WO2014192682A1

WO2014192682A1 - Activating agent containing cellobiose lipid as active ingredient and collagen production enhancer

Info

Publication number: WO2014192682A1
Application number: PCT/JP2014/063808
Authority: WO
Inventors: 周子山下; 周平山本
Original assignee: 東洋紡株式会社
Priority date: 2013-05-31
Filing date: 2014-05-26
Publication date: 2014-12-04
Also published as: TW201501727A

Abstract

[Problem] To provide: an activating agent and a collagen production enhancer, each of which has excellent activating effect and anti-aging effect on cells and has safety that is sufficient to withstand usage for a long period of time; and a cosmetic, a quasi-pharmaceutical product, a pharmaceutical product, a food and a drink, each of which contains the activating agent or the collagen production enhancer as an active ingredient. [Solution] A cell activation agent which is characterized by containing a cellobiose lipid that is a kind of a glycolipid-type biosurfactant.

Description

Activator and collagen production promoter containing cellobiose lipid as active ingredients

The present invention relates to an activator comprising cellobiose lipid as an active ingredient, and in particular, by activating various cells, it improves cell metabolism, regeneration and repair functions, and is effective in preventing aging and preventing hair growth and hair loss. The present invention relates to cosmetics, quasi drugs, pharmaceuticals, and foods and drinks containing cellobiose lipid. Furthermore, the present invention relates to a collagen production promoter, which is a cosmetic, quasi-drug, pharmaceutical, food and beverage containing a biosurfactant effective for anti-aging, skin texture adjustment, skin wrinkle prevention / control by collagen production. It is about goods.

Various diseases are deeply related to a decrease in the division rate and cell function of all dividing cells. For example, the dermis and epidermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as elastin, collagen, and hyaluronic acid that support these extracellular skin structures. In particular, fibroblasts mainly control the synthesis and degradation of collagen and hyaluronic acid. In young skin, skin properties such as flexibility are ensured by maintaining the homeostasis of these skin tissue interactions, and the skin is glossy, tightened, transparent, and maintained moist. . However, ultraviolet rays, dryness, stress, etc. cause a decrease in the function of the extracellular matrix and fibroblasts in particular, and as a result, the skin properties such as skin flexibility decline, and the skin loses its gloss, firmness, and transparency, Symptoms such as roughening, wrinkles and dullness occur. It is also known that the function of fibroblasts declines as skin characteristics decline, and the turnover rate of collagen and hyaluronic acid decreases.

Searching for an activator and an anti-aging agent at the cellular level has been conducted. For example, as an activator derived from an animal system, connective tissue hydrolyzate, thymus / spleen-derived water-soluble protein, bovine placenta extract and the like are known. Examples of plant-derived activators include sesame seeds, sanjak, capsicum, touki, dokudami, bakumondo, almond, dandelion, elderberry, nematode, senburi, sakuhakuhi, tonin, carrot, hop, muguge, yokuinin, turmeric genus, An extract of the genus Hyanaseri is known. Some of these are used in quasi-drugs and cosmetics as activators and anti-aging agents, but their effects are not sufficient due to the large individual differences, and they exhibit satisfactory effects. No cell activation or anti-aging effect has been obtained.

Glycolipids are amphiphilic substances having both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids, and have a function as surfactants. There are various amphiphiles in the living body, and they are involved in the exchange of substances, energy, and information at various interfaces and play a major role in the formation of ecological order.

In recent years, cellobiose lipid (CL), which is a kind of glycolipid-based biological surfactant (biosurfactant), has been reported to show antifungal activity and has attracted attention (Non-Patent Document 1). Cellobiose lipid is known to be produced by Ustilago maydis (Ustyago Maydis) and Cryptococcus humicola (Cryptococcus Fumicola) (Non-patent Document 2). Pseudozyma flocculosa (Pseudozyma floculosa) is also known to produce a kind of CL called floculosine (Non-patent Document 3). Furthermore, Ustilago esculenta has been found to be able to produce CL in large quantities (Patent Document 1). Since CL produced by these microorganisms all exhibits excellent antifungal activity, it is expected to be put to practical use as an antifungal agent.

