KR101980361B1 - Collagen hydrolysate having high concentration of collagen tripeptide and uses thereof - Google Patents

Abstract

The present invention relates to a collagen hydrolyzate containing collagen tripeptide and its use, and more particularly, to a collagen hydrolyzate prepared using collagen hydrolase that generates a large number of collagen tripeptides, and to an active ingredient thereof. It relates to a composition for preventing hair loss and hair growth.
The collagen hydrolyzate of the present invention has a significantly higher content of collagen tripeptide than the conventionally reported collagen hydrolyzate, and has an excellent absorption rate in the body, and shows excellent hair loss prevention, hair growth promoting ability, skin wrinkle improvement and skin moisturizing effect.

Description

Collagen hydrolysate having high concentration of collagen tripeptide and uses approximately}

The present invention relates to a collagen hydrolyzate containing a high content of collagen tripeptide and its use, and more particularly, to a collagen hydrolyzate prepared using a collagen hydrolase that specifically produces a collagen tripeptide, and It relates to a composition for promoting hair growth and skin condition comprising as an active ingredient.

Collagen is an animal's fibrous protein composed of about 18 amino acids such as proline, oxyproline, glycine, and glutamic acid, which is a special structural protein that accounts for 30% of the 5,000 kinds of proteins that make up the human body. In particular, the skin, bones, tendons are present in a lot, especially in the dermal layer of the skin 70% collagen is composed of collagen plays a very important role as a skin component. Collagen is a three-stranded form of amino acids are combined in the form of a polypeptide, the molecular weight is very large, about 300,000.

Low-molecular collagen is broken down by collagen degrading enzymes and lowered to molecular weight of 5,000. Also called collagen peptide. When the low-molecular collagen enters the human body, it is further broken down by proteolytic enzymes in the human body and finally absorbed into the human body in the form of amino acids. do. Since most proteins have molecular weight of 12,000 ~ 70,000, collagen has a molecular weight of 300,000, which is very difficult to absorb. Therefore, low molecular weight collagen is extracted, separated and purified to facilitate digestion and absorption in the human body. It is made in the form of a peptide having a molecular weight of 5,000 or less.

Collagen tripeptide is a small collagen (molecular weight: 200-500) that is linked to three amino acids (glycine-x-y), and is known to easily penetrate the skin due to its small molecular weight. On the other hand, if more than four amino acids are linked, the molecular structure is large and cannot penetrate the skin. Collagen tripeptides contribute to shortening the time to repair damaged collagen tissue by reconnecting to each other immediately after skin penetration. In addition, even when ingested higher absorption in the body than low molecular collagen has the advantage of maximizing the bioefficiency of collagen.

Generally, collagen is mainly made of cows, pigs, fish, squids, etc., and can be hydrolyzed by enzymes to obtain collagen peptide compositions. However, it is difficult to obtain a high content tripeptide composition by the protease which is generally used, so that the content of tripeptide is very low. Therefore, there is a steady need for an efficient production method for producing a high content of tripeptide collagen hydrolyzate.

On the other hand, the hair has different cycles and grows and falls through the anagen, the catagen, and the telogen. This cycle is repeated over three to six years, with an average of 50 to 100 hairs falling out daily. In general, alopecia refers to an abnormally large number of hairs falling out due to shortening of the growth phase hair and more degenerative or resting hairs during these cycles.

The causes of hair loss are discussed, such as hyperthermia of male hormone action, excessive sebum secretion, poor blood circulation, hypoxia caused by peroxide, bacteria, genetic factors, aging, stress. One of its causes, testosterone, a type of male hormone, is activated by dihydrotestoserone (DHT) by an enzyme called 5α-reductase, which induces a protein that binds to specific receptors and causes hair loss. Happens. In addition, by this mechanism, the sebum may be overproduced, and acne or seborrheic dermatitis may occur, causing hair loss with inflammation on the scalp.

Alopecia is known to be caused by disease, malnutrition, aging, hormonal imbalance, and the like. Although much research has been conducted, the mechanisms for underlying hair loss are not well known. Efforts have also been made to treat hair loss. Nevertheless, so far only two drugs (finasteride and minoxidil) have been approved by the Food and Drug Administration (U.S.A) for hair loss treatment. Finasteride, a 5α-reductase inhibitor, has been used to promote hair growth in androgenetic alopecia in men. Minoxidil, an anti-hypertensive agent, can promote hair growth by opening ATP-sensitive K-channels. However, the effects of drugs are limited and temporary because of unpredictable effects and side effects. There is a need for better new treatments that prevent hair loss and promote hair growth. In the case of preparations such as minoxidil and trichosaccharide, which are known to prevent hair loss and promote hair growth and hair growth, there are no obvious efficacy, human stability, and skin irritation. As a problem of side effects is on the rise, it is urgent to develop a composition in which safety and efficacy are secured.

Accordingly, the present inventors have endeavored to prepare a collagen hydrolyzate having high content of collagen tripeptides and having excellent absorption in the body. When the collagen is hydrolyzed by a novel collagen hydrolase prepared using Bacillus subtilis, It was found that the hydrolyzate having a high content of peptide could be prepared, and the hair growth promoting effect and skin condition improvement effect were found to be remarkably superior to the conventional collagen hydrolyzate, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for preparing a foodstuff comprising: (a) mixing and heat-treating young (fish scales) with water;

(b) preparing a collagen degrading enzyme by centrifuging the Bacillus subtilis strain culture solution, concentrating the centrifuged supernatant, and purifying using ion exchange chromatography; And

(c) the collagen degrading enzyme is added to the mixture of step (a) and hydrolyzed at 25 to 45 ° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 70% by weight relative to the total hydrolyzate. To provide a collagen hydrolyzate, characterized in that it is included.

It is another object of the present invention to provide a food composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

Another object of the present invention to provide a cosmetic composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles, improving skin elasticity, preventing skin aging, and skin moisturizing, including the collagen hydrolyzate as an active ingredient.

Another object of the present invention is to provide a food composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and skin moisturizing, including the collagen hydrolyzate as an active ingredient.

The present invention to achieve the above object (a) mixing the young (fish scales) with water and heat treatment;

(b) preparing a collagen degrading enzyme by centrifuging the Bacillus subtilis strain culture solution, concentrating the centrifuged supernatant, and purifying using ion exchange chromatography; And

(c) the collagen degrading enzyme is added to the mixture of step (a) and hydrolyzed at 25 to 45 ° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 70% by weight relative to the total hydrolyzate. To provide a collagen hydrolyzate, characterized in that it is included.

It is another object of the present invention to provide a food composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

In order to achieve the other object of the present invention, the present invention provides a cosmetic composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

In order to achieve the other object of the present invention, the present invention provides a cosmetic composition for skin wrinkle improvement, skin elasticity improvement, skin aging and skin moisturizing comprising the collagen hydrolyzate as an active ingredient.

In order to achieve the another object of the present invention, the present invention provides a food composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and skin moisturizing comprising the collagen hydrolyzate as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of (a) mixing the young (fish scales) with water and heat treatment;

(b) preparing a collagen degrading enzyme by centrifuging the Bacillus subtilis strain culture solution, concentrating the centrifuged supernatant, and purifying using ion exchange chromatography; And

(c) the collagen degrading enzyme is added to the mixture of step (a) and hydrolyzed at 25 to 45 ° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 70% by weight relative to the total hydrolyzate. It provides a collagen hydrolyzate, characterized in that it is included.

In the present invention, the step (a) is a process of heat treating at a high temperature by mixing the young to extract the collagen with water, the step of extracting the collagen contained in the young with water. The ratio of young to water added in step (a) is not particularly limited, but preferably young: water may be mixed in a weight ratio of 2: 8.

