TW202246302A - Ceramide synthase gene expression promoting agent, skin improving agent, functional food for skin improvement, cosmetic, and usage method for dipeptide - Google Patents

Abstract

Provided is a ceramide synthase gene expression promoting agent in which a prescribed dipeptide is employed as a main ingredient for bringing about a function, and which is capable of increasing the expression level of a ceramide synthase gene. A dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main ingredient for bringing about a function. The present invention is also characterized in that the ceramide synthase gene is the ceramide synthase 3 (CERS3) gene.

Description

神經醯胺合成酵素基因表現促進劑、膚質改善劑、膚質改善用機能食品及化妝料以及二肽之使用方法Ceramide synthesizing enzyme gene expression promoter, skin quality improving agent, functional food and cosmetics for skin quality improvement, and usage method of dipeptide

本發明係有關於一種神經醯胺合成酵素基因表現促進劑、膚質改善劑、膚質改善用機能食品及化妝料以及二肽之使用方法。The invention relates to a ceramide synthesizing enzyme gene expression promoter, a skin quality improving agent, a functional food and cosmetic material for skin quality improvement, and a method for using dipeptide.

美觀健康的肌膚係表現個人外表魅力的重要因素之一。因此，歷來不分男女均著重肌膚的保養。Good-looking and healthy skin is one of the most important factors in expressing one's personal appearance. Therefore, both men and women have always paid attention to skin care.

尤其是為了維持稚嫩的肌膚，關鍵在於緊緻感及充分濕潤。從而，主掌肌膚保濕之成分便極為重要，作為此種保濕成分可舉出例如神經醯胺。In particular, in order to maintain tender skin, the key is firmness and sufficient moisture. Therefore, the component responsible for moisturizing the skin is extremely important, and such a moisturizing component includes, for example, ceramide.

神經醯胺係於細胞內經生合成之鞘脂質的一種，係形成人類皮膚表皮層的表面之角質層的主成分。且神經醯胺可抑制水分過度蒸散，並且具有作為對微生物的障壁之作用，為了使肌膚呈現水嫩、細嫩狀態，則需使肌膚的神經醯胺量維持適當量。Ceramide is one of the sphingolipids that are synthesized in cells, and is the main component of the stratum corneum, which forms the surface of the epidermis of human skin. And ceramide can inhibit excessive evaporation of water, and has the effect of acting as a barrier to microorganisms. In order to make the skin tender and tender, it is necessary to maintain an appropriate amount of ceramide in the skin.

然而，神經醯胺容易隨著年齡增長而消失。However, ceramides tend to disappear with age.

因此，為促進皮膚表皮細胞中神經醯胺的生合成，有人提出一種以包含菸鹼酸及/或菸鹼醯胺之菌體培養物作為有效成分的神經醯胺合成促進劑。Therefore, in order to promote the synthesis of ceramide in skin epidermal cells, someone proposes a ceramide synthesis accelerator that uses a bacterial culture containing niacin and/or niacinamide as an active ingredient.

而且，根據此種神經醯胺合成促進劑，藉由在皮膚表層內部活化表皮細胞本身的神經醯胺合成而改善皮膚阻隔功能，可望改善粗澀肌膚並改善各種皮膚疾病。 [先前技術文獻] [專利文獻] Furthermore, according to such a ceramide synthesis accelerator, the barrier function of the skin is improved by activating the ceramide synthesis of the epidermal cells themselves inside the skin surface layer, which is expected to improve rough skin and improve various skin diseases. [Prior Art Literature] [Patent Document]

[專利文獻1]日本特開平10-259135號公報[Patent Document 1] Japanese Patent Application Laid-Open No. 10-259135

[發明所欲解決之課題][Problem to be Solved by the Invention]

然而，上述習知以包含菸鹼酸或菸鹼醯胺之菌培養物作為有效成分的神經醯胺合成促進劑係以外用劑形式提出，對於口服攝取時之效果仍舊不明。However, the above-mentioned ceramide synthesis accelerators that contain bacterial cultures containing nicotinic acid or nicotinamide as active ingredients are proposed in the form of external preparations, and the effect of oral ingestion is still unknown.

又，在促進神經醯胺的生合成時，作為產生功能用之主要成分，提供更多元的選擇，在擴大使用者的選擇上屬較佳者。In addition, when promoting the synthesis of ceramide, as the main component for producing functions, it provides more choices, which is better in expanding the choices of users.

本發明係有鑑於所述實情而完成者，茲提供一種採用既定的二肽作為產生功能用之主要成分，而能夠使神經醯胺合成酵素基因的表現量增大之神經醯胺合成酵素基因表現促進劑。The present invention is accomplished in view of the above facts, and hereby provides a ceramide synthetase gene expression that can increase the expression level of the ceramide synthetase gene by using a predetermined dipeptide as the main component for producing the function. Accelerator.

又，於本發明中，同樣地提供一種含有具有神經醯胺合成酵素基因表現促進機能之既定的二肽的膚質改善劑、膚質改善用機能食品或化妝料，還有該二肽之使用方法。 [解決課題之手段] Also, in the present invention, a skin quality improving agent, a functional food or cosmetic for skin quality improvement, and a use of the dipeptide are provided in the same manner, which contain a predetermined dipeptide having a function of promoting the expression of the ceramide synthetase gene. method. [Means to solve the problem]

為解決上述習知課題，於本發明之神經醯胺合成酵素基因表現促進劑中，係含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分。In order to solve the above-mentioned conventional problems, in the ceramide synthetase gene expression accelerator of the present invention, the dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main component for producing the function.

又，於本發明之神經醯胺合成酵素基因表現促進劑中，其特徵為前述神經醯胺合成酵素基因為神經醯胺合成酵素3(CERS3)基因。Also, in the ceramide synthetase gene expression accelerator of the present invention, it is characterized in that the aforementioned ceramide synthetase gene is a ceramide synthetase 3 (CERS3) gene.

又，於本發明之膚質改善劑、膚質改善用機能食品或化妝料中，係含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分。Moreover, the dipeptide represented by Gly-Ile and/or Ile-Gly is contained in the skin quality improving agent, functional food or cosmetic for skin quality improvement of the present invention as the main component for producing the function.

又，於本發明中，作為神經醯胺合成酵素基因表現促進劑中的產生功能用之主要成分，或以改善膚質為目的之相關成分，係使用Gly-Ile及/或Ile-Gly表示之二肽。 [發明之效果] Also, in the present invention, as the main component for the production function of the ceramide synthetase gene expression promoter, or the related components for the purpose of improving skin quality, it is represented by Gly-Ile and/or Ile-Gly dipeptide. [Effect of Invention]

根據本發明之神經醯胺合成酵素基因表現促進劑，由於含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分，可提供一種採用既定的二肽作為產生功能用之主要成分，而能夠使神經醯胺合成酵素基因的表現量增大之神經醯胺合成酵素基因表現促進劑。According to the ceramide synthetase gene expression accelerator of the present invention, since the dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main component for producing the function, it can provide a kind of agent that adopts the predetermined dipeptide as the producing function. The main component is a ceramide synthetase gene expression accelerator that can increase the expression of the ceramide synthetase gene.

又，前述神經醯胺合成酵素基因若為神經醯胺合成酵素3(CERS3)基因，能以主要於皮膚或角質細胞大量表現的神經醯胺合成酵素3為標的，而能夠作成更有效的神經醯胺合成酵素基因表現促進劑。In addition, if the aforementioned ceramide synthetase gene is the ceramide synthetase 3 (CERS3) gene, it is possible to target the ceramide synthetase 3 mainly expressed in a large amount in skin or keratinocytes, and to produce a more effective ceramide synthetase. Amine synthase gene expression enhancer.

又，於本發明之膚質改善劑、膚質改善用機能食品或化妝料中，由於含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分，而能夠作成可使神經醯胺合成酵素基因的表現量增大之膚質改善劑、膚質改善用機能食品或化妝料。Also, in the skin quality improving agent of the present invention, the functional food for skin quality improvement or cosmetics, since the dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main component for producing functions, it can be made A skin quality improving agent, a functional food or a cosmetic material for improving the skin quality by increasing the expression level of the ceramide synthesizing enzyme gene.