Cellobiose lipid is industrially used in a wide range of fields such as detergents and cosmetics, and is used as a liposome-forming agent (see Patent Document 2), an emulsifier / solubilizer (see Patent Document 3), a protein separation carrier (patent) There are reports such as low molecular weight organogel (refer to Patent Document 5).

Cellobiose is highly biodegradable, has low toxicity, and is environmentally friendly. Therefore, it is expected to be put to practical use as a cosmetic and pharmaceutical material. However, at present, no effect on cell of cellobiosespirid has been found, and no effect or efficacy has been reported.

JP 2004-254595 A JP 2006-028069 A JP 2007-181789 A JP 2006-219455 A JP 2012-176904 A

Giving activation and anti-aging to mammals, particularly humans, is a very important issue. Various activators and anti-aging agents derived from animals and plants have been found, but in fact, they have not been sufficiently effective and stable enough to be industrially usable. Agents are being explored. An object of the present invention is to provide an activator and an anti-aging agent that have excellent activation and anti-aging effects on cells and have safety sufficient to withstand long-term use, and cosmetics / pharmaceuticals comprising these as active ingredients To provide quasi-drugs, pharmaceuticals, and food and drinks.

As a result of diligent efforts to achieve the above object, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention. That is, this invention consists of the following structures.

1. A cell activating agent comprising cellobiose lipid.
2. 2. The cell activation agent according to 1, wherein the cell activation is activation of skin cells.
3. 2. The cell activation agent according to 1, wherein the cell activation is activation of fibroblasts, hair matrix cells, or hair papilla cells.
4). 2. The cell activation agent according to 1, wherein the cell activation is promotion of collagen production.
5. 5. The cell activator according to any one of 1 to 4 above, which comprises cellobiose lipid having a structure represented by the following general formula (I) or (II).

(In formula (I), R1 represents hydrogen or a hydroxyl group, n1 has 12 or 14 carbon atoms, and R2, R3, and R4 of sugar 1 in formula I are an acetyl group or a hydroxyl group. Is shown.)

(In formula (II), R1 and R2 each represent hydrogen or a hydroxyl group. N1 represents an alkylene group having 11 or 12 carbon atoms, and n2 represents an alkylene group having 2 or 4 carbon atoms.)

6). An anti-aging agent comprising the cell activator according to any one of 1 to 5 above.
7). A skin protecting agent comprising the cell activating agent according to any one of 1 to 5 above.
8). A rough skin improving agent comprising the cell activating agent according to any one of 1 to 5 above.
9. A skin texture regulator comprising the cell activating agent according to any one of 1 to 5 above.
10. A skin wrinkle preventing / suppressing agent comprising the cell activating agent according to any one of 1 to 5 above.

According to the present invention, it is possible to expect a cell activation action and collagen production action excellent in safety, and cosmetics and quasi-drugs (skin external preparations, bath preparations, hair restorers, etc.), pharmaceuticals, and foods and drinks containing cellobiose lipid as an active ingredient. Can be provided.

It is a graph which shows the cell activation effect | action of CL using a normal human skin fibroblast. It is a graph which shows the collagen production promotion effect of CL using a normal human skin fibroblast.

[Activator]
In this specification, “activation” is intended to maintain or enhance cell function or cell activity. As a result, it is possible to help maintain cell function and cell activity homeostasis, and to suppress aging of cells. Therefore, “activator” is synonymous with “cell activator” and has utility as “anti-aging agent”.

