WO2005023214A1

WO2005023214A1 - Composition for skin whitening

Info

Publication number: WO2005023214A1
Application number: PCT/KR2004/002254
Authority: WO
Inventors: Jae-Kwan Hwang; Ji-Eun Kim
Original assignee: Biocare Co., Ltd.
Priority date: 2003-09-06
Filing date: 2004-09-06
Publication date: 2005-03-17
Also published as: KR20050026716A; KR100571058B1

Abstract

The present invention relates to a composition for skin whitening, which contains phloroglucinol compound having inhibitory activity for melanin formation as an effective component. The composition for skin whitening containing phloroglucinol compound according to the present invention may be used safely since it shows no side effects on skin. In addition, the composition according to the present invention shows a superior effect to inhibit pigmentation on skin by restraining melanin generation, so it is very effective in skin whitening, e.g., to improve chloasma and freckles.

Description

COMPOSITION FOR SKIN WHITENING

TECHNICAL FIELD The present invention relates to a composition for skin whitening, and more

particularly to a composition for skin whitening comprising phloroglucinol compound

that has excellent stability without side effects on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin generation.

BACKGROUND ART Most people want to have white and fine skin. The color of human skin is

determined by the density and the distribution of melanin beneath skin and affected by

the environmental or physiological factors such as ultraviolet rays of the sun, fatigue, or

stress as well as the genetic factor. Melanin is formed as follows: tyrosin is converted into 3,4-dihydroxyphenylalanine (DOPA) or dopaquinone by tyrosinase, followed by

non-enzymatic oxidation to generate melanin. However, the mechanism inducing

melanin synthesis before tyrosinase works is not clarified yet in detail while the process

through which melanin is made is disclosed. When the melanin synthesis is excessively performed beneath skin, skin

becomes dark, and chloasma and freckles can be generated. Accordingly, the skin

whitening may be achieved by inhibiting the melanin synthesis: the skin becomes

lightened and hyperpigmentation such as chloasma, freckles, etc. due to ultraviolet rays or hormonic and genetic factors may be improved. Conventionally, tyrosinase inhibitors such as hydroquinone, ascorbic acid, kojic acid, arbutin, glutathione, etc. have been used as skin whitening agents in skin

whitening products such as ointments or cosmetics for improving hyperpigmentation

such as chloasma, freckles, etc. However, although hydroquinone provides a whitening effect, its use should be

restricted to the minimum since it seriously irritates skin and induces skin cancer.

Ascorbic acid is easy to be oxidized and the cosmetics mixed with the ascorbic acid

have problems of discoloration and change of scent. In addition, kojic acid and arbutin

have problems of safety and stability. Moreover, glutathione has limitations due to bad

smell and low absorptiveness into skin. As mentioned above, conventional skin whitening agents have many problems from the viewpoint of safety, stability, discoloration, or the like when used over

effective concentration in cosmetics and medical supplies, and also show insufficient

effects of skin whitening. Therefore, whitening agents from natural products having no side effect on skin and excellent whitening effect as well as safety are needed to

develop.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention is designed to solve the problems of the

prior art, and an object of the present invention is to provide a composition for skin

whitening containing whitening component from natural products, which has excellent stability without side effect on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin generation. In order to accomplish the object, the present invention provides a composition for skin whitening comprising phloroglucinol compound as an effective component

selected from the group consisting of a compound having the structure of chemical

formula 1, a compound having the structure of chemical formula 2, and a mixture

thereof: Chemical Formula 1

Chemical Formula 2

In the composition for skin whitening of the present invention, the content of the

phloroglucinol compound is preferably 0.00001 to 5.0wt% on the basis of total weight of the composition, and the phloroglucinol compound may be extracted from leaves of

Baeckea frutescens. Hereinafter, the composition for skin whitening according to the present invention will be described in detail. The phloroglucinol compounds included in the composition for skin whitening

according to the present invention as an effective component are shown as following

chemical formulas 1 and 2. Chemical formula 1

Chemical formula 2

As a result of screening new whitening agent to restrain melanin synthesis from

natural medicinal herbs using B16 mouse melanoma cell which can screen even the

material restraining induction of melanin synthesis itself, the inventors have found that

Baeckea frutescens extracts show excellent skin whitening effect. After selecting

