PKM2 (Pyruvate kinase isozyme type M2) - binding affinity of Iridin to the active pocket of PKM2 was -6.9 kcal/mol (molecular docking) [3].
PI3K/AKT signaling pathway - Iridin reduces expression of p-AKT and p-PI3K [1][2].
JAK/STAT signaling pathway - Iridin downregulates p-JAK1, p-STAT1, p-STAT3 [1][3].
NF-κB pathway - Iridin downregulates p-p65 [1][3].

In AGS gastric cancer cells, Iridin treatment (0, 50, 100, and 200 μM for 48 h) decreased cell growth, inhibited cell proliferation in a concentration-dependent manner, and promoted G2/M phase cell cycle arrest by attenuating the expression of Cdc25C, CDK1, and Cyclin B1 proteins [2].
In AGS cells, Iridin treatment (0, 50, 100, and 200 μM for 48 h) triggered apoptotic cell death, verified by increased cleaved Caspase-3 and cleaved PARP protein expression, and confirmed by increased apoptotic cell death fraction shown in APC/Annexin V and propidium iodide staining [2].
Iridin increased the expression of extrinsic apoptotic pathway proteins including Fas, FasL, and cleaved Caspase-8 in AGS cells, but did not show variations in intrinsic apoptotic pathway proteins such as Bax and Bcl-xL [2].
Iridin showed inhibition of PI3K/AKT signaling pathways by downregulation of p-PI3K and p-AKT proteins in AGS cells [2].
In RAW264.7 macrophages, Iridin treatment at concentrations of 12.5, 25, and 50 μM significantly inhibited the productions of TNF-α, IL-1β, MCP-1, and ROS, and suppressed the levels of glucose uptake and lactic acid in LPS-treated RAW264.7 cells [3].
Iridin (12.5-50 μM) increased OCR levels (indicative of oxidative phosphorylation) but decreased ECAR levels (indicative of glycolysis) in LPS-treated macrophages [3].
Iridin (12.5-50 μM) reduced the levels of NO in culture medium of LPS-treated RAW264.7 cells (IC50 on LPS-induced NO release was 24.8 μM) [3].
Iridin reduced the mRNA expressions of M1 markers iNOS and TNF-α but elevated the expressions of IL-10 and Arg-1 in macrophages of LPS-treated mice [3].
Iridin downregulated the expressions of PKM2 and its downstream proteins (p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2) in LPS-exposed RAW264.7 cells [3].
In TRAIL-resistant gastric cancer cells, the combination of TRAIL and iridin (mentioned as irigenin in this context - note: the study refers to irigenin, not iridin) activated caspase-8/9/3 and PARP, hyper-expressed FADD, DR5, and BAX, and inhibited the expression of cFLIB, Bcl-2, and Survivin [1].
In RAW264.7 cells, Iridin (12.5, 25, 50 μM) pretreatment for 2 h followed by LPS for 16 h significantly decreased TNF-α, IL-1β, MCP-1, and NO levels in a dose-dependent manner compared to control [1].

In ICR mice (24±2 g body weight), intranasal exposure with 20 mg/kg LPS to induce acute lung injury, followed by oral administration of Iridin (20, 40, and 80 mg/kg) once daily for 5 days (drugs given once daily for 3 days), showed that Iridin inhibited LPS-induced acute lung injury as well as inflammatory cytokine production [3].
Iridin treatment (20-80 mg/kg) in LPS-challenged mice reduced the levels of iNOS and TNF-α in serum but increased serum IL-10 levels, reduced the number of total cells and neutrophils in BALF, decreased lung indexes and pathologic scores in a dose-dependent manner, and reduced the mRNA expressions of iNOS and TNF-α while increasing IL-10 and Arg-1 mRNA expressions in macrophages [3].
Iridin (20-80 mg/kg) downregulated the protein expressions of PKM2, p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2 in lung tissues of LPS-treated mice [3].

Molecular docking analysis of Iridin with PKM2 target protein was tested using Autodock Tools (version 1.5.6). The active sites of PKM2 were determined as: center x = 6.527, center y = 1.534, center z = 10.683; size x = 20, size y = 20, size z = 20. The parameter exhaustiveness was set as 20 with others as default values. The highest scoring conformation was selected and analyzed. The binding affinity of Iridin to the active pocket of PKM2 was -6.9 kcal/mol, and Iridin formed hydrogen bonding interaction with PKM2 at TYP 390 and ASP 354 sites, and had Pi-Pi interaction at PHE 26 [3].
For surface plasmon resonance analysis, three serial concentrations of Iridin sample and a negative control (saline buffer) were respectively added to biotin-labeled and desalted PKM2 solutions (50 μM). The interaction between Iridin and PKM2 was performed at room temperature following the ForteBio manufacturer's protocol. Affinity curves rose more steeply with the increase of Iridin concentration until it reached an inflection point, demonstrating that Iridin was directly bound to the PKM2 protein [3].

