HK1181313A - Laminin-332 production accelerating composition - Google Patents

Description

Laminin 332 production promoting composition

Technical Field

The present invention relates to a laminin 332 production promoting composition including 1 or 2 or more compounds selected from the group consisting of D-alanine and D-hydroxyproline and derivatives and/or salts thereof, and a method of inhibiting and/or ameliorating skin damage including the step of administering the compounds.

Background

Laminins are known to be 3 peptide chain proteins consisting of an alpha peptide chain, a beta peptide chain, and a gamma peptide chain, and there are 5 alpha peptide chains, 3 beta peptide chains, and 3 gamma peptide chains in combination, and at least 15 isomers. In particular, laminin 332(α 3 β 3 γ 2, laminin 5 in the past nomenclature) is present in a large amount in the basement membrane that separates the epidermis and dermis, and is thought to play an important role in the structure and function of the skin (non-patent document 1). Knockout mice of laminin 332 exhibited separation between epidermis and dermis, and showed the same symptoms as epidermolysis bullosa, which is a junction of human genetic diseases forming blisters, and it was shown that laminin 332 plays a crucial role in the anchorage between epidermis and dermis (non-patent document 2). Further, a purified sample of laminin 332 was added to a skin equivalent model in which keratinocytes were cultured on a collagen gel embedded in human fibroblasts, thereby enhancing basement membrane formation (non-patent document 3). Plasmin, an active proteolytic enzyme produced in epidermal cells, cleaves the amino-and carboxyl-terminal peptides of the α 3 protein subunit of laminin 332, as well as the amino-terminal peptide of the β 3 protein subunit. The cleavage fragments include recognition sites for cell matrix adhesion molecules and binding sites for collagen type 7, respectively. Therefore, laminin 332 digested by fibrin has a reduced ability to fix to keratinocytes. Further, laminin 332 digested by fibrin has a reduced affinity for type 7 collagen. Therefore, it is considered that skin aging due to ultraviolet irradiation and other causes is related to the degradation of laminin 332 by plasmin and the function of the basement membrane thereof (non-patent document 4).

Thus, by promoting the production of laminin 332, the possibility of inhibiting and/or ameliorating skin aging due to ultraviolet irradiation and other causes can be created. With respect to factors that promote the production of laminin 332, HIF1 (non-patent document 5) and Smad4 (non-patent document 6) have recently been reported to induce transcription of genes of α 3 protein subunits. HIF1 is a transcriptional regulator that responds to environmental stimuli such as hypoxia and mechanical stimuli, and is also upregulated by inflammatory cytokines. Smad4 is a transcriptional regulator of signaling for TGF β. Since these factors are all proteins having a large molecular weight, the activity thereof is regulated by modification such as phosphorylation and association with other protein subunits. Therefore, it is impossible to administer these factors directly to the living body so that they reach epidermal cells in the skin tissue for promoting the production of laminin 332. Furthermore, any of the above factors is regulated by a wide range of factors, and is a factor that has a large influence on biological functions such as inflammatory reaction in addition to promoting the production of laminin 332, and therefore cannot be used safely on a daily basis.

Documents of the prior art

Non-patent document

Non-patent document 1: sugawara et al, Exp Dermatol.17(6), 473-80(2008)

Non-patent document 2: aberdam et al, nat. Genet.6, 299, (1994)

Non-patent document 3: amano, s., SOFW j., 134: 10(2008)

Non-patent document 4: amano, s., j.investig.dermotol.symp.proc.14: 2-7(2009)

Non-patent document 5: fitsialos, g, et al, j.cell sci., 121: 2992(2008)

Non-patent document 6: zboralski, d. et al, BMC Cancer, 8: 215(2008)

Disclosure of Invention

Problems to be solved by the invention

Therefore, there is a need to develop a composition that promotes the production of laminin 332, which composition can be used daily, is stable, and is safe.

Means for solving the problems

The present invention provides a composition for promoting laminin 332 production, which contains 1 or 2 or more compounds selected from the group consisting of D-alanine, D-hydroxyproline, and derivatives and/or salts thereof.

The laminin 332 production promoting composition of the present invention is used for inhibiting and/or improving the skin condition according to circumstances.

In the laminin 332 production promoting composition of the present invention, the skin conditions include, but are not limited to, photoaging, wrinkles, rough skin, fine wrinkles and dryness.

The laminin 332 production promoting composition of the present invention is used as a pharmaceutical product according to circumstances.

