Cosmetics based on naturally derived melanin-coated pigments

A melanin-coated pigment obtained by coating a carrier with a readily alkali-soluble natural melanin, and a cosmetic containing the pigment are provided. The melanin-coated pigment is highly safe and has high coloring and high saturation tone, and a cosmetic which contains it is also expected to have ultraviolet-ray-absorbing and anti-oxidizing effects.

TECHNICAL FIELD 
The present invention relates to a melanin-coated pigment obtained by 
coating a carrier with a readily alkali-soluble natural melanin, and a 
cosmetic containing the pigment. 
BACKGROUND ART 
Melanin is a dark brown or black pigment which is known to have 
ultraviolet-ray-absorbing and other properties. 
Sepia melanin, extracted from the ink bags of cuttlefish, is known as a 
black pigment which is dispersed in a system and used in cosmetics in the 
same manner as carbon black and black iron oxide (Japanese Published 
Unexamined Patent Application No. 53211/80). 
U.S. Pat. No. 4,961,754 discloses a powder prepared by modifying the 
surface of an inorganic powder with a synthetic melanin produced by an 
oxidative polymerization reaction of a compound having an indole skeleton, 
and cosmetics containing the powder. 
Powders prepared by modifying the powder surface with a polymerized 
synthetic melanin are not suitable for use in cosmetics in respect of 
safety because of skin irritation due to residual low molecular weight 
compounds, free monomers and oxidizing catalysts such as potassium iodide 
and hydrogen peroxide from the synthesis process. 
Further, sepia melanin is insoluble in acids or alkalis and is poorly 
soluble in solvents, which makes its separation and purification 
difficult, and thus it is not suitable for use in cosmetics, either. 
It has been discovered that melanin adsorbed on a carrier can be used for 
adding color, particularly to cosmetics, as a flesh- or gray-colored 
pigment which differs from the color of melanin. 
DISCLOSURE OF THE INVENTION 
The present invention provides a melanin-coated pigment obtained by coating 
a carrier with a readily alkali-soluble natural melanin, and cosmetics 
containing the pigment. 
As the carrier, any powder which is insoluble in water, oil, and the like 
and can adsorb melanin may be used. Preferred examples are inorganic and 
organic pigments. 
The inorganic pigment consists of an inorganic compound. Examples of the 
inorganic pigment include titanium dioxide, talc, sericite, mica, 
micaceous titanium, montmorillonite, kaolin, alumina, magnesium oxide, 
cesium oxide, silica, muscovite, barium sulfate, aluminum silicate, 
aluminum magnesium silicate, sodium magnesium silicate, calcium silicate, 
iron oxide, calcium carbonate, pumice powder, and ultramarine. 
The organic pigment consists of an organic compound. A number of dyes and 
pigments may be used as the organic pigment. In addition, as the inorganic 
or organic powder may be used resins such as nylon 6, nylon 6.6, nylon 
powder, and silicon resin, as well as cellulose, crystalline cellulose, 
silk powder, and the like. 
These carriers have a particle diameter of 0.01-100 microns and may be used 
alone or in combination. 
The readily alkali-soluble natural melanin according to the present 
invention refers to alkali-soluble melanins derived from microorganisms 
and plants. Preferred examples include melanins produced by microorganisms 
belonging to the genus Aureobasidium, Aspergillus, and Streptomyces 
(hereinafter referred to as "microbial melanin"), which can be obtained by 
known methods, for example, the methods described in FEMS Microbiology 
Letters, 9, 237-240, 1980; Trans. Br. Mycol. Soc., 70 (3), 453-455, 1978; 
and Mol. Gen. Genet., 200, 422-432, 1985. 
The readily alkali-soluble natural melanin to be used in the present 
invention not only dissolves in aqueous solutions of alkalis such as 
aqueous ammonia and aqueous solutions of sodium hydroxide, potassium 
hydroxide and calcium hydroxide, but is also soluble in organic solvents 
such as triethylamine, triethanolamine, ammonia-containing methanol, 
ethanol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, and 
glycerin, and may be subjected to nonaqueous treatment. 
The melanin-coated pigment according to the present invention can be 
prepared by applying a known method of adsorbing a component of a solution 
on a carrier. For example, it can be obtained by the following method. 
The above-mentioned melanin is dissolved in an alkali solution such as 
0.01-6.0N sodium hydroxide, potassium hydroxide or ethanolamine. A carrier 
is added thereto and the mixture is stirred. To the resulting colored 
suspension is gradually added an acidic solution such as 0.01-6.0N 
hydrochloric acid with stirring, until the melanin is sufficiently 
adsorbed on the carrier. By lowering the pH to 1-4 the supernatant becomes 
clear. The solution is subjected 2-5 times to filtration and washing with 
a 5- to 10-fold amount of water. Then, the residue is dried and the dried 
product is crushed to obtain a powdery melanin-coated pigment 0.05-50 
.mu.m in size. 
The carrier is coated with 0.01-50 w/w % of the readily alkali-soluble 
natural melanin. 
The thus obtained melanin-coated pigment can be used as a flesh- or 
gray-colored pigment in paint, ink, cosmetics, and the like. When used in 
cosmetics, it is expected to be also effective in absorbing ultraviolet 
rays. 
A melanin-coated pigment having the desired tint may be obtained by 
experimentally adjusting the type of carrier and the amount of the melanin 
coating. Such experiments may be easily carried out by a person skilled in 
the art. 
The malanin-coated pigment of the present invention may be employed in 
cosmetics in any desired form, for example, solutions, emulsions, powders, 
jellies, and solid preparations. Examples of such cosmetics include 
skin-care products such as lotions, emulsions, creams, soap and pack; 
make-up products such as lipsticks, foundations, eye-shadows, and 
eye-liners; and hair-care products such as shampoos and rinses. 