For example, “activation, anti-aging” in skin cells refers to skin wrinkles and sagging by reducing functional deterioration and metabolic abnormalities of skin cells that accompany the accumulation of structural changes in the underlying membrane due to aging and photoaging. This refers to the prevention and improvement of curing, etc., and maintaining the state of elasticity and youthful healthy skin. In addition, for example, in fibroblasts, the effects of external factors such as ultraviolet rays, significant air drying, excessive skin washing, stress, smoking, etc., and prevention of decreased fibroblast proliferation due to aging, skin elasticity or It refers to the reduction of elasticity, the prevention or improvement of skin wrinkles or sagging. Furthermore, for example, in hair papilla cells or hair matrix cells, it refers to the suppression of hair loss by maintaining the hair cycle by suppressing the functional decline of hair papilla cells or hair matrix cells due to aging, stress, hormone balance, etc. .

[Promoting collagen production]
In this specification, “collagen production promotion” refers to an effect of increasing the production amount of collagen produced by cells. Collagen IV and VII are involved in the support and adhesion of epidermal cells at the boundary between the epidermis and dermis. Damage caused by aging and ultraviolet rays, especially collagen IV and VII, is reduced by ultraviolet rays, the epidermis and dermis support function is reduced, the selective permeation function is weakened, and harmful components easily affect the skin, thus generating wrinkles Is done. The extracellular matrix that makes up the dermis is made from fibroblasts in the dermis, and is composed of fibrous proteins such as collagen and elastin and polysaccharides called acidic mucopolysaccharides such as hyaluronic acid. It is directly related to sex, metabolism and vitality. When the activity of fibroblasts is lowered, particularly from aging and photoaging, collagen biosynthesis is decreased and denatured elastin is increased. Furthermore, the increase in matrix metalloproteinase MMP-1 which degrades collagen type I, which is the main component of the dermis, increases the degradation of extracellular matrix components such as collagen, causing scratches in the dermis and the epidermis-dermis boundary Is destroyed and the dermis is accelerated. Eventually, the elasticity, freshness and vitality of the skin are lost, wrinkles, wrinkles and rough skin occur, resulting in skin aging. For this reason, an anti-aging action is expected due to the effect of “collagen production promotion”.

[Cellobiose lipid]
Cellobiose lipid is a kind of glycolipid type biosurfactant having surface-active ability and emulsifying ability produced by living organisms. “Biosurfactant” is a general term for substances having surface-active ability and emulsifying ability produced by living organisms, and not only exhibits excellent surface activity and high biodegradability, but also has various physiological functions. Therefore, there is a possibility of developing different behaviors and functions from synthetic surfactants.

Cellobiose lipid (hereinafter sometimes referred to as CL) has various variations in structure, and the difference in these molecular structures is based on the difference in producing microorganisms. In the formula (I), R1 represents hydrogen or a hydroxyl group, and n1 represents 12-14. Furthermore, R2, R3 and R4 of sugar 1 in formula (I) are acetyl groups or hydroxyl groups. These can be obtained as a mixture, but a single CL compound may be used after purification and separation. Moreover, in order to improve water solubility, you may use an alkali treatment and use as a sodium salt. In formula (II), R1 and R2 each represent hydrogen or a hydroxyl group. n1 represents 11 to 12, and n2 represents 2 to 4. These can be obtained as a mixture, but may be used as a mixture even if a single CL compound is used after purification and separation. Moreover, in order to improve water solubility, you may use an alkali treatment and use as a sodium salt.

[Production of cellobiose lipid]
Cellobiose lipid can be obtained by extracting and purifying a culture solution of cellobiose lipid-producing bacteria. Examples of the CL-producing bacteria include microorganisms belonging to the genus Cryptococcus and Ustilago and having the ability to produce cellobiose lipids. Microorganisms of the genus Cryptococcus mainly produce cellobiose lipids of the above structural formula (I), and microorganisms of the genus Ustilago mainly produce cellobiose lipids of the structural formula (II).

The method for producing cellobiose lipid is not particularly limited, but a fermentation method using a known biosurfactant-producing microorganism may be arbitrarily selected. For example, cellobiose lipid can be cultured by culturing Cryptococcus humicola or Ustilago esculenta according to a conventional method. As the cellobiose lipid-producing microorganism, in addition to the above, Ustilago maydis, Pseudozyma flocculosa, Pseudozyma graminicola, and the like can be used. The biosurfactant-producing microorganism is not particularly limited and can be appropriately selected depending on the purpose.

