WO1997020539A1 - Cosmetic preparation - Google Patents
Cosmetic preparation Download PDFInfo
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- WO1997020539A1 WO1997020539A1 PCT/JP1996/003571 JP9603571W WO9720539A1 WO 1997020539 A1 WO1997020539 A1 WO 1997020539A1 JP 9603571 W JP9603571 W JP 9603571W WO 9720539 A1 WO9720539 A1 WO 9720539A1
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- Prior art keywords
- skin
- sod
- zinc
- manganese
- bacteria
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/27—Zinc; Compounds thereof
Definitions
- the present invention relates to a cosmetic composition, and more specifically, to maintain and enhance a healthy flora resident in the skin, and to provide a suboxyxide dismutase (SOD) -like active substance secreted by the bacteria resident in the skin.
- SOD suboxyxide dismutase
- SOD derived from natural products purified from animals, plants, and microorganisms, or SOD derived from DNA recombinants are not suitable for quality control, such as storage stability in pharmaceuticals, from the viewpoint of production cost and storage stability in pharmaceuticals. Had problems.
- the purpose of the present invention is to grow most of the SOD or SOD-like active substance on the skin of healthy people instead of directly blending it into cosmetics so that the SOD or SOD-like active substance can always be stably expressed on the skin It.
- a cosmetic composition comprising at least one of a metal ion, a metal salt and a metal substance.
- the action of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and the function of producing and secreting Superoxide jism Cosmetic composition containing trace amounts of at least one of manganese and zinc, which are metal ions, and salts thereof, which have an effect of enhancing SOD-like active substances Is provided.
- Figure 1 shows the effect of metal ions (zinc and manganese) on the development of Staphylococcus epidermides.
- Figure 2 shows the effect of metal ions (zinc and manganese) on the growth of Staphylococcus aureus.
- the present invention has conducted intensive studies to increase the extracellular SOD-like specific activity of Staphylococcus' epidermides, and to reduce the metal ions manganese and zinc even in poor nutrient media.
- the present inventors have found that by adding the same, the same bacterium can be grown in a short time and the extracellular SOD-like active substance can be maintained for a long period of time, and the present invention has been accomplished.
- the present invention is a cosmetic containing a substance capable of growing Staphylococcus epidermides resident on the skin in a short period of time and increasing the extracellular SOD-like specific activity.
- this cosmetic is a highly stable and safe cosmetic that achieves skin conditioning by normalizing the skin flora and enhancing the activity of its extracellular secretory SOD-like active substance.
- the Staph I Loco Tsu dregs-E Biderumi death is established growth on the surface of the skin due to the high antioxidant capacity, and secrete the SOD-like active substances in the cells outside the body, the 0 2 on the skin caused Ri by such as on the results of ultraviolet rays It is thought to play a role in eliminating skin injuries by erasing, increasing skin health, and preventing other bacterial infections. In fact, skin with injuries such as atopic dermatitis (rough skin) or dry skin loses the balance of the bacterial bacterial flora and belongs to the genus Staphylococcus aureus. Harmful bacteria are found at a high rate. S.
- phylococcus as the dominant bacterium ⁇ Epidermides inhibits the growth of the above harmful bacteria, so that a slight skin injury can cause selective growth of Staphylococcus epidermides.
- Providing a cosmetic composition that can be used to reduce the number of pathogenic bacteria and improve skin damage can be expected.
- metal ions for example, Mn, Zn2 +
- metal salts for example, manganese chloride, zinc chloride, zinc oxide, zinc stearate, paraffin
- the cosmetic according to the second aspect of the present invention has an effect of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and producing and secreting the cells.
- One of the metal ions manganese and zinc or their salts (eg, manganese chloride and zinc oxide) that have the effect of enhancing superoxide 'dismutase (SOD) -like active substances In trace amounts.
- the manganese ion preferably has a concentration in the range of 0.1 to 100 mM, more preferably 0.1 to 10 mM.
- the zinc ion preferably has a concentration in the range of 0.01 to 5 mM, and more preferably 0.1 to 1 mM.
- the microorganism used to prove the effectiveness of the present invention was Staphylococcus epider, a representative resident bacterium inhabiting the skin of healthy humans, as used in the following examples.
- a force that is a mide or a skin Staphylococcus ⁇ P. aureus is a representative of skin harmful bacteria.
- the bacterium used to carry out the present invention can be used irrespective of any bacterial species and strains, including those that can pass through the skin.
- the minimum growth medium used in the present invention is defined as a medium to which a minimum concentration of metal capable of growing bacteria is added.
- the method of culturing bacteria used to prove the effectiveness of the present invention is as follows: using the above strains and medium, the bacteria are grown under aerobic conditions at 37 ° C under a rotary culture (120 rpm). ).
- the sample preparation method used to prove the effectiveness of the present invention is as follows. First, bacteria sufficiently grown in the above medium were separated into bacterial cells and culture supernatant at 4 ° C for 10 minutes using a cooling high-speed centrifuge. Subsequently, the SOD activity of the culture supernatant was measured and concentrated as follows. Add cold acetate to the culture supernatant to a final concentration of 75%, leave it in a freezer at 120 ° C, separate and collect the formed precipitate with a cooling centrifuge, and dissolve in a small amount of purified water Then, freeze drying was performed to obtain an acetone powder sample. The powder was dissolved in purified water and used as an extracellular secretory component sample.
- NBT Nitrole tetrazolium
- SOD activity is determined from this inhibition rate. Using this method, SOD-like active substances secreted outside the cells can be measured with high sensitivity by acetonitrile concentration treatment of the culture supernatant sample.
- the basal medium used in the series of experiments was Davis's medium (sinium sodium hydrogen phosphate decahydrate, 8 g; potassium dihydrogen phosphate, 2 g; sodium citrate, 0.5 g). g; magnesium sulfate heptahydrate, 0.1 g; ammonium sulfate, 1 g; glucose, 0.5 g / liter of distilled water (pH 7)) plus 0.1% of yeast extract and 1% of carbonated calcium was used.
- the inoculum was set at 1 ⁇ 10 5 cells / ml, and the culture was performed in a thermostat at 37 ° C.
- the metals used in the experiments were copper sulfate, zinc sulfate, manganese chloride, zinc oxide, and ferrous sulfate.
- the concentrations were 100 / M, 1 mM, and 10 mM, respectively, and were added before the start of culture. After culturing for 24 hours, the culture solution was applied to a standard agar plate, and the number of viable cells was counted by a standard method. The results are shown in Table 1.
- Table 1 Development of Staphylococcal 'Epidermides'
- Example 1 the SOD-like active substance in the culture supernatant of Staphylococcus aureus + Ipidermides was measured.As a result, the experimental group to which zinc sulfate and manganese chloride were added showed higher values than the control group.
- the harmful pathogen Staphylococcus aureus differs from the useful resident bacterium Staphylococcus epidermides by adding zinc oxide and chloride. No particular promotion of growth was observed with cancer, but rather a tendency toward inhibition.
- Sample preparation method For sample preparation, culture was performed on a 100 ml flask at the scale of a 50 ml culture solution, and 5 ml was sampled each hour. The culture was immediately centrifuged (10,000 rpm, 4 ° C, 10 minutes) to separate cells and supernatant. Cool the collected supernatant with ice, add acetone at 20 ° C with stirring to a final concentration of 75%, and leave the mixture in ice for 2 hours. Thereafter, the precipitate was collected by centrifugation again (10, OOOrpnu 4 ° C, 10 minutes). Dissolve the precipitate in a small amount of purified water, freeze at 180 ° C and freeze-dry to remove acetate.
- Protein quantification method Lowry method was used for quantification of protein. .
