JP3878612B2 - Composition for suppressing active oxygen, and external preparation for skin and cosmetic containing the composition - Google Patents
Composition for suppressing active oxygen, and external preparation for skin and cosmetic containing the composition Download PDFInfo
- Publication number
- JP3878612B2 JP3878612B2 JP2004056122A JP2004056122A JP3878612B2 JP 3878612 B2 JP3878612 B2 JP 3878612B2 JP 2004056122 A JP2004056122 A JP 2004056122A JP 2004056122 A JP2004056122 A JP 2004056122A JP 3878612 B2 JP3878612 B2 JP 3878612B2
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- JP
- Japan
- Prior art keywords
- composition
- chitosan
- comparative example
- active oxygen
- skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- 229960005542 ethidium bromide Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 150000002222 fluorine compounds Chemical class 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229940054190 hydroxypropyl chitosan Drugs 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- XUGNVMKQXJXZCD-UHFFFAOYSA-N isopropyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)C XUGNVMKQXJXZCD-UHFFFAOYSA-N 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000010702 perfluoropolyether Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- DQAKJEWZWDQURW-UHFFFAOYSA-N pyrrolidonecarboxylic acid Chemical compound OC(=O)N1CCCC1=O DQAKJEWZWDQURW-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 150000003377 silicon compounds Chemical class 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- QPQANCNBWQXGTQ-UHFFFAOYSA-N trihydroxy(trimethylsilylperoxy)silane Chemical compound C[Si](C)(C)OO[Si](O)(O)O QPQANCNBWQXGTQ-UHFFFAOYSA-N 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000010497 wheat germ oil Substances 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Landscapes
- Cosmetics (AREA)
Description
本発明は、活性酸素抑制用の組成物、特にシワ、シミ等の皮膚老化や、肌荒れ、炎症性ニキビ等の皮膚細胞損傷への影響性が高いヒドロキシラジカルを抑制する組成物と、その組成物を配合する皮膚外用剤、化粧料に関する。 The present invention relates to a composition for suppressing active oxygen, particularly a composition for suppressing hydroxy radicals having high influence on skin aging such as wrinkles and spots, rough skin, and skin cell damage such as inflammatory acne, and a composition thereof. The present invention relates to an external preparation for skin and a cosmetic.
活性酸素は、酸素分子等が高エネルギー放電、酵素反応等により、或いは電離してラジカルを持つようになった状態のものであり、酸素自体がラジカルを持つものの他、OHの酸素がラジカルを持ついわゆるヒドロキシラジカルも広義の活性酸素に含まれる。 Active oxygen is a state in which oxygen molecules have radicals by high energy discharge, enzymatic reaction, etc., or ionization, and oxygen itself has radicals, and OH oxygen has radicals. So-called hydroxy radicals are also included in the broad sense of active oxygen.
このような活性酸素種の中でも、ヒドロキシラジカルは紫外線や電磁波等により皮膚で発生し、皮膚に対して悪影響を及ぼすことが知られている。具体的には、ニキビ、アトピー性皮膚炎、肌荒れ等の炎症性の皮膚トラブルや、光老化によって起こるシワやシミ等に深く関与していることが指摘されている。ヒドロキシラジカルは細胞障害性や過酸化脂質発生が高い一方で、皮膚損傷反応は極めて短時間で進行するため、短時間でほぼ完全にヒドロキシラジカルを抑制する技術が求められている。 Among such active oxygen species, it is known that hydroxy radicals are generated in the skin by ultraviolet rays, electromagnetic waves or the like, and have an adverse effect on the skin. Specifically, it has been pointed out that it is deeply involved in inflammatory skin troubles such as acne, atopic dermatitis, and rough skin, and wrinkles and spots caused by photoaging. Hydroxyl radicals have high cytotoxicity and lipid peroxide generation, but the skin damage reaction proceeds in a very short time. Therefore, a technique for suppressing hydroxy radicals almost completely in a short time is required.
従来、酸素ラジカルの生成を抑制するものとしてSOD(スーパーオキシドディムスターゼ)や、抗酸化剤であるビタミンE(トコフェロール)等が知られている。またビタミンC(アスコルビン酸)に活性酸素の抑制作用があるとする下記特許文献1のような特許出願もなされている。
しかし、これらのSOD、ビタミンE、ビタミンC等は、ヒドロキシラジカル抑制効果が低く、安定性、持続性も弱いという問題があった。 However, these SOD, vitamin E, vitamin C, and the like have a problem that the hydroxy radical suppressing effect is low, and stability and sustainability are weak.
一方、植物からの抽出液が活性酸素の抑制作用を有することも指摘されており、たとえば下記特許文献2乃至4のように、植物エキスを配合した活性酸素抑制用の化粧料等に関する特許出願もなされている。
しかし、この特許文献2乃至4のように、植物エキスを配合したものであっても、活性酸素抑制効果が十分ではなく、安定性、持続性も弱いものであった。 However, even if a plant extract is blended as in Patent Documents 2 to 4, the effect of suppressing active oxygen is not sufficient, and the stability and sustainability are weak.
本発明は、このような従来の問題を解決するためになされたもので、安定性、持続性が高く、短時間でほぼ完全にヒドロキシラジカル等の活性酸素を抑制することのできる活性酸素抑制効果に優れた組成物、及びその組成物を配合した皮膚外用剤、化粧料を提供することを課題とする。 The present invention has been made to solve such conventional problems, and has a high stability and sustainability, and an active oxygen suppressing effect capable of suppressing active oxygen such as hydroxy radicals almost completely in a short time. It is an object of the present invention to provide an excellent composition, a skin external preparation and a cosmetic containing the composition.
本発明者等は、このような課題を解決すべく鋭意研究したところ、キチン若しくはキトサンの誘導体と、アミノ酸との共存、複合化、とりわけ平均分子量1000〜5000000の特定のキチン若しくはキトサンの誘導体と、シトルリン、テアニンのようなアミノ酸とを共存、複合化させることにより、ヒドロキシラジカル等の活性酸素の抑制効果が効果的、相乗的に高まり、さらに皮膚細胞への作用性が高まって、上記問題が解決されることを見出し、本発明を完成するに至った。 The present inventors have such problems Toko filtrate that to the intensive study to solve the, and derivatives of chitin or chitosan, coexistence with amino acids, conjugated, especially derivatives of specific chitin or chitosan with an average molecular weight from 1000 to 5,000,000 Together with amino acids such as citrulline and theanine, the effect of suppressing active oxygen such as hydroxy radicals is effectively and synergistically increased, and the action on skin cells is further increased. Has been found to be solved, and the present invention has been completed.
すなわち、請求項1記載の発明は、活性酸素抑制用の組成物に係る発明であり、アミノ酸と、キチン若しくはキトサンの誘導体とを含有することを特徴とする。キチン若しくはキトサンの誘導体としては、部分ミリストイル化キトサンピロリドンカルボン酸塩、カルボキシメチルキチン、キトサンピロリドンカルボン酸塩の少なくともいずれかが使用される。また、アミノ酸としては、シトルリン、テアニンの少なくともいずれかが使用される。 That is, the first aspect of the present invention is an invention relating to compositions for active oxygen suppressing, characterized in that it contains the amino acid, and a derivative of chitin or chitosan. Derivatives of chitin or chitosan, partially myristoylated chitosan pyrrolidone carboxylate, carboxymethyl chitin, at least one of chitosan pyrrolidone carboxylate salt are used. Also, as the amino acid, citrulline, at least one of theanine is used.
