JP6779627B2 - How to make callus for Queen of the Night, and external and internal skin preparations containing the extract as an active ingredient - Google Patents
How to make callus for Queen of the Night, and external and internal skin preparations containing the extract as an active ingredient Download PDFInfo
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- JP6779627B2 JP6779627B2 JP2016011683A JP2016011683A JP6779627B2 JP 6779627 B2 JP6779627 B2 JP 6779627B2 JP 2016011683 A JP2016011683 A JP 2016011683A JP 2016011683 A JP2016011683 A JP 2016011683A JP 6779627 B2 JP6779627 B2 JP 6779627B2
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- Medicines Containing Plant Substances (AREA)
Description
本発明は月下美人の組織・器官を材料としたカルス（脱分化細胞）の誘導方法、カルス培養増殖方法及び培養したカルスの抽出物を含有する、抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などに優れた新規な皮膚外用剤や内用剤などに関する。 The present invention contains a method for inducing callus (dedifferentiated cells) using tissues and organs of Tsukishita Bijin, a method for culturing and proliferating callus, and an extract of cultured callus, and has an antioxidant effect, an collagenase activity inhibitory effect, and a whitening effect. , A novel skin external agent or internal agent excellent in phyllagrin production promoting effect, anti-inflammatory effect, cell proliferation effect, collagen production promoting effect, hyaluronic acid production promoting effect and the like.
皮膚は生体の最外層に位置し、紫外線等の影響により活性酸素が発生しやすい臓器であり、絶えずその酸素ストレスに曝されている。一方、皮膚細胞内には活性酸素消去酵素が存在しており、その能力を超える活性酸素が発生しないかぎり活性酸素の傷害から皮膚細胞を防衛している。ところが、皮膚細胞内の活性酸素消去酵素の活性は加齢とともに低下することが知られており、活性酸素による傷害がその防御反応を凌駕したとき、皮膚は酸化され、細胞機能が劣化して老化してゆくと考えられる。また、皮膚以外の臓器においても、その活性酸素消去能を越える活性酸素に曝されたとき、機能低下が起こり老化したり、ガンや心筋梗塞など様々な生活習慣病が発症すると考えられる。そこで、活性酸素による傷害からの防御を目的として活性酸素消去剤や抗酸化剤が検討され、ＳＯＤやカタラーゼ等の活性酸素消去酵素、ＳＯＤ様活性物質などの活性酸素消去剤や抗酸化剤を配合した食品、化粧品、医薬部外品及び医薬品などが開発されている（特許文献１，２参照）。 The skin is located in the outermost layer of the living body, and is an organ that easily generates active oxygen due to the influence of ultraviolet rays and the like, and is constantly exposed to the oxygen stress. On the other hand, active oxygen scavenging enzyme exists in the skin cell, and protects the skin cell from the damage of the active oxygen unless the active oxygen exceeding its capacity is generated. However, it is known that the activity of active oxygen scavenging enzyme in skin cells decreases with aging, and when the damage caused by active oxygen exceeds the defense reaction, the skin is oxidized, the cell function deteriorates and aging. It is thought that it will continue. In addition, it is considered that when an organ other than the skin is exposed to active oxygen exceeding its active oxygen scavenging ability, functional deterioration occurs and aging occurs, and various lifestyle-related diseases such as cancer and myocardial infarction develop. Therefore, active oxygen scavengers and antioxidants have been studied for the purpose of protection from damage caused by active oxygen, and active oxygen scavengers such as SOD and catalase, and active oxygen scavengers and antioxidants such as SOD-like active substances are blended. Foods, cosmetics, non-pharmaceutical products, pharmaceuticals, etc. have been developed (see Patent Documents 1 and 2).
皮膚は、紫外線、乾燥、寒冷、熱、薬物等の様々な物理的及び化学的ストレスに日々曝されている。その結果、皮膚の機能低下が引き起こされ、様々な皮膚の老化現象が顕在化する。皮膚の老化現象の一つに、しわがある。しわには、表皮性のしわと、真皮性のしわの二種類が存在することが知られている。表皮性のしわは小じわと呼ばれ、皮膚の乾燥により、表皮角質層中の水分量が低下することによって一時的に生じるしわである。小じわの改善方法としては、保湿効果を有する化粧品の使用が一般的である。一方、真皮性のしわは、太陽光線に含まれる紫外線や加齢によって形成されるしわである。その形成メカニズムとしては、紫外線や加齢による真皮線維芽細胞におけるコラーゲン合成能の低下や、マトリックスメタロプロテアーゼ（ＭＭＰ）の増加によるコラーゲンの分解促進が挙げられる。 The skin is exposed daily to various physical and chemical stresses such as UV, dry, cold, heat and drugs. As a result, skin dysfunction is caused, and various skin aging phenomena become apparent. Wrinkles are one of the skin aging phenomena. It is known that there are two types of wrinkles: epidermal wrinkles and dermal wrinkles. Epidermal wrinkles are called fine wrinkles, which are temporary wrinkles caused by a decrease in the amount of water in the stratum corneum of the epidermis due to dry skin. As a method for improving fine wrinkles, it is common to use cosmetics having a moisturizing effect. On the other hand, dermal wrinkles are wrinkles formed by ultraviolet rays contained in sunlight and aging. The formation mechanism includes a decrease in collagen synthesis ability in dermal fibroblasts due to ultraviolet rays and aging, and an increase in matrix metalloproteinase (MMP) to promote collagen degradation.
乾燥に起因する表皮性のしわと真皮性のしわでは、組織学的形態、発症メカニズム、治療方法が異なり、紫外線や加齢により生じる真皮性のしわは、保湿効果を有する化粧品の使用によっては改善できない。 Epidermal wrinkles caused by dryness and dermal wrinkles have different histological morphology, onset mechanism, and treatment method, and dermal wrinkles caused by ultraviolet rays and aging can be improved by using cosmetics having a moisturizing effect. Can not.
これまでに、紫外線によって生じる真皮性のしわを改善することを目的として、加水分解アーモンドを有効成分とする皮膚のしわ形成防止・改善剤（特許文献３）、ジョチョウケイ、テンキシ及びキセンウの抽出物を有効成分とする紫外線照射に起因するしわの改善剤（特許文献４）が報告されている。 So far, for the purpose of improving dermal wrinkles caused by ultraviolet rays, an agent for preventing / improving skin wrinkles containing hydrolyzed almonds as an active ingredient (Patent Document 3), extracts of Jochokei, Tenxi and Kisenu A wrinkle improving agent (Patent Document 4) caused by irradiation with ultraviolet rays containing the active ingredient has been reported.
また、真皮には線維芽細胞やコラーゲンが存在し、Ｉ型コラーゲンが全体の８０％を占める。Ｉ型コラーゲンのほかにはＩＩＩ、Ｖ、ＸＩＩ及びＸＩＶ型コラーゲンの存在が知られている。しわやたるみの原因の一つとして、Ｉ型コラーゲンの減少が挙げられる。従って、Ｉ型コラーゲンの生成を促進させることが、しわ・たるみの予防・改善に有効であると考えられる。また、Ｉ型コラーゲンの生成促進は皮膚の創傷治癒の改善にも有効である。 In addition, fibroblasts and collagen are present in the dermis, and type I collagen accounts for 80% of the total. In addition to type I collagen, the presence of type III, V, XII and XIV collagen is known. One of the causes of wrinkles and sagging is a decrease in type I collagen. Therefore, promoting the production of type I collagen is considered to be effective in preventing and improving wrinkles and sagging. In addition, promotion of type I collagen production is also effective in improving skin wound healing.
また、線維芽細胞はコラーゲンなどのタンパク質を産生して真皮結合組織を形成し、皮膚のはりを保っている。この結合組織が収縮力を失い、さらに弾力性を失う結果として皮膚のしわやたるみが発生すると考えられている。 In addition, fibroblasts produce proteins such as collagen to form dermal connective tissue and maintain skin elasticity. It is believed that this connective tissue loses its contractile force and further loses its elasticity, resulting in wrinkles and sagging of the skin.
コラーゲンは、哺乳動物組織の約１/３を占める主要な構造タンパク質であり、軟骨、骨、腱、及び皮膚を含む多くのマトリックス組織の必須な成分である。マトリックスメタロプロテアーゼ（ＭＭＰ）に属するコラゲナーゼ（ＭＭＰ−１など）により１箇所を切断されると、通常の組織内では安定なコラーゲン分子は、変性して一本鎖のゼラチンとなり、他の様々なプロテアーゼにより分解されるようになる。その結果、マトリックス組織の構造の完全性が失われてしまう。 Collagen is a major structural protein that makes up about one-third of mammalian tissue and is an essential component of many matrix tissues, including cartilage, bone, tendons, and skin. When one site is cleaved by collagenase (such as MMP-1) belonging to matrix metalloproteinase (MMP), collagen molecules that are stable in normal tissues are denatured into single-stranded gelatin, and various other proteases. Will be decomposed by. As a result, the structural integrity of the matrix structure is lost.
一般に、しみ、そばかす、日焼けなどに見られる皮膚の色素沈着は、ホルモンの異常や紫外線の刺激により、皮膚内に存在するメラノサイトがメラニン色素を過剰に生成し、これが皮膚内に沈着することが原因と考えられている。このような色素沈着を防ぐ方法の一つに、メラニンの過剰な生成を抑制する方法が知られている。従来、色素沈着の治療には、内用や外用などにおいて、アスコルビン酸（ビタミンＣ）等が用いられてきた。 In general, skin pigmentation seen in age spots, freckles, sunburn, etc. is caused by excessive production of melanin pigment by melanocytes present in the skin due to hormonal abnormalities and UV stimulation, which are deposited in the skin. It is believed that. As one of the methods for preventing such pigmentation, a method for suppressing excessive production of melanin is known. Conventionally, ascorbic acid (vitamin C) and the like have been used for the treatment of pigmentation for internal and external use.
加齢とともに表皮細胞の増殖・分裂能は低下し、表皮層自体は薄くなる（非特許文献１参照）。生体因子であるＥｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ（ＥＧＦ／上皮細胞成長因子）や女性ホルモン（エストロゲン）は皮膚の表皮細胞増殖に働きかけるが、加齢と共にその分泌は低下する。このような加齢による表皮細胞代謝機能の低下は、皮膚のターンオーバー速度を遅らせ、肌荒れや皮膚の老化の原因となる。また、角層表面から剥がれ落ちる角層細胞が滞留することで、表皮内メラニンの排泄がスムーズに行われなくなり、色素沈着や肌のくすみの原因となる。さらに表皮の創傷治癒が遅くなることなども知られている。これらの現象の進行を防止あるいは改善するために、表皮細胞の増殖を促進させる成分の探索や、多くの皮膚外用剤の提案がなされてきた。 With aging, the ability of epidermal cells to proliferate and divide decreases, and the epidermal layer itself becomes thinner (see Non-Patent Document 1). The biological factors Epidermal Growth Factor (EGF / epidermal growth factor) and female hormone (estrogen) act on the proliferation of epidermal cells of the skin, but their secretion decreases with aging. Such a decrease in epidermal cell metabolic function due to aging slows down the turnover rate of the skin and causes rough skin and aging of the skin. In addition, the retention of stratum corneum cells that peel off from the surface of the stratum corneum prevents smooth excretion of melanin in the epidermis, which causes pigmentation and dullness of the skin. It is also known that wound healing of the epidermis is delayed. In order to prevent or improve the progression of these phenomena, the search for components that promote the proliferation of epidermal cells and the proposal of many external preparations for skin have been made.