- a 0.4 ml sample containing 10 to 100 g of protein is placed in a test tube, and an alkaline copper solution (1 ml of 1% copper sulfate and 1 ml of 2% rossiel salt is mixed with 2% carbonic acid) Sodium, added to 100 ml of 0.1 N sodium hydroxide)
- Add 0.2 ml of 1 N phenol reagent stir, and leave at room temperature for 30 minutes. Thirty minutes later, colorimetric determination was performed at 750 nm.
- Bovine serum albumin (BSA, SIGMA) was used as the standard solution.
- Measuring method of SOD-like active substance Take 50 /] sample in a test tube, dissolve X0D (xanthine oxidase) solution in 0.5M ammonium sulfate, 1 mM EDTA-2Na solution at 20 ° C Dissolve immediately before use, and dilute so that the change in 0D560nm per minute is 0.0185), add 50/1, and keep in a 25 ° C water bath for 10 minutes without stirring. After heating to 25 ° C, solution A (0.
- Sample Nos. 1 and 2 are manganese / zinc combination and calcium carbonate Sample No. 3 was prepared as a control to examine the irritation properties of Pem. As a result of the judgment by the naked eye, there was no red spot or irritation, and no problem was found in safety.
- Lipid peroxide was quantified by the TBA (thiobarbituric acid) method shown below.
- 0.1 ml was taken out from the sample solution, 2.0 ml of TBA reagent (0.2% tiobarbituric acid, 5.0% acetic acid pH3.5) was added, heated at 100 ° C for 60 minutes, and then cooled with water.
- the final metabolite MDA malonaldehyde
- the cosmetics of the present invention containing appropriate amounts of the metal ions zinc and manganese, the healthy growth of useful normal flora of skin and the secretion of SOD-like active substances from the cells are promoted, and the growth of skin harmful bacteria It is expected to be effective in preventing skin cells, preventing active oxygen damage to skin cells, and preventing skin cell aging.
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Abstract
A cosmetic preparation which contains zinc ions and/or manganese ions that are an ingredient serving as a nourishment for useful indigenous dermal bacteria and accelerating the secretion of active substances functioning like SODs derived from indigenous bacteria, is harmless or less harmful to the skin, and functions to indirectly inhibit skin oxidation.
Description
明 細 書 化粧料組成物 技術分野 Description Cosmetic composition Technical field
本発明は化粧料組成物に関し、 更に詳し く は、 健全な皮膚常在菌 叢の維持 · 増進をはかり、 その皮膚常在細菌が分泌するス—バーオ キサイ ド . ジスムターゼ(SOD) 様活性物質を高めるこ とによ り皮膚 に大敵である皮膚表面の活性酸素を分解除去し、 整肌、 皮膚老化防 止に役立つ化粧料組成物、 特に皮膚外用剤と しての利用が期待され る化粧料組成物に関する。 背景技術 The present invention relates to a cosmetic composition, and more specifically, to maintain and enhance a healthy flora resident in the skin, and to provide a suboxyxide dismutase (SOD) -like active substance secreted by the bacteria resident in the skin. By increasing the amount, it decomposes and removes active oxygen on the surface of the skin, which is a great enemy of the skin, and is useful for skin conditioning and preventing skin aging, especially cosmetics expected to be used as external preparations for skin Composition. Background art
生体にとって有害な活性酸素は 0 2 を代謝系に用いる生物に於い て恒常的に産生されるが、 活性酸素の一種であるスーパーォキサイ ドア二オン ( 0 2 ― ) の不均化反応を触媒する SOD は代表的な抗酸 化酵素であり、 この酵素活性により細胞はその酸素傷害から守られ ている といわれる。 皮膚の健康科学においても、 この活性酸素が太 陽光紫外線による皮膚脂質酸化に介在し、 皮膚老化の加速、 また、 肌の ト ラブルへの関与が注目され、 SOD 活性物質の化粧料、 或いは 皮膚外用剤への応用が提案されてきた (例えば特開平 1 - 96107 号 公報参照) 。 Although harmful active oxygen to living organisms is produced constitutively produced at the organism using 0 2 in the metabolic system, Super O wherein the door two on a kind of active oxygen - disproportionation of (0 2) Catalytic SOD is a typical antioxidant enzyme, and it is said that this enzyme activity protects cells from oxygen damage. In the skin health science, this active oxygen mediates the oxidation of skin lipids by solar ultraviolet rays, accelerates skin aging, and is also concerned with the involvement of skin troubles. Application to agents has been proposed (see, for example, JP-A-1-96107).
しかし、 動物、 あるいは、 植物、 微生物から精製される天然物由 来 SOD 、 あるいは、 DNA 組替体由来 SOD は、 製造コス 卜の上から、 また、 製剤の中での保存安定性など品質管理上からも問題を残して いた。 However, SOD derived from natural products purified from animals, plants, and microorganisms, or SOD derived from DNA recombinants are not suitable for quality control, such as storage stability in pharmaceuticals, from the viewpoint of production cost and storage stability in pharmaceuticals. Had problems.
最近、 皮膚表面に生息する皮膚常在細菌で好気的に生育する菌は
酸素に強く 、 また、 直接太陽の紫外線をう けるこ とから活性酸素に 暴露される こ とが多 く 高い抗酸化能をもつているこ とが示唆されて いる。 実際、 代表的好気性常在細菌で最優勢菌スタフ イ ロコ ッカス • ェピデル ミ デスは、 その培養液、 或いは、 その培養菌体破砕物が 抗酸化性を示すこ とから、 それらを配合する化粧品、 外用剤の提案 もなされている (例えば特開平 6 - 271851号公報参照) 。 しかし、 製剤と しての不安定性において問題が残っていた。 発明の開示 Recently, bacteria that grow aerobically among the skin-resident bacteria that live on the skin surface It is strongly resistant to oxygen, and it is suggested that it is highly exposed to active oxygen because it is directly exposed to the sun's ultraviolet rays and has high antioxidant ability. In fact, Staphylococcus epidermides, the most predominant aerobic resident bacterium, is used in cosmetics containing the culture fluid or the crushed bacterial cells that exhibit antioxidant properties. External preparations have also been proposed (see, for example, JP-A-6-271851). However, problems remained with the instability of the formulation. Disclosure of the invention
前述のごと く 、 SOD 又は培養抗酸化性物質を化粧料に直接配合す ると、 臭いや色さ らに使用時の SOD 活性の保存安定性など品質管理 上の問題解決が課題と して残っていた。 このよう に SOD および抗酸 化物質の整肌作用に永年注目 されてきたにもかかわらず、 化粧料へ の配合応用はまだ解決すべき点が多 く残されている。 As mentioned above, if SOD or cultured antioxidants are directly incorporated into cosmetics, the problem of quality control, such as the odor and color, and the storage stability of SOD activity during use remains to be solved. I was Although SOD and antioxidants have long paid attention to skin conditioning, there are still many issues to be solved in their application to cosmetics.
本発明の目的は、 SOD 又は SOD 様活性物質を常に皮 Itの上で安定 的に発現確保できるよう、 直接化粧料に配合するのではな く 、 健常 人の皮) 上に最も多く生育している有用な皮膚常在菌の SOD 活性を 効率よ く 分泌させる作用物質を配合するこ とにより、 皮膚の老化防 止効果及び整肌作用を示す安価で安全な皮膚抗酸化性化粧料を提供 するこ とにある。 The purpose of the present invention is to grow most of the SOD or SOD-like active substance on the skin of healthy people instead of directly blending it into cosmetics so that the SOD or SOD-like active substance can always be stably expressed on the skin It. Provide an inexpensive and safe skin antioxidant cosmetic that exhibits an effective anti-aging effect on the skin and a skin-regulating effect by incorporating an active substance that efficiently secretes the SOD activity of useful bacteria resident in the skin. It is here.
