JP5235439B2 - HMG-CoA reductase production promoter - Google Patents
HMG-CoA reductase production promoter Download PDFInfo
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- JP5235439B2 JP5235439B2 JP2008025866A JP2008025866A JP5235439B2 JP 5235439 B2 JP5235439 B2 JP 5235439B2 JP 2008025866 A JP2008025866 A JP 2008025866A JP 2008025866 A JP2008025866 A JP 2008025866A JP 5235439 B2 JP5235439 B2 JP 5235439B2
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Description
本発明は、ＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、セリンパルミトイルトランスフェラーゼ産生促進剤、アクアポリン３産生促進剤、インボルクリン産生促進剤、ＨＭＧ−ＣｏＡ還元酵素産生促進剤、及び、アデノシン三リン酸産生促進剤に関する。 The present invention relates to a type IV collagen production promoter, transglutaminase-1 production promoter, serine palmitoyltransferase production promoter, aquaporin 3 production promoter, involucrin production promoter, HMG-CoA reductase production promoter, and adenosine three The present invention relates to a phosphoric acid production promoter.
表皮は、角化細胞の分裂とその後の分化により、常に新しい角質細胞を作り出すことで、外界からの種々の刺激から皮膚を守る防御機能を有する。特に、角化細胞の分化過程において、有棘層から顆粒層にかけてインボルクリン等のタンパク質が発現し、酵素トランスグルタミナーゼ−１の作用によって架橋され、角化細胞を包み込む不溶性の細胞膜様構造体であるコーニファイドエンベロープ（ＣＥ）を形成し、角質細胞の細胞骨格及び構造の安定性に寄与する。
しかし、様々な要因で表皮におけるトランスグルタミナーゼ−１或いはインボルクリンの産生量が減少すると、コーニファイドエンベロープ（ＣＥ）形成が不完全な状態となり、角化が正常に行われなくなる。その結果、角質バリア機能及び皮膚の保湿機能が低下し、肌荒れや乾燥肌等の皮膚症状を呈するようになると考えられる。
このようなことから、角化細胞の表皮におけるインボルクリンやトランスグルタミナーゼ−１の産生を高め、コーニファイドエンベロープの形成を促進して角化を正常化することによれば、乾燥や紫外線等の外部刺激に伴う皮膚バリア機能の低下を抑制し、肌の乾燥や肌荒れなど、様々な皮膚症状を予防・改善することができると考えられる。
The epidermis has a protective function of protecting the skin from various stimuli from the outside by constantly creating new keratinocytes by dividing and subsequent differentiation of keratinocytes. In particular, in the differentiation process of keratinocytes, a protein such as involucrin is expressed from the spinous layer to the granule layer, is crosslinked by the action of the enzyme transglutaminase-1, and is an insoluble cell membrane-like structure that wraps around keratinocytes. It forms a fied envelope (CE) and contributes to the stability of the keratinocyte cytoskeleton and structure.
However, if the amount of transglutaminase-1 or involucrin produced in the epidermis is reduced due to various factors, the formation of a confined envelope (CE) becomes incomplete and keratinization cannot be performed normally. As a result, it is considered that the keratin barrier function and the skin moisturizing function are lowered, and skin symptoms such as rough skin and dry skin are exhibited.
For this reason, by increasing the production of involucrin and transglutaminase-1 in the epidermis of keratinocytes and promoting the formation of a cornified envelope to normalize keratinization, external stimuli such as drying and ultraviolet rays can be used. It is considered that various skin symptoms such as dry skin and rough skin can be prevented and ameliorated by suppressing the decrease in the skin barrier function associated with.
また、角層の構造はレンガとモルタルに例えられ、約１５層ほどに積み重なった角層細胞を細胞間脂質が繋ぎ止める形で、強固なバリア膜を形成している。角質の細胞間脂質は、外部からの異物の侵入や、内部からの水分の蒸発を防ぐ働き（バリア機能、及び水分保持機能）を有し、約５０％のセラミド、約３０％のコレステロール、約２０％の遊離脂肪酸等から構成される。セラミドは、表皮細胞の角化の過程において、セリンとパルミトイル−ＣｏＡを基に、セラミド合成の律速酵素として知られるセリンパルミトイルトランスフェラーゼ（ＳＰＴ）をはじめとした酵素の働きにより生成されることが知られている。また、コレステロールは、ＨＭＧ−ＣｏＡ還元酵素（ＨＭＧＣＲ）をはじめとした酵素の働きにより生成されることが知られている。
最近の研究において、加齢、或いはバリア障害として知られるアトピー性皮膚炎患者で、この細胞間脂質の減少や組成変化が報告されており（非特許文献１参照）、バリア機能の維持、改善にセラミドやコレステロールが重要であることが広く認識されるようになった。例えば、バリア機能を改善する方法として、セラミドを外部から補う方法（非特許文献２参照）や、皮膚内部においてセラミド産生能を高める方法（非特許文献３参照）などが種々検討されている。
このようなことから、セラミド合成の律速酵素として知られるセリンパルミトイルトランスフェラーゼや、コレステロールの律速酵素として知られるＨＭＧ−ＣｏＡ還元酵素の産生を促進することによれば、細胞間脂質の構成成分であるセラミドやコレステロールの産生能を高め、皮膚のバリア機能や水分保持機能を向上させ、乾燥肌、肌荒れ、小じわ、アトピー性皮膚炎など、様々な皮膚症状を予防・改善することができると考えられる。
The stratum corneum structure is compared to bricks and mortar, and forms a strong barrier membrane in such a way that intercellular lipids connect the stratum corneum cells stacked in about 15 layers. The keratin intercellular lipid has a function (barrier function and water retention function) to prevent the invasion of foreign substances from the outside and the evaporation of moisture from the inside (barrier function and water retention function), about 50% ceramide, about 30% cholesterol, about It is composed of 20% free fatty acids and the like. Ceramide is known to be produced by the action of enzymes such as serine palmitoyltransferase (SPT), which is known as the rate-limiting enzyme for ceramide synthesis, based on serine and palmitoyl-CoA in the process of keratinization of epidermal cells. ing. Cholesterol is known to be produced by the action of enzymes such as HMG-CoA reductase (HMGCR).
In a recent study, in atopic dermatitis patients known as aging or barrier disorders, this intercellular lipid decrease and composition change have been reported (see Non-Patent Document 1), and maintenance and improvement of barrier function have been reported. It has become widely recognized that ceramide and cholesterol are important. For example, as a method for improving the barrier function, various methods such as a method for supplementing ceramide from the outside (see Non-Patent Document 2) and a method for enhancing the ability to produce ceramide in the skin (see Non-Patent Document 3) have been studied.
Therefore, by promoting the production of serine palmitoyltransferase, which is known as the rate-limiting enzyme for ceramide synthesis, and HMG-CoA reductase, which is known as the rate-limiting enzyme for cholesterol, ceramide which is a component of intercellular lipids It is considered that the production of cholesterol and cholesterol can be enhanced, the skin barrier function and moisture retention function can be improved, and various skin symptoms such as dry skin, rough skin, fine lines, and atopic dermatitis can be prevented and improved.
また、ＩＶ型コラーゲンは、肌の表皮と真皮との間に存在する基底膜に多く含まれ、平面的な網目状のネットワークを形成し、基底膜の構造を支えていると考えられている。このＩＶ型コラーゲンの産生を高めることによれば、基底膜の構造を保持する機能を高め、皮膚バリア機能の強化を図ることができると考えられる。
また、アクアポリン３（ＡＱＰ３）は、表皮細胞における水の通り道として知られる膜タンパク質である。アクアポリン３の産生を高めることによれば、水分の通り道をケアすることにより、肌の潤いを高め、皮膚のバリア機能の強化を図ることができると考えられる。
また、アデノシン３リン酸（ＡＴＰ）は、生体内で用いられるエネルギー成分である。ＡＴＰの産生を高めることによれば、肌細胞に活力を与え、結果的にくすんだ肌や疲れた肌の改善など、様々な皮膚症状の予防・改善に繋がると考えられる。
In addition, type IV collagen is abundant in the basement membrane that exists between the epidermis and dermis of the skin, and is thought to form a planar network and support the structure of the basement membrane. By enhancing the production of this type IV collagen, it is considered that the function of maintaining the structure of the basement membrane can be enhanced and the skin barrier function can be enhanced.
Aquaporin 3 (AQP3) is a membrane protein known as a water passage in epidermal cells. According to the production of aquaporin 3, it is considered that the moisture of the skin can be increased and the barrier function of the skin can be enhanced by taking care of the passage of moisture.
Adenosine triphosphate (ATP) is an energy component used in vivo. Increasing the production of ATP is thought to give vitality to skin cells and consequently prevent or improve various skin symptoms such as improvement of dull skin and tired skin.
