- Extra Domain A (EDA) of fibronectin, specifically the C-C' loop residues LEU46, PHE47, PRO48, GLU58, LEU59, and GLN60 (no IC50/Ki values reported) [1].
- Modulates expression of pro-apoptotic proteins (FADD, DR5, Bax) and anti-apoptotic proteins (c-FLIP, Bcl-2, Survivin); no direct binding affinity or IC50 reported [2].

Extra Domain A (EDA) contains LEU46, PHE47, PRO48, GLU58, LEU59, and GLN60 in its CC loop. Irisin preferentially targets the α9β1 and α4β1 integrin binding sites on EDA. The epithelial-to-mesenchymal transition that follows is prevented by isisin's binding to the CC loop of EDA, which prevents it from interacting with cell surface integrins [1].

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- In human lung carcinoma A549 and NCI-H522 cells, Irigenin inhibited cell proliferation with IC50 values of 58 μM (A549) and 61 μM (NCI-H522) after 24 h treatment (MTT assay) [1].
- Irigenin (10, 25, 50 μM) dose-dependently inhibited wound closure in A549 and NCI-H522 cells after 24 h. At 50 μM, wound closure was reduced to ~25% in A549 and ~23% in NCI-H522 compared to DMSO control (~90% closure) [1].
- Irigenin (10, 25, 50 μM) significantly reduced Matrigel invasion in A549 and NCI-H522 cells. At 50 μM, invasion was inhibited by 80% in both cell lines compared to control [1].
- Western blot analysis showed that Irigenin (10, 25, 50 μM) increased E-cadherin expression and decreased vimentin, Snail, and Zeb-1 expression in a dose-dependent manner in A549 and NCI-H522 cells [1].
- Irigenin had no effect on migration, invasion, or EMT marker expression in EDA-negative T47D breast carcinoma cells even at concentrations effective in A549 cells [1].
- EDA knockdown in A549 cells (using shRNA) rendered the cells unresponsive to Irigenin treatment in migration, invasion, and EMT assays [1].
- In gastric cancer cell lines (MKN-28, BGC-823, MGC-803, SGC-7901, ACP-01), Irigenin alone showed dose-dependent cytotoxicity, with BGC-823 and SGC-7901 being more sensitive; no cytotoxicity was observed in normal gastric epithelial GES-1 cells up to 40 μM [2].
- Co-treatment of Irigenin (5, 10, 20 μM) with TRAIL (100 ng/ml) for 24 h markedly enhanced cytotoxicity in BGC-823 and SGC-7901 cells compared to either agent alone [2].
- Irigenin (10 μM) plus TRAIL (100 ng/ml) significantly increased sub-G1 population, nuclei chromatin damage (DAPI staining), and cytoplasmic histone-associated DNA fragmentation in SGC-7901 cells [2].
- Caspase-3 activity (DEVDase) was significantly increased by Irigenin plus TRAIL combination compared to single agents [2].
- Flow cytometry (Annexin V/PI) showed that Irigenin (10 μM) plus TRAIL (100 ng/ml) markedly induced apoptosis in SGC-7901 cells [2].
- Western blot: Irigenin (5, 10, 20 μM) alone dose-dependently increased cleaved caspase-8, -9, -3, and PARP; combination with TRAIL further enhanced these cleavages. Irigenin increased FADD, DR5, and Bax expression, and decreased c-FLIP, Bcl-2, and Survivin expression; DR4 and Mcl-1 were unchanged [2].
- siRNA knockdown of FADD, DR5, or Bax significantly reduced apoptosis (sub-G1 and Annexin V/PI) and restored cell viability induced by Irigenin plus TRAIL, confirming their involvement [2].
- Irigenin (10, 20 μM) increased ROS generation (DCF-DA staining) in SGC-7901 cells; combination with TRAIL further enhanced ROS. Pre-treatment with N-acetyl-cysteine (NAC, ROS scavenger) attenuated Irigenin plus TRAIL-induced apoptosis and caspase activation [2].

