JP5276813B2 - Elastin-degrading peptide, method for producing elastin and its enzyme-degrading peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a zymolytic peptide of an elastin obtained by using a pig aorta blood vessel as a raw material, and a method for preparing a pig aorta blood vessel-origin elastin and its zymolytic peptide. <P>SOLUTION: The elastin zymolytic peptide of this invention originates in a pig aorta blood vessel, and the amino acid composition can be made into one similar to a human elastin. Further, a pig aorta blood vessel exists abundantly as a raw material, and since it is discarded conventionally, it is advantageous in cost. Moreover, the method of this invention for preparing the elastin and the zymolytic peptide is a method in which the elastin and its zymolytic peptide are prepared efficiently from a pig aorta blood vessel. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to an enzyme-degrading peptide of elastin obtained from porcine aortic blood vessels, and a method for producing the elastin and the enzyme-degrading peptide. Such elastin enzyme-degrading peptides are useful not only as health food materials but also as cosmetic materials and medical materials.

Elastin is a protein that exists together with connective tissue components such as collagen in the skin, blood vessels, lungs, tendons, etc., and in addition to maintaining the structure of the tissue, it plays a role of giving elasticity that can respond to various shape changes of the tissue Yes. About 95% of the amino acid residues of elastin are nonpolar amino acids, which are characteristically contain desmosine and isodesmosine, and have a characteristic that the hydroxyproline content is lower than that of collagen. Since it has a cross-linked structure with desmosine and isodesmosine, it has strong resistance to acids and alkalis.
Elastin is present so as to be entangled with collagen in the dermis portion of the skin, and elasticity is given to the skin due to its elasticity and the elasticity is maintained, and the decrease in elastin causes skin wrinkles and tarmi. Therefore, it is known that elastin and its degradation peptide are useful as a topical or oral skin aging prevention / improving agent (see Patent Documents 1 and 2, etc.).
In addition, since it is effective for maintaining the elasticity of blood vessels and healing wounds, it is also attracting attention in the medical field.
JP 2001-72572 A JP 2006-143671 A

Elastin and its degradation peptide are substances used in the fields of cosmetics, food, medicine and the like, and bovine ligaments, fish skin and arterial spheres have been used as raw materials for elastin.
More specifically, bovine ligament has been conventionally known as a tissue having a high content of elastin, and elastin has been produced from bovine ligament. However, bovine spongiform encephalopathy (BSE) -infected cattle have been discovered in Japan, and elastin peptides derived from cattle have been avoided.
As an alternative, fish-derived elastin has been used (Patent Document 3). However, the elastin content of fish skin is low. Even in the case of an arterial sphere with a high elastin content, the amount of elastin that can be collected from one animal is small, so that elastin and elastin-degrading peptides derived from fish arterial spheres are expensive and are not sufficient as an alternative raw material for bovine ligament.
In addition, fish-derived elastin is far from the composition of elastin in human skin and blood vessels compared to elastin from mammalian tissues such as bovine ligaments, so there are doubts about its effectiveness as a cosmetic or functional ingredient. there were.
JP-A-2005-343851

From the above problems, there has been a demand for a raw material that is abundant, has no BSE problem, has a composition similar to that of human elastin, and has a high elastin concentration.
Therefore, as a result of studying raw materials that satisfy the above conditions, the present inventors recalled that pigs that are the same mammals as humans and have no concerns about BSE are used as raw materials. However, the neck ligament used as a raw material for bovine elastin is the neck ligament. However, since the neck is short in pigs, the neck ligament was too small to be used as a raw material. Therefore, when the site | part which can become a raw material was examined in a pig, it discovered that the aortic blood vessel was a structure | tissue with the highest total yield of elastin. Moreover, most of the aortic blood vessels in pigs are currently discarded, except that some are used for food, and using them as raw materials also leads to effective utilization of unused livestock resources. .
However, in comparison with the term ligament, the porcine aortic blood vessels are contaminated with various tissues and components other than elastin, and even if the method of purifying elastin from the bovine term ligament is applied as it is, high purity elastin can be obtained. I could not do it.
In addition, when elastin is purified from porcine tissues / organs, there is a problem that a bad odor (animal odor) is generated and the working environment is deteriorated. As a result, the purified elastin and its decomposed peptide may also have a bad odor. There was a need for high-quality elastin with low content.

