JP2010155820A - Skin ameliorating agent, and vascular ameliorating agent, and pharmaceutical composition, food, feed and cosmetic comprising the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a skin ameliorating agent, and a vascular ameliorating agent including an elastin peptide extracted from bulbus arteriosus of fishes as an active ingredient, and to provide a pharmaceutical composition, food, feed, and cosmetic comprising the same. <P>SOLUTION: The skin ameliorating agent, and vascular ameliorating agent include the elastin peptide extracted from the bulbus arteriosus of the fishes as the active ingredient, and have, based on 1,000 residues, the sum total of glycine, alanine, valine, and proline contents of ≥650 residues; the sum total of aspartic acid and asparagine contents of 10-35 residues; the sum total of glutamic acid and glutamine contents of 20-50 residues; the sum total of lysine, histidine and arginine contents of 20-50 residues; the sum total of desmosine and isodesmosine contents of ≥0.3 residue; and the hydroxyproline content of ≤10 residues. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a skin improving agent, a blood vessel improving agent, and pharmaceutical compositions, foods, feeds and cosmetics containing them, and more specifically, a skin improving agent containing elastin peptide extracted from arterial spheres of fish as an active ingredient. , A vascularity improving agent, and a pharmaceutical composition, food, feed and cosmetics containing them.

Hyaluronic acid constitutes an extracellular matrix together with collagen, elastin and the like, and plays a role such as water retention, lubricity and flexibility of the skin. There is a report that hyaluronic acid in the skin is synthesized in vivo and at the same time undergoes enzymatic degradation by hyaluronidase, which is its degrading enzyme, and the half-life of hyaluronic acid in the skin is about 1 day (non-patented). Reference 1). However, when hyaluronidase activity, which is a degrading enzyme, increases due to various external stresses such as aging and exposure to ultraviolet rays, the amount of hyaluronic acid degradation increases, so the tissue flexibility and lubricity are gradually lost. Cause joint pain, sagging skin, wrinkles, etc. Therefore, inhibiting the activity of hyaluronidase that degrades hyaluronic acid does not reduce hyaluronic acid in the living body and suppresses sagging and wrinkles.

Melanin is a pigment present in the skin and plays a role in protecting the skin and tissues from ultraviolet rays. However, excessive melanin production in the skin causes stains, buckwheat, spots, etc. due to overcoloring. Such overcoloring depends on the activity of an enzyme that produces melanin, and tyrosinase is known as an enzyme involved in the production of such melanin. Melanin is formed through the intermediates of dopa (DOPA, 3,4-dihydroxyphenylalanine), dopaquinone, 5,6-dihydroxyindophenol, etc., using tyrosine as a starting material by the action of tyrosinase biosynthesized in pigment cells Is done. Therefore, it is considered that by inhibiting the activity of tyrosinase, the production of melanin can be suppressed and spots, buckwheat, spots, etc. can be suppressed.

Kojic acid, L-ascorbic acid and the like have been reported as substances having a high tyrosinase inhibitory activity. Moreover, the search of the substance which has a hyaluronidase inhibitory activity and a tyrosinase inhibitory activity from a natural product is also performed, for example, a redfish or a redfish extract (refer patent document 1), a tenninka extract (refer patent document 2), a Hortonella plant, etc. Extract (refer to Patent Document 3), extract of Ammarok (refer to Patent Document 4), extract of a genus plant (refer to Patent Document 5), extract of fruit body (refer to Patent Document 6), genus Iwabenkei Plants (see Patent Document 7), Chapeu de Kouro extracts (see Patent Document 8), and the like have been reported.

Among substances that are known to have a skin improvement effect, those that are not derived from plants and that have been confirmed to be safe with food experience include, for example, gelatin derived from animal proteins and / or degradation products of collagen as active ingredients A collagen peptide-containing cosmetic composition containing as a cosmetic active ingredient a collagen peptide having an average molecular weight of 200 to 1500 obtained by subjecting a collagen component-containing raw material to a subcritical water treatment as a cosmetic active ingredient (see Patent Documents 9 and 10) ) Etc. are known.

On the other hand, elastin is a substance contained in skin and the like as much as collagen. Elastin is a major component of elastic fibers and is an insoluble protein widely distributed in vertebrate connective tissue. In the living body, it is distributed in many tissues that require elasticity and stretchability, such as arterial walls, ligaments, lungs, skin, etc., and functions to give elasticity. The elastin content in blood vessels and interfacial ligaments accounts for 50% or more of the total dry weight. Elastin is present in the dermis connective tissue together with collagen as a component involved in skin elasticity, and the reduction and degeneration of elastin in the skin due to aging, ultraviolet rays, etc. are a cause of skin sagging and wrinkles. . For this reason, elastin is currently used in many products, mainly in the cosmetics and health food fields. In addition, since elastin is a constituent of blood vessels as described above, degeneration of elastin is considered to be involved in various vascular diseases, and therefore its use in the pharmaceutical field is also being studied.

Conventionally, elastin has been made from an elastic tissue derived from mammals such as bovine ligaments, but due to the occurrence of cattle spongiform encephalopathy (BSE) and domestic animal plagues such as swine and foot-and-mouth disease, elastin derived from mammals from a safety standpoint. Tend to be shunned. Therefore, elastin derived from marine resources such as fish has attracted attention. In fish, elastin is abundant in tissue called arterial spheres. An arterial sphere is a fish-specific tissue, a type of arterial trunk that moves from the heart to the blood vessels, and a part of the aortic wall developed. Since the arterial sphere is rich in elastic fibers and has a spongy structure inside, the arterial sphere constantly expands and contracts due to the contraction of the heart. In some areas, this arterial sphere is used as a delicacy.

Regarding elastin, reports that porcine and equine-derived elastin have improved skin and whitening effects (see Patent Document 11), and animal-derived (bovine ligament) elastin has improved vascular conditions There is a report (see Patent Document 12).

