JP2020063219A - Dermis spots improver - Google Patents
Dermis spots improver Download PDFInfo
- Publication number
- JP2020063219A JP2020063219A JP2018197298A JP2018197298A JP2020063219A JP 2020063219 A JP2020063219 A JP 2020063219A JP 2018197298 A JP2018197298 A JP 2018197298A JP 2018197298 A JP2018197298 A JP 2018197298A JP 2020063219 A JP2020063219 A JP 2020063219A
- Authority
- JP
- Japan
- Prior art keywords
- macrophages
- macrophage
- fibroblasts
- rice bran
- melanosomes
- 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.)
- Granted
Links
Abstract
Description
本発明は、真皮シミ改善剤に関し、更に詳しくは、マクロファージを所望の部位に誘引し、所望の部位でマクロファージによるメラノソームの貪食を促進することによって、真皮シミを予防・改善する発明に関する。 The present invention relates to a dermal spot improving agent, and more particularly to an invention for preventing or improving dermal spots by attracting macrophages to a desired site and promoting phagocytosis of melanosomes by macrophages at the desired site.
私たちの肌は常に外的環境から様々なストレスを受けている。特に紫外線による皮膚への傷害は、シミ、そばかす、日焼けの大きな原因になっている。
紫外線照射などにより、基底層で産生されたメラニンは、表皮細胞に移行して角層に到達し、垢となって排出される。しかしながら、シミ部位の皮膚臨床所見では真皮においてもメラニンが検出されており、ターンオーバーが活発ではない真皮にメラニンが長期間留まり、改善しにくいシミの原因となっていた。
Our skin is constantly under various stresses from the external environment. In particular, skin damage caused by ultraviolet rays is a major cause of spots, freckles, and sunburn.
Melanin produced in the basal layer by irradiation with ultraviolet rays migrates to the epidermal cells, reaches the stratum corneum, and is excreted as dirt. However, in the clinical findings of the skin at the spot, melanin was also detected in the dermis, and melanin remained in the dermis where turnover was not active for a long period of time, which caused stains that were difficult to improve.
従来の美白剤は、メラニン産生に影響を及ぼす酵素であるチロシナーゼを阻害する物質や、表皮のターンオーバーを活性化する物質を有効成分として配合したものが主流であった。しかしながら、これらの美白剤は表皮のメラニンに作用するものであり、真皮のメラニンに対して作用を及ぼすものはなかった。 Most conventional whitening agents are those containing, as an active ingredient, a substance that inhibits tyrosinase, which is an enzyme that affects melanin production, and a substance that activates turnover of the epidermis. However, these whitening agents act on the melanin of the epidermis, and none act on the melanin of the dermis.
一方、マクロファージは貪食能が高い細胞と定義され、体に生じた廃棄物の処理が主な役割であり、古くなった細胞や死滅した細胞の処理を行っていることが知られている。この性質を利用して、真皮に存在するメラニンを除去することを目的としたマクロファージ活性化剤の提案がされている（特許文献１）。一方で、真皮の細胞である線維芽細胞はメラニンを集める性質があることが分かってきた（特許文献２）。先行特許は、真皮に単体で存在するメラニン（メラノソームを含む。以下同じ。）の貪食を促進するものであるため、特定の線維芽細胞に貪食されたメラニンを効率的に除去することは困難であった。 On the other hand, macrophages are defined as cells with high phagocytosis, and it is known that the treatment of waste generated in the body is the main role, and that treatment of old cells and dead cells is performed. Utilizing this property, a macrophage activator for removing melanin present in the dermis has been proposed (Patent Document 1). On the other hand, it has been found that fibroblasts, which are dermal cells, have a property of collecting melanin (Patent Document 2). Since the prior patent promotes the phagocytosis of melanin (including melanosomes. The same applies hereinafter) that exists alone in the dermis, it is difficult to efficiently remove melanin phagocytosed by specific fibroblasts. there were.
マクロファージは走化性因子、例えばＭＣＰ−１（ＣＣＬ２）の濃度勾配に従って移動して、死滅した細胞や菌、メラニンなどの異物を貪食することが知られている（非特許文献１、２）。しかし、化粧料に直接ＭＣＰ−１を配合しても、皮膚表層から深層への拡散による濃度勾配が形成されるのみで、標的部位であるメラニンを貪食した特定の線維芽細胞に対して、マクロファージを選択的に誘引することはできない。 It is known that macrophages migrate according to the concentration gradient of chemotactic factors such as MCP-1 (CCL2) and phagocytose foreign substances such as dead cells, bacteria and melanin (Non-patent Documents 1 and 2). However, even if MCP-1 is directly added to a cosmetic, a concentration gradient due to diffusion from the skin surface layer to the deep layer is formed, and macrophages are generated against specific fibroblasts that have phagocytosed melanin, which is a target site. Cannot be selectively attracted.