  From these problems, the present inventors can effectively remove contaminating proteins such as collagen coexisting with elastin using porcine aortic blood vessels as a raw material, resulting in high-quality elastin and producing off-flavors. When various purification conditions were examined, it was found that the intended purpose could be achieved by purification under specific conditions. The present invention is based on such knowledge, and provides a porcine aortic blood vessel-derived elastin enzymatically degrading peptide having high purity, high quality and little off-flavor, and a method for producing porcine aortic blood vessel-derived elastin and its enzymatically degrading peptide.

The present invention made to solve the above problems is an elastin degrading peptide that is an enzymatic degradation product of elastin derived from porcine aortic blood vessels. In particular, the content of desmosine and / or isodesmosine in the amino acid composition (molar ratio) is 0.06% or more, and the ratio of the sum of desmosine content and isodesmosine content / hydroxyproline content ratio (molar ratio) is 0.1 or more. Elastin degrading peptides that are preferably 0.22 or more, more preferably 0.27 or more are preferred.
The method for producing elastin of the present invention comprises treating porcine aortic blood vessels with 0.05-0.5N alkaline aqueous solution under heating to obtain porcine aortic blood vessel-derived elastin, and further converting the elastin with a protease. This is a method for producing an elastin enzymatic degradation peptide comprising enzymatic degradation.
Furthermore, the skin improving agent of the present invention contains the aforementioned elastin degrading peptide, and particularly preferably contains collagen and / or a collagen degrading peptide.

Since the elastin degrading peptide of the present invention is derived from porcine aortic blood vessels and is made from mammalian tissue, its amino acid composition can be close to that of human elastin. In addition, porcine aortic blood vessels are abundant as raw materials and are conventionally discarded, which is advantageous in terms of cost.
In addition, according to the method for producing elastin and elastin-degrading peptide of the present invention, not only high-purity elastin and elastin-degrading peptide can be obtained, but also off-flavor generated during production can be prevented or suppressed, and the working environment can be improved. Furthermore, the odor of the obtained elastin and the elastin degrading peptide can be reduced.
Furthermore, the skin-improving agent of the present invention contains the above-mentioned elastin-degrading peptide and, if necessary, collagen and / or collagen-degrading peptide, and is taken orally or administered (applied) externally. Thus, it is possible to maintain elasticity by giving elasticity to the skin, and to prevent and improve skin wrinkles and tarmi.

As described above, the elastin degrading peptide of the present invention is a peptide obtained by enzymatic degradation of porcine aortic blood vessel elastin and can be prepared by various production methods. It is preferable to prepare accordingly.
That is, the method for producing elastin according to the present invention is to obtain elastin by treating a porcine aortic blood vessel with a 0.05-0.5N alkaline aqueous solution under heating. As described above, since elastin has alkali resistance, when porcine aortic blood vessels are treated with an alkaline solution, collagen is gelatinized and solubilized, and other contaminants (eg, proteins) are also solubilized. Porcine elastin can be selectively obtained by collecting insoluble substances.
However, as described above, when refining raw materials from livestock tissues and organs such as pigs, there is a problem that a bad odor (animal odor) is generated and the working environment is deteriorated. As a result, the purified elastin and its degradation peptide also have a bad odor. There is a problem that remains.

Therefore, various studies have been made to solve the problem. As shown in Example 1 described later, when a porcine aortic blood vessel is treated with an alkaline aqueous solution of 0.05 to 0.5 N under heating, an unpleasant odor is reduced. It has become clear that elastin purification can be improved.
In such a method, the porcine aortic blood vessel as a raw material can be obtained from a processing plant (and a slaughterhouse) for processing pork meat. In particular, the porcine aortic blood vessel is a part that has been disposed of in the past, and can effectively use waste. The kind of pig (for example, Berkshire breed, ternary breeding, etc.) is not particularly limited, and may be any kind.
Furthermore, the content of elastin decreases dramatically with aging. For example, in the case of humans, it rapidly decreases after the age of 20. Cattle are usually shipped / killed in about two and a half years, while pigs are usually shipped / killed in about six months. Therefore, pigs are younger than cattle, and they are considered to be desirable raw materials with abundant elastin content.