このように、ＩＶ型コラーゲン、トランスグルタミナーゼ−１、セリンパルミトイルトランスフェラーゼ、アクアポリン３、インボルクリン、ＨＭＧ−ＣｏＡ還元酵素、及び、アデノシン三リン酸の産生を促進することのできる物質は、非常に有用であることが考えられる。しかしながら、従来においては、前記したような産生促進作用を有し、かつ安全性が高く、そのため、皮膚外用剤、美容用飲食品、研究用試薬などの成分として広く利用が可能な優れた物質は、未だ見出されていないのが現状である。 Thus, substances that can promote the production of type IV collagen, transglutaminase-1, serine palmitoyltransferase, aquaporin 3, involucrin, HMG-CoA reductase, and adenosine triphosphate are very useful. It is possible. However, in the past, it has a production promoting action as described above, and is highly safe. Therefore, an excellent substance that can be widely used as a component for external preparations for skin, cosmetic foods and drinks, research reagents, etc. The current situation has not yet been found.
本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。 即ち、本発明は、第一に、優れたＩＶ型コラーゲン産生促進作用を有し、かつ安全性の高いＩＶ型コラーゲン産生促進剤を提供することを目的とする。
また、本発明は、第二に、優れたトランスグルタミナーゼ−１産生促進作用を有し、かつ安全性の高いトランスグルタミナーゼ−１産生促進剤を提供することを目的とする。
また、本発明は、第三に、優れたセリンパルミトイルトランスフェラーゼ産生促進作用を有し、かつ安全性の高いセリンパルミトイルトランスフェラーゼ産生促進剤を提供することを目的とする。
また、本発明は、第四に、優れたアクアポリン３産生促進作用を有し、かつ安全性の高いアクアポリン３産生促進剤を提供することを目的とする。
また、本発明は、第五に、優れたインボルクリン産生促進作用を有し、かつ安全性の高いインボルクリン産生促進剤を提供することを目的とする。
また、本発明は、第六に、優れたＨＭＧ−ＣｏＡ還元酵素産生促進作用を有し、かつ安全性の高いＨＭＧ−ＣｏＡ還元酵素産生促進剤を提供することを目的とする。
また、本発明は、第七に、優れたアデノシン三リン酸産生促進作用を有し、かつ安全性の高いアデノシン三リン酸産生促進剤を提供することを目的とする。
An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the first object of the present invention is to provide a type IV collagen production promoter that has an excellent type IV collagen production promoting action and is highly safe.
A second object of the present invention is to provide a transglutaminase-1 production promoter that has an excellent transglutaminase-1 production promoting action and is highly safe.
A third object of the present invention is to provide a serine palmitoyltransferase production promoter that has excellent serine palmitoyltransferase production promoting activity and is highly safe.
A fourth object of the present invention is to provide an aquaporin 3 production promoter that has an excellent aquaporin 3 production promoting action and is highly safe.
In addition, a fifth object of the present invention is to provide an involucrin production promoter that has an excellent involucrin production promoting action and is highly safe.
A sixth object of the present invention is to provide a highly safe HMG-CoA reductase production accelerator having an excellent HMG-CoA reductase production promoting action and high safety.
A seventh object of the present invention is to provide an adenosine triphosphate production promoter that has an excellent adenosine triphosphate production promoting action and is highly safe.
本発明者らは、前記課題を解決するために鋭意検討を行った結果、ローヤルゼリー抽出物が、優れたＩＶ型コラーゲン産生促進作用、トランスグルタミナーゼ−１産生促進作用、セリンパルミトイルトランスフェラーゼ産生促進作用、アクアポリン３産生促進作用、インボルクリン産生促進作用、ＨＭＧ−ＣｏＡ還元酵素産生促進作用、及び、アデノシン三リン酸産生促進作用を有することを見出し、本発明を完成するに至った。
ローヤルゼリーは、更年期障害の改善、老化防止、免疫力向上等の目的で、従来から栄養補助食品等に配合され、使用されているが、従来の技術においては、ローヤルゼリーがどのような作用を介してそれらの効果を発揮するかについて、何ら解明がなされていなかった。本発明は、ローヤルゼリー抽出物が、前記したような具体的な各作用を有していることを見出したことに基づくものであり、これにより、各作用に基づくローヤルゼリー抽出物の、新たな利用の可能性をも提供することができる。
As a result of intensive studies to solve the above problems, the present inventors have found that royal jelly extract has excellent type IV collagen production promoting effect, transglutaminase-1 production promoting effect, serine palmitoyltransferase production promoting effect, aquaporin 3 production promotion action, involucrin production promotion action, HMG-CoA reductase production promotion action, and adenosine triphosphate production promotion action were found, and the present invention was completed.
Royal jelly has been conventionally used in dietary supplements for the purpose of improving menopause, preventing aging, improving immunity, etc., but in conventional technology, royal jelly No elucidation has been made as to whether these effects are exhibited. The present invention is based on the discovery that the royal jelly extract has the specific actions as described above, and thus, the new use of the royal jelly extract based on each action. Possibilities can also be provided.
本発明は、本発明者らの前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするＩＶ型コラーゲン産生促進剤である。
＜２＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするトランスグルタミナーゼ−１産生促進剤である。
＜３＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするセリンパルミトイルトランスフェラーゼ産生促進剤である。
＜４＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするアクアポリン３産生促進剤である。
＜５＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするインボルクリン産生促進剤である。
＜６＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするＨＭＧ−ＣｏＡ還元酵素産生促進剤である。
＜７＞ ローヤルゼリー抽出物を有効成分として含有することを特徴とするアデノシン三リン酸産生促進剤である。
The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A type IV collagen production promoter comprising a royal jelly extract as an active ingredient.
<2> A transglutaminase-1 production promoter comprising a royal jelly extract as an active ingredient.
<3> A serine palmitoyltransferase production promoter comprising a royal jelly extract as an active ingredient.
<4> An aquaporin 3 production promoter characterized by containing a royal jelly extract as an active ingredient.
<5> An involucrin production promoter comprising a royal jelly extract as an active ingredient.
<6> A HMG-CoA reductase production promoter comprising a royal jelly extract as an active ingredient.
<7> An adenosine triphosphate production promoter comprising a royal jelly extract as an active ingredient.
本発明によると、従来における諸問題を解決することができ、前記目的を達成することができる。
即ち、本発明によると、第一に、優れたＩＶ型コラーゲン産生促進作用を有し、かつ安全性の高いＩＶ型コラーゲン産生促進剤を提供することができる。
また、本発明によると、第二に、優れたトランスグルタミナーゼ−１産生促進作用を有し、かつ安全性の高いトランスグルタミナーゼ−１産生促進剤を提供することができる。
また、本発明によると、第三に、優れたセリンパルミトイルトランスフェラーゼ産生促進作用を有し、かつ安全性の高いセリンパルミトイルトランスフェラーゼ産生促進剤を提供することができる。
また、本発明によると、第四に、優れたアクアポリン３産生促進作用を有し、かつ安全性の高いアクアポリン３産生促進剤を提供することができる。
また、本発明によると、第五に、優れたインボルクリン産生促進作用を有し、かつ安全性の高いインボルクリン産生促進剤を提供することができる。
また、本発明によると、第六に、優れたＨＭＧ−ＣｏＡ還元酵素産生促進作用を有し、かつ安全性の高いＨＭＧ−ＣｏＡ還元酵素産生促進剤を提供することができる。
また、本発明によると、第七に、優れたアデノシン三リン酸産生促進作用を有し、かつ安全性の高いアデノシン三リン酸産生促進剤を提供することができる。
According to the present invention, conventional problems can be solved, and the object can be achieved.
That is, according to the present invention, firstly, a highly safe IV type collagen production promoter having an excellent type IV collagen production promoting action and high safety can be provided.
In addition, according to the present invention, secondly, it is possible to provide a transglutaminase-1 production promoter that has an excellent transglutaminase-1 production promoting action and is highly safe.
In addition, according to the present invention, thirdly, a highly safe serine palmitoyltransferase production promoter having an excellent serine palmitoyltransferase production promoting action and high safety can be provided.
In addition, according to the present invention, fourthly, an aquaporin 3 production promoter having an excellent aquaporin 3 production promoting action and high safety can be provided.
In addition, according to the present invention, fifthly, it is possible to provide an involucrin production promoter having an excellent involucrin production promoting action and high safety.
In addition, according to the present invention, sixthly, it is possible to provide a highly safe HMG-CoA reductase production promoter having an excellent HMG-CoA reductase production promoting action and high safety.
In addition, according to the present invention, seventhly, it is possible to provide an adenosine triphosphate production promoter having an excellent adenosine triphosphate production promoting action and high safety.
（ＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、セリンパルミトイルトランスフェラーゼ産生促進剤、アクアポリン３産生促進剤、インボルクリン産生促進剤、ＨＭＧ−ＣｏＡ還元酵素産生促進剤、アデノシン三リン酸産生促進剤）
本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、セリンパルミトイルトランスフェラーゼ（以下、ＳＰＴ）産生促進剤、アクアポリン３（以下、ＡＱＰ３）産生促進剤、インボルクリン産生促進剤、ＨＭＧ−ＣｏＡ還元酵素（以下、ＨＭＧＣＲ）産生促進剤、及び、アデノシン三リン酸（以下、ＡＴＰ）産生促進剤は、ローヤルゼリー抽出物を含有してなり、更に必要に応じてその他の成分を含有してなる。
前記ローヤルゼリー抽出物が含有する、前記各作用（ＩＶ型コラーゲン産生促進作用、トランスグルタミナーゼ−１産生促進作用、ＳＰＴ産生促進作用、ＡＱＰ３産生促進作用、インボルクリン産生促進作用、ＨＭＧＣＲ産生促進作用、及び、ＡＴＰ産生促進作用）を発揮する物質の詳細については不明であるが、前記ローヤルゼリー抽出物がこのような優れた作用を有することは、従来には知られておらず、本発明者らによる新たな知見である。
(Type IV collagen production promoter, transglutaminase-1 production promoter, serine palmitoyltransferase production promoter, aquaporin 3 production promoter, involucrin production promoter, HMG-CoA reductase production promoter, adenosine triphosphate production promoter )
Type IV collagen production promoter, transglutaminase-1 production promoter, serine palmitoyltransferase (hereinafter referred to as SPT) production promoter, aquaporin 3 (hereinafter referred to as AQP3) production promoter, involucrin production promoter, and HMG-CoA reduction The enzyme (hereinafter referred to as HMGCR) production promoter and the adenosine triphosphate (hereinafter referred to as ATP) production promoter contain a royal jelly extract, and further contain other components as necessary.
Each of the above-mentioned actions (type IV collagen production promoting action, transglutaminase-1 production promoting action, SPT production promoting action, AQP3 production promoting action, involucrin production promoting action, HMGCR production promoting action, and ATP contained in the royal jelly extract The details of the substance exhibiting the production promoting action) are unknown, but it has not been known so far that the royal jelly extract has such an excellent action, and new findings by the present inventors It is.
前記ローヤルゼリーは、ミツバチ科（Ａｐｉｄａｅ）ミツバチ属（Ａｐｉｓ）に属する昆虫の咽頭腺からの分泌物であり、女王蜂のエネルギー源として供給されるため、高タンパクで、ビタミン、ミネラル、アミノ酸等の様々な栄養素を含むことが広く知られている。前記ローヤルゼリーは、前記昆虫を飼育することにより常法に従い採取することもできるし、また、市販品として入手することもできる。 The royal jelly is a secretion from the pharyngeal gland of an insect belonging to the genus Apidae and is supplied as an energy source for the queen bee, so it has a high protein, various vitamins, minerals, amino acids and the like. It is widely known to contain nutrients. The royal jelly can be collected according to a conventional method by breeding the insects, or can be obtained as a commercial product.
抽出原料となる前記ローヤルゼリーは、採取した生の状態で（生ローヤルゼリー）溶媒抽出に供してもよいし、乾燥した状態で溶媒抽出に供してもよい。また、乾燥した後に、そのままの状態で又は粗砕機等を用いて粉砕した状態で、溶媒抽出に供することができる。前記乾燥は、例えば、天日で行ってもよいし、通常使用される乾燥機を用いて行ってもよい。なお、前記ローヤルゼリーは、ヘキサン、ベンゼン等の非極性溶媒によって脱脂等の前処理を施してから抽出原料として使用してもよい。脱脂等の前処理を行うことにより、前記ローヤルゼリーの極性溶媒による抽出処理を、効率よく行うことができる。 The royal jelly used as an extraction raw material may be subjected to solvent extraction in a collected raw state (raw royal jelly) or may be subjected to solvent extraction in a dried state. Moreover, after drying, it can use for solvent extraction as it is, or in the state grind | pulverized using the crusher etc. The drying may be performed, for example, in the sun or using a commonly used dryer. The royal jelly may be used as an extraction raw material after pretreatment such as degreasing with a nonpolar solvent such as hexane or benzene. By performing pretreatment such as degreasing, the royal jelly can be efficiently extracted with a polar solvent.
前記ローヤルゼリーの抽出物は、ローヤルゼリーの抽出に一般に用いられる方法を利用することによって、容易に得ることができる。また、前記ローヤルゼリーの抽出物としては、市販品を使用してもよい。なお、前記ローヤルゼリーの抽出物には、前記ローヤルゼリーの抽出液、該抽出液の希釈液若しくは濃縮液、該抽出液の乾燥物、又は、これらの粗精製物若しくは精製物のいずれもが含まれる。 The royal jelly extract can be easily obtained by utilizing a method generally used for royal jelly extraction. Moreover, you may use a commercial item as an extract of the said royal jelly. The royal jelly extract includes the royal jelly extract, a diluted or concentrated solution of the extract, a dried product of the extract, or a crude product or a purified product thereof.
前記抽出に用いる溶媒としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、水、親水性有機溶媒、又は、これらの混合溶媒を、室温又は溶媒の沸点以下の温度で用いることが好ましい。前記ローヤルゼリーに含まれる前記各作用を示す成分は、極性溶媒を抽出溶媒とする抽出処理によって、容易に抽出することができる。 There is no restriction | limiting in particular as a solvent used for the said extraction, According to the objective, it can select suitably, For example, water, a hydrophilic organic solvent, or these mixed solvents are room temperature or the temperature below the boiling point of a solvent. It is preferable to use it. The components having the above-mentioned actions contained in the royal jelly can be easily extracted by an extraction process using a polar solvent as an extraction solvent.
前記抽出溶媒として使用し得る水としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等の他、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、ろ過、イオン交換、浸透圧の調整、緩衝化等が含まれる。したがって、前記抽出溶媒として使用し得る水には、精製水、熱水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。 The water that can be used as the extraction solvent is not particularly limited and can be appropriately selected depending on the purpose. For example, pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc. In addition, those subjected to various treatments are included. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
前記抽出溶媒として使用し得る親水性有機溶媒としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３−ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２〜５の多価アルコール；スクワラン、ホホバ油等の天然オイルなどが挙げられ、該親水性有機溶媒と水との混合溶媒なども用いることができる。なお、前記水と前記親水性有機溶媒との混合溶媒を使用する際には、低級アルコールの場合は水１０質量部に対して１〜９０質量部、低級脂肪族ケトンの場合は水１０質量部に対して１〜４０質量部を混合したものを使用することが好ましい。また、多価アルコールの場合は水１０質量部に対して１〜９０質量部を混合したものを使用することが好ましい。 The hydrophilic organic solvent that can be used as the extraction solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include lower ones having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol. Alcohols; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin; natural oils such as squalane and jojoba oil; A mixed solvent of a water-soluble organic solvent and water can also be used. In addition, when using the mixed solvent of the said water and the said hydrophilic organic solvent, 1-90 mass parts with respect to 10 mass parts of water in the case of a lower alcohol, and 10 mass parts of water in the case of a lower aliphatic ketone. It is preferable to use what mixed 1-40 mass parts with respect to this. Moreover, in the case of polyhydric alcohol, it is preferable to use what mixed 1-90 mass parts with respect to 10 mass parts of water.
抽出原料である前記ローヤルゼリーから、前記各作用を有する抽出物を抽出するにあたって、特殊な抽出方法を採用する必要はなく、室温又は還流加熱下で、任意の抽出装置を用いて抽出することができる。
具体的には、抽出溶媒を満たした処理槽内に、前記抽出原料を投入し、更に必要に応じて時々攪拌しながら、３０分〜４時間静置して可溶性成分を溶出した後、ろ過して固形物を除去し、得られた抽出液から抽出溶媒を留去し、乾燥することにより抽出物を得ることができる。抽出溶媒量は通常、抽出原料の５〜１５倍量（質量比）である。抽出条件は、抽出溶媒として水を用いた場合には、通常５０〜９５℃にて１〜４時間程度である。また、抽出溶媒として水とエタノールとの混合溶媒を用いた場合には、通常４０〜８０℃にて３０分間〜４時間程度である。なお、溶媒で抽出することにより得られる抽出液は、抽出溶媒が安全性の高いものであれば、そのまま本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤の有効成分として用いることができる。
When extracting the extract having the above actions from the royal jelly, which is an extraction raw material, it is not necessary to adopt a special extraction method, and it can be extracted using any extraction device at room temperature or under reflux heating. .
Specifically, the extraction raw material is put into a processing tank filled with an extraction solvent, and further, while stirring occasionally as necessary, it is allowed to stand for 30 minutes to 4 hours to elute soluble components, followed by filtration. The solid can be removed, and the extract can be obtained by evaporating the extraction solvent from the resulting extract and drying. The amount of extraction solvent is usually 5 to 15 times (mass ratio) of the extraction raw material. The extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Moreover, when using the mixed solvent of water and ethanol as an extraction solvent, it is about 30 minutes-about 4 hours at 40-80 degreeC normally. In addition, if the extraction liquid obtained by extracting with a solvent has a high safety, the type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter of the present invention, It can be used as an active ingredient of AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter.
