JPS6314707A - External preparation for skin - Google Patents

Abstract

PURPOSE:An external preparation for skin having increased preventing and improving effects on chapped skin, containing tropoelastin, a precursor protein of elastin. CONSTITUTION:An external preparation containing 0.0001-10wt%, preferably 0.01-5wt% tropoelastin. The external preparation is useful as a cosmetic, quasi-drug or drug to be applied to human skin. The tropoelastin used is tropoelastin obtained by technology of genetic engineering, namely, one obtained by preparing mRNA to code tropoleastic from tissue (e.g. aorta) of human, bovine, swine, sheet, chicken or mouse producing a large amount of elastin, by integrating cDNA corresponding to the mRNA into a manifestation vector to make a tropoelastin manifestation vector and by using Escherichia coli, Bacillus subtilis or yeast which is transformed with the manifestation vector.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a skin preparation for external use containing tropoelastin, a precursor protein of elastin, and particularly to a skin preparation containing tropoelastin obtained by genetic engineering technology. Regarding external preparations.

[Problems to be solved by the prior art and the invention 1-1] Elastin (mature elastin), 1. Like collagen and acid mucopolymerase, it is a component of animal binding &I]H, It is present in elastic organs such as the lungs, aorta, ligaments, and skin. , two straw elastin is
Because it is a high-molecular protein with a complex crosslinked structure and several hundreds of thousands of molecules, it is insoluble in water and difficult to extract and purify. Generally, elastin is extracted and separated by a method in which tissue is extracted under harsh conditions such as treatment with a strong alkali or high temperature, and elastin is separated as a decomposed product. As a method for obtaining elastin with a relatively high molecular weight, there is a hot oxalic acid treatment method by Partridge et al. (formerly Ochem, J.
61, 11.1955), and α-elastin [molecular weight approximately 70 KDa (kilodaltons)] and β-elastin [molecular weight approximately 10 KDa] are known as water-soluble proteins.
J≧2 lower] is obtained. Recently, research on the physiological effects of elastin using α-elastin has been progressing, and it has become clear that α-elastin has the ability to inhibit platelet aggregation, which is a factor in arteriosclerosis.

Tropoelastin is a precursor of elastin protein, and is a water-soluble protein immediately translated from messenger RNA. Unlike adult W1 elastin, it does not contain desmosine, isodesmosine, or lysinonorleucine as residues, nor does it form crosslinks with them. Isolation of tropoelastin from living organisms is very difficult, and a method for isolating it from the Mi tissue of young animals fed a copper-deficient diet to inhibit cross-linking enzymes (Smith; D, W, et al., formerly och.
em, Riophys.

Res, Commun, 31, 309.1968), but the amount of I-roboelastin that can be isolated is very small.

For this reason, research into the molecular structure of tropoelastin has not made much progress, and only the molecular weight and amino acid composition have been clarified. Therefore, the application of tropoelastin to industry has not been considered at all so far.

The present inventors elucidated the structure of tropoelastin using DNA chemistry and succeeded in mass-producing tropoelastin using a microorganism using recombinant 1) NA technology.

Therefore, the present inventors investigated the characteristics of tropoelastin obtained in large quantities and found that tropoelastin has an excellent effect on preventing and improving skin roughness.

Therefore, an object of the present invention is to provide a skin preparation for external use that has excellent effects on preventing and improving skin roughness.

[Means for solving problems]

The above object can be achieved by an external skin preparation containing tropoelastin, particularly a skin external preparation containing tropoelastin obtained by genetic engineering techniques.

The present invention will be explained in more detail below.

First, we will discuss the method for producing cDNA for the messenger RNA encoding tropoelastin.

In humans, cows, pigs, sheep, chickens, or mice, tissues that produce a large amount of elastin (sometimes referred to as plus tissues), for example, the aorta of each of the above animals, are homogenized with guanidyl thiocyanate to obtain cells. The RNA fraction is obtained by disruption and cesium chloride equilibrium density gradient ultracentrifugation. Subsequently, by affinity chromatography supporting oligo dT cellulose, polyA-bearing RNA (polyA
3 RNA) is isolated.

Using this polyA''I?NA as a template, a known method, preferably the method of Okayama-Be, rg (Mo1.Cel.
l.

Biol, 2.16+, 1982), cDNA
Get the library. The Okayama-Berg method is carried out as follows. That is, the polyA part of polyA'' RNA is adsorbed to the polyT part of the Okayama-Rerg vector, and cDNA is synthesized by reacting with reverse transcriptase.

After adding oligo dC to the 3' end of the cDNA using terminal deoxynucleotidyl transferase, the vector DNA is cut with restriction enzyme ll1ndIII. After ligating an oligo-dG linker and circularizing the vector, the RNA portion is replaced with DNA using r) NA polymerase to obtain a cDNA-containing plasmid. Using these plasmids, the calcium chloride method (Strjk, Po et al., J. Bacteriol, I38.1033°197
Transform E. coli using a method such as 9).

Plasmid-receiving bacteria are obtained by selecting ampicillin-specific strains on a plate medium supplemented with ampicillin.

On the other hand, the above-mentioned plus tissue, that is, the tissue that produces a lot of elastin, and the tissue that does not produce much elastin (hereinafter sometimes referred to as minus tissue), for example, the skin of each of the animals mentioned above, are prepared, and the cells of each are extracted by the method described above. Isolate polyA″ RNA. Polynucleotide kinase and γ-ff2pATP are used to convert the 5′OH
Rahel with 32p and use this as a probe.

Next, colony hybridization method (Hanah
an, D, et al., Gene, 10.63.1980
), a colony is selected from the cDNA library described above that has complementarity with the probe derived from the plus tissue and has no complementarity with the probe derived from the minus tissue.

Plasmid DNA was obtained from the colonies selected in this way, and the dideoxynucleotide method (Messing, J.
Methods in Enzymology 101
+20+1983) etc. to determine the base sequence.

The amino acid sequence is deduced from the base sequence, and it is determined that an amino acid sequence unique to elastin is present in the amino acid sequence.Next, the above cDNA is inserted into an appropriate expression vector.

As the expression vector, expression vector systems developed for E. coli hyphae, Bacillus subtilis system, and yeast system can be used.

Expression vector systems for E. coli hyphae include pOMPo or pOP95-13 (0, Mercerau-Puija
lon et al., Nature.

275.505.1978), pKK223-3 (H,
A, de Boer et al., Proc, Natl, 8 cad.
, sci, [IsA, 80.21.1983) or p
DR540 (mouth, R, Ru5se1, etc., Gene, 20
, 231. 1982), ptrpLl (J, C, Edn.
+an etc., Nature+ 291 + 503+
1981) or pBN37 (T, H, Fraser et al., Proc, Natl, Acad, Sci, ll5A.

75.5936.1978), pKT279 (K, Ta
lmadge etc., Proc', Natl, Acad, S
ci, ll5A, 77.3988.1980) or p
KT287 (K, Talmadge et al., Proc, Na
tl, Acad, Sci, USA.

77, 3369.1980), pMH621 (T, Ma
niatis, Mo1e-cular Cloning
, Co1d Spring Harbor Lab,
omp promoter system represented by (1982), pIN
-1 or prM-T (Y, Masui et al., Expert
mental Manipulation of Gen
e Pression + M on E, InouyefJi
), p15. ^ academic Press, 1983
), pMCR-545 (T, Miki et al., Mol.

The recA promoter system typified by Gen. Genet, 183, 25. 1981) can be used.

As an expression vector for Bacillus subtilis, penP (D, Henner et al., 2nd
Tnt, Conference of Genetic
s and Biotechnology of Bac
illi, 1983), pK using the α-amylase gene promoter and signal peptide gene region.
TI+93 (1, Pa I va etc., Gene, 22
, 229. 1983) Expression vector using the promoter and signal peptide gene of openicillinase (Chang et al., S., Phylum 01e-cular Clone
ing and Gene Regulation i
n Bacilli.

p159. Academic Press, 1982)
etc. can be used.

As a yeast expression vector, pGl' using the glutaraldehyde 3-phosphate dehydrogenase promoter is used.
DI or pGPI12 (G, 8, Bitter et al., G
pM with α1 factor promoter and signal peptide gene, such as ene+32, 263.1984)
Fα8(A.

Miyaj ima et al., Gene, 37,155.19
85), AA) 15 or 8H5 (G, Ammerer) using the promoter of alcohol dehydrogenase.
, Methods in [Enzymology
(R, Wue etc. W) Vollol, p192+Acad
emic Press, 1983), YEp51 or YRp52 (J,R
.

Broach etc., lixperimental Man
ipulation of GeneExpressi
on (M, Inouye m) p83. Academy
cPress.

1983), expression vector using GAI, 7 promoter (Japanese Patent Application Laid-open No. 60-248181), pAT77 or C using repressible acid phosphatase promoter.
SaipAl'1F12 (^, former yanohara etc.,
Proc, Natl, 8cad, sci, ll5A,
80+L1983) etc. can be used. Tropoelastin cDNA is endonuclease Ba1
A suitable cloning site is created by partial digestion or the like, and inserted into the cloning site of various expression vector groups to create a tropoelastin cDNA expression vector.

To construct a tropoelastin expression vector, the 5' end of the cDNA is ligated to the promoter of the expression vector system or to the cloning site downstream of the signal peptide. At this time, for those with a signal peptide moiety,
(coding frames), and in expression vector systems that do not have a translation initiation coton (ATG, etc.), care should be taken to ensure that the ATG of the cDNA is located near the downstream of the cloning site.

The tropoelastin expression vector thus obtained is introduced into a host (E. coli, Bacillus subtilis, or yeast). For transformation, conventional methods can be used.

Transformation of E. coli was performed using the calcium treatment method (Mandel
et al., J. Mo1. Bio+, 53.159.1970)
This can be done according to the following. Transformation of Bacillus subtilis was performed using the protoplast method (S, Chang et al., Mo+, Gen, G
enet, 168, 111.1979).

