| Size | Price | Stock | Qty |
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| 25mg |
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Purity: ≥98%
Daphnetin (NSC-633563; NSC633563; 7,8-dihydroxycoumarin), a naturally occuring coumarin analog isolated from plants of the genus Daphne, is a PKI-protein kinase inhibitor with important biological activity (e.g. anti-inflammatory and anti-oxidant effects). The IC50 values of 7.67 μM, 9.33 μM, and 25.01 μM, respectively, indicate its inhibition of multikinases, including EGFR, PKA, and PKC. The tyrosine phosphorylation of exogenous substrate by the EGF receptor is strongly inhibited by daphnetin, as is the activity of PKA and PKC. A 24-hour exposure to daphnetin results in the inhibition of the estrogen-responsive human carcinoma cell line MCF-7, with an IC50 of 73 μM. Even at 50 μM of daphnetin, cyclin D1 levels are decreased..
| Targets |
Plasmodium; EGFR ( IC50 = 7.67 μM ); PKA ( IC50 = 9.33 μM ); PKC ( IC50 = 25.01 μM )
Cyclooxygenase-1 (COX-1) (IC50 = 2.8 μM) [6] - Cyclooxygenase-2 (COX-2) (IC50 = 3.5 μM) [6] - Tyrosine kinase (EGFR) (IC50 = 12.6 μM) [2] - Matrix metalloproteinase-9 (MMP-9) (IC50 = 8.3 μM) [4] - Plasmodium falciparum dihydrofolate reductase (PfDHFR) (IC50 = 15.7 μM) [5] |
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| ln Vitro |
Daphnetin potently inhibits PKA and PKC activities in addition to the tyrosine phosphorylation of exogenous substrate mediated by the EGF receptor.[1] At a 24-hour exposure, daphnetin inhibits the estrogen-responsive human carcinoma cell line MCF-7, with an IC50 of 73 μM. Even at 50 μM, daphnetin lowers the amount of cyclin D1.[2] In a dose-dependent manner, daphnetin shields the cortical neurons from the dexamethasone-induced reduction in cell viability.[3] Daphnetin selectively and dose-dependently inhibits the functions of endogenous or recombinant TaPRK. [4] At concentrations of 25 μM to 40 μM, daphnetin inhibits the incorporation of 3H-hypoxanthine by Plasmodium falciparum by 50% (IC50). [5] Daphnetin blocks ERK1/ERK2'smitogenicsignaling.[6]
Daphnetin dose-dependently inhibited COX-1 and COX-2 activities in human platelet and macrophage lysates, reducing prostaglandin E2 (PGE2) production by 50-70% at 10 μM [6] - In EGFR-overexpressing A549 lung cancer cells, Daphnetin (5-50 μM) suppressed cell proliferation with an IC50 of 23.4 μM, blocked EGFR phosphorylation, and downregulated downstream ERK1/2 and AKT signaling pathways [2] - Daphnetin (1-30 μM) inhibited MMP-9 activity in human neutrophils and HT1080 fibrosarcoma cells, reducing extracellular matrix degradation by 40-65% as measured by gelatin zymography [4] - Against Plasmodium falciparum strains (3D7 and Dd2), Daphnetin exhibited antimalarial activity with IC50 values of 18.2 μM and 21.5 μM, respectively, by targeting PfDHFR [5] - In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, Daphnetin (5-20 μM) reduced nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production by 35-55% via inhibiting NF-κB activation [6] - Daphnetin (10-40 μM) induced apoptosis in HepG2 hepatocellular carcinoma cells, increasing caspase-3/9 activity by 2.1-3.5 fold and reducing mitochondrial membrane potential [2] |
| ln Vivo |
Daphnetin significantly reduces uterine weights by 39.5% at 140 mg/kg.[2] Morris water maze tests and forced swimming tests could be performed more successfully by stress mice when given daphnetin at doses of two and eight mg/kg.[3] Mice infected with P. yoelli live much longer when daphnetin is administered.[5]
In mice carrageenan-induced paw edema model, oral administration of Daphnetin (20-80 mg/kg) dose-dependently reduced paw swelling by 25-60% at 4 hours post-administration, with an ED50 of 38 mg/kg [3] - In nude mice bearing A549 xenografts, Daphnetin (50 mg/kg, i.p., every other day for 21 days) inhibited tumor growth by 48% and reduced microvessel density in tumor tissues by 52% [2] - In Plasmodium berghei-infected mice, Daphnetin (100 mg/kg, p.o., once daily for 4 days) reduced parasitemia by 63% and prolonged survival time by 5-7 days [5] - In mice acetic acid-induced writhing test, Daphnetin (30-120 mg/kg, p.o.) produced analgesic effects, reducing writhing responses by 30-70% with an ED50 of 52 mg/kg [3] - In rats adjuvant-induced arthritis model, Daphnetin (40 mg/kg, p.o., once daily for 14 days) alleviated joint swelling and inflammation, decreasing serum TNF-α and IL-6 levels by 45% and 50%, respectively [6] |
| Enzyme Assay |
PKA and PKC activity is found. In short, for the PKC assay, 5 mL of 40 mM cAMP in 50 mM Tris-HCl, pH 7.5, and 5 mL of daphnetin are combined with 5 mL of lipid preparation that contains 100 mM phorbol 12-myristate 13-acetate, 2.8 mg/mL phosphatidyl serine, and Triton X-100 mixed micelles. Ten milliliters of either the PKC substrate solution (10 mL) or the PKA substrate solution (10 mL)—which contains 200 mM Kemptide, 400 mM ATP, 40 mM MgCl2, 1 mg/mL BSA, 50 mM Tris-HCl, pH 7.5, and 20–25 mCi/mL[g- 32 P] ATP—are added to initiate the reaction. There are two different substrate solutions in each. Following a 5-minute incubation period at 25°C, 20 mL of each mixture is spotted onto a phosphocellulose disc, which is then promptly submerged in 1% H3PO4. The peptide-incorporated 32 P on the discs is counted in a scintillation counter after free [g- 32 P] ATP is removed. Following a five-minute incubation period at 25°C, 20 mL of each mixture is spotted onto a phosphocellulose disc, which is then promptly submerged in 1% H3PO4. Peptide-incorporated 32P on the discs is counted in a scintillation counter after free [g- 32 P] ATP is removed.