抽出により得られる前記ローヤルゼリーの抽出液は、該抽出液の希釈液若しくは濃縮液、該抽出液の乾燥物、又はこれらの粗精製物若しくは精製物を得るため、常法に従って希釈、濃縮、乾燥、精製等の処理を施してもよい。なお、得られる前記ローヤルゼリーの抽出液は、そのままでも前記ＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤の有効成分として使用することができるが、濃縮液又はその乾燥物としたものの方が利用しやすい。抽出液の乾燥物を得るにあたっては、常法を利用することができ、また、吸湿性を改善するためにデキストリン、シクロデキストリン等のキャリアーを添加してもよい。また、抽出原料である前記ローヤルゼリーは特有の匂いと味を有しているため、前記ローヤルゼリーの抽出物に対しては、生理活性の低下を招かない範囲で、脱色、脱臭等を目的とする精製を行うことも可能であるが、例えば皮膚化粧料に添加する場合などには大量に使用するものではないから、未精製のままでも実用上支障はない。なお、精製は、具体的には、活性炭処理、吸着樹脂処理、イオン交換樹脂処理等によって行うことができる。 The royal jelly extract obtained by extraction is diluted, concentrated, dried according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or purified product thereof. Treatment such as purification may be performed. The obtained royal jelly extract can be used as it is as it is, the type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, Although it can be used as an active ingredient of an ATP production promoter, a concentrated solution or a dried product thereof is easier to use. In obtaining the dried extract, a conventional method can be used, and carriers such as dextrin and cyclodextrin may be added to improve hygroscopicity. In addition, since the royal jelly, which is an extraction raw material, has a characteristic odor and taste, the royal jelly extract is purified for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in physiological activity. However, since it is not used in large quantities, for example, when it is added to skin cosmetics, there is no practical problem even if it is not purified. Specifically, purification can be performed by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like.
以上のようにして得られる前記ローヤルゼリー抽出物は、優れたＩＶ型コラーゲン産生促進作用、トランスグルタミナーゼ−１産生促進作用、ＳＰＴ産生促進作用、ＡＱＰ３産生促進作用、インボルクリン産生促進作用、ＨＭＧＣＲ産生促進作用、及び、ＡＴＰ産生促進作用を有し、これらの作用に基づき、本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤の有効成分として好適に利用可能なものである。 The royal jelly extract obtained as described above has an excellent type IV collagen production promoting action, transglutaminase-1 production promoting action, SPT production promoting action, AQP3 production promoting action, involucrin production promoting action, HMGCR production promoting action, And ATP production promoting action, and based on these actions, the type IV collagen production promoting agent, transglutaminase-1 production promoting agent, SPT production promoting agent, AQP3 production promoting agent, involculin production promoting agent, HMGCR of the present invention It can be suitably used as an active ingredient of a production promoter and an ATP production promoter.
なお、前記各剤中の前記ローヤルゼリー抽出物の含有量としては、特に制限はなく、目的に応じて適宜選択することができ、また、前記各剤は、前記ローヤルゼリー抽出物そのものであってもよい。 In addition, the content of the royal jelly extract in each agent is not particularly limited and can be appropriately selected according to the purpose, and each of the agents may be the royal jelly extract itself. .
また、前記各剤中に含まれ得る、前記ローヤルゼリー抽出物以外のその他の成分としても、本発明の効果を損なわない範囲内であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記ローヤルゼリー抽出物を所望の濃度に希釈等するための、生理食塩液などが挙げられる。また、前記各剤は、前記その他の成分として、ＩＶ型コラーゲン産生促進作用、トランスグルタミナーゼ−１産生促進作用、ＳＰＴ産生促進作用、ＡＱＰ３産生促進作用、インボルクリン産生促進作用、ＨＭＧＣＲ産生促進作用、及び、ＡＴＰ産生促進作用を有する、前記ローヤルゼリー抽出物以外の天然抽出物等を含んでいてもよい。前記各剤中の前記その他の成分の含有量にも、特に制限はなく、目的に応じて適宜選択することができる。 Further, other components other than the royal jelly extract that can be contained in each agent are not particularly limited as long as the effects of the present invention are not impaired, and may be appropriately selected according to the purpose. Examples thereof include a physiological saline solution for diluting the royal jelly extract to a desired concentration. In addition, each of the agents includes, as the other components, type IV collagen production promoting action, transglutaminase-1 production promoting action, SPT production promoting action, AQP3 production promoting action, involucrin production promoting action, HMGCR production promoting action, and A natural extract other than the royal jelly extract having an ATP production promoting effect may be included. There is no restriction | limiting in particular also in content of the said other component in each said agent, According to the objective, it can select suitably.
また、前記各剤は、必要に応じて製剤化することにより、粉末状、顆粒状、錠剤状等、任意の剤形とすることができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯臭剤等を用いることができる。 Moreover, each said agent can be made into arbitrary dosage forms, such as a powder form, a granular form, and a tablet form, by formulating as needed. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.
前記ＩＶ型コラーゲン産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、ＩＶ型コラーゲン産生促進作用を発揮する。
前記トランスグルタミナーゼ−１産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、トランスグルタミナーゼ−１産生促進作用を発揮する。
前記ＳＰＴ産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、ＳＰＴ産生促進作用を発揮する。
前記ＡＱＰ３産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、ＡＱＰ３産生促進作用を発揮する。
前記インボルクリン産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、インボルクリン産生促進作用を発揮する。
前記ＨＭＧＣＲ産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、ＨＭＧＣＲ産生促進作用を発揮する。
前記ＡＴＰ産生促進剤は、有効成分として含有されるローヤルゼリー抽出物の作用により、ＡＴＰ産生促進作用を発揮する。
The type IV collagen production promoter exerts a type IV collagen production promotion effect by the action of a royal jelly extract contained as an active ingredient.
The transglutaminase-1 production promoter exerts a transglutaminase-1 production promotion effect by the action of a royal jelly extract contained as an active ingredient.
The SPT production promoter exerts an SPT production promoting action by the action of a royal jelly extract contained as an active ingredient.
The AQP3 production promoter exerts an AQP3 production promoting action by the action of a royal jelly extract contained as an active ingredient.
The involucrin production promoter exerts an involucrin production promoting action by the action of a royal jelly extract contained as an active ingredient.
The said HMGCR production promoter exhibits HMGCR production promotion effect | action by the effect | action of the royal jelly extract contained as an active ingredient.
The ATP production promoter exerts an ATP production promoting action by the action of a royal jelly extract contained as an active ingredient.
本発明のＩＶ型コラーゲン産生促進剤によると、優れたＩＶ型コラーゲン産生促進作用を通じて、例えば、肌の表皮と真皮との間に存在する基底膜の構造を保持する機能を高め、皮膚バリア機能の強化を図ることが可能となる。ただし、本発明のＩＶ型コラーゲン産生促進剤は、これらの用途以外にもＩＶ型コラーゲン産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the type IV collagen production promoter of the present invention, through an excellent type IV collagen production promotion effect, for example, the function of maintaining the structure of the basement membrane existing between the epidermis and dermis of the skin is enhanced, and the skin barrier function is improved. It is possible to strengthen. However, the type IV collagen production-promoting agent of the present invention can be used for all purposes that are meaningful for exerting the effect of promoting type IV collagen production in addition to these uses.
本発明のトランスグルタミナーゼ−１産生促進剤によると、優れたトランスグルタミナーゼ−１産生促進作用を通じて、例えば、角化細胞を包み込む不溶性の細胞膜様構造体であるコーニファイドエンベロープ（ＣＥ）の形成を促進して角化を正常化することにより、乾燥や紫外線等の外部刺激に伴う皮膚バリア機能の低下を抑制し、肌の乾燥や肌荒れなど、様々な皮膚症状を予防・改善することが可能となる。ただし、本発明のトランスグルタミナーゼ−１産生促進剤は、これらの用途以外にもトランスグルタミナーゼ−１産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the transglutaminase-1 production promoting agent of the present invention, through the excellent transglutaminase-1 production promoting action, for example, the formation of a cornified envelope (CE) that is an insoluble cell membrane-like structure that wraps around keratinocytes is promoted. By normalizing keratinization, it is possible to suppress a decrease in skin barrier function due to external stimulation such as dryness and ultraviolet rays, and to prevent and improve various skin symptoms such as dry skin and rough skin. However, the transglutaminase-1 production promoter of the present invention can be used for all uses that are meaningful for exerting a transglutaminase-1 production promoting action in addition to these uses.
本発明のＳＰＴ産生促進剤によると、優れたＳＰＴ産生促進作用を通じて、例えば、角質の細胞間脂質の構成成分であるセラミドの産生能を高め、皮膚のバリア機能や水分保持機能を向上させ、乾燥肌、肌荒れ、小じわ、アトピー性皮膚炎など、様々な皮膚症状を予防・改善することが可能となる。ただし、本発明のＳＰＴ産生促進剤は、これらの用途以外にもＳＰＴ産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the SPT production promoter of the present invention, through an excellent SPT production promoting action, for example, the ability to produce ceramide, which is a component of keratin intercellular lipid, is enhanced, the skin barrier function and the water retention function are improved, and the dryness is improved. Various skin symptoms such as skin, rough skin, fine lines, and atopic dermatitis can be prevented and ameliorated. However, the SPT production promoter of the present invention can be used for all purposes that are meaningful for exerting the SPT production promoting action in addition to these uses.
本発明のＡＱＰ３産生促進剤によると、優れたＡＱＰ３産生促進作用を通じて、例えば、表皮細胞における水分の通り道をケアし、肌の潤いを高め、皮膚のバリア機能の強化を図ることが可能となる。ただし、本発明のＡＱＰ３産生促進剤は、これらの用途以外にもＡＱＰ３産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the AQP3 production promoter of the present invention, for example, through the excellent AQP3 production promoting action, it is possible to care for the passage of moisture in the epidermis cells, increase the moisture of the skin, and enhance the barrier function of the skin. However, the AQP3 production promoter of the present invention can be used for all purposes that are meaningful for exerting the AQP3 production promoting action in addition to these uses.