又，於本發明中，作為神經醯胺合成酵素基因表現促進劑中產生功能用之主要成分，及以改善膚質為目的之相關成分，由於係使用Gly-Ile及/或Ile-Gly表示之二肽，可獲致Gly-Ile及/或Ile-Gly表示之二肽所產生之增大神經醯胺合成酵素基因的表現量所衍生的效果。Also, in the present invention, as the main component for producing functions in the ceramide synthetase gene expression promoter, and the related components for the purpose of improving skin quality, it is represented by Gly-Ile and/or Ile-Gly The dipeptide can obtain the effect derived from increasing the expression level of the ceramide synthesis enzyme gene produced by the dipeptide represented by Gly-Ile and/or Ile-Gly.

[實施發明之形態][Mode of Implementing the Invention]

本發明係提供一種採用既定的二肽作為產生功能用之主要成分，而能夠使神經醯胺合成酵素基因的表現量增大之神經醯胺合成酵素基因表現促進劑。The present invention provides a ceramide synthesizing enzyme gene expression accelerator which adopts a predetermined dipeptide as the main component for producing functions and can increase the expression of the ceramide synthesizing enzyme gene.

本實施形態之神經醯胺合成酵素基因表現促進劑的用途不特別限定，理當可為了對食品或化妝料賦予改善膚質之機能而作為添加原料使用，或作為改善膚質用之外用劑使用，亦可作為例如試驗研究用試劑使用。The use of the ceramide synthetase gene expression accelerator of this embodiment is not particularly limited, and it can be used as an additive raw material for imparting a skin quality improvement function to food or cosmetics, or used as an external preparation for skin quality improvement, It can also be used, for example, as a reagent for experimental research.

作為本實施形態之神經醯胺合成酵素基因表現促進劑的特徵，該促進劑係含有Gly-Ile(甘胺酸-異白胺酸)及/或Ile-Gly(異白胺酸-甘胺酸)表示之二肽作為產生功能用之主要成分。As a feature of the ceramide synthetase gene expression accelerator of this embodiment, the accelerator contains Gly-Ile (glycine-isoleucine) and/or Ile-Gly (isoleucine-glycine ) represents the dipeptide as the main component for producing functions.

Gly-Ile或Ile-Gly係甘胺酸與異白胺酸以胜肽鍵鍵結而成之二肽。Gly-Ile或Ile-Gly的來源不特別限定，可為既定的素材或食材的乾燥物或萃取物、取自既定的蛋白質的消化物，或藉由胜肽合成等，只要不違反目的，則其來源可為任意者。此外，於本說明書中除非特別合先敘明，否則胺基酸序列係以左側為N末端而記載。Gly-Ile or Ile-Gly is a dipeptide in which glycine and isoleucine are bonded by peptide bonds. The source of Gly-Ile or Ile-Gly is not particularly limited, and it can be a dry or extract of a given material or food material, a digest of a given protein, or by peptide synthesis, etc., as long as it does not violate the purpose, then Its source may be arbitrary. In addition, in this specification, unless otherwise stated first, the amino acid sequence is described with the left side being the N-terminus.

茲參照實驗結果並追加說明，根據本案發明人等致力研究，闡明Gly-Ile或Ile-Gly可促進神經醯胺合成酵素基因的表現，維持或增加皮膚中的神經醯胺量之效果可期。With reference to the experimental results and additional explanations, according to the inventors of this case, they have made great efforts to study and clarified that Gly-Ile or Ile-Gly can promote the expression of ceramide synthesis enzyme genes, and the effect of maintaining or increasing the amount of ceramide in the skin can be expected.

此外，咸認對肌膚帶來良好影響的二肽，已知有Gly-Leu或Leu-Gly，惟本案所提案之二肽係結構與此等物質完全不同的物質，且由後述試驗結果闡明，與Gly-Leu或Leu-Gly相比具有更高的促進神經醯胺合成酵素基因表現之效果。In addition, Gly-Leu or Leu-Gly are known dipeptides that have a good effect on the skin, but the dipeptide proposed in this case has a completely different structure from these substances, and it is clarified by the test results described later. Compared with Gly-Leu or Leu-Gly, it has a higher effect of promoting the expression of ceramide synthesis enzyme gene.

且於本案中，亦提供一種膚質改善劑。本實施形態之膚質改善劑，其特徵為含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分。於本案中，膚質改善劑可經口服攝取，又可作為皮膚外用劑使用。皮膚外用劑理當意指醫藥品、準醫藥品或化妝品(亦包含藥用化妝品)，但不限縮於此等範圍，只要是外用於皮膚之用劑則包含於此概念。And in this case, a skin quality improving agent is also provided. The skin quality improving agent of this embodiment is characterized in that it contains dipeptide represented by Gly-Ile and/or Ile-Gly as the main component for producing the function. In this case, the skin quality improving agent can be ingested orally, and can also be used as an external preparation for the skin. External skin preparations should refer to pharmaceuticals, quasi-drugs, or cosmetics (including cosmeceuticals), but are not limited to these scopes, as long as they are externally applied to the skin, they are included in this concept.

且於本案中，亦提供一種屬此皮膚外用劑之一樣態的化妝料。亦即，本實施形態之化妝料，其特徵為含有Gly-Ile及/或Ile-Gly表示之二肽作為有效成分。And in this case, also provide a kind of cosmetic material which belongs to the form of this skin external preparation. That is, the cosmetic of this embodiment is characterized by containing dipeptides represented by Gly-Ile and/or Ile-Gly as active ingredients.

於本案中，化妝料除所謂的彩妝用化妝品外，亦包含基礎化妝品或護髮素、香水、牙膏、洗髮精、潤絲精、用於清洗身體等之香皂或沐浴乳、入浴劑等化妝製品，且包含標榜預防效果等的藥用化妝品。In this case, in addition to so-called makeup cosmetics, cosmetics also include basic cosmetics or hair conditioner, perfume, toothpaste, shampoo, conditioner, soap or body wash, bath preparation, etc. Products, including cosmeceuticals that advertise preventive effects, etc.

此外，彩妝用化妝品可舉出粉底、眉部化妝品(眉筆)、睫毛膏、眼影、眼線、口紅、光澤劑、腮紅(cheek)、白粉或指甲油等；作為基礎化妝品，可舉出例如化妝水、乳液、洗面乳、潔面劑、美容液或乳霜等。此外，針對上述皮膚外用劑或化妝料等之說明係為了供理解本發明而舉出相當於此等之物品等的一例者，各用詞的解讀非限定於此等列舉之物品等。惟，於本案申請人將本案權利化之際，不妨害將本發明限定於此等物品等。In addition, cosmetics for make-up include foundation, eyebrow cosmetics (eyebrow pencil), mascara, eye shadow, eyeliner, lipstick, gloss, blush (cheek), powder or nail polish, etc.; as basic cosmetics, for example, Lotion, lotion, facial cleanser, cleanser, beauty serum or cream, etc. In addition, the description of the above-mentioned external preparations for skin, cosmetics, etc. is an example of articles corresponding to these for understanding the present invention, and the interpretation of each term is not limited to these listed articles. However, when the applicant of this case patents this case, it does not hinder the limitation of the present invention to these articles and the like.

且於本案中，亦提供一種Gly-Ile及/或Ile-Gly表示之二肽於神經醯胺合成酵素基因表現促進劑中作為產生功能用之主要成分之使用方法。具有神經醯胺合成酵素基因表現促進效果之成分，迄此已有數種提案，惟，迄今仍未有人提出含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分的神經醯胺合成酵素基因表現促進劑。And in this case, it also provides a dipeptide represented by Gly-Ile and/or Ile-Gly as a method of using a dipeptide represented by ceramide synthase gene expression as a main component for producing functions. There have been several proposals for ingredients that have the effect of promoting the expression of ceramide synthetase enzyme genes, but so far no one has proposed that the dipeptides represented by Gly-Ile and/or Ile-Gly be used as the main ingredient for producing functions. Accelerator of amide synthetase gene expression.

又，針對使用者，提出可引起神經醯胺合成酵素基因表現促進作用或效果之新穎成分，在增加其選擇上具顯著意義。In addition, for users, it is significant to increase their choices by proposing novel ingredients that can induce the expression-promoting action or effect of the ceramide synthesis enzyme gene.

且於本案中，亦提供一種Gly-Ile及/或Ile-Gly表示之二肽於機能食品中作為以改善膚質為目的之相關成分之使用方法。已有人提出數種藉由口服攝取來改善膚質之成分，惟，迄今仍未有人提出含有Gly-Ile及/或Ile-Gly表示之二肽作為改善膚質為目的之相關成分的機能食品。And in this case, a method of using a dipeptide represented by Gly-Ile and/or Ile-Gly in functional food as a related component for the purpose of improving skin quality is also provided. Several ingredients for improving skin quality through oral intake have been proposed, but so far no one has proposed a functional food containing dipeptides represented by Gly-Ile and/or Ile-Gly as related ingredients for the purpose of improving skin quality.