本発明に従えば、 金属イオン、 金属塩及び金属物質のうちの少な く と も一種を含んでなる化粧料組成物が提供される。 According to the present invention, there is provided a cosmetic composition comprising at least one of a metal ion, a metal salt and a metal substance.
本発明に従えば、 また、 皮膚有用常在細菌叢の好気性最優勢菌ス タフ イ ロコ ッ カス · ェピデル ミ デスの発育を増進する作用と、 当該 菌体が生産分泌するスー パーォキサイ ド · ジスム夕一ゼ(SOD) 様活 性物質を高める作用を有する、 金属イオンであるマンガン及び亜鉛 並びにそれらの塩の少な く と も一種を微量含んでなる化粧料組成物
が提供される。 図面の簡単な説明 According to the present invention, the action of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and the function of producing and secreting Superoxide jism Cosmetic composition containing trace amounts of at least one of manganese and zinc, which are metal ions, and salts thereof, which have an effect of enhancing SOD-like active substances Is provided. BRIEF DESCRIPTION OF THE FIGURES
以下、 図面を参照して本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail with reference to the drawings.
図 1 は、 スタフ イ ロ コ ッ カス · ェピデルミ デスの発育に及ぼす金 属イオン (亜鉛とマンガン) の影響を示す図である。 Figure 1 shows the effect of metal ions (zinc and manganese) on the development of Staphylococcus epidermides.
図 2 は、 スタフ ィ ロコ ッ カス · ァゥ レウスの発育に及ぼす金属ィ オン (亜鉛とマンガン) の影響を示す図である。 発明を実施するための最良の形態 Figure 2 shows the effect of metal ions (zinc and manganese) on the growth of Staphylococcus aureus. BEST MODE FOR CARRYING OUT THE INVENTION
本発明は上記問題を解決するために鋭意研究を重ね、 スタフ イ ロ コ ッ カス ' ェピデルミ デスの菌体外 SOD 様比活性を高め、 また貧栄 養培地においても金属イオンであるマンガンと亜鉛を添加するこ と により同菌を短時間で増殖させ、 菌体外 SOD 様活性物質を長期間維 持させるこ とができる こ とを見出 し本発明をするに至った。 In order to solve the above-mentioned problems, the present invention has conducted intensive studies to increase the extracellular SOD-like specific activity of Staphylococcus' epidermides, and to reduce the metal ions manganese and zinc even in poor nutrient media. The present inventors have found that by adding the same, the same bacterium can be grown in a short time and the extracellular SOD-like active substance can be maintained for a long period of time, and the present invention has been accomplished.
すなわち、 本発明は皮膚上に常在するスタフ イ ロ コ ッカス ' ェピ デルミ デスを短期間で増殖させ、 菌体外 SOD 様比活性を高めるこ と のできる物質を配合した化粧料である。 また、 この化粧料は、 皮膚 細菌叢の正常化及びその菌体外分泌 SOD 様活性物質の活性を高める こ とにより整肌を達成する安定性の高い、 安全な化粧料である。 特に、 健康なヒ トの皮膚の上で好気性菌と して最優勢のものは、 ス夕フ イ ロコ ッカス . ェピデルミ デスで 1 X 1 0 4 個 Z cm 2 程度で安 定的常在皮膚細菌叢を形成している。 このスタフ ィ ロコ ッ カス · ェ ビデルミ デスは高い抗酸化能により皮膚表面に定着増殖し、 SOD 様 活性物質を菌体外に分泌し、 その結果紫外線などによ り生じた皮膚 上の 0 2 を消去して皮膚傷害を防止し、 皮膚の健康度を増し、 他 細菌の感染を防止する役割などを果た していると考えられている。
実際、 ア ト ピー性皮膚炎等の傷害をもつ皮膚 (荒れた肌) あるいは 乾燥した肌では皮膚細菌フローラのバラ ンスか崩れ、 スタ フ イ ロコ ッカス · ァゥ レウスゃ ミ ク ロコ ッカス属に属する有害菌が高い割合 で見られる。 優勢菌と してのス夕フ ィ ロコ ッカス ■ ェピデル ミ デス は、 上記有害菌の増殖を抑制するので、 轻度の皮膚傷害はスタ フィ ロコ ッ カス . ェピデル ミ デスを選択的に増殖させる こ とのできる化 粧料を提供するこ とによ って、 病原菌の増殖を抑制し皮膚傷害の改 善効果を期待するこ と も可能となる。 That is, the present invention is a cosmetic containing a substance capable of growing Staphylococcus epidermides resident on the skin in a short period of time and increasing the extracellular SOD-like specific activity. In addition, this cosmetic is a highly stable and safe cosmetic that achieves skin conditioning by normalizing the skin flora and enhancing the activity of its extracellular secretory SOD-like active substance. In particular, those in the aerobic bacteria on the skin of healthy human of the most dominant, vinegar evening off Lee Loco Kkasu. Cheap Joteki in 1 X 1 0 about four Z cm 2 in Epiderumi Death resident skin Form a bacterial flora. The Staph I Loco Tsu dregs-E Biderumi death is established growth on the surface of the skin due to the high antioxidant capacity, and secrete the SOD-like active substances in the cells outside the body, the 0 2 on the skin caused Ri by such as on the results of ultraviolet rays It is thought to play a role in eliminating skin injuries by erasing, increasing skin health, and preventing other bacterial infections. In fact, skin with injuries such as atopic dermatitis (rough skin) or dry skin loses the balance of the bacterial bacterial flora and belongs to the genus Staphylococcus aureus. Harmful bacteria are found at a high rate. S. phylococcus as the dominant bacterium ■ Epidermides inhibits the growth of the above harmful bacteria, so that a slight skin injury can cause selective growth of Staphylococcus epidermides. Providing a cosmetic composition that can be used to reduce the number of pathogenic bacteria and improve skin damage can be expected.
本発明の第一の態様に係る化粧料では、 金属イオン (例えば M n , Z n 2 + ) 、 及び金属塩 (例えば塩化マ ンガン、 塩化亜鉛、 酸化亜鉛、 ステア リ ン酸亜鉛、 パラ フ ヱ ノ ールスルフ ォ ン酸亜鉛、 ラウ リ ン酸亜鉛、 マ ンガンノくィォレ ッ ト、 硫酸マ ンガン) のう ちの 一種以上を含有する。 In the cosmetic according to the first embodiment of the present invention, metal ions (for example, Mn, Zn2 + ) and metal salts (for example, manganese chloride, zinc chloride, zinc oxide, zinc stearate, paraffin) are used. Contains one or more of zinc norsulfonate, zinc laurate, manganese sorbet, and manganese sulfate.
本発明の第二の態様に係る化粧料は、 皮膚有用常在細菌叢の好気 性最優勢菌スタフ ィ ロコ ッ カス · ェピデル ミ デスの発育を増進する 作用と、 当該菌体が生産分泌するスーパーオキサイ ド ' ジスム夕 ー ゼ(SOD) 様活性物質を高める作用を有する、 金属イオンであるマン ガン及び亜鉛又はそれらの塩 (例えば、 塩化マ ンガン、 及び酸化亜 鉛) のいずれか一つを微量含有する。 前記マ ンガンイ オ ンは、 0, 1 〜100mM の濃度範囲であるのが好ま しく 、 0. 1 〜10mMの濃度範囲で あるのが更に好ま しい。 一方、 前記亜鉛イオンは、 0. 01〜 5 mMの濃 度範囲であるのが好ま し く 、 0. 1 〜 1 mMの濃度範囲であるのが更に 好ま しい。 The cosmetic according to the second aspect of the present invention has an effect of enhancing the growth of Staphylococcus epidermides, the most aerobic bacterium of the useful skin flora, and producing and secreting the cells. One of the metal ions manganese and zinc or their salts (eg, manganese chloride and zinc oxide) that have the effect of enhancing superoxide 'dismutase (SOD) -like active substances In trace amounts. The manganese ion preferably has a concentration in the range of 0.1 to 100 mM, more preferably 0.1 to 10 mM. On the other hand, the zinc ion preferably has a concentration in the range of 0.01 to 5 mM, and more preferably 0.1 to 1 mM.