The laminin 332 production promoting composition of the present invention is used as a skin external agent according to circumstances.

The laminin 332 production promoting composition of the present invention is used as a food according to circumstances.

The present invention provides a method for inhibiting and/or improving skin conditions, which comprises the step of administering a laminin 332 production promoting composition comprising 1 or 2 or more compounds selected from the group consisting of D-alanine and D-hydroxyproline and derivatives and/or salts thereof.

Skin conditions inhibited and/or improved by the methods of the present invention include, but are not limited to, photoaging, wrinkles, rough skin, fine wrinkles, and dryness.

In the method of the present invention, the laminin 332 production-promoting composition is a pharmaceutical product in some cases.

In the method of the present invention, the laminin 332 production promoting composition is an external skin preparation according to circumstances.

In the method of the present invention, the laminin 332 production-promoting composition is a food composition according to circumstances.

In the present specification, "salts" of D-alanine and D-hydroxyproline refer to: any salt including metal salts, amine salts and the like without impairing the effect of D-alanine and D-hydroxyproline on promoting the production of laminin 332. The aforementioned metal salt may include alkali metal salts, alkaline earth metal salts, and the like. The aforementioned amine salt may include triethylamine salt, benzylamine salt and the like.

In the present specification, "derivatives" of D-alanine and D-hydroxyproline mean: a compound in which D-alanine and D-hydroxyproline molecules are covalently bonded to an arbitrary atomic group at an amino group, a carboxyl group, or a side chain, without impairing the effect of D-alanine and D-hydroxyproline on promoting the production of laminin 332. The optional group includes, but is not limited to, a protecting group such as N-phenylacetyl, 4' -Dimethoxytrityl (DMT), a biopolymer such as a protein, a peptide, a sugar, a lipid, a nucleic acid, etc., a synthetic polymer such as polystyrene, polyethylene, a polyvinyl compound, a polyester, etc., and a functional group such as an ester group. The ester group may include an aliphatic ester such as a methyl ester, an ethyl ester, or an aromatic ester.

Among amino acids, there are the L-form and the D-form which are optical isomers, natural proteins are proteins in which L-amino acids are bonded by peptide bonds, and only L-amino acids are used except cell walls of bacteria, and therefore, it is considered that only L-amino acids exist and only L-amino acids are used in mammals including humans. (Gentianzhong Chengyao et al, protein nuclease, 50: 453-460(2005), Lehninger's new biochemistry [ supra ] pp 2-147(1993) Guanchuan bookstore, Huppe. Biochemical original paperwork pp 22 21-30(1991) Wanshan). Therefore, conventionally, the L-amino acid has been used mainly as an amino acid, and the L-amino acid has been used industrially.

As examples of the exceptional use of D-amino acids, there are cases where they are used as a raw material for producing antibiotics in bacteria; and an example of a dietary supplement in which D-amino acid is directly used in the form of DL-amino acid mixture in order to save the cost of separating and extracting only L-amino acid from a mixture of L-amino acid and D-amino acid obtained in the same amount as when chemically synthesizing amino acid. However, there has never been an example of using only D-amino acids as a substance having physiological activity in the industry.

D-serine and D-aspartic acid have been studied more rapidly because of the high proportion of D-bodies. D-serine is locally present in the brain, hippocampus, and is known to be a regulator of NMDA receptors in the brain. It was confirmed that D-aspartic acid is localized in the testis and pineal gland and is involved in controlling the secretion of hormones (Japanese patent laid-open No. 2005-3558). The physiological role of D-alanine and D-hydroxyproline in the skin is not clear.

As shown in the following examples, the effect of D-alanine and D-hydroxyproline on increasing the amount of laminin 332 produced has not been known. Therefore, the laminin 332 production promoting composition of the present invention including D-alanine and/or D-hydroxyproline is a novel invention.

In recent years, it has been reported that, when ddY mice were allowed to freely take 10mM aqueous solution of D-amino acid for two weeks and then the concentration of D-amino acid in each organ was measured, the results were as follows: in the pineal body, the concentration of D-amino acid is 3 to 1000pmol relative to 1 pineal body as a secretion organ; in brain tissue, the D Amino acid concentration is 2-500nmol relative to 1 gram wet weight (Morikawa, A. et al, Amino Acids, 32: 13-20 (2007)). Based on this, the lower limit of the daily intake of D-alanine and D-hydroxyproline contained in the composition of the present invention described below was calculated.

