JP2013103927A - Melanocyte differentiation induction inhibitor and method for using the same - Google Patents
Melanocyte differentiation induction inhibitor and method for using the same Download PDFInfo
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- JP2013103927A JP2013103927A JP2011250946A JP2011250946A JP2013103927A JP 2013103927 A JP2013103927 A JP 2013103927A JP 2011250946 A JP2011250946 A JP 2011250946A JP 2011250946 A JP2011250946 A JP 2011250946A JP 2013103927 A JP2013103927 A JP 2013103927A
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- melanocytes
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Abstract
Description
本発明は、例えば、幹細胞からメラノサイトへの分化誘導抑制剤及び当該分化誘導抑制剤を含有する色素異常症改善若しくは治療用又はシミ改善用組成物に関する。 The present invention relates to, for example, an agent for suppressing differentiation induction from stem cells to melanocytes and a composition for improving or treating dysplasia containing the agent for suppressing differentiation, or for improving stains.
メラノサイトは、メラニン色素を合成し、動物の皮膚の色や毛色を決めている細胞である。メラノサイトは、脊椎動物発生の基本となる神経管から派生する神経堤細胞に由来する。神経堤細胞は、神経管から真皮内に左右対称に腹側に遊走し、増殖しながら成熟する。最終的に、神経堤細胞は毛包に定着した後、さらに分化し、且つ成熟することで、メラノサイトへと分化し、且つ成熟する。 Melanocytes are cells that synthesize melanin pigments and determine the skin color and hair color of animals. Melanocytes are derived from neural crest cells derived from the neural tube that is the basis of vertebrate development. Neural crest cells migrate to the ventral side symmetrically from the neural tube into the dermis and mature while proliferating. Finally, neural crest cells settle into hair follicles, and further differentiate and mature to differentiate into melanocytes and mature.
また、毛包内では、成熟したメラノサイトとともに、バルジ領域付近に未分化な色素幹細胞が存在する。当該未分化色素幹細胞は、自己増殖するとともに、必要に応じて未熟なメラノブラストを経て、成熟したメラノサイトを表皮や毛母に供給していると考えられている。供給された成熟メラノサイトは周辺のケラチノサイトにメラノソームを移送することで、皮膚や毛に色素を供給している。このように、メラノサイトには様々な分化段階が存在する。 In the hair follicle, undifferentiated pigment stem cells are present in the vicinity of the bulge region together with mature melanocytes. The undifferentiated pigment stem cells are considered to be self-proliferating and supplying mature melanocytes to the epidermis and hair matrix through immature melanoblast as necessary. The supplied mature melanocytes supply pigments to the skin and hair by transferring melanosomes to the surrounding keratinocytes. Thus, there are various differentiation stages in melanocytes.
メラノサイトの分化及び増殖並びにメラニン合成機構の異常は、様々な色素異常症やシミの原因となる。例えば、神経堤細胞の遊走に異常が起きると、表皮層へ移動するはずの神経堤細胞が真皮に残存することとなる。当該真皮への残存は、生後様々な刺激で遅発性に顕在化してくる対称性真皮メラノサイトーシスを引き起こす。本症の病変は褐色調で左右対称性である。当該疾患は圧倒的に女性に多く、また10歳代後半から20歳代に発症が多い。従って、対称性真皮メラノサイトーシスに対する女性ホルモンの関与が考えられている。しかしながら、その原因は未だ不明な点が多い。 Melanocyte differentiation and proliferation as well as abnormalities in the mechanism of melanin synthesis cause various pigment abnormalities and spots. For example, when an abnormality occurs in the migration of neural crest cells, neural crest cells that should move to the epidermis layer remain in the dermis. Remaining in the dermis causes symmetric dermal melanocytosis that is manifested late by various stimuli after birth. The lesion is brown and symmetrical. The disease is overwhelmingly common in women, and it often develops in the late 10s to 20s. Therefore, the involvement of female hormones in symmetric dermal melanocytosis is considered. However, the cause is still unclear.
また、肝斑は、圧倒的に女性に多く、特に30歳代の女性に多く発症するシミである。肝斑の臨床症状は左右対称性の扁平な褐色斑であり、頬や口周囲及び前額部に生じる。肝斑の発症は、妊娠2〜3ヶ月に始まり、漸次高度となり、分娩後月経開始とともに徐々に消退する。しかしながら、肝斑が長期に持続して閉経後に及ぶこともある。さらに、女性ホルモンを含有する経口避妊薬により肝斑が誘発されることが知られている。 In addition, melasma is a stain that occurs predominantly in women, especially in women in their 30s. The clinical manifestations of melasma are symmetrical, brown brown spots that occur around the cheeks, around the mouth and forehead. The onset of melasma begins in the second or third month of pregnancy, gradually increases in severity, and gradually disappears with the onset of menstruation after delivery. However, melasma may persist for a long time and extend after menopause. Furthermore, it is known that melasma is induced by oral contraceptives containing female hormones.
ところで、メラノサイトの増殖や分化に働く代表的な外的要因としては、紫外線が挙げられる。紫外線は、表皮細胞に働きかけ、各種のメラノサイト増殖及び分化促進因子の分泌を促す。また、紫外線はメラノサイトに直接作用し、各種メラノサイト活性化因子に対する受容体の発現を向上させることも知られている。老人性色素斑は、この紫外線によるメラノサイトの増加及びメラニン合成の亢進が長年繰り返されることによって引き起こされる。日本人では、60歳台になると、ほぼ100％の人に老人性色素斑が認められ、頻度に男女差はない。顔、肩、背、手等の日光に比較的当たりやすい部位が老人性色素斑の好発部位であり、褐色から黒褐色の平らな斑を生じる。 By the way, a typical external factor that acts on the proliferation and differentiation of melanocytes includes ultraviolet rays. Ultraviolet rays act on epidermal cells and promote the proliferation of various melanocytes and the secretion of differentiation promoting factors. It is also known that ultraviolet rays act directly on melanocytes and improve the expression of receptors for various melanocyte activators. Senile pigment spots are caused by many years of repeated increase in melanocytes and increased melanin synthesis caused by ultraviolet rays. In Japanese, almost 100% of people have senile pigment spots at the age of 60, and there is no gender difference in frequency. Sites that are relatively easily exposed to sunlight, such as the face, shoulders, back, hands, etc., are the most frequent sites of senile pigment spots, and produce brown to black-brown flat spots.
以上のように、メラノサイトの発生、分化及び増殖並びにメラニン合成に異常が生じると、様々な色素異常症及びシミを生じる。これらの疾患は直接外面の表現系として現れるため、患者の精神的負担が重く、QOL(生活の質)を妨げる要因の一つとなっている。従って、これらの疾患を根本的に改善するためにメラノサイト制御因子の同定が不可欠である。 As described above, when abnormalities occur in the generation, differentiation and proliferation of melanocytes and melanin synthesis, various dyschromia and spots are generated. Since these diseases appear directly as an external expression system, the patient's mental burden is heavy and is one of the factors that hinder quality of life (QOL). Therefore, the identification of melanocyte regulators is essential to fundamentally improve these diseases.
一方、これまでにメラノサイトを制御する美白剤として、メラニン合成に関わる酵素であるチロシナーゼの活性阻害効果に着目した美白剤が多く開発されている。チロシナーゼ活性阻害効果を有する物質としては、例えばコウジ酸、ハイドロキノン、L-アスコルビン酸等がよく知られている。また、バショウ属植物の抽出液(特許文献１)やヤブコウジ科植物エンベリアの抽出物(特許文献２）等の天然成分を有効成分とするチロシナーゼ活性阻害及びメラニン生成抑制剤が開発されている。しかしながら、これらチロシナーゼ阻害剤は、主に分化後のメラノサイト(チロシナーゼを発現し、且つメラニン合成能を有する)に作用しているに過ぎず、根本的にメラノサイトを制御するものとは言えない。 On the other hand, as a whitening agent for controlling melanocytes, many whitening agents have been developed that focus on the activity inhibitory effect of tyrosinase, an enzyme involved in melanin synthesis. As substances having an inhibitory effect on tyrosinase activity, for example, kojic acid, hydroquinone, L-ascorbic acid and the like are well known. In addition, tyrosinase activity inhibitors and melanin production inhibitors containing natural ingredients such as an extract of a genus Pseudomonas plant (Patent Document 1) and an extract of an Ambudiaceae plant Embellia (Patent Document 2) have been developed. However, these tyrosinase inhibitors mainly act on differentiated melanocytes (express tyrosinase and have melanin synthesis ability) and cannot be said to fundamentally control melanocytes.