In the method for producing elastin of the present invention, porcine aortic blood vessels are treated with an alkaline aqueous solution. The alkaline substance is not particularly limited, and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide and alkaline earth metal hydroxides such as calcium hydroxide are used. To alkali metal hydroxides, in particular sodium hydroxide.
A 0.05 to 0.5 N solution is used as the alkaline aqueous solution. If it is less than 0.05N, the decomposition of the porcine aortic blood vessels is slow, and if it exceeds 0.5N, the nasty smell of the elastin preparation becomes strong during the treatment and is not preferable.

  The alkaline aqueous solution is used in an amount of 0.5 to 3 times (weight ratio), preferably 1 to 2 times (weight ratio) with respect to the wet weight of the porcine aortic blood vessel as a raw material. Moreover, when using an alkali metal hydroxide as an alkaline substance, it is preferable to use it in the ratio of 0.005-0.025 mol with respect to 100 weight part (wet weight) of pig aortic blood vessels.

  The treatment of porcine aortic blood vessels with such an alkaline aqueous solution is performed under heating, and is usually performed at about 50 to 95 ° C, preferably about 80 to 90 ° C. The treatment time can be appropriately adjusted depending on the alkali concentration of the alkaline aqueous solution and the degree of decomposition of collagen and other contaminants, but is usually about 0.5 to 5 hours, preferably about 1 to 2 hours.

  By the above treatment, the collagen that is the main component of the porcine aortic blood vessels is gelatinized, and other contaminants are also decomposed and solubilized, so that the insoluble part of the reaction solution is recovered, washed with water, washed with salt water as necessary, Porcine aortic blood vessel-derived elastin can be obtained by acid washing.

  In addition, after completion | finish of said alkaline aqueous solution process, it is preferable to neutralize a reaction liquid using a conventional acid (For example, hydrochloric acid, a sulfuric acid, phosphoric acid, an acetic acid, etc.). By such neutralization treatment, the degree of purification of the purified elastin can be increased (see Example 1 below).

The elastin-degrading peptide can be obtained by enzymatically degrading the porcine aortic blood vessel-derived elastin thus obtained with a protease.
The protease used here is not particularly limited as long as it is an enzyme capable of degrading elastin. For example, bromelain, papain, trypsin, pepsin, elastase and the like can be mentioned, but preferably a microorganism-derived alkaline protease (for example, Alkalase, Novozyme FM, Protease P, etc. are all trade names).
The amount of the enzyme used can be appropriately selected according to the enzyme activity, reaction temperature, reaction time, etc., but the enzyme is used in an amount of 1: 0.005-0.05 (weight ratio) relative to the substrate (elastin). ), Preferably 1: 0.01 (weight ratio).
The molecular weight of the elastin-degrading peptide can be appropriately adjusted according to the amount of enzyme used, reaction temperature, reaction time, purpose of use of the peptide, and the like. For example, the average molecular weight is about 300 to 8000, preferably about 500 to 5000, more preferably Is adjusted to about 1000 to 3000.

The method for preparing the elastin-degrading peptide will be described in more detail. Porcine aortic blood vessel-derived elastin is mixed with a buffer solution or purified water adjusted to the optimum pH of the protease to be used, and then a predetermined amount of protease is added to the protease. Incubate at an optimum temperature for an appropriate time (for example, about 24 hours) to enzymatically degrade elastin. After decomposition, heat (for example, at 90 ° C. for about 10 minutes) to inactivate the enzyme, allow to cool, filter to remove insoluble matter, and perform treatment with diatomaceous earth, activated carbon, etc. as necessary Thus, an elastin enzymatic degradation peptide solution is obtained. In the above treatment, when alkaline protease is used as the protease, the reaction solution is neutralized and desalted with a conventional acid agent (eg, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, etc.) after completion of the enzymatic reaction. Is preferred. By this operation, the off-flavor of the obtained elastin enzymatic degradation peptide can be remarkably reduced.
The elastin enzyme-degrading peptide thus obtained can be further purified by a conventional method such as ultrafiltration, gel filtration, ion exchange column chromatography, reverse phase high performance liquid chromatography and the like.
Furthermore, the obtained elastin enzyme-degraded peptide solution can be pulverized by drying by a conventional method such as freeze-drying or ventilation drying.