従来、アトピー性皮膚炎、接触性皮膚炎、湿疹、乾癬等の皮膚疾患による肌荒れや炎症、並びに健常人の肌荒れ、ニキビに対する皮膚外用剤の有効成分として、抗炎症、抗アレルギー作用を有するステロイド剤や保湿効果を有するワセリンや尿素などが用いられてきた。しかしながら、ステロイド剤は副作用が強く、保湿剤はその効果が必ずしも十分ではないことから、より安全性の高い優れた有効成分が望まれていた。 Conventionally, steroids having anti-inflammatory and anti-allergic effects as active ingredients of external skin preparations for rough skin and inflammation caused by skin diseases such as atopic dermatitis, contact dermatitis, eczema, and psoriasis, as well as rough skin and acne in healthy people. Vaseline and urea, which have a moisturizing effect, have been used. However, since steroids have strong side effects and moisturizers do not always have sufficient effects, an excellent active ingredient with higher safety has been desired.
皮膚の炎症反応において、炎症に関わる様々な細胞の炎症部位への遊走および活性化に関わるサイトカイン類が明らかになってきている。このうち、ＩＬ−１αは表皮角化細胞や浸潤してきた炎症細胞により産生され、炎症反応に関与するＮＦκＢ（Ｎｕｃｌｅａｒ Ｆａｃｔｏｒ κＢ）やＭＡＰキナーゼなどの活性化を引き起こし、一連の炎症反応の引き金を引く重要な役割を担っているものと考えられている（特許文献５）。 In the inflammatory reaction of the skin, cytokines involved in the migration and activation of various cells involved in inflammation to the inflamed site have been clarified. Of these, IL-1α is produced by epidermal keratinocytes and infiltrated inflammatory cells, triggers activation of NFκB (Nuclear Factor κB) and MAP kinase involved in the inflammatory reaction, and triggers a series of inflammatory reactions. It is considered to play an important role (Patent Document 5).
角質層の保湿性に重要な役割を果たしているのがＮＭＦであることは古くから知られており、これまでＮＭＦ成分は保湿剤の開発に応用されてきた。角質層におけるＮＭＦの減少は、その保湿性を低下させ乾燥を招く。その結果として乾燥性のカユミが引き起こされる。近年、ＮＭＦの主体をなすアミノ酸は、ケラトヒアリン顆粒の主成分であるフィラグリンというタンパク質の分解により産生されることが明らかとなった（非特許文献２）。すなわち、表皮顆粒層において合成されたプロフィラグリンはケラトヒアリン顆粒に蓄積された後、顆粒層上層から角質層に至る過程で脱リン酸、加水分解を経てフィラグリンに分解される。さらにフィラグリンは角質層上層に至る過程でアミノ酸に分解されＮＭＦの主体となる。一方、乾燥肌を呈する病態とフィラグリンに関する研究が進められ、老人性乾皮症やアトピー性皮膚炎などの角質層中ではアミノ酸が減少していることが知られているが、それらの皮膚ではフィラグリンの発現が低下していることが明らかにされている（非特許文献３）。したがって、角質層の保湿性維持の目的でＮＭＦの産生を高めるためにはケラチノサイトにおけるフィラグリンあるいはプロフィラグリンの生成促進が重要であると考えられるようになり、特許文献６、７などの植物成分を用いたフィラグリンあるいはプロフィラグリンの生成促進剤が報告されている。 It has long been known that NMF plays an important role in the moisturizing properties of the stratum corneum, and NMF components have been applied to the development of moisturizers. A decrease in NMF in the stratum corneum reduces its moisturizing properties and leads to dryness. The result is dry scabies. In recent years, it has been clarified that the amino acid that is the main component of NMF is produced by the decomposition of a protein called filaggrin, which is the main component of keratohyalin granules (Non-Patent Document 2). That is, profilaggrin synthesized in the epidermal granule layer is accumulated in keratohyalin granules, and then decomposed into filaggrin through dephosphorylation and hydrolysis in the process from the upper layer of the granule layer to the stratum corneum. Furthermore, filaggrin is decomposed into amino acids in the process of reaching the upper stratum corneum and becomes the main component of NMF. On the other hand, research on the pathophysiology of dry skin and filaggrin has been advanced, and it is known that amino acids are reduced in the stratum corneum such as senile xerosis and atopic dermatitis, but filaggrin is found in those skins. It has been clarified that the expression of is reduced (Non-Patent Document 3). Therefore, in order to increase the production of NMF for the purpose of maintaining the moisturizing property of the stratum corneum, it has become important to promote the production of filaggrin or profilaggrin in keratinocytes, and plant components such as Patent Documents 6 and 7 are used. There have been reports of filaggrin or profilaggrin production promoters.
従来より、皮膚中には真皮・表皮のいずれにもヒアルロン酸があることが知られている。ヒアルロン酸はグルクロン酸とＮ―アセチルグルコサミンが交互に結合した高分子で、その分子量は数百万にも及び、皮膚の弾力性又は粘弾性、保水性に深く関与している事が明らかとなっている。しかしながらこのヒアルロン酸は加齢と共に減少することが報告されており（特許文献１１）、これにより老化に伴う皮膚の保水力の減少を引き起こし、乾燥肌などの原因となると考えられている。 Conventionally, it has been known that hyaluronic acid is present in both the dermis and the epidermis in the skin. Hyaluronic acid is a polymer in which glucuronic acid and N-acetylglucosamine are alternately bound, and its molecular weight reaches several million, and it has been clarified that it is deeply involved in skin elasticity, viscoelasticity, and water retention. ing. However, it has been reported that this hyaluronic acid decreases with aging (Patent Document 11), which causes a decrease in the water retention capacity of the skin with aging, and is considered to cause dry skin and the like.
一方で、月下美人抽出物には、コラーゲン生成促進及び分解抑制の作用や、フィラグリン生成促進剤、ケラチノサイト分裂促進剤、ケラチノサイトのＡＴＰ産生促進剤としての効果が知られている（特許文献８、特許文献９、特許文献１０）。 On the other hand, the Tsukishita Bijin extract is known to have an action of promoting collagen production and suppressing decomposition, and an effect of a filaggrin production promoter, a keratinocyte division promoter, and a keratinocyte ATP production promoter (Patent Document 8, Patent Document 8, Patent Document 9, Patent Document 10).
しかし、月下美人のカルスには、抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果において高い効果を有することは知られていなかった。 However, the callus of Tsukishita Bijin has high effects in antioxidant effect, collagenase activity inhibitory effect, whitening effect, filaggrin production promoting effect, anti-inflammatory effect, cell proliferation effect, collagen production promoting effect, and hyaluronic acid production promoting effect. It was not known.
本発明は、安定的に原料を供給でき、かつ安全で安定性に優れ、抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などに優れた新規な皮膚外用剤や内用剤などを提供することを課題とする。 The present invention can stably supply raw materials, is safe and has excellent stability, and has an antioxidant effect, a collagenase activity inhibitory effect, a whitening effect, a filaggrin production promoting effect, an anti-inflammatory effect, a cell proliferation effect, and a collagen production promoting effect. An object of the present invention is to provide a new external preparation for skin or internal preparation having an excellent effect of promoting hyaluronic acid production.
本発明者らは、この問題を解決すべく、鋭意研究を重ねた結果、特定の濃度のオーキシンとサイトカイニンを含む培地で月下美人をカルス化・大量培養することに成功し、その抽出物に、抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などが優れていることを見出した。さらに、その抽出物を含有する外用剤が、安全で安定であり、抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などが優れており、多機能性美容剤となりうることを見出し、本発明を完成するに至った。 As a result of intensive research to solve this problem, the present inventors have succeeded in callusizing and mass-culturing the lunar beauty in a medium containing a specific concentration of auxin and cytokinin, and using the extract as the extract. , Antioxidant effect, collagenase activity inhibitory effect, whitening effect, phyllagulin production promoting effect, anti-inflammatory effect, cell proliferation effect, collagen production promoting effect, hyaluronic acid production promoting effect and the like were found to be excellent. Furthermore, the external preparation containing the extract is safe and stable, and has an antioxidant effect, a collagenase activity inhibitory effect, a whitening effect, a phyllagulin production promoting effect, an anti-inflammatory effect, a cell proliferation effect, a collagen production promoting effect, and hyaluronic acid. We have found that it has an excellent production promoting effect and can be a multifunctional beauty agent, and have completed the present invention.
すなわち、本発明は、以下の（１）〜（６）からなる。
（１）月下美人のカルスであって、オーキシン及びサイトカイニンを含有する培地で培養することを特徴とする月下美人のカルス。
（２）月下美人のカルスであって、オーキシンがインドール酢酸、インドール酪酸、α―ナフタレン酢酸、２，４−ジクロロフェノキシ酢酸及び２，６−ジクロロ安息香酸から一種又は二種以上選択され、サイトカイニンがゼアチン、フルフリルアミノプリン（カイネチン）、ベンジルアデニン、イソペンテニルアデニン及びジメチルアミノプリンから一種又は二種以上選択されることを特徴とする、（１）記載の月下美人のカルス。
（３）月下美人のカルスの製造方法であって、オーキシン及びサイトカイニンを含有する培地で培養することを特徴とし、オーキシンがインドール酢酸、インドール酪酸、α―ナフタレン酢酸、２，４−ジクロロフェノキシ酢酸及び２，６−ジクロロ安息香酸から一種又は二種以上選択され、サイトカイニンがゼアチン、フルフリルアミノプリン（カイネチン）、ベンジルアデニン、イソペンテニルアデニン及びジメチルアミノプリンから一種又は二種以上選択され、それぞれ１０−10〜１０−2Ｍの濃度でＭＳ培地及び／又はＢ５培地を用いて培養することを特徴とする月下美人のカルスの製造方法。
（４）月下美人のカルス及び／又はその抽出物を含有することを特徴とする皮膚外用剤。
（５）月下美人のカルス及び／又はその抽出物を含有することを特徴とする医薬品。
（６）月下美人のカルス及び／又はその抽出物を含有することを特徴とする食品。
That is, the present invention comprises the following (1) to (6).
(1) A queen of the night callus, which is characterized by being cultured in a medium containing auxin and cytokinin.
(2) Callus of the beauty of the moon, auxin is selected from one or more of indole acetic acid, indole butyric acid, α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid and 2,6-dichlorobenzoic acid, and cytokinin. The callus of the young beauty according to (1), wherein one or more of zeatin, flufurylaminopurine (kinetin), benzyladenine, isopentenyladenine and dimethylaminopurine are selected.
