Abstract:
Disclosed is a skin-whitening agent which comprises as its effective ingredient a group of substances capable of forming chemical complexes with melanin monomers. Boron-containing compounds and organelles originated from animals, plants and microorganisms make the invention feasible as they commonly suppress pigmentation through a novel action mechanism where melanin monomers are trapped by chemical complex formation.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to an entirely novel type of skin-whitening agent which suppresses melanin polymer formation through trapping of melanin monomers, in particular, to a skin-whitening agent characterized in that it comprises as its effective ingredient a group of substances which bear the property of forming chemical complexes with melanin monomers. 
     2. Description of Prior Art 
     Various researches have been carried out for skin-whitening agents which may eliminate pigmented spots on human skin such as melasma and freckles, as well as enhancing throughout the whole skin the ability of retaining both beauty and whiteness. It is said that several factors such as melanin, carotene, blood stream level, skin thickness and skin transparency are involved in the color tone of healthy human skin. Among these factors, the level of eumelanin and pheomelanin are also known to be a major cause of pigmentation. Researches for skin-whitening cosmetics have also long been carried out: Such cosmetics are directed at suppressing the formation of melanin, a cause of melasma and freckles, as well as to enhance throughout the whole skin the ability of retaining both beauty and whiteness. 
     Recently, as to the process of forming melanin polymer in animal and human, in addition to the conventionally known pathway wherein tyrosinase participates, the presence and role of further critical pathways after DOPAchrome and their regulating enzymes are currently being clarified: Such pathways are those which proceed via two distinct types of melanin monomers, i.e. DHICA (5,6-dihydroxyindole-2-carboxylic acid) and DHI (dihydroxy-indole), while these two melanin monomers regulating enzymes are those including DOPAchrome tautomerase and DHICA-oxidase. 
     Various tyrosinase inhibitory compounds including kojic acid have been used as a means of suppressing the formation of melanin: Such compounds however have the disadvantage of being unsatisfactory in efficacy or lacking rapid effectiveness. Thus, there have been great expectations for the development of skin-whitening agents which are capable of achieving consistently high efficacy through any novel action mechanisms. 
     SUMMARY OF THE INVENTION 
     In view of the foregoing, the object of the present invention is to provide an entirely novel type of skin-whitening agent which bears any action mechanism different to those hitherto known. 
     The present inventor first directed his attention to the fact that one of the chemicals, i.e. boronophenylalanine (abbreviated &#34;BPA&#34; hereinafter), as tested in neutron capture therapy, previously established by the inventor as an effective therapy for malignant melanoma, specifically accumulated in melanoma cells. 
     On the basis of this evidence, the present inventor then energetically pursued investigations, resulting in the discovery of an entirely novel action mechanism where a series of boron-containing compounds including BPA and natural substances from organelles including CV (coated vesicles) form chemical complexes in cells with melanin monomers such as DHICA, DHI and DOPA and suppress the formation of melanin polymers in pigment cells to reduce pigmentation. The present invention was completed on the basis of these findings. 
     In particular, the present invention provides a skin-whitening agent characterized in that it comprises as its effective ingredient a group of substances which form chemical complexes with melanin monomers and trap them from the pathway of melanin polymer synthesis. 
     Further, the present invention provides a skin-whitening agent of the same type, wherein the group of substances are boron-containing compounds and/or natural substances originated from animals, plants and microorganisms. 
     Still further, the present invention provides a skin-whitening agent of the same type, wherein the boron-containing compound is one or more members selected from the group consisting of but not limited to boronophenylalanine, boroglycine, borodimethylglycine, potassium borotartrate, boric acid, dihydroxyphenylborane, borobetaine and tetrasodium borate. 
     Still further, the present invention provides a skin-whitening agent of the same type, wherein the natural substances are extracts of CV, melanosomes, lysosomes and/or other organelles originated from animals, plants and microorganisms. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The formation mechanism for chemical complexes of the above described effective ingredients and melanin monomers, clarified by the present inventor, will be illustrated below using BPA as an example. ##STR1## 
     Examples of boron-containing compounds which are favorably incorporated as effective ingredients according to the present invention are one or more of the following: boronophenylalanine, boroglycine, borodimethylglycine, potassium borotartrate, boric acid, dihydroxy-phenylborane, borobetaine and tetrasodium borate. 
     Examples of natural substances originated from animals, plants and microorganisms are melanosomes as organelles, such as melanosomes, CV, lysosomes and organelle extracts thereof, which are prepared by the method as reported by Wilczck and Mishima (Melanoma Res 3:255-262,1993). 
     There are provided no special restrictions as to forms of the skin-whitening agent according to the present invention, as long as they are acceptable for external use: The skin-whitening agent according to the present invention is provided in extensive uses in conventional forms acceptable in pharmaceuticals and cosmetics, for example, poultice, plaster, paste, cream, ointment, tincture, aerosol, emulsion, lotion, milk, essence, gel, facial pack, powder, foundation, sunlight-shielding agent and bath salt. 
     Further, in the preparation of skin-whitening agents according to the present invention, the use of conventional skin-whitening compounds with action mechanisms different from that of the effective ingredient according to the present invention, in particular, ascorbic acid, arbutin and kojic acid, results in a remarkable dual effect which arithmetically or synergistically enhances the skin-whitening effect in the skin-whitening agent according to the present invention to render it very efficacious to intractable pigmentations. 
     In addition to these skin-whitening compounds, a variety of conventional effective ingredients, for example, peripheral vasodilators such as cepharanthine, vitamin E, vitamin E nicotinate, nicotinic acid, nicotinic acid amide, benzyl nicotinate, ginger tincture and mentha tincture, refrigerants such as camphor, menthol and mentha oil, antiseptics such as hinokitiol, benzalkonium chloride and undecylenic acid, anti-inflammatories such as adrenal cortex hormone, ε-aminocaproic acid, lysozyme chloride, glycyrrhizin and allantoin, and a variety of extracts originated from animals, plants and microorganisms such as placental extract, glycyrrhiza extract, lithospermum root extract and lactic acid bacteria-fermented extract, can be suitably used to meet respective final uses, as long as they do not hinder the attainment of the objectives of the present invention. 
     Still further, in the skin-whitening agent according to the present invention, in addition to the above described conventional effective ingredients, a variety of conventional moisture-retaining agents, antiseptics, antioxidants, chelating agents, pH regulating agents, flavoring agents, coloring agents, UV-absorbents and scattering agents can be used if necessary, as long as they do not hinder the attainment of the objectives of the present invention. 
    
