JP2016222567A - Cytoprotective agents of protecting cells from formyl group-containing substances - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cytoprotective agents containing a γ-glutamic-acid oligomer that protects cells from formyl group-containing substances, such as 2.4-nonadienal, 2-hydroxynonenal, and acrolein, which cause carbonylation and/or oxidation denaturation mainly to proteins, and oxidation disorders to the various biogenic components including lipids and nucleic acids; and to provide external preparations for anti-skin-aging that contain the cytoprotective agents.SOLUTION: By including a γ-glutamic acid oligomer obtained by hydrolysis of γ-polyglutamic acid, as a cytoprotective agent of protecting cells from a carbonyl compound mainly composed of a formyl group, cells can be protected from disorders caused by formyl group-containing substances, such as 2.4-nonadienal, 2-hydroxynonenal, and acrolein, which generate in vivo or in an environment.SELECTED DRAWING: None

Description

  The present invention relates to a cytoprotective agent characterized by containing a γ-glutamic acid oligomer to protect cells from cell damage caused by a formyl group-containing substance.

  Conventionally, various anti-aging agents that suppress aging have been developed. Such biological aging is known to occur due to two biological factors. One is physiological aging, and the other is photoaging that occurs in exposed areas such as ultraviolet rays. Physiological aging is influenced by active oxygen generated in the living body, and is related to degeneration of collagen fibers, which are the main components of blood vessels and skin, and a decrease in activity due to alteration of enzymes deeply related to physiological functions. Photo-aging is limited to the skin because it is caused by UV exposure.

  In physiological aging, removal of reactive oxygen species is mainly studied, and removal agents for superoxide anion, hydroxyl radical, singlet oxygen, peroxynitrite and the like are proposed. In recent years, attention has been paid to the participation of formyl group-containing substances such as 2-hydroxynonenal, acrolein, and 2,4-nonadienal as aging factors other than active oxygen, and a removal agent has been proposed (Patent Document 1).

JP2008-24600

  The problem of the present invention is that, as described above, a formyl group-containing substance such as 2-hydroxynonenal, acrolein, or 2,4-nonadienal involved in skin aging binds to an amino group of a protein and functions as an enzyme. It will be lost and cells will die. An object of the present invention is to provide an ingredient for protecting cells from such a formyl group-containing substance and to provide a skin anti-aging external preparation containing the ingredient.

  In view of the above circumstances, as a result of intensive studies by the present inventors, cells were removed from formyl group-containing substances such as 2-hydroxynonenal, acrolein and 2,4-nonadienal, in which γ-glutamic acid oligomers cause skin aging. The inventors have found that the protective action is remarkable and have completed the present invention.

  That is, the present invention comprises a γ-glutamic acid oligomer obtained by hydrolyzing γ-polyglutamic acid with an enzyme or an acid, and protects cells from a formyl group-containing substance such as 2-hydroxynonenal, acrolein or 2,4-nonadienal. It is a protective agent.

  When the cytoprotective agent of the present invention is applied to the skin by blending γ-glutamic acid oligomer obtained by hydrolyzing γ-polyglutamic acid with an enzyme or acid into a cosmetic as a cytoprotective agent from a formyl group-containing substance, The skin can be expected to have an excellent cosmetic effect.

  The present invention comprises a γ-glutamic acid oligomer obtained by hydrolyzing γ-polyglutamic acid using an enzyme or an acid. Polyglutamic acid, which is a polymer of glutamic acid serving as a substrate, is known as a viscous substance produced by Bacillus natto and the like, and is blended in order to have excellent safety as a natural product and to provide a characteristic feeling of use of cosmetics.

  Low molecular weight polyglutamic acid obtained by hydrolyzing polyglutamic acid with an enzyme or an acid can increase the permeability to the skin by reducing the molecular weight. And hyaluronic acid is protected by suppressing the activity of hyaluronidase which decomposes hyaluronic acid in the skin, and the decrease in the moisture retention ability of the skin is suppressed. Furthermore, it has been reported that glutamic acid oligomers act on epidermal cells and improve the ability to synthesize hyaluronic acid.

  Moreover, since the compounding quantity with respect to various skin external preparations of the cytoprotective agent which concerns on this invention can be fluctuate | varied according to the embodiment of a skin external preparation, the usage form of a skin external preparation, etc., it is not specifically limited. In principle, it is sufficient that an effective amount is present, but in general, in an amount of 0. 01-100 mass% can be utilized, Preferably it is 1.0-20 mass%, More preferably, it is 3.0-10.0 mass%. In particular, it can be assumed that the powdery preparation and the like prepared at the time of use are used at a high blending ratio including 100% by mass of the cytoprotective agent according to the present invention.

