KR102283526B1 - Cosmetic composition comprising seaweed fermented extract - Google Patents
Cosmetic composition comprising seaweed fermented extract Download PDFInfo
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- KR102283526B1 KR102283526B1 KR1020180170688A KR20180170688A KR102283526B1 KR 102283526 B1 KR102283526 B1 KR 102283526B1 KR 1020180170688 A KR1020180170688 A KR 1020180170688A KR 20180170688 A KR20180170688 A KR 20180170688A KR 102283526 B1 KR102283526 B1 KR 102283526B1
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- South Korea
- Prior art keywords
- fermented
- extract
- present
- seaweed
- cosmetic composition
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
본 발명은 해조류 발효 추출물을 유효성분으로 함유하는 조성물에 관한 것으로, 보다 상세하게는 본 발명에 따른 조성물은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 이용하며, 상기 해조류 발효 추출물은 항산화, 주름개선, 미백 등 다양한 효과를 나타내어 기능성 화장품 또는 건강기능식품 소재로서 유용하게 활용될 수 있다.The present invention relates to a composition containing a fermented seaweed extract as an active ingredient, and more particularly, the composition according to the present invention uses a fermented seaweed extract fermented with Enterococcus faecium MSS2, and the fermented seaweed extract is an antioxidant. , wrinkle improvement, whitening, etc., it can be usefully used as a functional cosmetic or health functional food material.
Description
본 발명은 해조류 발효 추출물을 유효성분으로 함유하는 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition containing a fermented seaweed extract as an active ingredient.
최근 의학의 발달과 생활환경의 개선에 의해 인간의 수명이 크게 연장됨에 따라 노화에 대한 관심이 높아져 많은 노화 관련 연구가 이루어지고 있다. 그 중에서도 경제적, 사회적 여유가 생기게 되면서 외모에 대한 관심이 많아져 젊고 깨끗한 피부를 갖고 싶어 함에 따라 피부의 노화 현상을 지연 또는 방지할 수 있는 소재 개발에 많은 관심이 집중되고 있다.Recently, as the lifespan of human beings is greatly extended due to the development of medicine and the improvement of the living environment, interest in aging has increased, and many studies related to aging are being conducted. Among them, as economic and social leeway is created, interest in appearance increases, and as people want to have young and clean skin, much attention is being paid to the development of materials that can delay or prevent skin aging.
노화로 인해 피부의 구성 성분인 표피, 진피 및 피하조직의 두께가 얇아지고 피부에 탄력을 주는 세포외 기질(Extracellular matrix, ECM) 성분이 변화하게 되는데, 그중 세포외 기질(ECM)의 70~80%를 차지하는 콜라겐은 나이가 들면서 그 생성이 급격하게 저하되어 피부가 탄력을 잃고 주름이 과도하게 형성되면서 노인성 피부로 변화되어 간다. Due to aging, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. Collagen, which occupies the %, rapidly decreases with age, and the skin loses elasticity and wrinkles are excessively formed, changing to senile skin.
나이가 들면서 피부 결합조직 내의 콜라겐 함량이 줄어드는 것은 콜라겐 합성의 저하와 분해의 촉진에 의한 것이다. 따라서 콜라겐 생합성의 저하와 콜라겐 분해 촉진은 피부 주름 형성의 가장 큰 원인이라 할 수 있다. 콜라겐 생합성 과정은 전사 수준(Transcription level)과 해독 후 수준(Post-translation level)에 관여하는 많은 인자(Factor)들에 의해 조절을 받아 변화를 일으키며, 콜라겐 분해는 자외선 등에 의해 콜라겐을 분해하는 콜라게네이즈(Collagenase)와 같은 기질금속단백질 분해 효소들(Matrix metalloproteases, MMP)의 발현의 촉진으로 콜라겐 분해가 촉진되어 콜라겐 함량이 줄어든다. 아울러 외부 환경에 의해 콜라겐의 변형이 가속화되면서 피부는 주름이 많아지고 깊어지게 된다. The decrease in collagen content in the skin connective tissue with age is due to the decrease in collagen synthesis and promotion of degradation. Therefore, the decrease in collagen biosynthesis and the promotion of collagen decomposition are the biggest causes of skin wrinkle formation. The collagen biosynthesis process is regulated by many factors involved in the transcription level and post-translation level, and the collagen breakdown is collagen that breaks down collagen by ultraviolet rays, etc. Collagen decomposition is promoted by promotion of expression of matrix metalloproteases (MMP) such as collagenase, thereby reducing collagen content. In addition, as the transformation of collagen is accelerated by the external environment, the skin becomes wrinkled and deep.
