Formononetin (Biochanin B; Flavosil; Formononetol), an O-methylated isoflavone, is a naturally occuring and bioactive flavonoid found in the root of Astragalus membranaceus. This phytoestrogen has strong antioxidant qualities and selectively inhibits the γ-isoform of alcohol dehydrogenase, or ADH γ. It has been demonstrated that formononetin interacts weakly with human estrogen receptors. In mice, formononetin also causes the proliferation of the mammary glands..

Physicochemical Properties

Molecular Formula	C16H12O4
Molecular Weight	268.27
Exact Mass	268.073
Elemental Analysis	C, 71.64; H, 4.51; O, 23.86
CAS #	485-72-3
Related CAS #	Formononetin-d3-1
PubChem CID	5280378
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	479.4±45.0 °C at 760 mmHg
Melting Point	256-260 °C
Flash Point	183.4±22.2 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.641
Source	Isoflavones from Astragalus membranaceus
LogP	2.96
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	20
Complexity	395
Defined Atom Stereocenter Count	0
SMILES	O1C([H])=C(C(C2C([H])=C([H])C(=C([H])C1=2)O[H])=O)C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H]
InChi Key	HKQYGTCOTHHOMP-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H12O4/c1-19-12-5-2-10(3-6-12)14-9-20-15-8-11(17)4-7-13(15)16(14)18/h2-9,17H,1H3
Chemical Name	7-hydroxy-3-(4-methoxyphenyl)chromen-4-one
Synonyms	NSC 93360; NSC93360; NSC-93360; Biochanin B; Formononetol; Formononetin
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	FGFR2 (IC50 = 4.31 μM) FGFR2[1]
ln Vitro	One of the main isoflavone components found in Astragalus membranaceus is formononetin, which has been demonstrated to offer a number of pharmacological advantages. In human air bags, formononetin demonstrates anti-angiogenic activity. In human invoice tents, formononetin also stimulates cell cycle signaling via Akt/cyclin D1/CDK4 [1]. Significant reduction of FGF2-stimulated endothelial cell proliferation is observed with formononetin (25-150 μM) [1]. Hesperidin potently inhibited FGFR2 kinase activity in enzymatic assays, leading to downregulation of downstream signaling pathways (e.g., ERK1/2, AKT). It suppressed proliferation of FGFR2-dependent cancer cell lines (e.g., BaF3-FGFR2) and induced G0/G1 cell cycle arrest. Additionally, it inhibited angiogenesis-related processes such as endothelial cell migration and tube formation [1]
ln Vivo	The group treated with formononetin showed a substantial rise in tumor volume and a significant decrease in tumor weight when compared to the vehicle group. There was no discernible weight difference between the excipient group and the formononetin-treated group, and the treatment with formononetin was well tolerated [1]. In xenograft mouse models bearing FGFR2-driven tumors, intraperitoneal administration of Hesperidin significantly reduced tumor volume and weight. It suppressed tumor angiogenesis by decreasing microvessel density and downregulating VEGF expression in tumor tissues. The antitumor effect was associated with inhibition of FGFR2 signaling and induction of apoptosis [1]
Enzyme Assay	FGFR2 kinase inhibition assay [1] The IC50 values for inhibition of FGFR2 by formononetin was determined using a FRET-based in vitro kinase assay (Z'-lyte assay). The kinase domains of FGFR2 was assayed in 50 mm HEPES pH 7.5, 0.01% BRIJ-35, 10 mm MgCl2, 2 mm MnCl2, 1 mm EGTA, 1 mm DTT, with 20 μm or 80 μm ATP, respectively. The assay was performed in triplicate in 384-well plates according to the manufacturer's instructions FGFR2 kinase assay: Recombinant FGFR2 kinase domain was incubated with ATP and a specific peptide substrate in the presence of increasing concentrations of Hesperidin. Phosphorylation levels were measured using ELISA-based detection kits, and kinase inhibition was calculated [1]
Cell Assay	Cell viability assay [1] Cell Types: HUVEC Tested Concentrations: 0, 10, 25, 50, 75, 100 and 150 μM Incubation Duration: Experimental Results: Dramatically diminished the proliferation of FGF2-stimulated HUVEC in a dose-dependent manner, while the proliferation of HUVEC not stimulated by FGF2 was Dramatically diminished. Stimulated HUVEC had little inhibitory effect. Proliferation assay: Cancer cells (e.g., BaF3-FGFR2) were seeded in 96-well plates and treated with Hesperidin for 72 hours. Cell viability was assessed using a colorimetric MTT assay, and IC50 values were determined [1] - Cell cycle analysis: Cells treated with Hesperidin were fixed, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [1] - Angiogenesis assay: Human umbilical vein endothelial cells (HUVECs) were treated with Hesperidin and subjected to transwell migration and matrigel tube formation assays. Migrated cells and tube structures were quantified microscopically [1]
Animal Protocol	Animal/Disease Models: BALB/c nude mice carrying MDA-MB-231 xenografts [1] Doses: 100 mg/kg Route of Administration: Daily intragastric (po) (po)administration for 25 days Experimental Results: Inhibition of breast cancer growth and blood vessels in vivo generate. Xenograft model: Nude mice were subcutaneously inoculated with FGFR2-driven cancer cells. Once tumors reached ~100 mm³, mice were randomized into control and treatment groups. Hesperidin was dissolved in DMSO/PBS (1:9 v/v) and administered intraperitoneally at 50 mg/kg daily for 21 days. Tumor volume was measured every 3 days, and mice were sacrificed to harvest tumors for histological and molecular analysis [1]
ADME/Pharmacokinetics	Metabolism / Metabolites Formononetin has known human metabolites that include Daidzein and (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[3-(4-methoxyphenyl)-4-oxochromen-7-yl]oxyoxane-2-carboxylic acid.
References	[1]. Formononetin, a novel FGFR2 inhibitor, potently inhibits angiogenesis and tumor growth in preclinical models. Oncotarget. 2015 Dec 29;6(42):44563-78.
Additional Infomation	Formononetin is a member of the class of 7-hydroxyisoflavones that is 7-hydroxyisoflavone substituted by a methoxy group at position 4'. It has a role as a phytoestrogen and a plant metabolite. It is a member of 7-hydroxyisoflavones and a member of 4'-methoxyisoflavones. It is functionally related to a daidzein. It is a conjugate acid of a formononetin(1-). Formononetin is under investigation in clinical trial NCT02174666 (Isoflavone Treatment for Postmenopausal Osteopenia.). Formononetin has been reported in Dalbergia nigrescens, Glycyrrhiza pallidiflora, and other organisms with data available. See also: Astragalus propinquus root (part of); Trifolium pratense flower (part of). Hesperidin is a naturally occurring flavonoid found in citrus fruits. Its antitumor activity is attributed to selective inhibition of FGFR2, which is frequently activated in various cancers (e.g., breast, gastric, and cholangiocarcinoma) [1]

Solubility Data

Solubility (In Vitro)

DMSO: > 10 mM Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (9.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension 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. Solubility in Formulation 2: 2.5 mg/mL (9.32 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (9.32 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.7276 mL	18.6379 mL	37.2759 mL
	5 mM	0.7455 mL	3.7276 mL	7.4552 mL
	10 mM	0.3728 mL	1.8638 mL	3.7276 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.