KR102536371B1 - Composition Comprising Lactobacillus rhamnosus, Culture Fluid, or Culture Fluid Extract thereof with Anti-Inflammatory, Moisturizing, Skin Barrier, and Skin Cell Regeneration Property as Active Ingredient - Google Patents
Composition Comprising Lactobacillus rhamnosus, Culture Fluid, or Culture Fluid Extract thereof with Anti-Inflammatory, Moisturizing, Skin Barrier, and Skin Cell Regeneration Property as Active Ingredient Download PDFInfo
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- KR102536371B1 KR102536371B1 KR1020200172270A KR20200172270A KR102536371B1 KR 102536371 B1 KR102536371 B1 KR 102536371B1 KR 1020200172270 A KR1020200172270 A KR 1020200172270A KR 20200172270 A KR20200172270 A KR 20200172270A KR 102536371 B1 KR102536371 B1 KR 102536371B1
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- skin
- inflammatory
- lactobacillus rhamnosus
- extract
- moisturizing
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Abstract
본 발명은 락토바실러스 람노서스 균주, 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 조성물에 관한 것이다.
본 발명의 조성물은 세포 독성이 없고, 항염증 및 피부 보습에 우수한 효과가 있으며, 피부 장벽을 강화하고, 피부 상처 재생을 촉진하는 우수한 효과가 있으므로 화장품과 식품 분야에서 유용하게 이용 가능하다.The present invention relates to a composition for anti-inflammatory, skin moisturizing, strengthening skin barrier, and promoting skin regeneration comprising a Lactobacillus rhamnosus strain, a culture medium thereof, or an extract thereof as an active ingredient.
The composition of the present invention has no cytotoxicity, has excellent anti-inflammatory and skin moisturizing effects, strengthens the skin barrier, and promotes skin wound regeneration, so it can be usefully used in the cosmetic and food fields.
Description
본 발명은 락토바실러스 람노서스 균주, 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 조성물에 관한 것이다.The present invention relates to a composition for anti-inflammatory, skin moisturizing, strengthening skin barrier, and promoting skin regeneration comprising a Lactobacillus rhamnosus strain, a culture medium thereof, or an extract thereof as an active ingredient.
염증 반응은 세포 또는 조직의 손상이나 박테리아, 곰팡이, 바이러스, 다양한 종류의 알레르기 유발물질 등의 외부감염원에 감염되었을 때 국소 혈관과 체액 중 각종 염증 매개인자 및 면역세포가 관련되어, 효소 활성화, 염증 매개물질 분비, 체액 침윤, 세포 이동, 조직 파괴 등 일련의 복합적인 생리적 반응과 홍반, 부종, 발열, 통증 등 외적 증상을 나타낸다. 평상시의 염증 반응은 외부감염원을 제거하고 손상된 조직을 재생하여 생명체 기능회복작용을 하지만, 항원이 제거되지 않거나 내부물질이 원인이 되어 염증 반응이 과도하거나 지속해서 일어나면 오히려 점막 손상을 촉진하고, 그 결과 일부에서는 암 발생 등의 질환을 이끈다. Inflammatory response is associated with various inflammatory mediators and immune cells in local blood vessels and body fluids when cells or tissues are damaged or infected with external infectious agents such as bacteria, fungi, viruses, and various types of allergens, activating enzymes and mediating inflammation. It shows a series of complex physiological reactions such as substance secretion, body fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, edema, fever, and pain. Normally, the inflammatory response removes external infectious agents and regenerates damaged tissues to restore life function. In some cases, it leads to diseases such as cancer.
염증의 발생 원인으로서는 다양한 생화학적인 현상이 관여하고 있는 것으로 알려져 있는데, 특히 산화질소(Nitric Oxide; NO)를 발생시키는 효소인 산화질소 합성 효소(Nitric Oxide Synthase; NOS)와 프로스타글란딘의 생합성과 관련된 효소들이 염증 반응을 매개하는 데 있어서 가장 중요한 역할을 하는 것으로 알려져 있다. 따라서 L-아르기닌으로부터 산화질소를 생성시키는 효소인 산화질소 합성효소(NOS)나, 아라키돈산으로부터 프로스타글란딘 등을 합성하는데 관련된 효소인 사이클로옥시게나아제(COX)의 활성 저해를 통해 염증을 차단하는 것이 염증 치료제 개발에 목표가 되고 있다. It is known that various biochemical phenomena are involved in the cause of inflammation. In particular, nitric oxide synthase (NOS), an enzyme that generates nitric oxide (NO), and enzymes related to the biosynthesis of prostaglandins are known to be involved. It is known to play the most important role in mediating the inflammatory response. Therefore, blocking inflammation by inhibiting the activity of nitric oxide synthase (NOS), an enzyme that generates nitric oxide from L-arginine, or cyclooxygenase (COX), an enzyme involved in synthesizing prostaglandins from arachidonic acid, is It is aimed at developing therapies.
또한, 일반적으로 피부에 사용되는 보습용 화장료 조성물은 사람의 모발이나 피부 등에 수분을 일정량 유지함으로써 부드럽고 생기있게 하며, 갈라짐, 건조함 등의 손상을 방지해주는 역할을 한다. 즉, 피부 보습용 화장료 조성물은 피부나 모발에 일정량 이상의 수분을 공급하거나 수분이 유지함으로써 피부나 모발을 미화하고, 건강하게 유지하기 위한 목적으로 사용된다. 피부는 수분 유지에 필수적인 기능을 하는 기관으로, 수분 조절에 대략 2/3 정도의 비중을 담당하는 것으로 알려져 있다. 따라서, 화장품 분야에서는 수분의 공급, 유지, 즉 보습과 관련된 기능성 연구가 활발하게 진행되고 있다. 또한, 최근에는 천연물 등으로부터 얻은 생리 활성 물질을 화장료에 도입하여 피부 자체의 고유 방어 기능을 더욱 강화함으로써 피부의 보습력을 증가시키는 화장품의 개발이 활발하게 진행되는 추세이다.In addition, moisturizing cosmetic compositions generally used for skin maintain a certain amount of moisture in human hair or skin to make them soft and lively, and to prevent damage such as splitting and dryness. That is, the cosmetic composition for moisturizing the skin is used for the purpose of beautifying the skin or hair and keeping them healthy by supplying or maintaining a certain amount of moisture to the skin or hair. The skin is an organ that plays an essential role in maintaining moisture, and is known to be responsible for about 2/3 of the weight in moisture control. Therefore, in the field of cosmetics, functional studies related to the supply and maintenance of moisture, that is, moisturizing, are being actively conducted. In addition, recently, the development of cosmetics that increase the moisturizing power of the skin by introducing physiologically active substances obtained from natural products into cosmetics to further strengthen the skin's own defense function is a trend that is actively progressing.