本発明をよ り よ く 説明するため、 以下に実施例を記述する。 Examples are described below to better explain the present invention.
( 1 ) 本発明の実効性を証明するため用いる微生物は、 以下の実 施例に用いられたごと く 、 健常人の皮膚に生息する代表的皮膚常在 菌であるスタフ イ ロコ ッ カス . ェピデル ミ デスである力、、 又は、 皮
膚有害菌の代表と してスタフ ィ ロ コ ッ カス ■ ァゥ レウス菌である。 しかし、 本発明を実施するため用いる細菌は、 皮膚に存在する細菌 と して通過性のものも含めていずれの菌種、 菌株を問わず用いるこ とが出来る。 (1) The microorganism used to prove the effectiveness of the present invention was Staphylococcus epider, a representative resident bacterium inhabiting the skin of healthy humans, as used in the following examples. A force that is a mide or a skin Staphylococcus ■ P. aureus is a representative of skin harmful bacteria. However, the bacterium used to carry out the present invention can be used irrespective of any bacterial species and strains, including those that can pass through the skin.
( 2 ) 本発明の実効性を証明するため用いた培地は、 通常の富栄 養培地だけでな く 、 金属イオ ン要求性を調べるため最小発育培地も 用いた。 すなわち、 最小発育培地では、 エネルギー源と してグルコ ース及び酵母エキスを添加し、 pH調製のため炭酸カルシウムを添加 したものを基礎培地と した。 細菌の金属要求性は知られているが、 あらかじめ SOD の活性中心となるマ ンガンをはじめとする数種類の 金属イ オ ンについて基礎培地に添加し、 発育阻害の起こ らない濃度 を検討した。 したがって、 本発明において用いる最小発育培地は細 菌類の生育可能な最少濃度の金属を添加したものと定義される。 (2) As a medium used to prove the effectiveness of the present invention, not only a normal nutrient medium but also a minimum growth medium was used to examine the requirement for metal ions. That is, in the minimum growth medium, glucose and yeast extract were added as energy sources, and calcium carbonate was added to adjust the pH, which was used as the basal medium. Although the metal requirement of bacteria is known, several kinds of metal ions, such as manganese, which are the active centers of SOD, were added to the basal medium in advance, and the concentration at which growth was not inhibited was examined. Therefore, the minimum growth medium used in the present invention is defined as a medium to which a minimum concentration of metal capable of growing bacteria is added.
( 3 ) 本発明の実効性を証明するため用いた細菌の培養法は、 上 記菌株及び培地を用い、 細菌を 37°Cの好気的条件下でロータ リ ーシ ヱ一力一 (120rpm) で培養した。 (3) The method of culturing bacteria used to prove the effectiveness of the present invention is as follows: using the above strains and medium, the bacteria are grown under aerobic conditions at 37 ° C under a rotary culture (120 rpm). ).
( 4 ) 本発明の実効性を証明するため用いた試料調製法は、 以下 の通りである。 まず、 上記培地で十分に発育した菌を、 冷却高速遠 心機で ΙΟΟΟΟΓΡΠΚ 10分、 4 °Cにて菌体と培養上清に分離。 ついで、 培養上清の SOD 活性測定にあたり以下のよう に濃縮した。 培養上清 に冷ァセ ト ンを最終濃度 75%となるよう添加して一 20°C冷凍庫に静 置し、 形成した沈澱物を冷却遠心分離機によって分離回収し、 少量 の精製水に溶解し凍結乾燥を行い、 アセ ト ン粉末試料を得た。 この 粉末体を精製水で溶解したものを菌体外分泌性成分試料と した。 (4) The sample preparation method used to prove the effectiveness of the present invention is as follows. First, bacteria sufficiently grown in the above medium were separated into bacterial cells and culture supernatant at 4 ° C for 10 minutes using a cooling high-speed centrifuge. Subsequently, the SOD activity of the culture supernatant was measured and concentrated as follows. Add cold acetate to the culture supernatant to a final concentration of 75%, leave it in a freezer at 120 ° C, separate and collect the formed precipitate with a cooling centrifuge, and dissolve in a small amount of purified water Then, freeze drying was performed to obtain an acetone powder sample. The powder was dissolved in purified water and used as an extracellular secretory component sample.
( 5 ) 本発明の実効性を証明するため用いた SOD 様活性物質の測 定と して、 ニ ト ロブル一テ トラゾリ ゥム(NBT) 法を用いた。 こ の測 定法は数ある測定法の中で比較的簡便で、 感度が高く 、 ngオーダー
まで検出が可能である。 キサンチンォキシダ一ゼによ り 〇 2 ― を発 生させ、 〇 2 ― によるニ ト ロブルーテ トラ ゾリ ゥムの発色度を波長(5) As a measurement of the SOD-like active substance used to prove the effectiveness of the present invention, the Nitrole tetrazolium (NBT) method was used. This measurement method is relatively simple among many measurement methods, has high sensitivity, and is on the order of ng. Detection is possible up to. Ri by the xanthine O Kishida one zero 〇 2 - the raised departure, 〇 2 - wavelength the color of the two-door Roburute tiger sledding ©-time by
560nm の吸光度で測定する ものである。 SOD の存在により 02 ― が 不均化される と NBT の発色阻害が起こ り、 この阻害率から SOD 活性 が求められる。 この方法を用いて菌体外に分泌される SOD 様活性物 質が培養上清試料のァセ ト ン濃縮処理によ り高い感度の測定が可能 と つに。 It is measured at an absorbance of 560 nm. 0 The presence of SOD 2 - is Ri Once disproportionation coloring inhibition of NBT is to put, SOD activity is determined from this inhibition rate. Using this method, SOD-like active substances secreted outside the cells can be measured with high sensitivity by acetonitrile concentration treatment of the culture supernatant sample.
実施例 Example
以下、 実施例及び実施例によ り本発明をさ らに詳細に説明するが- 本発明の範囲をこれらの実施例に限定する ものでないこ とはいう ま でもない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Examples. However, it is needless to say that the scope of the present invention is not limited to these Examples.
