JP2007045747A - Agent for regulating production of skin aging-associated component, and external agent for preventing skin aging formulated with the agent for regulating production of skin aging-associated component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production-regulating agent for regulating the production of a component such as a protein and a pigment associated with the skin aging by improving ability for synthesizing a protein constituting an extracellular matrix such as collagen and laminin, or inhibiting melanin-synthetic ability by acting on a skin cell; and an external agent for preventing skin aging formulated with such the production-regulating agent and having wrinkle-preventing effects and effects for inhibiting pigmentation or the like by formulating such the production-regulating agent, to promote the synthesis of the protein constituting the extracellular matrix such as the collagen and the laminin, to inhibit the production of melanin, thereby to prevent the formation of the wrinkle, and liver spot/freckle on the skin caused by aging, or to reduce the formed wrinkle, and the liver spot/freckle to achieve the smooth, fresh and young skin by allowing a molecular weight-reduced component to act on the skin cell directly. <P>SOLUTION: The agent for regulating the production of the skin aging-associated component comprises a hydrolyzed product of blue-green alga. The agent for regulating the production of the component associated with the skin aging, and having activities for promoting the synthesis of the extracellular matrix-constituting protein, and inhibiting the production of the melanin comprises the hydrolyzed product of the blue-green alga. The blue-green alga in the agent for regulating the production of a component associated with the skin aging is preferably the one of the genus Arthrospira. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention improves the ability to synthesize proteins constituting the extracellular matrix such as collagen and laminin by acting on skin cells, or suppresses melanin synthesis ability. A production regulator that regulates the production of ingredients such as pigments;
It is related with the external preparation for skin aging prevention which mix | blends such a production regulator and has the inhibitory effect of the wrinkle suppression effect, pigmentation, etc. which were excellent.

  In today's aging society, awareness of skin aging symptoms such as wrinkles and blemishes that increase with aging has increased, and the trend of improving QOL has been added to prevent and improve skin aging symptoms such as wrinkles and blemishes. The desire to do so is increasing. In order to meet such demands, research on aging has been actively conducted in addition to skin.

  The mechanism of wrinkles and spots, which are symptoms of skin aging, has not yet been fully elucidated, but in general for wrinkles, collagen, which makes up most of the extracellular matrix that makes up the dermis, is aging and long-term UV rays It is thought to be due to a decrease or degeneration due to exposure.

  According to Non-Patent Document 1 below, after collagen is produced as procollagen in fibroblasts existing in the dermis, a three-helical structure is formed, and after proline and lysine groups are hydroxylated, extracellular It is reported that the N-terminal and C-terminal propeptides of procollagen are cleaved after being secreted, and the respective helical structural chains are polymerized to form collagen fibers. The collagen fibers formed in this way greatly affect the flexibility and elasticity of the dermis, and the decrease or modification of collagen leads to a decrease in skin flexibility and elasticity, leading to the generation of wrinkles. It is.

Suzuki Masato, Development of anti-aging, whitening and moisturizing cosmetics, 2001, p. 135-137

  On the other hand, in recent years, the epidermal basement membrane present at the boundary between the epidermis and dermis of the skin has been damaged by external factors such as ultraviolet rays, oxidation, and drying, and the epidermal basement membrane structure is fragmented and multiplexed as described below. It is reported in Non-Patent Document 2 and the like. The epidermal basement membrane plays an important role in the differentiation and maintenance of the epidermal cells. Accumulation of this damage to the basement membrane structure causes structural abnormalities in the extracellular matrix of the dermis, leading to the generation of wrinkles and sagging .

Kaoru Amano, Journal of Cosmetic Engineers, Vol. 35, No. 1, 2001, p. 1-7

  As means for improving skin aging symptoms, collagen originating from pigs, cattle, fish and the like is blended in various cosmetics, focusing on the collagen content in skin and its function. However, these cosmetics only apply collagen to the skin surface, and since collagen is a polymer compound, it is difficult to be absorbed by the skin.

  In addition, laminin, which is a component of the extracellular matrix as well as collagen, has recently been reported to be a protein that is related to the maintenance of skin flexibility, elasticity and wrinkles, and promotes laminin synthesis. The following patent application 1 has been filed as a synthesis accelerator. However, with such cosmetics, it is difficult to improve skin problems such as wrinkles to the extent that they can satisfy skin problems due to permeability to the skin and stability problems.

