JP2001302690A - Skin-permeable tripeptide, skin-permeable collagen peptide, skin-permeable care preparation and highly absorbable food - Google Patents

Abstract

(57) [Problem] To provide a skin-penetrating tripeptide, a skin-penetrating collagen peptide, a skin-penetrating external preparation and a highly absorbent food having high skin permeability and a high moisturizing effect. The skin-penetrating tripeptide has a Gly-XY amino acid sequence obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme.
Consisting of a tripeptide of The skin-permeable collagen peptide, the skin-permeable external preparation and the superabsorbent food contain a skin-permeable tripeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a skin-penetrating tripeptide, a skin-penetrating collagen peptide, a skin-penetrating external preparation, and a skin-penetrating tripeptide obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme. Related to absorbent foods.

[0002]

2. Description of the Related Art Collagen and gelatin or decomposition products obtained by treating them with acid or heat are used in various industrial fields.
It is used as a material in the area. For example, it has been used as various edible jelly materials utilizing a high quality protein having a gelling ability, as well as a material for photographic printing paper or a capsule material for medicine utilizing its protective colloid property. Furthermore, the amino acids that make up the collagen molecules often have hydrophilic side chains, and can absorb a lot of water molecules, so the moisturizing effect is high,
Based on the experience of good familiarity with the skin, it has come to be widely used as a moisturizer and a system stabilizer mainly for many external preparations such as cosmetics.

[0003]

In addition to collagen and gelatin, those commonly used as humectants include polyhydric alcohols such as glycerin and propylene glycol, urea, lanolin, hyaluronic acid, and vitamin E.
In addition to these, ceramides have recently been attracting attention.However, polyhydric alcohol has discomfort such as stickiness, urea is irritating which is thought to be caused by high osmotic pressure, and lanolin is contact dermatitis Side effects such as have been reported.

[0004] In recent years, dry skin, sensitive skin, rough skin, atopic dermatitis, and other external factors such as dryness, ultraviolet rays, and stress, and internal factors such as aging of the skin due to aging.
Furthermore, dry eyes and the like due to prolonged use of computers are increasing, and these are closely related to the moisturizing of the skin and cornea, so the development of more functional and safe moisturizers was required. .

[0005] In view of the above-mentioned circumstances, the present inventors have conducted intensive studies on collagen derivatives. As a result, the collagenase enzyme alone or immobilized on various carriers is allowed to directly act on a raw material containing a collagen component or a gelatin component to perform a specific enzymatic degradation, thereby achieving the characteristic amino acids inherent in collagen. It has been found that a peptide composition having a sequence of (Gly-XY) n and having a molecular weight range of 1,000 or less can be obtained in high yield (see JP-A-7-82299).

Further, the inventors of the present invention have conducted intensive studies and found that a peptide composition having a molecular weight in the range of about 1,000 to 20,000 can be prepared by slightly modifying the above-mentioned production process. Was. This peptide composition has an effect of stabilizing various physiologically active substances and biologically active substances, similarly to conventional collagen and gelatin and their degradation products (JP-A-9-176196, JP-A-11-176196).
-12196), and has reached the stage of practical application particularly as a vaccine stabilizer.

However, conventionally, it has been known that a water-soluble substance and a component having a high molecular weight are hardly percutaneously absorbed due to the barrier function of the stratum corneum of the skin. JP-A-9-176196,
Even with the peptide composition having a molecular weight of 20,000 or less shown in JP-A-11-12196, no moisturizing effect inside the skin could be expected.

The present invention has been made in order to solve the above problems, and has high skin permeability and a high moisturizing effect. It is intended to provide an agent and a highly absorbent food.

[0009]

Means for Solving the Problems The present inventors have found that the amino acid sequence obtained by specifically decomposing a collagen component or a gelatin component using a collagenase enzyme has a Gly-
The XY tripeptide was found to have high skin permeability.

That is, the skin-penetrating tripeptide of the present invention comprises a tripeptide having an amino acid sequence of Gly-XY obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme. It is characterized by. In the Gly-XY formula of the amino acid sequence, X and Y are any amino acid residues other than glycine (Gly);
For example, proline and hydroxyproline.

Further, the skin-penetrating collagen peptide according to the present invention is obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme, and contains the skin-penetrating tripeptide. The skin-penetrating collagen peptide according to the present invention preferably contains 10% or more of the skin-penetrating tripeptide.

