JP5459826B2 - Elastin production promoter and vascular endothelial cell growth promoter in dermal fibroblasts - Google Patents

Description

The present invention relates to a growth-promoting agent of elastin production-enhancing agents and vascular endothelial cells in the skin fibroblasts, more particularly, elastin proportion can reach the blood while retaining activity at high dermal fibroblasts about the production promoter and growth-promoting agent of the vascular endothelial cells.

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 large numbers in tissues that require elasticity and elasticity, such as arterial walls, ligaments, lungs, and skin, and plays various roles such as maintaining elasticity and regulating cell functions in vivo. The elastin content in blood vessels and interfacial ligaments accounts for 50% or more of the total dry weight and about 2% in the skin. It is known that elastin in a living body is reduced or denatured by factors such as ultraviolet rays and aging, and such changes in the skin cause wrinkles and sagging of the skin and a decrease in elasticity. The skin of mice genetically incapable of forming elastin is not elastic (see Non-Patent Document 1), and the elasticity of the skin and wrinkle formation may occur due to structural changes in elastic fibers after UV irradiation ( Non-Patent Document 2) has been reported. For this reason, it is considered that a substance that promotes the production of elastin maintains the elasticity and firmness of the skin and leads to prevention and improvement of wrinkles.

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.

Examples of conventionally known elastin production-promoting substances include Narcoulis genus plant extract (see Patent Document 1), Rhizopus genus plant extract (see Patent Document 2), Gummiaceae Hippophae plant extract (Patent Document) 3) and oligopeptides such as Leu-Glu-His-Ala (see Patent Document 4).

Further, examples of conventionally known vascular endothelial cell proliferating active substances include plant extracts such as yamakiko extract (see Patent Document 5) and buckwheat extract (see Patent Document 6), water-soluble polymers or derivatives thereof. And a peptide complex in which two or more peptide chains are bonded to each other (see Patent Document 7).

JP-T-2001-513509 Japanese Patent Laid-Open No. 2005-60341 JP 2005-22993 A JP 2008-74788 A Japanese Patent Laid-Open No. 1994-025001 JP 2001-199898 A JP 2008-044915 A

Yanagisawa H. et al. Others, “Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo”, Nature, Nature Publishing Group (UK), 68, 68, p. 168-171 Imoka G. Others, “Degree of Ultraviolet-Induced Tortureity of Elastic Fibers in Rat Skin Is Age Dependent”, J. Org. Invest. Dermatol. , Nature Publishing Group (UK), Vol. 105, No. 2 (August 1995), p. 254-258

However, since plant extracts such as those described in Patent Documents 1 to 3, 5 and 6 contain various components, allergic symptoms such as rash and eczema appear depending on the sensitivity of the recipient. There is a fear. Moreover, utilization may be restrict | limited from the unique smell, the coloring at the time of a mixing | blending, and a physical property. In addition, in the case of peptides or derivatives thereof such as those described in Patent Documents 4 and 7, there is a high possibility that in the case of oral ingestion, there is a high possibility that they will be degraded in the process of digestion and absorption in vivo and lose their structure. The effect may not be obtained.

The present invention has been made in view of such circumstances, and there is no risk of allergic symptoms and the like, and there is a high ratio that can reach the blood while maintaining activity, a vascular function improving agent, and a pharmaceutical composition containing these The object is to provide food, food, feed and cosmetics.

In light of the above problems, the present inventors have conducted extensive studies on a novel skin-improving agent and vascular function-improving agent. As a result, the dipeptide Pro-Gly (L-prolylglycine) has elastin production-promoting activity in skin fibroblasts. And it has been found that it has the activity of promoting the proliferation of vascular endothelial cells, and the present invention has been completed.

According to the first aspect of the present invention that meets the above-mentioned purpose, the dipeptide Pro-Gly, an amino group in the Pro residue, and a carboxyl group in the Gly residue are each harmed with an acid, a base, and a salt that are harmless to a living body. Is a reaction product of one or both of the salt of Pro-Gly and the amino group in the Pro residue and the carboxyl group in the Gly residue with another compound that is harmless to the living body, and is enzymatically degraded in the living body elastin production in the skin fibroblasts of containing Mukoto wherein as an active ingredient one or more compounds selected from the inductive member or Ranaru group Pro-Gly which can generate a receive free Pro-Gly it is intended to solve the above problems by providing a prompting Susumuzai.

In the second aspect of the present invention, the dipeptide Pro-Gly, one or both of the amino group in the Pro residue and the carboxyl group in the Gly residue form a salt with an acid and base that are harmless to the living body, respectively. A reaction product of a salt of Gly and / or one or both of an amino group in a Pro residue and a carboxyl group in a Gly residue with another compound that is harmless to the living body, and is freed by enzymatic degradation in the living body providing growth prompting Susumuzai vascular endothelial cells characterized by containing Mukoto as an active ingredient only one or more compounds selected from the inductive member or Ranaru group Pro-Gly which can generate a Pro-Gly By doing so, the above-mentioned problems are solved.

