- Tyrosinase [1].
- NF‑κB pathway (inhibits IκB‑α phosphorylation) [2].
- HIF‑1α (downregulates HIF‑1α expression) [2].

- In a RAW 264.7 mouse monocyte macrophage cell line stimulated with RANKL (30 ng/mL) and P. gingivalis lipopolysaccharide (1 μg/mL), Velutin at concentrations of 0.5, 1, and 2 μM showed no cytotoxicity (Alamar Blue assay, p > 0.05) and did not induce apoptosis (caspase 3/7 assay, p > 0.05). DAPI staining revealed no alteration in nuclear morphology compared to control (p > 0.05). Velutin reduced osteoclast differentiation in a dose‑dependent manner as assessed by TRAP staining and total TRAP activity (p < 0.05 for large osteoclasts with ≥10 nuclei; p < 0.001 for total TRAP activity). Western blot analysis showed that Velutin (2 μM) decreased RANKL/Pg‑LPS‑induced IκB‑α phosphorylation and HIF‑1α protein levels after 30 min stimulation (p < 0.001) [2].
- In a zebrafish embryo model, Velutin at 30 and 300 μg/mL reduced melanocyte development (early melanogenesis) in a dose‑dependent manner when treated from 5 to 30 h post‑fertilization (hpf). Velutin also efficiently inhibited melanin synthesis in a PTU‑reversal assay (pre‑treatment with PTU from 12 hpf, then velutin from 36 to 56 hpf) in a dose‑dependent manner, mimicking the effect of the microwave‑assisted aglycone extract of mistletoe [1].

- In zebrafish embryos, Velutin (30 and 300 μg/mL in 1% DMSO/E3 medium) inhibited early melanocyte development when administered from 5 to 30 hpf, and also prevented melanin synthesis when applied after PTU washout from 36 to 56 hpf, both in a dose‑dependent manner. No significant cell death was observed at up to 300 μg/mL as measured by acridine orange (AO) staining (p < 0.001 compared to control) [1].
- In RAW 264.7 cells, Velutin (2 μM) reduced osteoclast formation and downregulated HIF‑1α expression via the NF‑κB pathway under RANKL/Pg‑LPS stimulation [2].

- Tyrosinase inhibition assay: The inhibitory effect on tyrosinase activity was assessed by measuring dopachrome produced from the catalytic reaction of tyrosinase. Briefly, 50 μL of Velutin‑containing extract (or ascorbic acid as control) at concentrations ranging from 0.05 to 5 mg/mL was mixed with 50 μL of mushroom tyrosinase (50 U/mL) in phosphate buffer (pH 6.8) in a 96‑well plate and incubated for 30 min at room temperature. Then, 50 μL of 1 mM L‑DOPA was added and further incubated for 10 min at 37 °C. After mixing with 50 μL of 400 ng/mL FITC solution, fluorescence intensity was measured. The aglycone‑rich extract containing Velutin completely inhibited tyrosinase activity at 5 mg/mL [1].
- Western blot analysis for NF‑κB and HIF‑1α: RAW 264.7 cells were stimulated with RANKL (30 ng/mL) and Pg‑LPS (1 μg/mL) and treated with Velutin (2 μM) for 30 min. Cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Lysates were centrifuged at 13,000 rpm for 20 min, and protein concentrations determined. Lysate (10 μg protein/lane) was separated by SDS‑PAGE, transferred to nitrocellulose membrane, blocked with 5% BSA/PBS‑Tween, and probed with antibodies against HIF‑1α, IκBα, p‑IκBα, and β‑actin. Bands were detected using enhanced chemiluminescence (ECL) [2].

- Cell viability assay (Alamar Blue): RAW 264.7 cells were seeded in 96‑well plates (5×10³ cells/well) and treated with Velutin at 0.5, 1, and 2 μM for 5 days with or without RANKL (30 ng/mL) and Pg‑LPS (1 μg/mL). After 5 days, 10% Alamar Blue was added, incubated for 4 h, and the absorbance measured at 570 nm and 600 nm. No significant reduction in metabolic activity was observed at any concentration (p > 0.05) [2].
- Total protein assay (BCA): Cells were cultured in 96‑well plates with or without Velutin for 5 days. Cells were washed with PBS and lysed in buffer containing 90 mM trisodium citrate, 10 mM NaCl, 0.1% Triton X‑100 (pH 4.8). Total protein was measured using BCA assay at 562 nm. No significant difference from control (p > 0.05) [2].
- Caspase‑3/7 activity assay: RAW 264.7 cells were treated with Velutin (0.5–2 μM) for 5 days. Caspase‑Glo 3/7 reagent (100 μL) was added to each well, incubated for 1 h at room temperature, and luminescence measured. No significant increase in caspase‑3/7 activity was observed compared to control (p > 0.05) [2].
- DAPI staining: Cells treated with Velutin for 5 days were stained with DAPI. Fluorescence microscopy revealed no fragmented or condensed nuclei, indicating no apoptosis (p > 0.05) [2].
- TRAP assay (qualitative and quantitative): For histological TRAP staining, cells were fixed and stained with a TRAP buffer (pH 5.0) containing naphthol AS‑MX phosphate and Fast Red Violet LB salt. For quantitative TRAP, cells were lysed in citrate buffer (pH 4.8) with 0.1% Triton X‑100, then substrate (p‑nitrophenyl phosphate) and tartaric acid buffer (40 mM L‑tartaric acid, pH 4.0) were added. Absorbance was measured at 405 nm. Velutin significantly reduced TRAP activity in a dose‑dependent manner (p < 0.001) [2].
- Zebrafish embryo melanocyte development assay: Embryos were treated with Velutin at 0, 30, and 300 μg/mL in 1% DMSO/E3 medium from 5 to 30 hpf, then bright‑field images were taken at 30 hpf. Velutin reduced pigment cell development dose‑dependently [1].
- Zebrafish embryo melanin synthesis (PTU‑reversal) assay: Embryos were pre‑treated with 200 μM PTU from 12 hpf, washed with 1% DMSO/E3 at 36 hpf, then exposed to Velutin at 0, 30, and 300 μg/mL from 36 to 56 hpf. Bright‑field images at 56 hpf showed dose‑dependent inhibition of melanin synthesis [1].