(3) A method for producing callus of Tsukishita Bijin, which is characterized by culturing in a medium containing auxin and cytokinin. And one or more selected from 2,6-dichlorobenzoic acid, and one or more cytokinins selected from zeatin, flufurylaminopurine (kinetin), benzyladenin, isopentenyladenin and dimethylaminopurine, each 10 A method for producing callus of Tsukishita Bijin, which comprises culturing using MS medium and / or B5 medium at a concentration of −10 to 10 −2 M.
(4) An external preparation for skin, which contains callus and / or an extract thereof of Queen of the Night.
(5) A pharmaceutical product containing callus and / or an extract thereof of Queen of the Night.
(6) A food containing queen of the night callus and / or an extract thereof.
本発明の月下美人のカルス又はその抽出物は、優れた抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などを有しており、医薬品、医薬部外品、化粧品、食品の分野などにおいて貢献できるものである。 The curls of Tsukishita Bijin or its extract of the present invention has excellent antioxidant effect, collagenase activity inhibitory effect, whitening effect, phyllagrin production promoting effect, anti-inflammatory effect, cell proliferation effect, collagen production promoting effect, hyaluronic acid production promoting effect. It has effects and can contribute to the fields of pharmaceuticals, quasi-drugs, cosmetics, foods, etc.
以下に、本発明の詳細について述べる。 The details of the present invention will be described below.
本発明に用いる月下美人とは、サボテン科クジャクサボテンの原種（Ｅｐｉｐｈｙｌｌｕｍ ｏｘｙｐｅｔａｌｕｍ）及びその近縁種を用いることができる。また、園芸品種を用いることができる。 As the queen of the night used in the present invention, the original species (Epiphyllum oxipetalum) of the cactaceae Epiphyllum oxypetalum and related species thereof can be used. In addition, horticultural varieties can be used.
本発明に用いるカルスとは、分化していない状態の植物細胞又は分化していない状態の植物細胞塊のことをいう。 The callus used in the present invention refers to a plant cell in an undifferentiated state or a plant cell mass in an undifferentiated state.
本発明の月下美人のカルスは、前記の月下美人の植物体や植物細胞などから誘導することができる。更に、得られたカルスを増殖させる２段階の培養で製造することもできる。
一例としては、カルスを誘導する培養では、月下美人の葉、茎、根などの組織を３０〜９５％エタノール水溶液、０．１〜５％次亜塩素酸ナトリウム水溶液などによってその植物体の表面を殺菌してから特定の培地にてカルスを誘導し、培養することができる。
The callus of the queen of the night of the present invention can be derived from the plant body or plant cell of the queen of the night. Furthermore, it can also be produced by a two-step culture in which the obtained callus is propagated.
As an example, in a callus-inducing culture, tissues such as leaves, stems, and roots of Tsukishita Bijin are treated with a 30-95% ethanol solution, 0.1-5% sodium hypochlorite solution, etc. Callus can be induced and cultured in a specific medium after sterilization.
カルスを誘導する培地は、ムラシゲ・スクーグ（ＭＳ培地）、リンスマイヤー・スクーグ、ホワイト、ニッチ、ガンボーグ（Ｂ５培地）、ＷＰＭ（Ｗｏｏｄｙ Ｐｌａｎｔ Ｍｅｄｉｕｍ）などの、植物組織培養に一般的に用いられる培地成分に炭素源及び植物ホルモンを添加して、１２１℃、１５分間の条件で蒸気加熱滅菌して使用することができる。中でも、有効性の面から、ＭＳ培地、Ｂ５培地が好ましく、ＭＳ培地が最も好ましい。 The medium for inducing callus is a medium component commonly used for plant tissue culture, such as Murashige and Skoog (MS medium), Rinsemeyer skoog, White, Nitch, Gambog (B5 medium), and WPM (Woody Plant Medium). A carbon source and a plant hormone can be added to the mixture, and the medium can be sterilized by steam heating at 121 ° C. for 15 minutes. Among them, MS medium and B5 medium are preferable, and MS medium is most preferable from the viewpoint of effectiveness.
また、炭素源はグルコース、フルクトースなどの単糖又はそれらを構成糖とするショ糖、マルトースなどの二糖類やオリゴ糖を培地全量に対して０．１〜１０％の範囲で含有することができるが、中でもショ糖を０．５〜５％の範囲で含有することが好ましい。 Further, the carbon source can contain monosaccharides such as glucose and fructose, or disaccharides such as sucrose and maltose having them as constituent sugars, and oligosaccharides in the range of 0.1 to 10% with respect to the total amount of the medium. However, it is preferable to contain sucrose in the range of 0.5 to 5%.
植物ホルモンとしては、オーキシン類、サイトカイニンなどを培地全量に対して１０−１０〜１０−２ Ｍの濃度範囲で単独、又は組み合わせて含有する。オーキシン類としては、インドール酢酸、インドール酪酸、α―ナフタレン酢酸、２，４−ジクロロフェノキシ酢酸、２，６−ジクロロ安息香酸が挙げられ、また、サイトカイニンとしては、ゼアチン、フルフリルアミノプリン（カイネチン）、ベンジルアデニン、イソペンテニルアデニン、ジメチルアミノプリンなどが挙げられる。中でも、オーキシン類としてはインドール酢酸及びα―ナフタレン酢酸が好ましく、サイトカイニンとしてはフルフリルアミノプリン（カイネチン）及びベンジルアデニンが好ましく、α―ナフタレン酢酸とベンジルアデニンとを組み合わせて含有することが最も好ましい。
また、オーキシン類やサイトカイニンの含有量は、１０−１０〜１０−２Ｍが好ましく、１０−６〜１０−４Ｍがより好ましい。
As plant hormones, auxins, cytokinins and the like are contained alone or in combination in a concentration range of 10-10 to 10-2 M with respect to the total amount of the medium. Examples of auxins include indole acetic acid, indole butyric acid, α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, and 2,6-dichlorobenzoic acid, and examples of cytokinins include zeatin and flufurylaminopurine (kinetin). , Benzylaminopurine, isopentenyladenine, dimethylaminopurine and the like. Among them, indoleacetic acid and α-naphthalene acetic acid are preferable as auxins, flufurylaminopurine (kinetin) and benzyladenine are preferable as cytokinins, and α-naphthalene acetic acid and benzyladenine are most preferably contained in combination.
The content of auxin and cytokinin is preferably 10 -10 to 10 -2 M, more preferably 10 -6 ~10 -4 M.
さらには、ジベレリン、アブシジン酸、ブラシノステロイドなどを培地に添加することもできる。 Furthermore, gibberellin, abscisic acid, brassinosteroids and the like can be added to the medium.
カルスの誘導に使用する培地に加える成分の内、加熱によって分解する物質は、それ以外の成分を蒸気加熱滅菌した後に別途、０．２μｍなどのフィルターを使用した濾過滅菌をして添加することができる。培地のｐＨは調製時に水酸化ナトリウムや水酸化カリウムなどの希アルカリ溶液と塩酸溶液によってｐＨ４〜７．５、好ましくは５．５〜６とすることができる。 Of the components added to the medium used to induce callus, substances that decompose by heating can be added by separately filtering and sterilizing the other components using a filter such as 0.2 μm after steam heat sterilization. it can. The pH of the medium can be adjusted to pH 4 to 7.5, preferably 5.5 to 6 by using a dilute alkaline solution such as sodium hydroxide or potassium hydroxide and a hydrochloric acid solution at the time of preparation.
カルス化に用いる植物体としては、上述のとおり、月下美人の葉、茎、根などの組織があげられるが、種子や子葉が好ましく、発芽したばかりの子葉（例えば高さが０．５〜１０ｃｍ程度）が最も好ましい。 As described above, the plants used for callus formation include tissues such as leaves, stems, and roots of the beautiful moon, but seeds and cotyledons are preferable, and freshly germinated cotyledons (for example, height is 0.5 to 0.5 to). (About 10 cm) is most preferable.
子葉を使用する場合は、月下美人の果実から種子を取り出し、例えばホルモンを添加していないＭＳ寒天培地などに播種して無菌的に培養することにより、子葉を得ることができる。より具体的には、月下美人の果実表面を滅菌し、果実から種子を無菌的に取り出し、例えばホルモンを添加していないＭＳ寒天培地に直接播種して無菌的に培養することが子葉を効率よく得る点で好ましい。果実から取り出し、種子の状態で保存したものよりも果実から取り出し、すぐに播種するほうが子葉の発生率の面で好ましい。 When cotyledons are used, cotyledons can be obtained by taking out seeds from the fruits of Tsukishita Bijin, sowing them on, for example, MS agar medium to which no hormone is added, and culturing them aseptically. More specifically, it is efficient to sterilize the fruit surface of Tsukishita Bijin, aseptically remove seeds from the fruit, sow directly on MS agar medium without adding hormones, and aseptically cultivate the leaflets. It is preferable because it can be obtained well. It is preferable in terms of the incidence of cotyledons to be taken out from the fruit and sown immediately rather than taken out from the fruit and stored in the state of seeds.
カルス誘導は固体培地でも、液体培地でも可能であるが、通常、前記培地に対して、０．４〜２％の寒天や０．１〜０．５％のゲルライトなどを添加することによって固化した固体培地上で培養するのが好ましい。培養は１５〜３０℃、好ましくは２３〜２７℃の温度で培養するのが好ましい。その際、明所で培養することが好ましい。培養については、１０〜２４時間明期、好ましくは１６〜２４時間明期で培養するのが好ましい。通常、培養５〜６０日後に、０．１〜３０ｍｍ径の細胞塊としてカルスを得ることができる。 Callus induction can be carried out in either a solid medium or a liquid medium, but it is usually solidified by adding 0.4 to 2% agar, 0.1 to 0.5% gellite, or the like to the medium. It is preferable to culture on a solid medium. The culture is preferably carried out at a temperature of 15 to 30 ° C., preferably 23 to 27 ° C. At that time, it is preferable to culture in a bright place. As for the culture, it is preferable to culture in the light period of 10 to 24 hours, preferably in the light period of 16 to 24 hours. Usually, after 5 to 60 days of culturing, callus can be obtained as a cell mass having a diameter of 0.1 to 30 mm.