Since the skin improving agent provided by the first aspect of the present invention and the vascular function improving agent provided by the second aspect of the present invention are dipeptides, most of them are absorbed from the intestinal tract without being decomposed to amino acids. In addition, it can reach the blood while maintaining its activity, can be supplied in a large amount at a low cost, and a salt or a derivative can be easily obtained as required. Therefore, it can respond easily to various dosage forms and applications. Furthermore, since it has no immunogenicity and toxicity, it can provide a safe skin-improving agent and vascular function-improving agent with no side effects.

According to the present invention, a skin improvement agent having an activity to promote elastin production in dermal fibroblasts, which solves the problems of the prior art relating to side effects, inherent odors, safety, etc. and can be applied to a wide range of uses, vascular endothelial cells It is possible to provide a vascular function improving agent having growth promoting activity, and a pharmaceutical composition, food, feed and cosmetics containing them.

Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.
Skin improvement agents and vascular function improving agent according to an embodiment of the present invention, the dipeptide Pro-Gly (L-prolyl-glycine), one or more compounds selected from its salts and its derivatives thereof or Ranaru group Is included as an active ingredient. The dipeptide Pro-Gly used for the skin improving agent and vascular function improving agent is a dipeptide composed of L-proline and glycine.

As the salt of Pro-Gly, use any salt in which one or both of the amino group in the Pro residue and the carboxyl group in the Gly residue form a salt with an acid and base that are harmless to the living body, respectively. Can do. Specific examples of amino group salts include (1) hydrochlorides, sulfates, phosphates, diphosphates, hydrobromides, nitrates and other inorganic acid salts, (2) acetates, propionates , Maleate, fumarate, tartrate, malonate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, salicylate, oxalate, stearate, Examples include organic acid salts such as ascorbate, malate, adipate, and gluconate. Specific examples of the salt of the carboxyl group include (1) metal salts such as sodium salt, potassium salt, magnesium salt and calcium salt, and (2) ammonium salt, pyridinium salt and organic base such as salt with basic amino acid. Salt. Alternatively, a so-called inner salt (Zwitter ion) may be formed.

The derivative of Pro-Gly is a reaction product of one or both of the amino group in the Pro residue and the carboxyl group in the Gly residue with another compound that is harmless to the living body, and is capable of enzymatic degradation in the living body. Any derivative can be used as long as it can produce free Pro-Gly. Specific examples of the derivative include N-acylamide, O-alkyl ester and the like.

Regardless of the origin of the dipeptide Pro-Gly used as a raw material for the skin improving agent and the vascular function improving agent, each of the compounds selected from the group consisting of the above-mentioned dipeptide Pro-Gly, a salt thereof and a derivative thereof is used alone. You may use, and it can also use combining arbitrary 2 or more types.
The dipeptide Pro-Gly is also contained in seafood, livestock, poultry, whales and other meats, soybeans and other hydrolysates, which contain a large amount of animal and vegetable proteins. Using these as raw materials, for example, an extract containing dipeptide Pro-Gly can be obtained by a conventionally known extraction method such as water extraction, hot water extraction, hot water extraction, alcohol extraction, or supercritical extraction.

When the extract 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. If necessary, the dipeptide Pro-Gly may be isolated using any known method such as column chromatography, reprecipitation, recrystallization, or a combination thereof.

The extract containing the dipeptide Pro-Gly thus obtained may be used as a solution as it is, or by using any known method such as recrystallization, spray drying or freeze drying, Depending on the form, it may be used in the form of a solid having any suitable form such as crystals, powder, granules and the like. Alternatively, it may be reacted with any of an appropriate acid, base and compound and used as an appropriate salt or derivative. Also in this case, the extract containing the dipeptide Pro-Gly may be used as it is in a solution, or may be used in the form of a solid having any suitable form such as crystals, powders, granules and the like.

If necessary, a pure product of dipeptide Pro-Gly can also be used as a raw material for skin improvers and skin improvers, or as a raw material for salt or derivative synthesis. The origin of the pure product is not particularly limited, and it may be used after purchasing a commercially available product and further purifying as necessary, or using a product synthesized by any known method. Good. The synthesis method is not particularly limited, and any known synthesis method such as an organic chemical synthesis method (liquid phase and solid phase synthesis) or a genetic engineering synthesis method can be used.

When the synthesized dipeptide Pro-Gly is used as a solution, the solution is adjusted to a concentration and pH suitable for the desired application. Alternatively, when the dipeptide Pro-Gly is used as a solid, any known method such as recrystallization, spray drying or freeze drying may be used, and crystals, powders, granules, etc., depending on the application and the form of the composition It can be a solid having any suitable form of The dipeptide Pro-Gly may be reacted with any suitable acid, base and compound and used as a suitable salt or derivative. Also in this case, the extract containing the dipeptide Pro-Gly may be used as it is in a solution, or may be used in the form of a solid having any suitable form such as crystals, powders, granules and the like.

The dipeptide Pro-Gly (including salts or derivatives unless otherwise specified, the same applies hereinafter) has elastin production promoting activity 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.