As shown in the following examples, D-alanine of the present invention has an effect of promoting the production of laminin 332 in cultured human epidermal keratinocytes at a concentration of 0.1 to 1. mu.M. Therefore, the amount of D-alanine contained in the dermatological disease remedy, the external preparation for skin, and the food composition of the present invention may be any amount as long as the D-alanine in the concentration range satisfies the condition that D-alanine can reach fibroblasts in living skin tissues. When the composition of the present invention is an external preparation, the content of D-alanine in the total amount of the composition of the present invention may be 0.000015 wt% to 50 wt% or a maximum weight concentration range that can be incorporated. That is, the content of D-alanine in the composition for external use is preferably 0.00003 to 30% by weight, and most preferably 0.0003 to 3% by weight. When the composition of the present invention is an oral preparation, the content of D-alanine may be in the range of 0.00001 to 100% by weight. The content of D-alanine in the composition of the present invention is preferably 0.00002 to 80% by weight, and most preferably 0.0002 to 60% by weight when the composition is an oral preparation. The lower limit of the daily intake of D-alanine contained in the composition of the present invention may be 0.01ng, preferably 0.1ng, more preferably 1ng, per 1kg body weight.

As shown in the following examples, D-hydroxyproline of the present invention has an effect of promoting the production of laminin 332 in human epidermal keratinocytes cultured at a concentration of 0.1 to 1. mu.M. Therefore, the amount of D-hydroxyproline contained in the dermatological disease-ameliorating agent, the external preparation for skin and the food composition of the present invention may be any amount as long as the D-hydroxyproline satisfies the condition that the D-hydroxyproline in the concentration range can reach fibroblasts of living skin tissues. When the composition of the present invention is an external preparation, the content of D-hydroxyproline may be 0.000015 to 50% by weight or may be in the maximum weight concentration range in the total amount of the composition of the present invention. That is, the content of D-hydroxyproline in the composition for external use is preferably 0.00003 to 30% by weight, and most preferably 0.0003 to 3% by weight. When the composition of the present invention is an oral preparation, the content of D-hydroxyproline may be in the range of 0.00001 to 100% by weight. The content of D-hydroxyproline in the composition of the present invention is preferably 0.00002 to 80% by weight, and most preferably 0.0002 to 60% by weight when the composition is an oral preparation. The lower limit of the daily intake amount of D-hydroxyproline contained in the composition of the present invention may be 0.01ng, preferably 0.1ng, more preferably 1ng, per 1kg body weight.

The composition of the present invention may contain 1 or 2 or more pharmaceutically acceptable additives without impairing the effect of promoting the production of laminin 332 by D-alanine and D-hydroxyproline, in addition to D-alanine and D-hydroxyproline, and salts of D-alanine and D-hydroxyproline, and/or derivatives capable of releasing D-alanine in vivo by a drug metabolizing enzyme or the like. The additives include, but are not limited to, diluents and bulking agents, binders and binders, lubricants, glidants, plasticizers, disintegrants, carrier solvents, buffers, coloring materials, flavors, sweeteners, preservatives and stabilizers, adsorbents, and other pharmaceutical additives known to those skilled in the art.

The composition of the present invention may be prepared by using only D-alanine and D-hydroxyproline, salts of D-alanine and D-hydroxyproline, and/or derivatives capable of releasing D-alanine and D-hydroxyproline in vivo by a drug metabolizing enzyme or the like as active ingredients, but other ingredients used in cosmetics, external skin preparations such as pharmaceuticals including quasi drugs, and the like may be appropriately blended as necessary within a range not impairing the effects of the present invention. Examples of the other components (optional components) include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, humectants, perfumes, various medicinal components, preservatives, pH regulators, and neutralizers.

The dosage form of the composition for promoting the production of laminin 332 (hereinafter referred to as "skin condition improving agent") used for the purpose of inhibiting and/or improving the skin condition of the present invention may be any dosage form used in conventional quasi-drug compositions and pharmaceutical compositions, and includes, for example, external preparations such as ointments, creams, lotions, masks, gels, and patches, oral preparations such as powders, granules, soft capsules, and tablets, and nasal preparations such as nasal spray, and injections.

The dosage form of the external preparation for skin of the present invention may be any one of conventional external preparations for skin, and includes, for example, ointment, cream, milky lotion, face toilet, pack, gel, patch and the like.