- Zebrafish embryo protocol for melanogenesis inhibition: Wild‑type zebrafish (AB strain) embryos were raised in E3 medium (5 mM NaCl, 0.33 mM MgSO₄, 0.33 mM CaCl₂, 0.17 mM KCl) at 28.5 °C. Velutin was dissolved in DMSO and diluted in E3 medium to a final concentration of 1% DMSO. For melanocyte development assay, embryos were treated with Velutin at 0, 30, and 300 μg/mL from 5 to 30 hpf (five embryos per treatment in 2 mL E3 medium per well in 24‑well plates). For melanin synthesis assay, embryos were first treated with 200 μM PTU from 12 hpf, dechorionated at 36 hpf, washed twice with 1% DMSO/E3, then treated with Velutin (0, 30, 300 μg/mL) from 36 to 56 hpf. At endpoints, embryos were anesthetized with 0.02% tricaine and mounted in 3% methylcellulose for bright‑field imaging using a stereoscope [1].
- Zebrafish embryo toxicity (AO staining) protocol: Embryos were pre‑treated with PTU from 12 hpf, then treated with Velutin (0, 30, 300 μg/mL) from 36 to 52 hpf. Live embryos were stained with 3 μg/mL acridine orange in E3 medium for 20 min in the dark, washed twice, anesthetized, and imaged using a confocal microscope (488 nm excitation, 415‑735 nm emission). The number of AO‑positive cells was counted. Valproic acid (16.6 μg/mL) was used as positive control. No significant cell death was observed with Velutin up to 300 μg/mL [1].

- In RAW 264.7 cells, Velutin at concentrations up to 2 μM showed no cytotoxicity as measured by Alamar Blue assay (p > 0.05), no increase in caspase‑3/7 activity (p > 0.05), and no nuclear morphological changes by DAPI staining (p > 0.05). Similarly, under RANKL/Pg‑LPS stimulation, Velutin up to 2 μM did not affect cell metabolic activity or total protein levels (p > 0.05) [2].
- In zebrafish embryos, Velutin at 300 μg/mL did not cause significant cell death as quantified by acridine orange staining. The ratio of AO‑positive cells in velutin‑treated embryos compared to vehicle control was not significantly different (p < 0.001 for comparison with positive control, no significant difference from control) [1].

[1]. Velutin, an Aglycone Extracted from Korean Mistletoe, with Improved Inhibitory Activity against Melanin Biosynthesis. Molecules. 2019 Jul 12;24(14).

[2]. Cytotoxicity and potential anti-inflammatory activity of velutin on RAW 264.7 cell line differentiation: Implications in periodontal bone loss. Arch Oral Biol. 2017 Nov;83:348-356.

Verutin is a dimethoxyflavonoid, a derivative of luteolin, with its 7' and 3' hydroxyl groups replaced by methoxy groups. It possesses various activities including anti-inflammatory, phytometabolic, melanin-inhibiting, antibacterial, antioxidant, and anti-allergic effects. Verutin is a dimethoxyflavonoid and a dihydroxyflavonoid, with its function related to 4',5,7-trihydroxy-3'-methoxyflavonoid. Verutin has been reported in Ajania fastigiata, Capsicum annuum, and several other organisms with relevant data. See also: Acai (partial).

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- Velutin is an aglycone flavonoid generated by microwave‑assisted hydrolysis of homoflavoyadorinin B glycoside from Korean mistletoe. The microwave‑assisted aglycone extract showed improved tyrosinase inhibition and antioxidant activity compared to the glycoside extract. Velutin was identified as a major inhibitory component of this extract [1].
- In the context of periodontal inflammation, Velutin inhibits osteoclast differentiation and downregulates HIF‑1α via the NF‑κB pathway, suggesting potential for treating inflammatory bone disorders such as periodontitis [2].
- Velutin (5‑hydroxy‑2‑(4‑hydroxy‑3‑methoxyphenyl)‑7‑methoxychromen‑4‑one) has a molecular formula C₁₇H₁₄O₆ and molar mass 314.29 g/mol [2].

C17H14O6

314.2895

314.079

25739-41-7

5464381

White to yellow solid

1.4±0.1 g/cm3

567.5±50.0 °C at 760 mmHg

213.1±23.6 °C

0.0±1.6 mmHg at 25°C

1.646

2.06

2

6

3

23

476

0

O1C(=C([H])C(C2=C(C([H])=C(C([H])=C12)OC([H])([H])[H])O[H])=O)C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])O[H]

ROCUOVBWAWAQFD-UHFFFAOYSA-N

InChI=1S/C17H14O6/c1-21-10-6-12(19)17-13(20)8-14(23-16(17)7-10)9-3-4-11(18)15(5-9)22-2/h3-8,18-19H,1-2H3

5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxychromen-4-one

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~159.09 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)