本発明のインボルクリン産生促進剤によると、優れたインボルクリン産生促進作用を通じて、例えば、角化細胞を包み込む不溶性の細胞膜様構造体であるコーニファイドエンベロープ（ＣＥ）の形成を促進して角化を正常化することにより、乾燥や紫外線等の外部刺激に伴う皮膚バリア機能の低下を抑制し、肌の乾燥や肌荒れなど、様々な皮膚症状を予防・改善することが可能となる。ただし、本発明のインボルクリン産生促進剤は、これらの用途以外にもインボルクリン産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the involucrin production promoter of the present invention, through excellent involucrin production promoting action, for example, the formation of a cornified envelope (CE), which is an insoluble cell membrane-like structure enveloping keratinocytes, is promoted to normalize keratinization. By doing so, it is possible to prevent the skin barrier function from being lowered due to external stimuli such as dryness and ultraviolet rays, and to prevent and improve various skin symptoms such as dry skin and rough skin. However, the involucrin production-promoting agent of the present invention can be used for all uses other than these uses that are meaningful for exerting an involucrin production-promoting action.
本発明のＨＭＧＣＲ産生促進剤によると、優れたＨＭＧＣＲ産生促進作用を通じて、例えば、角質の細胞間脂質の構成成分であるコレステロールの産生能を高め、皮膚のバリア機能や水分保持機能を向上させ、乾燥肌、肌荒れ、小じわ、アトピー性皮膚炎など、様々な皮膚症状を予防・改善することが可能となる。ただし、本発明のＨＭＧＣＲ産生促進剤は、これらの用途以外にもＨＭＧＣＲ産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the HMGCR production promoter of the present invention, through an excellent HMGCR production promoting action, for example, the ability to produce cholesterol, which is a constituent component of keratin intercellular lipid, is enhanced, the skin barrier function and the water retention function are improved, and the drying is performed. Various skin symptoms such as skin, rough skin, fine lines, and atopic dermatitis can be prevented and ameliorated. However, the HMGCR production promoter of the present invention can be used for all purposes that are meaningful for exerting the HMGCR production promoting action in addition to these uses.
本発明のＡＴＰ産生促進剤によると、優れたＡＴＰ産生促進作用を通じて、例えば、肌細胞に活力を与え、結果的にくすんだ肌や疲れた肌の改善など、様々な皮膚症状を予防・改善することが可能となる。ただし、本発明のＡＴＰ産生促進剤は、これらの用途以外にもＡＴＰ産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 According to the ATP production promoter of the present invention, through an excellent ATP production promoting action, for example, skin cells are revitalized, and as a result, various skin symptoms such as improvement of dull skin and tired skin are prevented and improved. It becomes possible. However, the ATP production promoter of the present invention can be used for all purposes that are meaningful for exerting an ATP production promoting action in addition to these uses.
なお、本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物（例えば、マウス、ラット、ハムスター、イヌ、ネコ、ウシ、ブタ、サルなど）に対して適用することもできる。 The type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter of the present invention are Applicable to animals, but as long as each effect is achieved, it applies to animals other than humans (eg, mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) You can also
本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤は、優れた作用を有するとともに、皮膚に適用した場合の使用感と安全性に優れているため、例えば、皮膚外用剤に配合するのに好適である。ここで、前記皮膚外用剤としては、その区分に制限はなく、皮膚化粧料、医薬部外品、医薬品などを幅広く含むものであり、具体的には、例えば、軟膏、クリーム、乳液、美容液、ローション、パック、ファンデーション、リップクリーム、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディシャンプーなどが挙げられる。 The type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter of the present invention have excellent effects. In addition, since it has excellent usability and safety when applied to the skin, it is suitable, for example, for blending into an external preparation for skin. Here, the external preparation for skin is not limited in its category, and includes a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, etc. Specifically, for example, ointments, creams, milky lotions, cosmetic liquids , Lotions, packs, foundations, lip balms, bath salts, hair nicks, hair lotions, soaps, body shampoos and the like.
また、本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤は、優れた作用を有するとともに、経口的に摂取した場合の安全性にも優れているため、例えば、美容用飲食品に配合するのに好適である。ここで、前記美容用飲食品としては、その区分に制限はなく、経口的に摂取される一般食品、健康食品、保健機能食品、医薬部外品、医薬品などを幅広く含むものである。 Further, the type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter of the present invention are excellent. Since it has an effect | action and is also excellent in the safety | security when ingested orally, it is suitable for mix | blending with cosmetics food / beverage products, for example. Here, the cosmetic foods and drinks are not limited in their classification, and include a wide range of foods such as general foods, health foods, health functional foods, quasi drugs, and pharmaceuticals that are taken orally.
また、本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤は、優れた作用を有するので、ＩＶ型コラーゲン、トランスグルタミナーゼ−１、ＳＰＴ、ＡＱＰ３、インボルクリン、ＨＭＧＣＲ、及び、ＡＴＰの機能の研究や、ＩＶ型コラーゲン、トランスグルタミナーゼ−１、ＳＰＴ、ＡＱＰ３、インボルクリン、ＨＭＧＣＲ、及び、ＡＴＰに関連する疾患の研究のための試薬としても好適に利用可能である。 Further, the type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter of the present invention are excellent. Study of the functions of type IV collagen, transglutaminase-1, SPT, AQP3, involucrin, HMGCR, and ATP, type IV collagen, transglutaminase-1, SPT, AQP3, involucrin, HMGCR, and It can also be suitably used as a reagent for the study of diseases related to ATP.
以下、本発明の製造例及び実施例を説明するが、本発明は、これらの製造例及び実施例に何ら限定されるものではない。 EXAMPLES Hereinafter, although the manufacture example and Example of this invention are demonstrated, this invention is not limited to these manufacture examples and Examples at all.
（製造例１：ローヤルゼリー抽出物（液状）の製造）
ヨーロッパミツバチ（Ａｐｉｓ ｍｅｌｉｆｉｃａ Ｌ．）の咽頭部から分泌されるローヤルゼリー（生の状態のもの）１ｋｇに５０ｖ／ｖ％エタノール９．５Ｌを加えて、１０℃にて５時間撹拌した。これを５０００ｒｐｍで３０分間遠心分離し、得られた上澄液を集め、冷所にて１ヶ月間放置した。これをろ過し、ろ液に５０ｖ／ｖ％エタノールを追加し、全量を１０Ｌとした。
(Production Example 1: Production of Royal Jelly Extract (Liquid))
9.5 L of 50 v / v% ethanol was added to 1 kg of royal jelly (raw state) secreted from the pharynx of European honey bees (Apis melicica L.), and the mixture was stirred at 10 ° C. for 5 hours. This was centrifuged at 5000 rpm for 30 minutes, and the resulting supernatant was collected and allowed to stand in a cold place for 1 month. This was filtered, and 50 v / v% ethanol was added to the filtrate to make the total volume 10 L.
（製造例２：ローヤルゼリー抽出物（液状）の製造）
ヨーロッパミツバチ（Ａｐｉｓ ｍｅｌｉｆｉｃａ Ｌ．）の咽頭部から分泌されるローヤルゼリー（乾燥させた状態のもの）１ｋｇに５０ｖ／ｖ％エタノール９．５Ｌを加えて、１０℃にて５時間撹拌した。これを５０００ｒｐｍで３０分間遠心分離し、得られた上澄液を集め、冷所にて１ヶ月間放置した。これをろ過し、ろ液に５０ｖ／ｖ％エタノールを追加し、全量を１０Ｌとした。
(Production Example 2: Production of Royal Jelly Extract (Liquid))
9.5 L of 50 v / v% ethanol was added to 1 kg of royal jelly (in a dried state) secreted from the pharynx of European honey bees (Apis melicica L.), and the mixture was stirred at 10 ° C. for 5 hours. This was centrifuged at 5000 rpm for 30 minutes, and the resulting supernatant was collected and allowed to stand in a cold place for 1 month. This was filtered, and 50 v / v% ethanol was added to the filtrate to make the total volume 10 L.
（製造例３：ローヤルゼリー抽出物（凍結乾燥品）の製造）
ヨーロッパミツバチ（Ａｐｉｓ ｍｅｌｉｆｉｃａ Ｌ．）の咽頭部から分泌されるローヤルゼリー（生の状態のもの）１ｋｇに５０ｖ／ｖ％エタノール９．５Ｌを加えて、１０℃にて５時間撹拌した。これを５０００ｒｐｍで３０分間遠心分離し、得られた上澄液を集め、冷所にて１ヶ月間放置した。これをろ過し、ろ液に５０ｖ／ｖ％エタノールを追加し、全量を１０Ｌとした。これを真空凍結乾燥し、凍結乾燥物を得た。
(Production Example 3: Production of royal jelly extract (freeze-dried product))
9.5 L of 50 v / v% ethanol was added to 1 kg of royal jelly (raw state) secreted from the pharynx of European honey bees (Apis melicica L.), and the mixture was stirred at 10 ° C. for 5 hours. This was centrifuged at 5000 rpm for 30 minutes, and the resulting supernatant was collected and allowed to stand in a cold place for 1 month. This was filtered, and 50 v / v% ethanol was added to the filtrate to make the total volume 10 L. This was freeze-dried in vacuo to obtain a freeze-dried product.