In the step (a), the temperature conditions for extracting collagen from the young may be 80 to 120 ° C, preferably 85 to 110 ° C, more preferably 90 to 100 ° C, but the present invention is not limited thereto. Depends on the time of collagen extraction and the ratio of young and water. The time for heat treatment in step (a) may vary depending on the temperature conditions for extracting collagen, but may be 1 hour to 10 hours, preferably 2 hours to 9 hours, and more preferably 3 hours to 8 hours. It is not limited.

Step (b) is a step of preparing a hydrolase to hydrolyze collagen, it is possible to prepare a hydrolyzate having a particularly high content of collagen tripeptide compared to the enzymes conventionally used for hydrolyzing collagen in the art. This step is to prepare the enzyme. The present inventor has filed with the Korean Intellectual Property Office for the method for preparing collagen degrading enzyme in step (b) (Application No. KR10-2015-0124453), and the patent document may be referred to in the present invention.

In the present invention, the Bacillus subtilis may be characterized as a strain having accession number KCTC12866BP, but is not limited thereto.

According to one embodiment of the present invention, the present inventors performed ion exchange chromatography under various conditions in order to isolate collagen degrading enzyme contained in Bacillus subtilis strain culture. The ion exchange chromatography of step (b) may use cation exchange chromatography, in which case it was confirmed that the recovery of the target enzyme is the best when using sodium chloride (NaCl) in a concentration of 0.1 to 0.3M. Therefore, when the ion exchange chromatography of step (b) is cation exchange chromatography, it is preferable to use sodium chloride (NaCl) of 0.1 to 0.3M.

According to another embodiment of the present invention, the ion exchange chromatography of step (b) in the present invention may use anion exchange chromatography, in this case when using sodium chloride (NaCl) at a concentration of 0.09 to 0.155M It was confirmed that the recovery rate of the target enzyme was the best. Therefore, when the ion exchange chromatography of step (b) is anion exchange chromatography, it is preferable to use sodium chloride (NaCl) of 0.09 to 0.155M.

In the present invention, step (c) is a step of hydrolyzing collagen by mixing the collagen extract prepared in step (a) and the collagen degrading enzyme prepared in step (b).

In one embodiment of the present invention, the collagen degrading enzyme prepared in step (b) was confirmed to maintain the enzyme activity for the longest time at 25 to 45 ℃ temperature conditions. If the temperature condition exceeds 45 ℃, the initial enzyme activity may temporarily increase, but the loss of activity over time is too large, there is a problem that the effect of hydrolyzing collagen does not appear sufficiently, on the contrary, if it is less than 25 ℃ There is a problem that the enzyme activity is too low to hydrolyze collagen.

Therefore, the hydrolysis temperature condition in step (c) is preferably 25 to 45 ℃, more preferably 27 to 42 ℃, even more preferably 30 to 40 ℃, most preferably 35 to 40 ℃ have.

In the present invention, the collagen hydrolyzate obtained after the step (c) may further include a step of removing foreign substances through centrifugation, ion exchange chromatography, filtration, and concentrating. Removing and concentrating the foreign matter can be used without limitation the methods commonly used in the art.

In the present invention, the collagen hydrolyzate prepared according to the above method is characterized in that the content of the collagen tripeptide is remarkably improved as compared to the collagen hydrolyzate prepared using the tagase.

Collagen tripeptide is a small collagen (molecular weight of 200 ~ 500) that is linked to three amino acids (glycine-x-y) and is known to easily penetrate the skin due to its small molecular weight. On the other hand, if more than four amino acids are linked, the molecular structure is large and cannot penetrate the skin. Collagen tripeptides contribute to shortening the time to repair damaged collagen tissue by reconnecting to each other immediately after skin penetration. In addition, even when ingested higher absorption in the body than the general low molecular weight collagen has the advantage of maximizing the bioefficiency of collagen. Therefore, the higher the content of collagen tripeptide in the collagen hydrolyzate, the higher the bioavailability can maximize the physiological activity of collagen.

According to one embodiment of the invention, the collagen hydrolyzate of the present invention was confirmed that the content of collagen tripeptide is about 53% by weight relative to the total hydrolyzate. The collagen hydrolyzate prepared using the tagase was confirmed to contain little collagen tripeptide (Example 4 and Comparative Example). Since collagen is not easy to be absorbed into the body by itself, it is most preferable to use it by hydrolysis in the form of a small peptide. It is very good in that it is significantly higher than the hydrolyzate.

Specifically, the collagen hydrolyzate in the present invention may be characterized in that the collagen tripeptide is contained in 20 to 70% by weight relative to the total hydrolyzate, preferably 30 to 70% by weight, 40 to 70% by weight, It may be 50 to 70% by weight, 30 to 60% by weight, 40 to 60% by weight, and most preferably 50 to 60% by weight, but is not limited thereto.

The present invention also provides a composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

As used herein, "hair loss" refers to a phenomenon in which hair falls off the scalp or a condition in which hair is coarse or thin, and "hair loss prevention" refers to preventing and suppressing hair loss as described above, and "hair growth". "Promoting" means not only promoting the production of new hair, but also allowing the existing hair to grow healthy.

In general, "hair growth" occurs in the growth phase and is promoted by induction from the resting phase to the growth phase and the delay from the growth phase to the regression phase. The hair cycle can be divided into three main stages, known as the growing, degenerative and resting phases. In the growth phase, hair growth occurs as hair follicles grow deep into the skin with rapid proliferation of cells. The next phenomenon is the degenerative phase, which is a transitional period in which the disruption of cell division is prominent, during which hair follicles gradually degenerate and hair growth ceases. In the next phase, the resting phase, the degenerative hair follicles contain germs with densely packed dermal papilla cells. The onset of new growth phase phenomena at rest is induced by rapid cell proliferation in the embryo, swelling of the dermal papilla and the synthesis of basement membrane elements.

Therefore, it is necessary to prevent hair loss, that is, to prevent hair loss or to induce hair regrowth, i.e., to promote hair growth by promoting or extending the growth phase, and the composition according to the present invention has the effect of preventing the existing hair from falling out. At least one of the effects of thickening already existing hair and the like, and creating new hair.

Compared with the conventional hydrolyzate, the composition of the present invention comprising a collagen hydrolyzate having a significantly higher content of collagen tripeptide as an active ingredient has high absorption and transdermal absorption rate of the collagen hydrolyzate and has a fast absorption rate, thus preventing hair loss and promoting hair growth. Very excellent.

Specifically, according to an embodiment of the present invention, the in vivo absorption of collagen hydrolyzate prepared using the collagen hydrolyzate according to the present invention and a hydrolase which is generally used to hydrolyze collagen in the art. As a result of the evaluation, it was confirmed that the collagen hydrolyzate according to the present invention was significantly high in AUClast, Cmax, and short in Tmax, so that the absorption in the body was very good. In the case of collagen hydrolyzate according to the present invention, the content of collagen tripeptide is significantly high, so it can be determined that absorption in the body is very fast and the absorption rate is excellent.

According to another embodiment of the present invention, the composition comprising the collagen hydrolyzate according to the present invention as an active ingredient promotes the proliferation of hair follicle cells in the degenerative hair loss animal model, inhibits the progression of the degenerative phase and maintains the growth stage. It was confirmed that the hair growth effect was very excellent. On the other hand, the hair growth promoting effect of the composition according to the present invention was confirmed to be excellent to the same extent as minoxidil (Minoxidil) used as a positive control group.

The collagen hydrolyzate according to the present invention is considered to have an excellent effect because the content of collagen tripeptide is very high and the amount of collagen-derived peptide absorbed into the body is high.