有色米由来の抽出物には、各種の効果が見出されている。例えば、親水性の溶媒で抽出された有色米の抽出物にメラニン生成抑制作用があることが知られている（特許文献３）。また、米ヌカ米胚芽等から得られたスフィンゴ糖脂質によりマクロファージの貪食能を促進する効果が報告されている（特許文献４）。
しかしながら、メラニンを効率的に除去するためには、マクロファージの貪食能を促進するだけでなく、マクロファージが所望する部位、即ち線維芽細胞がメラニンを集めている部位にまで素早くマクロファージを誘引させる必要がある。
Various effects have been found in extracts derived from colored rice. For example, it is known that an extract of colored rice extracted with a hydrophilic solvent has a melanin production suppressing action (Patent Document 3). In addition, it has been reported that glycosphingolipids obtained from rice bran rice germ and the like promote the phagocytic ability of macrophages (Patent Document 4).
However, in order to efficiently remove melanin, it is necessary not only to promote the phagocytic ability of macrophages but also to quickly attract macrophages to a desired site of macrophages, that is, a site where fibroblasts collect melanin. is there.
本発明は、このような問題を解決するためになされたものであり、真皮に存在するメラニンによるシミ（以下、「真皮シミ」という場合がある。）の改善することを課題とするものである。 The present invention has been made in order to solve such a problem, and an object thereof is to improve spots due to melanin present in the dermis (hereinafter, sometimes referred to as "dermis spots"). .
有色米ヌカの抽出物を用いることにより、上記問題を解決した。 The above problem was solved by using an extract of colored rice bran.
本発明によれば、真皮に存在するメラニンを蓄積した線維芽細胞に、マクロファージを誘引させ、メラニンを貪食させることにより、真皮に存在するメラニンによるシミを改善することができる。 ADVANTAGE OF THE INVENTION According to this invention, the fibroblast which accumulate | stored the melanin which exists in a dermis is made to attract a macrophage, and a melanin is phagocytosed, The stain by the melanin existing in a dermis can be improved.
本発明の有色米ヌカ抽出物は、イネ科（Ｐｏａｃｅａｅ）イネ属（Ｏｒｙｚａ）イネ（学名：Ｏｒｙｚａ ｓａｔｉｖａ）のヌカから抽出することができる。ここで、有色米とは玄米中の種皮、糊粉層またはでんぷん層の一部または全部に色素を含有する品種の米であり、これらの米の色調により、黒米、赤米等が挙げられる。黒米は、中国原産の古代米の一種で、紫黒米、紫米とも呼ばれる。その玄米の表面は黒色で、果皮・種皮および胚芽の部分に紫黒色系色素（アントシアニン系）を含む。赤米は、野生稲の大部分が赤米であることから、赤米は米のルーツとされる。玄米の色が赤褐色で果皮、種皮の部分に赤色系色素（タンニン系）を含む。ヌカ層を有する状態の紫玄米は紫黒色を呈し、褐色を呈する一般的な白米の玄米とは外観によって容易に識別が可能である。また、紫玄米を搗精する際に生じるヌカも、一般的な白米のヌカとは外観によって容易に識別が可能である。 The colored rice bran extract of the present invention can be extracted from rice bran of the Poaceae family (Oryza) rice (scientific name: Oryza sativa). Here, the colored rice is rice of a variety that contains a pigment in a part or all of the seed coat, the aleurone layer, or the starch layer in brown rice, and examples thereof include black rice and red rice depending on the color tone of these rice. Black rice is a type of ancient rice originating in China and is also called purple black rice or purple rice. The surface of the brown rice is black, and a purple-black pigment (anthocyanin type) is contained in the pericarp, seed coat and embryo. Red rice is the root of rice because most of the wild rice is red rice. Brown rice has a reddish brown color and contains a red pigment (tannin type) in the pericarp and seed coat. Purple brown rice in the state of having a nuka layer has a purple black color, and can be easily distinguished from brown rice, which is an ordinary brown rice, by its appearance. In addition, the nuka that occurs during the polishing of purple brown rice can be easily distinguished from the general white rice nuka by its appearance.
本発明の有色米ヌカ抽出物の調製も特に限定されるものではない。例えば、水、種々適当な有機溶媒、及びこれらの混合溶媒のいずれかに投入し、低温下及び／又は加温下で抽出された物が使用できる。 The preparation of the colored rice bran extract of the present invention is not particularly limited. For example, a substance extracted by adding water, various suitable organic solvents, or a mixed solvent thereof and extracting at low temperature and / or under heating can be used.
有色米ヌカ抽出物の各剤の組成物全体に対する配合量は、有効量であれば特に限定されないが、乾燥質量に換算して０．００１〜１０質量％が好ましく、より好ましくは０．００１〜１．０質量％である。 The compounding amount of each agent of the colored rice bran extract with respect to the entire composition is not particularly limited as long as it is an effective amount, but it is preferably 0.001 to 10% by mass in terms of dry mass, and more preferably 0.001 to 10% by mass. It is 1.0 mass%.