The skin-penetrating external preparation according to the present invention is characterized in that it contains 0.0005% by weight or more of the skin-penetrating tripeptide. The skin-penetrating external preparation according to the present invention may be an external preparation for atopic dermatitis, a dry skin (xeroderma), a baby, a sensitive skin or a skin care for nursing care, or a cosmetic. Quasi-drugs or pharmaceuticals. As cosmetics, for example, lotion,
Examples include basic cosmetics such as milky lotions and serums, makeup cosmetics such as foundations, hair cosmetics, cleansing cosmetics, lip cosmetics, oral cosmetics, nail cosmetics, eyeliner cosmetics, bath cosmetics, suntan / sunscreen cosmetics, and the like. As pharmaceuticals or quasi-drugs, for example,
In addition to eye drops for dry eyes, medicated cosmetics, hair restorers and the like can be mentioned.

The skin-penetrating tripeptide according to the present invention comprises
It has high skin permeability and high moisturizing effect. The topical skin permeable external preparation according to the present invention is useful for improving the moisturizing power of skin or eyes with reduced moisturizing power. It also helps heal diseases such as tape rash and hemorrhoids.

The external preparation for skin penetration according to the present invention may contain urea. In this case, it is particularly useful for improving the moisturizing power of the skin having reduced moisturizing power.

The superabsorbent food according to the present invention is characterized in that it contains 0.0005% by weight or more of the skin-penetrating tripeptide. The superabsorbent food according to the present invention can be positioned as a functional food, and may be a beverage such as a health drink or a dietary supplement. Examples of healthy drinks include, for example, nutritional drinks.When the superabsorbent food according to the present invention is applied to nutritional drinks, not only nutritional supplementation but also contributes to improving the moisturizing power in the mouth and throat, It is also expected to protect the gastrointestinal mucosa and inner wall.

The highly absorbent food according to the present invention is excellent in digestibility and absorption.

The external preparation for skin penetration or the highly absorbent food according to the present invention may be a DDS preparation in which a drug is bound to the skin-penetrating tripeptide. The drug to bind is
For example, it is a physiologically active substance such as a vaccine. Such DDS preparations are useful as preparations that absorb through the skin and mucous membranes.

The collagen component or gelatin component which is the starting material of the skin-penetrating tripeptide according to the present invention comprises:
It is prepared from bones, hides and tendons of cows, pigs, birds, whales and the like, or fish skins such as salmon and sharks as raw materials. The collagenase enzyme is Cl
It is derived from bacteria such as ostridium histolyticum and Streptomyces parvulus, actinomycetes, lactic acid bacteria, yeast and fungi, and uses an enzyme that specifically cleaves the amino group side of glycine in the amino acid sequence (Gly-XY) n specific to collagen. In addition, these enzyme genes are incorporated into specific vectors by genetic engineering and produced by other cells or animals such as lactic acid bacteria and yeast, and are enzymes obtained by genetic recombination. Collagenase having similar substrate specificity A similar enzyme may be used.

An important consideration when using the collagenase enzyme in the present invention is the purity of the collagenase enzyme. Normally, collagenase enzymes prepared from various cells may be contaminated with other proteases (proteases). If a large amount of this impure enzyme is contained, proteins other than the gelatin component or collagen component in the raw material will be degraded, and the gelatin or collagen component itself will be non-specifically decomposed by the impure enzyme, so that it will be purified. The quality of the peptide composition is reduced.

The collagenase enzyme may be used in a free form, or may be used as an immobilized enzyme in which the collagenase enzyme is bound to various carriers by a physical adsorption method or a chemical bonding method. In addition, the method of enzymatic degradation by collagenase enzyme, (a) batch method, (b) column method or (c)
There is a method combining these and the like.

Various combinations of the production line by the methods (a) to (c) and the form of the collagenase enzyme to be used can be adopted. In detail, the method may be carried out according to the method described in Japanese Patent Application Laid-Open No. 7-82299, but it is not necessary to dare to use this method. Rather, it suffices that care is taken so as to maintain the specificity, purity, and the like described in the columns of collagen and collagenase as raw materials.