且於本案中，亦提供一種膚質改善用機能食品，尤其係提供一種可藉由口服攝取而發揮優良皮膚保濕效果的膚質改善用機能食品。And in this case, a functional food for improving skin quality is also provided, especially a functional food for improving skin quality that can exert an excellent skin moisturizing effect through oral intake.

機能食品係包含所有不含醫藥品成分且咸認有助於保持增進健康的食品之概念，例如除營養輔助食品或健康輔助食品、營養調性食品外，亦包含所謂的營養補充品等一般食品，或如特定保健用食品或營養機能食品、機能性標示食品之保健機能食品。Functional food is a concept that includes all foods that do not contain pharmaceutical ingredients and are believed to help maintain and improve health. For example, in addition to nutritional supplements, health supplements, and nutritionally adjusted foods, it also includes general foods such as so-called nutritional supplements. , or health functional food such as food for specific health use, nutritional functional food, or functionally labeled food.

又，作成營養補充品狀時，其劑形不特別限定，可選擇錠劑、膠囊劑、細粒劑、丸劑、片劑、液劑、膠凍狀等各種劑形。Also, when it is in the form of a nutritional supplement, the dosage form is not particularly limited, and various dosage forms such as lozenges, capsules, fine granules, pills, tablets, liquids, and jelly can be selected.

此外，針對上述外用劑、化妝料或機能食品之說明係為了供理解本發明而舉出相當於此等之物品等的一例者，各用詞的解讀非限定於此等列舉之物品等。惟，於本案申請人將本案權利化之際，不妨害將本發明限定於此等物品等。In addition, the description of the above-mentioned external preparations, cosmetics, or functional foods is an example of articles corresponding to these for understanding the present invention, and the interpretation of each term is not limited to these listed articles. However, when the applicant of this case patents this case, it does not hinder the limitation of the present invention to these articles and the like.

本實施形態之神經醯胺合成酵素基因表現促進劑、膚質改善劑、膚質改善用機能食品或化妝料等所含有之作為產生功能用之成分的Gly-Ile及/或Ile-Gly的濃度，只要是可產生前述功能的濃度則不特別限定。如特舉一例，膚質改善用機能食品所含之Gly-Ile或Ile-Gly的濃度，可取例如每日可攝取20~600μg之Gly-Ile或Ile-Gly的量，Gly-Ile與Ile-Gly的總量可取每日可攝取20~600μg的量。附帶一提，若為以每日1粒為基準而攝取之膚質改善用機能食品，亦能以1粒所含之Gly-Ile及/或Ile-Gly的量取20~600μg，或若以每日2粒為基準則每粒取10~300μg的方式，適宜調整1粒、1膠囊或一包等攝取單位量所含之Gly-Ile及/或Ile-Gly的量。Concentration of Gly-Ile and/or Ile-Gly contained in the ceramide synthesizing enzyme gene expression promoter, skin texture improving agent, functional food for skin texture improvement, cosmetics, etc. of this embodiment as a functional component , is not particularly limited as long as it is a concentration that can produce the aforementioned functions. As a specific example, the concentration of Gly-Ile or Ile-Gly contained in functional foods for skin quality improvement, for example, the amount of Gly-Ile or Ile-Gly that can be ingested 20-600 μg per day, Gly-Ile and Ile-Gly The total amount of Gly can be taken as 20~600μg per day. Incidentally, if it is a functional food for skin quality improvement that is ingested on the basis of 1 capsule per day, 20~600 μg of Gly-Ile and/or Ile-Gly contained in 1 capsule can also be taken, or if Take 2 capsules per day as the basis, take 10~300μg per capsule, and adjust the amount of Gly-Ile and/or Ile-Gly contained in intake units such as 1 capsule, 1 capsule or a pack.

又，就化妝料而言，可使其含有0.03~200μg/g的Gly-Ile或Ile-Gly，或將Gly-Ile與Ile-Gly的總濃度定為0.03~200μg/g。神經醯胺合成酵素基因表現促進劑或膚質改善劑中之Gly-Ile或Ile-Gly的濃度，可分別採用適於實驗等中使用的濃度，或適合作為供製造食品或化妝料之原料的濃度。Also, cosmetics may contain 0.03-200 μg/g of Gly-Ile or Ile-Gly, or the total concentration of Gly-Ile and Ile-Gly may be 0.03-200 μg/g. The concentration of Gly-Ile or Ile-Gly in the ceramide synthetizing enzyme gene expression accelerator or the skin quality improving agent can be respectively adopted in a concentration suitable for use in experiments, etc., or suitable as a raw material for the manufacture of food or cosmetics concentration.

又，本實施形態之神經醯胺合成酵素基因表現促進劑、膚質改善劑、膚質改善用機能食品或化妝料等，除作為產生功能用之成分的Gly-Ile及/或Ile-Gly外，亦可包含摻合劑形或食品形態等之賦形劑或添加劑、輔助成分等。In addition, the ceramide synthetase gene expression promoter, skin quality improving agent, skin quality improving functional food or cosmetics of this embodiment, except Gly-Ile and/or Ile-Gly as components for producing functions , may also contain excipients or additives, auxiliary ingredients, etc. in the form of admixtures or food.

作為賦形劑，例如劑形為固形劑時，可採用乳糖、結晶纖維素或澱粉等。As an excipient, for example, when the dosage form is a solid preparation, lactose, crystalline cellulose or starch can be used.

又，添加劑可舉出例如安定劑、界面活性劑、可溶化劑、塑化劑、甜味劑、抗氧化劑、著香劑、著色劑、保存劑、無機填充劑等。Moreover, examples of additives include stabilizers, surfactants, solubilizers, plasticizers, sweeteners, antioxidants, flavoring agents, colorants, preservatives, and inorganic fillers.

以下就本實施形態之神經醯胺合成酵素基因表現促進劑、膚質改善劑、膚質改善用機能食品及化妝料以及二肽之使用方法，邊參照實驗過程或結果邊進一步加以說明。In the following, the method of using the ceramide synthetizing enzyme gene expression promoter, skin quality improving agent, functional food and cosmetics for skin quality improving, and dipeptide will be further explained with reference to the experimental process or results.

[1.神經醯胺合成酵素基因的表現試驗] 皮膚係由表皮及真皮構成，表皮可分類為基底層、棘狀層、顆粒層及角質層。在位於皮膚最外層的角質層細胞間，由神經醯胺等的脂質所構成之層狀構造係主掌作為保持體內的水分或防止細菌及病毒的入侵之物理障壁之作用。 [1. Expression test of ceramide synthesis enzyme gene] The skin is composed of epidermis and dermis, and the epidermis can be classified into basal layer, spinous layer, granular layer and stratum corneum. Among the stratum corneum cells located in the outermost layer of the skin, the layered structure composed of lipids such as ceramide is mainly used as a physical barrier to maintain water in the body or prevent the invasion of bacteria and viruses.

於本探討中，係藉由探討神經醯胺合成酵素基因的表現量的變化，來評估Gly-Ile或Ile-Gly(下稱本案二肽)之表皮細胞的改善效果。又，比較對照係使用Gly-Leu或Leu-Gly(下稱比較二肽)，亦探討與本案二肽的差異。In this study, the effect of improving the epidermal cells of Gly-Ile or Ile-Gly (hereinafter referred to as the dipeptide in this case) is evaluated by investigating the changes in the expression level of the ceramide synthesizing enzyme gene. In addition, Gly-Leu or Leu-Gly (hereinafter referred to as comparative dipeptide) was used as the comparison control system, and the difference with the dipeptide of this case was also investigated.

人類表皮角化細胞(HaCaT細胞)係使用Dulbecco’s Modified Eagle Medium (DMEM)(高葡萄糖)(含1%青黴素-鏈黴素及10%胎牛血清(FBS))，於φ10cm培養皿中進行前培養至滿盤。Human epidermal keratinocytes (HaCaT cells) were pre-cultured in a φ10cm dish using Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) to full plate.

其後，以磷酸緩衝生理食鹽水(PBS)洗淨，再懸浮於培養基後，以1.0×10 ⁵cells/well的濃度播種於24孔孔盤中，並於CO ₂恆溫箱(37℃,5%CO ₂)中培養過夜。 Thereafter, they were washed with phosphate-buffered saline (PBS), resuspended in culture medium, seeded in 24-well plates at a concentration of 1.0×10 ⁵ cells/well, and placed in a CO ₂ incubator (37°C, 5 %CO ₂ ) overnight.