    
     EXAMPLE 
     The present invention will be explained hereinafter by disclosing several Experiments and Formulations. Such disclosure is however only illustrative of preferred embodiments according to the present invention, and not intended in any way to limit the scope of the present invention. 
     Experiment 1 
     Test of Inhibition on the Formation of Melanochrome from DOPAchrome 
     (a) Method 
     Preparation of 5 mM DOPAchrome solution as reagent 
     Solution A: 10 mM DOPA 
     Solution B: 20 mM aqueous sodium metaperiodate solution 
     Equi-volumes of Solutions A and B are mixed to obtain a solution of vermilion color. The solution is prepared immediately before use. 
     Procedure 
     One milliliter aliquots of 1,000 mM phosphate buffer were mixed first with either boronophenylalanine, potassium borotartrate, dihydroxylphenylborane, boroglycine, tetrasodium borate or boric acid to give respective concentrations given in Tables 1 to 7, then with pure water to bring the total volumes to 1.8 ml. The resultant solutions were then placed in 37° C. water bath, quickly mixed after adding 0.2 ml aliquots of a fresh 5 mM DOPAchrome, and allowed to react while stirring. After 30 minute reaction in the water bath, the absorbance of reaction mixtures were immediately measured at 500 nm, and color tone was observed macroscopically. 
     (b) Results 
     Results are shown in Tables 1 to 6. Dark melanochrome was formed and the formation of dark fine sediment was initiated after 30 minutes in reaction mixtures which had no effective ingredients according to the present invention. Conversely, reaction mixtures which contained any of the effective ingredients according to the present invention turned brown but caused no sedimentation as checked by macroscopic observation. These results indicated that the ingredients according to the present invention do suppress the formation of melanochrome in a concentration-dependent manner. 
     Further, similar results were obtained by measurement of absorbance at 500 nm as those found by macroscopic observation. Still further, noticeable bluish dark sediment was observed in reaction mixtures with no effective ingredients according to the present invention after standing overnight, while reaction mixtures with any of the effective ingredients according to the present invention remained transparent brown and contained no sedimentation. 
     (c) Consideration 
     Formation of melanochromes was suppressed in reaction mixtures containing any of the effective ingredients according to the present invention. This can be explained by the mechanism in which the effective ingredients according to the present invention form chemical complexes with melanin monomers which have been formed from DOPAchrome, thus this melanin monomer trapping inhibits the polymerization of DOPAchrome. 
     