Cosmetics according to the present invention may comprise various 
components which are generally used in cosmetics, for example, fats and 
oils, hydrocarbons, waxes, fatty acids, synthetic esters, alcohols, 
surfactants, thickeners, moisturizers, preservatives, fragrances, 
pigments, ultraviolet absorbers, chemicals, and water, which are selected 
depending upon the form of cosmetics. 
Examples of the fats and oils are jojoba oil, castor oil, olive oil, 
soybean oil, coconut oil, palm oil, cacao butter, mink oil, turtle oil, 
fatty acid diethanolamide, and shea butter. 
Examples of the hydrocarbons are liquid paraffin, solid paraffin, vaseline, 
microcrystalline wax, and squalane. 
Examples of the waxes are beeswax, Japan wax, lanolin, carnauba wax, 
candelilla wax, and hydrocarbon wax. 
Examples of the fatty acids are myristic acid, palmitic acid, stearic acid, 
oleic acid, and isostearic acid. 
Examples of the synthetic esters are isopropyl myristate, isopropyl 
palmitate, butyl oleate, myristyl myristate, octyldecyl myristate, 
propylene glycol monostearate, myristyl lactate, isostearyl malate, 
glycerin monostearate, distearyldimethyl ammonium chloride, and 
acetyltributyl citrate. 
The fats and oils, hydrocarbons, waxes, fatty acids and synthetic esters 
are usually contained in the cosmetics in a total amount of 0.01-70 w/w %. 
Examples of the alcohols are ethanol, 1,3-butylene glycol, propylene 
glycol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, and 
denatured alcohol. The alcohols are contained in the cosmetics in an 
amount of 0.01-60 w/w %. 
Examples of the surfactants are polyoxyethylene (60) hardened castor oil, 
sodium lauryl sulfate, polyoxyethylene glyceryl pyroglutamate isostearate, 
sodium alkylbenzene sulfonate, polyoxyethylene (10) stearyl ether, dialkyl 
sulfosuccinate, cetyl pyridinium bromide, n-octadecyl trimethylammonium 
chloride, monoalkyl phosphate, N-acylglutamic acid, sucrose fatty acid 
ester, glyceryl isostearate, polyoxyethylene (20) sorbitan monostearate, 
sorbitan sesquioleic acid ester, polyoxyethylene (20) sorbitan monooleate, 
sorbitan sesquioleate, glycerin monostearate, polyoxyethylene cetyl ether 
phosphate, polyoxyethylene lauryl ether sodium sulfate, 
polyoxyethylene-reduced lanolin, polyoxyethylene (20) sorbitan 
tristearate, polyoxyethylene (20) sorbitan trioleate, and soybean 
phospholipid. The surfactants are contained in the cosmetics in an amount 
of 0.01-40 w/w %. 
Examples of the thickeners are carboxyvinyl polymer, methylpolysiloxane, 
dextran, carboxymethyl cellulose, carrageenin, hydroxymethyl cellulose, 
vinyl acetate resin emulsion, and polyacrylic acid ester emulsion. The 
thickeners are contained in the cosmetics in an amount of 0.01-5 w/w %. 
Examples of the moisturizers are glycerin, propylene glycol, 1,3-butylene 
glycol, pyrrolidonecarboxylic acid, lactic acid, hyaluronic acid, 
collagen, N-acetyl-L-glutamine, triethanolamine, citric acid, and sodium 
citrate. The moisturizers are contained in the cosmetics in an amount of 
0.01-30 w/w %. 
Examples of the preservatives are benzoic acid, salicylic acid, 
dehydroacetic acid or salts thereof, phenols such as p-hydroxybenzoate, 
2,4,4'-trichloro-2'-hydroxydiphenyl ether, and 
3-trifluoromethyl-4,4'-dichlorocarbanilide. The preservatives are 
contained in the cosmetics in an amount of 0.01-0.5 w/w %. 
Any fragrances which are conventionally used in cosmetics may be used. 
Examples of the pigments to be added to the cosmetics are titanium dioxide, 
talc, sericite, mica, micaceous titanium, montmorillonite, kaolin, 
alumina, magnesium oxide, cesium oxide, silica, muscovite, barium sulfate, 
aluminum silicate, aluminum magnesium silicate, sodium magnesium silicate, 
calcium silicate, iron oxide, calcium carbonate, pumice powder, 
ultramarine, nylon 6, nylon 6.6, nylon powder, cellulose, crystalline 
cellulose, and silk powder. The pigments are contained in the cosmetics in 
an amount of 0.01-90 w/w %. 
Examples of the ultraviolet absorbers are ethyl diisopropyl cinnamate, 
methyl diisopropyl cinnamate, glyceryl mono(2-ethylhexylate) di(p-methoxy) 
cinnamate, p-aminobenzoic acid, ethyl p-aminobenzoate, 
4-tert-butyl-4'-methoxy-dibenzoylmethane, and 2-ethylhexyl 
p-dimethylaminobenzoate. The ultraviolet absorbers are contained in the 
cosmetics in an amount of 0.01-10 w/w %. 
Examples of the chemicals are vitamin E, vitamin C or a derivative thereof, 
licorice extract, dipotassium glycyrrhizinate, and placenta liquid. The 
chemicals are contained in the cosmetics in an amount of 0.01-5 w/w %. 
The melanin-coated pigment according to the present invention may be used 
alone, or a mixture of two or more pigments may be used. The pigment is 
contained in the cosmetics in an amount of 0.01-90% by weight. 
The cosmetics containing the melanin-coated pigment according to the 
present invention can be prepared in the same manner as in known methods 
for the production of cosmetics, except that a portion or all of the 
pigments used is replaced by the pigment of the present invention. 
The microbial melanin to be used in the present invention is highly safe 
and has a wide band of absorption in the ultraviolet region. The pigment 
coated with the microbial melanin has high coloring and saturation tones, 
and the cosmetics which contain the pigment have an excellent 
ultraviolet-ray-blocking effect.

BEST MODE FOR CARRYING OUT THE INVENTION 
Examples, test examples and comparative examples are provided below. The 
percentages indicated in the Examples are % by weight. 