抽出溶媒としては、特に限定はされないが例えば、水；メチルアルコール、エチルアルコール等の低級１価アルコール；グリセリン、プロピレングリコール、1,3-ブチレングリコール等の液状多価アルコール；アセトン、メチルエチルケトン等のケトン；酢酸エチルなどのアルキルエステル；ベンゼン、ヘキサン等の炭化水素；ジエチルエーテル等のエーテル類；ジクロルメタン、クロロホルム等のハロゲン化アルカン等の１種または２種以上を用いることができる。就中、水、エチルアルコール、1,3-ブチレングリコールの１種または２種以上の混合溶媒が特に好適である。 The extraction solvent is not particularly limited, and examples thereof include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone. One or more of alkyl esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; halogenated alkanes such as dichloromethane and chloroform. Above all, one or a mixture of two or more of water, ethyl alcohol and 1,3-butylene glycol is particularly suitable.
以上のような条件で得られる有色米ヌカ抽出物は、抽出された溶液のまま用いても良いが、さらに必要により濾過等の処理をして、濃縮、粉末化したものを適宜使い分けて用いることができる。また、市販品を用いることができ、丸善製薬社製の紫玄米ヌカエキスＢＧ等を用いることができる。 The colored rice bran extract obtained under the above conditions may be used as it is as an extracted solution, but if necessary, it may be subjected to a treatment such as filtration, concentrated, and powdered to be used properly. You can Moreover, a commercially available product can be used, and purple brown rice bran extract BG manufactured by Maruzen Pharmaceutical Co., Ltd. can be used.
本発明におけるマクロファージ誘引剤とは有効成分がマクロファージに直接的に作用して、マクロファージを誘引するものをさし、マクロファージ誘引活性剤とは有効成分が細胞等に作用して、マクロファージを間接的に誘引するものをさす。 The macrophage attractant in the present invention refers to one in which the active ingredient directly acts on the macrophage to attract the macrophage, and the macrophage attractant activator means that the active ingredient acts on the cell or the like to indirectly act on the macrophage. The thing that attracts you.
以下、本発明における有色米ヌカ抽出物の調製、効果試験等の実施例を示すが、ここに記載された実施例に限定されないのは言うまでもない。 Hereinafter, examples of the preparation of the colored rice bran extract according to the present invention, an effect test, and the like will be shown, but it goes without saying that the examples are not limited thereto.
＜試料の調製＞
有色米ヌカ抽出物は、丸善製薬社製の紫玄米ヌカエキスＢＧ、米ヌカ抽出物は、丸善製薬社製のコメヌカ抽出液ＢＧ、アントシアニンは、和光純薬製の塩化デルフィニジンを用いた。
<Preparation of sample>
The colored rice bran extract was purple brown rice bran extract BG manufactured by Maruzen Pharmaceutical Co., Ltd., the rice bran extract was rice bran extract BG manufactured by Maruzen Pharmaceutical Co., Ltd., and the anthocyanins used were delphinidin chloride manufactured by Wako Pure Chemical Industries.
＜メラノソームの調製＞
ヒト由来メラノーマ細胞ＨＭ３ＫＯを５％ＣＯ２下、３７℃のインキュベーター内で、１０％ＦＢＳを含むＤ−ＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社製Ｇｉｂｃｏ）を用いて培養した。
１００％コンフルエント近くになりメラニン産生が進んだ細胞を、トリプシンを用いて７．０×１０６ｃｅｌｌｓ/ ｔｕｂｅになるようにエッペンドルフチューブに回収、遠心（１０００ｇ、４℃、３分）によって細胞ペレットを作成した。その後ペレットをＰＢＳ（−）にて洗浄した。
<Preparation of melanosomes>
Human-derived melanoma cells HM3KO were cultured in a 37 ° C. incubator under 5% CO 2 using D-MEM medium (Gibco manufactured by Invitrogen) containing 10% FBS.
Cells with nearly 100% confluency and advanced melanin production were collected in an Eppendorf tube using trypsin so that the cell pellet became 7.0 × 10 6 cells / tube, and the cell pellet was centrifuged (1000 g, 4 ° C., 3 minutes). Created. Thereafter, the pellet was washed with PBS (-).