In addition, yeast transformation can be performed using the spheroplast method (A, t
Linnen et al., Proc, Natl, 8cad, Sc
i.

11sA, 75.1929.1978) and the lithium chloride method (11, Ito et al., J. Bacteriol, 1
53, 163.1983).

Transformants are obtained by selection on a plate using a selection marker (eg, ampicillin resistance) incorporated into the respective expression vector.

The obtained transformant is cultured as follows.

For E. coli or Bacillus subtilis, use TYG medium (tryptone 0.5%, yeast extract 0.5%, grape 110.1%
, KzHPO4,0,1%) or LB medium (tryptone 1%, yeast extract 0.5%, salt 1%), a medium in which E. coli or Bacillus subtilis is generally grown, with a selection marker for the tropoelastin expression vector. Culture is performed using a medium containing an appropriate amount of a certain antibiotic (ampicillin, kanamycin, etc.). Antibiotics are not necessarily required for short-term culture.

Culture is performed at a temperature of 25 to 45°C with shaking. The transformed strain of "plate medium" 2 was cultured with shaking in the above medium of 5 to 100 mβ overnight, transferred to a medium of 50 to 10100000,
Culture is carried out for 10 to 30 hours.

For yeast, minimal medium [0.7% yeast nitrogen source hese (no amino acids), 2% g/:1-s, 20
Using various nucleic acids and amino acids of about 71 mg,
A medium is prepared and used that excludes selection markers (for example, uracil, ■, -leucine, etc.) possessed by various expression vectors. Culture is performed at a temperature of 25 to 35°C with shaking.

The transformed strain in plate medium 2 is cultured with shaking in a 5-100 ml medium for 1-2 days, transferred to a 50-1,000,000 ml medium, and cultured for 1-5 days.

Tropoelastin obtained by culture is collected as follows.

For tropoelastin produced using a secretion vector system, the culture solution is centrifuged to obtain a supernatant. In addition, for other vector systems, the bacterial cells are obtained, and after washing the bacterial cells with water, the bacterial cells are destroyed using a cell destruction device such as ultrasonication, French press, X press, Hue press, Dounce tube, etc. , obtain the supernatant by centrifugation.

These supernatants were concentrated by acetone precipitation, ultrafiltration, freeze-drying, etc., partially purified by gel filtration ion exchange chromatography using an open column, and then I
Purify by high performance fluid chromatography using IPLc, FPLC, etc.

The amino acid sequence of the tropoelastin thus obtained differs slightly depending on the species of organism from which the messenger RNA is extracted and the cells from which it is extracted. However, all tropoelastins satisfy the following three conditions in terms of their amino acid sequences.

(1)■,-valine-L-proline-glycine-■,-
It has multiple pentapeptide regions represented by valine-glycine in its entire amino acid sequence.

(2) L-proline-14-alanine-■, -alanine-I7-alanine-I, -alanine-■, -lysine-L
-Alanine - Contains an octapeptide region represented by L-alanine in its amino acid sequence.

(3) The amino acid sequence does not contain residues formed by post-transcriptional modifications such as hydroxyproline, desmosine, isodesmosine, and lysinonorleucine.

The skin external preparation of the present invention contains the above-mentioned tropoelastin at 0.
0001-10% by weight, preferably 0.0001-10% by weight. Contains in an amount of ~5% by weight.

The external skin preparation of the present invention refers to one that can be applied to human skin and used as cosmetics, quasi-drugs, or pharmaceuticals.

In addition to the above-mentioned essential ingredients, the external skin preparation of the present invention may optionally include various ingredients commonly used in cosmetics, pharmaceuticals, etc.
That is, an aqueous component, a powder component, an oil component, a surfactant, a humectant, a thickener, a preservative, an ultraviolet absorber, a coloring agent, an antioxidant, a fragrance, a drug, etc. can be blended.

Typical ingredients and their typical examples will be explained below, but the ingredients that can be included in the external preparation of the present invention are not limited to the following.

Examples of pearl luster imparting agents include natural fish M foil, mica-
Examples include titanium oxide composites and bismuth oxychloride. In addition, treated powders obtained by making these powders hydrophilic or lipophilic can also be used as pearl luster imparting agents.

Examples of synthetic esters include isopropyl myristate, cetyl octoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate. , lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexylate, dipentaerythritol fatty acid ester, N-alkyl glycol monoisostearate, neopentyl glycol capriate, dimalate Isostearyl, glyceryl di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexylate, trimethylolpropane triisostearate, pentaneerythritol tetra-2-ethylhexylate, glyceryl tri-2-ethylhexylate, trimethylol triisostearate Propane, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate,
Glycerin trimyristate, tri-2-heptylundecanoic acid glyceride, castor oil fatty acid methyl ester, oleic acid oil, cetostearyl alcohol, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid -2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl cepatate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, cepatic acid diisopropyl,
Examples include 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, and the like.

Examples of the synthetic resin emulsion include acrylic resin emulsion, polyethyl acrylate emulsion, acrylic resin liquid, polyacrylic alkyl ester emulsion, and the like.

Examples of organic amines include monoethanolamine, jetanolamine, triethanolamine, morpholine, triisopropanolamine, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1 - Examples include property tools.

Examples of inorganic powders include talc, titanium oxide, kaolin, anhydrous silicic acid, silicate, zinc oxide, calcium carbonate, magnesium carbonate, red iron oxide, yellow iron oxide, chromium oxide, carbon blank, ultramarine blue, mica, and sericite. , nylon powder, polyethylene powder, cellulose powder, acrylic resin, titanium dioxide, iron oxide, etc.

Examples of inorganic pigments include talc, kaolin, calcium carbonate, zinc white, titanium dioxide, red iron oxide, yellow iron oxide,
Black iron oxide, ultramarine, titanium coati sodomite, bismuth oxychloride, hengara, caking pigment, gunjo pink,
Chromium hydroxide, titanium mica, yellow iron oxide, chromium oxide, aluminum oxide, mucobalt oxide, dark blue, black iron oxide, carbon blank, silicic anhydride, magnesium silicate, bentonite, mica, zirconium oxide, magnesium oxide, zinc oxide , titanium oxide, light calcium carbonate, heavy calcium carbonate, light magnesium carbonate, heavy magnesium carbonate, calamine, and the like.

Examples of lipophilic nonionic surfactants include sorbitan monooleate, sorbicun monoisostearate,
Sorbicum monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, diglycerol pent-2-ethylhexylate, sorbicun, tetra-2
- Sorbitan fatty acid esters such as diglycerol sorbitan ethylhexylate, glyceryl monocottonseed oil fatty acid, glyceryl monoerucate, glyceryl sesquioleate, glyceryl monostearate, glyceryl pyroglutamate α, α′-oleate, glyceryl monostearate malic acid, etc. glycerin polyglycerin fatty acids, propylene glycol fatty acid esters such as propylene glycol monostearate, hydrogenated castor oil derivatives,
Examples include glycerin alkyl ether.

Examples of the hydrophilic nonionic surfactant include POE sorbitan fatty acid esters such as POE sorbitan monooleate, POE-sorbitan monostearate, POE-sorbitan monooleate, and POE sorbitan tetraoleate, POE sorbitan monolaurate, P.O.F.
, sorbitol monooleate, POE sorbitolpentaoleate, POE turpinto monostearate, etc.
PO such as OE sorbitol fatty acid esters, POE glycerin monostearate, POE glycerin monoisostearate, POF, glycerin triisostearate, etc.
E glycerin fatty acid esters, POE monooleate, POE distearate, P(')B monooleate,
POE fatty acid esters such as ethylene glycol distearate, P.

E lauryl ether, POE oleyl ether, POE
Stearyl ether, POE behenyl ether, POE
2-octyl dodecyl ether, POE alkyl ethers such as POE cholestanol ether, POE octyl phenyl ether, POE nonylphenyl ether,
POE alkylphenyl ethers such as POE dinonylphenyl ether, Pluronic types such as Pluronic, POE-POP cetyl ether, POI!・POP
2-decyltetradecyl ether, POE-POP
Monobutyl ether, POE-POP hydrogenated lanolin,
POE-P such as POIE/POP glycerin ether
TetraPO such as OP alkyl ethers and Tetronic
E-tetra POP ethylenediamine condensates, POE castor oil, POE hydrogenated castor oil, POE hydrogenated castor oil monoisostearate, POE hydrogenated castor oil twisostearate, POE hardened castor monopyroglutamic acid monoisostearic acid diester OE hardened hardened Castor Simaleic Acid POE Castor Oil Hard Oil Superified Castor Oil Derivative OE
POE beeswax/lanolin derivatives such as sorbit beeswax, alkanolamides such as coconut oil fatty acid jetanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide, POE propylene glycol fatty acid ester, POE alkylamine, POE fatty acid amide, sucrose Examples include fatty acid ester, POE nonylphenyl formaldehyde condensate, alkyl ethoxydimethylamine oxide, trioleyl phosphoric acid, and the like.

Examples of anionic surfactants include soap bases, fatty acid bases such as sodium laurate and sodium palmitate, higher alkyl sulfate salts such as sodium lauryl sulfate and lauryl sulfate, POE lauryl sulfate triethanolamine, and POE lauryl sulfate. Alkyl ether sulfate salts such as sodium sulfate, N-acyl sarcosinates such as lauroyl sarcosinate, N
- Myristoyl-N-methyltaurine sodium, higher fatty acid amide sulfonates such as coconut oil fatty acid methyltauride sodium, lauryl methyltauride sodium, POF, sodium oleyl ether phosphate, PO
Phosphate ester salts such as E-stearyl ether phosphate, sulfosuccinates such as sodium di-2-ethylhexyl sulfosuccinate, sodium monolauroyl monoethanolamide polyoxyethylene sulfosuccinate, sodium lauryl polypropylene glycol sulfosuccinate, linear dodecylbenzene sulfone acid sodium, linear dodecylbenzenesulfonic acid triethanolamine,
Alkylbenzenesulfonic acid salts such as linear dodecylbenzenesulfonic acid, monosodium N-lauroylglutamate, disodium N-stearoylglutamate,
N-acyl glutamates such as monosodium N-myristoyl-L-glutamate, higher fatty acid ester sulfate salts such as hydrogenated coconut oil fatty acid glycerol sodium sulfate, sulfated oils such as funnel oil, POE alkyl ether carboxylic acids, POE Alkyl allyl ether carboxylate, α-olefin sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate salt, higher fatty acid alkylolamide sulfate salt, sodium lauroyl monoethanolamide succinate, N-valmitoyl Examples include ditriethanolamine aspartate and sodium caseinate.