  The elastin derived from porcine aorta blood vessels obtained by the above production method and the enzyme-decomposed peptide of elastin, which is an enzyme degradation product thereof, are 0.06% in amino acid composition (molar ratio) of desmosine and / or isodesmosine which are characteristic amino acids of elastin. Further, the sum of desmosine (Des) content and isodesmosine (Ide) content / ratio (molar ratio) of hydroxyproline (Hyp) content is 0.1 or more, more preferably 0.22 or more, more preferably It had the characteristic of being 0.27 or more.

The skin improving agent of the present invention contains the porcine aortic blood vessel-derived elastin degrading peptide as an active ingredient, and preferably further contains collagen and / or a collagen degrading peptide.
The origin of the collagen is not particularly limited, but pig-derived collagen is preferably used. The method for producing collagen is already well known, and commercially available collagen may be used. The collagen degrading peptide is a peptide obtained by enzymatic degradation of collagen using the above-mentioned protease, and a peptide having a molecular weight of about 150 to 3000, preferably about 250 to 2500 is used. A commercially available collagen-degrading peptide may be used.
The amount of collagen and / or collagen-degrading peptide to be used with respect to the elastin-degrading peptide is not particularly limited. Preferably it is used in a ratio of 1 to 50 times.

The skin-improving agent of the present invention may be an agent in any dosage form of oral ingestion (administration) or external administration (application).
Examples of the form of oral intake (administration) include food (health food material) and pharmaceutical products.
As food (health food material), it is ingested by humans as it is in various forms such as solid or food and drink.
As a solid, the above-mentioned components are mixed with necessary components in the formulation such as appropriate physiologically acceptable additives (for example, carriers, excipients, diluents, etc.) as necessary, It can be obtained by preparing it in an appropriate dosage form, and examples of the form include tablets, powders, granules, capsules and the like.

Examples of the food and drink include beverages (for example, drinks, lactic acid bacteria beverages, milk beverages, coffee beverages, tea beverages, green tea beverages, juices), confectionery (for example, biscuits, cookies, candy, snacks, ramunes). Confectionery, etc.), seasoning liquids (eg, sauce), meat products (eg, ham, sausage, etc.), fish products (eg, kamaboko, chikuwa), dairy products (eg, butter, cheese, etc.) Add to and ingest.
Such food and drink can be prepared according to a conventional method except that an active ingredient is added in an appropriate process in the preparation stage. In addition, conventional additives may be added to such foods and drinks as necessary. Examples of such additives include vitamins (for example, vitamin C, vitamin A, vitamin E, etc.), minerals (For example, zinc, copper, manganese, etc.), physiologically active substances, sweeteners, acidulants, antioxidants, fragrances, salts, excipients, colorants and the like.

  Moreover, as a pharmaceutical, each said component is mixed with components necessary for formulation such as appropriate physiologically acceptable additives (for example, carriers, excipients, diluents, etc.) as necessary, It can be obtained by preparing it in an appropriate dosage form, and examples of the form include tablets, powders, granules, capsules and the like.

Moreover, as an external preparation, for example, forms, such as cream, an emulsion, a lotion, an ointment, and packs, can be taken. These preparations can be prepared according to conventional methods in cosmetic production.
Conventional ingredients such as a humectant and an antioxidant may be added to the preparation as necessary.

In the skin improving agent of the present invention, when it is in the form of oral ingestion (administration), 0.1 to 10 g, preferably 0.5 to 7 g, more preferably 1 to 10 g of the elastin degrading peptide of the present invention per day. 5 g may be taken once or divided into several times. Further, as a more preferable form, when collagen and / or collagen-degrading peptide is added, the component is adjusted so as to take 0.5 to 10 g, preferably 1 to 7 g, more preferably 5 g per day. That's fine.
On the other hand, when it is used as an external preparation, a preparation containing about 1 to 10%, preferably about 2 to 5%, of the elastin degrading peptide of the present invention may be applied once or several times a day. Further, as a more preferable form, when collagen and / or a collagen-degrading peptide is added, a preparation containing the component in an amount of 1 to 10%, preferably about 2 to 5% may be used.