EXAMPLE 1 
Preparation of Melanin-Coated Pigment 
Streptomyces aureofaciens (ATCC 10762) cells were inoculated into 300 ml of 
a seed medium (2.0% starch, 0.5% peptone, 0.9% corn steep liquor, 0.1% 
CaCO.sub.3, pH 5.5) in a 2-liter Erlenmeyer flask, and subjected to 
shaking culture (rotation: 200 rpm) at 30.degree. C. for 3 days. 
The resulting seed culture was inoculated into 30 liters of a production 
medium (5.0% starch, 6.0% soybean powder, 0.75% lysine hydrochloride, 50 
mg/l Mg.sub.2 SO.sub.4.7H.sub.2 O, 50 mg/l CuSO.sub.4.5H.sub.2 O, 50 mg/l 
ZnSO.sub.4.7H.sub.2 O, 0.1% CaCO.sub.3, 4.0% soybean oil) in a 30-liter 
jar fermenter, and stirring culturing with aeration (rotation: 425 rpm, 
aeration: 10 l/min) was carried out at 27.degree. C. for 10 days. 
To 10 liters of the resulting culture was added 40 g of sodium hydroxide, 
the mixture was subjected to centrifugation (8,000 rpm, 30 minutes), and 
the supernatant was separated. To the obtained cells was added 1 liter of 
0.1N aqueous sodium hydroxide, followed by centrifugation (8,000 rpm, 30 
minutes) to obtain a supernatant. This supernatant was combined with the 
previously separated supernatant, and 150 ml of 10N hydrochloric acid was 
added thereto, followed by centrifugation (8,000 rpm, 30 minutes). To the 
obtained residue was added 1 liter of 1N sodium hydroxide, and the mixture 
was stirred and then filtered to obtain a filtrate. To the filtrate was 
added 100 ml of 10N hydrochloric acid, and the mixture was subjected to 
centrifugation (8,000 rpm, 30 minutes). The obtained residue was subjected 
three times to a washing procedure consisting of addition of 1 liter of 
purified water and centrifugation (8,000 rpm, 30 minutes) to obtain 60 g 
of crude microbial melanin. 
To 1.4 g of the obtained crude microbial melanin was added 50 ml of 6N 
hydrochloric acid, and the mixture was heated at 110.degree. C. for 48 
hours for hydrolysis. The reaction mixture was dissolved in 100 ml of 6N 
sodium hydroxide, and the solution was filtered through a glass fiber 
filter paper. To the filtrate was added 200 ml of 6N hydrochloric acid, 
and a precipitate was separated by sedimentation. After 1 liter of water 
was added to the sediment, a precipitate was again separated by 
sedimentation and was freeze-dried to obtain 1 g of purified melanin. The 
purified melanin (1 g) was dissolved uniformly in 3,000 g of 0.1N aqueous 
sodium hydroxide. Then, 100 g of a carrier (titanium dioxide, mica, talc, 
nylon powder or sericite) was added thereto and uniformly dispersed, and 
400 g of 1N hydrochloric acid was slowly added thereto with stirring. The 
mixture was filtered, washed with water, and dried, and the dried product 
was crushed to obtain a melanin-coated pigment 5 .mu.m in size. 
EXAMPLE 2 
Preparation of Melanin-Coated Pigment 
The purified melanin (0.1 g) obtained in Example 1 was dissolved uniformly 
in 100 ml of 0.1N aqueous potassium hydroxide. Then, 5 g of titanium 
dioxide was added to the solution and dispersed uniformly therein, and 12 
g of 1 N sulfuric acid was slowly added thereto with stirring to adjust 
the pH to 7. Fifty milliliters of purified water containing 0.5 g of 
barium chloride was added to the mixture with stirring, and then 3 g of 1N 
sulfuric acid was slowly added thereto. The resulting mixture was 
filtered, washed with water and dried, and the dried product was crushed 
to obtain a melanin-coated pigment 5 .mu.m in size. 
EXAMPLE 3 
Non-Aqueous Preparation of Melanin-Coated Pigment 
The purified melanin (0.1 g) obtained in Example 1 was dissolved uniformly 
in 100 ml of methanol containing 0.5 g of triethylamine. Then, 5 g of 
titanium dioxide was added to the solution and dispersed uniformly 
therein. The solvent was distilled off, and the dried product was crushed 
to obtain a melanin-coated pigment 5 .mu.m in size. 
EXAMPLE 4 
Non-Aqueous Preparation of Melanin-Coated Pigment (Hydrophobic Treatment 
with a Silicon Derivative) 
To a uniform dispersion of 0.1 g of the purified melanin obtained in 
Example 1, 0.5 g of triethylamine, 100 ml of methanol and 5 g of titanium 
dioxide was added 20 ml of isopropyl alcohol containing 0.5 g of 
methylhydrogen polysiloxane to obtain a uniform solution. The solvent was 
distilled off, and the dried product was heated at 160.degree. C. for one 
hour and then crushed to obtain a hydrophobic melanin-coated pigment 5 
.mu.m in size. 
EXAMPLE 5 
Preparation of a Solid-Type Foundation 
A solid-type foundation having the following composition was prepared using 
the melanin-coated pigments obtained in Example 1. 
______________________________________ 
(2) Melanin-coated mica 55.0% 
(3) Melanin-coated talc 22.0% 
(4) Iron oxide 1.0% 
(5) Liquid paraffin 6.0% 
(6) Lanolin 4.0% 
(7) Sorbitan sesquioleic ester 
1.5% 
(8) Fragrance 0.7% 
(9) p-Hydroxybenzoate 0.8% 
______________________________________ 
A uniform solution of the ingredients (6)-(9) in the ingredient (5) was 
added to the ingredients (1)-(4) while they were being mixed in a blender. 
The resulting mixture was crushed, and the obtained powder was subjected 
to compression molding to prepare a solid-type foundation. 