A substance that promotes the production of elastin in skin fibroblasts that produce elastin in the skin has potential as a skin improvement agent. For example, Patent Documents 13 to 15 propose an elastin production promoter containing a plant extract, and Patent Document 16 proposes an elastin production promoter containing a synthetic peptide.

JP 2007-332081 A JP 2006-199678 A JP 2003-095857 A Japanese Patent Laid-Open No. 2003-081749 JP 2003-055184 A JP 2002-241300 A JP 2002-020301 A Japanese Patent Laid-Open No. 9-286738 JP-A-10-265401 JP 2006-342107 A JP 2002-205913 A JP 2007-045722 A JP 2002-293747 A JP 2005-22993 A Japanese Patent Laid-Open No. 2005-60341 JP 2008-74788 A

Tammi R. Other books, “Degradation of Newly Magnified High Molecular Mass Hyaluronan in the Epidemials and Dermal Compartments of Human Skin in Organ Culture.” Invest. Dermatol. , Nature Publishing Group (UK), Vol. 97, No. 1 (July 1991), p. 126-130

However, many organic compounds known so far as substances exhibiting high hyaluronidase inhibitory activity and tyrosinase inhibitory activity have risks such as side effects. For example, kojic acid has safety problems such as suspected carcinogenicity, and L-ascorbic acid and its derivatives have poor stability in solution and have problems such as coloring and decomposition.
In addition, the plant extracts described in Patent Documents 1 to 10 are made from expensive herbal medicines, which are difficult to secure in a stable and large amount throughout the year, have not been confirmed to be safe, and have unique plant characteristics. When it is used as food due to the smell, it has a problem that the flavor may be impaired.

Glycyrrhizic acid, sodium cromoglycate, baicalin, indomethacin, aspirin, etc. used in medicines (anti-inflammatory analgesics, antiallergic agents, etc.) have been confirmed to have high hyaluronidase activity inhibitory activity. There are problems such as there are side effects.

Regarding water-soluble collagen peptides and elastin peptides obtained by decomposing animal-derived collagen and elastin, hyaluronidase inhibitory activity, tyrosinase inhibitory activity, and skin whitening effect have been reported, whereas fish-derived elastin The inhibitory activity of hyaluronidase and tyrosinase has not been reported yet. In addition, the use of proteins derived from elastic tissues of animals as a raw material tends to be avoided from a safety standpoint after the occurrence of cattle spongiform encephalopathy (BSE) and livestock plague such as swine and foot-and-mouth disease.

Moreover, about the elastin peptide derived from fish, it has not been reported until now to improve skin function by promoting the production of elastin in dermal fibroblasts.

The present invention has been made in view of such circumstances, and includes a skin improving agent, an vascular improving agent containing elastin peptide extracted from arterial spheres of fish as an active ingredient, and a pharmaceutical composition, food, feed and cosmetic containing them. The purpose is to provide.

The first aspect of the present invention that meets the above-mentioned object is to solve the above-mentioned problems by providing a skin improving agent characterized by containing an elastin peptide extracted from arterial spheres of fish as an active ingredient.
The “elastin peptide” means a polypeptide and an oligopeptide obtained by fragmenting a polypeptide chain of elastin or a mixture thereof.

In the first aspect of the present invention, the total content of glycine, alanine, valine and proline is 650 residues / 1000 residues or more, and the total content of aspartic acid and asparagine is 10 residues / 1000 residues or more 35 residues / 1000 residues or less, the total content of glutamic acid and glutamine is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less, and the total content of lysine, histidine and arginine is 20 residues / 1000 residues It is preferably 50 residues / 1000 residues or less, the sum of desmosine and isodesmosine content is preferably 0.3 residues / 1000 residues or more, and the hydroxyproline content is preferably 10 residues / 1000 residues or less.

1st aspect of this invention WHEREIN: It is preferable that the ratio of those whose molecular weight is 1000 or less among the said elastin peptides is 70% or more.
In the present invention, “%” means “% by weight” unless otherwise specified.

In the first aspect of the present invention, the skin improving agent may have hyaluronidase inhibitory activity.

In the first aspect of the present invention, the skin improving agent may have tyrosinase inhibitory activity.

In the first embodiment of the present invention, the skin improving agent may have elastin production promoting activity in dermal fibroblasts.

The second aspect of the present invention solves the above-mentioned problems by providing a blood vessel improving agent characterized by containing an elastin peptide extracted from arterial spheres of fish as an active ingredient.

In the second embodiment of the present invention, the total content of glycine, alanine, valine and proline is 650 residues / 1000 residues or more, and the total content of aspartic acid and asparagine is 10 residues / 1000 residues or more 35 residues / 1000 residues or less, the total content of glutamic acid and glutamine is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less, and the total content of lysine, histidine and arginine is 20 residues / 1000 residues It is preferably 50 residues / 1000 residues or less, the sum of desmosine and isodesmosine content is preferably 0.3 residues / 1000 residues or more, and the hydroxyproline content is preferably 10 residues / 1000 residues or less.

2nd aspect of this invention WHEREIN: It is preferable that the ratio of those whose molecular weight is 1000 or less among the said elastin peptides is 70% or more.

In the second aspect of the present invention, the blood vessel improving agent may have an activity of decreasing the b / a value of the acceleration pulse wave and increasing the d / a value.
The “acceleration pulse wave” is a second-order differential wave of the fingertip volume pulse wave, and reflects vascular resistance and blood vessel responsiveness to blood flow, and is therefore used for so-called estimation of blood vessel age.

In the second aspect of the present invention, the blood vessel improving agent may have smooth muscle growth inhibitory activity.

In the second embodiment of the present invention, the blood vessel improving agent may have endothelin-1 production inhibitory activity in vascular endothelial cells.

