Estrogen receptor alpha (ERα). Genistin downregulates ERα protein expression, inhibits its nuclear translocation and DNA binding activity, thereby suppressing ERα signaling in breast cancer cells. No specific IC₅₀ or Kᵢ values were reported. [3]
Genistin itself has weak direct biological activity, but its aglycone metabolite genistein is a well-characterized multi-target agent. Genistein inhibits protein-tyrosine kinases (including EGFR) and topoisomerase II (DNA topoisomerase, ATP-hydrolysing). It also binds to estrogen receptors (ERα and ERβ) as a phytoestrogen with selective estrogen receptor modulator (SERM) properties. Competitive binding assays using recombinant human ERα have demonstrated that genistein competes with 17β-estradiol for receptor binding. Additionally, genistein acts as an autophagy inducer. No specific IC₅₀ values for genistin itself were reported in the available literature.

Anti-adipogenic and anti-lipogenic activity in 3T3-L1 cells: In 3T3-L1 preadipocytes, genistin (25–100 μM, 48 h) showed no cytotoxicity. During adipocyte differentiation, genistin (50 and 100 μM) significantly inhibited lipid accumulation (21.7% and 69.2% reduction, respectively). It dose-dependently suppressed the protein and mRNA expression of adipogenic transcription factors (C/EBPα, PPARγ, aP2/FABP4) and lipogenic enzymes (ACL, ACC1, FAS). Genistin also activated AMPKα phosphorylation and inhibited SREBP-1c mRNA expression. [1]
Anti-cancer activity in breast cancer cells: In MCF-7 (ERα-positive) breast cancer cells, genistin (50–150 μM, 24 h) exhibited concentration-dependent cytotoxicity, with stronger effects than in MDA-MB-231 (ERα-negative) cells. In MCF-7 cells, genistin (100 μM) induced apoptosis with sub-G1 phase accumulation (21.8–42.1% at 24–48 h) and TUNEL-positive cells (10.7–20.4% at 48 h). It activated caspase-8, caspase-9, and PARP cleavage, and downregulated anti-apoptotic (Bcl-2, Bcl-xL, Survivin), proliferative (Cyclin D1, COX-2), angiogenic (VEGF), and metastatic (MMP-9) proteins and genes. [3]
Mechanism of ERα modulation in breast cancer cells: In MCF-7 cells, genistin (50–150 μM, 24 h) decreased ERα protein levels in nuclear extracts while increasing them in cytoplasmic extracts, indicating inhibition of nuclear translocation. ERα-DNA binding activity was suppressed in a concentration- and time-dependent manner. Immunocytochemistry confirmed reduced nuclear ERα localization. [3]
Effect on ERα knockdown and overexpression: In ERα siRNA-transfected MCF-7 cells, genistin (150 μM)-induced apoptosis and PARP cleavage were significantly reduced, and cell viability increased. Conversely, in ERα-overexpressing MDA-MB-231 cells, genistin (150 μM) enhanced apoptosis and PARP cleavage, and reduced cell viability, confirming that genistin’s pro-apoptotic effects are mediated through ERα signaling. [3]
ERα is negatively regulated by genistin. In breast cancer cells, genistin also efficiently inhibits ER nuclear translocation and DNA binding activity. Furthermore, GS efficiently reduces the levels of oncogene markers and promotes apoptosis in MCF-7 cells [3].

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The biological activity of genistin is primarily mediated through its aglycone metabolite genistein after deglycosylation. Genistein inhibits serum-stimulated growth of ER+ breast cancer cells (MCF-7 and T47D), with IC₅₀ values of 7.6 and 8.7 μg/mL by dye exclusion assay, and 9.4 and 7 μg/mL by MTT assay, respectively. At concentrations up to 20 μg/mL over an 8-hour incubation period, genistein does not alter mitochondrial MTT reduction. Genistein also induces G2 phase arrest in human and murine cell lines. Competitive binding assays to recombinant human ERα show that genistein effectively displaces [³H]estradiol from the receptor, with binding affinity influenced by sulfation at the 4′ and 7 positions.

The in vivo activity of genistin is attributed to its metabolite genistein. In an induced breast cancer rat model (DMBA-induced), administration of genistein at 0.2 mg/kg body weight for 100 days affected renal mineral composition, demonstrating systemic bioavailability and biological activity. In studies on lipid metabolism, genistein administration in rats fed a high-fat diet significantly reduced hepatic PPARγ protein expression compared to high-fat diet alone or high-fat diet with BPA groups. In ovariectomized hamsters, genistin administration increased appetite and body weight, consistent with its estrogenic activity.

Competitive estrogen receptor binding assay: The binding affinity of genistein (the active metabolite of genistin) to recombinant human estrogen receptor α (ERα) was determined using a competitive radioligand binding assay. In single-point competitive binding assays, 0.8 nM of [2,4,6,7-³H]estradiol was incubated with 0.8 nM recombinant receptor protein plus varying molar excess of unlabelled test compounds (including genistein and its sulfated metabolites). The ability of the test compound to displace the radiolabeled estradiol from the receptor was measured. Results showed that genistein effectively competed with estradiol for ERα binding, with the 4′-sulphate and 7-sulphate metabolites exhibiting different binding profiles. M3 muscarinic receptor binding assay: Genistin has also been used as a standard in receptor binding studies, including assays involving the M3 muscarinic acetylcholine receptor, where commercially obtained genistin (purity >98%, HPLC grade) served as a reference standard.

