Natural flavonoid; tyrosinase i

Baicalein (1), 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-Hydroxykaempferol (14), which are naturally occurring flavonoids from a set of 14 hydroxy-flavones tested, exhibited high inhibitory effects on tyrosinase with respect to L-DOPA, while each of the 5,6,7-trihydroxyflavones 1, 6, 13 or 14 acted as a cofactor to monophenolase. Moreover, 6-hydroxykaempferol (14) showed the highest activity and was a competitive inhibitor of tyrosinase compared to L-DOPA. 5,6,7-Trihydroxyflavones 1, 6, 13 or 14 showed also high antioxidant activities. Hence, we conclude that the 5,6,7-trihydroxy-flavones are useful as good depigmentation agents with inhibitory effects in addition to their antioxidant properties [1].

Tyrosinase inhibition assays [1]
Tyrosinase inhibition assays were performed according to a modified method described by Kubo. Mushroom tyrosinase (EC 1. 14. 18. 1, Sigma Product T3824 with an activity of 3320 units/mg) was used for the bioassay in this study. The tested compounds were first dissolved in DMSO and diluted 25 times with water in each experiment before use. The activity was expressed as the sample concentration that gave a 50% inhibition in the enzyme activity (IC50). For the measurement of diphenolase inhibition, a sample solution (0.1 mL) and 1.5 M l-DOPA solution (1.5 mL, 0.1M phosphate buffer, pH 6.8) were mixed with H2O (0.4 mL) and preincubated at 25 °C for 5 min. Then, tyrosinase solution (0.5 mL, 125 units) was added and the formation of dopachrome was monitored at 475 nm from 0.5 to 4 min using a Hitachi UV-3210 spectrophotometer. For the measurement of monophenolase inhibition, a sample solution (0.1 mL) and 2 m l-tyrosine solution (1 mL) were mixed with of 0.1 M phosphate buffer (0.9 mL, pH 6.8), and pre-incubated at 25 °C for 5 min. Then, tyrosinase solution (0.5 mL, 250 units) was added and the dopachrome formed was monitored at 475 nm for 20 min.

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Chelation ability of 5,6,7-trihydroxyflavones towards copper ions [1]
A solution of test compounds (10 mM) was prepared in DMSO. Then 50 μM solution was prepared in a cuvette containing phosphate buffer (0.1 M, pH 6.8) and the sample absorbance was recorded in the 200-540 nm range using Shimadzu UV-1600 spectrophotometer. A repeated scan was recorded after addition of 100 μM CuSO4 and a third spectrum after addition of 250 μM EDTA. To check if 5,6,7-trihydroxyflavones can chelate copper in the enzyme, the UV-visible spectra (200-540 nm) was measured. A mixture containing 0.1 M phosphate buffer (1.5 mL, pH 6.8), H2O (0.4 mL), a test solution (0.1 mL) and tyrosinase solution (0.5 mL, 125 units/mL), was incubated at 25 °C for 30 min, and then the spectra was recorded.

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ABTS radical scavenging activity [1]
ABTS radical scavenging activity was determined according to a modification of the method of reference. In brief, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt, ABTS, 19 mg) was reacted with potassium persulfate (3.3 mg) overnight in the dark at room temperature. The working solution was prepared by diluting it with water to get an absorbance around 0.70 at 734 nm. Test sample (30μL in DMSO) was reacted with diluted ABTS (2.97 mL) and absorbance at 734 nm was recorded within 30 min. Trolox® was used as a positive control, and the control was prepared without the samples. The activity was expressed as the concentration of sample necessary to give a 50% reduction in the original absorbance (EC50).

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SOD (superoxide dismutase)-like activity [1]
SOD (superoxide dismutase)-like activity was measured by the nitrite method. In brief, a mixture of the sample solution (0.01 mL), EDTA-phosphate buffer solution (pH 8.2, 0.6 mL), reagent A (0.5 mM xanthine and 10 mM hydroxylammonium chloride in buffer, 0.2 mL) and xanthine-oxidase solution (10 mU/mL in buffer) was incubated at 37 °C for 30 min, and then coloring reagent was added (25 μM N-(1-naphthyl)ethylenediamine, 2 mM sulfanilic acid and 16.7% acetic acid, 2 mL). The resulting mixture was allowed to stand for 30 min at room temperature and the optical absorbance was measured at 550 nm. SOD-like activity was expressed as a value of EC50 against the production of superoxide anion from hypoxanthine-xanthine-oxidase.

[1]. Inhibitory effects of 5,6,7-trihydroxyflavones on tyrosinase. Molecules. 2007 Jan 29;12(1):86-97.

6-Hydroxykaempferol has been reported in Grindelia hirsutula, Achillea virescens, and other organisms with data available.

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We have disclosed considerable information about the structure-activity relationships with respect of the number and relative position of the hydroxyl groups of baicalein (1) for enhancing the inhibitory activity against tyrosinase, and found that 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-hydroxykaemferol (14) all exhibited high inhibitory effects towards tyrosinase. Moreover, 14 showed the highest activity among the tested flavonoids, and 14 was a competitive inhibitor of tyrosinase with respect to l-DOPA. 5,6,7-Trihydroxyflavones 1, 6, 13 and 14 also showed high antioxidant activities. Hence, we may conclude that the 5,6,7-trihydroxyflavones are useful as good depigmentation agents with inhibitory effects on tyrosinase in addition to their antioxidant properties.[1]

C15H10O7

302.24

302.043

4324-55-4

5281638

Typically exists as solid at room temperature

1.799g/cm3

669.3ºC at 760 mmHg

>320 ºC (ethanol )

258.4ºC

1.823

1.988

5

7

1

22

480

0

C1=C(C=CC(=C1)O)C2=C(C(=O)C3=C(C=C(C(=C3O)O)O)O2)O

LFPHMXIOQBBTSS-UHFFFAOYSA-N

InChI=1S/C15H10O7/c16-7-3-1-6(2-4-7)15-14(21)13(20)10-9(22-15)5-8(17)11(18)12(10)19/h1-5,16-19,21H

3,5,6,7-tetrahydroxy-2-(4-hydroxyphenyl)chromen-4-one

6-Hydroxykaempferol; 4324-55-4; 3,5,6,7-tetrahydroxy-2-(4-hydroxyphenyl)chromen-4-one; DTXSID30415163; DTXCID20366014; SCHEMBL674949; CHEMBL455504; LMPK12112860;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

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.)