또, 세포 내에서 티로시네이즈에 의해 타이로신으로부터 도파(dopa), 도파퀴논(dopaquinone)으로 변환되어 도파크롬(dopachrome) 등을 거처 생성되어지는 멜라닌은 피부에 존재하여 자외선 등으로부터 신체를 보호하는 중요한 기능을 가지고 있다. 그러나 멜라닌이 과잉 생산됨으로써 기미, 주근깨 등을 형성하고, 피부노화를 촉진하며, 피부암 유발에 중요한 작용을 하는 것으로 알려져 있으며, 또한 과실류나 채소류등의 식품에서의 과잉 축적으로 인해 갈변화 현상을 일으키는 주요 원인으로 작용하기도 한다. In addition, melanin, which is converted from tyrosine into dopa and dopaquinone by tyrosinase in the cell and produced through dopachrome, is present in the skin and is important to protect the body from UV rays. has a function. However, due to excessive production of melanin, it is known to form spots and freckles, promote skin aging, and play an important role in skin cancer induction. It can also act as a cause.
한편, 우리나라는 삼면이 바다로 둘러싸여 있어 풍부한 해양생물 자원을 보유하고 있으며, 근래 국내연안에서 생산되는 해조류의 총생산량은 약 80만톤에 이르고 있으며, 해조류는 진화 과정이 육상 생물계와는 많은 점에서 차이가 있으므로 유상 식물에는 존재하지 않는 다양한 생리 활성을 지니는 화합물을 포함한다.On the other hand, Korea has abundant marine biological resources because it is surrounded by the sea on three sides. Therefore, it contains compounds with various physiological activities that are not present in oily plants.
이러한 해조류를 이용한 기능성 화장품 소재 개발에 대한 관심이 높아졌으나, 아직까지 해조류를 첨가하고 특정 균주로 상온 발효한 해조류 발효 추출물의 기능성 화장품 소재에 대한 상용화는 부족한 실정이며, 해조류 발효 추출물을 이용한 기능성 화장품 소재 개발이 여전히 필요하다.Although interest in the development of functional cosmetic materials using these seaweeds has increased, commercialization of functional cosmetic materials using seaweed fermented extracts added with seaweed and fermented at room temperature with a specific strain is still insufficient. Development is still needed.
상기와 같은 문제점을 해결하기 위해, 본 발명의 목적은 특정 균주로 발효한 해조류 발효 추출물을 이용한 기능성 화장료 조성물을 제공하는 데에 있다.In order to solve the above problems, an object of the present invention is to provide a functional cosmetic composition using a fermented seaweed extract fermented with a specific strain.
또한, 본 발명의 다른 목적은 특정 균주로 발효한 해조류 발효 추출물을 이용한 건강기능식품 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a health functional food composition using a fermented seaweed extract fermented with a specific strain.
본 발명은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 유효성분으로 함유하는 화장료 조성물을 제공한다.The present invention provides a cosmetic composition comprising a fermented seaweed extract fermented with Enterococcus faecium MSS2 as an active ingredient.
또한, 본 발명은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 유효성분으로 함유하는 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition comprising a fermented seaweed extract fermented with Enterococcus faecium MSS2 as an active ingredient.