次に、カルスを新しい培地に移植して培養することによってカルスを増殖させることができる。本培養は固体培養、液体培養のどちらでも良いが、経済性、大量生産性を考えて液体培養で行うのが好ましい。培養はカルスを誘導する際の培地と同じか、又は塩濃度を減ずるなどの改変を加えた培地で培養を行うことができる。塩濃度を減ずる改変を加えた培地とは培地成分中の硝酸カリウム、硝酸ナトリウム、硝酸アンモニウム、リン酸二水素ナトリウム、塩化カルシウム、硫酸マグネシウムなどの主要無機塩のうち１種又は２種以上の成分をペクチンの生産に影響を及ぼさない程度に減じた培地であることが好ましい。また、培地中に公知の添加剤として知られるカザミノ酸などの有機酸、アミノ酸などの窒素源、ココナッツミルク、酵母エキス、ポリペプトンを０．０１〜１０ｇ／Ｌの濃度で加えることもできる。 The callus can then be grown by transplanting the callus into a new medium and culturing it. The main culture may be either solid culture or liquid culture, but it is preferable to perform the main culture in liquid culture in consideration of economy and mass productivity. The culture can be carried out in the same medium as the medium used for inducing callus, or in a medium modified such as by reducing the salt concentration. What is a modified medium that reduces the salt concentration? Pectin is one or more of the major inorganic salts such as potassium nitrate, sodium nitrate, ammonium nitrate, monosodium dihydrogen phosphate, calcium chloride, and magnesium sulfate in the medium components. It is preferable that the medium is reduced to such an extent that it does not affect the production of. Further, an organic acid such as casamino acid known as a known additive, a nitrogen source such as an amino acid, coconut milk, yeast extract, and polypeptone can be added to the medium at a concentration of 0.01 to 10 g / L.
カルスを増殖させる培養の培養温度、光条件、培養期間は、カルス誘導時の培養条件と同様の条件で、培養を２〜５回繰り返すことによって数個の細胞から数百個の細胞によって作られる細胞塊として培養細胞が得られる。液体培養時の酸素供給は往復及び旋回振とうや、培養液への直接通気又はフォローファイバーを用いた間接的な方法のいずれの方法でも可能である。旋回振とう培養では振とう数を５０回転／分以上、２００回転／分以下とすることやフラスコ容器あたりの培地量を容量の５％以上、３０％以下として気液界面を増大することが好ましく、培養液へ直接通気する場合では培養液あたりの通気量が毎分５容積％以上、２０容積％以下とすることが好適である。酸素供給量の増大は細胞の増殖速度を早めることに対して有効である。 The culture temperature, light conditions, and culture period of the culture in which the crows are propagated are the same as the culture conditions at the time of culturing, and the culture is repeated 2 to 5 times to be produced by several to several hundred cells. Cultured cells are obtained as cell clusters. Oxygen supply during liquid culture can be performed by either reciprocating and swirling shaking, direct ventilation to the culture solution, or an indirect method using follow fibers. In the swirling shaking culture, it is preferable to increase the gas-liquid interface by setting the number of shakes to 50 rpm or more and 200 rpm or less and the amount of medium per flask container to be 5% or more and 30% or less of the volume. In the case of direct aeration to the culture medium, it is preferable that the aeration rate per minute is 5% by volume or more and 20% by volume or less per minute. Increasing the oxygen supply is effective in accelerating the growth rate of cells.
本発明に用いる月下美人のカルス抽出物とは、前記の月下美人のカルスから抽出溶媒によって抽出したものである。その抽出方法は特に限定されず、例えば、加熱抽出したものであっても良いし、常温抽出したものであっても良い。また、培養したカルスを生のまま抽出しても良いし、乾燥や凍結乾燥したカルス、冷凍保存したカルスを使用することができる。 The queen of the night callus extract used in the present invention is extracted from the queen of the night callus with an extraction solvent. The extraction method is not particularly limited, and for example, it may be extracted by heating or may be extracted at room temperature. In addition, the cultured callus may be extracted raw, or dried or freeze-dried callus or frozen-preserved callus can be used.
抽出溶媒としては、例えば、水、低級アルコール（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）、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、グリセリンなど）、ケトン類（アセトン、メチルエチルケトンなど）、アセトニトリル、エステル類（酢酸エチル、酢酸ブチルなど）、炭化水素類（ヘキサン、ヘプタン、流動パラフィンなど）、エーテル類（エチルエーテル、テトラヒドロフラン、プロピルエーテルなど）が挙げられる。好ましくは、水、低級アルコール及び液状多価アルコールなどの極性溶媒が良く、特に好ましくは、水、エタノール、１，３−ブチレングリコール及びプロピレングリコールが良い。これらの溶媒は一種でも二種以上を混合して用いても良い。 Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol), 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.) Can be mentioned. Polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferred, and water, ethanol, 1,3-butylene glycol and propylene glycol are particularly preferred. These solvents may be used alone or in admixture of two or more.
上記抽出物は、抽出した溶液のまま用いても良く、必要に応じて、濃縮、希釈及び濾過処理、活性炭などによる脱色、脱臭処理などをして用いても良い。更には、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥などの処理を行い、乾燥物として用いても良い。 The above extract may be used as it is in the extracted solution, or may be used after concentration, dilution and filtration treatment, decolorization with activated carbon or the like, deodorization treatment and the like, if necessary. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze drying and the like, and used as a dried product.
本発明の皮膚外用剤や内用剤は、上記カルス及び／又はその抽出物をそのまま使用しても良く、これらの効果を損なわない範囲内で、化粧品、医薬部外品、医薬品又は食品などに用いられる成分である油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、金属石鹸、ｐＨ調整剤、防腐剤、香料、保湿剤、粉体、紫外線吸収剤、増粘剤、色素、酸化防止剤、美白剤、キレート剤、賦形剤、皮膜剤、甘味料などの成分を配合することもできる。 As the skin external preparation or internal preparation of the present invention, the above-mentioned curls and / or extracts thereof may be used as they are, and within the range not impairing these effects, they may be used in cosmetics, non-pharmaceutical products, pharmaceuticals, foods, etc. Oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, etc. Ingredients such as thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, film agents, and sweeteners can also be blended.
本発明は、医薬品、医薬部外品、化粧品又は食品などに用いることができ、その剤型としては、例えば、化粧水、クリーム、マッサージクリーム、乳液、ゲル剤、エアゾール剤、パック、洗浄剤、浴用剤、ファンデーション、打粉、口紅、軟膏、パップ剤、ペースト剤、プラスター剤、エッセンス、散剤、丸剤、錠剤、注射剤、坐剤、乳剤、カプセル剤、顆粒剤、液剤（チンキ剤、流エキス剤、酒精剤、懸濁剤、リモナーデ剤などを含む）、飲料などが挙げられる。 The present invention can be used for pharmaceuticals, non-pharmaceutical products, cosmetics, foods, etc., and the dosage forms thereof include, for example, lotions, creams, massage creams, emulsions, gels, aerosols, packs, cleaning agents, and the like. Bathing agents, foundations, dusting powders, lipsticks, ointments, poultices, pastes, plasters, essences, powders, pills, tablets, injections, suppositories, emulsions, capsules, granules, liquids (tinki agents, flow extracts) Agents, alcoholic agents, suspending agents, limonade agents, etc.), beverages and the like.
本発明に用いる上記抽出物の含有量は、外用の場合、全量に対し、固形物に換算して０．０００１重量％以上が好ましく、０．００１〜１０重量％がより好ましい。さらに、０．０１〜５重量％が最も好ましい。０．０００１重量％未満では十分な効果は望みにくい。１０重量％を越えて含有した場合、効果の増強は認められにくく不経済である。一方、内用の場合、投与量は年齢、体重、症状、治療効果、投与方法、処理時間などにより異なるが、通常、成人１人当たりの１日の量としては、乾燥物に換算して、５ｍｇ以上が好ましく、１０ｍｇ〜５ｇがより好ましい。さらに、１００ｍｇ〜１ｇが最も好ましい。 In the case of external use, the content of the extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 to 10% by weight, in terms of solid matter, based on the total amount. Further, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it is contained in an amount exceeding 10% by weight, the enhancement of the effect is hardly recognized and it is uneconomical. On the other hand, in the case of internal use, the dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but usually, the daily dose per adult is 5 mg in terms of dry matter. The above is preferable, and 10 mg to 5 g is more preferable. Further, 100 mg to 1 g is most preferable.
次に本発明を詳細に説明するため、実施例として本発明に用いる月下美人カルスの抽出物の製造例、実験例及び処方例を挙げるが、本発明はこれに限定されるものではない。製造例に示す％とは重量％を、実施例に示す含有量の部とは重量部を示す。 Next, in order to explain the present invention in detail, examples of production, experimental examples, and prescription examples of the extract of Tsukishita Bijin Callus used in the present invention will be given, but the present invention is not limited thereto. The% shown in the production example means% by weight, and the content part shown in the example means a weight part.
製造例Ａ 月下美人のカルス
月下美人の果実から種子を無菌的に取り出し、ホルモンを添加していないＭＳ寒天培地に播種して無菌的に培養し、子葉を得る。この子葉の切片を１０−５Ｍのα―ナフタレン酢酸及び１０−５Ｍのベンジルアデニンを含むＭＳ寒天培地に置床し、明期１６時間でカルスを誘導した。一ヶ月後、このカルスを前記のＭＳ液体培地７５ｍＬに入れて明期２４時間で２週間、１００ｒｐｍで回転振とう培養し、その懸濁培養細胞を調製した。この培養細胞を１０−５Ｍのα―ナフタレン酢酸及び１０−５Ｍのベンジルアデニンを含むＭＳ液体培地０．７５Ｌに接種し、明期２４時間で２週間、１００ｒｐｍで回転振とう培養した。得られた培養細胞を精製水で十分に洗浄し、カルスを得た（湿潤物）。
Production Example A Callus of Tsukishita Bijin The seeds are aseptically taken out from the fruit of Tsukishita Bijin, sown on MS agar medium to which no hormone is added, and aseptically cultured to obtain cotyledons. The sections of the cotyledons were plated on MS agar medium containing 10 -5 M of α- naphthalene acetic acid and 10 -5 M benzyladenine was induced calli light period of 16 hours. One month later, the callus was placed in 75 mL of the above-mentioned MS liquid medium and cultured with rotary shaking at 100 rpm for 2 weeks at 24 hours in the light period to prepare suspended cultured cells. The cultured cells were inoculated into MS liquid medium 0.75L containing 10 -5 M of α- naphthalene acetic acid and 10 -5 M benzyladenine, 2 weeks photoperiod 24 h, followed by rotary shaking culture at 100 rpm. The obtained cultured cells were thoroughly washed with purified water to obtain callus (wet product).
製造例１ 月下美人カルスの熱水抽出物
製造例Ａで得たカルス２０ｇに精製水２００ｍＬを加え、９５〜１００℃で２時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して熱水抽出物を０．２ｇ得た。
Production Example 1 Hot water extract of Shimobijin Callus 200 mL of purified water was added to 20 g of callus obtained in Production Example A, extracted at 95-100 ° C for 2 hours, filtered, the filtrate concentrated, and freeze-dried. 0.2 g of hot water extract was obtained.
製造例２ 月下美人カルスの５０％エタノール抽出物
製造例Ａで得たカルス２０ｇに５０％エタノール水溶液２００ｍＬを加え、常温で１〜７日間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して５０％エタノール抽出物を０．２ｇ得た。
Production Example February 50% ethanol extract of Shimobijin callus Add 200 mL of 50% ethanol aqueous solution to 20 g of callus obtained in Production Example A, extract at room temperature for 1 to 7 days, filter, concentrate the filtrate, and freeze. Drying gave 0.2 g of 50% ethanol extract.