                       TABLE 1______________________________________Inhibition on the formation ofmelanochrome from DOPAchrome of BPA            Macroscopic observation            Absorbance         After overnight Concentration            at a               standing at of BPA     wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.19      Dark     Dark                      Fine     Bluish dark                      sedimentation                               sedimentation1     0.25       0.80      Brown    Brown2     0.50       0.70      Brown    Brown3     1.00       0.58      Brown    Brown4     2.50       0.43      Brown    Brown5     5.00       0.33      Brown    Brown6     7.50       0.27      Brown    Brown7     10.00      0.24      Brown    Brown______________________________________ 
    
     
                       TABLE 2______________________________________Inhibition on the formation of melanochrome fromDOPAchrome of potassium borotartrate            Macroscopic observation Concentration            Absorbance         After overnight of potassium            at a               standing at borotartrate            wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.245     Dark Fine                               Dark                      sedimentation                               Bluish dark-                               sedimentation1     0.25       1.127     Brown    Brown2     0.50       1.119     Brown    Brown3     1.00       1.045     Brown    Brown4     2.50       0.806     Brown    Brown5     5.00       0.635     Brown    Brown6     7.50       0.553     Brown    Brown7     10.00      0.480     Brown    Brown______________________________________ 
    
     
                       TABLE 3______________________________________Inhibition on the formation of melanochrome fromDOPAchrome of dihydroxyphenylborane            Macroscopic observation Concentration            Absorbance         After overnight of dihydroxy-            at a               standing at phenylborane            wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.305     Dark Fine                               Dark                      sedimentation                               Bluish dark-                               sedimentation1     0.25       0.924     Brown    Brown2     0.50       0.771     Brown    Brown3     1.00       0.602     Brown    Brown4     2.50       0.381     Brown    Brown5     5.00       0.283     Brown    Brown6     7.50       0.240     Brown    Brown7     10.00      0.172     Brown    Brown______________________________________ 
    
     
                       TABLE 4______________________________________Inhibition on the formation of melanochromefrom DOPAchrome of boroglycine            Macroscopic observation            Absorbance         After overnight Concentration            at a               standing at of boroglycine            wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.144     Dark Fine                               Dark                      sedimentation                               Bluish dark-                               sedimentation1     0.50       0.873     Brown    Brown2     1.00       0.817     Brown    Brown3     2.00       0.703     Brown    Brown4     5.00       0.380     Brown    Brown5     8.00       0.217     Brown    Brown______________________________________ 
    
     
                       TABLE 5______________________________________Inhibition on the formation of melanochromefrom DOPAchrome of tetrasodium borate            Macroscopic observation Concentration            Absorbance         After overnight of tetrasodium            at a               standing at borate     wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.269     Dark Fine                               Dark                      sedimentation                               Bluish dark-                               sedimentation1     0.25       1.098     Brown    Brown2     0.50       0.988     Brown    Brown3     1.00       0.749     Brown    Brown4     2.50       0.523     Brown    Brown5     5.00       0.329     Brown    Brown6     7.50       0.227     Brown    Brown7     10.00      0.153     Brown    Brown______________________________________ 
    
