JP7560148B2 - Pharmaceutical composition for promoting hyaluronic acid synthesis and method for producing pharmaceutical composition for promoting hyaluronic acid synthesis containing pomegranate seed extract - Google Patents
Pharmaceutical composition for promoting hyaluronic acid synthesis and method for producing pharmaceutical composition for promoting hyaluronic acid synthesis containing pomegranate seed extract Download PDFInfo
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- JP7560148B2 JP7560148B2 JP2022118522A JP2022118522A JP7560148B2 JP 7560148 B2 JP7560148 B2 JP 7560148B2 JP 2022118522 A JP2022118522 A JP 2022118522A JP 2022118522 A JP2022118522 A JP 2022118522A JP 7560148 B2 JP7560148 B2 JP 7560148B2
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- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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Description
本発明は、ヒアルロン酸合成促進用医薬組成物、及びヒアルロン酸合成促進用医薬組成物の製造方法に関し、特に、ザクロ種子エキスを含有するヒアルロン酸合成促進用医薬組成物、及びヒアルロン酸合成促進用医薬組成物の製造方法に関する。 The present invention relates to a pharmaceutical composition for promoting hyaluronic acid synthesis and a method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis, and in particular to a pharmaceutical composition for promoting hyaluronic acid synthesis that contains pomegranate seed extract and a method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis.
ザクロは果実全体に対して種子がかなりの割合を占める果実であるが、原則として、搾汁後に残る種子は廃棄されている。ザクロ種子を利用した少ない例として、ザクロ種子エキスを用いたサプリメント提案システムであって、提案サプリメント演算手段が、希望事項情報が美容の場合には、提案サプリメントとして、マルチビタミン、マルチミネラル、ビタミンＡ、ビタミンＢ１、ビタミンＢ２、ビタミンＢ６、ビタミンＢ１２、ビタミンＣ、ビタミンＥ、ビタミンＰ、ナイアシン、ビオチン、コラーゲン、エラスチン、ポリフェノール、パントテン酸、ヒアルロン酸、アセチルグルコサミン、イノシトール、コエンザイムＱ１０、αカロチン、βカロチン、大豆サポニン、ロイヤルゼリー、ツイントース、葉酸、バラ花弁エキス、ザクロ種子エキス、ブラックコホッシュエキス、大豆イソフラボン、葛イソフラボンスクワレン、ビール酵母、コロハエキス、カルシウム、マグネシウム、銅、鉄、亜鉛、マンガン、とから成る群の少なくとも１つを出力するサプリメント提案システムが知られている（特許文献１）。 Pomegranate is a fruit in which seeds make up a significant proportion of the entire fruit, but as a rule, the seeds remaining after squeezing the juice are discarded. A few examples of using pomegranate seeds include a supplement suggestion system using pomegranate seed extract, in which the suggested supplement calculation means outputs at least one of the following suggested supplements when the desired item information is beauty: multivitamins, multiminerals, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin P, niacin, biotin, collagen, elastin, polyphenols, pantothenic acid, hyaluronic acid, acetylglucosamine, inositol, coenzyme Q10, alpha-carotene, beta-carotene, soybean saponin, royal jelly, twin tose, folic acid, rose petal extract, pomegranate seed extract, black cohosh extract, soybean isoflavones, kudzu isoflavone squalene, brewer's yeast, fenugreek extract, calcium, magnesium, copper, iron, zinc, and manganese (Patent Document 1).
しかしながら、現在のところ多くの場合、ザクロ種子は原則として、廃棄物として処理されており、前記特許文献以外は、有効に利用されていないのが現状である。一方で、廃棄物の有効利用が見直されている。 However, currently, in most cases, pomegranate seeds are generally disposed of as waste, and apart from the above-mentioned patent documents, they are not effectively utilized. On the other hand, effective utilization of waste materials is being reconsidered.
また、近年、アトピー性皮膚炎（AD）が、角化関連遺伝子であるフィラグリン（FLG）の機能欠失変異によって生じることが判明し、皮膚バリア機能の重要性が注目されている。かかる状況下、廃棄物たるザクロ種子が種々の疾患に対して効果的であれば、より有益となる。 In addition, in recent years, it has been discovered that atopic dermatitis (AD) is caused by a loss-of-function mutation in the keratinization-related gene filaggrin (FLG), and the importance of skin barrier function has been drawing attention. Under these circumstances, it would be even more beneficial if pomegranate seeds, a waste product, were effective against various diseases.
したがって、本発明の目的は、廃棄されるザクロ種子の有効活用及び付加価値化をめざしてヒアルロン酸合成を促進する作用を有する組成物を提供することにある。 Therefore, the object of the present invention is to provide a composition that promotes hyaluronic acid synthesis, aiming at effective utilization and added value of discarded pomegranate seeds.
上記目的を達成するために、本発明者らは、ザクロ種子の有効活用について鋭意検討した結果、本発明を見出すに至った。 To achieve the above objective, the inventors conducted extensive research into the effective use of pomegranate seeds, and came up with the present invention.
すなわち、本発明のヒアルロン酸合成酵素の発現促進用医薬組成物は、ザクロ種子エキスを含有するヒアルロン酸合成酵素の発現促進用医薬組成物であって、前記医薬組成物は、角化膜形成関連遺伝子を増加する医薬組成物であり、前記ヒアルロン酸合成酵素は、HAS-1、HAS-2又はHAS３であって、前記角化膜形成関連遺伝子は、ｈ-LOR遺伝子、又はｈ-INV遺伝子であることを特徴とするヒアルロン酸合成酵素の発現促進用医薬組成物。
That is, the pharmaceutical composition for promoting the expression of hyaluronic acid synthase of the present invention is a pharmaceutical composition for promoting the expression of hyaluronic acid synthase containing pomegranate seed extract , wherein the pharmaceutical composition is a pharmaceutical composition for increasing a cornified membrane formation-related gene, the hyaluronic acid synthase being HAS-1, HAS-2 or HAS3, and the cornified membrane formation-related gene being the h-LOR gene or the h-INV gene .
また、本発明の医薬部外品は、本発明の医薬組成物を有効成分とすることを特徴とする。 The quasi-drug of the present invention is characterized in that it contains the pharmaceutical composition of the present invention as an active ingredient.
また、本発明のヒアルロン酸合成酵素の発現促進用医薬組成物の製造方法は、本発明のヒアルロン酸合成酵素の発現促進用医薬組成物の製造方法であって、ザクロ種子を粉砕して得た粉砕物を溶媒中に浸漬した後、上清を分取する工程と、前記上清を分取することによりザクロ種子エキスを得る工程と、得られたザクロ種子エキスを有効的な量に調整する工程と、を有することを特徴とするザクロ種子エキスを含有することを特徴とする。 In addition, the manufacturing method of the pharmaceutical composition for promoting the expression of hyaluronic acid synthase of the present invention is a manufacturing method of the pharmaceutical composition for promoting the expression of hyaluronic acid synthase of the present invention, which is characterized by containing pomegranate seed extract, comprising the steps of: immersing the pulverized product obtained by pulverizing pomegranate seeds in a solvent and then separating the supernatant; obtaining a pomegranate seed extract by separating the supernatant; and adjusting the obtained pomegranate seed extract to an effective amount.
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法の好ましい実施態様において、溶媒が、酢酸エチル、酢酸エチル-ヘキサン、酢酸エチル-メタノール、エタノール、メタノール、ヘキサン、水からなる群から選択される少なくとも１種であることを特徴とする。 In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法の好ましい実施態様において、エタノールで抽出したザクロ種子エタノールエキスを、さらに、酢酸エチルと水で分配して、前記酢酸エチル層を減圧濃縮して、酢酸エチル画分としてザクロ種子エキスを得ることを特徴とする。 In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the pomegranate seed ethanol extract extracted with ethanol is further partitioned with ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain pomegranate seed extract as an ethyl acetate fraction.
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法の好ましい実施態様において、さらに、前記酢酸エチル画分を、ヘキサンとメタノールで分配して、酢酸エチル-ヘキサン画分及び酢酸エチル-メタノール画分として、ザクロ種子エキスを得ることを特徴とする。 In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the ethyl acetate fraction is further partitioned with hexane and methanol to obtain an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction as a pomegranate seed extract.
本発明のヒアルロン酸合成促進用医薬組成物によれば、従来廃棄物として処理されていたザクロ種子を有効利用することが可能であるという有利な効果を奏する。また、本発明のヒアルロン酸合成促進用医薬組成物によれば、ヒアルロン酸合成を促進する作用を有し、保湿機能の改善に有効であるという有利な効果を奏する。 The pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention has the advantageous effect of making it possible to effectively utilize pomegranate seeds, which have conventionally been treated as waste. In addition, the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention has the advantageous effect of promoting hyaluronic acid synthesis and being effective in improving moisturizing function.