Examples of cationic surfactants include stearyltrimethylammonium chloride, alkyltrimethylammonium salts such as lauryltrimethylammonium chloride J4, distearyldimethylammonium chloride dialkyldimethylammonium salts, and poly(N,N'-dimethyl-3,5-chloride). methylenepiperidinium), alkylpyridinium salts such as cetylpyridinium chloride, alkyl quaternary ammonium salts, alkyldimethylbenzylammonium salts, alkylisoquinolinium salts, dialkylmorphonium salts, POE alkylamines, al:toluamine salts, polyamines Fatty acid derivatives, amyl alcohol fatty acid derivatives, benzalkonium chloride, henzethonium chloride, cationic polymers, acrylics.

Examples of double-sided surfactants include 2-undecyl-N
, N, N-(hydroxyethylcarboxymethyl)-2
- imidacillin double-faced surfactants such as imidazolinium hydroxide, 2-cocoyl-2-imidazolinium hydroxide-1-carpoxyethyloxy disodium salt,
Betaine surfactants such as 2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryldimethylaminoacetic acid betaine, alkylbetaine, amidobetaine, sulfobetaine, N-
Examples include amino acid salts such as lauryl β-alanine and N-stearyl β-alanine.

As a UV absorber, paraaminobenzoic acid (hereinafter referred to as PA
abbreviated as R^), PABA monoglycerin ester, N
, N-dipropoxy PAII^ethyl ester, N
, N-jethoxy PAIIA ethyl ester, N, N-
Dimethyl PABA ethyl ester, N,N-dimethyl P
Benzoic acid-based UV absorbers such as ABA butyl ester, N,N-dimethyl PAB methyl ester, homomenthyl-N
- Anthranilic acid UV absorbers such as acetylanthranilate, anthranilate, menthyl salicylate,
Salicylic acid UV absorbers such as homomenthyl salicylate, octyl salicylate, phenyl salicylate, pendyl salicylate, p-isopropanol phenyl salicylate, octyl cinnamate, ethyl-4-isopropyl cinnamate, methyl-2,5-diisopropyl cinnamate, ethyl -2,4-diisopropylcinnamate, methyl-2,4-diisopropylcinnamate, propyl-p-methoxycinnamate, isopropyl-p-methoxycinnamate, isoamyl-p-methoxycinnamate, octyl-p-methoxycinnamate (2-ethylhexyl-p-methoxycinnamate)
, 2-ethoxyethyl-p-methoxycinnamate, cyclohexyl-p-methoxycinnamate, ethyl-α
-Cinnamic acid UV absorbers such as cyano-β-phenylcinnamate, 2-ethylhexyl-α-cyano-β-phenylcinnamate, glyceryl mono-2-ethylhexanoyl dip-paramethoxycinnamate, 2,4 -dihydroxybenzophenone, 2.2'-dihydroxydi-4=
Methoxybenzophenone, 2,2'-dihydroxy-4
, 4'-dimethoxyhensiphenone, 2°2', 4.4
'-Tetrahydrokidibenzophenone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-
Methoxy-4'-methylbenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate,
4-phenylbenzophenone, 2-ethylhexyl-4
Benzophenone ultraviolet absorbers such as '-phenyl-benzophenone-2-carboxylate, 2-hydroxy-4-n-octoxybenzophenone, 4-hydroxy-3-carboxyhenzophenone, 3-(4'-methylbenzylidene)- d, 1-camphor, 3-benzylidene-d, 1-camphor, urocanic acid, urocanic acid ethyl ester, 2-phenyl-5-methylbenzoxazole, 2゜2'-hydroxy-5-methylphenylbenzotriazole, 2 -(2'-hydroxy-5'- also-
octylphenyl)benzotriazole, 2-(2'-
Hydroxy-5'-methylphenylbenzotriazole, dibenzaladine, dianisoylmethane, 4-methoxy-4'-t-butyldibenzoylmethane, 5-(3,3
-dimethyl-2-norbornylidene)-3-pentane-
2-one etc. are mentioned.

Examples of clay minerals include montmorillonite, zakounite, nontronite, saponite, hectorite, vermiculite, vegum, bentonite, silicate, fluorosilicate, magnesium, aluminum,
Examples include natural and synthetic water-swellable clay minerals such as synthetic hectorite (laponite).

Examples of amino acids include glycine, alanine, valine,
Neutral amino acids such as leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, proline, hydroxyproline, -11 opa, acidic amino acids such as aspartic acid, glutamic acid, asparagine, glutamine, and araginine. , histidine, lysine, and other basic amino acids. Further, examples of amino acid derivatives include sodium acylsarcosine (sodium lauroylsarcosine), acylglutamate, sodium acyl β-alanine, glutathione, and pyrrolidone carboxylic acid.

Examples of natural water-soluble polymers include gum arabic, gum tragacanth, galactan, guar gum, carob gum, gum karaya, carrageenan, pectin, agar, quince seed (quince), algae colloid (cassava extract), starch (rice, corn, potato, wheat). ), plant polymers such as glycyrrhizic acid, microbial polymers such as xanthan gum, dextran, succinoglucan, pullulan, and animal polymers such as collagen, casein, albumin, and gelatin.

Examples of semi-synthetic water-soluble polymers include starch-based polymers such as carboxymethyl starch and methylhydroxypropyl starch, methylcellulose, nitrocellulose, ethylcellulose, methylhydroxypropylcellulose, hydroxyethylcellulose, sodium cellulose sulfate, hydroxypropylcellulose, Examples include cellulose polymers such as sodium carboxymethyl cellulose (CMC), crystalline cellulose, and cellulose powder, and alginic acid polymers such as sodium alginate and propylene glycol alginate ester.

Examples of synthetic water-soluble polymers include vinyl polymers such as polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, carboxyvinyl polymer (Carbobol), and polyethylene glycol 20.000.
4,000,000.600,000 isomers, acrylic polymers such as sodium polyacrylate, polyethyl acrylate, and polyacrylamide, polyethyleneimine,
Examples include cationic polymers.

Examples of inorganic water-soluble polymers include bentonite, AlMg silicate (vegum), laponite, hectorite, and silicic anhydride.

On the moisturizing side, for example, polyethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, xylitol, sorbitol, maltitol,
chondroitin sulfate, hyaluronic acid, mucoitin sulfate,
caronic acid, atelocollagen, cholesteryl-12-
Hydroxystearate, sodium lactate, bile salts,
Examples include dl-pyrrolidone carboxylate, short chain soluble collagen, diglycerin (EO) PO adduct, and the like.

Examples of polyhydric alcohols include ethylene glycol, propylene glycol, trimethylene glycol,
, 2-butylene glycol, 1゜3-butylene gelcol, tetramethylene gelcol, 2,3-butylene gelcol, pentamethylene glycol, 2-butylene-1,4-di-tyl, hexylene glycol , dihydric alcohols such as octylene glycol, trihydric alcohols such as glycerin, trimethylolpropane, 1,2,6-hexanetriol, tetrahydric alcohols such as pentaerythritol, pentahydric alcohols such as xylitol, sorbitol. , hexahydric alcohols such as mannitol, polyhydric alcohol polymers such as diethylene glycol, dipropylene glycol, l-diethylene glycol, polypropylene glycol, tetraethylene glycol, diglycerin, polyethylene glycol, triglycerin, tetraglycerin, polyglycerin, ethylene glycol Monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monophenyl ether, ethylene glycol monohexyl alcohol, ethylene glycol mono2
- methylhexyl ether, ethylene glycol isoamyl ether, ethylene glycol benzyl ether,
Divalent alcohol alkyl ethers such as ethylene glycol isopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol butyl ether , diethylene glycol methyl ethyl ether, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monobutyl ether, propylene glycol isopropyl ether, dipropylene glycol methyl ether, dipropylene glycol ethyl Ether, dihydric alcohol alkyl ethers such as dipropylene glycol butyl ether, ethylene glycol methyl ether acetate, ethylene glycol monoethyl ether acetate, ethylene glycol monobutyl ether acetate,
ethylene glycol monophenyl ether acetate,
Ethylene glycol diadihate, ethylene glycol disuccinate, diethylene glycol monoethyl ether astate, diethylene glycol 1,000 butyl ether acetate-1-, propylene glycol monomethyl ether acetate-1-, propylene glycol monoethyl ether acetate, propylene glycol monopropyl ether acetate, dihydric alcohol ether esters such as propylene glycol monophenyl ether acetate, glycerin monoalkyl ethers such as xyl alcohol, serakyl alcohol, batyl alcohol, sorbitol, maltitol, maltotriose, mannitol, sucrose, erythritol, glucose, Sugar alcohols such as fructose, starch decomposing sugar, maltose, xylyl--s, starch decomposing sugar reducing alcohol, glycolide, tetrahydrofurfuryl ether lycerin ether phosphate, POP POIE pentane erythritol ether, and the like.

Examples of thickeners include gum arabic, carrageenan,
Karaya gum, tragacanth gum, carob gum, quince seed (quince), casein, dextrin, gelatin, sodium pectate, sodium alaginate,
Methyl cellulose, ethyl cellulose, CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, PVA, PVM.