- In a nude mouse xenograft model bearing SGC-7901 gastric tumors, Irigenin (10 mg/kg, intraperitoneal injection once daily for 21 days) alone slightly inhibited tumor growth. Combination of Irigenin (10 mg/kg i.p.) and TRAIL (100 μg/kg intratumoral injection, given 24 h after Irigenin) significantly reduced tumor volume and tumor weight compared to either agent alone. H&E staining showed decreased tumor cell density in the combination group [2].

- Molecular docking simulations using AutoDock 4.2 were performed to evaluate binding of Irigenin to the C-C' loop of Fibronectin EDA (PDB ID: 1J8K). The docking binding energy (ΔG) was -10.04 Kcal/mol. Hydrogen bonding analysis showed six hydrogen bonds between Irigenin and EDA residues (HIS44, ILE43) with bond lengths of 1.81–2.43 Å [1].
- Molecular Dynamics (MD) simulations of Irigenin-EDA complex were carried out using GROMACS 4.6 with GROMOS 43a1 force field for 100 ns. The binding free energy calculated by MM-PBSA was -221.602 ± 35.657 kJ/mol. Energy decomposition per residue identified key interacting residues: LEU46 (-6.9 kJ/mol), PHE47 (-3.6 kJ/mol), PRO48 (-7.8 kJ/mol), GLU58 (-4.1 kJ/mol), LEU59 (-6.1 kJ/mol), and GLN60 (-3.7 kJ/mol) [1].
- Caspase-3 (DEVDase) activity assay: Cell lysates (20 μg) were incubated in reaction buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 1% NP-40, 10% glycerol) with 5 μM DEVD-pNA substrate for 2 h at 37°C, and absorbance at 405 nm was measured [2].

- Cell proliferation (MTT assay): Cells were seeded in 24-well plates, treated with various concentrations of Irigenin for 24 h, then incubated with 0.1 mg/ml MTT for 4 h at 37°C in dark. Formazan crystals were dissolved in DMSO and absorbance measured at 565 nm with background at 650 nm [1].
- Wound healing assay: Cells grown to confluence in 6-well plates were scratched with a 200 μl pipette tip, washed with PBS, photographed (T0), then treated with Irigenin (10, 25, 50 μM) or DMSO for 24 h. Wounds were photographed at T24, and percentage wound closure was calculated using ImageJ software [1].
- Transwell invasion assay: Cells suspended in DMEM were seeded into Matrigel-coated transwell inserts (8 μm pore size) containing Irigenin (10, 25, 50 μM) or IST-9 antibody. Lower chambers contained 10% FBS as chemoattractant. After 24 h, non-invaded cells were removed from the upper membrane surface, invaded cells on the lower side were fixed in 3.7% formaldehyde, stained with crystal violet, and counted in five fields [1].
- Western blotting: Cells treated with Irigenin were lysed in RIPA buffer, protein concentrations determined by BCA method. Equal protein amounts were separated on SDS-PAGE (10-12%), transferred to PVDF membranes, blocked with 5% skim milk, incubated with primary antibodies (anti-E-cadherin, anti-vimentin, anti-Snail, anti-Zeb-1, anti-β-actin, etc.) overnight at 4°C, then with IR-tagged secondary antibodies, and scanned using an infrared scanner [1].
- DAPI staining for nuclei condensation: Cells fixed with 1% paraformaldehyde for 30 min, washed with PBS, stained with DAPI solution for 5 min, and analyzed by fluorescence microscopy [2].
- Cytoplasmic histone-associated DNA fragmentation assay: Performed using a kit (not specified) after treatment with Irigenin and/or TRAIL [2].
- Flow cytometry for sub-G1: Cells resuspended in PBS, fixed with 95% ethanol (while vortexing), incubated for 1 h at 4°C, washed, resuspended in sodium citrate buffer (pH 8.4) with RNase, incubated at 37°C for 30 min, then stained with propidium iodide (50 μg/ml) for 30 min at room temperature, and analyzed by flow cytometer [2].
- Flow cytometry for apoptosis (Annexin V/PI): Cells stained using Annexin V/PI double staining kit according to manufacturer's protocol after treatment with Irigenin and/or TRAIL for 24 h [2].
- ROS measurement: Cells treated as indicated, then incubated with 5 μM DCF-DA at 37°C for 30 min, and fluorescence was analyzed and captured using a fluorescent microscope [2].
- siRNA transfection: Cells were transfected with siRNA oligonucleotides (30 nmol/l) targeting FADD, DR5, or Bax (or scrambled control) using lipofectamine 2000 following manufacturer's recommendations [2].