また、これまでに、海草類抽出物に由来するチロシナーゼ阻害剤が幾つか報告されている。例えば、特許文献３は、アイヌワカメを始めとするAlaria属の海藻が、マッシュルーム由来チロシナーゼに対する阻害効果を有することを記載する。しかしながら、上記の美白剤(コウジ酸、ハイドロキノン、L-アスコルビン酸等)や天然成分(バショウ属植物の抽出液、ヤブコウジ科植物エンベリアの抽出物等)と同様に、特許文献３において、当該海草類抽出物がメラノサイトの分化自体に及ぼす影響に関しては検討されていなかった。 In addition, several tyrosinase inhibitors derived from seaweed extracts have been reported so far. For example, Patent Document 3 describes that seaweeds of the genus Alaria including Ainu wakame have an inhibitory effect on mushroom-derived tyrosinase. However, in the same manner as in the above-mentioned whitening agents (kojic acid, hydroquinone, L-ascorbic acid, etc.) and natural ingredients (extracts of the genus Ganoderma genus, extract of embeliaceae plant Embellia, etc.), Patent Document 3 discloses the extraction of seaweeds. The effect of the product on the differentiation of melanocytes has not been studied.
上述のように、メラノサイトには様々な分化段階(神経堤細胞、色素幹細胞、メラノブラスト等)が存在しており、その分化異常により様々な色素異常症やシミが引き起こされる。よって、このメラノサイトの分化自体を制御する因子の同定が根本的な色素異常症及びシミの解決のために重要である。しかしながら、従来においては、メラノサイト制御因子の探索には、主にメラノーマ細胞(メラノサイトの癌化により生じる)、最終分化後のメラノサイト及びマッシュルーム由来チロシナーゼ等を用いて行うスクリーニング系が用いられていた。そのため、メラノサイトの分化を制御する因子の探索は従来において行われていなかった。 As described above, melanocytes have various differentiation stages (neural crest cells, pigment stem cells, melanoblasts, etc.), and various pigment abnormalities and spots are caused by the abnormal differentiation. Therefore, identification of factors that control the differentiation itself of melanocytes is important for the resolution of fundamental dysplasia and spots. However, in the past, screening systems that use melanoma cells (generated by canceration of melanocytes), melanocytes after final differentiation, mushroom-derived tyrosinase, and the like have been used to search for melanocyte regulatory factors. Therefore, the search of the factor which controls the differentiation of a melanocyte has not been performed conventionally.
上述のように、メラノサイトの分化自体を制御する因子の同定が根本的な色素異常症及びシミの解決のために重要である。 As described above, the identification of factors that control the differentiation of melanocytes themselves is important for the resolution of fundamental dysplasia and spots.
そこで、本発明は、上述した実情に鑑み、幹細胞からメラノサイトへの分化抑制活性を有する素材を見出し、これまでにない根本的な色素異常症改善又は治療用組成物及びシミ改善用組成物を提供することを目的とする。 Therefore, in view of the above-mentioned circumstances, the present invention finds a material having an activity of suppressing differentiation from stem cells to melanocytes, and provides an unprecedented radical pigmentation improvement or treatment composition and a stain improvement composition. The purpose is to do.
上記課題を解決するため鋭意検討を行った結果、幹細胞からメラノサイトへの分化誘導系を用いることにより、メラノサイトの発生、分化、増殖及びメラニン合成の全てを試験管内(in vitro)で再現できることを確認し、さらに本誘導系をスクリーニング系として用いることにより、チガイソ科に属するアイヌワカメ(学名：Alaria praelonga Kjellman)の抽出物がメラノサイトの分化を抑制する優れた効果を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, it was confirmed that the generation, differentiation, proliferation and melanin synthesis of melanocytes can be reproduced in vitro by using a differentiation induction system from stem cells to melanocytes. Furthermore, by using this induction system as a screening system, it was found that an extract of Ainu sea turtle (scientific name: Alaria praelonga Kjellman) belonging to the family Chigaiso has an excellent effect of suppressing melanocyte differentiation and completed the present invention. It came to do.
すなわち、本発明は、以下を包含する。
（１）アイヌワカメの抽出物を有効成分として含有するメラノサイト分化誘導抑制剤。
（２）前記抽出物が水、低級アルコール、液状多価アルコール及び無機酸から成る群より選択される溶媒により抽出された抽出物である、（１）記載のメラノサイト分化誘導抑制剤。
That is, the present invention includes the following.
(1) A melanocyte differentiation induction inhibitor containing an extract of Ainu Wakame as an active ingredient.
(2) The melanocyte differentiation induction inhibitor according to (1), wherein the extract is an extract extracted with a solvent selected from the group consisting of water, lower alcohols, liquid polyhydric alcohols, and inorganic acids.
（３）低級アルコールがエタノールである、（２）記載のメラノサイト分化誘導抑制剤。
（４）液状多価アルコールが1,3-ブチレングリコールである、（２）記載のメラノサイト分化誘導抑制剤。
(3) The melanocyte differentiation induction inhibitor according to (2), wherein the lower alcohol is ethanol.
(4) The melanocyte differentiation induction inhibitor according to (2), wherein the liquid polyhydric alcohol is 1,3-butylene glycol.
（５）無機酸が塩酸である、（２）記載のメラノサイト分化誘導抑制剤。
（６）（１）〜（５）のいずれか１記載のメラノサイト分化誘導抑制剤を含有する色素異常症改善又は治療用組成物。
(5) The melanocyte differentiation induction inhibitor according to (2), wherein the inorganic acid is hydrochloric acid.
(6) A composition for improving or treating dysplasia containing the melanocyte differentiation induction inhibitor according to any one of (1) to (5).
（７）（１）〜（５）のいずれか１記載のメラノサイト分化誘導抑制剤を含有するシミ改善用組成物。
（８）幹細胞をメラノサイト分化誘導系において候補物質と接触させる工程と、該候補物質非存在下での該幹細胞と比較して、該候補物質と接触した該幹細胞においてメラニン合成量又はメラノサイトマーカー遺伝子の発現が調節されていることにより、該候補物質がメラノサイト分化誘導調節剤であると判定する工程とを含む、メラノサイト分化誘導調節剤のスクリーニング方法。
(7) A stain improving composition comprising the melanocyte differentiation induction inhibitor according to any one of (1) to (5).
(8) a step of bringing a stem cell into contact with a candidate substance in a melanocyte differentiation induction system, and the amount of melanin synthesis or melanocyte marker gene in the stem cell in contact with the candidate substance compared to the stem cell in the absence of the candidate substance A method for screening a melanocyte differentiation-inducing regulator, comprising the step of determining that the candidate substance is a melanocyte differentiation-inducing regulator by regulating expression.
本発明によれば、幹細胞からメラノサイトへの分化誘導を顕著に抑制することでメラノサイトの分化を根本的に制御することができる。従って、本発明は、メラノサイトが関与する色素異常症及びシミの治療、予防及び改善の分野において大きく貢献できるものであり、医学、医薬品、医薬部外品、美容及び健康分野への応用が可能である。 According to the present invention, differentiation of melanocytes can be fundamentally controlled by remarkably suppressing differentiation induction from stem cells to melanocytes. Therefore, the present invention can greatly contribute to the field of treatment, prevention and improvement of dysplasia and stains involving melanocytes, and can be applied to the fields of medicine, pharmaceuticals, quasi drugs, beauty and health. is there.
以下、本発明を詳細に説明する。
上述のように、従来のメラノーマ細胞や最終分化後のメラノサイト及びマッシュルーム由来チロシナーゼ等を用いて行う従来のスクリーニング系ではなく、未分化な幹細胞からメラノサイトへの分化誘導系をスクリーニング系として用いていることにより、メラノサイトの分化自体を抑制する素材の探索を行ったところ、チガイソ科に属するアイヌワカメ(学名：Alaria praelonga Kjellman)の抽出物が、幹細胞からメラノサイトへの分化誘導抑制活性を有することを見出した。
Hereinafter, the present invention will be described in detail.
As mentioned above, the screening system uses differentiation induction system from undifferentiated stem cells to melanocytes instead of the conventional screening system that uses conventional melanoma cells, terminally differentiated melanocytes and mushroom-derived tyrosinase, etc. As a result of searching for a material that suppresses differentiation of melanocytes, it was found that an extract of Ainu sea turtle (scientific name: Alaria praelonga Kjellman) belonging to the family Chigaiso has activity to suppress differentiation induction from stem cells to melanocytes. .