한편, 화장품 소재는 안전성, 효능, 물성 등이 균형을 이루어야 한다. 또한, 경제성 확보도 중요한 요인으로서 화장품용 소재는 다양한 개발 방법, 천연물 추출과 미생물 등에 의한 발효를 이용하여 피부 투과, 노화 억제, 생리활성, 안전성 및 물성을 최적화하여 개발하게 된다. 화장품과 의약품의 경계가 무너지면서 화장품의 안정성과 의약품의 효과성을 겸비한 복합 기능성 원료 및 바이오 소재를 통해 다양한 코스메슈티컬 제품들이 높은 성장세를 이어가고 있다.On the other hand, cosmetic materials must balance safety, efficacy, and physical properties. In addition, securing economic feasibility is also an important factor, and cosmetic materials are developed by optimizing skin permeation, aging inhibition, physiological activity, safety and physical properties using various development methods, natural product extraction and fermentation by microorganisms. As the boundary between cosmetics and pharmaceuticals is collapsing, various cosmeceutical products continue to grow rapidly through complex functional raw materials and bio-materials that combine the stability of cosmetics and the effectiveness of pharmaceuticals.
이러한 배경 하에서, 본 발명자들은 항염증 또는 피부 보습에 효과가 우수한 유산균 소재에 관하여 연구를 수행하였으며, 항염증 또는 피부 보습 효과와 더불어 피부 상처 재생, 및 피부 장벽 강화 활성을 갖는 유산균 소재를 찾기 위해 예의 연구노력한 결과, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주가 갖는 우수한 피부 상태 개선 효과를 규명하여, 본 발명을 완성하게 되었다.Under this background, the present inventors have conducted research on lactic acid bacteria materials that are effective in anti-inflammatory or skin moisturizing, and are diligent to find lactic acid bacteria materials having anti-inflammatory or skin moisturizing effects, skin wound regeneration, and skin barrier strengthening activity. As a result of research efforts, the present invention was completed by identifying the excellent skin condition improving effect of the Lactobacillus rhamnosus mesopharm001 strain.
따라서, 본 발명의 주된 목적은 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진 활성을 갖는 락토바실러스 람노서스 균주, 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 조성물을 제공하는 데 있다.Accordingly, a main object of the present invention is to provide a composition comprising a Lactobacillus rhamnosus strain, a culture thereof, or an extract thereof having anti-inflammatory, skin moisturizing, skin barrier strengthening, and skin regeneration promoting activities as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 한 양태에 따르면, 본 발명은 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 화장료 조성물을 제공한다.According to one aspect of the present invention, the present invention provides anti-inflammatory, skin moisturizing, skin barrier strengthening, And it provides a cosmetic composition for promoting skin regeneration.
본 발명자들은 항염증 및 피부 보습에 뛰어난 효과가 있는 여러 가지 천연물질들을 대상으로 그 효과를 탐색하였고, 유산균 소재들이 갖는 피부 상태 개선 효과에 주목하여 우수한 유산균 소재를 발굴하고자 연구노력한 결과, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)를 유제품에서 분리 동정하였고, 동 균주의 배양액이 항염증, 피부 보습, 피부장벽 강화, 및 피부 상처 재생에 우수한 효과가 있음을 규명하였고, 피부에 적용 시에도 안전성에 문제가 없음을 확인하였다(도 1 참조).The present inventors searched for the effects of various natural substances that have excellent effects on anti-inflammatory and skin moisturizing, and as a result of research efforts to discover excellent lactic acid bacteria materials by paying attention to the skin condition improvement effect of lactic acid bacteria materials, Lactobacillus rhamno The Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) was isolated and identified from dairy products, and it was found that the culture solution of the strain had excellent effects on anti-inflammatory, skin moisturizing, skin barrier strengthening, and skin wound regeneration. Even when applied, it was confirmed that there was no problem with safety (see FIG. 1).
또한, 상기 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주는 부다페스트 조약 하의 국제기탁기관인 한국생명공학연구원(KCTC)에 국제기탁하여 수탁번호 KCTC 14350BP를 부여받았다. 본 발명에서 용어 "락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주", "mesopharm001 균주", 및 "mesopharm001"는 서로 혼용하여 사용할 수 있다. In addition, the Lactobacillus rhamnosus mesopharm001 strain was internationally deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC), an international depository under the Budapest Treaty, and received accession number KCTC 14350BP. In the present invention, the terms " Lactobacillus rhamnosus mesopharm001 strain", "mesopharm001 strain", and "mesopharm001" may be used interchangeably.
본 발명에서 사용되는 용어 "추출물"은 락토바실러스 람노서스 균주 또는 그 배양액의 추출 처리에 따라 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 본 발명의 상기 추출물은 바람직하게 추출 후 건조 분말 형태로 제조되어 사용될 수 있다.The term "extract" used in the present invention refers to an extract obtained by extraction of a Lactobacillus rhamnosus strain or its culture, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or mixtures thereof, and extracts of all formulations that can be formed using the extract itself and the extract. The extract of the present invention may be preferably prepared and used in the form of a dry powder after extraction.
상기 락토바실러스 람노서스 균주 또는 그 배양액의 추출에 있어서, 추출하는 방법은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 상기 추출방법의 비제한적인 예로는, 열수 추출법, 초음파 추출법, 여과법, 환류 추출법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2종 이상의 추출방법을 병용하여 수행될 수 있다. In the extraction of the Lactobacillus rhamnosus strain or its culture, the extraction method is not particularly limited, and can be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination with two or more extraction methods.
본 발명에서 상기 락토바실러스 람노서스 균주 또는 그 배양액을 추출하는데 사용되는 추출 용매의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물; 메탄올, 에탄올, 프로필알코올, 부틸알코올 등의 C1 내지 C4의 저급 알코올; 글리세린, 부틸렌글리콜, 프로필렌글리콜 등의 다가 알코올; 및 메틸아세테이트, 에틸아세테이트, 아세톤, 벤젠, 헥산, 디에틸에테르, 디클로로메탄 등의 탄화수소계 용매; 또는 이들의 혼합물을 사용할 수 있으며, 바람직하게, 물, 저급알코올, 1,3-부틸렌글리콜, 에틸아세테이트를 단독으로 사용하거나 2종 이상 혼합하여 사용할 수 있다. In the present invention, the type of extraction solvent used to extract the Lactobacillus rhamnosus strain or its culture medium is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.
본 발명에서 열수 추출 또는 냉침 추출한 추출물은 부유하는 고체 입자를 제거하기 위해 여과, 예를 들어 나일론 등을 이용해 입자를 걸러내거나 냉동여과법 등을 이용해 여과한 후, 그대로 사용하거나 이를 동결건조, 열풍건조, 분무건조 등을 이용해 건조하여 사용할 수 있다.In the present invention, the extract obtained by hot water extraction or cold brewing is filtered to remove floating solid particles by filtering out the particles using, for example, nylon or by using cryofiltration, and then used as it is or freeze-dried, hot-air-dried, It can be used after being dried using spray drying or the like.