さらに、請求項2記載の発明は、請求項1記載の活性酸素抑制用の組成物を含有することを特徴とする皮膚外用剤の発明である。さらに、請求項3記載の発明は、請求項1記載の活性酸素抑制用の組成物を含有することを特徴とする化粧料の発明である。このような皮膚外用剤や化粧料は、炎症性皮膚部位の予防,軽減用として使用することができ、或いは光老化によって起るシワ又はシミの予防,軽減用として使用することができる。 Further, the invention of claim 2 is an invention of the skin external agent characterized by containing the composition for the active oxygen inhibition of claim 1 Symbol placement. Further, the invention of claim 3 wherein is a cosmetic composition of the invention which is characterized by containing a composition for active oxygen inhibition of claim 1 Symbol placement. Such external preparations for skin and cosmetics can be used for prevention and reduction of inflammatory skin sites, or can be used for prevention and reduction of wrinkles or spots caused by photoaging.
本発明によって、安定性,持続性が高く、短時間でほぼ完全にヒドロキシラジカル等の活性酸素を抑制することのできる活性酸素抑制効果に優れた組成物、及びその組成物を配合した皮膚外用剤、化粧料を提供することが可能となった。 According to the present invention, a composition having high stability and sustainability and capable of suppressing active oxygen such as hydroxy radicals almost completely in a short time, and a composition for external application to skin containing the composition. It became possible to provide cosmetics.
本発明の組成物に含まれるアミノ酸としては、上述のようにシトルリン、テアニンの少なくともいずれかが用いられる。 The amino acids contained in the composition of the present invention, Shitoruri down as described above, at least one of Te cyanine is used.
キチン、キトサンの誘導体としては、上記アミノ酸との相乗的作用より、上述のように平均分子量1000〜5000000のものを用いるのが好ましく、たとえば、カルボキシメチルキチン、カルボキシメチルキトサン、カルボキシブチルキトサン、ヒドロキシプロピルキトサン、N−ジカルボキシメチルキトサン、サクシニル化キトサン、リン酸化キチン、リン酸化キトサン、部分加水分解キチン、硫酸化キチン、硫酸化キトサン、キチンやキトサンのグリコール酸塩やアスコルビン酸塩やピロリドンカルボン酸塩等の塩類等が挙げられる。本発明においては、これらのうち、カルボキシメチルキチン、キトサンピロリドンカルボン酸塩が用いられる。 Chitin, derivatives of chitosan down, from the synergistic effect of the above amino acids, rather is preferable to use those having an average molecular weight from 1,000 to 5,000,000, as described above, For example other, carboxymethyl chitin, carboxymethyl chitosan, carboxy Butyl chitosan, hydroxypropyl chitosan, N-dicarboxymethyl chitosan, succinylated chitosan, phosphorylated chitin, phosphorylated chitosan, partially hydrolyzed chitin, sulfated chitin, sulfated chitosan, glycolate and ascorbate of chitin and chitosan And salts such as pyrrolidone carboxylate . Among these, carboxymethyl chitin and chitosan pyrrolidone carboxylate are used in the present invention.
又、炭素数8〜20のアシル基等の脂肪酸基を0.1〜50.0%部分導入したようなものも用いられる。アミノ基や水酸基に炭素数8〜20の脂肪酸基を0.1〜50.0%部分導入した物質は相乗的作用がさらに高まるため好ましい。具体的には部分ミリストイル化キトサンピロリドンカルボン酸塩(製品名PM−キトサン:ピアス株式会社)や部分アセチルミリストイル化キトサンピロリドンカルボン酸塩、部分ミリストイル化キチン乳酸塩、部分ミリストイル化四級化キトサングリコ−ル酸塩等を利用するのが好ましい。本発明においては、これらのうち、部分ミリストイル化キトサンピロリドンカルボン酸塩が用いられる。
ここで、「部分導入」とは構成単糖1残基当たりにアシル基等の脂肪酸基がどの程度導入されているかを示すもので、「脂肪酸基を0.1〜50.0%部分導入した」とは、たとえばキトサンの場合であれば、構成単糖であるヘキソサミン1000残基に、脂肪酸基が1〜500個導入されていることを意味する。このように炭素数8〜20のアシル基等の脂肪酸基が部分導入されたキチン、キトサンの誘導体は、両親媒性を示すものである。
Further, those in which 0.1 to 50.0% of a fatty acid group such as an acyl group having 8 to 20 carbon atoms is partially introduced are also used. 0.1 to 50.0% parts introduced material fatty acid group having 8-20 carbon atoms to an amino group and a hydroxyl group is not preferable to further increase the synergistic effect. Concrete in part myristoylated chitosan pyrrolidone carboxylate (product name PM- Chitosan: Pierce Co.) and partial acetyl-myristoyl chitosan pyrrolidone carboxylate salt, partially myristoylated chitin lactate, partially myristoylation quaternized chitosan glycolate -It is preferable to use a sulfate or the like. Of these, partially myristoylated chitosan pyrrolidone carboxylate is used in the present invention.
Here, “partial introduction” indicates how much fatty acid groups such as acyl groups are introduced per residue of the constituent monosaccharide. “Partial introduction of 0.1 to 50.0% of fatty acid groups” For example, in the case of chitosan, 1 to 500 fatty acid groups are introduced into 1000 residues of hexosamine, which is a constituent monosaccharide. Thus, chitin and chitosan derivatives into which fatty acid groups such as acyl groups having 8 to 20 carbon atoms are partially introduced exhibit amphipathic properties.
上記の場合の平均分子量は、たとえばデータモジュールGPC用カートリッジを連結させたGPC−HPLC〔ゲル濾過クロマトグラフィーカラム:東ソー(株)製TSK−gel−G3000WXL+TSK−gel−G2500PWXL、溶媒:0.4 M酢酸−酢酸Na緩衝液(pH=4.8)〕分析により分子量分布を明らかにすることによって測定される。分子量スタンダードとしては、キトサンオリゴ糖(分子量:413,1006)、デキストラン硫酸塩(分子量:5000 、8000) 及びプルラン分子量スタンダードが用いられる。
また、粘度測定法等からも平均分子量を求めることが可能である。
The average molecular weight in the above case is, for example, GPC-HPLC (gel filtration chromatography column: TSK-gel-G3000WXL + TSK-gel-G2500PWXL manufactured by Tosoh Co., Ltd.) connected with a data module GPC cartridge, solvent: 0.4 M acetic acid-acetic acid Na buffer (pH = 4.8)] determined by revealing the molecular weight distribution by analysis. As molecular weight standards, chitosan oligosaccharide (molecular weight: 413,1006), dextran sulfate (molecular weight: 5000, 8000) and pullulan molecular weight standard are used.
The average molecular weight can also be obtained from a viscosity measurement method or the like.
キトサンは、天然多糖であるキチンの高脱アセチル化物であり、脱アセチル化度50〜100%を示す脱アセチル化キチンを元に誘導体を合成するのが好ましい。アミノ酸と、キチン、キトサンの誘導体の共存比は、特に限定されないが, アミノ酸100容量に対してキチン、キトサンの誘導体の容量比が0.1〜20容量であることが望ましい。 Chitosan is a highly deacetylated product of chitin, which is a natural polysaccharide, and it is preferable to synthesize derivatives based on deacetylated chitin that exhibits a degree of deacetylation of 50 to 100%. And amino acids, coexistence ratio of chitin, chitosan down the derivative is not particularly limited, chitin the amino acids 1 00 capacity, it is desirable that the volume ratio of chitosan down the derivative is 0.1 to 20 volume.