     
                       TABLE 6______________________________________Inhibition on the formation of melanochromefrom DOPAchrome of boric acid            Macroscopic observation Concentration            Absorbance         After overnight of boric   at a               standing at acid       wavelength                      On       ambientControl (mg/ml)    of 500 nm measurement                               temperature______________________________________Control 0          1.226     Dark Fine                               Dark                      sedimentation                               Bluish dark-                               sedimentation1     0.25       0.955     Brown    Brown2     0.50       0.759     Brown    Brown3     1.00       0.602     Brown    Brown4     2.50       0.417     Brown    Brown5     5.00       0.279     Brown    Brown6     7.50       0.223     Brown    Brown7     10.00      0.172     Brown    Brown______________________________________ 
    
     Experiment 2 
     Whitening Effect on Cultured Melanoma Cells 
     (a) Method 
     Procedure 
     1×10 5  cells of B16F10 cells were seeded in commonly used medium which was replaced after 8 hours with those containing either boronophenylalanine, potassium borotartrate, dihydroxyphenylborane, boroglycine, tetrasodium borate and boric acid at respective concentrations given in Tables 7 to 12, followed by cultivation. Cultured medium was changed 3 days after the beginning of cultivation, and 6 days after the cells were collected and subjected to macroscopic observation. 
     (b) Results 
     Results are shown in Tables 7 to 12. The B16F10 cells cultured in medium with any of the effective ingredients according to the present invention marked a more noticeable whitening in comparison with those which had been cultured in medium containing no ingredients according to the present invention. 
     
                       TABLE 7______________________________________Skin-whitening effect of BPA on cultured melanoma cellsConcentration of BPA(μg/ml)           Macroscopic observation______________________________________Control   0              Unchanged1        500             Slightly whitened2       1000             Whitened______________________________________ 
    
     
                       TABLE 8______________________________________Skin-whitening effect of borotartaric acid on cultured melanoma cellsConcentration ofborotartaric acid(μg/ml)          Macroscopic observation______________________________________Control   0             Unchanged1       200             Slightly whitened2       400             Whitened______________________________________ 
    
     
                       TABLE 9______________________________________Skin-whitening effect of dihydroxyphenylboraneon cultured melanoma cellsConcentration ofdihydroxyphenylborane(μg/ml)           Macroscopic observation______________________________________Control  0               Unchanged1       200              Slightly whitened2       500              Whitened______________________________________ 
    
     
                       TABLE 10______________________________________Skin-whitening effect of boroglycine on cultured melanoma cellsConcentration ofboroglycine(μg/ml)          Macroscopic observation______________________________________Control  0              Unchanged1       200             Slightly whitened2       500             Whitened______________________________________ 
    
     
                       TABLE 11______________________________________Skin-whitening effect of tetrasodium borate on cultured melanoma cellsConcentration oftetrasodium borate(μg/ml)          Macroscopic observation______________________________________Control  0              Unchanged1       100             Slightly whitened2       200             Whitened______________________________________ 
    
     
                       TABLE 12______________________________________Skin-whitening effect of boric acid on cultured melanoma cellsConcentration ofboric acid(μg/ml)          Macroscopic observation______________________________________Control  0              Unchanged1       200             Slightly whitened2       400             Whitened______________________________________ 
    
     Example of Formulations 
     Following are examples of formulation according to the present invention. In respective formulations, the wording &#34;appropriate&#34; shall refer to the amount of specified ingredient which is to bring the total amounts in respective formulations up to 100% by weight. 
     Formulation 
     