製造例３ 月下美人カルスのエタノール抽出物
製造例Ａで得たカルス２０ｇにエタノール２００ｍＬを加え、常温で１〜７日間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥してエタノール抽出物を０．１８ｇ得た。
Production Example March Ethanol Extract of Callus Shimobijin Add 200 mL of ethanol to 20 g of callus obtained in Production Example A, extract at room temperature for 1 to 7 days, filter, concentrate the filtrate, freeze-dry and extract ethanol. 0.18 g of the product was obtained.
製造例４ 月下美人花、月下美人葉・茎の熱水抽出物
月下美人の花２０ｇ又は、月下美人の葉・茎混合物の乾燥物２０ｇに精製水４００ｍＬを加え、９５〜１００℃で２時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して熱水抽出物をそれぞれ、５．９ｇ、３．６ｇ得た。
Production example April Queen of the Night flower, hot water extract of queen of the night leaf / stem Add 400 mL of purified water to 20 g of queen of the night flower or 20 g of dried product of queen of the night leaf / stem mixture, and add 400 mL of purified water to 95-100 ° C. After extraction for 2 hours in the above, the mixture was filtered, the filtrate was concentrated, and lyophilized to obtain 5.9 g and 3.6 g of hot water extracts, respectively.
製造例５ 月下美人花、月下美人葉・茎の５０％エタノール抽出物
月下美人の花２０ｇ又は、月下美人の葉・茎混合物の乾燥物２０ｇに５０％エタノール水溶液４００ｍＬを加え、常温で１〜７日間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥して５０％エタノール抽出物をそれぞれ、１．８ｇ、０．８ｇ得た。
Production example May 50% ethanol extract of queen of the night, queen of the night leaves and stems Add 400 mL of 50% ethanol aqueous solution to 20 g of queen of the night flowers or 20 g of dried queen of the night leaves and stems at room temperature. After 1 to 7 days of extraction, the filtrate was filtered, and the filtrate was concentrated and lyophilized to obtain 1.8 g and 0.8 g of 50% ethanol extracts, respectively.
製造例６ 月下美人花、月下美人葉・茎のエタノール抽出物
月下美人の花２０ｇ又は、月下美人の葉・茎混合物の乾燥物２０ｇにエタノール水溶液４００ｍＬを加え、常温で１〜７日間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥してエタノール抽出物をそれぞれ、０．４ｇ、０．３ｇ得た。
Production example 6 Queen of the Night flower, queen of the night leaf / stem ethanol extract Add 400 mL of ethanol solution to 20 g of queen of the night flower or 20 g of dried queen of the night leaf / stem mixture, and 1 to 7 at room temperature. After extraction for days, the mixture was filtered, the filtrate was concentrated, and lyophilized to obtain 0.4 g and 0.3 g of ethanol extracts, respectively.
実験例１ 活性酸素消去作用
フリーラジカル捕捉除去作用の評価を行った。陽性対照としてはアスコルビン酸を用いた。フリーラジカルのモデルとしては、安定なフリーラジカルであるα，α−ジフェニル−β−ピクリルヒドラジル（以下ＤＰＰＨとする）を用い、試料と一定の割合で一定時間反応させ、減少するラジカルの量を波長５１７ｎｍの吸光度の減少量から測定した。
Experimental Example 1 Active oxygen scavenging action Free radical capture and removal action was evaluated. Ascorbic acid was used as a positive control. As a model of free radicals, stable free radicals α, α-diphenyl-β-picrylhydrazil (hereinafter referred to as DPPH) are used, and the amount of radicals decreases after reacting with a sample at a constant ratio for a certain period of time. Was measured from the amount of decrease in absorbance at a wavelength of 517 nm.
フリーラジカル捕捉除去作用の測定方法
各試料を、最終濃度０．００５〜０．０２ｍｇ／ｍＬ（アスコルビン酸は０．０１ｍｇ／ｍＬ）となるように加えた０．１Ｍ酢酸緩衝液（ｐＨ５．５）２ｍＬに無水エタノール２ｍＬ及び０．５ｍＭ ＤＰＰＨ無水エタノール溶液１ｍＬを加えて反応液とした。その後、３７℃で３０分間反応させ、水を対照として波長５１７ｎｍの吸光度（Ａ）を測定した。また、ブランクとして試料の代わりに精製水を加えた反応液を用いて吸光度（Ｂ）を測定した。フリーラジカル補足除去率が５０％のときの試料の濃度（ＩＣ５０）を算出した。
フリーラジカル補足除去率（％）＝（１−Ａ／Ｂ）×１００
Method for measuring free radical capture and removal action 0.1 M acetate buffer (pH 5.5) added to each sample so as to have a final concentration of 0.005 to 0.02 mg / mL (for ascorbic acid: 0.01 mg / mL). To 2 mL, 2 mL of absolute ethanol and 1 mL of 0.5 mM DPPH absolute ethanol solution were added to prepare a reaction solution. Then, the reaction was carried out at 37 ° C. for 30 minutes, and the absorbance (A) at a wavelength of 517 nm was measured using water as a control. In addition, the absorbance (B) was measured using a reaction solution containing purified water instead of the sample as a blank. The concentration of the sample (IC 50 ) when the free radical capture removal rate was 50% was calculated.
Free radical supplement removal rate (%) = (1-A / B) x 100
これらの試験結果を表１に示した。本発明の抽出物は、安定で優れたフリーラジカル補足除去作用を有していることが認められた。これは、従来技術である月下美人の花や葉・茎と比較して顕著に高い効果である。なお、アスコルビン酸は、１００℃、１時間の熱処理で失活するが、本発明の抽出物は、活性に変化はなかった。 The results of these tests are shown in Table 1. It was found that the extract of the present invention has a stable and excellent free radical capture and removal action. This is a significantly higher effect than the flowers, leaves and stems of the Queen of the Night, which is a conventional technique. Ascorbic acid was inactivated by heat treatment at 100 ° C. for 1 hour, but the activity of the extract of the present invention did not change.
実験例２ コラゲナーゼ活性阻害試験
試料液５０μＬ（最終濃度が５ｍｇ／ｍＬ）に酵素液として５０Ｕ／ｍＬのコラゲナーゼＴｙｐｅ ＩＶ（シグマ製）水溶液を５０μＬ加えた。基質溶液として０．３９ｍｇ／ｍＬのＰｚ−ペプタイド（Ｐｚ−Ｐｒｏ−Ｌｅｕ−Ｇｌｙ−Ｐｒｏ−Ｄ−Ａｒｇ−ＯＨ、シグマ製）を含む２０ｍＭ塩化カルシウム入りトリス塩酸緩衝液（ｐＨ７．１）を４００μＬ加えて混合し、３７℃、３０分反応させた後、２５ｍＭクエン酸０．５ｍＬを加えて反応を停止させた。酢酸エチル２．５ｍＬを加え、酢酸エチル層について３２０ｎｍにおける吸光度を測定した。また、各試料の阻害作用は、次の式から求められる阻害率で算出した。なお、対照には試料の代わりに精製水を用い、ブランクとしてコラゲナーゼの代わりに２０ｍＭ塩化カルシウム入りトリス塩酸緩衝液（ｐＨ７．１）を用いた。
阻害率（％）＝〔１−（Ｃ−Ｄ）／（Ａ−Ｂ）〕×１００
Ａ：対照の３２０ｎｍにおける吸光度（Ｏ．Ｄ．３２０）
Ｂ：対照ブランクのＯ．Ｄ．３２０
Ｃ：試料のＯ．Ｄ．３２０
Ｄ：試料ブランクのＯ．Ｄ．３２０
Experimental Example 2 Collagenase activity inhibition test 50 μL of 50 U / mL collagenase Type IV (manufactured by Sigma) aqueous solution was added as an enzyme solution to 50 μL of the sample solution (final concentration: 5 mg / mL). Add 400 μL of Tris-hydrochloric acid buffer (pH 7.1) containing 20 mM calcium chloride containing 0.39 mg / mL Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH, manufactured by Sigma) as a substrate solution. After mixing and reacting at 37 ° C. for 30 minutes, 0.5 mL of 25 mM citric acid was added to stop the reaction. 2.5 mL of ethyl acetate was added, and the absorbance of the ethyl acetate layer at 320 nm was measured. The inhibitory effect of each sample was calculated by the inhibition rate obtained from the following formula. Purified water was used as a control instead of the sample, and Tris-hydrochloric acid buffer (pH 7.1) containing 20 mM calcium chloride was used as a blank instead of collagenase.
Inhibition rate (%) = [1- (CD) / (AB)] × 100
A: Absorbance at 320 nm of the control (OD320)
B: O.D. of the control blank. D. 320
C: Sample O. D. 320
D: Sample blank O.D. D. 320
これらの実験結果を表２に示した。その結果、本発明の月下美人カルスの抽出物は優れたコラゲナーゼ活性阻害作用を示した。
実験例３ メラニン生成抑制試験
対数増殖期にあるＢ１６マウスメラノーマ細胞を６０ｍｍ ｄｉｓｈに３×１０４個播種し、各試料（最終濃度１０μｇ／ｍＬ）を含むＥａｇｌｅｓ’ＭＥＭ（１０％牛胎児血清含有）培地にて、３７℃、５％ＣＯ２条件下で５日間培養した。次に、細胞をｄｉｓｈから剥離し、超音波破砕した後、４Ｎ ＮａＯＨを加え６０℃で２時間の処理を行い、分光光度計でＯ．Ｄ．４７５ｎｍを測定した。尚、超音波処理後の細胞破砕液についてＬｏｗｒｙの方法（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，１９３，２６５−２７５，１９５１）にてタンパク定量し、タンパク量当りのメラニン量を算出、試料未添加のメラニン生成量をコントロールとし、コントロールに対する試料添加時のメラニン生成量の値からメラニン生成抑制率を算出した。
Experimental Example 3 Melanin production suppression test Eagles'MEM (containing 10% fetal bovine serum) containing 3 × 10 4 B16 mouse melanoma cells in the logarithmic growth phase and each sample (final concentration 10 μg / mL) was seeded in a 60 mm dish. The cells were cultured in medium at 37 ° C. under 5% CO 2 conditions for 5 days. Next, the cells were exfoliated from the dish, ultrasonically crushed, 4N NaOH was added, and the cells were treated at 60 ° C. for 2 hours. D. 475 nm was measured. The cell crushed solution after the ultrasonic treatment was quantified by the Lowry method (J. Biol. Chem., 193,265-275,1951), the amount of melanin per protein amount was calculated, and the amount of melanin without sample was added. Using the amount of production as a control, the melanin production suppression rate was calculated from the value of the amount of melanin produced when the sample was added to the control.
これらの試験結果を表３に示した。本発明の抽出物は、優れたメラニン生成抑制作用を有していることが認められた。 The results of these tests are shown in Table 3. It was found that the extract of the present invention has an excellent melanin production inhibitory effect.