  Hereinafter, although the present invention will be described in more detail based on examples and test examples, the present invention is not limited to such examples.

Example 1
Examination of production method of elastin derived from porcine aortic blood vessel 100 g (wet weight) of porcine aortic blood vessel was dispersed in 200 g of sodium hydroxide aqueous solution having the concentration shown in Table 1-1 and treated at 90 ° C. for the time shown in Table 1, The unpleasant odor and the purity of the resulting elastin were examined.
In the table, the degree of purification of elastin refers to the sum of the content of desmosine (Des), which is an amino acid specific to elastin, and the content of isodesmosine (Ide), and the content of hydroxyproline (Hyp), an amino acid specific to collagen. Using the ratio as a guide, high means the ratio is about 0.3, medium means about 0.2, and low means about 0.1.
In addition, the measurement of off-flavor was carried out by a sensory test (1 point: no odor, 2 points: slightly odor, 3 points: odor, 4 points: bad odor), and the average score was rounded off. Based on.

  As the reaction conditions for the alkali treatment on porcine aortic blood vessels, when considering the problem of elastin purification and off-flavor, as shown in Table 1, the concentration of sodium hydroxide aqueous solution is preferably 0.05 to 0.5N, It was found that the reaction time is preferably about 0.5 to 5 hours. More preferably, it has been found that the concentration of the sodium hydroxide aqueous solution is 0.125 to 0.5 N and the reaction time is about 0.5 to 2 hours, most preferably 0.125 N and the reaction time is about 1 to 2 hours.

  After performing the alkali treatment in Experiment No. 2 of Table 1-1, when the treatment solution was neutralized with 1N hydrochloric acid, the purity of the obtained elastin was improved as shown in Table 1-1. It turns out to get.

  In addition, 30 g of elastin obtained by performing the alkali treatment in Experiment No. 4 in Table 1-I was added to 50 ml of water, and under conditions of 60 ° C. and pH 8.0, enzyme alcalase (trade name, alkaline protease, manufactured by Novozyme) ) When processing and enzymatic decomposition progressed (about 240 minutes), heat at 90 ° C for 10 minutes to inactivate the enzyme, then neutralize with 0.1N hydrochloric acid, then remove insoluble matter with mesh The filter was pressed using diatomaceous earth (filter aid, Cell Pure S1000). When the nasty smell of the obtained elastin enzymatic degradation peptide was measured in the same manner as described above, as shown in Table 1-III, by performing the above-described hydrochloric acid neutralization treatment, the nasty smell of the resulting elastin enzymatic degradation peptide was It became clear to reduce.

Example 2
1000 kg of water was added to 500 kg (wet weight) of fresh porcine aortic blood vessels, 5 kg of sodium hydroxide flakes were added, and the mixture was stirred at 90 ° C. for 1 hour, and then the alkaline solution was discharged. Next, 500 kg of 50 ° C. water was added, and the mixture was stirred for 15 minutes for washing, and the washing water was discharged. Further, 500 kg of 50 ° C. water is added, and the mixture is stirred for 15 minutes. The washing water is discharged, 400 kg of 50 ° C. water is added, 300 ml of 10% hydrochloric acid is added, and the mixture is neutralized by stirring for 1 hour. After discharging, 400 kg of water at 50 ° C. and 16 kg of sodium chloride were added and stirred for 1 hour. Next, the saline solution was discharged, 500 kg of cold water was added, and the mixture was stirred for 15 minutes. Further, 500 kg of cold water was added, stirred for 15 minutes, the cold water was discharged, and insoluble matter (elastin) was collected. The obtained insoluble matter was pulverized with a chopper to obtain 125 kg of pulverized product. The same operation was repeated to obtain a total of 250 kg of pulverized material.
500 kg of water was added to 250 kg of the obtained pulverized product and heated to 60 ° C. with stirring. After confirming that the temperature reached 60 ° C., 2.5 L of enzyme alcalase was added, and Brix measurement was performed while adjusting the pH to 8.0. When enzymatic degradation progressed (about 360 minutes, Brix 9.5), 90 ° The enzyme was inactivated by heating at 10 ° C. for 10 minutes. The enzyme treatment solution was filtered through a mesh to remove insoluble matters. The filtrate is filtered through a filter filled with diatomaceous earth (trade name: Celpure S1000), treated with activated carbon, further filtered through a filter filled with diatomaceous earth (Celpure S300), heated to 90 ° C, and maintained at 70 ° C until Brix 50 is reached. Concentrated. Next, after heat sterilization at 90 ° C., the product was dried with a ventilator, further pulverized, pulverized, and sieved (40 mesh) to obtain an elastin-degraded peptide powder.