In the second aspect of the present invention, the blood vessel improving agent may have tissue plasminogen activator (t-PA) production promoting activity in vascular endothelial cells.

In the second aspect of the present invention, the blood vessel improving agent may have vascular endothelial cell proliferation activation activity.

According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising any one or both of the skin improving agent according to the first aspect of the present invention and the vascular improving agent according to the second aspect of the present invention. The present invention solves the above problems.

According to a fourth aspect of the present invention, there is provided a food containing either or both of the skin improving agent according to the first aspect of the present invention and the blood vessel improving agent according to the second aspect of the present invention. Is a solution.

According to a fifth aspect of the present invention, there is provided the above-mentioned problem by providing a feed containing either or both of the skin improving agent according to the first aspect of the present invention and the blood vessel improving agent according to the second aspect of the present invention. Is a solution.

According to a sixth aspect of the present invention, there is provided the above-mentioned problem by providing a cosmetic comprising one or both of the skin improving agent according to the first aspect of the present invention and the blood vessel improving agent according to the second aspect of the present invention. Is a solution.

According to the present invention, a skin improving agent having one or both of hyaluronidase inhibitory activity and tyrosinase inhibitory activity, which solves the problems of the prior art relating to side effects, inherent odor, safety, etc. and can be applied to a wide range of uses, vascular elasticity A blood vessel improving agent having one or more of an improving action, a smooth muscle growth inhibitory activity in blood vessels, an endothelin-1 production inhibitory activity, a tissue plasminogen activator production promoting activity and a vascular endothelial cell growth promoting activity, and these Pharmaceutical compositions, foods, feeds and cosmetics can be provided.

It is a figure which shows the standard waveform of an acceleration pulse wave. 2 is a graph showing measurement results of hyaluronidase inhibitory activity for the elastin peptide obtained in Example 1. 2 is a graph showing measurement results of hyaluronidase inhibitory activity for the elastin peptide obtained in Example 2. 2 is a graph showing measurement results of tyrosinase inhibitory activity for the elastin peptide obtained in Example 1. 2 is a graph showing measurement results of smooth muscle growth inhibitory activity for the elastin peptide obtained in Example 1. 2 is a graph showing measurement results of elastin production promoting activity for the elastin peptide obtained in Example 1. It is a graph which shows the measurement result of endothelin-1 production promotion activity about the elastin peptide obtained in Example 1. It is a graph which shows the measurement result of the tissue plasminogen activator (t-PA) production promotion activity about the elastin peptide obtained in Example 1. 2 is a graph showing measurement results of vascular endothelial cell proliferation promoting activity for the elastin peptide obtained in Example 1. FIG.

Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.
The skin improving agent and blood vessel improving agent according to one embodiment of the present invention contain an elastin peptide extracted from a fish arterial sphere (hereinafter simply referred to as “elastin peptide”) as an active ingredient.

An arterial sphere is an organ peculiar to fish, is connected to the ventricle through a valve, and is involved in blood flow regulation of blood sent from the ventricle to the aorta. There is no particular limitation on the origin of the arterial sphere used as a raw material, and arterial spheres derived from any fish species can be used, but it is necessary to have a certain size in order to collect arterial spheres from the heart. Therefore, the arterial sphere used as a raw material is preferably derived from large fish such as bonito, tuna, swordfish, cod, yellowtail, yellowtail, salmon, trout, etc. More preferably, it is derived from tuna, cod, hamachi, salmon.

The elastin peptide is prepared, for example, by the following method.
First, in order to remove blood from the arterial sphere used as a raw material, it is washed with running water and then pulverized. The pulverization can be performed by any known means such as a homogenizer or a food cutter. Then, by removing lipids, soluble proteins and collagen from the pulverized arterial spheres, an insoluble protein mixture based on elastin can be obtained, but pretreatment for facilitating further washing of the raw material and subsequent processing. It is preferable to perform an immersion treatment using an alkaline solution.

Since the treatment conditions with the alkaline solution vary depending on the fish species, it is preferable to determine in advance, but when using bonito-derived arterial spheres, the alkaline solution used is sodium hydroxide or calcium hydroxide, preferably Is a solution of sodium hydroxide. The concentration of the alkaline solution is 0.01N to 0.1N, preferably 0.02N. The immersion temperature is 20 ° C. or less, and the immersion period is several days to 2 weeks, preferably 1 week. Moreover, during immersion, it is preferable to change an alkaline solution twice or more per day. After immersion, neutralization is performed after removing excess alkali by washing with running water. Any acid used in the art can be used for neutralization.

The removal of lipid and collagen from the alkali-treated raw material can be performed, for example, by adding distilled water and heating at a high temperature (for example, 95 ° C.). By heating at a high temperature, the helical structure of collagen collapses, the trimer dissociates, and water-soluble tropocollagen (gelatin) is released. When the supernatant is separated and removed by any known method such as filtration, centrifugation, and decantation, an insoluble protein mixture is obtained.

The elastin peptide can be obtained by subjecting the insoluble protein mixture thus obtained to fragmentation of the polypeptide chain and solubilization treatment for improving water solubility. At this time, the insoluble protein mixture may be further fragmented prior to the solubilization treatment.

The solubilization treatment can be performed using any known method, and specific examples include enzymatic degradation using a proteolytic enzyme. For the enzymatic degradation, any proteolytic enzyme used in the production of foods, pharmaceuticals and cosmetics can be used, but those having a high titer, such as Alcalase 2.4L FG (manufactured by Novoenzyme), protin AC-10F (Yamato) Kasei), protease N “Amano” G, and pepsin (Amano Enzyme) are preferred. Degradation conditions are appropriately determined according to the enzyme used, arterial sphere, desired molecular weight distribution, and the like. The addition amount of enzyme (weight ratio of enzyme to substrate) is a normal amount used for proteolysis in the art, and is, for example, 1: 5000 to 1: 10000. Moreover, although these enzymes can also be used independently, it is preferable to use combining 2 or more types. After the reaction, the enzymatic decomposition reaction is terminated by heat deactivation of the enzyme.