The food composition of the present invention may contain seasonings, coloring materials, preservatives, and other ingredients acceptable for food, in addition to D-alanine and D-hydroxyproline, salts of D-alanine and D-hydroxyproline, and/or derivatives capable of releasing D-alanine and D-hydroxyproline in vivo by a drug metabolizing enzyme or the like, without impairing the effect of D-alanine and D-hydroxyproline on promoting the production of laminin 332.

The food composition of the present invention may be used in conventional food compositions such as candies, cookies, miso, French salad dressing, mayonnaise, French bread, soy sauce, yogurt, powdery food spread on rice, seasoning of seasoning natto, and moromi black vinegar, but is not limited to the above examples.

Drawings

FIG. 1 is a graph showing the effect of D-alanine on KC cells.

FIG. 2 is a graph showing the effect of D-alanine on laminin 332 production in KC cells.

FIG. 3 is a graph showing the effect of D-alanine on HaCaT cells.

FIG. 4 is a graph showing the effect of D-hydroxyproline on HaCaT cells.

FIG. 5 is a graph showing the effect of D-alanine on laminin 332 production in HaCaT cells.

FIG. 6 is a graph showing the effect of D-hydroxyproline on laminin 332 production in HaCaT cells.

FIG. 7 is a graph showing the effect of various concentrations of D-alanine on laminin 332 production in HaCaT cells.

FIG. 8 is a graph showing the effect of various concentrations of D-hydroxyproline on laminin 332 production in HaCaT cells.

FIG. 9 is a graph showing the effect of various concentrations of D-aspartic acid on laminin 332 production in HaCaT cells.

FIG. 10 is a graph showing the effect of various concentrations of D-asparagine on laminin 332 production in HaCaT cells.

FIG. 11 is a graph showing the effect of various concentrations of D-proline on laminin 332 production in HaCaT cells.

FIG. 12 is a graph showing the effect of various concentrations of D-serine on laminin 332 production in HaCaT cells.

Detailed Description

The following examples of the present invention are given for illustrative purposes only and do not limit the scope of the present invention. The scope of the present invention is defined only by the description of the claims.

All documents referred to in this specification are incorporated in their entirety by reference.

Example 1

1. Promotion of laminin 332 production by addition of alanine and hydroxyproline

1-1. materials and methods

(1) Cells

Cells HaCaT cells derived from cells of human epidermis (H.Hans et al, Experimental Cell Research 239: 399(1998)) and KC cells derived from human keratinocytes (Sanguan pure pharmaceutical Co., Ltd., manufacturer: LONZA Walkersville Inc.) were used. The cells were 4X 10 per well⁴The cells were inoculated in a 24-well cell culture plate, and used in a medium (hereinafter referred to as "normal medium") containing 0.1% BSA in a cell culture medium (D-MEM (1g/L glucose) or Wako pure chemical industries, and at 37 ℃ with 5% CO₂And culturing under saturated steam condition for 24 hr.

(2) Adding alanine and hydroxyproline

Thereafter, the cells were transferred to the medium to which 1. mu.M of L-or D-alanine, 0.5. mu.M of each of L-and D-alanine, 1. mu.M of L-or D-hydroxyproline, and 0.5. mu.M of each of L-and D-hydroxyproline were added to the common medium, and cultured for 24 hours. The normal medium without addition of alanine and hydroxyproline served as a negative control. The D-hydroxyproline used in this example was cis-4-D-hydroxyproline.

(3) Quantification of laminin 332 production

After completion of the culture, the medium was withdrawn, centrifuged at 3000rpm for 5 minutes, and the supernatant was assayed for the concentration of laminin 332 according to the ELISA method (Amano, S. et al, J.Immunol. methods, 224: 161 (1999)). The ELISA method was carried out by a double antibody sandwich ELISA method using BM165 monoclonal to the α 3 peptide chain of laminin 5 and a biotin conjugate of 6F12 monoclonal to the β 3 peptide chain of laminin 5, and was detected by horseradish peroxidase-labeled avidin D (Vector Labs, Inc., trade name A-2004). PBS was used as control.

(4) Quantification of viable cell number

After the medium was recovered, the cells were washed with PBS, and alamarBlue (trademark, Biosource International) was added so that the final concentration reached 10%. After 2 hours, the fluorescence intensity of alamarBlue liquid was measured at an excitation wavelength of 544nm and a fluorescence wavelength of 590nm according to Ahmed S.A. et al, (J.Immunol.method.170, 211-224(1994)), and the manufacturer's instructions.

1-2. results

(1) Quantification of KC viable cell number

FIG. 1 shows the results of experiments investigating the effect of alanine addition on the proliferation of KC cells. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 3 times under the same condition.