本発明のヒアルロン酸合成促進用医薬組成物は、ザクロ種子エキスを含有することを特徴とする。ザクロ種子エキスは、ザクロの種子由来のエキスである。本発明に適用するザクロ種子エキスは、ザクロの種子由来である限り、総てのザクロ種子エキスを対象とする。 The pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized by containing pomegranate seed extract. Pomegranate seed extract is an extract derived from pomegranate seeds. The pomegranate seed extract applied to the present invention covers all pomegranate seed extracts as long as they are derived from pomegranate seeds.
また、対象となる細胞としては、特に限定されないが、特に有効に作用するのは、皮膚の細胞である。したがって、本明細書において主として角化細胞を含め皮膚の細胞について説明するが、本発明は、これに限定される意図ではない。 The target cells are not particularly limited, but the cells that act particularly effectively are skin cells. Therefore, although the present specification will mainly describe skin cells, including keratinocytes, the present invention is not intended to be limited thereto.
また、本発明のヒアルロン酸合成促進用医薬組成物の好ましい実施態様において、前記医薬組成物は、ヒアルロン酸合成酵素の発現増加によりヒアルロン酸合成を促進する医薬組成物であり、前記ヒアルロン酸合成酵素は、皮膚等の細胞の水分保湿機能を向上させるという観点から、HAS-1、HAS-2又はHASー3であることを特徴とする。すなわち、皮膚は生体を外界から保護する働きや水分損失防止機能を担っており、皮膚を正常な状態を保つことは非常に重要であり、皮膚のバリア機能および水分保湿機能に関与する主要な調整剤として、角化膜（ＣＥ）やヒアルロン酸（ＨＡ）が挙げられるが、今回本発明により、ヒアルロン酸の合成を促進することが可能であることが判明したものである。 In a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the pharmaceutical composition promotes hyaluronic acid synthesis by increasing the expression of hyaluronic acid synthase, and the hyaluronic acid synthase is HAS-1, HAS-2 or HAS-3 from the viewpoint of improving the moisture retention function of cells such as skin. That is, the skin protects the body from the outside world and prevents moisture loss, and it is very important to maintain the skin in a normal state. Major regulators involved in the barrier function and moisture retention function of the skin include cornified epithelium (CE) and hyaluronic acid (HA), and it has now been discovered that the synthesis of hyaluronic acid can be promoted by the present invention.
なお、HAS-1、HAS-2又はHAS-3は、ヒアルロン酸合成酵素の略号（HASは、Hyaluronic Acid Synthaseの略。）を意味し、また、HAS-1遺伝子、HAS-2遺伝子又はHAS-3遺伝子は、当該ヒアルロン酸合成酵素を発現する遺伝子を意味する。 Note that HAS-1, HAS-2, and HAS-3 are abbreviations for hyaluronic acid synthase (HAS is an abbreviation for hyaluronic acid synthase), and the HAS-1 gene, HAS-2 gene, and HAS-3 gene are genes that express the hyaluronic acid synthase.
また、本発明のヒアルロン酸合成促進用医薬組成物の好ましい実施態様において、さらに、前記医薬組成物は、ザクロ種子エキスには角化膜形成を促進する作用も有するという観点から、角化膜形成関連遺伝子を増加することを特徴とする。すなわち、今回本発明により、角化膜形成関連遺伝子を増加することにより、ひいては、角化膜形成を促進することが可能であることが判明したものである。 In a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the pharmaceutical composition is further characterized by increasing cornified membrane formation-related genes, from the viewpoint that pomegranate seed extract also has the effect of promoting cornified membrane formation. In other words, it has been found that the present invention makes it possible to promote cornified membrane formation by increasing cornified membrane formation-related genes.
また、本発明のヒアルロン酸合成促進用医薬組成物の好ましい実施態様において、前記角化膜形成関連遺伝子は、角化膜を構成するタンパク質であるロリクリンおよびインボルクリンを増加するという観点から、ｈ-LOR遺伝子、又はｈ-INV遺伝子であることを特徴とする。 In a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the cornified membrane formation-related gene is the h-LOR gene or the h-INV gene, from the viewpoint of increasing loricrin and involucrin, which are proteins that constitute the cornified membrane.
また、本発明のヒアルロン酸合成促進用医薬組成物の好ましい実施態様において、ザクロ種子を粉砕して得た粉砕物を、酢酸エチル、酢酸エチル-ヘキサン、酢酸エチル-メタノール、エタノール、メタノール、水、ヘキサンからなる群から選択される少なくとも1種の溶媒に浸漬して、上清を分取して前記ザクロ種子エキスを得たことを特徴とする。例えば、振とう抽出させることができる。振とう抽出において、例えば、約4℃等の低温室にてローテーターにセットして回転させながら抽出することができる。また、本発明のヒアルロン酸合成促進用医薬組成物の好ましい実施態様において、前記溶媒は、エタノール抽出物に高い活性が認められるという観点から、エタノールであることを特徴とする。 In a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the pomegranate seeds are crushed, the crushed product is immersed in at least one solvent selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, and hexane, and the supernatant is separated to obtain the pomegranate seed extract. For example, shaking extraction can be performed. In shaking extraction, for example, extraction can be performed while rotating by setting the extract on a rotator in a low-temperature room at about 4°C. In a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, the solvent is ethanol, from the viewpoint that high activity is observed in ethanol extracts.
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法の好ましい実施態様において、食品への応用・適用を想定する観点から、エタノールで抽出したザクロ種子エタノールエキスを、さらに、酢酸エチルと水で分配して、前記酢酸エチル層を減圧濃縮して、酢酸エチル画分としてザクロ種子エキスを得ることを特徴とする。 In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis according to the present invention, from the viewpoint of assuming application to food, the pomegranate seed ethanol extract extracted with ethanol is further partitioned with ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain pomegranate seed extract as an ethyl acetate fraction.
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法の好ましい実施態様において、活性成分を同定するという観点から、さらに、前記酢酸エチル画分を、ヘキサンとメタノールで分配して、酢酸エチル-ヘキサン画分及び酢酸エチル-メタノール画分として、ザクロ種子エキスを得ることを特徴とする。 In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis according to the present invention, from the viewpoint of identifying the active ingredient, the ethyl acetate fraction is further partitioned with hexane and methanol to obtain an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction as a pomegranate seed extract.
なお、酢酸エチル-ヘキサン画分（ＰＳＥ－ＥＡ－Ｈ）、及び酢酸エチル-メタノール画分（ＰＳＥ－ＥＡ－Ｍｅ）とは、以下の通りである。すなわち、エタノールで抽出したザクロ種子エタノール抽出物（ＰＳＥ）を、分液漏斗を用いて酢酸エチルと水で分配し、酢酸エチル層の溶媒をエバポレーターにより減圧濃縮し、酢酸エチル画分（PSE-EA）として、水層は溶媒を凍結乾燥し、水画分（PSE-W）とした場合、PSE-EA をヘキサンとメタノールで分配後、溶媒をそれぞれエバポレーターで減圧濃縮し、得られた画分を、酢酸エチル-ヘキサン画分（PSE-EA-H）及び酢酸エチル-メタノール画分（PSE-EA-Me）とすることができる。 The ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me) are as follows. That is, if pomegranate seed ethanol extract (PSE) extracted with ethanol is partitioned between ethyl acetate and water using a separatory funnel, and the solvent in the ethyl acetate layer is concentrated under reduced pressure using an evaporator to obtain the ethyl acetate fraction (PSE-EA), and the solvent in the water layer is freeze-dried to obtain the water fraction (PSE-W), PSE-EA is partitioned between hexane and methanol, and the solvents are then each concentrated under reduced pressure using an evaporator to obtain the fractions obtained as the ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me).
また、本発明のヒアルロン酸合成促進用医薬組成物の製造方法は、ザクロ種子を粉砕して得た粉砕物を溶媒中に浸漬した後、上清を分取する工程と、前記上清を分取することによりザクロ種子エキスを得る工程と、得られたザクロ種子エキスを有効的な量に調整する工程と、を有することを特徴とするザクロ種子エキスを含有することを特徴とする。 The method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized by comprising the steps of: immersing the pulverized product obtained by pulverizing pomegranate seeds in a solvent, separating the supernatant; obtaining a pomegranate seed extract by separating the supernatant; and adjusting the obtained pomegranate seed extract to an effective amount, which contains a pomegranate seed extract.
ここで、まず、ザクロ種子エキスの調製方法について説明する。まず、ザクロ種子を準備する。ザクロ種子は、必要に応じて洗浄し、乾燥する。乾燥は十分に行なうのが好ましい。後の粉砕を均質に行なうためである。 Here, we will first explain how to prepare pomegranate seed extract. First, pomegranate seeds are prepared. If necessary, the pomegranate seeds are washed and dried. It is preferable to dry them thoroughly, so that the subsequent grinding can be performed uniformly.