The solubilization treatment can also be performed by an acid decomposition method in which an insoluble protein mixture is heated in an inorganic acid solution. Examples of the acid to be used include any inorganic acid, but oxalic acid is preferable, and the concentration and heating temperature are preferably 0.25 N and 90 ° C. After the solubilization treatment, neutralization is performed with an alkali, and sodium hydroxide and calcium hydroxide are preferable as the alkali used at this time. In particular, when oxalic acid is used, neutralization with calcium hydroxide is essential in order to completely remove it.

Alternatively, the solubilization treatment can also be performed by an alkali-ethanol method in which an insoluble protein mixture is treated with an alkaline aqueous ethanol solution. The solution used at this time is preferably a 1N sodium hydroxide 80% ethanol solution, and the treatment temperature is preferably room temperature.

When the elastin peptide obtained as described above is used as a solution, the solution is adjusted to a pH suitable for a desired application, and desalting is performed if necessary. Desalting can be performed by any method such as an ultrafiltration method or an ion exchange method.
Moreover, when an insoluble matter exists, it can remove using arbitrary methods, such as filtration, centrifugation, and a decantation. In the case of removal by filtration, an adsorbent such as activated carbon, bentonite, or celite or a filter aid may be added as necessary to remove impurities. In particular, when used as a solution, it is preferable to perform sterilization filtration with a membrane filter or the like.
The elastin peptide thus obtained may be used as a solution as it is, or may be used in the form of a powder obtained by further performing spray drying or freeze drying after concentration.

The proportion of the elastin peptide obtained as described above having a molecular weight of 1000 or less is 70% (% by weight, the same applies hereinafter) or more, preferably 75% or more, more preferably 80% or more. Each of the skin improving agent and the blood vessel improving agent having the above conditions exhibits high activity and is excellent in absorption characteristics at the time of administration. The molecular weight of the elastin peptide and its abundance ratio (weight ratio) can be determined using any known method such as size exclusion chromatography (gel filtration chromatography) or mass spectrometry.
If the proportion of the elastin peptide having a molecular weight of 1000 or less is less than 70%, problems such as a decrease in absorption efficiency or expression of mutagenicity may occur.

Elastin peptide has hyaluronidase inhibitory activity. As described above, with the increase in hyaluronidase activity caused by various external stresses such as aging and exposure to ultraviolet rays, the amount of degradation of hyaluronic acid, which plays the role of water retention, lubricity, and flexibility of the skin, increases. , Tissue flexibility and lubricity are lost, causing skin sagging and wrinkles. Accordingly, by inhibiting the activity of hyaluronidase, sagging and wrinkles of the skin can be suppressed and skin function can be improved.

The elastin peptide has tyrosinase inhibitory activity. Tyrosinase is involved in the production of melanin in the skin, and the increase in tyrosinase activity causes pigmentation on the skin such as spots and buckwheat. Therefore, by inhibiting tyrosinase activity, abnormal pigmentation on the skin can be suppressed and skin function can be improved. As described above, the elastin peptide can be used as a skin improving agent having a skin improving effect.

Furthermore, the elastin peptide has an activity to promote the production of elastin in dermal fibroblasts. As described above, elastin is present in the dermis connective tissue together with collagen as a component involved in skin elasticity, and the reduction and degeneration of elastin in the skin due to aging, ultraviolet rays, etc. are a cause of skin sagging and wrinkles. It is. Therefore, by promoting the production of elastin in dermal fibroblasts, it is possible to suppress skin sagging and wrinkles associated with aging and improve skin function.

Furthermore, the elastin peptide has the activity of decreasing the b / a value of the acceleration pulse wave and increasing the d / a value. The acceleration pulse wave is a second-order differential wave of the fingertip volume pulse wave, and can be obtained by a calculation process of the measured fingertip volume pulse wave. The blood flow expelled from the heart by one heart contraction causes changes in the internal pressure and volume of the artery, but the relationship between the internal pressure and volume measured at a specific part of the artery depends on the individual from the heart to the part. The total effect of arterial characteristics. Therefore, the volume pulse wave measured at the fingertip is affected by the synthesis and resonance of the projected wave and the reflected wave generated in the process of propagating from the aorta to the peripheral artery. From the waveform analysis of the fingertip volume pulse wave, It is possible to comprehensively comprehend the manner in which a person pulsates from the heart, the vascular properties, the state of the vascular wall, the viscosity of blood, and the like.

A standard waveform of the acceleration pulse wave is shown in FIG. A standard acceleration pulse wave has five element waves called a, b, c, d, and e waves. It has been confirmed that the waveform of the acceleration pulse wave shows a strong correlation with age. As the age increases, that is, as the artery ages, the b wave becomes shallower (displacement from the baseline increases in the positive direction), while d It is known that the waves are deep (displacement from the baseline increases in the negative direction). Therefore, with arterial aging, the b / a value increases and the d / a value decreases.
The fact that elastin peptide has the activity of decreasing b / a value and increasing d / a value means that elastin peptide has the activity of improving arteries to a young age state To do.

The elastin peptide has smooth muscle growth inhibitory activity. Factors that cause atherosclerosis due to aging include a decrease in vasorelaxant factor produced from vascular endothelial cells, an increase in contraction factor, and excessive proliferation of smooth muscle cells in the vascular media. It is done. Thus, the elastin peptide can prevent atherosclerosis due to smooth muscle proliferation and improve the elasticity of the arterial wall. As described above, the elastin peptide can be used as a blood vessel improving agent having an effect of improving aged blood vessels.