実験例 1 ス夕フイ ロコ ッカス . ェピデル ミ デスの発育に及ぼす 金属ィォンの影響について : Experimental Example 1 Influence of metal ions on the growth of S. huirococcus epidermides:
一連の実験に用いた基礎培地は、 デイ ビス培地 ( リ ン酸水素ニナ ト リ ウム 12水和物、 8 g ; リ ン酸二水素カ リ ウム、 2 g ; クェン酸 ナ ト リ ウム、 0.5 g ; 硫酸マグネシウム 7水和物、 0.1 g ; 硫酸ァ ンモニァ、 1 g ; グルコース、 0.5 g /蒸留水 1 リ ッ トル ( pH 7 ) ) に酵母エキス 0.1%及び炭酸力ルシゥムを 1 %添加したものを用い た。 接種菌 1 X 105 個/ mlと し、 培養は 37°Cの恒温槽で行った。 実 験に用いた金属は硫酸銅、 硫酸亜鉛、 塩化マンガン、 酸化亜鉛、 硫 酸第一鉄であり、 濃度はそれぞれ 100 / M, 1 mM, 10mMと し、 培養 開始前に添加した。 24時間の培養後標準寒天プレー トに培養液を塗 布し定法により生菌数を計測した。 その結果を表 1 に示す。
表 1 : スタ フ イ ロ コ ッ カ ス ' ェピデルミ デスの発育 The basal medium used in the series of experiments was Davis's medium (sinium sodium hydrogen phosphate decahydrate, 8 g; potassium dihydrogen phosphate, 2 g; sodium citrate, 0.5 g). g; magnesium sulfate heptahydrate, 0.1 g; ammonium sulfate, 1 g; glucose, 0.5 g / liter of distilled water (pH 7)) plus 0.1% of yeast extract and 1% of carbonated calcium Was used. The inoculum was set at 1 × 10 5 cells / ml, and the culture was performed in a thermostat at 37 ° C. The metals used in the experiments were copper sulfate, zinc sulfate, manganese chloride, zinc oxide, and ferrous sulfate. The concentrations were 100 / M, 1 mM, and 10 mM, respectively, and were added before the start of culture. After culturing for 24 hours, the culture solution was applied to a standard agar plate, and the number of viable cells was counted by a standard method. The results are shown in Table 1. Table 1: Development of Staphylococcal 'Epidermides'
及ぼす金属イ オ ンの影響 添加金属イ オ ン 添加濃度 ( mM) Effect of metal ion on concentration of added metal ion (mM)
1 0 1 0. 1 硫酸銅 1 0 1 0.1 Copper sulfate
硫酸亜鉛 Zinc sulfate
酸化亜鉛 + Zinc oxide +
塩化マ ンガン + + 十 + 硫酸第一鉄 注) 発育状態の表示法の意味 ; + + : 著し く増加、 + : 増加、 Manganese chloride + + tens + ferrous sulfate Note) Meaning of the expression of the growth status;
+ Z— : 増減な し、 一 : 低下、 — : 死減 + Z—: No increase / decrease, One: Decline, —: Death
表 1 に示すごと く 、 塩化マ ンガン及び硫酸亜鉛、 酸化亜鉛、 硫酸 第一鉄に発育促進効果が見られた。 As shown in Table 1, manganese chloride and zinc sulfate, zinc oxide, and ferrous sulfate had a growth promoting effect.
実験例 2 皮膚から分離した菌に対する酸化亜鉛と塩化マ ンガン の生育促進及び阻害効果について : + + Experimental Example 2 Growth promotion and inhibition effects of zinc oxide and manganese chloride on bacteria isolated from skin: + +
実験例 1 においてスタフ イ ロコ ッカス · ェ +一ピデルミ デス培養上清 の S OD 様活性物質を測定した結果、 硫酸亜鉛及び塩化マ ンガンを添 加した実験群が対照群よ り高い値を示したため、 この 2価金属ィォ In Example 1, the SOD-like active substance in the culture supernatant of Staphylococcus aureus + Ipidermides was measured.As a result, the experimental group to which zinc sulfate and manganese chloride were added showed higher values than the control group. The divalent metal
+ + + +
ンに注目 した。 このと き硫酸亜鉛に対して化粧品に広く 用いられて いる酸化亜鉛を用い、 硫酸第一鉄は酸化して変色し、 皮膚に悪影響 を及ぼす可能性があるので除外した。 したがって酸化亜鉛及び塩化 マンガンを添加した培地を用いてスタフ ィ ロ コ ッ カス · ェピデルミ デスの増殖曲線を検討した。 両金属濃度は 1 00 Mと し、 接種菌量 は 1 X 1 0 s 個 Zmlと した。 結果を図 1 に示す。 Attention. At this time, zinc oxide, which is widely used in cosmetics, was used for zinc sulfate, and ferrous sulfate was oxidized and discolored, and was excluded because it could adversely affect the skin. Therefore, the growth curve of Staphylococcus epidermides was examined using a medium supplemented with zinc oxide and manganese chloride. The concentration of both metals was 100 M, and the inoculum volume was 1 × 10 s / ml. The results are shown in Figure 1.
図 1 に示すごと く 、 酸化亜鉛及び塩化マ ンガンとの混合物におい てスタフ ィ ロコ ッカス · ェピデルミ デスの增殖率に顕著な増大が認 められた。 As shown in FIG. 1, a remarkable increase in the growth rate of Staphylococcus epidermides was observed in the mixture with zinc oxide and manganese chloride.
また、 スタフ ィ ロ コ ッ カス · ェピデルミ デス以外の皮膚常在菌に ついても同様な実験を行った。 代表的なものと してスタフ ィ ロコ ッ
カス · ァウ レウス IDD671株を用いて前述と同様な実験を行い、 菌数 の変化は波長 595nm の吸光度で測定した。 結果は図 2 に示す。 In addition, similar experiments were carried out on skin-resident bacteria other than Staphylococcus epidermides. A typical example is the staff The same experiment as described above was performed using Cassaureaus IDD671 strain, and the change in the number of bacteria was measured by the absorbance at a wavelength of 595 nm. The results are shown in FIG.
図 2 に示すごと く 、 有害病原菌であるスタ フ イ ロ コ ッ カ ス ' ァゥ レウスは、 有用常在細菌スタフ ィ ロ コ ッ カス · ェピデルミ デスと異 なり、 添加された酸化亜鉛、 塩化マ ンガンによって特段の生育促進 は認められず、 むしろ、 阻害傾向が認められた。 As shown in Figure 2, the harmful pathogen Staphylococcus aureus differs from the useful resident bacterium Staphylococcus epidermides by adding zinc oxide and chloride. No particular promotion of growth was observed with cancer, but rather a tendency toward inhibition.
実 例 3 スタフ ィ P コ ッ カス ' ェピデルミ デスの菌体外 SOD 様 活性に及ばす酸化亜鉛、 塩化マ ンガンの影響 : Example 3 Influence of zinc oxide and manganese chloride on extracellular SOD-like activity of Staphy P coccus' Epidermides:
次に、 実験例 1 で見出された菌体外 SOD 様比活性の酸化亜鉛、 塩 化マ ンガンの添加で増加する現像が培養時間と関係あるか以下のご と く 調べた。 培養は実験例 1 と同様な条件で行い、 SOD 様活性は時 間経過を追って測定した。 各実験群につき試料数は 3 と し、 平均値 と標準偏差を算出 した。 以下に実験例 3 の実験方法についてさ らに 詳細説明する。 Next, the following studies were conducted to determine whether the development of extracellular SOD-like specific activity, zinc oxide and chlorinated manganese, which was found in Experimental Example 1, was related to the culture time. The culture was performed under the same conditions as in Experimental Example 1, and the SOD-like activity was measured over time. The number of samples in each experimental group was 3, and the average and standard deviation were calculated. Hereinafter, the experimental method of Experimental Example 3 will be described in more detail.