従って、本発明に係るメラノサイト分化誘導抑制剤は、アイヌワカメの抽出物を有効成分として含有するものである。本発明に係るメラノサイト分化誘導抑制剤によれば、幹細胞からメラノサイトへの分化を抑制することで、メラノサイトの分化を制御することができる。また、本発明に係るメラノサイト分化誘導抑制剤によれば、メラノサイトの分化を制御することで、メラノサイトが関連する色素異常症及びシミ等の疾患を根本的に予防、改善及び治療することができる。さらに、幹細胞からメラノサイトへの分化を抑制するための研究用試薬として、本発明に係るメラノサイト分化誘導抑制剤を使用することもできる。 Therefore, the melanocyte differentiation induction inhibitor according to the present invention contains an Ainu turtle extract as an active ingredient. According to the melanocyte differentiation induction inhibitor according to the present invention, the differentiation of melanocytes can be controlled by suppressing the differentiation from stem cells to melanocytes. In addition, according to the melanocyte differentiation induction inhibitor according to the present invention, by controlling the differentiation of melanocytes, it is possible to fundamentally prevent, improve and treat diseases such as dysplasia associated with melanocytes and blemishes. Furthermore, the melanocyte differentiation induction inhibitor according to the present invention can also be used as a research reagent for suppressing the differentiation of stem cells into melanocytes.
本発明においては、先ずアイヌワカメの抽出物を準備する。アイヌワカメを溶媒抽出に供することで、アイヌワカメの抽出物を得ることができる。アイヌワカメの抽出物の溶媒抽出においては、アイヌワカメ本体をそのまま使用してもよく、あるいは、乾燥、粉砕、細切等の処理を行ったアイヌワカメ調製物を使用することもできる。 In the present invention, first, an Ainu Wakame extract is prepared. An Ainu-wakame extract can be obtained by subjecting Ainu-wakame to solvent extraction. In the solvent extraction of the Ainuwakame extract, the Ainuwakame body may be used as it is, or an Ainuwakame preparation that has been subjected to treatments such as drying, pulverization, and shredding can also be used.
抽出に使用する溶媒としては、例えば、水(熱水)、低級アルコール類(メタノール、エタノール、1-プロパノール、2-プロパノール、1-ブタノール、2-ブタノール等）、液状多価アルコール(1,3-ブチレングリコール、プロピレングリコール、グリセリン等）、ケトン類(アセトン、メチルエチルケトン等)、アセトニトリル、エステル類(酢酸エチル、酢酸ブチル等）、炭化水素類(ヘキサン、ヘプタン、流動パラフィン等)、エーテル類(エチルエーテル、テトラヒドロフラン、プロピルエーテル等)、無機酸(塩酸等)等が挙げられる。水、低級アルコール、液状多価アルコール及び無機酸が好ましく、水、エタノール、1,3-ブチレングリコール及び塩酸が特に好ましい。これらの溶媒は単独で、又は2種以上を混合して用いても良い。また、これら溶媒に酸やアルカリを添加してpH調整を行うこともできる。特に、エタノール又は1,3-ブチレングリコールを溶媒として使用する場合には、例えば50〜100％(v/v)、好ましくは50〜60％(v/v)の濃度のエタノール又は1,3-ブチレングリコールを使用することができる。また、塩酸を溶媒として使用する場合には、例えば0.01〜1N、特に0.1Nの塩酸を使用することができる。 Examples of the solvent used for extraction include water (hot water), lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3 -Butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl) Ether, tetrahydrofuran, propyl ether, etc.), inorganic acids (hydrochloric acid, etc.) and the like. Water, lower alcohol, liquid polyhydric alcohol and inorganic acid are preferable, and water, ethanol, 1,3-butylene glycol and hydrochloric acid are particularly preferable. These solvents may be used alone or in combination of two or more. Further, the pH can be adjusted by adding acid or alkali to these solvents. In particular, when ethanol or 1,3-butylene glycol is used as a solvent, for example, ethanol or 1,3-butyl alcohol having a concentration of 50 to 100% (v / v), preferably 50 to 60% (v / v) is used. Butylene glycol can be used. When hydrochloric acid is used as a solvent, for example, 0.01 to 1N, particularly 0.1N hydrochloric acid can be used.
上述の溶媒を用いて、アイヌワカメを溶媒抽出に供する。アイヌワカメに対する溶媒の割合は、例えば0.5〜50％(w/v)、好ましくは1〜20％(w/v)が挙げられる。溶媒抽出方法は、特に限定されないが、加熱による抽出が好ましい。例えば、アイヌワカメ乾燥物に水を加え、95〜100℃における熱水抽出を行うことで、アイヌワカメ抽出物を得ることができる。あるいは、アイヌワカメ乾燥物に無機酸(例えば、塩酸)を加え、4℃程度で抽出を行うことで、アイヌワカメ抽出物を得ることができる。また、アイヌワカメ乾燥物に低級アルコール又は液状多価アルコール(例えば、エタノール、1,3-ブチレングリコール等)を添加し、常温(例えば5〜35℃)で抽出を行うことで、アイヌワカメ抽出物を得ることができる。 Ainu seaweed is subjected to solvent extraction using the solvent described above. The ratio of the solvent to Ainu Wakame is, for example, 0.5 to 50% (w / v), preferably 1 to 20% (w / v). The solvent extraction method is not particularly limited, but extraction by heating is preferable. For example, an Ainu turtle extract can be obtained by adding water to a dried Ainu turtle and performing hot water extraction at 95 to 100 ° C. Alternatively, an Ainu wakame extract can be obtained by adding an inorganic acid (for example, hydrochloric acid) to the dried Ainu wakame and performing extraction at about 4 ° C. Ainu seaweed extract is obtained by adding lower alcohol or liquid polyhydric alcohol (eg, ethanol, 1,3-butylene glycol, etc.) to dried Ainu seaweed and performing extraction at room temperature (eg, 5 to 35 ° C.). Can be obtained.
溶媒抽出後、得られた溶媒相自体をアイヌワカメ抽出物とすることができる。あるいは、必要に応じて、得られた溶媒相を、濃縮、希釈、濾過、乾燥等の処理及び活性炭等による脱色処理、脱臭処理等に供して、得られた生成物をアイヌワカメ抽出物とすることができる。特に、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理に供し、得られた乾燥物をアイヌワカメ抽出物として用いることが好ましい。 After solvent extraction, the obtained solvent phase itself can be used as an Ainu Wakame extract. Alternatively, the obtained solvent phase is subjected to a treatment such as concentration, dilution, filtration, and drying, and a decolorization treatment using activated carbon, a deodorization treatment, or the like, if necessary, and the resulting product is used as an Ainu Wakame extract. be able to. In particular, it is preferable that the extracted solution is subjected to a treatment such as concentration to dryness, spray drying, freeze drying and the like, and the obtained dried product is used as an Ainu Wakame extract.
このようにして得られたアイヌワカメ抽出物を本発明に係るメラノサイト分化誘導抑制剤の有効成分とする。本発明に係るメラノサイト分化誘導抑制剤は、医薬、医薬部外品、化粧料又は飲食品として使用することができる。また、上述のアイヌワカメ抽出物を、医薬、医薬部外品、化粧料又は飲食品の製造のために使用することもできる。特に、本発明に係る分化誘導抑制剤は、色素異常症改善若しくは治療用組成物又はシミ改善用組成物として、又は当該組成物の製造のために使用することができる。 The Ainu Wakame extract thus obtained is used as an active ingredient of the melanocyte differentiation induction inhibitor according to the present invention. The melanocyte differentiation induction inhibitor according to the present invention can be used as a medicine, a quasi drug, a cosmetic, or a food or drink. Moreover, the above-mentioned Ainu Wakame extract can also be used for the manufacture of pharmaceuticals, quasi drugs, cosmetics or foods and drinks. In particular, the differentiation induction inhibitor according to the present invention can be used as a dysplasia improvement or treatment composition or a stain improvement composition, or for the production of the composition.
ここで、色素異常症としては、例えば対称性真皮メラノサイトーシス、肝斑、老人性色素斑、雀卵斑、Riehl黒皮症、摩擦黒皮症、遺伝性対側性色素異常症、Addison病、光線性花弁状色素斑、色素異常性固定紅斑等が挙げられる。 Here, examples of dyschromia include symmetric dermal melanocytosis, liver spots, senile pigment spots, sparrow egg spot, Riehl melanosis, trichodermatosis, hereditary contralateral dyschromia, Addison disease , Photo-petal pigment spots, pigmented abnormal erythema, and the like.