The elastin peptide has endothelin-1 production inhibitory activity. It is well known that blood vessels harden or atherosclerosis develops as blood vessels age. These phenomena involve a decrease in the endothelium-dependent vasorelaxation effect due to a decrease in the ability to produce nitric oxide (NO) and prostacyclin in vascular endothelial cells, and an increase in the vasoconstriction effect by angiotensin and endothelin-1. It is thought that there is.

Endothelin-1 is a strong vasoconstrictor peptide consisting of 21-residue amino acids produced by vascular endothelial cells, and has a strong and vasoconstrictive action as well as a continuous and strong pressor action, and acts directly on smooth muscle. Have been shown to constrict blood vessels. Endothelin production is enhanced by vascular endothelial damage, and its overproduction is one of the etiologies such as hypertension, pulmonary blood pressure, diabetes, arteriosclerosis, renal failure, myocardial infarction, angina, cerebral vasospasm and cerebral infarction. It is considered to be one. In patients suffering from hypertension, arteriosclerosis, and acute renal failure, it has been reported that the amount of endothelin-1 in the blood is significantly higher than that of healthy individuals (Marushige et al., Medical History, 51- 54, 1992). Therefore, if the production of endothelin-1 can be suppressed, diseases such as hypertension can be treated or prevented.

The elastin peptide has a tissue plasminogen activator (tissue plasminogen activator: t-PA) production promoting activity. It is considered that the increase in blood coagulation and the decrease in the fibrinolytic system due to a decrease in the function of vascular endothelial cells are also involved in the progress of vascular sclerosis and atherosclerosis accompanying aging of blood vessels.
Tissue plasminogen activator (t-PA) produced in vascular endothelial cells activates plasminogen and converts it into plasmin, thereby degrading fibrin and dissolving thrombus.
In Japan, the prevalence of cardiovascular diseases such as cerebral infarction and ischemic heart disease in the elderly is increasing with the westernization of eating habits, and it is important to normalize the fibrinolytic system for its prevention. Therefore, it is considered that thrombotic diseases can be treated or prevented by promoting t-PA production and preventing thrombus formation.

In addition, elastin peptide has a proliferation promoting activity on vascular endothelial cells. Vascular endothelial cells are cells that make up the lining of blood vessels, produce physiologically active substances that control blood pressure, blood coagulation, and fibrinolytic system, and maintain the vascular environment. However, vascular endothelial cells are constantly damaged by aging and accumulation of oxidative stress, and their functions are reduced. The progression of arteriosclerosis and thrombosis is thought to be due to a decline in endothelial function, and repairing vascular endothelial cells and enhancing their growth-promoting action are important for the prevention and improvement of these vascular diseases. It is believed that there is.

The elastin peptide is fractionated using a known method such as column chromatography, and each activity is assayed for each fraction, whereby a fraction having one or more of the above-mentioned activities is singly or arbitrarily selected. Two or more may be used in combination, or the elastin peptide may be used as it is without separation and purification. In any case, the elastin peptide may have both a skin improvement effect and an effect of improving aged blood vessels.

By mixing elastin peptide with a carrier or the like, it can be used as a pharmaceutical composition having one or both of an effect of improving skin and an effect of improving aging blood vessels. Examples of the dosage form of the pharmaceutical composition for humans or animals include oral, rectal, parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.), and the dosage is the formulation of the pharmaceutical composition. However, in the case of humans, the active ingredient generally contained in the formulation is difficult to determine and uniquely determined depending on the administration method, purpose of use, and age, weight, and symptoms of the subject to be applied. The amount is preferably 0.1 to 2000 mg / kg per day for an adult. Of course, since the dosage varies depending on various conditions, an amount smaller than the above dosage may be sufficient or may be necessary beyond the range.

When prepared as an orally administered preparation, it can be prepared in the form of tablets, granules, powders, capsules, coatings, liquids, suspensions, etc. It can be prepared in the form of drops, suppositories and the like. Any known method can be used for formulation. For example, an elastin peptide and a pharmaceutically acceptable carrier or diluent, a stabilizer, and other desired additives can be blended to obtain the above desired dosage form.

As a food containing one or both of a skin improving agent and a blood vessel improving agent, elastin peptide prepared as it is as a food, added to other foods, or normally used in foods or health foods such as capsules and tablets It can take any form.

When ingested or administered in foods, mix with excipients, extenders, binders, thickeners, emulsifiers, colorants, flavors, food additives, seasonings, etc. Accordingly, it can be formed into a powder, granule, tablet or the like. Moreover, it can be ingested by preparing a food as a functional food having a skin improvement effect by appropriately mixing it in a food material and preparing a food.

The feed containing one or both of the skin improving agent and the blood vessel improving agent can take various forms such as a prepared elastin peptide as it is or a blended feed. When mixed in feed and administered to animals such as livestock, it can be mixed in advance with feed ingredients to prepare a feed with functionality. It can also be added to the feed for administration. That is, a skin improvement agent containing an elastin peptide as an active ingredient is safe by adding it to livestock such as pigs, chickens, cows, horses, sheep, and feed such as fish, pets (dogs, cats, birds), It can be used as a functional feed having one or both of a skin improvement effect and an effect of improving aging blood vessels.

Cosmetics containing a skin improving agent can take various forms such as those obtained by blending elastin peptide in lotion or cream.
In order to mix and administer to cosmetics etc., it can mix with liquid or creamy cosmetics suitably, and can prepare as functional cosmetics etc. In that case, solubilizers, stabilizers, emulsifiers and the like well known in the preparation of cosmetics and the like can be used.

Next, examples carried out for confirming the effects of the present invention will be described.