본 발명에 따른 조성물은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 이용하며, 상기 해조류 발효 추출물은 항산화, 주름개선, 미백 등 다양한 효과를 나타내어 기능성 화장품 또는 건강기능식품 소재로서 유용하게 활용될 수 있다.The composition according to the present invention uses a fermented seaweed extract fermented with Enterococcus faecium MSS2, and the fermented seaweed extract exhibits various effects such as antioxidant, wrinkle improvement, and whitening, and is useful as a functional cosmetic or health functional food material. can be
도 1은 본 발명에 따른 해조류 발효 추출물 제조공정을 도시한 것이고,
도 2는 본 발명에 따른 해조류 발효 추출물의 티로시네이즈 억제 효과를 나타낸 것이고,
도 3은 본 발명에 따른 해조류 발효 추출물의 DPPH 라디칼 소거 효과를 나타낸 것이고,
도 4 및 도 5는 각각 본 발명에 따른 해조류 발효 추출물의 콜라게네이즈 억제 효과 및 엘라스테이즈 억제 효과를 나타낸 것이고,
도 6 및 도 7은 각각 본 발명에 따른 해조류 발효 추출물의 총 페놀 함량 분석 및 총 플라보노이드 함량 분석를 나타낸 것이고,
도 8은 본 발명에 따른 해조류 발효 추출물의 발효 시간별 pH 변화를 나타낸 것이고,
도 9 및 도 10은 각각 본 발명에 따른 해조류 발효 추출물 처리에 따른 멜라닌 함량 분석 및 세포성 티로시네이즈 활성 분석을 나타낸 것이고,
도 11 및 도 12는 각각 본 발명에 따른 해조류 발효 추출물 처리에 따른 멜라노마 세포 및 섬유아세포의 생존률을 나타낸 것이고,
도 13은 본 발명에 따른 해조류 발효 추출물 처리에 따른 티로시네이즈 및 TRP-1의 단백질 발현 수준 분석을 나타낸 것이고,
도 14는 본 발명에 따른 해조류 발효 추출물 처리에 따른 MMP-1 및 MMP-12의 단백질 발현 수준 분석을 나타낸 것이다.1 shows a process for preparing a fermented seaweed extract according to the present invention;
2 shows the tyrosinase inhibitory effect of the fermented seaweed extract according to the present invention;
3 shows the DPPH radical scavenging effect of the fermented seaweed extract according to the present invention;
4 and 5 show the collagenase inhibitory effect and the elastase inhibitory effect of the fermented seaweed extract according to the present invention, respectively;
6 and 7 respectively show the analysis of the total phenol content and the total flavonoid content of the fermented seaweed extract according to the present invention;
8 shows the change in pH of the fermented seaweed extract according to the fermentation time according to the present invention;
9 and 10 show an analysis of melanin content and an analysis of cellular tyrosinase activity, respectively, according to the treatment of the fermented seaweed extract according to the present invention;
11 and 12 show the survival rates of melanoma cells and fibroblasts, respectively, according to the treatment with the fermented seaweed extract according to the present invention;
13 shows the analysis of the protein expression level of tyrosinase and TRP-1 according to the treatment of the fermented seaweed extract according to the present invention;
14 shows the analysis of protein expression levels of MMP-1 and MMP-12 according to the treatment of the fermented seaweed extract according to the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자는 해조류를 이용한 기능성 화장품 소재 개발을 위해 연구노력한 결과, 톳과 같은 해조류에 상온 발효가 가능한 특정 균주인 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물이 탁월한 항산화, 미백 및 주름개선 효과를 나타내는 것을 발견함으로써 본 발명을 완성하였다.As a result of research efforts for the development of functional cosmetic materials using seaweed, the present inventors have found that the fermented seaweed extract fermented with Enterococcus faecium MSS2, a specific strain capable of fermenting seaweed at room temperature on seaweeds such as seaweed, has excellent antioxidant, whitening and anti-wrinkle effects. The present invention was completed by finding that
본 발명에서 "추출물"은 추출 방법, 추출 용매, 추출된 성분 또는 추출물의 형태를 불문하고, 천연물의 성분을 뽑아냄으로써 얻어진 물질을 모두 포함하는 것이며, 또한 천연물의 성분을 뽑아내어 얻어진 물질을 추출 후 다른 방법으로 가공 또는 처리하여 얻어질 수 있는 물질을 모두 포함하는 광범위한 개념이다.In the present invention, "extract" includes all substances obtained by extracting the components of a natural product, regardless of the extraction method, extraction solvent, extracted component, or the form of the extract, and also after extracting the material obtained by extracting the component of the natural product It is a broad concept encompassing all substances that can be obtained by processing or processing in other ways.
본 발명에서 "화장료"는 피부 노화 등의 예방 또는 개선의 효과를 가지는 기능성 화장료를 포함할 수 있고, 상기 기능성 화장료는 화장품법에 정의된 바에 따라 특정 효능과 효과가 강조된 화장료로 피부의 주름 개선에 도움을 주거나 피부의 기능 약화로 인한 건조함을 방지하는 것 등을 포함할 수 있다.In the present invention, "cosmetics" may include functional cosmetics having an effect of preventing or improving skin aging, etc., and the functional cosmetics are cosmetics emphasizing specific efficacy and effects as defined in the Cosmetics Act, helping to improve wrinkles on the skin It may include giving or preventing dryness due to weakening of the skin's function.
본 발명에서 "건강기능식품"은 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미한다. 여기서 '기능성'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. In the present invention, "health functional food" refers to a food manufactured and processed using raw materials or ingredients useful for the human body. Here, the term 'functionality' refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body.
