| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 100mg |
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| Other Sizes |
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| Targets |
EphA receptors
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| ln Vitro |
Among the 21 phenolics screened by an ELISA-binding assay, just urolithin C, urolithin D, and ellagic acid succeeded to inhibit the EphA2-ephrin-A1 binding. Urolithin D, the most active, was a competitive and reversible antagonist of EphA receptors able to discriminate between EphA and EphB receptors, showing intra-classes selectivity. Molecular modeling and structure-activity relationships shed light on the binding mode and selective activity of urolithin D. This catabolite blocked EphA2 phosphorylation mediated by ephrin-A1, while lacking cytotoxicity and anti-proliferative effects, and was inactive on the EphA2 kinase assay.
Conclusion: The mechanisms behind the cancer preventive properties of foods rich in flavan-3-ols and caffeoylquinic acids are not associated with metabolic pathways directly linked to the Eph-ephrin system. However, the ellagitannin-derived colonic metabolite urolithin D was able to exert remarkable and selective EphA-ephrin-A inhibition, which might impact on prostate cancer prevention [1].
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| Enzyme Assay |
EphA2 kinase activity [1]
The kinase activity of the EphA2 kinase domain was evaluated with the “EphA2 kinase assay/inhibitor assay kit” following manufacturer instructions. EphA2 kinase activity is not inhibited by Urolithin D/UroD [1] The observed inhibition of the EphA2 phosphorylation could also be linked to a direct inhibition of the kinase domain. To rule out this hypothesis, 30 μM of UroD were incubated with EphA2 kinase domain, a substrate peptide, and appropriate reagents. Under these conditions, 100 nM of dasatinib, used as reference compound, completely blocked EphA2 kinase activity whereas UroD was inactive, thus confirming its action as protein-protein inhibitor (Fig. 8). EGFR kinase activity [1] In order to partially assess the specific interaction of Urolithin D/UroD with Eph-receptors, we performed a functional assay on another tyrosine-kinase receptor (EGFR). UroD 30 μM was completely inactive when tested against the phosphorylation of EGF receptors induced by EGF in PC3 cells whereas the EGFR kinase inhibitor gefitinib (10 μM), used as reference compound, completely abolished response to EGF (Fig. 9). |
| Cell Assay |
Phosphorylation of EphA2 and EGFR in cells [1]
EphA2-, and EGFR-phosphorylation was measured in cell lysates using DuoSet® IC Sandwich ELISA following manufacturer's protocol. Briefly, 96-well ELISA high binding plates were incubated overnight with 100 μL/well of the specific capture antibody diluted in sterile PBS at the proper working concentrations. Next day, the wells were washed and blocked for 1 h and 100 μL/well of lysates (10 μg/well) were added for 2 h. Then, wells were incubated with the specific detection antibody and the phosphorylation level was revealed utilizing a standard HRP format and tetra-methylbenzidine through a colorimetric reaction read at 450 nm. Each step was performed at room temperature and followed by the washing of each well. LDH and MTT assays [1] Cytotoxicity was evaluated with CytoTox 96® Non-Radioactive Cytotoxicity Assay, following the manufacturer's protocol. Cells were seeded in 96-well plates (100 μL/well) at a density of 105 cells/mL and the day after treated with compounds or lysis buffer for 2 or 15 h. After incubation, released LDH in culture supernatants was measured using a 30-min coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of colour formed is proportional to the number of lysed cells and quantified by an ELISA plate reader at 492 nm. The results were expressed as the ratio between absorbance of the cells treated with the compounds and the cells treated with lysis buffer. Cell viability was evaluated using the MTT colorimetric assay. Cells were seeded in 96-well plates at a density of 105cells/mL and the day after treated with compounds or 1% DMSO for 15 or 72 h. MTT was added at the final concentration of 1 mg/mL and incubated for 2 h. The resulting formazan crystals were solubilized with DMSO 100 μL/well. The absorbance was measured at 550 nm using an ELISA plate reader and the results were expressed as the ratio between absorbance of the cell treated with the compounds and untreated cells. |
| References | |
| Additional Infomation |
Urolithin D is a hydroxycoumarin.
Urolithin D has been reported in Punica granatum with data available. In conclusion, the results of this study suggest that the mechanisms behind the reported cancer preventive properties of foods rich in flavan-3-ols and caffeoylquinic acids are not associated with metabolic pathways directly linked to the Eph-ephrin system. On the contrary, UroD, an ellagitannin-derived colonic metabolite, was able to exert selective EphA-ephrin-A inhibition, which could impact on prostate cancer prevention. This selectivity feature could be of particular interest because intestinal cells express the Eph-ephrin system, where it regulates the renewal of the epithelium through the activity of stem cells located at the bottom of the Lieberkühn crypts 52 by regulating cell migration patterns on the crypt-villus axis. Notably, this process is mediated by EphB receptors interacting with ephrin-Bs. The lack of activity of UroD on EphB receptors could be useful to avoid interference with intestinal layer renewal. However, as the large intestine is the site where UroD reaches its highest concentration in human tissues, studying the ability of UroD to inhibit colon cancer cell migration deserves further, more detailed studies on the putative multiple effects of this ellagitannin metabolite in cancer prevention.[1] |
| Molecular Formula |
C13H8O6
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|---|---|
| Molecular Weight |
260.20
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| Exact Mass |
260.032
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| CAS # |
131086-98-1
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| PubChem CID |
5482042
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| Appearance |
White to off-white solid powder
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| Density |
1.8±0.1 g/cm3
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| Boiling Point |
634.1±55.0 °C at 760 mmHg
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| Flash Point |
253.0±25.0 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.801
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| LogP |
1.07
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
19
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| Complexity |
372
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NEZDQSKPNPRYAW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H8O6/c14-8-2-1-5-6-3-9(15)10(16)4-7(6)13(18)19-12(5)11(8)17/h1-4,14-17H
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| Chemical Name |
3,4,8,9-tetrahydroxybenzo[c]chromen-6-one
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| Synonyms |
Urolithin D; 131086-98-1; 3,4,8,9-Tetrahydroxybenzo[c]chromen-6-one; CHEMBL6338; 3,4,8,9-Tetrahydroxy-6H-benzo[c]chromen-6-one; 6H-Dibenzo(b,d)pyran-6-one, 3,4,8,9-tetrahydroxy-; 6H-Dibenzo[b,d]pyran-6-one, 3,4,8,9-tetrahydroxy-; Urolithin-D;
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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) |
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 | 3.8432 mL | 19.2160 mL | 38.4320 mL | |
| 5 mM | 0.7686 mL | 3.8432 mL | 7.6864 mL | |
| 10 mM | 0.3843 mL | 1.9216 mL | 3.8432 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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