Example 1: Production of bonito-derived elastin peptide powder Arterial spheres (100 g) were collected from fresh bonito, washed with running water and ground. As a pretreatment of the raw material, it was immersed in a 0.02N aqueous sodium hydroxide solution for 1 week in a refrigerator. After immersion, excess alkali was removed by running water washing, and running water washing was performed until the discharged liquid became neutral. To this was added 3 volumes of distilled water, heated to 95 ° C., and the supernatant was removed to remove lipids and collagen. The residue was cut into pieces with a food cutter, and Protin AC-10F (Yamato Kasei 0.5%) and Protease N “Amano” G (Amano Enzyme) 0.1% were added at 1% of the base mass for 10 hours. Decomposition was performed. Heat deactivation was performed at a temperature of 85 ° C. or higher, and the residue was separated by filtration and centrifugation. Thereafter, the extract clarified by microfiltration was spray-dried to obtain water-soluble elastin peptide powder (8 g).

Example 2: An elastin peptide powder was obtained in the same manner as in Example 1 using arterial spheres collected from fresh hamachi as a raw material for producing hamachi-derived elastin peptide powder.

Example 3: Elastin peptide powder was obtained in the same manner as in Example 1 using arterial spheres collected from fresh tuna as a raw material for producing tuna-derived elastin peptide powder.

The amino acid analysis results of the elastin peptides obtained according to Examples 1 to 3 are shown in Table 1 below.

For any elastin peptide, the total of glycine, alanine, valine and proline content per 1000 residues is 650 residues or more, the sum of aspartic acid and asparagine content is 10 to 35 residues, glutamic acid and glutamine content The total of lysine, histidine and arginine content is 20 to 50 residues, the sum of desmosine and isodesmosine content is 0.3 residues or more, and the hydroxyproline content is It turns out that it is 10 residues or less.

The results of protein, lipid, moisture, and ash content analysis of the elastin peptides obtained in Examples 1 to 3 are shown in Table 2 below.

Table 3 below shows the molecular weight measurement results of the elastin peptides obtained in Examples 1 to 3 (measured by size exclusion chromatography using the Japan Food Analysis Center: TSKgel G2500PWXL column). In Table 3, “%” means wt%.

As for any elastin peptide, it can be seen that the proportion occupied by those having a molecular weight of 1000 or less is 70% or more.

Example 4: Measurement of hyaluronidase inhibitory activity Hyaluronidase (manufactured by Wako Pure Chemical Industries, Ltd.) 16.5 U in 50 μl of elastin peptide solution at a predetermined concentration dissolved in 100 mM acetate buffer (pH 4.0), then 33 μl of 0.5 mM CaCl ₂ ( Wako Pure Chemical Industries, Ltd.) was added and pre-warmed at 37 ° C. for 5 minutes. Hyaluronic acid was added to this solution to a final concentration of 0.4 mg / ml and reacted at 37 ° C. for 10 minutes, and then 33 μl of 0.4N NaOH was added to stop the reaction. 33 μl of 0.8 M borate buffer (pH 9.1) was added to the reaction solution, heated at 100 ° C. for 3 minutes, and then ice-cooled. 1 ml of p-dimethylaminobenzaldehyde reagent was added to this solution (sample) and reacted at 37 ° C. for 20 minutes, and then the absorbance at λ = 585 nm was measured. Hyaluronidase inhibitory activity was determined by the following formula.

Hyaluronidase inhibitory activity (%) = [(A−B) − (C−D)] / (A−B) × 100
here,
A = absorbance of control (prepared in the same manner as the sample except for no elastin peptide) B = absorbance of blank (prepared in the same manner as the sample except for no elastin peptide and hyaluronidase) C = absorbance of the sample D = sample blank ( It means the absorbance of each sample prepared except that it does not contain hyaluronidase.

2 and 3, it can be seen that the elastin peptides obtained in Examples 1 and 2 show hyaluronidase inhibitory activity in a concentration-dependent manner. In addition, the elastin peptide obtained in Example 3 was also confirmed to have the same concentration-dependent hyaluronidase inhibitory activity.

Example 5: Measurement of tyrosinase inhibitory activity To 100 μl of 0.1 M phosphate buffer (pH 6.8), 20 μl of a predetermined concentration of elastin peptide solution and 20 μl of 500 U / ml tyrosinase solution were added and preliminarily maintained at 35 ° C. for 5 minutes. Warmed up. 60 μl of 0.05% L-tyrosine solution was added to this solution (sample) and reacted at 35 ° C. for 15 minutes, and then the absorbance at λ = 490 nm was measured. Hyaluronidase inhibitory activity was determined by the following formula.

Tyrosinase inhibitory activity (%) = [(A−B) − (C−D)] / (A−B) × 100
here,
A = absorbance of control B = absorbance of blank C = absorbance of sample D = absorbance of sample blank. The definitions of “control”, “blank”, and “sample blank” are the same as those in the fourth embodiment.

As shown in FIG. 4, it can be seen that the elastin peptide obtained in Example 1 exhibits tyrosinase inhibitory activity in a concentration-dependent manner. In addition, the elastin peptides obtained in Examples 2 and 3 were also confirmed to have concentration-dependent tyrosinase inhibitory activity.

Example 6: Examination of effect of elastin peptide on acceleration pulse wave 46 subjects (30 men, average age 43.8 ± 11.5 years, 16 women, average age 42.3 ± 11.8 years) The supplement containing the elastin peptide obtained in Examples 1 to 3 was administered, and the acceleration pulse wave was measured before administration and every month after the start of administration. Table 4 shows the composition of the administered supplement (requested for preparation from Senbe Daikando Pharmaceutical Co., Ltd.). The supplement was provided in a chuck seal aluminum pouch (with desiccant enclosed, 250 mg × 150 tablets).

Two tablets of elastin peptide-containing supplement were ingested with water or lukewarm water before going to bed. The elastin peptide intake per day was 360 mg (assuming a protein content of 90%).