（製造例４：ローヤルゼリー抽出物（凍結乾燥品）の製造）
ヨーロッパミツバチ（Ａｐｉｓ ｍｅｌｉｆｉｃａ Ｌ．）の咽頭部から分泌されるローヤルゼリー（乾燥させた状態のもの）１ｋｇに５０ｖ／ｖ％エタノール９．５Ｌを加えて、１０℃にて５時間撹拌した。これを５０００ｒｐｍで３０分間遠心分離し、得られた上澄液を集め、冷所にて１ヶ月間放置した。これをろ過し、ろ液に５０ｖ／ｖ％エタノールを追加し、全量を１０Ｌとした。これを真空凍結乾燥し、凍結乾燥物を得た。
(Production Example 4: Production of royal jelly extract (freeze-dried product))
9.5 L of 50 v / v% ethanol was added to 1 kg of royal jelly (in a dried state) secreted from the pharynx of European honey bees (Apis melicica L.), and the mixture was stirred at 10 ° C. for 5 hours. This was centrifuged at 5000 rpm for 30 minutes, and the resulting supernatant was collected and allowed to stand in a cold place for 1 month. This was filtered, and 50 v / v% ethanol was added to the filtrate to make the total volume 10 L. This was freeze-dried in vacuo to obtain a freeze-dried product.
（参考例１：ＩＶ型コラーゲン産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりＩＶ型コラーゲン産生促進作用を試験した。
( Reference Example 1: Type IV collagen production promoting action test)
Using the royal jelly extract of Production Example 3 as a test sample, the type IV collagen production promoting effect was tested by the following test method.
ヒト正常線維芽細胞（ＮＢ１ＲＧＢ）を１０％ＦＢＳ含有ダルベッコＭＥＭを用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１．６×１０５ｃｅｌｌｓ／ｍＬの濃度に上記培地で希釈した後、９６ｗｅｌｌマイクロプレートに１ｗｅｌｌ当たり１００μＬずつ播種し、一晩培養した。培養終了後、培地を抜き、０．２５％ＦＢＳ含有ダルベッコＭＥＭに溶解した被験試料を各ｗｅｌｌに１５０μＬ添加し、３日間培養した。培養後、各ｗｅｌｌの培地中のＩＶ型コラーゲン量をＥＬＩＳＡ法により測定した。結果を表１に示す。
ＩＶ型コラーゲン産生促進作用の計算方法は以下のとおりである。
ＩＶ型コラーゲン産生促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時のＩＶ型コラーゲン量
Ｂ：被験試料無添加時のＩＶ型コラーゲン量
After normal human fibroblasts (NB1RGB) were cultured using Dulbecco's MEM containing 10% FBS, the cells were collected by trypsin treatment. The collected cells were diluted with the above medium to a concentration of 1.6 × 10 5 cells / mL, then seeded at 100 μL per well on a 96-well microplate, and cultured overnight. After completion of the culture, the medium was removed, 150 μL of a test sample dissolved in 0.25% FBS-containing Dulbecco MEM was added to each well, and the cells were cultured for 3 days. After culture, the amount of type IV collagen in the medium of each well was measured by ELISA. The results are shown in Table 1.
The calculation method of the type IV collagen production promoting action is as follows.
Type IV collagen production promotion rate (%) = A / B × 100
A: Type IV collagen when test sample is added B: Type IV collagen when no test sample is added
表１の結果から、ローヤルゼリー抽出物が、高いＩＶ型コラーゲン産生促進作用を有することが認められた。 From the result of Table 1, it was recognized that the royal jelly extract has a high type IV collagen production promoting effect.
（参考例２：トランスグルタミナーゼ−１産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりトランスグルタミナーゼ−１産生促進作用を試験した。
( Reference Example 2: Transglutaminase-1 production promoting action test)
Using the royal jelly extract of Production Example 3 as a test sample, the transglutaminase-1 production promoting effect was tested by the following test method.
ヒト正常新生児皮膚表皮角化細胞（ＮＨＥＫ）をヒト正常新生児表皮角化細胞用培地（ＫＧＭ）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１×１０５ｃｅｌｌｓ／ｍＬの濃度になるようにＫＧＭで希釈した後、９６ｗｅｌｌプレートに１ｗｅｌｌ当たり１００μＬずつ播種し、２日間培養した。培養終了後、ＫＧＭで溶解した被験試料を各ｗｅｌｌに１００μＬ添加し、２４時間培養した。培養終了後、培地を抜き、細胞をプレートに固定させ細胞表面に発現したトランスグルタミナーゼ−１の量をモノクローナル抗ヒトトランスグルタミナーゼ−１抗体を用いたＥＬＩＳＡ法により測定した。結果を表２に示す。
トランスグルタミナーゼ−１産生促進作用の計算方法は以下のとおりである。
トランスグルタミナーゼ−１産生促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時の波長４０５ｎｍにおける吸光度
Ｂ：被験試料無添加時（コントロール）の波長４０５ｎｍにおける吸光度
Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 5 cells / mL, then seeded on a 96-well plate at 100 μL per well, and cultured for 2 days. After completion of the culture, 100 μL of a test sample dissolved in KGM was added to each well and cultured for 24 hours. After completion of the culture, the medium was removed, the cells were fixed on a plate, and the amount of transglutaminase-1 expressed on the cell surface was measured by ELISA using a monoclonal anti-human transglutaminase-1 antibody. The results are shown in Table 2.
The calculation method of the transglutaminase-1 production promoting action is as follows.
Transglutaminase-1 production promotion rate (%) = A / B × 100
A: Absorbance at a wavelength of 405 nm when a test sample is added B: Absorbance at a wavelength of 405 nm when no test sample is added (control)
表２の結果から、ローヤルゼリー抽出物が、高いトランスグルタミナーゼ−１産生促進作用を有することが認められた。 From the results of Table 2, it was confirmed that the royal jelly extract has a high transglutaminase-1 production promoting action.
（参考例３：セリンパルミトイルトランスフェラーゼ産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりセリンパルミトイルトランスフェラーゼ（ＳＰＴ）産生促進作用を試験した。
( Reference example 3: Serine palmitoyltransferase production promoting action test)
Using the royal jelly extract of Production Example 3 as a test sample, serine palmitoyltransferase (SPT) production promoting effect was tested by the following test method.
正常ヒト新生児包皮表皮角化細胞（ｎｏｒｍａｌ ｈｕｍａｎ ｅｐｉｄｅｒｍｉｓ ｋｅｒａｔｉｎｏｃｙｔｅ；ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（ＥｐｉＬｉｆｅ−ＫＧ２）において、３７℃、５％ＣＯ２下で前培養し、トリプシン処理により細胞を集めた。
ＥｐｉＬｉｆｅ−ＫＧ２を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２下で一晩培養した。２４時間後に培養液を捨て、ＥｐｉＬｉｆｅ−ＫＧ２で必要濃度に溶解した被験試料を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２下で２４時間（製造例１〜２のローヤルゼリー抽出物を被験試料とした場合）、又は、４８時間（製造例３〜４のローヤルゼリー抽出物を被験試料とした場合）培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ；Ｃａｔ．ｎｏ．３１１−０２５０１）にてｔｏｔａｌ ＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるようにｔｏｔａｌＲＮＡを調製した。
このｔｏｔａｌＲＮＡを鋳型とし、ＳＰＴ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Ｓｍａｒｔ Ｃｙｃｌｅｒ（登録商標）（Ｃｅｐｈｅｉｄ社）を用いて、ＴａＫａＲａ ＳＹＢＲ（登録商標）ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ−ＰＣＲ Ｋｉｔ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）（ｃｏｄｅ Ｎｏ．ＲＲ０６３Ａ）によるリアルタイム２ Ｓｔｅｐ ＲＴ−ＰＣＲ反応により行った。ＳＰＴの発現量は、被験試料無添加、被験試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに被験試料無添加の補正値を１００とした時の被験試料添加の補正値を算出した。結果を表３に示す。
ＳＰＴｍＲＮＡ発現促進作用の計算方法は以下の通りである。
ＳＰＴｍＲＮＡ発現促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時の補正値
Ｂ：被験試料無添加時の補正値
Normal human neonatal foreskin keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes in an 80 cm 2 flask at 37 ° C. under 5% CO 2. Cells were collected by trypsinization.
Epilife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 . After 24 hours, the culture solution was discarded, and 2 mL of the test sample dissolved in EpiLife-KG2 to the required concentration was added to each petri dish at 37 ° C. under 5% CO 2 for 24 hours (the royal jelly extract of Production Examples 1 and 2 was removed). Cultured for 48 hours (when the royal jelly extract of Production Examples 3 to 4 was used as the test sample). After culturing, the culture solution is discarded, total RNA is extracted with ISOGEN (NIPPON GENE; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and total RNA is adjusted to 200 ng / μL. Was prepared.