  The application range of the external preparation for skin according to the present invention is not particularly limited. That is, according to the effect which the active ingredient of this invention has, it can apply to all the skin external preparations which can utilize each effect.

  For example, the cytoprotective agent according to the present invention can be blended with various skin external preparation bases and applied to creams, emulsions, lotions, packs, face wash, and the like. Moreover, the various skin external preparations can be applied in various forms such as lotion, emulsion, ointment, sol, gel, powder, spray, and solid.

  Furthermore, since the cytoprotective agent according to the present invention has a low molecular weight and is an oligomer of acidic amino acid, it has a negative charge. Therefore, it is possible to quickly deliver the target product to the deep part of the skin by applying a skin external preparation containing a cytoprotective agent from a carbonyl compound to the skin and performing an iontophoresis treatment.

  Hereinafter, embodiments of the present invention will be described in detail.

[Example 1: Production of γ-glutamic acid oligomer]
In 0.01M phosphate buffer adjusted to pH 7.1, γ-polyglutamic acid (molecular weight 10,000 to 100,000) is dissolved to a concentration of 10 mg / mL, and 20 μg / mL of γ-glutamic acid depolymerase is respectively added. Added and allowed to react at 25 ° C. The α-amino group exposed by enzymatic degradation of γ-polyglutamic acid by γ-glutamic acid depolymerase was measured with a TNBS monitor (Tokyo Chemical Industry, T2024), and an appropriate enzymatic degradation time was examined. As a result, 4.65 wt% of a 5- to 7-mer γ-glutamic acid oligomer was obtained in a reaction time of about 30 hours.

  The quantitative analysis of the γ-glutamic acid oligomer was performed by amino acid analysis by HTLC (high performance thin layer chromatography) and HPLC (high performance liquid chromatography). HTLC is a ninhydrin reagent using a silica gel plate (TLC-Plastic sheets silica 60 F254, Merck), n-Butyl alcohol: Aceticacid: H2O = 3: 2: 1 (v / v) as a developing solvent. The color was detected and detected.

  Detection of γ-glutamic acid oligomer by amino acid analysis was performed using a HPLC apparatus (LC-9A, manufactured by Shimadzu Corporation) and a strongly acidic cation exchange resin column Shim-pack ISC-07 as a column for amino acid analysis. Was determined by fluorescence measurement using ο-phthalaldehyde as excitation wavelength = 348 nm and fluorescence wavelength = 450 nm. The turbidity of a complex obtained by adding cetablone (cetyltrimethylammonium bromide; CET) to the γ-glutamic acid polymer was measured at 660 nm, and the concentration of the γ-glutamic acid polymer was calculated.

[Example 2: Protection against human skin cell death by 2,4-nonadienal]
Subsequently, human skin cells produced by 2,4-nonadienal using the γ-glutamic acid oligomer (weight molecular weight 660 to 1950) produced in Example 1 and γ-polyglutamic acid (weight molecular weight 10,000 to 100,000) as a comparison target. The protective effect against death was compared.

  As a preliminary preparation, human skin keratinocytes HaCaT were cultured in a medium containing 10% fetal bovine serum of Dulbecco's modified Eagle's medium in a 24-well multiplate under conditions of 5% carbon dioxide, 37 ° C and 100% humidity. Cultured. Then, 60 μM 2,4-nonadienal is added, and γ-glutamic acid oligomer or γ-polyglutamic acid is neutralized with pH, respectively, and administered to the cells so that the concentration is 3%, 6%, or 10% by weight. And cultured for 72 hours. Each sample was performed with n = 4. The group not treated with 2,4-nonadienal and not administered with γ-glutamic acid was cultured in the same manner as a control.

In order to measure formazan generated by tetrazolium reduction due to intracellular mitochondrial dehydrogenase activity, measurement was performed using (phenyl) -5- (2-disulphophenyl) -2H-tetrazolium, monosodium salt as a redox indicator. The cell viability was calculated by measuring the absorbance at 450 nm with a microplate reader. The survival rate of the other groups was expressed with 100% as the group not treated with 2,4-nosylenal.

  The results are shown in Table 1. In the results, the group treated with 2,4-nonadienal dropped to 22.7% compared to the untreated group, but the survival rate was slightly lower in the untreated γ-polyglutamic acid group than the untreated group. High, a slight protective effect was observed from the damage of 2,4-nonedenal to cells. On the other hand, the survival rate of the γ-glutamic acid oligomer increased in a concentration-dependent manner, and it was confirmed that it exhibited a strong protective action against damage of 2,4-nonedenal to cells.

[実施例３：イオン導入効率の比較]

[Example 3: Comparison of ion introduction efficiency]

  The ion introduction efficiency was compared between the γ-glutamic acid oligomer (weight molecular weight 660 to 1950) produced in Example 1 and γ-polyglutamic acid (weight molecular weight 10,000 to 200,000) as a comparison target.

