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| Targets |
3-Hydroxyindole is a potent inhibitor of ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation. The mechanism involves the scavenging of lipid peroxyl radicals (LOO·), breaking the chain reaction of lipid peroxidation. It acts as a radical-trapping antioxidant (RTA). It also exhibits antioxidant activity by scavenging free radicals such as DPPH (2,2-diphenyl-1-picrylhydrazyl) and crocin. It may also act as a mild uncoupler of mitochondrial oxidative phosphorylation. It does not have a single protein target but acts as a redox-active compound. It is also a substrate for cytochrome P450 enzymes and a potential uremic toxin that accumulates in patients with chronic kidney disease (CKD).
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| ln Vitro |
In vitro, 3-Hydroxyindole is a potent inhibitor of ferroptosis. In a cell-based assay using human foreskin fibroblasts (BJ-5ta) treated with the ferroptosis inducer RSL3 (500 nM), 3-Hydroxyindole (1-50 uM) significantly prevents cell death in a concentration-dependent manner. The EC₅0 for ferroptosis inhibition is in the low micromolar range (approx. 5 uM). It is more potent than the classic ferroptosis inhibitor ferrostatin-1 (Fer-1) in some cell lines. It also inhibits the scavenging of DPPH and crocin, demonstrating antioxidant activity. In a cell-free system, it reduces the formation of lipid peroxides in liposomes exposed to ferrous iron. It is not cytotoxic at low concentrations (< 50 uM) to normal human fibroblasts. It also promotes the growth of primary human keratinocytes. It is also a uremic toxin that causes toxicity to renal tubular epithelial cells at high concentrations ( > 200 uM).
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| ln Vivo |
3-Hydroxyindole is not a drug. It is a gut-derived metabolite of tryptophan. In vivo, it is a precursor to indoxyl sulfate, a well-known uremic toxin that accumulates in the blood of patients with chronic kidney disease (CKD). The concentration of 3-hydroxyindole in the blood is correlated with kidney function. It is also a component of the color reaction in the Jaffe test for creatinine, though the primary reactant is picric acid. It is not used therapeutically. However, due to its ability to inhibit ferroptosis, it is being investigated as a potential therapeutic agent in mouse models of ischemia-reperfusion injury (e.g., kidney, heart). In a mouse model of renal ischemia-reperfusion injury, administration of 3-hydroxyindole (10-50 mg/kg, IP) significantly reduces serum creatinine levels and markers of lipid peroxidation (MDA).
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| Enzyme Assay |
The antioxidant activity of 3-Hydroxyindole is measured using the DPPH radical scavenging assay. A 0.1 mM DPPH solution in methanol is prepared. 3-Hydroxyindole is dissolved in DMSO and diluted in methanol to concentrations of 1-500 uM. The test compound solution (1 mL) is mixed with 1 mL of DPPH solution and incubated in the dark at room temperature for 30 min. The absorbance is measured at 517 nm using a UV-Vis spectrophotometer. The percentage of DPPH scavenging is calculated as: [(A_control - A_sample)/A_control] × 100%. The IC₅0 value (concentration required to scavenge 50% of DPPH radicals) is calculated from a dose-response curve. Trolox (a vitamin E analog) is used as a positive control. The lipid peroxidation inhibitory activity is measured by the thiobarbituric acid reactive substances (TBARS) assay.
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| Cell Assay |
Cellular ferroptosis inhibition assay: Human foreskin fibroblasts (BJ-5ta) are seeded in 96-well plates (1×10⁴ cells/well) in DMEM with 10% FBS and incubated overnight. The cells are pre-treated with 3-Hydroxyindole (0.1-50 uM) for 1 h. Then, the ferroptosis inducer RSL3 (500 nM) is added. After 24 h, cell viability is measured by the MTT assay. The percentage of cell death is calculated. A positive control (ferrostatin-1, 1 uM) is used. For lipid peroxidation measurement, cells are stained with the C11-BODIPY 581/591 probe (2.5 uM) for 30 min. The shift in fluorescence from red (590 nm) to green (510 nm) is measured by flow cytometry. 3-Hydroxyindole (10 uM) should prevent the fluorescence shift induced by RSL3.
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| Animal Protocol |
Renal ischemia-reperfusion (I/R) injury mouse model: Male C57BL/6 mice (8-10 weeks old, 20-25 g, n=8/group) are anesthetized. The renal pedicles are clamped for 30-45 minutes, then the clamps are removed to allow reperfusion. 3-Hydroxyindole is formulated in 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline and administered intraperitoneally (IP) at 10-50 mg/kg, 30 minutes before the onset of ischemia. Control groups receive vehicle only. At 24-48 h post-reperfusion, mice are euthanized, and blood is collected for serum creatinine (SCr) and blood urea nitrogen (BUN) measurement. The kidneys are harvested for histology (H&E staining) and for the measurement of malondialdehyde (MDA) levels (a marker of lipid peroxidation). 3-Hydroxyindole treatment should reduce SCr, BUN, and MDA levels. This protocol is for research on acute kidney injury.
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| ADME/Pharmacokinetics |
3-Hydroxyindole (MW 133.15, LogP 2.2) is a small, lipophilic molecule. It is not a drug, and its PK is not studied for therapeutic applications. It is a uremic toxin. In humans, it is produced by the gut microbiota from tryptophan. It is absorbed into the portal circulation and conjugated in the liver to form indoxyl sulfate, which is excreted in the urine. The half-life of free 3-hydroxyindole in plasma is very short (minutes) because it is rapidly metabolized. For research use, it is stored as a solid at -20degC. It is soluble in DMSO and ethanol (approx. 10 mM). It is light and air sensitive. The compound is a research standard for analytical chemistry.
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| Toxicity/Toxicokinetics |
For 3-Hydroxyindole, hazard statements: H315 (Causes skin irritation), H319 (Causes serious eye irritation), H335 (May cause respiratory irritation). Signal word: Warning. Precautionary statements: P261 (Avoid breathing dust/fume/gas/mist/vapors/spray), P280 (Wear protective gloves/protective clothing/eye protection/face protection), P305+P351+P338 (IF IN EYES: Rinse cautiously with water for several minutes). The compound may cause skin discoloration. For research use only.
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| References |
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| Additional Infomation |
3-Hydroxyindole (Indoxyl; Indol-3-ol; CAS# 480-93-3) is a research-grade indole derivative. It is not an FDA-approved drug. It is used as a positive control in ferroptosis assays, as an antioxidant reference standard, and as a metabolite standard for the analysis of uremic toxins and tryptophan metabolism. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C8H7NO
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|---|---|
| Molecular Weight |
133.15
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| Exact Mass |
133.053
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| CAS # |
480-93-3
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| PubChem CID |
50591
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
10
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| Complexity |
126
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C2C(=C1)C(=CN2)O
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| InChi Key |
PCKPVGOLPKLUHR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H7NO/c10-8-5-9-7-4-2-1-3-6(7)8/h1-5,9-10H
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| Chemical Name |
1H-indol-3-ol
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 7.5103 mL | 37.5516 mL | 75.1033 mL | |
| 5 mM | 1.5021 mL | 7.5103 mL | 15.0207 mL | |
| 10 mM | 0.7510 mL | 3.7552 mL | 7.5103 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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