JP2014001148A - Fibroblast proliferation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fibroblast proliferation promoter, an anti-ageing agent, a wrinkle and sagging ameliorating agent and a humectant each exhibiting an excellent fibroblast proliferation-promoting action and comprising an ingredient having stability and high safety against skin as an active ingredient.SOLUTION: A fibroblast proliferation promoter, an anti-ageing agent, a wrinkle and sagging ameliorating agent and a humectant each comprise acidic mucopolysaccharide produced by a marine bacterium, or its physiologically acceptable salt or a derivative as an active ingredient.

Description

  The present invention relates to a fibroblast growth promoter, an anti-aging agent, a wrinkle / sagging improver, and a moisturizer containing an acidic mucopolysaccharide produced by marine bacteria as an active ingredient.

  The skin epidermis and dermis are composed of epidermal cells, fibroblasts and extracellular matrix, which form connective tissue and play an important role in the skin. By maintaining the constancy of the skin tissue interaction, moisture retention, flexibility, elasticity and the like are ensured, and the skin is maintained in a fresh state with a firm appearance, luster and texture.

  The influence of certain external factors such as aging, ultraviolet rays, and air drying reduces fibroblast proliferation ability, reduces the production of extracellular matrix, and reduces elasticity due to aging crosslinking. . As a result, the skin is less moisturized and elastic, loses tension and gloss, and exhibits skin aging symptoms such as rough skin, wrinkle formation, dullness, and loss of texture. Thus, it is thought that the change accompanying the skin aging is deeply related to the decrease in the proliferative ability of fibroblasts.

  Based on these findings, various components and extracts that have a highly safe natural product-derived fibroblast growth-promoting action for the purpose of preventing or improving skin aging by growing fibroblasts. Things have been proposed.

  For example, lotus germ extract (Patent Document 1), moon peach extract (Patent Document 2), cacao bean extract (Patent Document 3), and other plants or extracts thereof (Patent Document 4, Patent Document 5) Etc. have been proposed.

JP 2002-29980 A JP 2002-3390 A JP 2008-208053 A JP 2011-195533 A JP 2008-94739 A Japanese Patent Application No. 2012-57185

  However, few specific compounds that promote fibroblast proliferation in the extract have been identified.

  For this reason, there is a strong demand for a natural component having a fibroblast growth promoting effect by further development of unused resources.

  Therefore, the present invention finds a highly safe natural product produced by marine microorganisms, which has been delayed until now, and has a fibroblast proliferation promoting action, and uses it as an active ingredient for fibroblast proliferation. An object is to provide an accelerator, an anti-aging agent, a wrinkle / sagging agent, and a moisturizing agent.

  In order to solve the above-mentioned problems, the present inventors have conducted earnest research on what has a fibroblast proliferation promoting action from among safe polysaccharides produced by marine microorganisms rich in unused resources. Fibroblast proliferation promoter and anti-aging agent effective for prevention or improvement of skin aging by using acidic mucopolysaccharide produced by bacteria having fibroblast proliferation promoting action and using it as an active ingredient The present inventors have found that a wrinkle / sag improver and a humectant can be obtained, and the present invention has been completed.

  That is, the present invention contains an acidic mucopolysaccharide produced by a marine bacterium or a physiologically acceptable salt or derivative thereof as an active ingredient, a fibroblast proliferation promoter, an anti-aging agent, wrinkle / sag The present invention relates to an improving agent and a humectant.

  The fibroblast proliferation promoter, anti-aging agent, wrinkle / sagging agent, and moisturizing agent of the present invention are the acidic mucopolysaccharide represented by the following formula (1) or a physiologically acceptable salt or derivative thereof ( (Hereinafter referred to as “the present acidic mucopolysaccharide”) as an active ingredient.

（上記の構造式において、GlcNAcpはピラノース型Ｎ-アセチルグルコサミン残基を、GlcUApはピラノース型グルクロン酸残基を、Gluはグルタミン酸を、ＤはＤ型を、ＬはＬ型を、ｎはゲル濾過クロマトグラフィーで測定した平均分子量がプルランを標準として１００万〜１５０万であることを示す繰り返しの数をそれぞれ表す。）

(In the above structural formula, GlcNAcp is a pyranose type N-acetylglucosamine residue, GlcUAp is a pyranose type glucuronic acid residue, Glu is glutamic acid, D is D type, L is L type, and n is gel filtration. Represents the number of repetitions indicating that the average molecular weight measured by chromatography is 1 to 1.5 million with pullulan as a standard.)