本発明における皮膚外用剤又は化粧料に対する組成物の配合量は、特に限定されるものではないが、外用剤等の全量中、乾燥固形物重量で0.0005〜5質量%が好ましい。0.0005質量%未満では本発明の効果が充分に得られない可能性があり、一方、5質量%を越えても、その増量に見合った効果の向上は認められないからである。この観点からは、0.001 〜3質量%がより好ましい。 Although the compounding quantity of the composition with respect to the external preparation for skin or cosmetics in this invention is not specifically limited, 0.0005-5 mass% is preferable at the dry solid weight in the whole quantity, such as an external preparation. If the amount is less than 0.0005% by mass, the effect of the present invention may not be sufficiently obtained. On the other hand, if the amount exceeds 5% by mass, an improvement in the effect commensurate with the increase is not recognized. From this viewpoint, 0.001 to 3 mass% is more preferable.
皮膚外用剤の形態は特に限定されるものではなく、たとえば、ローション、エッセンス、ジェル、乳液、乳化ローション及びクリーム等にすることができる。より具体的には、低分子界面活性剤を含まない炎症予防軽減用のスキンケアクリーム、乳液状の乳化ファンデーション、乳液状の化粧下地、乳化型マスカラ等の製剤として提供することができる。 The form of the external preparation for skin is not particularly limited and can be, for example, lotion, essence, gel, emulsion, emulsified lotion, cream and the like. More specifically, it can be provided as a preparation such as a skin care cream for preventing or reducing inflammation, an emulsion emulsion foundation, an emulsion makeup base, an emulsion mascara, etc., which does not contain a low molecular surfactant.
また、皮膚外用剤の形態に応じ、上記必須成分以外に化粧料や外用剤で一般的に用いられる成分を本発明の効果を阻害しない範囲で配合できる。たとえばブチレングリコール、ジプロピレングリコール、グリセリン等の多価アルコール類、セタノール、ベヘニルアルコール等の高級アルコール類、流動パラフィン、スクワラン等の非極性油剤類、パルミチン酸イソプロピル、ミリスチン酸イソプロピル等のエステル系油剤類、小麦胚芽油やオリーブ油等の植物油類、トリメチルシロキシケイ酸、メチルフェニルポリシロキサン等のシリコン化合物類、パーフルオロポリエーテル等のフッ素化合物類が挙げられる。また、保湿柔軟化剤、抗酸化剤、収斂剤、美白剤、抗菌剤、抗炎症剤、紫外線吸収剤類、紫外線散乱剤、ビタミン類、酵素等の医薬部外品原料規格、化粧品種別配合成分規格、化粧品原料基準、日本薬局方、食品添加物公定書規格等の成分等が挙げられる。 Moreover, according to the form of a skin external preparation, the component generally used with cosmetics and an external preparation other than the said essential component can be mix | blended in the range which does not inhibit the effect of this invention. For example, polyhydric alcohols such as butylene glycol, dipropylene glycol and glycerin, higher alcohols such as cetanol and behenyl alcohol, nonpolar oils such as liquid paraffin and squalane, ester oils such as isopropyl palmitate and isopropyl myristate, Examples include vegetable oils such as wheat germ oil and olive oil, silicon compounds such as trimethylsiloxysilicic acid and methylphenylpolysiloxane, and fluorine compounds such as perfluoropolyether. Moisturizing and softening agents, antioxidants, astringents, whitening agents, antibacterial agents, anti-inflammatory agents, UV absorbers, UV scattering agents, vitamins, enzymes, etc. Ingredients such as standards, cosmetic raw material standards, Japanese pharmacopoeia, official standards for food additives, etc.
以下、本発明の実施例について説明する。 Examples of the present invention will be described below.
(実施例1)
本実施例では、シトルリンと部分ミリストイル化キトサンピロリドンカルボン酸塩とを混合して活性酸素抑制用の組成物を調整した。具体的にはシトルリン1%、及び部分ミリストイル化キトサンピロリドンカルボン酸塩0.01%を含む溶液を調製し、これを活性酸素抑制用の組成物とした。部分ミリストイル化キトサンピロリドンカルボン酸塩としては、ピアス株式会社製のPM−キトサンを用いた。
Example 1
In this example, citrulline and partially myristoylated chitosan pyrrolidone carboxylate were mixed to prepare a composition for suppressing active oxygen. Specifically, a solution containing 1% citrulline and 0.01% partially myristoylated chitosan pyrrolidone carboxylate was prepared and used as a composition for suppressing active oxygen. As the partially myristoylated chitosan pyrrolidone carboxylate, PM-chitosan manufactured by Pierce Co., Ltd. was used.
このようにして調製した活性酸素抑制用組成物のヒドロキシラジカルの消去活性を測定し、消去作用を試験した。一方、シトルリン1%のみを含む溶液(比較例1−1)、及び部分ミリストイル化キトサンピロリドンカルボン酸塩0.01%のみを含む溶液(比較例1−2)を調製し、同様にヒドロキシラジカル消去活性を測定して、上記混合物である活性酸素抑制用の組成物と比較した。 The scavenging activity of the hydroxy radical scavenging activity of the thus prepared active oxygen suppressing composition was measured and tested for scavenging action. On the other hand, a solution containing only 1% citrulline (Comparative Example 1-1) and a solution containing only 0.01% partially myristoylated chitosan pyrrolidone carboxylate (Comparative Example 1-2) were prepared, and similarly, hydroxyl radical scavenging activity was obtained. It was measured and compared with the composition for suppressing active oxygen which is the above mixture.
反応組成は、40mM K-Pi buffer(pH 7.4)、0.26mMアスコルビン酸、0.15mM FeEDTA 、0.6mM 過酸化水素、2mM サリチル酸と、上記シトルリン、部分ミリストイル化キトサン等の目的とする溶質を含み、全量を400mL とし、アスコルビン酸−過酸化水素系によりヒドロキシラジカルを発生させた。25℃で90分間保温した後、水酸化されたサリチル酸を誘導体化して発色させ、510nm の吸光度を測定することにより、ラジカルの検出を行った。コントロールには、試料の代わりに精製水を添加し、ブランクは、アスコルビン酸、FeEDTA、過酸化水素を除いた溶液を用いて測定した。 The reaction composition contains 40mM K-Pi buffer (pH 7.4), 0.26mM ascorbic acid, 0.15mM FeEDTA, 0.6mM hydrogen peroxide, 2mM salicylic acid and the target solutes such as citrulline and partially myristoylated chitosan. Was made into 400 mL, and a hydroxy radical was generated by an ascorbic acid-hydrogen peroxide system. After incubating at 25 ° C. for 90 minutes, hydroxylated salicylic acid was derivatized to develop color, and the absorbance was detected at 510 nm to detect radicals. For the control, purified water was added instead of the sample, and the blank was measured using a solution excluding ascorbic acid, FeEDTA, and hydrogen peroxide.