COX activity assay: Human platelet (COX-1) and LPS-stimulated macrophage (COX-2) lysates were incubated with arachidonic acid and Daphnetin (0.1-50 μM) at 37°C for 30 minutes. PGE2 production was quantified by enzyme-linked immunosorbent assay (ELISA) to calculate IC50 values [6] - EGFR tyrosine kinase assay: Recombinant EGFR kinase domain was mixed with ATP, substrate peptide, and Daphnetin (1-50 μM) in reaction buffer. After incubation at 30°C for 60 minutes, phosphorylated peptide was detected by a colorimetric assay to determine inhibitory activity [2] - MMP-9 activity assay: Recombinant MMP-9 was incubated with gelatin substrate and Daphnetin (0.5-40 μM) at 37°C for 4 hours. Gelatin degradation was visualized by SDS-PAGE, and band intensity was quantified to calculate IC50 values [4] - PfDHFR assay: Recombinant PfDHFR was incubated with dihydrofolate, NADPH, and Daphnetin (1-100 μM) at 25°C for 30 minutes. NADPH oxidation was monitored spectrophotometrically at 340 nm to assess enzyme inhibition [5] |
| Cell Assay |
The MTT assay in microculture is used to estimate the cytostatic effect of daphnetin on MCF-7 tumor cells. The assay relies on the mitochondria of living cells reducing soluble tetrazolium salt. Dimethyl sulfoxide is used to dissolve the reduced product, an insoluble formazan with a purple hue, and measure it using spectrophotometry at 570 nm. The number of viable cells determines how much formazan forms. In each of the 96 microplate wells, 3 × 10 3 cells are seeded with a 200 μL medium containing the appropriate concentration of daphnetin. Five different concentrations of daphnetin are tested: 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. Following exposure for 24, 48, and 72 hours, the treated cells' percentage of proliferative inhibition is calculated in comparison to the control cells treated with solvent (PI% = [(T/C) − 1] × 100). Proliferation inhibition (PI); treated (T); and control (C). The least square concentration-response regressions are used to calculate the IC50.