Baeckea frutescens as a raw material for the research, active material for whitening was separated by bioassay-directed fractionation. As a result, it was found that the phloroglucinol compounds of aforementioned chemical formulas 1 and 2 have very

strong inhibitory effect on formation of melanin and skin whitening effect. Therefore, if

the phloroglucinol compound having the structure of chemical formula 1 and/or 2 is added and mixed into the composition for skin whitening such as cosmetic composition

(e.g., ointment for external use, essence, nutritious cream, softening lotion, nutritious

lotion, pack, skin, lotion, foundation, gel, foam cleansing, soap, etc.) and pharmaceutical composition (e.g., ointment for external use), the high-potency of skin whitening without any special side effect can be obtained. The phloroglucinol compounds having the structures of chemical formulas 1 and 2 as effective components of the composition for skin whitening according to the

present invention may be extracted from Baeckea frutescens.

Baeckea frutescens is a plant belonging to Myrtaceae, whose leaves are in a

shape of needle and have some incense. For a long time, it has been called "tea tree" in

Sountheast Asia, and its leaves have been eaten universally instead of tea to remove

fever and to relax febrile disease, dysuria, lechopyra, paramenia, abdominal pain, and so on (Jantan, I. et al, Flavour and Fragrance Journal, 13, 245, 1998).

It was reported that Baeckea frutescens, especially its leaves, contains large

amount of essential oil, and some monoterpenoids and sesquiterpenoids compounds

including eugenol, α-pinene, β-pinene, 1,8-cineole, limonene, β-caryophyllene,

α-humulene, etc. were isolated ( Jantan, I. et al, Flavour and Fragrance Journal, 13, 245,

1998). Particularly, it was reported that BF-1 and BF-2, phloroglucinol materials

isolated from Baeckea frutescens, have killing effect on leukaemia cell L 1210

(Fujimoto Y. et al, Phytochemistry, 41(3), 923-5, 1996). Moreover, according to KR application No. 10-2000-0050229, it was reported that Baeckea frutescens extracts have

antibacterial activities against Streptococcus mutans and Porphyromonas gingivalis,

which may induce dental caries and periodontitis in oral cavity.

The aforementioned reports about physiological activity of Baeckea frutescens and isolated materials are not related to whitening activity of the present invention. The

inventors of the present invention have first discovered the whitening effect of phloroglucinol compounds having the structures of the chemical formulas 1 and 2. The phloroglucinol compounds having whitening effects according to the present invention

may be isolated from other plants as well as Baeckea frutescens leaves, and may be chemically synthesized.

The phloroglucinol compounds of the present invention as effective

components for skin whitening may be isolated and purified from organic extracts of

Baeckea frutescens or oil obtained by compressing it. Solvents capable of extracting the effective components are at least one selected from water, methanol, ethanol, propanol,

isopropanol, butanol, acetone, ether, benzene, chroloform, ethylacetate,

methylenechloride, hexane, cyclohexane, petroleumether, and so on. Isolation and purification of phloroglucinol compounds from Baeckea frutescens extracts may be

conducted using column chromatography filled with various synthetic resins such as

silica gel or active alumina, HPLC, etc., alone or in combination thereof. However, methods for extraction, isolation and purification are not limited herein.

The phloroglucinol compounds having the structures of chemical formulas 1

and 2 as whitening components according to the present invention may be used alone or

in combination thereof in various compositions for skin whitening such as ointments for

external use or cosmetics, wherein the phloroglucinol compounds may be used in forms

of high-purified compounds or Baeckea frutescens extracts which are not purified.

The content of the phloroglucinol compounds in the composition for skin whitening of

the present invention is preferably 0.00001 to 5.0wt% on the basis of the total weight of the composition in consideration of whitening effect and economical efficiency.

