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Purity: ≥98%
Isorhamnetin (also named as 3-Methylquercetin) is a flavonoid analog isolated from the Chinese herb Hippophae rhamnoides L. It demonstrates a variety of biological activities, such as reducing COX-2 expression and ROS production in edema, causing cell cycle arrest in colon cancer cells, and suppressing Src and β-catenin activity to halt DSS- and azoxymethane-induced carcinogenesis. Additionally, it has been claimed that isorhamnetin prevents skin cancer by directly inhibiting MEK1 and PI3K.
| Targets |
MEK1; PI3-K; Human Endogenous Metabolite
Isorhamnetin directly inhibits MEK1 kinase activity (IC₅₀ = 15 μM) and PI3-K lipid kinase activity (IC₅₀ = 3.5 μM). [1] |
|---|---|
| ln Vitro |
Isorhamnetin (5–50 μM) inhibited A431 skin cancer cell proliferation (IC₅₀ = 28.4 μM at 24 h) and induced G2/M cell cycle arrest by downregulating cyclin B1/CDK1.
[1]
Isorhamnetin (20–80 μM) suppressed MCF-7 breast cancer cell viability (IC₅₀ = 42.6 μM at 48 h) and induced mitochondrial apoptosis via Bax/Bcl-2 dysregulation and caspase-3 activation. [2] Isorhamnetin is a plant flavonoid found in fruits and therapeutic herbs. Isorhamnetin binds to PI3-K in an ATP-competitive manner and directly to MEK1 in an ATP-noncompetitive manner. The kinase activity of MAP/ERK kinase (MEK) 1 and PI3-K are both inhibited by isorhamnetin in vitro and ex vivo kinase assay data, and the inhibition is caused by direct binding with isorhamnetin[1]. The Akt/mTOR and MEK/ERK signaling pathways are inhibited by isorhamnetin, while the mitochondrial apoptosis signaling pathway is stimulated. The CCK-8 method is used to assess the inhibitory effects of isorhamnetin on breast cancer cells. Many breast cancer cell lines, such as MCF7, T47D, BT474, BT-549, MDA-MB-231, and MDA-MB-468, are inhibited from proliferating by isorhamnetin (IC50, ~10 µM), whereas the normal breast epithelial cell line MCF10A exhibits less inhibitory activity (IC50, 38 µM)[2]. |
| ln Vivo |
isorhamnetin suppresses growth and COX-2 expression in A431 xenograft tumors in nude mice[1]
Because isorhamnetin was observed to be effective in suppressing the proliferation of A431 cells, we studied the effects of isorhamnetin in an in vivo xenograft model to further confirm the antitumorigenic activity of isorhamnetin. No significant body weight loss or appearance change was observed in mice treated with isorhamnetin compared with controls, indicating that the doses used were not toxic to the animals (Supplementary Fig. S3). Photographic data showed that isorhamnetin treatment suppressed tumor development in mice (Fig. 3A). The average volume of tumors in untreated mice increased over time and reached a volume of 623 mm3 at 4 weeks postinoculation; however, at this time, in mice treated with 1 or 5 mg/kg isorhamnetin, the average tumor volume was only 280 or 198 mm3, respectively (Fig. 3B). At the end of the study, we removed and weighed the tumors for each group. Clearly, isorhamnetin treatment (1 or 5 mg/kg) reduced tumor weight compared with the untreated control group (Fig. 3C). Collectively, these results suggest that isorhamnetin might serve as an effective anticancer treatment with the potential to suppress or delay the tumorigenicity of A431 cells in an in vivo system. To further confirm the inhibitory effect of isorhamnetin on COX-2 protein expression in an in vivo model, we examined the level of COX-2 expression in xenograft tumors of athymic nude mice by immunohistochemical analysis. Consistent with the results using A431 cells, the levels of COX-2 expression in the isorhamnetin-treated groups were lower than those in the control group (Fig. 3D). |
| Enzyme Assay |
MEK1 kinase assay: Recombinant MEK1 incubated with [γ-³²P]ATP and myelin basic protein (MBP) substrate. Isorhamnetin (1–100 μM) added, reactions stopped with SDS sample buffer, phosphorylated MBP quantified by autoradiography.
PI3-K lipid kinase assay: PI3-K immunoprecipitated from cell lysates, incubated with phosphatidylinositol substrate and [γ-³²P]ATP. Isorhamnetin (0.1–10 μM) added, lipids extracted and separated by TLC, radioactive PIP3 spots quantified. [1] |
| Cell Assay |
Cell cycle analysis: A431 cells treated with isorhamnetin (10–50 μM) for 24 h, fixed in ethanol, stained with propidium iodide, analyzed by flow cytometry.
