JP6922064B1 - Hair growth and hair restorer - Google Patents
Hair growth and hair restorer Download PDFInfo
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- JP6922064B1 JP6922064B1 JP2020209107A JP2020209107A JP6922064B1 JP 6922064 B1 JP6922064 B1 JP 6922064B1 JP 2020209107 A JP2020209107 A JP 2020209107A JP 2020209107 A JP2020209107 A JP 2020209107A JP 6922064 B1 JP6922064 B1 JP 6922064B1
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Abstract
【課題】毛乳頭細胞増殖促進剤、発毛促進因子発現亢進剤、脱毛誘導因子発現抑制剤、育毛剤、及び頭髪化粧料を提供する。【解決手段】本発明の毛乳頭細胞増殖促進剤、発毛促進因子発現亢進剤、発毛抑制因子抑制剤、脱毛誘導因子発現抑制剤、育毛剤、及び頭髪化粧料は、いずれもシーバックソーンの果実加工物を有効成分とすることを特徴とする。【選択図】なしPROBLEM TO BE SOLVED: To provide a hair papilla cell growth promoter, a hair growth promoter expression enhancer, a hair loss inducer expression inhibitor, a hair restorer, and a hair cosmetic. SOLUTION: The hair papilla cell growth promoter, hair growth promoter expression enhancer, hair growth inhibitor suppressant, hair loss inducer expression inhibitor, hair restorer, and hair cosmetic of the present invention are all sea buckthorn. It is characterized in that the processed fruit product of the above is used as an active ingredient. [Selection diagram] None
Description
本発明は、毛乳頭細胞増殖促進剤、発毛促進因子発現亢進剤、脱毛誘導因子発現抑制剤、育毛剤、及び頭髪化粧料に関する。 The present invention relates to a hair papillary cell proliferation promoter, a hair growth promoter expression enhancer, a hair loss inducer expression inhibitor, a hair restorer, and a hair cosmetic.
高齢化が進み、またストレスの多い現代社会において、男女を問わず、頭部毛髪（頭髪）の脱毛や薄毛の問題を抱える人が増えてきており、手軽に使用でき、しかも安全な育毛剤のニーズが高まっている。 In today's stressful and aging society, more and more men and women are having problems with hair loss and thinning of their head hair, making it an easy-to-use and safe hair restorer. Needs are increasing.
毛髪は、成長期、退行期及び休止期からなる周期的なヘアサイクル (毛周期）に従って成長と脱落を繰り返している。このヘアサイクルのうち、休止期から成長期にかけての新たな毛包が形成されるステージが発毛に最も重要であると考えられており、この毛包上皮系細胞の増殖分化に重要な役割を果たしているのが、毛乳頭細胞であると考えられている。毛乳頭細胞は、毛根近傍にある外毛根鞘細胞とマトリックス細胞とからなる毛包上皮系細胞の内側にあって、基底膜に包まれている毛根の根幹部分に位置する細胞であり、毛包上皮系細胞に作用してその増殖を促進する等、毛包上皮系細胞の増殖分化及び毛髪の形成に重要な 役割を担っている。 Hair repeats growth and shedding according to a periodic hair cycle (hair cycle) consisting of a growth phase, a catagen phase, and a resting phase. Of this hair cycle, the stage where new hair follicles are formed from the telogen to the growth phase is considered to be the most important for hair growth, and plays an important role in the proliferation and differentiation of these hair follicle epithelial cells. It is believed that telogen cells play a role. Hair follicle cells are cells located inside hair follicle epithelial cells consisting of outer hair follicle sheath cells and matrix cells near the hair root, and are located at the root of the hair follicle surrounded by the basal membrane. It plays an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation, such as acting on epithelial cells to promote their proliferation.
このように、毛乳頭細胞は毛包上皮系細胞の増殖分化及び毛髪の形成において重要な役割を果たしており、毛乳頭細胞の増殖を促進することで、薄毛や脱毛症を予防または改善できると考えられる。従来、毛乳頭細胞増殖促進作用を有する植物抽出物として、例えば、オウギ抽出物、オウレン抽出物、カシュウ抽出物（以上、特許文献１）、クマノギク抽出物（特許文献２）、クロレラ抽出物、柚子果実抽出物、エイジツ果実抽出物、イチョウ葉抽出物（以上、特許文献３）、及びブラックジンジャー抽出物（特許文献４）等が知られている。 Thus, dermal papilla cells play an important role in the proliferation and differentiation of hair follicle epithelial cells and hair formation, and it is considered that thinning hair and alopecia can be prevented or ameliorated by promoting the proliferation of dermal papilla cells. Be done. Conventionally, as plant extracts having a hair papilla cell proliferation promoting action, for example, Ougi extract, Ouren extract, Kashu extract (above, Patent Document 1), Kumanogiku extract (Patent Document 2), Chlorella extract, Yuzu Fruit extracts, agetsu fruit extracts, ginkgo biloba extracts (Patent Document 3), black ginger extracts (Patent Document 4) and the like are known.
ヘアサイクルのうち、休止期から成長期に移行する際、毛乳頭細胞はコンドロイチン硫酸プロテオグリカンの一種であるバーシカンを特異的に発現することが知られている（非特許文献１）。従来、このバーシカンに着目した育毛料の研究もされており、例えば、インドトチノキエキスは、バーシカンの発現を亢進する作用を有しており、その作用に基づいて発毛・育毛効果を発揮することが知られている（特許文献５）。 It is known that dermal papilla cells specifically express versican, which is a kind of chondroitin sulfate proteoglycan, during the transition from the telogen phase to the anagen phase in the hair cycle (Non-Patent Document 1). Conventionally, research on hair growth agents focusing on this versican has also been conducted. For example, Indian horse chestnut extract has an action of enhancing the expression of versican, and exerts a hair growth / hair growth effect based on the action. Is known (Patent Document 5).
線維芽細胞増殖因子(Fibroblast growth factors、FGF)は、線維芽細胞に対して細胞増殖活性を有するだけでなく、様々な細胞に対して細胞増殖・分化活性を発揮する形態形成因子、組織障害に作用する組織修復因子、生体の恒常性を維持する代謝調節因子等として重要な役割を果たす多機能性分泌因子である。このようなFGFとしては、例えば、線維芽細胞増殖因子−７（以下、「FGF-7」とも称する）が挙げられる。男性型脱毛症患者の毛乳頭細胞ではFGF-7の発現が低下していることが知られている（非特許文献１参照)。そのため、FGF-7の発現を亢進させることで脱毛症や薄毛を予防または改善できると考えられる。従来、FGF-7発現亢進作用を有する天然由来のものとして、例えばローヤルゼリー等が知られている（特許文献６)。 Fibroblast growth factors (FGF) are morphogenetic factors and tissue disorders that not only have cell proliferation activity against fibroblasts but also exert cell proliferation and differentiation activity against various cells. It is a multifunctional secretory factor that plays an important role as a tissue repair factor that acts, a metabolic regulator that maintains the homeostasis of the living body, and the like. Examples of such FGF include fibroblast growth factor-7 (hereinafter, also referred to as "FGF-7"). It is known that the expression of FGF-7 is decreased in the dermal papilla cells of androgenetic alopecia patients (see Non-Patent Document 1). Therefore, it is considered that alopecia and thinning hair can be prevented or ameliorated by increasing the expression of FGF-7. Conventionally, for example, royal jelly and the like are known as naturally derived substances having an action of enhancing FGF-7 expression (Patent Document 6).