細胞ペレットに対し、１ｍＬのｃｏｌｄ ｌｙｓｉｓ ｂｕｆｆｅｒ（１％ ｏｃｔｙｌｐｈｅｎｏｘｙ ｐｏｌｙ （ｅｔｈｙｌｅｎｅｏｘｙ）ｅｔｈａｎｏｌ（ＩＧＥＰＡＬ ＣＡ−６３０）、０．０１％ ＳＤＳ含有０．１Ｍ Ｔｒｉｓ−ＨＣｌ溶液ｐＨ７．５）を添加した。これを１０分ごとに攪拌しながら、２０分室温にて静置した。この分散溶液を遠心分離し（１０００ｇ、４℃、３分）、不要物を沈殿させ、メラノソームを含む上清を回収した。回収した上清を再度、遠心分離し（１０００ｇ、４℃、３分）、上清を回収した。この上清を遠心分離し（２００００ｇ、４℃、３分）、得られた沈殿をメラノソームリッチ画分とした。上清を吸引除去し、メラノソームのペレットをＰＢＳにて２度洗浄した。（２００００ｇ、４℃、３分）これにＰＢＳを添加し、５０回以上ピペッティングすることによってメラノソームを分散させた。このメラノソーム懸濁液をメラノソームとした。 To the cell pellet, 1 mL of cold lysis buffer (1% octylphenoxy poly (ethyleneoxy) ethanol (IGEPAL CA-630), 0.01 M SDS-containing 0.1 M Tris-HCl solution pH 7.5) was added. This was left standing at room temperature for 20 minutes while stirring every 10 minutes. This dispersion solution was centrifuged (1000 g, 4 ° C., 3 minutes) to precipitate unnecessary substances, and a supernatant containing melanosomes was collected. The recovered supernatant was centrifuged again (1000 g, 4 ° C., 3 minutes), and the supernatant was recovered. This supernatant was centrifuged (20,000 g, 4 ° C., 3 minutes), and the obtained precipitate was used as a melanosome-rich fraction. The supernatant was removed by suction, and the melanosome pellet was washed twice with PBS. (20000 g, 4 ° C., 3 minutes) PBS was added thereto, and the melanosomes were dispersed by pipetting 50 times or more. This melanosome suspension was designated as melanosome.
＜アントシアニン量の調製＞
紫玄米に含まれるアントシアニンの量は収穫年度や品種によって異なるが、各紫玄米１ｇあたりのアントシアニン量が、朝紫は１−６ｍｇ、おくのむらさきは０−０．５ｍｇであった。
これらの数値より、紫玄米に含まれる平均のアントシアニン量を算出し、０．０３ｍｇ／ｍLのアントシアニンを１０００ｐｐｍ紫玄米ヌカ抽出物の比較例として用いた。
<Preparation of anthocyanin amount>
The amount of anthocyanins contained in purple brown rice varies depending on the harvest year and variety, but the amount of anthocyanins per 1 g of each purple brown rice was 1-6 mg for morning purple and 0-0.5 mg for Okunomurasaki.
The average amount of anthocyanins contained in purple brown rice was calculated from these values, and 0.03 mg / mL of anthocyanins was used as a comparative example of 1000 ppm purple brown rice bran extract.
−マクロファージの貪食能確認試験−
線維芽細胞を１．０×１０５ｃｅｌｌ／ｍＬおよび単球細胞を１．０×１０６ｃｅｌｌ／ｍＬになるように５％ＦＢＳを含むＲＰＭＩ１６４０培地（Ｉｎｖｉｔｒｏｇｅｎ社製Ｇｉｂｃｏ）にそれぞれ懸濁し、各細胞を７０μＬずつｃｕｌｔｕｒｅ-Ｉｎｓｅｒｔ ｉｎ μ-Ｄｉｓｈ ３５ ｍｍ, ｈｉｇｈ, ｉｂｉＴｒｅａｔ（ｉｂｉｄｉ）のウェルに播種した。５％のＣＯ２下、３７℃のインキュベーター内で２４時間培養した。培養後線維芽細胞のウェルにはメラノソーム１０μＬを添加し、単球細胞のウェルには、単球細胞をマクロファージに分化させるために、３．２ｍＭ ＰＭＡをそれぞれ添加した。５％のＣＯ２下、３７℃のインキュベーター内で７２時間培養した。各ウェル内をＰＢＳ（―）で洗浄した後に、インサートを除去して共培養を開始し、マクロファージの貪食能について確認を行った。
-Macrophage phagocytosis confirmation test-
Each of the fibroblasts was suspended in RPMI1640 medium (Gibco manufactured by Invitrogen) containing 5% FBS at 1.0 × 10 5 cells / mL and monocyte cells at 1.0 × 10 6 cells / mL, and each suspension was prepared. 70 μL of the cells were seeded in each well of culture-Insert in μ-Dish 35 mm, high, ibiTreat (ibidi). It was cultured for 24 hours in an incubator at 37 ° C. under 5% CO 2 . After the culturing, 10 μL of melanosomes were added to the wells of fibroblasts, and 3.2 mM PMA was added to the wells of the monocyte cells in order to differentiate the monocyte into macrophages. The cells were cultured for 72 hours in a 37 ° C. incubator under 5% CO 2 . After washing the inside of each well with PBS (-), the insert was removed and co-culture was started to confirm the phagocytic ability of macrophages.
＜分析条件＞
画像解析ソフト（ＷｉｎＲＯＯＦ：三谷商事株式会社）を用いて、メラノソームによって黒く見える部分とそれ以外を二値化し、メラノソームの存在する面積を算出した。
<Analysis conditions>
Using an image analysis software (WinROOF: Mitani Shoji Co., Ltd.), the area where the melanosomes appear black and the other areas were binarized, and the area where the melanosomes existed was calculated.
