JP5548561B2 - Skin fibroblast growth promoter and skin collagen production promoter - Google Patents

Description

  The present invention relates to a dermal fibroblast proliferation promoter and a skin collagen production promoter containing apicin contained in royal jelly as an active ingredient.

  Generally, royal jelly is a jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and the greater vagina by female honeybees 3 to 15 days after emergence, and contains specific proteins, fatty acids, minerals, etc. It is known that Royal jelly is known to have various physiological actions such as a blood pressure lowering action, an antitumor action, and an antibacterial action. Thus, royal jelly has been used not only for health foods with high nutritional value but also for uses such as pharmaceuticals.

  Royal jelly extract contains fatty acids peculiar to royal jelly such as decenoic acid, lipids composed of esters thereof, special water-soluble proteins contained only in royal jelly, amino acids, carbohydrates, minerals and the like. Among them, proteins account for 10% or more in royal jelly, but their detailed functions and physiological actions have not been fully elucidated.

  Conventionally, apicin is known as a glycoprotein having a molecular weight of 350 kD as a protein contained in royal jelly (Non-patent Document 1). Apisin is known to have a proliferative action on human blood cell monocytic cell lines (Non-patent Document 1).

Bee Science 19 (1): 15-22 (1998)

This invention is made | formed by discovering that an apicin exhibits a skin fibroblast proliferation promoting effect and a skin collagen production promoting effect.
It is an object of the present invention is to provide a cosmetic, skin fibroblasts growth promoting agent can be used in various applications, such as pharmaceuticals and skin collagen production promoter.

In order to achieve the above object, the skin fibroblast growth promoter according to claim 1 contains , as an active ingredient, apicin isolated from royal jelly or apicin obtained by a genetic engineering technique. , it applied pharmaceuticals, or as a research reagent, characterized in Rukoto.
According to a second aspect of the invention, the dermal fibroblast proliferation promoting agent according to claim 1, separating from the royal jelly, and characterized in that it comprises the step of separating the components by using a liquid chromatography To do.

The skin collagen production promoter according to claim 3 contains apicin isolated from royal jelly or apicin obtained by a genetic engineering technique as an active ingredient, and is applied as a skin external preparation, cosmetic, pharmaceutical, or research reagent. It is characterized by that.
According to a fourth aspect of the invention, the skin collagen production promoter according to claim 3, separating from the royal jelly, characterized in that it comprises a step of separating the components by a liquid chromatography.

According to the present invention, it is possible to provide a cosmetic, skin fibroblasts growth promoting agent can be used in various applications, such as pharmaceuticals and skin collagen production promoter.

The test result regarding the growth promotion effect | action of the human skin fibroblast of an apicin is shown. The test result regarding the collagen production promotion effect of human skin fibroblasts of apicin is shown.

(First embodiment)
Hereinafter, a first embodiment in which the dermal fibroblast proliferation promoter of the present invention is embodied will be described.
The skin fibroblast proliferation promoter of this embodiment contains apicin as an active ingredient. Apicin is a water-soluble glycoprotein contained in royal jelly, and is a hetero-oligomeric protein having a molecular weight of about 350 kDa, in which two molecules of two types of 58 kDa are associated with each other.

  Apicin can be obtained using royal jelly as a raw material. As the raw royal jelly, any of raw royal jelly and royal jelly powder (FD-RJ) obtained by drying and pulverizing raw royal jelly by freeze drying or the like can be used. The production area of the raw royal jelly and the dried royal jelly used in this embodiment is not particularly limited. For example, any of Asian countries such as China, Taiwan, and Japan, European countries, North American countries, South American countries such as Brazil, and Oceania countries But you can.

  Apicin is a water-soluble glycoprotein having a molecular weight of about 350 kD, and can be extracted and purified by using known extraction, separation and purification methods using royal jelly as an extraction raw material. Specifically, an extraction process using a solvent from an extraction raw material, a separation process using ultrafiltration and dialysis, and a separation / purification process using chromatography.