ヒドロキシラジカル消去率は、以下の計算式に従い、算出した。
ヒドロキシラジカル消去率 (%)=〔1−(試料OD−ブランクOD)/(コントロールOD−ブランクOD)〕×100
この式において、ODとは、510nm における吸光度を示す。生成したヒドロキシラジカルに対する消去率を表1及び図1に示す。
The hydroxy radical elimination rate was calculated according to the following formula.
Hydroxy radical scavenging rate (%) = [1- (sample OD−blank OD) / (control OD−blank OD)] × 100
In this formula, OD indicates absorbance at 510 nm. Table 1 and FIG. 1 show the erasure rate for the generated hydroxy radical.
表1及び図1からも明らかなように、シトルリンのみを含む溶液の比較例1−1では約17%の消去活性が得られ、部分ミリストイル化キトサンのみを含む溶液の比較例1−2では約3%の消去活性が得られた。これに対して、両方を含む溶液の場合は約28%の消去活性が得られた。この結果から、シトルリンと部分ミリストイル化キトサンとの双方を含む液の場合には、シトルリンのみを含む溶液の消去活性の数値と、部分ミリストイル化キトサンのみを含む溶液の消去活性の数値とを合算した以上の消去活性が得られることがわかった。このことから、シトルリンと部分ミリストイル化キトサンとの双方を含有させることで、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is clear from Table 1 and FIG. 1, about 17% scavenging activity was obtained in Comparative Example 1-1 of the solution containing only citrulline, and about Comparative Example 1-2 of the solution containing only partially myristoylated chitosan. An erase activity of 3% was obtained. In contrast, in the case of a solution containing both, an erasing activity of about 28% was obtained. From this result, in the case of a liquid containing both citrulline and partially myristoylated chitosan, the value of the erasing activity of the solution containing only citrulline and the value of the erasing activity of the solution containing only partially myristoylated chitosan were combined. It was found that the above erasing activity can be obtained. From this, it was confirmed that the inclusion of both citrulline and partially myristoylated chitosan synergistically increases the hydroxy radical scavenging activity in each case.
(実施例2)
本実施例では、シトルリンの濃度を2%とし、部分ミリストイル化キトサンピロリドンカルボン酸塩の濃度を0.05%とした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1と同じである。
そして、本実施例の活性酸素抑制用の組成物について、実施例1と同様にヒドロキシラジカル消去活性を測定した。一方、シトルリン2%のみを含む溶液(比較例2−1)、及び部分ミリストイル化キトサンピロリドンカルボン酸塩0.05%のみを含む溶液(比較例2−2)を調製し、同様にヒドロキシラジカル消去活性を測定して、上記混合物である本実施例の活性酸素抑制用の組成物と比較した。
(Example 2)
In this example, the concentration of citrulline was 2%, and the concentration of partially myristoylated chitosan pyrrolidone carboxylate was 0.05%. A composition for suppressing active oxygen was prepared by mixing the two, and the preparation method is the same as in Example 1.
And the hydroxy radical scavenging activity was measured like Example 1 about the composition for active oxygen suppression of a present Example. On the other hand, a solution containing only citrulline 2% (Comparative Example 2-1) and a solution containing only 0.05% partially myristoylated chitosan pyrrolidone carboxylate (Comparative Example 2-2) were prepared, and similarly, hydroxyl radical scavenging activity was obtained. It was measured and compared with the composition for suppressing active oxygen of the present example, which is the above mixture.
生成したヒドロキシラジカルに対する消去率を表2に示す。
表2からも明らかなように、シトルリンのみを含む溶液の比較例2−1では約22%の消去活性が得られ、部分ミリストイル化キトサンのみを含む溶液の比較例2−2では約29%の消去活性が得られた。これに対して、両方を含む溶液の場合は約57%の消去活性が得られた。この結果から、シトルリンと部分ミリストイル化キトサンピロリドンカルボン酸塩との双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is clear from Table 2, the scavenging activity of about 22% was obtained in Comparative Example 2-1 of a solution containing only citrulline, and about 29% of Comparative Example 2-2 of a solution containing only partially myristoylated chitosan. Erase activity was obtained. On the other hand, in the case of a solution containing both, an erasing activity of about 57% was obtained. From this result, in the case of the liquid containing both citrulline and the partially myristoylated chitosan pyrrolidone carboxylate, it was confirmed that the hydroxy radical scavenging activity in each case increased synergistically.
(実施例3)
本実施例では、キトサン誘導体として実施例2の部分ミリストイル化キトサンピロリドンカルボン酸塩に代えて、濃度0.1 %のカルボキシメチルキチンを用いた。シトルリンの濃度は実施例2と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1、2と同じである。
本実施例の活性酸素抑制用の組成物について、実施例1、2と同様にヒドロキシラジカル消去活性を測定し、シトルリン2%のみを含む溶液(比較例3−1)、及びカルボキシメチルキチン0.1 %のみを含む溶液(比較例3−2)のヒドロキシラジカル消去活性を同様に測定して本実施例の活性酸素抑制用の組成物と比較した。
(Example 3)
In this example, carboxymethyl chitin having a concentration of 0.1% was used as the chitosan derivative instead of the partially myristoylated chitosan pyrrolidone carboxylate of Example 2. The concentration of citrulline was the same as in Example 2. Both were mixed to prepare a composition for suppressing active oxygen. The preparation method was the same as in Examples 1 and 2.
About the composition for active oxygen suppression of a present Example, hydroxy radical scavenging activity was measured like Example 1, 2, and the solution (comparative example 3-1) containing only 2% of citrulline, and carboxymethyl chitin 0.1% The hydroxy radical scavenging activity of the solution containing only the salt (Comparative Example 3-2) was measured in the same manner and compared with the composition for suppressing active oxygen of this Example.
生成したヒドロキシラジカルに対する消去率を表3に示す。
表3からも明らかなように、比較例3−1では約22%の消去活性が得られ、比較例3−2では約8%の消去活性が得られた。これに対して、両方を含む溶液の場合は約35%の消去活性が得られた。この結果から、シトルリンとカルボキシメチルキチンとの双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As apparent from Table 3, about 22% of the erasing activity was obtained in Comparative Example 3-1, and about 8% of the erasing activity was obtained in Comparative Example 3-2. On the other hand, in the case of a solution containing both, an erasing activity of about 35% was obtained. From this result, in the case of a liquid containing both citrulline and carboxymethylchitin, it was confirmed that the hydroxy radical scavenging activity in each case increased synergistically.
(実験例4)
本実施例では、キトサン誘導体として実施例2の部分ミリストイル化キトサンピロリドンカルボン酸塩に代えて、濃度0.1 %のキトサンピロリドンカルボン酸塩を用いた。シトルリンの濃度は実施例2、3と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至3と同じである。
本実施例の活性酸素抑制用の組成物について、実施例1乃至3と同様にヒドロキシラジカル消去活性を測定し、シトルリン2%のみを含む溶液(比較例4−1)、及びキトサンピロリドンカルボン酸塩0.1 %のみを含む溶液(比較例4−2)のヒドロキシラジカル消去活性を同様に測定して本実施例の活性酸素抑制用の組成物と比較した。
(Experimental example 4)
In this example, a chitosan pyrrolidone carboxylate having a concentration of 0.1% was used in place of the partially myristoylated chitosan pyrrolidone carboxylate of Example 2 as a chitosan derivative. The concentration of citrulline was the same as in Examples 2 and 3. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 3.