Cell proliferation/viability (MTT): 3T3-L1 preadipocytes (文献[1]) or MCF-7 and MDA-MB-231 breast cancer cells (文献[3]) were seeded in 96-well plates and treated with various concentrations of genistin (0–150 μM) for 24–48 h. MTT solution was added, incubated for 2 h, and formazan was dissolved in lysis buffer. Absorbance was measured at 570 nm to calculate cell viability. [1,3]
Real-time cell proliferation analysis (RTCA): MCF-7 and MDA-MB-231 cells were seeded in E-plates, and after 20 h, treated with genistin (50 or 100 μM). Cell index was monitored for 72 h using the xCELLigence system. [3]
Lipid accumulation (Oil Red O staining): 3T3-L1 cells were differentiated in MDI medium with genistin (25–100 μM) for 7 days. Cells were fixed, stained with Oil Red O, and dye was extracted with isopropanol. Absorbance at 500 nm was measured to quantify lipid accumulation. [1]
Cell cycle analysis (flow cytometry): MCF-7 cells treated with genistin (50–150 μM, 24–48 h) were fixed in ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine sub-G1 phase accumulation. [3]
Apoptosis detection (TUNEL assay): MCF-7 cells treated with genistin (50–150 μM, 48 h) were fixed, permeabilized, and stained with TUNEL reaction mixture. DNA damage was analyzed by flow cytometry. [3]
Western blot analysis: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against C/EBPα, PPARγ, FABP4, AMPKα, p-AMPKα, ACL, ACC1, FAS, ERα, cleaved PARP, cleaved caspase-8/9, Bcl-2, Bcl-xL, Survivin, Cyclin D1, COX-2, VEGF, MMP-9, and GAPDH/β-actin. [1,3]
RT-PCR: Total RNA was extracted using TRIzol, reverse transcribed to cDNA, and amplified with specific primers for C/EBPα, PPARγ, aP2, SREBP-1c, ACC1, ACL, FAS, Bcl-2, Bcl-xL, Cyclin D1, MMP-9, and ERα. GAPDH was used as an internal control. [1,3]
Electrophoretic mobility shift assay (EMSA): Nuclear extracts from MCF-7 cells treated with genistin (50–150 μM, 6–24 h) were incubated with a biotinylated ERα oligonucleotide probe. Complexes were separated on native polyacrylamide gels, transferred to nylon membranes, cross-linked, and detected by chemiluminescence. [3]
Immunocytochemistry: MCF-7 cells treated with genistin (100 μM, 24 h) were fixed, permeabilized, blocked, and stained with ERα antibody. Localization was visualized by confocal microscopy. [3]
Cell proliferation assay (MTT and dye exclusion): Human breast cancer cells (MCF-7 and T47D, ER+) were cultured and treated with varying concentrations of genistein. Cell viability was assessed using MTT reduction assay and dye exclusion assay. IC₅₀ values were calculated as 7.6 μg/mL (MCF-7, dye exclusion), 8.7 μg/mL (T47D, dye exclusion), 9.4 μg/mL (MCF-7, MTT), and 7 μg/mL (T47D, MTT). Colorectal cancer cell studies: Genistin (along with other isoflavones) has been used as a research tool in studies involving human colorectal cancer cell lines including HCT-116, SW480, HT-29, and LoVo, as well as HCT-116 p53−/− cells.

Rat breast cancer model: Female Sprague-Dawley rats (40 days old) were divided into experimental groups. Mammary gland cancer was induced using DMBA (7,12-dimethyl-1,2-benz(a)anthracene). Genistein was administered at a dose of 0.2 mg/kg body weight per day (0.1 mg/mL, twice daily) in feed for a duration of 100 days. Three forms of genistein were tested: nanoparticles (size: 92±41 nm), microparticles (size: 587±83 nm), and macroparticles (normal, classical form). At the end of the experiment, kidneys were collected for analysis of elemental composition. Rat PBPK model development: An oral rat PBPK (physiologically based pharmacokinetic) model for genistein was built using PK-Sim® software based on in vitro ADME input data. The model was qualified as it predicted plasma concentration values within 2-fold of measured in vivo PK values.