  In the extraction process, as a solvent used for extraction, for example, water, a hydrophilic organic solvent, and a mixed solution of water / hydrophilic organic solvent are used. Among these, water is preferable from the viewpoint of excellent extraction efficiency of apicin. Water is preferably used as a buffer solution to which various acid salts are added in order to further increase the extraction efficiency. Examples of the buffer solution include a phosphate buffer solution and an acetate buffer solution. Examples of the hydrophilic organic solvent include lower alcohols such as ethanol, methanol, and isopropanol, ketones such as acetone and methyl ethyl ketone, and ethyl acetate.

  As the chromatography, known chromatography such as liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. However, since apicin is a protein component, liquid chromatography is preferably applied. As liquid chromatography, column chromatography can be used, for example, More specifically, high performance liquid chromatography (HPLC) and open column chromatography can be mentioned. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (normal phase / reverse phase chromatography), adsorption chromatography, and molecular exclusion chromatography (gel filtration / molecular sieve chromatography). Since apicin has an isoelectric point of 4.4 to 5.4 and is an acidic protein, an anion exchange resin is preferably used as ion exchange chromatography. Examples of the anion exchange resin include DEAE cellulose and Q sepharose. By appropriately combining them, the apicin component can be separated and purified by a known method of use.

  The dermal fibroblast proliferation promoter of this embodiment has the effect of promoting the proliferation of the fibroblasts of the skin or prolonging the life. By promoting the proliferation of dermal fibroblasts, it supports the extracellular matrix produced by dermal fibroblasts, such as collagen that contributes to the elasticity of the skin, hyaluronic acid, a moisturizing component, and collagen fibers. An increase in contributing elastin is expected. It is known that fibroblasts decrease with aging or when the skin is exposed to ultraviolet rays and active oxygen. This also reduces the extracellular matrix such as collagen brought about by fibroblasts, resulting in aging phenomena such as a decrease in dermis thickness, the occurrence of wrinkles and sagging of the skin, disappearance of the beam, and a decrease in moisture retention. When the proliferation of skin fibroblasts is promoted by the dermal fibroblast proliferation promoter of this embodiment, it is expected to prevent wrinkles and sagging, to prevent the disappearance of beams, and to improve the moisture retention of the skin. It is effective in preventing and improving skin aging. Therefore, the skin fibroblast proliferation promoter of this embodiment is expected to be applied as an anti-aging or improving agent for skin.

  It is also known that when skin tissue is damaged, fibroblasts migrate and proliferate from the damaged site, and the damaged site is repaired by synthesis of the extracellular matrix. When the proliferation of skin fibroblasts is promoted by the dermal fibroblast proliferation promoter of this embodiment, it is expected that damage repair of skin tissue is promoted and healing is accelerated. Therefore, it is expected that the skin fibroblast proliferation promoter of this embodiment can be applied as a skin wound healing promoter.

Specific blending forms of the dermal fibroblast growth promoter of this embodiment include skin external preparations, cosmetics, pharmaceuticals, research reagents, and foods and drinks as reference examples for the purpose of obtaining the above-described effects. (Hereinafter, the configuration of the food and drink is a reference example) . Among these, the dermal fibroblast proliferation promoter of the present embodiment is preferably applied to the skin surface (skin) as a skin external preparation or cosmetic. When applying the skin fibroblast proliferation promoter of the present embodiment as an external preparation for skin, cosmetics, and foods and drinks, in order to distinguish it from conventional products, the above-mentioned actions and effects, for example, promotion of proliferation of skin fibroblasts, It is preferable to indicate that the purpose is to obtain effects such as prevention of wrinkles and sagging, prevention of beam disappearance, improvement of skin moisture retention, prevention and improvement of aging, and promotion of skin wound healing.

  When applying the dermal fibroblast proliferation promoter of this embodiment to cosmetics, it can manufacture by mix | blending with a cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. By applying such cosmetics to the skin, it is possible to obtain a skin fibroblast proliferation promoting action. Cosmetic bases are generally blended in common with cosmetics. For example, oil, purified water, and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-seborrheic agents. At least one selected from an agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately blended.