  The fibroblast proliferation promoter comprising the present acidic mucopolysaccharide produced by marine bacteria as an active ingredient promotes the proliferation of fibroblasts without showing cytotoxicity to fibroblasts. The acidic mucopolysaccharide also has a wrinkle / sag improving effect and a moisturizing effect.

  The fibroblast proliferation promoter of the present invention contains the present acidic mucopolysaccharide produced by marine bacteria, and thus exhibits a high safety, high moisturizing and excellent fibroblast proliferation promoting action. Therefore, it has usefulness not found in conventional polysaccharides as a fibroblast growth promoter, anti-aging agent, wrinkle / sagging agent, and moisturizer.

FIG. 1 shows the effect of the acidic mucopolysaccharide on the proliferation of human dermal fibroblasts.

  The present invention will be described below with reference to embodiments, but the present invention is not limited to the following embodiments.

  This acidic mucopolysaccharide has a melanin production inhibitory effect of mouse melanoma, and its derivative is known to have an antiviral effect, but there is a report that it was used as a fibroblast proliferation promoter for skin. To date, nothing is known.

  In the present invention, the fibroblast proliferation promoter is a composition effective for preventing or treating skin aging by promoting the proliferation of skin fibroblasts. The fibroblast proliferation promoter of the present invention is a skin external preparation composition or an oral composition, and is a cosmetic or quasi-drug, and a pharmaceutical cosmetic such as a lotion, emulsion, cream, pack, soap, etc. And external preparations for skin such as lotions, emulsions, creams, ointments and the like as pharmaceuticals, and oral preparations such as cosmetic foods and drinks.

The skin external preparation composition for promoting fibroblast proliferation of the present invention comprises the acidic mucopolysaccharide as an active ingredient and various components generally used in skin external preparations, such as oils, surfactants, and UV absorbers. , Alcohol, moisturizer, preservative, bactericidal agent, colorant, powder, fragrance, thickener, buffering agent, etc., according to the dosage form, prepared by appropriately blending within the range not impairing the effects of the present invention. The
Further, the oral composition for promoting fibroblast proliferation of the present invention comprises the present acidic mucopolysaccharide as an active ingredient and various components generally used in oral compositions such as excipients, antiseptics. It is prepared by blending an agent, a colorant, a fragrance, and the like in accordance with the dosage form, as appropriate, within a range not impairing the effects of the present invention.

  In blending the acidic mucopolysaccharide with the topical skin composition for promoting fibroblast proliferation of the present invention, it is preferable to consider the fibroblast proliferation promoting action of these compounds and the skin keratin permeability, etc. In general, these compounds may be added as active ingredients at least 0.001% by weight or more, preferably about 0.01 to 20.0% by weight. It is not always necessary to isolate and use the active ingredient, and a crude product containing the compound of the present invention can be used as long as the effects of the present invention are not impaired.

The dosage form of the skin external preparation composition for promoting fibroblast proliferation according to the present invention is arbitrary, for example, a solubilizing system such as lotion, an emulsifying system such as emulsion or cream, or an ointment or dispersion. be able to.
Moreover, the dosage form of the oral preparation for promoting the proliferation of fibroblasts of the present invention is arbitrary. For example, it can take a dosage form such as a tablet, a capsule, a liquid, a syrup, a powder, and a jelly.
Thus, a desired fibroblast proliferation promoting effect, for example, generation of wrinkles such as a face can be prevented, and a fibroblast proliferation promoting agent that can be used to improve wrinkles that have already been generated is provided.

(Active ingredient)
As the acidic mucopolysaccharide used in the present invention, those prepared by various methods can be used as long as they are purified to such an extent that they can be used as pharmaceuticals. Various methods are known as methods for preparing the present acidic mucopolysaccharide. For example, it can be obtained by collecting and purifying polysaccharides produced by culturing marine microorganisms in a medium prepared with seawater containing sucrose as a carbon source, peptone as a nitrogen source, and yeast extract.

  The microorganism used for production of the present acidic mucopolysaccharide can be used without particular limitation as long as it can produce the polysaccharide. Preferably, marine microorganisms, more preferably marine microorganisms capable of producing the present acidic mucopolysaccharide are used. Specific examples include bacteria belonging to the genus Covetia, and more specifically, strains belonging to the genus Cobecia and deposited with a deposit number of FERM P-21297 or mutants thereof. This strain is a marine bacterium isolated from seawater by the present inventors in the Seto Inland Sea, and its taxonomic characteristics are described in Patent Document 6.

  As a basic medium for culturing microorganisms that produce this acidic mucopolysaccharide, microorganisms that can produce polysaccharides can grow, and at least a carbon source, a nitrogen source, various inorganic salts, and trace elements Those containing an appropriate amount of are used. More preferably, as the basic medium, one capable of growing microorganisms belonging to the genus Cobetia is used. Examples of the carbon source include sugars such as glucose, fructose, galactose, and sucrose, and molasses and molasses. One or two or more carbon sources can be used alone or in combination. Nitrogen sources include compounds such as nitrates and ammonium salts, and natural products such as peptone, yeast extract and amino acids. One or two or more nitrogen sources can be used alone or in combination. Examples of the inorganic salt include phosphate, magnesium salt, potassium salt and the like. One or two or more inorganic salts can be used alone or in combination. In the case of a solid medium, agar is used.

  Culture conditions such as the medium to be used, the pH of the medium, the additive to the medium, and the culture temperature can be set according to the conditions normally used for culturing microorganisms.

  The culture conditions in the present invention are not limited as long as the pH during culture is a range in which microorganisms can grow and produce polysaccharides, but a pH in the range of 6 to 7.5 is usually preferable. The culture temperature is not limited as long as microorganisms grow and produce polysaccharides, but a range of 25 ° C. to 30 ° C. is good for producing polysaccharides. The culture period varies depending on the culture pH and temperature, but usually 2 to 5 days is appropriate.

  By culturing microorganisms using the above-described medium and microorganisms using a conventional method, the present acidic mucopolysaccharide, which is an active ingredient of the present invention, is efficiently produced.

  The acidic mucopolysaccharide used in the present invention has a constituent sugar molar ratio of N-acetyl-D-glucosamine: D-glucuronic acid: L-glutamic acid of 2: 1: 1 (molar concentration ratio) and gel filtration chromatography. It is preferable that the average molecular weight measured by lithography is 100 to 1,500,000 with pullulan as a standard. Cellulose acetate membrane electrophoresis or high performance liquid chromatography can be used for the analysis of the constituent components. For analysis of this component, polysaccharide was hydrolyzed with 2M trifluoroacetic acid (TFA) or 4N-HCl at 100 ° C. for 12 hours, and TFA or HCl was removed with a rotary evaporator as a sample. Analyzes of sexual sugars, uronic acids, organic acids, amino acids and amino sugars. In addition to this, an enzymatic method can be used for analysis of constituent organic acids.

  The molecular weight of the present acidic mucopolysaccharide can be measured by gel filtration chromatography. Specifically, high-performance liquid chromatography (manufactured by Shimadzu Corporation) using Asahipak GFA-7M (manufactured by Showa Denko) as a column, 0.1M-NaCl as a mobile phase, and pullulan (Shodex STANDARD with a known molecular weight). KIT P-82 (manufactured by Showa Denko KK) can be measured using a molecular weight retention time standard curve prepared as a standard sample.

(Method for producing active ingredient)
Next, a method for producing the present acidic mucopolysaccharide, which is an active ingredient of the fibroblast growth promoter of the present invention, will be described with reference to preferred embodiments. In addition, the production method of this acidic mucopolysaccharide is not limited to the following production method.
The method for producing the present acidic mucopolysaccharide as one preferred embodiment includes (a) a step of culturing a microorganism in a medium to produce a polysaccharide, and (b) a step of extracting and recovering the produced present acidic mucopolysaccharide. And have.