試料調製法 : 試料の調整は培養は 100mlのフ ラ ス コ に 50mlの培養 液のスケールで行い、 各時間毎に 5 mlずつサ ンプ リ ング した。 培養 液はただちに遠心分離(10, 000rpm、 4 °C、 10分) して菌体と上清に 分けた。 集めた上清を氷冷し、 一 20°Cのアセ ト ンを終濃度 75 %にな るよう に撹拌しながら添加して氷冷中で 2 時間放置する。 そのあと 再び遠心分離(10, OOOrpnu 4 °C、 10分) して沈澱物を収集した。 沈 澱物を少量の精製水に溶解し、 一 80°Cで凍結させ凍結乾燥を行い、 アセ ト ンを除去する。 得られた乾燥粉末を精製水で溶解したものを サ ンプルと して用い、 タ ンパク定量及び S0D 様活性の測定を行った, タ ンパク定量法 : タ ンパク質の定量には Lowry 法を用いた。 タ ン パク質を 10~100 gを含む試料 0.4mlを試験管にと り、 アルカ リ 銅溶液 ( 1 %硫酸銅 l mlと 2 %ロ ッ シ エル塩 l mlの混合液を 2 %炭 酸ナ ト リ ウム、 0.1 N水酸化ナ ト リ ウム 100mlに添加したもの) を
2 ml添加して撹拌し、 室温で 10分間放置する。 その後直ちに 1 Nフ エ ノ 一ル試薬を 0.2ml添加撹拌した後、 室温で 30分間放置する。 30 分後に 750nmで比色定量した。 牛血清アルブミ ン (BSA, SIGMA社製) を標準液と した。 Sample preparation method: For sample preparation, culture was performed on a 100 ml flask at the scale of a 50 ml culture solution, and 5 ml was sampled each hour. The culture was immediately centrifuged (10,000 rpm, 4 ° C, 10 minutes) to separate cells and supernatant. Cool the collected supernatant with ice, add acetone at 20 ° C with stirring to a final concentration of 75%, and leave the mixture in ice for 2 hours. Thereafter, the precipitate was collected by centrifugation again (10, OOOrpnu 4 ° C, 10 minutes). Dissolve the precipitate in a small amount of purified water, freeze at 180 ° C and freeze-dry to remove acetate. Using the obtained dried powder dissolved in purified water as a sample, protein quantification and S0D-like activity were measured. Protein quantification method: Lowry method was used for quantification of protein. . A 0.4 ml sample containing 10 to 100 g of protein is placed in a test tube, and an alkaline copper solution (1 ml of 1% copper sulfate and 1 ml of 2% rossiel salt is mixed with 2% carbonic acid) Sodium, added to 100 ml of 0.1 N sodium hydroxide) Add 2 ml, stir, and leave at room temperature for 10 minutes. Immediately thereafter, add 0.2 ml of 1 N phenol reagent, stir, and leave at room temperature for 30 minutes. Thirty minutes later, colorimetric determination was performed at 750 nm. Bovine serum albumin (BSA, SIGMA) was used as the standard solution.
SOD 様活性物質の測定法 : 試験管に試料を 50 / 】 をと り X0D (キサ ンチンォキシダ一ゼ) 液(X0Dを 0.5M硫酸ア ンモニゥム、 EDTA— 2 Na 1 mM溶液に溶解して 20°Cに保存しておき、 使用直前に溶解し、 1 分あたりの 0D560nm の変化が 0.0185になるよ うに希釈したもの) を 50/ 1 添加し、 撹拌せずに 25°C水浴下に 10分間おく 。 25°Cに加温 しておいて A液(0. ImMキサンチン、 0.025mM ニ ト ロブルーテ トラゾ リ ウ ム、 0. lmM EDTA— 2 Na, 50mM炭酸ナ ト リ ウ ム緩衝液 (ρΗΙΟ.2) を 2.85ml添加後、 直ちに撹拌し、 25°C水浴下で 5 分間おいた後、 塩 化銅溶液(0.2% CuCl 2 , H20)を 50 1 添加して撹拌し、 560nm で 比色定量する。 対照には試料の代わり に精製水を添加し、 ブラ ンク は試料及び X0D 液を除いた 0.2M硫酸ア ンモニゥム溶液のみで測定 した。 SOD 活性は吸光度変化が 50%阻害されるときを 1 単位 (U) と定義し、 以下の計算式によ って算出 した。 Measuring method of SOD-like active substance: Take 50 /] sample in a test tube, dissolve X0D (xanthine oxidase) solution in 0.5M ammonium sulfate, 1 mM EDTA-2Na solution at 20 ° C Dissolve immediately before use, and dilute so that the change in 0D560nm per minute is 0.0185), add 50/1, and keep in a 25 ° C water bath for 10 minutes without stirring. After heating to 25 ° C, solution A (0. ImM xanthine, 0.025 mM nitrobluete trazolyme, 0.1 lmM EDTA- 2 Na, 50 mM sodium carbonate buffer (ρΗΙΟ.2) After stirring for 2 minutes in a 25 ° C water bath, add 50 1 of copper chloride solution (0.2% CuCl 2 , H 20 ), stir and colorimetrically measure at 560 nm. For the control, purified water was added instead of the sample, and the blank was measured only with the sample and the 0.2 M ammonium sulfate solution excluding the X0D solution.The SOD activity was 1 when the change in absorbance was inhibited by 50%. Unit (U) was defined and calculated by the following formula.
S0D(U/ml) = [(対照 0D—ブラ ン ク 0D)バ試料 0D ブラ ン ク 0D) - 1] x 20 結果を表 2 に示す。
S0D (U / ml) = [(control 0D-blank 0D) bar sample 0D blank 0D)-1] x 20 The results are shown in Table 2.
表 2 : 菌体外 SOD 様比活性 ( U/mg夕 ンパク) 経時変化に 及ぼす金属ィォンの影響 培養 (時間) Table 2: Extracellular SOD-like specific activity (U / mg protein) Effect of metal ion on time course Culture (time)
添加金属' 19 26 無添加 (対照) 11.2± 4. 16.2土し 37.9± 4.6 酸化亜鉛 12.9± 2. *13.2± 1. 36.9± 4.6 塩化マ ンガン '29.0± 2. *37.8± 3. *49.2土 3.2 ¾合物 *23.5± 3. 25.5± 7. 34.5士 1.0 (酸化亜鉛 +塩化マンガン) Additive metal '19 26 No additive (control) 11.2 ± 4.16.2 Soil 37.9 ± 4.6 Zinc oxide 12.9 ± 2 * 13.2 ± 1.36.9 ± 4.6 Manganese chloride '29 .0 ± 2 * 37.8 ± 3 * 49.2 Sat 3.2 Compound * 23.5 ± 3.25.5 ± 7.34.5% 1.0 (zinc oxide + manganese chloride)
*' : Davis 培地に酵母エキス(0· \ % ) と炭酸カ ルシウム ( 1 %) を加えたものを基礎培地 (ρΗ7.0)と した。 金属濃度 : 0. ImM *2 : 平均値土標準偏差 ( n 二 35 * ': Basal medium (ρΗ7.0) was prepared by adding yeast extract (0%) and calcium carbonate (1%) to Davis medium. Metal concentration: 0. ImM * 2 : Average soil standard deviation (n-35
* P く 0.05 ** P < 0.01 U : 活性単位 * P <0.05 ** P <0.01 U: Activity unit
表 2のごと く 、 酸化亜鉛及び塩化マ ンガンを単独あるいは混合し 添加する と、 培養初期において顕著な菌体 + +外 SOD 比活性の増大が認 められる結果を得た。 As shown in Table 2, when zinc oxide and manganese chloride were added alone or as a mixture, a remarkable increase in the specific bacterial cell +/- outside SOD activity was observed in the initial stage of the culture.
実施例 1 皮膚刺激性テス ト (安全性について) : Example 1 Skin irritation test (safety):
以上の結果にも とずいて、 マ ンガン及び亜鉛を配合した化粧料の 刺激性テス トを行った。 化粧料の配合例と して以下 (表 3 ) の溶液 を濾紙に 20 / 1 浸み込ませ、 成人男子 ( n 二 1 ) の前腕内側部皮膚 上にテープで閉鎖貼布し、 24時間後の皮膚刺激性について観察を行 い、 表 4 に示す結果を得た。 Based on the above results, an irritation test was conducted on cosmetics containing manganese and zinc. As an example of the composition of the cosmetic, the following (Table 3) solution was soaked in filter paper 20/1, and closed and taped on the inner skin of the forearm of an adult male (n21), and 24 hours later The skin irritation was observed, and the results shown in Table 4 were obtained.