Examples include pvp, sodium polyacrylate, carboxyvinyl polymer, locust bean gum, guar gum, tamarind gum, dialkyldimethylammonyl 1, cellulose sulfate, xanthan gum, silicate AIMghentonite, and hectorite.

Examples of oil include avocado oil, camellia oil, turdle oil, macadamia nut oil, corn oil, mink oil,
Olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil , liquid oils and fats such as Shinakiri oil, Japanese tung oil, jojoba oil, germ oil, triglycerin, glycerin trioctanoate, glycerin triisopalmitate,
Cocoa butter, coconut oil, horse tallow, hydrogenated coconut oil, palm oil, beef tallow, mutton tallow, hydrogenated beef tallow, palm kernel oil, liver fat, beef bone tallow, Japanese oak kernel oil, hydrogenated oil, beef leg tallow, Japanese oak, hydrogenated castor Solid fats and oils such as oil, beeswax, candelilla wax, cotton wax, carnauba wax, paiberry wax, privet wax, spermaceti wax, monk wax, bran wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugar cane wax, lanolin fatty acid isopropyl, hexyl laurate, reduction Lanolin, Jojo Hallow, Hard Lanolin, Sera Sokurotsu, PO
Waxes such as E lanolin alcohol ether, POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty acid polyethylene glycol, POE hydrogenated lanolin alcohol ether, liquid paraffin, osikelite, squalene, pristane, paraffin, ceresin, squalane, petrolatum, microcrystalline wax Hydrocarbons such as

Examples of silicon compounds include linear polysiloxanes such as dimethylpolysiloxane, methylphenylpolysiloxane, and methylhydrodienepolysiloxane, decamethylpolysiloxane, dodecamethylpolysiloxane, tetramethyltetrasilicone resin, and silicone rubber. It will be done.

The formulation for external use on the skin of the present invention can be in any form, for example, a solubilized type such as a lotion, an emulsified type such as a milky lotion, a cream, an ointment, a dispersion, and the like.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to this.

Example-1-2c, Preparation of DNA (1) The aorta around the heart was separated from 50 15-day-old chicken eggs, crushed with liquid nitrogen, and then homogenized by adding an aqueous solution of guanidium thiocyanate. did. The obtained homogenate was subjected to the method of Chirgwin et al.
RN by cesium chloride equilibrium density gradient ultracentrifugation according to Ochem, 18, 5294.1979).
A was isolated. Poly A containing poly A was extracted from the RNA fraction by affinity chromatography supporting oligo dT cellulose (manufactured by Pharmacia, Type 7).
” RNA was isolated.

(2) cDNA using commercially available Okayama-Barb vector
cDNA by cloning system (manufactured by Amajam)
A library was created.

That is, Tris-HCl buffer (pl+ 8.3),
Magnesium chloride, potassium chloride, dithiothreitol, dATP, dGTP, dTTP and α-32pd
Add polyA'' obtained in (1) above to an aqueous solution containing CTP.
About 10 μg of RNA and about 5 μg of Okayama-Hague vector primer were added, and a reverse transcription reaction was performed at 37° C. for 30 minutes in the presence of reverse transcriptase (manufactured by Seikagaku Corporation). The vector plasmid containing the synthesized cDNA was recovered by ethanol precipitation. After drying this precipitate, it was dissolved in an aqueous solution containing Tris-HCl buffer (pH 6, 9), sodium cacodylate, cobalt chloride, dithiothreitol, bovine serum albumin, and α-''PdCTP, and the terminal deoxynucleotide derived from bovine thymus was dissolved. Dyltransferase was added and reacted at 37°C for 30 minutes to add about 15 to 30 oligocytosine residues per end to the cDNA.The resulting cDNA-containing vector was digested with restriction enzymes
After digestion with l[I, heku-ku containing oligocytosine-added cDNA was recovered by ethanol precipitation.

Buffer containing commercially available oligo dG tail linker DNA (
Tris hydrochloric acid pl+8.0, magnesium chloride, IEDT
The oligocytosine-added cDNA-containing vector was dissolved in A, NAI') containing ammonium sulfate, E. coli DNA ligase was added, and the mixture was reacted at 16°C for 15 hours. Then dATP, dCTP, dGTP, dTT
P, NAD, E. coli DNA ligase, E. coli DNA polymerase, and E. coli RNA5e H were added and reacted for 1 hour at room temperature to obtain a cDNA-containing plasmid.

Using the obtained plasmid, the calcium chloride method (Man
rl et al., J. Mo14io1.53.159.19
70), E. coli M2S strain was transformed to obtain an ampicillin-resistant strain that grew in LB medium containing 30 μg/m of ampicillin.

(3) Poly A'' RNA was isolated from the skin tissue of a 15-day old chicken using the same method as described in (1) above. This poly A'' RNA and the poly A'' RNA obtained in (1) above were isolated. ”
RNA and E. coli alkaline phosphatase treatment (25°C) in Tris-HCl buffer at pl+8.0.
1 hour) to remove the 5'-terminal phosphate and inactivate the enzyme. Then, T4 polynucleotide kinase and γ-"P-
ATP was added and the phosphorylation reaction was carried out at 37°C for 1 hour.
32p labeled poly A'' RNA (probe) was obtained.

(4) Using the probe obtained in (3) above,
Regarding the transformed strain obtained in 2), the method of Hanahan et al. (Hanahan D, et al., Gene, 10.
Colony hybridization was performed according to 63.1980).

Colonies showing complementarity to the aorta-derived probe but not to the skin-derived probe were selected, and 6 clones out of 1000 colonies were obtained. When plasmid DNA was extracted from these clones and a restriction enzyme map was created using the restriction enzyme Pvorl, a common DNA fragment was found among the three clones. Regarding CDNA with this common DNA fragment,
Dideoxynucleotide enzymatic method using M13mp11 phage (Messing, J., Method in
Enzymo1ogy+Vol, lo1+P2O, Ac
DNA according to Academic Press, 1983)
The base sequence was determined. The base sequence of the cDNA thus determined and the amino acid sequence deduced from it are shown in FIG. The amino acid sequence shown in Figure 1 contains a pentapeptide (Val-Pro-Gly-V
There were multiple at-Gly; underlined in FIG. 1) and multiple crosslinking peripheral sequences (boxed in FIG. 1). Therefore, it was shown that the obtained cDNA was that of tropoelastin.

In this example, a tropoelastin expression vector was constructed using a pKK223-3 expression vector (manufactured by Pharmacia) that uses a tac promoter and an rrnB terminator (see FIG. 2).

(1) The cDNA portion encoding the tropoelastin protein was excised from the Okayama-Barb vector ρE1,2 into which the elastin cDNA prepared in Example 1 was cloned. Approximately 10 ng of pE12 DNA was digested with H4nd m and further partially digested with Ba131. 37
When reacted at ℃ for 5 minutes, an average of about 300 bp was digested. This l'')NA was treated with T4 DNA polymerase large fragment, and C1al linker (pCA
TCGATG) was ligated with T4 DNA ligase. The obtained DNA fragment was inserted into the m-restriction enzyme site Nde.
Digested with 1 and C1al, and electrophoresed on 1% agarose gel (Sigma type ■) to obtain approximately 1.5 kbr.
) NA fragments were extracted at 70°C and isolated by ethanol precipitation.

(2) A plasmid was prepared by converting the BamH1 site of pBR322 (approximately 5 ng) to Char, this plasmid was digested with C1aI and Ndel, and a 1.9 kb DNA fragment was separated by 1% agarose gel electrophoresis (1
) was isolated in the same manner.

(3) The r) NA fragments obtained in (1) and (2) above were combined with T4 DNA polymerase, and the calcium treatment method (Mandel et al., J, Mo1. Riol, 53.1
59゜1970) was used to introduce 12 strains of E. coli into E. coli JM]05 [42]. 30μg7m7! I-B plates containing ampicillin (1% tryptone, 0.5% yeast extract, 1% Na
A transformed strain that grows in CI) was obtained. The obtained transformed strain was subjected to alkaline extraction method (Birnboim et al., Nucl.
, 8c,'+d Ros, 7, 1513.1979) to extract plasmid DNA pEI, 12.

(4) pEl, 12 to 1lind If -P
The vu II fragment was isolated and subcloned into the old ndllll-3mar site of M13mplO (manufactured by Amajam). This single-stranded DNA was used to perform the dideoxynucleotide enzymatic method (Messing, Method i).
n Enzymology, of, 101. D according to p. 20° Academic Press, 1983)
The NA base sequence was determined.

(5) The site partially digested with Ba131 in pEl, 12 (Clar site) is located at many sites in the cDNA, and a C1al linker is attached to the 5' end of the cDNA as shown in Figure 1, resulting in CATCG-ATG-GTC. -GGT-Me
t Val Gly ... A table-bound version was also obtained (pEL12-A).

(6) Obtain the CIaI and Ndel fragments into pEl, 12- and insert blank 1 into the EcoRT site of pKK223-3.
Inserted with enF Rization [operations are from (1) above]
(3)] Transform in the same manner as (3),
After extracting the DNA, plasmid plasmid 22 in which cDNA in the transcriptional direction was bound downstream of the tac promoter was obtained.

(7) 30μg/m7 of the obtained transformed strain! 1. containing ampicillin and 1mM TPTG. r3 medium 500
mA' was inoculated and cultured with shaking at 30°C for 24 hours. After culturing, the bacterial cells were collected by centrifugation and added to a LOM buffer solution (50mM Tris-HCl pH 7.5, 50mM EDT8,
-Mercaptoethanol to 1mM and Sonifier
1 at 4℃ using an ultrasonic homogenizer (manufactured by Planson).
After treatment for 5 minutes to disrupt the cells, a supernatant was obtained by centrifugation.

