JP5990058B2 - Estrogen receptor β activator - Google Patents
Estrogen receptor β activator Download PDFInfo
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- JP5990058B2 JP5990058B2 JP2012173179A JP2012173179A JP5990058B2 JP 5990058 B2 JP5990058 B2 JP 5990058B2 JP 2012173179 A JP2012173179 A JP 2012173179A JP 2012173179 A JP2012173179 A JP 2012173179A JP 5990058 B2 JP5990058 B2 JP 5990058B2
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Description
本発明は、エストロゲン受容体βに対して選択性の高い、エストロゲンβ受容体活性化剤に関する。 The present invention relates to an estrogen β receptor activator having high selectivity for estrogen receptor β.
女性は閉経を迎えると血液中のエストロゲン濃度が著しく減少し、皮膚全体の機能に変化が生じることが知られている。特に、真皮においてはコラーゲン線維が著しく減少し、その結果しわやたるみといった皮膚老化現象が目立つようになる（非特許文献１、２）。
閉経後の女性に対して女性ホルモン補充療法（ＨＲＴ）を行うとしわ、血行が改善すること、エストロゲンを塗布するとしわ、弾力性、水分量が改善することが報告されている（非特許文献３、４）。また、エストロゲンを塗布すると皮脂腺からの皮脂の分泌が抑制されることが報告されている（非特許文献５）。
It is known that when women reach menopause, the concentration of estrogen in the blood decreases significantly, and the function of the entire skin changes. In particular, collagen fibers are remarkably reduced in the dermis, and as a result, skin aging phenomena such as wrinkles and sagging become noticeable (Non-patent Documents 1 and 2).
It has been reported that female hormone replacement therapy (HRT) is performed on postmenopausal women, blood circulation is improved, and when estrogen is applied, wrinkles, elasticity, and water content are improved (Non-patent Document 3). 4). In addition, it has been reported that the application of estrogen suppresses the secretion of sebum from the sebaceous glands (Non-patent Document 5).
エストロゲンは２種類のエストロゲン受容体（ＥＲα、ＥＲβ）に結合し、これらが協調して働くことでその作用を発揮することが知られている。エストロゲンは初めに、ＥＲα、ＥＲβのｌｉｇａｎｄ ｂｉｎｄｉｎｇ ｄｏｍａｉｎ（ＬＢＤ）に結合して立体構造を変化させ、それに続いて二量体（ＥＲα／α、ＥＲβ／β、ＥＲα／βの三種類の組み合わせ）を形成する（非特許文献６、７）。二量体を形成したＥＲは核内へ移行し、ゲノム上に存在するエストロゲン応答配列に結合し、そこで様々な共役因子をリクルートした後に、標的遺伝子の転写活性を制御する（非特許文献８、９）。
ＥＲαとＥＲβの標的遺伝子は、大部分が同一であることが報告されている（非特許文献１０）。ところが最近になって、いくつかの相違する標的遺伝子の組み合わせに起因して、ＥＲαとＥＲβがそれぞれ異なる生理機能を有していることが示唆されている（非特許文献１１）。例えば、ＥＲαは、乳癌細胞の増殖を活性化する一方で、ＥＲβは、その増殖を抑制するといった正反対の役割を果たしていることが報告されている（非特許文献１２）。また、ＥＲβは、皮膚の光老化を防御する機能を有することが報告されている（非特許文献１３）。
このようなことから、ＥＲβは安全にエストロゲンの作用を発揮できる理想的な分子であり、ＥＲβを選択的に活性化できれば、エストロゲン減少に起因する皮膚老化症状の予防・改善等に有用であると考えられる。
It is known that estrogen binds to two types of estrogen receptors (ERα, ERβ) and exerts its action when they work in concert. Estrogen first binds to the binding binding domain (LBD) of ERα and ERβ and changes its conformation, followed by dimer (three combinations of ERα / α, ERβ / β and ERα / β). (Non-Patent Documents 6 and 7). The ER that formed the dimer moves into the nucleus, binds to an estrogen response element existing on the genome, and after recruiting various coupling factors there, the transcriptional activity of the target gene is controlled (Non-Patent Document 8, 9).
It has been reported that the target genes of ERα and ERβ are mostly the same (Non-patent Document 10). However, recently, it has been suggested that ERα and ERβ have different physiological functions due to several different combinations of target genes (Non-patent Document 11). For example, it has been reported that ERα activates proliferation of breast cancer cells, while ERβ plays the opposite role of suppressing its proliferation (Non-patent Document 12). In addition, ERβ has been reported to have a function of protecting the skin from photoaging (Non-patent Document 13).
For these reasons, ERβ is an ideal molecule that can safely exert the action of estrogen. If ERβ can be selectively activated, it is useful for prevention and improvement of skin aging symptoms caused by estrogen reduction. Conceivable.
一方、ホウトウ（Ｐａｕｌｏｗｎｉａ ｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．）はゴマノハグサ科の高木で、鼻炎、結膜炎、やけど等の治療に、カチラン（Ｃｒｏｔａｌａｒｉａ ｆｅｒｒｕｇｉｎｅａ Ｇｒａｈ．）はマメ科の草木で、咳止めや耳鳴り等の治療に、キョウヨウヨウ（Ｐｏｐｕｌｕｓ ａｄｅｎｐｏｄａ Ｍａｘｉｍ．）はヤナギ科の植物で、虫歯や打撲等の治療に、キンセンソウ（Ｄｅｓｍｏｄｉｕｍ ｓｔｙｒａｃｉｆｏｌｉｕｍ（Ｏｓｂ．）Ｗｅｒｒ）はマメ科の植物で、尿路感染や結石、胆石、腎炎浮腫、でき物、黄疸等の治療に、ロウバイ（Ｃｈｉｍｏｎａｎｔｈｕｓ ｐｒａｅｃｏｘ（Ｌ．）Ｌｉｎｋ）はロウバイ科の低木で、解熱、鎮咳、鎮痛、麻疹、百日咳等の治療に、ミゾコウジュ（Ｓａｌｖｉａ ｐｌｅｂｅｉａ Ｒ．Ｂｒｏｗｎ）はシソ科の草で、吐血、痔等の治療に、ウケツ（Ｓｔｅｎｏｌｏｍａ ｃｈｕｓａｎｕｍ（Ｌ．）Ｃｈｉｎｇ）はイノモトソウ科の植物で、肝炎、急性気管支炎、やけど等の治療に、シナフジ（Ｗｉｓｔｅｒｉａ ｓｉｎｅｎｓｉｓ Ｓｗｅｅｔ）はマメ科のつる性の木本で、下剤等に、テンオウゴン（Ｓｃｕｔｅｌｌａｒｉａ ａｍｏｅｎａ Ｃ．Ｈ．Ｗｒｉｇｈｔ）はシソ科の植物で、消炎、解熱、気管支炎、風邪等の治療に、それぞれ有用なことが知られている。
しかしながら、これらの植物とエストロゲン受容体活性化との関係については知られていない。
On the other hand, Baotou (Paulonia fortunei (Sem.) Hemsl.) Is a tree of the scorpion family, and is used for the treatment of rhinitis, conjunctivitis, burns, etc. For the treatment, Populus adenpoda Maxim. Is a plant of the willow family, for the treatment of caries and bruises, etc., for the treatment of genus sempirium (Osb. For the treatment of nephritis edema, product, jaundice, etc., Chimonanthus praecox (L. Link) is a shrub of the department of the family Lepidoptera, and for the treatment of antipyretic, antitussive, analgesic, measles, pertussis, etc. eia R. Brown) is a grass of the family Lamiaceae, and is used for the treatment of bloody bleeds, hemorrhoids, etc., and Stem. (Wisteria sinensis Sweet) is a legume vine of wood, laxatives, Tenougon (Scutellaria amoena C.H. Wright) is a Labiatae plant for treating anti-inflammatory, antipyretic, bronchitis, cold, etc. , Each known to be useful.
However, the relationship between these plants and estrogen receptor activation is not known.
本発明は、エストロゲン受容体βに対して選択性の高い、エストロゲン受容体β活性化剤を提供することに関する。 The present invention relates to providing an estrogen receptor β activator that is highly selective for estrogen receptor β.
本発明者は、ＥＲβの分子構造内にあるｌｉｇａｎｄ ｂｉｎｄｉｎｇ ｄｏｍａｉｎ（ＬＢＤ）を利用した評価系及びＤＮＡ上に存在するエストロゲン応答配列を利用した転写活性化能評価系を構築し、エストロゲン受容体のうちＥＲβの活性を選択的に高める物質について鋭意研究したところ、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴンに優れたエストロゲン受容体β活性化作用があることを見出した。 The present inventor has constructed an evaluation system using ligand binding domain (LBD) in the molecular structure of ERβ and a transcription activation ability evaluation system using an estrogen response element present on DNA. As a result of diligent research on substances that selectively enhance the activity of ERβ, it was found that Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, Utsutsu, Synafuji, Tenougon have excellent estrogen receptor β activation activity. .
すなわち、本発明は、次の（１）〜（５）に係るものである。
（１）ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴン及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とするエストロゲン受容体β活性化剤。
（２）ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ及びそれらの抽出物からなる群より選択される少なくとも１種を含有する皮膚外用剤。
（３）ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ウケツ、シナフジ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とする皮膚老化予防又は改善剤。
（４）ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とする皮脂分泌抑制剤。
（５）ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とするニキビ改善剤。
That is, the present invention relates to the following (1) to (5).