EXAMPLE 6 
Preparation of a Liquid Foundation 
A liquid foundation having the following composition was prepared using the 
melanin-coated pigments obtained in Example 1. 
______________________________________ 
(1) Melanin-coated titanium dioxide 
3.0% 
(2) Melanin-coated mica 2.0% 
(3) Melanin-coated nylon powder 
0.5% 
(4) Iron oxide 1.0% 
(5) Liquid paraffin 3.0% 
(6) Beeswax 2.0% 
(7) Carnauba wax 0.2% 
(8) Squalane 3.0% 
(9) Polyoxyethylene (20) sorbitan monooleate 
0.5% 
(10) Sorbitan sesquioleate 1.0% 
(11) Glycerin monostearate 3.0% 
(12) Polyoxyethylene cetyl ether phosphate 
0.2% 
(13) 1,3-Butylene glycol 12.0% 
(14) Carboxyvinyl polymer (1% aqueous 
10.0% 
solution) 
(15) Triethanolamine 0.1% 
(16) Fragrance 0.3% 
(17) p-Hydroxybenzoate 0.2% 
(18) Purified water 58.0% 
______________________________________ 
The ingredients (1)-(4), (12) and (13) were mixed and uniformly dispersed 
using a 3-roll mill (pigment portion). The ingredients (5)-(11), (16) and 
(17) were heated for dissolution at 80.degree. C. and the ingredients 
(14), (15) and (18) which had been uniformly mixed by heating at 
80.degree. C. and the pigment portion were added thereto. The resulting 
mixture was emulsified with a homomixer, and then cooled to obtain a 
liquid foundation. 
EXAMPLE 7 
Preparation of a Lipstick 
A lipstick having the following composition was prepared using the 
melanin-coated pigments obtained in Example 1. 
______________________________________ 
(1) Hydrocarbon wax 5.0% 
(2) Candelilla wax 2.0% 
(3) Glyceryl isostearate 35.0% 
(4) Liquid paraffin 45.0% 
(5) Melanin-coated titanium dioxide 
3.0% 
(6) Melanin-coated mica 6.7% 
(7) Organic pigment (Pigment Red 3 
3.0% 
aluminum lake) 
(8) Fragrance 0.3% 
______________________________________ 
The ingredients (4)-(7) were mixed and kneaded with a 3-roll mill to obtain 
a uniform mixture, and the ingredients (1)-(3) and (8) were added thereto, 
followed by heating for dissolution until the temperature reached 
85.degree. C. The mixture was stirred to obtain a uniform mixture, which 
was then charged into a container to prepare a lipstick. 
EXAMPLE 8 
Preparation of an Eye-Liner 
An eye-liner having the following composition was prepared using the 
melanin-coated pigments obtained in Example 1. 
______________________________________ 
(1) Ultramarine 2.0% 
(2) Melanin-coated talc 4.0% 
(3) Melanin-coated mica 4.0% 
(4) Vinyl acetate resin emulsion 
45.0% 
(5) Glycerin 5.0% 
(6) Polyoxyethylene (20) sorbitan monooleate 
1.0% 
(7) Carboxymethyl cellulose 15.0% 
(10% aqueous solution) 
(8) Acetyltributyl citrate 1.0% 
(9) Purified water 22.7% 
(10) Fragrance 0.2% 
(11) Dehydroacetic acid 0.1% 
______________________________________ 
The ingredients (5), (6) and (9) were heated for dissolution, and the 
ingredients (1)-(3) were added thereto, followed by mixing using a colloid 
mill to obtain a uniform mixture. The ingredients (4), (6)-(8), (10) and 
(11) were added thereto and the mixture was uniformly dispersed with a 
homomixer to obtain an eye-liner. 
EXAMPLE 9 
Preparation of an Eyebrow Pencil 
An eyebrow pencil having the following composition was prepared using the 
melanin-coated pigments obtained in Example 1. 
______________________________________ 
(1) Melanin-coated titanium dioxide 
3.0% 
(2) Melanin-coated talc 22.0% 
(3) Melanin-coated sericite 22.4% 
(4) Melanin-coated mica 15.0% 
(5) Ultramarine 2.0% 
(6) Japan wax 25.0% 
(7) Vaseline 7.0% 
(8) Squalane 3.0% 
(9) p-Hydroxybenzoate 0.3% 
(10) Vitamin E 0.3% 
______________________________________ 
The ingredients (1)-(5) were uniformly mixed using a blender (pigment 
portion). The ingredients (6)-(10) were heated for dissolution and stirred 
to obtain a uniform mixture. The pigment portion was added thereto, and 
the mixture was kneaded and formed into a stick to obtain an eyebrow 
pencil. 
EXAMPLE 10 
Preparation of a Mascara 
A mascara having the following composition was prepared using the 
melanin-coated pigment obtained in Example 1. 
______________________________________ 
(1) Melanin-coated mica 10.0% 
(2) Polyacrylic acid ester emulsion 
27.0% 
(3) Solid paraffin 7.0% 
(4) Lanolin 7.0% 
(5) Sorbitan sesquioleate 3.0% 
(6) p-Hydroxybenzoate 0.3% 
(7) Fragrance 0.2% 
(8) Purified water 45.5% 
______________________________________ 
The ingredient (8) was dispersed in the ingredient (1) with a homomixer, 
and the dispersion was uniformly mixed with the ingredient (2). The 
mixture was added to a mixture of the ingredients (3)-(7) which had been 
heated at 70.degree. C. for dissolution, and the resulting mixture was 
emulsified and dispersed with a homomixer to obtain a mascara. 
EXAMPLE 11 
Preparation of Sunscreen Emulsion 
A sunscreen emulsion having the following composition was prepared using 
the melanin-coated pigment obtained in Example 1. 