次に、ザクロ種子を粉砕する。粉砕の方法は特に限定されず、ボールミル、ハンマーミル、ローラーミル、ロッドミル、サンプルミル、スタンプミル、ディスインテグレーター、乳鉢、冷却装置付きブレンダーなどの公知の粉砕機を用いることができる。なお、粉砕時における発熱により、ザクロ種子組成物の分解等が発生することも考えられることより、粉砕時間を数秒とし、十数回繰り返すことができる。 Next, the pomegranate seeds are pulverized. The pulverization method is not particularly limited, and known pulverizers such as a ball mill, hammer mill, roller mill, rod mill, sample mill, stamp mill, disintegrator, mortar, and blender with a cooling device can be used. Note that, since heat generated during pulverization may cause decomposition of the pomegranate seed composition, the pulverization time is set to several seconds, and the process may be repeated a dozen times.
次いで、ザクロ種子を粉砕し粉砕物を得た後、各種溶媒に前記粉砕物を浸漬する。この場合の溶媒は、特に限定されず、所望とする効果に対応して適宜溶媒を設定することができる。また、本発明のザクロ種子エキスの製造方法の好ましい実施態様において、溶媒が、酢酸エチル、酢酸エチル-ヘキサン、酢酸エチル-メタノール、エタノール、メタノール、ヘキサン、水からなる群から選択される少なくとも１種であることを特徴とする。溶媒としては、酢酸エチル、酢酸エチル-ヘキサン、酢酸エチル-メタノール、エタノール、メタノール、水、へキサン、酢酸エチル、クロロホルム、アセトンなどの極性、非極性溶媒を問わず挙げることができる。好ましくは、酢酸エチル、酢酸エチル-ヘキサン、酢酸エチル-メタノール、メタノール、エタノール、水等を挙げることができる。 Next, the pomegranate seeds are pulverized to obtain a pulverized product, and the pulverized product is immersed in various solvents. The solvent in this case is not particularly limited, and can be appropriately selected according to the desired effect. In a preferred embodiment of the method for producing a pomegranate seed extract of the present invention, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water. Examples of the solvent include ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, hexane, ethyl acetate, chloroform, acetone, and other polar and non-polar solvents. Preferred examples include ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, methanol, ethanol, and water.
浸漬は、緩やかな攪拌下で行なうことができる。各種溶媒に前記粉砕物を浸漬して各種溶液を得る。各種溶液について、溶液の状態に応じて攪拌を行い、場合によりそのまま溶液を放置しても良い。攪拌する場合には、特に限定されないが、１０時間～４８時間、好ましくは、およそ1日（24時間）攪拌を持続させることができる。 The immersion can be carried out under gentle stirring. The ground material is immersed in various solvents to obtain various solutions. The various solutions are stirred depending on the state of the solution, and in some cases the solution may be left as is. When stirring, the stirring can be continued for 10 to 48 hours, preferably for about one day (24 hours), although there are no particular limitations.
その後、上清を分取することによりザクロ種子エキスを得ることができる。必要に応じて、上清を蒸発乾固する。蒸発乾固は、エバポレーターを用いて、２０℃～６０℃、好ましくは、３７℃～４０℃の温浴上で行なうことができる。蒸発乾固することにより、ザクロ種子エキスを長期間保存することができる。 Then, the supernatant can be separated to obtain pomegranate seed extract. If necessary, the supernatant can be evaporated to dryness. The evaporation to dryness can be carried out using an evaporator in a warm bath at 20°C to 60°C, preferably 37°C to 40°C. By evaporating to dryness, the pomegranate seed extract can be stored for a long period of time.
ザクロ種子中に含まれる成分は、ザクロ種子を極性の異なる溶媒を用いて抽出することにより、その物性により振り分けられる。したがって、使用した溶媒により、ザクロ種子エキスの成分の種類及び含有量は異なる。 The components contained in pomegranate seeds are extracted using solvents of different polarity and are separated based on their physical properties. Therefore, the type and content of components in pomegranate seed extract varies depending on the solvent used.
また、本発明の医薬部外品は、本発明の組成物を有効成分とすることを特徴とする。本発明に適用可能な医薬部外品については、本発明のヒアルロン酸合成促進用医薬組成物を有効成分とする限り、特に限定されない。 The quasi-drug of the present invention is characterized by having the composition of the present invention as an active ingredient. There are no particular limitations on the quasi-drugs applicable to the present invention, so long as they have the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention as an active ingredient.
＜有効量＞
本発明による組成物は、有効的な量のザクロ種子エキス、及び適当な投与形態の形で調製される。
<Effective amount>
The compositions according to the present invention are prepared by providing an effective amount of pomegranate seed extract and a suitable dosage form.
本発明の組成物におけるザクロ種子エキスの投与量は、投与対象患者の病態及びその重篤度、投薬形態、選択した投与経路及び１日当たりの投与回数等により変更することができる。 The dosage of pomegranate seed extract in the composition of the present invention can be varied depending on the pathology and severity of the patient to be treated, the dosage form, the selected administration route, and the number of doses per day, etc.
本発明の組成物におけるザクロ種子エキスの投与量は、マウスで実施する時、1000 mg/kg/day投与量に設定することができ、ヒトにおいては感受性の相違等により、更に低い量であることが好ましい。 The dosage of pomegranate seed extract in the composition of the present invention can be set at 1000 mg/kg/day when tested in mice, and a lower dosage is preferable in humans due to differences in sensitivity, etc.
また、投与形態は、経口剤(タブレット、カプセル、被膜タブレット、顆粒、溶液、シロップ)、塗布のための軟膏やクリームなどを挙げることができる。投与対象患者は、皮膚の水分補給及びバリア機能という観点から、女性、男性、大人、子供等を問わず、対象とすることが可能である。 The dosage form may be oral (tablets, capsules, coated tablets, granules, solutions, syrups), or topical ointments or creams. From the standpoint of skin hydration and barrier function, the target patients may be men, women, adults, children, etc.
投与形態には、従来の他の成分、例えば、安定保存剤、甘味料、着色剤、芳香料などを含むことができる。 The dosage form may contain other conventional ingredients, such as preservatives, sweeteners, colorants, flavorings, etc.
＜急性毒性試験＞
ザクロ種子エキスの含有成分については、リノレン酸も含まれる成分であるため毒性は認められないと考えられている。
<Acute toxicity test>
As for the ingredients contained in pomegranate seed extract, including linoleic acid, it is believed that there is no toxicity.
なお、略号については、以下の通りである。
・BMP：Bone Morphogenetic Proteins
・CE：Cornified Envelope
・CSF：Colony-stimulating factors
・COL：Collagen
・COL1A1：Type I collagen α1 chain
・DEPC：Diethylpyrocarbonate
・DMSO：Dimethyl Sulfoxide
・ECM：Extracellular Matrix
・EGF：Epidermal Growth Factor
・ELN：Elastin
・FBS：Fetal Bovine Serum
・F1：PSE-Ethyl Acetate-Hexane-Fraction 1 画分
・F2：PSE-Ethyl Acetate-Hexane-Fraction 2 画分
・F3：PSE-Ethyl Acetate-Hexane-Fraction 3 画分
・F4：PSE-Ethyl Acetate-Hexane-Fraction 4 画分
・HA：Hyaluronic Acid
・HABP：Hyaluronic Acid Binding Protein
・HAS：Hyaluronic Acid Synthase
・HIF-1α：Hypoxia-inducible factor-1α
・HYAL：Hyaluronidase
・IL：Interleukins
・INV：Involcrin
・KGF：Keratinocyte Growth Factor
・LOR：Loricrin
・MAPK：Mitogen-activated protein kinase
・miRNA：micro RNA
・MMP：Matrix metalloproteinase
・NF-κB：Nuclear factor-kappa B
・NMF：Natural moisturizing factor
・Nrf-2：Nuclear factor erythroid 2-related factor 2
・PBS：Phosphate Buffered Saline
・PC：Positive Control
・PSE：Punica gtanatum Seed Ethanol extract（ザクロ種子エタノール抽出物）
・PSE-Bu-Me：PSE-Butanol-Methanol 画分
・PSE-EA：PSE-Ethyl Acetate 画分
・PSE-EA-H：PSE-Ethyl Acetate-Hexane 画分
・PSE-EA-Me：PSE-Ethyl Acetate-Methanol 画分
・PSE-W：PSE-Water 画分
・ROS：Reactive oxygen species
・RT-PCR：Reverse Transcription-Polymerase Chain Reaction
・SASP：Senescence-associated secretory phenotype
・TGF-β：Transforming Growth Factor-１
・TIMP:TISSUE INHIBITORS OF MMPS
・TGM：Transglutaminase
・TNF-α：Tumor Necrosis Factor-α
・UV：Ultraviolet
・VC：Vitamin C
The abbreviations are as follows:
・BMP：Bone Morphogenetic Proteins
・CE: Cornified Envelope
・CSF：Colony-stimulating factors
・COL: Collagen
・COL1A1：Type I collagen α1 chain
・DEPC：Diethylpyrocarbonate
・DMSO: Dimethyl Sulfoxide
・ECM: Extracellular Matrix
・EGF：Epidermal Growth Factor
・ELN: Elastin
・FBS: Fetal Bovine Serum
・F1: PSE-Ethyl Acetate-Hexane-Fraction 1 fraction ・F2: PSE-Ethyl Acetate-Hexane-Fraction 2 fraction ・F3: PSE-Ethyl Acetate-Hexane-Fraction 3 fraction ・F4: PSE-Ethyl Acetate-Hexane-Fraction 4 fraction ・HA: Hyaluronic Acid
・HABP：Hyaluronic Acid Binding Protein
・HAS：Hyaluronic Acid Synthase
・HIF-1α: Hypoxia-inducible factor-1α
・HYAL: Hyaluronidase
・IL: Interleukins
・INV: Involcrin
・KGF: Keratinocyte Growth Factor
・LOR: Loricrin
・MAPK: Mitogen-activated protein kinase
・miRNA: microRNA
・MMP：Matrix metalloproteinase
・NF-κB: Nuclear factor-kappa B
・NMF：Natural moisturizing factor
・Nrf-2：Nuclear factor erythroid 2-related factor 2
・PBS：Phosphate Buffered Saline
・PC: Positive Control
・PSE: Punica granatum Seed Ethanol Extract
・PSE-Bu-Me: PSE-Butanol-Methanol fraction ・PSE-EA: PSE-Ethyl Acetate fraction ・PSE-EA-H: PSE-Ethyl Acetate-Hexane fraction ・PSE-EA-Me: PSE-Ethyl Acetate-Methanol fraction ・PSE-W: PSE-Water fraction ・ROS: Reactive oxygen species
・RT-PCR：Reverse Transcription-Polymerase Chain Reaction
・SASP：Senescence-associated secretory phenotype
・TGF-β：Transforming Growth Factor-1
・TIMP:TISSUE INHIBITORS OF MMPS
・TGM: Transglutaminase
・TNF-α：Tumor Necrosis Factor-α
・UV: Ultraviolet
・VC: Vitamin C
以下では本発明の一実施例を説明するが、本発明は、下記の実施例に限定して解釈されるものではない。 One embodiment of the present invention is described below, but the present invention should not be interpreted as being limited to the following embodiment.