(1) An estrogen receptor β activator comprising, as an active ingredient, at least one selected from the group consisting of Baotou, Katachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, Uketsu, Synafuji, Tenougon and extracts thereof.
(2) A skin external preparation containing at least one selected from the group consisting of Baotou, Cachilan, Kyoyouyou, Ginseng, and extracts thereof.
(3) A skin aging preventive or ameliorating agent comprising as an active ingredient at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Bucket, Synapse and extracts thereof.
(4) A sebum secretion inhibitor comprising as an active ingredient at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, and extracts thereof.
(5) An acne-improving agent comprising as an active ingredient at least one selected from the group consisting of Baotou, Kachiran, Kyoyoujo, Ginseng, Wintersweet, Mizokouju, and extracts thereof.
本発明のエストロゲン受容体β活性化剤等は、エストロゲン受容体βを選択的に活性化し、また、乳癌発症・悪化の危険性がなく安全であることから、皮膚の老化症状の予防又は改善や皮脂腺からの皮脂分泌抑制効果等を発揮し得る医薬品、医薬部外品、化粧品として、或いはこれらへ配合するための素材又は製剤として有用である。 The estrogen receptor β activator of the present invention selectively activates estrogen receptor β, and is safe without the risk of developing or worsening breast cancer. It is useful as a pharmaceutical, a quasi-drug, a cosmetic that can exert an effect of suppressing sebum secretion from the sebaceous glands, or as a material or preparation for blending into these.
本明細書において、「非治療的」とは、医療行為、すなわち治療による人体への処理行為を含まない概念である。 In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.
本明細書において、「改善」とは、疾患、症状又は状態の好転、疾患、症状又は状態の悪化の防止又は遅延、あるいは疾患又は症状の進行の逆転、防止又は遅延をいう。 As used herein, “improvement” refers to improvement of a disease, symptom or condition, prevention or delay of worsening of the disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom.
本明細書において、「予防」とは、個体における疾患若しくは症状の発症の防止又は遅延、あるいは個体の疾患若しくは症状の発症の危険性を低下させることをいう。 As used herein, “prevention” refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing an individual's disease or symptom.
本明細書において、ホウトウとは、ゴマノハグサ科キリ属のＰａｕｌｏｗｎｉａ ｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．（和名ココノエギリ、キリ）を指し、カチランとは、マメ科タヌキマメ属のＣｒｏｔａｌａｒｉａ ｆｅｒｒｕｇｉｎｅａ Ｇｒａｈ．（和名ナンバンタヌキマメ、キョウレイソウ）を指し、キョウヨウヨウとは、ヤナギ科ポプラ属のＰｏｐｕｌｕｓ ａｄｅｎｐｏｄａ Ｍａｘｉｍ．を指し、キンセンソウとは、マメ科ヌスビトハギ属のＤｅｓｍｏｄｉｕｍ ｓｔｙｒａｃｉｆｏｌｉｕｍ（Ｏｓｂ．）Ｗｅｒｒを指し、ロウバイとは、ロウバイ科ロウバイ属のＣｈｉｍｏｎａｎｔｈｕｓ ｐｒａｅｃｏｘ（Ｌ．）Ｌｉｎｋ（和名ロウバイ）を指し、ミゾコウジュとは、シソ科サルビア（アキギリ）属のＳａｌｖｉａ ｐｌｅｂｅｉａ Ｒ．Ｂｒｏｗｎ（和名ミゾコウジュ）を指し、ウケツとは、イノモトソウ科ステノロマ属のＳｔｅｎｏｌｏｍａ ｃｈｕｓａｎｕｍ（Ｌ．）Ｃｈｉｎｇ（和名ダイヨウキンカソウ）を指し、シナフジとは、マメ科フジ属のＷｉｓｔｅｒｉａ ｓｉｎｅｎｓｉｓ Ｓｗｅｅｔ（和名シナフジ、シトウ）を指し、テンオウゴンとは、シソ科タツナミソウ属のＳｃｕｔｅｌｌａｒｉａ ａｍｏｅｎａ Ｃ．Ｈ．Ｗｒｉｇｈｔ（和名オウゴン）を指す。 In this specification, Baotou refers to Paulownia fortunei (Seem.) Hemsl. (Japanese name Cocono Egiri, Kiri) refers to Cachiran, which refers to the genus Ranunculaceae Crotalaria ferruginea Grah. (Japanese name Nanbantanukimeme, Kyoreiso), and Kyoyouyou refers to Populus adenpoda Maxim. Ginseng refers to Desmodium styracifolium (Osb.) Werr of the genus Leguminosae, and Loby refers to Chimonanthus praecox (L.) Link (Japanese name Loby), Salvia plebeia R. of the genus Salvia (Akigiri) Brown refers to Stenoroma chusanum (L.) Ching of the genus Stenoroma family (L.) Chin (Japanese name Daikikinkaso), and Sinafuji refers to Wisteria sinensis Sweet of the legume family. (Sinafuji, Sitou) and Tenougon refers to Scutellaria amoena C. belonging to the genus Lamiaceae. H. It points to Wright (Japanese name Ogon).
斯かる植物は、いずれの任意の部位、例えば全草、葉、茎、芽、花、蕾、木質部、樹皮、地衣体、根、根茎、仮球茎、球茎、塊茎、種子、果実、菌核若しくは樹脂等、又はそれらの組み合わせを使用することができるが、ホウトウ（Ｐａｕｌｏｗｎｉａ ｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．）は花、カチラン（Ｃｒｏｔａｌａｒｉａ ｆｅｒｒｕｇｉｎｅａ Ｇｒａｈ．）は全草、キョウヨウヨウ（Ｐｏｐｕｌｕｓ ａｄｅｎｐｏｄａ Ｍａｘｉｍ．）は根皮、樹皮或いは葉、キンセンソウ（Ｄｅｓｍｏｄｉｕｍ ｓｔｙｒａｃｉｆｏｌｉｕｍ（Ｏｓｂ．）Ｗｅｒｒ）は枝葉、ロウバイ（Ｃｈｉｍｏｎａｎｔｈｕｓ ｐｒａｅｃｏｘ（Ｌ．）Ｌｉｎｋ）は花蕾、ミゾコウジュ（Ｓａｌｖｉａ ｐｌｅｂｅｉａ Ｒ．Ｂｒｏｗｎ）は全草、ウケツ（Ｓｔｅｎｏｌｏｍａ ｃｈｕｓａｎｕｍ（Ｌ．）Ｃｈｉｎｇ）は全草或いは根状茎、シナフジ（Ｗｉｓｔｅｒｉａ ｓｉｎｅｎｓｉｓ Ｓｗｅｅｔ）は茎葉、テンオウゴン（Ｓｃｕｔｅｌｌａｒｉａ ａｍｏｅｎａ Ｃ．Ｈ．Ｗｒｉｇｈｔ）は根、をそれぞれ用いるのが好ましい。
斯かる植物は、そのまま又は乾燥・粉砕等して用いることができる。また、上記部位は、そのまま抽出工程に付されてもよく、又は粉砕、切断若しくは乾燥された後に抽出工程に付されてもよい。該抽出物は天然成分由来であり安全性も高い。
Such a plant can be any part, for example, whole grass, leaves, stems, buds, flowers, buds, wood parts, bark, lichens, roots, rhizomes, corms, corms, tubers, seeds, fruits, mycorrhiza or Resin or the like, or a combination thereof may be used, but flowers such as Paulownia fortunei (Seem.) Hemsl., Flowers of Cachiran (Crotariaria ferruginea Grah.), Whole plants, Populus adenpoxaM. Skin, bark or leaves, ginseng (Desmodium styracifolium (Osb.) Werr) is a branch and leaf, Chimanthus praecox (L.) is a flower bud, Salvia plebeia R. Stenoloma chusanum (L.) Ching) is Zenkusa or Nejokuki, Shinafuji (Wisteria sinensis Sweet) is foliage, Teng scutellaria root (Scutellaria amoena C.H.Wright) is preferably used roots, respectively.
Such a plant can be used as it is or after being dried, ground or the like. Moreover, the said site | part may be attached | subjected to an extraction process as it is, or may be attached | subjected to an extraction process, after grind | pulverizing, cut | disconnecting, or drying. The extract is derived from natural ingredients and has high safety.
抽出物を得る抽出手段は、具体的には、固液抽出、液液抽出、浸漬、煎出、浸出、還流抽出、超音波抽出、マイクロ波抽出、攪拌等の手段を用いることができる。抽出時間を短縮する場合には、攪拌を伴う固液抽出が望ましい。この固液抽出の好適な条件の一例としては、１００〜４００ｒ／ｍｉｎで１〜３０分間の攪拌が挙げられる。
抽出物の酸化を防止するため、煮沸脱気や窒素ガス等の不活性ガスを通気して溶存酸素を除去しつつ、いわゆる非酸化的雰囲気下で抽出する手段を併用してもよい。
Specific examples of the extraction means for obtaining the extract include solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, and stirring. In order to shorten the extraction time, solid-liquid extraction with stirring is desirable. Examples of suitable conditions for this solid-liquid extraction include stirring at 100 to 400 r / min for 1 to 30 minutes.
In order to prevent the oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.