         ______________________________________CreamIngredients         Amounts (% by weight)______________________________________BPA                 1.00Sodium hyaluronate  2.00Polyethylene glycol 400               3.00Polyoxyethylene cetylether (EO 25)               5.00Stearic acid        5.00Avocado oil         1.00Almond oil          10.00Sodium dl-pyrrolidonecarboxylate solution               5.00Parahydroxybenzoate 0.70Disodium edetate    0.01Refined water       Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________CreamIngredients         Amounts (% by weight)______________________________________Melanosome          2.0Boric acid          0.5Polyethyleneglycol monostearate (EO 40)               2.0Self-emulsifying glyceryl monostearate               5.0Stearic acid        5.0Behenyl alcohol     1.0Liquid paraffin     10.0Glyceryl trioctanoate               10.0Glycerine           5.0Ethylparaben        0.1Refined water       Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________Milky lotionIngredients          Amounts (% by weight)______________________________________Dihydroxyphenylborane                4.002-Octyldodecanol     3.00Polyoxyethylene cetylether (EO 25)                0.50Polyoxyethylene oleylether (EO 20)                1.00Stearic acid         0.50Shea butter          0.50Avocado oil          4.004-tert-Butyl-4&#39;-methoxy-dibenzoyl methane                5.00Parahydroxybenzoate  0.20Quince seed extract  5.00Xanthan gum          0.14Disodium edetate     0.01Refined water        Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________MilkIngredients         Amounts (% by weight)______________________________________Tetrasodium borate  0.50Glycol salicylate   0.10Butylalcohol        3.50Arbutin             2.00Coconut fatty acid monoethanolamine               2.00Stearic acid        0.50Myristic acid       0.50Avocado oil         4.00Octyl methooxycinnamate               2.00Natural vitamin E   0.04Parahydroxybenzoate 0.20Sodium hyaluronate  5.00Scutellaria root extract               0.14Disodium edetate    0.01Refined water       Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________Facial lotionIngredients   Amounts (% by weight)______________________________________BPA           1.00Boroglycine   0.25Ethanol       15.00Ethylparaben  0.10Citric aid    0.10Sodium citrate         0.301,3-Butylene glycol         4.00Disodium edetate         0.01Refined water Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________Cream facial packIngredient           Amounts (% by weight)______________________________________Borodimethylglycine  3.00Polyethylene glycol 1500                5.00Stearic acid diethanolamide                5.00Stearic acid         5.00Myristic             5.00Coconut oil          15.00Natural vitamin E    0.04Parahydroxybenzoate  0.20Sodium dl-pyrrolidone carboxylate solution                5.00Disodium edetate     0.01Refined water        Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________OintmentIngredients        Amounts (% by weight)______________________________________Borobetaine        1.00Phenyl salicylate  0.40Sodium hydroxymethoxybenzophenone              1.00sulfonateIsoamyloctyl gallate              2.00Coconut fatty acid monoethanolamide              5.00Petrolatum         10.00Stearic acid       5.00Oleic acid         1.00Olive oil          10.00Parahydroxybenzoate              0.30Carrageenan        5.00Disodium edetate   0.01Refined water      Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________PoulticeIngredients       Amounts (% by weight)______________________________________Potassium borotartrate             0.50Boroglycine       0.10Allantoin         0.10Stearic acid diethanolamide             3.00Polyacrylic acid  27.00Ethanolic glycyrrhiza extract             0.10Aqueous scutellaria extract             0.05Disodium edetate  0.05Methoxycinnamate  4.00Sodium polyacrylate             7.00Aluminum chloride 0.30Concentrated glycerin             20.00Titanium oxide    4.00Refined water     Appropriate______________________________________ 
    
     Formulation 
     
         ______________________________________EssenceIngredients           Amounts (% by weight)______________________________________Lysosome              1.00Urocanic acid         0.50Isopropanol           0.50Benzylalcohol         0.05Aqueous keffiran solution                 1.50Coconut fatty acid monoethanolamide                 2.00Stearic acid          0.50Linolenic acid        0.50Avocado oil           2.00Turtle oil            3.00Natural vitamin E     0.04Parahydroxybenzoate   1.00One percent of aqueous carboxylvinylpolymer                 5.00solutionPlacental extract     0.14Disodium edetate      0.01Refined water         Appropriate______________________________________ 
    
     It was confirmed that the skin-whitening agents with these formulations exhibited similar efficacies as those shown in the above described Experiments. 
     The present invention provides a novel skin-whitening agent comprising as its effective ingredient a group of substances which form chemical complexes with melanin monomers. This skin-whitening agent is highly safe for humans and superior in skin-whitening effect. 
     While there has been described what is at present considered to be the preferred embodiments of the invention, it will be understood the various modifications may be made therein, and it is intended to cover in the appended claims all such modifications as fall within the true spirit and scope of the invention.