본 발명의 화장료 조성물에서, 상기 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물은 산화질소의 생성을 억제하여 항염증 활성을 갖는 것을 특징으로 한다. 본 발명의 실시예에서는 본 균주의 배양액 추출물이 농도 의존적으로 산화질소의 생성을 억제하여 항염증 활성을 갖는 것을 확인하였다(도 2 참조). In the cosmetic composition of the present invention, the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP), its culture medium, or its extract is characterized in that it has anti-inflammatory activity by inhibiting the production of nitric oxide. In an embodiment of the present invention, it was confirmed that the culture extract of this strain had anti-inflammatory activity by inhibiting the production of nitric oxide in a concentration-dependent manner (see FIG. 2).
또한, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물을 포함하는 본 발명의 화장료 조성물은 히알루론산 합성을 촉진하여 피부 보습 활성을 갖는 것을 특징으로 한다. 상기 용어 "피부 보습"은 피부에 수분감을 증가시켜주고, 촉촉한 상태를 유지하는 것을 의미한다. 또한, 히알루론산(HA, Hyaluronan, Hyaluronic acid)은 주로 표피 각질형성세포와 진피 섬유아세포에 의해 합성되며, 수분과 결합하여 세포가 정상적으로 활동할 수 있는 환경을 조성하는 중요한 역할을 하는 물질이다. 피부에서 히알루론산의 감소는 피부 탄력 저하 및 수분 함유량 감소의 직접적인 원인 중 하나이다. 따라서 본 발명의 바람직한 실시예에서는 인간 섬유아세포 내에서 히알루론산의 생성량을 확인하였다. 그 결과, 본 균주의 배양액 추출물이 농도 의존적으로 히알루론산 합성을 증가시키고, 양성대조군인 레티노익산(retinoic acid, RA) 처리군과 비교하여도 50㎍/㎖ 및 100㎍/㎖ 처리군에서 각각 105%, 및 125% 개선된 히알루론산 합성 증가 효과가 있는 것을 확인한 바 있다(도 3 참조). In addition, the cosmetic composition of the present invention comprising a Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP), a culture solution thereof, or an extract thereof is characterized in that it has skin moisturizing activity by promoting hyaluronic acid synthesis. The term "moisturizing the skin" means to increase the feeling of moisture in the skin and maintain a moist state. In addition, hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is synthesized mainly by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with moisture. A decrease in hyaluronic acid in the skin is one of the direct causes of a decrease in skin elasticity and a decrease in moisture content. Therefore, in a preferred embodiment of the present invention, the amount of hyaluronic acid produced in human fibroblasts was confirmed. As a result, the culture extract of this strain increased hyaluronic acid synthesis in a concentration-dependent manner, and compared to the positive control group, retinoic acid (RA) treatment group, 50 μg / ml and 100 μg / ml treatment group respectively 105 %, and it was confirmed that there was an effect of increasing hyaluronic acid synthesis improved by 125% (see FIG. 3).
또한, 본 발명의 화장료 조성물은 필라그린 유전자 및 히알루론산 합성효소(hyaluronan synthase-3) 유전자의 발현을 증가시켜 피부 장벽 강화 활성을 갖는 것을 특징으로 한다. 필라그린은 표피 과립층에서 케라토히알린과립(keratohyalin granule)을 형성하는 단백질인 프로필라그린(profilaggrin)의 형태로 존재하다가, 각질형성세포의 최종분화과정에서 필라그린으로 분해된 후에 각질세포막을 형성할 때 케라틴 필라멘트를 응집하여 각질세포의 단단하고 편평한 구조를 형성함으로써 피부 장벽에서 벽돌(brick)의 역할을 한다. 본 발명의 바람직한 실시예에서는 본 균주의 추출물 처리군이 양성대조군인 레티노익산(retinoic acid, RA) 처리군과 비교하여 필라그린(filaggrin) 유전자의 mRNA 발현을 각각 151%(50μg/mL) 및 188%(100μg/mL) 증가시키고, 히알루론산 합성효소(hyaluronan synthase-3) 유전자의 mRNA 발현량을 각각 233%(50μg/mL) 및 295%(100μg/mL) 증가시켜 피부 장벽 강화에 효과적임을 확인하였다(도 4 참조). In addition, the cosmetic composition of the present invention is characterized in that it has skin barrier strengthening activity by increasing the expression of filaggrin gene and hyaluronan synthase-3 gene. Filaggrin exists in the form of profilagrin, a protein that forms keratohyalin granules in the granular layer of the epidermis. It acts as a brick in the skin barrier by forming a hard and flat structure of keratinocytes by aggregating keratin filaments. In a preferred embodiment of the present invention, the extract treatment group of this strain compared to the retinoic acid (RA) treatment group, which is a positive control, increased the mRNA expression of the filaggrin gene by 151% (50 μg / mL) and 188, respectively. % (100μg/mL) and increased the mRNA expression of the hyaluronan synthase-3 gene by 233% (50μg/mL) and 295% (100μg/mL), respectively, confirming that it is effective in strengthening the skin barrier (see FIG. 4).
또한, 본 발명의 화장료 조성물은 피부 상처의 재생을 촉진하는 것을 특징으로 하는데, 본 발명의 바람직한 실시예에서는 본 균주의 배양액 추출물 처리에 의해서 피부 상처의 재생이 촉진되는 것을 확인할 수 있었으며, 양성대조군인 레티노익산(retinoic acid, RA) 처리군과 비교하여도, 50, 및 100㎍/㎖ 농도에서 각각 105%(50μg/mL) 및 120%(100μg/mL) 개선된 상처 수복율을 확인한 바 있다(도 5 참조). In addition, the cosmetic composition of the present invention is characterized by promoting the regeneration of skin wounds. In a preferred embodiment of the present invention, it was confirmed that the regeneration of skin wounds was promoted by treatment with the culture solution extract of this strain, and the positive control Compared to the retinoic acid (RA) treatment group, 105% (50 μg/mL) and 120% (100 μg/mL) improved wound repair rates were confirmed at concentrations of 50 and 100 μg/mL, respectively ( see Figure 5).
본 발명의 화장료 조성물은 식용 가능한 유산균 소재에서 유래하여 인체에 해가 없으며, 그 첨가로 인해 화장품의 물질의 품질을 손상하지 않으므로 화장료 조성물에 포함되어 사용되는데 적합하다. 특히, 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진과 관련된 기능이 있으므로, 기능성 화장료 조성물에 포함되어 사용되는 것이 바람직하다.The cosmetic composition of the present invention is derived from edible lactic acid bacteria, is harmless to the human body, and does not impair the quality of cosmetics due to its addition, so it is suitable for being included in the cosmetic composition. In particular, since it has functions related to anti-inflammatory, skin moisturizing, skin barrier enhancement, and skin regeneration promotion, it is preferably included in functional cosmetic compositions and used.