In KC cells, the relative value of the fluorescence intensity of alamarBlue (trademark) of the negative control (the same applies hereinafter) was 50. The fluorescence intensities of KC cells cultured in the medium containing 1. mu.M L-alanine, the medium containing 1. mu.M D-alanine, and the medium containing 0.5. mu.M L-and D-alanine were 40, 45, and 50, respectively. In KC cells, the fluorescence intensity of alamarBlue (trade mark) of cells cultured in alanine-supplemented medium was not significantly different in Tukey-Kramer assay compared to the negative control. Thus, it was shown that L-and D-alanine were not cytotoxic to KC cells.

(2) Production of laminin 332 by KC cells

FIG. 2 shows the results of an experiment to investigate the effect of alanine addition on laminin 332 production in KC cells. The ordinate of the graph in fig. 2 shows the quotient of the fluorescence intensity of alamarBlue (trademark) proportional to the number of cells in each well divided by the absorbance of ELISA measurement proportional to the concentration of laminin 332 in the culture supernatant of each well (hereinafter referred to as "relative value of laminin 332 concentration per cell number"). The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 3 times under the same condition. In addition, in Tukey-Kramer test, asterisks (. sup.) -indicate that p is less than 1%.

Relative values of laminin 332 concentration per cell count, negative control was 0.35. The relative values of the laminin 332 concentration per cell number of KC cells cultured in the medium supplemented with 1. mu.M L-alanine, the medium supplemented with 1. mu.M D-alanine, and the medium supplemented with 0.5. mu.M L-and D-alanine, respectively, were 0.40, 0.50, and 0.45. Comparison between the addition of 1. mu.M D-alanine and the negative control resulted in a significant difference of less than 1% p in the Tukey-Kramer test. Thus, it was demonstrated that the addition of D-alanine can promote the production of laminin 332 in KC cells.

(3) Quantification of HaCaT viable cell number

Fig. 3 and 4 show experimental results investigating the effect of alanine and hydroxyproline addition on the proliferation of HaCaT cells. Hereinafter, D-hydroxyproline (D-Hyp) means cis-4-D-hydroxyproline. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 3 times under the same condition.

In HaCaT cells, the fluorescence intensity of alamarBlue (trademark) as a negative control was 300. All of the fluorescence intensities of HaCaT cells cultured in the medium containing 1. mu.M L-alanine, the medium containing 1. mu.M D-alanine, and the medium containing 0.5. mu.M each of L-and D-alanine were 250 (FIG. 3). The fluorescence intensity of HaCaT cells cultured in the medium containing 1. mu.M L-hydroxyproline, the medium containing 1. mu.M D-hydroxyproline, and the medium containing 0.5. mu.M each of L-and D-hydroxyproline was 280 (FIG. 4). In HaCaT cells, the fluorescence intensity of alamarBlue (trademark) of cells cultured in a medium supplemented with alanine and D-hydroxyproline compared to the negative control was also not significantly different in Tukey-Kramer assay. Thus, it was shown that L-and D-alanine and L-and D-hydroxyproline are also not cytotoxic to HaCaT cells.

(4) Production of laminin 332 by alanine-added HaCaT cells

FIG. 5 shows the results of experiments investigating the effect of alanine addition on laminin 332 production in HaCaT cells. The vertical axis of the graph of fig. 5 shows the relative value of the laminin 332 concentration per unit cell number in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 3 times under the same condition. In addition, in the Scheffe's F-test, asterisks (. + -.) indicate that p is less than 1%. Asterisks indicate p is less than 5%.

Relative values of laminin 332 concentration per cell count, negative control was 0.04. The relative values of the laminin 332 concentration per cell number of HaCaT cells cultured in the medium supplemented with 1. mu.M L-alanine, the medium supplemented with 1. mu.M D-alanine, and the medium supplemented with 0.5. mu.M each of L-and D-alanine were 0.07, 0.11, and 0.10, respectively. P was less than 1% in the Scheffe's F-test between the addition of 1. mu.M D-alanine and the negative control, p was less than 5% in the Scheffe's F-test between the addition of 0.5. mu.M D-and L-alanine each in combination with the negative control, and p was less than 5% in the Scheffe's F-test between 1. mu.M D-alanine and 1. mu.M L-alanine, all with significant differences. Thus, it was demonstrated that the addition of D-alanine also promotes the production of laminin 332 in HaCaT cells, as in KC cells.