Using this total RNA as a template, the expression level of SPT and GAPDH mRNA as an internal standard was measured. Detection is performed using a real-time PCR apparatus Smart Cycler (registered trademark) (Cepheid), TaKaRa SYBR (registered trademark) PrimeScript ™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). It went by. The expression level of SPT is determined based on the total RNA preparation prepared from the cells cultured with no test sample added and with the test sample added, and a correction value is obtained from the GAPDH value. The correction value for the test sample addition was calculated. The results are shown in Table 3.
The calculation method of the SPT mRNA expression promoting action is as follows.
SPT mRNA expression promotion rate (%) = A / B × 100
A: Correction value when test sample is added B: Correction value when test sample is not added
表３の結果から、ローヤルゼリー抽出物が、高いセリンパルミトイルトランスフェラーゼ（ＳＰＴ）産生促進作用を有することが認められた。 From the results of Table 3, it was confirmed that the royal jelly extract has a high serine palmitoyltransferase (SPT) production promoting action.
（参考例４：アクアポリン３産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりアクアポリン３（ＡＱＰ３）産生促進作用を試験した。
( Reference Example 4: Aquaporin 3 production promoting action test)
The royal jelly extract of Production Example 3 was used as a test sample, and aquaporin 3 (AQP3) production promoting action was tested by the following test method.
正常ヒト新生児包皮表皮角化細胞（ｎｏｒｍａｌ ｈｕｍａｎ ｅｐｉｄｅｒｍｉｓ ｋｅｒａｔｉｎｏｃｙｔｅ；ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（ＥｐｉＬｉｆｅ−ＫＧ２）において、３７℃、５％ＣＯ２下で前培養し、トリプシン処理により細胞を集めた。
ＥｐｉＬｉｆｅ−ＫＧ２を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２下で一晩培養した。２４時間後に培養液を捨て、ＥｐｉＬｉｆｅ−ＫＧ２で必要濃度に溶解した被験試料を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２下で２４時間（製造例１〜２のローヤルゼリー抽出物を被験試料とした場合）、又は、４８時間（製造例３〜４のローヤルゼリー抽出物を被験試料とした場合）培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ；Ｃａｔ．ｎｏ．３１１−０２５０１）にてｔｏｔａｌＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるようにｔｏｔａｌＲＮＡを調製した。
このｔｏｔａｌＲＮＡを鋳型とし、ＡＱＰ３及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Ｓｍａｒｔ Ｃｙｃｌｅｒ（登録商標）（Ｃｅｐｈｅｉｄ社）を用いて、ＴａＫａＲａ ＳＹＢＲ（登録商標）ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ−ＰＣＲ Ｋｉｔ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）（ｃｏｄｅ Ｎｏ．ＲＲ０６３Ａ）によるリアルタイム２ Ｓｔｅｐ ＲＴ−ＰＣＲ反応により行った。ＡＱＰ３の発現量は、被験試料無添加、被験試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに被験試料無添加の補正値を１００とした時の被験試料添加の補正値を算出した。結果を表４に示す。
ＡＱＰ３ｍＲＮＡ発現促進作用の計算方法は以下の通りである。
ＡＱＰ３ｍＲＮＡ発現促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時の補正値
Ｂ：被験試料無添加時の補正値
Normal human neonatal foreskin keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes in an 80 cm 2 flask at 37 ° C. under 5% CO 2. Cells were collected by trypsinization.
Epilife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 . After 24 hours, the culture solution was discarded, and 2 mL of the test sample dissolved in EpiLife-KG2 to the required concentration was added to each petri dish at 37 ° C. under 5% CO 2 for 24 hours (the royal jelly extract of Production Examples 1 and 2 was removed). Cultured for 48 hours (when the royal jelly extract of Production Examples 3 to 4 was used as the test sample). After culture, the culture solution is discarded, total RNA is extracted with ISOGEN (NIPPON GENE; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and total RNA is adjusted to 200 ng / μL. Prepared.
Using this total RNA as a template, the expression levels of AQP3 and GAPDH as an internal standard were measured. Detection is performed using a real-time PCR apparatus Smart Cycler (registered trademark) (Cepheid), TaKaRa SYBR (registered trademark) PrimeScript ™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). It went by. The expression level of AQP3 is calculated based on the total RNA preparation prepared from cells cultured without addition of the test sample and with the addition of the test sample, and a correction value is obtained from the value of GAPDH. The correction value for the test sample addition was calculated. The results are shown in Table 4.
The calculation method of the AQP3 mRNA expression promoting action is as follows.
AQP3 mRNA expression promotion rate (%) = A / B × 100
A: Correction value when test sample is added B: Correction value when test sample is not added
表４の結果から、ローヤルゼリー抽出物が、高いアクアポリン３（ＡＱＰ３）産生促進作用を有することが認められた。 From the results in Table 4, it was confirmed that the royal jelly extract has a high aquaporin 3 (AQP3) production promoting action.
（参考例５：インボルクリン産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりインボルクリン産生促進作用を試験した。
( Reference Example 5: Involucrin production promoting test)
Using the royal jelly extract of Production Example 3 as a test sample, the involucrin production promoting effect was tested by the following test method.
ヒト正常新生児皮膚表皮角化細胞（ＮＨＥＫ）をヒト正常新生児表皮角化細胞用培地（ＫＧＭ）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を１×１０５ｃｅｌｌｓ／ｍＬの濃度になるようにＫＧＭで希釈した後、４８ｗｅｌｌプレートに１ｗｅｌｌ当たり２００μＬずつ播種し、一晩培養した。培養終了後、培地を抜き、ＫＧＭで溶解した被験試料を各ｗｅｌｌに２００μＬ添加し、４８時間培養した。培養終了後、培地を抜き、細胞をプレートに固定させ細胞表面に発現したインボルクリンの量をモノクローナル抗ヒトインボルクリン抗体を用いたＥＬＩＳＡ法により測定した。結果を表５に示す。
インボルクリン産生促進作用の計算方法は以下のとおりである。
インボルクリン産生促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時の波長４０５ｎｍにおける吸光度
Ｂ：被験試料無添加時（コントロール）の波長４０５ｎｍにおける吸光度
Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 5 cells / mL, then seeded on a 48-well plate at 200 μL per well, and cultured overnight. After completion of the culture, the medium was removed, 200 μL of a test sample dissolved in KGM was added to each well, and cultured for 48 hours. After completion of the culture, the medium was removed, the cells were fixed on a plate, and the amount of involucrin expressed on the cell surface was measured by ELISA using a monoclonal anti-human involucrin antibody. The results are shown in Table 5.
The calculation method of the involucrin production promoting action is as follows.
Involucrin production promotion rate (%) = A / B × 100
A: Absorbance at a wavelength of 405 nm when a test sample is added B: Absorbance at a wavelength of 405 nm when no test sample is added (control)
表５の結果から、ローヤルゼリー抽出物が、高いインボルクリン産生促進作用を有することが認められた。 From the result of Table 5, it was recognized that the royal jelly extract has a high involucrin production promoting effect.
（実施例６：ＨＭＧ−ＣｏＡ還元酵素産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりＨＭＧ−ＣｏＡ還元酵素（ＨＭＧＣＲ）産生促進作用を試験した。
(Example 6: HMG-CoA reductase production promoting action test)
Using the royal jelly extract of Production Example 3 as a test sample, the HMG-CoA reductase (HMGCR) production promoting action was tested by the following test method.
正常ヒト新生児包皮表皮角化細胞（ｎｏｒｍａｌ ｈｕｍａｎ ｅｐｉｄｅｒｍｉｓ ｋｅｒａｔｉｎｏｃｙｔｅ；ＮＨＥＫ）を８０ｃｍ２フラスコで正常ヒト表皮角化細胞長期培養用増殖培地（ＥｐｉＬｉｆｅ−ＫＧ２）において、３７℃、５％ＣＯ２下で前培養し、トリプシン処理により細胞を集めた。
ＥｐｉＬｉｆｅ−ＫＧ２を用いて３５ｍｍシャーレ（ＦＡＬＣＯＮ）に４０×１０４ｃｅｌｌｓ／２ｍＬ／シャーレずつ播き、３７℃、５％ＣＯ２下で一晩培養した。２４時間後に培養液を捨て、ＥｐｉＬｉｆｅ−ＫＧ２で必要濃度に溶解した被験試料を各シャーレに２ｍＬずつ添加し、３７℃、５％ＣＯ２下で２４時間培養した。培養後、培養液を捨て、ＩＳＯＧＥＮ（ＮＩＰＰＯＮ ＧＥＮＥ；Ｃａｔ．ｎｏ．３１１−０２５０１）にてｔｏｔａｌＲＮＡを抽出し、それぞれのＲＮＡ量を分光光度計にて測定し、２００ｎｇ／μＬになるようにｔｏｔａｌＲＮＡを調製した。
このｔｏｔａｌＲＮＡを鋳型とし、ＨＭＧＣＲ及び内部標準であるＧＡＰＤＨのｍＲＮＡの発現量を測定した。検出はリアルタイムＰＣＲ装置Ｓｍａｒｔ Ｃｙｃｌｅｒ（登録商標）（Ｃｅｐｈｅｉｄ社）を用いて、ＴａＫａＲａ ＳＹＢＲ（登録商標）ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ−ＰＣＲ Ｋｉｔ（Ｐｅｒｆｅｃｔ Ｒｅａｌ Ｔｉｍｅ）（ｃｏｄｅ Ｎｏ．ＲＲ０６３Ａ）によるリアルタイム２ Ｓｔｅｐ ＲＴ−ＰＣＲ反応により行った。ＨＭＧＣＲの発現量は、被験試料無添加、被験試料添加でそれぞれ培養した細胞から調製した総ＲＮＡ標品を基にして、ＧＡＰＤＨの値で補正値を求め、さらに被験試料無添加の補正値を１００とした時の被験試料添加の補正値を算出した。結果を表６に示す。
ＨＭＧＣＲｍＲＮＡ発現促進作用の計算方法は以下の通りである。
ＨＭＧＣＲｍＲＮＡ発現促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料添加時の補正値
Ｂ：被験試料無添加時の補正値
Normal human neonatal foreskin keratinocytes (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermis keratinocytes in an 80 cm 2 flask at 37 ° C. under 5% CO 2. Cells were collected by trypsinization.