본 발명의 화장료 조성물은 스킨로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 맛사지크림, 영양크림, 모이스처크림, 핸드크림, 파운데이션, 에센스, 영양에센스, 마스크팩, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션 및 바디클린저에서 선택된 어느 하나의 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, foundation, essence, nutrient essence, mask pack, It may be prepared in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.
본 발명의 상기 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한 담체를 1종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면 활성제, 보습제, 저급 알코올, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as typical ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , A chelating agent, a colorant, a preservative, a flavoring agent, etc. may be appropriately mixed, but is not limited thereto.
본 발명의 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 화장료 조성물의 제형에 따라 다양하다.Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.
본 발명의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체 성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화아연 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. This may be used, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 등이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로하드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, low carbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.
본 발명의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제 등이 이용될 수 있으며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일 등이 이용될 수 있고, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브 오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan Can be used, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 제형이 현탁액인 경우에는, 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타하이드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a suspension, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 제형이 비누인 경우에는, 담체 성분으로서 지방산의 알칼리 금속 염, 지방산 헤미에스테르 염, 지방산 단백질 히드롤리제이트, 이세티오네이트, 라놀린 유도체, 지방족 알코올, 식물성 유, 글리세롤, 당 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. are used as carrier components. It may be, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 화장료 조성물에서, 상기 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 균주, 그 배양액, 또는 그 추출물은 구체적으로는 건조 중량으로, 화장료 조성물 총 중량의 0.001 중량% 내지 50 중량%로 포함될 수 있으며, 보다 구체적으로는 0.005 중량% 내지 10 중량%로 포함될 수 있다. 상기 범위 내에서 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진에 대한 우수한 효능을 나타내는 이점이 있으며, 조성물의 제형이 안정화되는 이점이 있다.In the cosmetic composition of the present invention, the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) strain, its culture medium, or its extract is specifically dry weight, 0.001% to 50% by weight of the total weight of the cosmetic composition It may be included in % by weight, and more specifically, it may be included in 0.005% by weight to 10% by weight. There is an advantage of exhibiting excellent efficacy for anti-inflammatory, skin moisturizing, skin barrier strengthening, and skin regeneration promotion within the above range, and there is an advantage that the formulation of the composition is stabilized.
본 발명의 다른 양태에 따르면, 본 발명은 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides anti-inflammatory, skin moisturizing, skin barrier strengthening, And it provides a food composition for promoting skin regeneration.
본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물은 항염증 및 피부 보습에 우수한 활성을 갖는 동시에 피부 장벽 강화 및 피부 상처 재생에도 우수한 효과를 나타내어 피부 상태 개선을 위한 식품 조성물로 유용하게 사용될 수 있다. 본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물와 "항염증", "피부 보습", "피부 장벽 강화" 및 "피부 상처 재생"에 대한 효과는 상기에서 설명한 바와 같다. 상기 식품 조성물은 건강기능식품의 형태로 사용될 수 있으나, 이에 제한되는 것은 아니다. The Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP), its culture medium, or its extract of the present invention has excellent activity in anti-inflammatory and skin moisturizing, and at the same time shows excellent effects in strengthening the skin barrier and regenerating skin wounds. It can be usefully used as a food composition for improving skin conditions. Lactobacillus rhamnosus mesopharm001 strain of the present invention (Accession No. KCTC 14350BP), its culture, or its extract and its effects on "anti-inflammatory", "skin moisturizing", "skin barrier strengthening" and "skin wound regeneration" is as described above. The food composition may be used in the form of health functional food, but is not limited thereto.
본 발명의 식품 조성물은 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물과, 이들의 가공물의 형태로 포함될 수 있다. 또한, 상기 조성물은 유효성분 이외에 식품학적으로 허용 가능한 식품 보조첨가제를 포함할 수 있다.The food composition of the present invention may be included in the form of a Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP), a culture thereof, or an extract thereof, and a processed product thereof. In addition, the composition may include food additives acceptable food additives in addition to the active ingredient.
본 발명에 있어서, "식품 보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품 보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품 보조첨가제의 종류가 제한되는 것은 아니다.In the present invention, "food supplement additive" means a component that can be added to food supplementally, as added to prepare a health functional food of each formulation, can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.
본 발명의 식품 조성물에는 건강기능식품이 포함될 수 있다. 본 발명에 있어서, "건강기능식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미하며, 본 발명에서는 "항염증", "피부 보습", "피부 장벽 강화" 및 "피부 상처 재생"을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법으로 제조 가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. The food composition of the present invention may include health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining useful effects for health purposes such as adjusting nutrients for the structure and function of the human body or physiological actions, and in the present invention, "anti-inflammatory", "skin moisturizing", "skin barrier" "strengthening" and "regenerating skin wounds". The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation.
또한, 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 식품 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식용 유산균을 원료로 하여 식품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나며, 피부에 염증 발생을 줄여주고, 보습 효과를 개선하며, 피부 상처 재생을 촉진하고, 피부 장벽을 강화하는 효과를 증진시키기 위한 보조제로 섭취할 수 있다.In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and unlike general medicines, it has the advantage of using edible lactic acid bacteria as a raw material and has no side effects that may occur when taking food for a long time, has excellent portability, and is effective on the skin. It can be taken as an adjuvant to reduce inflammation, improve moisturizing effect, promote skin wound regeneration, and enhance the effect of strengthening the skin barrier.
본 발명의 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있고, 기능성 식품 등 당업계에 알려진 용어와 혼용하여 사용할 수 있다. 아울러 본 발명의 건강기능식품은 당업자의 선택에 따라 식품에 포함될 수 있는 적절한 기타 보조 성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP), 그 배양액, 또는 그 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다. 또한, 동물을 위한 사료로 이용되는 식품도 포함된다.There is no limit to the form that the health functional food of the present invention can take, and it may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as functional foods. In addition, the health functional food of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., prepared by adding the Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) according to the present invention, its culture medium, or its extract to juice, tea, jelly and juice prepared as a main component can do. Also included are foods used as feed for animals.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(1) 본 발명은 락토바실러스 람노서스 균주, 그 배양액, 또는 그 추출물을 유효성분으로 포함하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 조성물을 제공한다.(1) The present invention provides a composition for anti-inflammatory, skin moisturizing, strengthening skin barrier, and promoting skin regeneration comprising a Lactobacillus rhamnosus strain, its culture medium, or an extract thereof as an active ingredient.