実施例１
まず、溶媒として、エタノールを用いて、ザクロ種子エキスの調製を試みた。ザクロ種子を粉砕し、100%エタノールで振とう抽出した。これらを濃縮乾固して再溶解し、ザクロ種子エタノール抽出物として実験に供した。さらに、エタノールで抽出したザクロ種子エタノール抽出物（以下、ＰＳＥともいう。）を、分液漏斗を用いて酢酸エチルと水で分配し、酢酸エチル層の溶媒をエバポレーターにより減圧濃縮し、酢酸エチル画分（PSE-EA）として、水層は溶媒を凍結乾燥し、水画分（PSE-W）とした場合、PSE-EA をヘキサンとメタノールで分配後、溶媒をそれぞれエバポレーターで減圧濃縮し、得られた画分を、酢酸エチル-ヘキサン画分（以下、PSE-EA-Hともいう。）及び酢酸エチル-メタノール画分（以下、PSE-EA-Meともいう。）とした。具体的には、以下の通りである。
Example 1
First, we tried to prepare a pomegranate seed extract using ethanol as a solvent. Pomegranate seeds were crushed and extracted by shaking with 100% ethanol. These were concentrated to dryness and redissolved, and used as a pomegranate seed ethanol extract for the experiment. Furthermore, the pomegranate seed ethanol extract (hereinafter also referred to as PSE) extracted with ethanol was partitioned with ethyl acetate and water using a separatory funnel, and the solvent of the ethyl acetate layer was concentrated under reduced pressure using an evaporator to obtain an ethyl acetate fraction (PSE-EA), and the solvent of the water layer was freeze-dried to obtain a water fraction (PSE-W). After partitioning PSE-EA with hexane and methanol, the solvents were each concentrated under reduced pressure using an evaporator, and the obtained fractions were named an ethyl acetate-hexane fraction (hereinafter also referred to as PSE-EA-H) and an ethyl acetate-methanol fraction (hereinafter also referred to as PSE-EA-Me). Specifically, the procedure is as follows.
＜PSE（Punica granatum Seed Ethanol extract）の調製方法＞
乾燥ザクロ種子をミルサーで粉末化し、10 倍量 100% エタノール（和光純薬工業株式会社製）を加え、4℃、24 時間で低温振盪抽出を行った。24 時間後、室温、3,000 rpm で 20 分間遠心分離を行い、上清を回収した。上清を減圧濃縮して得られた濃縮物をザクロ種子エタノール抽出物（PSE : Punica granatum Seed Ethanol extract）とした。
<Preparation method of PSE (Punica granatum Seed Ethanol extract)>
Dried pomegranate seeds were powdered in a mill, and 10 volumes of 100% ethanol (Wako Pure Chemical Industries, Ltd.) were added and low-temperature shaking extraction was performed at 4°C for 24 hours. After 24 hours, the mixture was centrifuged at room temperature at 3,000 rpm for 20 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure, and the resulting concentrate was used as pomegranate seed ethanol extract (PSE: Punica granatum Seed Ethanol extract).
＜酢酸エチル-ヘキサン画分（PSE-EA-H）及び酢酸エチル-メタノール画分（PSE-EA-Me）の調整方法＞
PSE 2.00gを、分液漏斗を用いて酢酸エチル（100ml×3）とMiliQ 水(100 ml）で分配し、酢酸エチル層の溶媒をエバポレーターにより減圧濃縮し、酢酸エチル画分（PSE-EA）とした。水層は溶媒を凍結乾燥し、水画分（PSE-W）とした。さらにPSE-EAをヘキサン（100ml×3）とメタノール（100ml）で分配後、溶媒をそれぞれエバポレーターで減圧濃縮し、酢酸エチル-ヘキサン画分（PSE-EA-H）と酢酸エチル-メタノール画分（PSE-EA-Me）を得た。収量の多いPSE-EA-Hをヘキサン（100ml）、ヘキサンと酢酸エチルの混合物（ヘキサン:酢酸エチル=９:１の場合（ヘキサン９０ｍＬ、酢酸エチル１０ｍＬ）、ヘキサン:酢酸エチル=８:２の場合（ヘキサン８０ｍＬ、酢酸エチル２０ｍＬ）、ヘキサン:酢酸エチル=６:４の場合（ヘキサン４０ｍＬ、酢酸エチル６０ｍＬ））、酢酸エチル（100ml）、メタノール（100ml）の順でカラム（30φ×300mm）に流し、分配した。下記の式より、画分の収率を算出し、画分調製のフローチャートを図３３に示した。
<Preparation method of ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me)>
2.00g of PSE was partitioned between ethyl acetate (100ml x 3) and MiliQ water (100ml) using a separatory funnel, and the solvent in the ethyl acetate layer was concentrated under reduced pressure using an evaporator to obtain the ethyl acetate fraction (PSE-EA). The solvent in the water layer was freeze-dried to obtain the water fraction (PSE-W). PSE-EA was further partitioned between hexane (100ml x 3) and methanol (100ml), and the solvents were then concentrated under reduced pressure using an evaporator to obtain the ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me). The PSE-EA-H, which had a high yield, was passed through a column (30φ×300 mm) in the following order and distributed: hexane (100 ml), a mixture of hexane and ethyl acetate (hexane:ethyl acetate=9:1 (hexane 90 ml, ethyl acetate 10 ml), hexane:ethyl acetate=8:2 (hexane 80 ml, ethyl acetate 20 ml), hexane:ethyl acetate=6:4 (hexane 40 ml, ethyl acetate 60 ml)), ethyl acetate (100 ml), and methanol (100 ml). The fraction yield was calculated from the following formula, and a flowchart of fraction preparation is shown in FIG. 33.
収率（％）＝（画分の質量（ｇ）/ PSEの質量（ｇ））×１００ Yield (%) = (mass of fraction (g) / mass of PSE (g)) x 100
調製した抽出物は実験に使用するまで-20℃で冷凍保存した。 The prepared extracts were stored frozen at -20°C until use in experiments.
次に、ヒアルロン酸（ＨＡ）の合成及び角化膜（ＣＥ）の形成に関して、作用メカニズムを解明するために反定量的RT-PCR法を用いて評価し、RT-PCRにて発現増加が確認された遺伝子については定量的リアルタイムPCRにて評価を行った。具体的には、CE 形成に関与する遺伝子としてLOR、INV、TGM-1の発現を評価し、PSE およびその画分の添加における皮膚の水分保湿機能の評価を行うため、HAに着目し、 HAを合成するHAS-1、HAS-2、HAS-3の遺伝子発現の評価を行った。 Next, in order to clarify the mechanism of action regarding the synthesis of hyaluronic acid (HA) and the formation of the cornified epithelium (CE), we used inverse quantitative RT-PCR to evaluate, and genes whose expression was confirmed to be increased by RT-PCR were evaluated by quantitative real-time PCR. Specifically, we evaluated the expression of LOR, INV, and TGM-1, which are genes involved in CE formation, and to evaluate the skin's moisture retention function when PSE and its fractions were added, we focused on HA and evaluated the gene expression of HAS-1, HAS-2, and HAS-3, which synthesize HA.