表 3 : 化粧料配合例 サンプル番号 Table 3: Example of cosmetic formulation
配合例 3 (対照) 塩化マ ンガン(0. ImM) + Formulation Example 3 (Control) Manganese chloride (0. ImM) +
酸化亜鉛(0.1m M) + Zinc oxide (0.1 mM) +
CaC03( 1 % ) 一 CaC0 3 (1%) one
グ リ セ リ ン ( 5 % ) + + Glycerin (5%) + +
精製水 (計 10ml) サンプル番号 1 , 2 は、 マ ンガン と亜鉛の配合剤及び炭酸カ ルシ
ゥムの刺激性について調べるため、 サンプル番号 3 は対照と して作 成した。 肉眼による判定の結果は、 赤斑及び刺激性は全く な く 、 安 全性について問題は認められなかった。 Purified water (total 10 ml) Sample Nos. 1 and 2 are manganese / zinc combination and calcium carbonate Sample No. 3 was prepared as a control to examine the irritation properties of Pem. As a result of the judgment by the naked eye, there was no red spot or irritation, and no problem was found in safety.
表 4 : 皮膚刺激試験成績 (24時間貼布) 酸色精塩香ェ防ポグ Table 4: Skin irritation test results (24-hour patch)
化料腐素製化夕ソリリ Sake
サンプル番号剤水亜セォマルノ 除去 30分後 除去 24時間後 除去 48時間後 Sample No.Mizuseo Maruno Removal 30 minutes after removal 24 hours after removal 48 hours after removal
鉛キ一ンリ Lead-free
1 1
2 ルガシン 2 Lugasin
3 (対照) ェン 次に、 こ の発明にかかる化粧料の処方例を例示する。 配合割合は ン 3 (Control) Next, a formulation example of the cosmetic according to the present invention will be exemplified. The mixing ratio is
すべて重量%で示す。 なお、 この発明は以下の処方例にかかる化粧 料になんら限定されないのは言う までもない。 All are indicated by weight%. It goes without saying that the present invention is not limited to cosmetics according to the following formulation examples.
実施例 2 化粧水の組成 Example 2 Composition of lotion
表 5 組 成 重量 (%) Table 5 Composition Weight (%)
0. 002 0. 002
0. 0008 0. 0008
5. 0 5.0
8. 0 8.0
1 . 3 13
ビタ ンモノ ラ ウ レ一卜 Vitamin Mono Laulet
適直
Proper
実施例 3 カ ラ ミ ンロー シ ョ ンの組成 Example 3 Composition of Karamin Lotion
油水 Oil and water
相相 組 成 Phase composition
エタ ノ ール Ethanol
グリ セ塩酸オ精ェセセソグミ リ ン Glycerate HCl
酸化亜鉛化化製ルチスタクリリ Luchistacryli made by zinc oxide
塩化マ ンガ亜水ルチギマセスノビン Manga chloride subwater Ruchigimasesunobin
炭酸カルシ鉛パウルチスタンリ ム Lead calcium calcium carbonate
ベンカ ラ 組ルルテガンンンド Benka La Gumi Lulte Gandundo
7J 、) ノ 7J)) No
カ ンフ ァ ー 酸デアンミ Deamimi camprate
テジリ Teziri
精製水 ルン Purified water Rune
酸卜 矢 i^ ίR Acid arrow i ^ ίR
ί Ί ^ A フ 十 ノ π · ノ ョ Π幺 A A Ί ^ A
^ レリ 1iE nX !一 ^ Leri 1iE nX! One
o o
組 成 Composition
塩化マ ンガン 1 OOO O 酸化亜鉛 Mangan chloride 1 OOO O Zinc oxide
ジプロ ピ レ ング リ コ ―ル Dipro Pilling Records
精製水 実施例 5 スキ ンク リ ームの組成 Purified water Example 5 Composition of skin cream
表 8 成 重量 (%) Table 8 Weight (%)
30.0 30.0
6. 06.0
3. 03.0
4. 04.0
0. 0002 0. 000080. 0002 0. 00008
5. 0
実験例 4 スタフ イ ロ コ ッ カス ' ェピデルミ デスの菌体外分泌 S 0Dの UVA によるスク ワ レン過酸化反応に及ぼす抗過酸化作用につい て : 5.0 Experimental Example 4 Anti-peroxidative effects of UVA on squalene peroxidation of extracellular secretory S 0D of Staphylococcus' Epidermides:
さ らに、 塩化マンガン(0. ImM) と酸化亜鉛(0. lmM) を添加して培 養したス夕フ ィ ロコ ッ カス · ェピデルミ デスの菌体外分泌 SOD (0.1 6U/mg) の過酸化脂質抑制作用についてスク ワ レンに UVA 照射させ る系を用いて検討した。 In addition, peroxidation of extracellular SOD (0.16 U / mg) of S. phylococcus epidermides cultivated by adding manganese chloride (0. ImM) and zinc oxide (0.1 lmM) The lipid-suppressing effect was examined using a system that irradiates squalene with UVA.
対照と しては、 当該金属イオンを添加せず培養した、 同菌株培養 上清液のァセ ト ン粉末を同重量添加したものを用いた。 この中には SOD 活性がほとんど認められなかった。 As a control, a culture supernatant of the same strain, which had been cultured without the addition of the metal ion, to which was added the same weight of an acetone powder was used. SOD activity was hardly observed in these.
過酸化脂質の定量は以下に示す TBA (チォバルビツール酸) 法に て行った。 1.0 %スク ワ レンを 10mg/mlの ト リ ト ン X— 100 で可溶 化した水溶液 1.8ml に試料液 0.2ml を添加し、 37°Cで紫外線 (東芝 FL20S - BL.352nm 0.416mW/cm2 ) を 48時間照射した。 試料液より 0.1ml を取り出 し、 TBA 試薬 (0.2 %チォバルビッ一ル酸、 5.0 % 酢酸 pH3, 5 ) 2.0ml を添加し 100 °Cで 60分間加熱した後水冷した。 さ らに、 n —ブタノ ール 2. Oiiil で最終代謝産物である MDA (マロ ン ジアルデヒ ド) を抽出 し、 530nm における吸光度を測定した。 Lipid peroxide was quantified by the TBA (thiobarbituric acid) method shown below. Add 0.2 ml of the sample solution to 1.8 ml of an aqueous solution prepared by solubilizing 1.0% squalene with 10 mg / ml of Triton X-100, and apply ultraviolet light at 37 ° C (Toshiba FL20S-BL.352 nm 0.416 mW / cm 2 ) was irradiated for 48 hours. 0.1 ml was taken out from the sample solution, 2.0 ml of TBA reagent (0.2% tiobarbituric acid, 5.0% acetic acid pH3.5) was added, heated at 100 ° C for 60 minutes, and then cooled with water. Furthermore, the final metabolite MDA (malonaldehyde) was extracted with n-butanol 2.Oiii and the absorbance at 530 nm was measured.