(2) 본 발명의 조성물은 세포 독성이 없고, 항염증 및 피부 보습에 우수한 효과가 있으며, 피부 장벽을 강화하고, 피부 상처 재생을 촉진하는 우수한 효과가 있으므로 화장품과 식품 분야에서 유용하게 이용 가능하다.(2) The composition of the present invention has no cytotoxicity, has excellent anti-inflammatory and skin moisturizing effects, strengthens the skin barrier, and promotes skin wound regeneration, so it can be usefully used in the cosmetics and food fields. .
(3) 또한, 본 발명의 조성물은 인체에 매우 안전할 뿐만 아니라, 안정성도 매우 탁월하다.(3) In addition, the composition of the present invention is not only very safe to the human body, but also has excellent stability.
도 1은 락토바실러스 람노서스 균주의 배양액 추출물 처리에 따른 인간각질형성세포(HaCaT)의 세포생존능을 측정하여 나타낸 그래프이다.
도 2는 대식세포주인 RAW 264.7 세포에서 락토바실러스 람노서스 균주의 배양액 추출물 처리에 따른 산화질소 생성량 변화를 측정하여 나타낸 그래프이다.
도 3은 인간각질형성세포(HaCaT)에서 락토바실러스 람노서스 균주의 배양액 추출물 처리에 따른 히알루론산의 생성량 변화를 측정하여 나타낸 그래프이다.
도 4는 인간각질형성세포(HaCaT)에서 락토바실러스 람노서스 균주의 배양액 추출물 처리에 따른 필라그린 및 히알루론산 합성효소(hyaluronan synthase-3) 유전자의 mRNA 발현 변화를 측정하여 나타낸 그래프이다.
도 5는 인간각질형성세포(HaCaT)에서 락토바실러스 람노서스 균주의 배양액 추출물 처리에 따른 피부 재생 효과를 MTT(methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) 분석 방법과 디프 퀵 스테인(Diff quic stain) 방법을 이용하여 세포염색 후 현미경으로 확인하고 정상세포와 비교하여 나타낸 그래프이다.Figure 1 is a graph showing the measurement of cell viability of human keratinocytes (HaCaT) according to the treatment of the culture extract of the Lactobacillus rhamnosus strain.
Figure 2 is a graph showing the measurement of the change in the amount of nitric oxide production according to the treatment of the culture extract of the Lactobacillus rhamnosus strain in the macrophage cell line RAW 264.7 cells.
Figure 3 is a graph showing the change in the amount of hyaluronic acid production according to the treatment of the culture extract of the Lactobacillus rhamnosus strain in human keratinocytes (HaCaT).
Figure 4 is a graph showing changes in mRNA expression of filaggrin and hyaluronan synthase-3 genes according to the treatment of culture extracts of Lactobacillus rhamnosus strains in human keratinocytes (HaCaT).
Figure 5 is a MTT (methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) analysis method and Diff quic stain for the skin regeneration effect according to the treatment of the culture extract of the Lactobacillus rhamnosus strain in human keratinocytes (HaCaT) ) method after staining the cells, confirming them under a microscope and comparing them with normal cells.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 락토바실러스 람노서스 균주 배양액 추출물의 제조Example 1. Preparation of Lactobacillus rhamnosus strain culture extract
MRS 브로쓰(Difco) 100mL에 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)를 접종하고 호기성 조건 하에 30℃ 항온기에서 OD 600nm에서 1.0이 될 때까지 배양하여 배양물을 획득하였다. 배양액 10㎖를 4℃에서 4,000rpm으로 20분간 원심분리하여 상층액만을 모았다. 이렇게 얻은 락토바실러스 람노서스 균주 배양액을 감압농축기(Rotary evaporator N-1000, EYELA, Japan)를 이용하여 55℃에서 감압 농축한 후, 동결건조하여 분말형태로 제조하였다. Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) was inoculated in 100 mL of MRS broth (Difco) and cultured under aerobic conditions in a thermostat at 30 ° C until an OD of 600 nm was 1.0 to obtain a culture. 10 ml of the culture was centrifuged at 4° C. at 4,000 rpm for 20 minutes to collect only the supernatant. The obtained Lactobacillus rhamnosus strain culture solution was concentrated under reduced pressure at 55° C. using a rotary evaporator (Rotary evaporator N-1000, EYELA, Japan), and then freeze-dried to prepare a powder form.
실시예 2. 세포독성 시험(MTTAssay)Example 2. Cytotoxicity test (MTTAssay)
실시예 1에서 준비한 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물의 인간각질형성세포(HaCaT)에 대한 세포 독성을 시험하였다. The cytotoxicity of the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) prepared in Example 1 to human keratinocytes (HaCaT) was tested.
세포 독성은 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 비색정량분석법(colorimetric assay)을 사용하여 측정하였다. MTT(Methyl thiazol tetrazolium) 분석은 세포 생존 능력을 측정하기 위해 사용되는 것으로, MTT가 보라색을 생성하는 포마잔 염색으로 바꾸는 것이다. 인큐베이션 72시간 후, 배지의 부피는 1㎖로 감소하였고, 1㎎/㎖ MTT의 100㎕는 각 벽에 첨가되었다. 이후, 인간각질형성세포(HaCaT)를 37℃에서 2시간 동안 5%의 CO2 및 95%의 O2 존재 하에 배양하였다. 기질-함유 배지는 제거되었고, 1㎖의 DMSO는 포마잔 결정을 용해하기 위해 각 벽에 첨가되었다. 또한, 상온에서 30분 동안 오비탈 셰이커로 플레이트를 흔들었다. 100㎕의 부분 표본의 흡수율은 마이크로플레이트 리더(Molecular Devices E09090; 샌프란시스코, CA, 미국)를 사용하여 570㎚에서 흡수율을 측정함으로써 정량화하였다. Cytotoxicity was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Methyl thiazol tetrazolium (MTT) assay is used to measure cell viability, in which MTT converts to formazan staining, which produces a purple color. After 72 hours of incubation, the volume of the medium was reduced to 1 ml and 100 μl of 1 mg/ml MTT was added to each wall. Thereafter, human keratinocytes (HaCaT) were cultured at 37° C. for 2 hours in the presence of 5% CO 2 and 95% O 2 . The substrate-containing medium was removed and 1 ml of DMSO was added to each wall to dissolve the formazan crystals. Additionally, the plate was shaken on an orbital shaker for 30 minutes at room temperature. The absorbance of 100 μl aliquots was quantified by measuring the absorbance at 570 nm using a microplate reader (Molecular Devices E09090; San Francisco, CA, USA).