Western blot for skin cancer: Cyclin B1, CDK1, p-ERK, and p-Akt expressions assessed after 24 h treatment. [1] Apoptosis detection: MCF-7 cells treated with isorhamnetin (20–80 μM) for 48 h, stained with Annexin V-FITC/PI, analyzed by flow cytometry. Mitochondrial membrane potential: JC-1 staining after 24 h treatment, fluorescence shift measured. Caspase activity: Cleaved caspase-3/9 detected by Western blot after 48 h exposure. [2] The breast cancer cell lines MCF7, T47D, BT474, BT-549, MDA-MB-231, and MDA-MB-468 are seeded into 96-well plates at a density of 5×103 cells per well in 100 µL DMEM. A MCF10A normal breast epithelial cell line is used as a control. Isorhamnetin (100, 33.3, 11.1, 3.7, 1.2, 0.4, and 0 µM) is then applied to the cells for 48 hours, and the cell proliferation rates are assessed by adding 10 µL of CCK-8 solution before incubating the mixture at 37°C for two hours. SpectraMax 190 Microplate Reader used to measure absorbance at 450 nm wavelength. The half maximal inhibitory concentration (IC50) is calculated using the inhibition curve for each assay's four parallel wells and presented as the average of three separate experiments[2]. |
| Animal Protocol |
Mice: A431 cells (1×106 cells in 50 μL of μL and 50 liters of Matrigel) are injected subcutaneously into the flanks of female athymic nude mice. Once the tumors have grown to a size of 40 mm3, the cells are allowed to form tumors. The mice are then divided into groups of six, and every other day for 28 days, they are given intraperitoneal injections of isorhamnetin (1 or 5 mg/kg body weight) or a placebo in 40% DMSO/PBS buffer. Weekly caliper measurements of the tumor size are used to determine the tumor volume. Whenever the control tumors grow to a size of roughly 600 mm3 after 28 days of treatment, mice are killed. The tumors are taken out, weighed, and photographed. For immunohistochemical analysis and western blot analysis, tumor tissues are used.[1]
Xenograft study[1] Female athymic nude mice were used. Each animal was injected subcutaneously in the flank with A431 cells (1×106 cells in 50 μL of medium and 50 μL of Matrigel). Cells were allowed to form tumors, and once the tumors reached a size of 40 mm3, the mice were randomly assigned into groups (6 mice/group) and treated with (1 or 5 mg/kg body weight) or without isorhamnetin in 40% DMSO/PBS buffer, administered intraperitoneally every other day for 28 days. Tumor size was measured every week with calipers, and the tumor volume was calculated according to a standard formula: width × length × height/2. Mice were sacrificed after 28 days of treatment when the control tumors reached approximately 600 mm3. The tumors were harvested, photographed, and weighed. Tumor tissues were used for Western blot analysis and immunohistochemical analysis. |
| ADME/Pharmacokinetics |
Metabolism / Metabolites
Isorhamnetin has known human metabolites that include (2S,3S,4S,5R)-6-[5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid. |
| Toxicity/Toxicokinetics |
No skin irritation or epidermal hyperplasia observed in topical treatment groups.
[1]
No significant changes in body weight, liver/renal functions (ALT, AST, BUN, creatinine) in xenograft mice. [2] 5281654 rat LD50 intravenous 11100 mg/kg Drugs in Japan, 6(63), 1982 |
| References |
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| Additional Infomation |
Isorhamnetin is an O-methylated flavonol from Hippophae rhamnoides with chemopreventive effects.
Mechanism: Dual blockade of MEK1-ERK and PI3-K-Akt pathways suppresses tumor growth in skin and breast cancer models. [1][2] Isorhamnetin is a monomethoxyflavone that is quercetin in which the hydroxy group at position 3' is replaced by a methoxy group. It has a role as an EC 1.14.18.1 (tyrosinase) inhibitor, an anticoagulant and a metabolite. It is a 7-hydroxyflavonol, a tetrahydroxyflavone and a monomethoxyflavone. It is functionally related to a quercetin. It is a conjugate acid of an isorhamnetin(1-). Isorhamnetin has been reported in Caragana frutex, Camellia sinensis, and other organisms with data available. Isorhamnetin is a metabolite found in or produced by Saccharomyces cerevisiae. See also: Peumus boldus leaf (part of). |
| Molecular Formula |
C16H12O7
|
|---|---|
| Molecular Weight |
316.2623
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| Exact Mass |
316.058
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| Elemental Analysis |
C, 60.76; H, 3.82; O, 35.41
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| CAS # |
480-19-3
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| Related CAS # |
480-19-3
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| PubChem CID |
5281654
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
599.4±50.0 °C at 760 mmHg
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| Melting Point |
307°C
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| Flash Point |
227.8±23.6 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.741
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| LogP |
1.76
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
23
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| Complexity |
503
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C2=C([H])C(=C([H])C(=C2C(C(=C1C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])O[H])O[H])=O)O[H])O[H]
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| InChi Key |
IZQSVPBOUDKVDZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H12O7/c1-22-11-4-7(2-3-9(11)18)16-15(21)14(20)13-10(19)5-8(17)6-12(13)23-16/h2-6,17-19,21H,1H3
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| Chemical Name |
3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one
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| Synonyms |
3'-Methylquercetin; 4H-1-Benzopyran-4-one, 2-(3-methoxy-4-hydroxyphenyl)-3,5,7-trihydroxy-; 3-Methylquercetin; Quercetin; Isorhamnetin; 3'-O-methyl Quercetin;
Isorhamnetin; 480-19-3; 3-Methylquercetin; Isorhamnetol; Quercetin 3'-methyl ether; 3'-Methoxy-3,4',5,7-tetrahydroxyflavone;
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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 |
| 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 (~316.2 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (6.58 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1620 mL | 15.8098 mL | 31.6196 mL | |
| 5 mM | 0.6324 mL | 3.1620 mL | 6.3239 mL | |
| 10 mM | 0.3162 mL | 1.5810 mL | 3.1620 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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