Therefore, the present invention provides a food composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

In addition to containing the collagen hydrolyzate as an active ingredient, such a food composition may contain various flavors or natural carbohydrates, etc. as additional components, as in general food compositions. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food, or may be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition to the collagen hydrolyzate, which is an active ingredient, the food composition may include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese, chocolate), pectic acid, and the like. Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.

Collagen hydrolyzate, the active ingredient of the present invention, is a natural substance and has almost no toxicity and side effects, and thus can be used safely even when taken for a long time in order to prevent hair loss and promote hair growth.

The food composition of the present invention may be a health functional food for hair loss prevention and hair growth promotion comprising collagen hydrolyzate as an active ingredient.

The health functional food of the present invention can be produced and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing hair loss and promoting hair growth.

In the present invention, "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and nutrients for the structure and function of the human body. It is meant to be consumed for the purpose of regulating or obtaining a useful effect for health use such as physiological action.

The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.

Examples of the items listed in the "Food Additive Reduction" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes. For example, the health functional food in the form of tablets is a mixture of a collagen hydrolyzate, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives, granulated in a conventional manner, and then compressed with a lubricant and the like. Or the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary.

Hard capsules among the health functional foods in the form of capsules may be prepared by filling a mixture of a mixture of additives, such as a collagen hydrolyzate, which is the active ingredient of the present invention, in a conventional hard capsule, and the soft capsules may contain a collagen hydrolyzate, etc. The mixture mixed with the additive of may be prepared by filling in a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The health functional food in the form of a cyclic form may be prepared by molding a mixture of a collagen hydrolyzate, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method. It may be avoided, or the surface may be coated with materials such as starch, talc.

The health functional food in the form of granules can be prepared into a granular mixture of a collagen hydrolyzate, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method. And the like.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

The present invention also provides a pharmaceutical composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

The pharmaceutical composition according to the present invention may contain the collagen hydrolyzate of the present invention alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Can be. Preferably the pharmaceutical composition of the present invention may be administered orally. For example, the pharmaceutical composition of the present invention can be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin needle or by applying it directly to the skin.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients, milling them, adding suitable auxiliaries and then processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

When the pharmaceutical composition of the present invention is used as an external skin pharmaceutical composition, it is a skin external agent having the effect of preventing hair loss, promoting hair growth, and improving the scalp, such as cream, gel, patch, spray, ointment, warning agent, lotion, linen agent, and pasta. It may be prepared and used as a pharmaceutical composition in the form of a topical or external preparation of cataplasma, but is not limited thereto.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol which is administered in multiple doses for a long time. . The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of the glycoprotein fraction of the invention may be about 0.01 ug to 1,000 mg, most preferably 0.1 ug to 100 mg per kg of patient body weight per day. However, the dose of the therapeutic agent candidate of the present invention may be adjusted to the patient in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition. Since the effective dosage is determined, one of ordinary skill in the art, in light of this, will determine the appropriate effective dosage according to the specific use of the pharmaceutical composition of the present invention as a pharmaceutical preparation for preventing hair loss and promoting hair growth. Could be. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

The present invention also provides a cosmetic composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate as an active ingredient.

The cosmetic composition according to the present invention may include not only collagen hydrolyzate, which is an active ingredient, but also components commonly used in cosmetic compositions, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes. And a carrier.

In addition, the composition of the present invention can be used in addition to the above-described collagen hydrolyzate, in addition to the hair loss prevention agent and / or hair regrowth agent that has been conventionally used as long as it does not impair the action of hair loss prevention and hair growth promotion by collagen hydrolyzate.

In addition, the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, Hair Treatment, Hair Cream, Nutritional Cream, Hair Moisture Cream, Hair Massage Cream, Hair Wax, Hair Aerosol, Hair Pack, Hair Nutrition Pack, Hair Soap, Hair Cleansing Foam, Hair Oil, Hair Dryer, Hair Preservative, Hair Dye, Hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, hair mousse and hair spray can be prepared in various forms, but is not limited thereto.

According to one embodiment of the present invention, the collagen hydrolyzate of the present invention was found to have a very good effect of improving the skin condition, such as skin wrinkle improvement effect, skin moisturizing effect. On the other hand, the skin condition improvement effect was remarkably superior to the collagen hydrolyzate produced by the previously reported collagen hydrolase. This is because the collagen hydrolyzate according to the present invention is composed of a high content of collagen tripeptide, the bioavailability was high, it could be determined that the physiological activity of the collagen was more exerted.

The skin condition improvement effect of the collagen hydrolyzate according to the present invention can be more clearly identified through specific examples.

Accordingly, the present invention provides a cosmetic composition for improving skin wrinkles, improving skin elasticity, preventing skin aging and moisturizing the skin including the collagen hydrolyzate as an active ingredient.

In addition, the present invention provides a food composition for skin wrinkle improvement, skin elasticity improvement, skin anti-aging and skin moisturizing comprising the collagen hydrolyzate as an active ingredient.

The cosmetic composition and the food composition described above may be applied as it is.

The present invention relates to a collagen hydrolyzate containing a high amount of collagen tripeptide and its use. The collagen hydrolyzate of the present invention has a significantly higher content of collagen tripeptide compared to the previously reported collagen hydrolyzate, and thus has an excellent absorption rate in the body. It has excellent hair loss prevention, hair growth promotion, wrinkle improvement and moisturizing effect.