(8) On the other hand, the decapeptide Pro-A Ia-A Ia- which is part of the amino acid sequence of tropoelastin
A Ia-Ala-Lys-Ala-A
la-Ala-Lys was chemically synthesized using Peptide Synthesizer Model 430A (manufactured by Abrite Biosystems), and the method of Kagan et al.
an et al., Tn Methods of llormon
eRadioimmunoassay, B. M. Ja
ffe and Il. R. Edited by Behyman, p328
Academjc Press, 1979), about 10 molecules of decapeptide were bound to each molecule of BSA (bovine serum albumin) using glutaraldehyde. 10 mg of this BSA derivative I in 20 weeks.
It was injected subcutaneously into white male rabbits in divided doses. After confirming that antibodies were produced by antigen-antibody reaction, blood was obtained and antiserum was obtained by a conventional method.

(9) Transfer the supernatant obtained in (7) above to Sephadex G-10.
0 column chromatography to obtain a fraction that reacts with the antibody prepared in (8) above. This fraction was concentrated by ultrafiltration and subjected to FPLC (manufactured by Pharmacia) Mo
It was purified by high performance liquid chromatography using noQ Color 1 to obtain a fraction that reacts with the antibody. This fraction was freeze-dried to obtain tropoelastin.

(10) The obtained tropoelastin showed a single band in SDS polyacrylamide gel electrophoresis, and the molecular weight was about 3.
It was 0000 Daltons. Furthermore, when the N-terminus was determined by Eman decomposition, Val - G) y - V
The sequence was confirmed to be al-Pro-Gay, and we were able to confirm that the target tropoelastin was produced by E. coli.

IIL Bacillus subtilis 11 - Expression vector - α I Bacillus subtilis α -
pTUB228 (K. Ohmura et al., J.B.
jochem. 95, 87. A Bacillus subtilis expression vector was created using the following method (1984).

(1) IlindTIT the Cla1 site of pEL]2
Prepare the plasmid pEI, 12-1, and insert the approximately 3 kb llindTII-old ndrllr)NA fragment containing the elastin cDNA into the old nd11 of pTUB22B.
It was inserted into one site. As a result, a Bacillus subtilis expression vector pEL112 was obtained in which elastin cDNA was linked downstream of the α-amylase signal peptide via the old ndT11 site.

(2) pEL112 was purified using the method of Wilson et al. (Wi
Ison et al., J.

Bacteriol. 95, 1439. 1968)
Bacillus subtilis
) was introduced into Marburg168 strain.

At this time, the transformed strain was selected based on kanamycin resistance.

(3) The transformed strain was cultured in L and G medium in the same manner as in Example 2 above. After culturing, 1.
``1 boelastin was isolated.

Example --- 4 One hole yeast expression - Current status: Freshly produced yeast, plasmid using galactose monophosphate uridyl transferase gene GAL 7 promoter (■
) (Japanese Patent Application Laid-Open No. 60-248181) to create a yeast expression vector.

(1) Change C1ar of plEL]2 to the plant end, and add the DNA fragment containing the NdeI-digested cDNA to the plant end, and convert 13g1ll of plasmid (1) to the plant end.
It was ligated to the eT-digested 2μori-containing I''NA fragment. As a result, a yeast expression vector was obtained in which an ATG derived from the C1al linker was present at 20 bp downstream of the GAI and 7 transcription start sites.

(2) The obtained expression vector was expressed using the lithium chloride method (11,
Ito et al., J. Bacteriol, 153.163.
(1983) by yeast IU, (Saccharomyce
S cerevisiael M'FB-1 strain (M.

Tajima et al., Mo1. Co11.8io1.6,2
46.1986). At this time, uracil non-auxotrophy was used to select the transformed strain.

(3) The transformed strain was grown on minimal medium (0.66% fermentation Lυ source base, 0.5% casamino acids, 0.002% I, -1 to
Lyptophan, 0.004% adenine, 2% galactose) 500m+8 was cultured with shaking at 30°C for 2 days. After culturing, the cells were harvested, the cells were disrupted in the same manner as in Example 2, and tropoelastin was separated and purified.

Example-y (1) A lotion according to the present invention was prepared from the following ingredients.

(Weight %) Tropoelastin (Niwato Tsuyu &) 0.5 Glycerin 10 Polyoxyethylene isostearyl alcohol (POE 15 mol) 0.5
Fragrance Optimal Purified Water Residue (Production Method) Tropoelastin, glycerin, and polylioxyethylene isostearyl alcohol were added to purified water, stirred and dissolved at room temperature, and then filtered.

(2) Skin roughness improvement effect example 5 The skin roughness improvement effect was measured using a human body panel using the lotion obtained in (1) and a control lotion prepared without tropoelastin (replaced with purified water). The test was conducted.

(Test method) A replica of the skin surface morphology of a healthy female person (face) is taken using silicone resin and observed under a microscope (17x magnification).

Using the lotion of Example 5 (1) and the control lotion, applied to the left and right half of the face of 24 people (skin roughness panel) who were judged to have a skin roughness rating of 1.2 as shown in Table 1 based on the condition of skin marks and peeling of the stratum corneum. I applied this lotion once a day.

(Evaluation method) One week later, a replica was taken again and the condition of the skin was evaluated according to the criteria in Table 1.

The results are shown in Table-2.

Table 1 Judgment criteria for the replica method (18* Scale is also visible to the naked eye) Table 2 Skin quality evaluation 1 week after using the lotion From the results of Table 2, it was determined that the lotion containing tropoelastin was The facial area where the lotion was used had a remarkable effect on improving rough skin compared to the facial area where the control lotion was used.

(3) Skin improvement effect after sunburn (test method) Using Toshiba 4O-3E lamp (tJVB) 6 lights, 30c
3 on each side of the left and right forearms of 20 male panelists from a distance of n+.
The c111 corner is irradiated with 8W4 ultraviolet rays for 6 minutes to create an artificial sunburn. Since sunburn causes rough skin, from the day of irradiation, the lotion obtained in Example 5 (1) above and a control lotion containing no tropoelastin were applied twice a day to the left and right sides for 10 days. We investigated its effect on improving rough skin.

(Evaluation method) The improvement of rough skin after sunburn was observed on the 10th day using the replica method, and evaluated according to the criteria shown in Table 1 above.

The results are shown in Table-3.

Table 3 Evaluation of skin quality 10 days after using the lotion The use of the tropoelastin-containing lotion was found to have a significant effect on improving rough skin after sunburn.

Example-” A lotion according to the present invention was prepared from the following ingredients.

(Weight%) Tropoelastin (Vivzo*P%) 0.000
1 glycerin 3.0], 3-
Butylene glycol 4,0 ethanol
8.0 Polyoxyethylene oleyl full: J-B (POE 15 MoB)
0. 5 Methylparaben 0
．． 1 Citric acid O1 Old sodium citric acid O0 ■ Loofah extract 0.3 Fragrance
Appropriate amount of purified water Remainder (manufacturing method) Dissolve citric acid, sodium citrate, glycerin, tropoelastin, 1,3-butylene glycol, and loofah extract in purified water. Separately, polyoxytylene oleyl alcohol, fragrance, and methylparaben were dissolved in ethanol, and this was added to the above-mentioned purified aqueous solution to solubilize and filter to obtain a lotion.

- side υ A cream was prepared from the following ingredients.

Below is the margin (% by weight) (Manufacturing method) Phase A was dissolved by heating and kept at 75°C, and then added to Phase B, which was heated and dissolved at 75°C, with stirring.

After processing with a homomixer to make emulsified particles fine, the mixture was rapidly cooled while stirring to obtain cream.

Example −β− An emulsion was prepared from the following ingredients.

(% by weight) Stearic acid 1.5 Cetyl alcohol 0.5 Isopropyl myristate 4.0 Squalane
5.0 min wax
2.0 Polyoxyethylene (10 mol) Monooleic acid ester It l, Q Stearic acid monoglyceride 1.0 4y'P
r-V Jllllfl liquid (5% aqueous solution)
20.0 Propylene glycol
5.0 Ethyl alcohol 3.0 Fragrance Appropriate amount Ultraviolet absorber Appropriate amount Purified water
Residue (manufacturing method) Add tropoelastin and propylene glycol to purified water, dissolve with heat, and keep at 70°C (aqueous phase).

Add fragrance and UV absorber to ethyl alcohol and dissolve (alcohol phase). Among the remaining components, the components other than the quince seed extract are mixed, dissolved by heating, and kept at 70° C. (oil phase). Pre-emulsify by adding the oil phase to the water phase and homogeneously emulsify using a homomixer. While stirring, add the alcohol phase and quince seed extract. Thereafter, the mixture was cooled to 30° C. while stirring to obtain a milky lotion.

Example-J- Punk was prepared from the following ingredients.

(Weight %) Polyester Nylalco-1 Misaki (Saponification degree asχ: Polymerization degree 20
0'0) 1 5. 0 polyethylene glycol
3.0 propylene glycol
7.0 ethanol 10
．． 0 Methylparaben 0.1 Tropoelastin (mouse-LU 5.0 Sodium chondroitin sulfate 0.05 Fragrance
Appropriate amount of purified water Remainder (manufacturing method) Add polyethylene glycol, propylene glycol, methylparaben, tropoelastin, and sodium chondroitin sulfate to purified water and dissolve with stirring. Next, polyvinyl alcohol was added and dissolved by heating, and ethanol in which the fragrance had been dissolved was added and dissolved with stirring to obtain a bank.

Example - A scalp treatment (scalp cosmetic) was prepared from four or less ingredients.

(Weight %) Δ (Production method) Phase C dissolved at 75°C was added to Phase B maintained at 75°C with stirring, and then Phase D, which had been sufficiently stirred and dissolved at room temperature, was added and stirred. The mixture was cooled to 50°C, and a human phase dissolved in a small amount of purified water was added thereto, and the mixture was further stirred and cooled to 30°C to obtain a scalp treatment.

Example □□11- A water-absorbing ointment was prepared from the following ingredients.

