WO2007083904A1

WO2007083904A1 - C-kit activation inhibitor, skin whitening compound and composition for skin whitening containing the same

Info

Publication number: WO2007083904A1
Application number: PCT/KR2007/000227
Authority: WO
Inventors: Ho-Jeoung Kim; Mu-Hyun Jin; Byong-Jun Kim; Sang-Jin Kang
Original assignee: Lg Household & Health Care Ltd.
Priority date: 2006-01-18
Filing date: 2007-01-12
Publication date: 2007-07-26
Also published as: JP5583346B2; JP2009523786A

Abstract

Disclosed are a c-Kit activation inhibitor, a skin whitening compound and a composition for skin whitening comprising the skin whitening compound as an active ingredient. The c-Kit activation inhibitor of the present invention is a flavone derivative selected from the group consisting of the compounds represented by Formulas 1 to 10. The flavone derivative inhibits activity of the c-Kit associated with melanin synthesis, melanocyte differentiation and maturity, etc. Accordingly, the above-mentioned flavone derivatives are useful as skin whitening compound, and the cosmetic compositions comprising the flavone derivatives as an active ingredient can be highly effectively used for skin whitening, for example treating melasma, freckles, etc.

Description

C-KIT ACTIVATION INHIBITOR, SKIN WHITENING COMPOUND AND

COMPOSITION FOR SKIN WHITENING CONTAINING THE SAME

TECHNICAL FIELD

The present invention relates to an activation inhibitor of c-Kit associated with

melanin synthesis, melanocyte differentiation and maturity, etc., a skin whitening

compound having skin whitening effect such as treatment of melasma or freckles, and a

composition for skin whitening comprising the skin whitening compound as an active

ingredient.

BACKGROUND ART

It has been known that c-Kit, which is a receptor belonging to a class III receptor

tyrosine kinase (RTK), is associated with survival, proliferation and differentiation of

melanocyte. The number of the melanocyte is increased in skins when the skins are

exposed to UV rays. In this reaction, the c-Kit plays an important role. It was

confirmed that increased stem cell factors (SCFs) in a hair follicle 'niche,' which

generates and differentiates melanocyte, grow and differentiate melanocyte to migrate

out of the niche in the case of a Kl 4 (keratin promoter)-steel factor transgenic mouse,

and that mice have white hair and white skin if the mice are treated with ACK2 which is

an antibody against c-Kit in an embryonic step (Nature 416, 854-860, 2002).

Form the beginning, there have been many more attempts to study c-Kit as a target of an anti-cancer drug than as a whitening compound. Imatinib (Gleevec,

STI-571, Novatis, East Hanover, NJ, USA) has been widely known as an anti-cancer drug for treating leukemia, which targets Bcr-Abl kinase. However, it was revealed

that a level of melanin pigment is decreased in skins of 6 patients who receive a

prescription of imatinib, indicating that the imatinib inhibits activity of c-Kit in addition

to the Bcr-Abl. Accordingly, it might be seen that SCF/c-Kit plays an essential role in

growing and sustaining human melanocyte (Cancer 98, 2483-7, 2003).

Meanwhile, it was reported that SCF, which is a ligand of c-kit, is more

excessively expressed in a lesion region of melasma than a normal skin region (Korean J.

Dermatol. 2005; 43 (8): 1046-1052), and that SCF is excessively expressed in excessive

melanotic conditions such as UVB-melanosis, lentigo senilis, dermatofibroma and Caffe

aure macule (Pigment Cell Research 17:96- 110. 2004).

If c-Kit is activated, the activated c-Kit activates MAP kinase, and the activated MAP kinase sequentially phosphorylates a microphthalmia-associated transcription factor (Mitf), which is a helix-loop-helix/leucine zipper protein, into an activated state.

The activated Mitf stimulates transcription of melanin-synthesizing enzymes such as

tyrosinase, Tyrp-2, etc. to synthesize melanin pigment. SCF, secreted from

keratinocyte when exposed to UV-rays, stimulates differentiation of precursor melanocyte into mature melanocyte harboring c-Kit, and also stimulates synthesis of

melanin pigment by stimulating transcription of enzymes associated with the melanin

synthesis.