抽出のための溶剤には、極性溶剤、非極性溶剤のいずれをも使用することができる。溶剤の具体例としては、例えば、水；メタノール、エタノール、プロパノール、ブタノール等のアルコール類；プロピレングリコール、ブチレングリコール等の多価アルコール類；アセトン、メチルエチルケトン等のケトン類；酢酸メチル、酢酸エチル等のエステル類；テトラヒドロフラン、ジエチルエーテル等の鎖状及び環状エーテル類；ポリエチレングリコール等のポリエーテル類；スクワラン、ヘキサン、シクロヘキサン、石油エーテル等の炭化水素類；トルエン等の芳香族炭化水素類；ジクロロメタン、クロロホルム、ジクロロエタン等のハロゲン化炭化水素類；及び超臨界二酸化炭素；ピリジン類；油脂、ワックス等その他オイル類等の有機溶剤；ならびにこれらの混合物が挙げられる。好適には、水、アルコール類、アルコール−水混合液、炭化水素類が挙げられ、アルコール−水混合液、炭化水素類がより好ましい。アルコール類としては炭素数１〜５のアルコール類が好ましく、エタノールがより好ましい。炭化水素類としてはヘキサンが好ましい。 As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate and ethyl acetate Esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane and chloroform And halogenated hydrocarbons such as dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as oils and fats, other oils such as wax; and mixtures thereof. Preferable examples include water, alcohols, alcohol-water mixtures and hydrocarbons, with alcohol-water mixtures and hydrocarbons being more preferred. As the alcohol, an alcohol having 1 to 5 carbon atoms is preferable, and ethanol is more preferable. Hexane is preferred as the hydrocarbon.
抽出のための溶剤としてアルコール−水混合液を使用する場合には、アルコール類と水との配合割合（容量比）としては、０．００１〜１００：９９．９９９〜０が好ましく、５〜９５：９５〜５がより好ましく、２０〜８０：８０〜２０がさらに好ましく、３０〜７０：７０〜３０がさらにより好ましく、４０〜６０：６０〜４０がなお好ましい。エタノール水溶液の場合、エタノール濃度が４０〜６０容量％であることが好ましい。 When an alcohol-water mixture is used as a solvent for extraction, the blending ratio (volume ratio) of alcohols and water is preferably 0.001 to 100: 99.999-0, preferably 5 to 95. : 95-5 is more preferable, 20-80: 80-20 are more preferable, 30-70: 70-30 are still more preferable, 40-60: 60-40 are still more preferable. In the case of an ethanol aqueous solution, the ethanol concentration is preferably 40 to 60% by volume.
溶剤の使用量としては、それぞれの植物（乾燥質量換算）１ｇに対して１〜１００ｍＬ、更に８〜５０ｍＬが好ましく、抽出時間としては、１分間〜１００日間が好ましく、１分間〜３日間がより好ましい。このときの抽出温度は、０℃〜溶媒沸点、より好ましくは２０〜１００℃、さらに好ましくは４０〜９０℃である。 As a usage-amount of a solvent, 1-100 mL with respect to 1 g of each plant (dry mass conversion), Furthermore, 8-50 mL is preferable, As extraction time, 1 minute-100 days are preferable, and 1 minute-3 days are more preferable. The extraction temperature at this time is 0 ° C. to the boiling point of the solvent, more preferably 20 to 100 ° C., and further preferably 40 to 90 ° C.
斯くして得られる植物抽出物は、抽出液や画分をそのまま用いてもよく、適宜な溶媒で希釈した希釈液として用いてもよく、或いは濃縮エキスや乾燥粉末としたり、ペースト状に調製したものでもよい。また、凍結乾燥し、用時に、通常抽出に用いられる溶剤、例えば水、エタノール、プロピレングリコール、ブチレングリコール、水・エタノール混液、水・プロピレングリコール混液、水・ブチレングリコール混液等の溶剤で希釈して用いることもできる。また、リポソーム等のベシクルやマイクロカプセル等に内包させて用いることもできる。
また、植物の抽出物は市販品を使用することもできる。
The plant extract thus obtained may be used as it is as the extract or fraction, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or dry powder, or prepared in a paste form. It may be a thing. Also, freeze-dry and dilute with a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, water / ethanol mixture, water / propylene glycol mixture, water / butylene glycol mixture, etc. It can also be used. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.
Moreover, the plant extract can also use a commercial item.
本発明の植物の抽出物は、食品上・医薬品上許容し得る規格に適合し本発明の効果を発揮するものであれば粗精製物であってもよく、さらに得られた粗精製物を公知の分離精製方法を適宜組み合わせてこれらの純度を高めてもよい。精製手段としては、有機溶剤沈殿、遠心分離、限界濾過膜、高速液体クロマトグラフやカラムクロマトグラフ等が挙げられる。 The plant extract of the present invention may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention, and the obtained crude product is publicly known. These separation and purification methods may be combined as appropriate to increase their purity. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.
後記実施例で示すとおり、本発明の植物又はそれらの抽出物は、ＥＲβの分子構造内にあるｌｉｇａｎｄ ｂｉｎｄｉｎｇ ｄｏｍａｉｎ（ＬＢＤ）を利用したｉｎ ｖｉｔｒｏ評価系において優れたエストロゲン受容体β活性化作用を示した。
当該評価系は、ＥＲαとＥＲβのｌｉｇａｎｄ ｂｉｎｄｉｎｇ ｄｏｍａｉｎ（ＬＢＤ）の配列の違い（Ｊ Ｅｎｄｏｃｒｉｎｏｌ １６３：３７９−３８３（１９９９））を利用した系であって、リガンドのＬＢＤへの結合、ＥＲの活性化を判別することによりＥＲβの選択的な活性化評価を可能にしたものである。
また、本発明の植物又はそれらの抽出物は、エストロゲン応答配列（Ｅｓｔｒｏｇｅｎ Ｒｅｓｐｏｎｓｅ Ｅｌｅｍｅｎｔ：ＥＲＥ、ＡＧＧＴＣＡｎｎｎＴＧＡＣＣＴ）を利用したＥＲβを介する転写活性化能評価系において標的遺伝子の転写を活性化させる作用を示した。
そして、本発明の植物又はそれらの抽出物は、ＥＲβを活性化することが有用と考えられる各種疾病や症状の予防、改善又は治療に有効であると考えられる。例えば、ＥＲβが皮膚の光老化を防御する機能を有することが報告されていることから（非特許文献１３）、本発明の植物又はそれらの抽出物は、皮膚のシワ、タルミ（弛緩）、弾力性やハリの低下等の皮膚の老化症状の予防、改善又は治療に有効であると考えられる。
また、後記参考例に示すように、ＥＲβの活性化によりハムスター耳介部の皮脂腺の大きさが減少するため、本発明の植物又はそれらの抽出物は皮脂の分泌抑制やニキビの改善に有効であると考えられる。
As shown in the Examples below, the plant of the present invention or an extract thereof exhibits an excellent estrogen receptor β activation effect in an in vitro evaluation system using a ligand binding domain (LBD) in the molecular structure of ERβ. It was.
This evaluation system is a system that utilizes the difference in the binding binding domain (LBD) of ERα and ERβ (J Endocrinol 163: 379-383 (1999)), which binds ligand to LBD and activates ER. This makes it possible to selectively evaluate ERβ activation.
In addition, the plant of the present invention or an extract thereof showed an action of activating transcription of a target gene in an ERβ-mediated transcriptional activation evaluation system utilizing an estrogen response element (ERE, AGGTCAnnnTGACCT). .
And it is thought that the plant of this invention or those extracts is effective in prevention, improvement, or treatment of various diseases and symptoms considered to activate ERβ. For example, since it has been reported that ERβ has a function of protecting the skin from photoaging (Non-patent Document 13), the plant of the present invention or an extract thereof can be used for skin wrinkles, tarmi (relaxation), elasticity. It is considered to be effective for the prevention, improvement or treatment of skin aging symptoms such as decreased sex and elasticity.
In addition, as shown in Reference Examples below, ERβ activation reduces the size of the sebaceous glands in the hamster pinna, so that the plants of the present invention or their extracts are effective in suppressing sebum secretion and improving acne. It is believed that there is.
従って、本発明の植物又はその抽出物は、ＥＲβの選択的な活性化のために或いは皮膚の老化症状の予防、改善又は治療や、皮脂の分泌抑制のために使用することができる。当該使用は、ヒト若しくは非ヒト動物、又はそれらに由来する検体における使用であり得、また治療的使用であっても非治療的使用であってもよい。 Therefore, the plant of the present invention or an extract thereof can be used for selective activation of ERβ, prevention, improvement or treatment of skin aging symptoms, and suppression of sebum secretion. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.
また、本発明の植物又はそれらの抽出物は、エストロゲン受容体β活性剤、皮膚外用剤、皮膚老化予防又は改善剤、皮脂分泌抑制剤、ニキビ改善剤（以下、エストロゲン受容体β活性剤等）として使用することができ、さらにこれらの剤を製造するために使用することができる。このとき、当該エストロゲン受容体β活性化剤等には、本発明の植物及びそれらの抽出物からなる群より選択される少なくとも１種を単独で、又はこれ以外に、必要に応じて適宜選択した担体等の、配合すべき後述の対象物において許容されるものを使用してもよい。なお、当該製剤は配合すべき対象物に応じて常法により製造することができる。 Further, the plant of the present invention or an extract thereof includes an estrogen receptor β activator, an external preparation for skin, a skin aging preventive or ameliorating agent, a sebum secretion inhibitor, an acne improving agent (hereinafter referred to as an estrogen receptor β activator) And can be used to produce these agents. At this time, as the estrogen receptor β activator, at least one selected from the group consisting of the plant of the present invention and an extract thereof was used alone or in addition to this, as needed. You may use what is accept | permitted in the below-mentioned target object which should mix | blend, such as a support | carrier. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.
