PPAR-γ/α
Estrogen Receptor α (ERα) (Ki = 1.2 μM, determined by ligand binding assay) [5]
- Estrogen Receptor β (ERβ) (Ki = 0.3 μM, determined by ligand binding assay) [5]
- Tyrosine Kinase (IC50 = 25 μM, determined by kinase activity assay) [1]
- Cyclooxygenase-2 (COX-2) (IC50 = 38 μM, determined by COX activity assay) [4]

Daidzein inhibits the expression of the iNOS protein and mRNA as well as the generation of NO in a dose-dependent manner. Additionally, daizenein prevents the activation of another crucial transcription factor for iNOS, signal transducer and activator of transcription 1 (STAT-1). (Source: ) Daidzein (1 μM and 10 μM) significantly increases the amount of DNA, protein, and alkaline phosphatase activity in cells; these increases are approximately 1.4-, 1.5-, and 2.0-fold, respectively.[2] Daidzein (2-50 mM) increases the viability of osteoblasts by about 1.4-fold. The alkaline phosphatase activity and osteocalcin synthesis of osteoblasts are increased by approximately 1.4 and 2.0 times, respectively, by daifzein (2-100 mM). Osteoblast differentiation is stimulated by daizenein at different stages (from osteoprogenitors to terminally differentiated osteoblasts).[3] Calcium contents, deoxyribonucleic acid (DNA), and alkaline phosphatase activity are all markedly increased in bone tissues by daidzein (1 μM and 10 μM). Cycloheximide (1 μM) totally inhibits the rise in calcium content and alkaline phosphatase activity in bone tissues caused by dainzocine.[4]

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Inhibited tyrosine kinase activity: 50 μM Daidzein reduced tyrosine phosphorylation of cellular proteins by ~70% in A431 cells [1]
- Suppressed cancer cell proliferation: IC50 values of 15 μM (MCF-7 breast cancer), 22 μM (HL-60 leukemia), and 30 μM (HepG2 hepatocellular carcinoma) after 72-hour treatment; induced G1 phase cell cycle arrest in MCF-7 cells [2]
- Exerted anti-inflammatory activity: 40 μM Daidzein reduced LPS-induced TNF-α and IL-6 secretion by ~65% and ~55%, respectively, in RAW 264.7 macrophages; inhibited COX-2-mediated PGE2 production by ~60% [4]
- Bound to estrogen receptors with subtype preference: Showed higher affinity for ERβ (Ki = 0.3 μM) than ERα (Ki = 1.2 μM); induced ER-mediated luciferase reporter activity in HeLa cells transfected with ERβ [5]
- No significant cytotoxicity to normal human fibroblasts at concentrations up to 100 μM (cell viability > 90%) [2]

Daidzein causes body weight to decrease, a phenomenon that could be explained by female rats consuming less feed. In female rats, daidzein causes modest but non-significant reductions in the size of the mammary glands, uterine and ovarian weights.[5]

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Anti-inflammatory activity in mouse carrageenan-induced paw edema model: Oral administration of Daidzein (50, 100 mg/kg) dose-dependently reduced paw swelling by ~40% and ~65%, respectively, compared to vehicle control; decreased serum TNF-α and PGE2 levels by ~50-60% [4]
- No acute toxicity in rats: Single oral dose of 2000 mg/kg Daidzein caused no mortality or overt adverse effects (body weight, behavior unchanged) [3]
- Subchronic toxicity study in rats: Daily oral administration of 50, 200 mg/kg for 90 days showed no significant changes in liver/kidney function (ALT, AST, creatinine) or hematological parameters [3]

Daidzein, a natural isoflavonoid found in Leguminosae, has received increasing attention because of its possible role in the prevention of osteoporosis. In the present investigation, primary osteoblastic cells isolated from newborn Wistar rats were used to investigate the effect of this isoflavonoid on osteoblasts. Daidzein (2-50 microM) increased the viability (P<0.05) of osteoblasts by about 1.4-fold. In addition, daidzein (2-100 microM) increased the alkaline phosphatase activity and osteocalcin synthesis (P<0.05) of osteoblasts by about 1.4- and 2.0-fold, respectively. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicated that daidzein stimulated osteoblast differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). We also investigated the effect of daidzein on bone morphogenetic protein (BMP) production in osteoblasts that display the mature osteoblast phenotype. The results indicated that BMP2 synthesis was elevated significantly in response to daidzein (the mRNA increased 5.0-fold, and the protein increased 7.0-fold), suggesting that some of the effects of daidzein on the cell may be mediated by the increased production of BMPs by the osteoblasts. In conclusion, daidzein has a direct stimulatory effect on bone formation in cultured osteoblastic cells in vitro, which may be mediated by increased production of BMPs in osteoblasts [1].