Subsequently, the above-described embodiment will be described in detail for each step.
It is desirable to use a medium suitable for the production of the present acidic mucopolysaccharide, which is an active ingredient of the above-described fibroblast growth promoter of the present invention, and in this case, sucrose is used as the carbon source, peptone is used as the nitrogen source, and yeast extract is used. It is more desirable.
(A) Step of culturing microorganisms in medium to produce polysaccharides The microorganisms are cultured using the medium prepared as described above to produce the desired polysaccharides, and the present acidic mucopolysaccharides are obtained. Therefore, it is preferable to use a bacterium belonging to the genus Cobetia as a microorganism. More preferably, it is desirable to use a strain belonging to the genus Cobetia and deposited with a deposit number of FERM P-21297 or a mutant thereof. The acidic mucopolysaccharide can be efficiently produced by using the strain or a mutant thereof as a microorganism.
And it becomes possible to obtain this acidic mucopolysaccharide of high purity by a high yield by passing through the extraction and collection | recovery process demonstrated below.

(B) Step of extracting and recovering the produced predetermined polysaccharide As a method for extracting the acidic mucopolysaccharide from the culture solution obtained by the above production method, a method known in the art should be used. Can do. For example, after sterilizing the culture solution as it is or at a high temperature, the microbial cells are removed by centrifugation, and after adding or concentrating this, add 2-3 times the amount of ethanol, isopropanol, or acetone, This causes precipitation. After this precipitate is dissolved again in water or 1-15% sodium chloride solution, precipitation with alcohol or the like is repeated 2-3 times, dialyzed with water, and dried using a spray dryer or a freeze dryer. This acidic mucopolysaccharide is obtained. In addition, electrodialysis and ultrafiltration can be used. For further purification, various types of chromatography such as ion exchange and gel filtration, precipitation with quaternary ammonium salts, salting out, and the like can be used.

  Thus, the acidic mucopolysaccharide will be produced and recovered. The acidic mucopolysaccharide obtained by the present invention can be used for various applications without any particular limitation, but preferably the above-mentioned fibroblast growth promoter, anti-aging agent, wrinkle / sagging agent, and humectant Is preferably used.

(上記の構造式において、GlcNAcpはピラノース型Ｎ-アセチルグルコサミン残基を、GlcUApはピラノース型グルクロン酸残基を、Gluはグルタミン酸を、ＤはＤ型を、ＬはＬ型を、ｎはゲル濾過クロマトグラフィーで測定した平均分子量がプルランを標準として１００万〜１５０万であることを示す繰り返しの数をそれぞれ表す。) Here, the present acidic mucopolysaccharide has the following structural units.

(In the above structural formula, GlcNAcp is a pyranose type N-acetylglucosamine residue, GlcUAp is a pyranose type glucuronic acid residue, Glu is glutamic acid, D is D type, L is L type, and n is gel filtration. (The number of repetitions indicating that the average molecular weight measured by chromatography is 1 to 1.5 million with pullulan as a standard)

  In the present invention, at least 0.001% by weight or more, preferably 0.01 to 20% by weight of the present acidic mucopolysaccharide is added to the fibroblast proliferation promoter as an active ingredient. The fibroblast growth promoter containing this acidic mucopolysaccharide as an active ingredient should be in an appropriate form such as cream, emulsion, lotion, etc. by using a commonly used skin external preparation base material. Can do. In addition, this fibroblast growth promoter contains optional additives such as UV absorbers, UV scattering agents, melanin synthesis inhibitors such as arbutin, other medicinal ingredients, thickeners, plasticizers, coloring agents, and fragrances. Can be made. The acidic mucopolysaccharide, which is an active ingredient of the fibroblast growth promoter of the present invention, has neither toxicity nor irritation to the skin and no side effects. In addition, since the polysaccharide generally has a moisturizing effect and the acidic mucopolysaccharide contains glutamic acid, which is known as a natural moisturizing factor, the present polysaccharide containing the acidic mucopolysaccharide as an active ingredient. The fibroblast proliferation promoter of the invention has a preferable moisturizing effect in addition to the fibroblast proliferation promoting effect.