Fermentation media for producing cellobiose lipids include yeast extracts, N sources such as peptone, C sources such as glucose and fructose, and inorganic nitrogen sources such as sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, etc. A medium having a general composition composed of inorganic salts can be used.

Fermentation conditions such as pH and temperature, culture time, and the like can be arbitrarily set, and the culture solution after fermentation can be used as it is as the biosurfactant of the present invention. In addition, it is possible to appropriately add any operation such as filtration, centrifugation, extraction, purification, sterilization and the like to the culture solution after fermentation, and the obtained extract can be diluted, concentrated and dried. .

There is no restriction | limiting in particular as an inorganic nitrogen source, Although it can select suitably according to the objective, For example, ammonium nitrate, urea, sodium nitrate, ammonium chloride, ammonium sulfate, etc. are mentioned.

There is no particular limitation on the method for recovering and purifying cellobiose lipid, and it can be appropriately selected according to the purpose. For example, it can be recovered by centrifuging the culture solution to recover the oil, and extracting and concentrating with an organic solvent such as ethyl acetate.

As an extraction solvent, use an organic solvent such as water, alcohols, ketones, diethyl ether, dioxane, acetonitrile, esters, xylene, benzene, chloroform, alone or in any combination of two or more kinds. A combination of the respective solvent extracts can also be used.

There are no particular limitations on the extraction method, but usually it may be in the range of the boiling point of the solvent from room temperature to normal pressure. After extraction, it is filtered or adsorbed, decolored and purified using an ion exchange resin to form a solution. , Paste, gel, and powder. In many cases, it can be used as it is, but if necessary, further purification treatment such as deodorization and decolorization may be added as long as the effect is not affected. As a purification treatment means such as deodorization and decolorization, an activated carbon column or the like may be used, and a normal means generally applied depending on the extracted substance may be arbitrarily selected. If necessary, a cellobiose lipid with high purity can be obtained by purification using a silica gel column. *

Although the cellobiose lipid obtained as described above can be used as an activator as it is, it is preferably used by being blended in cosmetics, quasi drugs, pharmaceuticals, and foods. The concentration of the cellobiose lipid is appropriately selected depending on the degree of absorption, the degree of action, the product form, the frequency of use, etc., and is not particularly limited, but is usually 0.00001 wt% to 0.1 wt%, preferably It is 0.0005 wt% to 0.05 wt%, more preferably 0.0001 wt% to 0.01 wt%.

The usage form of the cellobiose lipid to be blended with the activator is arbitrary. For example, cellobiose lipid can be used as an extract from a culture solution or as a purified high-purity product. Since cellobiose lipid is highly hydrophobic, it is preferably dissolved in a nonionic surfactant, lower alcohol or polyhydric alcohol. Alternatively, a sodium salt of cellobiose lipid that is easily dissolved in water may be used.

[Skin protectant]
Fibroblasts are activated by using cellobiose lipid which is an active ingredient of the activator according to the present invention. Fibroblast activation normalizes epidermal cells and restores or restores skin barrier function and turnover, and is influenced by external factors such as ultraviolet rays, significant air drying, excessive skin washing, stress, and smoking. It is possible to prevent or improve a decrease in elasticity or elasticity of the skin due to aging, and to protect the skin from wrinkles or sagging of the skin.

[Skin roughening agent]
Activation of fibroblasts can be promoted by using cellobiose lipid which is an active ingredient of the activator according to the present invention. With the activation of fibroblasts, the production of natural moisturizing factors such as hyaluronic acid is promoted, leading to improvement of rough and dry skin.

The activator according to the present invention is more preferably carried out in the form of a composition by blending cellobiose lipid, which is an active ingredient, into cosmetics, quasi drugs, pharmaceuticals, and foods.