  When the dermal fibroblast proliferation promoter of this embodiment is applied to a food or drink, the dermal fibroblast proliferation promoter can be used as a food or drink itself or by blending it with various food materials or beverage materials. . The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, and solid, and the dosage form is any of tablets, capsules, granules, and drinks. May be. Among these, a capsule is preferable because hygroscopicity is suppressed. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  When the dermal fibroblast proliferation promoter of this embodiment is used as a pharmaceutical material or medicine, it can be administered by application to the skin, taken (orally ingested), subcutaneous injection, intravascular administration, and transdermal administration. Any administration method can be employed. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive. In addition, the dermal fibroblast proliferation promoter of the present embodiment may be applied as an experimental / research reagent in the form of a dermal fibroblast proliferation promoting reagent. It is suitably used in fields such as elucidation of physiological mechanisms related to skin fibroblasts or research and development of treatment methods for various symptoms.

  The dose of the dermal fibroblast growth promoter is not particularly limited, but from the viewpoint of exerting excellent efficacy, the concentration of apicin contained in the royal jelly (approximately 20 as solid content) is used as the concentration of apicin as the active ingredient. It is preferable to administer at a concentration (ratio) higher than (mass%).

The effects exhibited by this embodiment will be described below.
(1) The dermal fibroblast proliferation promoter of this embodiment exhibits an excellent dermal fibroblast proliferation promoting action. Therefore, it can be preferably applied to the fields of external preparations for skin, cosmetics, pharmaceuticals, research reagents, and foods and drinks for the purpose of demonstrating the dermal fibroblast proliferation promoting action.

  (2) Preferably, in the dermal fibroblast growth promoter of the present embodiment, apicin, which is an active ingredient, is separated from royal jelly as a naturally derived raw material. Therefore, it can be safely applied to fields such as external preparations for skin, cosmetics, pharmaceuticals, research reagents, and foods and drinks. Moreover, apicin can be administered at a higher concentration (ratio) than when royal jelly is administered as it is.

  (3) Preferably, in the dermal fibroblast growth promoter of this embodiment, the concentration (ratio) of apicin as an active ingredient is administered at a concentration higher than the concentration (ratio) of apicin contained in royal jelly. The Therefore, various superior effects related to the promotion of dermal fibroblast proliferation can be exhibited.

(Second Embodiment)
Hereinafter, a second embodiment in which the skin collagen production promoter of the present invention is embodied will be described. The skin collagen production promoter of this embodiment contains apicin as an active ingredient. The method for obtaining apicin is the same as in the first embodiment.

  The skin collagen production promoter of this embodiment has an action of promoting collagen production in the skin, particularly in fibroblasts. It is known that fibroblasts decrease with aging, or skin collagen decreases by denaturation with ultraviolet rays and active oxygen. This causes aging phenomena such as a reduction in the thickness of the dermis, the occurrence of wrinkles and sagging of the skin, and the disappearance of the beam. When skin collagen production is promoted by the skin collagen production promoter of this embodiment, it is expected to prevent wrinkles and sagging and to prevent the disappearance of a beam, and exhibits excellent skin aging prevention and improvement effects. . Therefore, the skin collagen production promoter of this embodiment is expected to be applied as an anti-aging or improving agent for skin.

  In addition, when skin tissue is damaged, it is known that fibroblasts synthesize collagen at the damaged site, thereby repairing the damaged site. When skin collagen production is promoted by the skin collagen production promoter of the present embodiment, it is expected to promote damage repair of skin tissue and accelerate healing. Therefore, the skin collagen production promoter of this embodiment is expected to be applicable as a skin wound healing promoter.

  As a specific blending form of the skin collagen production promoter of the present embodiment, it can be applied as a skin external preparation, cosmetics, pharmaceuticals, research reagents, foods and drinks for the purpose of obtaining the above-mentioned effects. it can. Among these, it is preferable that the skin collagen production promoter of this embodiment is applied to the skin surface (skin) as a skin external preparation or cosmetic. When applying the skin collagen production promoter of this embodiment as an external preparation for skin, cosmetics, and foods and drinks, in order to distinguish it from conventional products, the above actions and effects, for example, promotion of collagen production in skin, prevention of wrinkles and sagging It is preferable to attach a sign indicating that the purpose is to obtain effects such as prevention of loss of beam, prevention / improvement of aging, and promotion of skin wound healing. Other specific blending forms are the same as those in the first embodiment.