さらに、本発明に係るメラノサイト分化誘導抑制剤を医薬、医薬部外品、化粧料又は飲食品として利用する場合には、その取扱い説明書又はパッケージに、アイヌワカメ抽出物を含有し、メラノサイト分化誘導抑制活性を有することを特徴とする旨を表示することができる。 Further, when the melanocyte differentiation induction inhibitor according to the present invention is used as a medicine, quasi-drug, cosmetic or food / drink, it contains an Ainu wakame extract in its instruction manual or package, and induces melanocyte differentiation. The fact that it has the inhibitory activity can be displayed.
本発明に係るメラノサイト分化誘導抑制剤を、医薬又は医薬部外品として使用する場合には、剤形としては、特に限定されるものではないが、例えば、内服剤(錠剤、カプセル剤等)、外用剤(軟膏(クリーム)、水剤、エキス剤、ローション剤、乳剤等)並びに注射剤が挙げられる。当該医薬又は医薬部外品には、アイヌワカメ抽出物の他に、助剤、安定化剤、湿潤剤、乳化剤、吸収促進剤及び界面活性剤等の薬学的に許容される担体を任意に組合せて配合することができる。 When the melanocyte differentiation induction inhibitor according to the present invention is used as a medicine or quasi-drug, the dosage form is not particularly limited, but for example, internal use (tablets, capsules, etc.), External preparations (ointments (creams), liquids, extracts, lotions, emulsions, etc.) and injections are mentioned. In addition to the Ainu wakame extract, the drug or quasi-drug is optionally combined with a pharmaceutically acceptable carrier such as an auxiliary agent, stabilizer, wetting agent, emulsifier, absorption enhancer and surfactant. Can be blended.
本発明に係るメラノサイト分化誘導抑制剤を化粧料として使用する場合には、剤形としては、特に限定されるものではないが、例えば、クリーム、ローション、フォーム、エッセンス、ファンデーション、パック、スティック及びパウダー等が挙げられる。当該化粧料には、アイヌワカメ抽出物の他に、化粧料成分として一般に使用されている油分、界面活性剤、紫外線吸収剤、アルコール類、キレート剤、pH調整剤、防腐剤、増粘剤、色素類、香料等を任意に組合せて配合することができる。 When the melanocyte differentiation induction inhibitor according to the present invention is used as a cosmetic, the dosage form is not particularly limited. For example, cream, lotion, foam, essence, foundation, pack, stick and powder Etc. In addition to Ainu Wakame extract, the cosmetics include oils, surfactants, ultraviolet absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, which are commonly used as cosmetic ingredients. Pigments, fragrances and the like can be combined in any combination.
本発明に係るメラノサイト分化誘導抑制剤におけるアイヌワカメ抽出物の配合量は、特に限定されるものではないが、乾燥物として計算して、全組成の0.00001〜10重量％とすることが好ましく、0.0001〜1重量％とすることが最も好ましい。全組成の0.00001重量％未満とすると、メラノサイト分化誘導抑制効果が十分に発揮されにくい場合がある。 The compounding amount of Ainu Wakame extract in the melanocyte differentiation induction inhibitor according to the present invention is not particularly limited, but is preferably 0.00001 to 10% by weight of the total composition, calculated as a dry product, 0.0001 Most preferred is ˜1 wt%. If it is less than 0.00001% by weight of the total composition, the melanocyte differentiation induction suppressing effect may not be sufficiently exerted.
以上に説明した本発明に係るメラノサイト分化誘導抑制剤を、ヒトを含めた動物に適用することで、幹細胞からメラノサイトへの分化誘導を抑制でき、また色素異常症又はシミを治療、予防又は改善することができる。本発明に係るメラノサイト分化誘導抑制剤の幹細胞からメラノサイトへの分化誘導抑制活性は、下記の本発明に係るスクリーニング方法に準じて評価することができる。 By applying the melanocyte differentiation induction inhibitor according to the present invention described above to animals including humans, differentiation induction from stem cells to melanocytes can be suppressed, and pigmentation disorders or spots can be treated, prevented or improved. be able to. The activity of inhibiting differentiation induction from stem cells to melanocytes of the melanocyte differentiation induction inhibitor according to the present invention can be evaluated according to the screening method according to the present invention described below.
一方、本発明に係るスクリーニング方法は、幹細胞をメラノサイト分化誘導系において候補物質と接触させる工程と、候補物質非存在下での該幹細胞と比較して、該候補物質と接触した該幹細胞においてメラニン合成量又はメラノサイトマーカー遺伝子の発現が調節されていることにより、該候補物質がメラノサイト分化誘導調節剤であると判定する工程とを含む、メラノサイト分化誘導調節剤のスクリーニング方法である。本発明に係るスクリーニング方法では、メラノサイト分化誘導系において各種候補物質存在下で幹細胞の分化誘導を行った後、メラニン合成量と細胞数とを定量し、該候補物質が細胞数当たりのメラニン合成量を促進した場合にメラノサイト分化誘導促進剤として、またメラニン合成量を抑制した場合にメラノサイト分化誘導抑制剤として候補物質を選択するものである。あるいは、メラノサイトマーカー遺伝子の発現を指標として、候補物質が当該遺伝子の発現を促進した場合にメラノサイト分化誘導促進剤として、また当該遺伝子の発現を抑制した場合にメラノサイト分化誘導抑制剤として候補物質を選択することができる。 On the other hand, the screening method according to the present invention comprises a step of contacting a stem cell with a candidate substance in a melanocyte differentiation induction system, and a melanin synthesis in the stem cell in contact with the candidate substance as compared with the stem cell in the absence of the candidate substance. And a step of determining that the candidate substance is a melanocyte differentiation-inducing regulator by regulating the amount or expression of the melanocyte marker gene. In the screening method according to the present invention, after inducing differentiation of stem cells in the presence of various candidate substances in the melanocyte differentiation induction system, the amount of melanin synthesis and the number of cells are quantified, and the candidate substance is the amount of melanin synthesis per number of cells. The candidate substance is selected as a melanocyte differentiation-inducing promoter when the selenium is promoted, and as a melanocyte differentiation-inducing inhibitor when the amount of melanin synthesis is suppressed. Alternatively, using the expression of the melanocyte marker gene as an index, select the candidate substance as a melanocyte differentiation induction promoter when the candidate substance promotes the expression of the gene, and as a melanocyte differentiation induction inhibitor when the gene expression is suppressed can do.
本発明に係るスクリーニング方法で使用する幹細胞としては、メラノサイトに分化する幹細胞であればいずれのものであってよく、例えば胚性幹細胞(ES細胞）；骨髄、血液、皮膚、脂肪、脳、肝臓、膵臓、腎臓、筋肉やその他の組織に存在する未分化な状態の細胞(総称して、体性幹細胞と称する)；遺伝子導入等により人工的に作製された幹細胞等が挙げられる。これら幹細胞は、初代培養細胞、継代培養細胞、凍結細胞のいずれであってもよい。好ましくは、ES細胞又は骨髄、血液、皮膚若しくは脂肪組織由来の幹細胞を使用することができる。また、幹細胞の分化の方向性及び分化の過程等について同等の特性を持っていれば、全ての哺乳動物由来の幹細胞を使用することができる。例えば、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物由来の幹細胞を使用することができる。 Stem cells used in the screening method according to the present invention may be any stem cells that differentiate into melanocytes, such as embryonic stem cells (ES cells); bone marrow, blood, skin, fat, brain, liver, Examples include undifferentiated cells existing in pancreas, kidney, muscle and other tissues (collectively referred to as somatic stem cells); stem cells artificially prepared by gene transfer and the like. These stem cells may be primary cultured cells, subcultured cells, or frozen cells. Preferably, ES cells or stem cells derived from bone marrow, blood, skin or adipose tissue can be used. In addition, stem cells derived from all mammals can be used as long as they have equivalent characteristics regarding the direction of differentiation of the stem cells and the process of differentiation. For example, stem cells derived from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs can be used.
一方、本発明に係るスクリーニング方法において、候補物質としては、例えば、核酸、ペプチド、タンパク質、合成化合物、微生物の培養上清、植物や海洋生物由来の天然成分、植物抽出物、動物組織抽出物等が挙げられる。 On the other hand, in the screening method according to the present invention, examples of candidate substances include nucleic acids, peptides, proteins, synthetic compounds, culture supernatants of microorganisms, natural components derived from plants and marine organisms, plant extracts, animal tissue extracts, etc. Is mentioned.