[Skin texture regulator]
“Skin texture” refers to irregularities formed by depressions (skins) and swells (skins) on the surface of the epidermis. “Skin texture” means that the width of the skin groove is narrow and the skin is flat and aligned, and it is determined by the width and spacing of the skin groove. They are composed of the thickness and hardness of the epidermis and the elasticity of the intercellular matrix. If this component becomes non-uniform with aging or photoaging, the regularity of the texture is impaired. Collagen is involved in the elasticity of the intercellular matrix. By using cellobiose lipid which is an active ingredient for promoting collagen production according to the present invention, the ability to produce collagen is activated, the elasticity of the intercellular matrix is improved, and the texture is adjusted to a normal state. When the texture is adjusted, the light scattering effect is enhanced, and the dullness of the skin disappears and the skin tone becomes brighter.

[Skin Wrinkle Prevention / Inhibitor]
“Skin wrinkle prevention / suppression” refers to the effect of preventing wrinkle formation associated with aging and photoaging. By using the cellobiose lipid according to the present invention, wrinkle is prevented and suppressed by promoting the ability of cells to produce collagen. The generation of wrinkles is due to damage to the dermis cells accompanying irradiation of ultraviolet rays, destruction of the structure of the dermis composed of elastin and collagen due to the effects of aging, and a decrease in production thereof.
[Usage form of the present invention]

When implemented in the form of cosmetics, quasi-drugs, and pharmaceuticals, it is preferable to use external preparations. However, since biosurfactants can be taken orally, they are not limited to external preparations, and may be internal preparations and foods and drinks.

The dosage form is not limited and can be various such as ampules, capsules, powders, granules, pills, tablets, solids, liquids, gels, bubbles, emulsions, creams, ointments, sheets, mousses, bath preparations, etc. .

Cosmetics, quasi-drugs, and pharmaceuticals include, for example, basic and cosmetic preparations for internal and external use, lotions, emulsions, creams, ointments, lotions, oils, packs, facial cleansers and skin cleansers, shampoos, rinses, Hair treatments, hair creams, pomades, hair sprays, hair preparations, permanents, hair nickings, hair dyes, hair cosmetics such as hair growth and hair restorations, foundations, white powder, funny, lipstick, blusher, eye shadow, eyeliner, mascara Makeup cosmetics such as eyebrows and eyelashes, cosmetics for finishing such as beauty nails, perfumes, bath preparations, toothpastes, mouth fresheners, gargles, liquid odors and deodorants, sanitary products, sanitary cotton And wet tissue.

Examples of the food and drink include beverages such as soft drinks, carbonated drinks, nutrition drinks, fruit drinks, and lactic acid drinks, various forms of health nutrition supplements, health functional foods, tablets, capsules, drinks, troches, and the like. .

The activator according to the present invention is suitably applied to humans, but can also be applied to animals other than humans as long as each effect can be expected.

In addition to cellobiose lipid, which is an active ingredient, the activator according to the present invention includes ingredients used in cosmetics, quasi-drugs, pharmaceuticals, foods and drinks, as long as the effects of the present invention are not impaired as necessary. Additives can be used in combination.

For example, fats and oils, waxes, mineral oils, fatty acids, alcohols, esters, metal soaps, gums and water-soluble polymer compounds, surfactants, vitamins, amino acids, whitening agents, moisturizers , Hair restorer, animal or plant, herbal extracts or extracts, microbial culture metabolites, α-hydroxy acids, inorganic pigments, UV absorbers, astringents, antioxidants, anti-inflammatory agents, bactericides / disinfectants, hair Agents, fragrances, pigments / colorants, sweeteners, nutrition enhancers, hormones, sequestering agents, pH adjusters, chelating agents, antiseptic / antibacterial agents, cooling agents, stabilizers, emulsifiers, animal / plant properties Protein and its degradation products, animal and plant polysaccharides and their degradation products, animal and plant glycoproteins and their degradation products, blood flow promoters, anti-inflammatory agents, antiallergic agents, cell activators, keratolytic agents, wound treatment agents, foam increase Agent, thickener, oral preparation Deodorant and deodorizing agent, bittering agent, seasonings, such as enzymes and the like.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