  The dose of the skin collagen production promoter is not particularly limited, but from the viewpoint of exerting excellent efficacy, the concentration of apicin as the active ingredient is the concentration of apicin contained in the royal jelly (about 20% by mass as the solid content). ) Is preferably administered at a higher concentration (ratio).

The effects exhibited by this embodiment will be described below.
(1) The skin collagen production promoter of this embodiment exhibits an excellent skin collagen production promoting action. Therefore, it can be preferably applied to the fields of external preparations for skin, cosmetics, pharmaceuticals, research reagents, foods and drinks, etc. for the purpose of exerting a skin collagen production promoting effect.

  (2) Preferably, in the skin collagen production promoter of this embodiment, apicin, which is an active ingredient, is separated from royal jelly as a naturally derived raw material. Therefore, it can be safely applied to fields such as external preparations for skin, cosmetics, pharmaceuticals, research reagents, and foods and drinks. Moreover, apicin can be administered at a higher concentration (ratio) than when royal jelly is administered as it is.

  (3) Preferably, in the skin collagen production promoter of the present embodiment, the concentration of apicin that is an active ingredient is administered at a concentration (ratio) higher than the concentration (ratio) of apicin contained in royal jelly. Therefore, various superior effects related to the promotion of skin collagen production can be exhibited.

In addition, you may change the said embodiment as follows.
-Apicin which is an active ingredient in the above-described embodiment may be any one of those roughly extracted from royal jelly, roughly purified, and purified.

  -Apicin which is an active ingredient in the embodiment is preferably obtained by extracting royal jelly as an extraction raw material. However, in addition to the extraction method from natural products, it may be obtained by a known genetic engineering technique. In order to obtain the apicin of this embodiment by a genetic engineering technique, it can be produced using a known in vitro protein synthesis system. Specifically, it is obtained by incorporating a gene consisting of a base sequence encoding apicin into any known vector, introducing the vector into a host to produce a transformant, and culturing the transformant. Can do.

  Examples are given below to describe the above embodiments more specifically, but the present invention is not limited to these examples. In this example, the effects of apicin on the promotion of human skin fibroblast proliferation and collagen production were tested and examined.

(1) Preparation of apicin Chinese raw royal jelly (raw RJ) was diluted 2-fold with distilled water and dialyzed against a 50 mM sodium phosphate buffer (pH 6.2) with a dialysis membrane with a molecular weight cut off of 10000 for 48 hours. went. Thereafter, the mixture was centrifuged (15000 × g, 60 minutes, 4 ° C.), and the resulting supernatant was used as a royal jelly soluble protein. This is added to a DEAE-Cellulofine A-500 column (manufactured by Seikagaku Corporation) and the non-adsorbed fraction is eluted with the same buffer, and then the NaCl concentration is linearly increased from 0 to 0.3M. Thus, the adsorbed fraction was eluted and separated from the royal jelly. The flow rate was 90 mL / hour, and the protein was detected by ultraviolet absorption at 280 nm. The protein concentration was measured using a DC protein assay (Bio-Rad Laboratories). The fraction eluted as described above was used as an apicin-containing fraction (apicin content of about 80% or more (in the solid content)) in the following test using fibroblasts. In addition, freeze-dried raw royal jelly from China was used as a comparison target.

(2) Human skin fibroblasts NB1RGB strain was used as human skin fibroblasts. The NB1RGB strain was purchased from RIKEN. The NB1RGB strain was cultured in DMEM containing 10% FBS, 100 units / mL penicillin and 0.1 mg / mL streptomycin under the conditions of 37 ° C. and 5% CO ₂ . Cell passage was performed by trypsin treatment every 3-4 days.