______________________________________ 
(1) Melanin-coated talc 0.5% 
(2) Soybean phospholipid 0.1% 
(3) 1,3-Butylene glycol 5.0% 
(4) 2-Ethylhexyl p-dimethylaminobenzoate 
0.1% 
(5) Liquid paraffin 2.0% 
(6) Glycerin monostearate 1.0% 
(7) Carboxyvinyl polymer 10.0% 
(1% aqueous solution) 
(8) Triethanolamine 0.1% 
(9) p-Hydroxybenzoate 0.2% 
(10) Fragrance 0.2% 
(11) Purified water 80.8% 
______________________________________ 
The ingredients (1)-(3) were mixed and uniformly dispersed with a 3-roll 
mill (pigment portion). The ingredients (4)-(6) were heated at 80.degree. 
C. for dissolution (oil phase). The ingredients (7)-(11) were heated at 
80.degree. C. for dissolution, the pigment portion was added thereto, and 
the resulting mixture was stirred to obtain a uniform mixture (water 
phase). The water phase was added to the oil phase, and the mixture was 
emulsified with a homomixer and then cooled to obtain a sunscreen 
emulsion. 
EXAMPLE 12 
Preparation of a Sunscreen Cream 
A sunscreen cream having the following composition was prepared using the 
melanin-coated pigment obtained in Example 1. 
______________________________________ 
(1) Melanin-coated sericite 1.0% 
(2) Beeswax 2.0% 
(3) Carnauba wax 0.2% 
(4) Cetanol 3.0% 
(5) Liquid paraffin 3.0% 
(6) 1,3-Butylene glycol 5.0% 
(7) POE (20) sorbitan tristearate 
1.5% 
(8) Glycerin monostearate 3.0% 
(9) Carboxyvinyl polymer 10.0% 
(1% aqueous solution) 
(10) Triethanolamine 0.1% 
(11) p-Hydroxybenzoate 0.2% 
(12) Fragrance 0.3% 
(13) Purified water 70.7% 
______________________________________ 
The ingredients (2)-(7) were heated at 80.degree. C. for dissolution (oil 
phase). The ingredients (1) and (8)-(13) were heated at 80.degree. C. for 
dissolution and stirred to obtain a uniform mixture (water phase). The 
water phase was added to the oil phase, and the mixture was emulsified 
with a homomixer and then cooled to obtain a sunscreen cream. 
EXAMPLE 13 
Preparation of a Sunscreen Lotion 
A sunscreen lotion having the following composition was prepared using the 
melanin-coated pigment obtained in Example 1. 
______________________________________ 
(1) Melanin-coated nylon powder 
0.5% 
(2) Glycerin 1.0% 
(3) 1,3-Butylene glycol 5.0% 
(4) Citric acid 0.1% 
(5) Sodium citrate 0.08% 
(6) POE (60) hardened castor oil 
1.0% 
(7) p-Hydroxybenzoate 0.2% 
(8) Fragrance 0.2% 
(9) Ethanol 5.0% 
(10) Purified water 86.92% 
______________________________________ 
The ingredients (1)-(5) and (10) were heated at 80.degree. C. for 
dissolution and stirred to obtain a uniform mixture (water phase). The 
ingredients (6)-(9) were heated at 80.degree. C. for dissolution (oil 
phase). The water phase was added to the oil phase with stirring, and the 
resulting uniform mixture was cooled to obtain a sunscreen lotion. 
EXAMPLE 14 
Preparation of a Sunscreen Cream 
A sunscreen cream having the following composition was prepared using the 
melanin-coated pigments obtained in Example 1. 
______________________________________ 
(1) Melanin-coated titanium dioxide 
2.4% 
(2) Melanin-coated talc 1.2% 
(3) Stearic acid 1.0% 
(4) Cetanol 0.5% 
(5) Shea butter 0.05% 
(6) Castor oil 0.7% 
(7) Methylpolysiloxane 1.0% 
(8) Liquid paraffin 1.0% 
(9) Natural vitamin E 0.05% 
(10) Glycerin monostearate 1.0% 
(11) POE (20) sorbitan trioleate 
0.2% 
(12) Soybean phospholipid 0.2% 
(13) 2-Ethylhexyl p-dimethylaminobenzoate 
3.0% 
(14) 4-tert-Butyl-4'-methoxydibenzoylmethane 
2.0% 
(15) Triethanolamine 0.7% 
(16) p-Hydroxybenzoate 0.2% 
(17) 1,3-Butylene glycol 4.2% 
(18) Carboxyvinyl polymer 0.2% 
(19) Ethanol 7.0% 
(20) Collagen 0.05% 
(21) N-Acetyl-L-glutamine 0.01% 
(22) Plant (hamamelis) extract 
0.7% 
(23) Purified water 72.64% 
______________________________________ 
The ingredients (1), (2) , (6) , (11) , (12) and (17) were mixed and 
uniformly dispersed with a 3-roll mill (pigment portion). The ingredients 
(3)-(5) , (7)-(10), (13) and (14) were heated at 80.degree. C. for 
dissolution (oil phase). The ingredients (15)-(18), (21) and (23) were 
heated at 80.degree. C. for dissolution, the pigment portion was added 
thereto, and the resulting mixture was stirred to obtain a uniform mixture 
(water phase). The water phase was added to the oil phase, and the mixture 
was emulsified with a homomixer and then cooled, followed by mixing with 
the ingredients (19), (20) and (22) to obtain a sunscreen cream. 
EXAMPLE 15 
Preparation of a Sunscreen Cream 
A sunscreen cream having the following composition was prepared using the 
melanin-coated pigment obtained in Example 2. 
______________________________________ 
(1) Melanin-coated barium sulfate 
1.0% 
(2) Beeswax 2.2% 
(3) Cetanol 3.0% 
(4) Liquid paraffin 3.0% 
(5) 1,3-Butylene glycol 5.0% 
(6) POE (20) sorbitan tristearate 
1.5% 
(7) Glycerin monostearate 3.0% 
(8) Carboxyvinyl polymer 10.0% 
(1% aqueous solution) 
(9) Triethanolamine 0.2% 
(10) p-Hydroxybenzoate 0.2% 
(11) Fragrance 0.3% 
(12) Purified water 70.6% 
______________________________________ 
The ingredients (2)-(6) were heated at 80.degree. C. for dissolution (oil 
phase). The ingredients (1) and (7)-(12) were heated at 80.degree. C. for 
dissolution and stirred to obtain a uniform mixture (water phase). The 
water phase was added to the oil phase, and the mixture was emulsified 
with a homomixer, and then cooled to obtain a sunscreen cream. 