本発明に係るスクリーニング方法において、幹細胞培養培地又は幹細胞からメラノサイトへの分化誘導培地、また、これら培地と同時に用いる添加剤としては、限定されるものではないが、例えば以下のものが挙げられる。 In the screening method according to the present invention, a stem cell culture medium, a differentiation induction medium from stem cells to melanocytes, and an additive used simultaneously with these mediums are not limited, and examples thereof include the following.
培地としては、増殖因子として白血球遊走阻止因子(LIF)の少なくともいずれか1種(好ましくは、これら増殖因子の全て)を添加した、細胞の生存に必要な成分(無機塩、炭水化物、ホルモン、必須アミノ酸、非必須アミノ酸、ビタミン等)を含む基本培地が挙げられる。当該基本培地としては、例えば、Dulbecco's Modified Eagle Medium(D-MEM)、Minimum Essential Medium(MEM)、RPMI 1640、Basal Medium Eagle(BME)、Dulbecco's Modified Eagle Medium：Nutrient Mixture F-12(D-MEM/F-12)、Glasgow Minimum Essential Medium(Glasgow MEM)、ハンクス液(Hank's balanced salt solution）、MCDB153培地等が挙げられる。また、必要に応じて、培地は、上皮細胞増殖因子(EGF)、腫瘍壊死因子(TNF)、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、フィブロネクチン、プロゲステロン、セレナイト、B27-サプリメント、N2-サプリメント、ITS-サプリメント、抗生物質等を含有してもよい。 As a medium, at least one of leukocyte migration inhibitory factor (LIF) (preferably all of these growth factors) is added as a growth factor, and components necessary for cell survival (inorganic salts, carbohydrates, hormones, essential) And basic media containing amino acids, non-essential amino acids, vitamins, etc.). Examples of the basic medium include Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Hank's balanced salt solution, MCDB153 medium and the like. If necessary, the medium may be epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 -It may contain supplements, N2-supplements, ITS-supplements, antibiotics, etc.
また、上記以外には、1〜20％の含有率でFBS(ウシ胎児血清)等の血清が培地に含まれることが好ましい。しかしながら、血清はロットの違いにより成分が異なり、その効果にバラツキがあるため、ロットチェックを行った後に使用することが好ましい。 In addition to the above, it is preferable that serum such as FBS (fetal bovine serum) is contained in the medium at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in its effects, it is preferably used after lot check.
なお、幹細胞培養又は幹細胞からメラノサイトへの分化誘導における培地の交換は2〜3日に1回行うことが好ましく、より好ましくは毎日行うことが好ましい。また、メラノサイト分化誘導系における幹細胞からメラノサイトへの分化誘導を24日以上行うことが好ましい。 The medium exchange in stem cell culture or differentiation induction from stem cells to melanocytes is preferably performed once every 2 to 3 days, more preferably daily. In addition, differentiation induction from stem cells to melanocytes in the melanocyte differentiation induction system is preferably performed for 24 days or more.
幹細胞培養又は幹細胞からメラノサイトへの分化誘導に使用する容器としては、例えば使い捨てのシャーレ、マイクロタイタープレート等が挙げられる。 Examples of the container used for stem cell culture or differentiation induction from stem cells to melanocytes include disposable petri dishes, microtiter plates, and the like.
幹細胞の培養(前培養)においては、未分化状態を保つために、マイトマイシンCで処理したMEF細胞(マウス胎児線維芽細胞)等をフィーダー細胞として使用する。培地は、LIF、L-グルタミン等を含有することが好ましい。例えば、培地含有容器底面上にフィーダー細胞をコンフルエントの状態まで培養し、その上に幹細胞を播種し、幹細胞を、所定の時間及び培養温度で培養する。培養後、フィーダー細胞から分離した幹細胞を回収し、メラノサイト分化誘導に使用する。 In stem cell culture (preculture), MEF cells (mouse fetal fibroblasts) treated with mitomycin C or the like are used as feeder cells in order to maintain an undifferentiated state. The medium preferably contains LIF, L-glutamine and the like. For example, feeder cells are cultured to a confluent state on the bottom surface of the medium-containing container, stem cells are seeded thereon, and the stem cells are cultured at a predetermined time and a culture temperature. After culturing, the stem cells separated from the feeder cells are collected and used for induction of melanocyte differentiation.
一方、メラノサイト分化誘導系としては、例えばマウスストローマ細胞であるST2細胞上への幹細胞の播種、及びFBS、DEX(デキサメタゾン)、bFGF(塩基性線維芽細胞増殖因子）、CT(コレラトキシン）、EDN3(エンドセリン3)等のメラノサイトの分化を促す因子を添加した培地の使用が挙げられる(Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T., Developmental Dynamics, 1999年, Vol. 216, Issue 4-5, pp. 450-458)。 On the other hand, as a melanocyte differentiation induction system, for example, seeding of stem cells on ST2 cells which are mouse stromal cells, FBS, DEX (dexamethasone), bFGF (basic fibroblast growth factor), CT (cholera toxin), EDN3 Examples include the use of media supplemented with factors that promote melanocyte differentiation such as (endothelin 3) (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T., Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458).
本発明に係るスクリーニング方法では、先ず幹細胞をメラノサイト分化誘導系において候補物質と接触させる。ここで、接触とは、幹細胞からメラノサイトへの分化に候補物質が影響を及ぼす状態を意味する。例えば、メラノサイト分化誘導用培地に候補物質を添加するだけでもよい。あるいは、候補物質をある種のキャリアー物質（タンパク質や脂質等）とともにメラノサイト分化誘導用培地に添加してもよい。また、幹細胞にマイクロインジェクション等で候補物質を直接導入してもよい。 In the screening method according to the present invention, stem cells are first contacted with a candidate substance in a melanocyte differentiation induction system. Here, the contact means a state in which the candidate substance affects the differentiation from stem cells to melanocytes. For example, the candidate substance may be simply added to the melanocyte differentiation induction medium. Alternatively, the candidate substance may be added to the melanocyte differentiation induction medium together with a certain kind of carrier substance (protein, lipid, etc.). In addition, candidate substances may be directly introduced into stem cells by microinjection or the like.
次いで、本発明に係るスクリーニング方法においては、候補物質非存在下での幹細胞と比較して、該候補物質と接触した該幹細胞においてメラニン合成量又はメラノサイトマーカー遺伝子の発現が調節されているか否かを決定する。メラニン合成及びメラノサイトマーカー遺伝子の発現は、幹細胞からメラノサイトへの分化の指標である。ここで、メラニン合成量の調節とは、幹細胞におけるメラニン合成量の低下又は増加を意味する。また、メラノサイトマーカー遺伝子の発現の調節とは、幹細胞におけるmRNAレベル又はタンパク質レベルでのメラノサイトマーカー遺伝子発現の低下又は増加を意味する。メラノサイトマーカー遺伝子としては、例えばMitf-M(メラノサイト特異的小眼球症関連転写因子：Melanocyte-specific Microphthalmia-associated transcription factor)(Tachibana M. Pigment Cell Res.(2000)13(4):230-40.)及びTyrp1(チロシナーゼ関連タンパク質1：Tyrosinase-related protein 1)(Sarangarajan R, Boissy RE. Pigment Cell Res. (2001) 14(6):437-44.)が挙げられる。 Next, in the screening method according to the present invention, it is determined whether or not the amount of melanin synthesis or the expression of the melanocyte marker gene is regulated in the stem cells in contact with the candidate substance, compared to the stem cells in the absence of the candidate substance. decide. Melanin synthesis and melanocyte marker gene expression are indicators of differentiation from stem cells to melanocytes. Here, the regulation of the amount of melanin synthesis means a decrease or increase in the amount of melanin synthesis in the stem cells. The regulation of the expression of the melanocyte marker gene means a decrease or increase in the expression of the melanocyte marker gene at the mRNA level or protein level in the stem cell. Examples of the melanocyte marker gene include Mitf-M (Melanocyte-specific Microphthalmia-associated transcription factor) (Tachibana M. Pigment Cell Res. (2000) 13 (4): 230-40. ) And Tyrp1 (Tyrosinase-related protein 1) (Sarangarajan R, Boissy RE. Pigment Cell Res. (2001) 14 (6): 437-44.).