図１は、共培養開始直後（０時間）、共培養開始から１３２時間後まで２４時間ごとに撮影した写真である（ＢＺ−Ｘ７００、ＫＥＹＥＮＣＥ、明視野１０倍レンズ）。共培養開始直後には左側にマクロファージ、右側にメラノソームを貪食した線維芽細胞が位置していたが、共培養開始２４時間後あたりからマクロファージが線維芽細胞の方に誘引され始めた。その後、マクロファージがメラノソームを貪食し始め、共培養開始１３２時間後では、画面上のメラノソームの量が減少していることが目視で明らかである。メラノソーム部分の面積を計測したところ、メラノソーム添加直後を１とすると、１３２時間後は０．３０２にまで減少した（図２）。これにより、マクロファージは線維芽細胞に取り込まれたメラノソームも貪食できることを確認した。 FIG. 1 shows photographs taken every 24 hours immediately after the start of co-culture (0 hour) and 132 hours after the start of co-culture (BZ-X700, KEYENCE, bright field 10 × lens). Immediately after the start of co-culture, macrophages were located on the left side and fibroblasts that phagocytosed melanosomes on the right side, but macrophages began to be attracted toward fibroblasts around 24 hours after the start of co-culture. After that, macrophages began to phagocytose the melanosomes, and 132 hours after the start of the co-culture, it is visually apparent that the amount of melanosomes on the screen has decreased. When the area of the melanosome portion was measured, assuming that 1 immediately after the addition of melanosome, it decreased to 0.302 after 132 hours (FIG. 2). This confirmed that macrophages can also phagocytose melanosomes taken up by fibroblasts.
ＣｙｔｏＳｅｌｅｃｔ ９６−ｗｅｌｌ Ｃｅｌｌ Ｍｉｇｒａｔｉｏｎ Ａｓｓａｙ、５μｍ、Ｆｌｕｏｒｏｍｅｔｒｉｃ Ｆｏｒｍａｔ（ＣＢＡ−１０５、セルバイオラボ）の下層のウェルに５％ＦＢＳを含むＲＰＭＩ１６４０培地および１０００ｐｐｍのスクリーニングサンプルを加えた。なお、ポジティブコントロールとしてＭＣＰ−１ １００ｎｇ／ｍＬ、ネガティブコントロールとしてＰＢＳ（―）を添加した。
上層のメンブレンチャンバーを下層のウェルにセットし、一晩ＦＢＳを除いたＲＰＭＩ１６４０培地で培養を行った単球細胞懸濁液（５．０×１０６ｃｅｌｌ／ｍＬ）を１００μＬ添加した。フタをして５％のＣＯ２下、３７℃のインキュベーター内で４時間インキュベートした。１５０μＬの Ｃｅｌｌ ｄｅｔａｃｈｍｅｎｔ Ｓｏｌｕｔｉｏｎを９６−ｗｅｌｌ Ｃｅｌｌ Ｈａｒｖｅｓｔｉｎｇ Ｔｒａｙに添加し、その上に単球細胞懸濁液を除いたメンブレンチャンバーをのせて５％のＣＯ２下、３７℃のインキュベーター内で３０分間インキュベートした。新しい９６ウェルプレートに下層に誘引された単球細胞懸濁液７５μＬおよびＤｅｔａｃｈｍｅｎｔ Ｓｏｌｕｔｉｏｎ７５μＬを合わせた。そこに４ｘＬｙｓｉｓ ｂｕｆｆｅｒ／Ｃｙｑｕａｎｔ ＧＲ Ｄｙｅ Ｓｏｌｕｔｉｏｎ（７５：１）を作製し、先ほどの誘引されたマクロファージを合わせた１５０μＬの懸濁液の中に５０μＬずつ添加した。この溶液１５０μＬを新しい９６ウェルプレートに移し、蛍光プレートリーダー（ｉｎｆｉｎｉｔｅ Ｆ２００Ｐｒｏ、ＴＥＣＡＮ）で４８０ｎｍ／５２０ｎｍで測定を行った。コントロールよりも誘引作用が高ければ、誘引作用があると言えるが、ポジティブコントロールであるＭＣＰ−１よりも高い誘引性作用があれば、著しく高い誘引作用があると言える。
RPMI1640 medium containing 5% FBS and a 1000 ppm screening sample were added to the lower wells of CytoSelect 96-well Cell Migration Assay, 5 μm, Fluorometric Metro Format (CBA-105, Cell Biolab). In addition, 100 ng / mL of MCP-1 was added as a positive control, and PBS (−) was added as a negative control.