As described above, the c-Kit is mainly associated with a signal transduction system which stimulates synthesis of melanin by UV-rays. Accordingly, if it is

possible to inhibit the c-Kit activity, differentiation and maturity of the melanocyte may

be inhibited, in addition to the anti-cancer effect. Generally, many persons hope to have a white and soft skin, but if melanin is

excessively synthesized in their skins, they have a dark skin tone, and also have

melasma, freckles, etc. Accordingly, if the synthesis of melanin pigment in skin is

inhibited, a skin tone may be brightened to thereby whiten skin, and cutaneous

hyperpigmentation such as melasma, freckles, lentigo senilis, dermatofibroma, Caffe

aure macule etc., which are caused by UV-rays, hormones and other genetic factors, may

be treated to thereby whiten skin.

Accordingly, conventional compounds such as hydroquinone, ascorbic acid,

kojic acid and glutathione, which have an inhibitory activity on tyrosinase, were mixed with skin-applicable compositions such as ointment or cosmetics, and then the resultant

compositions were used in the art to whiten skin, for example to treat melasma and

freckles. However, it was confirmed that the hydroquinone has some whitening effect,

but it is used in an extremely low amount due to severe irritation to skin. Also, the

ascorbic acid has a problem that smells and colors of an ascorbic acid-containing

cosmetic composition are easily changed due to its susceptibility to oxidation.

Meanwhile, thiol compounds such as glutathione, cysteine, etc. have an inherent

disgusted smell and a transdermal delivery-related problem, and their glycosides and derivatives are also difficult to use as mixing ingredients due to its high polarity.

DISCLOSURE OF INVENTION [Technical Problem]

Therefore, one object of the present invention is to provide a c-Kit activation inhibitor capable of inhibiting activity of c-Kit associated with melanin synthesis, anti-cancer activity, etc.

In addition, another object of the present invention is to provide a skin whitening

compound and a composition for skin whitening being able to be safely used without

any side effect on skin and having an excellent inhibitory effect on pigmentation.

[Technical Solution]

In order to accomplish the first object, the present invention provides a c-Kit

activation inhibitor which is a flavone derivative selected from the group consisting of

compounds represented by the following Formulas 1 to 10.

[Formula 1 ]

6-methoxyflavone

[Formula 2]

3,7-dihydroxyflavone

[Formula 3 ]

3,6-dihydroxyflavone

[Formula 4]

3-hydroxy-6-m ethylf lavone

[Formula 5]

3, 3'-dihydroxyf lavone

[Formula 6]

6,2',3'-trihydroxyflavone

[Formula 7]

6,4'-dimethoxy-3-hydroxyflavone

[Formula 8]

4 '-hydroxy-beta-naphthof lavone

[Formula 9]

3,7,3'-trihydroxyflavone

[Formula 10]

diosmetin

In addition, the present invention provides a skin whitening compound, one

flavone derivative, selected from the group consisting of compounds represented by the

Formulas 1 to 10, and a composition for skin whitening comprising any of the

compounds as an active ingredient.

In the composition for skin whitening, a content of the flavone derivatives

preferably ranges from 0.000001 to 10 % by weight, based on the total weight of the composition, and these flavone derivatives can be added to various formulations such as skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, soap, ointment, etc.

to give a skin whitening effect. Hereinafter, the present invention will be described in more detail.

The present invention provides a c-Kit inhibitor, one flavone derivative, selected from the group consisting of 6-methoxyflavone

(6-methoxy-2-phenyl-4H-chromen-4-one, C₁₆H₁₂O_3, CAS No. 26964-24-9) of Formula 1,

3,7-dihydroxyflavone (C_I5H_I0O₄, CAS NO. 492-00-2) of Formula 2,

3,6-Dihydroxyflavone (C₁₅H₁₀O_4, CAS No. 08238-41-1) of Formula 3,

3-hydroxy-6-methylflavone (6-methyl-3-hydroxyflavone, C₁₆H₁₂O₃, CAS No.