＜細胞の培養方法＞
＜試薬＞
・D-MEM（High Glucose）with L-Glutamine, Phenol Red, and Sodium Pyruvate： 富士フイルム 和光純薬株式会社
・Penicillin-Streptomycin Solution（×100）：和光純薬工業株式会社
・Phosphate Buffered Saline（PBS）
・Fetal Bovine Serum（FBS）：シグマ・アルドリッチ・ジャパン合同会社
・0.25%（w/v）Trypsin/EDTA with Phenol Red：和光純薬工業株式会社
・0.4 w/v% Trypan Blue Solution：和光純薬工業株式会社
・Dimethyl Sulfoxide（DMSO）：和光純薬工業株式会社
・1 mg/ml Insulin：Sigma-Aldrich
・4 mg/ml Transferrin：Sigma-Aldrich
・4 mM Ethanol Amine：和光純薬工業株式会社
・5 μM Sodium Selenite：和光純薬工業株式会社
<Cell culture method>
<Reagents>
・D-MEM (High Glucose) with L-Glutamine, Phenol Red, and Sodium Pyruvate: Fujifilm Wako Pure Chemical Industries, Ltd. ・Penicillin-Streptomycin Solution (x100): Wako Pure Chemical Industries, Ltd. ・Phosphate Buffered Saline (PBS)
・Fetal Bovine Serum (FBS): Sigma-Aldrich Japan, LLC ・0.25% (w/v) Trypsin/EDTA with Phenol Red: Wako Pure Chemical Industries, Ltd. ・0.4 w/v% Trypan Blue Solution: Wako Pure Chemical Industries, Ltd. ・Dimethyl Sulfoxide (DMSO): Wako Pure Chemical Industries, Ltd. ・1 mg/ml Insulin: Sigma-Aldrich
・4 mg/ml Transferrin: Sigma-Aldrich
・4 mM Ethanol Amine: Wako Pure Chemical Industries, Ltd. ・5 μM Sodium Selenite: Wako Pure Chemical Industries, Ltd.
＜試薬調製＞
・1 mg/ml Insulin
Insulinを100 mg秤量し、50mlのPBSに溶解した。この時、結晶を溶解するために濃塩酸を50μl添加した。0.22μmのフィルターでろ過滅菌し、1mlごとに分注し、冷凍保存したものを溶解し使用した。
<Reagent preparation>
・1 mg/ml Insulin
100 mg of insulin was weighed and dissolved in 50 ml of PBS. At this time, 50 μl of concentrated hydrochloric acid was added to dissolve the crystals. The solution was sterilized by filtration through a 0.22 μm filter, dispensed into 1 ml portions, and frozen. The solution was dissolved and used.
・4 mg/ml Transferrin
Apo-transferrin humanを100mg秤量し、25mlの PBSで溶解した。0.22μmのフィルターでろ過滅菌し、1mlごとに分注し、冷凍保存したものを溶解し使用した。
・4 mg/ml Transferrin
100 mg of human apo-transferrin was weighed out and dissolved in 25 ml of PBS. The solution was sterilized by filtration through a 0.22 μm filter, dispensed into 1 ml portions, and frozen. The solution was then dissolved and used.
・4 mg/ml Ethanol Amine
2-Aminoethaolを蒸留水で250倍希釈し、さらに25倍希釈した。0.22μmのフィ ルターでろ過滅菌し、1mlごとに分注し、冷凍保存したものを溶解し使用した。
・4 mg/ml Ethanol Amine
2-Aminoethaol was diluted 250-fold with distilled water, and then further diluted 25-fold. It was sterilized by filtration through a 0.22 μm filter, dispensed into 1 ml portions, and stored frozen. The solution was then dissolved and used.
・5μM Sodium Selenite
Sodium Seleniteを43.2mg秤量し、5mlの蒸留水で溶解し、50mM水溶液を調製した。その後、50mM水溶液を10,000倍希釈し、さらに100倍希釈した。その後0.22μmのフィルターでろ過滅菌し、1 mlごとに分注し、冷凍保存したものを溶解し使用した。
・5μM Sodium Selenite
43.2 mg of sodium selenite was weighed out and dissolved in 5 ml of distilled water to prepare a 50 mM aqueous solution. The 50 mM aqueous solution was then diluted 10,000 times and then further diluted 100 times. The solution was then sterilized by filtration through a 0.22 μm filter, dispensed into 1 ml portions, and frozen for storage. The solution was then dissolved and used.
・2×ITES/D-MEM（2% FBS）
D-MEM培地（1%量のPenicillin-Streptomycin Solution 添加済）18.8mlにFBSを 400μl、1mg/ml Insulin（I）、4mg/ml Transferrin（T）、4mg/ml Ethanol Amine（E）、5μM Sodium Selenite（S）をそれぞれ200μl添加し、転倒混和により軽く攪拌した。
・2xITES/D-MEM (2% FBS)
To 18.8 ml of D-MEM medium (containing 1% penicillin-streptomycin solution), 400 μl of FBS, 200 μl each of 1 mg/ml insulin (I), 4 mg/ml transferrin (T), 4 mg/ml ethanol amine (E), and 5 μM sodium selenite (S) were added and gently mixed by inversion.
＜培養条件＞
HaCaT細胞は1%量のPenicillin-Streptomycin Solutionを添加したD-MEMに10%量のFBSを添加した培養培地を用いて、37℃、5% CO2濃度条件下で培養を行った。
<Culture conditions>
HaCaT cells were cultured at 37°C under 5% CO2 concentration conditions using a culture medium consisting of D-MEM supplemented with 1% Penicillin-Streptomycin Solution and 10% FBS.
＜細胞の継代方法＞
10cmの培養ディッシュに80～90%サブコンフルエント状態に増殖した細胞にPBS を添加・除去し、細胞を洗浄した。その後、0.25%（w/v）Trypsin/EDTAを添加し、37℃、5% CO2濃度条件のもと、5分間インキュベートした。FBSでTrypsin 反応を停 止させ、D-MEMを添加した後、ピペッティングにより単細胞浮遊液とし、15 ml遠心 管に回収した。1500rpmで5分間遠心分離を行い、上清を除去した。沈殿物にD-MEM を添加し、ピペッティングによる懸濁の後、10% FBS存在下で細胞播種を行った。
<Cell passage method>
PBS was added to and removed from cells grown in a 10 cm culture dish to a subconfluent state of 80-90%, and the cells were washed. Then, 0.25% (w/v) Trypsin/EDTA was added and incubated at 37°C and 5% CO2 for 5 minutes. The Trypsin reaction was stopped with FBS, D-MEM was added, and a single cell suspension was obtained by pipetting and collected in a 15 ml centrifuge tube. The cells were centrifuged at 1500 rpm for 5 minutes and the supernatant was removed. D-MEM was added to the precipitate, suspended by pipetting, and the cells were seeded in the presence of 10% FBS.
＜培養方法＞
10cmの培養ディッシュに80～90％サブコンフルエント状態に増殖した細胞にPBS を添加・除去し、細胞を洗浄した。その後、0.25%（w/v）Trypsin/EDTAを添加し、37℃、5% CO2濃度条件のもと、5分間インキュベートした。FBSでTrypsin反応を停 止させ、D-MEMを添加した後、ピペッティングにより単細胞浮遊液とし、15 ml遠心 管に回収した。1,500rpmで5分間遠心分離を行い、上清を除去した。最終細胞密度が３．０×１０５cells/mlになるようにD-MEM（10%FBS）を添加し、ピペッティングにより懸濁した。6 well-plateに細胞懸濁液を1 mlずつ播種し、３７℃、５％ ＣＯ２濃度条件下で24時間前培養を行った。
<Culture method>
PBS was added to and removed from cells grown to 80-90% subconfluent in a 10 cm culture dish, and the cells were washed. Then, 0.25% (w/v) Trypsin/EDTA was added and incubated at 37°C and 5% CO2 for 5 minutes. The Trypsin reaction was stopped with FBS, D-MEM was added, and a single cell suspension was obtained by pipetting and collected in a 15 ml centrifuge tube. The cells were centrifuged at 1,500 rpm for 5 minutes and the supernatant was removed. D-MEM (10% FBS) was added so that the final cell density was 3.0 x 105 cells/ml, and the cells were suspended by pipetting. 1 ml of the cell suspension was seeded on a 6-well plate and pre-cultured for 24 hours at 37°C and 5% CO2 .
PSEおよびその画分はDMSOで溶解し、最終濃度の2倍濃度になるようにD-MEMにて希釈を行った。サンプルの濃度範囲は第2章の結果より、細胞毒性が確認されなか った濃度範囲とした。前培養を行った後、PBSにて洗浄を行い、2×ITES/D-MEM（2% FBS）と各サンプルを1ml/well添加し48時間および96時間培養を行った。96時間 培養のプレートは48時間培養後に培地交換を行った。Controlとして終濃度1% DMSO を加えたD-MEMを添加した。 PSE and its fractions were dissolved in DMSO and diluted with D-MEM to twice the final concentration. The concentration range of the samples was set to a range in which no cytotoxicity was confirmed based on the results in Chapter 2. After pre-incubation, the plates were washed with PBS, and 2xITES/D-MEM (2% FBS) and each sample were added at 1ml/well for 48 and 96 hours of incubation. For the 96-hour incubation plate, the medium was replaced after 48 hours of incubation. As a control, D-MEM with a final concentration of 1% DMSO was added.