The acceleration pulse wave was measured by an acceleration pulse wave measurement system Artett (Umedica Co., Ltd.) under the guidance of a nurse at our clinic. After resting for about 5 minutes, the test subject inserted an index finger or middle finger into the Artett sensor, and measured the acceleration pulse wave, which is the second derivative of the fingertip volume pulse wave and the fingertip volume pulse wave for 18 seconds. Waveform index and vascular aging deviation value were obtained from b wave and d wave related to vascular elasticity and resistance among the element waves constituting the acceleration pulse wave.

The acceleration pulse wave parameter and blood vessel aging deviation value of each subject (male group and female group) to which a supplement prepared from the elastin peptide obtained in Example 1 was administered were as shown in Table 5 and Table 6 below. It was. The results are shown as mean ± standard deviation. “P <0.05” and “p <0.01” represent that the significance probability p-value in the t-test is less than 5% and less than 1%, respectively.

In the male group, significant improvement was observed in the acceleration pulse wave (wave height ratio b / a, Waveform index I) and vascular aging deviation value from the third month of ingestion. In the female group, significant improvement was observed in the acceleration pulse wave (wave height ratio d / a) in May.
Similar results were obtained for the elastin peptides obtained in Example 2 and Example 3.

Example 7: Measurement of smooth muscle growth inhibitory activity Normal human aortic vascular smooth muscle cells (Kurabo) were used as cells, and Humedia-SG2, which is a dedicated medium, was used as the culture medium.

Smooth muscle cells were cultured until they became semi-confluent in a 25 cm ² flask, and the cells were collected by a conventional method. The collected cells were prepared to 2 × 10 ⁴ cells / ml using Humedia-SG2, and 0.1 ml each was seeded on a 96-well plate. The culture medium was removed 24 hours after cell seeding, and 0.1 ml of a medium in which the elastin peptide was dissolved was added (final elastin concentration 125, 250, 500, 1000, 2000 μg / ml). Four days after the start of culture (three days after the addition of elastin peptide), the culture supernatant was removed, the cell surface was washed with PBS, and a new medium was added in an amount of 100 μl / well. Furthermore, 10 μl / well of cellcountingkit-8 (Dojindo Laboratories) was added and held at 37 ° C. for 1 hour. After confirming that the dye was colored, 450 nm was measured using a microplate reader.

The results for the elastin peptide obtained in Example 1 are shown in FIG. A tendency of growth inhibition was observed in all of those added with elastin peptide, and cell growth rates of 90.2% and 82.2% with respect to the control (both p <0.01) in the group added with 1000 and 2000 μg / ml, respectively. Thus, it was suggested that there was an effect of suppressing smooth muscle proliferation.
Similar results were obtained for the elastin peptides obtained in Example 2 and Example 3.

Example 8: Measurement of elastin production promoting activity Elastin peptide was added to fibroblasts, and elastin in the culture supernatant produced from the fibroblasts was quantified. Fibroblast culture conditions were such that D-MEM (1% FBS, 50 units / ml penicillin G, containing 50 μg / ml streptomycin) was used as the culture medium, and the number of cells was adjusted to 2 × 10 ⁵ cells / ml. 100 μl each was seeded on the plate (2 × 10 ⁴ cells / well). After 24 hours, the culture medium was removed, 90 μl of culture medium was added to all wells, and elastin peptide dissolved in PBS was added at 10, 100, and 1000 μg / ml. The elastin peptide solution used was sterilized by passing through a 0.22 μm filter. Three days after the addition of elastin peptide, the culture supernatant was collected and used as a sample for measuring the amount of elastin produced from fibroblasts.
The amount of elastin produced from fibroblasts was quantified by ELISA.

FIG. 6 shows the relationship between the amount of elastin peptide added and the amount of elastin produced (relative to the control) for the elastin peptide obtained in Example 1. Elastin production is promoted in a concentration-dependent manner by the addition of elastin peptide, and the production promotion rates of 130% and 139%, respectively, are 100% and 139% of the control group (p <0.05 and p <0.01, respectively). Thus, the results suggesting that there is an elastin production promoting effect were obtained.
Similar results were obtained for the elastin peptides obtained in Example 2 and Example 3.

Example 9: Measurement of Endothelin-1 Production Inhibitory Activity Normal human umbilical vein endothelial cells (HUVEC) (Kurabo) were used as the cells, and Humedia-EG2, which is a dedicated medium, was used as the culture medium.
Vascular endothelial cells pre-cultured in a 25 cm ² flask were collected by a conventional method. The collected cells were seeded on a 24 well plate so as to be 1 × 10 ⁴ cells / well and cultured for 4 to 5 days until almost confluent. After removing the medium and washing with PBS, 0.9 ml of medium and the elastin peptide obtained in Example 1 dissolved in PBS, or 0.1 ml of PBS as a control (control) were added, and the final concentration of elastin peptide was determined. 0.1, 0.5, 1 and 5 μg / ml. The elastin peptide solution used was sterilized by passing through a 0.22 μm filter. After culturing at 37 ° C. and 5% CO ₂ for 24 hours, the culture supernatant was collected and used as a sample. The amount of endothelin-1 in the culture supernatant was quantified with Endothelin-1 (human) EIA Kit (assay designs).

As shown in FIG. 7, the production of endothelin-1 is suppressed in a concentration-dependent manner by the elastin peptide, and the production amount is 89.8% at 1 μg / ml, assuming that the production amount of endothelin-1 in the control is 100%. At 5 μg / ml, it was 88.1% (both p <0.001). From these results, it was confirmed that the elastin peptide obtained in Example 1 has endothelin production inhibitory activity.
Similar results were obtained for the elastin peptides obtained in Examples 2 and 3.