본 발명은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 유효성분으로 함유하는 화장료 조성물을 제공한다. The present invention provides a cosmetic composition comprising a fermented seaweed extract fermented with Enterococcus faecium MSS2 as an active ingredient.
상기 엔테로코쿠스 파에시움 MSS2은 기탁번호 KCTC 12795BP로 기탁된 기탁 균주일 수 있고, 상온 발효 균주이다.The Enterococcus faecium MSS2 may be a deposited strain deposited with accession number KCTC 12795BP, and is a room temperature fermentation strain.
상기 해조류 발효 추출물은 톳, 김, 다시마, 미역, 꼬시래기, 도박 및 큰실말로 이루어진 군에서 선택된 하나 이상의 해조류 발효 추출물일 수 있지만, 이에 한정되는 것은 아니다.The fermented seaweed extract may be one or more fermented extracts of seaweed selected from the group consisting of seaweed, seaweed, kelp, seaweed, seaweed, gambling, and fermented seaweed extract, but is not limited thereto.
상기 해조류 발효 추출물은 항산화, 보습, 미백, 주름개선 및 항노화로 이루어진 군에서 선택된 하나 이상의 기능성을 갖는다.The fermented seaweed extract has one or more functions selected from the group consisting of antioxidant, moisturizing, whitening, anti-wrinkle and anti-aging.
상기 해조류 발효 추출물은 해조류 물 추출물을 엔테로코쿠스파에시움 MSS2로 상온 발효하여 얻을 수 있다.The fermented seaweed extract may be obtained by fermenting a seaweed water extract with Enterococcus sphaecium MSS2 at room temperature.
본 발명에 따른 화장료 조성물은 식품 의약품 안전청에 등록된 화장품 원료 리스트 및 ICID(국제화장품 원료집)에 등재되어 있는 성분 중에서 1종 이상을 더 포함할 수 있다. 예를 들어, 상기 화장료 조성물은 유기 용매, 용해제, 농축제, 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 방향제, 계면활성제, 유화제, 충전제, 금속이온 봉쇄제, 방부제, 비타민, 차단제, 습윤화제, 염료 및 안료, 친수성 또는 친유성 활성제, 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품 분야에서 통상적으로 사용되는 보조제를 하나 이상 더 포함할 수 있다.The cosmetic composition according to the present invention may further include one or more of the ingredients listed in the list of cosmetic raw materials registered with the Food and Drug Administration and ICID (International Cosmetic Ingredients). For example, the cosmetic composition may include an organic solvent, a solubilizer, a thickener, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a fragrance, a surfactant, an emulsifier, a filler, a sequestering agent, a preservative, a vitamin, a blocking agent, It may further comprise one or more adjuvants commonly used in the cosmetic field, such as wetting agents, dyes and pigments, hydrophilic or lipophilic actives, or any other ingredients commonly used in cosmetics.
상기 비타민은 수용성 또는 유용성 비타민을 포함할 수 있고, 상기 수용성 비타민은 수용성 비타민으로서 화장품에 배합 가능한 것으로, 바람직하게는 비타민 B1, 비타민 B2, 비타민 B6, 피리독신, 염산피리독신, 비타민 B12, 판토텐산, 니코틴산, 니코틴산아미드, 엽산, 비타민C, 비타민 H 등을 들 수 있으며, 보다 바람직하게는 콜라겐 합성이 도움을 주는 비타민 C를 포함할 수 있다. 또한, 그들의 염 (티아민염산염, 아스코르빈산나트륨염 등)이나 유도체(아스코르빈산-2-인산나트륨염, 아스코르빈산-2-인산마그네슘염 등)도 포함될 수 있고, 상기 수용성 비타민은 미생물 변환법, 미생물의 배양물로부터의 정제법, 효소법 또는 화학 합성법 등의 통상의 방법에 의해 수득될 수 있다.The vitamin may include a water-soluble or oil-soluble vitamin, and the water-soluble vitamin is a water-soluble vitamin that can be formulated in cosmetics, preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, and nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like, and more preferably, vitamin C that helps collagen synthesis. In addition, salts thereof (thiamine hydrochloride, sodium ascorbate salt, etc.) or derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-phosphate magnesium salt, etc.) may be included, and the water-soluble vitamin is a microorganism transformation method , can be obtained by a conventional method such as purification from a culture of microorganisms, enzymatic method or chemical synthesis method.