For the composition for suppressing active oxygen of this example, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 3, and a solution containing only 2% citrulline (Comparative Example 4-1) and chitosan pyrrolidone carboxylate The hydroxy radical scavenging activity of a solution containing only 0.1% (Comparative Example 4-2) was measured in the same manner and compared with the composition for suppressing active oxygen of this Example.
生成したヒドロキシラジカルに対する消去率を表4に示す。
表4からも明らかなように、シトルリンのみを含む溶液の比較例4−1では約22%の消去活性が得られ、キトサンピロリドンカルボン酸塩のみを含む溶液の比較例4−2では約15%の消去活性が得られた。これに対して、両方を含む溶液の場合は約41%の消去活性が得られた。この結果から、シトルリンとキトサンピロリドンカルボン酸塩の双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is apparent from Table 4, about 22% of the scavenging activity was obtained in Comparative Example 4-1 of the solution containing only citrulline, and about 15% in Comparative Example 4-2 of the solution containing only chitosan pyrrolidone carboxylate. The erasing activity was obtained. On the other hand, in the case of a solution containing both, an erasing activity of about 41% was obtained. From this result, in the case of a liquid containing both citrulline and chitosan pyrrolidone carboxylate, it was confirmed that the hydroxy radical scavenging activity in each case increased synergistically.
(実験例5)
本実施例では、アミノ酸として実施例2のシトルリンに代えて濃度2%のテアニンを用いた。キトサン誘導体の種類、濃度は実施例2と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至4と同じである。
本実施例の活性酸素抑制用の組成物について、実施例1乃至4と同様にヒドロキシラジカル消去活性を測定し、テアニン2%のみを含む溶液(比較例5−1)、及び部分ミリストイル化キトサンピロリドンカルボン酸塩0.05%のみを含む溶液(比較例5−2)のヒドロキシラジカル消去活性を同様に測定して本実施例の活性酸素抑制用の組成物と比較した。
(Experimental example 5)
In this example, theanine having a concentration of 2% was used as an amino acid instead of citrulline of Example 2. The type and concentration of the chitosan derivative were the same as in Example 2. Both were mixed to prepare a composition for suppressing active oxygen. The preparation method was the same as in Examples 1 to 4.
For the composition for suppressing active oxygen of this example, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 4, and a solution containing only 2% of theanine (Comparative Example 5-1) and partially myristoylated chitosan pyrrolidone The hydroxy radical scavenging activity of a solution containing only 0.05% carboxylate (Comparative Example 5-2) was measured in the same manner and compared with the composition for suppressing active oxygen of this Example.
生成したヒドロキシラジカルに対する消去率を表5に示す。
表5からも明らかなように、比較例5−1では約20%の消去活性が得られ、比較例5−2では約28%の消去活性が得られた。これに対して、両方を含む溶液の場合は約56%の消去活性が得られた。この結果から、テアニンと部分ミリストイル化キトサンピロリドンカルボン酸塩の双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is clear from Table 5, about 20% erasing activity was obtained in Comparative Example 5-1, and about 28% erasing activity was obtained in Comparative Example 5-2. On the other hand, in the case of a solution containing both, an erasing activity of about 56% was obtained. From this result, in the case of a liquid containing both theanine and partially myristoylated chitosan pyrrolidone carboxylate, it was confirmed that the hydroxy radical scavenging activity in each case increased synergistically.
(実験例6)
本実施例では、アミノ酸として濃度2%のテアニンを用い、キトサン誘導体の種類、濃度は実施例3と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至5と同じである。
本実施例の活性酸素抑制用の組成物について、実施例1乃至5と同様にヒドロキシラジカル消去活性を測定し、テアニン2%のみを含む溶液(比較例6−1)、及びカルボキシメチルキチン0.1 %のみを含む溶液(比較例6−2)のヒドロキシラジカル消去活性を同様に測定して本実施例の活性酸素抑制用の組成物と比較した。
(Experimental example 6)
In this example, theanine having a concentration of 2% was used as the amino acid, and the type and concentration of the chitosan derivative were the same as those in Example 3. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 5.
About the active oxygen suppression composition of this example, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 5, and a solution containing only 2% of theanine (Comparative Example 6-1) and carboxymethyl chitin 0.1% The hydroxy radical scavenging activity of a solution containing only (Comparative Example 6-2) was measured in the same manner and compared with the composition for suppressing active oxygen of this Example.
生成したヒドロキシラジカルに対する消去率を表6に示す。
表6からも明らかなように、比較例6−1では約20%の消去活性が得られ、比較例6−2では約8%の消去活性が得られた。これに対して、両方を含む溶液の場合は約33%の消去活性が得られた。この結果から、テアニンとカルボキシメチルキチンの双方を含む液の場合には、それぞれのヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is clear from Table 6, about 20% erasing activity was obtained in Comparative Example 6-1 and about 8% erasing activity was obtained in Comparative Example 6-2. In contrast, in the case of a solution containing both, an erasing activity of about 33% was obtained. From this result, in the case of a liquid containing both theanine and carboxymethylchitin, it was confirmed that the scavenging activity of each hydroxy radical increased synergistically.
(実験例7)
本実施例では、アミノ酸として濃度2%のテアニンを用い、キトサン誘導体の種類、濃度は実施例4と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至5と同じである。
本実施例の活性酸素抑制用の組成物について、実施例1乃至6と同様にヒドロキシラジカル消去活性を測定し、テアニン2%のみを含む溶液(比較例7−1)、及びキトサンピロリドンカルボン酸塩0.1 %のみを含む溶液(比較例7−2)のヒドロキシラジカル消去活性を同様に測定して本実施例の活性酸素抑制用の組成物と比較した。
(Experimental example 7)
In this example, theanine having a concentration of 2% was used as the amino acid, and the type and concentration of the chitosan derivative were the same as those in Example 4. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 5.
About the composition for suppressing active oxygen of this example, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 6, and a solution containing only 2% theanine (Comparative Example 7-1) and chitosan pyrrolidone carboxylate The hydroxy radical scavenging activity of a solution containing only 0.1% (Comparative Example 7-2) was measured in the same manner and compared with the composition for suppressing active oxygen of this example.
生成したヒドロキシラジカルに対する消去率を表7に示す。
表7からも明らかなように、比較例7−1では約20%の消去活性が得られ、比較例7−2では約15%の消去活性が得られた。これに対して、両方を含む溶液の場合は約40%の消去活性が得られた。この結果から、テアニンとキトサンピロリドンカルボン酸塩の双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇することが確認できた。 As is clear from Table 7, about 20% erasing activity was obtained in Comparative Example 7-1 and about 15% erasing activity was obtained in Comparative Example 7-2. On the other hand, in the case of a solution containing both, an erasing activity of about 40% was obtained. From this result, in the case of a liquid containing both theanine and chitosan pyrrolidone carboxylate, it was confirmed that the hydroxy radical scavenging activity in each case increased synergistically.
(比較例8)
実施例2、5のアミノ酸に代えて、濃度2%のアルギニンを用いた。キトサン誘導体の種類、濃度は実施例2、5と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至7と同じである。
比較例8の組成物について、実施例1乃至7と同様にヒドロキシラジカル消去活性を測定し(比較例8−1)、アルギニン2%のみを含む溶液(比較例8−2)、及び部分ミリストイル化キトサンピロリドンカルボン酸塩0.05%のみを含む溶液(比較例8−3)のヒドロキシラジカル消去活性を同様に測定して比較した。
(Comparative Example 8)
Instead of the amino acids of Examples 2 and 5, arginine at a concentration of 2% was used. The type and concentration of the chitosan derivative were the same as in Examples 2 and 5. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 7.