メラニン合成量の評価方法では、培養後、メラニン合成量と細胞数とを定量し、細胞数当たりのメラニン合成量を算出する。細胞数測定において、例えば市販品のCell Counting Kit-8(同仁化学研究所製)等の測定後も細胞を回収できるキットを用いることが望ましい。また、メラニンの定量方法としては、例えば分化誘導後に細胞数を測定した細胞を2N NaOHで60℃で2時間溶解し、溶解物を475nmの吸光度で測定する方法が挙げられるが、これに限定されるものではない。次いで、得られた細胞数当たりのメラニン合成量を、候補物質不在下で培養した幹細胞における細胞数当たりのメラニン合成量と比較する。 In the method for evaluating the amount of melanin synthesis, after the culture, the amount of melanin synthesis and the number of cells are quantified, and the amount of melanin synthesis per number of cells is calculated. In measuring the number of cells, it is desirable to use a kit that can collect cells even after measurement, such as a commercially available Cell Counting Kit-8 (manufactured by Dojindo Laboratories). Examples of the quantification method for melanin include, but are not limited to, a method in which the number of cells after differentiation induction is lysed with 2N NaOH at 60 ° C. for 2 hours, and the lysate is measured at an absorbance of 475 nm. It is not something. Next, the obtained amount of melanin synthesis per number of cells is compared with the amount of melanin synthesis per number of cells in stem cells cultured in the absence of a candidate substance.
一方、メラノサイトマーカー遺伝子発現の評価方法では、培養後の細胞からmRNA又はタンパク質を抽出する。次いで、得られたmRNA又はタンパク質中のメラノサイトマーカー遺伝子発現量を、候補物質不在下で培養した幹細胞における当該遺伝子発現量と比較する。mRNAレベルでは、例えばメラノサイトマーカー遺伝子に特異的なプライマーやプローブを用いたRT-PCR、定量PCRやノーザンブロッティングによって確認する方法が挙げられる。また、タンパク質レベルでは、例えばメラノサイトマーカー遺伝子によりコードされるタンパク質に特異的な抗体を用いたELISA、フローサイトメトリー、ウエスタンブロッテイング等の免疫学的方法が挙げられる。 On the other hand, in the method for evaluating melanocyte marker gene expression, mRNA or protein is extracted from the cultured cells. Next, the expression level of the melanocyte marker gene in the obtained mRNA or protein is compared with the expression level of the gene in the stem cells cultured in the absence of the candidate substance. At the mRNA level, for example, RT-PCR using a primer or probe specific for the melanocyte marker gene, quantitative PCR, or Northern blotting may be used. At the protein level, for example, immunological methods such as ELISA, flow cytometry, and Western blotting using an antibody specific for the protein encoded by the melanocyte marker gene can be mentioned.
候補物質不在下で培養した幹細胞に比べて、候補物質を接触させた幹細胞において、メラニン合成量又はメラノサイトマーカー遺伝子の発現が有意(例えば、1.5〜100倍、好ましくは2〜5倍)に増加した場合に、あるいは、有意(例えば、1/2〜1/1000、好ましくは1/4〜1/10)に低下した場合に、候補物質がメラノサイト分化誘導調節剤であると判定することができる。 Compared with stem cells cultured in the absence of the candidate substance, the amount of melanin synthesis or the expression of the melanocyte marker gene was significantly increased (for example, 1.5 to 100 times, preferably 2 to 5 times) in the stem cells contacted with the candidate substance In some cases, or when significantly decreased (for example, 1/2 to 1/1000, preferably 1/4 to 1/10), the candidate substance can be determined to be a melanocyte differentiation induction regulator.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
〔実施例１〕アイヌワカメからの溶媒抽出物の製造例
以下に、アイヌワカメを用いた溶媒抽出物の製造例を示す。
１．製造例１ アイヌワカメの熱水抽出物
アイヌワカメの乾燥物20gに精製水800mLを加え、95〜100℃で2時間抽出を行った。抽出後、抽出液を濾過に供し、得られた濾液を濃縮及び凍結乾燥に供することで、アイヌワカメの熱水抽出物を3.4g得た。
[Example 1] Production example of solvent extract from Ainu sea turtle A production example of a solvent extract using Ainu sea turtle is shown below.
1. Production Example 1 Ainu Wakame Hot Water Extract 800 ml of purified water was added to 20 g of dried Ainu Wakame and extracted at 95-100 ° C. for 2 hours. After extraction, the extract was subjected to filtration, and the obtained filtrate was concentrated and freeze-dried to obtain 3.4 g of a hot water extract of Ainu Wakame.
２．製造例２ アイヌワカメのHCl抽出物
アイヌワカメの乾燥物20gに0.1N HCl 800mlを加え、4℃で7日間抽出を行った。抽出後、抽出液を濾過に供し、得られた濾液を濃縮及び凍結乾燥に供することで、アイヌワカメのHCl抽出物を3.1g得た。
2. Production Example 2 Ainu Wakame's HCl Extract To 20 g of dried Ainu Wakame, 800 ml of 0.1N HCl was added, followed by extraction at 4 ° C for 7 days. After extraction, the extract was subjected to filtration, and the obtained filtrate was concentrated and freeze-dried to obtain 3.1 g of an Ainu Wakame HCl extract.
３．製造例３ アイヌワカメの50％エタノール抽出物
アイヌワカメの乾燥物20gに50(v/v)％エタノール400mLを加え、常温で7日間抽出を行った。抽出後、抽出液を濾過に供し、得られた濾液を濃縮乾固に供することで、アイヌワカメの50％エタノール抽出物を3.7g得た。
3. Production Example 3 Ainu Wakame 50% Ethanol Extract To 20 g of dried Ainu Wakame, 400 mL of 50 (v / v)% ethanol was added and extracted at room temperature for 7 days. After extraction, the extract was subjected to filtration, and the obtained filtrate was concentrated to dryness to obtain 3.7 g of a 50% ethanol extract of Ainu Wakame.
４．製造例４ アイヌワカメのエタノール抽出物
アイヌワカメの乾燥物100gにエタノール1Lを加え、常温で7日間抽出を行った。抽出後、抽出液を濾過に供し、得られた濾液を濃縮乾固に供することで、アイヌワカメのエタノール抽出物を6.7g得た。
4). Production Example 4 Ethanol extract of Ainu Wakame 1 L of ethanol was added to 100 g of dried Ainu Wakame and extracted at room temperature for 7 days. After extraction, the extract was subjected to filtration, and the obtained filtrate was concentrated to dryness to obtain 6.7 g of an Ainu wakame ethanol extract.
５．製造例５ アイヌワカメの50％1,3-ブチレングリコール抽出物
アイヌワカメの乾燥物20gに50％1,3-ブチレングリコール水溶液400mLを加え、常温で7日間抽出を行った。抽出後、抽出液を濾過に供することで、アイヌワカメの50％1,3-ブチレングリコール抽出物を370g得た。
5. Production Example 5 Ainu Wakame 50% 1,3-butylene glycol extract To 20 g of dried Ainu Wakame, 400 mL of 50% 1,3-butylene glycol aqueous solution was added, followed by extraction at room temperature for 7 days. After extraction, the extract was subjected to filtration to obtain 370 g of a 50% 1,3-butylene glycol extract of Ainu Wakame.
〔実施例２〕幹細胞からメラノサイトへのアイヌワカメ抽出物の分化誘導抑制効果の評価
以下に、実施例１において製造した製造例１〜５のアイヌワカメ抽出物を用いて、幹細胞からメラノサイトへの分化誘導抑制効果を評価し、該抽出物のメラノサイトへの分化誘導抑制剤としての効果について確認した。
[Example 2] Evaluation of inhibitory effect on differentiation induction of Ainu turtle extract from stem cells to melanocytes Differentiation from stem cells to melanocytes using the Ainu turtle extract of Production Examples 1 to 5 produced in Example 1 below The inhibitory effect on induction was evaluated, and the effect of the extract as an inhibitor of induction of differentiation into melanocytes was confirmed.
１．実験例１ アイヌワカメ抽出物のメラノサイトへの分化誘導抑制効果の評価(メラニン定量試験)
本実験例では、過去に山根らが開発したマウス胚性幹細胞(ES cell：embryonic stem cell)からのメラノサイト分化誘導系(Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T., Developmental Dynamics, 1999年, Vol. 216, Issue 4-5, pp. 450-458)を用いて、アイヌワカメ抽出物の効果を検討した。本誘導系を用いることで、メラノサイトの発生から成熟までの全ての段階において素材の評価を行うことが可能となる。以下に詳細を説明する。
1. Experimental Example 1 Evaluation of the inhibitory effect of Ainu wakame extract on differentiation induction into melanocytes (Melanin quantitative test)
In this experimental example, melanocyte differentiation induction system (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T.) from mouse embryonic stem cells (ES cells) developed by Yamane et al. , Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458), the effect of Ainu Wakame extract was examined. By using this induction system, it is possible to evaluate materials at all stages from the generation of melanocytes to maturity. Details will be described below.