The dipeptide Pro-Gly has vascular endothelial cell proliferation promoting activity. If vascular endothelial cells decrease due to oxidative stress increase due to aging or diabetes, etc., thrombosis associated with decrease in vascular relaxation factors such as NO and PGI ₂ produced from vascular endothelial cells and decrease in production of coagulation inhibitory factors And the narrowing of the inner wall of the blood vessel accompanying adhesion of platelets and mononuclear cells to the vascular endothelium. The dipeptide Pro-Gly can improve blood vessel function by promoting proliferation of vascular endothelial cells. As described above, the dipeptide Pro-Gly can be used as a vascular function improving agent.

By mixing the dipeptide Pro-Gly with a carrier or the like, it can be used as a pharmaceutical composition having one or both of a skin improvement effect and an effect of improving blood vessel function. 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, the dipeptide Pro-Gly and a pharmaceutically acceptable carrier or diluent, a stabilizer, and other desired additives can be blended to obtain the desired dosage form.

As foods containing one or both of skin improvers and vascular function improvers, dipeptide Pro-Gly prepared as it is, added to other foods, or foods such as capsules, tablets, etc. or health foods It can take any commonly used 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 in a food material and preparing a food.

The feed containing one or both of the skin improving agent and the vascular function improving agent can take various forms such as a dipeptide Pro-Gly prepared as it is or a feed formulated with the 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, the skin improvement agent containing dipeptide Pro-Gly as an active ingredient can be safely added to livestock such as pigs, chickens, cows, horses, sheep, and feeds such as fish, pets (dogs, cats, birds). Therefore, it can be used as a functional feed having one or both of a skin improvement effect and an effect of improving aged blood vessels.

Cosmetics containing a skin improving agent can take various forms such as those obtained by blending dipeptide Pro-Gly in a 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 Measurement of Elastin Production Promoting Activity Normal human dermal fibroblasts (DS Pharma) were suspended in D-MEM containing 1% FBS, and seeded in a 96-well plate so that the number of cells was 2 × 10 ⁴ . The cells were cultured for 24 hours in a CO ₂ incubator. Subsequently, the medium was replaced with a medium prepared such that the concentration of Pro-Gly (Kokusan Chemical Co., Ltd.) was 0.1, 1, 10 μg / ml, and the culture was performed. The culture supernatant after 3 days from the addition of Pro-Gly was collected, and the amount of elastin produced from the fibroblasts was quantified by ELISA. The control elastin production amount was set to 100, and the relative value of the elastin production amount in the Pro-Gly addition group was determined.

The measurement results are shown in Table 1. It can be seen that the cell group to which Pro-Gly was added at 0.1, 1, 10 μg / ml showed a significant elastin production promoting effect at a risk rate of less than 1% relative to the control. In addition, “*” in the table represents that the significance probability is less than 1% (p <0.01) with respect to the significance probability p value in the t test.

Example 2 Measurement of Vascular Endothelial Cell Growth Promoting Activity Normal human vascular endothelial cells HUVEC (Kurabo) were suspended in EG-2 medium and seeded in a 96-well plate so that the number of cells was 1 × 10 ³ . At the same time, Pro-Gly (Kokusan Chemical Co., Ltd.) dissolved in PBS was added to 1/10 of the culture solution. After culturing in a CO ₂ incubator for 4 days, Cell Counting Kit-8 (Dojindo Laboratories) was added and maintained at 37 ° C. for 2 hours. After confirming that the dye was colored, the absorbance at 450 nm was measured using a microplate reader. The contents of the wells to which only PBS was added were used as a control (control group). The absorbance of the control was taken as 100, and the relative value of the absorbance of the Pro-Gly added group was determined.

The results are shown in Table 2. Pro-Gly showed a growth-promoting effect on vascular endothelial cells in a concentration-dependent manner, and showed a significant vascular endothelial cell proliferation action at a risk rate of less than 1% in the group added with 50 or 100 ng / ml. In addition, “*” in the table represents that the significance probability is less than 1% (p <0.01) with respect to the significance probability p value in the t test.

Claims (2)

Dipeptide Pro-Gly, Pro-Gly salt in which one or both of amino group in Pro residue and carboxyl group in Gly residue form a salt with acid and base that are harmless to living body, respectively, in Pro residue It is a reaction product of one or both of the amino group and the carboxyl group in the Gly residue with other compounds that are harmless to the living body, and can generate free Pro-Gly upon enzymatic degradation in the living body elastin production prompting Susumuzai in skin fibroblasts, wherein the free Mukoto as an active ingredient one or more compounds selected from the inductive member or Ranaru group Pro-Gly. Dipeptide Pro-Gly, Pro-Gly salt in which one or both of amino group in Pro residue and carboxyl group in Gly residue form a salt with acid and base that are harmless to living body, respectively, in Pro residue It is a reaction product of one or both of the amino group and the carboxyl group in the Gly residue with other compounds that are harmless to the living body, and can generate free Pro-Gly upon enzymatic degradation in the living body growth prompting Susumuzai vascular endothelial cells characterized by containing Mukoto as an active ingredient only one or more compounds selected from the inductive member or Ranaru group Pro-Gly.