自播種HaCaT細胞24小時後，將包含達既定最終濃度的量之Gly-Ile、Ile-Gly、Gly-Leu、Leu-Gly之任一者的無血清之DMEM(高葡萄糖)(含1%青黴素-鏈黴素)各取1mL添加至各井孔中，於CO ₂恆溫箱(37℃,5%CO ₂)中培養48小時。 24 hours after seeding HaCaT cells, serum-free DMEM (high glucose) containing any one of Gly-Ile, Ile-Gly, Gly-Leu, and Leu-Gly (containing 1% penicillin) - Streptomycin) 1 mL each was added to each well, and cultured in a CO ₂ incubator (37° C., 5% CO ₂ ) for 48 hours.

其次，針對根據即時PCR之基因的轉錄表現量的變化進行試驗。首先，添加各二肽並回收經過48小時後的HaCaT細胞，使用RNeasy Mini kit(Qiagen)萃取出total RNA。Next, experiments were performed on changes in transcriptional expression levels of genes based on real-time PCR. First, HaCaT cells 48 hours after addition of each dipeptide were recovered, and total RNA was extracted using RNeasy Mini kit (Qiagen).

接著，以ReverTra Ace qPCR RT Master Mix with gDNA Remover(TOYOBO)，由萃取出之total RNA合成cDNA。以合成之cDNA為模板，藉由AriaMX(Agilent)裝置進行即時PCR。即時PCR反應中係使用THUNDERBIRD SYBR qPCR Mix(TOYOBO)。Then, cDNA was synthesized from the extracted total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Using the synthesized cDNA as a template, real-time PCR was performed with an AriaMX (Agilent) device. In the real-time PCR reaction, THUNDERBIRD SYBR qPCR Mix (TOYOBO) was used.

又，即時PCR反應中，神經醯胺合成酵素基因CERS2用引子係使用5’-CCGATTACCTGCTGGAGTCAG-3’(序列編號1)及5’-GGCGAAGACGATGAAGATGTTG-3’(序列編號2)，神經醯胺合成酵素基因CERS3用引子係使用5’-ACATTCCACAAGGCAACCATTG-3’(序列編號3)及5’-CTCTTGATTCCGCCGACTCC-3’(序列編號4)，神經醯胺合成酵素基因CERS4用引子係使用5’-GGAGGCCTGTAAGATGGTCA-3’(序列編號5)及5’-GAGGACCAGTCGGGTGTAGA-3’(序列編號6)，內標準β-肌動蛋白用引子則使用5’-GGGTCAGAAGGACTCCTATG-3’(序列編號7)及5’-GTAACAATGCCATGTTCAAT-3’(序列編號8)。作為即時PCR反應條件，係進行以下PCR反應40循環：於95℃、60秒的初始變性後，經95℃、15秒的變性及60℃、60秒的引子黏合/延長此兩步驟。Also, in the real-time PCR reaction, 5'-CCGATTACCTGCTGGAGTCAG-3' (SEQ ID NO: 1) and 5'-GGCGAAGACGATGAAGATGTTG-3' (SEQ ID NO: 2) were used as primers for the ceramide synthetase gene CERS2. The primers for CERS3 used 5'-ACATTCCACAAGGCAACCATTG-3' (SEQ ID NO: 3) and 5'-CTCTTGATTCCGCCGACTCC-3' (SEQ ID NO: 4), and the primers for ceramide synthetase gene CERS4 used 5'-GGAGGCCTGTAAGATGGTCA-3' ( SEQ ID NO: 5) and 5'-GAGGACCAGTCGGGTGTAGA-3' (SEQ ID NO: 6), the internal standard β-actin primers use 5'-GGGTCAGAAGGACTCCTATG-3' (SEQ ID NO: 7) and 5'-GTAACAATGCCATGTTCAAT-3' ( Serial number 8). As real-time PCR reaction conditions, the following PCR reaction was performed for 40 cycles: initial denaturation at 95°C for 60 seconds, followed by two steps of denaturation at 95°C for 15 seconds and primer binding/extension at 60°C for 60 seconds.

將本案二肽之Ile-Gly或Gly-Ile、比較二肽之Leu-Gly或Gly-Leu添加於屬人類表皮角化細胞株之一的HaCaT細胞，探討神經醯胺合成酵素基因的表現變化。圖1示出添加各二肽之HaCaT細胞的細胞生存率。Add Ile-Gly or Gly-Ile of the dipeptide of this case, and Leu-Gly or Gly-Leu of the comparison dipeptide to HaCaT cells, which is one of the human epidermal keratinocyte lines, to investigate the expression changes of the ceramide synthase gene. Fig. 1 shows the cell viability of HaCaT cells added with each dipeptide.

如圖1所示，若添加本案二肽或比較用二肽，與控制組相比可看出人類角化細胞HaCaT的細胞生存率上升之傾向。尤其是添加本案二肽時，於0.1μg/mL、10μg/mL、200μg/mL之任一種濃度下皆可確認相對於控制組之細胞生存率的上升。又，就Gly-Leu 10μg/mL、Ile-Gly 0.1及10μg/mL、Gly-Ile 200μg/mL，與控制組相比可確認細胞生存率顯著上升。由此等結果，示意本案二肽之Ile-Gly或Gly-Ile其角化細胞賦活化作用可期。As shown in Figure 1, when the dipeptide of this case or the dipeptide for comparison is added, the cell survival rate of human keratinocyte HaCaT tends to increase compared with the control group. In particular, when the dipeptide of the present invention was added, an increase in the cell survival rate relative to the control group was confirmed at any concentration of 0.1 μg/mL, 10 μg/mL, and 200 μg/mL. Also, for Gly-Leu 10 μg/mL, Ile-Gly 0.1 and 10 μg/mL, and Gly-Ile 200 μg/mL, it was confirmed that the cell survival rate was significantly increased compared with the control group. These results indicate that the Ile-Gly or Gly-Ile dipeptide of this case can activate keratinocytes.

圖2示出添加本案二肽之Ile-Gly或Gly-Ile、比較二肽之Leu-Gly或Gly-Leu之HaCaT細胞的神經醯胺合成酵素基因的表現變化。圖2(a)表示神經醯胺合成酵素CERS2基因的表現量相對於控制組的相對值，同樣地圖2(b)以相對值表示神經醯胺合成酵素CERS3基因、圖2(c)以相對值表示神經醯胺合成酵素CERS4基因的表現量。Fig. 2 shows the expression change of the ceramide synthase gene of the HaCaT cells added with the Ile-Gly or Gly-Ile of the dipeptide of the present case and the Leu-Gly or Gly-Leu of the comparison dipeptide. Figure 2(a) shows the expression level of ceramide synthetase CERS2 gene relative to the relative value of the control group, the same map 2(b) shows the relative value of ceramide synthetase CERS3 gene, and Figure 2(c) shows the relative value of ceramide synthetase CERS3 gene Indicates the expression level of the ceramide synthetase CERS4 gene.

有人報導神經醯胺合成酵素CERS2基因會大量表現於腎臟、肝臟及腸部等諸多細胞組織。又，神經醯胺合成酵素CERS3基因主要表現於皮膚、角質細胞。再者，神經醯胺合成酵素CERS4基因不限於皮膚，也會表現於白血球、心臟及肝臟。It has been reported that the ceramide synthetase CERS2 gene will be expressed in a large number in many cell tissues such as kidney, liver and intestine. Also, the ceramide synthetase CERS3 gene is mainly expressed in skin and keratinocytes. Furthermore, the ceramide synthase CERS4 gene is not limited to the skin, but also expressed in white blood cells, heart and liver.

於本探討中，係將各二肽添加至HaCaT細胞後，利用即時PCR進行神經醯胺合成酵素CERS2、CERS3及CERS4基因的表現解析。其結果如圖2(a)所示，CERS2基因的表現，與控制組相比於任一種濃度下皆無法確認顯著增加。In this study, each dipeptide was added to HaCaT cells, and the expression analysis of the ceramide synthetizing enzymes CERS2, CERS3, and CERS4 genes was performed by real-time PCR. As a result, as shown in FIG. 2( a ), no significant increase in the expression of the CERS2 gene was confirmed at any of the concentrations compared with the control group.

相對於此，神經醯胺合成酵素CERS3基因係如圖2(b)所示，藉由添加本案二肽之Ile-Gly或Gly-Ile，與控制組相比於任一種濃度下皆可確認表現量上升。In contrast, the ceramide synthetase CERS3 gene is shown in Figure 2(b), and by adding Ile-Gly or Gly-Ile of the dipeptide of this case, the expression can be confirmed at any concentration compared with the control group Volume rises.