The elastin-degrading peptide powder thus obtained (hereinafter simply referred to as the elastin peptide of the present invention) has the amino acid composition (molar ratio) shown in Table 2, and is an amino acid contained only in elastin, and the content of elastin. It contained 0.28% in total of desmosine (Des) and isodesmosine (Ide), which are index of The ratio of the sum of Des content + Ide content / Hyp content was 0.33.
Table 3 shows the results of comparing the contents (%) of Des, Ide and Hyp of the elastin peptide of the present invention and the conventional products (commercial products A and B). As shown in Table 3, the elastin peptide of the present invention contains Des and Ide at a higher level than conventional products, and also has a low Hyp content, so it is highly purified and is an excellent elastin-degrading peptide. It showed that.

In addition, when compared with the amino acid composition of the fish-derived elastin degradation product described in Patent Document 3, the elastin peptide of the present invention has a high Ala and Val content, while having a low Asp, His, Thr and Lis content. It can be clearly distinguished from the fish-derived elastin degradation product.
In addition, the elastin peptide of the present invention has a high His and Tyr content, while having a low Ile and Leu content, compared with the already known amino acid composition of bovine ligament-derived elastin degradation product. Can be distinguished from degradation products.

Example 3
The molecular weight of the elastin peptide of the present invention was measured by high performance liquid chromatography (HPLC) under the following conditions.
Column = Superdex
Peptide (GE Healthcare Bioscience)
Eluent = 0.1% TFA and 30% acetonitrile
HPLC condition = flow rate: 0.5 ml / min, detection: 240 nm, gradient mixed solvent As a result, the average molecular weight was about 2,500 (molecular weight range: 500 to 7,500). Elastin is a protein in which the precursor tropoelastin is intermolecularly cross-linked by desmosine, isodesmosine, etc., so it is difficult to specify the molecular weight, but tropoelastin is generally said to have a molecular weight of 68,000-70,000 Thus, elastin itself is presumed to be a fairly large protein exceeding 100,000, and is an insoluble protein in water. As described above, since the elastin peptide of the present invention has a molecular weight reduced to about 2,500, the solubility in water can be remarkably improved.
That is, as described above, elastin is a substantially insoluble protein. However, in the elastin peptide of the present invention, 36.7 g dissolves in 100 ml of distilled water at room temperature, and its solubility is 26.86%. all right. In other words, elastin peptide formation significantly improves the solubility, and accordingly, the absorbability can also be improved.

Test example 1
Prepare drinking water using the elastin peptide of the present invention and porcine-derived collagen-degrading peptide, and have 70 females in their 20s to 50s (10 people in each test group) drink, The test was conducted as follows.
The porcine collagen-degrading peptide (hereinafter referred to as collagen-degrading peptide) was purified from pig skin according to a conventional method. That is, insoluble collagen obtained from fresh pig skin was reacted with the enzyme trypsin at 30 to 80 ° C. for about 0.5 to 3 hours, and purified by ultrafiltration and diatomaceous earth filtration (average molecular weight of peptide: About 2500). As a comparative example, a fish-derived elastin-degrading peptide (commercially available product) was also used for the test.

Test example 1
One 100 ml of drinking water containing 5% (weight%, hereinafter the same) of the elastin peptide of the present invention was ingested daily for 20 days.
Test example 2
The test was conducted in the same manner as in Test Example 1 except that drinking water containing 5% of the elastin peptide of the present invention and 5% of the collagen degrading peptide was used.
Test example 3
The test was conducted in the same manner as in Test Example 1 except that drinking water containing 0.1% of the elastin peptide of the present invention and 5% of the collagen degrading peptide was used.
Comparative Example 1
The test was conducted in the same manner as in Test Example 1 except that drinking water containing 5% fish-derived elastin-degrading peptide was used.
Comparative Example 2
The test was conducted in the same manner as in Test Example 1 except that mere drinking water (drinking water containing no elastin peptide of the present invention) was used.
Comparative Example 3
The test was conducted in the same manner as in Test Example 1 except that drinking water containing 0.1% of the elastin peptide of the present invention was used.
Comparative Example 4
The test was conducted in the same manner as in Test Example 1 except that drinking water containing 5% collagen-degrading peptide was used.