그 결과, 도 1에 나타난 바와 같이, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물은 인간각질형성세포(HaCaT)에 독성을 나타내지 않고, 오히려 농도 의존적으로 생장을 촉진하는 것을 확인할 수 있었다.As a result, as shown in FIG. 1, the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) culture extract does not exhibit toxicity to human keratinocytes (HaCaT), but rather promotes growth in a concentration-dependent manner. could confirm that
실시예 3. 산화질소 생성 억제를 통한 항염증 활성Example 3. Anti-inflammatory activity through inhibition of nitric oxide production
본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물이 갖는 항염증 효과를 확인하기 위해, 염증 유발에 관여하는 대표적인 세포 독성물질인 일산화질소(nitric oxide, NO)의 생성 억제율을 측정하였다.In order to confirm the anti-inflammatory effect of the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) of the present invention, nitric oxide (NO), a representative cytotoxic substance involved in inducing inflammation, Production inhibition rate was measured.
먼저, 대식세포주인 RAW 264.7 세포를 한국세포주은행(KTCC, Seoul, Korea)에서 분양받아 사용하였고, 10% FBS(Fetal bovine serum)를 함유한 DMEM(Dulbecco’s modified Eagle’s medium)에 10% FBS, 100㎍/㎖ 페니실린 및 100㎍/㎖ 스트렙토마이신을 첨가한 배지에 상기 세포를 접종하여 37℃, 5% CO2 조건에서 배양하였다.First, RAW 264.7 cells, a macrophage cell line, were purchased and used from Korea Cell Line Bank (KTCC, Seoul, Korea), and DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum) was mixed with 10% FBS and 100 μg. The cells were inoculated into a medium supplemented with /ml penicillin and 100 μg/ml streptomycin, and cultured at 37°C and 5% CO 2 conditions.
구체적으로, 상기 RAW 264.7 세포를 1×106 cells/mL의 농도(in DMEM)로 96 웰 플레이트에 접종하고 24시간 배양 후, 실시예 1에서 제조된 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물을 각각 10, 50, 및 100㎍/㎖ 농도로 희석한 시료를 첨가하였고, 내독소로 알려진 LPS(1μg/ml)를 함유한 새로운 배지를 처리하여 24시간 배양하였다. 그 후, 세포 배양 상등액 100㎕와 Griess reagent[1%(w/v) sulfanilamide, 0.1%(w/v) naphtylethylenediamine in 2.5%(v/v) phosphoric acid] 100㎕를 혼합하여 96 웰 플레이트에서 10분 동안 반응시키고, ELISA 리더를 사용하여 540nm에서 흡광도를 측정하여 생성된 산화질소의 양을 측정하였다. 비교실험을 위해, 염증억제제로 사용되고 있는 덱사메타손(Dexamethasone) 10㎕ 처리군을 양성대조군으로 하였다.Specifically, the RAW 264.7 cells were inoculated into a 96-well plate at a concentration of 1×10 6 cells/mL (in DMEM) and cultured for 24 hours, followed by Lactobacillus rhamnosus mesopharm001 prepared in Example 1 strain ( Accession No. KCTC 14350BP) culture medium extracts diluted to concentrations of 10, 50, and 100 μg/ml, respectively, were added, and a new medium containing LPS (1 μg/ml) known as an endotoxin was treated and cultured for 24 hours. Then, 100 μl of the cell culture supernatant was mixed with 100 μl of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid], and 10 μl was prepared in a 96-well plate. After reacting for minutes, the absorbance was measured at 540 nm using an ELISA reader to measure the amount of nitric oxide produced. For comparative experiments, 10 μl of dexamethasone used as an inflammation inhibitor was used as a positive control group.
생성된 아질산염(nitrite)의 농도는 아질산나트륨(sodium nitrite)을 DMEM 배지에 용해한 표준 곡선을 이용하여 계산하였다. LPS를 처리한 대조군과 LPS를 처리하지 않은 대조군에서 생성된 아질산염의 생성량 차이를 기준으로 각 시료의 일산화질소 생성 저해 활성을 확인하였다.The concentration of nitrite produced was calculated using a standard curve in which sodium nitrite was dissolved in DMEM medium. Based on the difference in the amount of nitrite produced in the control group treated with LPS and the control group not treated with LPS, the nitric oxide production inhibitory activity of each sample was confirmed.
실험 결과, 본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물은 농도의존적으로 산화질소의 생성을 억제하는 효과는 나타내었는데, 구체적으로, 시료 무처리 음성 대조군과 비교하여 각각 8%(10㎍/㎖), 45%(50㎍/㎖) 및 59%(100㎍/㎖) 감소된 수준으로 산화질소 생성을 억제하는 것으로 관찰되었으며, 양성대조군인 덱사메타손 처리군과 비교하여도 각각 13%(50㎍/㎖) 및 35%(100㎍/㎖) 감소된 수준으로 산화질소의 생성을 억제하여 우수한 항염증 효과가 있는 것으로 관찰되었다(도 2 참조).As a result of the experiment, the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) of the present invention showed a concentration-dependent inhibitory effect on the production of nitric oxide. Specifically, compared with the negative control group without sample treatment It was observed that nitric oxide production was inhibited by 8% (10 μg/ml), 45% (50 μg/ml), and 59% (100 μg/ml), respectively, compared to the positive control group, dexamethasone-treated group. However, it was observed that there was an excellent anti-inflammatory effect by suppressing the production of nitric oxide to a level reduced by 13% (50 μg/ml) and 35% (100 μg/ml), respectively (see FIG. 2).
실시예 4. 피부 보습 활성Example 4. Skin moisturizing activity
본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물이 갖는 피부 보습 효과를 검증하기 위해 이들이 히알루론산(Hyaluronan) 합성 촉진에 미치는 영향을 실험하였다.In order to verify the skin moisturizing effect of the Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) culture extract of the present invention, their effect on promoting hyaluronan synthesis was tested.
인간각질형성세포(HaCaT)를 24 well 세포배양 접시에 1㎖의 배양액을 넣고 약 1×105의 농도로 세포를 접종한 후, 37℃, 5% CO2 환경에서 24시간 동안 배양하였다. 그 후, 실시예 1에 따라 준비된 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물을 각각 10, 50, 및 100㎍/㎖를 포함한 배지로 교체하여 24시간 배양하였다. 이후 배양액을 회수(harvest)하여 4℃, 7,500rpm 환경에서 5분간 원심분리(centrifuge)한 뒤, ELISA 방법을 이용하여 히알루론산(Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA)의 발현량 변화를 확인하였다. 비교실험을 위해 피부 보습 효과가 우수한것으로 알려진 병풀 추출물과 레티노익산(retinoic acid, RA)을 각각 양성대조군으로 사용하였다. Human keratinocytes (HaCaT) were put in 1 ml of the culture medium in a 24 well cell culture dish, and the cells were inoculated at a concentration of about 1×10 5 , and then cultured for 24 hours at 37° C., 5% CO 2 environment. Then, the culture medium extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) prepared according to Example 1 was replaced with a medium containing 10, 50, and 100 μg/ml, respectively, and cultured for 24 hours. Afterwards, the culture solution was harvested, centrifuged for 5 minutes at 4°C and 7,500 rpm, and the expression of hyaluronic acid (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was performed using the ELISA method. Quantity change was confirmed. For comparative experiments, centella asiatica extract and retinoic acid (RA), which are known to have excellent skin moisturizing effects, were used as positive controls, respectively.