  As a test method, first, (skin layer + epidermis: keratinocytes + dermis: fibroblasts) is prepared as a human skin reconstructed tissue. Specifically, in a modified Bronoff diffusion chamber, human skin fibroblasts OUMS-36 are seeded in a type I collagen gel, and then a type IV collagen-containing reconstituted basement membrane Matrigel is applied thereon. Human skin keratinocytes HaCaT were overlaid, and then air lifted to cause dry cell death gently on the surface layer cells to form a stratum corneum, thereby preparing a three-dimensional skin tissue model.

  Subsequently, the pad of the modified Bronov diffusion chamber including the three-dimensional skin tissue model is prepared by placing the pad impregnated with the sample sample on the modified Bronov diffusion chamber containing the three-dimensional skin tissue model and contacting the negative electrode terminal therewith. A positive electrode terminal was connected to the bottom of the Boyden double diffusion chamber to form an electrical circuit. A multimeter was interposed in the circuit.

  Ions are introduced for 8 minutes at an ellipsoidal waveform of 1500 Hz and 0.4 mA using the ion introducer having both electrode terminals. After that, a 3D skin tissue model is cut out from the housing, EDTA / collagenase is gently acted, each layer separated into an epidermis layer and a dermis layer is rinsed three times with PBS (−), pulverized, and then cooled and centrifuged. Collect the supernatant. Further, the precipitate was suspended in PBS (−), centrifuged again, and the supernatant was collected again and combined with the first supernatant.

  The supernatant was quantitatively analyzed by HTLC and HPLC. As for the γ-glutamic acid oligomer, the epidermis 36.7 ± 8.0 μg / g tissue, the dermis 9.1 ± 3.3 μg / g tissue, the high molecular γ-glutamic acid oligomer Then, results of epidermis 8.5 ± 2.4 μg / g tissue and dermis 1.7 ± 0.2 μg / g tissue were obtained (see Table 2). From this result, it was found that the γ-glutamic acid oligomer of the present invention has a significantly improved ion introduction efficiency than γ-polyglutamic acid.

Examples of the present invention are given below.
<Prescription Example 1> Lotion (Ingredient name) (mass%)
γ-glutamic acid oligomer 2.0
Glycerin 5.0
Polyoxyethylene sorbitan monolaurate (20E0) 1.5
Ethanol 8.0
Triethyl citrate 2.0
Preservative / Antioxidant Appropriate amount of purified water Balance

<Formulation example 2> Cosmetic cream (ingredient name) (mass%)
γ-glutamic acid oligomer 5.0
Beeswax 2.0
Stearyl alcohol 5.0
Stearic acid 8.0
Squalane 10.0
Self-emulsifying glyceryl monostearate 3.0
Polyoxyethylene cetyl ether (20E.0) 1.0
Glycerin 5.0
Potassium hydroxide 0.3
Perfume Appropriate amount of preservative / antioxidant Appropriate amount of purified water

<Formulation example 3> Emulsion (component name) (mass%)
γ-glutamic acid oligomer 1.0
Squalane 8.0
Vaseline 2.0
Beeslow 0.5
Sorbitan sesquioleate 0.8
Polyoxyethylene oleyl ether (20E.0) 1.2
Carboxyvinyl polymer 0.2
Glycerin 1.5
Potassium hydroxide 0.1
Ethanol 7.0
Perfume Appropriate amount of preservative / antioxidant Appropriate amount of purified water

<Formulation example 4> Packing agent (component name) (mass%)
γ-glutamic acid oligomer 0.5
Vinyl acetate resin emulsion 15.0
Polyvinyl alcohol 10.0
Jojoba oil 3.0
Glycerin 5.0
Titanium oxide 8.0
Kaolin 7.0
Ethanol 5.0
Perfume Appropriate amount of preservative / antioxidant Appropriate amount of purified water

<Formulation example 5> Ointment (component name) (mass%)
γ-glutamic acid oligomer 0.01
Tocopherol acetate 0.5
Octyl paradimethylaminobenzoate 4.0
Butylmethoxybenzoylmethane 4.0
Stearyl alcohol 18.0
Owl 20.0
Glycerin monostearate 0.3
Vaseline 33.0
Perfume Appropriate amount of preservative / antioxidant Appropriate amount of purified water

Claims (3)

A cytoprotective agent that protects against cell damage from a formyl group-containing substance, comprising a γ-glutamic acid oligomer. The cytoprotective agent according to claim 1, wherein the formyl group-containing substance is 2,4-nonadienal. The cytoprotective agent according to claim 1 or 2, wherein the molecular weight of the γ-glutamic acid oligomer is 450 to 9000.