Example 10: Measurement of tissue plasminogen activator (t-PA) production inhibitory activity Normal human umbilical vein endothelial cells (HUVEC) (Kurabo) were used as the culture medium, and the dedicated culture medium HuMedia-EG2 (Kurabo) was used as the culture medium. ) Was used.
Vascular endothelial cells pre-cultured in a 25 cm ² flask were collected by a conventional method. The collected cells were seeded on a 24 well plate so as to be 1 × 10 ⁴ cells / well and cultured for 4 to 5 days until almost confluent. After removing the medium and washing twice with PBS, 0.9 ml of measurement medium (RPMI1640 (containing 5% FBS)) and the elastin peptide obtained in Example 1 dissolved in PBS, or PBS as a control (control) 0.1 ml was added to make final concentrations of elastin peptide of 10, 100, 1000 μg / ml. The elastin peptide solution used was sterilized by passing through a 0.22 μm filter. After culturing at 37 ° C. under 5% CO ₂ for 24 hours, the culture supernatant was recovered. After collection, the mixture was centrifuged at 4 ° C. and 2000 g for 10 minutes, and the supernatant was used as a sample. The amount of t-PA in the culture supernatant was quantified with an Assay Max Human Tissue-Type Plasminogen Activator ELISA Kit (ASSAYPRO).

As shown in FIG. 8, the elastin peptide promotes t-PA production in a concentration-dependent manner, and its production amount is 105.2% and 100 μg at 10 μg / ml when the control (control) t-PA amount is 100%. It was 106.2% (p <0.05) at / ml, and 113.1% (p <0.01) at 1000 μg / ml. From these results, it was confirmed that the elastin peptide obtained in Example 1 has t-PA production promoting activity.
Similar results were obtained for the elastin peptides obtained in Examples 2 and 3.

Example 11: Measurement of proliferation promoting activity on vascular endothelial cells Normal human umbilical vein vascular endothelial cells (HUVEC) (Kurabo) were used, and the culture medium used was Humedia-EG2, which is a dedicated medium.
Vascular endothelial cells pre-cultured in a 25 cm ² flask were collected by a conventional method. The collected cells were suspended in EG-2 medium and seeded at 1 × 10 ³ cells in a 96-well plate. At the same time, a test product in which the elastin peptide obtained in Example 1 was dissolved in PBS was added to 1/10 of the culture solution. After culturing in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 3 days, the absorbance at 450 nm was measured using a cell counting kit-8. Only PBS was added as a control. The absorbance of the control was taken as 100, and the relative value of the elastin peptide addition group was determined.

As shown in FIG. 9, the elastin peptide obtained in Example 1 showed an effect of promoting the proliferation of vascular endothelial cells in a concentration-dependent manner, and was 108% in the group added with 12.5 μg / ml and 110% at 25 μg / ml relative to the control. At 50 μg / ml, it was 115% (p <0.01).
Similar results were obtained for the elastin peptides obtained in Examples 2 and 3.

Claims (18)

A skin improver comprising an elastin peptide extracted from an arterial sphere of fish as an active ingredient. The total content of glycine, alanine, valine and proline is 650 residues / 1000 residues or more,
The total aspartic acid and asparagine content is 10 residues / 1000 residues or more and 35 residues / 1000 residues or less,
The total glutamic acid and glutamine content is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less,
The total content of lysine, histidine and arginine is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less,
The sum of desmosine and isodesmosine content is 0.3 residues / 1000 residues or more,
The skin improving agent according to claim 1, wherein the hydroxyproline content is 10 residues / 1000 residues or less. The skin improving agent according to any one of claims 1 and 2, wherein a proportion of the elastin peptide having a molecular weight of 1000 or less is 70% or more. The skin improvement agent of any one of Claim 1 to 3 which has a hyaluronidase inhibitory activity. The skin improving agent according to any one of claims 1 to 4, which has tyrosinase inhibitory activity. The skin improving agent according to any one of claims 1 to 5, which has elastin production promoting activity in skin fibroblasts. A blood vessel improving agent comprising an elastin peptide extracted from a fish arterial sphere as an active ingredient. The total content of glycine, alanine, valine and proline is 650 residues / 1000 residues or more,
The total aspartic acid and asparagine content is 10 residues / 1000 residues or more and 35 residues / 1000 residues or less,
The total glutamic acid and glutamine content is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less,
The total content of lysine, histidine and arginine is 20 residues / 1000 residues or more and 50 residues / 1000 residues or less,
The sum of desmosine and isodesmosine content is 0.3 residues / 1000 residues or more,
8. The blood vessel improving agent according to claim 7, wherein the hydroxyproline content is 10 residues / 1000 residues or less. The blood vessel improving agent according to any one of claims 7 and 8, wherein a proportion of the elastin peptide having a molecular weight of 1000 or less is 70% or more. The blood vessel improving agent according to any one of claims 7 to 9, which has an activity of decreasing the b / a value of the acceleration pulse wave and increasing the d / a value. The blood vessel improving agent according to any one of claims 7 to 10, which has smooth muscle growth inhibitory activity. The blood vessel improving agent according to any one of claims 7 to 11, which has endothelin-1 production inhibitory activity in vascular endothelial cells. The blood vessel improving agent according to any one of claims 7 to 12, which has a tissue plasminogen activator (t-PA) production promoting activity in vascular endothelial cells. The blood vessel improving agent according to any one of claims 7 to 13, which has a vascular endothelial cell proliferation activation activity. A pharmaceutical composition comprising any one or both of the skin improving agent according to any one of claims 1 to 6 and the vascular improving agent according to any one of claims 7 to 14. A food comprising any one or both of the skin improving agent according to any one of claims 1 to 6 and the vascular improving agent according to any one of claims 7 to 14. A feed comprising one or both of the skin-improving agent according to any one of claims 1 to 6 and the vascular-improving agent according to any one of claims 7 to 14. Cosmetics containing any one or both of the skin improvement agent of any one of Claims 1-6, and the blood vessel improvement agent of any one of Claims 7-14.