(Weight%) Vaseline 40.0 Stearyl Alcohol 18.0 Mokuro
20.0 polyoxyethylene (1
0 mol) Monooleic acid ester 0
．． 25 Stearic acid monoglyceride 0.25
2.0 Purified water (manufacturing method) Add tropoelastin to purified water and keep at 70°C (aqueous phase). Other components are mixed and dissolved at 70°C (oil phase). The aqueous phase was added to the oil phase, uniformly emulsified using a homomixer, and then cooled to obtain a water-absorbing ointment.

When used on the skin and scalp, the external skin preparations of Examples 6 to 11 all had excellent skin-improving effects, little skin irritation and sensitization, and excellent stability over time, so they could be used suitably. .

Example-1-'L An emulsified foundation was prepared from the following ingredients in a conventional manner.

(Heavy wall%) Stearic acid 1.2 Palmitic acid 0.8 Cetyl alcohol 2.0 Liquid paraffin
12.0 Cetyl Isooctanate
5.0 dimethylpolysiloxane (6cs)
3.0Decamethylcyclopentasiloxane 3
．． 0 Preservatives 0.3 Antioxidants 0.05 Fragrances
0.3 Purified water 46.5 Propylene glycol 5.0 Polyethylene glycol 5.0 Tropoelastin C=Sotri*Ice) 0.5 Sodium hyaluronate
0.1 potassium hydroxide
0.2 Titanium dioxide 6.
0 Red iron oxide 0.32 Yellow iron oxide 0.75 Black iron oxide 0.2 Talc
5.0 kaolin
2.73 Dispersant
Example 0.05-13- A makeup base was prepared from the following ingredients in a conventional manner.

(Weight%) Stearic acid 0.5 Isostearic acid 0.5 Squalane
8 Glyceryl triisooctanate 2 Vaseline l Cetostearyl alcohol 0.5 POE-added glyceryl monostearate 1.0 Diglyceryl diisostearate 1.0 Preservative
0.3 antioxidant
0.05 Fragrance 0.3 Purified water 68.151.3
-Butylene glycol 10.0 Glycerin 1.0 Tropoelastin and Watori 4) 0.5 Polyacrylic acid
0.1 potassium hydroxide
O0 ■ Ethyl alcohol
5.0 Example 14 A powder leaf amplifier was prepared from the following ingredients in a conventional manner.

(Weight%) Mica 47.29 Talc 20.0 Kaolin
5. 〇Titanium dioxide
10.0 iron oxide
5.0 Sorbitan Sesquiolate
1.5 Squalane 5.0
Trimeko Low + Ltn Vantorisos 5y rate
5.0 preservative
0.7 Fragrance 0.5 Tropoelastin Cζ・N'd) 0.
Old Example-1-5- Mascara was prepared from the following ingredients in a conventional manner.

(% by weight) Liquid polybutene (average molecular weight 1000) IO Mic 11 Crystalline wax 8 Sorbitan monostearate 1 Polyquaternary soethylene (l
i, 0.20 solu) Solhitan monoster 7 rate
1 pile of ice 23.99
1:1 Boelastin (Shimaru 4) 0.01 Iron oxide blank 10 Daitosol (Note 1)
20 Varnish Dyne (Note 2) 2
0 Nylon fiber 6 Preservative
Appropriate amount of fragrance
Appropriate amount (Note 1) Trade name of polyethyl acrylate emulsion manufactured by Daito Kasei KK (Note 2) Trade name of polyvinyl acetate emulsion manufactured by Blockchain Chemical KK Example-Ll A transparent lipstick was prepared from the following ingredients in a conventional manner.

(% by weight) Purified water 89.999 Glycerin 5 Tropoelastin and filtrate) 0.001 Polyvinyl alcohol 1 Monohenzylidene-D-sorbitol 2 Dihenzylidene-D-sorbitol 2 Dye Appropriate amount Preservative Appropriate amount Fragrance
Example of Appropriate Amount An emulsified lipstick was prepared from the following ingredients in a conventional manner.

(Weight%) Castor oil 50 Isostearic acid diglyceride 10 Candelilla wax
8 Microcrystalline wax
10P, O, F, (20) P, O, P, (20)
2 Tetra Tesyl Octate Sil D - Te + 1
4 Red No. 202
Titanium monoxide 1 Red iron oxide 2.95 Refined ice
10 Sodium pyrrolidone carboxylate 11.3-Butylene glycol
1.995 hyaluronic acid
0. , O05 tropoelastin (human $)
0.05-Example'' - An emulsified enamel was prepared from the following ingredients in a conventional manner.

(Weight%) 5% human Xib Pi; 1 methyl cellulose/10 water
Propylene glycol 0.1 Tropoelastin (17) 0.03 Nitrocellulose (A) 12 Alkyd resin (hersatinocic acid glycityl ether modified (2) 1) 6 Sucrose henzoate 6 Acetyl tributyl citrate 5 Camphor
1.5-n-butyl acetate
32.4 Ethyl acetate 10 Toluene 15 Red No. 202
0.2 titanium dioxide
0.8 Bentone 3B, (Note 1) 1 (Note 1) Organically modified montmorillonite clay, trade name 1119, manufactured by NL Chemica 1. A water-based enamel was prepared from the following ingredients by a conventional method.

(% by weight) Polyvinyl acetate monopolymer (711%) (effective content 50%)
8 Styrene, Methyl MEC 1 root, Phthyl acrylate 2 Ethylhexyl 7 Root, Methacrylic acid (1,000 zum ratio 19:60:10:6:
Microemma copolymer (effective content: 40%) 60% purified ice
16.99710 Boelastin (pig origin)
0.003 Polyquine ethylene sorbitan monooleate 1 Laponite X1. G 1 isopropyl alcohol 5 ethylene chloride; t
Monoethi BI-chill
5 pigments (red No. 202, yellow iron oxide, titanium oxide; 4:2:1)
2 Mica titanium 1 Antifoaming agent
Appropriate amount of preservative
Appropriate amount f'L-LO- A hair blow lotion was prepared from the following ingredients.

(Weight%) (1) Dimethylpolysiloxane (5cs) 5(
2) Liquid paraffin 3 (3) Dipropylene glycol 5 (4) Polyoxyethylene glycol (60) Hardened castor? Mountain 2 (5) Tropoelastin and Watori ψ powder) 0.5 (6) Ethanol (95%) 30 (7) Fragrance Appropriate amount (8) TM Cyste 7lylsimethylammonium light 0.3 (9) ion exchange water
54.2 Component (3) Component (IO2) was added to the solution of (4) and emulsified. Ingredients (5
) to (9) were dissolved and mixed, and the above emulsion was added.

One side go-so A hair mousse was prepared from the following ingredients.

(Heavy-walled %) (1) Cystea chloride 1/1 mth: Toammonium chloride 1 0.5 (2) Dipropylene glycol 5 (3) Polyoxyethylene (40)
Dimethylpolysiloxane 3 (4) Coconut oil 1'1 (5) Polyoxyethylene (80) Hardened castor
1 (6) Acidic acrylic acid polymer 3 (Product name Plus Size L-53) (7) l-n +f Knee 7 Suti 7 (:!-So)lli powder) 2 (8) Fragrance Appropriate amount (9) Peptide Hydrolyzate (MW700) 1
(10) Ion exchange water 83.5 components (1) to (10) were dissolved and mixed to prepare a stock solution.

To 96 parts by weight of the stock solution, 4 parts by weight of propellant gas (+, PG, 25°C, pressure 4.0 kg>4) was added and filled into an aerosol can.

Example-2-2- A hair liquid was prepared from the following ingredients in a conventional manner.

(Weight %) (1) Polyoxybutyrene (40) buty) [ether 1 [10(2) polyoxybutylene 11pyrene (70) Decagrise Jan [ether 10(3) tropoelastin to I noodle powder] 5 (4 ) Fragrance appropriate amount (5) Pantothenic acid ethyl ether 0.5 (6)
Ethanol (95%) 50 (7) Ion exchange water 24.5 ingredients (
1) to (7) were dissolved and mixed.

Example - is A hairspray was prepared from the following ingredients in a conventional manner.

(weight V%) (1) Copolymer of 7-IJIL acid and methacrylic acid and their alkyl esters 5(2) Meth-IL
Phenyl Messiloxane 20cs
1 (3) POE (4) Oleyl ether 0,5 (4) Ethyl alcohol (99%)
76.5(5) Tropoelastin was dissolved with stirring to obtain a stock solution. Injection gas (Freon 11/12 = 40 parts by weight for 50 parts by weight of stock solution)
A hair spray was obtained by adding 150 parts by weight of /60 and filling it into an aerosol can.

子hi】”− A hair tonic was prepared from the following ingredients.

(Weight%) (1) Propylene glycol 3.0 (2)
Pantothenyl ethyl ether 1.0(3) Diphenhydramine hydrochloride 0.1(4) Salicylic acid
0.1 (5) Tropoelastin (
:! - Ancient times) 0.5 (6) 7! −Menthol
0.1 (7) Fragrance
0.5 (8) Ethanol 40.0 (9)
Purified water remainder (manufacturing method) Component (9), component (1) (2) (3) (4)
(5) was stirred and mixed (Part A). Other ingredients were stirred and mixed (Part B).

Part B was added to Part A and mixed with stirring to obtain a liquid hair tonic.

Example-'L5- A scalp treatment was prepared from the following ingredients.

(Weight%) (1) Liquid paraffin 15.0 (2)
Vaseline 2.0 (3) Setanol 2.0 (4) Polyethylene glycol 1500 7.0 (5) Stearic acid
2.5 (6) POE (6) So1
1 Hitane Monostearate
1.0(7) Glyceryl monostearate 1.0(8
) Caustic potash 1.0 (9) Tropoelastin *1) 3.0 (10) Quinoline Yellow Appropriate amount (11) Ethylparaben 0.1 (12) Fragrance
0,5(13) Purified water
Residue (manufacturing method) Ingredient (13) and Ingredient (4) (8) (9) (10)
was heated and dissolved and kept at 70°C (Part A). Other ingredients were mixed, heated and melted and kept at 70°C (Part B). Part B was added to Part A, mixed with stirring, and then emulsified using a homomixer.