Table 4 shows the results of the above test examples. In addition, the determination of the effect was evaluated as follows based on the report of each subject.
1: No effect 2: Somewhat effective 3: Some effect 4: Somewhat strong effect 5: Some strong effect

As shown in Table 4, the drinking water containing 5% of the elastin peptide of the present invention is compared with the drinking water containing 5% fish-derived elastin degrading peptide, drinking water not containing elastin peptide and drinking water containing 5% collagen degrading peptide. The gloss was significantly superior.
In addition, the drinking water containing 0.1% elastin peptide of the present invention + 5% collagen degrading peptide is synergistically superior to the drinking water containing 0.1% elastin peptide of the present invention or the drinking water containing 5% collagen degrading peptide. Showed the effect.
Furthermore, drinking water containing 5% elastin peptide + 5% collagen degrading peptide of the present invention has a very remarkable effect, and synergistically compared to drinking water containing 5% elastin peptide or 5% collagen degrading peptide. Excellent effect.

Test example 4
A cell culture solution using an elastin-degrading peptide was prepared, normal human skin fibroblasts were cultured, and cell growth promoting action and collagen production promoting action were examined.
That is, the elastin peptide of the present invention is added to a cell culture medium (DMEM, 5% FBS and 0.1% gentamicin added, all Sigma) so as to be 5 or 50 ng / ml, and human skin is used using this culture liquid (100 μl). Derived fibroblasts NHDF (Normal Human Dermal
Fibroblasts (Neonatal Skin), Sanko Junyaku, number of cells: 5000) were cultured for 5 days at 37 ° C. and CO ₂ 5.0%.
After the culture, the number of cells was measured by Cell Counting Kit-8 (DOJINDO). Further, the amount of collagen was measured using a collagen stain kit (collagen workshop), and the amount of collagen produced outside the cells was measured. Each action was evaluated by comparison with the case where no elastin peptide was added (control).
The result is shown in FIG. As shown in FIG. 1, depending on the amount of the elastin peptide of the present invention added, both the number of cells and the amount of collagen increase, and it is found that the addition of 50 ng / ml of the elastin peptide of the present invention increases significantly. It was. That is, it was found that the elastin peptide of the present invention has a fibroblast proliferation promoting action and a collagen production promoting action.

Test Example 5
A feed for mice using the elastin peptide of the present invention was prepared and administered to hairless mice, and the effect on mouse skin viscoelasticity was examined.
Based on the basic feed (AIN-93G, Oriental yeast), a feed in which 1/4 of the protein mass was replaced with the elastin peptide of the present invention was prepared, and male hairless mice (Hos: HR-1, 6 weeks old) Administration by free intake was performed (elastin group). In addition, as a control, mice were allowed to ingest a feed containing no elastin peptide (control group). The breeding environment is as follows.
Water: Free intake temperature / humidity with automatic water supply device: 21-23 ° C, 55-60%
Light / dark time: 12 hours each The breeding period was 8 weeks, during which time ultraviolet light (UVB) was irradiated by an ultraviolet irradiation device (total 540 mJ / cm ² ) to promote photoaging of the skin. CUTOMETER (MPA580, C + K electronic) was used to measure skin viscoelasticity, and the return elastic modulus was used as an index of viscoelasticity. The results were expressed as relative values with the viscoelasticity before administration being 100.
The result is shown in FIG. As shown in FIG. 2, since the used mice were young, the viscoelasticity of the skin of all the mice was improved with breeding. However, in the elastin group (the elastin peptide intake group of the present invention), It was revealed that the skin viscoelasticity was significantly improved after the 6th week of breeding compared with the control group.