35mmシャーレにMMC(mitomycin C)処理済みのMEF細胞(Mouse embryonic fibroblast)をコンフルエントの状態で培養し、その上にマウスES細胞を10×104〜20×104個播種し、37℃において5％CO2インキュベーターで前培養した。使用した培地は、DMEMにchemicon社製のES細胞用添加因子(L-グルタミン液、2-メルカプトエタノール液、ヌクレオシド液、非必須アミノ酸液及びESGRO)を推奨濃度で添加した後、FBSを15％添加したものであった。 MMC (mitomycin C) -treated MEF cells (Mouse embryonic fibroblast) are cultured in a confluent state on a 35 mm dish, and 10 × 10 4 to 20 × 10 4 mouse ES cells are seeded thereon, and 5 ° C. at 37 ° C. Pre-cultured in a% CO 2 incubator. The medium used was the addition of chemicon additive factors for ES cells (L-glutamine solution, 2-mercaptoethanol solution, nucleoside solution, non-essential amino acid solution and ESGRO) to DMEM at the recommended concentration, and then 15% FBS. It was what was added.
次いで、ST2細胞を24wellプレート上でコンフルエントになるまで培養し、そこにMEF細胞から分離した上述の培養ES細胞を250〜500個播種した。当該培養物を、α-MEMに10％ウシ胎児血清、100nM デキサメタゾン、20pM 塩基性線維芽細胞増殖因子、10pM コレラトキシン及び100ng/mL エンドセリン3を添加した分化誘導培地で培養し、ES細胞をメラノサイトへ分化誘導した。顕微鏡観察により、誘導後6日目にはST2細胞上に形成されたES細胞のコロニーが広がり始め、18日目前後でコロニーの周りにメラノサイトが出現した。誘導後24日目までに、出現したメラノサイトがさらにメラニン合成を行う様子が観察された。以上より、本誘導系を用いることにより、メラノサイトの発生、分化及びメラニン合成の全てを再現できることを確認した。 Subsequently, ST2 cells were cultured until they became confluent on a 24-well plate, and 250-500 cultured ES cells separated from MEF cells were seeded there. The culture is cultured in a differentiation induction medium supplemented with 10% fetal bovine serum, 100 nM dexamethasone, 20 pM basic fibroblast growth factor, 10 pM cholera toxin and 100 ng / mL endothelin 3 in α-MEM, and the ES cells are melanocytes. Differentiation was induced. Microscopic observation revealed that ES cell colonies formed on ST2 cells began to spread on the 6th day after induction, and melanocytes appeared around the colonies around 18th day. By the 24th day after induction, it was observed that the melanocytes that emerged further synthesize melanin. From the above, it was confirmed that the generation of melanocytes, differentiation and melanin synthesis can all be reproduced by using this induction system.
本誘導系において、上述の分化誘導培地に各濃度(12.5、25、50μg/mL)でアイヌワカメ抽出物を継続して添加し、24日間の分化誘導を実施した。その後、Cell Counting Kit-8を用いて相対細胞数を定量した。一方、細胞数定量後、細胞をPBS(-)で3回洗浄した後、2N NaOHを用いて60℃で2時間溶解し、溶解物について475nmの吸光度を測定した。その結果、細胞数当たりのメラニン合成量は濃度依存的に減少した。
これらの試験結果を以下の表１に示す。
In this induction system, Ainu wakame extract was continuously added to the above-described differentiation-inducing medium at each concentration (12.5, 25, 50 μg / mL) to induce differentiation for 24 days. Then, the relative cell number was quantified using Cell Counting Kit-8. On the other hand, after quantifying the number of cells, the cells were washed 3 times with PBS (−) and then dissolved in 2N NaOH at 60 ° C. for 2 hours. The absorbance of the lysate was measured at 475 nm. As a result, the amount of melanin synthesis per cell number decreased in a concentration-dependent manner.
The test results are shown in Table 1 below.
表１に示すように、アイヌワカメ抽出物(製造例１〜５)の全てに、顕著なメラノサイト分化誘導抑制効果が認められた。以上より、アイヌワカメ抽出物が極めて優れた幹細胞からのメラノサイト分化誘導抑制効果を有することが明らかとなり、アイヌワカメ抽出物のメラノサイト分化誘導抑制剤としての効果を確認した。 As shown in Table 1, all of the Ainu Wakame extract (Production Examples 1 to 5) exhibited a remarkable effect of suppressing differentiation induction of melanocytes. From the above, it was revealed that the Ainu wakame extract has an excellent inhibitory effect on melanocyte differentiation induction from stem cells, and the effect of the Ainu wakame extract as a melanocyte differentiation induction inhibitor was confirmed.
２．実験例２ アイヌワカメ抽出物のメラノサイトへの分化誘導抑制効果の評価(遺伝子発現解析)
試験例１と同様に、マウスES細胞からメラノサイトへの分化誘導を実施し、メラノサイトへの分化効率についてメラノサイトの特異的マーカー遺伝子(Mitf-M及びTyrp1）の発現を指標に評価した。
2. Experimental Example 2 Evaluation of the inhibitory effect of Ainu wakame extract on differentiation induction into melanocytes (gene expression analysis)
In the same manner as in Test Example 1, differentiation induction from mouse ES cells into melanocytes was performed, and the efficiency of differentiation into melanocytes was evaluated using the expression of melanocyte specific marker genes (Mitf-M and Tyrp1) as an index.
具体的には、上述の分化誘導培地に50μg/mLの濃度で実施例１で製造したアイヌワカメ抽出物を継続して添加し、24日間の分化誘導を実施した。分化誘導24日目において、細胞を回収し、PBS(-)にて2回洗浄し、Trizol Reagent(Invitrogen)によって細胞からRNAを抽出した。次いで、2-STEPリアルタイムPCRキット(Applied Biosystems)を用いて、抽出したRNAをcDNAに逆転写した後、ABI7300(Applied Biosystems)により、得られたcDNAをテンプレートとして、下記の各プライマーセットを用いたリアルタイムPCR(95℃：15秒間、60℃：30秒間、40cycles)を実施した。その他の操作は定められた方法に従って実施した。
Mitf-M用のプライマーセット：
5'-TGCCTTGTTTATGGTGCCTTCT-3'(配列番号１)、及び
5'-TCCCTCTACTTTCTGTAATTCCAATTC-3'(配列番号２)
Tyrp1用のプライマーセット：
5'-CAACGCTATGCTGAGGACTATGA-3'(配列番号３)、及び
5'-GCGGCTATCAGACCATGGA-3'(配列番号４)
GAPDH用のプライマーセット：
5'-TGCACCACCAACTGCTTAGC-3'(配列番号５)、及び
5'-TCTTCTGGGTGGCAGTGATG-3'(配列番号６)
Specifically, the Ainu wakame extract produced in Example 1 was continuously added to the above-described differentiation induction medium at a concentration of 50 μg / mL, and differentiation induction was performed for 24 days. On the 24th day of differentiation induction, the cells were collected, washed twice with PBS (−), and RNA was extracted from the cells with Trizol Reagent (Invitrogen). Next, using 2-STEP real-time PCR kit (Applied Biosystems), the extracted RNA was reverse-transcribed to cDNA, and then the obtained cDNA was used as a template by ABI7300 (Applied Biosystems), and the following primer sets were used. Real-time PCR (95 ° C .: 15 seconds, 60 ° C .: 30 seconds, 40 cycles) was performed. Other operations were carried out in accordance with established methods.