(3) Examination of human skin fibroblast proliferation promoting action and collagen production promoting action NB1RGB strain was seeded at a density of 10,000 cells / well in a 96-well plate, and was cultivated under conditions of 37 ° C. and 5% CO _2. Cultured overnight. Cells were washed twice with serum-free DMEM and replaced with this medium. The test samples, apicin and royal jelly, were dissolved in DMEM and filtered. Apicin was added so that the concentration in the culture solution was 20 μg / mL, 100 μg / mL, and 300 μg / mL, and royal jelly was added so that the solid content concentration was 100 μg / mL. In addition, vitamin C glycoside (AA-2G, manufactured by Hayashibara Biochemical Laboratories) essential as a reducing agent in the production of collagen by fibroblasts was added simultaneously with each test sample at a concentration of 250 μM. A group to which no vitamin C glycoside was added was used as a control group. Moreover, only the vitamin C glycoside is added, and each test sample is not added to the 0 μg / mL group. Cell viability and collagen production were measured 96 hours after adding each test sample. The cell viability was calculated from the amount of formazan produced by reduction with intracellular dehydrogenase using Cell Counting kit-8 (WST-8) (Dojin Kagaku). The cell viability of each test sample was determined as a value (%) when the control was 100%. Formazan was measured by ultraviolet absorption at 492 nm. The amount of collagen produced was determined by quantifying the amount of type I collagen secreted into the cell culture supernatant using the Procollagen type I C-peptide (PIP) EIA Kit (manufactured by Takara). The PIP EIA kit was developed using a monoclonal antibody specific for type I procollagen C-terminal propeptide (PIP) and measured by ultraviolet absorption at 450 nm. The collagen production amount of each test sample was determined as a value (%) when the control was 100%. The result of cell viability is shown in FIG. 1, and the result of collagen production is shown in FIG. All experimental results were expressed as mean ± standard deviation, and a significance level of 5% or less was considered significant. Statistical analysis was performed by analysis of variance (Dunnet method).

(4) Results After adding apicin and examining the fibroblast proliferation action from day 1 to day 5, the apicin addition group has a tendency to proliferate compared to the control group from day 1. On the 4th day, the most difference from the control group was observed (data not shown). From this, the cell viability and collagen production amount on the 4th day after addition of the test sample were measured.

  As shown in FIG. 1, regarding the growth of fibroblasts, the control group to which no vitamin C glycoside was added and the 0 μg / mL group to which only the vitamin C glycoside was added without adding each test sample. There was no difference in results. In the presence of vitamin C glycoside, significant cell growth promoting action was observed by adding 100 to 300 μg / mL of apicin. An increase of about 1.3-fold was observed compared to the 0 μg / mL group to which only vitamin C glycoside was added by adding 300 μg / mL of apicin. In the royal jelly 100 μg / mL administration group, no fibroblast proliferation action was observed.

  As shown in FIG. 2, regarding collagen production, the 0 μg / mL group in which each test sample was not added and only the vitamin C glycoside was added was compared with the control group in which no vitamin C glycoside was added. About 1.6 times increase in collagen production was observed. It was confirmed that the amount of collagen produced tends to increase in a concentration-dependent manner by the addition of apicin in the presence of vitamin C glycoside (p value <0.1). In the royal jelly 100 μg / mL administration group, it was confirmed that collagen production tended to be accelerated as compared with the 0 μg / mL group to which only vitamin C glycoside was added (p value <0.1).

  Next, the technical idea that can be grasped from the above embodiment and other examples will be described below. (A) The dermal fibroblast proliferation promoter and skin collagen production promoter administered at a concentration higher than the concentration of apicin contained in royal jelly. (B) A skin wound healing promoter comprising apicin as an active ingredient.

Claims (4)

The Apishin obtained by Apishin or genetic engineering techniques isolated from royal jelly containing as an active ingredient, a skin external preparation, cosmetics, pharmaceuticals, or applied as a research reagent dermal fibroblast cell growth promoting agent characterized Rukoto . It said step of separating from royal jelly, dermal fibroblast proliferation promoting agent according to claim 1, characterized in that it comprises a step of separating the components by a liquid chromatography. The Apishin obtained by Apishin or genetic engineering techniques isolated from royal jelly containing as an active ingredient, a skin external preparation, cosmetics, pharmaceuticals, or skin collagen enhancing agent characterized Rukoto applied as research reagents. It said step of separating from royal jelly, skin collagen production promoter according to claim 3, characterized in that it comprises a step of separating the components by a liquid chromatography.