Epilife-KG2 was used to seed 40 × 10 4 cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO 2 . After 24 hours, the culture solution was discarded, and 2 mL of the test sample dissolved in EpiLife-KG2 to the required concentration was added to each dish and cultured at 37 ° C. under 5% CO 2 for 24 hours. After culture, the culture solution is discarded, total RNA is extracted with ISOGEN (NIPPON GENE; Cat. No. 311-02501), the amount of each RNA is measured with a spectrophotometer, and total RNA is adjusted to 200 ng / μL. Prepared.
Using this total RNA as a template, the expression levels of HMGCR and GAPDH, which is an internal standard, were measured. Detection is performed using a real-time PCR apparatus Smart Cycler (registered trademark) (Cepheid), TaKaRa SYBR (registered trademark) PrimeScript ™ RT-PCR Kit (Perfect Real Time) (code No. RR063A). It went by. The expression level of HMGCR is determined based on the total RNA preparation prepared from cells cultured without addition of the test sample and with the addition of the test sample, and a correction value is obtained from the value of GAPDH. The correction value for the test sample addition was calculated. The results are shown in Table 6.
The calculation method of the HMGCR mRNA expression promoting action is as follows.
HMGCR mRNA expression promotion rate (%) = A / B × 100
A: Correction value when test sample is added B: Correction value when test sample is not added
表６の結果から、ローヤルゼリー抽出物が、高いＨＭＧ−ＣｏＡ還元酵素（ＨＭＧＣＲ）産生促進作用を有することが認められた。 From the results in Table 6, it was confirmed that the royal jelly extract has a high HMG-CoA reductase (HMGCR) production promoting action.
（参考例７：アデノシン三リン酸産生促進作用試験）
前記製造例３のローヤルゼリー抽出物を被験試料として用い、下記の試験方法によりアデノシン三リン酸（ＡＴＰ）産生促進作用を試験した。
( Reference Example 7: Adenosine triphosphate production promoting action test)
Using the royal jelly extract of Production Example 3 as a test sample, the adenosine triphosphate (ATP) production promoting action was tested by the following test method.
正常ヒト新生児包皮表皮角化細胞（ＮＨＥＫ）を正常ヒト表皮角化細胞長期培養用増殖培地（ＥｐｉＬｉｆｅ−ＫＧ２）を用いて培養した後、トリプシン処理により細胞を回収した。回収した細胞を２．０×１０５ｃｅｌｌｓ／ｍＬの濃度にＥｐｉＬｉｆｅ−ＫＧ２で希釈した後、コラーゲンコートした９６ｗｅｌｌプレートに１ｗｅｌｌ当たり１００μＬずつ播種し、一晩培養した。培養終了後、培地を抜き、ＥｐｉＬｉｆｅ−ＫＧ２で溶解した被験試料を各ｗｅｌｌに１００μＬ添加し、２時間培養した。ＡＴＰ産生促進作用は、ホタルルシフェラーゼ発光法を用いて細胞内のＡＴＰ量を測定した。すなわち、培養終了後、『「細胞の」ＡＴＰ測定試薬』（東洋ビーネット社）を各ｗｅｌｌに１００μＬずつ添加した。反応後、化学発光量を測定した。結果を表７に示す。
ＡＴＰ産生促進作用の計算方法は以下のとおりである。
ＡＴＰ産生促進率（％）＝Ａ／Ｂ×１００
Ａ：被験試料を添加した細胞での化学発光量
Ｂ：被験試料を添加しない細胞での化学発光量
Normal human neonatal foreskin keratinocytes (NHEK) were cultured using a normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with EpiLife-KG2 to a concentration of 2.0 × 10 5 cells / mL and then seeded at 100 μL per well on a collagen-coated 96-well plate and cultured overnight. After completion of the culture, the medium was removed, and 100 μL of a test sample dissolved in EpiLife-KG2 was added to each well, followed by culturing for 2 hours. For ATP production promoting action, the amount of ATP in the cells was measured using a firefly luciferase luminescence method. That is, after completion of the culture, 100 [mu] L of "" Cellular "ATP measurement reagent" (Toyo B-Net) was added to each well. After the reaction, the amount of chemiluminescence was measured. The results are shown in Table 7.
The calculation method of the ATP production promoting action is as follows.
ATP production promotion rate (%) = A / B × 100
A: Chemiluminescence amount in cells to which test sample was added B: Chemiluminescence amount in cells to which test sample was not added
表７の結果から、ローヤルゼリー抽出物が、高いアデノシン三リン酸（ＡＴＰ）産生促進作用を有することが認められた。 From the result of Table 7, it was recognized that the royal jelly extract has a high adenosine triphosphate (ATP) production promoting action.
本発明のＩＶ型コラーゲン産生促進剤、トランスグルタミナーゼ−１産生促進剤、ＳＰＴ産生促進剤、ＡＱＰ３産生促進剤、インボルクリン産生促進剤、ＨＭＧＣＲ産生促進剤、及び、ＡＴＰ産生促進剤は、優れたＩＶ型コラーゲン産生促進作用、トランスグルタミナーゼ−１産生促進作用、ＳＰＴ産生促進作用、ＡＱＰ３産生促進作用、インボルクリン産生促進作用、ＨＭＧＣＲ産生促進作用、及び、ＡＴＰ産生促進作用を有し、かつ安全性にも優れるので、例えば、皮膚外用剤、美容用飲食品中の成分や、研究用の試薬として好適に利用可能である。 The type IV collagen production promoter, transglutaminase-1 production promoter, SPT production promoter, AQP3 production promoter, involucrin production promoter, HMGCR production promoter, and ATP production promoter of the present invention are excellent type IV Collagen production promoting action, transglutaminase-1 production promoting action, SPT production promoting action, AQP3 production promoting action, involucrin production promoting action, HMGCR production promoting action, and ATP production promoting action, and also excellent in safety For example, it can be suitably used as a skin external preparation, a component in cosmetic foods and beverages, or a research reagent.
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| JP2011162476A (en) * | 2010-02-09 | 2011-08-25 | Maruzen Pharmaceut Co Ltd | SERINE PALMITOYLTRANSFERASE mRNA EXPRESSION PROMOTER AND AQUAPORIN 3 mRNA EXPRESSION PROMOTER |
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| JPH10120561A (en) * | 1996-10-18 | 1998-05-12 | Dokutaazu Kosumeteikusu:Kk | Skin preparation for improving sputum for external use |
| JP2001240529A (en) * | 2000-03-01 | 2001-09-04 | Pola Chem Ind Inc | Cosmetics for skin care |
| JP2003128553A (en) * | 2001-08-15 | 2003-05-08 | Rohto Pharmaceut Co Ltd | Ophthalmic composition |
| JP4456796B2 (en) * | 2001-09-27 | 2010-04-28 | 株式会社林原生物化学研究所 | Method for producing collagen production enhancer and use thereof |
| JP2003171258A (en) * | 2001-12-06 | 2003-06-17 | Katakura Chikkarin Co Ltd | Cosmetic composition |
| WO2003068215A1 (en) * | 2002-02-14 | 2003-08-21 | Ajinomoto Co., Inc. | Drugs for mitochondrial diseases |
| JP2005200370A (en) * | 2004-01-16 | 2005-07-28 | Nikko Chemical Co Ltd | Nitrogen monoxide production promotor and its utilization |
| JP4768238B2 (en) * | 2004-07-01 | 2011-09-07 | 丸善製薬株式会社 | Profilagrin mRNA expression promoter |
| US7348034B2 (en) * | 2005-03-07 | 2008-03-25 | Access Business Group International Llc | Plant based formulations for improving skin moisture, texture, and appearance |
| FR2892410B1 (en) * | 2005-10-21 | 2010-10-15 | Fabre Pierre Dermo Cosmetique | NOVEL UNSATURATED FATTY HYDRO ACIDS AND THEIR DERMO COSMETOLOGICAL USE |
| KR20100023910A (en) * | 2007-06-04 | 2010-03-04 | 가부시끼가이샤 하야시바라 세이부쓰 가가꾸 겐꾸조 | Novel fractionation product of royal jelly, method for production of the same, and use of the same |
| JP2009107931A (en) * | 2007-10-26 | 2009-05-21 | Kracie Home Products Kk | Epidermal function improving agent |
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