mg/mL (41.28 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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