Metabolic profiling in rats: Following oral administration of genistin (350 mg/kg) to SD rats, a total of 64 metabolites were identified in urine and plasma using UHPLC-LTQ-Orbitrap mass spectrometry. Major metabolic pathways included methylation, hydrogenation, hydroxylation, glucosylation, glucuronidation, sulfonation, acetylation, ring-cleavage, and composite reactions. Metabolites were identified using multiple metabolite templates (genistin, genistein, daidzin, daidzein, puerarin, dihydrogenistein, tetrahydrogenistein, dihydrodaidzein, equol, and O-desmethylangolensin). [2]
Metabolite detection: Biological samples were pretreated by solid-phase extraction (SPE) using C18 cartridges. Plasma and urine were collected at multiple time points (0.5, 1, 1.5, 2, 4 h for plasma; 0–24 h for urine) after oral administration. Chromatographic separation was performed on a C18 column with a gradient of acetonitrile and 0.1% formic acid. MS detection was conducted in positive and negative ion modes with a resolution of 30,000. [2]
The pharmacokinetics of genistin are closely tied to its aglycone metabolite genistein, as genistin is rapidly hydrolyzed to genistein in the intestine. Genistein exhibits low oral bioavailability, which is the major challenge for its development as a chemopreventive agent. Following oral administration, genistein undergoes extensive first-pass metabolism, including glucuronidation and sulfation in the intestine and liver. The compound is a substrate for the breast cancer resistance protein (BCRP) transporter, which limits its oral absorption. Human PBPK models for genistein have been developed using a bottom-up approach, incorporating human-specific physiological parameters and human in vitro ADME data. These models were used to predict plasma concentrations from the in vivo NOAEL (No Observed Adverse Effect Level) for genistein. Intrinsic hepatic clearance and unbound fraction in plasma were identified as sensitive parameters impacting predicted Cmax values. Sensitivity and uncertainty analyses indicated moderate confidence in the developed PBPK models.

Cytotoxicity in 3T3-L1 cells: Genistin at concentrations up to 100 μM for 48 h showed no significant cytotoxicity in 3T3-L1 preadipocytes, as determined by CCK-8 assay and live/dead cell imaging. [1]
Cytotoxicity in breast cancer cells: Genistin exhibited concentration-dependent cytotoxicity in MCF-7 cells (IC₅₀ not specified), with greater sensitivity in ERα-positive cells compared to ERα-negative MDA-MB-231 cells. [3]
Acute toxicity: In mice, the intraperitoneal LD₅₀ of genistin is reported to be >2 mg/kg. Developmental and reproductive toxicity: Genistein (the active metabolite) is listed as a possible human reproductive or developmental toxin by the European Chemicals Agency (ECHA), with a LOW_MODERATE concern rating for developmental and reproductive toxicity according to EWG Skin Deep. General safety: The compound is classified with low cancer concern and low allergies/immunotoxicity concerns. Safety precautions include avoiding inhalation of dust and contact with skin and eyes.

[1]. Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent. Molecules. 2020;25(9):2042.

[2]. A Comprehensive Screening and Identification of Genistin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-HRMS Analysis. Molecules. 2018;23(8):1862.

[3]. Genistin attenuates cellular growth and promotes apoptotic cell death breast cancer cells through modulation of ERalpha signaling pathway [published online ahead of print, 2020 Oct 16]. Life Sci. 2020;263:118594.

Genistein 7-O-β-D-glucoside is a 7-hydroxyisoflavone 7-O-β-D-glucoside. It is functionally related to genistein. It is the conjugate acid of genistein 7-O-β-D-glucoside (1-). Genistein has been reported in sage, soybeans, and other organisms with relevant data.

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Source and natural occurrence: Genistin is naturally abundant in soybeans, tofu, fava beans, kudzu, and lupin. It is one of the primary isoflavone glycosides in soy products. Metabolism and bioactivation: Genistin is hydrolyzed by intestinal β-glucosidases to release genistein, the bioactive aglycone. This conversion is essential for its biological effects, as the glycosylated form has limited direct activity. Clinical applications: Genistein has been investigated in clinical trials as an alternative to classical hormone therapy to help prevent cardiovascular disease in postmenopausal women. It also exhibits antihelmintic activity and has been identified as the active ingredient in Felmingia vestita, a plant traditionally used against worms. Regulatory status: Genistin is used as a reference standard for analytical purposes and is not approved as a therapeutic drug. The EWG Skin Deep database indicates low use restrictions for this ingredient.

C21H20O10

432.38

432.105

C, 58.34; H, 4.66; O, 37.00

529-59-9

5281377

White to off-white solid powder

1.6±0.1 g/cm3

788.9±60.0 °C at 760 mmHg

254ºC

280.7±26.4 °C

0.0±2.9 mmHg at 25°C

1.717

0.79

6

10

4

31

675

5

C1=CC(=CC=C1C2=COC3=CC(=CC(=C3C2=O)O)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O

ZCOLJUOHXJRHDI-CMWLGVBASA-N

InChI=1S/C21H20O10/c22-7-15-18(26)19(27)20(28)21(31-15)30-11-5-13(24)16-14(6-11)29-8-12(17(16)25)9-1-3-10(23)4-2-9/h1-6,8,15,18-24,26-28H,7H2/t15-,18-,19+,20-,21-/m1/s1

5-hydroxy-3-(4-hydroxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one

CS 4240; Genistin; 529-59-9; Genistine; Genistoside; Genistein 7-glucoside; CS-4240; Glucosyl-7-genistein; NSC 5112; NSC5112; NSC-5112; Genistein glucoside

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 (~231.28 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 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 (5.78 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 (5.78 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.)