実験例４ フィラグリン生成促進試験
ＮＭＦ（天然保湿因子）の前駆物質であるフィラグリン生成への影響をフィラグリン遺伝子（ＦＬＧ）のｍＲＮＡ発現量を指標として評価した。すなわち、ケラチノサイト由来ＨａＣａＴ細胞を６ｗｅｌｌプレートに１ｗｅｌｌあたり５×１０４個播種し、１０％ＦＢＳを含むＤＭＥＭ培養液にて、３７℃、５％ＣＯ２条件下で４日間培養した。次に、各試料（最終濃度１０μｇ／ｍＬ）を添加したＤＭＥＭ培養液にて、２４時間培養した後、総ＲＮＡの抽出を行った。細胞からの総ＲＮＡの抽出はＴＲＩＺＯＬ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて行い、総ＲＮＡ量は分光光度計（ＮａｎｏＤｒｏｐ）を用いて２６０ｎｍにおける吸光度により求めた。ｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ−ＰＣＲ法により行った。リアルタイムＲＴ−ＰＣＲ法には、ＳｕｐｅｒＳｃｒｉｐｔＩＩＩ Ｐｌａｔｉｎｕｍ Ｔｗｏ−Ｓｔｅｐ ｑＲＴ−ＰＣＲ Ｋｉｔ ｗｉｔｈ ＳＹＢＲ Ｇｒｅｅｎ（Ｉｎｖｉｔｒｏｇｅｎ）を用いた。すなわち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：３０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従い、ＦＬＧ ｍＲＮＡ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求めた。ＦＬＧ発現量は、コントロールのＦＬＧ ｍＲＮＡの発現量に対する試料添加群のＦＬＧ ｍＲＮＡの発現量の比率として算出した。なお、ＦＬＧ用のプライマーは、以下に示したものを使用した。
Experimental Example 4 Filaggrin production promotion test The effect of NMF (natural moisturizing factor) on the production of filaggrin, which is a precursor, was evaluated using the mRNA expression level of the filaggrin gene (FLG) as an index. That is, 5 × 10 4 cells derived from keratinocytes were seeded on a 6-well plate per 1 well, and cultured in DMEM culture medium containing 10% FBS for 4 days under 37 ° C. and 5% CO 2 conditions. Next, total RNA was extracted after culturing for 24 hours in DMEM culture medium to which each sample (final concentration 10 μg / mL) was added. Extraction of total RNA from cells was performed using a TRIZOL Reagent (Invitrogen), and the total amount of RNA was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The mRNA expression level was measured by the real-time RT-PCR method based on the total RNA extracted from the cells. As the real-time RT-PCR method, SuperScriptIII Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen) was used. That is, after a reverse transcription reaction of 500 ng of total RNA, a PCR reaction (95 ° C: 15 seconds, 60 ° C: 30 seconds, 40 cycles) was performed. For other operations, the expression level of FLG mRNA was determined as a ratio to the expression level of GAPDH mRNA, which is an internal standard, according to a predetermined method. The FLG expression level was calculated as the ratio of the expression level of FLG mRNA in the sample addition group to the expression level of the control FLG mRNA. The primers shown below were used as the primers for FLG.
ＦＬＧ用のプライマーセット
ＧＧＡＴＣＡＣＴＴＧＧＡＴＡＴＡＧＡＣＣＡＣＡＡＣＡ（配列番号１）
ＴＧＡＧＣＣＡＡＣＴＴＧＡＡＴＡＣＣＡＴＣＡＧＡ（配列番号２）
ＧＡＰＤＨ用のプライマーセット
ＴＧＣＡＣＣＡＣＣＡＡＣＴＧＣＴＴＡＧＣ（配列番号３）
ＴＣＴＴＣＴＧＧＧＴＧＧＣＡＧＴＧＡＴＧ（配列番号４）
Primer set for FLG GGATCACTTGGATATAGACCACAACA (SEQ ID NO: 1)
TGAGCCAACTTTGATATACCATCAGA (SEQ ID NO: 2)
Primer set for GAPDH TGCACCACCAACTGCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGTGCAGTGATG (SEQ ID NO: 4)
これらの試験結果を表４に示した。その結果、本発明の抽出物にはフィラグリン生成促進効果が認められた。 The results of these tests are shown in Table 4. As a result, the extract of the present invention was found to have a filaggrin production promoting effect.
実験例５ ＩＬ−１α産生阻害試験
界面活性剤であるｓｏｄｉｕｍ ｄｏｄｅｃｙｌ ｓｕｌｆａｔｅ（ＳＤＳ）曝露によるケラチノサイトにおける炎症性サイトカイン、ＩＬ−１α発現亢進に対する抑制作用を指標に抗炎症効果を評価した。すなわち、ケラチノサイト由来ＨａＣａＴ細胞を、６０ｍｍ ｄｉｓｈに１×１０５個播種し、１０％ＦＢＳを含むＤＭＥＭ培養液にて、３７℃、５％ＣＯ２条件下で培養した。コンフルエントな状態になったところで、２０μｇ／ｍＬのＳＤＳおよび１０μｇ／ｍＬの試料を添加した２％ＦＢＳを含むＤＭＥＭ培養液にてさらに６時間培養した後、総ＲＮＡの抽出を行った。細胞からの総ＲＮＡの抽出はＴＲＩＺＯＬ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて行い、総ＲＮＡ量は分光光度計（ＮａｎｏＤｒｏｐ）を用いて２６０ｎｍにおける吸光度により求めた。ｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ−ＰＣＲ法により行った。リアルタイムＲＴ−ＰＣＲ法には、ＳｕｐｅｒＳｃｒｉｐｔＩＩＩ Ｐｌａｔｉｎｕｍ Ｔｗｏ−Ｓｔｅｐ ｑＲＴ−ＰＣＲ Ｋｉｔ ｗｉｔｈ ＳＹＢＲ Ｇｒｅｅｎ（Ｉｎｖｉｔｒｏｇｅｎ）を用いた。すなわち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：３０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従い、ＩＬ−１α ｍＲＮＡ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求めた。尚、ＩＬ−１α用のプライマーは、以下に示したものを使用した。
Experimental Example 5 IL-1α production inhibition test The anti-inflammatory effect was evaluated using the inhibitory effect on the upregulation of IL-1α, an inflammatory cytokine in keratinocytes, by exposure to sodium dodecyl sulfate (SDS), which is a surfactant. That is, 1 × 10 5 keratinocyte-derived HaCaT cells were seeded on a 60 mm dish and cultured in a DMEM culture medium containing 10% FBS under 37 ° C. and 5% CO 2 conditions. When the confluent state was reached, total RNA was extracted after further culturing for 6 hours in DMEM culture medium containing 2% FBS supplemented with 20 μg / mL SDS and 10 μg / mL sample. Extraction of total RNA from cells was performed using a TRIZOL Reagent (Invitrogen), and the total amount of RNA was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The mRNA expression level was measured by the real-time RT-PCR method based on the total RNA extracted from the cells. As the real-time RT-PCR method, SuperScriptIII Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen) was used. That is, after a reverse transcription reaction of 500 ng of total RNA, a PCR reaction (95 ° C: 15 seconds, 60 ° C: 30 seconds, 40 cycles) was performed. For other operations, the expression level of IL-1α mRNA was determined as a ratio to the expression level of GAPDH mRNA, which is an internal standard, according to a predetermined method. As the primer for IL-1α, the one shown below was used.
ＩＬ−１αのプライマーセット
ＡＴＴＧＴＡＴＧＴＧＡＣＴＧＣＣＣＡＡＧＡＴＧＡ（配列番号５）
ＡＧＴＴＴＣＣＣＡＧＡＡＧＡＡＧＡＧＧＡＧＧＴＴ（配列番号６）
ＧＡＰＤＨ用のプライマーセット
ＴＧＣＡＣＣＡＣＣＡＡＣＴＧＣＴＴＡＧＣ（配列番号３）
ＴＣＴＴＣＴＧＧＧＴＧＧＣＡＧＴＧＡＴＧ（配列番号４）
IL-1α primer set ATTGTATGTGACTGCCCAAGATGA (SEQ ID NO: 5)
AGTTTCCCAGAAGAAGAGGAGGTT (SEQ ID NO: 6)
Primer set for GAPDH TGCACCACCAACTGCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGTGCAGTGATG (SEQ ID NO: 4)
試料の抗炎症効果は、試料のＳＤＳ曝露によるＨａＣａＴ細胞のＩＬ−１α ｍＲＮＡ発現量の増加を抑制する割合として、以下の計算式にて算出した。
ＩＬ−１α産生阻害率（％）＝（試料未添加ＳＤＳ添加のＩＬ−１α ｍＲＮＡ発現量−試料・ＳＤＳ添加のＩＬ−１αｍＲＮＡ発現量）／（試料未添加ＳＤＳ添加のＩＬ−１αｍＲＮＡ発現量−コントロールのＩＬ−１αｍＲＮＡ発現量）×１００
The anti-inflammatory effect of the sample was calculated by the following formula as a ratio of suppressing an increase in IL-1α mRNA expression level of HaCaT cells due to SDS exposure of the sample.
IL-1α production inhibition rate (%) = (IL-1α mRNA expression level without sample added SDS-IL-1α mRNA expression level with sample / SDS added) / (IL-1α mRNA expression level with sample-free SDS added-control IL-1α mRNA expression level) × 100
これらの試験結果を表５に示した。その結果、本発明の抽出物にはＩＬ−１α産生抑制効果が認められた。 The results of these tests are shown in Table 5. As a result, the extract of the present invention was found to have an effect of suppressing IL-1α production.
実験例６ 細胞増殖促進試験
ケラチノサイト由来ＨａＣａＴ細胞を９６ｗｅｌｌプレートに１ｗｅｌｌあたり５×１０３個播種し、各試料（最終濃度１μｇ／ｍＬ）を添加した０．１％ＦＢＳを含むＤＭＥＭ培養液にて、３７℃、５％ＣＯ２条件下で３日間培養した。細胞数の測定は、ＭＴＴ法により行った。すなわち、培養終了後、培養液を除き、５００μｇ／ｍＬの濃度にて、ＭＴＴ（３−［４，５−ｄｉｍｅｔｈｙｌｔｈｉａｚｏｌ−２−ｙｌ］−２，５−ｄｉｐｈｅｎｙｌ ｔｅｔｒａｚｏｌｉｕｍ ｂｒｏｍｉｄｅ）を溶解させたＤＭＥＭに培地を入れ替え、２時間培養した後、１５０μＬのｉｓｏｐｒｏｐａｎｏｌに細胞を溶解させ、マイクロプレートリーダーを用いて５７０及び６３０ｎｍにおける吸光度を測定した。細胞数は、５７０ｎｍの吸光度値から、６３０ｎｍの吸光度値を引いた値にて算出し、試料未添加の細胞数をコントロールとし、コントロールに対する試料添加時の細胞数から試料の細胞増殖促進効果を評価した。
Experimental Example 6 Cell Proliferation Promotion Test In a DMEM culture medium containing 0.1% FBS in which 5 × 10 3 keratinocytes-derived HaCaT cells were seeded per 96-well plate and each sample (final concentration 1 μg / mL) was added. The cells were cultured at 37 ° C. under 5% CO 2 conditions for 3 days. The cell number was measured by the MTT method. That is, after the completion of the culture, the culture medium was removed, and the medium was dissolved in DMEM in which MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl terrazolium medium) was dissolved at a concentration of 500 μg / mL. After culturing for 2 hours, the cells were lysed in 150 μL of isopropanol, and the absorbance at 570 and 630 nm was measured using a microplate reader. The number of cells is calculated by subtracting the absorbance value at 630 nm from the absorbance value at 570 nm, the number of cells without sample addition is used as a control, and the cell growth promoting effect of the sample is evaluated from the number of cells at the time of sample addition to the control. did.