본 발명에 따른 화장료 조성물은 본 발명이 속하는 기술 분야에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있다. 예를 들어, 스킨로션, 스킨소프너, 스킨토너, 겔, 크림, 로션, 밀크로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 팩, 마사지 크림 및 스프레이 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다. 더욱 상세하게는, 영양 크림, 마사지 크림, 에센스, 아이 크림, 선로션, 선크림, 메이크업 프라이머, 메이크업 베이스, 비비크림, 클렌징크림, 클렌징폼, 클렌징 워터, 팩, 스틱상 제품, 밤(Balm) 타입 제품, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the technical field to which the present invention pertains. For example, skin lotions, skin softeners, skin toners, gels, creams, lotions, milk lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays. and the like, but is not limited thereto. More specifically, nourishing cream, massage cream, essence, eye cream, sun lotion, sun cream, makeup primer, makeup base, BB cream, cleansing cream, cleansing foam, cleansing water, pack, stick product, balm type It may be prepared in the form of a product, spray or powder.
본 발명에 따른 화장료 조성물은 단독 또는 중복으로 도포하여 사용하거나, 본 발명 이외의 다른 화장료 조성물과 중복 도포하여 사용할 수 있다. 또한, 본 발명에 따른 화장료 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 피부 상태 또는 취향에 따라 그 사용횟수를 달리할 수 있다.The cosmetic composition according to the present invention may be used alone or by being applied in duplicate, or may be used by overlapping with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention can be used according to a conventional method of use, and the number of times of use can be varied according to the skin condition or taste of the user.
또한, 본 발명은 자연 건조한 해조류에 물을 첨가하고 100~130℃에서 추출하는 단계; 상기 추출된 추출물에 엔테로코쿠스 파에시움 MSS2을 접종하고 상온 발효하는 단계; 및 상기 발효된 발효물을 원심분리 하여 상층액을 회수하고 필터로 여과하여 균체와 해조류 분말을 제거한 발효물을 제조하는 단계를 포함하는, 해조류 발효 추출물의 제조방법을 제공한다.In addition, the present invention comprises the steps of adding water to naturally dried seaweed and extracting it at 100 ~ 130 ℃; Inoculating the extracted extract with Enterococcus faecium MSS2 and fermenting at room temperature; and centrifuging the fermented fermented product to recover the supernatant and filtering it with a filter to prepare a fermented product from which cells and algae powder are removed.
상기 추출 단계에서는 물 100 중량부에 대하여 해조류를 0.1 내지 10 중량부로 포함하여 추출하며, 바람직하게는 121℃에서 10~20분 동안 추출할 수 있다.In the extraction step, 0.1 to 10 parts by weight of seaweed is extracted based on 100 parts by weight of water, and the extraction may be preferably performed at 121° C. for 10 to 20 minutes.
상기 발효 단계에서는 추출물에 엔테로코쿠스 파에시움 MSS2을 접종하고 상온 발효를 수행하며, 바람직하게는 15~30℃에서 48~72시간 동안 발효할 수 있다.In the fermentation step, the extract is inoculated with Enterococcus faecium MSS2 and fermented at room temperature, preferably fermented at 15 to 30° C. for 48 to 72 hours.
상기 원심분리는 800~1200 rpm에서 10~20분 동안 수행하여 상층액을 회수할 수 있다.The centrifugation may be performed at 800 to 1200 rpm for 10 to 20 minutes to recover the supernatant.
상기 제조방법은 제조된 발효물을 -50℃에서 5~10 mmHg 조건 하에서 동결 건조하여 분말 형태로 사용할 수 있다.The preparation method can be used in powder form by freeze-drying the prepared fermented product at -50°C under 5 to 10 mmHg conditions.
또한, 본 발명은 엔테로코쿠스 파에시움 MSS2로 발효한 해조류 발효 추출물을 유효성분으로 함유하는 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition comprising a fermented seaweed extract fermented with Enterococcus faecium MSS2 as an active ingredient.
본 발명에 따른 건강기능식품은 분말, 과립, 정제, 캡슐, 시럽 또는 음료 등으로 제조될 수 있고, 본 발명에 따른 해조류 발효 추출물 이외에 다른 식품 또는 식품 첨가물과 함께 제조될 수 있다. 즉, 상기 건강기능식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하고, 건강기능식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 또한, 상기 건강기능식품 제조 시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있으며 통상적인 방법에 따라 적절하게 사용될 수 있다. The health functional food according to the present invention may be prepared as powder, granule, tablet, capsule, syrup or beverage, and may be prepared together with other foods or food additives in addition to the fermented seaweed extract according to the present invention. That is, the health functional food can be manufactured by a method commonly used in the art, and can be manufactured without limitation as long as it is a formulation recognized as a health functional food. In addition, when manufacturing the health functional food, it can be prepared by adding raw materials and components commonly added in the art, and can be appropriately used according to a conventional method.