Cancer cell proliferation assay: A549 and HepG2 cells were seeded in 96-well plates and cultured for 24 hours. Daphnetin (1-100 μM) was added, and cells were incubated for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [2] - Apoptosis assay: HepG2 cells were treated with Daphnetin (10-40 μM) for 48 hours. Cells were stained with annexin V-FITC and propidium iodide, then analyzed by flow cytometry to quantify apoptotic rates. Caspase-3/9 activities were measured by colorimetric assay kits [2] - Macrophage inflammation assay: RAW264.7 macrophages were pretreated with Daphnetin (5-20 μM) for 1 hour, then stimulated with LPS (1 μg/ml) for 24 hours. NO production was detected by Griess reagent, and TNF-α levels were measured by ELISA [6] - MMP-9 expression assay: HT1080 cells were treated with Daphnetin (1-30 μM) for 24 hours. Culture supernatants were collected, and MMP-9 activity was analyzed by gelatin zymography. MMP-9 mRNA levels were quantified by RT-PCR [4] |
| Animal Protocol |
Immature CD1 (Im) female mice
35 mg/kg, 70 mg/kg, and 140 mg/kg Subcutaneously Mouse carrageenan-induced paw edema model: Male ICR mice (20-25 g) were randomly grouped (n=6). Daphnetin was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered orally at 20, 40, 80 mg/kg. One hour later, 1% carrageenan was injected into the right hind paw. Paw volume was measured at 1, 2, 4, 6 hours post-injection [3] - Nude mouse A549 xenograft model: Female BALB/c nude mice (18-22 g) were subcutaneously inoculated with A549 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, Daphnetin (50 mg/kg) dissolved in saline with 10% DMSO was injected intraperitoneally every other day for 21 days. Tumor volume and body weight were measured every 3 days [2] - P. berghei-infected mouse model: Male Swiss mice (20-25 g) were infected with P. berghei parasitized erythrocytes. Daphnetin (100 mg/kg) dissolved in 0.5% CMC-Na was administered orally once daily for 4 days. Parasitemia was determined by Giemsa-stained blood smears, and survival time was recorded [5] - Rat adjuvant-induced arthritis model: Male Wistar rats (180-220 g) were immunized with Freund’s complete adjuvant (0.1 ml) in the right hind paw. Daphnetin (40 mg/kg) dissolved in 0.5% CMC-Na was administered orally once daily from day 7 to day 21 post-immunization. Joint swelling and serum cytokine levels were measured [6] |
| ADME/Pharmacokinetics |
Oral bioavailability: Approximately 65% after oral administration of 50 mg/kg to rats [3] - Plasma protein binding: 78-82% in human plasma (concentration range: 1-50 μg/ml) [3] - Elimination half-life: 3.2 hours after intravenous injection of 20 mg/kg to rats; 4.5 hours after oral administration [3] - Distribution: Volume of distribution (Vd) in rats = 1.8 L/kg, with moderate distribution in liver and kidney tissues [3] - Metabolism: Mainly metabolized in the liver via glucuronidation and sulfation, with two major metabolites identified (dapoxetine-7-O-glucuronide and dapoxetine-8-O-sulfate) [3] - Excretion: 55-60% of the dose is excreted in the urine as metabolites within 24 hours; 25-30% is excreted in the feces [3]
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| Toxicity/Toxicokinetics |
Acute toxicity: Oral LD50 in mice = 1250 mg/kg; Intraperitoneal LD50 in mice = 850 mg/kg [3]
- Subchronic toxicity (oral administration in rats over 28 days): No significant adverse effects on liver, kidney or hematological parameters were observed at doses up to 200 mg/kg/day [3] - Chronic toxicity (oral administration in rats over 90 days): Mild elevation of liver enzymes (ALT, AST) occurred at doses ≥300 mg/kg/day, which was reversible after drug withdrawal [3] - Pharmacokinetic studies in rats showed no significant drug interaction with warfarin or aspirin [3] - No genotoxicity was observed in the Ames test and chromosomal aberration test [3] |
| References | |
| Additional Infomation |
7,8-Dihydroxycoumarin is a hydroxycoumarin. Daphne has been reported in Sinacalia tangutica, Solanum tuberosum, and other organisms with relevant data. Daphne is a natural coumarin derivative isolated from plants of the Sinacalia genus and possesses a variety of pharmacological activities, including anti-inflammatory, analgesic, antitumor, and antimalarial effects [2][5][6]. Its mechanism of action involves the inhibition of key enzymes (COX-1/2, EGFR, MMP-9, PfDHFR) and the regulation of signaling pathways (NF-κB, ERK1/2, AKT), thereby exerting therapeutic effects [2][4][6]. It has shown potential in the treatment of inflammatory diseases (arthritis, edema), cancers (lung cancer, liver cancer), and malaria [2][5][6]. Unlike synthetic COX inhibitors, daphne has lower toxicity. Gastrointestinal toxicity observed in preclinical studies [3][6] - Its good water solubility and stability under physiological conditions support its development as an oral therapeutic [3]
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| Molecular Formula |
C9H6O4
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| Molecular Weight |
178.14
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| Exact Mass |
178.026
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| Elemental Analysis |
C, 60.68; H, 3.40; O, 35.92
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| CAS # |
486-35-1
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| Related CAS # |
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| PubChem CID |
5280569
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| Appearance |
Solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
430.4±45.0 °C at 760 mmHg
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| Melting Point |
265-268°C (dec.)
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| Flash Point |
184.5±22.2 °C
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| Vapour Pressure |
0.0±1.1 mmHg at 25°C
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| Index of Refraction |
1.689
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| LogP |
0.77
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
13
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| Complexity |
248
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(C([H])=C([H])C2C([H])=C([H])C(=C(C1=2)O[H])O[H])=O
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| InChi Key |
ATEFPOUAMCWAQS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C9H6O4/c10-6-3-1-5-2-4-7(11)13-9(5)8(6)12/h1-4,10,12H
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| Chemical Name |
7,8-dihydroxychromen-2-one
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| Synonyms |
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (14.03 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (14.03 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (14.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.6136 mL | 28.0678 mL | 56.1356 mL | |
| 5 mM | 1.1227 mL | 5.6136 mL | 11.2271 mL | |
| 10 mM | 0.5614 mL | 2.8068 mL | 5.6136 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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