실험 결과, 도 3을 참조하면, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 배양액 추출물의 처리로 인해 히알루론산의 생성이 농도 의존적으로 증가하는 것을 관찰할 수 있었다. 구체적으로 시료 무처리군과 비교하여 본 발명의 균주 배양액 추출물 처리군은 각각 110%(10㎍/㎖), 141%(50㎍/㎖), 및 168%(100㎍/㎖) 개선된 히알루론산 합성 증가 효과를 나타내었고, 병풀 추출물 처리 양성대조군과 비교하여도 각각 105%(10㎍/㎖), 126%(50㎍/㎖), 및 138%(100㎍/㎖) 개선된 히알루론산 합성 증가 효과를 나타내었다. As a result of the experiment, referring to FIG. 3, it was observed that the production of hyaluronic acid increased in a concentration-dependent manner due to the treatment of the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP). Specifically, compared to the sample untreated group, the strain culture extract treatment group of the present invention improved hyaluronic acid by 110% (10 μg/ml), 141% (50 μg/ml), and 168% (100 μg/ml), respectively. The synthetic increase effect was shown, and hyaluronic acid synthesis increased by 105% (10 μg/ml), 126% (50 μg/ml), and 138% (100 μg/ml), respectively, compared to the positive control group treated with centella asiatica extract. showed effect.
또한, 단일 화합물인 레티노익산(retinoic acid, RA) 처리군과 비교하여도 50㎍/㎖ 및 100㎍/㎖ 처리군에서 각각 105%, 및 125% 개선된 히알루론산 합성 증가 효과가 있는 것으로 확인되었다. In addition, compared to the retinoic acid (RA) treatment group, which is a single compound, the 50 μg/ml and 100 μg/ml treatment groups were found to have 105% and 125% improvement in hyaluronic acid synthesis, respectively. .
상기와 같은 결과를 통해, 본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP) 또는 이의 배양액 추출물은 히알루론산의 합성을 증가시켜 피부 보습에 효과가 있음을 확인할 수 있었다.Through the above results, it was confirmed that the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) or its culture extract of the present invention is effective in moisturizing the skin by increasing the synthesis of hyaluronic acid.
실시예 5. 피부 장벽 강화(HAS-3 및 Filaggrin 유전자 발현 분석)Example 5. Skin barrier enhancement (HAS-3 and Filaggrin gene expression analysis)
락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물이 필라그린(Filaggrin, FLG, 각질층 형성 인자) 및 HAS-3(Hyaluronan synthase-3, 피부보습 인자) 유전자 발현에 미치는 영향을 실험하였다.Effect of Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) culture medium extract on filaggrin (FLG, stratum corneum forming factor) and HAS-3 (Hyaluronan synthase-3, skin moisturizing factor) gene expression was experimented with.
먼저, 100mm 세포 배양 접시에 10% FBS를 첨가한 DMEM 배지를 첨가하고, 인간각질형성세포(HaCaT)를 1×106 세포 농도로 접종하여 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 24시간 배양 후, 상기 락토바실러스 람노서스 균주의 배양액 추출물이 각각 50, 및 100㎍/㎖ 농도가 되도록 DMEM 배지에 희석하여 제조한 희석용액을 첨가하여 24시간 동안 동일 조건에서 추가로 배양하였다. 이후 Trizol reagent(invitrogen, USA)를 이용하여 세포를 회수하여 RNA를 분리하였다. RT-PCR(Step One Plus, Applied Biosystems, 미국) 방법을 이용하여 필라그린 및 HAS-3(Hyaluronan synthase-3) 유전자의 mRNA 발현량을 확인하였다. 사용한 프라이머는 다음과 같다:First, DMEM medium supplemented with 10% FBS was added to a 100 mm cell culture dish, and human keratinocytes (HaCaT) were inoculated at a cell concentration of 1×10 6 and cultured for 24 hours in a 37° C., 5% CO 2 incubator. . After culturing for 24 hours, a diluted solution prepared by diluting the culture extract of the Lactobacillus rhamnosus strain in DMEM medium to a concentration of 50 and 100 μg/ml, respectively, was added and further cultured under the same conditions for 24 hours. Thereafter, cells were collected using Trizol reagent (Invitrogen, USA) and RNA was isolated. The mRNA expression levels of filaggrin and hyaluronan synthase-3 (HAS-3) genes were confirmed using RT-PCR (Step One Plus, Applied Biosystems, USA) method. The primers used were:
- Filaggrin : 정방향 5′-AAG CTT CAT GGT GAT GCG AC-3′, 역방향 5′-TCA AGC AGA AGA GGA AGG CA-3′- Filaggrin: forward 5′-AAG CTT CAT GGT GAT GCG AC-3′, reverse 5′-TCA AGC AGA AGA GGA AGG CA-3′
- HAS-3: 정방향 5′-CCC AGC CAG ATT TGT TGA TG-3′, 역방향 5′-AGT GGT CAC GGG TTT CTT CC-3′- HAS-3: forward 5′-CCC AGC CAG ATT TGT TGA TG-3′, reverse 5′-AGT GGT CAC GGG TTT CTT CC-3′
- GAPDH: 정방향 5'-ACC ACA GTC CAT GCC ATC AC-3', 역방향 5'-CCA CCA CCC TGT TGC TGT AG-3'.- GAPDH: forward 5'-ACC ACA GTC CAT GCC ATC AC-3', reverse 5'-CCA CCA CCC TGT TGC TGT AG-3'.
상기와 같은 프라이머를 이용하여 RT-PCR 수행하고, 그 결과를 도 4에 정리하였다. 비교실험을 위해 레티노익산(retinoic acid, RA)을 양성대조군으로 사용하였다. RT-PCR was performed using the above primers, and the results are summarized in FIG. 4 . For comparative experiments, retinoic acid (RA) was used as a positive control.
실험 결과, 시료 무처리군과 비교하여 본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물을 처리한 시험군에서는 필라그린(filaggrin) 유전자의 mRNA 발현량이 각각 151%(50μg/mL) 및 188%(100μg/mL) 증가하였고, 히알루론산 합성효소(hyaluronan synthase-3)의 mRNA 발현량이 각각 233%(100μg/mL) 및 295%(250μg/mL) 증가한 것으로 관찰되었다(도 4 참조). As a result of the experiment, in the test group treated with the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) of the present invention compared to the sample untreated group, the mRNA expression level of the filaggrin gene was 151, respectively. It was observed that % (50 μg/mL) and 188% (100 μg/mL) increased, and the mRNA expression level of hyaluronan synthase-3 increased by 233% (100 μg/mL) and 295% (250 μg/mL), respectively. (see FIG. 4).