After emulsifying, the mixture was cooled to 30° C. while stirring to obtain a milky scalp treatment.

Example-Yu-W A pre-shampoo preparation was prepared from the following ingredients.

(Weight%) (1) Polyvinyl alcohol 2.0 (2)
Quen # 0.5 (3)
oxyethylene (10) oleyl ether
1.0 (4) Dipropylene glycol 10.0 (5) Hydroxyethyl cellulose 0.5 (6) Xanthan gum
0.5 (7) Fragrance 0.5 (8) Purified water Remaining (9)
1.0 (Production method) Components (5) and (6) were wet-dispersed in tropoelastin (1.0 (manufacturing method)).
Part A). Other components were mixed, heated and dissolved, and then cooled to normal temperature (room temperature) (Part B). Part A was added to Part B and mixed with stirring to obtain a slimy pre-shampoo.

An example - 1 A hair colorant was prepared from the following ingredients in a conventional manner.

The following margin (% by weight) [Caustic soda (10% aqueous solution) 1.0 Phase (manufacturing method) Phase B, C, and D were sequentially added to phase A and mixed by stirring.

Example 28 A conventional hair dye was prepared from the following ingredients.

The following is a margin: (74) (% by weight) B [Hydroxyethyl cellulose 0.5 parts [Citric acid 1.5 parts (manufacturing method) Part B was gradually added to Part A with stirring to prepare a viscous liquid. Dissolve 0 parts of each dye in this viscous liquid, and adjust the pH in part D.
was adjusted to 3.2 to 3.5 to obtain a reddish brown hair dye.

Example 29 A water-soluble gel hair conditioner was prepared from the following ingredients in a conventional manner.

(% of heavy walls) Total 100.0 (Production method) Phase A and phase B were stirred and dissolved at 70 to 90°C, then mixed and cooled at a constant temperature of 0°C.

Second 1””- An oxidative hair dye was prepared from the following ingredients in a conventional manner.

(% by weight) 1st 'N1 Polyoxyethylene (2) Oleyl ether 14 Oleic acid
12 Propylene glycol 10 Ethanol 8 Paraphenylenediamine Lusorcin 0.5 Metaaminophenol 0.2 Sodium edetate 0.1 Thioglycolic acid
0.1 tropoelastin (2 vMd > 1.

Ammonia water Adjusted to pHlo Fragrance
0.1 Purified water Other ingredients were sequentially added to the purified water in an amount to make the whole 100χ and dissolved with stirring to obtain the first agent.

Part 2: Hydrogen peroxide (30%) 20% Phosphate buffer Adjusted to pH 4 Purified water
Other ingredients were sequentially added to the purified water in amounts to make the whole 100χ and dissolved with stirring to obtain a second agent.

, Example 31 A hair dye was prepared from the following ingredients in a conventional manner.

(% by weight) 1st polyoxyethylene (5) noni 11 phenyl ether IL 15 oleic acid 1
0 Propylene glycol 10 Ethanol 8 Paraphenylenediamine Lusorcin 0.3 Sodium edetate O31000-glycolic acid 0.1 Aqueous ammonia
p Old 0 adjusted fragrance 0.1 Purified water 100 parts Adjusted 2nd agent: Hydrogen peroxide solution (30%) 20 Tropoelastin Cv-R'f, > 0.5 Phosphate buffer pH 4 adjusted purified water
100 parts The first and second parts of Preparation Examples 30 and 31 were mixed in equal volumes, applied to gray hair, and applied to gray hair.
When the hair was left to stand for 0 minutes and then washed off, the hair was sufficiently dyed and no irritation to the scalp was observed during or after use.

Example-32 An unwanted hair bleaching agent was prepared from ingredients below 1 so.

[1 agent] (wt%) (Ammonia water reagent special grade (28%) 5.0 phase Dissolve phase A by heating and keep it at 70°C, and add it to phase B, which was dissolved by heating at 70°C, with stirring. After processing with a homomixer to make the emulsified particles fine, the mixture was rapidly cooled while stirring to obtain a cream.Furthermore, phase C was added thereto and stirred to obtain a uniform one-drug cream.

[Part 2] (% by weight) Phase A was dissolved by heating and kept at 70°C, and then added to Phase B, which had been heated and dissolved at 70°C, with stirring. After processing with a homomixer to make the emulsified particles fine, the mixture is rapidly cooled while stirring.
A cream was obtained.

When I mixed equal amounts of agents 1 and 2 of this Example 32, applied it to areas with unwanted hair on my hands and feet, and washed it off after about 15 minutes, the unwanted hair was evenly bleached and my skin did not get rough after use. However, it was desirable.

Further, no irritation was observed during use.

(Weight%) (1) Tropoelastin (shift 9 * $) 7 (
2) 1,3-butylene glycol 5(3)
Undenatured ethanol 5 (4) Methylparahen 0.3 (5) Propyl paraben 0.03 (6) Fragrance Appropriate amount (7) Ion exchange water Amount to make the whole 100χ (
Manufacturing method) Dissolve component (1) in component (7). Add to this the ingredient (2
) to (6) were mixed and dissolved, and the mixture was stirred and mixed.

Example 34 A hair treatment was prepared from the following ingredients.

(Weight %) (1) Ethylene oxide impact strength of tare alcohol, 5 (2) Stearyl alcohol
2.0 (3) Stear trimethylammonium chloride (27%) 1.4 (4) Stear 1 halide trimethyl ammonium chloride (73%) 0.8 (5) Propylene glycol 3.0 (6) Tropoelastin (
2V) Q*1 1.5 (7) Fragrance Appropriate amount (8) Ion exchange water Amount to make the whole 100χ (
Manufacturing method) Components (1) to (4) were weighed out and dissolved in a humidified manner at about 80°C (A phase). Phase A was added to component (8) at about 80°C and mixed with stirring (Phase B). In phase B, sequential components (5) (6)
(7) was added and mixed with stirring (phase C). Cool phase C (
30°C).

seconds l A hair conditioner was prepared from the following ingredients.

(Weight%) (1) Contribution to tropoelastin 1) 0.8
(2) Stearyltrimethylammonium tetrachloride (60
%) 2.0 (3) Liquid paraffin 1
．． 4(4) 1-oxyethylenecetyl I-te11
(20,000B) 1.2 (5) Cetanol 2.4 (6) Fragrance
Appropriate amount (7) Citric acid Appropriate amount (8) Ion-exchanged water Amount to make the whole 100χ (manufacturing method) Components (3), (4), and (5) were weighed out and dissolved by heating to about 80°C (A phase). Phase A was added to component (8) at 80° C. and mixed with stirring (phase B). In phase B, component (7)
(6) were added one after another and mixed with stirring (phase C). C phase,
The mixture was stirred and cooled to about 40°C.

Example 36 Hair cream J was prepared from the following ingredients in a conventional manner.

(Weight %) (1) Tropoelasti shearing bird association, i) 0.
4(2) Stearic acid 13.0(3
) Setanol 2.0 (4) Edelengrigol cyste 7 rate
4.0 (5) Silicone oil KF9t
,,3 D(J O,2(6) Triethanolamine 1.6(7) Propylene glycol
8.0(8) Edetate
0.1 (9) Perfume Appropriate amount (10) Ion-exchanged water Amount to make the total 1007 (Production method) Components (2) to (5) were weighed and dissolved by heating at about 80°C (Phase A). Ingredients (6), (7), (8), and (10) were heated to about 80° C., and then component (1) was added (phase B).

Gradually add phase A to phase B, stir, and mix the ingredients (9) at about 60°C.
), about 4! The mixture was prepared by stopping stirring at 0°C.

-Example 7- A permanent waving agent was prepared from the following ingredients.

First agent; (wt%) 7-simonium oglycolic acid
12.5 (50% as thioglycolic acid) Ammonia water reagent special grade (28%) 1.5 Ammonium bicarbonate 3.5 Tropoelastin (:l
-Wato is God! t 0.5 stear chloride 1 haletrimethyl 7 ammonium (80%) 0.1 polyoxyethylene (20 moles (unit) oleyl l-te + L
O,5 Perfume 0.1 Purified Water 81.3 Polyoxyethylene (20 mol) Oleyl ether was heated and melted, perfume was added and stirred, and then added to purified water and stirred and dissolved. Next, the other components were sequentially added and dissolved with stirring to obtain a transparent first agent.

Second agent; (wt%) Sodium bromate 5.0 Sodium phosphate dibasic 0.1 Potassium monophosphate
0.3 Purified water
94.6 Other ingredients were sequentially added to the purified water and dissolved with stirring to obtain a transparent second agent.

80 rrl of the first agent was applied to human hair by winding and applying with a rond, and the hair was covered with a cap and left for 10 minutes. After removing the cap, performing an intermediate wash with water and wiping off excess water with a towel, 100 ml of the second agent was applied in two portions and left for 15 minutes. Next, I removed the ronde, washed it with water, and then towel-dried it, resulting in moist, supple, and bouncy waves. Drying it with a hairdryer gives it a shiny, wavy finish.

-Example-3''- A permanent waving agent was prepared from the following ingredients.

First agent; (wt%) Thiocryl 7-9ymonium
13.0 (50 as thiocrylic acid)
%) Diammonium dithiodiglycolic acid
10.0 (40% using cythiosyglyco-1 as acid) Ammonia water reagent special grade (28%) 1.
0 Monoethanolamine 1.0 Ammonium bicarbonate 2.5 Tropoelastin (Relationship 1> 1.0 St7lyltrimethychloride + 17 ammonium
0.5 polyoxyethylene (15 molar force)
Oleyl I-tit 0.5 fragrance
0.2 Edetate tetrasodium 0.1 Purified water
70.2 Polyoxyethylene (15 mol) Oleyl ether was heated and melted, a fragrance was added and stirred, and then added to purified water and dissolved with stirring. Next, the other ingredients were sequentially added and dissolved with stirring to obtain a transparent first agent.