  This acidic mucopolysaccharide has a fibroblast growth-promoting action and a moisturizing effect, and is also excellent in safety, so it is formulated in beauty foods, health foods, functional foods, etc. It is also suitable to do.

  When the present acidic mucopolysaccharides are blended in cosmetic foods and drinks, the amount of active ingredients in them can be appropriately changed in consideration of the purpose of use, symptoms, sex, etc. In consideration of the correct intake, it is preferable that the intake of the present acidic mucopolysaccharide per day for an adult is about 1 to 1000 mg.

  The cosmetic food / beverage product of the present invention may be a blend of the present acidic mucopolysaccharides in any food / beverage product that does not impede its activity, or a nutritional supplement food / drink comprising the present acidic mucopolysaccharides as the main component. It may be a product.

  The fibroblast proliferation promoter, skin external preparation and cosmetic food and drink of the present invention described above are suitably applied to humans, but as long as the respective effects are exerted, other than humans It can also be applied to animals.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all to these Examples. Unless otherwise specified, percentages (%) are based on weight.

Example 1
A medium prepared with seawater having a composition of 0.5% peptone, 0.1% yeast extract and 3% sucrose was sterilized in an autoclave at a temperature of 121 ° C. for 15 minutes, and the strain (FERM P-21297) From the slant culture, one platinum loop is inoculated into the above-mentioned sterilized medium (10 ml) in a test tube, and statically cultured at a temperature of 25 ° C. for 24 hours, and then this preculture solution is placed in a 500 ml Erlenmeyer flask. The medium was inoculated into 100 ml (121 ° C., sterilized for 15 minutes) and statically cultured at 25 ° C. for 3 days. After culturing, the culture broth is centrifuged, filtered through GF-75 filter paper (manufactured by Advantec) using a filter aid (Radiolite # 500), and the hollow fiber having a molecular weight of 50,000 cut from the culture filtrate from which the cells are removed. The polymer fraction obtained by a membrane filtration apparatus equipped with a UF membrane module (manufactured by Spectrum) was concentrated, desalted and recovered in a short time, treated with activated carbon, and pulverized by freeze drying. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering.
The polysaccharide fraction obtained in this manner was confirmed by homogeneity using cellulose acetate membrane electrophoresis, and by using DEAE-cellulose ion exchange column chromatographic analysis, chemical analysis, and nuclear magnetic resonance analysis, The mucopolysaccharide was confirmed.

(Example 2)
Until the preculture, the same treatment as in Example 1 was performed, and then this preculture was inoculated into a 500 ml Erlenmeyer flask to 100 ml of sterilized medium (121 ° C, 15 minutes) prepared from seawater having the above composition, and 25 ° C. The shaking culture was performed at the temperature of 3 days. Next, the culture solution is centrifuged and then filtered through a GF-75 filter paper (manufactured by Advantec) using a filter aid (Radiolite # 500), and a hollow fiber UF membrane having a molecular weight of 50,000 cuts from the culture filtrate from which the cells are removed. The polymer fraction obtained by a membrane filtration apparatus equipped with a module (manufactured by Spectrum) was concentrated, desalted and recovered in a short time, treated with activated carbon, and powdered by freeze drying. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering. The polysaccharide fraction obtained in this manner was confirmed by homogeneity using cellulose acetate membrane electrophoresis, and by using DEAE-cellulose ion exchange column chromatographic analysis, chemical analysis, and nuclear magnetic resonance analysis, The mucopolysaccharide was confirmed.

(Example 3)
Until the pre-culture, the same treatment as in Example 1 was performed, and 250 ml of agar plate medium obtained by adding 1.5% of agar to the medium described in Example 2 was spread on a flat plate (18 × 26 cm) and smeared with the pre-culture solution. The culture was performed at a temperature of 25 ° C. for 4 days. The sticky matter generated on the surface of the agar plate is scraped off, suspended in 1% phenol solution, centrifuged, and filtered through GF-75 filter paper using a filter aid (Radiolite # 500) to remove the cells. Fraction precipitated by adding ethanol to the supernatant was collected, dissolved in water, ethanol added again to collect the precipitated fraction, dissolved in water, and dialyzed. Subsequently, it was treated with activated carbon, and a polysaccharide fraction was obtained by freeze-drying. The polysaccharide fraction obtained in this manner was confirmed by homogeneity using cellulose acetate membrane electrophoresis, and by using DEAE-cellulose ion exchange column chromatographic analysis, chemical analysis, and nuclear magnetic resonance analysis, The mucopolysaccharide was confirmed.

Example 4
(Cell proliferation test using cultured human fibroblast cell lines)
Using this acidic mucopolysaccharide obtained in Example 1, the effect on cell proliferation on fibroblasts was examined, and compared with the case where no sample was added (control) and the case where sodium hyaluronate (HA) was added. The cell growth rate in the sample addition system was determined.

(Cell culture)
(1) Pre-cultured human fibroblasts (Kurabo Co., Ltd.) were seeded in each well of a 96-well multiwell plate at a cell number of 1.0 × 10 ⁴ cells / well. 24 hours after sowing, the medium was replaced with DMEM + 10% FBS medium containing a test substance at a predetermined concentration. The concentration of the test substance was set to 0 mg / ml (control), 2.5 mg / ml, and 5.0 mg / ml. Moreover, sodium hyaluronate (HA; 5.0 mg / ml, molecular weight: 1 million, manufactured by Wako Pure Chemical Industries, Ltd.) was also used as a comparative sample. Three wells were assigned for each sample concentration and subjected to the assay (N = 3). Two plates for counting cell proliferation were used as a set for assaying each sample. After the medium change, the culture was continued for 72 hours.
(2) The cell viability was measured by measuring the number of cells in each well (relative value) using an MTT method and measuring the absorbance at 570-630 nm with a microplate reader (manufactured by BIO RAD). The value (relative value) of each sample was determined when the value of the control containing no test substance was 100. These results are shown in FIG. 1 (mean value ± SE).

  As is clear from FIG. 1, the HA did not confirm the growth promotion of fibroblasts compared with the control, but the acidic mucopolysaccharide showed the action of promoting the growth of fibroblasts.

(Prescription Example 1)
Using the acidic mucopolysaccharide obtained in Example 1, a lotion shown in Formulation Example 1 was prepared by a conventional method. Moreover, the lotion shown in the comparative example 1 was prepared as a comparative example.

(Example 5)
(Keratin moisture retention test)
Using the lotion prepared in Formulation Example 1 and Comparative Example 1, a keratin moisture retention test was performed.

(Evaluation test)
After arbitrarily extracting the monitors of 5 healthy people in their 20s to 50s, the subjects were acclimatized for 60 minutes in an artificial environment room with a room temperature of 25 ° C and a humidity of 50%. Two squares were written, and the electrical conductivity was measured three times in each square with a skin keratin moisture meter (SKICON-200EX manufactured by IBS). Thereafter, 20 μl of the lotion prepared in Formulation Example 1 and Comparative Example 1 was applied to each square. The electrical conductivity (μS) was measured before coating and 3 hours after coating, and the average value of the increase in retention of keratin moisture was determined. The greater the amount of moisture in the skin, the greater the value of electrical conductivity (μS). Using this, the amount of retained keratin water was evaluated and shown in Table 2 (mean value ± SE).

The results are shown in Table 2.

  As shown in Table 2, the keratin water retention amount of the subjects when the present acidic mucopolysaccharide is blended is clearly larger than that of Comparative Example 1 in which all the subjects do not blend the present acidic mucopolysaccharide. It was. From these results, it can be understood that the amount of keratin moisture is suitably maintained by blending the acidic mucopolysaccharide with the lotion.

  As is apparent from FIG. 1 and Table 2, the addition of the acidic mucopolysaccharides promotes fibroblast proliferation and retains keratinous water, and the acidic mucopolysaccharide exhibits excellent fibroblast proliferation and keratinous moisture. It has a retention action and is useful as a fibroblast growth promoter, anti-aging agent, wrinkle / sagging agent, and moisturizer.