The upper membrane chamber was set in the lower well, and 100 μL of a monocyte suspension (5.0 × 10 6 cells / mL) cultured overnight in RPMI1640 medium without FBS was added. It was covered and incubated for 4 hours in a 37 ° C. incubator under 5% CO 2 . 150 μL of Cell detachment Solution was added to 96-well Cell Harvesting Tray, and the membrane chamber except the monocyte cell suspension was placed thereon and incubated at 37 ° C. for 30 minutes under 5% CO 2 . . A new 96-well plate was combined with 75 μL of the attracted monocyte cell suspension and 75 μL of Detachment Solution in the lower layer. 4 × Lysis buffer / Cyquant GR Dye Solution (75: 1) was prepared there, and 50 μL was added to each 150 μL suspension containing the attracted macrophages. 150 μL of this solution was transferred to a new 96-well plate, and the measurement was performed at 480 nm / 520 nm with a fluorescence plate reader (infinite F200Pro, TECAN). It can be said that if the attracting action is higher than the control, there is an attracting action, but if the attracting action is higher than that of the positive control MCP-1, it can be said that there is a remarkably high attracting action.
図３に示したマクロファージ誘引試験結果より、米ヌカ抽出物およびアントシアニンには誘引作用がなく、紫玄米ヌカ抽出物には著しく高い誘引作用が認められた。 From the macrophage attraction test results shown in FIG. 3, the rice bran extract and anthocyanin had no attractant action, and the purple brown rice bran extract had a remarkably high attractant action.
以上の結果より、有色米である紫玄米のヌカ抽出物にマクロファージ誘引作用があることが見出されたが、有色米ではない通常の米ヌカ抽出物には誘引作用がないことが分かった。また、アントシアニンには誘引作用が見られなかった。このことから、本誘引効果は、アントシアニン以外の成分によるものであることが分かった。
そこで、次に有色米ヌカ抽出物によって、メラノソームを取り込んでいる線維芽細胞部分にマクロファージを誘引できるかを確認した。
From the above results, it was found that the Nuka extract of purple brown rice, which is a colored rice, has a macrophage attracting action, but the normal rice Nuka extract that is not a colored rice has no attracting action. In addition, anthocyanins did not show any attracting action. From this, it was found that this attractive effect was due to components other than anthocyanins.
Therefore, it was next confirmed whether the colored rice bran extract could attract macrophages to the fibroblast portion that had taken up melanosomes.
線維芽細胞を１．０×１０５ｃｅｌｌ／ｍＬおよび単球細胞を１．０×１０６ｃｅｌｌ／ｍＬになるように５％ＦＢＳを含むＲＰＭＩ１６４０培地（Ｉｎｖｉｔｒｏｇｅｎ社製Ｇｉｂｃｏ）に懸濁し、それぞれの細胞を７０μＬずつｃｕｌｔｕｒｅ-Ｉｎｓｅｒｔ ｉｎ μ-Ｄｉｓｈ ３５ ｍｍ, ｈｉｇｈ, ｉｂｉＴｒｅａｔ（ｉｂｉｄｉ）のウェルに播種した。５％のＣＯ２下、３７℃のインキュベーター内で２４時間培養した。培養後線維芽細胞のウェルにはメラノソーム１０μＬを添加し、単球細胞のウェルには、単球細胞をマクロファージに分化させるために、３．２ｍＭ ＰＭＡをそれぞれ添加した。５％のＣＯ２下、３７℃のインキュベーター内で７２時間培養した。各ウェル内をＰＢＳ（―）で洗浄した後にインサートを除去して共培養を開始した。なお、７２時間培養後に共培養を開始したのは、メラノソームを添加した線維芽細胞が特定の線維芽細胞にメラノソームを移送終了するのが概ね７２時間後であることからである。共培養開始直後に紫玄米ヌカ抽出物１０００ｐｐｍおよびコントロールとして同量のＰＢＳ（−）を培地に添加後試料添加による濃度勾配ができないよう培地を十分攪拌した。培養直後からタイムラプス動画撮影を行った。コントロールの結果と比較して、誘引されたマクロファージの数が多い、あるいは早さが早ければ、誘引活性化作用があると判断できる。 Fibroblasts were suspended in RPMI1640 medium (Gibco manufactured by Invitrogen) containing 5% FBS at 1.0 × 10 5 cells / mL and monocyte cells at 1.0 × 10 6 cells / mL, and the respective suspensions were prepared. 70 μL of the cells were seeded in each well of culture-Insert in μ-Dish 35 mm, high, ibiTreat (ibidi). It was cultured for 24 hours in an incubator at 37 ° C. under 5% CO 2 . After the culturing, 10 μL of melanosomes were added to the wells of fibroblasts, and 3.2 mM PMA was added to the wells of the monocyte cells in order to differentiate the monocyte into macrophages. The cells were cultured for 72 hours in a 37 ° C. incubator under 5% CO 2 . After washing the inside of each well with PBS (-), the insert was removed and co-culture was started. The reason why co-culture was started after 72 hours of culturing was that it was approximately 72 hours after the transfer of the melanosomes to the specific fibroblasts by the melanosome-added fibroblasts. Immediately after the start of co-culture, 1000 ppm of purple brown rice bran extract and the same amount of PBS (-) as a control were added to the medium, and then the medium was sufficiently stirred so that a concentration gradient due to the addition of the sample could not be obtained. Immediately after culturing, time-lapse movie shooting was performed. If the number of macrophages that are attracted is larger or faster than the control results, it can be judged that there is an action of activating attraction.