JP 2003-137767 A

  On the other hand, it is considered that stains are biosynthesized by melanin pigments due to hormonal abnormalities and stimulation of ultraviolet rays contained in sunlight, and these are abnormally deposited in the skin. The melanin pigment is biosynthesized in melanin-producing granules (melanosomes) in melanocytes (melanocytes) existing in the basal layer of the epidermis in the skin, and the biosynthesized melanin diffuses to adjacent epidermis cells. The following can be considered as the melanin production reaction in the melanocytes. That is, tyrosine, which is an amino acid, becomes dopaquinone by the action of the enzyme tyrosinase, and the process in which the reaction proceeds enzymatically or non-enzymatically and finally black melanin is generated is the melanin pigment generating process. Therefore, it is important for the suppression of melanin production to suppress the action of tyrosinase, which is the first step of the melanin pigment production reaction.

  As a result, for the purpose of preventing and improving pigmentation, stains, freckles, etc. after sunburn, ascorbine has been used for external preparations for skin, such as emulsions, lotions, creams, gels, packs, cleaning agents, foundations, and ointments. Contains whitening ingredients such as acids and glutathione. For example, applications such as the following Patent Document 2 and Patent Document 3 have been filed for blending ascorbic acid, and applications such as the following Patent Document 4 have been filed for blending glutathione.

JP 2003-104864 A JP 2003-73252 A JP-A-5-301811

  However, in cosmetics containing these whitening ingredients, the effects of the whitening ingredients are not sufficient, or sufficient effects cannot be obtained due to deterioration in the preparation, and skin troubles such as spots can be satisfied. It is difficult to improve to the extent.

  The present invention solves the above-mentioned problems, and promotes the synthesis of proteins constituting the extracellular matrix such as collagen and laminin by directly acting on low-molecular components on skin cells, Or skin aging that suppresses the production of melanin, thereby preventing the generation of wrinkles, spots and freckles in the skin due to aging, or reducing the generated wrinkles, spots and freckles, realizing a smooth, fresh and youthful skin It is an object to provide an external preparation for prevention.

  As a result of intensive studies to solve the above problems, the present inventors have found that cyanobacterial hydrolyzate has an excellent effect of promoting the synthesis of proteins constituting extracellular matrix such as collagen and laminin and an effect of suppressing melanin production. As a result, the present invention has been completed.

  That is, the invention according to claim 1 relating to the production regulator of skin aging-related components contains a hydrolyzate of cyanobacteria. The invention according to claim 2 relating to the production regulator of a skin aging-related component having an action of promoting the synthesis of extracellular matrix-constituting protein and inhibiting the production of melanin is characterized by containing a hydrolyzate of cyanobacteria.

  Furthermore, the invention described in claim 3 is characterized in that in the production regulator of skin aging-related components according to claim 1 or 2, the cyanobacteria belong to the genus Orthospira. Further, the invention according to claim 4 relating to the external preparation for preventing skin aging is characterized by blending the production regulator of the skin aging-related component according to any one of claims 1 to 3.

According to the present invention, it has an excellent promoting effect on proteins constituting an extracellular matrix such as collagen and laminin, an excellent melanin production suppressing effect, and promotes the synthesis of extracellular matrix constituting proteins that are highly safe for the human body and It has become possible to provide a production regulator of a skin aging-related component having a melanin production-inhibiting action, and an external preparation for preventing skin aging containing the production regulator.

  The production regulator for skin aging-related components of the present invention contains a cyanobacterial hydrolyzate as described above. The term “containing” as used herein may mean that the production regulator of the skin aging-related component of the present invention comprises only a hydrolyzate of cyanobacteria, and the production regulator of the skin aging-related component includes cyanobacteria. It means that things other than the hydrolyzate may be contained.

  Examples of the cyanobacteria used for the production regulator of skin aging-related components of the present invention include, for example, the genus Arthrospira, Spirulina, Halosspirulina, and the like. Examples include the genus, the genus Nostock, and the genus Synocococcus.

  Among these cyanobacteria, algae of the order of uremo, more preferably Orthospira, Spirulina, and Halospirulina are used. All three algae are commonly referred to as “spirulina” and are widely used as health foods because of their extremely high protein content.

  Examples of orthospira cyanobacteria include Arthrospira PCC8005, Arthrospira maxima, Arthrospira fusiformis, Arthrospira FACHB438, Arthrospira FACHB439, Arthrospira Etc. can be used.

Examples of the cyanobacteria belonging to the genus Spirulina include Spirulina MSIS4,
Spirulina strain CCC, Spirulina subsala, etc. can be used. Furthermore, as the cyanobacteria belonging to the genus Halospirulina, Halosspirulina CCC, Halospilulina MPI, Halospilulina tapeticola, and the like can be used.

In the present invention, as described above, a hydrolyzate of cyanobacteria can be preferably used.
As a result of intensive studies by the present inventors, the algae inhabiting each lake of Ye Kharr Lake and Twin Taung Lake in Myanmar are particularly excellent in proteins that constitute an extracellular matrix such as collagen and laminin. It has been found that an excellent accelerating effect and an excellent melanin production suppressing effect are observed.

When the partial homology search of the base sequence of 16S rRNA gene was performed about these algae, the homology was as follows.
[Algae of Ye Kharr Lake]
Homology with Arthrospira PCC8005: 98.8%
Homology with Arthrospira maxima: 99.3%
Homology with Arthrospira fusiformis: 99.3%
Homology with Arthrospira plantensis: 98.8%
Arthrospira sp. Homology: 98.4%
Lyngbya sp. Homology: 94.2%
Anabena sp. Homology: 88.2%
Homology with Oscillatoria spongeliae: 89.7%
Homology with Spirulina strainCCC: 91.0%
Homology with Halospirulina tapeticola: 89.2%

[Algae of Lake Twin Taung]
Homology with Arthrospira PCC8005: 99.1%
Homology with Arthrospira maxima: 99.5%
Homology with Arthrospira fusiformis: 99.5%
Homology with Arthrospira plantensis: 99.1%
Arthrospira sp. Homology: 98.8%
Lyngbya sp. Homology: 94.7%
Anabena sp. Homology: 89.2%
Homology with Oscillatoria spongeliae: 90.0%
Homology with Spirulina strainCCC: 91.6%
Homospirillina tapeticola homology: 90.1%

As is clear from the above results, the algae of Lake Ye Kharr and the lake of Twin Taung are the genus of the genus Euremo, Ringvia, Nenjumo, Anabena, Osciltoria spongeriae, Compared with homologous algae of the genus Orthospira, compared with the homologues of the genus Spirulina and the genus Halopirulina, the algae of Lake Ye Kharr and Lake Twin Taung (Twintang) These algae can be recognized as cyanobacteria belonging to the genus Orthospira.

Incidentally, the homology between the algae of Lake Ye Kharr and the lake of Twin Taung was 99.0%. These two types of cyanobacteria are called Myanmar Spirulina. The partial homology of the base sequence of the 16S rRNA gene was determined by amplifying the genetic region of the 16S rRNA gene from the obtained Spirulina DNA using a 16S rRNA gene-specific primer by PCR, and then ABI PRISM 310 Genetic Analyzer (manufactured by PE Biosysytems). The base sequence was determined by a thermal sequencing method using a biological search software BLAST of NCBI (National Center for Biotechnology Information).
The base sequences of the primers used in the PCR method are as follows.
(16S rRNA Forward)
5'-AGAGTTTGATCCCTGCTCAG-3 '
(16S rRNA Reverse)
5'-AAAGGAGGTGATCCAGCC-3 '
As a DNA polymerase, AmpliTaq Gold DNA polymerase (PE Biosystems) was used.
Furthermore, the temperature conditions are as follows.
(1) 95 ° C. for 10 minutes (2) 95 ° C. for 1 minute (3) 47 ° C. to 56 ° C. for 1 minute (4) 72 ° C. for 1 minute (5) 72 ° C. for 10 minutes In addition, the denaturation of (2) to (4), Each cycle of annealing and polymerase extension was performed for 30 cycles. In the annealing of (3), optimum conditions were obtained in the temperature range of 47 ° C. to 56 ° C. and used as temperature conditions.

  The skin aging-related component production regulator of the present invention includes a cyanobacterial hydrolyzate having only an action of promoting the synthesis of extracellular matrix constituent proteins such as collagen and laminin, and a cyanobacterial having only an inhibitory action on the production of melanin. Any hydrolyzate is included. However, the orthospira cyanobacteria, especially the above-mentioned Myanmar spirulina, can be used particularly suitably because both the action of promoting the synthesis of extracellular matrix constituent proteins and the action of inhibiting the production of melanin are recognized. . In addition to the above-mentioned collagen and laminin, there are proteins such as elastin and proteoglycan as proteins constituting the extracellular matrix, and it is recognized that skin aging can be prevented by promoting the synthesis of these proteins. It is done.

  The cyanobacterial hydrolyzate of the present invention is composed of a polypeptide, an amino acid, an amine, and the like that are produced by hydrolysis of a protein that constitutes the algae. It is preferable that the hydrolyzate contains, for example, a peptide mixture having an average molecular weight of 100 to 20000.

  Hydrolysis is performed by, for example, allowing an acid, alkali, enzyme, or the like to act on a processed product such as cyanobacteria or a powder thereof. Examples of the acid include inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, and nitric acid, and organic acids such as p-toluenesulfonic acid (PTS) and methanesulfonic acid. As the acid, a water-soluble compound that generates an acid in the system after addition may be used. Although the usage-amount of an acid changes with the density | concentrations and kind of acid to be used, it is used at about 1 to 60 weight%, for example. The average molecular weight of the hydrolyzed product can be adjusted by appropriately selecting the amount, concentration, and type of acid used.

  Examples of the alkali include hydroxides and carbonates of alkali metals or alkaline earth metals such as ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, calcium carbonate, and magnesium carbonate. The alkali varies depending on the concentration and type of alkali to be used, but is used at about 1 to 60% by weight, for example. The average molecular weight of the hydrolyzed product can be adjusted by appropriately selecting the amount, concentration, and type of alkali used.

  In the hydrolysis with the acid and alkali, for example, an acid or alkali is added to a dispersion suspension of cyanobacteria or a processed product thereof in water, and the mixture is heated under an atmosphere of normal pressure, reduced pressure, or increased pressure. By stirring or refluxing. The processing temperature at the time of hydrolysis is, for example, 40 to 110 ° C, preferably 70 to 100 ° C, and particularly preferably 75 to 95 ° C. The treatment time is, for example, 2 to 12 hours, preferably about 5 to 10 hours. The pressure, temperature, and reaction time during hydrolysis are appropriately selected depending on the average molecular weight of the peptide mixture produced by hydrolysis. After the hydrolysis treatment with an acid or alkali, it is preferable to carry out a neutralization treatment promptly to obtain a hydrolyzate comprising a peptide mixture having a desired average molecular weight. Furthermore, treatments such as deammonia treatment by vacuum distillation operation, desalting treatment using an electrodialyzer, activated carbon treatment, filtration treatment and the like are performed as necessary. For example, after the hydrolysis treatment with alkali, it is preferable to perform deammonia treatment by vacuum distillation operation. This is because insufficient removal of ammonia causes subsequent discoloration.

  The enzyme used for hydrolysis is preferably a protease that hydrolyzes protein (alkaline protease or the like) or peptidase. For example, Rhizopus genus, Aspergillus genus, Mucor genus, Bacilus genus, Pseudomonas (Pseudomonas) genus, Streptococcus (Streptococcus) genus, Escherichia (Escherichia) genus-derived enzymes, trypsin, renin, pancreatin and other animal-derived enzymes, papain, bromelain, ficin and other plant-derived enzymes be able to. Preferably, enzymes derived from the genus Rhizopus, Aspergillus, and Bacilus are used, and these refined products and crude products are used alone or in combination. Hydrolysates (peptide mixtures) with different properties can be obtained depending on the enzyme used. The enzyme reaction conditions are not particularly limited, and are appropriately selected according to the type of enzyme used. The amount of the enzyme to be added is preferably such that the hydrolyzate has an average molecular weight within the above range. The active unit of the proteolytic enzyme is 10 to 50000 units, preferably 100 to 30000, based on 1 g of dry weight of cyanobacteria. It is appropriately selected from the unit range.

  The hydrolyzate thus obtained can be further subjected to conventional separation, concentration and purification means. Cyanobacteria usually contain abundant water-soluble pigments such as phycocyanin (blue pigment) and fat-soluble pigments such as chlorophyll and carotene. The cyanobacterial hydrolyzate used in the present invention can be subjected to a pigment removal treatment on the hydrolyzate obtained by the above method. As a method for removing the dye, separation and purification means such as centrifugation, filtration, solvent extraction, adsorption treatment with activated carbon, and the like can be used. The filtration treatment can be performed using, for example, filter paper, filter cloth, membrane filter, or the like.