  In addition, the mouse skin after breeding was collected, and the desmosine content in the skin was measured. The result is shown in FIG. As shown in FIG. 3, desmosine, an amino acid peculiar to elastin, was increased in the elastin group as compared with the control group. That is, it was found that the elastin peptide of the present invention is absorbed into the body after ingestion and is effective in suppressing “wrinkles” and “sagging” caused by aging.

Test Example 6
A rat diet using an elastin-degrading peptide was prepared and administered to spontaneously hypertensive rats, and the effect on rat blood vessels was tested.
That is, an aqueous solution containing 50% of the elastin peptide of the present invention was prepared and administered by gavage to male spontaneously hypertensive rats (SHR / Ism, 18 weeks old) so that the peptide was 4 g / kg body weight / day. (Elastin group). As a control, the same amount of water was forcibly orally administered to rats (control group). The breeding environment is as follows.
Feed: Free intake of AIN-93G (Oriental yeast) Water supply: Free intake temperature / humidity by automatic water supply device: 21-23 ° C, 55-60%
Light / dark time: 12 hours each The breeding period was 10 weeks. After the breeding, blood was collected from the rats and various factors in the blood were measured. In addition, the thoracic aorta was collected after the breeding, and the desmosine content in the coronary artery was measured.
The results are shown in FIG. 4 (blood test) and FIG. 5 (desmosine content in the coronary artery). As shown in FIG. 4, the collected blood is involved in the onset of VCAM-1 (Vascular cell adhesion molecule-1), which is an adhesion factor expressed in vascular endothelial cells that have undergone inflammatory stimulation, and arteriosclerosis. MCP-1 (Monocyte chemoattractant protein-1), which is considered to be an adhesion factor, decreased, and TIMP-1 (Tissue inhibitor of Metalloproteinases-1), an inhibitor of extracellular matrix degradation, increased. Further, as shown in FIG. 5, when the content of desmosine in the coronary artery was measured, the content of desmosine was increased in the elastin group as compared with the control group. From these facts, it was found that the elastin peptide of the present invention suppresses arteriosclerosis and is effective for blood vessel maintenance.

Formulation Example 1
Production of capsules Hard capsules were filled with 500 mg of the elastin peptide of the present invention according to a conventional method to produce capsules.
Formulation Example 2
Manufacture of fruit juice beverage Citric acid (0.2 parts by weight), orange fruit juice (35 parts by weight) and sugar (5 parts by weight) are heated and dissolved in purified water (29.8 parts by weight), and the elastin peptide of the present invention is further dissolved. 30 parts by weight of a 10% aqueous solution was added, and after cooling, the container was filled and sterilized by heating at 85 ° C. for 30 minutes to produce a fruit juice drink.
Production Example 3
Production of skin lotion 1 part by weight of elastin peptide of the present invention, 5 parts by weight of ethanol, 2.5 parts by weight of glycerol, 1 part by weight of collagen-degrading peptide, 0.2 part by weight of sodium pyrrolidonecarboxylate, 0.2 part by weight of sodium hyaluronate A lotion was prepared by mixing 0.1 parts by weight of methylparaben and 90 parts by weight of purified water.

It is a figure which shows the fibroblast proliferation promotion effect and collagen production promotion effect of the elastin peptide of this invention. It is a figure which shows the skin viscoelasticity improvement effect of the mouse | mouth which ingested the elastin peptide of this invention. It is a figure which shows the desmosine content increase effect in the skin of the mouse | mouth which ingested the elastin peptide of this invention. It is a figure which shows the tendency of VCAM-1, MCP-1 and TIMP-1 in the blood of the rat which administered the elastin peptide of this invention. It is a figure which shows the desmosine content increase effect in the coronary artery of the rat which administered the elastin peptide of this invention.

Claims (3)

Elastin degradation product of porcine aortic blood vessel elastin, the content of isodesmosine and / or desmosine in the amino acid composition (molar ratio) is 0.06% or more and the sum of desmosine content and isodesmosine content / ratio of hydroxyproline content ( An elastin-degrading peptide having a molar ratio of 0.1 or more and an average molecular weight of 1000 to 3000 . A skin improving agent comprising the elastin degrading peptide according to claim 1. The skin improving agent according to claim 2, comprising collagen and / or a collagen-degrading peptide.

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