当該エストロゲン受容体β活性剤等は、それ自体、ＥＲβの選択的活性化、皮膚の老化症状の予防、改善又は治療、皮脂の分泌抑制効果を発揮する、ヒト若しくは動物用の医薬品、医薬部外品、化粧料、食品又は飼料であってもよく、又は当該医薬品等に配合して使用される素材又は製剤であってもよい。ここで、食品には、エストロゲンβ受容体の活性化或いは皮膚の老化症状の予防、改善又は治療や、皮脂の分泌抑制、ニキビ改善等の生理機能をコンセプトとし、必要に応じてその旨を表示した飲食品、機能性飲食品、病者用飲食品、特定保健用食品等が包含される。 The estrogen receptor β activator itself has a selective activation of ERβ, prevention, amelioration or treatment of skin aging symptoms, a sebum secretion inhibitory effect, a human or veterinary drug, quasi-drug Product, cosmetics, food or feed, or a material or preparation used in combination with the pharmaceutical product. Here, food is based on the concept of physiological functions such as activation of estrogen β receptor or prevention, improvement or treatment of skin aging symptoms, suppression of sebum secretion, improvement of acne, etc. Food / beverage products, functional food / beverage products, food / beverage products for the sick, food for specified health use, and the like are included.
上記医薬品（医薬部外品も含む）の剤形は、例えば注射剤、坐剤、吸入薬、経皮吸収剤、各種外用剤、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤等の何れでもよく、また投与形態も、経口投与（内用）、非経口投与（外用、注射）の何れであってもよい。
このような種々の剤型の医薬製剤を調製するには、本発明のエストロゲン受容体β活性剤等を単独で、又は他の薬学的に許容される賦形剤、結合剤、増量剤、崩壊剤、界面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、嬌味剤、香料、被膜剤、担体、希釈剤等を適宜組み合わせて用いることができる。
The dosage form of the above-mentioned pharmaceutical (including quasi-drugs) may be any of injections, suppositories, inhalants, transdermal absorption agents, various external preparations, tablets, capsules, granules, powders, syrups, etc. Moreover, the administration form may be any of oral administration (internal use) and parenteral administration (external use, injection).
In order to prepare pharmaceutical preparations of such various dosage forms, the estrogen receptor β activator of the present invention alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrations. Agents, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents and the like can be used in appropriate combinations.
これらの投与形態のうち、好ましい形態は経口投与でありエストロゲン受容体β活性剤等を含む製剤中の本発明の植物又はそれらの抽出物の含有量（抽出物の乾燥物換算）は、一般的に０．００００１質量％以上、好ましくは０．０００１質量％以上、そして１０質量％以下、好ましくは１質量％以下が挙げられる。例えば０．００００１〜１０質量％とするのが好ましく、０．０００１〜１質量％とするのがより好ましい。 Among these dosage forms, the preferred form is oral administration, and the content of the plant of the present invention or the extract thereof in a preparation containing an estrogen receptor β activator or the like (dry matter equivalent of the extract) is generally 0.00001 mass% or more, preferably 0.0001 mass% or more, and 10 mass% or less, preferably 1 mass% or less. For example, it is preferable to set it as 0.00001-10 mass%, and it is more preferable to set it as 0.0001-1 mass%.
上記食品の形態は、固形、半固形又は液状であり得、例えば、パン類、ケーキ類、麺類、菓子類、ゼリー類、冷凍食品、アイスクリーム類、乳製品、飲料等の各種食品の他、上述した経口投与製剤と同様の形態（錠剤、カプセル剤、シロップ等）が挙げられる。
種々の形態の食品を調製するには、本発明のエストロゲン受容体β活性剤等を単独で、又は他の食品材料や、溶剤、軟化剤、油、乳化剤、防腐剤、香科、安定剤、着色剤、酸化防止剤、保湿剤、増粘剤等を適宜組み合わせて用いることができる。当該食品中の本発明の植物又はそれらの抽出物の含有量（抽出物の乾燥物換算）は、一般的に０．０１質量％以上、好ましくは０．１質量％以上、より好ましくは１質量％以上、そして１００質量％以下、９５質量％以下が挙げられる。例えば０．０１〜１００質量％とするのが好ましく、０．１〜１００質量％とするのがより好ましく、更に好ましくは１〜１００質量％とするのが好ましい。
The form of the food may be solid, semi-solid or liquid, for example, various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages, Examples include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.
To prepare various forms of food, the estrogen receptor β activator of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, Colorants, antioxidants, humectants, thickeners, and the like can be used in appropriate combination. The content of the plant of the present invention or the extract thereof in the food (in terms of dry matter of the extract) is generally 0.01% by mass or more, preferably 0.1% by mass or more, more preferably 1% by mass. % Or more, 100 mass% or less, and 95 mass% or less. For example, it is preferably 0.01 to 100% by mass, more preferably 0.1 to 100% by mass, and still more preferably 1 to 100% by mass.
また、上記飼料としては、例えば牛、豚、鶏、羊、馬等に用いる家畜用飼料、ウサギ、ラット、マウス等に用いる小動物用飼料、マグロ、ウナギ、タイ、ハマチ等に用いる魚介類用飼料、犬、猫、小鳥、リス等に用いるペットフード等が挙げられる。
尚、飼料を製造する場合には、本発明のエストロゲン受容体β活性剤等の他に、牛、豚、羊等の肉類、蛋白質、穀物類、ぬか類、粕類、糖類、野菜、ビタミン類、ミネラル類等一般に用いられる飼料原料、更に一般的に飼料に使用されるゲル化剤、保型剤、ｐＨ調整剤、調味料、防腐剤、栄養補強剤等を組み合わせて用いることができる。
また、飼料中の、本発明の植物又はそれらの抽出物の含有量は、その使用形態により異なるが、乾燥物換算で、通常０．０００１質量％以上、好ましくは０．００１質量％以上、より好ましくは０．０１質量％以上、そして２０質量％以下、好ましくは１０質量％以下、より好ましくは５質量％以下が挙げられる。例えば０．０００１〜２０質量％であり、０．００１〜１０質量％が好ましく、０．０１〜５質量％がより好ましい。
Examples of the above feed include livestock feed used for cattle, pigs, chickens, sheep, horses, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for tuna, eel, Thailand, Hamachi, etc. Pet foods used for dogs, cats, small birds, squirrels and the like.
In addition, when producing feed, in addition to the estrogen receptor β activator of the present invention, meat such as cattle, pigs and sheep, proteins, grains, bran, potatoes, sugars, vegetables, vitamins In addition, feed materials generally used for minerals and the like, and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutrient reinforcements and the like generally used for feed can be used in combination.
Further, the content of the plant of the present invention or the extract thereof in the feed varies depending on the use form, but is usually 0.0001% by mass or more, preferably 0.001% by mass or more, in terms of dry matter. Preferably 0.01 mass% or more and 20 mass% or less, Preferably it is 10 mass% or less, More preferably, 5 mass% or less is mentioned. For example, it is 0.0001-20 mass%, 0.001-10 mass% is preferable, and 0.01-5 mass% is more preferable.
本発明のエストロゲン受容体β活性剤等の投与量又は摂取量は、対象者の状態、体重、性別、年齢又はその他の要因に従って変動し得るが、経口投与又は摂取の場合成人１人当たり、植物抽出物（乾燥物換算）として、１日あたり０．０１ｍｇ〜１００ｍｇ／ｋｇとすることが好ましく、特に０．１ｍｇ〜１０ｍｇ／ｋｇとするのが好ましい。 The dose or intake of the estrogen receptor β activator of the present invention may vary according to the subject's condition, body weight, sex, age or other factors, but in the case of oral administration or intake, plant extraction per adult It is preferable to set it as 0.01 mg-100 mg / kg per day as a thing (dry matter conversion), and it is preferable to set it as 0.1 mg-10 mg / kg especially.
上記化粧料（医薬部外品も含む）のは、皮膚外用剤、洗浄剤、メイクアップ化粧料等の形態とすることができ、使用方法に応じて、ローション、乳液、ゲル、クリーム、軟膏剤、粉末、顆粒等の種々の剤型で提供することができる。このような種々の剤型の化粧料は、本発明の植物及びそれらの抽出物からなる群より選択される少なくとも１種と、皮膚化粧料に配合され得る、油性成分、保湿剤、粉体、色素、乳化剤、可溶化剤、洗浄剤、紫外線吸収剤、増粘剤、薬剤（例えば、抗炎症剤、殺菌剤、酸化防止剤、ビタミン類、脂肪代謝促進作用又は脱共役蛋白質発現促進作用が知られている薬物或いは天然物）、香料、樹脂、防菌防黴剤、植物抽出物、アルコール類等を適宜組み合わせることにより調製することができる。 The cosmetics (including quasi-drugs) can be in the form of external preparations for skin, cleaning agents, makeup cosmetics, etc., depending on the method of use, lotion, emulsion, gel, cream, ointment , Powders, granules and the like. Such cosmetics in various dosage forms include at least one selected from the group consisting of the plant of the present invention and extracts thereof, and oily ingredients, moisturizers, powders, which can be blended in skin cosmetics. Pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs (for example, anti-inflammatory agents, bactericides, antioxidants, vitamins, fat metabolism promoting action or uncoupling protein expression promoting action are known Prepared drugs or natural products), fragrances, resins, antibacterial and antifungal agents, plant extracts, alcohols, and the like.