More specifically, one example of the method for producing the skin-penetrating tripeptide according to the present invention is to use gelatin or collagen as a starting material to improve the enzyme recovery as shown in the following Examples. And a bioreactor method using a batch method or a column method using an immobilized collagenase enzyme. That is, the collagenase enzyme is bound to various carriers, for example, chitopearl, by a physical adsorption method or a chemical bonding method, and a solubilized gelatin solution or decomposition occurs at a constant temperature in a normal system packed in a column for chromatography. Enzymatic degradation is carried out through denatured collagen at moderate temperatures, preferably at 40-45 ° C. At this time, the rate of feeding the collagen raw material is appropriately selected depending on the activity of the immobilized enzyme and the required degree of decomposition.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples do not limit the scope of the present invention. In addition, the method of incorporating the skin-penetrating tripeptide according to the present invention into an external preparation or a food can be carried out according to a known operation method.

[0024]

[Example] 50 g of high-purity gelatin (Miyagi Chemical Industry Co., Ltd.)
With 1,000 ml of 20 mM Tris-HCl buffer (pH 7.4) /0.1 M
After dissolving in NaCl while heating, the mixture was cooled to 50 ° C. The immobilized enzyme for enzymatic degradation was 100 mg of collagenase enzyme (a high-purity purified from type IV, manufactured by Washington, Inc.).
g of the particle carrier. The amount of binding to the carrier was calculated by measuring the change in absorbance at 280 nm before and after binding, and the binding rate was 99% or more. At the time of use, this immobilized enzyme is filled into a double column type bioreactor with a pH sensor
Washing and equilibration were performed well with a Tris-HCl buffer (pH 7.4) /0.1 M NaCl buffer. The pH is measured by a system in which a pH sensor installed between the two columns detects a change and the concentrated Tris buffer flows in from the connected tube.

The high-purity collagen obtained in the above steps is
The column was applied to the column of the double collagenase enzyme immobilization prepared in the above step, and the enzyme was decomposed by the column method. During this time, the flow rate was controlled at 20-60 ml / min, and the column temperature was controlled at 39 ± 1 ° C. The enzyme reaction ending solution that came out of the last (second column) column was fractionated, and then collected.
Filtration was carried out with a 45 μm filter.

The filtrate is spray-dried (spray dryer)
After that, the collagen peptide having a tripeptide content of at least 10% or more was purified by a gel filtration method using a gel filter (trade name: Superdex 30 pg, manufactured by Pharmacia). The purified collagen peptide showed a peak at a molecular weight of about 280 due to the tripeptide. The tripeptide content can be increased by purification, and a collagen peptide having a tripeptide content of 80% or more can be obtained.

The collagen peptide obtained in the above step,
That is, after a collagen peptide having a tripeptide content of 10% or more was lyophilized, each NH2-terminal amino acid and the second amino acid from the NH2-terminal were assayed by the Edman degradation method. As a result, it was found that 97.5% of the amino acids on the NH2 terminal side were glycine, respectively, and it was proved that the amino acid at the NH2-terminal side had a characteristic amino acid sequence (Gly-XY) n structure.

<Skin penetration test (investigation by microautoradiography)> 10 mg of a tripeptide contained in the collagen peptide obtained in the above step labeled with a radioactive isotope: tritium (3H) was precisely weighed.
0.3 g of a liquid obtained by mixing propylene glycol and glycerin in a weight ratio of 6: 4 to this precisely weighed product, purified water 1.
A transdermal preparation was prepared by adding 49 g and 0.2 g of ethanol. The preparation for transdermal administration was applied with a spatula to the rat on the previous day, which was shaved under ether anesthesia, while dropping a small amount at the center of the back. 8, 24, and 72 hours after application of the sample, microautoradiograms were prepared using the excised skin, and the radioactivity distribution in the skin was examined using an optical microscope. As a result, tripeptides labeled with tritium
It was observed everywhere in the stratum corneum, granular layer, spiny layer, epidermis and basal layer, and furthermore, it was observed that it reached the dermis. Thus, it was confirmed that the tripeptide contained in the collagen peptide obtained in the example had high skin permeability. Therefore, this tripeptide is a skin-penetrating tripeptide, and the collagen peptide obtained in the examples is a skin-penetrating collagen peptide.