- Xenograft model in nude mice: SGC-7901 cells (2 × 10^6 in 200 μl) were subcutaneously injected into the flank of 4-week-old male nude mice. When tumor volume reached ~50 mm^3, mice were randomized into 4 groups (n=?. not specified) and treated once daily for 21 days: control (vehicle only); TRAIL alone (100 μg/kg intratumoral injection); Irigenin alone (10 mg/kg intraperitoneal injection); combination group (Irigenin 10 mg/kg i.p., followed 24 h later by TRAIL 100 μg/kg intratumoral injection). Tumor size was measured weekly using formula: volume = 0.5 × length × width. At the end, tumor weight was measured, and tumors were fixed for H&E staining [2].

- Irigenin showed no cytotoxicity to normal human gastric epithelial GES-1 cells up to 40 μM as measured by MTT assay [2].

[1]. Irigenin, a novel lead from Western Himalayan chemiome inhibits Fibronectin-Extra Domain A induced metastasis in Lung cancer cells. Sci Rep. 2016 Nov 16;6:37151.

[2]. Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells. Biochem Biophys Res Commun. 2018 Feb 12;496(3):998-1005.

Irisin is a hydroxyisoflavone with a structure in which isoflavones are substituted with hydroxyl groups at the 5', 7', and 3' positions and with methoxy groups at the 6', 4', and 5' positions. It is a plant metabolite. Irisin belongs to the 4'-methoxyisoflavone class of compounds and is functionally related to isoflavones. It has been reported to be found in iris (Iris tectorum), iris (Iris milesii), and other organisms with relevant data.

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- Irigenin is a flavonoid from Iris kashmiriana and Belamcanda chinensis [1][2].
- In silico screening predicted that Irigenin follows Lipinski's Rule of Five (molecular weight ≤500, clogP ≤4.5, H-bond donors ≤5, H-bond acceptors ≤10) and has favorable ADME properties; it was also predicted non-mutagenic and non-carcinogenic using Pre-ADMET tool [1].
- Irigenin blocks the C-C' loop of Fibronectin EDA, preventing its interaction with α9β1 and α4β1 integrins, thereby inhibiting Epithelial-Mesenchymal Transition (EMT) and metastasis in lung cancer cells [1].
- In gastric cancer, Irigenin sensitizes TRAIL-induced apoptosis by upregulating FADD, DR5, and Bax, and downregulating c-FLIP, Bcl-2, and Survivin, partially through ROS generation [2].
- The combination of Irigenin and TRAIL suppressed tumor growth in a gastric cancer xenograft model without obvious systemic toxicity reported [2].

C18H16O8

360.3148

360.085

C, 60.00; H, 4.48; O, 35.52

548-76-5

5464170

Off-white to light yellow solid powder

1.461g/cm3

646.1ºC at 760mmHg

189-192ºC

237.7ºC

1.649

2.602

3

8

4

26

549

0

O1C([H])=C(C(C2C(=C(C(=C([H])C1=2)O[H])OC([H])([H])[H])O[H])=O)C1=C([H])C(=C(C(=C1[H])OC([H])([H])[H])OC([H])([H])[H])O[H]

TUGWPJJTQNLKCL-UHFFFAOYSA-N

InChI=1S/C18H16O8/c1-23-13-5-8(4-10(19)17(13)24-2)9-7-26-12-6-11(20)18(25-3)16(22)14(12)15(9)21/h4-7,19-20,22H,1-3H3

5,7-dihydroxy-3-(3-hydroxy-4,5-dimethoxyphenyl)-6-methoxychromen-4-one

Irigenin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~277.54 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 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 (6.94 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 (6.94 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.)