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Irisolidone is natural product of the flavonoid class with hepatoprotective activity. It is the major isoflavone isolated from Pueraria lobata flowers.
| Targets |
Sp1 transcription factor (inhibition of Sp1 binding to the Sp1-II site on the JC virus promoter) [1].
Volume-regulated anion channels (VRACs) (IC50 = 9.8 ± 0.2 μM for VRAC currents in HEK293 cells; Emax = 88.5% ± 0.5%) [3]. |
|---|---|
| ln Vitro |
Irisolidone is an isoflavone metabolite that prevents human glial cells from binding Sp1, thereby suppressing the production of the JC virus gene [1]. Endothelial cell growth is inhibited by irisolidone [3].
Irisolidone significantly inhibited JC virus early promoter activity in a dose-dependent manner in U87MG human glioma cells and primary cultured human fetal astrocytes. The IC50 was approximately 7.5 μM, and promoter activity was inhibited by more than 90% in the presence of 50 μM irisolidone [1]. Irisolidone inhibited the transcriptional activity of both MH1 and Mad-1 type JC virus early promoters in U87MG and U373MG glial cell lines [1]. Mutation of the Sp1 binding site downstream of the TATA box (Sp1-II) on the JC virus promoter dramatically diminished the inhibitory activity of irisolidone (only 30% inhibition compared to wild type), while mutations of pentanucleotide, TATA, AP1, NF-1, NF-κB, Sp1-I, or Sp1-III sites did not significantly alter the inhibition [1]. Treatment with irisolidone (20 μM for 24 h) significantly reduced Sp1 binding to the Sp1-II site in a dose-dependent manner in U87MG cells, as shown by electrophoretic mobility shift assay (EMSA) [1]. Irisolidone (100 μM) inhibited hypotonic extrusion-induced VRAC currents by >80% in HEK293 cells, with a mean inhibition of 88.5% ± 0.5% [3]. Irisolidone inhibited VRAC currents with an IC50 of 9.8 ± 0.2 μM and Emax of 88.5% ± 0.5% in HEK293 cells [3]. Irisolidone (100 μM) significantly inhibited endogenous VRAC currents in human umbilical vein endothelial cells (HUVECs) [3]. Irisolidone significantly inhibited the growth of HUVEC cells at concentrations of 30 or 100 μM for 48 h (p < 0.05) [3]. |
| Enzyme Assay |
Electrophoretic Mobility Shift Assay (EMSA) for Sp1 binding: Nuclear extracts were prepared from U87MG human glioma cells treated with irisolidone. A 32P-labeled oligonucleotide probe harboring the Sp1-II sequence (5'-CCGAGGCCGCCTCCGCCTCCAAGCTTAC-3' sense) was incubated with 6 μg of nuclear extract in a buffer containing 12.5% glycerol, 12.5 mM Hepes (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM DTT, and 1 μg poly(dI-dC) at 4°C for 30 min. The binding products were resolved on 5% polyacrylamide gel and visualized by autoradiography. For competition assays, non-radioactive oligonucleotides were added in molar excess before adding the labeled probe. For supershift assays, antibodies against Sp1 or IRF were incubated with the nuclear extract for 30 min on ice prior to adding the probe [1].
Whole-cell patch clamp electrophysiology for VRAC currents: HEK293 or HUVEC cells were used. Pipettes had resistances of 2-3 MΩ. Currents were recorded using an amplifier and software. External solution (in mM): 160 NaCl, 6 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 8 glucose, pH 7.4. Hypotonic external solution (HYPO): 105 NaCl, 6 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 8 glucose. Internal solution (in mM): 140 CsCl, 5 EGTA, 2 CaCl2, 1 MgCl2, 10 HEPES, 4 ATP, pH 7.3. An eight-channel perfusion pencil was used for single-cell drug application. A -100 mV gap-free voltage protocol was used to record whole-cell VRAC currents. Drug effects were quantified by measuring current amplitude changes at -100 mV. Current-voltage (I-V) relationships were determined using step pulses between -100 and +100 mV in 10 mV increments from a holding potential of 0 mV [3]. |
| Cell Assay |
Transient transfection and luciferase reporter assay for JC virus promoter activity: U87MG or U373MG cells (2×10^5 in 60 mm dishes) were transfected with 4 μg of JC virus reporter plasmid (pMH1long-luc or Mad-1 promoter construct), 1 μg of pRSV β-gal, and pUC19 plasmid to a total of 10 μg DNA using a standard calcium phosphate method. After 48 h, cells were harvested, and luciferase activity was measured and normalized to β-galactosidase activity (determined by ONPG assay). To test inhibition, various concentrations of irisolidone were added to the transfected cells 24 h before harvesting [1].