1 is a view showing the results of separation and purification using cationic ion resin of collagen degrading enzyme prepared according to an embodiment of the present invention. A is the result of SDS-PAGE analysis of purified collagenase (M: protein size marker, conc: concentrated sample, wash: wash, E1 ~ E5: elution samples 1-5). B shows the peak of the collagenase purification according to the change of buffer concentration in the AKTA primer (arrow is indicated for each elution sample).
Figure 2 is a diagram showing the results of separation and purification using anionic ion resin of the collagen degrading enzyme prepared by the embodiment of the present invention. A is the result of SDS-PAGE analysis of purified collagenase (M: protein size marker, P1: peak 1). B shows the peak of the purification of collagenase according to the change of buffer concentration in the AKTK primer (arrow is indicated for each elution sample).
Figure 3 is a graph showing the results of the enzyme activity according to the temperature of the collagen degrading enzyme prepared by the embodiment of the present invention.
Figure 4 is a graph showing the results of changes in enzyme activity according to the time and temperature of the collagen degrading enzyme prepared by the embodiment of the present invention.
Figure 5 is a graph showing the enzyme activity results for each pH of the collagen degrading enzyme prepared by the embodiment of the present invention.
FIG. 6 is HPLC data comparing collagen tripeptide productivity of collagenase and general protease prepared according to an embodiment of the present invention.
Figure 7 is a production process for producing a collagen hydrolyzate having a high collagen tripeptide content using the collagenase produced by the embodiment of the present invention.
8 is HPLC data analyzing the collagen tripeptide (CTP) content of the hydrolyzate produced using the collagen degrading enzyme prepared according to the embodiment of the present invention.
Figure 9 is a graph evaluating the effect of the administration of the test substance on the growth and degenerative ratio of hair follicles in the animal model induced degenerative hair loss with Dexamethasone ((A) 9 days epilation, (B) 11 days epilation, (C) 14 days of epilation, (D) 17 days of epilation, anagen: growth, catagen: degenerative, Nor: depilatory normal group without treatment, Control: dexamethasone treatment and depilation after solvent treatment, BP-CH: dexamethasone treatment and hair removal after administration of the collagen hydrolyzate of the present invention, Minox: dexamethasone treatment and hair removal positive control group after minoxidil).
10 is a result showing the effect of the administration of the test substance on the hair growth progression of the test animal in the animal model induced degenerative hair loss with Dexamethasone.
11 is a result of observing hair follicle growth of animal model skin tissue induced by dexamethasone through tissue staining (Dex: dexamethasone).
12 is a result of observing the effect of wrinkle improvement with a replica plate using silicone and EPON resin after administering each substance to the animal model induced wrinkles by UV irradiation (N: Normal, C: Control, P: Positive control , COL17, COL34: 17 mg / kg or 34 mg / kg of collagen hydrolyzate produced by general collagenase, CTP17, CTP34: 17 mg / kg or 34 mg / kg of collagen hydrolyzate of the present invention).
Figure 13 shows the results of the Masson's Trichrome staining and the skin of SKH-1 hairless mice after the end of the experiment (N: Normal, C: Control, P: Positive control, COL17, COL34: to normal collagenase 17 mg / kg or 34 mg / kg administered group, CTP17, CTP34: 17 mg / kg or 34 mg / kg administered group of the present invention).
14 shows the results of quantifying the skin expression of SKH-1 hairless mice after the experiment was completed, and confirming the amount of protein expression of Collagen 1A (A) and MMP-1 (B) in the tissues by Western blot (N: Normal, C: Control, P: Positive control, COL17, COL34: Collagen hydrolyzate produced by normal collagenase 17 mg / kg or 34 mg / kg dose group, CTP17, CTP34: Collagen hydrolyzate of the present invention 17 mg / kg or 34 mg / kg dose group).
15 is a result of checking the difference between the oil content (A), moisture (B) of the skin of the mouse at the 5th and 10th weeks of the experiment (N: Normal, C: Control, P: Positive control, COL17, COL34: General 17 mg / kg or 34 mg / kg administration group of collagen hydrolyzate produced by collagenase, CTP17, CTP34: 17 mg / kg or 34 mg / kg administration group of the collagen hydrolyzate of the present invention).
16 is a result of evaluating the skin moisture content after administration of each substance to Balb-c mice induced skin drying (N: Normal, C: Control, P: Positive control, CTP17: Collagen hydrolyzate of the present invention) 17 mg / kg, COL34: 34 mg / kg of collagen hydrolyzate prepared using a previously reported hydrolase).
17 is a result of measuring the number of scratches due to itching after administration of each substance to Balb-c mice induced skin drying (N: Normal, C: Control, P: Positive control, CTP17: Collagen of the present invention Hydrolyzate 17 mg / kg, COL34: group administered with 34 mg / kg collagen hydrolyzate prepared using a previously reported hydrolase).
18 is a result of administering each substance to Balb-c mice induced skin drying, and after the experiment is finished, the skin tissue of each mouse is collected and the amount of the skin moisturizing factor protein is confirmed through Western blot and quantified. (A: Collagen 1A, B: AQP3, C: HAS2, N: Normal, C: Control, P: Positive control, CTP17: Collagen hydrolyzate 17 mg / kg of the present invention, COL34: A previously reported hydrolase Collagen hydrolyzate prepared using 34 mg / kg administration group)

Hereinafter, the present invention will be described in detail.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Preparation of Collagen Hydrolase

For the method of preparing the collagen hydrolyzate in the present invention and the collagen hydrolyzate prepared by the method, Korean Patent Application No. 10-2014-0158019 may be referred.

That is, Bacillus subtilis strain (Accession No. KCTC12866BP) was incubated in 100 ml of mTB medium (yeast extract 2.4%, tryptone 1.2%, glycerol 1%, KH2PO4 2.31%, H2HPO4 12.54%), and then centrifuged at 6500 rpm for 15 minutes. Separated. The supernatant was carefully transferred to a new tube and concentrated 5 times using a 30 kDa filter. The concentrated supernatant was purified by ion exchange chromatography using an AKTA prime apparatus.

Purification conditions used were A buffer (50 mM Tris-HCl, pH 7.5) was used as the binding buffer and sloped using B buffer (50 mM Tris-HCl, pH 7.5, 0.5M NaCl). The flow rate was flowed at 5 ml / min. Purification was carried out using cation exchange chromatography and anion exchange chromatography. As the ion resin, SP sepharose resin (GE Healthcare, new-Jersey, USA), a cation resin, and Q sepharosse resin (GE healthcar, new-Jersey, USA) respectively.

In the first cation exchange chromatography, 0.5M NaCl was inclined and purified protein was obtained for each fraction. As shown in FIG. 1B, the protein purity of the fractions (E1 to E5) in which the protein peaks appear in the purification profile was confirmed on SDS-PAGE, and the protein expected to be the target enzyme was included in E1 and E2. It was confirmed that there was the most collagenase (arrows in Fig. 1A).

NaCl concentrations of E1 to E5 are E1: 0.1-0.2M, E2: 0.2-0.3M, E3: 0.3-0.4M, E4: 0.4-0.5M, E5: 0.5M. The concentration of NaCl that can be separated was found to be 0.1-0.3M, and the recovery rate of the target enzyme was the best at the NaCl concentration of 0.1-0.2M which is the E1 part. Next, E1 was purified by anion exchange chromatography.

The buffer used was 0.25M NaCl and was inclined in the same manner as cation exchange chromatography. As shown in FIG. 2B, the anion exchange resin in the purification profile showed that a protein peak appeared in 0.09 to 0.115 M NaCl (arrow of FIG. 2B).

As a result of confirming the purification degree of this fraction on SDS-PAGE, it was confirmed that the target enzyme was mostly purified (P1 of FIG. 2A). Therefore, the anion exchange chromatography showed the best recovery of the target enzyme in NaCl of 0.09 ~ 0.155M. Thereafter, the purified new collagen degrading enzyme was named BP.

<Example 2>

Experiment of Enzymatic Reaction of BP

The titer was measured by performing an enzymatic reaction at a temperature of 20 ~ 70 ℃ in the optimum temperature experiment. As shown in FIG. 3, the experimental results have enzymatic activity at 30-55 ° C., and show the highest specific activity and relative activity at 50 ° C., and activity drops sharply from 60 ° C. Confirmed.

Temperature stabilization experiments were conducted for actual mass production. The temperature was adjusted to 30 ° C, 35 ° C, 40 ° C, and 50 ° C, and samples of each temperature were sampled for each hour from 0 to 12 hours to confirm their remaining activity.

As shown in FIG. 4, as a result, the lower the temperature, the more stable the enzyme was observed, and it was confirmed that the activity of the enzyme remained about 59.3% after 12 hours at 35 ° C and 40 ° C. From these results, it was desirable to set the production process of the collagen tripeptide at 35 ° C.

Next, the optimum pH was examined. Donpi gelatin substrates were prepared for each pH (50 mM citrate-Na ₂ HPO _{4 (} pH 5.0-6.0), 50 mM Tris-HCl (pH 6.0-9.0), 50 mM Na ₂ CO ₃ -Na ₂ HCO ₃ ( pH 9.0 ~ 10.0)) and then determine the optimum pH by measuring the activity of the enzyme. BP showed the highest activity at pH 7.4 and high activity in the neutral zone up to pH 6 ~ 10. However, as shown in Figure 5, it was confirmed that the activity of the enzyme is similarly reduced toward the acidic or basic.

Hereinafter, a process of preparing collagen tripeptides using the collagen degrading enzyme (BP) will be described.

< Example  3>

BP Collagen Tripeptide Productivity Verification

Each of the four samples was prepared by uniformly mixing the weight ratio of young and water in a reactor equipped with a stirrer at a ratio of 20:80 in a pre-treated fish (fish scales) at a ratio of 20:80 for 5 hours. BP (manufacturer: Amicogen), Alcalase 2.4L FG (manufacturer: Novozyme, vendor: Biosis), Flavorzyme 1,000L (manufacturer: Novozyme, vendor: Biosis), Collupμlin MG (manufacturer: Novozyme) (Commercially available from Daejong Corp.) was used to compare collagen tripeptide productivity.