骨形成タンパク（Bone Morphogenetic protein、BMP)は、骨、軟骨、腱、及び骨中に存在するその他の組織を形成させるタンパク質である。このタンパク質には特有の誘導活性があり、骨中に存在することから、骨修復過程の重要な制御因子であり、骨組織の正常な維持に関与していると示唆されている。このタンパク質のサブファミリーの一つである骨形成タンパク−４（以下「BMP-4」とも称する）は、ヘアサイクルの休止期から成長期に移行するのを阻害する作用を有することが知られている。このため、BMP-4の発現を抑制することで、ヘアサイクルの成長期を促し、脱毛症や薄毛を予防または改善できると考えられる。 Bone morphogenetic protein (BMP) is a protein that forms bone, cartilage, tendons, and other tissues present in bone. This protein has a unique inducing activity and is present in bone, suggesting that it is an important regulator of the bone repair process and is involved in the normal maintenance of bone tissue. Bone morphogenetic protein-4 (hereinafter also referred to as "BMP-4"), which is one of the subfamilies of this protein, is known to have an action of inhibiting the transition from the resting phase of the hair cycle to the anagen phase. There is. Therefore, by suppressing the expression of BMP-4, it is considered that the growth phase of the hair cycle can be promoted and alopecia and thinning hair can be prevented or ameliorated.
また、トランスフォーミング増殖因子ベータ（Transforming growth factor-β、TGF-β）は、毛乳頭細胞や毛母細胞の活動を抑制する因子として知られている。その発現産生が高まると、毛母細胞の分裂・増殖が抑制されて、成長期の毛髪が退行期に移行する。つまり、TGF-βの影響で、髪は十分に成長することなく退行期・休止期に入り、短く細いまま抜け落ちることが繰り返され（退行期への早期移行）、これが脱毛症や薄毛の原因となっていることが知られている。特に、TGF-β2は、細胞死をもたらす自殺酵素カスパーゼを活性化し、そのカスパーゼ（カスパーゼ３、カスパーゼ９）が毛母細胞を死（アポトーシス）に導き、これが脱毛現象をもたらすことが知られている。このため、TGF-β2を始めとするTGF-βの発現を抑制することで、ヘアサイクルの退行期への早期移行を阻止し、脱毛症や薄毛を予防または改善できると考えられる。なお、ユリアニア科植物の抽出物であるクアチャララーテエキスにカスパーゼ３及び９の活性抑制作用があることが知られている（非特許文献２参照）。 In addition, transforming growth factor-β (TGF-β) is known as a factor that suppresses the activity of hair papilla cells and hair matrix cells. When the expression production is increased, the division and proliferation of hair matrix cells are suppressed, and the growing hair shifts to the catagen phase. In other words, due to the influence of TGF-β, hair enters the catagen / telogen phase without sufficient growth, and repeatedly falls out short and thin (early transition to the catagen phase), which is the cause of alopecia and thinning hair. It is known that it has become. In particular, it is known that TGF-β2 activates the suicide enzyme caspase that causes cell death, and the caspase (caspase 3, caspase 9) leads to death (apoptosis) of hair matrix cells, which causes a hair loss phenomenon. .. Therefore, by suppressing the expression of TGF-β such as TGF-β2, it is considered that the early transition of the hair cycle to the catagen phase can be prevented and alopecia and thinning hair can be prevented or ameliorated. It is known that quacharalate extract, which is an extract of Anacardioideae, has an activity-suppressing effect on caspases 3 and 9 (see Non-Patent Document 2).
本発明は、毛乳頭細胞増殖促進剤、発毛促進因子発現亢進剤、脱毛誘導因子発現抑制剤、育毛剤、及び頭髪化粧料を提供することを目的とする。好ましくは天然に由来し、生体に安全な物質を有効成分とする毛乳頭細胞増殖促進剤、発毛促進因子発現亢進剤、脱毛誘導因子発現抑制剤、育毛剤、及び頭髪化粧料を提供することを目的とする。 An object of the present invention is to provide a hair papilla cell proliferation promoter, a hair growth promoter expression enhancer, a hair loss inducer expression inhibitor, a hair restorer, and a hair cosmetic. Provided are a hair papillary cell proliferation promoter, a hair growth promoter expression enhancer, a hair loss inducer expression inhibitor, a hair restorer, and a hair cosmetic containing a substance naturally derived and safe for the living body as an active ingredient. With the goal.
本発明者らは、上記課題を解決すべく鋭意検討を重ねていたところ、シーバックソーンの果実に、毛乳頭細胞増殖促進作用、バーシカン及び線維芽細胞増殖因子−７（FGF7）といった発毛促進因子の発現を亢進する作用、骨形成タンパク−４（BMP-4）及びトランスフォーミング増殖因子ベータ２（TGF-β2）といった脱毛誘導因子の発現を抑制する作用があることを見出し、当該果実加工物が育毛剤の有効成分として、また頭髪化粧料の育毛成分として有用であることを確認した。 As a result of diligent studies to solve the above problems, the present inventors have found that the fruits of Seaback Thorn have a hair-papillary cell growth-promoting effect and hair growth-promoting effects such as versican and fibroblast growth factor-7 (FGF7). We found that it has the effect of enhancing the expression of factors and the effect of suppressing the expression of hair loss-inducing factors such as bone-forming protein-4 (BMP-4) and transforming growth factor beta 2 (TGF-β2). Was confirmed to be useful as an active ingredient in hair growth agents and as a hair growth ingredient in hair cosmetics.
本発明はかかる知見に基づいて、さらに検討を重ねて本発明を完成するに至った。
本発明は下記の実施形態を有するものである。
項１．シーバックソーンの果実加工物を有効成分とする毛乳頭細胞増殖促進剤。
項２．シーバックソーンの果実加工物を有効成分とする、バーシカン及びFGF7からなる群から選択される少なくとも１種の発毛促進因子の発現亢進剤。
項３．シーバックソーンの果実加工物を有効成分とする、BMP-4及びTGF-β2からなる群から選択される少なくとも１種の脱毛誘導因子の発現抑制剤。
項４．シーバックソーンの果実加工物を含有する育毛剤。
項５．シーバックソーンの果実加工物を含有する頭髪化粧料。
Based on such findings, the present invention has been further studied to complete the present invention.
The present invention has the following embodiments.
Item 1. A hair papilla cell proliferation promoter containing processed sea buckthorn fruit as an active ingredient.
Item 2. An expression enhancer for at least one hair growth-promoting factor selected from the group consisting of versican and FGF7, which comprises a processed fruit product of sea buckthorn as an active ingredient.