  The solvent used for the solvent extraction is not particularly limited as long as the colored pigment can be dissolved, and for example, esters such as alcohol, ketone, ether, and ethyl acetate can be used. Of these, alcohols are preferable, and lower alcohols such as ethyl alcohol are preferably used from the viewpoint of cosmetic use. Solvent extraction is performed by adding the solvent and, for example, a method of standing for a predetermined time or a method of performing a heating reflux operation. The fat-soluble dye can also be removed by allowing it to stand after adding the extraction solvent. On the other hand, cyanobacteria contain a large amount of water-soluble pigments, and when these water-soluble pigments are intended to be removed, the pigments are not sufficiently removed by standing, and heating and refluxing operations may be performed. preferable.

  For the adsorption treatment, for example, a porous substance such as activated carbon can be used. Adsorption is performed, for example, by adding activated carbon and stirring for 10 minutes to 5 hours, preferably 30 minutes to 3 hours.

  These dye removing means are used alone or in combination. Algae generally have a high pigment content, so that it is usually necessary to combine two or more of the above means or repeat the removal operation in order to sufficiently remove the pigment. The fat-soluble dye can be removed at a relatively low temperature, but when removing the water-soluble dye, it is preferable to perform an operation such as reflux under heating.

  The treatment temperature in the dye removal step can be appropriately selected depending on the treatment means, and is, for example, 0 to 120 ° C., preferably 0 to 100 ° C., more preferably 0 to 85 ° C., particularly about 4 to 75 ° C. The removal treatment of the water-soluble pigment is often performed at a low temperature, but it can also be performed at room temperature to 85 ° C. within a range in which contamination with various bacteria can be prevented. The pigment removal may be performed in any atmosphere under reduced pressure, increased pressure, or normal pressure. By removing the pigment, a pale yellow processing solution is obtained.

  By removing the pigment by the above method, it is advantageous in that the odor can be removed at the same time. The pigment removal step may be provided in any of the preceding step and the subsequent step of the hydrolysis treatment, but it is preferable to provide the hydrolysis treatment step after the pigment removal step.

  The blending amount of the spirulina hydrolyzate in the skin antiaging agent of the present invention is usually preferably 0.0001 to 50% by weight as a dry solid content. If the amount is less than 0.0001% by weight, the effect of the present invention may not be sufficiently obtained. On the other hand, if the amount exceeds 50% by weight, an improvement in the effect commensurate with the increase is not recognized. In this respect, 0.001 to 10% by weight is particularly preferable.

  The anti-aging skin external preparation of the present invention requires various optional ingredients that are generally used in cosmetics or used in skin external preparations such as quasi-drugs and pharmaceuticals as long as the effects of the present invention are not impaired. It can mix | blend suitably according to it. Examples of such optional components include purified water, ethanol, oily components, humectants, thickeners, preservatives, emulsifiers, medicinal components, powders, ultraviolet absorbers, dyes, fragrances, and emulsion stabilizers. Can do.

  The form of the anti-aging skin external preparation of the present invention is not limited as long as it is applicable to the skin, such as liquid, emulsion, ointment, cream, gel, aerosol, soap, etc. A skin external preparation for anti-aging of desired form can be prepared by blending agent components and the like. Moreover, the anti-aging skin external preparation of this invention is applicable in various fields, such as a pharmaceutical, a quasi-drug, or cosmetics.

  The anti-aging skin external preparation of the present invention can be used for the prevention and improvement of wrinkles, spots and freckles due to aging. The aging symptoms shown here are exemplary, and the application of the skin aging-related component production regulator of the present invention and the anti-aging skin external preparation containing the production regulator is limited to these aging symptoms. It is not a thing.

  Examples of the present invention will be described below.

Example 1
Myanmar Spirulina (Species of the cyanobacterium Oremospira spp. Inhabiting Lake Ye Kharr, Myanmar) dried at 60 ° C, 5 parts by volume of pure water were added to 1 part by volume of powder and 2 days at 4 ° C. After being allowed to stand, centrifugation and filtration were repeated and dried at 60 ° C. to obtain Spirulina powder from which the blue pigment was removed. 5 parts by volume of ethyl alcohol was added to 1 part by volume of the obtained dry powder, transferred to a four-necked flask, and heated to reflux for 4 hours. After taking out from the system, filtration was repeated until no color was seen in the filtrate, and a crude Spirulina extract was obtained. Furthermore, the dried powder of the crude extract of Spirulina was obtained by drying at 60 degreeC.