6971-18-2) of Formula 4, 3,3'-dihydroxyflavone (C₁₅H₁₀O₄, CAS No. 55977-09-8) of

Formula 5, 6,2',3'-trihydroxyflavone (C₁₅Hi₀O_5, CAS No. 108238-47-7) of Formula 6,

6,4'-dimethoxy-3-hydroxyfiavone (C₁₇H₁₄O_5, CAS No. 93176-02-4) of Formula 7,

4'-hydroxy-β -naphthoflavone (C₁₉H₁₂O_3, CAS No. 98166-72-4) of Formula 8,

3,7,3'-trihydroxyflavone (C₁₅H₁₀Os) of Formula 9 and diosmetin (5,7-dihydroxy-2-

(3-hydroxy-4-methoxy-phenyl)-chromen-4-one, C₁₆H₁₂O₆, CAS No. 520-34-3) of

Formula 10.

In particular, the diosmetin of Formula 10 is a compound that has been reported

as an ingredient of plants including Acacia farnesiana, Achillea asiatica, Arnica

longifolia, Artemisia rutifolia, Artemisia vulgaris, Caleriana chionophila, Capsella

bursa-pastoris, Citrus limon, Cnidium monnieri, Cyperus alopecuroides, Gentiana

barbata, Hieracium compositum, Lnaria macroura, Mentha spicata, Origaganum vulgare,

Pedalium murex, Petroselinum crispum, Rosmarinus officinalis, Salvia candidissima, Salvia nutans, Salvia reptans, Soroseris hookeriana, Stemodia viscose, Tanacetum vulgare, Toddalia floribunda, Thymbra capitata, Thymus hirtus, Thymus vulgaris,

Verbena bipinnatifida, Valeriana cardamines, Valeriana eriophylla, Valeriana fedtschenkoi, Valeriana laxiflora, Vicia truncatula, Xanthorrhoea hastile durch and

Xanthorrhoea hastila. It has been known that the diosmetin has an anti-bacterial effect

(Planta Medica, 70 (6), 2004, 509-514), and anti-allergic effect (Bioorg. Med. Chem, 10

(10), 2002, 3123-3128), anti-inflammation effect (J. haram. Pharmacol., 50 (9), 1998,

1069-1074), etc.

The inventors have studied about compounds that can inhibit the activity of the

c-KIT associated with melanin synthesis, melanocyte differentiation and maturity, etc.,

and have found that the above-mentioned flavone derivatives show a very potent

inhibitory effect on the c-Kit, on which the present invention is based. That is to say, the above-mentioned flavone derivatives inhibit the activity of the

c-Kit, and therefore they can be used as multifunctional skin whitening compounds that

inhibit synthesis of enzymes associated with melanocyte differentiation and maturity

and melanin synthesis, as well as an anti-cancer effect. Particularly, the c-Kit

activation inhibitor such as the above-mentioned flavone derivatives can be useful to

attain an excellent skin whitening effect even if it is used in a small amount since it acts

at the beginning of signal transduction for the melanin synthesis.

In the present invention, the term "skin whitening effect " is generally intended

to include effects of improving undesirable conditions associated with the skin colors,

for example treating melasma and freckles, improving lentigo senilis, dermatofibroma,

and cutaneous hyperpigmentation, as well as an effect of making skin whiter.

The above-mentioned flavone derivatives of Formulas 1 to 10 are commercially available. However, it is more preferable to use an extract of Chrysanthemum

morifolium {Chrysanthemum morifolium petals) as the diosmetin of Formula 10. A method for extracting diosmetin from Chrysanthemum morifolium will be illustrated as

described below, but the scope of the present invention is not limited to the subject to be

extracted or the extraction methods as described in the present invention.