For the composition of Comparative Example 8, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 7 (Comparative Example 8-1), a solution containing only 2% of arginine (Comparative Example 8-2), and partial myristoylation The hydroxy radical scavenging activity of a solution containing only 0.05% chitosan pyrrolidone carboxylate (Comparative Example 8-3) was similarly measured and compared.
生成したヒドロキシラジカルに対する消去率を表8に示す。
表8からも明らかなように、比較例8−2では約16%の消去活性が得られ、比較例8−3では約28%の消去活性が得られた。これに対して、両方を含む比較例8−1では約41%の消去活性が得られた。この結果から、アルギニンと部分ミリストイル化キトサンピロリドンカルボン酸塩の双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇しないことが確認できた。 As is apparent from Table 8, about 16% erasing activity was obtained in Comparative Example 8-2, and about 28% erasing activity was obtained in Comparative Example 8-3. On the other hand, about 41% of erasing activity was obtained in Comparative Example 8-1 including both. From this result, it was confirmed that in the case of a liquid containing both arginine and partially myristoylated chitosan pyrrolidone carboxylate, the hydroxy radical scavenging activity in each case does not increase synergistically.
(比較例9)
比較例8と同様のアルギニンを用い、キトサン誘導体の種類、濃度は実施例3、6と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至7と同じである。
比較例9の組成物について、実施例1乃至7と同様にヒドロキシラジカル消去活性を測定し(比較例9−1)、アルギニン2%のみを含む溶液(比較例9−2)、及びカルボキシメチルキチン0.1 %のみを含む溶液(比較例9−3)のヒドロキシラジカル消去活性を同様に測定して比較した。
(Comparative Example 9)
The same arginine as in Comparative Example 8 was used, and the types and concentrations of chitosan derivatives were the same as in Examples 3 and 6. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 7.
For the composition of Comparative Example 9, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 7 (Comparative Example 9-1), a solution containing only 2% of arginine (Comparative Example 9-2), and carboxymethyl chitin The hydroxy radical scavenging activity of a solution containing only 0.1% (Comparative Example 9-3) was similarly measured and compared.
生成したヒドロキシラジカルに対する消去率を表9に示す。
表9からも明らかなように、比較例9−2では約16%の消去活性が得られ、比較例9−3では約9%の消去活性が得られた。これに対して、両方を含む比較例9−1では約22%の消去活性が得られた。この結果から、アルギニンとカルボキシメチルキチンの双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇しないことが確認できた。 As is apparent from Table 9, about 16% of the erasing activity was obtained in Comparative Example 9-2, and about 9% of the erasing activity was obtained in Comparative Example 9-3. On the other hand, in Comparative Example 9-1 including both, an erasing activity of about 22% was obtained. From this result, it was confirmed that in the case of a liquid containing both arginine and carboxymethyl chitin, the hydroxy radical scavenging activity in each case alone does not increase synergistically.
(比較例10)
比較例8、9と同様のアルギニンを用い、キトサン誘導体の種類、濃度は実施例4、7と同じとした。この両者を混合して活性酸素抑制用の組成物を調製したが、その調製方法は実施例1乃至7と同じである。
比較例10の組成物について、実施例1乃至7と同様にヒドロキシラジカル消去活性を測定し(比較例10−1)、アルギニン2%のみを含む溶液(比較例10−2)、及びキトサンピロリドンカルボン酸塩0.1 %のみを含む溶液(比較例10−3)のヒドロキシラジカル消去活性を同様に測定して比較した。
(Comparative Example 10)
The same arginine as in Comparative Examples 8 and 9 was used, and the type and concentration of the chitosan derivative were the same as in Examples 4 and 7. Both were mixed to prepare a composition for suppressing active oxygen, and the preparation method was the same as in Examples 1 to 7.
For the composition of Comparative Example 10, the hydroxy radical scavenging activity was measured in the same manner as in Examples 1 to 7 (Comparative Example 10-1), a solution containing only 2% of arginine (Comparative Example 10-2), and chitosan pyrrolidone carboxylic The hydroxy radical scavenging activity of a solution containing only 0.1% of the acid salt (Comparative Example 10-3) was measured and compared in the same manner.
生成したヒドロキシラジカルに対する消去率を表10に示す。
表10からも明らかなように、比較例10−2では約16%の消去活性が得られ、比較例10−3では約15%の消去活性が得られた。これに対して、両方を含む比較例10−1では約28%の消去活性が得られた。この結果から、アルギニンとキトサンピロリドンカルボン酸塩の双方を含む液の場合には、それぞれ単独の場合におけるヒドロキシラジカルの消去活性が相乗的に上昇しないことが確認できた。 As is clear from Table 10, about 16% erasing activity was obtained in Comparative Example 10-2, and about 15% erasing activity was obtained in Comparative Example 10-3. On the other hand, in Comparative Example 10-1 including both, an erasing activity of about 28% was obtained. From this result, it was confirmed that in the case of a liquid containing both arginine and chitosan pyrrolidone carboxylate, the hydroxy radical scavenging activity in each case alone does not increase synergistically.
以上の実施例2乃至7及び比較例8乃至10の結果をまとめると次表11のとおりである。表11において、○は相乗効果が認められたこと、△は必ずしも相乗効果が認められなかったこと、△−○はほぼ相乗効果が認められたことを示す。
すなわち、表11に示すように、アミノ酸であるシトルリン若しくはテアニンと、キトサン若しくはキチン誘導体との混合物のヒドロキシラジカル消去活性は、それぞれ単独のヒドロキシラジカル消去活性に比べて相乗的に上昇したが、アルギニンとキトサン若しくはキチン誘導体との混合物のヒドロキシラジカル消去活性は、それぞれ単独のヒドロキシラジカル消去活性に比べて相乗的に上昇しなかった。 That is, as shown in Table 11, the hydroxy radical scavenging activity of a mixture of the amino acids citrulline or theanine and chitosan or chitin derivative increased synergistically compared to the single hydroxy radical scavenging activity, but arginine and The hydroxy radical scavenging activity of the mixture with chitosan or chitin derivative did not increase synergistically compared to the single hydroxy radical scavenging activity.
(実施例8)
本実施例では、アミノ酸とキトサン又はキチン誘導体との混合物のDNA損傷抑制作用について試験した。被検物質である濃度0.2 %のシトルリン及び濃度0.05%の部分ミリストイル化カルボキシメチルキトサン、20mM Tris-Hcl(pH7.4)、80ng/μL のpUC18プラスミドDNA、2mM 50μM EDTA−Na−Fe(III) 、8mM過酸化水素となるように全量が20uLの溶液を調整し、25℃で保温した。2時間後、反応溶液にゲルローディングバッファー(0.25%BPB,1mMEDTA,30%グリセロール)を1/2 当量加え、エチジウムブロマイド含有1%アガロースゲル、及びTBE(×0.5)を用いて電気泳動した。泳動後、アガロースゲルに紫外線照射を行い、泳動結果の写真を撮影した。各バンドの大きさはATTO Lanc & Spot Analyzer により定量した。
(Example 8)
In this example, a DNA damage inhibitory action of a mixture of an amino acid and chitosan or chitin derivative was tested. The test substances 0.2% citrulline and 0.05% partially myristoylated carboxymethyl chitosan, 20 mM Tris-Hcl (pH 7.4), 80 ng / μL pUC18 plasmid DNA, 2 mM 50 μM EDTA-Na-Fe (III) A solution with a total amount of 20 uL was adjusted to 8 mM hydrogen peroxide and kept at 25 ° C. After 2 hours, 1/2 equivalent of gel loading buffer (0.25% BPB, 1 mM EDTA, 30% glycerol) was added to the reaction solution, and electrophoresis was performed using 1% agarose gel containing ethidium bromide and TBE (× 0.5). After the electrophoresis, the agarose gel was irradiated with ultraviolet rays, and a photograph of the electrophoresis result was taken. The size of each band was quantified by ATTO Lanc & Spot Analyzer.