尤其是添加Gly-Ile，可看出表現量隨濃度提高而顯著增加；若為200μg/mL的濃度，與控制組相比，表現量增達3倍，如此極為耐人尋味。Especially with the addition of Gly-Ile, it can be seen that the expression level increases significantly with the increase of the concentration; if the concentration is 200 μg/mL, compared with the control group, the expression level increases by 3 times, which is very intriguing.

又，添加Ile-Gly，可使CERS3基因的表現量增加，且以0.1μg/mL的濃度使用時可看出顯著差異，惟其特徵為隨著濃度上升，表現量下降。Also, the addition of Ile-Gly can increase the expression level of CERS3 gene, and a significant difference can be seen when it is used at a concentration of 0.1 μg/mL, but its characteristic is that the expression level decreases as the concentration increases.

此外，藉由添加比較二肽之Leu-Gly或Gly-Leu，與控制組相比亦可確認最大約1.6倍的表現量上升。惟，可看出其表現量不若本案二肽顯著增加。In addition, by adding Leu-Gly or Gly-Leu of the comparative dipeptide, a maximum increase of about 1.6-fold compared with the control group was confirmed. However, it can be seen that its expression level is not as significantly increased as that of the dipeptide in this case.

又，雖省略數據，但在本案發明人等所進行的研究經驗上，添加0.03μg/g左右的濃度的Ile-Gly或Gly-Ile，顯然與CERS3基因的表現量，與控制組相比亦顯著增加。Also, although the data is omitted, in the research experience conducted by the inventors of this case, the addition of Ile-Gly or Gly-Ile at a concentration of about 0.03 μg/g is obviously related to the expression level of the CERS3 gene, compared with the control group. significantly increased.

神經醯胺合成酵素CERS4基因係如圖2(c)所示，藉由添加Gly-Ile而確認表現量顯著上升。As shown in Figure 2(c) of the ceramide synthetase CERS4 gene line, it was confirmed that the expression level was significantly increased by adding Gly-Ile.

由此等結果示意，本案二肽其細胞賦活化作用可期，具有亢進神經醯胺合成酵素CERS3及CERS4基因的表現之作用。此作用亦示意，尤其在本案二肽中，Gly-Ile較高。如此，本案二肽可看出促進神經醯胺合成酵素基因的表現之效果，對皮膚的阻隔功能發揮重要作用的促進神經醯胺合成之效果可期。These results indicate that the dipeptide of this case can be expected to activate cells, and has the function of promoting the expression of ceramide synthetase enzymes CERS3 and CERS4 genes. This effect also shows that, especially in the dipeptide of this case, Gly-Ile is higher. In this way, the dipeptide in this case can be seen to promote the expression of ceramide synthesis enzyme gene, and the effect of promoting ceramide synthesis, which plays an important role in the barrier function of the skin, can be expected.

又，其顯示Gly-Ile及/或Ile-Gly表示之二肽為膚質改善劑中產生功能用之主要成分、機能食品中供改善膚質之相關成分，或可作為化妝料中供改善膚質之有效成分而利用。In addition, it shows that the dipeptide represented by Gly-Ile and/or Ile-Gly is the main component for producing functions in skin quality improving agents, related components for improving skin quality in functional foods, or can be used as cosmetics for improving skin quality. Use the effective ingredients of the quality.

[2.神經醯胺合成酵素基因的表現試驗(低用量)] 其次，就本案二肽之Gly-Ile或Ile-Gly之神經醯胺合成酵素基因的表現，對於在低於上述濃度的濃度下能否誘導表現，根據同樣手法進行確認。 [2. Expression test of ceramide synthesis enzyme gene (low dosage)] Next, with regard to the expression of the ceramide synthesizing enzyme gene of Gly-Ile or Ile-Gly of the dipeptide in this case, whether the expression can be induced at a concentration lower than the above-mentioned concentration was confirmed by the same method.

於本探討中，係以含有至少0.00038μg/mL的Gly-Ile與至少0.00097μg/mL的Ile-Gly之兩種二肽的總濃度為0.00135μg/mL的試樣溶液S1、含有至少0.00076μg/mL的Gly-Ile與至少0.00195μg/mL的Ile-Gly之兩種二肽的總濃度為0.00271μg/mL的試樣溶液S2(試樣溶液S1的2倍濃度溶液)，及含有至少0.00152μg/mL的Gly-Ile與至少0.0039μg/mL的Ile-Gly之兩種二肽的總濃度為0.00542μg/mL的試樣溶液S3(試樣溶液S2的2倍濃度溶液)作為受試液，自播種HaCaT細胞24小時後，將構成上述任一受試液之最終濃度之無血清的DMEM(高葡萄糖)(含1%青黴素-鏈黴素)各以1mL添加至各井中，於CO ₂恆溫箱(37℃，5%CO ₂)中培養48小時。 In this study, sample solution S1 containing at least 0.00038 μg/mL of Gly-Ile and at least 0.00097 μg/mL of Ile-Gly dipeptides with a total concentration of 0.00135 μg/mL of sample solution S1, containing at least 0.00076 μg The total concentration of the two dipeptides of Gly-Ile/mL and at least 0.00195 μg/mL of Ile-Gly is 0.00271 μg/mL sample solution S2 (2 times concentration solution of sample solution S1), and contains at least 0.00152 The total concentration of the two dipeptides of Gly-Ile of μg/mL and Ile-Gly of at least 0.0039 μg/mL is 0.00542 μg/mL sample solution S3 (2 times concentration solution of sample solution S2) as the test solution, 24 hours after seeding HaCaT cells, add 1 mL of serum-free DMEM (high glucose) (containing 1% penicillin-streptomycin) to each well to form the final concentration of any of the above test solutions, and place in a _CO2 incubator (37°C, 5% CO ₂ ) for 48 hours.

首先，圖3(a)示出添加各試樣溶液S1~S3之HaCaT細胞的細胞生存率。如圖3(a)所示，各試樣溶液S1~S3，與控制組相比，未特別看出顯著差異。First, FIG. 3( a ) shows the cell viability of HaCaT cells to which each sample solution S1 to S3 was added. As shown in FIG. 3( a ), no significant difference was particularly seen in each of the sample solutions S1 to S3 compared with the control group.

圖3(b)示出添加各試樣溶液S1~S3後之HaCaT細胞之神經醯胺合成酵素基因的表現變化。如圖3(b)所示，CERS2基因的表現，與控制組相比，於任一種濃度下皆未確認顯著差異。另一方面，經報導表現於皮膚中之CERS3基因或CERS4基因，與控制組相比表現上升約2~4倍左右。又，CERS3基因，與CERS4基因相比較，可看出更顯著的表現上升。Fig. 3(b) shows the expression changes of the ceramide synthetase gene of HaCaT cells after adding each sample solution S1-S3. As shown in FIG. 3( b ), no significant difference was confirmed at any concentration in the expression of the CERS2 gene compared with the control group. On the other hand, it is reported that the expression of CERS3 gene or CERS4 gene expressed in the skin is about 2-4 times higher than that of the control group. In addition, the expression of CERS3 gene was significantly increased compared with that of CERS4 gene.

由以上結果示意，本案二肽之Gly-Ile或Ile-Gly未看出細胞毒性，且具有亢進神經醯胺合成酵素CERS3及CERS4基因的表現之作用。從而，本案二肽之Gly-Ile或Ile-Gly，當兩種二肽的總濃度為0.00135μg/mL以上時，對皮膚的阻隔功能發揮重要作用的促進神經醯胺合成之效果可期。附帶一提，較佳為例如藉由將含有至少0.00038μg/mL的Gly-Ile與至少0.00097μg/mL的Ile-Gly之兩種二肽的總濃度定為0.03μg/mL，能更可靠地發揮效果。The above results indicated that Gly-Ile or Ile-Gly of the dipeptide in this case did not show cytotoxicity, and had the function of enhancing the expression of ceramide synthase CERS3 and CERS4 genes. Therefore, when the total concentration of the dipeptide Gly-Ile or Ile-Gly of the present case is above 0.00135 μg/mL, the effect of promoting the synthesis of ceramide that plays an important role in the barrier function of the skin can be expected. Incidentally, it is preferable to set the total concentration of two dipeptides containing at least 0.00038 μg/mL of Gly-Ile and at least 0.00097 μg/mL of Ile-Gly to 0.03 μg/mL, for example, so that more reliable play an effect.