Chrysanthemum morifolium, commercially available as a medicinal herb, was

purchased, ground into small pieces, and the ground substance was extracted with 5-20

times volume of methanol at 50-100 ^°C for 1-5 hours in an extractor having a reflux

cooler. The residue was extracted through a filter cloth using the same method, and

then the remaining extract was filtered once or more. The resultant extracts were

collected, concentrated under a reduced pressure, and then freeze-dried or spray-dried to

obtain a dry extract. In order to confirm the presence of an active ingredient in the

methanol extract of the Chrysanthemum morifolium, the methanol extract was

suspended in 10 times volume of water, and fractionated with an equivalent amount of

hexane to remove non-polar compounds, and the remaining aqueous layer was then

fractionated with an equivalent amount of butanol solvent to obtain a butanol fraction of

the Chrysanthemum morifolium. An active ingredient was separated from the resultant

butanol fraction of the Chrysanthemum morifolium with a silica gel column

chromatography using mixed chloroform/methanol solvent mixture as an eluent, and the

obtained active ingredient was purified using a preparative HPLC under the same

conditions. The purified active ingredient was confirmed to be diosmetin using

nuclear magnetic resonance (NMR) and mass spectrometry (Mass).

In order to give a skin whitening effect, the above-mentioned pure flavone derivatives of Formulas 1 to 10 or the diosmetin extracts can be added in an effective

amount to various compositions such as an ointment and cosmetic products including a skin, a lotion, a cream, a foundation, an essence, a gel, a pack, a foam cleansing, a soap,

etc., and the flavone derivatives or the diosmetin extracts can be used alone or in combination thereof.

In consideration of a skin whitening effect and economical efficiency, an amount

of the added flavone derivatives preferably ranges from 0.000001 to 10 % by weight,

more preferably from 0.0001 to 1 % by weight, based on the total weight of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a H¹ -NMR spectrum of diosmetin extracted and

purified according to one embodiment of the present invention.

FIG. 2 is a graph showing a C¹³ NMR spectrum of diosmetin extracted and

purified according to one embodiment of the present invention.

FIG. 3 is a graph showing a mass spectrometry spectrum of diosmetin extracted

and purified according to one embodiment of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in

detail referring to the accompanying drawings. However, the description proposed

herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the

invention. The preferred embodiments of the present invention will be described in detail for the purpose of better understandings, as apparent to those skilled in the art.

Evaluation of Flavone Derivatives of Formulas 1 to 9

The commercially available flavone derivative compounds of Formulas 1 to 9

were purchased and used. Each of the flavone derivative compounds was adjusted to a

final concentration of 1 uM and added to each well of a 384-well plate, and c-Kit RTK

and ATP were also added to each well of the 384-well plate, and then subject to a

primary reaction at a room temperature. Then, biotinylated-poly[Glu:Tyr] (4:1) as a

substrate was added, and then subject to a secondary enzyme reaction.

A capture buffer containing donor beads coated with streptavidin and acceptor

beads to which antibody (P-Tyr-100) binds was added, and then subject to a tertiary

reaction to bind to the substrate. Phosphorylation levels of the substrates were

determined by measuring AlphaScreen signal using a Fusion™ microplate analyzer.

Also, their inhibitory effects were compared using Tyrphostin A51 known as c-Kit

inhibitor in the prior art.

In order to measure the AlphaScreen signal to an accurate level, each test sample

was tested three times. Here, in order to determine inhibition by c-Kit, DMSO was

used as a positive control instead of the test samples, and DMSO and a buffer were used

instead of the test samples and the enzyme as a positive control, respectively.

The inhibition (%) on c-Kit was calculated from the detected signal according to the following Equation 1. The results are listed in the following Table 1.

Equation 1 Inhibition (%) = (Mean Value of Test Samples - Mean Value of Negative Control) / (Mean Value of Positive Control - Mean Value of Negative Control) X 100

Table 1

As listed in Table 1, it was revealed that the flavone derivative compounds of

Formulas 1 to 9 exhibit a potent inhibitory activity on the c-Kit in a lower concentration

than the Tyrphostin A51 known as the c-Kit activation inhibitor.

On the basis of compositions and their contents as listed in the following Table 2,

some of the above-mentioned flavone derivative compounds were added to a cream, and

then evaluated for a whitening effect.

Table 2

In order to inspect skin whitening effects of the creams produced on the basis of

the above-mentioned compositions, tests on inhibition of pigmentation and treatment of

melasma were carried out, as follows.