[0029]

[Formulation Example 1] (Lotion) 0.5 g of skin-penetrating collagen peptide and PEG 1500 according to Examples
Was dissolved in 50 ml of purified water, and 2.0 g of propylene glycol and 2.0 g of glycerin were added and mixed well. To this, 0.1 g of paraben and 10 g of ethanol
And purified water is added to bring the total amount to 10
0 g and mixed well. As a result, a skin lotion having high permeability to skin, high moisturizing property, good spread at the time of use, and no stickiness was obtained.

[0030]

[Formulation Example 2] (Emulsion) 0.5 g of skin-penetrating collagen peptide according to Example and propylene glycol 5.0
g, PEG 3.0 g and triethanolamine
0 g was added to 70 g of purified water, and the temperature was adjusted to 70 ° C. to prepare an aqueous phase. 2.0 g of stearic acid, 1.0 g of cetyl alcohol, 2.0 g of petrolatum, 5.0 squalane
g and glycerol tri-2-ethylhexanoate 2.0 g were dissolved at 75 ° C., and sorbitan monooleate 2.0 g and paraben 0.1
g and adjusted to 70 ° C. The oil phase was added to the previously prepared aqueous phase and mixed well with stirring. After that, the emulsified particles were homogenized with a homomixer, followed by degassing, filtration and cooling. As a result, an emulsion which was highly permeable to the skin, highly moisturizing, and spread well when used and was not sticky was obtained.

[0031]

Formulation Example 3 (Moisturizing serum) 2.0 g of skin-penetrating collagen peptide according to the example, 8.0 g of sorbitol,
5.0 g of propylene glycol and PEG 1500
7.0 g was dissolved in 69.7 g of purified water. Next, POE oleyl alcohol ether was added to 7.0 g of ethanol.
1.0 g, 0.2 g of olive oil and 0.1 of paraben
g was dissolved, and the above aqueous phase was added and dissolved. As a result, a moisturizing essence having high permeability to the skin, high moisturizing properties, and good spreadability during use was obtained.

[0032]

[Formulation Example 4] (Hand cream) 0.5 g of skin-penetrating collagen peptide and 15.0 of glycerin according to the example
g, 3.0 g of propylene glycol and 0.2 g of potassium hydroxide were dissolved in 66.2 g of purified water, and heated to 70 ° C. 3.0 g of stearic acid, 3.0 g of stearic acid monoglyceride, 3.0 g of petrolatum,
6.0 g of liquid paraffin and 0.1 g of paraben were mixed and adjusted to 70 ° C. The oil phase was added to the previously prepared aqueous phase, and the mixture was thoroughly stirred and mixed. The homogenized particles were homogenized with a homomixer, followed by degassing, filtration, and cooling. This allows
A hand cream with high permeability to the skin, high moisturizing properties, and good spread during use was obtained.

[0033]

Formulation Example 5 (Lotion for dry skin) Purified water was added to 1.0 g of skin-penetrating collagen peptide, 1.0 g of glycerin and 0.1 g of paraben to dissolve the total amount to 100 g, and mixed well. did. As a result, a skin lotion for dry skin which was highly permeable to skin, highly moisturizing, had good spread at the time of use, and was non-sticky was obtained. By using this lotion, the dry skin was improved.

[0034]

[Formulation Example 6] (Cream for dry skin) Hydrophilic ointment 9
To 5.0 g, 5.0 g of the skin-penetrating collagen peptide according to the example was added and mixed well. As a result, a cream for dry skin having high permeability to the skin, high moisturizing property, and good spreadability during use was obtained. By continuing to use this cream, dry skin was improved as in the case of the above-mentioned lotion. In addition, there was a tendency that healing was accelerated by applying this cream to the vicinity of the affected part of hemorrhoids.

[0035]

[Formulation Example 7] (Crea containing urea) 0.5 g of skin-penetrating collagen peptide and glycerin 5.0 according to the example
g, 4.5 g of dipropylene glycol and 1.0 g of triethanolamine were dissolved in 32.9 g of purified water,
The temperature was adjusted to 70 ° C. In addition, 3.0 g of stearic acid,
After heating and dissolving 5.0 g of cetyl alcohol, 5.0 g of petrolatum, 10.0 g of squalane and 7.0 g of glycerol tri-2-ethylhexanoate, 3.0 g of propylene glycol monostearate, PO
E (20) 3.0 g of cetyl alcohol ether and 0.1 g of paraben were added, and the mixture was adjusted to 70 ° C. This oil phase was added to the previously prepared aqueous phase, and 20.0 g of urea was further added, followed by thorough stirring and mixing. After stirring and mixing, the emulsified particles were homogenized with a homomixer, and degassed, filtered, and cooled. As a result, a cream having high permeability to the skin, high moisturizing properties, and good spreadability at the time of use was obtained. The use of this cream improved keratosis such as rough fingers and elbows, knees and heels.

[0036]

Formulation Example 8 (Eye drops for dry eye) Purified water is added to 0.05 g of skin-penetrating collagen peptide, 0.28 g of sodium chloride, 0.02 g of potassium chloride and 0.025 g of paraben according to the example, and these are dissolved. ,
The total amount was adjusted to 50 g and mixed well. This allows
A highly moisturizing dry eye drop was obtained.

[0037]

[Formulation Example 9] (Nutrient drink) 500 mg of skin-penetrating collagen peptide according to the example, 500 mg of taurine, 20 mg of nicotinamide, 50 mg of anhydrous caffeine,
Vitamin B 65mg, Vitamin B1 nitrate 5mg, Vitamin B2 phosphate 5mg, Carnitine chloride 1
00 mg, paraben 10 mg and ethanol 0.1
Purified water was added to 5 g to make 100 ml, and mixed.
Thereby, in addition to the nutritional supplement effect, an energy drink that contributes to improving the moisturizing power of the mouth and throat was obtained.

[0038]

According to the skin-penetrating tripeptide, the skin-penetrating collagen peptide, the skin-penetrating external preparation and the highly absorbent food of the present invention, the skin-penetrating or body-penetrating material has an excellent moisturizing effect. ing. In particular, the skin-permeable external preparation of the present invention is excellent in use feeling. Further, the highly absorbent food of the present invention is excellent in digestion and absorption and is useful.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) A61K 9/00 A61P 17/16 4C084 38/00 27/02 4H045 A61P 17/16 C12P 21/06 27/02 A23L 2 / 00J // C12P 21/06 A61K 37/18 37/02 (72) Inventor Yasuo Sakai 2-7-1, Wakabayashi-ku, Wakabayashi-ku, Sendai F-term in Miyagi Kagaku Kogyo Co., Ltd. 4B017 LC03 LK15 4B018 LB08 MD20 MD69 ME14 4B064 AG01 CA21 CB06 DA03 DA10 4C076 AA07 AA12 AA16 AA95 CC18 CC40 EE41A FF32 FF34 4C083 AA122 AB032 AC012 AC022 AC072 AC102 AC112 AC122 AC132 AC182 AC242 AC392 AC442 AC482 AC542 AC061 AC662 A0412 MA02 MA58 MA63 ZA331 ZA332 ZA891 ZA892 4H045 AA10 BA12 CA40 EA01 EA20 FA70

Claims (12)

[Claims] 1. A skin-penetrating tripeptide having an amino acid sequence of Gly-XY, which is obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme. 2. A skin-penetrating collagen peptide obtained by specifically decomposing a gelatin component or a collagen component using a collagenase enzyme and containing the skin-penetrating tripeptide according to claim 1. 3. A skin-permeable external preparation comprising the skin-penetrating tripeptide of claim 1 in an amount of 0.0005% by weight or more. 4. The skin-penetrating external preparation according to claim 3, which is a drug, a quasi-drug or a cosmetic. 5. The skin-penetrable external preparation according to claim 4, which is an external preparation for atopic dermatitis, dry skin, baby, sensitive skin or nursing care. 6. An external preparation for skin penetration according to claim 4, which is an eye drop for dry eye. 7. The external preparation for skin penetration according to claim 4, wherein urea is incorporated. 8. The skin-penetrating external preparation according to claim 4, which is a DDS preparation comprising a drug bound to the skin-penetrating tripeptide. 9. A superabsorbent food comprising the skin-penetrating tripeptide according to claim 1 in an amount of 0.0005% by weight or more. 10. The superabsorbent food according to claim 9, which is a beverage. 11. The superabsorbent food according to claim 9, which is a dietary supplement. 12. The superabsorbent food according to claim 9, which is a DDS preparation comprising a drug bound to the skin-penetrating tripeptide.