Cell viability was assessed using MTT assay. AGS cells (5×10⁴) and HaCaT cells (1×10⁴) were seeded in 48-well plates and treated with different concentrations of Iridin (0, 12.5, 25, 50, 100, and 200 μM) for 48 h at 37°C. After incubation, 50 μL of 0.5% (w/v) MTT dissolved in 1× PBS was added to each well and incubated for about 3 h at 37°C. Formazan precipitates formed after incubation were dissolved in 300 μL of DMSO, and absorbance of transformed dye was measured at 540 nm using a microplate reader. Cell viability was expressed as a percentage of proliferation versus Iridin-untreated group [2].
For cell cycle distribution analysis, AGS cells were incubated with or without Iridin at 50, 100, and 200 μM concentrations for 48 h at 37°C. Cells were washed with ice-cold PBS, trypsinized, collected, and pelleted by centrifugation (3000 rpm for 4 min). Pellets were washed twice with ice-cold PBS and fixed in 70% ice-cold ethanol for 1 h at -20°C. Cell suspension was centrifuged, washed in PBS, and re-suspended in 400 μL of PBS containing 50 μg/mL propidium iodide and 0.1 mg/mL RNase A, then incubated in the dark for 15 min at room temperature. Flow cytometry analysis was performed with approximately 10,000 cells sorted per sample [2].
For apoptosis detection, AGS cells were seeded on 60-mm plates at 1×10⁵ cells and incubated with specified concentrations of Iridin (0, 50, 100, and 200 μM) for 48 h. Cells were harvested using trypsinization for 3 min, washed with PBS, and resuspended in binding buffer. Cells were stained at room temperature with APC/annexin V and propidium iodide for 15 min in the dark before binding buffer addition. Flow cytometry analysis was performed with approximately 10,000 cells sorted per sample [2].
For Western blotting, AGS cells were seeded at 3×10⁵ in 60-mm plates and treated with specified concentrations of Iridin (0, 50, 100, and 200 μM) for 48 h. Harvested cells were lysed in RIPA buffer consisting of protease inhibitor and phosphatase inhibitor cocktail for 30 min on ice. Protein quantification was performed using BCA assay. Proteins (8-15% SDS-PAGE) were separated and transferred onto PVDF membranes using a semi-dry transfer method. Membranes were blocked using bovine serum albumin with TBS-T (pH 7.4) at 4°C overnight with 1:1000 dilution of respective primary antibody. Membranes were washed five times with TBS-T for 10 min intervals and incubated for 3 h at RT with HRP-conjugated secondary antibody (1:1000 to 1:5000 dilution). Blots were developed under an ECL detection system, and protein quantification was analyzed using ImageJ software [2].
For RAW264.7 cell viability, cells were inoculated into 96-well plates at 1×10⁴/well and incubated with different concentrations of Iridin (0, 6.25, 12.5, 25, 50, 100, 150, and 200 μM) for 24 h. CCK-8 diluted with complete medium (1:10 proportion) was added (100 μL/well) and incubated at 37°C for 2 h, and absorbance at A450 nm was measured [3].
For glucose uptake assay, RAW264.7 cells were inoculated into 24-well plates at 1×10⁵/well, pre-treated with Iridin for 2 h and then with LPS for 16 h. After culturing with sugar-free medium for 1 h, cells were treated with 2-NBDG Fluorescent Dyes (final concentration 20 μM) in the dark at 37°C for 10 min. Fluorescence value was detected by a fluorescent microplate reader (excitation: 465 nm; emission: 540 nm). Cellular OCR and ECAR were measured by Seahorse Bioscience Analyzer [3].
For detection of lactate and cytokines, RAW264.7 cells were inoculated into 24-well plates at 1×10⁵/well, pre-treated with Iridin (or activator, agonist) for 2 h and then with LPS for 16 h. Cell culture medium was collected and levels of lactate, TNF-α, IL-1β, and MCP-1 were measured by ELISA, while NO levels were detected by Griess assay [3].
For phagocytic activity, RAW264.7 macrophages were inoculated into 96-well plates (1×10⁵ cells/well) and cultured overnight for 12 h. Cells were pre-treated with Iridin (or activator, agonist) for 2 h and then exposed to LPS for 16 h. After LPS stimulus, supernatant was removed and 100 μL of 0.075% neutral red solution was added to each well and incubated for an additional 4 h. After washing three times in PBS, 100 μL of lysis buffer (0.01% glacial acetic acid:ethanol = 1:1 v/v) was added. After 2 h incubation at 4°C, absorbance was measured at 540 nm [3].
For intracellular ROS and NO detection, 1×10⁵ RAW264.7 cells were inoculated into 24-well plates and cultured overnight for 12 h. After pre-treatment with Iridin (or activator, agonist) for 2 h and exposure to LPS for 16 h, cells were treated with DCFH2-DA (10 μM) for 30 min and intracellular ROS production was measured by flow cytometry. For NO detection, cells were incubated with DAF-FM (5 μM) for 1 h before flow cytometry analysis [3].