これらの実験結果を表６に示した。その結果、本発明の抽出物は、ケラチノサイトに対して優れた細胞増殖促進作用を示した。 The results of these experiments are shown in Table 6. As a result, the extract of the present invention showed an excellent cell growth promoting effect on keratinocytes.
実験例７ コラーゲン生成促進効果
Ｉ型コラーゲン（ＣＯＬ１Ａ）のｍＲＮＡ発現量の測定を行った。ヒト皮膚線維芽細胞（ＮＢ１ＲＧＢ）を６０ｍｍ ｄｉｓｈに１×１０５個播種し、１０％ＦＢＳを含むＤＭＥＭ培養液にて、３７℃、５％ＣＯ２条件下で培養した。コンフルエントな状態になったところで、ＣＯＬ１Ａ ｍＲＮＡ発現量測定では各試料を最終濃度１μｇ／ｍＬを添加したＤＭＥＭ培養液にて、２４時間培養した後、総ＲＮＡの抽出を行った。細胞からの総ＲＮＡの抽出はＴＲＩＺＯＬ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて行い、総ＲＮＡ量は分光光度計（ＮａｎｏＤｒｏｐ）を用いて２６０ｎｍにおける吸光度により求めた。ｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ−ＰＣＲ法により行った。リアルタイムＲＴ−ＰＣＲ法には、ＳｕｐｅｒＳｃｒｉｐｔＩＩＩ Ｐｌａｔｉｎｕｍ Ｔｗｏ−Ｓｔｅｐ ｑＲＴ−ＰＣＲ Ｋｉｔ ｗｉｔｈ ＳＹＢＲ Ｇｒｅｅｎ（Ｉｎｖｉｔｒｏｇｅｎ）を用いた。すなわち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：３０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従い、ＣＯＬ１Ａ ｍＲＮＡの発現量を、内部標準であるβ―ａｃｔｉｎ ｍＲＮＡの発現量に対する割合として求めた。ＣＯＬ１Ａ発現量は、コントロールのＣＯＬ１Ａ ｍＲＮＡの発現量に対する試料添加群のＣＯＬ１Ａ ｍＲＮＡの発現量の比率として算出した。なお、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
Experimental Example 7 Collagen production promoting effect The mRNA expression level of type I collagen (COL1A) was measured. Human skin fibroblasts (NB1RGB) were seeded in 1 × 10 5 pieces on a 60 mm dish and cultured in DMEM culture medium containing 10% FBS under 37 ° C. and 5% CO 2 conditions. When the state became confluent, in the measurement of COL1A mRNA expression level, each sample was cultured in DMEM culture medium to which the final concentration of 1 μg / mL was added for 24 hours, and then total RNA was extracted. Extraction of total RNA from cells was performed using a TRIZOL Reagent (Invitrogen), and the total amount of RNA was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). The mRNA expression level was measured by the real-time RT-PCR method based on the total RNA extracted from the cells. As the real-time RT-PCR method, SuperScriptIII Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen) was used. That is, after a reverse transcription reaction of 500 ng of total RNA, a PCR reaction (95 ° C: 15 seconds, 60 ° C: 30 seconds, 40 cycles) was performed. For other operations, the expression level of COL1A mRNA was determined as a ratio to the expression level of β-actin mRNA, which is an internal standard, according to a predetermined method. The COL1A expression level was calculated as the ratio of the COL1A mRNA expression level of the sample addition group to the COL1A mRNA expression level of the control. The primers used to measure the expression level of each gene are as follows.
ＣＯＬ１Ａ用のプライマーセット
ＡＧＧＡＣＡＡＧＡＧＧＣＡＴＧＴＣＴＧＧＴＴ（配列番号７）
ＴＴＧＣＡＧＴＧＧＴＡＧＧＴＧＡＴＧＴＴＣＴＧ（配列番号８）
β―Ａｃｔｉｎ用のプライマーセット
ＣＡＣＴＣＴＴＣＣＡＧＣＣＴＴＣＣＴＴＣＣ（配列番号９）
ＧＴＧＴＴＧＧＣＧＴＡＣＡＧＧＴＣＴＴＴＧ（配列番号１０）
Primer set for COL1A AGGACAAGAGGCCATGTCGGTT (SEQ ID NO: 7)
TTGCAGTGGTAGGTGATGTTTCTG (SEQ ID NO: 8)
Primer set for β-Actin CACTCTTCCAGCCTTCCTCC (SEQ ID NO: 9)
GTGTTGGGCGTACAGGTCTTG (SEQ ID NO: 10)
これらの実験結果を表７に示した。その結果、本発明の抽出物は、優れたＣＯＬ１Ａ発現促進効果（コラーゲン生成促進効果）が認められた。 The results of these experiments are shown in Table 7. As a result, the extract of the present invention was found to have an excellent COL1A expression promoting effect (collagen production promoting effect).
実験例８ ヒアルロン酸生成促進効果
ＨａＣａＴ細胞を６０ｍｍ ｄｉｓｈに１×１０５個播種し、１０％ＦＢＳを含むＤＭＥＭ培地にて３７℃、５％ ＣＯ２条件下で４日間培養した。ＰＢＳ（−）にて２回洗浄後、試料を最終濃度として１０μｇ／ｍＬとなるように添加したＦＢＳを含まないＤＭＥＭにてさらに２４時間培養し、総ＲＮＡを抽出した。総ＲＮＡの抽出には、ＲＮＡｉｓｏ Ｐｌｕｓ（タカラバイオ）を用いた。細胞から抽出した総ＲＮＡをもとに、ＲＴ−ＰＣＲ法によりＨＡＳ１およびＨＡＳ３ ｍＲＮＡ発現量の測定を行った。ＲＴ−ＰＣＲ法にはＰｒｉｍｅＳｃｒｉｐｔ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（タカラバイオ）及びＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用い、ＨＡＳ１及びＨＡＳ３のプライマーは、以下に示したものを使用した。ＰＣＲ反応は、９５℃：２分の初期変性を行った後、９５℃：１５秒、６０℃：６０秒を１ｃｙｃｌｅとして４０ｃｙｃｌｅ行った。また、内部標準としては、ＧＡＰＤＨを用いた。その他の操作は、定められた方法に従い、ＨＡＳ１及びＨＡＳ３ ｍＲＮＡの発現量を、内部標準であるＧＡＰＤＨ ｍＲＮＡ発現量に対する割合として求めた。
Experimental Example 8 Hyaluronic acid production promoting effect HaCaT cells were seeded in 1 × 10 5 seeds on a 60 mm dish and cultured in DMEM medium containing 10% FBS for 4 days under 37 ° C. and 5% CO 2 conditions. After washing twice with PBS (−), the sample was cultured in DMEM containing no FBS added to a final concentration of 10 μg / mL for another 24 hours, and total RNA was extracted. RNAiso Plus (Takara Bio) was used for the extraction of total RNA. Based on the total RNA extracted from the cells, the expression levels of HAS1 and HAS3 mRNA were measured by the RT-PCR method. PrimeScript RT Master Mix (Takara Bio) and SYBR Select Master Mix (Life Technologies) were used for the RT-PCR method, and the primers shown below were used as the primers for HAS1 and HAS3. The PCR reaction was carried out by performing initial denaturation at 95 ° C. for 2 minutes, and then performing 40 cycles with 95 ° C.: 15 seconds and 60 ° C.: 60 seconds as 1 cycle. In addition, GAPDH was used as an internal standard. For other operations, the expression levels of HAS1 and HAS3 mRNA were determined as a ratio to the expression level of GAPDH mRNA, which is an internal standard, according to a predetermined method.
ＨＡＳ１用のプライマーセット
ＡＡＴＧＴＧＧＡＧＣＧＧＧＣＴＴＧＴＣ（配列番号１１）
ＡＧＧＣＣＴＡＧＡＧＧＡＣＣＧＣＴＧＡＴ（配列番号１２）
ＨＡＳ３用のプライマーセット
ＧＡＣＡＴＧＧＣＣＣＣＣＡＡＧＣＡ（配列番号１３）
ＴＣＣＣＣＴＴＣＣＣＴＣＣＣＴＴＡＣＣ（配列番号１４）
ＧＡＰＤＨ用のプライマーセット
ＴＧＣＡＣＣＡＣＣＡＡＣＴＧＣＴＴＡＧＣ（配列番号３）
ＴＣＴＴＣＴＧＧＧＴＧＧＣＡＧＴＧＡＴＧ（配列番号４）
Primer set for HAS1 AATGTGGAGGCGGCTTGTC (SEQ ID NO: 11)
AGGCCTAGAGGACCGCTGAT (SEQ ID NO: 12)
Primer set for HAS3 GACATGGCCCCCAAGCA (SEQ ID NO: 13)
TCCCCTTCCCCTCCCTTACC (SEQ ID NO: 14)
Primer set for GAPDH TGCACCACCAACTGCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGTGCAGTGATG (SEQ ID NO: 4)
これらの実験結果を表８、９に示した。その結果、本発明の抽出物は、優れたＨＡＳ１、ＨＡＳ３発現促進効果（ヒアルロン酸生成促進効果）が認められた。 The results of these experiments are shown in Tables 8 and 9. As a result, the extract of the present invention was found to have an excellent HAS1 and HAS3 expression promoting effect (hyaluronic acid production promoting effect).
処方例１ 化粧水
処方 含有量（部）
１．製造例１の抽出物 １．０
２．１，３‐ブチレングリコール ８．０
３．グリセリン ２．０
４．キサンタンガム ０．０２
５．クエン酸 ０．０１
６．クエン酸ナトリウム ０．１
７．エタノール ５．０
８．パラオキシ安息香酸メチル ０．１
９．ポリオキシエチレン硬化ヒマシ油（４０Ｅ．Ｏ．） ０．１
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１〜６及び１１と、成分７〜１０をそれぞれ均一に溶解し、両者を混合し濾過して製品とする。
Prescription example 1 Toner prescription content (part)
1. 1. Extract of Production Example 1 1.0
2.1,3-butylene glycol 8.0
3. 3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40EO) 0.1
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water to make the total amount 100, and both are mixed and filtered to obtain a product.