一方、被検物質を加えない状態で同様に電気泳動を行い、泳動結果の写真を撮影し、各バンドの大きさを同様に定量した。被検物質を加えた場合と加えない場合の定量値から、DNA損傷の抑制率を求めた。抑制率は次式で示される。
抑制率(%)=〔1−(被検物質を加えた場合のバンドの定量値)/(被検物質を加え ない場合のバンドの定量値)〕×100
On the other hand, electrophoresis was performed in the same manner without adding a test substance, a photograph of the electrophoresis result was taken, and the size of each band was similarly quantified. The inhibition rate of DNA damage was determined from the quantitative values when the test substance was added and when it was not added. The inhibition rate is expressed by the following equation.
Inhibition rate (%) = [1- (quantitative value of the band when the test substance is added) / (quantitative value of the band when the test substance is not added)] × 100
この抑制率を求めることで、過酸化水素から発生するヒドロキシラジカルによってpUC18プラスミドDNAが損傷される程度が、上記被検物質によりどの程度抑制されるかが確認できる。結果を表12に示す。 By obtaining this inhibition rate, it can be confirmed how much the degree to which the pUC18 plasmid DNA is damaged by hydroxy radicals generated from hydrogen peroxide is inhibited by the test substance. The results are shown in Table 12.
(実施例9)
本実施例では、被検物質として濃度0.2 %のテアニン及び濃度0.05%のカルボキシメチルキチンの混合物を用いた。実施例8と同様の方法でDNA損傷抑制作用について試験した。本実施例では、表12に示すように抑制率は45%であり、実施例8と同様に非常に優れた結果が得られた。
Example 9
In this example, a mixture of 0.2% concentration of theanine and 0.05% concentration of carboxymethyl chitin was used as the test substance. A DNA damage inhibitory action was tested in the same manner as in Example 8. In this example, as shown in Table 12, the inhibition rate was 45%, and as in Example 8, a very excellent result was obtained.
(比較例11)
被検物質として濃度0.2%のシトルリンを用いた。実施例8、9と同様の方法でDNA損傷抑制作用について試験した。比較例11では、表12に示すように抑制率は16%であり、ある程度の抑制作用は認められたが、実施例8に比べると抑制率は劣っていた。
(Comparative Example 11 )
Citrulline having a concentration of 0.2% was used as a test substance. The DNA damage inhibitory action was tested in the same manner as in Examples 8 and 9. In Comparative Example 11 , as shown in Table 12, the inhibition rate was 16%, and a certain degree of inhibition was observed, but the inhibition rate was inferior compared to Example 8.
(比較例12)
被検物質として濃度0.2%のテアニンを用いた。実施例8、9と同様の方法でDNA損傷抑制作用について試験した。比較例12では、表12に示すように抑制率は29%であり、ある程度の抑制作用は認められたが、実施例9に比べると抑制率は劣っていた。
(Comparative Example 12 )
Theanine having a concentration of 0.2% was used as a test substance. The DNA damage inhibitory action was tested in the same manner as in Examples 8 and 9. In Comparative Example 12 , the inhibition rate was 29% as shown in Table 12, and a certain degree of inhibition was observed, but the inhibition rate was inferior to Example 9.
(比較例13)
被検物質として濃度0.2%のシステインを用いた。実施例8、9と同様の方法でDNA損傷抑制作用について試験した。比較例13では、表12に示すように抑制率は5%以下であり、抑制作用はほとんど認められなかった。
(Comparative Example 13 )
Cysteine having a concentration of 0.2% was used as a test substance. The DNA damage inhibitory action was tested in the same manner as in Examples 8 and 9. In Comparative Example 13 , as shown in Table 12, the inhibition rate was 5% or less, and almost no inhibition effect was observed.
(比較例14)
被検物質として濃度0.2%のアルギニンを用いた。実施例8、9と同様の方法でDNA損傷抑制作用について試験した。比較例14では、表12に示すように抑制率は5%以下であり、抑制作用はほとんど認められなかった。
(Comparative Example 14 )
Arginine with a concentration of 0.2% was used as a test substance. The DNA damage inhibitory action was tested in the same manner as in Examples 8 and 9. In Comparative Example 14 , as shown in Table 12, the inhibition rate was 5% or less, and almost no inhibition effect was observed.
(処方例1)
本処方例は、化粧料の一例としてのエッセンスの処方例であり、その組成は次のとおりである。
成分 配合量(質量%)
(1) シトルリン 2.0
(2) 部分ミリストイル化キトサンピロリドン
カルボン酸塩1%水溶液(製品名:PM−キトサン) 20.0
(3) アセチルシトルリン 0.5
(4) グリセリン 10.0
(5) ヒドロキシエチルセルロース 0.5
(6) メチルパラベン 0.1
(7) 精製水 残量
(Prescription Example 1)
This prescription example is a prescription example of an essence as an example of a cosmetic, and its composition is as follows.
Ingredient Amount (% by mass)
(1) Citrulline 2.0
(2) Partially myristoylated chitosan pyrrolidone carboxylate 1% aqueous solution (Product name: PM-chitosan) 20.0
(3) Acetylcitrulline 0.5
(4) Glycerin 10.0
(5) Hydroxyethyl cellulose 0.5
(6) Methylparaben 0.1
(7) Purified water remaining
(1) 〜(3) の成分を均一に加熱処理し、超音波処理又は高圧乳化処理したものを凍結乾燥させることにより複合化させる。この複合化したものを、(4) 〜(7) の成分を混合、攪拌した攪拌溶解物に徐々に添加、溶解することにより、エッセンスを調製した。 The components (1) to (3) are uniformly heat-treated, and are subjected to sonication or high-pressure emulsification treatment to be lyophilized to be combined. Essence was prepared by gradually adding and dissolving this complexed product into the stirred and melted product obtained by mixing and stirring the components (4) to (7).
(処方例2)
本処方例は、化粧料の一例としてのローションの処方例であり、その組成は次のとおりである。
成分 配合量(質量%)
(1) テアニン 1.5
(2) アセチルヒスチジン 0.5
(3) カルボキシメチルキトサン 0.2
(4) 部分ミリストイル化キトサンピロリドン
カルボン酸塩1%水溶液(製品名:PM−キトサン) 10.0
(5) ブチレングリコール 10.0
(6) グリセリン 5.0
(7) クエン酸 0.01
(8) メチルパラベン 0.2
(9) 精製水 残量
(Prescription example 2)
This formulation example is a formulation example of a lotion as an example of cosmetics, and the composition thereof is as follows.