＜RT-PCR法＞
PCR法はDNA配列上の特定の領域（目的のDNA領域）を１対のプライマーと耐熱性 DNAポリメラーゼを用いて増幅させる方法である。PCR法では「１.熱変性→２．アニ ーリング→３．伸長反応」という3段階の温度変化による反応を繰り返すことによって、理論的にはDNA量を指数関数的に増幅させることが出来る。PCR法を用いることで、微量のDNAから特定のDNA領域のみを迅速かつ簡便に増幅させることができる。 RNAを鋳型としてDNAを合成（逆転写）した後に、PCR法によりDNAを増幅させる操作である。
<RT-PCR method>
PCR is a method to amplify a specific region (target DNA region) on a DNA sequence using a pair of primers and a heat-resistant DNA polymerase. In PCR, the amount of DNA can theoretically be exponentially amplified by repeating a three-step reaction with temperature change: 1. Thermal denaturation → 2. Annealing → 3. Extension reaction. PCR can quickly and easily amplify only a specific DNA region from a small amount of DNA. After synthesizing DNA (reverse transcription) using RNA as a template, the DNA is amplified by PCR.
＜試薬＞
・ISOGENII：ニッポンジーン
・DEPC water
・2-Propanol：和光純薬工業株式会社
・75% Ethanol：関東化学株式会社
・ReverTra Ace：東洋紡株式会社
・25 mM Oligo dT primer：東洋紡株式会社
・Taq DNA polymerase：東洋紡株式会社
・KOD-plus-Ver.2：東洋紡株式会社
・Dimethyl Sulfoxide（DMSO）：和光純薬株式会社
・MilliQ
・Agarose Basic：タカラバイオ株式会社
・エチジウムブロマイド溶液：ナカライテスク株式会社
・Gene Ladder 100（0.1-2 kbp）：ニッポンジーン
・Boric Acid：和光純薬工業株式会社
・bromophenol blue：ナカライテスク株式会社
・キシレンシアノール：ナカライテスク株式会社
・Tris（hydroxymethyl）aminomethane：和光純薬工業株式会社
・Glycerol：和光純薬工業株式会社
<Reagents>
・ISOGENII: Nippon Gene・DEPC water
・2-Propanol: Wako Pure Chemical Industries, Ltd. ・75% Ethanol: Kanto Chemical Co., Ltd. ・ReverTra Ace: Toyobo Co., Ltd. ・25 mM Oligo dT primer: Toyobo Co., Ltd. ・Taq DNA polymerase: Toyobo Co., Ltd. ・KOD-plus-Ver.2: Toyobo Co., Ltd. ・Dimethyl Sulfoxide (DMSO): Wako Pure Chemical Industries, Ltd. ・MilliQ
・Agarose Basic: Takara Bio Inc. ・Ethidium bromide solution: Nacalai Tesque Inc. ・Gene Ladder 100 (0.1-2 kbp): Nippon Gene Co., Ltd. ・Boric Acid: Wako Pure Chemical Industries, Ltd. ・Bromophenol blue: Nacalai Tesque Inc. ・Xylene cyanol: Nacalai Tesque Inc. ・Tris (hydroxymethyl) aminomethane: Wako Pure Chemical Industries, Ltd. ・Glycerol: Wako Pure Chemical Industries, Ltd.
＜試薬調製＞
・5×TBE buffer
Tris 27g、Boric Acid 13.75g、および 0.5M EDTA（pH8.0）10mlに蒸留水加え、全量を500mlとした。実験使用時は蒸留水にて1×TBEに希釈し利用した。
<Reagent preparation>
・5×TBE buffer
Distilled water was added to 27 g of Tris, 13.75 g of boric acid, and 10 ml of 0.5 M EDTA (pH 8.0) to make up a total volume of 500 ml. When used in experiments, the solution was diluted with distilled water to 1x TBE.
・TE buffer
MillQ 49.4 mlに対して0.5M EDTA（pH8.0）100μlおよび1M Tris-HCl 500μlを添加し混合した。
・TE buffer
To 49.4 ml of MillQ, 100 μl of 0.5 M EDTA (pH 8.0) and 500 μl of 1 M Tris-HCl were added and mixed.
・6×Loading Dye
bromophenol blue 15mg、キシレンシアノール 15mg、0.5M EDTA（pH8.0）1.2 ml、およびGlycerol 1.8mlに蒸留水を加え、全量を10mlとし、混合した。混合したものを1mlずつ分注し、-20℃で冷凍保存したものを溶解し利用した。
・6×Loading Dye
Distilled water was added to 15 mg of bromophenol blue, 15 mg of xylene cyanol, 1.2 ml of 0.5 M EDTA (pH 8.0), and 1.8 ml of glycerol to make a total volume of 10 ml, and mixed. The mixture was dispensed into 1 ml aliquots, frozen and stored at -20°C, and then dissolved and used.
＜primer＞
・GAPDH：ファスマック株式会社
・Loricrin：ファスマック株式会社
・Involcrin：ファスマック株式会社
・Transglutaminase-1：ファスマック株式会社
・Hyaluronic Acid Synthase-1：ファスマック株式会社
・Hyaluronic Acid Synthase-2：ファスマック株式会社
・Hyaluronic Acid Synthase-3：ファスマック株式会社
＜primer＞
・GAPDH: FASMAC Corporation ・Loricrin: FASMAC Corporation ・Involcrin: FASMAC Corporation ・Transglutaminase-1: FASMAC Corporation ・Hyaluronic Acid Synthase-1: FASMAC Corporation ・Hyaluronic Acid Synthase-2: FASMAC Corporation ・Hyaluronic Acid Synthase-3: FASMAC Corporation
表１にprimer の塩基配列（RT-PCR）を示す。 Table 1 shows the primer sequences (RT-PCR).
＜機器＞
・Nano Drop 2000：Thermo
・Thermal Cycler（My Cycler）：Bio Rad
・Thermal Cycler（Mini Amp Plus）：Thermo
・Mupid-2 plus：タカラバイオ株式会社
・AE-6933FXCF：ATTO 株式会社
<Equipment>
・Nano Drop 2000: Thermo
・Thermal Cycler (My Cycler): Bio Rad
・Thermal Cycler (Mini Amp Plus): Thermo
・Mupid-2 plus: Takara Bio Inc. ・AE-6933FXCF: ATTO Inc.
＜方法＞
＜Total RNA の抽出＞
48時間及び96時間培養後、PBSを添加・除去し細胞を洗浄した。ISOGENIIを700μl/well添加した後、ピペッティングにより懸濁し1.5 mlチューブに回収した。DEPC waterを300μl加え、ボルテックスミキサーを用いて15秒間激しく攪拌した。室温15分間静置後、12,000×g、15分間遠心分離を行い、上清から500μlを新たな 1.5mlチューブに回収した。そこに同量（500μl）の2-Propanolと20 mgグリコーゲン溶液1μl加え転倒混和した。室温10分間静置後、12,000×g、15分間遠心分離を行い、上清を除去した。沈殿物に75% Ethanol 300μl/tubeを加え、転倒混和により洗浄した後、8,000×g、3分間遠心分離を行い、上清を除去した。この操作を2 回繰り返し、上清をできる限り除去した後、チューブ内の液体を揮発させるためにチューブ状にサランラップ（登録商標）をかけて20-30分間静置した。チューブを氷上へ移し、20 μl/tubeのDEPC waterを加え、ピペッティングにより沈殿物を溶解したものを Total RNA溶液とした。Nano Drop 2000を用いてTotal RNA溶液の濃度および純度を測定した。
Methods
<Extraction of total RNA>
After 48 and 96 hours of incubation, PBS was added and removed to wash the cells. ISOGENII was added at 700μl/well, suspended by pipetting, and collected in a 1.5 ml tube. 300μl of DEPC water was added and vigorously stirred for 15 seconds using a vortex mixer. After standing at room temperature for 15 minutes, the cells were centrifuged at 12,000×g for 15 minutes, and 500μl of the supernatant was collected in a new 1.5 ml tube. The same amount (500μl) of 2-Propanol and 1μl of 20 mg glycogen solution were added thereto and mixed by inversion. After standing at room temperature for 10 minutes, the cells were centrifuged at 12,000×g for 15 minutes, and the supernatant was removed. 75% Ethanol 300μl/tube was added to the precipitate, washed by inversion, centrifuged at 8,000×g for 3 minutes, and the supernatant was removed. This procedure was repeated twice, and the supernatant was removed as much as possible. The tubes were then wrapped in Saran Wrap (registered trademark) and left to stand for 20-30 minutes to volatilize the liquid in the tubes. The tubes were then transferred onto ice, and 20 μl/tube of DEPC water was added. The precipitate was dissolved by pipetting to give the total RNA solution. The concentration and purity of the total RNA solution were measured using Nano Drop 2000.