Test of Inhibitory Effect on Pigmentation

20 healthy men and women were selected, and aluminum foil having two

grooved lines, each having 6 holes with a diameter of 7 mm, was attached to both

forearms of their arms. Then, the forearms were irradiated with a radiation intensity of

60 mJ/cm² using a IOOOW ORIEL solar simulator located at a distance of 10 cm from

the arms. Before the irradiation, sites to be irradiated were washed thoroughly with an aqueous 70 % ethanol solution. The base compositions prepared according to

Preparative example 1-3 and Comparative example 1 were applied in pairs to the same

grooved lines two times daily during a period from 3 days before the irradiation to 3 weeks after the irradiation. Pigmentation rates on the base compositions of the preparative examples and the

comparative examples were evaluated with the naked eye, and then the base

compositions of the preparative examples were evaluated in two level, namely to be

effective and not to be effective, compared to the base compositions of the comparative

examples. The results are listed in the following Table 3.

Table 3

As listed in Table 3, it was revealed that the flavone derivative-containing

creams of Preparative examples 1 -3 have a whitening effect but no side effect in at least

10 of the 20 subjects.

Test of Effect on Treatment of Melasma

10 healthy women having melasma were selected, and then the base composition

prepared according to Preparative example 1 was applied to their faces with melasma

two times daily for three weeks.

After the experiment was completed, medical specialists valuated pigmentations

with the naked eye, and the subjects to be tested evaluated treatment of melasma on the basis of their subjective judgments, and therefore the cream of Preparative example 1 was evaluated in two level, namely to be effective and not to be effective on the

treatment of melasma. The results are listed in the following Table 4. Table 4

As listed in Table 4, it was revealed that the 6-Methoxyflavone-containing cream

prepared according to Preparative examples 1 has an effect on the treatment of melasma

but no side effect in at least 6 of the 10 subjects.

Preparation and Evaluation of Diosmetin Extract of Formula 10

Example 1

Dry Chrysanthemum morifolium commercially available as a medicinal herb was

purchased and ground into small pieces, and 10 kg of the ground substance was heated

to 70 ^°C for 3 hours in an extractor provided with a reflux cooler and extracted with

150L of methanol. The resultant extract was filtered through a filter cloth, and the

remaining extract was then filtered once or more. The resultant extracts were collected

and concentrated under a reduced pressure to obtain 1.2 kg of a dry extract.

Example 2

1 kg of the Chrysanthemum morifolium methanol extract prepared in Example 1

was suspended in 1OL of water and fractionated with 1OL of hexane solvent three times to remove a hexane fraction. Then, the remaining aqueous layer was fractionated with

1OL of butanol solvent three times to obtain a butanol fraction. The butanol fraction was concentrated under a reduced pressure to obtain 125 g of a dry substance. Example 3

1 kg of the Chrysanthemum morifolium methanol extract prepared in Example 1

was suspended in 1OL of water and fractionated with 1OL of ethylacetate solvent three

times to obtain an ethylacetate fraction. Then, the ethylacetate fraction was

concentrated under a reduced pressure to obtain 230 g of a dry substance.

Example 4

100 g of the soluble Chrysanthemum morifolium butanol fraction prepared in

Example 2 was subject to silica gel column chromatography using mixed

chloroform/methanol solvent as an eluent to obtain an active fraction. Then, the

resultant active fraction was purified with preparative HPLC to obtain 1.2 g of an active ingredient. The purified active ingredient was confirmed to be diosmetin, represented

by Formula 10, through nuclear magnetic resonance (NMR) and mass spectrometry

(Mass) (see FIGs. 1 to 3), as follows.

Molecular Formula: C₁₆H₁₂O₆

Molecular Weight: 300.27

Melting Point: 260-264 ^°C

UV ( λ ax, nm) (MeOH): 272, 342

IH-NMR (DMSO-d6) σ : 3H (6.69,s), 6H (6.13,s), 8H (6.39,s), 2Η (7.41, S),

5¹H (7.06,d,8.6Hz), 6'H (7.51,d,8.6Hz), 5-OH (12.89,br), 7-OH (lO.O.br), 4'-OMe

(3.85,s)

Inhibitory Effect on c-Kit Activity

Each concentrations of the methanol extract of Example 1 and the soluble butanol fraction of Example 2 were adjusted to 20 ug/ml, and a final concentration of

the diosmetin of Example 4 were adjusted to 0.3 uM, and then evaluated in the same

manner as the evaluation method of the above-mentioned flavone derivative compounds

of Formulas 1 to 9. The results are listed in the following Table 5.