EXAMPLE 16 
Preparation of a Sunscreen Emulsion 
A sunscreen emulsion having the following composition was prepared using 
the melanin-coated pigment obtained in Example 3. 
______________________________________ 
(1) Melanin-coated titanium dioxide 
2.0% 
(2) Soybean phospholipid 0.2% 
(3) 1,3-Butylene glycol 3.0% 
(4) 2-Ethylhexyl p-dimethylaminobenzoate 
1.0% 
(5) Liquid paraffin 2.0% 
(6) Glycerin monostearate 1.0% 
(7) Carboxyvinyl polymer 20.0% 
(1% aqueous solution) 
(8) Triethanolamine 0.5% 
(9) p-Hydroxybenzoate 0.2% 
(10) Fragrance 0.1% 
(11) Purified water 70.0% 
______________________________________ 
The ingredients (1)-(3) were mixed and uniformly dispersed-with a 3-roll 
mill (pigment portion). The ingredients (4)-(6) were heated at 80.degree. 
C. for dissolution (oil phase). The ingredients (7)-(11) were heated at 
80.degree. C. for dissolution, the pigment portion was added thereto, and 
the resulting mixture was stirred to obtain a uniform mixture (water 
phase). The water phase was added to the oil phase, and the mixture was 
emulsified with a homomixer, and then cooled to obtain a sunscreen 
emulsion. 
EXAMPLE 17 
Preparation of a Lipstick 
A lipstick having the following composition was prepared using the 
hydrophobic melanin-coated pigment obtained in Example 4. 
______________________________________ 
(1) Candelilla wax 10.0% 
(2) Solid paraffin 15.0% 
(3) Purified lanolin 10.0% 
(4) Liquid paraffin 33.0% 
(5) Castor oil 20.0% 
(6) Hydrophobic melanin-coated titanium 
3.0% 
dioxide 
(7) Organic pigment 8.7% 
(Pigment Red 3 aluminum lake) 
(8) Fragrance 0.3% 
______________________________________ 
The ingredients (5)-(7) were mixed and kneaded with a 3-roll mill to obtain 
a uniform mixture, and the ingredients (1)-(4) and (8) were added thereto, 
followed by heating for dissolution until the temperature reached 
85.degree. C. The mixture was stirred to obtain a uniform mixture, which 
was then charged into a container and cooled to obtain a lipstick. 
TEST EXAMPLE 1 
The ultraviolet-ray-absorbing effect of the microbial melanin upon 
ultraviolet irradiation is indicated below. 
1) Survival curve of L929 fibroblasts upon ultraviolet (UVB) irradiation 
Mouse-derived L929 fibroblasts were inoculated into wells of a 6-well 
microplate (product of Iwaki Glass Co.) to 2.times.10.sup.4 cells/well, 
with 2 ml of MEM medium (product of Nissui Seiyaku Co.) containing 10% 
fetal calf serum, and were cultured in a 5% CO.sub.2 incubator at 
37.degree. C. for 3 days. After the culturing, the MEM medium was removed, 
and the cells were washed with PBS (-) [composition: 8,000 mg of sodium 
chloride, 200 mg of potassium chloride, 1,150 mg of anhydrous sodium 
monohydrogenphosphate, 200 mg of anhydrous potassium dihydrogenphosphate, 
and 1 l of purified water (pH 7.5); the same shall apply hereinafter] 
(product of Takara Shuzo Co., Ltd.). Then, 1 ml of PBS (-) was poured into 
each well, followed by irradiation with UVB (wavelength: 300 nm; the same 
shall apply hereinafter) with varied intensity. 
The UV lamp used was FL20S.E (maximum wavelength: 300 nm, product of 
TOSHIBA Co.), and the UVB intensity was measured using an ultraviolet 
intensity meter UVR-1 (Light receiver unit UVR-25, Tokyo Kogaku Kikai 
Co.). 
After removal of the PBS (-), 2 ml of MEM medium was added to each well, 
followed by culturing in a 5% CO.sub.2 incubator at 37.degree. C. for 3 
days. The protein content was determined, and the survival rate of the 
cells upon UVB irradiation was calculated according to the equation given 
below. 
The protein content was determined by suspending the cells in the plate by 
treatment with 0.05% trypsin, subjecting them to ultrasonic disruption for 
30 seconds in a microtube (1.5 ml, product of Eppendorf Co.) in the 
presence of 1 ml of PBS (-) and then to centrifugation (9,000 rpm, 20 
minutes), and measuring the protein content of the supernatant using a 
Protein Measuring Kit (product of Bio-Rad Co.). 
##EQU1## 
FIG. 1 shows the survival curve of L929 fibroblasts upon irradiation with 
UVB. 
2) UVB-absorbing effect of microbial melanin 
Microbial melanin (0.1 g) was completely dissolved in 100 ml of a 0.1% 
aqueous solution of sodium hydroxide, and then one PBS tablet (product of 
Takara Shuzo Co., Ltd.) was dissolved in the solution. The solution was 
adjusted to pH 7 with hydrochloric acid, and was sterilized using an 
autoclave (121.degree. C. 15 minutes) to prepare a microbial 
melanin.phosphate buffer solution (microbial melanin/PBS). 
L929 fibroblasts were cultured in the same manner as in 1) above, and then 
washed with PBS (-). The microbial melanin/PBS appropriately diluted with 
PBS (-) was poured into wells in an amount of 1 ml/well, followed by 
irradiation with UVB giving a 50% survival rate and UVB giving a 4% 
survival rate. 