(Example 2)
1 part by volume of the dry powder obtained in Example 1 and 5 parts by volume of a 10% strength by weight aqueous sodium hydroxide solution were added and heated to reflux at 85 ° C. for 6 hours. The mixture was moved out of the system and subjected to deammonia treatment by vacuum distillation under reduced pressure, and 10% by weight hydrochloric acid was immediately added to neutralize the treatment solution (pH 6 to 7.5). By repeating the filtration operation, a transparent liquid was collected, and then desalted using an electrodialyzer (4 μS or less). Furthermore, the activated carbon treatment was performed by the following method. That is, 1% by weight of activated carbon was added, stirred for 2 hours, and allowed to stand, followed by filtration with filter paper, followed by filtration with a φ0.2 μm membrane filter to obtain a spirulina hydrolyzate.

Example 3
In a four-necked flask, 1 part by volume of the dry powder obtained in Example 1 and 5 parts by volume of 4N hydrochloric acid were placed and heated to reflux at 85 ° C. for 8 hours. It moved out of the system and neutralized the processing liquid by adding 4N sodium hydroxide aqueous solution (pH 6-7.5). By repeating the filtration operation, a transparent liquid was collected, and then desalted using an electrodialyzer (4 μS or less). Furthermore, the activated carbon treatment was performed by the following method. That is, 1% by weight of activated carbon was added, stirred for 2 hours, and allowed to stand, followed by filtration with filter paper, followed by filtration with a φ0.2 μm membrane filter to obtain a spirulina hydrolyzate.

Example 4
5 parts by volume of pure water was added to 1 part by volume of the dry powder obtained in Example 1, adjusted to pH 9.5, transferred to a four-necked flask, and proteolytic enzyme [alkaline protease (manufactured by Nagase Sangyo Co., Ltd.): titer [50,000 units] 0.002 parts by volume were added, and the enzyme treatment was performed by holding at 37 ° C. for 2 hours. After moving out of the system and adjusting the treatment solution to pH 11 or higher using 4N aqueous sodium hydroxide solution, ammonia was removed by vacuum distillation operation under reduced pressure. By repeating the filtration operation, a transparent liquid was collected, and then desalted using an electrodialyzer (4 μS or less). Furthermore, the activated carbon treatment was performed by the following method. That is, 1% by weight of activated carbon was added, stirred for 2 hours, and allowed to stand, followed by filtration with filter paper, followed by filtration with a φ0.2 μm membrane filter to obtain a spirulina hydrolyzate.

(Test Example 1)
[Measurement of collagen synthesis ability in normal human skin fibroblasts]
Normal human skin fibroblasts (manufactured by Cascade Biologics) are placed in Dulbecco's modified Eagle medium (manufactured by Sigma) containing 10% by volume fetal calf serum (manufactured by Sigma) so that the cell density becomes 10 × 10 ⁴ cells / ml. 1 ml each in a 24-well microplate (manufactured by IWAKI), and cultured in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide) at 37 ° C. for 48 hours.

After culturing for 48 hours, the medium in the microplate was removed, and no fetal calf serum was added in advance so that the spirulina hydrolyzate (Example 2) had a final concentration of 0.01% to 1.0% by weight. Dulbecco's modified Eagle medium (manufactured by Sigma) was added and cultured in a CO ₂ incubator (95% by volume air, 5% by volume carbon dioxide) at 37 ° C. After 48 hours, the culture supernatant was collected.

Collagen synthesis ability was measured using the culture supernatant obtained as described above.
Collagen synthesis ability was performed using a procollagen type I C-peptide EIA kit (Procollagen type I C-peptide (PIP) EIA Kit: manufactured by Takara Bio Inc.). Procollagen type I C-peptide is a free peptide that is cleaved by endopeptidase when type I procollagen, which is a precursor of type I collagen synthesized in cells, is secreted outside the cell. It is a biochemical index that reflects the amount.

  That is, appropriately diluted culture supernatant and peroxidase-labeled anti-PIP monoclonal antibody were added to a microplate coated with anti-PIP monoclonal antibody, and reacted at 37 ° C. for 3 hours. Thereafter, washing was performed, a substrate solution for peroxidase was added to cause color development, 1N sulfuric acid was added, and the absorbance at 450 nm was measured. A standard curve was created from the simultaneously measured PIP standard solution, and the PIP concentration was determined from the absorbance of the test substance. Collagen synthesis promoting effect of Spirulina hydrolyzate was shown as a value (ratio) when the collagen synthesis ability when cultured only in Dulbecco's modified Eagle medium, that is, when the PIP amount was 100. The results are shown in Table 1.