Moreover, in case of adding Baeckea frutescens extracts, its content is preferably 0.0001 to 50.0wt% of dry weight on the basis of total weight of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of

the present invention will be more fully described in the following detailed description, taken accompanying drawings. In the drawings: FIG. 1 is a schematic view showing a method of extracting the phloroglucinol

compounds of the present invention from Baeckea frutescens leaves;

FIG. 2 is C-NMR spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2;

FIG. 3 is 1H-NMR spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2;

FIG. 4 is mass spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2;

FIG. 5 is HMBC spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2; FIG. 6 is HMQC spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2;

FIG. 7 is H-H COSY spectrum of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2; FIG. 8 is NOESY spectrum of the phloroglucinol compound of the present

invention (BF-2J) having the structure of the chemical formula 2;

FIG. 9 illustrates "C-Η correlation obtained from HMBC and HMQC spectra of the phloroglucinol compound of the present invention (BF-2J) having the structure of the chemical formula 2; FIG. 10 is a photograph showing inhibiting effect of the phloroglucinol

compounds of the present invention (BF-2 and BF-2J) on melanin formation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The detailed description of the present invention referring to the embodiments is provided hereinafter. However, the embodiments according to the present invention

can be modified in various ways and should not be understood that the present invention

is restricted to the embodiments described below. The embodiments of the present

invention are provided for illustration purpose only to a person having ordinary skill in

the art.

In embodiments and experimental examples below, % means wt% unless vol%

is denoted specifically. FIG. 1 illustrates a method of extracting and purifying the phloroglucinol

compounds having the structures of chemical formulas 1 (hereinafter, referred to as

"BF-2") and 2(hereinafter, referred to as "BF-2J") from Baeckea frutescens.

Example 1 : extraction and purification l,000g of ground material of Baeckea frutescens was put into 75% methanol

solution 4000ml, extracted twice at room temperature while stirred for 2 days, and then filtered using filter paper (Whatman No.2). Solvent in filtered extracts was removed

by concentrating under reduced pressure using rotary vacuum concentrator at 50 ^°C to

obtain 210g of methanol extract. Subsequently, the methanol extract was suspended in purified water 1000ml, and then chloroform 1000ml was added to it three times repeatedly to obtain

chloroform-soluble fraction 51g. After that, the chloroform-soluble fraction was treated using silica gel column

(6 x 50cm) with solvent system of hexane, ethyl acetate, chloroform and methanol of

10 : 1 : 1 : 1 (v/v) respectively to obtain fraction A. The active fraction A was

separated again using silica gel column with solvent mixture of hexane, methylene

chloride and ethyl acetate (12 : 0.2 : 0.4 (v/v) repectively) to obtain two fractions of

A- LI (0.74g) and A-rV(0.19g) having activity for skin whitening.

Subsequently, A- II , an active fraction for skin whitening, 0.74g was purified

using silica gel column with a mixture of hexane, methylene chloride and ethyl acetate

(12 : 0.2 : 0.2 (v/v) respectively) to obtain 420mg of BF-2 as a single substance.

In order to analyze the structure of BF-2 separated in the above example 1, ¹³C-NMR spectrum at 150MHz and 1H-NMR spectrum at 600MHz (solvent: CDC1₃)

were measured and the result was summarized in Table 1.

Table 1

BF-2 structure is the chemical formula 1 described above, which was

determined by consideration of ¹³C-NMR and H-NMR spectra in Table 1 and the existing research reports (Fujimoto, Y. et al, Phytochemistry, 41(3), 923-5, 1996).

Example 2 : extraction and purification

In order to separate a single substance from the active fraction A- IV obtained in

the example 1 described above, Preparative HPLC was used. The separation condition

used is as follows:

Instruments: Recycling Prep HPLC(LC-908, JAI, Japan) Column: JAIGEL GS-310 column Detector: UV detector(254nm) Pump flow: 3.5ml/min Solvent: 100% methanol

73mg of the active fraction A-IV-2 was obtained by the above separation condition.

The active fraction A-IV-2 was purified again using RP-18 (LiChroprep, RP-18,

Merck) column (1.5 X 50cm) with 85% methanol finally to obtain 20mg of BF-2J as a

single substance. In order to analyze the structure of BF-2J, ¹³C-NMR spectrum at 150MHz and

¹ H-NMR spectrum at 600MHz (solvent: CDC1₃) were measured and shown in FIG. 2

and FIGig. 3 respectively. Moreover, HMBC and HMQC spectra was measured to

analyze the correlation of ¹³C-1H and shown in FIG. 5 and FIG. 6. The ¹³C-NMR

spectrum and correlation of ¹³C-1H of the compound were summarized in Table 2.