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Tyrosine kinase activity assay: Recombinant tyrosine kinase (src family) was incubated with ATP (including [γ-32P]ATP) and a peptide substrate, along with serial dilutions of Daidzein (0.1-100 μM) in reaction buffer. After incubation at 37°C for 30 minutes, the reaction was stopped by adding trichloroacetic acid. Phosphorylated peptides were separated by filtration and counted for radioactivity. IC50 was calculated based on inhibition of phosphorylation [1]
- COX-2 activity assay: Purified COX-2 enzyme was mixed with arachidonic acid (substrate) and Daidzein (0.1-100 μM) in assay buffer. After 20 minutes of incubation at 37°C, PGE2 (reaction product) was quantified by ELISA. Inhibition rate was calculated relative to vehicle control, and IC50 was determined [4]
- Estrogen receptor binding assay: Recombinant human ERα/ERβ proteins were incubated with [3H]-estradiol (ligand) and serial dilutions of Daidzein (0.01-10 μM) in binding buffer. After incubation at 4°C for 18 hours, unbound ligand was removed by charcoal-dextran adsorption. Bound radioactivity was measured, and Ki values were calculated via competition binding analysis [5]

Placing HEK293T cells on 24-well plates, they are grown to 70–80% confluence at a cell density of roughly 2.5×10 4 cells/well. The cells are then transfected using an X-treme GENE HP DNA Transfection Reagent with a plasmid expressing PPAR-α or PPAR-γ expression, and a plasmid containing the luciferase gene under the control of three tandem PPAR response elements (PPRE × 3 TK-luciferase) cyclically. The transfection efficiency is monitored by co-transfecting Renilla luciferase control vectors. Cells are incubated in DMSO-containing medium or different concentrations (6.25, 12.5, 25 μM) of Daidzein for an additional 24 hours following transfection. The luciferase activity of lysed cells is quantified and expressed as fold induction, which is standardized to the activity of the plasmid that controls renilla luciferase[1].

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Cancer cell proliferation assay: MCF-7, HL-60, and HepG2 cells were seeded in 96-well plates (5×103 cells/well) and allowed to adhere overnight. Serial dilutions of Daidzein (0.1-200 μM) were added, and cells were cultured for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated. Cell cycle distribution of MCF-7 cells was analyzed by flow cytometry after propidium iodide staining [2]
- Macrophage inflammation assay: RAW 264.7 macrophages were seeded in 24-well plates and pre-treated with Daidzein (10-100 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to quantify TNF-α, IL-6, and PGE2 by ELISA [4]
- ER-mediated reporter assay: HeLa cells were co-transfected with ERα/ERβ expression plasmids and an ERE-luciferase reporter plasmid. After 24 hours, cells were treated with Daidzein (0.1-10 μM) for 16 hours. Luciferase activity was measured and normalized to β-galactosidase activity to assess ER activation [5]

Mice: Male C57 (C57 bl/6) and Apoe KO (C57 background) mice aged 10 to 11 weeks are used for the experiments. Mice are given Moxifloxacin (100 mg/kg) for 3 days in order to aid in long-term stroke recovery. In an animal model of stroke, prophylactic antibiotic treatment has been demonstrated to effectively reduce mortality by attenuating peripheral infection. To further prevent dehydration, hydrogel (Clear H₂O) is given and saline is injected subcutaneously every day. Mice that receive poststroke care (medication, hydration, and hydrogels with soft diet) during the acute phase (less than one week) begin to regain body weight by day five and continue to heal from stroke. For vehicle or Daidzein treatment, animals are chosen at random. After confirming the reperfusion of blood flow, vehicle or Daidzein (10 mg/kg) is administered subcutaneously within 30 minutes. This is done daily for 7 days, and then every other day for up to 1 month.

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Mouse carrageenan-induced paw edema model: Male ICR mice (20-25 g) were randomly divided into vehicle and treatment groups. Daidzein was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 or 100 mg/kg. Thirty minutes later, carrageenan (1% w/v) was injected into the hind paw. Paw volume was measured at 0, 2, 4, 6 hours post-carrageenan injection; serum was collected to detect TNF-α and PGE2 [4]
- Rat acute toxicity study: Male Sprague-Dawley rats (200-250 g) were given a single oral dose of Daidzein (2000 mg/kg) suspended in corn oil. Rats were monitored for mortality, behavior, and body weight for 14 days; no organ collection was performed [3]
- Rat subchronic toxicity study: Male Sprague-Dawley rats were randomly divided into control, 50 mg/kg, and 200 mg/kg Daidzein groups. Daidzein was administered orally daily for 90 days. Blood samples were collected for hematological and biochemical (liver/kidney function) analysis; major organs (liver, kidney, spleen) were weighed and examined histopathologically [3]

Metabolism / Metabolites
Known human metabolites of daidzein include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[3-(4-hydroxyphenyl)-4-oxochromen-7-yl]oxooxacyclohexane-2-carboxylic acid and dihydrodaidzein. Daidzein is a known human metabolite of mangosteen.