Ingredient Amount (% by mass)
(1) Theanine 1.5
(2) Acetylhistidine 0.5
(3) Carboxymethyl chitosan 0.2
(4) Partially myristoylated chitosan pyrrolidone carboxylate 1% aqueous solution (Product name: PM-chitosan) 10.0
(5) Butylene glycol 10.0
(6) Glycerin 5.0
(7) Citric acid 0.01
(8) Methylparaben 0.2
(9) Purified water remaining
(1) 〜(4) の成分を均一に加熱処理し、超音波処理又は高圧乳化処理したものを凍結乾燥させることにより複合化させる。この複合化したものを、(5) 〜(9) の成分を混合、攪拌した攪拌溶解物に徐々に添加、溶解することにより、ローションを調製した。 The components (1) to (4) are uniformly heat-treated, and are subjected to sonication or high-pressure emulsification treatment to be lyophilized to be combined. A lotion was prepared by gradually adding and dissolving this complexed product to the stirred and melted product obtained by mixing and stirring the components (5) to (9).
(処方例3)
本処方例は、化粧料の一例としての乳化ファンデーションの処方例であり、その組成は次のとおりである。
成分 配合量(質量%)
(1) 流動パラフィン 2.0
(2) ミリスチン酸イソプロピル 3.0
(3) スクワラン 7.0
(4) ワセリン 2.0
(5) セタノール 1.5
(6) 部分ミリストイル化キトサン乳酸塩 0.2
(7) キトサンピロリドンカルボン酸塩 0.1
(8) N−ジカルボキシメチルキトサン 0.1
(9) 黄酸化鉄 4.0
(10)タルク 5.5
(11)酸化チタン 5.0
(12)シトルリン 0.5
(13)テアニン 1.0
(14)グリセリン 5.0
(15)メチルパラベン 0.1
(16)精製水 残量
(Prescription Example 3)
This prescription example is a prescription example of an emulsified foundation as an example of a cosmetic, and its composition is as follows.
Ingredient Amount (% by mass)
(1) Liquid paraffin 2.0
(2) Isopropyl myristate 3.0
(3) Squalane 7.0
(4) Petrolatum 2.0
(5) Cetanol 1.5
(6) Partially myristoylated chitosan lactate 0.2
(7) Chitosan pyrrolidone carboxylate 0.1
(8) N-dicarboxymethyl chitosan 0.1
(9) Yellow iron oxide 4.0
(10) Talc 5.5
(11) Titanium oxide 5.0
(12) Citrulline 0.5
(13) Theanine 1.0
(14) Glycerin 5.0
(15) Methylparaben 0.1
(16) Remaining purified water
加熱処理した上記(6) 〜(13)の成分に、(1) 〜(5) の成分を添加し、ホモミキサー処理(8500rpm,20分処理)による乳化により乳化ファンデーションを調製した。 The components (1) to (5) were added to the heat-treated components (6) to (13), and an emulsion foundation was prepared by emulsification by homomixer treatment (8500 rpm, treatment for 20 minutes).
(処方例4)
本処方例は、化粧料の一例としての乳化マスカラの処方例であり、その組成は次のとおりである。
成分 配合量(質量%)
(1) 流動パラフィン 5.0
(2) ミツロウ 15.0
(3) ベヘニルアルコール 1.0
(4) ワセリン 2.0
(5) セタノール 1.5
(6) シトルリン 1.0
(7) 部分ミリストイル化キトサン
ピロリドンカルボン酸塩1%水溶液
(製品名:PM−キトサン〔ピアス株式会社製〕) 20.0
(8) グリセリン 1.0
(9) メチルパラベン 0.2
(10)黒酸化鉄 5.0
(11)タルク 2.5
(12)微粒子酸化チタン 1.0
(13)ナイロン末 4.0
(14)ヒドロキシエチルセルロース 0.5
(15)精製水 残量
(Prescription Example 4)
This formulation example is a formulation example of an emulsified mascara as an example of a cosmetic, and the composition thereof is as follows.
Ingredient Amount (% by mass)
(1) Liquid paraffin 5.0
(2) Beeswax 15.0
(3) Behenyl alcohol 1.0
(4) Petrolatum 2.0
(5) Cetanol 1.5
(6) Citrulline 1.0
(7) Partially myristoylated chitosan pyrrolidone carboxylate 1% aqueous solution (Product name: PM-chitosan [Pierce Co., Ltd.]) 20.0
(8) Glycerin 1.0
(9) Methylparaben 0.2
(10) Black iron oxide 5.0
(11) Talc 2.5
(12) Fine particle titanium oxide 1.0
(13) Nylon powder 4.0
(14) Hydroxyethyl cellulose 0.5
(15) Purified water remaining
加熱処理した上記(6) 〜(15)の成分に、加熱溶解させた上記(1) 〜(5) の油相組成のも+−のを添加し、ホモミキサー処理による乳化によりpH4.8 の弱酸性の耐水性O/W型エマルションを調製した。 To the heat-treated components (6) to (15), the oil phase composition (+) of (1) to (5) dissolved by heating is added, and the mixture is adjusted to pH 4.8 by emulsification by homomixer treatment. A weakly acidic water-resistant O / W emulsion was prepared.
本発明の組成物は、活性酸素抑制用、特にシワ、シミ等の皮膚老化や、肌荒れ、炎症性ニキビ等の皮膚細胞損傷への影響性が高いヒドロキシラジカルを抑制する皮膚膚外用剤、化粧料に広く適用することができる。 The composition of the present invention is an external skin skin preparation for suppressing active oxygen, especially a skin skin aging agent such as wrinkles and stains, and a skin radical agent that suppresses hydroxy radicals having a high effect on skin cell damage such as rough skin and inflammatory acne. Can be widely applied to.
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JP4954531B2 (en) * | 2005-10-31 | 2012-06-20 | 一丸ファルコス株式会社 | Peroxisome proliferator-responsive receptor activator |
JP4981327B2 (en) * | 2006-02-14 | 2012-07-18 | 焼津水産化学工業株式会社 | UV absorber |
US8119780B2 (en) | 2006-06-02 | 2012-02-21 | Synedgen, Inc. | Chitosan-derivative compounds and methods of controlling microbial populations |
FR2913885B1 (en) * | 2007-03-22 | 2012-07-20 | Univ Paris Descartes | USE OF CITRULLINE FOR THE TREATMENT OF PATHOLOGIES ASSOCIATED WITH INCREASED CARBONYLATION OF PROTEINS |
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JP2010006724A (en) * | 2008-06-25 | 2010-01-14 | Hoyu Co Ltd | Hair strengthening agent, hair cosmetic composition containing the hair strengthening agent and hair strengthening method using the same |
JP2010202574A (en) * | 2009-03-03 | 2010-09-16 | Hamamatsu Photonics Kk | Production-enhancing agent for active oxygen species |
KR101698436B1 (en) * | 2010-08-11 | 2017-01-20 | (주)아모레퍼시픽 | Theanine derivatives, preparation method thereof and composition of the skin external application for anti-acne containing the same |
KR101346661B1 (en) * | 2010-11-15 | 2014-02-06 | 부경대학교 산학협력단 | Cosmetic composition for preventing skin aging comprising chitooligosaccharides |
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KR102529681B1 (en) * | 2020-11-10 | 2023-05-08 | (주)인코돈바이오코스메틱 | Cosmetic composition for improving acne of skin comprising glutathionyl genistein |
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