Table 2

In ¹³C-NMR spectrum, chemical shifts of C-ll and C-4 was shifted to low

magnetic field by Δ 25.9ppm and Δ32.9ppm respectively relative to BF-2(Fujimoto,

Y. et al, Phytochemistry, 41(3), 923-5, 1996) because of isotope effect of carbonyl

carbon C-9, and β proton of H-14 was severely shifted to low magnetic field because of

the same effect. Moreover, FAB-MS was measured for mass analysis as shown in FIG.

4. The molecular formula of the active substance was determined as C₂₄H ₆O₃(M⁺= 372) by FAB-MS.

Table 3

As a consequence of examining all the analysis results, the isolated BF-2J has

the structure of the chemical formula 2 as above. This compound is concluded as a material isolated by the example 2 from nature for the first time, which has been named

as BF-2J. The correlation of C-H in BF-2J obtained by HMBC spectrum is shown in FIG.

9.

Experimental example : Inhibitory effect on melanin formation

The whitening effect was tested by adding the methanol extract, the chloroform fraction, BF-2 and BF-2J obtained from the extraction and purification examples to a

culture medium of B-16 mouse melanoma cell (Loten, R., Lotan, D., Cancer Research.

40, 3345-3350, 1980). The final concentration of each sample was prepared to be

lO g/πtC or 20μg/m£, and arbutin, a commercial whitening agent, was used as a

comparative material. Each sample was added to a culture medium of B-16 mouse

melanoma cell, and then cultured for 3 days. Next, the cultured cell was treated with

trypsin, separated from the culture plate, and then centrifuged. The isolated cells were

treated by freezing-thawing procedure three times followed by adding perchlonic acid in order to be 0.5N of final concentration, and then extracted twice. After that, the extract

was washed with 1ml of a mixture of cooled ethanol and ether (3:1 v/v) twice followed by washing with ether once. 1ml of IN sodium hydroxide was added here and the mixture was boiled for 10 minutes for melting melanin. Then, the absorbance at

400nm was measured using spectrophotometer. The amount of generated melanin was indicated as the absorbance per unit number of cells (10⁶ cells). Then, the inhibition ratio (%) was obtained by calculating the amount of melanin generated relative to the

control group, and the results are described in Table 4.

Table 4

Referring to Table 4, methanol extract, chloroform-soluble fraction, BF-2 and BF-2J show excellent inhibitory abilities against melanin formation even at low concentration of lOppm and 20ppm. Meanwhile, while arbutin, currently used as a

whitening agent, shows about 57% of inhibitory ability against melanin formation, BF-2

and BF-2J, effective components, show inhibitory abilities of 62% and 89% respectively at 20ppm, i.e., BF-2 and BF-2J show more inhibitory effects than arbutin. FIG. 10 is a

photograph of whitening effect of BF-2 and BF-2J compared with arbutin against mouse

melanoma cells. It is shown that BF-2 and BF-2J of 20ppm have much better

whitening effects than arbutin of 300ppm. Moreover, as BF-2 and BF-2J have high activity for skin whitening without any effect on cell proliferation, it is shown that the effective components for skin whitening of the present invention are safe as skin

whitening agents without toxicity to cells. Hereinafter, the composition for skin whitening was prepared by adding

methanol extract, BF-2 and BF-2J to ointment for external use, essence, nutritious cream, softening lotion, nutritious lotion and pack, and then the effect of inhibiting

pigmentation was tested by applying it to the testees.

Embodiment 1 Ointments for external use were manufactured with the components and

contents in Tables 5 ~ 7 as described below, in which methanol extract, BF-2 or BF-2J

was added as an effective component.

Comparative example 1

Ointments for external use were manufactured with the components and

contents in Tables 5 ~ 7 as described below without adding any effective component for whitening.

Table 5

Table 6

Table 7

Embodiment 2

Essences were manufactured with the components and contents in Tables 8 ~ 10

as described below, in which methanol extract, BF-2 or BF-2J was added as an effective

component.

Comparative example 2 Essences were manufactured with the components and contents in Tables 8 ~ 10 as described below without adding any effective component for whitening.

Table 8

Embodiment 3

Nutritious creams were manufactured with the components and contents in

Tables 11 ~ 13 as described below, in which methanol extract, BF-2 or BF-2J was added as an effective component.

Comparative example 3

Nutritious creams were manufactured with the components and contents in Tables 11 ~ 13 as described below without adding any effective component for

whitening.