[3.機能食品的製造] 其次，進行含有Gly-Ile及Ile-Gly表示之二肽作為膚質改善用之產生功能成分，例如為機能性食品時之相關成分之機能食品的製造。 [3. Manufacture of functional food] Next, the production of functional foods containing dipeptides represented by Gly-Ile and Ile-Gly as functional ingredients for skin quality improvement, such as related ingredients in functional foods.

供試驗用之機能食品的劑形係採硬膠囊。具體而言，係以200mg的粉末糊精為基材，添加藉由胜肽合成而得之Gly-Ile及/或Ile-Gly並進行混合，將所得混合粉體填充於以明膠為皮膜原料之膠囊的本體並實施封蓋而得到硬膠囊狀的機能食品A。The dosage form of the functional food used in the test is a hard capsule. Specifically, 200 mg of powdered dextrin is used as the base material, Gly-Ile and/or Ile-Gly obtained by peptide synthesis are added and mixed, and the resulting mixed powder is filled in a film with gelatin as the raw material. The main body of the capsule is sealed and the functional food A of hard capsule shape is obtained.

[4.機能食品中之Gly-Ile及Ile-Gly的測定] 其次，測定所得機能食品A之1粒錠劑中所含之本案二肽的含量。 [4. Determination of Gly-Ile and Ile-Gly in functional foods] Next, the content of the dipeptide of the present invention contained in one lozenge of the obtained functional food A was measured.

(4-1.2種二肽的回收試驗) 首先，為了探討此次實施之試驗方法在測定機能食品A之Gly-Ile或Ile-Gly的含量上是否妥適，而進行試驗，此試驗係確認預先添加於仿機能食品A所調製之試樣食品中之已知量的Gly-Ile及Ile-Gly的回收率。 (4-1.2 recovery test of dipeptides) First of all, in order to investigate whether the test method implemented this time is suitable for determining the content of Gly-Ile or Ile-Gly in functional food A, a test is carried out. This test is to confirm that it is added to the sample prepared in imitation functional food A Recovery of known amounts of Gly-Ile and Ile-Gly in food.

將2種胜肽標準品(Gly-Ile、Ile-Gly)混合溶液(500μg/mL)分別以20, 50, 100, 150μL(測定溶液中的最終濃度：20, 50, 100, 150ng/mL)添加於1粒不含混合粉體的膠囊中。其後，添加10mL的超純水，進行超音波萃取(38kHz、200W)40分鐘。此外，於萃取操作中係劇烈搖晃3~4次以使萃取溶媒均勻分散。萃取後，進行離心分離(3500rpm、10min)，將上清液置入25mL的量瓶中。對殘渣進而以8mL、5mL的超純水與上述同樣地進行2次超音波萃取、離心分離。組合共3次之萃取液，以超純水定容而調成萃取溶液(n=3)。對上述萃取溶液100μL添加20μL的內標準溶液(2μg/mL)，進而加入380μL的乙腈，經攪拌、離心分離(3500rpm、10min)後，將上清液以0.20μm PTFE膜過濾。濾液係以超純水稀釋4倍，供予LC-MS分析。添加之標準品的回收率之算式係示於式1，分析條件則示於表1。

Add 2 kinds of peptide standard (Gly-Ile, Ile-Gly) mixed solution (500μg/mL) to 20, 50, 100, 150μL respectively (the final concentration in the assay solution: 20, 50, 100, 150ng/mL) Add to 1 capsule without mixing powder. Thereafter, 10 mL of ultrapure water was added, and ultrasonic extraction (38 kHz, 200 W) was performed for 40 minutes. In addition, during the extraction operation, shake vigorously 3~4 times to disperse the extraction solvent evenly. After the extraction, centrifugation (3500 rpm, 10 min) was carried out, and the supernatant was placed in a 25 mL measuring flask. The residue was further subjected to ultrasonic extraction and centrifugation twice in the same manner as above with 8 mL and 5 mL of ultrapure water. A total of 3 extracts were combined, and the volume was adjusted with ultrapure water to prepare an extraction solution (n=3). Add 20 μL of internal standard solution (2 μg/mL) to 100 μL of the above extraction solution, and then add 380 μL of acetonitrile, stir and centrifuge (3500 rpm, 10 min), and filter the supernatant with a 0.20 μm PTFE membrane. The filtrate was diluted 4 times with ultrapure water for LC-MS analysis. The calculation formula for the recovery rate of the added standard is shown in Formula 1, and the analysis conditions are shown in Table 1.

據LC-MS分析的結果，未由不含混合粉體之硬膠囊(膠囊本體及帽部)驗出2種二肽。從而，可確認膠囊中不含2種二肽。According to the results of LC-MS analysis, the two dipeptides were not detected from the hard capsules (capsule body and cap) that did not contain mixed powder. Therefore, it was confirmed that the two kinds of dipeptides were not contained in the capsule.

其次，為了驗證試樣溶液之前處理方法或定量分析值的真度，而對膠囊中添加有2種已知量之二肽作為標準品的試樣食品進行分析，算出添加回收率。表2示出添加回收率。

Next, in order to verify the authenticity of the previous treatment method or quantitative analysis value of the sample solution, the sample food with two known dipeptides added to the capsule as a standard was analyzed to calculate the addition recovery rate. Table 2 shows the addition recoveries.

由表2可知，添加標準品之試樣食品，以任一種添加量，2種胜肽的平均回收率皆為97.8%~104.2%的範圍內。從而，研判本實驗之前處理方法及定量分析方法為妥適者。It can be seen from Table 2 that the average recoveries of the two peptides are all in the range of 97.8%~104.2% for the sample food added with the standard substance at any amount added. Therefore, the processing method and quantitative analysis method before this experiment were determined to be appropriate.

(4-2.機能食品A所含之2種二肽的定量分析) 其次，進行機能食品A所含之2種二肽的定量分析。對1粒機能食品A添加10mL的超純水，進行超音波萃取(38kHz、200W)40分鐘。此外，於萃取操作中係劇烈搖晃3~4次以使萃取溶媒均勻分散。 (4-2. Quantitative analysis of two dipeptides contained in functional food A) Next, the quantitative analysis of the two dipeptides contained in functional food A was carried out. Add 10mL of ultrapure water to one functional food A, and perform ultrasonic extraction (38kHz, 200W) for 40 minutes. In addition, shake vigorously 3~4 times during the extraction operation to disperse the extraction solvent evenly.

萃取操作後，進行離心分離(3500rpm、10min)，將上清液置入25mL的量瓶中。對殘渣進而以8mL、5mL的超純水與上述同樣地進行2次超音波萃取、離心分離。組合共3次之萃取液，以超純水定容而調成萃取溶液(n=3)。After the extraction operation, centrifugal separation (3500 rpm, 10 min) was performed, and the supernatant was placed in a 25 mL measuring flask. The residue was further subjected to ultrasonic extraction and centrifugation twice in the same manner as above with 8 mL and 5 mL of ultrapure water. A total of 3 extracts were combined, and the volume was adjusted with ultrapure water to prepare an extraction solution (n=3).

對上述萃取溶液100μL添加20μL的內標準溶液(2μg/mL)，進而加入380μL的乙腈，經攪拌、離心分離(3500rpm、10min)後，將上清液以0.20μm PTFE膜過濾。濾液係以超純水稀釋4倍，供予LC-MS分析。分析條件係如表1所示。Add 20 μL of internal standard solution (2 μg/mL) to 100 μL of the above extraction solution, and then add 380 μL of acetonitrile, stir and centrifuge (3500 rpm, 10 min), and filter the supernatant with a 0.20 μm PTFE membrane. The filtrate was diluted 4 times with ultrapure water for LC-MS analysis. The analytical conditions are shown in Table 1.

首先，根據使用標準品之分析結果來確認Gly-Ile及Ile-Gly的溶出時間。又，如表3所示，2種二肽標準品係於5ng/mL至200ng/mL的檢量線範圍顯示良好的線性(R ²=0.999)。

First, the dissolution time of Gly-Ile and Ile-Gly was confirmed based on the analysis results using standard products. Also, as shown in Table 3, the two dipeptide standard strains showed good linearity in the calibration curve range from 5 ng/mL to 200 ng/mL (R ² =0.999).

進而，將使用檢量線之機能食品A的二肽含量示於表4。確認1粒機能食品A中，Gly-Ile含有26.25±0.38μg、Ile-Gly含有22.57±0.48μg。

Furthermore, the dipeptide content of the functional food A using the calibration line is shown in Table 4. It was confirmed that in one functional food A, 26.25±0.38 μg of Gly-Ile and 22.57±0.48 μg of Ile-Gly were contained.