当該化粧料の全量中の、本発明の植物又はそれらの抽出物は、抽出物の乾燥物換算で、通常０．０００１質量％以上、好ましくは０．００１質量％以上、より好ましくは０．０１質量％以上、そして２０質量％以下、好ましくは１０質量％以下、より好ましくは５質量％以下が挙げられる。例えば０．０００１〜２０質量％であり、０．００１〜１０質量％が好ましく、０．０１〜５質量％がより好ましい。 The plant of the present invention or the extract thereof in the total amount of the cosmetic is usually 0.0001% by mass or more, preferably 0.001% by mass or more, and more preferably 0.01% in terms of dry matter of the extract. More than mass% and 20 mass% or less, Preferably it is 10 mass% or less, More preferably, 5 mass% or less is mentioned. For example, it is 0.0001-20 mass%, 0.001-10 mass% is preferable, and 0.01-5 mass% is more preferable.
上述した実施形態に関し、本発明においては以下の態様が開示される。
＜１＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴン及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とするエストロゲン受容体β活性化剤。
＜２＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ及びそれらの抽出物からなる群より選択される少なくとも１種を含有する皮膚外用剤。
＜３＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ウケツ、シナフジ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とする皮膚老化予防又は改善剤。
＜４＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とする皮脂分泌抑制剤。
＜５＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を有効成分とするニキビ改善剤。
With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An estrogen receptor β activator comprising, as an active ingredient, at least one selected from the group consisting of Baotou, Cachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, Uketsu, Synafuji, Tenougon and extracts thereof.
<2> A skin external preparation containing at least one selected from the group consisting of Baotou, Cachilan, Kyoyouyou, Ginseng, and extracts thereof.
<3> A skin aging preventive or ameliorating agent comprising, as an active ingredient, at least one selected from the group consisting of Baotou, Kachiran, Kyouyo, Ginseng, Wintersweet, Uketsu, Synafuji, and extracts thereof.
<4> A sebum secretion inhibitor containing as an active ingredient at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, and extracts thereof.
<5> An acne-improving agent comprising as an active ingredient at least one selected from the group consisting of Baotou, Kachiran, Kyouyo, Ginseng, Wintersweet, Mizokouju, and extracts thereof.
＜６＞ エストロゲン受容体β活性化剤を製造するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴン及びそれらの抽出物からなる群より選択される少なくとも１種の使用。
＜７＞ 皮膚外用剤を製造するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ及びそれらの抽出物からなる群より選択される少なくとも１種の使用。
＜８＞ 皮膚老化予防又は改善剤を製造するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ウケツ、シナフジ及びそれらの抽出物からなる群より選択される少なくとも１種の使用。
＜９＞ 皮脂分泌抑制剤を製造するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種の使用。
＜１０＞ ニキビ改善剤を製造するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種の使用。
＜１１＞ エストロゲン受容体β活性化に使用するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴン及びそれらの抽出物からなる群より選択される少なくとも１種。
＜１２＞ 皮膚に外用するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ及びそれらの抽出物からなる群より選択される少なくとも１種。
＜１３＞ 皮膚老化予防又は改善に使用するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ウケツ、シナフジ及びそれらの抽出物からなる群より選択される少なくとも１種。
＜１４＞ 皮脂分泌抑に使用するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種。
＜１５＞ ニキビ改善に使用するための、ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種。
＜１６＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ、ウケツ、シナフジ、テンオウゴン及びそれらの抽出物からなる群より選択される少なくとも１種を、それらを必要とする対象に投与又は摂取するエストロゲン受容体β活性化方法。
＜１７＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ウケツ、シナフジ及びそれらの抽出物からなる群より選択される少なくとも１種を、それらを必要とする対象に投与又は摂取する皮膚老化予防又は改善方法。
＜１８＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を、それらを必要とする対象に投与又は摂取する皮脂分泌抑制方法。
＜１９＞ ホウトウ、カチラン、キョウヨウヨウ、キンセンソウ、ロウバイ、ミゾコウジュ及びそれらの抽出物からなる群より選択される少なくとも１種を、それらを必要とする対象に投与又は摂取するニキビ改善方法。
<6> For producing an estrogen receptor β activator, at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, Utsutsu, Synafuji, Tenougon and extracts thereof Use of.
<7> Use of at least one selected from the group consisting of Baotou, Kachiran, Kyoyoujo, Ginseng, and extracts thereof for producing a skin external preparation.
<8> Use of at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Uketsu, Synafuji, and extracts thereof for producing an agent for preventing or improving skin aging.
<9> Use of at least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju and extracts thereof for producing a sebum secretion inhibitor.
<10> Use of at least one selected from the group consisting of Baotou, Cachilan, Kyoyouyou, Ginseng, Wintersweet, Mizokouju and extracts thereof for producing an acne improving agent.
<11> At least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju, Utsutsu, Synafuji, Tenougon and extracts thereof for use in activating estrogen receptor β.
<12> At least one selected from the group consisting of Baotou, Kachiran, Kyoyoujo, Ginseng, and extracts thereof for external use on the skin.
<13> At least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Uketsu, Synafuji, and extracts thereof for use in preventing or improving skin aging.
<14> At least one selected from the group consisting of Baotou, Kachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju and extracts thereof for use in suppressing sebum secretion.
<15> At least one selected from the group consisting of Baotou, Kachiran, Kyoyoujou, Ginseng, Wintersweet, Oyster, and extracts thereof for use in improving acne.
<16> Administer or ingest at least one selected from the group consisting of Baotou, Kachiran, Kyoyou, Ginseng, Wintersweet, Mizokouju, Uketsu, Synafuji, Tenougon and extracts thereof to a subject in need thereof Estrogen receptor β activation method.
<17> Prevention or improvement of skin aging by administering or ingesting at least one selected from the group consisting of Baotou, Kachiran, Kyoyou, Ginseng, Wintersweet, Bucket, Synapse and extracts thereof to a subject in need thereof Method.
<18> A method for inhibiting sebum secretion, comprising administering or ingesting at least one selected from the group consisting of Baotou, Cachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju and extracts thereof to a subject in need thereof.
<19> A method for improving acne, which comprises administering or ingesting at least one selected from the group consisting of Baotou, Cachiran, Kyoyouyou, Ginseng, Wintersweet, Mizokouju and extracts thereof to a subject in need thereof.
参考例１ 皮脂の分泌抑制
＜ハムスター耳介部への化合物の塗布＞
６週齢のＳｙｒｉａｎ ｈａｍｓｔｅｒ雄（日本ＳＬＣ製）を使用し、１０％グリセリン含有エタノール溶媒のみを塗布するコントロール群を５匹、１００ｎＭのエストラジオール（和光純薬社製、以下同じ）を塗布するＥｓｔｒａｄｉｏｌ（Ｅ２）群を５匹、１６０ｎＭの１，３，５−トリス（４−ヒドロキシフェニル）−４−プロピル−１Ｈ−ピラゾール（ＰＰＴ、ＥＲα選択的アゴニスト、Ｔｏｃｒｉｓ社製、以下同じ）を塗布するＰＰＴ群を５匹、７００ｎＭの２，３−ビス（４−ヒドロキシフェニル）プロピオニトリル（ＤＰＮ、ＥＲβ選択的アゴニスト、Ｔｏｃｒｉｓ社製、以下同じ）を塗布するＤＰＮ群５匹を用意し、それぞれの溶液１００μｌを１日１回、２週間連続して耳介部に塗布した（ＰＰＴ、ＤＰＮの塗布濃度はエストラジオールのＥＣ５０の値に基づいて設定した）。その後、J Invest Dermatol 124:1127−1133, 2005に記載の方法に準じて耳介部を回収し、凍結組織包埋剤（ＯＣＴコンパウンド、 サクラファインテックジャパン）に包埋後、使用するまで−８０℃にて保存した。
Reference Example 1 Inhibition of Sebum Secretion <Application of Compound to Hamster Auricle>
Using a 6-week-old male Syrian hamster (manufactured by SLC, Japan), 5 control groups to which only 10% glycerin-containing ethanol solvent was applied, and 100 nM estradiol (manufactured by Wako Pure Chemical Industries, Ltd., the same shall apply hereinafter) E2) 5 groups, PPT group to which 160 nM 1,3,5-tris (4-hydroxyphenyl) -4-propyl-1H-pyrazole (PPT, ERα selective agonist, manufactured by Tocris, the same applies below) is applied 5 DPN groups to which 700 nM 2,3-bis (4-hydroxyphenyl) propionitrile (DPN, ERβ selective agonist, manufactured by Tocris, the same applies below) is prepared, and 100 μl of each solution is prepared. Was applied to the auricle region once a day for 2 weeks in succession (PPT and DPN application concentrations were Set based on EC value of radiol). Thereafter, the auricle was collected according to the method described in J Invest Dermatol 124: 1127-1133, 2005, embedded in a frozen tissue embedding agent (OCT compound, Sakura Finetech Japan), and used until it was used. Stored at ° C.