(5) Production of laminin 332 by HaCaT cells with hydroxyproline addition

FIG. 6 shows the results of experiments investigating the effect of hydroxyproline addition on laminin 332 production in HaCaT cells. The vertical axis of the graph of fig. 6 shows the relative value of the laminin 332 concentration per unit cell number in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 3 times under the same condition.

Relative values of laminin 332 concentration per cell count, negative control was 0.04. The relative values of the laminin 332 concentration per cell number of HaCaT cells cultured in the medium supplemented with 1. mu.M L-hydroxyproline, the medium supplemented with 1. mu.M D-hydroxyproline, and the media supplemented with 0.5. mu.M each of L-and D-hydroxyproline were 0.05, 0.065, and 0.06, respectively.

Example 2

2. Comparison of amounts of laminin 332 produced by addition of various amino acids

2-1. materials and methods

HaCaT cells were used as the cells, and they were cultured in the same manner as in example 1. Then, the cells were transferred to a medium supplemented with 10nM, 100nM and 1000nM each of the amino acids L-or D-alanine, L-or D-hydroxyproline, L-or D-aspartic acid, L-or D-asparagine, L-or D-proline, L-or D-serine in the normal medium and cultured for 24 hours. The normal medium without the addition of the amino acid was used as a negative control. The D-hydroxyproline used in this example was cis-4-D-hydroxyproline. The amount of laminin 332 produced was measured in the same manner as in example 1. In the following experiments, since cytotoxicity due to addition of amino acids was not observed in all concentrations, the amounts of laminin 332 produced by cells were compared under each experimental condition.

2-2. results

(1) Adding alanine

Fig. 7 shows the results of experiments investigating the effect of adding various concentrations of alanine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 7 is shown as the concentration of laminin 332 (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.7 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in media supplemented with 10nM, 100nM, and 1000nM L-alanine, and D-alanine, were 1.7ng/mL and 1.7ng/mL, 2.3ng/mL and 3.4ng/mL, and 2.8ng/mL and 4.8ng/mL, respectively. From the above results, it was shown that D-alanine promoted the production of laminin 332 in concentrations above 100 ng/mL.

(2) Addition of hydroxyproline

Fig. 8 shows the results of experiments investigating the effect of various concentrations of hydroxyproline addition on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 8 is shown as the concentration of laminin 332 (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.5 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in the medium supplemented with 10nM, 100nM and 1000nM L-hydroxyproline and the medium supplemented with D-hydroxyproline were 1.5ng/mL and 1.5ng/mL, 2.3ng/mL and 3.3ng/mL, and 2.7ng/mL and 4.4ng/mL, respectively. From the above results, it was shown that D-hydroxyproline promotes the production of laminin 332 in a concentration of 100ng/mL or more.

(3) Adding aspartic acid

Fig. 9 shows the results of experiments investigating the effect of adding various concentrations of aspartic acid on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 9 shows the concentration of laminin 332 (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.7 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in the medium supplemented with 10nM, 100nM and 1000nM L-aspartic acid and the medium supplemented with D-aspartic acid were 1.8ng/mL and 1.9ng/mL, and 2.0ng/mL and 1.7ng/mL, respectively. From the above results, it was shown that L-and D-aspartic acid did not promote the production of laminin 332.

(4) Adding asparagine

Fig. 10 shows the results of experiments investigating the effect of adding various concentrations of asparagine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 10 shows the concentration of laminin 332 (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.5 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in media supplemented with 10nM, 100nM, and 1000nM L-asparagine and D-asparagine were 1.6ng/mL and 1.5ng/mL, 1.5ng/mL and 1.5ng/mL, and 1.6ng/mL and 1.5ng/mL, respectively. From the above results, it was shown that L-and D-asparagine did not promote the production of laminin 332.

(5) Addition of proline

Fig. 11 shows the results of experiments investigating the effect of adding various concentrations of proline on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 11 shows the concentration of laminin 332 (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.8 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in the medium supplemented with 10nM, 100nM and 1000nM L-proline and the medium supplemented with D-proline were 1.8ng/mL and 2.0ng/mL, 1.9ng/mL and 1.9ng/mL, and 1.9ng/mL, respectively. From the above results it was shown that L-and D-proline do not promote the production of laminin 332.

(6) Addition of serine

FIG. 12 shows the results of experiments investigating the effect of adding various concentrations of serine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 12 shows the laminin 332 concentration (ng/mL) in the culture supernatant of each well. The error bars for each experimental condition represent the standard deviation of the measured values of the experimental results repeated 4 times under the same condition.