Male ICR mice (24±2 g body weight) were raised in Zhejiang animal Centre. Mice were divided into six groups: (1) control group; (2) model group (intranasally exposed with 20 mg/kg LPS); (3) Dexamethasone-treated group (LPS-challenged mice treated with 10 mg/kg Dex); (4-6) Iridin-treated groups (LPS-challenged mice orally administered with 20, 40, and 80 mg/kg of Iridin once a day for 5 days). Drugs were given once daily for 3 days. Seven days after LPS exposure, all mice were asphyxiated by CO₂ and blood and lung were collected. The number of total cells and neutrophils in bronchoalveolar lavage fluid was counted by automatic blood cell analyzer [3].

In HaCaT human keratinocyte cells, Iridin treatment did not affect cell viability up to 200 μM concentration [2]. In RAW264.7 cells, when treated with Iridin at concentrations less than 100 μM, Iridin did not affect cell proliferation. There were no significant differences in cell viabilities between the control group and LPS+Iridin-treated groups (P > 0.05) [3].

[1]. The Effects of Iridin and Irigenin on Cancer: Comparison with Well-Known Isoflavones in Breast, Prostate, and Gastric Cancers. Int J Mol Sci. 2025;26(6):2390. Published 2025 Mar 7.

[2]. Iridin Induces G2/M Phase Cell Cycle Arrest and Extrinsic Apoptotic Cell Death through PI3K/AKT Signaling Pathway in AGS Gastric Cancer Cells. Molecules. 2021;26(9):2802. Published 2021 May 10.

[3]. Iridin Prevented Against Lipopolysaccharide-Induced Inflammatory Responses of Macrophages via Inactivation of PKM2-Mediated Glycolytic Pathways. J Inflamm Res. 2021;14:341-354. Published 2021 Feb 5.

[4]. Isoflavones of two Iris species.  Phytochemistry 23(11):2703-2704.

Irisin is a glycosyl isoflavone formed by the substitution of a iris aglycone at the 7-position with a β-D-glucopyranose residue via a glycosidic bond. It is a plant metabolite. Irisin is a hydroxy isoflavone, belonging to the monosaccharide derivative family, and is a member of 4'-methoxyisoflavone and 7-hydroxyisoflavone 7-O-β-D-glucoside. It is functionally related to iris aglycone. Irisin has been reported in iris (Iris tectorum), iris (Iris milesii), and other organisms with relevant data. See also: Root (part) of Iris versicolor.

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Iridin is a glycoside flavonoid monomer and its aglycone is irigenin [1][2]. The chemical structure of Iridin consists of a 7-glucoside of irigenin [2]. Iridin is classified as an isoflavone (glycoside form) and is found in the Iris family, Belamcanda chinensis, Iris kumaonesis, and Iris florentina [1]. Iridin has been reported to induce an extrinsic pathway by exhibiting Fas-mediated apoptotic cell death in AGS cells by regulating the PI3K/AKT signaling pathway [1]. Iridin binds to the active site of PKM2 to inhibit the expression of PKM2, which can downregulate the JAK/STAT signaling pathway [1]. In AGS gastric cancer cells, Iridin induced G2/M phase cell cycle arrest and extrinsic apoptotic cell death through PI3K/AKT signaling pathway [2]. The half-maximal inhibitory concentration (IC50) of Iridin in AGS cells was 161.3 μM [2]. Iridin is a natural isoflavone that exerts anticancer, antioxidant, and anti-inflammatory effects [3]. Iridin alleviated LPS-induced inflammation through suppressing PKM2-mediated pathways, including JAK/STATs and NF-κB pathways, and could be a potential candidate for the prevention of inflammatory diseases [3].

C24H26O13

522.4554

522.137

491-74-7

5281777

White to off-white solid

1.6±0.1 g/cm3

833.5±65.0 °C at 760 mmHg

208℃

284.1±27.8 °C

0.0±3.2 mmHg at 25°C

1.660

-0.08

6

13

7

37

823

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1C([H])=C2C(C(C(C3=C([H])C(=C(C(=C3[H])OC([H])([H])[H])OC([H])([H])[H])O[H])=C([H])O2)=O)=C(C=1OC([H])([H])[H])O[H]

LNQCUTNLHUQZLR-OZJWLQQPSA-N

InChI=1S/C24H26O13/c1-32-13-5-9(4-11(26)22(13)33-2)10-8-35-12-6-14(23(34-3)19(29)16(12)17(10)27)36-24-21(31)20(30)18(28)15(7-25)37-24/h4-6,8,15,18,20-21,24-26,28-31H,7H2,1-3H3/t15-,18-,20+,21-,24-/m1/s1

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~191.40 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.79 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 (4.79 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 (4.79 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.)