図４（紫玄米ヌカ抽出物を添加）および図５（コントロールとしてＰＢＳ（−）を添加）は、共培養開始直後（０時間）、共培養開始から７２時間後まで２４時間ごとに撮影した写真である（ＢＺ−Ｘ７００、ＫＥＹＥＮＣＥ、明視野１０倍レンズ）。共培養開始直後には左側にマクロファージ、右側にメラノソームを貪食した線維芽細胞が位置していたが、紫玄米ヌカ抽出物を添加した方では、共培養開始２４時間後あたりからマクロファージが線維芽細胞の方に誘引され始めた。一方、コントロールであるＰＢＳ（−）を添加した方では、共培養開始２４時間後にはまだマクロファージは線維芽細胞の方に誘引されていない。また、共培養開始７２時間後を比較すると、明らかに有色米である紫玄米ヌカ抽出物を添加した方が、マクロファージが多く誘引されている。
この結果より、有色米ヌカ抽出物にはマクロファージ誘引活性化作用があることが確認され、また、線維芽細胞の方に誘引されるマクロファージの様子をリアルタイムで確認することができた。
Fig. 4 (added purple brown rice bran extract) and Fig. 5 (added PBS (-) as control) are photographs taken every 24 hours immediately after the start of co-culture (0 hour) and 72 hours after the start of co-culture. (BZ-X700, KEYENCE, bright field 10 × lens). Immediately after the start of co-culture, macrophages were located on the left side, and fibroblasts that phagocytosed melanosomes were located on the right side. Began to be attracted to. On the other hand, in the control (PBS (-))-added, macrophages were not yet attracted to fibroblasts 24 hours after the start of co-culture. In addition, comparing 72 hours after the start of co-culture, more macrophages were attracted when the purple brown rice bran extract, which is obviously colored rice, was added.
From these results, it was confirmed that the colored rice bran extract had a macrophage attracting activation effect, and the state of macrophages attracted to fibroblasts could be confirmed in real time.
本発明によれば、シミ部位等の所望部位にマクロファージを誘引させることができ、マクロファージにより真皮に落ち込んだメラニン（メラノソーム含む）を貪食させることによって、できたシミを改善することができる。また、本発明のシミ改善剤を用いれば、メラニンが真皮に落ち込んだ後停滞する前にメラニンを貪食させれば、シミ予防も期待できる。
According to the present invention, macrophages can be attracted to a desired site such as a stain site, and the resulting stain can be improved by phagocytosing melanin (including melanosomes) that has fallen into the dermis by the macrophage. In addition, if the spot-improving agent of the present invention is used, if melanin is phagocytosed after it has fallen into the dermis and before it becomes stagnant, it can be expected to prevent spots.
Claims (4)
（２） 被験物質を培地に添加する工程
（３） メラノソームを貪食した線維芽細胞を含む線維芽細胞に対するマクロファージの
移動速度及び／又は数を指標とし、
コントロールと比較してマクロファージの移動速度が速い被験物質及び／又は
マクロファージの数が多い被験物質を有効物質と判定する工程
を含むマクロファージの誘引活性化剤のスクリーニング方法。
(1) A step of isolating a macrophage group and a fibroblast group containing fibroblasts that phagocytosed melanosomes and culturing them in the same culture system (2) a step of adding a test substance to a medium (3) a phagocytosis of melanosomes Using the migration speed and / or number of macrophages relative to fibroblasts including fibroblasts as an index,
A method for screening a macrophage attracting activator, comprising a step of determining a test substance having a higher macrophage migration rate and / or a test substance having a large number of macrophages as an effective substance as compared with a control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018197298A JP7184593B2 (en) | 2018-10-19 | 2018-10-19 | Dermal blemish improver |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018197298A JP7184593B2 (en) | 2018-10-19 | 2018-10-19 | Dermal blemish improver |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020063219A true JP2020063219A (en) | 2020-04-23 |
| JP7184593B2 JP7184593B2 (en) | 2022-12-06 |
Family
ID=70386774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018197298A Active JP7184593B2 (en) | 2018-10-19 | 2018-10-19 | Dermal blemish improver |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7184593B2 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000044485A (en) * | 1998-07-27 | 2000-02-15 | Real:Kk | Active oxygen species scavenger and skin cosmetic |
| JP2004345969A (en) * | 2003-05-20 | 2004-12-09 | Real:Kk | Tyrosinase inhibitor and bleaching cosmetic using the same |
| JP2011126850A (en) * | 2009-12-21 | 2011-06-30 | Maruzen Pharmaceut Co Ltd | INHIBITOR AGAINST EXPRESSION AND ELEVATION OF STEM CELL FACTOR (SCF) mRNA, AND INHIBITOR AGAINST EXPRESSION AND ELEVATION OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) mRNA |
| JP2013249265A (en) * | 2012-05-30 | 2013-12-12 | Bloom Classic Co Ltd | Whitening agent and skin cosmetic |
| JP2015010070A (en) * | 2013-06-28 | 2015-01-19 | 富士フイルム株式会社 | Melanin decomposition accelerator and melanosome protein decomposition accelerator |
-
2018
- 2018-10-19 JP JP2018197298A patent/JP7184593B2/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000044485A (en) * | 1998-07-27 | 2000-02-15 | Real:Kk | Active oxygen species scavenger and skin cosmetic |