Reaction conditions (temperature, pH, enzyme use, reaction time) of each enzyme are as follows.

    BP: 35 ° C., pH 7.4, 30 unit / g (young), 12 h

    Alcalase 2.4L FG: 60 ° C, pH 7.0, 30 unit / g (young), 12 hours

    -Flavorzyme 1,000L: 55 ℃, pH 6.0, 30 unit / g (young), 12 hours

    Collupμin MG: 60 ° C, pH 7.0, 30 unit / g (child), 12 hours

After the enzyme treatment, heat treatment at 80 ℃ 30 minutes to inactivate the enzyme. Next, comparative analysis of the four enzymes confirmed the collagen tripeptide production of BP.

The collagen hydrolyzate was analyzed using HPLC (gilson), Superdex ^TM Peptide 10 / 300GL column, mobile phase (10 mM Tris-Cl, pH 7.4), 0.15 M NaCl, 5 mM CaCl ₂ , flow rate: 0.5 ml Collagen tripeptide (CTP) was analyzed under the condition of / min. As a standard of CTP, glycine-proline-hydroxyproline (GPH) consisting of three amino acids was used.

As shown in FIG. 6, in the case of the CTP standard, peaks were formed at about 55 minutes. As a result of comparing the degradation activity of the four enzymes, it was found that in the case of collagen hydrolysates hydrolyzed using BP, the peaks appeared late and collagen was low in molecular weight. It can be seen that the content of CTP is high. The collagen hydrolyzate hydrolyzed with Alcalase 2.4 L FG had higher molecular weight than the collagen hydrolyzate hydrolyzed with other Flavorzyme 1000 L or collupμin MG, but showed little CTP content. As a result, it was found that BP is very effective for collagen tripeptide production.

<Example 4>

Using BP Collagen Hydrolyzate  Produce

In order to produce collagen hydrolyzate containing collagen tripeptide, a manufacturing method using BP was established. The pretreated broiler (fish scales) was uniformly mixed with the weight ratio of the broth and water in a reactor equipped with a stirrer at a ratio of 20:80 and heat-treated at about 90 ° C. for 5 hours.

The heat-treated solution was corrected to pH 7.4 in solution by adding 10% NaOH at 35 ° C. BP was added to the calibrated solution and reacted at 35 ° C. for 12 hours after adding about 30 units / g (young). After enzymatic reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 30 minutes. Enzyme-treated collagen peptide solution was removed using an ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion column purification.

The liquid was concentrated to Brix 35% using a vacuum pressure concentrator, and then decolorized and deodorized through activated carbon tablets. Activated charcoal purified liquid was microfiltration (membrane filtration) and then powdered with a spray dryer to perform a quality test and through the packaging step to prepare a collagen hydrolyzate containing tripeptide content (Fig. 7).

Comparative Example 1

Using general hydrolase Collagen Hydrolyzate  Produce

Collagen tripeptide productivity was compared using Alcalase 2.4 L FG (manufacturer: Novozyme, vendor: Biosis), which is typically used for low molecular weight collagen digestion for comparison with the new BP. The pretreated broiler (fish scales) was uniformly mixed with the weight ratio of the broth and water in a reactor equipped with a stirrer at a ratio of 20:80 and heat-treated at about 90 ° C. for 5 hours.

The heat-treated solution was corrected to pH 7.5 in solution by adding 10% NaOH at 55 ° C. Alcalase enzyme was added to the calibrated solution, and about 30 units / g (young) were treated at 35 ° C. for 12 hours. After enzymatic reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 30 minutes.

Enzyme-treated collagen peptide solution was removed using an ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion column purification. The liquid was concentrated to Brix 35% using a vacuum pressure concentrator, and then decolorized and deodorized through activated carbon tablets. Activated charcoal tablets were sterilized by filter press filtration and membrane filtration, and then powdered with a spray dryer and analyzed for quality after collagen tripeptide (CTP) content.

CTP content was carried out in the same manner as in Example 2. Samples were analyzed for collagen hydrolyzate at the end of final formulation prepared using BP and Alcalase.

As shown in FIG. 8, in the case of the CTP content, it was confirmed that the peak appeared at the same position as the CTP standard in the collagen hydrolyzate prepared using BP, and in the same position in the collagen hydrolyzate prepared using Alcalase. There was no peak.

The quantitative comparison of CTP content of collagen hydrolysates prepared using BP and Alcalase showed 53.1% for BP and 0% for Alcalase. These results show that BP can be used to prepare collagen hydrolyzate with high content of collagen tripeptides.

Example 5

BP use  Collagen Hydrolyzate and Alcalase use  Collagen Hydrolyzate  Absorbance Comparison

<5-1> Animal Experiment

After anesthetizing male Sprague-Dawley (SD) rats with ether, the right carotid artery was cannulated with polyethylene tubing, and then the cannula was pulled back and forth using infusion harness to allow rats to move freely and the surgical site was sutured. After fasting cannulated rats for 12 hours or more, and freely supplying water, the collagen hydrolyzate (BP-CH) using BP and the collagen hydrolyzate of Example 4 and Comparative Example 1 were prepared in each of 6 rats. Collagen hydrolyzate (Alcalase-CH) using Alcalase was administered dissolved in distilled water at a dose of 500 mg / kg (5 mL / kg). Plasma was isolated by blood collection and centrifugation at time after administration (15, 30, 45, 60, 90, 120, 180, 240, and 360 minutes). Plasma samples were stored in a -20 ° C freezer until analysis, and quantitatively analyzed by simultaneous analysis of the concentration of hydroxyproline (Hydroxyproline, Hyp) in plasma by LC-MS / MS.

<5-2> LC-MS / MS method

* Sample pretreatment: Add 50 μL of internal standard solution (Gabapentin 0.1 μg / mL) to 50 μL of plasma, add 50 μL of 5% trichloroacetic acid, mix, and centrifuge for 10 min at 13,000 rpm. 2 μL of the diluted solution was injected into LC-MS / MS for analysis.

* Analytical device: Agilent 1260 HPLC system and Agilent6460 triple-quadrupole mass spectrometer

Column: Zorbax SB-Aq, particle size 1.8μ, 2.0 mm, i.d. × 100 mm, l.

Column temperature: 30 ° C

Mobile phase: 0.1% formic acid (A) acetonitrile (B) gradient elution

Flow rate: 0.25 mL / min

* Injection volume: 2 μL

<5-3> Absorbency Evaluation Method

After preparing the plasma concentration-time curves for each target component in each group, the area under the plasma concentration-time curve (AUC) was calculated to compare the absorbance. AUC was obtained by the linear trapezoidal method. Extrapolation in the terminal phase was difficult, so the total AUC up to infinity was not obtained, but AUClast up to 6 hours, the last time of blood collection, was obtained. The peak blood concentration (Cmax) and the peak blood concentration arrival time (Tmax) were obtained by reading directly on the graph. The AUC, Cmax and Tmax values of each group were examined by one-way ANOVA analysis and Duncan's post hoc test.

<5-4> Absorption test result of collagen hydrolyzate

AUC and Cmax levels of blood free Hyp were significantly different between BP-CH and Alcalase-CH groups. This means that the absorption rate of Hyp in the blood was high. In addition, the Tmax value was faster in the BP-CP administration group than in the Alcalase-CH group. This result indicates that Hyp was rapidly absorbed after ingesting BP-CH, and the short time spent in the blood showed that the absorption from blood to tissue occurred quickly (Table 1).

Through this experiment, it was confirmed that the absorption efficiency of collagen hydrolyzate using BP was higher than that of collagen hydrolyzate using Alcalase by examining the change of blood collagen-specific amino acid Hyp.

< Example  6>

Collagen Using BP Hydrolyzate  Hair loss prevention and hair growth promoting effect

<6-1> Experimental Animal Diet

 The collagen hydrolyzate was prepared by the same method as the method of preparing collagen hydrolase of Example 4, but the collagen hydrolyzate (BP-CH) having 28% CTP content was prepared by treating BP enzyme with 17 units / g. Used for.

<6-2> Establishment of experimental and degenerative alopecia animal models

The experimental animal was a C57BL / 6 mouse, a melanin pigment was present in the hair follicles and the skin color was changed according to the amount of the melanin pigment, and the hair color could be identified by the skin color (growth: black, degenerative and resting phase: pink). At 7 weeks of age, it turns into a resting period. Because of these characteristics, 35 eyes were obtained from 6-week-old C57BL / 6 female mice (17 g-20 g) from Daehan Biolink (Chungbuk).

The test group consisted of dexamethasone treated group, dexamethasone treated group, dexamethasone treated group and collagen hydrolysate (BP-CH) treated group using BP, solvent control and positive control group (3% Minoxidil) treated group. Each group consisted of eight animals. Solvent control and collagen hydrolyzate were orally administered once every 10 days for 2,000 mg / 10 ml / kg, and the positive control Minoxidil was also applied daily.

The back of the 7-week-old test animal was removed with a hair using an animal hair remover and a hair remover Nikrin (Ildong Pharmaceutical) and was quickly washed with water. After 7 days of hair removal, the sample and minoxidil were administered once daily for 10 days. 0.1% Dexamethasone was applied to the back of test animals for 5 days at 1 ml / day starting from 9th day of depilation. The test animals were sacrificed by anesthesia with ether on the last sample and the day after Minoxidil administration.

<6-3> Determination of hair loss stage and maintenance of growth stage

The clinical symptoms and death of test animals were checked once daily. Photographs were taken once a week to visually identify growth induction effects. Photographs were taken once every three days to observe the effects of degenerative stage suppression. The photograph was taken to determine the ratio of growth stage and degeneration stage by measuring the rate of change of color from gray to black of the total epilation of the test animals.

The results are shown in Table 1, FIG. 9 and FIG. 10.

The numerical value in the above table means the rate of progression of the retrograde phase at the site where the retrogenital phase is induced by hair removal. In other words, the higher the ratio, the higher the rate of progression of the degenerative stage, and the lower the ratio, the more the regression stage is suppressed. In the above table, BP-CH means the animal group ingested collagen hydrolyzate of the present invention.

As shown in Table 2, FIG. 10 and FIG. 11, the ratio of the growth period and the degenerative phase on the 9th day of hair removal was not different between the normal control group and the test substance administration group, but the minoxidil-coated group was statistically significant compared to the solvent control group treated with dexamethasone only. The ratio of the ratio was high and the ratio of the age was reduced.

At 11, 14, and 17 days of hair removal, the solvent control group remained pink until its back was pink and the test was completed. From the 11th day of hair removal, the growth rate of the test group and the minoxidil group increased significantly.

<6-3> histological examination

After conducting the experiment according to Example <5-1>, it was intended to observe the hair follicle state by collecting the hair removal site of the test animal.

That is, the test animals were sacrificed with ethyl ether, anesthetized, and the skin tissue was removed, stored in a 10% neutral formalin solution, paraffin embedded and sliced to a thickness of 5 μm to prepare tissue slides, and stained with Hematoxylin & Eosin Staining.

The results are shown in FIG. 11.

As shown in FIG. 11, in the solvent control group induced by the dexamethasone treatment, the epidermal layer was thinned and no hair follicles were formed. The epidermis of the group treated with dexamethasone and ingested collagen hydrolyzate (BP-CH) was thickened and developed as hair follicles developed. The minoxidil-coated group also showed thickening of the epidermal layer and development of hair follicles.

That is, it was found that the collagen hydrolyzate according to the present invention has an excellent effect of promoting hair growth by promoting hair follicle cell growth in a degenerative hair loss state.

<Example 7>

Collagen Using BP Hydrolyzate  Wrinkle improvement effect

SKH-1 hairless mouse-based, 6-week-old female (female) used in this test was imported and sold from Samtako BIOKOREA (Osan-si, Gyeonggi-do, Korea). Animal History (Dongnam Chemical Research Institute: Animal Facility Registration No. 412) A healthy animal that has undergone quarantine, purifying and rearing for one week at a farming environment with a temperature (22 ± 3) ℃, relative humidity (50 ± 10)%, and lighting time of 12 hours (07:00 to 19:00). It was.

As shown in Table 3, the experiment was divided into a total of seven groups of 10 animals in each group. Except for the normal group, the wrinkle-inducing group was subjected to UVB irradiation for 10 weeks, and in the irradiation group, the collagen hydrolyzate by the control group, the positive control group (retinoic acid 2 mg / kg, ip), and the normal collagen degrading enzyme (Alcalase) (COL) and collagen hydrolyzate (CTP) sample administrations according to the present invention were administered orally at 17 mg / kg and 34 mg / kg for 14 weeks, respectively. The feed was used for solid animal feed (Samtako BIOKOREA, Korea) and the drinking water was freely consumed.

In the case of positive control, 0.05% of retinoic acid was intraperitoneally referred to Belinda M method (The journal of investigative dermatology, 112 (3); 271-278, 1999.).

The ultraviolet irradiation device (East-West Science, UV-1000) was emitted by a Sunlamp emitting UVB in the cabinet, and the light source was irradiated at a height of 302 nm and UVB intensity of 0.3 mW / cm ² .

The amount of UV irradiation was measured by a UV-radiometer, the mouse was placed in a cage for ultraviolet irradiation, three times a week every other day on the back area, for a total of 10 weeks [0 weeks: 60 mJ / m ² (1 MED), 1 Note: 120 mJ / m ² (2 MED), 2-3 weeks: 180 mJ / m ² (3 MED), 4-5 weeks: 240 mJ / m ² (4 MED) 5-10 weeks: 240 mJ / m ² (4 MED)].

<7-1> Morphological Observation of Skin Wrinkles

For skin wrinkle observation, the animals were lightly anesthetized with ether for 5 to 14 weeks, photographed on the back surface with a digital camera, and then dorsal skin wrinkles with SILFLO (Flexico developments LTD. Tokyo, Japan) silicone rubber impression material. After replicating with microscope, the wrinkles were compared and observed for each experimental group.

 The result of visual observation of the shape of the skin wrinkles is shown in FIG. 12. SKH-1 hairless mouse skin was observed on the wrinkled skin pattern of simulated plate made using SILFLO. UVB-irradiated control group (group C) had thick wrinkles, wide intervals, deep wrinkles, and retinoic. P group and COL 17, 34 mg / kg group, CTP 17, 34 mg / kg group were less wrinkled or thinner and thinner than group C. (FIG. 12).

 In consideration of this, despite the 10-week UVB irradiation collagen hydrolyzate administration according to the present invention was determined to be effective on SKH-1 hairless mouse epidermal keratinocytes to improve and prevent skin wrinkles.

<7-2> Histological observation of skin

The excised skin tissues were fixed in a 10% neutral formalin solution at room temperature for 24 hours, washed, dehydrated, cleared, and infiltrated in a conventional manner, then embedded in paraffin, sliced into 4 μm thick overnight in a Bouin solution at room temperature. After soaking and Masson'trichrome staining, the amount and shape of the collagen fibers in the dermis were observed under an optical microscope.

In addition, the cut skin tissue was placed in 10% formalin to fix the tissue, washed with water, and then dehydrated sequentially with 60% to 100% alcohol and embedded in paraffin to make a block. Tissue sections of 5 μm thickness were prepared using a rotary microtome, stained with Hematoxylin & Eosin, and observed with an optical microscope (Nikon Co., Tokyo, Japan).

 When UVB is irradiated excessively, the epidermis becomes coarse and the structure of normal collagen is destroyed, resulting in an irregular array of collagen and decreased expression of elastic fibers.

As a result of observing the change and loss amount and shape of the elastic fibers in the dermal layer of the skin, the control group (C group) was able to confirm the denatured elastic fibers, positive control group and general collagen hydrolyzate (COP group), according to the present invention, In the collagen hydrolyzate-administered group (CTP group), it was confirmed that the elastic fibers were densified compared to the C group by 14 weeks of sample treatment (FIG. 13).

 In addition, as a result of observing the amount and form of collagen in the dermis, UVB-only group C irradiated collagen fibers, resulting in irregular arrangements, and a decrease in collagen content compared to normal (N group). However, all of the positive control group (P group) treated with retinoic acid (C group), COL group and CTP treated group for 14 weeks showed relatively dense and regular collagen density compared to group C. In particular, the CTP-administered group showed better results than the COL group in the amount and density of collagen in the dermis (FIG. 13).

<7-3> Expression of collagen and collagenase protein expression in skin tissue (Collagen 1A, MMP-1)

Excessive exposure to UV light causes a lot of MMP-1 expression and excess free radicals, which significantly impair antioxidant activity. In addition, it acts on keratinocytes to increase inflammatory cytokine secretion, inhibits collagen synthesis in fibroblasts in the dermis, promotes enzyme activity and forms excess wrinkles. Therefore, the present inventors administered the normal collagen hydrolyzate (COL group) and the collagen hydrolyzate (CTP group) according to the present invention to the SKH-1 haireless mouse irradiated with UVB for 14 weeks, and then collagen synthesis factor Collagen 1A and collagen decomposition. Protein expression of the enzyme MMP-1 was confirmed by Western blot.

For protein analysis, the mouse skin tissue collected after the end of the experiment was put in 200 μL per 20 mg of cell lysis buffer, and the tissue was crushed using a homogenizer, followed by centrifuge at 800 rpm for 10 seconds to use only the supernatant. After 24 hours ice incubation, centrifuge at 14,000 rpm, 4 ℃, 20 min. The protein was quantified using Bradford assay2) and separated by size using SDS-PAGE (Poly acrylamide Gel Electrophoresis). After transfer to PVDF (Polyvinylidene fluoride) membrane using Semi-dry transfer system (Bio-Rad, USA), treatment with Blocking buffer (5% skim milk, 1 × TBST buffer) containing 5% skim milk for 1 hour It was. After washing for 10 min and 3 times with 1 × TBST buffer, after treatment with Collagen 1A, MMP-1, and MMP-13 as primary antibodies, the reaction was carried out at 4 ° C. overnight, followed by washing for 10 min and 3 times with 1 × TBST buffer. After the reaction with the membrane using a Western Blot detection kit (Abfrontier, WEST SAVE GOLD), the expression level was observed using the Chemi-Doc (BIORAD XRS system) equipment, and compared with the GAPDH.

The results for this are shown in FIG. 14.

The control group (group C) irradiated with UVB significantly decreased the expression level of Collagen 1A (p <0.05), which is a collagen synthesis factor, whereas the expression levels of Collagen 1A were all increased in the sample administration group. In particular, it was confirmed that the p group and the COL 34, CTP 34 administration group to which retinoic acid was administered increased to a level of p <0.05 (Fig. 14A).

 As a result of confirming the protein expression of MMP-1, which is known as an enzyme that degrades collagen, it was confirmed that the expression level of MMP-1 was increased in the C group irradiated with UVB. On the other hand, the expression level of MMP-1 was significantly decreased (p <0.05) in the CTP 17 and 34 groups, which was much better than the positive control group and the COL group (FIG. 14B).

 Taken together, the results showed that collagen hydrolyzate increased and collagen synthase decreased due to excessive UV irradiation. Collagen hydrolyzate according to the present invention was treated for 14 weeks. The amount of expression was increased, and the expression of MMP-1 was decreased, and thus it was judged to be excellent in suppressing skin wrinkle formation.

<7-4> Skin moisturizing of skin tissue

When the skin tissue is damaged by UV irradiation, the oil and water content of the skin is reduced. To determine whether the content of oil and water in the skin tissue of the mouse irradiated with ultraviolet light for 10 weeks, and whether the collagen hydrolyzate of the present invention can recover the reduction of oil and water.

Corneometer CM 820 (Courage-hazake, Koln, Germany), a skin oil / moisture content measuring device, was administered after 5 weeks and 10 weeks of normal collagen hydrolyzate (COL group) and collagen hydrolyzate (CTP group) according to the present invention. Oil / moisture content was measured. After stabilizing at 21.5 ± 2 ° C. and 40 ± 5% relative humidity for 30 minutes or longer, a certain area was marked to measure moisture content.

The results are shown in FIG. 15.

As can be seen in Figure 15, due to 10 weeks of ultraviolet irradiation, the oil content and moisture content of the control (C) group was confirmed to be significantly reduced compared to the normal control group (N) not irradiated with ultraviolet rays. On the other hand, in weeks 5 and 10, the animal group treated with the collagen hydrolyzate according to the present invention showed higher oil and water content than the normal control group, and the effect was that the amount of oil / moisture increased as the content of the collagen hydrolyzate increased. Increased. In particular, it was confirmed that the skin moisturization of the rat administered the collagen hydrolyzate (CTP group) according to the present invention is very excellent compared to the normal collagen hydrolyzate (COL group) (FIG. 15A, B).

<Example 8>

Collagen Using BP Hydrolyzate Skin hydration  Improvement effect

With reference to the KFDA guidelines (Health functional food test guideline, beauty-related functional test, 2004.), the experiment was divided into a total of five groups, each 10 dogs as shown in Table 4. The normal group (Group N) used Balb-C mice and the skin dry model was set to NC / Nga Tnd mouse which increased the water release of the epidermis, which is useful for moisturizing experiments. To induce dry skin, Okawa et al. Refer to the method (Dermatol Sci. 66 (2): 136-43, 2012.) and moisten cotton pad (2 x 2cm) in a 1: 1 mixture of acetone and ether for 1 week before dissection, and put it on the back for 15 seconds. After placing it, the skin was washed with distilled water for 30 seconds to induce dry skin.

 In the normal control group (Normal group, N group) that did not induce skin dryness and the negative control group (Control group, C group) that induced skin dryness and administered only solvent, 0.9% saline was orally administered for 4 weeks, and the positive control group (P Group NAG used N-acetyl glucosamine (NAG), which has been approved by the Ministry of Food and Drug Safety to improve skin drying and water retention. The intake dose was orally administered 17 mg / kg of N-acetyl glucosamine.

 On the other hand, despite the low content of CTP17 in Example 7-4, skin moisturizing was better than COL34, compared with the collagen hydrolyzate of the present invention prepared according to Example 4 17 mg / kg (CLT17 group) 34 mg / kg of collagen hydrolyzate (COL34 group) prepared according to Example 1 were orally administered for 4 weeks each. Feed was fed using experimental animal solid feed (Samtako BIOKOREA, Korea), and drinking water was freely consumed.

<8-1> Skin moisture content measurement

The stratum corneum of healthy epidermis contains 15 ~ 20% of moisture, and when moisture of epidermis falls below 10%, skin becomes dry, shine and elasticity disappear, and wrinkles increase. This soft, moist feeling of the skin is maintained by the moisture present in the stratum corneum of the epidermis.

 Moisture content of the stratum corneum is determined by lipids produced and secreted from the epidermis, sebaceous membranes that are adherents, and Natural Moisture Factor (NMF), a water-soluble component present in the stratum corneum. It is closely related to the change of content of.

The effects and characteristics of 4 weeks of administration of collagen hydrolysate 17 mg / kg (CTP 17) or collagen hydrolyzate 34 mg / kg (COL 34) prepared by the conventionally reported collagen degrading enzyme according to the present invention One week before the end of the experiment, the skin moisture content measuring device, Corneometer CM 820 (Courage-hazake, Koln, Germany) was used to confirm the results. In the constant temperature and humidity room (temperature 21.5 ± 2 ℃, relative humidity 40 ± 5%) was allowed to stabilize for more than 30 minutes, and then marked a certain area to measure the moisture content.

The results are shown in FIG. 16.

Balb-C mice in the normal control group (N group) retained the water content of the epidermal layer, whereas the control group (group C) induced skin drying decreased water content compared to the normal control group. On the other hand, the positive control group, CTP 17, COL 34 group showed the effect of increasing the skin moisture content, of which CTP 17 showed the most excellent effect.

In the case of CTP, despite the administration of 17 mg / kg collagen hydrolyzate of the collagen hydrolyzate according to the present invention has a high content of collagen tripeptide, as it shows a better skin moisturizing effect than COL 34 mg / kg It was found to be significantly better than the degradation product.

<8-2> Effects on Itching due to Skin Drying

The skin is inherent in various immune substances and is the first place to react to foreign substances. Damage to the skin barrier increases transdermal moisture loss and decreases water binding, resulting in dry skin and itching and scratching. Therefore, itching effect was evaluated by the administration of each substance in a water-dry animal model.

 Scratching behavior was observed to scratch the animals for 10 minutes. The same group was placed in one cage and counted how many times each mouse scratched. If the continuous operation lasting longer than 2 seconds lasted for n seconds, it counted n times, and each short operation within 2 seconds was counted once.

The results are shown in FIG. 17.

As a result of confirming the number of scratches for 10 minutes after inducing skin dryness, the number of scratches increased at a p <0.05 significance level in the control group (group C) that caused the dryness, whereas the number of scratches in the group treated with each sample was mostly increased. Decreased (p <0.05).

On the other hand, itching effect from skin dryness was the best in CTP 17, which was superior to the positive control group as well as COL 34.

<8-3> Skin moisturizing factor protein recovery effect (Collagen 1A, AQP3, HAS2)

For protein analysis, the skin tissue was placed in a cell lysis buffer, the tissue was crushed with a homogenizer and centrifuge. Proteins were quantified by Bradford assay, followed by SDS-PAGE (Poly acrylamide Gel Electrophoresis). After transfer to PVDF (Polyvinylidene fluoride) membrane using semi-dry transfer system (Bio-Rad, USA), the expression level of Collagen 1A, AQP3, HAS2 protein was measured using Chemi-Doc (BIORAD XRS system, USA). Observations were made and compared with GAPDH.

(i) Collagen is an important component that participates in the regulation of functions such as cell shape, differentiation, migration, protein synthesis, and in the healing of wounds. Dry skin can have a negative effect on skin physiology, including a decrease in the amount of collagen protein in the skin, resulting in wrinkles.

(ii) Aquaporin-3 (AQP3) is known to play the most important role in hydration of the skin. Dry skin reduces AQP3 expression and has been reported to affect trans epidermal water loss (TEWL). Increased aquaporine 3 (AQP3), an aquaglyceroporin present in cell membranes, is known to preserve skin moisture and eventually prevent aging (J. Lipid Res. 46: 2706, 2005.) Therefore, aquapotine3 (AQP3) present in cell membranes Confirmation is one of the key factors to check skin moisture level (Biology of the cell. 97 (7): 479-486 2005.)

(iii) Hyaluronic acid (HA) is a glycosaminoglycan of macromolecules in extracellular connective tissues and is known to act as a barrier against water evaporation. However, it is known that the content of hyaluronic acid, which acts as a water barrier, decreases rapidly with age. Among the external environmental factors, especially ultraviolet rays have a negative effect on the maintenance of constant hyaluronic acid of the skin. The decrease in the content of hyaluronic acid due to these intrinsic or extrinsic factors becomes a factor of decreased skin elasticity, rough skin and wrinkles. Therefore, maintaining the content of hyaluronic acid in the skin is a very important factor in maintaining the skin while moisturizing and skin. Hyaluronic acid is continuously synthesized by hyaluronic acid synthase (HAS). Therefore, if the gene expression of HAS is increased, the amount of hyaluronic acid in the skin is increased to have a positive effect on not only skin moisturizing but also elasticity, wrinkles and the like.

Thus, the present inventors attempted to evaluate the effect of collagen hydrolyzate according to the present invention on the reduction of skin moisturizing factors by skin drying through Western blot.

The results are shown in FIG. 18.

As can be seen in Figure 18, Collagen 1A, AQP3 and HAS was confirmed that significantly reduced than the normal control by the skin dry induction. On the other hand, in the positive control group, CTP 17 and COL 34 administration group, the amount of each protein was recovered significantly compared to Control (C group). Among them, the CTP 17 group showed a significantly better skin moisturizing factor recovery effect than the positive control group (P group), and showed a significantly better effect than the COL 34 group despite the half dose of the COL 34 group.

In other words, the collagen hydrolyzate of the present invention containing a high amount of collagen tripeptides has a very good absorption in the body, it can be seen that it can exhibit an excellent effect even at a small dosage.

As described above, since the collagen hydrolyzate of the present invention exhibits an excellent skin wrinkle improvement and skin moisturizing effect, it may have an effect of preventing skin aging and improving skin elasticity.

The present invention relates to a collagen hydrolyzate containing a high amount of collagen tripeptide and its use. The collagen hydrolyzate of the present invention has a significantly higher content of collagen tripeptide compared to the previously reported collagen hydrolyzate, and thus has an excellent absorption rate in the body. It has high industrial applicability due to excellent hair loss prevention, hair growth promotion, skin wrinkle improvement and moisturizing effect.

Claims (9)

(a) mixing the young (fish scales) with water and heat-treating;
(b) preparing collagen degrading enzyme by centrifuging Bacillus subtilis strain culture of Accession No. KCTC12866BP, concentrating the centrifuged supernatant, and purifying using ion exchange chromatography; And
(c) the collagen degrading enzyme is added to the mixture of step (a) and hydrolyzed at 25 to 45 ° C. for 5 to 15 hours, and the collagen tripeptide is 20 to 70% by weight relative to the total hydrolyzate. Collagen hydrolyzate, characterized in that it is contained as.
delete The collagen hydrolyzate of claim 1, wherein the ion exchange chromatography of step (b) uses cation exchange chromatography, and sodium chloride (NaCl) is used at a concentration of 0.1 to 0.3 M.
The collagen hydrolyzate of claim 1, wherein the ion exchange chromatography of step (b) uses anion exchange chromatography, and sodium chloride (NaCl) is used at a concentration of 0.09 to 0.155M.
A food composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate according to any one of claims 1, 3 and 4 as an active ingredient.
A pharmaceutical composition for preventing hair loss and promoting hair growth, comprising the collagen hydrolyzate according to any one of claims 1, 3 and 4 as an active ingredient.
A cosmetic composition for preventing hair loss and promoting hair growth comprising the collagen hydrolyzate according to any one of claims 1, 3 and 4 as an active ingredient.
Cosmetic composition for skin wrinkle improvement, skin elasticity improvement, or skin moisturizing comprising the collagen hydrolyzate according to any one of claims 1, 3 and 4. as an active ingredient.
A food composition for skin wrinkle improvement, skin elasticity improvement, or skin moisturizing, comprising the collagen hydrolyzate according to any one of claims 1, 3, and 4 as an active ingredient.

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