Item 3. An agent for suppressing the expression of at least one hair loss-inducing factor selected from the group consisting of BMP-4 and TGF-β2, which comprises a processed sea buckthorn fruit as an active ingredient.
Item 4. A hair restorer containing processed sea buckthorn fruit.
Item 5. Hair cosmetics containing processed sea buckthorn fruit.
本発明によれば、シーバックソーンの果実加工物を含有した、毛乳頭細胞増殖促進剤、バーシカン及びFGF7からなる群から選択される少なくとも１種の発毛促進因子の発現亢進剤、BMP-4及びTGF-β2からなる群から選択される少なくとも１種の脱毛誘導因子の発現抑制剤、育毛剤、並びに頭髪化粧料を提供することができる。 According to the present invention, BMP-4, an agent containing a processed sea buckthorn fruit, at least one hair growth-promoting factor selected from the group consisting of a hair papillary cell proliferation-promoting agent, versican and FGF7. And an agent for suppressing the expression of at least one hair loss inducer selected from the group consisting of TGF-β2, a hair restorer, and a hair cosmetic can be provided.
本発明の毛乳頭細胞増殖促進剤は、有効成分であるシーバックソーンの果実加工が有する毛乳頭細胞増殖促進作用により、毛乳頭細胞の増殖を促進することで頭髪の太毛化を促し、薄毛（特に男性の薄毛）を予防又は改善することができる。 The hair papillary cell proliferation promoting agent of the present invention promotes the proliferation of hair papillary cells by the hair papillary cell proliferation promoting action possessed by the fruit processing of Seaback Thorn, which is an active ingredient, thereby promoting thickening of the hair and thinning hair. (Especially men's thinning hair) can be prevented or ameliorated.
本発明の発毛促進因子発現亢進剤は、有効成分であるシーバックソーンの果実加工が有するバーシカン発現亢進作用及び／又はFGF7発現亢進作用により、当該発毛促進因子の発現を亢進することで、薄毛（特に女性の薄毛）や脱毛症等を予防又は改善することができる。 The hair growth-promoting factor expression-enhancing agent of the present invention enhances the expression of the hair-growth-promoting factor by enhancing the expression of versican and / or FGF7, which is possessed by the fruit processing of sea buckthorn, which is an active ingredient. It is possible to prevent or improve thinning hair (particularly female thinning hair) and alopecia.
本発明の脱毛誘導因子発現抑制剤は、有効成分であるシーバックソーンの果実加工が有するBMP-4発現抑制作用及び／又は及びTGF-β2発現抑制作用により、当該脱毛誘導因子の発現を抑制することで、ヘアサイクルを休止期から成長期に促したり、退行期への早期移行を食い止めることで、薄毛や壮年性脱毛症等を予防又は改善することができる。 The hair loss-inducing factor expression inhibitor of the present invention suppresses the expression of the hair loss-inducing factor by the BMP-4 expression-suppressing action and / or the TGF-β2 expression-suppressing action of the fruit processing of Seaback Thorn, which is an active ingredient. Therefore, by promoting the hair cycle from the resting period to the growing period and stopping the early transition to the degeneration period, thinning hair, alopecia of the middle age, and the like can be prevented or ameliorated.
本発明の育毛剤は、有効成分であるシーバックソーンの果実加工が有する毛乳頭細胞増殖促進作用、バーシカン及びFGF7からなる群から選択される少なくとも１種の発毛促進因子の発現亢進作用、BMP-4及びTGF-β2からなる群から選択される少なくとも１種の脱毛誘導因子の発現抑制作用により、育毛や養毛を促進し、薄毛や脱毛症を予防又は改善することができる。 The hair restorer of the present invention has a hair papillary cell proliferation promoting action possessed by fruit processing of sea buckthorn, which is an active ingredient, an action promoting the expression of at least one hair growth promoting factor selected from the group consisting of versican and FGF7, and BMP. By the expression-suppressing action of at least one hair loss-inducing factor selected from the group consisting of -4 and TGF-β2, hair growth and hair growth can be promoted, and thinning hair and alopecia can be prevented or ameliorated.
本発明の頭髪化粧料は、育毛成分として含有するシーバックソーンの果実加工が有する毛乳頭細胞増殖促進作用、バーシカン及びFGF7からなる群から選択される少なくとも１種の発毛促進因子の発現亢進作用、BMP-4及びTGF-β2からなる群から選択される少なくとも１種の脱毛誘導因子の発現抑制作用により、育毛や養毛を促進し、薄毛や脱毛症を予防又は改善することができる。 The hair cosmetic of the present invention has a hair papillary cell proliferation promoting action possessed by the fruit processing of Seaback Thorn contained as a hair growth component, and an action promoting the expression of at least one hair growth promoting factor selected from the group consisting of versican and FGF7. , BMP-4 and TGF-β2 can promote hair growth and hair growth, and prevent or ameliorate thinning hair and alopecia by the action of suppressing the expression of at least one hair loss-inducing factor selected from the group.
（１）シーバックソーンの果実加工物
シーバックソーン（Sea buckthorn）は、サジー（沙棘）又はシーベリー（Sea berry）とも称されるグミ科の植物（学名：Hippophae rhamnoides）である。その果実部は、従来から、栄養価が高い果物として食されている。また果実やその中に含まれている種子から採られるオイル（シーバックソーンオイル：果実油、種子油、コンプリート油）には、抗炎症作用、抗菌作用、日焼け止め作用、抗酸化作用、血圧降下作用、血中コレステロール低下作用、血栓除去作用、免疫刺激作用、エモリエント作用、瘢痕形成作用などの効果効能が知られている。さらに果皮にも、フラボノイド、カロテノイド、植物ステロール及びアミノ酸等の活性物質が多量に含まれており、その栄養価の高さが注目されている。
(1) Processed fruit of sea buckthorn Sea buckthorn is a plant of the family Elaeagnaceae (scientific name: Hippophae rhamnoides), which is also called sea buckthorn or sea berry. The fruit part has traditionally been eaten as a highly nutritious fruit. In addition, the oils (seaback thorn oil: fruit oil, seed oil, complete oil) obtained from the fruits and the seeds contained therein have anti-inflammatory, antibacterial, sunscreen, antioxidant, and blood pressure lowering effects. It is known to have effects such as action, blood cholesterol lowering action, blood clot removing action, immunostimulating action, emollient action, and scar forming action. Further, the pericarp also contains a large amount of active substances such as flavonoids, carotenoids, plant sterols and amino acids, and its high nutritional value is attracting attention.