Primer set for Mitf-M:
5'-TGCCTTGTTTATGGTGCCTTCT-3 '(SEQ ID NO: 1), and
5'-TCCCTCTACTTTCTGTAATTCCAATTC-3 '(SEQ ID NO: 2)
Primer set for Tyrp1:
5'-CAACGCTATGCTGAGGACTATGA-3 '(SEQ ID NO: 3), and
5'-GCGGCTATCAGACCATGGA-3 '(SEQ ID NO: 4)
Primer set for GAPDH:
5'-TGCACCACCAACTGCTTAGC-3 '(SEQ ID NO: 5), and
5'-TCTTCTGGGTGGCAGTGATG-3 '(SEQ ID NO: 6)
各細胞のメラノサイトへの分化誘導効率については、抽出物を添加せずに分化誘導した細胞におけるメラノサイトマーカー遺伝子(Mitf-M及びTyrp1）の発現量を内部標準であるGAPDH mRNAの発現量に対する割合として算出したメラノサイト特異的マーカー遺伝子相対発現量の値を100とし、これに対し、アイヌワカメ抽出物を添加して分化誘導した各細胞におけるメラノサイト特異的マーカー遺伝子相対発現量の値を算出し、評価した。
その結果を下記の表２に示す。
Regarding the differentiation induction efficiency of each cell into melanocytes, the expression level of the melanocyte marker genes (Mitf-M and Tyrp1) in the cells induced to differentiate without adding an extract as a ratio to the expression level of GAPDH mRNA as an internal standard The calculated value of the relative expression level of the melanocyte-specific marker gene was set to 100. On the other hand, the value of the relative expression level of the melanocyte-specific marker gene in each cell induced to differentiate by adding the Ainu wakame extract was calculated and evaluated. .
The results are shown in Table 2 below.
表２に示すように、アイヌワカメ抽出物(製造例１〜５)は顕著なメラノサイト分化誘導抑制効果を示した。一方で、従来の美白剤であるアルブチンは、メラノサイト分化誘導抑制効果を示さなかった。なお、製造例３の抽出物もまた製造例１の抽出物と同様の顕著なメラノサイト分化誘導抑制効果を示した。 As shown in Table 2, Ainu Wakame extract (Production Examples 1 to 5) showed a remarkable effect of suppressing melanocyte differentiation induction. On the other hand, arbutin, which is a conventional whitening agent, did not show an inhibitory effect on melanocyte differentiation induction. In addition, the extract of Production Example 3 also showed a remarkable melanocyte differentiation induction inhibitory effect similar to that of Production Example 1.
〔実施例３〕アイヌワカメ抽出物の処方例
実施例１で製造したアイヌワカメ抽出物について、処方例として下記の製剤化を行った。
Example 3 Ainu Wakame Extract Formulation Example The Ainu wakame extract produced in Example 1 was formulated as a formulation example as follows.
１．処方例１ ローション
[製造方法]
成分1〜6及び12と、成分7〜11をそれぞれ均一に溶解した後、双方を混合し、濾過することで製品とした。
[Production method]
Components 1 to 6 and 12 and components 7 to 11 were uniformly dissolved, and then both were mixed and filtered to obtain a product.
２．比較処方例１ 従来のローション
処方例１において、アイヌワカメ抽出物を精製水に置き換えたものを従来のローションとした。
2. Comparative Formulation Example 1 Conventional Lotion In Formulation Example 1, a product obtained by replacing the Ainu Wakame extract with purified water was used as a conventional lotion.
３．処方例２ クリーム
［製造方法］
成分2〜9を加熱溶解して混合し、70℃に保ち油相とした。また、成分1及び成分11〜14を加熱溶解して混合し、75℃に保ち水相とした。次いで、油相に水相を加えて乳化して、かき混ぜながら冷却し、45℃で成分10を加え、さらに30℃まで冷却することで製品とした。
[Production method]
Ingredients 2-9 were dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. In addition, Component 1 and Components 11 to 14 were heated and dissolved and mixed, and kept at 75 ° C. to obtain an aqueous phase. Next, an aqueous phase was added to the oil phase to emulsify, and the mixture was cooled while stirring. The component 10 was added at 45 ° C., and further cooled to 30 ° C. to obtain a product.
４．比較処方例２ 従来のクリーム
処方例２において、アイヌワカメ抽出物を精製水に置き換えたものを従来のクリームとした。
4). Comparative Formulation Example 2 Conventional Cream In Formulation Example 2, Ainu Wakame extract was replaced with purified water to obtain a conventional cream.
５．処方例３ 錠剤
［製造方法］
成分1〜5を混合し、次いで10％の水を結合剤として加えて、押出し造粒に供した後、乾燥させた。成形した顆粒に成分6を加えて混合し、打錠した。1錠を0.52gとした。
[Production method]
Components 1-5 were mixed, then 10% water was added as a binder, subjected to extrusion granulation and then dried. Ingredient 6 was added to the molded granules, mixed and tableted. One tablet was 0.52 g.
６．比較処方例３ 従来の錠剤
処方例３において、アイヌワカメ抽出物を精製水に置き換えたものを従来の錠剤とした。
6). Comparative Formulation Example 3 Conventional Tablet In Formulation Example 3, the Ainu Wakame extract was replaced with purified water to obtain a conventional tablet.
７．処方例４ 飲料
［製造方法］
成分1〜4を成分5の一部の水に撹拌溶解した。次いで、成分5の残りの水を加えて混合した。
[Production method]
Ingredients 1 to 4 were dissolved in a part of the ingredient 5 water with stirring. The remaining water of component 5 was then added and mixed.
８．比較処方例４ 従来の飲料
処方例４において、アイヌワカメ抽出物を水に置き換えたものを従来の飲料とした。
8). Comparative Formulation Example 4 Conventional Beverage In Formulation Example 4, Ainu Wakame extract was replaced with water to obtain a conventional beverage.
〔実施例４〕アイヌワカメ抽出物を含有する製剤のシミ及びくすみの改善作用の評価
実施例３で製造した処方例１のローション及び処方例２のクリーム並びに比較処方例１のローション及び比較処方例２のクリームを用いて、シミ及びくすみに悩む女性30人(18才〜50才)を対象に2ヶ月間の使用試験を行った。
使用後、シミ及びくすみの改善作用をアンケートにより判定した。
結果を表７に示す。
[Example 4] Evaluation of spot and dullness improving effect of preparation containing Ainu Wakame extract Extract of Preparation Example 1 lotion and Cream of Formulation Example 2 prepared in Example 3 and Lotion of Comparative Formulation Example 1 and Comparative Formulation Example Using the cream of No. 2, 30 women (18 to 50 years old) suffering from spots and dullness were subjected to a 2-month use test.
After use, the effect of improving spots and dullness was determined by a questionnaire.
The results are shown in Table 7.
表７に示すように、処方例１のローション及び処方例２のクリームは、比較処方例１のローション及び比較処方例２のクリームと比較して、シミ及びくすみの予防改善効果に優れていた。なお、試験期間中、皮膚トラブルは一人もなく、安全性においても問題がなかった。また、処方例１のローション及び処方例２のクリームの処方成分の劣化についても問題がなかった。 As shown in Table 7, the lotion of Formulation Example 1 and the cream of Formulation Example 2 were superior in the effect of preventing and improving spots and dullness compared to the lotion of Comparative Formulation Example 1 and the cream of Comparative Formulation Example 2. During the test period, there were no skin problems and no safety problems. Moreover, there was no problem also about deterioration of the prescription component of the lotion of prescription example 1 and the cream of prescription example 2.
また、同様に、処方例３の錠剤及び処方例４の飲料についても、経口摂取による使用試験を行ったところ、安全で優れたシミ及びくすみの予防改善作用を示した。なお、試験期間中、体調を崩した被験者は一人もなく、安全性においても問題がなかった。また、処方例３の錠剤及び処方例４の飲料の処方成分の劣化についても問題がなかった。 Similarly, the tablets of Formulation Example 3 and the beverage of Formulation Example 4 were tested for use by oral ingestion, and showed a safe and excellent anti-smudge and dullness-improving action. During the test period, there were no subjects who were unwell, and there was no problem in safety. Moreover, there was no problem about deterioration of the prescription component of the tablet of prescription example 3 and the drink of prescription example 4.
本発明に係るメラノサイト分化誘導抑制剤によれば、色素異常症及びシミを治療、予防及び改善することができる。例えば、本発明において見出されたアイヌワカメ抽出物を用いることにより根本からの色素異常症及びシミの解決に繋がる。本発明で見出されたアイヌワカメ抽出物を経口投与、皮膚への直接注入、塗布、貼付等により導入することで、組織に存在する未分化細胞のメラノサイトへの分化を抑制させることができる。 According to the melanocyte differentiation induction inhibitor according to the present invention, it is possible to treat, prevent, and improve dyschromia and stains. For example, the use of the Ainu wakame extract found in the present invention leads to the solution of pigmentation disorders and stains from the root. By introducing the Ainu Wakame extract found in the present invention by oral administration, direct injection into the skin, application, sticking, etc., differentiation of undifferentiated cells present in the tissue into melanocytes can be suppressed.
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