[Example 1: Production of cellobiose lipid (CL) using Cryptococcus humicola]
Inoculum culture was performed by inoculating Cryptococcus humicola NBRC 10251 colonies into a seed medium (50 mL / 500 mL Sakaguchi flask) for 1 loop. Cultured overnight at 27 ° C. The obtained culture broth was used as an inoculum. The seed medium composition was 10 g / L glucose, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.03 g / L sodium dihydrogen phosphate 0.1 g / L yeast extract. The culture was carried out by inoculating 60 mL of the above-mentioned inoculum into 6 L (10 L-jar) of the production medium and using 10 L-jar under the conditions of 27 ° C., 530 rpm (stirring rotation), and 3 L / min (Air). The composition of the production medium was 10 g / L glucose, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.03 g / L sodium dihydrogen phosphate 0.1 g / L yeast extract. The culture solution cultured for 6 days was centrifuged (7500 rpm, 20 min) to separate the cells and the supernatant. The cells were added with an amount of ethyl acetate: acetone = 4: 1 and stirred, followed by CL extraction. The precipitate was separated into a supernatant and the supernatant was dried with an evaporator to obtain a powder. In order to further remove lipid impurities from the obtained powder, hexane (300 g) was added and stirred, followed by glass filter filtration and evaporation to remove hexane. Thereby, a mixture of CL was obtained.

[Example 2: Production of cellobiose lipid (CL) using Ustilago esculenta]
Inoculum culture was performed by inoculating a colony of Ustilago esculenta NBRC 9887 into a seed medium (50 mL / 500 mL Sakaguchi flask) for 1 loop. Cultured overnight at 27 ° C. The obtained culture broth was used as an inoculum. Seed medium composition is 50 g / L glucose, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.025 g / L potassium dihydrogen phosphate 0.5 g / L dipotassium hydrogen phosphate, 8 g / L L Yeast extract. For the culture, 100 mL of the above inoculum was inoculated into 6 L (10 L-jar) of the production medium, and cultured using 10 L-jar under the conditions of 27 ° C., 530 rpm (stirring rotation), and 3 L / min (Air). Production medium composition is 50 g / L glucose, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.025 g / L potassium dihydrogen phosphate 0.5 g / L dipotassium hydrogen phosphate, 8 g / L Yeast extract. The culture solution cultured for 6 days was centrifuged (7500 rpm, 20 min) to separate the cells and the supernatant. The same amount of ethyl acetate: acetone = 4: 1 as the cells was added, and after sufficient stirring, CL extraction was performed. The precipitate was separated into a supernatant and the supernatant was dried with an evaporator to obtain a powder. In order to further remove lipid impurities from the obtained powder, hexane (300 g) was added, and after stirring, glass filter filtration and evaporation were performed to remove hexane. Thereby, a mixture of CL was obtained.

[Example 3: Na chloride of cellobiose lipid]
To 4 g of the CL obtained in Example 1 or 2, 20 mL of distilled water was added and stirred. 1N NaOH is added dropwise to this solution,
The preparation was made so that the pH was 7-8. For powdering, water was removed with an evaporator and ethanol was further added and dried.

[Example 4: Structural analysis of cellobiose lipid]
CLs obtained from Cryptococcus humicola and Ustilago esculenta were each identified by 1H-NMR measurement (proton nuclear magnetic resonance spectroscopy) at a resonance frequency of 500 MHz. The measuring apparatus used was an NMR instrument AVANCE 500 manufactured by BRUKER, and deuterated chloroform (CDCl 3) and deuterated methanol (CD ₃ OD) were used as solvents. As a result, it was found that all the powders obtained from these strains were CL. Formulas 3 and 4 show general formulas. Chemical formula 3 shows the main structure of CL derived from Cryptococcus humicola, and chemical formula 4 shows the main structure of CL obtained from Ustilago esculenta. For each representative structure, the assigned chemical shift is shown when the chloroform peak is 7.24 ppm in the measurement using deuterated chloroform and the methyl proton peak in methanol is 3.30 ppm in the measurement using deuterated methanol. The results are shown in Table 1 and Table 2. Table 1 shows n1 = 14 and R1 = OH in the formula (III), and Table 2 shows n1 = 12, R1 = OH and R2 = OH in the formula (IV).