＜cDNAの合成＞
0.2ml PCRチューブに各サンプルのTotal RNA 4μgと表２に示した溶液を加え、 液量全体が20μlになるように、DEPC waterを加え、Thermal Cyclerを用いて表３ の反応条件で逆転写反応を行った。表２は、逆転写反応液調製条件を示し、表３は、逆転写反応条件を示す。
<cDNA synthesis>
4 μg of total RNA from each sample and the solution shown in Table 2 were added to a 0.2 ml PCR tube, DEPC water was added to bring the total volume to 20 μl, and reverse transcription reaction was carried out using a Thermal Cycler under the reaction conditions shown in Table 3. Table 2 shows the conditions for preparing the reverse transcription reaction solution, and Table 3 shows the reverse transcription reaction conditions.
＜PCR反応による目的遺伝子の増幅＞
TE bufferで5倍希釈した各サンプルのcDNA溶液を使用した。Primerは、TE bufferで20 pmol/mlに調製し使用した。0.2ml PCRチューブに反応溶液全量が25μlになるように、下記の表４に従いそれぞれの反応液を混合した。それらをThermal Cyclerを用いて、表５の反応条件でPCR反応を行った。Internal ControlとしてGAPDHを用いて補正を行った。表４は、各遺伝子のPCR反応液条件を示す。また、表５は、各遺伝子のPCR反応条件を示す。
<Amplification of target genes by PCR reaction>
The cDNA solution of each sample was diluted 5-fold with TE buffer and used. Primers were adjusted to 20 pmol/ml with TE buffer and used. Each reaction solution was mixed in a 0.2 ml PCR tube according to Table 4 below so that the total volume of the reaction solution was 25 μl. PCR reactions were carried out using a Thermal Cycler under the reaction conditions in Table 5. Correction was performed using GAPDH as an internal control. Table 4 shows the PCR reaction solution conditions for each gene. Table 5 also shows the PCR reaction conditions for each gene.
＜電気泳動＞
Agarose Basic「TaKaRa」2gに1×TBE bufferを加え、電子レンジで加熱し溶解した。アガロース溶液をゲルメーカーに流し込み、室温で15分以上静置し2%アガロースゲルを作成した。各PCR産物：6×Loading Dye＝5：1で混合し、ゲルのウェルにサンプルおよび Gene Ladderをアプライした。100V、30-40分間電気泳動を行い、ゲルをエチジウムブロマイドで染色した。染色したゲルはゲル撮影装置にて増幅産物を確認した。得られた増幅バンドの強度は画像解析ソフト（CS Analyzer 4）を用いて数値化した。Controlとサンプル添加群間との統計学的有意差は Dunnett’s Testにより検定し、*p＜0.05として表した。
<Electrophoresis>
2 g of Agarose Basic "TaKaRa" was mixed with 1x TBE buffer and dissolved by heating in a microwave oven. The agarose solution was poured into a gel maker and left at room temperature for more than 15 minutes to create a 2% agarose gel. PCR products and 6x Loading Dye were mixed at a ratio of 5:1, and samples and gene ladders were applied to the wells of the gel. Electrophoresis was performed at 100V for 30-40 minutes, and the gel was stained with ethidium bromide. The stained gel was examined for amplified products using a gel camera. The intensity of the amplified bands obtained was quantified using image analysis software (CS Analyzer 4). Statistical differences between the control and sample-added groups were examined using Dunnett's test and expressed as *p<0.05.
＜リアルタイム PCR＞
＜試薬＞
・ISOGENII：ニッポンジーン
・DEPC water
・2-Propanol：和光純薬工業株式会社
・75% Ethanol：関東化学株式会社
・ReverTra Ace：東洋紡株式会社
・25 mM Oligo dT primer：東洋紡株式会社
・THUNDERBIRD SYBR qPCR Mix：東洋紡株式会社
・MilliQ
<Real-time PCR>
<Reagents>
・ISOGENII: Nippon Gene・DEPC water
・2-Propanol: Wako Pure Chemical Industries, Ltd. ・75% Ethanol: Kanto Chemical Co., Ltd. ・ReverTra Ace: Toyobo Co., Ltd. ・25 mM Oligo dT primer: Toyobo Co., Ltd. ・THUNDERBIRD SYBR qPCR Mix: Toyobo Co., Ltd. ・MilliQ
＜primer＞
・β-actin：ファスマック株式会社
・Loricrin：ファスマック株式会社
・Involcrin：ファスマック株式会社
・Hyaluronic Acid Synthase-2：ファスマック株式会社
＜primer＞
・β-actin: FASMAC Corporation ・Loricrin: FASMAC Corporation ・Involcrin: FASMAC Corporation ・Hyaluronic Acid Synthase-2: FASMAC Corporation
表６に、primer の塩基配列（リアルタイム PCR）を示す。 Table 6 shows the primer sequences (real-time PCR).
＜機器＞
・Nano Drop 2000：Thermo
・CFX96TM Real-Time System：Bio Rad
<Equipment>
・Nano Drop 2000: Thermo
・CFX96TM Real-Time System: Bio Rad
＜方法＞
＜Total RNAの抽出＞
48時間及び96時間培養後、PBSを添加・除去し細胞を洗浄した。ISOGENIIを700 μl/well添加した後、ピペッティングにより懸濁し 1.5mlチューブに回収した。DEPC water を300μl加え、ボルテックスミキサーを用いて15秒間激しく攪拌し た。室温15分間静置後、12,000×g、15分間遠心分離を行い、上清から500μlを 新たな1.5mlチューブに回収した。そこに同量（500μl）の2-Propanolと20mgグリコーゲン溶液1μl加え転倒混和した。室温10分間静置後、12,000×g、15分間遠心分離を行い、上清を除去した。沈殿物に75% Ethanol 300μl/tubeを加え、転倒混和により洗浄した後、8,000×g、3分間遠心分離を行い、上清を除去した。こ の操作を2回繰り返し、上清をできる限り除去した後、チューブ内の液体を揮発さ せるためにチューブ状にサランラップ（登録商標）をかけて20-30分間静置した。チューブを氷 上へ移し、20μl/tubeのDEPC waterを加え、ピペッティングにより沈殿物を溶解 したものをTotal RNA溶液とした。Nano Drop 2000を用いてTotal RNA溶液の濃度および純度を測定した。
Methods
<Total RNA extraction>
After 48 and 96 hours of incubation, PBS was added and removed to wash the cells. ISOGENII was added at 700 μl/well, suspended by pipetting, and collected in a 1.5 ml tube. 300 μl of DEPC water was added and vigorously stirred for 15 seconds using a vortex mixer. After standing at room temperature for 15 minutes, the cells were centrifuged at 12,000×g for 15 minutes, and 500 μl of the supernatant was collected in a new 1.5 ml tube. The same amount (500 μl) of 2-Propanol and 1 μl of 20 mg glycogen solution were added thereto and mixed by inversion. After standing at room temperature for 10 minutes, the cells were centrifuged at 12,000×g for 15 minutes, and the supernatant was removed. 75% Ethanol 300 μl/tube was added to the precipitate, washed by inversion, centrifuged at 8,000×g for 3 minutes, and the supernatant was removed. This procedure was repeated twice, and after removing as much of the supernatant as possible, the tubes were covered with Saran Wrap (registered trademark) and left to stand for 20-30 minutes to volatilize the liquid in the tubes. The tubes were then transferred onto ice, and 20 μl/tube of DEPC water was added. The precipitate was dissolved by pipetting to obtain a total RNA solution. The concentration and purity of the total RNA solution were measured using a Nano Drop 2000.
＜cDNAの合成＞
0.2ml PCRチューブに各サンプルのTotal RNA 4μgと表１に示した溶液を加え、液量全体が20μlになるようにDEPC waterを加え、Thermal Cyclerを用いて表２ の反応条件で逆転写反応を行った。
<cDNA synthesis>
Total RNA 4 μg of each sample and the solution shown in Table 1 were added to a 0.2 ml PCR tube, DEPC water was added to bring the total volume to 20 μl, and reverse transcription reaction was carried out using a Thermal Cycler under the reaction conditions shown in Table 2.
＜PCR反応による目的遺伝子の増幅＞
TE bufferで5倍希釈した各サンプルのcDNA溶液を使用した。Primerは、TE bufferで20pmol/mlに調製し使用した。0.2ml PCRチューブに反応溶液全量が20μlになるように、下記の表７に従いそれぞれの反応液を混合した。それらを CFX96TM Real-Time Systemにセットし、表８の反応条件でPCR反応を行った。表７は、リアルタイムPCRの反応液条件を示す。また、表８は、リアルタイムPCRの反応条件を示す。
<Amplification of target genes by PCR reaction>
The cDNA solution of each sample was diluted 5-fold with TE buffer and used. Primers were prepared at 20 pmol/ml with TE buffer and used. Each reaction solution was mixed in a 0.2 ml PCR tube according to Table 7 below so that the total reaction solution volume was 20 μl. These were then placed in the CFX96TM Real-Time System, and PCR reactions were carried out under the reaction conditions in Table 8. Table 7 shows the reaction solution conditions for real-time PCR. Table 8 also shows the reaction conditions for real-time PCR.