또한, 레티노익산(retinoic acid, RA)을 처리한 양성대조군과 비교하여도, 필라그린(filaggrin) 유전자의 mRNA 발현량이 각각 120%(50μg/mL) 및 149%(100μg/mL) 증가하였고, 히알루론산 합성효소(hyaluronan synthase-3)의 mRNA 발현량이 각각 133%(50μg/mL) 및 189%(100μg/mL) 증가한 것으로 관찰되었다.In addition, compared to the positive control group treated with retinoic acid (RA), the mRNA expression level of the filaggrin gene increased by 120% (50μg/mL) and 149% (100μg/mL), respectively, and hyaluronic acid It was observed that the mRNA expression level of hyaluronan synthase-3 was increased by 133% (50 μg/mL) and 189% (100 μg/mL), respectively.
실시예 6. 피부 재생 활성Example 6. Skin regeneration activity
락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물이 갖는 피부 재생 활성을 실험하였다. The skin regeneration activity of the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) was tested.
세포배양 100㎜ 디쉬에 1차 배양 피부 세포인 인간각질형성세포(HaCaT)가 80% 증식했을 때, 멸균된 tip(200ul)의 끝단을 이용하여 피부 세포에 상처를 주고, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물을 각각 10, 50, 및 100㎍/㎖ 농도로 주입하였다. 주입 48 시간 경과 후, 세포 재생 효과를 확인하였다. 세포 증식의 측정은 MTT(methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) 분석 방법과 디프 퀵 스테인(Diff quic stain) 방법을 이용하여 세포염색 후 현미경으로 확인한 후, 무처리군 대비 세포 재생율을 측정하였다. 또한, 비교실험을 위해 피부 재생 효과가 우수한 것으로 알려진 병풀 추출물과 레티노익산(retinoic acid, RA)을 각각 양성대조군으로 사용하였다. When human keratinocytes (HaCaT), which are primary cultured skin cells, proliferate by 80% in a 100 mm cell culture dish, the skin cells are wounded using the tip of a sterilized tip (200ul), and Lactobacillus rhamnosus ( Lactobacillus rhamnosus mesopharm001) strain (accession number KCTC 14350BP) was injected at concentrations of 10, 50, and 100 μg/ml, respectively. After 48 hours of injection, the cell regeneration effect was confirmed. Cell proliferation was measured using MTT (methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) analysis method and Diff quic stain method, after cell staining and microscopic confirmation, cell regeneration rate compared to untreated group was measured did In addition, for comparative experiments, centella asiatica extract and retinoic acid (RA), which are known to have excellent skin regeneration effects, were used as positive controls, respectively.
실험 결과, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물의 처리로 10, 50, 및 100㎍/㎖ 농도에서 시료 무처리군 대비 각각 122%, 137%, 및 157% 개선된 상처 수복율을 나타내었고, 이를 통해 피부 재생 효과가 있는 것을 확인할 수 있었다(도 5 참조). 또한, 양성대조군인 병풀 추출물 처리군과 비교하여도, 동일 용량에서 각각 120%, 123%, 및 133% 개선된 상처 수복율을 확인할 수 있었으며, 레티노익산(retinoic acid, RA) 처리군과 비교하여도, 50, 및 100㎍/㎖ 농도에서 각각 105%(50μg/mL) 및 120%(100μg/mL) 개선된 상처 수복율이 관찰되었다.As a result of the experiment, treatment of the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (Accession No. KCTC 14350BP) resulted in 122%, 137%, and 157%, respectively, compared to the sample untreated group at concentrations of 10, 50, and 100 μg/ml. % improved wound repair rate, and through this, it was confirmed that there was a skin regeneration effect (see FIG. 5). In addition, compared to the centella asiatica extract treatment group, which is a positive control group, it was confirmed that the wound repair rate was improved by 120%, 123%, and 133% at the same dose, respectively, compared to the retinoic acid (RA) treatment group. 105% (50 μg/mL) and 120% (100 μg/mL) improved wound repair rates were observed at the 50, and 100 μg/mL concentrations, respectively.
또한, 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물을 손상된 피부세포에 처리하는 경우, 재생된 피부세포의 형태적인 모양이 정상적인 피부세포와 동일하게 성장하는 것으로 관찰되었다. In addition, when the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) was treated with damaged skin cells, it was observed that the morphological shape of the regenerated skin cells grew the same as normal skin cells. .
상기와 같은 결과를 통해, 본 발명의 락토바실러스 람노서스(Lactobacillus rhamnosus mesopharm001) 균주(기탁번호 KCTC 14350BP)의 배양액 추출물은 세포 독성이 없고, 산화질소 생성 억제를 통해 항염증 활성에 기여하며, 피부 장벽 형성 및 피부 재생에 기여함으로써 피부 상태 개선에 우수한 효과가 있음을 확인할 수 있었다. Through the above results, the culture extract of the Lactobacillus rhamnosus mesopharm001 strain (accession number KCTC 14350BP) of the present invention has no cytotoxicity, contributes to anti-inflammatory activity through inhibition of nitric oxide production, and has a skin barrier It was confirmed that there is an excellent effect in improving skin conditions by contributing to formation and skin regeneration.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (5)
상기 조성물은 산화질소의 생성을 억제하여 항염증 활성을 갖는 것을 특징으로 하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition is anti-inflammatory, skin moisturizing, skin barrier strengthening, and a cosmetic composition for promoting skin regeneration, characterized in that it has anti-inflammatory activity by inhibiting the production of nitric oxide.
상기 조성물은 히알루론산 합성을 촉진하여 피부 보습 활성을 갖는 것을 특징으로 하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition is anti-inflammatory, skin moisturizing, skin barrier strengthening, and a cosmetic composition for promoting skin regeneration, characterized in that it has skin moisturizing activity by promoting hyaluronic acid synthesis.
상기 조성물은 필라그린 유전자 및 히알루론산 합성효소(hyaluronan synthase-3) 유전자의 발현을 증가시켜 피부 장벽 강화 활성을 갖는 것을 특징으로 하는 항염증, 피부 보습, 피부 장벽 강화, 및 피부 재생 촉진용 화장료 조성물.According to claim 1,
The composition has anti-inflammatory, skin moisturizing, skin barrier strengthening, and cosmetic composition for promoting skin regeneration, characterized in that it has skin barrier strengthening activity by increasing the expression of filaggrin gene and hyaluronan synthase-3 gene. .
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