本発明が対象とするシーバックソーンの果実（以下、単に「SBT果実」とも称する）は、可食部である果肉を含むものであればよく、その限りにおいて、果皮を含むものであっても、また果実内に含まれる種子を含むものであってもよい。好ましくは果肉と果皮を含むものである。
SBT果実の加工物は、前記SBT果実の加工物であればよく、例えばSBT果実から採取したエキスやオイルを挙げることができる。SBT果実のエキスには、SBT果実の絞汁及びその濃縮又は乾燥物；並びにSBT果実の溶媒抽出液及びその濃縮又は乾燥物が含まれる。またSBT果実のオイルには、SBT果実を低温圧搾して採取したオイル（果実油、コンプリート油）が含まれる。好ましくはSBT果実のエキスであり、当該エキスにはより好適なものとしてSBT果実の溶媒抽出エキスが含まれる。
The sea buckthorn fruit (hereinafter, also simply referred to as “SBT fruit”), which is the subject of the present invention, may be any fruit containing flesh, which is an edible portion, and to the extent that it contains pericarp. , It may also contain seeds contained in the fruit. It preferably contains flesh and pericarp.
The processed product of the SBT fruit may be any processed product of the SBT fruit, and examples thereof include extracts and oils collected from the SBT fruit. Extracts of SBT fruit include squeezed juice of SBT fruit and its concentrated or dried product; and solvent extract of SBT fruit and its concentrated or dried product. The oil of SBT fruit includes oil (fruit oil, complete oil) collected by cold pressing SBT fruit. It is preferably an extract of SBT fruit, and the extract includes a solvent-extracted extract of SBT fruit as more preferable.
SBT溶媒抽出エキスは、SBT果実を乾燥した後、そのまま又は粗砕若しくは粉砕し、溶媒を用いて抽出することにより得ることができる。乾燥は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。また、予めへキサ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、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 SBT solvent extract can be obtained by drying the SBT fruit and then extracting it as it is or by coarsely crushing or crushing it and extracting it with a solvent. Drying may be performed in the sun or using a commonly used dryer. Further, it may be used as an extraction raw material after pretreatment such as degreasing with a non-polar solvent such as hexane in advance. By performing pretreatment such as degreasing, extraction treatment with a polar solvent can be efficiently performed.
抽出溶媒は、制限されないものの、極性溶媒が好ましく、例えば、水、親水性有機溶媒等が挙げられる。これらは、単独又は２種以上を組み合わせて、室温又は溶媒の沸点以下の温度で使用することが好ましい。 The extraction solvent is not limited, but a polar solvent is preferable, and examples thereof include water and a hydrophilic organic solvent. These are preferably used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent.
抽出溶媒として使用し得る水は、可食性の水であればよく、例えば純水、水道水、井戸水、鉱泉水、鉱水、温泉 水、湧水、淡水等が挙げられ、またこれらの水に各種処理（例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧調整、緩衝化等）を施したものも含まれる。抽出溶媒として使用し得る親水性有機溶媒としては、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の低級脂肪族アルコール；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の多価アルコール等が挙げられる。好ましくはエタノール等の低級脂肪族アルコールである。２種以上の極性溶媒の混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には、水１０質量部に対して低級脂肪族アルコール１〜９０質量部、特に５〜２０質量部を混合することが好ましく、水と低級脂肪族ケトンとの混合液を使用する場合には、水 １０質量部に 対して低級脂肪族ケトン１〜４０質量部を混合することが好ましく、水と多価アルコールとの混合液を使用する場合には、水１０質量部に対して多価アルコール１０〜９０質量部を混合することが好ましい。好ましくは水とエタノール等の低級脂肪族アルコールとの混合液である。 The water that can be used as the extraction solvent may be edible water, and examples thereof include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, and fresh water. Treatments (eg, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc.) are also included. Hydrophilic organic solvents that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1, 3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin. A lower aliphatic alcohol such as ethanol is preferable. When a mixed solution of two or more polar solvents is used as the extraction solvent, the mixing ratio can be appropriately adjusted. For example, when a mixed solution of water and a lower fatty alcohol is used, it is preferable to mix 1 to 90 parts by mass, particularly 5 to 20 parts by mass of the lower fatty alcohol with respect to 10 parts by mass of water, and water. When using a mixed solution of water and a lower aliphatic ketone, it is preferable to mix 1 to 40 parts by mass of the lower aliphatic ketone with respect to 10 parts by mass of water, and a mixed solution of water and a polyhydric alcohol is used. In this case, it is preferable to mix 10 to 90 parts by mass of the polyhydric alcohol with 10 parts by mass of water. It is preferably a mixed solution of water and a lower aliphatic alcohol such as ethanol.
抽出処理は、本発明の効果を発揮するSBT果実抽出エキスが得られる限り、特に限定はされず、常法に従って行うことができる。例えば、抽出原料の５〜１５倍量 (質量比)の抽出溶媒に、抽出原料を浸漬し、常温又は還流加熱下で可溶性成分を抽出させた後、濾過して抽出残渣を除去することにより抽出液を得ることができる。得られた抽出液から溶媒を留去するとペースト状の濃縮物が得られ、この濃縮物をさらに乾燥すると乾燥物が得られる。なお、必要に応じて、斯くして得られるSBT果実抽出液、当該抽出液の濃縮又は乾燥物に対してさらに精製処理を施すこともできる。 The extraction treatment is not particularly limited as long as an SBT fruit extract that exhibits the effects of the present invention can be obtained, and can be carried out according to a conventional method. For example, the extraction material is immersed in an extraction solvent 5 to 15 times the amount (mass ratio) of the extraction material, the soluble component is extracted at room temperature or under reflux heating, and then filtered to remove the extraction residue. The liquid can be obtained. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product. If necessary, the SBT fruit extract thus obtained, the concentrated or dried product of the extract can be further purified.
斯くして調製されるSBT果実加工物は、毛乳頭細胞増殖促進作用を有することから、毛乳頭細胞増殖促進剤の有効成分として使用することができる。またSBT果実加工物は、バーシカン発現亢進作用及び／又はFGF7発現亢進作用を有することから、バーシカンやFGF7といった発毛促進因子の発現亢進剤の有効成分として使用することができる。またSBT果実加工物は、BMP-4発現抑制作用及び／又は及びTGF-β2発現抑制作用を有することから、BMP-4やTGF-β2といった脱毛誘導因子の発現抑制剤の有効成分として使用することができる。さらにSBT果実加工物は、前述する各種の作用を有することから、育毛剤の有効成分として、また頭髪化粧料の育毛成分として使用することができる。 Since the processed SBT fruit product thus prepared has a hair papilla cell proliferation promoting action, it can be used as an active ingredient of a hair papilla cell proliferation promoting agent. Further, since the processed SBT fruit product has a versican expression-enhancing effect and / or an FGF7 expression-enhancing effect, it can be used as an active ingredient of an expression-enhancing agent for hair growth-promoting factors such as versican and FGF7. In addition, since the processed SBT fruit has a BMP-4 expression-suppressing effect and / or a TGF-β2 expression-suppressing effect, it should be used as an active ingredient of an expression-suppressing agent for hair loss-inducing factors such as BMP-4 and TGF-β2. Can be done. Further, since the processed SBT fruit product has the above-mentioned various actions, it can be used as an active ingredient of a hair restorer and as a hair restorer component of a hair cosmetic.