Internal Controlとしてβ-actinを用いて補正を行った。リアルタイムPCRで測定した値はΔΔCt法およびPfaffl法にて算出した。Controlとサンプル添加群間との統計学的有意差はDunnett’s Testにより検定し、*p＜0.05として表した。 β-actin was used as an internal control for correction. Values measured by real-time PCR were calculated using the ΔΔCt method and the Pfaffl method. Statistical differences between the control and sample-added groups were tested using Dunnett's test and expressed as *p<0.05.
・ΔΔCt 法＝2＾(Ct目的遺伝子-CtHK遺伝子) HK：ハウスキーピング
・Pfaffl 法＝E目的遺伝子^(CtControl-CtSample)/EHK遺伝子^(CtControl-CtSample) E:1+（増幅 効率/100）
・ΔΔCt method = 2^(Ct target gene - Ct HK gene ) HK: Housekeeping ・Pfaffl method = E target gene ^ (Ct control - Ct sample ) / E HK gene ^ (Ct control - Ct sample ) E: 1+ (amplification efficiency / 100)
＜結果＞
PSEおよびその分画であるPSE-W、PSE-EA-H、PSE-EA-MeをHaCaT細胞添加した後、48時間および96時間培養し、RT-PCRを行った結果を図1～図16に示した。RT-PCRにてPSEおよびその分画であるPSE-W、PSE-EA-H、PSE-EA-Meの添加培養により増加が確認された HAS-2、h-LOR、h-INV 遺伝子に関してはリアルタイム PCR を行い、RT-PCR 同様 48 時間および 96 時間培養後、測定を行った。PSEおよび その分画であるPSE-W、PSE-EA-H、PSE-EA-MeのリアルタイムPCRの結果を図17～図24に示した。PSEの添加培養により48時間でHAS-2遺伝子の顕著な発現増加が確認された。また、96 時間培養にてCE形成に関与するh-LORおよびh-INVの遺伝子発現の増加が見られた（図17、図18）。HAS-2は皮膚の水分保湿に関与するヒアルロン酸を合成する酵素であり、PSEはCEの形成を促進し皮膚のバリア機能を改善するだけでなく、ヒアルロン酸合成に寄与し、皮膚の水分保湿効果を改善する 可能性が示唆された（図17、図18）。PSE-W、PSE-EA-H、PSE-EA-Meの添加培養により48時間でHAS-2遺伝子の増加が認められた（図19、図21、図23）ことから、PSEには皮膚の水分保湿に寄与する成分が複数含まれていることが考えられる。また、96時間でh-LORおよび h-INVの遺伝子発現増加が促進された（図20、図22、図24）。今回、PSE-W、PSE-EA-MeにおいてもCE形成関連遺伝子の増加が確認されたことから、PSE-EA-Hは翻訳の過程およびTGM-1の活性に関与し、CEの形成を促進している可能性が示された。
<Results>
HaCaT cells were cultured for 48 and 96 hours after addition of PSE and its fractions PSE-W, PSE-EA-H, and PSE-EA-Me, and the results of RT-PCR are shown in Figures 1 to 16. RT-PCR confirmed that the expression of HAS-2, h-LOR, and h-INV genes was increased by culture with PSE and its fractions PSE-W, PSE-EA-H, and PSE-EA-Me. Real-time PCR was performed for the HAS-2, h-LOR, and h-INV genes, and measurements were performed after 48 and 96 hours of culture, as with RT-PCR. The results of real-time PCR for PSE and its fractions PSE-W, PSE-EA-H, and PSE-EA-Me are shown in Figures 17 to 24. A significant increase in the expression of the HAS-2 gene was confirmed after 48 hours of culture with PSE. In addition, an increase in the expression of the h-LOR and h-INV genes, which are involved in CE formation, was observed after 96 hours of culture (Figures 17 and 18). HAS-2 is an enzyme that synthesizes hyaluronic acid, which is involved in skin moisture retention, and it was suggested that PSE not only promotes CE formation and improves the skin's barrier function, but also contributes to hyaluronic acid synthesis and improves the skin's moisture retention effect (Fig. 17, Fig. 18). The addition of PSE-W, PSE-EA-H, and PSE-EA-Me to the culture increased the HAS-2 gene expression at 48 hours (Fig. 19, Fig. 21, Fig. 23), suggesting that PSE contains multiple components that contribute to skin moisture retention. In addition, the gene expression of h-LOR and h-INV was promoted at 96 hours (Fig. 20, Fig. 22, Fig. 24). In this study, the increase in CE formation-related genes was also confirmed in PSE-W and PSE-EA-Me, suggesting that PSE-EA-H may be involved in the translation process and TGM-1 activity to promote CE formation.
PSE-EA-Hをさらに分画し、F1～F4の4検体に分配した後、PSE同様リアルタイムPCRを行った。なお、PSE-EA-Hをさらに分画し、F1～F4が得られたとは、F1はヘキサンと酢酸エチルの8：2の混合物、F2はヘキサンと酢酸エチルの6：4の混合物、F3は酢酸エチル 100%、F4はメタノール100%でカラムを流した後、得られたものという意味である。その結果を図25～図32 に示した。F1～F4画分すべての検体で 48時間及び96時間培養でHAS-2遺伝子の増加が認められた（図25～図32）。F1 画分において96時間培養でh-LOR遺伝子の有意な発現増加が見られた（図26）。さらに、F1～F3画分において96時間培養でh-INV遺伝子の有意な増加が認められた（図26、図28、図30）ことから、PSE-EA-HにおけるCE形成促進能はF1～F3画分に含まれる成分に起因していることが示された。また、ヒアルロン酸合成酵素が3種類存在しており、そのうちのHAS-2がもっとも皮膚のヒアルロン酸合成の活性化に寄与していることが判明した。ヒアルロン酸合成の活性の強さに限っては、HAS-2>HAS-3>HAS-1となることが判明し、HAS-1は主に胚発生（発生初期：胎児期）に関与しており、表皮細胞においての発現は低くてもとりわけ問題ではない。 PSE-EA-H was further fractionated and divided into four samples, F1 to F4, and real-time PCR was performed in the same manner as for PSE. Note that PSE-EA-H was further fractionated to obtain F1 to F4, meaning that F1 was obtained after running the column with a mixture of 8:2 hexane and ethyl acetate, F2 with a mixture of 6:4 hexane and ethyl acetate, F3 with 100% ethyl acetate, and F4 with 100% methanol. The results are shown in Figures 25 to 32. In all samples from fractions F1 to F4, an increase in the HAS-2 gene was observed after 48 and 96 hours of culture (Figures 25 to 32). In the F1 fraction, a significant increase in h-LOR gene expression was observed after 96 hours of culture (Figure 26). Furthermore, a significant increase in the h-INV gene was observed in the F1 to F3 fractions after 96-hour culture (Fig. 26, Fig. 28, Fig. 30), indicating that the ability of PSE-EA-H to promote CE formation is due to components contained in the F1 to F3 fractions. It was also found that there are three types of hyaluronan synthase, of which HAS-2 is the enzyme that contributes most to activating hyaluronan synthesis in the skin. In terms of the strength of hyaluronan synthesis activity, it was found that the order was HAS-2>HAS-3>HAS-1, and HAS-1 is mainly involved in embryonic development (early development: fetal stage), and its low expression in epidermal cells is not a particular problem.
また、本研究の結果から、HAS関連遺伝子は48時間培養で、CE形成関連遺伝子は96時間培養で比較的顕著な増加が確認された。つまり、培養時間により調節される遺伝子が異なり、HAS関連遺伝子はCE形成関連遺伝子より早い段階で調節されている可能性が示唆された。 Furthermore, the results of this study confirmed a relatively significant increase in HAS-related genes after 48 hours of culture, and in CE formation-related genes after 96 hours of culture. This suggests that the genes regulated differ depending on the culture time, and that HAS-related genes may be regulated at an earlier stage than CE formation-related genes.
本発明によると、医療業界の分野への貢献はもとより、廃棄物の有効利用等、社会的貢献度が高く、広範囲において利用価値が高い。 This invention not only contributes to the medical industry, but also contributes greatly to society by making effective use of waste materials, making it highly useful in a wide range of areas.
Claims (6)
6. The method of claim 5, further comprising partitioning the ethyl acetate fraction with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.
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| JP2017523173A (en) | 2014-07-22 | 2017-08-17 | エイチエルサイエンス カンパニー，リミテッド | A composition for improving skin containing pomegranate concentrate as an active ingredient |
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| JP2015017081A (en) | 2013-03-12 | 2015-01-29 | 株式会社東洋新薬 | Aging inhibitor |
| JP2017523173A (en) | 2014-07-22 | 2017-08-17 | エイチエルサイエンス カンパニー，リミテッド | A composition for improving skin containing pomegranate concentrate as an active ingredient |
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