As is clear from Table 1, spirulina hydrolyzate was found to promote the collagen synthesis ability of normal human skin fibroblasts.

(Test Example 2)
[Measurement of laminin synthesis ability in normal human skin fibroblasts]
Normal human skin fibroblasts (manufactured by Cascade Biologics) were placed in Dulbecco's modified Eagle medium (manufactured by Sigma) containing 10% by volume fetal bovine serum (manufactured by ICN) with a cell density of 10 × 10 ^4.
1 ml / ml, inoculated into a 24-well microplate (IWAKI) at 1 ml each, and in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide) at 37 ° C. for 48 hours. Cultured. After culturing for 48 hours, the medium in the microplate was removed, and Dulbecco containing no fetal calf serum was added in advance so that the spirulina hydrolyzate (Example 2) had a final concentration of 0.01 to 1.0% by weight. Modified Eagle's medium (manufactured by Sigma) was added and cultured in a CO ₂ incubator (95% by volume air, 5% by volume carbon dioxide) at 37 ° C. After 48 hours, the culture supernatant was collected.

  The laminin synthesis ability was measured using the culture supernatant obtained as described above. Laminin synthesis ability was performed using a laminin EIA kit (Laminin EIA Kit: manufactured by Takara Bio Inc.). That is, an appropriately diluted culture supernatant was added to a microplate coated with anti-human laminin monoclonal antibody and allowed to react at 25 ° C. for 1 hour. After washing, a peroxidase-labeled anti-laminin monoclonal antibody was added and reacted at 25 ° C. for 1 hour. Thereafter, washing was performed, a substrate solution for peroxidase was added to cause color development, 1N sulfuric acid was added, and the absorbance at 450 nm was measured. A standard curve was created from the simultaneously measured human laminin standard solution, and the laminin concentration was determined from the absorbance of the test substance. The laminin synthesis promoting effect of Spirulina hydrolyzate was shown as a value (ratio) when the amount of laminin was 100 when cultured only in Dulbecco's modified Eagle medium. The results are shown in Table 2.

  As is clear from Table 2, spirulina hydrolyzate was found to promote the ability of normal human skin fibroblasts to synthesize laminin.

(Test Example 3)
[Effect of hydrolyzed spirulina degradation products on skin changes caused by long-term ultraviolet irradiation in hairless mice]
Hairless mice (Skh / HR-1) females (6 weeks old) were used as experimental animals, and a low dose of UVB (95 mJ / cm ² / day) was applied to Toshiba FL20S • E lamp 5 times a week for 10 weeks. The method of forming wrinkles by irradiating the film was used. The coated sample was a 20% 1,3-butylene cricol solution containing 10% hydrolyzed spirulina degradation product, the blank was 20% 1,3-butylene cricol, and a sample was prepared for 4 weeks for 6 animals in each group. After the application, the degree of wrinkles on the back of the hairless mouse was determined with the naked eye based on the following criteria.

  Table 3 shows the criteria for the naked eye determination of each group, and the determination results were averaged and shown in Table 4.

  As is clear from Table 4, it was shown that by applying a hydrolyzed spirulina degradation product, the average score of wrinkles was lowered and wrinkles induced by ultraviolet rays were improved.

(Test Example 4)
[Improvement effect on small wrinkles]
As described above, a cream containing hydrolyzed spirulina hydrolyzate having the effect of promoting collagen synthesis in normal human fibroblasts and the effect of improving wrinkles induced in the back skin of mice by ultraviolet rays was prepared by the following production method. The effect of improving small wrinkles, which are skin aging symptoms, was examined.

Squalene, cetyl isooctanoate, and microcrystalline wax are heated and dissolved, and then clay mineral and POE glycerol triisostearic acid ester (surfactant) are added and kept at 70 ° C. to uniformly disperse and dissolve to obtain an oily gel It was.
Next, the hydrolyzed spirulina degradation product was dissolved in purified water to a predetermined concentration, heated to 70 ° C., and then slowly added to the oily gel with sufficient stirring. After uniformly mixing with a homomixer, the mixture was deaerated under reduced pressure and cooled to room temperature to obtain a cream. The composition and blending ratio of the cream thus obtained are as follows.