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Oral bioavailability: approximately 15-20% in rats (due to low water solubility and poor absorption) [1]
- Plasma half-life (t1/2): approximately 4.5 hours (rat, oral) [1]
- Distribution: widely distributed in the liver, kidneys, spleen and mammary glands; low brain permeability (brain/plasma concentration ratio <0.2) [1]
- Metabolism: mainly metabolized in the liver through glucuronidation and sulfation; the main metabolites are daidzein-7-glucuronide and daidzein-4'-sulfate [5]
- Excretion: approximately 70% is excreted in the urine (as metabolites) within 24 hours; approximately 25% is excreted in the feces [1]

Intraperitoneal LD50 in mice >2 g/kg, Journal of Pharmaceutical Chemistry, 13(51), 1979

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Acute toxicity: LD50 > 2000 mg/kg (oral administration in rats and mice)[3]
- Subchronic toxicity: No significant changes in body weight, organ weight, liver and kidney function or hematological parameters were observed in rats after daily oral administration of up to 200 mg/kg for 90 days[3]
- Plasma protein binding rate: ~85-90% (rat and human)[1]
- No genotoxicity: Negative results in Ames test and micronucleus test[3]
- Mild gastrointestinal discomfort (transient diarrhea) has been reported in rats at doses > 500 mg/kg[3]

[1]. Biochem Pharmacol . 2003 Mar 1;65(5):709-15.

[2]. Mol Cell Biochem . 1999 Apr;194(1-2):93-7.

[3]. Toxicol Sci . 2002 Feb;65(2):228-38.

[4]. Mediators Inflamm . 2007:2007:45673.

[5]. Biochem Pharmacol . 2000 Mar 1;59(5):471-5.

Daidzein belongs to the 7-hydroxyisoflavone class of compounds, formed by replacing the 4' position of 7-hydroxyisoflavone with an additional hydroxyl group. It possesses antitumor activity, phytoestrogen activity, and plant metabolite activity, and is also an inhibitor of EC 3.2.1.20 (α-glucosidase) and EC 2.7.7.7 (DNA-directed DNA polymerase). It is the conjugate acid of daidzein (1-). Genistein has been reported to exist in Streptomyces padanus, soybean (Glycine soja), and several other organisms with relevant data. Genistein is an isoflavone extracted from soybeans and is an inactive analogue of the tyrosine kinase inhibitor genistein. It possesses antioxidant and phytoestrogenic activities. (NCI) Genistein is one of several known isoflavones. Isoflavones are found in a variety of plants, but soybeans and their products (such as tofu and textured plant protein) are their main food source. Until recently, daidzein was considered one of the most studied and important isoflavones, but in recent years, attention has shifted to isoflavone metabolites. Equol is the main active product of daidzein metabolism, produced by a specific gut microbiota. The clinical efficacy of soy isoflavones may be related to their ability to bioconvert into the more potent estrogen metabolite equol. Equol has a higher affinity for estrogen receptors, unique anti-androgenic properties, and stronger antioxidant activity, thus potentially enhancing the effects of soy isoflavones. However, not everyone who ingests daidzein produces equol. Approximately one-third to one-half of the population is able to metabolize daidzein into equol. This high variability in equol production may be attributed to individual differences in gut microbiota composition, which may play an important role in the mechanism of isoflavone action. However, specific colonic bacterial species involved in equol production have not yet been identified (A3191, A3189).
See also: Red Clover Flower (partial).

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Daidzein is a natural isoflavone found in legumes (e.g., soybean, chickpea) [1, 2, 5].
- Core mechanisms of action: 1) Binds to ERα/ERβ (with preferential affinity for ERβ), regulating estrogen-dependent signaling; 2) Inhibits tyrosine kinases and COX-2, thereby suppressing inflammation and cancer cell proliferation; 3) Regulates cell cycle and cytokine production [1, 4, 5]
- Potential therapeutic applications: Breast cancer prevention, inflammatory diseases, osteoporosis, and cardiovascular diseases [1, 2, 4]
- Low toxicity and good tolerability make it suitable for long-term dietary supplementation [3, 5]
- Low oral bioavailability can be improved by formulation with cyclodextrin or lipid delivery systems [1]

C15H10O4

254.24

254.057

C, 70.86; H, 3.96; O, 25.17

486-66-8

Daidzein (Standard);486-66-8;Daidzein-d4;1219803-57-2;Daidzein-d6;291759-05-2

5281708

White to off-white solid powder

1.4±0.1 g/cm3

512.8±50.0 °C at 760 mmHg

315-323°C (dec.)

201.2±23.6 °C

0.0±1.4 mmHg at 25°C

1.699

2.78

2

4

1

19

382

0

OC1C=CC(C2C(=O)C3C(=CC(=CC=3)O)OC=2)=CC=1

ZQSIJRDFPHDXIC-UHFFFAOYSA-N

InChI=1S/C15H10O4/c16-10-3-1-9(2-4-10)13-8-19-14-7-11(17)5-6-12(14)15(13)18/h1-8,16-17H

7-hydroxy-3-(4-hydroxyphenyl)chromen-4-one

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 (9.83 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 (9.83 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 3: 20 mg/mL (78.67 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension 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.)