（２）毛乳頭細胞増殖促進剤、発毛促進因子発現促進剤、脱毛誘導因子発現抑制剤、育毛剤
本発明の毛乳頭細胞増殖促進剤、発毛促進因子発現促進剤、脱毛誘導因子発現抑制剤、及び育毛剤は、前述するSBT果実加工物を有効成分として含有することを特徴とする。
これらの製剤は、SBT果実加工物のみからなるものであってもよいし、SBT果実加工物を他の成分を配合して所望の形態に製剤化したものであってもよい。製剤化は、制限されないものの、具体的には、SBT果実加工物に加えて、生体に安全な、例えば薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、液状、乳液状、クリーム状、フォーム状、ゼリー状等の任意の剤形に調製することができる。
(2) Hair papillary cell proliferation promoter, hair growth promoter expression promoter, hair loss inducer expression inhibitor, hair restorer The hair papilla cell proliferation promoter of the present invention, hair growth promoter expression promoter, hair loss inducer expression inhibitor The agent and the hair restorer are characterized by containing the above-mentioned processed SBT fruit product as an active ingredient.
These preparations may consist only of the processed SBT fruit product, or may be a product prepared by blending the processed SBT fruit product with other components into a desired form. The formulation is not limited, but specifically, in powder form according to a conventional method, using a biosafe, for example, a pharmaceutically acceptable carrier or any other auxiliary agent, in addition to the processed SBT fruit product. It can be prepared in any dosage form such as granule, liquid, milky liquid, cream, foam, jelly and the like.
本発明の毛乳頭細胞増殖促進剤は、SBT果実加工物が有する毛乳頭細胞増殖促進作用を通じて、毛乳頭細胞の増殖を活性化し、より大きな毛球部の形成を促して、毛髪の形成 （特に太毛化）を促進することができる。本発明の毛乳頭細胞増殖促進剤のSBT果実加工物の配合量は、毛乳頭細胞増殖促進作用を発揮する有効量であればよく、１００質量％を限度として適宜設定することができる。なお、SBT果実加工物やそれを含有する毛乳頭細胞増殖促進剤が有する毛乳頭細胞増殖促進作用は、後述する実験例１に記載するように、ヒト毛乳頭細胞を用いた細胞培養法により評価測定することができる。本発明の毛乳頭細胞増殖促進剤によれば、ヘアサイクルにおいて、成長期から退行期及び休止期へと移行するのを抑止して、成長期を延長させることが期待される。このため、本発明の毛乳頭細胞増殖促進剤によれば、毛髪の伸長や太毛化（育毛）を促すことで薄毛（特に男性の薄毛）や脱毛症を予防したり改善することができる。本発明の毛乳頭細胞増殖促進剤が好適に対象とする薄毛や脱毛症は、制限されないが、例えば、男性型脱毛症、女性型脱毛症等が挙げられる。 The hair papillary cell proliferation promoting agent of the present invention activates the proliferation of hair papillary cells through the hair papillary cell proliferation promoting action of the processed SBT fruit product, promotes the formation of larger hair bulbs, and forms hair (particularly). (Thickening) can be promoted. The blending amount of the processed SBT fruit product of the hair papilla cell proliferation promoting agent of the present invention may be an effective amount that exerts the hair papilla cell proliferation promoting action, and can be appropriately set up to 100% by mass. The hair papillary cell proliferation promoting action of the processed SBT fruit product and the hair papillary cell proliferation promoting agent containing the same was evaluated by a cell culture method using human hair papilla cells as described in Experimental Example 1 described later. Can be measured. According to the hair papillary cell proliferation promoter of the present invention, it is expected to suppress the transition from the anagen phase to the catagen phase and the telogen phase in the hair cycle to prolong the anagen phase. Therefore, according to the hair papillary cell proliferation promoter of the present invention, thinning hair (particularly male thinning hair) and alopecia can be prevented or improved by promoting hair elongation and thickening (hair growth). The thinning hair and alopecia that the hair papilla cell proliferation promoter of the present invention preferably targets are not limited, and examples thereof include androgenetic alopecia and female pattern baldness.
本発明の発毛促進因子発現促進剤は、SBT果実加工物が有するバーシカン発現亢進作用を通じてヘアサイクルの休止期から成長期への移行を促進したり、及び／又は、SBT果実加工物が有するFGF7発現亢進作用を通じて、毛乳頭細胞におけるFGF7の発現や産生を促進し、薄毛や脱毛症等を予防したり改善することができる。本発明の発毛促進因子発現促進剤のSBT果実加工物の配合量は、バーシカン発現亢進作用進作用及び／又はFGF7発現亢進作用を発揮する有効量であればよく、１００質量％を限度として適宜設定することができる。なお、SBT果実加工物やそれを含有する発毛促進因子発現促進剤が有するバーシカン発現亢進作用やFGF7の発現亢進作用は、後述する実験例２に記載するように、ヒト毛乳頭細胞におけるバーシカンやFGF7の発現量を測定することで評価することができる。本発明の発毛促進因子発現促進剤が好適に対象とする薄毛や脱毛症は、制限されないが、例えば、男性型脱毛症、女性型脱毛症等が挙げられる。 The hair growth-promoting factor expression-promoting agent of the present invention promotes the transition of the hair cycle from the telogen phase to the anagen phase through the versican expression-enhancing action of the SBT fruit processed product, and / or the FGF7 of the SBT fruit processed product. Through the expression-enhancing action, the expression and production of FGF7 in hair papilla cells can be promoted, and thinning hair, alopecia and the like can be prevented or ameliorated. The blending amount of the processed SBT fruit product of the hair growth-promoting factor expression-promoting agent of the present invention may be an effective amount that exerts the versican expression-promoting action and / or the FGF7 expression-promoting action, and is appropriately limited to 100% by mass. Can be set. As described in Experimental Example 2 described later, the versican expression-enhancing effect and the FGF7 expression-enhancing effect of the processed SBT fruit product and the hair growth-promoting factor expression-promoting agent containing the SBT fruit processed product are described in the versican in human hair papilla cells. It can be evaluated by measuring the expression level of FGF7. The thinning hair and alopecia that the hair growth-promoting factor expression-promoting agent of the present invention preferably targets are not limited, and examples thereof include androgenetic alopecia and female pattern baldness.
本発明の脱毛誘導因子発現抑制剤は、SBT果実加工物が有するBMP-4発現抑制作用を通じてヘアサイクルの休止期から成長期への移行を促進したり、及び／又は、SBT果実加工物が有するTGF-β2発現抑制作用を通じてヘアサイクルの退行期への早期移行を阻止することで、薄毛や脱毛症等を予防したり改善することができる。本発明の脱毛誘導因子発現抑制剤のSBT果実加工物の配合量は、BMP-4発現抑制作用及び／又はTGF-β2発現抑制作用を発揮する有効量であればよく、１００質量％を限度として適宜設定することができる。なお、SBT果実加工物やそれを含有する脱毛誘導因子発現抑制剤が有するBMP-4発現抑制作用や、TGF-β2発現抑制作用は、後述する実験例３に記載するように、ヒト毛乳頭細胞におけるBMP-4やTGF-β2の発現量を測定することで評価することができる本発明の脱毛誘導因子発現抑制剤が好適に対象とする薄毛や脱毛症は、制限されないが、例えば、男性型脱毛症、女性型脱毛症等が挙げられる。 The hair loss-inducing factor expression inhibitor of the present invention promotes the transition of the hair cycle from the telogen phase to the anagen phase through the BMP-4 expression inhibitory action of the processed SBT fruit product, and / or has the processed SBT fruit product. By preventing the early transition of the hair cycle to the telogen phase through the TGF-β2 expression inhibitory action, thinning hair, alopecia, etc. can be prevented or ameliorated. The blending amount of the processed SBT fruit product of the hair loss-inducing factor expression inhibitor of the present invention may be an effective amount that exerts the BMP-4 expression inhibitory effect and / or the TGF-β2 expression inhibitory effect, and is limited to 100% by mass. It can be set as appropriate. The BMP-4 expression-suppressing action and the TGF-β2 expression-suppressing action of the processed SBT fruit product and the hair loss-inducing factor expression-suppressing agent containing the same are described in human hair papilla cells as described in Experimental Example 3 described later. The thinning hair and alopecia that are preferably targeted by the hair loss-inducing factor expression inhibitor of the present invention, which can be evaluated by measuring the expression level of BMP-4 and TGF-β2 in, are not limited, but for example, male pattern baldness. Alopecia, female pattern baldness, etc. can be mentioned.
本発明の育毛剤は、SBT果実加工物が有する毛乳頭細胞増殖促進作用を通じて、育毛や養毛を促進し、薄毛や脱毛症を予防又は改善することができる。また、本発明の育毛剤 は、SBT果実加工物が有するバーシカン発現亢進作用、FGF7発現亢進作用、BMP-4発現抑制作用、及び／又は、TGF-β2発現抑制作用を通じて、ヘアサイクルを制御し、薄毛や脱毛症を予防又は改善することができる。本発明の育毛剤のSBT果実加工物の配合量は、前記の作用を１または２以上発揮する有効量であればよく、１００質量％を限度として適宜設定することができる。 The hair restorer of the present invention can promote hair growth and hair growth through the hair papillary cell proliferation promoting action of the processed SBT fruit product, and can prevent or ameliorate thinning hair and alopecia. In addition, the hair restorer of the present invention controls the hair cycle through the versican expression-enhancing effect, FGF7 expression-enhancing effect, BMP-4 expression-suppressing effect, and / or TGF-β2 expression-suppressing effect of the processed SBT fruit product. It can prevent or improve thinning hair and alopecia. The blending amount of the processed SBT fruit product of the hair restorer of the present invention may be an effective amount that exerts the above-mentioned action by 1 or 2 or more, and can be appropriately set up to 100% by mass.
（３）頭髪化粧料
本発明の頭髪化粧料は、SBT果実加工物を育毛成分として含有するものである。
SBT果実加工物は、前述するように、毛乳頭細胞増殖促進作用、バーシカン発現亢進作用、FGF7発現亢進作用、BMP-4発現抑制作用、及び／又は、TGF-β2発現抑制作用を有しており、育毛成分として、頭髪化粧料に配合するのに好適である。
本発明の頭髪化粧料は、本発明の効果を奏することを限度として、その製造に際して、頭髪化粧料の原料としてSBT果実加工物をそのまま配合してもよいし、SBT果実加工物を製剤化して毛乳頭細胞増殖促進剤、バーシカン発現亢進剤、FGF7発現亢進剤、BMP-4発現抑制剤、TGF-β2発現抑制剤、及び／又は育毛剤として調製したものを配合してもよい。頭髪化粧料におけるSBT果実加工物やこうした各種製剤の配合割合は、前記の作用を１または２以上発揮する有効量であればよく、その使用目的、性別、年齢等を考慮して適宜調整することができる。好適な配合割合は、SBT果実加工物（好ましくはSBT果実抽出物）の量として約 ０.００１〜１０質量％の範囲から適宜設定することができる。
(3) Hair Cosmetics The hair cosmetics of the present invention contain a processed SBT fruit product as a hair growth component.
As described above, the processed SBT fruit product has a hair papilla cell proliferation promoting effect, a versican expression enhancing effect, an FGF7 expression enhancing effect, a BMP-4 expression suppressing effect, and / or a TGF-β2 expression suppressing effect. As a hair growth ingredient, it is suitable for blending in hair cosmetics.
The hair cosmetic of the present invention may be blended with the processed SBT fruit as it is as a raw material of the hair cosmetic in the production thereof, as long as the effect of the present invention is exhibited, or the processed SBT fruit is formulated. A hair papilla cell proliferation promoter, a versican expression enhancer, an FGF7 expression enhancer, a BMP-4 expression inhibitor, a TGF-β2 expression inhibitor, and / or a hair growth agent may be blended. The blending ratio of the processed SBT fruit product and these various preparations in the hair cosmetics may be an effective amount that exerts the above-mentioned action of 1 or 2 or more, and should be appropriately adjusted in consideration of the purpose of use, gender, age, etc. Can be done. The suitable blending ratio can be appropriately set from the range of about 0.001 to 10% by mass as the amount of the processed SBT fruit product (preferably SBT fruit extract).
本発明の頭髪化粧料の形態は特に限定されるものではなく、例えば、その具体例として は、ヒトの頭髪化粧料として通常使用される形態のもの、例えばヘアローション、ヘアトニック、ヘアクリーム、ヘアリキッド、シャンプー、リンス、ポマード等が挙げられる。 The form of the hair cosmetic of the present invention is not particularly limited, and for example, a specific example thereof is a form commonly used as a human hair cosmetic, such as a hair lotion, a hair tonic, a hair cream, and a hair. Examples include liquids, shampoos, conditioners, and pomades.
本発明の頭髪化粧料は、SBT果実加工物とともに、用途に応じた任意の生理活性物質や助剤、例えば収斂剤、殺菌剤、抗菌剤、紫外線吸収剤、保湿剤、細胞賦活剤、消炎剤、抗アレルギー剤、抗酸化剤、活性酸素消去剤等を併用することができる。
以上、本明細書において、「含む」及び「含有する」の用語には、「からなる」及び「から実質的になる」という意味が含まれる。
The hair cosmetic of the present invention, together with the processed SBT fruit product, is an arbitrary physiologically active substance or auxiliary agent according to the application, for example, an astringent agent, a bactericidal agent, an antibacterial agent, an ultraviolet absorber, a moisturizer, a cell activator, and an anti-inflammatory agent. , Anti-allergic agents, antioxidants, active oxygen scavengers and the like can be used in combination.
As described above, in the present specification, the terms "including" and "containing" include the meanings of "consisting of" and "consisting of substantially".
以下、本発明の構成及び効果について、その理解を助けるために、実験例を用いて本発明を説明する。但し、本発明はこれらの実験例によって何ら制限を受けるものではない。以下の実験は、特に言及しない限り、室温（２５±５℃）、及び大気圧条件下で実施した。なお、特に言及しない限り、以下に記載する「％」は「質量％」、「部」は「質量部」を意味する。 Hereinafter, the present invention will be described with reference to experimental examples in order to help understanding the structure and effects of the present invention. However, the present invention is not limited by these experimental examples. The following experiments were performed under room temperature (25 ± 5 ° C.) and atmospheric pressure conditions, unless otherwise noted. Unless otherwise specified, "%" described below means "mass%" and "parts" means "parts by mass".
製造例 シーバックソーン果実加工物の調製方法
シーバックソーン（Hippophae rhamnoides）の果実（果皮を含む生の全果実を細断したもの）１０ｇを入れた容器に、５０容量％のエタノール水溶液を、約１００ｍＬを加える。暗所室温条件下で7日間浸漬、熟成した後、固形分を濾過して除去し抽出液を採取する。この工程で生じるオリや沈殿を濾過した。斯くして調製した抽出液を、そのまま、以下の実験例に使用した。
Production example Sea buckthorn Fruit processed
実験例１ 毛乳頭細胞の増殖促進作用の評価
製造例で調製したシーバックソーン果実抽出物（以下、「SBT果実エキス」と称する）について、正常ヒト毛乳頭細胞を用いて、毛乳頭細胞増殖促進作用を評価した。
Experimental Example 1 Evaluation of proliferation promoting action of hair papilla cells Sea buckthorn fruit extract (hereinafter referred to as "SBT fruit extract") prepared in the production example was used to promote proliferation of hair papilla cells using normal human hair papilla cells. The effect was evaluated.
（１）実験方法
（ａ）使用培地
基礎培地：牛胎児血清（FBS）を含まないDMEM培地（Life Technologies社製）
栄養培地：10％FBS含有DMEM培地（Life Technologies社製）
いずれの培地にも、抗生物質(Penicillin 100 units/ml、Streptomycin 100μg/ml、Amphotericin B 250ng/ml)を配合した。
（ｂ）細胞培養
正常ヒト毛乳頭細胞を、栄養培地を入れた96wellプレートに、7000 cells/wellの割合になるように播種し、１日培養した後、栄養培地を、各種濃度のSBT果実エキス（乾燥残分として16.3ppm、81.5ppm）含有基礎培地（被験例）、SBT果実エキスを含まない基礎培地（Negative control：NC）（陰性対照）、またはSBT果実エキスを含まない栄養培地（Positive control：PC）（陽性対照）に交換し、それぞれ４日間培養した。培養は全て37℃-5%CO2条件下で行った。各系列ともｎ＝８で実験を行った。
インジケーター色素としてalamarBlue（登録商標）試薬(コスモバイオ製)を用いて、製品説明に従って、細胞増殖を測定した。具体的には、４日間培養した後、alamarBlue（登録商標）試薬を各ウエルに添加し、37℃、5％CO2条件下でインキュベートし、細胞の代謝活性によりレサズリンをレゾルフィンに変換させて、その蛍光強度（Ex 550 nm / Em 590 nm）を測定した。陰性対照の測定値を１００％として、各被験例及び陽性対照の細胞増殖率（％）を算出した。
(1) Experimental method (a) Medium used Basic medium: DMEM medium containing no fetal bovine serum (FBS) (manufactured by Life Technologies)
Nutrition medium: DMEM medium containing 10% FBS (manufactured by Life Technologies)
Antibiotics (
(B) Cell culture Normal human dermal papilla cells were seeded on a 96-well plate containing a nutrient medium at a ratio of 7,000 cells / well, cultured for 1 day, and then the nutrient medium was used as an SBT fruit extract of various concentrations. (Test example) containing (16.3ppm, 81.5ppm as dry residue), basal medium without SBT fruit extract (Negative control: NC) (negative control), or nutritional medium without SBT fruit extract (Positive control) : PC) (positive control) was replaced, and each was cultured for 4 days. All cultures were performed under 37 ° C-5% CO 2 conditions. Experiments were performed with n = 8 for each series.
Cell proliferation was measured using alamarBlue (registered trademark) reagent (manufactured by Cosmo Bio) as an indicator dye according to the product description. Specifically, after culturing for 4 days, alamarBlue (registered trademark) reagent was added to each well, and the mixture was incubated at 37 ° C. under 5% CO 2 conditions to convert resazurin to resorphin by the metabolic activity of the cells. The fluorescence intensity (Ex 550 nm / Em 590 nm) was measured. The cell proliferation rate (%) of each test example and the positive control was calculated with the measured value of the negative control as 100%.
（２）実験結果
結果を図１に示す。図１に示すように、SBT果実エキスには、ヒト毛乳頭細胞の増殖を促進し亢進させる作用があることが確認された。
(2) Experimental results The results are shown in FIG. As shown in FIG. 1, it was confirmed that the SBT fruit extract has an effect of promoting and enhancing the proliferation of human dermal papilla cells.
実験例２ 毛乳頭細胞における発毛促進因子発現亢進作用の評価
製造例で調製したSBT果実エキスについて、正常ヒト毛乳頭細胞を用いて、発毛促進因子であるバーシカン及びFGF7の遺伝子発現亢進作用を評価した。
Experimental Example 2 Evaluation of hair growth-promoting factor expression-enhancing effect on hair papilla cells The SBT fruit extract prepared in the production example was subjected to gene expression-enhancing effects of hair growth-promoting factors versican and FGF7 using normal human hair papilla cells. evaluated.
（１）実験方法
毛乳頭細胞を、栄養培地を入れた24wellプレートに、30000cells/wellの割合になるように播種し、37℃、5％CO2条件下で１日培養した後、栄養培地を、乾燥残分として15ppmのSBT果実エキスを含有する基礎培地に交換し、それぞれ37℃、5％CO2条件下で４時間培養した。培養した後に毛乳頭細胞を回収し、毛乳頭細胞におけるバーシカン、及びFGF7の遺伝子発現量を解析した。具体的には、回収した毛乳頭細胞から、ISOGEN II（日本ジーン社）を用いて、製品説明書に従って、mRNAを抽出し、その後、SuperScript III（Thermo Fisher Scientific社）を用いてcDNAに逆転写し、リアルタイムPCRを実施して、毛乳頭細胞におけるバーシカン、及びFGF7の発現量を測定した。なお、各実験において、SBT果実エキスを添加しないで培養した毛乳頭細胞（陰性対照例：SBT濃度 0ppm）についても、バーシカン、及びFGF7の発現量を測定し、当該対照例の発現量に対する相対比として、SBT果実エキスの効果（遺伝子発現亢進作用）を評価した。
(1) Experimental method The dermal papilla cells were seeded on a 24-well plate containing a nutrient medium at a ratio of 30,000 cells / well , cultured for 1 day under 37 ° C. and 5% CO 2 conditions, and then the nutrient medium was used. , The medium was replaced with a basal medium containing 15 ppm of SBT fruit extract as a dry residue, and the cells were cultured for 4 hours under the conditions of 37 ° C. and 5% CO 2 respectively. After culturing, dermal papilla cells were collected, and the gene expression levels of versican and FGF7 in the dermal papilla cells were analyzed. Specifically, from the collected dermal papilla cells, mRNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.) according to the product description, and then reverse transcribed into cDNA using SuperScript III (Thermo Fisher Scientific Co., Ltd.). , Real-time PCR was performed to measure the expression levels of versican and FGF7 in hair papilla cells. In each experiment, the expression levels of versican and FGF7 were also measured for hair papilla cells cultured without adding SBT fruit extract (negative control example:
（２）実験結果
バーシカンの発現亢進作用を評価した結果を図２に、FGF7の発現亢進作用を評価した結果を図３に、それぞれ示す。図２及び３に示すように、SBT果実エキスには、バーシカン及びFGF7の発現を促進し亢進させる作用があることが確認された。
(2) Experimental Results Figure 2 shows the results of evaluating the expression-enhancing effect of versican, and FIG. 3 shows the results of evaluating the expression-enhancing effect of FGF7. As shown in FIGS. 2 and 3, it was confirmed that the SBT fruit extract has an action of promoting and enhancing the expression of versican and FGF7.
実験例３ 毛乳頭細胞における発毛抑制因子発現抑制作用の評価
製造例で調製したSBT果実エキスについて、正常ヒト毛乳頭細胞を用いて、発毛抑制因子であるBMP-4の遺伝子発現亢進作用を評価した。
Experimental Example 3 Evaluation of hair growth inhibitory factor expression inhibitory effect on hair papilla cells For the SBT fruit extract prepared in the production example, the gene expression enhancing effect of BMP-4, which is a hair growth inhibitory factor, was performed using normal human hair papilla cells. evaluated.
（１）実験方法
毛乳頭細胞を、栄養培地を入れた24wellプレートに、30000 cells/wellの割合になるように播種し、37℃、5％CO2条件下で１日培養した後、栄養培地を、乾燥残分として15ppmのSBT果実エキスを含有する基礎培地に交換し、それぞれ37℃、5％CO2条件下で４時間培養した。培養した後に毛乳頭細胞を回収し、毛乳頭細胞におけるBMP-4の遺伝子発現量を解析した。具体的には、回収した毛乳頭細胞から、ISOGEN II（日本ジーン社）を用いて、製品説明書に従って、mRNAを抽出し、その後、SuperScript III（Thermo Fisher Scientific社）を用いてcDNAに逆転写し、リアルタイムPCRを実施して、毛乳頭細胞におけるBMP-4の発現量を測定した。なお、SBT果実エキスを添加しないで培養した毛乳頭細胞（対照例：SBT濃度 0ppm）についても、BMP-4の発現量を測定し、当該対照例の発現量に対する相対比として、SBT果実エキスの効果（遺伝子発現抑制作用）を評価した。
(1) Experimental method The dermal papilla cells were seeded on a 24-well plate containing a nutrient medium at a ratio of 30,000 cells / well, cultured for 1 day under the conditions of 37 ° C. and 5% CO 2, and then the nutrient medium. Was replaced with a basal medium containing 15 ppm of SBT fruit extract as a dry residue, and cultured for 4 hours under the conditions of 37 ° C. and 5% CO 2 respectively. After culturing, dermal papilla cells were collected and the gene expression level of BMP-4 in the dermal papilla cells was analyzed. Specifically, from the collected dermal papilla cells, mRNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.) according to the product description, and then reverse transcribed into cDNA using SuperScript III (Thermo Fisher Scientific Co., Ltd.). , Real-time PCR was performed to measure the expression level of BMP-4 in hair papilla cells. The expression level of BMP-4 was also measured for hair papilla cells cultured without adding SBT fruit extract (control example:
（２）実験結果
BMP-4の発現抑制作用を評価した結果を図４に示す。図４に示すように、SBT果実エキスには、BMP-4の発現を抑制する作用があることが確認された。
(2) Experimental results
The results of evaluating the expression-suppressing effect of BMP-4 are shown in FIG. As shown in FIG. 4, it was confirmed that the SBT fruit extract has an effect of suppressing the expression of BMP-4.
実験例４ 毛乳頭細胞における脱毛誘導因子発現抑制作用の評価
製造例で調製したSBT果実エキスについて、正常ヒト毛乳頭細胞を用いて、脱毛誘導因子であるTGF-β2の遺伝子発現亢進作用を評価した。
Experimental Example 4 Evaluation of inhibitory effect on hair loss-inducing factor expression in hair papilla cells The SBT fruit extract prepared in the production example was evaluated for gene expression-enhancing effect of TGF-β2, which is a hair loss-inducing factor, using normal human hair papilla cells. ..
（１）実験方法
毛乳頭細胞を、栄養培地を入れた24wellプレートに、30000 cells/wellの割合になるように播種し、37℃、5％CO2条件下で１日培養した後、栄養培地を、乾燥残分として15ppmのSBT果実エキスを含有する基礎培地に交換し、それぞれ37℃、5％CO2条件下で４時間培養した。培養した後に毛乳頭細胞を回収し、毛乳頭細胞におけるTGF-β2の遺伝子発現量を解析した。具体的には、回収した毛乳頭細胞から、ISOGEN II（日本ジーン社）を用いて、製品説明書に従って、mRNAを抽出し、その後、SuperScript III（Thermo Fisher Scientific社）を用いてcDNAに逆転写し、リアルタイムPCRを実施して、毛乳頭細胞におけるTGF-β2の発現量を測定した。なお、SBT果実エキスを添加しないで培養した毛乳頭細胞（対照例：SBT濃度0ppm）についても、TGF-β2の発現量を測定し、対照例の発現量に対する相対比として、SBT果実エキスの効果（遺伝子発現抑制作用）を評価した。
(1) Experimental method The dermal papilla cells were seeded on a 24-well plate containing a nutrient medium at a ratio of 30,000 cells / well, cultured for 1 day under the conditions of 37 ° C. and 5% CO 2, and then the nutrient medium. Was replaced with a basal medium containing 15 ppm of SBT fruit extract as a dry residue, and cultured for 4 hours under the conditions of 37 ° C. and 5% CO 2 respectively. After culturing, dermal papilla cells were collected and the gene expression level of TGF-β2 in the dermal papilla cells was analyzed. Specifically, from the collected dermal papilla cells, mRNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.) according to the product description, and then reverse transcribed into cDNA using SuperScript III (Thermo Fisher Scientific Co., Ltd.). , Real-time PCR was performed to measure the expression level of TGF-β2 in hair papilla cells. The expression level of TGF-β2 was also measured for hair papilla cells cultured without adding SBT fruit extract (control example:
（２）実験結果
TGF-β2の発現抑制作用を評価した結果を図５に示す。図５に示すように、SBT果実エキスには、TGF-β2の発現を抑制する作用があることが確認された。
(2) Experimental results
The results of evaluating the expression-suppressing effect of TGF-β2 are shown in FIG. As shown in FIG. 5, it was confirmed that the SBT fruit extract has an effect of suppressing the expression of TGF-β2.
Claims (4)
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