(Prescription Example 1)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1.0%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Hydrolyzed Spirulina degradation product 5.0%
Water remaining

(Comparative Example 1)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1.0%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Water remaining

  20 female panels (33-52 years old) were randomly divided into two groups, one group with the cream of Formula 1 and the other group with the cream of Comparative Example 1 twice daily, morning and evening It was applied to the face after washing for 2 months. The skin condition before and after the test was compared and evaluated. The evaluation was performed according to the criteria shown in Table 5 for the degree of improvement of small wrinkles by cream application by visual inspection.

  Table 6 shows the total evaluation score of the degree of fine wrinkle improvement of each panel in each group.

  As is clear from Table 6, the improvement in small wrinkles was high in the panel using Formulation Example 3, and it became clear that the cream containing the hydrolyzed spirulina degradation product was effective in improving small wrinkles.

(Test Example 5)
[Melanin production inhibition test]
Mouse-derived B16 melanoma cultured cells were used. B16 melanoma cells are suspended in Dulbecco's modified Eagle medium (Sigma) containing 10% fetal calf serum (ICN), seeded on a 6-well microplate (IWAKI), and CO ₂ incubator (95% by volume air). (5 vol% carbon dioxide) and cultured at 37 ° C. for 24 hours.

After culturing for 24 hours, the medium in the microplate was removed, and 10% fetal bovine serum (Spirulina hydrolyzate (Example 2) added in advance to a final concentration of 0.01% to 1.0% by weight ( Dulbecco's modified Eagle medium (manufactured by ICN) (Sigma) was added and cultured in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide) at 37 ° C for 7 days. After removing the medium and washing once with 1 ml of PBS, 0.2 ml of 3N sodium hydroxide solution was added to completely lyse the cells. The absorbance at 405 nm of this cell lysate was measured. A calibration curve was created using synthetic melanin (manufactured by Sigma) as standard melanin, and the amount of melanin was determined. The amount of protein in the cell lysate was determined using a protein assay (Bio-Rad). The amount of melanin produced was compared by the amount of melanin per protein amount. The melanin synthesis inhibitory effect of Spirulina hydrolyzate was shown as a value (ratio) when the amount of melanin produced when cultured only in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (ICN) was taken as 100. The results are shown in Table 7.

  As is apparent from Table 7, it can be seen that spirulina hydrolyzate suppresses the amount of melanin produced by cultured mouse B16 melanoma cells.

(Test Example 6) Whitening effect test As described above, a lotion containing spirulina hydrolyzate, which is an excellent melanin synthesis inhibitor, was prepared by the following production method, and the whitening effect was examined.
A lotion base containing POE (20) oleyl alcohol ether (surfactant), methylcellulose and quince seed (thickener) and ethanol was prepared, and a predetermined concentration of spirulina hydrolyzate was added. The composition and blending ratio of the lotion of the obtained Formulation Example 2 are as follows.

(Prescription example 2)
Composition ratio (wt%)
POE (20) oleyl alcohol ether 0.5%
Methylcellulose 0.2%
Quince Seed 0.1%
Ethanol 10%
Spirulina hydrolyzate 3.0%
Water remaining

(Comparative Example 2)
Composition ratio (wt%)
POE (20) oleyl alcohol ether 0.5%
Methylcellulose 0.2%
Quince Seed 0.1%
Ethanol 10%
Water remaining

  20 female panels (29-54 years old) were randomly divided into two groups, one group with the skin lotion of Prescription Example 2 and the other group with the skin lotion of Comparative Example 2 every morning and night It was applied to the face twice after washing the face for 2 months. The skin condition before and after the test was compared and evaluated. In the evaluation, the skin beautifying effect by applying the skin lotion was evaluated according to the criteria shown in Table 8 by naked eye determination.

Table 9 shows the results of the whitening effect test.

  As is clear from Table 9, the effectiveness of the skin using the formulation example 2 was confirmed for skin dullness, and the skin lotion containing the hydrolyzed spirulina degradation product can prevent and improve skin dullness, It became clear that it was beautiful skin.

Claims (4)

  A production regulator for skin aging-related components, comprising a hydrolyzate of cyanobacteria.   A production regulator of a skin aging-related component having an action of promoting synthesis of extracellular matrix-constituting protein and suppressing melanin production, comprising a hydrolyzate of cyanobacteria.   The production regulator of a skin aging-related component according to claim 1 or 2, wherein the cyanobacteria are Orthospira.   An external preparation for preventing skin aging, comprising the production regulator for skin aging-related components according to any one of claims 1 to 3.