The cells were washed with PBS (-), and after removal of the PBS (-), 2 ml 
of MEM medium was added thereto, and culturing was carried out in the same 
manner as in 1) above. The protein content was determined, and the 
survival rate of the cells upon UVB irradiation was calculated. 
The results are shown in FIG. 2. 
From the above results, it can be seen that the survival rate of the cells 
upon UVB irradiation increased until the melanin concentration reached 
0.025%, and thereafter, the survival rate was only slightly lowered. 
3) UVB-absorbing effect of microbial melanin film 
Microbial melanin (0.1 g) was completely dissolved in 100 ml of a 0.1% 
aqueous solution of sodium hydroxide. The solution was adjusted to pH 7 
with hydrochloric acid, and then sterilized using an autoclave 
(121.degree. C., 15 minutes) to prepare a 1% aqueous solution of microbial 
melanin. 
Ten grams of the 1% aqueous solution of microbial melanin and 10 g of 
acrylic resin (product of Mitsubishi Petrochemical Co., Ltd.) were 
dissolved in ethanol to make a total amount of 100 g. Ten grams of the 
obtained solution was poured uniformly onto the cover of a 6-well 
microplate and allowed to dry overnight at 45.degree. C. whereby a 0.1% 
microbial melanin film was prepared. 
L929 fibroblasts were cultured in the same manner as in 1) above, and the 
MEM medium was removed. After the cells were washed with PBS (-), 1 ml of 
PBS (-) was poured into each well, and the above microbial melanin film 
was laid thereon, followed by irradiation with UVB with varied intensity. 
The cells were washed with PBS (-), and after removal of the PBS (-), 2 ml 
of MEM medium was added thereto, and culturing was carried out in the same 
manner as in 1) above. The protein content was determined, and the 
survival rate of the cells upon UVB irradiation was calculated. 
As a control, the same procedure as described above was repeated except 
that the cells were not irradiated with UVB, and the survival rate of the 
cells was calculated. 
The results are shown in FIG. 3. 
From the above results, it is apparent that the survival rate of the cells 
upon UVB irradiation at 104.4 mJ/cm.sup.2 and 212.4 mJ/cm.sup.2 increases. 
TEST EXAMPLE 2 
The microbial melanin used in the present invention was examined for safety 
by the human 48-hour closed patch test [Nippi Kaishi, 80(5), 301-314, 
1970], the single oral lethal toxicity test [No. 118 of Evaluation and 
Registration Division, Ministry of Health and Welfare (Feb. 15, 1984), 
Guidelines on Toxicity Tests Required for Application Approval for 
Production (or Import) of Medicines (1)] and the photosensitization test 
(Nishinihon Hifukagakukaishi, vol. 42, No. 5). 
The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Test Result 
______________________________________ 
Human 48-hr closed patch test 
Negative (44 cases) 
24 males, 20 females 
Single oral lethal toxicity test 
LD.sub.50 2000 mg/kg or more*.sup.1 
Photosensitization test 
Negative*.sup.2 
______________________________________ 
*.sup.1 :Test animal: CD1 (ICR) mice 
*.sup.2 :Test animal: Hartley guinea pigs 
Positive control substance: TCSA (3,3',4',5tetrachlorosalicylanilide) 
From the above results, it can be seen that the microbial melanin is highly 
safe and can be safely used in cosmetics. 
TEST EXAMPLE 3 
The different color tones of the microbial melanin-coated pigments of the 
present invention, depending on the type of carrier (pigment) and the 
coating concentration of the microbial melanin, were examined according to 
the same method as in Comparative Example 1 below, and were indicated by 
the values L, a and b. 
The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Coating concentration of 
Color microbial melanin (%) 
Carrier pigment 
tone 0.5 1.0 2.0 
______________________________________ 
Titanium Dioxide 
L 45.1 42.5 40.3 
a 8.7 9.1 9.1 
b 10.8 11.3 11.7 
Sericite L 43.73 41.0 36.29 
a 11.54 11.55 
10.97 
b 14.47 14.15 
13.11 
Talc L 40.22 37.28 
32.25 
a 10.30 9.91 
9.89 
b 10.86 10.42 
10.39 
Nylon powder L 42.86 27.87 
33.15 
a 10.70 11.01 
10.85 
b 14.51 14.54 
14.18 
______________________________________ 
From the above results, it can be seen that the color tone of the microbial 
melanin-coated pigment changes depending on the type of pigment used as 
the carrier. 
TEST EXAMPLE 4 
The ultraviolet-ray-blocking effect of cosmetics containing the 
melanin-coated pigment of the present invention are indicated by SPF (Sun 
Protection Factor) values below. 
The measurement of the SPF was carried out in accordance with the 
"Standards for SPF Measurement" of the Japan Cosmetic Industry 
Association. 
The sunscreen cream obtained in Example 14 above was used as the sample for 
SPF measurement in the present invention (listed as "sunscreen cream" in 
Table 3). 
A cream prepared by the same procedure as in Example 14 except that the 
ingredients (1) and (2) in the composition were not coated with melanin 
was used as the control sample for SPF measurement (listed as "control" in 
Table 3). 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
SPF measurement Measured 
sample SPF value 
______________________________________ 
Sunscreen cream 24 
Control 13 
______________________________________ 
From the above results, it can be seen that the cosmetics containing the 
melanin-coated pigment of the present invention exhibit a very high SPF 
value, and thus have an excellent ultraviolet-ray-blocking effect. 
COMATIVE EXAMPLE 1 
The color tone and tinctorial power of microbial melanin, synthetic 
melanin, sepia melanin, black iron oxide and carbon black (hereinafter 
referred to as black pigments) were compared. 
Titanium dioxide was kneaded with each of the above-mentioned black 
pigments to make dispersions, and the color tone and tinctorial power were 
measured in a disperse system. 