処方例２ クリーム
処方 含有量（部）
１．製造例２の抽出物 ０．５
２．スクワラン ５．５
３．オリーブ油 ３．０
４．ステアリン酸 ２．０
５．ミツロウ ２．０
６．ミリスチン酸オクチルドデシル ３．５
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ベヘニルアルコール １．５
９．モノステアリン酸グリセリン ２．５
１０．香料 ０．１
１１．パラオキシ安息香酸メチル ０．２５
１２．１，３‐ブチレングリコール ８．５
１３．精製水にて全量を１００とする
［製造方法］成分２〜９を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１１〜１３を加熱溶解して混合し、７５℃に保ち水相とする。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、更に３０℃まで冷却して製品とする。
Prescription example 2 Cream prescription content (part)
1. 1. Extract of Production Example 2 0.5
2. Squalene 5.5
3. 3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Behenyl alcohol 1.5
9. Glycerin monostearate 2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.25
12.1,3-butylene glycol 8.5
13. [Manufacturing method] Ingredients 2 to 9 having a total amount of 100 in purified water are dissolved by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1 and 11 to 13 are heated and dissolved and mixed to prepare an aqueous phase at 75 ° C. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 10 is added at 45 ° C., and the mixture is further cooled to 30 ° C. to obtain a product.
処方例３ 乳液
処方 含有量（部）
１．製造例１の抽出物 １．０
２．スクワラン ５．０
３．オリーブ油 ５．０
４．ホホバ油 ５．０
５．セタノール １．５
６．モノステアリン酸グリセリン ２．０
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ポリオキシエチレンソルビタンモノオレエート（２０Ｅ．Ｏ．） ２．０
９．香料 ０．１
１０．プロピレングリコール １．０
１１．グリセリン ２．０
１２．パラオキシ安息香酸メチル ０．２
１３．精製水にて全量を１００とする
［製造方法］成分２〜８を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１０〜１３を加熱溶解して混合し、７５℃に保ち水相とする。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９を加え、更に３０℃まで冷却して製品とする。
Prescription example 3 Emulsion prescription content (part)
1. 1. Extract of Production Example 1 1.0
2. Squalene 5.0
3. 3. Olive oil 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glycerin monostearate 2.0
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Polyoxyethylene sorbitan monooleate (20EO) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Ingredients 2 to 8 having a total amount of 100 in purified water are dissolved by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1 and 10 to 13 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 9 is added at 45 ° C, and the product is further cooled to 30 ° C to obtain a product.
処方例４ ゲル剤
処方 含有量（部）
１．製造例３の抽出物 ０．００１
２．エタノール ５．０
３．パラオキシ安息香酸メチル ０．１
４．ポリオキシエチレン硬化ヒマシ油（６０Ｅ．Ｏ．） ０．１
５．香料 適量
６．１，３‐ブチレングリコール ５．０
７．グリセリン ５．０
８．キサンタンガム ０．１
９．カルボキシビニルポリマー ０．２
１０．水酸化カリウム ０．２
１１．精製水にて全量を１００とする
［製造方法］成分２〜５と、成分１及び６〜１１をそれぞれ均一に溶解し、両者を混合して製品とする。
Prescription example 4 Gel preparation Prescription content (part)
1. 1. Extract of Production Example 3 0.001
2. Ethanol 5.0
3. 3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60EO) 0.1
5. Appropriate amount of fragrance 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Manufacturing method] Ingredients 2 to 5 and components 1 and 6 to 11 having a total amount of 100 in purified water are uniformly dissolved, and the two are mixed to obtain a product.
処方例５ パック
処方 含有量（部）
１．製造例１の抽出物 ０．１
２．製造例２の抽出物 ０．１
３．ポリビニルアルコール １２．０
４．エタノール ５．０
５．１，３‐ブチレングリコール ８．０
６．パラオキシ安息香酸メチル ０．２
７．ポリオキシエチレン硬化ヒマシ油（２０Ｅ．Ｏ．） ０．５
８．クエン酸 ０．１
９．クエン酸ナトリウム ０．３
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１〜１１を均一に溶解し製品とする。
Prescription example 5 pack prescription content (part)
1. 1. Extract of Production Example 1 0.1
2. Extract of Production Example 2 0.1
3. 3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5.1,3-butylene glycol 8.0
6. Methyl paraoxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20EO) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 11 having a total amount of 100 are uniformly dissolved in purified water to prepare a product.
処方例６ ファンデーション
処方 含有量（部）
１．製造例３の抽出物 １．０
２．ステアリン酸 ２．４
３．ポリオキシエチレンソルビタンモノステアレート（２０Ｅ．Ｏ．） １．０
４．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ２．０
５．セタノール １．０
６．液状ラノリン ２．０
７．流動パラフィン ３．０
８．ミリスチン酸イソプロピル ６．５
９．カルボキシメチルセルロースナトリウム ０．１
１０．ベントナイト ０．５
１１．プロピレングリコール ４．０
１２．トリエタノールアミン １．１
１３．パラオキシ安息香酸メチル ０．２
１４．二酸化チタン ８．０
１５．タルク ４．０
１６．ベンガラ １．０
１７．黄酸化鉄 ２．０
１８．香料 適量
１９．精製水にて全量を１００とする
［製造方法］成分２〜８を加熱溶解し、８０℃に保ち油相とする。成分１９に成分９をよく膨潤させ、続いて、成分１及び１０〜１３を加えて均一に混合する。これに粉砕機で粉砕混合した成分１４〜１７を加え、ホモミキサーで撹拌し７５℃に保ち水相とする。この油相に水相をかき混ぜながら加え、乳化する。その後冷却し、４５℃で成分１８を加え、かき混ぜながら３０℃まで冷却して製品とする。
Prescription Example 6 Foundation Prescription Content (Part)
1. 1. Extract of Production Example 3 1.0
2. Stearic acid 2.4
3. 3. Polyoxyethylene sorbitan monostearate (20EO) 1.0
4. Polyoxyethylene cetyl ether (20EO) 2.0
5. Cetanol 1.0
6. Liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium Carboxymethyl Cellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Appropriate amount of fragrance 19. [Manufacturing method] Ingredients 2 to 8 having a total amount of 100 in purified water are dissolved by heating and kept at 80 ° C. to prepare an oil phase. Ingredient 19 is well swollen with Ingredient 9, followed by Additions 1 and 10-13 and mixed uniformly. Ingredients 14 to 17 pulverized and mixed by a pulverizer are added thereto, and the mixture is stirred with a homomixer and kept at 75 ° C. to prepare an aqueous phase. The aqueous phase is added to this oil phase while stirring to emulsify. After that, it is cooled, component 18 is added at 45 ° C., and the product is cooled to 30 ° C. with stirring.
処方例７ 浴用剤
処方 含有量（部）
１．製造例１の抽出物 ５．０
２．製造例２の抽出物 １．０
３．炭酸水素ナトリウム ５０．０
４．黄色２０２号（１） 適量
５．香料 適量
６．硫酸ナトリウムにて全量を１００とする
［製造方法］成分１〜６を均一に混合し製品とする。
Prescription example 7 Prescription content for bath (part)
1. 1. Extract of Production Example 1 5.0
2. Extract of Production Example 2 1.0
3. 3. Sodium bicarbonate 50.0
4. Yellow No. 202 (1) Appropriate amount 5. Appropriate amount of fragrance 6. [Manufacturing method] Ingredients 1 to 6 with sodium sulfate to make the total amount 100 are uniformly mixed to prepare a product.
処方例８ 軟膏
処方 含有量（部）
１．製造例３の抽出物 ０．５
２．ポリオキシエチレンセチルエーテル（３０Ｅ．Ｏ．） ２．０
３．モノステアリン酸グリセリン １０．０
４．流動パラフィン ５．０
５．セタノール ６．０
６．パラオキシ安息香酸メチル ０．１
７．プロピレングリコール １０．０
８．精製水にて全量を１００とする
［製造方法］成分２〜５を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び６〜８を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら３０℃まで冷却して製品とする。
Prescription example 8 Ointment Prescription content (part)
1. 1. Extract of Production Example 3 0.5
2. Polyoxyethylene cetyl ether (30EO) 2.0
3. 3. Glycerin monostearate 10.0
4. Liquid paraffin 5.0
5. Cetanol 6.0
6. Methyl paraoxybenzoate 0.1
7. Propylene glycol 10.0
8. [Manufacturing method] Ingredients 2 to 5 having a total amount of 100 in purified water are dissolved by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1 and 6 to 8 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring.
処方例９ 散剤
処方 含有量（部）
１．製造例１の抽出物 ２０．０
２．乾燥コーンスターチ ３０．０
３．微結晶セルロース ５０．０
［製造方法］成分１〜３を混合し、散剤とする。
Prescription example 9 Powder prescription content (part)
1. 1. Extract of Production Example 1 20.0
2. Dried cornstarch 30.0
3. 3. Microcrystalline Cellulose 50.0
[Manufacturing method] Ingredients 1 to 3 are mixed to prepare a powder.
以上のことから、月下美人のカルスの抽出物は、優れた抗酸化効果、コラゲナーゼ活性阻害効果、美白効果、フィラグリン生成促進効果、抗炎症効果、細胞増殖効果、コラーゲン生成促進効果、ヒアルロン酸生成促進効果などを示し、これらを含有する皮膚外用剤又は内用剤などは特に有効である。 From the above, the extract of the curls of Tsukishita Bijin has an excellent antioxidant effect, collagenase activity inhibitory effect, whitening effect, filaggrin production promoting effect, anti-inflammatory effect, cell proliferation effect, collagen production promoting effect, hyaluronic acid production. An external preparation for skin or an internal preparation containing these is particularly effective because it exhibits a promoting effect.
Claims (8)
An external preparation for skin, which comprises the callus of the queen of the night and / or an extract thereof according to claim 1 .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016011683A JP6779627B2 (en) | 2016-01-25 | 2016-01-25 | How to make callus for Queen of the Night, and external and internal skin preparations containing the extract as an active ingredient |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016011683A JP6779627B2 (en) | 2016-01-25 | 2016-01-25 | How to make callus for Queen of the Night, and external and internal skin preparations containing the extract as an active ingredient |
Publications (2)
| Publication Number | Publication Date |
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| JP2017132694A JP2017132694A (en) | 2017-08-03 |
| JP6779627B2 true JP6779627B2 (en) | 2020-11-04 |
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| JP2016011683A Active JP6779627B2 (en) | 2016-01-25 | 2016-01-25 | How to make callus for Queen of the Night, and external and internal skin preparations containing the extract as an active ingredient |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP7162848B2 (en) * | 2017-12-20 | 2022-10-31 | Ｓｈｉｏｄａライフサイエンス株式会社 | Antioxidant or hair restorer manufacturing method, and antioxidant or hair restorer manufactured by said manufacturing method |
| CN114303944B (en) * | 2020-09-30 | 2023-08-11 | 伽蓝（集团）股份有限公司 | Callus culture medium and extract of rosa tenuifolia, preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003342156A (en) * | 2002-05-29 | 2003-12-03 | Nonogawa Shoji Kk | External preparation for skin for prevention of aging |
| JP5184813B2 (en) * | 2007-05-11 | 2013-04-17 | 日本メナード化粧品株式会社 | Collagen production promotion and degradation inhibitor |
| JP2011162515A (en) * | 2010-02-13 | 2011-08-25 | Nippon Menaade Keshohin Kk | Filaggrin production promoter |
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