| JP2004345969A (en) * | 2003-05-20 | 2004-12-09 | Real:Kk | Tyrosinase inhibitor and bleaching cosmetic using the same |
| JP2011126850A (en) * | 2009-12-21 | 2011-06-30 | Maruzen Pharmaceut Co Ltd | INHIBITOR AGAINST EXPRESSION AND ELEVATION OF STEM CELL FACTOR (SCF) mRNA, AND INHIBITOR AGAINST EXPRESSION AND ELEVATION OF BASIC FIBROBLAST GROWTH FACTOR (bFGF) mRNA |
| JP2013249265A (en) * | 2012-05-30 | 2013-12-12 | Bloom Classic Co Ltd | Whitening agent and skin cosmetic |
| JP2015010070A (en) * | 2013-06-28 | 2015-01-19 | 富士フイルム株式会社 | Melanin decomposition accelerator and melanosome protein decomposition accelerator |
Non-Patent Citations (2)
| Title |
|---|
| 有色米オリザノール成分の分析−黒米のオリザノールの特徴−, [ONLINE], 農研機構, [2022.06.17検索], イン, JPN6022025778, ISSN: 0004811680 * |
| 食品研究部門 研究成果展示会 2017年 展示内容, [ONLINE], 農研機構, [2022.06.17検索], インターネット, <, JPN6022025779, ISSN: 0004811681 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7184593B2 (en) | 2022-12-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108699519A (en) | The stem cell-derived allochthon containing the raised growth factor | |
| CN108473980A (en) | Including the topical compositions of tiny RNA tiger lily extract and the beautifying nursing method for reducing signs of skin aging | |
| CN101426934A (en) | Method of predicting skin spot formation and method of screening skin spot formation inhibitor with the use of spot site-increasing genes as indication | |
| JP7344591B2 (en) | Cosmetic composition containing exosomes derived from Galactomyces as an active ingredient | |
| CN103619318A (en) | Molecular signature for skin pigment spots, which is combined with the extracellular matrix | |
| EP3475697B1 (en) | Juvenile atopic dermatitis models | |
| Hou et al. | Regulation of the Paneth cell niche by exogenous L‐arginine couples the intestinal stem cell function | |
| Morales et al. | Interleukin-10 regulates goblet cell numbers through Notch signaling in the developing zebrafish intestine | |
| JP2009029788A (en) | Skin care composition for external use containing seawater-derived yeast culture | |
| DE10320602B4 (en) | A method for identifying a possible modification of at least one biological parameter using young and old living cells | |
| JP6630066B2 (en) | Enzyme activator | |
| KR20150084629A (en) | Luterial and Method for Isolating and Culturing the Same | |
| JP6824689B2 (en) | Screening method for dermis stain preventive / ameliorating agent and / or macrophage attractant | |
| JP7184593B2 (en) | Dermal blemish improver | |
| DE10320633A1 (en) | Method for identifying a possible modification of at least one biological parameter using living cells that have been exposed to a stimulus and living cells that have not been exposed to this stimulus | |
| JP6042904B2 (en) | Novel markers of papillary and reticulated fibroblasts and uses thereof | |
| CN102026631B (en) | Emollient composition for the preventive treatment of atopic dermatitis | |
| JP6810288B2 (en) | Screening method for dermis stain preventive / ameliorating agent and / or macrophage attractant | |
| EP2303928B1 (en) | Process for obtaining polysaccharides from jatoba (hymenea courbaril l) seeds | |
| JP2015197337A (en) | Screening method of material having horny plug formation suppressing effect | |
| JP2010099015A (en) | Method for screening agent for ameliorating pimple and agent for ameliorating atopic dermatitis | |
| KR102106813B1 (en) | Compositions for preventing skin damage and accelerating skin regeneration containing Allium cepa Extracts | |
| JP2017112998A (en) | Cell laminated bodies, biological graft materials, methods for evaluating differentiation state of epidermis, and methods of producing cell laminated bodies, as well as insulin-like growth factor-1 production promoters | |
| Chávez-Munguı́a et al. | Entamoeba histolytica: ultrastructure of trophozoites recovered from experimental liver lesions | |
| JP6113981B2 (en) | Screening method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210910 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20220624 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220628 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220804 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20221122 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20221124 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7184593 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |