JP6242575B2 - Dendritic elongation inhibitor - Google Patents
Dendritic elongation inhibitor Download PDFInfo
- Publication number
- JP6242575B2 JP6242575B2 JP2013001527A JP2013001527A JP6242575B2 JP 6242575 B2 JP6242575 B2 JP 6242575B2 JP 2013001527 A JP2013001527 A JP 2013001527A JP 2013001527 A JP2013001527 A JP 2013001527A JP 6242575 B2 JP6242575 B2 JP 6242575B2
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- Prior art keywords
- oil
- water
- polyoxyethylene
- acid
- extraction
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Description
本発明は、デンドライト伸長を抑制する製剤に関する。 The present invention relates to a preparation that suppresses dendrite elongation.
樹状突起の形成機構には、細胞内における低分子量Gタンパクが関与している。この低分子量Gタンパクは細胞の増殖、接着、遊走、分泌など様々な細胞活動を制御している。
また、低分子量Gタンパクはがん細胞における転移や浸潤などとも密接に関与していることが報告されている。（非特許文献１）
メラノサイトにおいても、デンドライトの形成には、低分子量Ｇタンパクの関与が報告されており、その活性化には炎症性サイトカインとして知られているプロスタグランジンＥ２による作用が報告されている。（非特許文献２）
一方で、色素沈着の詳細な発現機構は十分には解明されていないが、大まかには、次のような機構が考えられている。
皮膚の基底層に存在するメラニン細胞（メラノサイト）の小胞（メラノソーム）内でチロシナーゼを触媒として、チロシンからメラニンが生成されるとともに、成熟したメラノソームを形成する。同時にメラノサイトから樹状突起が形成され、メラノサイトの先端から周囲に存在する角化細胞（ケラチノサイト）へメラノソームが受け渡される。
そして、ケラチノサイトはメラノソームを受け取ることで皮膚が暗色化する。メラノサイトのデンドライトが形成、伸長されないとケラチノサイトへのメラノソームの転移が効率よく行われず、結果的に皮膚に沈着するメラニン色素の量が減少するものと考えられる。
また、メラノサイトを含めデンドライトの伸長を抑制することは神経細胞腫の治療などにおいて有用であることが知られている。（特許文献１）
メラノサイトの樹状突起形成阻害剤としては、ヒドランゲノールまたはその誘導体、酵母抽出物、キサントン誘導体等が知られている。（特許文献２〜４）
Low molecular weight G protein in the cell is involved in the formation mechanism of dendrites. This low molecular weight G protein controls various cellular activities such as cell proliferation, adhesion, migration, and secretion.
In addition, it has been reported that low molecular weight G protein is closely related to metastasis and invasion in cancer cells. (Non-Patent Document 1)
In melanocytes, the involvement of low molecular weight G protein has been reported in the formation of dendrites, and the action of prostaglandin E2, which is known as an inflammatory cytokine, has been reported in its activation. (Non-Patent Document 2)
On the other hand, although the detailed expression mechanism of pigmentation has not been fully elucidated, the following mechanism is considered roughly.
Melanin is produced from tyrosine and formed into mature melanosomes using tyrosinase as a catalyst in vesicles (melanosomes) of melanocytes (melanocytes) existing in the basal layer of the skin. At the same time, dendrites are formed from the melanocytes, and the melanosomes are transferred from the melanocyte tip to the keratinocytes existing around.
And keratinocytes darken the skin by receiving melanosomes. If the dendrites of melanocytes are not formed and elongated, melanosome transfer to keratinocytes is not efficiently performed, and as a result, the amount of melanin pigment deposited on the skin is considered to be reduced.
In addition, it is known that inhibiting dendrite elongation including melanocytes is useful in the treatment of neurocytomas. (Patent Document 1)
As melanocyte dendritic formation inhibitors, hydrangenol or its derivatives, yeast extracts, xanthone derivatives and the like are known. (Patent Documents 2 to 4)
ジャトバは、マメ科(Leguminosae)Hymenaea courbaril L.の果実である。ジャトバに関しては、テストステロン−５α−リダクターゼ阻害剤、ヒスタミン遊離抑制剤、ヒアルロニダーゼ阻害剤、美白作用、エラスターゼ阻害剤などについて検討された。（特許文献５〜８）
白朮（ビャクジュツ）は、漢方生薬の１種であり、キク科の植物であるオケラ（Atractylodes japonica）、オオバナオケラ（Atractylodes ovata）等のオケラ属の周皮を除いた根茎部分（ワビャクジュツ）、オオバナオケラ（Atractylodes ovata）の根茎部をいう。
美白、シワ改善等の目的で外用剤に用いられている。（特許文献９〜１０）。
また、イシゲは、褐藻植物門、褐藻網、ナガマツモ目、イシゲ科、イシゲ属の海藻で、学名Ishige okamurai Yendoといい、茨城以南の沿岸で広く分布する褐藻類である。食用には硬いので適さない。
すでに、美白、育毛、痩身或いは抗菌・防腐等の目的で皮膚外用剤や食品に利用されている。（特許文献１１〜１３）
Jatoba is the fruit of the leguminous family (Leguminosae) Hymenaea courbaril L. Regarding Jatoba, a testosterone-5α-reductase inhibitor, a histamine release inhibitor, a hyaluronidase inhibitor, a whitening action, an elastase inhibitor, and the like were examined. (Patent Documents 5 to 8)
Birch Jutsu is one of the herbal medicines. Rhizome excluding periwinks of the genus Ochera (Atractylodes japonica) and Atractylodes ovata (Actylodes ovata) This refers to the rhizome part of Atractylodes ovata.
It is used as an external preparation for the purpose of whitening and wrinkle improvement. (Patent Documents 9 to 10).
Ishige is a seaweed belonging to the genus Brown algae, Brown algae, Pleurotus, Ishigeaceae, and Ishige genus. The scientific name is Ishige okamurai Yendo. Not suitable for edible use.
Already used for skin external preparations and foods for the purposes of whitening, hair growth, slimming, antibacterial and antiseptic. (Patent Documents 11 to 13)
本発明の目的はデンドライト伸長を抑制する製剤を得ることにある。 An object of the present invention is to obtain a preparation that suppresses dendrite elongation.
本発明者らが鋭意検討した結果、ジャトバ、白朮、イシゲの抽出物が上記目的を達することがわかった。
ジャトバの実、白朮、イシゲを、抽出効率を考え、細切、乾燥、粉砕等の処理を行った後に抽出を行うことが好ましい。ジャトバの実、すなわち豆果は成熟すると非常に堅いので、必要な細切、粉砕等の処理を行う。
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。
前記抽出に用いる溶媒としては、水若しくは親水性有機溶媒又はこれらの混合液を用いる。
前記抽出溶媒として使用し得る水としては、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。従って、本発明において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。
前記親水性有機溶媒としては、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の多価アルコールなどが挙げられ、これら親水性有機溶媒と水との混合溶媒などを用いることができる。
なお、前記水と親水性有機溶媒との混合溶媒を使用する場合には、低級アルコールの場合は水１０質量部に対して１〜２０質量部、低級脂肪族ケトンの場合は水１０質量部に対して１〜１５質量部添加することが好ましい。多価アルコールの場合は水１０質量部に対して１〜２０質量部添加することが好ましい。
抽出に使用する有機溶媒の量は、原料となる植物（海藻）に対して望ましくは５〜１００倍量程度、さらに望ましくは１０〜５０倍量程度が良い。さらに抽出効率を上げるため、抽出溶媒中で撹拌やホモジナイズしてもよい。抽出温度としては、５℃程度から抽出溶媒の沸点以下の温度とするのが適切である。抽出時間は抽出溶媒の種類や抽出温度によっても異なるが、１時間〜１４日間程度とするのが適切である。
尚、抽出操作は１回のみの操作に限定されるものではない。抽出後の残渣に再度新鮮な溶媒を添加し、抽出操作を施すこともできるし、抽出溶媒を複数回抽出原料に接触させることも可能である。
必要ならば、その効果に影響のない範囲で更に脱臭、脱色等の精製処理を加えても良く、エバポレーターのような減圧濃縮装置や加熱による溶媒除去などにより、濃縮することができる。
また、この抽出物を合成吸着剤（ダイアイオンＨＰ２０やセファビースＳＰ８２５、アンバーライトＸＡＤ４、ＭＣＩｇｅｌＣＨＰ２０Ｐ等）やデキストラン樹脂（セファデックスＬＨ−２０など）、限外濾過等を用いてさらに精製することも可能である。
As a result of intensive studies by the present inventors, it has been found that extracts of jatoba, white birch and shigege can achieve the above object.
It is preferable to extract jatoba berries, white birch, and ishige after processing such as shredding, drying, and pulverization in consideration of extraction efficiency. Jatoba fruit, that is, legumes, is very hard when mature, and is processed as necessary, such as shredding and crushing.
Drying may be performed in the sun or using a commonly used dryer.
As the solvent used for the extraction, water, a hydrophilic organic solvent, or a mixture thereof is used.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. 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 in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
Examples of the hydrophilic organic solvent include lower 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 glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, it is 1-20 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, it is 10 mass parts of water. It is preferable to add 1-15 mass parts with respect to it. In the case of a polyhydric alcohol, it is preferable to add 1 to 20 parts by mass with respect to 10 parts by mass of water.
The amount of the organic solvent used for the extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times the amount of the plant (seaweed) as the raw material. Furthermore, in order to raise extraction efficiency, you may stir or homogenize in an extraction solvent. The extraction temperature is suitably about 5 ° C. to the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.
The extraction operation is not limited to a single operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material a plurality of times.
If necessary, purification treatment such as deodorization and decolorization may be further added within a range that does not affect the effect, and concentration can be performed by a vacuum concentrator such as an evaporator or solvent removal by heating.
Further, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephabies SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc. is there.
本発明の製剤は、経口、注射、外用のいずれでも薬効を発現するが、皮膚外用剤として用いるのが好ましい。皮膚外用剤には、皮膚化粧料、外用医薬部外品、医療用皮膚外用剤が含まれる。 The preparation of the present invention exhibits drug efficacy for oral, injection, and external use, but is preferably used as a skin external preparation. Skin external preparations include skin cosmetics, external quasi-drugs, and medical skin external preparations.
また、本発明の製剤には、上記成分の他に医薬品や化粧品の各種製剤において使用されている界面活性剤、油性成分、保湿剤、高分子化合物、紫外線吸収剤、抗炎症剤、殺菌剤、酸化防止剤、金属イオン封鎖剤、防腐剤、ビタミン類、色素、香料、水等を配合することができる。 In addition to the above ingredients, the preparations of the present invention include surfactants, oily ingredients, moisturizers, polymer compounds, ultraviolet absorbers, anti-inflammatory agents, bactericides, Antioxidants, sequestering agents, preservatives, vitamins, pigments, fragrances, water and the like can be blended.
上記界面活性剤としては、アニオン性、カチオン性、非イオン性、天然、合成のいずれの界面活性剤も使用できるが、皮膚に対する刺激性を考慮すると非イオン性のものを使用することが好ましい。非イオン性界面活性剤としては、例えばグリセリン脂肪酸エステル、プロピレングリコール脂肪酸エステル、ソルビタン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレンソルビット脂肪酸エステル、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンポリオキシプロピレングリコール、ポリオキシエチレンポリオキシプロピレンアルキルエーテル、ポリエチレングリコール脂肪酸エステル、ポリオキシエチレンヒマシ油、ポリオキシエチレン硬化ヒマシ油、アルキルグリコシド等が挙げられる。 As the surfactant, any of anionic, cationic, nonionic, natural, and synthetic surfactants can be used, but it is preferable to use a nonionic surfactant in consideration of irritation to the skin. Examples of the nonionic surfactant include glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbite fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkylglycoside and the like.
油性成分としては、油脂類、ロウ類、炭化水素類、高級脂肪酸類、高級アルコール類、エステル類、精油類、シリコーン油類などを挙げることができる。油脂類としては、例えば大豆油、ヌカ油、ホホバ油、アボガド油、アーモンド油、オリーブ油、カカオ油、ゴマ油、パーシック油、ヒマシ油、ヤシ油、ミンク油、牛脂、豚脂等の天然油脂、これらの天然油脂を水素添加して得られる硬化油及びミリスチン酸グリセリド、２−エチルヘキサン酸トリグリセリド等の合成トリグリセリド等が；ロウ類としては、例えばカルナバロウ、鯨ロウ、ミツロウ、ラノリン等が；炭化水素類としては、例えば流動パラフィン、ワセリン、パラフィンマイクロクリスタリンワックス、セレシン、スクワラン、ブリスタン等が；高級脂肪酸類としては、例えばラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、ベヘニン酸、オレイン酸、リノール酸、リノレン酸、ラノリン酸、イソステアリン酸等が；高級アルコール類としては、例えばラウリルアルコール、セチルアルコール、ステアリルアルコール、オレイルアルコール、ラノリンアルコール、コレステロール、２−ヘキシルデカノール等が；エステル類としては、例えばオクタン酸セチル、オクタン酸トリグリセライド、乳酸ミリスチル、乳酸セチル、ミリスチン酸イソプロピル、ミリスチン酸ミリスチル、ミリスチン酸オクチルドデシル、パルミチン酸イソプロピル、アジピン酸イソプロピル、ステアリン酸ブチル、オレイン酸デシル、イソステアリン酸コレステロール、ＰＯＥソルビット脂肪酸エステル等が；精油類としては、例えばハッカ油、ジャスミン油、ショウ脳油、ヒノキ油、トウヒ油、リュウ油、テレピン油、ケイ皮油、ベルガモット油、ミカン油、ショウブ油、パイン油、ラベンダー油、ベイ油、クローブ油、ヒバ油、バラ油、ユーカリ油、レモン油、タイム油、ペパーミント油、ローズ油、セージ油、メントール、シネオール、オイゲノール、シトラール、シトロネラール、ボルネオール、リナロール、ゲラニオール、カンファー、チモール、スピラントール、ピネン、リモネン、テルペン系化合物等が；シリコーン油類としては、例えばジメチルポリシロキサン等が挙げられる。これら上述の油性成分は一種又は二種以上を組み合わせて使用することができる。本発明においては、このうち特にミリスチン酸グリセリド、２−エチルヘキサン酸トリグリセリド、ラノリン、流動パラフィン、ワセリン、パラフィンマイクロクリスタリンワックス、スクワラン、ラウリン酸、ミリスチン酸、パルミチン酸、リノール酸、リノレン酸、イソステアリン酸、セチルアルコール、ステアリルアルコール、オレイルアルコール、コレステロール、オクタン酸セチル、オクタン酸トリグリセライド、ミリスチレン酸イソプロピル、ミリスチン酸オクチルドデシル、イソステアリン酸コレステロール、ＰＯＥソルビット脂肪酸エステル、ハッカ油、トウヒ油、ケイ皮油、ローズ油、メントール、シネオール、オイゲノール、シトラール、シトロネラール、ゲラニオール、ピネン、リモネン、ジメチルポリシロキサンを使用することが好ましい。 Examples of the oil component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils, and the like. Examples of the fats and oils include soybean oil, nutka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, coconut oil, mink oil, beef fat, pork fat and the like, and these Hardened oil obtained by hydrogenation of natural fats and oils and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; waxes include, for example, carnauba wax, whale wax, beeswax, lanolin and the like; hydrocarbons For example, liquid paraffin, petrolatum, paraffin microcrystalline wax, ceresin, squalane, bristan etc .; higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, Linolenic acid, lanolinic acid, isostearic acid, etc .; Examples of the class alcohols include lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol, etc .; examples of the esters include cetyl octanoate, triglyceride octanoate, myristyl lactate, cetyl lactate, Examples of essential oils include mint oil and jasmine. Oil, pepper brain oil, cypress oil, spruce oil, ryu oil, turpentine oil, cinnamon oil, bergamot oil, mandarin oil, ginger oil, pine , Lavender oil, bay oil, clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citronellal, borneol, linalool, geraniol, Camphor, thymol, spirantol, pinene, limonene, terpene compounds, etc .; examples of silicone oils include dimethylpolysiloxane. These oily components described above can be used alone or in combination of two or more. In the present invention, among these, myristic acid glyceride, 2-ethylhexanoic acid triglyceride, lanolin, liquid paraffin, petrolatum, paraffin microcrystalline wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanoate, isopropyl myristate, octyldodecyl myristate, cholesterol isostearate, POE sorbite fatty acid ester, peppermint oil, spruce oil, cinnamon oil, rose Oil, menthol, cineol, eugenol, citral, citronellal, geraniol, pinene, limonene, dimethylpolysiloxane It is preferable to use.
本発明の製剤には、さらに下記のような成分を配合することができるが、その成分もこれらに限定されるものではない。
色素類；黄色４号、青色１号、黄色２０２号等の厚生省令に定められたタール色素別表Ｉ及びIIの色素、クロロフィル、リボフラビン、クロシン、紅花、アントラキノン等の食品添加物として認められている天然色素等。
ビタミン類；ビタミンＡ、ビタミンＣ、ビタミンＤ、ビタミンＥ等。
その他；殺菌剤、防腐剤、その他製剤上必要な成分等。
The following components can be further added to the preparation of the present invention, but the components are not limited thereto.
Colors: Yellow color No. 4, Blue No. 1 and Yellow No. 202 are recognized as food additives such as tar color dyes I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments.
Vitamins; vitamin A, vitamin C, vitamin D, vitamin E, etc.
Others: bactericides, preservatives, other ingredients necessary for formulation.
本発明の製剤は、前記必須成分に必要に応じて前記任意成分を加え、常法に従って製造することができ、クリーム、乳液、化粧水等の形態とすることができる。 The preparation of the present invention can be produced according to a conventional method by adding the optional components to the essential components as necessary, and can be in the form of cream, emulsion, lotion or the like.
次に実施例（製造例）を挙げて本発明を詳細に説明する。 Next, an Example (manufacturing example) is given and this invention is demonstrated in detail.
製造例１
ジャトバの実（成熟した乾燥品）を２０メッシュ以下となるようにミルで粉砕し、そのうち、５０．０ｇをはかりとり、５０％エタノール水溶液４５０ｇを加え、撹拌しつつ１０時間抽出した。
これを遠心分離（２０００Ｇ、１５分間）し、その上澄みを濾過した。（Ｎｏ５Ｃ）
濾液をエバボレーターで濃縮したのち、凍結乾燥した。
Production Example 1
Jatoba fruit (mature dry product) was pulverized with a mill so as to be 20 mesh or less, and 50.0 g was weighed out, 450 g of 50% aqueous ethanol solution was added, and the mixture was extracted with stirring for 10 hours.
This was centrifuged (2000 G, 15 minutes), and the supernatant was filtered. (No5C)
The filtrate was concentrated with an evaporator and then freeze-dried.
製造例２
白朮（日本薬局方ビャクジュツ末栃本天海堂）を１００ｇに精製水５００mlを加え、２時間湯煎（水温９０℃）で抽出した。放冷したのち、濾過した（Ｎｏ５Ｃ）のち、凍結乾燥した。
Production Example 2
Shirakaba (Japanese Pharmacopoeia Byakujutsu Sue Tochimoto Tenkaido) was added to 100 g of purified water (500 ml) and extracted with a hot water bath (water temperature 90 ° C.) for 2 hours. After standing to cool, it was filtered (No5C) and then lyophilized.
製造例３
イシゲ５０ｇ（湿重量）に精製水５００ｇを加え、ミキサーを用いてホモジナイズした。これを遠心分離し濾液を得た。この濾液に４倍量のエタノール２Lを加え、ときどき撹拌しながら、２４時間後に濾過（Ｎｏ５Ｃ）し、これを凍結乾燥した。
Production Example 3
Purified water (500 g) was added to Ishige 50 g (wet weight) and homogenized using a mixer. This was centrifuged to obtain a filtrate. To this filtrate was added 2 L of 4-fold amount of ethanol, and filtered (No5C) after 24 hours with occasional stirring and lyophilized.
確認試験（メラノサイトのデンドライト伸長抑制試験）
４ｗｅｌｌ ラブテック２チェンバースライドシステム(nunc製)に正常ヒトメラニン細胞（クラボウ製）を７００cell/well播種する。２４時間培養した後、増殖因子を含まない、基礎培地であるDermaLife M(登録商標)(Lifeline Cell Technology製)へ交換する。
更に２４時間培養したのち、試験品および、樹状突起伸長因子としてＹ−２７６３２（和光純薬）を含むDermaLife Mへ交換し、２４時間培養した。（Ｙ−２７６３２は終濃度５０uMになるように濃度を調整した）
２４時間後、培地を除去しActi-Stain 488 phalloidin(Cytoskeleton,Inc)にて細胞骨格を染色し、DAPI（同仁化学）にて核を染色した。
染色後、画像解析ソフト(Image J)にてメラノサイトの樹状突起の長さを計測し、サンプルにおける樹状突起の長さを比較した。
Confirmation test (Dendrite extension suppression test of melanocytes)
Normal human melanocytes (manufactured by Kurabo Industries) are seeded at 700 cells / well on a 4-well Lovetech 2 chamber slide system (manufactured by nunc). After culturing for 24 hours, the medium is replaced with DermaLife M (registered trademark) (manufactured by Lifeline Cell Technology) which is a basal medium containing no growth factor.
After further culturing for 24 hours, the specimen was replaced with DermaLife M containing Y-27632 (Wako Pure Chemical Industries) as a dendritic elongation factor and cultured for 24 hours. (The concentration of Y-27632 was adjusted to a final concentration of 50 uM)
After 24 hours, the medium was removed, the cytoskeleton was stained with Acti-Stain 488 phalloidin (Cytoskeleton, Inc), and the nucleus was stained with DAPI (Dojin Chemical).
After staining, the length of dendrites of melanocytes was measured with image analysis software (Image J), and the length of dendrites in the samples was compared.
下記のＭＴＴ活性の確認試験を行い、製造例が細胞の存在に影響のないことを確認を行い、製造例が細胞の存在に影響のない範囲の確認試験（メラノサイトのデンドライト伸長抑制試験）の結果を以下の図１〜３に示す。
24well plateで、確認試験と同一条件で培養した後、培地を交換し、１/１０量の５mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideを添加してさらに４時間培養後、等量の0.04N HClを含むイソプロパノールを添加して完全に溶解する。この溶液をリファレンス波長630nm、測定波長560nmで測定して、コントロールに対する相対値として算出した。
The following confirmation test of MTT activity was performed, it was confirmed that the production example had no effect on the presence of cells, and the results of a confirmation test (melanocyte dendrite extension inhibition test) in which the production example did not affect the presence of cells Is shown in FIGS.
After culturing in a 24-well plate under the same conditions as the confirmation test, the medium is changed, and 1/10 amount of 5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide is added. Then, after further incubation for 4 hours, isopropanol containing an equal amount of 0.04N HCl is added and completely dissolved. This solution was measured at a reference wavelength of 630 nm and a measurement wavelength of 560 nm, and calculated as a relative value to the control.
製造例１〜３をそのまま外用剤としても利用できるが、これに他の原料を配合して外用剤を作成することも可能である。 Production Examples 1 to 3 can be used as they are as external preparations, but it is also possible to prepare external preparations by blending other raw materials with them.
外用剤１
オリーブ油 ０．５
ポリオキシエチレン（２０Ｅ．Ｏ．）ソルビタンモノステアレート ２．０
ポリオキシエチレン（６０Ｅ．Ｏ．）硬化ヒマシ油 ２．０
エタノール １０．０
精製水 ８０．０
製造例１ ０．３
パラオキシ安息香酸メチル ０．２
External preparation 1
Olive oil 0.5
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 2.0
Ethanol 10.0
Purified water 80.0
Production Example 1 0.3
Methyl paraoxybenzoate 0.2
外用剤２
オリーブ油 ０．５
ポリオキシエチレン（２０Ｅ．Ｏ．）ソルビタンモノステアレート ２．０
ポリオキシエチレン（６０Ｅ．Ｏ．）硬化ヒマシ油 ２．０
エタノール １０．０
精製水 ８０．０
製造例３ ０．３
パラオキシ安息香酸メチル ０．２
External preparation 2
Olive oil 0.5
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 2.0
Ethanol 10.0
Purified water 80.0
Production Example 3 0.3
Methyl paraoxybenzoate 0.2
外用剤３
Ａ
スクワラン ２０．０
オリーブ油 ２．０
ミンク油 １．０
ホホバ油 ５．０
ミツロウ ５．０
セトステアリルアルコール ２．０
グリセリンモノステアレート １．０
ソルビタンモノステアレート ２．０
Ｂ
精製水 ５１．０
製造例２ ０．７
ポリオキシエチレン（２０Ｅ．Ｏ．）ソルビタンモノステアレート ２．０
ポリオキシエチレン（６０Ｅ．Ｏ．）硬化ヒマシ油 １．０
グリセリン ５．０
Ｌ−アスコルビン酸−２−グルコシド ２．０
パラオキシ安息香酸メチル ０．１
External preparation 3
A
Squalane 20.0
Olive oil 2.0
Mink oil 1.0
Jojoba oil 5.0
Beeswax 5.0
Cetostearyl alcohol 2.0
Glycerol monostearate 1.0
Sorbitan monostearate 2.0
B
Purified water 51.0
Production Example 2 0.7
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
Glycerin 5.0
L-ascorbic acid-2-glucoside 2.0
Methyl paraoxybenzoate 0.1
外用剤４
Ａ
スクワラン ２０．０
オリーブ油 ２．０
ミンク油 １．０
ホホバ油 ５．０
ミツロウ ５．０
セトステアリルアルコール ２．０
グリセリンモノステアレート １．０
ソルビタンモノステアレート ２．０
Ｂ
精製水 ５１．０
製造例１ ０．７
ジプロピレングリコール ２．０
グリセリン ３．０
ポリオキシエチレン（２０Ｅ．Ｏ．）ソルビタンモノステアレート ２．０
ポリオキシエチレン（６０Ｅ．Ｏ．）硬化ヒマシ油 １．０
Ｌ−アスコルビン酸−２−グルコシド ２．０
パラオキシ安息香酸メチル ０．１
External preparation 4
A
Squalane 20.0
Olive oil 2.0
Mink oil 1.0
Jojoba oil 5.0
Beeswax 5.0
Cetostearyl alcohol 2.0
Glycerol monostearate 1.0
Sorbitan monostearate 2.0
B
Purified water 51.0
Production Example 1 0.7
Dipropylene glycol 2.0
Glycerin 3.0
Polyoxyethylene (20E.O.) sorbitan monostearate 2.0
Polyoxyethylene (60E.O.) hydrogenated castor oil 1.0
L-ascorbic acid-2-glucoside 2.0
Methyl paraoxybenzoate 0.1
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| CN108210375A (en) * | 2018-02-27 | 2018-06-29 | 泉州师范学院 | A kind of facial mask containing okamura ishige algae extract and preparation method thereof |
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| CN108210375A (en) * | 2018-02-27 | 2018-06-29 | 泉州师范学院 | A kind of facial mask containing okamura ishige algae extract and preparation method thereof |
| CN108210375B (en) * | 2018-02-27 | 2020-02-18 | 泉州师范学院 | A kind of facial mask containing iron nail extract and preparation method thereof |
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