＜オイルレッド染色（中性脂質の染色法）＞
ＯＣＴコンパウンドに包埋した凍結耳介部組織より、クリオスタットを用いて６μｍの厚さで凍結切片を作成し、スライドガラス上に貼り付けた（１匹に対して１スライド作成）。組織の固定を行うために、１０％ 中性緩衝ホルマリンに１分間浸漬し、その後ＯＣＴコンパウンドを除去するためにｄｉｓｔｉｌｌｅｄ ｗａｔｅｒ（Ｄ．Ｗ．）に浸漬した。次に６０％イソプロパノールに浸漬し、余分なイソプロパノールを除去した後、オイルレッド染色溶液を１００μＬマウントし５分間染色を行った。染色終了後、６０％ イソプロパノール、Ｄ．Ｗ．の順番で洗浄し、水溶性封入剤を用いて封入を行った。
<Oil red staining (neutral lipid staining method)>
From the frozen auricular tissue embedded in the OCT compound, a frozen section was prepared with a thickness of 6 μm using a cryostat and pasted on a glass slide (one slide was prepared for one animal). To fix the tissue, it was immersed in 10% neutral buffered formalin for 1 minute, and then immersed in a distilled water (DW) to remove the OCT compound. Next, it was immersed in 60% isopropanol to remove excess isopropanol, and then 100 μL of oil red staining solution was mounted and stained for 5 minutes. After dyeing, 60% isopropanol, D.I. W. Were washed in this order, and sealed with a water-soluble mounting medium.
＜オイルレッド染色陽性領域の抽出＞
皮脂腺においてオイルレッド染色陽性領域を抽出するために、顕微鏡下で組織像を取り込み、画像解析ソフトであるＩｍａｇｅ Ｐｒｏ Ｐｌｕｓ（Ｍｅｄｉａ Ｃｙｂｅｒｎｅｔｉｃｓ社製）を用いて画像解析を行った。取り込んだ画像からダクト部分を含む皮脂腺をランダムに５つ選択し、皮脂腺１つにおけるオイルレッド染色陽性領域（赤色）のピクセル数を算出した。
<Extraction of oil red staining positive area>
In order to extract an oil red staining positive region in the sebaceous gland, a tissue image was taken under a microscope, and image analysis was performed using Image Pro Plus (manufactured by Media Cybernetics), which is image analysis software. Five sebaceous glands including the duct portion were selected at random from the captured image, and the number of pixels of the oil red staining positive region (red) in one sebaceous gland was calculated.
＜オイルレッド染色後の組織像＞
オイルレッド染色後の組織像を図１に示した。基底細胞（最外層の細胞）付近の染色強度は弱く、成熟細胞の分化過程の進行（皮脂腺の中心に向かって分化は進行する）に伴い、染色強度が増強する傾向が認められた。コントロール群と比較しエストラジオール群、ＰＰＴ群、ＤＰＮ群で皮脂腺サイズの減少が視覚的にも確認された。
<Tissue image after oil red staining>
The tissue image after oil red staining is shown in FIG. The staining intensity in the vicinity of the basal cells (outermost layer cells) was weak, and the staining intensity tended to increase as the differentiation process of mature cells progressed (differentiation progressed toward the center of sebaceous glands). A decrease in sebaceous gland size was also visually confirmed in the estradiol group, the PPT group, and the DPN group as compared with the control group.
＜皮脂腺サイズの測定＞
各群の皮脂腺サイズをＩｍａｇｅ Ｐｒｏ Ｐｌｕｓを用いて解析した結果を図２に示した。統計学的有意差検定はＤｕｎｎｅｔｔ法を用いて解析した。その結果、コントロール群と比較してエストラジオール群、ＰＰＴ群、ＤＰＮ群のいずれも有意に皮脂腺サイズを縮小していた（ｐ＜０．０１：コントロールｖｓエストラジオール、コントロールｖｓＰＰＴ、ｐ＜０．０５：コントロールｖｓＤＰＮ）。また、有意差は認められなかったもののＰＰＴ群は、ＤＰＮ群よりも若干強く皮脂腺サイズを抑制していた。
このことから、ＥＲβの活性化は皮脂の分泌抑制に有効であることが示唆された。
<Measurement of sebaceous gland size>
The results of analyzing the sebaceous gland size of each group using Image Pro Plus are shown in FIG. Statistical significance test was analyzed using Dunnett method. As a result, the sebaceous gland size was significantly reduced in all of the estradiol group, the PPT group, and the DPN group as compared with the control group (p <0.01: control vs estradiol, control vsPPT, p <0.05: control). vsDPN). Although no significant difference was observed, the PPT group suppressed sebaceous gland size slightly more strongly than the DPN group.
This suggests that ERβ activation is effective in suppressing sebum secretion.
参考例２ 評価系の構築
ｐＢＩＮＤ−ＥＲα及びｐＧＬ４．３５［ｌｕｃ２Ｐ／９ＸＧＡＬ４ＵＡＳ／Ｈｙｇｒｏ］はＰｒｏｍｅｇａ社より購入した。ｐＢＩＮＤ−ＥＲβは、ｐＢＩＮＤ ｖｅｃｔｏｒにＥＲβのＬＢＤ（アミノ酸配列：２２２−５３１）をクローニングしプラスミドを構築した。
この評価系では、ＥＲのＬＢＤに被験物質が反応した場合にのみ、ｐＧＬ４．３５にコードされたホタルルシフェラーゼの転写活性が上昇する（詳細はＰｈａｒｍ Ｒｅｓ ２７：１４３９−１４６８（２０１０）参照）。
Reference Example 2 Construction of Evaluation System pBIND-ERα and pGL4.35 [luc2P / 9XGAL4UAS / Hygro] were purchased from Promega. For pBIND-ERβ, a plasmid was constructed by cloning the LBD of ERβ (amino acid sequence: 222-531) into pBIND vector.
In this evaluation system, the transcriptional activity of firefly luciferase encoded by pGL4.35 increases only when the test substance reacts with the LBD of ER (for details, see Pharm Res 27: 1439-1468 (2010)).
ＨＥＫ２９３細胞を９６−ｗｅｌｌ ｐｌａｔｅに４．０×１０4ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種し、その１２時間後に培養液（５％ チャコール処理血清を含むＤＭＥＭ）を交換し、ｐＢＩＮＤ−ＥＲα又はｐＢＩＮＤ−ＥＲβをｐＧＬ４．３５［ｌｕｃ２Ｐ／９ＸＧＡＬ４ＵＡＳ／Ｈｙｇｒｏ］とともに、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ２０００（Ｉｎｖｔｒｏｇｅｎ社）を用い、製品添付のプロトコールに従い細胞に導入した。導入２４時間後に、陰性対照である５０（ｖ／ｖ）％エタノール、陽性対照であるエストラジオール、ＰＰＴ又はＤＰＮをそれぞれ表１記載の終濃度となるように添加し、さらに２４時間培養を行った。
ホタルルシフェラーゼ（ＥＲ活性の指標）、及びウミシイタケルシフェラーゼ（ｐｌａｓｍｉｄの導入効率の指標）の活性測定には、Ｄｕａｌ−ＧｌｏTM Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）を用い、添付のプロトコールに従いルシフェラーゼ活性を測定した。ホタルルシフェラーゼの発光強度をウミシイタケルシフェラーゼの発光強度で除したものを、Ｒｅｌａｔｉｖｅ ｌｉｇｈｔ ｕｎｉｔ（ＲＬＵ）として算出した。それぞれの結合活性の値は陽性対照であるエストラジオールを終濃度１０ｎＭで添加した際のＲＬＵを１とした場合の相対値として示した。
結果を表１に示す。対ＥＲα、対ＥＲβの欄の数値は、それぞれｐＢＩＮＤ−ＥＲαを導入した細胞、ｐＢＩＮＤ−ＥＲβを導入した細胞に、各化合物を添加した際の結合活性を示している。結合活性増強作用の評価は陰性対照である５０（ｖ／ｖ）％エタノール水溶液を添加した際の結合活性値と比較して行った。
HEK293 cells were seeded on a 96-well plate at a cell density of 4.0 × 10 4 cells / well, and after 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) was replaced, and pBIND-ERα or pBIND- ERβ was introduced into cells together with pGL4.35 [luc2P / 9XGAL4UAS / Hygro] using Lipofectamine 2000 (Invitrogen) according to the protocol attached to the product. 24 hours after the introduction, 50 (v / v)% ethanol as a negative control and estradiol, PPT or DPN as a positive control were respectively added to the final concentrations shown in Table 1, and further cultured for 24 hours.
The activity of firefly luciferase (an index of ER activity) and Renilla luciferase (an index of efficiency of introduction of plasmid) was measured using Dual-Glo ™ Luciferase Assay System (Promega) and luciferase activity was measured according to the attached protocol. . The emission intensity of firefly luciferase divided by the emission intensity of Renilla luciferase was calculated as a relative light unit (RLU). The value of each binding activity was shown as a relative value when RLU was 1 when estradiol as a positive control was added at a final concentration of 10 nM.
The results are shown in Table 1. The numerical values in the column for ERα and ERβ indicate the binding activity when each compound is added to a cell into which pBIND-ERα has been introduced and a cell into which pBIND-ERβ has been introduced. The evaluation of the binding activity enhancing effect was performed by comparison with the binding activity value when a 50 (v / v)% ethanol aqueous solution as a negative control was added.
その結果、ｐＢＩＮＤ−ＥＲαを導入した細胞では陽性対照であるエストラジオール及びＥＲα選択的アゴニストであるＰＰＴによるＥＲαとの結合活性増強作用が認められた。他方、ｐＢＩＮＤ−ＥＲβを導入した細胞ではエストラジオール及びＥＲβ選択的アゴニストであるＤＰＮによるＥＲβとの結合活性増強作用が認められ、ＰＰＴによるＥＲβとの結合活性増強作用は認められなかった。このことから、当該評価系によりＥＲβとの結合活性を選択的に増強する物質の探索が可能であることが確認された。 As a result, in the cells into which pBIND-ERα was introduced, the positive activity estradiol and the ERα selective agonist, PPT, enhanced the binding activity with ERα. On the other hand, in the cells into which pBIND-ERβ was introduced, estradiol and the ERβ selective agonist DPN enhanced the binding activity with ERβ, and PPT did not enhance the binding activity with ERβ. From this, it was confirmed that the substance which selectively enhances the binding activity with ERβ can be searched by the evaluation system.