Table 5

As listed in Table 5, it was revealed that the test samples of Examples 1 and 2,

which are the diosmetin-containing crude extracts, have a high inhibitory effect on c-Kit,

and the test sample of Example 4, which is the purified diosmetin, has an excellent

inhibitory effect on c-Kit activity, which is comparable to that of Tyrphostin A51 known

as the c-Kit activation inhibitor.

Evaluation of Whitening Effect

In order to evaluate a whitening effect of the diosmetin-containing cosmetics,

creams were prepared on the basis of compositions and their contents listed in the following Table 2.

Table 6

The creams prepared as listed in Table 6 were evaluated for an inhibitory effect

on pigmentation in the same manner as in the above-mentioned Preparative examples

1-3. The results are listed in the following Table 7.

Table 7

As listed in Table 7, it was revealed that the diosmetin-containing cream of

Preparative example 4 has a whitening effect but no side effect on skin in 14 of the 20

subjects.

Effect on Treatment of Melasma

10 healthy women having melasma were selected, and then the base composition

prepared according to Preparative example 4 was applied to their faces with melasma

two times daily for three weeks.

After the experiment was completed, medical specialists valuated pigmentations with the naked eye, and the subjects to be tested evaluated treatment of melasma on the basis of their subjective judgments, and therefore the cream of Preparative example 4

was evaluated in two level, namely to be effective and not to be effective on the treatment of melasma. The results are listed in the following Table 8.

Table 8

As shown in the Table 8, it was revealed that the diosmetin-containing cream

prepared according to the preparative example 4 shows an effect on the treatment of

melasma in 7 of 10 subjects.

INDUSTRIAL APPLICABILITY

As described above, the flavone derivatives of the present invention are effective

c-Kit activation inhibitors. Particularly, the above-mentioned flavone derivatives have

no side effect on skin, and inhibit melanin synthesis, and differentiation and maturity of melanocyte, and therefore the composition containing the flavone derivatives the as

active ingredients can be highly effectively used to treat melasma and freckles, improve

cutaneous hyperpigmentations such as lentigo senilis, dermatofibroma, Caffe aure

macule, etc. and attain a skin whitening effect.

Claims

What is claimed is:

1. A c-Kit activation inhibitor which is a flavone derivative selected from

the group consisting of the following Formulas 1 to 10.

Formula 1

6-methoxyflavone

Formula 2

3 , 7-dihydroxyf lavone

Formula 3

3, 6-dihydroxyf lavone

Formula 4

3-hydroxy-6-methylflavone

Formula 5

3 , 3 '-dihydroxyf lavone

Formula 6

6 , 2 ' , 3 '-trihydroxyf lavone

Formula 7

6,4'-dimethoxy-3-hydroxyflavone

Formula 8

4 '-hydroxy-beta-naphthof lavone

Formula 9

3,7,3'-trihydroxyflavone

Formula 10

2. A composition for skin whitening comprising, as an active ingredient, at

least one flavone derivative selected from the group consisting of the compounds

represented by the Formulas 1 to 10 as defined in Claim 1.

3. A composition for skin whitening comprising a compound of the

following Formula 10 as an active ingredient.

Formula 10

4. A composition for skin whitening comprising a Chrysanthemum

morifolium {Chrysanthemum morifolium petals) extract as an active ingredient.

5. The composition for skin whitening according to any one of claims 2 to 4,

wherein the skin whitening is the treatment of melasma or cutaneous

hyperpigmentation.

6. The composition for skin whitening according to any one of claims 2 to 4,

wherein a content of the flavone derivative ranges from 0.000001 to 10 % by weight,

based on the total weight of the composition.

7. The composition for skin whitening according to any one of claims 2 to 4,

wherein the composition for skin whitening is one formulation selected from the group consisting of skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, soap

and ointment.