(In Formula (III), R1 represents hydrogen or a hydroxyl group, n1 has 12 or 14 carbon atoms, and R2, R3, and R4 of Sugar 1 in Formula I are an acetyl group or a hydroxyl group. Is shown.)

(In formula (IV), R1 and R2 each represent hydrogen or a hydroxyl group. N1 represents an alkylene group having 11 or 12 carbon atoms, and n2 represents an alkylene group having 2 or 4 carbon atoms.)

[Example 5: Cell activation effect of cellobiose lipid using normal human skin fibroblasts]
Normal human skin fibroblasts were seeded in a 48-well microplate so as to be 2.0 × 10 ⁴ per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. After culturing at 37 ° C. in a carbon dioxide concentration of 5% for 24 hours, CL-Na salt was added to the test medium to a final concentration of 100 μg / mL to 1 μg / mL, and further cultured for 72 hours.
Distilled water was provided as a solvent control. Next, 25 μL of the viable cell count measurement reagent SF was added and cultured for 3 hours, and the absorbance at 450 nm was measured with a microplate reader.
At the same time, the absorbance at 650 nm was measured as turbidity, and the cell activation effect was evaluated by the difference between the two measured values.

The evaluation results are shown in FIG. 1 as relative values with the cell activation effect in distilled water as 100.
As shown in FIG. 1, when CL-Na salt was added at a final concentration of 100 μg / mL to 10 μg / mL, human normal skin fibroblasts showed a higher cell activation effect than distilled water. In particular, when CL was added at a final concentration of 25 μg / mL, a significant cell activation effect of 30% or more than that of distilled water was observed. From this result, it is clear that CL has an excellent cell activating effect, and by applying CL to the skin, the metabolism and regeneration ability of skin cells are improved, and accordingly, the skin which is caused by aging, UV exposure, etc. It was suggested that wrinkles and sagging can be effectively improved.

[Example 6: Evaluation of collagen production ability by CL-Na salt]
The CL-Na salt powder described in Example 3 was dissolved in distilled water, and the final CL-Na salt concentrations were 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL, respectively. A cellobiose lipid aqueous solution of 1 μg / mL was prepared, and changes in collagen production in normal human skin fibroblasts were evaluated. As a control, only distilled water was used and evaluated in the same manner. The evaluation was performed according to the following procedure. Normal human skin fibroblasts were seeded in a 48-well microplate so that the number of normal human skin fibroblasts was 1.0 × 10 ⁴ per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 1% fetal bovine serum. After culturing at 37 ° C. in a carbon dioxide concentration of 5 vol% for 24 hours, after washing twice with PBS (−), the medium was replaced with a serum-free medium to which a sample of any concentration was added, and the conditions were the same for 3 days and 6 days. Incubated in From the culture supernatant, type I procollagen C-terminal peptide (Procollagen type I carboxyterminal propeptide: PIP) produced by human fibroblasts was measured with Procollagen type I C-peptide (PIP) EIA Kit (TaKaRa). The collagen production acceleration rate was determined by measuring a standard product with the above-mentioned ELISA kit, creating a calibration curve from the results, and obtaining the collagen production amount when the sample was added and the collagen production amount when no sample was added from the calibration curve.

2. From FIG. 2, when CL-Na salt was added at a final concentration of 100 μg / mL to 10 μg / mL, it showed a higher collagen production promoting effect than normal distilled water on human normal skin fibroblasts. In particular, when CL was added at a final concentration of 50 μg / mL, a significant collagen production promoting effect of 260% or more was observed compared to distilled water. From this result, it became clear that CL has an excellent collagen production promoting action.