実施例１ 各抽出物におけるＥＲβ結合活性の選択的増強作用
＜植物抽出物の調製＞
（製造例１）ホウトウ（Ｐａｕｌｏｗｎｉａ ｆｏｒｔｕｎｅｉ（Ｓｅｅｍ．）Ｈｅｍｓｌ．）抽出物の調製
ホウトウの花２００ｇに、１５倍量（３Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、８５℃、３時間で抽出し、不溶物をろ別後、抽出液を得、減圧濃縮し、ホウトウの抽出乾固物を得た。
この抽出固形分を５０（ｖ／ｖ）％エタノールに溶解し、ホウトウの５０（ｖ／ｖ）％エタノール抽出物を調製した。
Example 1 Selective enhancement of ERβ binding activity in each extract <Preparation of plant extract>
(Production Example 1) Preparation of Baotou (Paulonia fortunei (Seem.) Hemsl.) Extract 15 times (3 L) of a 50 (v / v)% ethanol-water mixed solution was added to 200 g of Baitou flowers, and 85 ° C. Extraction was performed in 3 hours, and the insoluble material was filtered off to obtain an extract, which was concentrated under reduced pressure to obtain a dried dried bait product.
This extracted solid was dissolved in 50 (v / v)% ethanol to prepare a 50 (v / v)% ethanol extract of potato.
（製造例２）カチラン（Ｃｒｏｔａｌａｒｉａ ｆｅｒｒｕｇｉｎｅａ Ｇｒａｈ．）抽出物の調製
カチランの全草１００ｇに、２０倍量（２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてカチランの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 2) Preparation of Cachiran (Crotalaria ferruginea Grah.) Extract To 100 g of the whole plant of Cachiran, a 20-fold amount (2 L) of a 50 (v / v)% ethanol-water mixed solution was added, and (1) and Similarly, a 50 (v / v)% ethanol extract of Cachiran was prepared.
（製造例３）キョウヨウヨウ（Ｐｏｐｕｌｕｓ ａｄｅｎｐｏｄａ Ｍａｘｉｍ．）抽出物の調製
キョウヨウヨウの根皮、樹皮、葉計１００ｇに、２０倍量（２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてキョウヨウヨウの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 3) Preparation of an extract of Populus adenpoda Maxim. 20 g (2 L) of 50 (v / v)% ethanol-water mixed solution was added to 100 g of the root bark, bark, and leaf meter of Kyouyou. In addition, a 50 (v / v)% ethanol extract of Kyouyo was prepared in the same manner as (1) above.
（製造例４）キンセンソウ（Ｄｅｓｍｏｄｉｕｍ ｓｔｙｒａｃｉｆｏｌｉｕｍ（Ｏｓｂ．）Ｗｅｒｒ）抽出物の調製
キンセンソウの枝葉２００ｇに、１０倍量（２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてキンセンソウの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 4) Preparation of extract of Desmodium styracifolium (Osb.) Werr 10 times (2 L) of a 50 (v / v)% ethanol-water mixed solution was added to 200 g of Ginseng branches and leaves. ) To prepare a 50 (v / v)% ethanol extract of snapdragons.
（製造例５）ロウバイ（Ｃｈｉｍｏｎａｎｔｈｕｓ ｐｒａｅｃｏｘ（Ｌ．）Ｌｉｎｋ）抽出物の調製
ロウバイの花蕾１８０ｇに、約１１倍量（２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてロウバイの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 5) Preparation of Extract of Chimanthanthus praecox (L. Link) To 180 g of flower buds of loquat, about 11 times (2 L) of 50 (v / v)% ethanol-water mixed solution was added, A waxy 50 (v / v)% ethanol extract was prepared in the same manner as 1).
（製造例６）ミゾコウジュ（Ｓａｌｖｉａ ｐｌｅｂｅｉａ Ｒ．Ｂｒｏｗｎ）抽出物の調製
ミゾコウジュの全草１００ｇに、１５倍量（１．５Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてミゾコウジュの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 6) Preparation of Extract of Salvia plebeia R. Brown To 100 g of whole plant of Mizokouju, 15 times (1.5 L) of 50 (v / v)% ethanol-water mixed solution was added, and the above ( In the same manner as in 1), 50 (v / v)% ethanol extract of Mizokouju was prepared.
（製造例７）ウケツ（Ｓｔｅｎｏｌｏｍａ ｃｈｕｓａｎｕｍ（Ｌ．）Ｃｈｉｎｇ）抽出物の調製
ウケツの全草、根状茎計２００ｇに、１１倍量（２．２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてウケツの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 7) Preparation of uketsu (Stenoloma chusanum (L.) Ching) Extract 11 times (2.2 L) of 50 (v / v)% ethanol-water in 200 g of whole ukes and roots The mixed solution was added, and a 50 (v / v)% ethanol extract of a bucket was prepared in the same manner as in (1) above.
（製造例８）シナフジ（Ｗｉｓｔｅｒｉａ ｓｉｎｅｎｓｉｓ Ｓｗｅｅｔ）抽出物の調製
シナフジの茎葉２００ｇに、１０倍量（２Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてシナフジの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 8) Preparation of Synapse (Wisteria sinensis Sweet) Extract 10 times (2 L) of 50 (v / v)% ethanol-water mixed solution was added to 200 g of Sinafuji stems and leaves, and the same as in (1) above. A 50 (v / v)% ethanol extract of synafuji was prepared.
（製造例９）テンオウゴン（Ｓｃｕｔｅｌｌａｒｉａ ａｍｏｅｎａ Ｃ．Ｈ．Ｗｒｉｇｈｔ）抽出物の調製
テンオウゴンの根２００ｇに、１５倍量（３Ｌ）の５０（ｖ／ｖ）％エタノール−水混合溶液を加え、上記（１）と同様にしてテンオウゴンの５０（ｖ／ｖ）％エタノール抽出物を調製した。
(Production Example 9) Preparation of Scutellaria amoena C.H. Wright extract To 200 g of roots of Tenougon, a 15-fold amount (3 L) of a 50 (v / v)% ethanol-water mixed solution was added. In the same manner as in (1), a 50 (v / v)% ethanol extract of Tenougon was prepared.
＜評価方法＞
ＨＥＫ２９３細胞を９６−ｗｅｌｌ ｐｌａｔｅに４．０×１０4 ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種し、その１２時間後に培養液（５％ チャコール処理血清を含むＤＭＥＭ）を交換し、ｐＢＩＮＤ−ＥＲα、もしくはｐＢＩＮＤ−ＥＲβをｐＧＬ４．３５［ｌｕｃ２Ｐ／９ＸＧＡＬ４ＵＡＳ／Ｈｙｇｒｏ］とともに、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ２０００（Ｉｎｖｔｒｏｇｅｎ社）を用い、製品添付のプロトコールに従い細胞に導入した。導入２４時間後に、陰性対照である５０（ｖ／ｖ）％エタノール水溶液、陽性対照であるエストラジオール（終濃度１０ｎＭ）、又は上記製造例１〜９で調製した植物抽出物を表２記載の終濃度となるように添加し、さらに２４時間培養を行った。
ルシフェラーゼ活性を上記と同様にして測定し、ホタルルシフェラーゼの発光強度をウミシイタケルシフェラーゼの発光強度で除したものを、Ｒｅｌａｔｉｖｅ ｌｉｇｈｔ ｕｎｉｔ（ＲＬＵ）として算出した。それぞれの活性値は陽性対照におけるＲＬＵを１とした場合の相対値として示した。結合活性増強作用の評価は陰性対照である５０（ｖ／ｖ）％エタノール水溶液を添加した際の結合活性値と比較して行った。
結果を表２に示す。
<Evaluation method>
HEK293 cells were seeded on a 96-well plate at a cell density of 4.0 × 10 4 cells / well, and after 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) was replaced, and pBIND-ERα or pBIND -ERβ was introduced into cells together with pGL4.35 [luc2P / 9XGAL4UAS / Hygro] using Lipofectamine 2000 (Invitrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol aqueous solution as a negative control, estradiol as a positive control (final concentration 10 nM), or the plant extract prepared in the above Production Examples 1 to 9 was used as the final concentration shown in Table 2. And further cultured for 24 hours.
The luciferase activity was measured in the same manner as described above, and the value obtained by dividing the luminescence intensity of firefly luciferase by the luminescence intensity of Renilla luciferase was calculated as a relative light unit (RLU). Each activity value is shown as a relative value when RLU in the positive control is 1. The evaluation of the binding activity enhancing effect was performed by comparison with the binding activity value when a 50 (v / v)% ethanol aqueous solution as a negative control was added.
The results are shown in Table 2.
＜結果＞
表２より、本発明の植物抽出物はＥＲαと比較してＥＲβとの結合活性増強作用が顕著に高いことが認められた。
このことから、上記植物抽出物は、ＥＲβと選択性高く結合し、活性化する作用を有していることが確認された。
<Result>
From Table 2, it was recognized that the plant extract of the present invention has a markedly higher binding activity enhancing activity with ERβ than ERα.