Site-directed mutagenesis of JC virus promoter: Base substitutions in the promoter region (Sp1-II site) were generated in the context of the 408 bp upstream sequence using a PCR-based site-directed mutagenesis kit. Oligonucleotides used for Sp1-II mutation: 5'-CGAGGCCGCCTCATTACTCAAGCTTA-3' and 5'-GTAAGCTTGGAAATATGAGGCGGCCTC-3'. Constructs with mutations were screened by restriction enzyme digestion and sequencing analysis [1]. BrdU cell proliferation assay in HUVECs: Cells were seeded in 96-well plates. Twelve hours after seeding, cells were treated with different concentrations of irisolidone (10, 30, and 100 μM). After 48 h incubation, cells were labeled with BrdU (100 μM) for 4 h. After fixation, cells were incubated with anti-BrdU antibody for 1 h, followed by secondary antibody for 30 min, then substrate (tetramethylbenzidine) for 30 min at room temperature. OD readings were done at 450 nm. Cell proliferation inhibition (%) was calculated as (1 - mean OD of treated cells / mean OD of control cells) × 100 [3]. |
| Animal Protocol |
Pharmacokinetic study in rats: Male Sprague-Dawley rats (220-250 g) were fasted for 12 h before the experiment. Irisolidone was dispersed in 0.5% carboxymethyl cellulose solution at 10.0 mg/mL and sonicated for 5 min to obtain a homogeneous suspension. A single oral dose of 100 mg/kg body weight was administered. Whole blood samples were collected from the suborbital vein at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h after administration. Blood was immediately centrifuged at 3500 rpm for 10 min at 4°C to obtain plasma [2].
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| ADME/Pharmacokinetics |
After a single oral dose of 100 mg/kg irisolidone to rats, a total of 15 metabolites (including parent irisolidone) were detected in plasma. Metabolic pathways included decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation [2].
The main metabolites in rat plasma were irisolidone-7-O-glucuronide (Ir-7G) and 6-hydroxybiochanin A-6-O-glucuronide (6-OH-BiA-6G), accounting for 55.8% and 31.5% of total metabolites, respectively. The parent irisolidone (Ir) accounted for only 2.8% [2]. Pharmacokinetic parameters for irisolidone (Ir) in rat plasma after oral administration of 100 mg/kg: Cmax = 0.843 ± 0.259 μmol/L; Tmax = 9.67 ± 0.816 h; T1/2 = 4.64 ± 3.43 h; AUC(0→t) = 6.38 ± 1.55 μmol·h/L; AUC(0→∞) = 6.40 ± 1.56 μmol·h/L [2]. Pharmacokinetic parameters for main metabolites: Ir-7G: Cmax = 10.7 ± 2.80 μmol/L, Tmax = 9.71 ± 8.18 h; 6-OH-BiA-6G: Cmax = 4.10 ± 1.83 μmol/L, Tmax = 15.3 ± 6.89 h; Te (tectorigenin, demethylated metabolite): Cmax = 0.918 ± 0.400 μmol/L, Tmax = 11.3 ± 1.03 h [2]. The plasma concentrations of conjugated metabolites (e.g., Ir-7G, 6-OH-BiA-6G) were much higher than that of the irisolidone aglycone, indicating extensive phase II metabolism [2]. |
| Toxicity/Toxicokinetics |
Irisolidone is described as a natural compound with low toxicity and fewer side effects in the body compared to synthetic agents, though no specific toxicity data (e.g., LD50) are provided in the given texts [1].
No specific in vitro or in vivo toxicity data (e.g., hepatotoxicity, nephrotoxicity, protein binding) for irisolidone were reported in the provided literature [1, 2, 3]. |
| References |
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| Additional Infomation |
Irisone belongs to the 4'-methoxyisoflavones class of compounds. It has been reported to exist in Iris pseudobulbus, Iris truncata, and other organisms with relevant data.
Irisolidone is the active aglycone metabolite of the prodrug kakkalide, which is found in Pueraria lobata flowers. Kakkalide is transformed into irisolidone by human intestinal bacteria, and irisolidone exhibits more potent hepatoprotective and anti-inflammatory activity than kakkalide [1]. Irisolidone inhibits JC virus gene expression at least in part by suppressing Sp1 binding to the Sp1-II site on the JC virus promoter region, which is important for basal JC virus promoter activity [1]. The inhibitory effect of irisolidone on JC virus may provide a new therapeutic modality for progressive multifocal leukoencephalopathy (PML) and other CNS disorders caused by this virus [1]. Irisolidone is a potent VRAC inhibitor, and VRAC inhibition by flavonoids like irisolidone might be responsible for their anti-angiogenic effects [3]. |
| Molecular Formula |
C17H14O6
|
|---|---|
| Molecular Weight |
314.2895
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| Exact Mass |
314.079
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| CAS # |
2345-17-7
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| PubChem CID |
5281781
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| Appearance |
White to off-white solid
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| Density |
1.402g/cm3
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| Boiling Point |
565.5ºC at 760mmHg
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| Melting Point |
192 ºC (methanol )
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| Flash Point |
212.3ºC
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| Vapour Pressure |
2.15E-13mmHg at 25°C
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| Index of Refraction |
1.645
|
| LogP |
2.888
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
23
|
| Complexity |
468
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O1C([H])=C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])C(C2C(=C(C(=C([H])C1=2)O[H])OC([H])([H])[H])O[H])=O
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| InChi Key |
VOOFPOMXNLNEOF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H14O6/c1-21-10-5-3-9(4-6-10)11-8-23-13-7-12(18)17(22-2)16(20)14(13)15(11)19/h3-8,18,20H,1-2H3
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| Chemical Name |
5,7-dihydroxy-6-methoxy-3-(4-methoxyphenyl)chromen-4-one
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~318.18 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.95 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.95 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 | 3.1818 mL | 15.9089 mL | 31.8177 mL | |
| 5 mM | 0.6364 mL | 3.1818 mL | 6.3635 mL | |
| 10 mM | 0.3182 mL | 1.5909 mL | 3.1818 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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