Collagen is produced by fibroblasts. However, while the activation activity of fibroblasts by CL in FIG. 1 is 100 to 130%, the collagen production rate by CL in FIG. 2 shows an extremely high value of 160% to 260%. Therefore, this result shows that not only the ability to produce collagen was improved with the ability to activate fibroblasts by CL, but also that collagen production was promoted by the action of CL on any pathway in the metabolic system of collagen. Is suggested.

[Example 7: Production example of serum]
A cosmetic liquid having the following composition was produced by a conventional method.
(Composition) (wt%)
Citric acid 0.01
Na citrate 0.04
CL-Na salt 0.5
1.3. Butylene glycol 5.0
Concentrated glycerin 2.5
1.2. Pentanediol 2.0
Phenoxyethanol 0.25
Total amount of purified water is 100

[Example 8: Production example of emulsion]
An emulsion having the composition shown below was produced by a conventional method.
(Composition) (wt%)
Glycerol ether 1.5
CL-Na salt 0.01
Sucrose fatty acid ester 1.5
Sorbitan monostearate 1.0
Squalane 7.5
Dipropylene glycol 5.0
Total amount of purified water is 100

[Example 9: Production example of cream]
A cream having the following composition was produced by a conventional method.
(Composition) (wt%)
ε-aminocaproic acid 0.2
CL-Na salt 0.1
Methyl paraoxybenzoate 0.5
Phenoxyethanol 0.2
1,3-butylene glycol 7.5
MEL 0.1
Cetanol 2.5
Behenyl alcohol 3.0
Squalane 5.0
Tri (caprylic / capric) glyceryl 15.0
Polyglyceryl pentastearate-10 0.95
Stearoyl lactate Na 0.3
Total amount of purified water is 100

[Example 10: Production example of face wash]
A face wash having the composition shown below was produced by a conventional method.
(Composition) (wt%)
Edetate disodium 0.05
ε-aminocaproic acid 0.2
Concentrated glycerin 1.5
CL-Na salt 0.1
Methyl paraoxybenzoate 0.15
1,3-butylene glycol 4.5
2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine (30% aqueous solution)
3.0
N-coconut oil fatty acid acyl-DL-alanine triethanolamine solution (30% aqueous solution)
30.0
Phenoxyethanol 0.5
Total amount of purified water is 100

According to the present invention, a biosurfactant-derived safety-enhanced cell activation effect, collagen production promoting effect, and anti-aging effect can be expected, and cosmetics and quasi-drugs containing these as active ingredients (skin external preparations, bath preparations, It is expected to make a significant contribution to the industry because it can provide hair restorers, food and drinks, and pharmaceuticals.

Claims

A cell activating agent comprising cellobiose lipid.
The cell activation agent according to claim 1, wherein the cell activation is activation of skin cells.
The cell activation agent according to claim 1, wherein the cell activation is activation of fibroblasts, hair matrix cells or hair papilla cells.
The cell activation agent according to claim 1, wherein the cell activation is promotion of collagen production.
The cell activating agent according to any one of claims 1 to 4, comprising a cellobiose lipid having a structure represented by the following general formula (I) or (II).

(In formula (I), R1 represents hydrogen or a hydroxyl group, n1 has 12 or 14 carbon atoms, and R2, R3, and R4 of sugar 1 in formula I are an acetyl group or a hydroxyl group. Is shown.)

(In formula (II), R1 and R2 each represent hydrogen or a hydroxyl group. N1 represents an alkylene group having 11 or 12 carbon atoms, and n2 represents an alkylene group having 2 or 4 carbon atoms.)
An anti-aging agent comprising the cell activator according to any one of claims 1 to 5.
A skin protecting agent comprising the cell activating agent according to any one of claims 1 to 5.
A skin roughness improving agent comprising the cell activating agent according to any one of claims 1 to 5.
A skin texture regulator comprising the cell activating agent according to any one of claims 1 to 5.
A skin wrinkle preventing / suppressing agent comprising the cell activating agent according to any one of claims 1 to 5.