Table 11

Embodiment 4 Softening lotions were manufactured with the components and contents in

Tables 14 ~ 16 as described below, in which methanol extract, BF-2 or BF-2J was

added as an effective component.

Comparative example 4

Softening lotions were manufactured with the components and contents in

Tables 14 ~ 16 as described below without adding any effective component for

whitening.

Table 14

Embodiment 5

Nutritious lotions were manufactured with the components and contents in

Tables 17 ~ 19 as described below, in which methanol extract, BF-2 or BF-2J was

added as an effective component.

Comparative example 5

Nutritious lotions were manufactured with the components and contents in

Tables 17 ~ 19 as described below without adding any effective component for

whitening.

Table 17

Table 18

Embodiment 6 Packs were manufactured with the components and contents in Tables 20 ~ 22

as described below, in which methanol extract, BF-2 or BF-2J was added as an effective component.

Comparative example 6 Packs were manufactured with the components and contents in Tables 20 ~ 22

as described below without adding any effective component for whitening.

Table 20

Table 21

Table 22

The process for testing the pigmentation inhibitory effect by the ointment for

external use, essence, nutritious cream, softening lotion, nutritious lotion and pack

manufactured as described above is as follows: First, an aluminum foil having two rows of six holes of 7mm diameter was

adhered to each forearm of twenty healthy men and women, and 60 mJ/cm² of light was

irradiated at 10cm distance from the arm by ORIEL solar simulator 1000W. Before the irradiation, the portion to be irradiated was washed with 70% aqueous ethanol solution. After the irradiation, the cosmetic compositions containing whitening

component according to the embodiments 1 to 6 and the cosmetic compositions not

containing whitening component according to the comparative examples 1 to 6 were

applied to the same row respectively in pairs three times a day. After applying for 3 weeks, the pigmentation degree was determined with a naked eye. Then, the degrees

of restraining pigmentation of the cosmetic compositions according to the embodiments were compared with those of the comparative examples respectively, and the result was

evaluated into 3 stages, i.e., remarkable effectiveness, effectiveness and ineffectiveness,

and was depicted in the following Table 23 along with the result of side effect

occurrence.

Table 23

As shown in Table 23, the cosmetic compositions containing whitening

component according to the present invention show skin whitening effect in at least 11

persons of 20 testees. Particularly, samples of embodiments 1-2 and 1-3 having lwt% of BF-2 and BF-2J respectively show excellent inhibitory effect of 75% and 80% respectively against pigmentation. It means that BF-2 and BF-2J are excellent skin

whitening agent for improving chloasma or freckles.

INDUSTRIAL APPLICABILITY As described above, the composition for skin whitening containing phloroglucinol compounds according to the present invention inhibits pigmentation on skin by restraining melanin generation, so it is very effective in skin whitening, e.g., to

improve chloasma and freckles. In addition, the composition of the present invention

may be used safely since it shows no side effects on skin.

Claims

WHAT IS CLAIMED IS:

1. A composition for skin whitening comprising phloroglucinol compound

as an effective component selected from the group consisting of a compound having the

structure of chemical formula 1, a compound having the structure of chemical formula 2, and a mixture thereof:

Chemical Formula 1

Chemical Formula 2

2. The composition for skin whitening according to claim 1, wherein the

content of the phloroglucinol compound is 0.00001 to 5.0wt% on the basis of total weight of the composition.

3. The composition for skin whitening according to claim 1, wherein the

phloroglucinol compound is extracted from Baeckea frutescens.

4. The composition for skin whitening according to claim 3, wherein the

phloroglucinol compound is extracted from Baeckea frutescens using a solvent selected

from the group consisting of methanol, chloroform, and a mixture thereof.

5. The composition for skin whitening according to claim 1, wherein the

composition has one form selected from the group consisting of ointment for external

use, essence, nutritious cream, softening lotion, nutritious lotion, and pack.

6. A composition for skin whitening comprising Baeckea frutescens extract

as an effective component.

7. The composition for skin whitening according to claim 6, wherein the Baeckea frutescens extract is extracted using a solvent selected from the group consisting of methanol, chloroform, and a mixture thereof.

8. The composition for skin whitening according to claim 6 or 7, wherein the content of the Baeckea frutescens extract is 0.0001 to 50.0wt% of dry weight on the

basis of total weight of the composition.