From this, it was confirmed that the plant extract has an action of binding and activating ERβ with high selectivity.
参考例３ ＥＲβを介する転写活性化能評価系の構築
ＥＲβは、ゲノム上に存在するＥＲＥ（J Steroid Biochem Mol Biol. 120, 172-9, (2010).）に結合し、標的遺伝子の転写を制御することが知られている。そこで、ＥＲＥを利用し、ＥＲβを介した転写活性化能の有無を評価できる系の構築を行った。
ｐＥＲＥ（ホタルルシフェラーゼ）はＳＡＢｉｏｓｃｉｅｎｃｅｓ社より購入した。ｐＲＬ−ＣＭＶ（ウミシイタケルシフェラーゼ）は、Ｐｒｏｍｅｇａ社より購入した。
Reference Example 3 Construction of Evaluation System for Transcriptional Activation Ability via ERβ ERβ binds to ERE (J Steroid Biochem Mol Biol. 120, 172-9, (2010).) Present on the genome and transcribes the target gene. It is known to control. Therefore, a system that can evaluate the presence of ERβ-mediated transcription activation ability using ERE was constructed.
pERE (firefly luciferase) was purchased from SABiosciences. pRL-CMV (Renilla luciferase) was purchased from Promega.
ＨＥＫ２９３細胞を９６−ｗｅｌｌ ｐｌａｔｅに４．０×１０4 ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種し、その１２時間後に培養液（５％ チャコール処理血清を含むＤＭＥＭ）を交換し、ｐＥＲＥ、ｐＲＬ−ＣＭＶ及びｐｃＤＮＡ３−ＥＲβをＬｉｐｏｆｅｃｔａｍｉｎｅ ２０００（Ｉｎｖｔｒｏｇｅｎ社）を用い、製品添付のプロトコールに従い細胞に導入した。導入２４時間後に、陰性対照として５０（ｖ／ｖ）％エタノール、陽性対照としてエストラジオール、ＰＰＴ又はＤＰＮをそれぞれ表３記載の終濃度となるように添加し、さらに２４時間培養を行った。ホタルルシフェラーゼ（転写活性化能の指標）、及びウミシイタケルシフェラーゼ（ｐｌａｓｍｉｄの導入効率の指標）の活性測定には、Ｄｕａｌ−ＧｌｏTM Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）を用い、添付のプロトコールに従い実施した。ホタルルシフェラーゼの発光強度をウミシイタケルシフェラーゼの発光強度で除したものを、Ｒｅｌａｔｉｖｅ ｌｉｇｈｔ ｕｎｉｔ（ＲＬＵ）として算出した。ＥＲβを介した転写活性化能は、陰性対照である５０（ｖ／ｖ）％エタノール水溶液を添加した際のＲＬＵと比較して評価した。 HEK293 cells were seeded on a 96-well plate at a cell density of 4.0 × 10 4 cells / well, and after 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) was replaced, and pERE, pRL-CMV and pcDNA3-ERβ was introduced into cells using Lipofectamine 2000 (Invitrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol as a negative control and estradiol, PPT, or DPN as positive controls were added so as to have final concentrations shown in Table 3, respectively, and further cultured for 24 hours. The activity of firefly luciferase (index of transcription activation ability) and Renilla luciferase (index of efficiency of introduction of plasmid) was measured using Dual-Glo ™ Luciferase Assay System (Promega) according to the attached protocol. The emission intensity of firefly luciferase divided by the emission intensity of Renilla luciferase was calculated as a relative light unit (RLU). The transcription activation ability via ERβ was evaluated by comparison with RLU when a 50 (v / v)% aqueous ethanol solution as a negative control was added.
その結果、ＰＰＴ又はＤＰＮを添加したところ、ＰＰＴよりもＤＰＮの方が、ＥＲβを介した転写活性化能が高かった。このことから、当該評価系はＥＲβを介した転写活性化能を評価するのに有用であることが確認された。 As a result, when PPT or DPN was added, DPN had a higher ability to activate transcription through ERβ than PPT. From this, it was confirmed that the said evaluation system is useful for evaluating the transcriptional activation ability via ERβ.
実施例２ 各抽出物におけるＥＲβを介した転写活性化能の評価
＜評価方法＞
ＨＥＫ２９３細胞を９６−ｗｅｌｌ ｐｌａｔｅに４．０×１０4 ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種し、その１２時間後に培養液（５％ チャコール処理血清を含むＤＭＥＭ）を交換し、ｐＥＲＥ、ｐＲＬ−ＣＭＶ及びｐｃＤＮＡ３−ＥＲβをＬｉｐｏｆｅｃｔａｍｉｎｅ ２０００（Ｉｎｖｔｒｏｇｅｎ社）を用い、製品添付のプロトコールに従い細胞に導入した。導入２４時間後に、陰性対照として５０（ｖ／ｖ）％エタノール、陽性対照としてエストラジオール、又は上記製造例１〜９で調製した各植物抽出物を表４記載の終濃度となるように添加し、さらに２４時間培養を行った。ルシフェラーゼ活性を上記と同様にして測定し、ホタルルシフェラーゼの発光強度をウミシイタケルシフェラーゼの発光強度で除したものを、Ｒｅｌａｔｉｖｅ ｌｉｇｈｔ ｕｎｉｔ（ＲＬＵ）として算出した。ＥＲβを介した転写活性化能は、陰性対照である５０（ｖ／ｖ）％エタノール水溶液を添加した際のＲＬＵと比較して評価した。
Example 2 Evaluation of transcription activation ability via ERβ in each extract <Evaluation method>
HEK293 cells were seeded on a 96-well plate at a cell density of 4.0 × 10 4 cells / well, and after 12 hours, the culture medium (DMEM containing 5% charcoal-treated serum) was replaced, and pERE, pRL-CMV and pcDNA3-ERβ was introduced into cells using Lipofectamine 2000 (Invitrogen) according to the protocol attached to the product. 24 hours after introduction, 50 (v / v)% ethanol as a negative control, estradiol as a positive control, or each plant extract prepared in the above Production Examples 1 to 9 was added so as to have a final concentration described in Table 4, The culture was further continued for 24 hours. The luciferase activity was measured in the same manner as described above, and the value obtained by dividing the luminescence intensity of firefly luciferase by the luminescence intensity of Renilla luciferase was calculated as a relative light unit (RLU). The transcription activation ability via ERβ was evaluated by comparison with RLU when a 50 (v / v)% aqueous ethanol solution as a negative control was added.
＜結果＞
表４より、本発明の植物抽出物にＥＲβを介した転写活性化能が認められた。以上より、本発明の植物抽出物はＥＲβと選択性高く結合し、標的遺伝子の転写を活性化する作用を有していることが確認された。
<Result>
From Table 4, the transcriptional activation ability via ERβ was recognized in the plant extract of the present invention. From the above, it was confirmed that the plant extract of the present invention has an action of binding to ERβ with high selectivity and activating transcription of the target gene.
実施例２ 植物抽出物の皮脂分泌抑制作用
＜評価方法＞
ハムスター初代培養皮脂腺細胞（クラボウ社）を２４−ｗｅｌｌ ｐｌａｔｅに１×１０5ｃｅｌｌｓ／ｗｅｌｌとなるよう播種し、３日間Ｈｕｍｅｄｉａ−ＢＧ（クラボウ社）で培養した後、陰性対照であるＤｉｍｅｔｈｙｌ ｓｕｌｆｏｘｉｄｅ（コントロール）、皮脂蓄積を抑制することが知られているａｌｌ ｔｒａｎｓ ｒｅｔｉｎｏｉｃ ａｃｉｄ（ａｔＲＡ）、アダパレン（ａｄａｐａｌｅｎｅ）、および上記製造例１で調製したホウトウ抽出物をそれぞれ１.０（ｖ／ｖ）％となるように添加したＨｕｍｅｄｉａ−ＢＤ（クラボウ社）中で更に３日間培養した。培養後、それぞれのｗｅｌｌをＰＢＳで２回洗浄し、１０μｇ／ｍｌ Ｎｉｌｅ Ｒｅｄ染色液を滴下し３０分３７℃、ＣＯ2 ５％の条件下で染色した。染色終了後にＰＢＳで２回洗浄し、４８５ｎｍの励起波長を当て、５６５ｎｍの波長の蛍光強度を測定した。
Example 2 Sebum secretion inhibitory action of plant extract <Evaluation method>
Hamster primary cultured sebaceous gland cells (Kurabo) were seeded on a 24-well plate to 1 × 10 5 cells / well, cultured for 3 days in Humdia-BG (Kurabo), then Dimethylsulfoxide (control) ), All trans retinoic acid (atRA), which is known to suppress sebum accumulation, and 1.0% (v / v)% of the potato extract prepared in Production Example 1 above, respectively. The cells were further cultured for 3 days in Humedia-BD (Kurabo) added as described above. After culturing, each well was washed twice with PBS, 10 μg / ml Nile Red staining solution was added dropwise, and the mixture was stained for 30 minutes at 37 ° C. and CO 2 5%. After staining, the plate was washed twice with PBS, applied with an excitation wavelength of 485 nm, and the fluorescence intensity at a wavelength of 565 nm was measured.
＜結果＞
ホウトウ抽出物は、ハムスター皮脂腺細胞における皮脂の産生を抑制した（表１）。
<Result>
Bait extract suppressed the production of sebum in hamster sebaceous gland cells (Table 1).
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