  The fibroblast proliferation promoter, anti-aging agent, wrinkle / sagging agent, and moisturizing agent according to the present invention are used in various dosage forms as described above. In addition, formulation examples of cosmetic liquids, creams, milky lotions, soaps, emulsifying foundations and bath preparations will be described as Formulation Examples 2 to 7, and formulation examples of beauty foods and beverages will be described as Formulation Examples 8 and 9.

Formulation Example 2 (Cosmetic liquid)
A cosmetic solution was prepared in the usual manner using the following components.

Formulation Example 3 (Cream)
The following component A and component B were heated and dissolved to prepare A solution and B solution, respectively. The B solution was added to the A solution to emulsify, and cooled with stirring to prepare a cream.

Formulation example 4 (milky lotion)
The following component A and component B were heated and dissolved to prepare A solution and B solution, respectively. The B solution was added to the A solution to emulsify, and cooled with stirring to prepare an emulsion.

Formulation Example 5 (soap)
The soap was manufactured by mixing the following components according to a conventional method for manufacturing soap.

Formulation Example 6 (Emulsification Foundation)
The following component A is sufficiently mixed and pulverized into powder part A, component B is liquid B, and component C is liquid C. After the liquid C is heated and stirred, A is added and homomixed, and the liquid B which has been heated and mixed is added and homomixed. While stirring, the mixture was cooled to room temperature to prepare an emulsified foundation.

Formulation Example 7 (Bath Agent)
A bath preparation was prepared in the usual manner using the following components.

Formulation Example 8 (tablet)
The following component A was sifted and mixed, and then component B was added and mixed. Thereafter, tableting was performed by a conventional method to obtain a tablet having a total amount of 600 mg.

Formulation example 9 (soft drink)
A soft drink was prepared by the following method using the following ingredients.

  The fibroblast proliferation promoter, anti-aging agent, wrinkle / sagging agent, and moisturizing agent of the present invention are a skin external preparation useful for prevention or improvement of skin aging symptoms associated with a decrease in fibroblast proliferation ability and It can be used for beauty foods and drinks.

  The above-mentioned FERM P-21297 strain used in the present example is the center of Japan National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center 1-5-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan 305-8586 Japan And deposited under the accession number FERM P-21297 on May 16, 2007.

Claims (10)

The following general formula (1)

(In the above structural formula, GlcNAcp is a pyranose type N-acetylglucosamine residue, GlcUAp is a pyranose type glucuronic acid residue, Glu is glutamic acid, D is D type, L is L type, and n is gel filtration. The average molecular weight measured by chromatography is the number of repetitions indicating that the average molecular weight is 1 million to 1.5 million with pullulan as a standard.) And the physiologically acceptable salt or derivative thereof. A fibroblast growth promoter characterized by containing as an active ingredient.   The fibroblast proliferation promoter according to claim 1, which is an external preparation for skin.   The fibroblast proliferation promoter according to claim 1, which is a cosmetic food or drink. The following general formula (1)

(In the above structural formula, GlcNAcp is a pyranose type N-acetylglucosamine residue, GlcUAp is a pyranose type glucuronic acid residue, Glu is glutamic acid, D is D type, L is L type, and n is gel filtration. The average molecular weight measured by chromatography is the number of repetitions indicating that the average molecular weight is 1 million to 1.5 million with pullulan as a standard.) And the physiologically acceptable salt or derivative thereof. An anti-aging agent characterized by containing as an active ingredient.   5. The anti-aging agent according to claim 4, which is a wrinkle / sagging agent.   The anti-aging agent according to claim 4 or 5, which is an external preparation for skin.   The anti-aging agent according to claim 4 or 5, which is a beauty food or drink. The following general formula (1)

(In the above structural formula, GlcNAcp is a pyranose type N-acetylglucosamine residue, GlcUAp is a pyranose type glucuronic acid residue, Glu is glutamic acid, D is D type, L is L type, and n is gel filtration. The average molecular weight measured by chromatography is the number of repetitions indicating that the average molecular weight is 1 million to 1.5 million with pullulan as a standard.) And the physiologically acceptable salt or derivative thereof. A moisturizer characterized by containing as an active ingredient.   The moisturizer according to claim 8, which is an external preparation for skin.   The moisturizer according to claim 8, which is a beauty food or drink.

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