The concentration of laminin 332 in the negative control was 1.7 ng/mL. The laminin 332 concentrations of HaCaT cells cultured in the medium supplemented with L-serine at 10nM, 100nM, and 1000nM, and the medium supplemented with D-serine were 1.8ng/mL and 1.8ng/mL, 1.9ng/mL and 1.8ng/mL, and 1.9ng/mL and 1.8ng/mL, respectively. From the above results, it was shown that L-and D-serine did not promote the production of laminin 332.

Conclusion

From the experimental results of examples 1 and 2, it was confirmed that D-alanine and D-hydroxyproline have the effect of promoting the production of laminin 332, and aspartic acid, asparagine, proline and serine do not have the effect of promoting the production of laminin 332. Thus, it is suggested that D-alanine and D-hydroxyproline can inhibit and/or improve the skin condition by promoting the production of laminin 332, which has an important role in the structure and function of basement membrane.

Example 3

Examples of formulations of the emulsion preparation, patch, tablet, soft capsule, granule, beverage, candy, biscuit, miso, french salad dressing, mayonnaise, french bread, soy sauce, yogurt, powdery food spread on rice, dressing of seasoning natto, fermented black vinegar, cream, body cream, gel, peel-off pack, impregnated pack, emulsion, lotion, and aerosol according to the present invention are shown below. These examples are given for the purpose of illustration and are not intended to limit the scope of the present invention.

COMPARATIVE EXAMPLE 1 (emulsion preparation)

COMPARATIVE EXAMPLE 2 (Patch)

COMPARATIVE EXAMPLE 3 (TABLET)

COMPARATIVE EXAMPLE 4 (TABLET)

COMPARATIVE EXAMPLE 5 (Soft Capsule)

COMPARATIVE EXAMPLE 6 (Soft Capsule)

COMPARATIVE EXAMPLE 7 (PARTICLES)

COMPARATIVE EXAMPLE 8 (DRINKS)

COMPARATIVE EXAMPLE 9 (candy)

COMPARATIVE EXAMPLE 10 (biscuit)

Preparation of blending example 10 (biscuit)

Slowly adding fine granulated sugar while stirring butter, adding egg and spice, and D-alanine or D-hydroxyproline, and stirring. After mixing thoroughly, add the shaken whole low gluten flour, stir at low speed, store in a refrigerator in blocks. Then, the mixture was molded and baked at 170 ℃ for 15 minutes to make a biscuit.

COMPATIVE EXAMPLE 11 (miso)

Preparation of blend example 11 (miso)

The rice koji and the salt are mixed well. Soaking cleaned semen glycines in 3 times of water overnight, removing water, adding new water, cooking, and sieving. The boiled broth (raw juice) was collected and dissolved to a concentration of 10% w/v D-alanine or D-hydroxyproline. Immediately mashing the cooked beans, adding rice koji mixed with salt, and adding the raw juice dissolved with D-alanine or D-hydroxyproline while uniformly mixing to have the hardness similar to that of clay. The kneaded dough is tightly packed into a barrel without a gap until each corner is filled up, and after the surface is flattened, the dough is covered with a wrapper and sealed. After 3 months, the container was replaced, the surface was leveled and covered with a wrap. In addition, instead of adding D-alanine or D-hydroxyproline to the raw juice, rice koji which produces D-alanine or D-hydroxyproline in a large amount may be used. The koji can be obtained by quantifying D-alanine or D-hydroxyproline by the method described in Japanese patent application laid-open No. 2008-185558. In addition, D-alanine, D-hydroxyproline or salts thereof may be added to commercially available miso.

COMPARATIVE EXAMPLE 12 (French salad dressing)

Preparation of blend example 12 (French salad dressing)

Adding sodium chloride and D-alanine or D-hydroxyproline into vinegar, stirring thoroughly and dissolving. Salad oil was added, stirred well and pepper was added.

Preparation example 13 (mayonnaise)

Preparation method of blending example 13 (mayonnaise)

Adding vinegar, sodium chloride, pepper, and D-alanine or D-hydroxyproline into egg yolk (at room temperature), and stirring with a whisk. Then, while adding salad oil little by little, stirring was continued until an emulsion was prepared. Finally adding granulated sugar and stirring.