その結果、 表 9 に示す如く 、 これら金属イオンにより誘導された 菌体外 S0D (0.16U/mg) は対照群と比較して有意 ( P く 0.01) に過 酸化脂質産生を抑制する傾向を示した。 このとき、 陽性対照と した ト コフ エロール ( 1 mgZml) を添加したときの値より低い効果であ るが濃度依存性が認められている。
表 9 し スタ Jイ ロ コ ッ カ ス ェ ピデル ミ デスの菌体外 SOD の UVA によるスク ワ レ ン過酸化抑制効果 添加試料 ( n = 3 ) 添加量 マロ ン ジアルデヒ ド 抑制率 As a result, as shown in Table 9, the extracellular S0D (0.16 U / mg) induced by these metal ions showed a tendency to significantly (P and 0.01) inhibit lipid peroxide production as compared to the control group. Was. At this time, the effect was lower than the value obtained when the tocopherol (1 mgZml) as a positive control was added, but concentration dependence was observed. Table 9 Squalene Peroxidation Inhibition by UVA of Extracellular SOD of Star J Ilococca Spidermides Additive Sample (n = 3) Addition Amount Malonaldehyde Inhibition
(mg/ml) (nmol/mlZ48時間) (%) 平均値土標準偏差 (mg / ml) (nmol / mlZ48 hours) (%) Mean Soil standard deviation
対照 (精製水) 1.0 43.5土 3. 1 0.0 金属ィォン非存在 1.0 41.9士 5.6 3.7 下培養上清 Control (purified water) 1.0 43.5 soil 3.1 0.0 No metal ion 1.0 41.9 5.6 3.7 Subculture supernatant
金属イオ ン存在 1.0 *27.0± 3.3 ' 37.9 下培養上清 Presence of metal ion 1.0 * 27.0 ± 3.3'37.9
陽性対照 Positive control
a ト コ フ エ ロール 1.0 1. 1 ± 0.2 ** 97.5 a Tocopherol 1.0 1.1 ± 0.2 ** 97.5
* : P < 0.01 P < 0.001 産業上の利用可能性 *: P <0.01 P <0.001 Industrial applicability
本発明による金属イオンの亜鉛とマンガンを適量含有する化粧料 を用いるこ とにより、 皮膚有用常在細菌叢の健全な育成と菌体から の SOD 様活性物質分泌が促進され、 皮膚有害菌の生育阻止、 皮膚細 胞の活性酸素傷害防止、 皮膚細胞老化防止などの効果が期待される
By using the cosmetics of the present invention containing appropriate amounts of the metal ions zinc and manganese, the healthy growth of useful normal flora of skin and the secretion of SOD-like active substances from the cells are promoted, and the growth of skin harmful bacteria It is expected to be effective in preventing skin cells, preventing active oxygen damage to skin cells, and preventing skin cell aging.
Claims
1 . 金属イオン、 金属塩及び金属物質のう ちの少な く と も一種を 含んでなる化粧料組成物。 1. A cosmetic composition comprising at least one of a metal ion, a metal salt and a metal substance.
2 . 皮膚有用常在細菌叢の好気性最優勢菌スタフ イ ロコ ッ カス ' ェピデルミ デスの発育を増進する作用と、 当該菌体が生産分泌する スー パ —ォキサイ ド . ジスム夕一ゼ(SOD) 様活性物質を高める作用 を有する、 金属イオンであるマンガン及び亜鉛並びにそれらの塩の 少なく と も一種を微量含んでなる化粧料組成物。 2. The effect of promoting the growth of Staphylococcus' epidermides, the most aerobic bacterium of the useful skin flora, and the superoxide. A cosmetic composition comprising at least a trace amount of manganese and zinc, which are metal ions, and a salt thereof, which has an action of enhancing the active substance.
3 . 0. 1 〜 100mM の濃度のマンガンイオンを含む請求の範囲第 2 項に記載の化粧料組成物。 3. The cosmetic composition according to claim 2, which contains manganese ions at a concentration of 3.0 to 100 mM.
4 . 0. 01〜 5 mMの濃度の亜鉛ィオンを含む請求の範囲第 2項に記 載の化粧料。
3. The cosmetic according to claim 2, comprising zinc ion at a concentration of 4.0 to 5 mM.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7/339898 | 1995-12-05 | ||
JP33989895A JP3608059B2 (en) | 1995-12-05 | 1995-12-05 | Cosmetics |
Publications (1)
Publication Number | Publication Date |
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WO1997020539A1 true WO1997020539A1 (en) | 1997-06-12 |
Family
ID=18331847
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/JP1996/003571 WO1997020539A1 (en) | 1995-12-05 | 1996-12-05 | Cosmetic preparation |
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JP (1) | JP3608059B2 (en) |
WO (1) | WO1997020539A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1155686A1 (en) * | 2000-05-18 | 2001-11-21 | L'oreal | Use og manganese in the treatment of wrinkles |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2791260B1 (en) * | 1999-03-26 | 2003-06-06 | Dior Christian Parfums | COSMETIC OR DERMATOLOGICAL COMPOSITIONS CONTAINING AT LEAST ONE SUBSTANCE FOR INCREASING THE FUNCTIONALITY AND / OR EXPRESSION OF CD44 MEMBRANE RECEPTORS OF SKIN CELLS |
JP4641150B2 (en) * | 2004-04-20 | 2011-03-02 | 伯東株式会社 | Antioxidant secretion promoter and method for producing antioxidant substance |
ES2316312B1 (en) * | 2008-06-20 | 2010-02-08 | Ignacio Umbert Millet | DERMATOLOGICAL PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF SKIN INFLAMMATION PATHOLOGIES, SUCH AS FOR EXAMPLE DERMATITIS, ATOPICA DERMATITIS, VITILIGO, AREATA ALOPECIA, ACNE, PSORIASIS AND PRURITO, AND COMBINATIONS OF THE SAME. |
KR101406808B1 (en) * | 2011-11-18 | 2014-06-12 | 가부시키가이샤 바이오제노믹스 | Skin care method, skin care composition, and dried microbial cell |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57134408A (en) * | 1981-02-13 | 1982-08-19 | Minato Sangyo Kk | Skin activating agent |
JPS60174711A (en) * | 1984-02-20 | 1985-09-09 | Kanebo Ltd | Anti-suntan cosmetic |
JPS6339808A (en) * | 1986-08-05 | 1988-02-20 | Risuburan Prod:Kk | Cosmetic composition containing mineral components |
JPH0597645A (en) * | 1991-10-09 | 1993-04-20 | Riyuuhoudou Seiyaku Kk | Cosmetic |
JPH05285370A (en) * | 1992-04-14 | 1993-11-02 | Kao Corp | Water and oil repellent powder and cosmetic containing the same |
JPH07149787A (en) * | 1993-11-29 | 1995-06-13 | Toyo Beauty Kk | Astringent and binding compound and preparation for medical and cosmetic use |
-
1995
- 1995-12-05 JP JP33989895A patent/JP3608059B2/en not_active Expired - Fee Related
-
1996
- 1996-12-05 WO PCT/JP1996/003571 patent/WO1997020539A1/en active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57134408A (en) * | 1981-02-13 | 1982-08-19 | Minato Sangyo Kk | Skin activating agent |
JPS60174711A (en) * | 1984-02-20 | 1985-09-09 | Kanebo Ltd | Anti-suntan cosmetic |
JPS6339808A (en) * | 1986-08-05 | 1988-02-20 | Risuburan Prod:Kk | Cosmetic composition containing mineral components |
JPH0597645A (en) * | 1991-10-09 | 1993-04-20 | Riyuuhoudou Seiyaku Kk | Cosmetic |
JPH05285370A (en) * | 1992-04-14 | 1993-11-02 | Kao Corp | Water and oil repellent powder and cosmetic containing the same |
JPH07149787A (en) * | 1993-11-29 | 1995-06-13 | Toyo Beauty Kk | Astringent and binding compound and preparation for medical and cosmetic use |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1155686A1 (en) * | 2000-05-18 | 2001-11-21 | L'oreal | Use og manganese in the treatment of wrinkles |
FR2809005A1 (en) * | 2000-05-18 | 2001-11-23 | Oreal | USE OF MANGANESE IN THE TREATMENT OF WRINKLES |
Also Published As
Publication number | Publication date |
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JPH09157128A (en) | 1997-06-17 |
JP3608059B2 (en) | 2005-01-05 |
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