[5.攝取試驗] 其次，針對前述[3.機能食品的製造]中所得之膠囊狀的機能食品A，進行健康成人之膚質改善效果的驗證。 [5. Intake test] Next, for the capsule-shaped functional food A obtained in the aforementioned [3. Manufacture of functional food], the effect of improving the skin quality of healthy adults was verified.

以20位40~50歲的一般健康女性為對象，分成測試品群10位、安慰劑群10位此2群來進行試驗。就攝取量，測試品群(機能食品A)、安慰劑群(安慰劑食品)皆為1日2錠。此外，安慰劑食品中不含Gly-Ile及Ile-Gly。攝取期間皆採4週。Taking 20 healthy women aged 40-50 as subjects, they were divided into 2 groups of 10 test products and 10 placebo groups for the test. In terms of intake, both the test product group (functional food A) and the placebo group (placebo food) are 2 tablets a day. In addition, Gly-Ile and Ile-Gly were not included in the placebo food. The intake period was 4 weeks.

又，於經過攝取期間後進行膚質的測定。具體而言，係針對肌膚的水分量、水分蒸散量、黏彈性進行測定。此外，測定部位為左腕。Moreover, the measurement of the skin quality was performed after the ingestion period passed. Specifically, the water content, water evaporation rate, and viscoelasticity of the skin are measured. In addition, the measurement site was the left wrist.

測定係使用Cutometer DUAL MPA580(Courage +Khazaka electronic GmbH)，測定水分、黏彈性各5次，以排除最低值與最高值之3次的平均作為測定值。又，黏彈性的分析係使用R2(總彈力)、R5(淨彈性)、R7(回復力)。The measurement system used Cutometer DUAL MPA580 (Courage + Khazaka electronic GmbH), measured moisture and viscoelasticity 5 times each, and took the average of 3 times excluding the lowest value and the highest value as the measured value. In addition, the analysis of viscoelasticity uses R2 (total elasticity), R5 (net elasticity), and R7 (recovery force).

算出標準誤差，為進行事先測定與事後測定的比較(群內比較)及群間比較，而分別進行有對應之t檢定、無對應之t檢定。顯著水準係定為5%，未達5%係判斷為有顯著差異(p＜0.05:**)，未達10%則判斷為有顯著傾向(p＜0.1:*)。Standard errors were calculated, and t-tests with and without correspondence were performed in order to perform comparisons between pre-measurement and post-measurement (intra-group comparison) and inter-group comparisons. The significance level is set at 5%, if it does not reach 5%, it is judged to have a significant difference (p<0.05:**), if it does not reach 10%, it is judged to have a significant tendency (p<0.1:*).

以編入本試驗的20位中，排除無法參加事後測定之1位的另19位作為解析對象者。具體而言，安慰劑群係10位女性，平均年齡為44.7歲；測試品群則為9位女性，平均年齡為45.0歲。Among the 20 people enrolled in this test, the other 19 people who were unable to participate in the subsequent measurement were excluded as the analysis objects. Specifically, the placebo group consisted of 10 women with an average age of 44.7 years; the test group consisted of 9 women with an average age of 45.0 years.

圖4表示水分量相關之試驗結果。水分量為角質層所含之水分量，係參考作為肌膚濕潤之指標的值。圖4(a)表示測試食品或安慰劑食品於攝取前後之水分量測定值的變化；圖4(b)則表示安慰劑群與測試品群之水分量的變化量。Fig. 4 shows the test results related to moisture content. The amount of water is the amount of water contained in the stratum corneum, and refers to a value that is an index of skin moisture. Fig. 4(a) shows the change of the measured value of the water content of the test food or the placebo food before and after ingestion; Fig. 4(b) shows the change of the water content of the placebo group and the test product group.

由圖4(a)及圖4(b)可知，測試品群可看出攝取後水分量顯著(p＜0.05)上升，在肌膚容易乾燥的季節環境，仍確認肌膚水分量提升。It can be seen from Figure 4(a) and Figure 4(b) that the water content of the test group can be seen to increase significantly (p<0.05) after ingestion, and the skin moisture content is still confirmed to increase in the seasonal environment where the skin is prone to dryness.

又，雖省略圖示，以受試者的面頰為對象進行同樣的水分量測定，就安慰劑群，於攝取前後，面頰的水分量未看出顯著變化，而測試品群則看出顯著上升(p＜0.05)。In addition, although the illustration is omitted, the same moisture content was measured on the cheeks of the test subjects. In the placebo group, there was no significant change in the moisture content of the cheeks before and after ingestion, but a significant increase was seen in the test group. (p<0.05).

圖5表示經皮水分蒸散量相關之試驗結果。經皮水分蒸散量為從皮膚表面蒸發的水分量，係作為阻隔功能之指標的值。圖5(a)表示測試食品或安慰劑食品於攝取前後之經皮水分蒸散量的變化；圖5(b)則表示安慰劑群與測試品群之經皮水分蒸散量的變化量。Fig. 5 shows the test results related to transdermal water evapotranspiration. The transepidermal water evapotranspiration is the amount of water evaporated from the skin surface, and is a value serving as an index of the barrier function. Fig. 5(a) shows the change of transepidermal water evapotranspiration before and after ingestion of test food or placebo food; Fig. 5(b) shows the change of transepidermal water evapotranspiration of placebo group and test product group.

由圖5(a)可知，相對於安慰劑群之經皮水分蒸散量於攝取後顯著上升，測試品群則未看出顯著變化，表示藉由攝取測試品，可維持肌膚阻隔功能。又，由圖5(b)可知，其顯示攝取測試品，與攝取安慰劑品時相比，可顯著抑制經皮水分蒸散量的變化量(p＜0.05)。It can be seen from Figure 5(a) that compared with the placebo group, the transepidermal water evapotranspiration increased significantly after ingestion, while the test group did not see a significant change, indicating that the skin barrier function can be maintained by ingesting the test product. Also, it can be seen from Fig. 5(b) that the ingestion of the test product significantly suppressed the change in transepidermal water evapotranspiration compared with the ingestion of the placebo product (p<0.05).

圖6表示肌膚的總彈力(R2)相關之試驗結果。總彈力係表示皮膚整體的彈力之指標。皮膚彈性可藉由真皮層的纖維質或保水成分來維持。圖6(a)表示測試食品或安慰劑食品於攝取前後之總彈力的變化；圖6(b)則表示安慰劑群與測試品群之總彈力的變化量。Fig. 6 shows the test results related to the total elasticity (R2) of the skin. The total elasticity is an index indicating the overall elasticity of the skin. Skin elasticity can be maintained by fibrous or water-retaining ingredients in the dermis. Figure 6(a) shows the change of the total elasticity of the test food or the placebo food before and after ingestion; Figure 6(b) shows the change of the total elasticity of the placebo group and the test product group.

由圖6(a)及圖6(b)可知，表示總彈力之R2，於安慰劑群中顯示減少傾向，且看出時間與群此二主因的顯著交互作用，安慰劑群與測試品群的變化量顯示顯著的群間差。由此，Gly-Ile及Ile-Gly可謂與肌膚的總彈力(R2)有關，具有活化真皮層之機能，並維持肌膚的緊緻感或彈性之效果。From Figure 6(a) and Figure 6(b), it can be seen that R2, which represents the total elasticity, shows a tendency to decrease in the placebo group, and it can be seen that there is a significant interaction between the two main causes of time and group, the placebo group and the test group The amount of change shows a significant difference between groups. Therefore, Gly-Ile and Ile-Gly can be said to be related to the total elasticity (R2) of the skin, have the function of activating the dermis, and maintain the firmness or elasticity of the skin.

圖7表示肌膚的黏彈性(R5(淨彈性))相關之試驗結果。黏彈性係作為皮膚的緊緻感或彈性之指標的值。圖7(a)表示測試食品或安慰劑食品於攝取前後之黏彈性的變化；圖7(b)則表示安慰劑群與測試品群的黏彈性的變化量。Fig. 7 shows the test results related to the viscoelasticity (R5 (net elasticity)) of the skin. Viscoelasticity is a value used as an indicator of firmness or elasticity of the skin. Fig. 7(a) shows the change of viscoelasticity of test food or placebo food before and after ingestion; Fig. 7(b) shows the change of viscoelasticity of placebo group and test product group.

由圖7(a)及圖7(b)可知，就測試品群，表示淨彈性之R5，與攝取前相比顯著(p＜0.05)上升，顯示攝取測試品可提升肌膚的彈性。又，可看出時間與群此二主因之顯著傾向的交互作用，且變化量亦顯示顯著傾向之群間差異。It can be seen from Figure 7(a) and Figure 7(b) that for the test product group, R5, which represents the net elasticity, increased significantly (p<0.05) compared with before ingestion, indicating that ingestion of the test product can improve skin elasticity. In addition, it can be seen that the interaction between time and the significant tendency of the two main causes of the group, and the amount of change also shows the difference between the groups of the significant tendency.

圖8表示肌膚的回復力R7(復原率)相關之試驗結果。回復力為表示吸引時皮膚的瞬間收縮率相對於延展長度的值，係與肌膚的緊緻感或鬆弛顯示高相關性。圖8(a)表示測試食品或安慰劑食品於攝取前後之回復力的變化；圖8(b)則表示安慰劑群與測試品群的回復力的變化量。Fig. 8 shows the test results related to the resilience R7 (recovery rate) of the skin. The recovery force is a value representing the instantaneous contraction rate of the skin at the time of suction relative to the stretching length, and it shows a high correlation with the firmness or relaxation of the skin. Figure 8(a) shows the change of the recovery power of the test food or the placebo food before and after ingestion; Figure 8(b) shows the change of the recovery power of the placebo group and the test product group.

由圖8(a)及圖8(b)可知，就測試品群，回復力R7，與攝取前相比顯著(p＜0.05)上升，顯示攝取測試品可提升肌膚的回復力。又，就回復力R7，可看出時間與群此二主因之顯著傾向的交互作用，且變化量亦顯示顯著傾向之群間差異。It can be seen from Figure 8(a) and Figure 8(b) that for the test product group, the recovery power R7 was significantly (p<0.05) higher than before ingestion, indicating that ingestion of the test product can improve the skin's recovery power. Also, regarding the resilience R7, we can see the interaction between time and the significant tendency of the two main causes of the group, and the variation also shows the difference between the groups of the significant tendency.

根據此等各攝取試驗的結果顯示，含有Gly-Ile及Ile-Gly表示之二肽作為膚質改善用之產生功能成分，例如為機能性食品時之相關成分的機能食品係有助於保持肌膚水分、抑制肌膚水分的蒸散並維持及提升肌膚的緊緻感或黏彈性，而能夠改善膚質。According to the results of these intake tests, functional foods containing dipeptides represented by Gly-Ile and Ile-Gly as functional ingredients for skin quality improvement, such as related ingredients in functional foods, help to maintain skin Moisture, inhibit the evaporation of skin moisture and maintain and enhance the firmness or viscoelasticity of the skin, thereby improving the skin texture.

諸如上述，根據本實施形態之神經醯胺合成酵素基因表現促進劑，由於含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分，可提供一種採用既定的二肽作為產生功能用之主要成分，而能夠使神經醯胺合成酵素基因的表現量增大之神經醯胺合成酵素基因表現促進劑。As mentioned above, according to the ceramide synthetase gene expression accelerator of the present embodiment, since the dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main component for producing the function, it can provide a method using a predetermined dipeptide as a A ceramide synthetase gene expression enhancer that can increase the expression of ceramide synthetase gene by producing the main component for function.

再者，於本實施形態之膚質改善劑や膚質改善用機能食品、化妝料中，由於含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分，而能夠作成使神經醯胺合成酵素基因的表現量增大，得以改善膚質的膚質改善劑、膚質改善用機能食品或化妝料。Furthermore, in the skin quality improving agent of the present embodiment, the functional food for skin quality improvement, and cosmetics, since the dipeptide represented by Gly-Ile and/or Ile-Gly is contained as the main component for producing the function, it can be made A skin quality improving agent, a functional food or a cosmetic for skin quality improvement by increasing the expression level of the ceramide synthesizing enzyme gene to improve the skin quality.

最終，上述各實施形態之說明僅為本發明之一例，本發明非限定於上述實施形態。因此，除上述各實施形態以外，只要為不悖離本發明技術思想之範圍，理當可因應設計等施予種種變更。Finally, the description of each of the above-mentioned embodiments is merely an example of the present invention, and the present invention is not limited to the above-mentioned embodiments. Therefore, in addition to the above-mentioned embodiments, as long as the scope of the technical idea of the present invention is not deviated, various changes can be made in accordance with the design and the like.

[圖1]為表示添加各二肽之HaCaT細胞的細胞生存率的說明圖。 [圖2]為表示添加各二肽之HaCaT細胞之神經醯胺合成酵素基因的表現變化的說明圖。 [圖3]為表示添加少量二肽之HaCaT細胞之神經醯胺合成酵素基因的表現變化的說明圖。 [圖4]為表示攝取測試食品時之水分量相關試驗結果的說明圖。 [圖5]為表示攝取測試食品時之經皮水分蒸散量相關試驗結果的說明圖。 [圖6]為表示攝取測試食品時之肌膚的總彈力R2相關試驗結果的說明圖。 [圖7]為表示攝取測試食品時之肌膚的黏彈性(R5(淨彈性))相關試驗結果的說明圖。 [圖8]為表示攝取測試食品時之肌膚的回復力R7相關試驗結果的說明圖。 [ Fig. 1 ] is an explanatory graph showing the cell viability of HaCaT cells to which each dipeptide was added. [ Fig. 2] Fig. 2 is an explanatory diagram showing changes in the expression of the ceramide synthetase gene in HaCaT cells to which each dipeptide was added. [ Fig. 3] Fig. 3 is an explanatory diagram showing changes in the expression of the ceramide synthetase gene in HaCaT cells to which a small amount of dipeptide was added. [ Fig. 4 ] It is an explanatory diagram showing the test results related to the water content when the test food was ingested. [ Fig. 5 ] It is an explanatory diagram showing the test results related to transdermal water evapotranspiration when the test food is ingested. [ Fig. 6 ] It is an explanatory diagram showing the test results related to the total elasticity R2 of the skin when the test food was ingested. [ Fig. 7 ] It is an explanatory diagram showing the test results related to the viscoelasticity (R5 (net elasticity)) of the skin when the test food was ingested. [ Fig. 8 ] It is an explanatory diagram showing the test results related to the resilience R7 of the skin when the test food was ingested.

                                 
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          ccgattacct gctggagtca g                                                 21
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          ggcgaagacg atgaagatgt tg                                                22
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          acattccaca aggcaaccat tg                                                22
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          ctcttgattc cgccgactcc                                                   20
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          ggaggcctgt aagatggtca                                                   20
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          gaggaccagt cgggtgtaga                                                   20
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          gggtcagaag gactcctatg                                                   20
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          gtaacaatgc catgttcaat                                                   20
          
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Claims (7)

一種神經醯胺合成酵素基因表現促進劑，其係含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分。A ceramide synthesizing enzyme gene expression promoter, which contains dipeptide represented by Gly-Ile and/or Ile-Gly as the main component for producing function. 如請求項1之神經醯胺合成酵素基因表現促進劑，其中前述神經醯胺合成酵素基因為神經醯胺合成酵素3(CERS3)基因。Such as the ceramide synthetase gene expression promoter of claim 1, wherein the aforementioned ceramide synthetase gene is the ceramide synthetase 3 (CERS3) gene. 一種膚質改善劑，其係含有Gly-Ile及/或Ile-Gly表示之二肽作為產生功能用之主要成分。A skin quality improving agent, which contains dipeptide represented by Gly-Ile and/or Ile-Gly as the main component for producing functions. 一種膚質改善用機能食品，其係含有Gly-Ile及/或Ile-Gly表示之二肽作為相關成分。A functional food for improving skin quality, which contains dipeptides represented by Gly-Ile and/or Ile-Gly as related components. 一種化妝料，其係含有Gly-Ile及/或Ile-Gly表示之二肽作為有效成分。A cosmetic containing dipeptide represented by Gly-Ile and/or Ile-Gly as an active ingredient. 一種Gly-Ile及/或Ile-Gly表示之二肽於神經醯胺合成酵素基因表現促進劑中作為產生功能用之主要成分的使用。A dipeptide represented by Gly-Ile and/or Ile-Gly is used in a ceramide synthase gene expression promoter as a main component for producing functions. 一種Gly-Ile及/或Ile-Gly表示之二肽於機能食品中作為以改善膚質為目的之相關成分的使用。A dipeptide represented by Gly-Ile and/or Ile-Gly is used in functional food as a relevant ingredient for the purpose of improving skin quality.

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