COMPATIVE EXAMPLE 14 (French bread)

Preparation of formulation example 14 (French bread)

Add 1g granulated sugar and dry yeast to warm water for pre-fermentation. Placing strong flour, weak flour, sodium chloride, 5g granulated sugar and D-alanine or D-hydroxyproline into a bowl, and adding yeast fermented before. After sufficient kneading, the pellets were formed, and the first fermentation in the main fermentation was conducted at 30 ℃. The dough was kneaded again, later shaped to an appropriate shape, and post-fermented using an electronic fermenter. After scoring the dough surface, it was baked in an oven at 220 ℃ for 30 minutes.

COMPARATIVE EXAMPLE 15 (Soy sauce)

Preparation of formulation example 15 (Soy sauce)

D-alanine or D-hydroxyproline or a salt thereof is added to commercially available soy sauce and sufficiently stirred. In addition, instead of adding D-alanine or D-hydroxyproline or a salt thereof, rice koji which produces a large amount of D-alanine or D-hydroxyproline may be used to brew soy sauce. The koji can be obtained by quantifying D-alanine or D-hydroxyproline by the method described in Japanese patent application laid-open No. 2008-185558.

COMPATIVE EXAMPLE 16 (yogurt)

Preparation method of formula 16 (yogurt)

Fermenting at 40-45 deg.C. Other commercially available strains may be used, or D-alanine, D-hydroxyproline, or salts thereof may be added to commercially available yogurt. In addition, instead of adding D-alanine or D-hydroxyproline or salts thereof, bacteria that produce large amounts of D-alanine or D-hydroxyproline may be used. In order to obtain such bacteria, D-alanine or D-hydroxyproline can be quantitatively determined and selected by the method described in Japanese patent application laid-open No. 2008-185558.

COMPARATIVE EXAMPLE 17 (powdery food sprinkled on rice)

COMPARATIVE EXAMPLE 18 (seasoning Natto sauce)

COMPATIVE EXAMPLE 28 (Natto)

Preparation of blend example 19 (Natto)

D-alanine or D-hydroxyproline or its salt is added to commercially available natto, followed by well stirring. Further, instead of adding D-alanine or D-hydroxyproline or salts thereof, natto may be produced using a bacterium that produces a large amount of D-alanine or D-hydroxyproline. In order to obtain the above-mentioned bacteria, D-alanine or D-hydroxyproline can be quantitatively determined and selected by the method described in Japanese patent application laid-open No. 2008-185558.

Blending example 20 (fermented glutinous black vinegar)

Preparation of blending example 20 (fermented glutinous Black Vinegar)

D-alanine or D-hydroxyproline or salts thereof are added to commercially available moromi black vinegar and then stirred well. Instead of adding D-alanine or D-hydroxyproline or salts thereof, vinegar, black vinegar, and mash may be prepared using a bacterium that produces a large amount of D-alanine or D-hydroxyproline. In order to obtain such bacteria, D-alanine or D-hydroxyproline can be quantitatively determined and selected by the method described in Japanese patent application laid-open No. 2008-185558.

COMPARATIVE EXAMPLE 21 (cream)

COMPARATIVE EXAMPLE 22 (cream for body)

COMPARATIVE EXAMPLE 23 (gelata)

Blending example 24 (peel-off type mask)

Blend example 25 (impregnated type mask)

COMPARATIVE EXAMPLE 26 (emulsion)

COMPARATIVE EXAMPLE 27 (emulsion)

COMPARATIVE EXAMPLE 28 (astringent)

COMPARATIVE EXAMPLE 29 (astringent)

Blend example 30 (stock solution of urea aerosol for external use)

Blend example 31 (Aerosol urea spray)

Blending example 31 (Aerosol Urea spray) filling method

An aerosol was prepared by filling a stock solution of an aerosol urea external preparation and dimethyl ether in a pressure-resistant aerosol aluminum can whose inner surface was treated by coating with teflon (registered trademark).

Claims (6)

1. A composition for promoting the production of laminin 332, characterized by containing 1 or 2 or more compounds selected from the group consisting of D-alanine and D-hydroxyproline, and derivatives and/or salts thereof.

2. Composition according to claim 1, characterized in that it is used for inhibiting and/or improving the condition of the skin.

3. The composition of claim 2, wherein the skin condition is selected from the group consisting of photoaging, wrinkles, rough skin, fine wrinkles and dryness.

4. Composition according to any one of claims 1 to 3, for use as a pharmaceutical product.

5. Composition according to any one of claims 1 to 3, characterized by being used as a skin external agent.

6. Composition according to any one of claims 1 to 3, characterized by being used as a food product.