That is, a composition of 0.04% black pigment, 1.96% titanium dioxide, 2.0% 
polyvinyl alcohol, 5.0% acrylic resin, 73.0% 1,3-butylene glycol and 18.0% 
purified water was thoroughly kneaded using a 3-roll mill, and the 
obtained uniform paste was used as the sample of the disperse system. 
The microbial melanin and the synthetic melanin were also examined in a 
coating system. 
That is, a composition of 2.0% titanium dioxide coated with 2% microbial 
melanin or synthetic melanin, 2.0% polyvinyl alcohol, 5.0% acrylic resin, 
73.0% 1,3-butylene glycol and 18.0% purified water was thoroughly kneaded 
using a 3-roll mill, and the obtained uniform paste was used as the sample 
of the coating system. 
Certain amounts of the above-mentioned samples were applied to a hiding 
power test paper having a white side and a black side, and were pressed to 
a thickness of 0.20 mm using a Doctor blade, followed by drying to form 
films. Each of the samples were subjected to the measurement using a color 
and color-difference meter (MINOLTA model CR-200), and the color tones 
were indicated by L, a and b. The tinctorial power was compared on the 
basis of the lightness. 
The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Black pigment in 
measurement sample 
System L a b 
______________________________________ 
Microbial melanin 
Disperse system 
78.7 4.3 4.6 
Coating system 
73.8 5.5 8.6 
Synthetic melanin 
Disperse system 
75.7 4.8 4.2 
Coating system 
88.3 5.8 7.2 
Sepia melanin Disperse system 
76.3 2.1 3.9 
Black iron oxide 
Disperse system 
49.5 7.9 9.7 
Carbon black Disperse system 
30.2 5.0 4.6 
______________________________________ 
L: Lightness [measured with a hiding power test paper (white 
a,b: Chromaticity 
From the above results, it is seen that the microbial melanin exhibits a 
color tone close to that of the synthetic melanin, which is different from 
the jet-black color exhibited by black iron oxide and carbon black, thus 
presenting a warm, natural tint. 
A cosmetic which contains the microbial melanin-coated pigment of the 
present invention provides a soft tint which is compatible with the color 
of skin. 
In addition, it can be seen that the color tone of titanium dioxide coated 
with the microbial melanin is characterized by high coloring and high 
saturation compared with dispersed products, and that when the microbial 
melanin is contained in a cosmetic, coating systems provide products of a 
higher stability as compared with disperse systems. 
COMATIVE EXAMPLE 2 
Purified water was added to 0.1 g of microbial melanin, synthetic melanin 
or sepia melanin and 0.1 g of sodium hydroxide to prepare 100 ml of 
solution, and the pH was adjusted to 7 with hydrochloric acid. 
To 0.1 g of dl-.alpha.-tocopherol was added ethanol to prepare 100 ml of 
solution. 
To 0.1 g of ascorbic acid was added purified water to prepare 100 ml of 
solution. 
To 5.02 mg of an 80% cumene hydroperoxide (hereinafter abbreviated to CPO) 
solution was added purified water to prepare 100 ml of solution (264 
nmol/ml). 
The microbial melanin solution, the ascorbic acid solution, the 
dl-.alpha.-tocopherol solution, the synthetic melanin solution or the 
sepia melanin solution was added to a system in which CPO was reacted with 
a methylene blue derivative in the presence of hemoglobin as a catalyst, 
using Determiner LPO (product of Kyowa Medex Co., Ltd.) as a reaction 
reagent, thereby producing an equimolar amount of methylene blue which 
developed the color of the system. The anti-oxidizing property of the 
microbial melanin, ascorbic acid, dl-.alpha.-tocopherol, the synthetic 
melanin and the sepia melanin was determined on the basis of the degree of 
inhibition of the production of methylene blue. 
That is, the test sample, the standard sample and the blank test sample 
shown in Table 5 were prepared, and in accordance with the directions for 
the use of the above-mentioned reagent which were annexed thereto, the 
absorbance of each of the reacted samples at a wavelength of 675 nm was 
measured with the blank test sample as a control, and the lipid peroxide 
elimination rate was calculated by the equation given below. 
TABLE 5 
______________________________________ 
Test Standard Blank test 
sample sample sample 
(S) (Std) (B) 
______________________________________ 
Solution of 0.5 ml -- -- 
microbial melanin, 
ascorbic acid, 
dl-.alpha.-tocopherol, 
synthetic melanin or 
sepia melanin 
CPO solution 0.1 ml 0.1 ml -- 
Purified water 
-- 0.5 ml 0.6 ml 
______________________________________ 
##STR1## 
In the equation, A.sub.s indicates the absorbance of the test sample, 
A.sub.std the absorbance of the standard sample, and A.sub.B the 
absorbance of the blank test sample. 
The lipid peroxide elimination rates of the microbial melanin, ascorbic 
acid, dl-.alpha.-tocopherol, the synthetic melanin and the sepia melanin 
at a concentration of 0.014% are shown in Table 6. 
TABLE 6 
______________________________________ 
Lipid peroxide elimination rate 
(%) 
______________________________________ 
Ascorbic acid 
97.4 
dl-.alpha.-tocopherol 
72.4 
Synthetic melanin 
90.2 
Sepia melanin 
57.7 
Microbial melanin 
48.5 
______________________________________ 
The anti-oxidizing property of the microbial melanin, ascorbic acid, 
dl-.alpha.-tocopherol, the synthetic melanin and the sepia melanin can be 
expressed as the lipid peroxide elimination rate. 
From the above results, it can be seen that the microbial melanin used in 
the present invention has an anti-oxidizing property. 
INDUSTRIAL APPLICABILITY 
The present invention provides a melanin-coated pigment which is highly 
safe and has high coloring and high saturation tone, and a cosmetic 
containing the pigment. 
The melanin-coated pigment can be used as a flesh-colored or gray-colored 
coloring pigment in paint, ink, cosmetics, and the like. When used in 
cosmetics, it is expected to be also effective for the absorption of 
ultraviolet rays and for anti-oxidation.