2nd Agent: Same as Example 37 - By the same method as Example 37, waves were obtained that were moist, supple, uniform, and elastic when wet. When I dried it with a hairdryer, I was able to get shiny and very firm waves.

Seconds ↓”− A permanent waving agent was prepared from the following ingredients.

First agent; (wt%) thioglyco-+>9 ammonium
2.0 (50% as thioglycolic acid)
) ■, -cysteine 6.5 Monoethanolamine 2.0 Ammonia water reagent special grade (28%) 1.5 Cetanol
0.3 Sodium lauryl sulfate
0.1 polyoxoederene (15mol IL)
Seven de-1 diether 0.1 Tropoelastin (Otsuzuyai Q> 0.7 Fragrance
0.08 Edetate tetrasodium 0.2 Purified ice
86.52 Heat 30% purified ice to about 80°C, add cetanol, sodium lauryl sulfate, and polyoxyethylene (15 mole adduct) cetyl ether, stir and dissolve, and slowly cool while stirring to form a viscous liquid. Created a cane gel. Add other ingredients to the remaining purified water (56.52%) and stir to dissolve.
This was stirred together with the previously prepared gel to obtain a viscous first agent.

2nd agent: This 1st agent was applied to the hair of the same person as in Example 37 by winding with a ronde using 80 mA, and then covered with camp and left for 20 minutes. After removing the camp and performing an intermediate wash with water and wiping off excess water with a towel, 100 rrl of the second agent was applied in two doses and left for 15 minutes. Next, I removed the ronde, washed it with water, and then towel-dried it, resulting in smooth, bouncy waves. Drying with a hair dryer will give you shiny, firm, uniform waves, and <Li[! The resistance was also very good.

Italy 4 units (1- A permanent waving agent was prepared from the following ingredients.

First agent; (wt%) ammonium thioglycolate
12.0 (50% as thiochlorocholic acid) Ammonia water reagent special grade (28%), 1.0 Ammonium bicarbonate 4.0 Tetrasodium edetate 0.1 Purified water
82.9 Other ingredients were added to purified water and dissolved with stirring to obtain a transparent first agent.

2nd agent; (wt%), sodium bromate 6.0 secondary phosphoric acid 1
-Rium 0.1 monopotassium phosphate
0.3 Tropoelastin
1.0 Purified water 92.6 Other ingredients were added to purified ice and dissolved with stirring to obtain a transparent second agent.

By the same method as in Example 37, smooth waves with good combability were obtained when wet. When dried, shiny and firm waves were obtained.

lp ``41- A permanent enamel agent was prepared from the following ingredients.

1st part: 2nd part same as Example 40; (wt%) hydrogen peroxide water reagent special grade (30%) 7.0 dibasic sodium phosphate 0.1 dibasic potassium phosphate
0.5 edetate, disodium
0.1 Tropoelastin (7″Fm and a half)
0.5 Purified water 91.
80 mρ of the first agent was applied to the hair of 8 people in a winding manner, and the hair was covered with a cap and left for 10 minutes. After removing the cap, washing with water, and wiping off excess water with a towel, apply 80mj of this second agent! Apply,
It was left for 5 minutes. Next, the lot was removed, washed with water, and then towel-dried to obtain smooth, elastic waves. When dried with a hair dryer, shiny and firm waves were obtained and the combability was good.

Example - "(- A liquid facial cleanser was prepared from the following ingredients.

(Weight%) (1) Potassium lauric acid soap 5.0 (2
) Potassium myristate soap 3.0 (3) Potassium oleate soap 3.0 (4) Coconut fatty acid cytanol amite
5.0 (5) Propylene glycol 10.
0 (6) Tropoelastin (2 v) + 1 ice) 0.5 (7
) Purified water 73.5 ingredients (1)
Component (4) in the mixed solution of (2) (3) (7)
Then, component (5) in which component (6) is dispersed is added, and the mixture is heated, mixed and dissolved. This was cooled using a heat exchanger to obtain a liquid facial cleanser. When I washed my face with this liquid facial cleanser, it lathered smoothly during use, did not feel stiff after use, and had an excellent feeling of use.

Examples A bar soap was prepared from the following ingredients.

(Weight%) (1) Soap base 98.6 (2)
Titanium dioxide 0.3 (3) Tropoelastin (chicken) 0.5 (4) Fragrance
1.0 Components (2), (3), and (4) were added to component (1) and kneaded to prepare a cleansing foam from the following components in a conventional manner.

(% by weight) (1) Stearic acid 10.0 (2)
Valmitic acid 10.0 (3) Myristic acid 12.0 (4) Lauric acid
4.0 (5) Peace wax
1.0 (6) Caustic potash
6.0(7) Propylene glycol
1O30(8) Polyethylene glycol 400
5.0 (9) Tropoelastin and Ditri91)
0.5 (lO) Fragrance 0.
5 (11) Purified water 36.5-
45 When a hair rinse was prepared from the following ingredients, a product with excellent usability was obtained.

(Weight %) Steer 1 hat trimedelammonium chloride
2.0 Sorbitan monooleate 1.4 Mineral oil
2.8 cetanol
2.0 polyoxyethylene (20) [1971
1 year old-F 0.3 Tropoelastin ヮ) 9*來) 1 Fragrance
Appropriate amount of pigment Appropriate amount of citric acid Appropriate amount of water
Feet side (lower = 8 rounds of body shampoo was prepared from the following ingredients by a conventional method, with the total number being 100).

Coconut oil potash soap 10.0 Coconut fatty acid methyl; [sodium taurite (30%)
10.0 laul]yldiethanolamide 5.01.3-Butylene glycol
3.0 Tropoelastin to Watori ml) 0.
5 Purified water 65.0 A product with excellent usability was obtained.

(% by weight) Sodium lauryl sulfate 10,000 nostearate ethylene glycolate 2 polyoxyethylene (7
5) Lanolin 0.25 Coconut oil fatty acid diethanolamide 5 Fragrance as appropriate Citric acid as appropriate Sodium chloride 0.75 Tropoelastin (% % > 0.8 Kathionized cellulose ether 0.5 Water
Foot side - L8 - (% by weight) Calcium diphosphate dihydrate 40.0 Glycerin 10.0 Sorbit
10.0 Calnexymethyl cellulose sodium
1.5 Sodium lauryl sulfate 1.5 Sankarin 0.1 Fragrance
1.0 Tropoelastin (%) 0.1 Purified water Balance The toothpaste of the present invention was obtained from the above composition by a conventional manufacturing method.

A comparative toothpaste was obtained in the same manner as in Example 48 except that tropoelastin was removed from the composition of Example 48.

Twenty patients with periodontal disease were divided into two groups of 10 patients each so that the severity of symptoms was the same in each group. Each group received 1 dose of the present invention and comparative toothpaste.
Brushing was performed for 3 minutes three times a day for 14 days. During this period, the use of other oral compositions was prohibited, and no particular instructions on brushing were given.

The effectiveness was evaluated using the following criteria.

Significant effect on periodontal disease: 3. Clear effect on periodontal disease: 2. Slight effect on periodontal disease: 1. No effect was observed: 0 The results are shown in one table, but the numbers in the table are the average values of 10 people.

Table-1 Toothpaste of the present invention Comparative toothpaste rating 1.8 0.7 As is clear from Table 4, the toothpaste of the present invention containing 0.1% tropoelastin was superior to the comparative toothpaste. It was effective against periodontal disease.

Example"j A toothpaste was prepared from the following ingredients in a conventional manner.

(weight V%) silicic anhydride 20.0 sorbitol 50.0 carrageenan 0.5 strength; I phoxymethylsebo-sodium
1.0 1-lium lauryl sulfate 1
．． 8 Saccharin sodium 0.08
Methyl paraoxybenzoate 0.2 fragrance
0.9 Tropoelastin (if”*ne) 1.0 Purified water
Residue side - Pasta for oral cavity was prepared from the following ingredients by a conventional method.

(% by weight) Vaseline 10.0 Propylene glycol 7.0 Stearyl alcohol 10.0 Polyethylene glycol 1
500 30.0 Hinokitiol
0.1 Mouse E G F
O,001ppm tropoelastin cut off? )
5.0 polyethylene glycol 400
A mouthwash was prepared from the remaining ingredients listed below in a conventional manner.

(Weight%) Ethyl alcohol 10.0 Saccharin sodium 0.05 Fragrance
0.8 Polyoxyethylene (20 mo; Misaki) Misaki bitan laurate 1.0 Tropoelastin (v
) 1 spring 2 1.0 purified water
A mouth freshener (spray type) was prepared by a conventional method from the remaining ingredients below.

(Weight%) Ethyl alcohol 40.0 Sankarin sodium 0.1 Sorbit
10.0 Fragrance 1
．． 0 Polyoxyethylene (60 mo+L) Cured Himazine-derived 0.7 Chlorhexidine hydrochloride 0.0
5 Tropoelastin (9) Book Q) 0.1 Purified water Remainder side 53 Chewing gum was prepared from the following ingredients by a conventional method.

(Weight%) Gumhose 25゜0 Calcium carbonate 2.0 Fragrance
1.0 Copper chlorophyll 0.05 Tropoelastin C citrate 0.1 Sorbit powder Balance

[Brief explanation of drawings]

Figure 1 shows the cD encoding chicken tropoelastin.
The base sequence of the NA fragment and the amino acid sequence assumed from it are shown. FIG. 2 is a flowchart showing the construction of an E. coli expression vector.

Claims (1)

[Claims] 1. A skin external preparation containing tropoelastin. 2. The above-mentioned tropoelastin is produced by Escherichia coli, Bacillus subtilis, or yeast transformed with an expression vector incorporating a cDNA for a messenger RNA encoding tropoelastin derived from human, bovine, pig, sheep, chicken, or mouse. The skin external preparation according to claim 1, which is tropoelastin.