본 발명에 따른 건강기능식품은 일반 약품과는 달리 해조류를 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 피부 주름 개선, 피부 탄력 증진 또는 피부 미백 효과를 증진시키기 위한 보조제로 섭취될 수 있다.The health functional food according to the present invention has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using seaweed as a raw material, unlike general drugs, and it is excellent in portability and has the effect of improving skin wrinkles, enhancing skin elasticity, or skin whitening effect. It can be taken as an adjuvant to enhance
본 발명에 따른 건강기능식품 조성물에서, 유효성분인 상기 해조류 발효 추출물의 혼합양은 그의 사용 목적, 예를 들어, 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다. In the health functional food composition according to the present invention, the mixing amount of the fermented seaweed extract as an active ingredient may be appropriately determined according to the purpose of its use, for example, preventive, health or therapeutic treatment.
본 발명에 따른 건강기능식품이 취할 수 있는 형태에는 제한이 없으며, 통상적인 의미의 식품을 모두 포함할 수 있고, 기능성 식품 등 당업계에 알려진 용어와 혼용 가능하다. 아울러 본 발명에 따른 천연 추출물을 유효성분으로 포함하는 건강기능식품은 당업자의 선택에 따라 식품에 함유될 수 있는 적절한 기타 보조성분과 공지의 첨가제를 혼합하여 제조할 수 있다. 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 본 발명에 따른 천연 추출물을 주성분으로 하여 제조한 즙, 차, 젤리 및 주스 등에 첨가하여 제조할 수 있다. 또한 동물을 위한 사료로 이용되는 식품도 포함할 수 있다.There is no limitation in the form that the health functional food according to the present invention can take, and it may include any food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food. In addition, the health functional food containing the natural extract according to the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained 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, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the natural extract according to the present invention as a main component to juice, tea, jelly, juice, and the like. It may also include food used as feed for animals.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
<실시예 1> 톳을 이용한 발효 추출물 제조<Example 1> Preparation of fermented extract using Tochigi
해조류 톳(H. fusiforme)을 상온에서 자연 건조하여 얻은 톳 건조물 0.5 g과 증류수(Distilled water) 50 ml을 잘 섞이게 혼합하여 혼합물을 제조하였다. 상기 혼합물 중에서 유효성분을 추출하기 위해 121℃에서 15분 정도 추출을 진행하였다. 상기 추출된 추출물에 엔테로코쿠스 파에시옴(Enterococcus Faecium) MSS2 균주(기탁번호: KCTC 12795BP) 1 X 106 CFU/ml를 접종하고 발효가 잘 일어나도록 진탕 배양기를 이용하여 30℃에서 50 rpm을 조건으로 72시간 동안 발효시켰다. 발효 과정을 거쳐서 수확한 톳(H. fusiforme) 액상 발효물은 원심 분리기를 이용하여 1000 rpm에서 10 ~ 20 분간 원심 분리시켜 상층액을 회수하였다. 상기 회수한 톳(H. fusiforme) 발효물을 필터 페이퍼(0.072 mm)를 이용하여 균체 및 톳(H. fusiforme) 분말을 제거하고, 얻어진 발효물은 -50℃, 5 ~ 10 mmHg 조건에서 동결 건조하여 분말 형태로 제조하였다(도 1의 공정도 참조).A mixture was prepared by mixing 0.5 g of dried seaweed ( H. fusiforme ) obtained by natural drying at room temperature and 50 ml of distilled water. In order to extract the active ingredient from the mixture, extraction was performed at 121° C. for about 15 minutes. Enterococcus Faecium MSS2 strain (Accession No.: KCTC 12795BP) 1
상기 제조한 분말 형태의 톳(H. fusiforme) 추출물 분말 10 mg/증류수 1 ml을 혼합하고 충분히 볼텍싱(vortexing) 하여 이하 실험의 화장품 원료로 사용하였다. 10 mg of H. fusiforme extract powder prepared above in powder form / 1 ml of distilled water were mixed and sufficiently vortexed to be used as a cosmetic raw material for the following experiments.
<실험예 1> 톳을 이용한 발효 추출물의 기능성 분석<Experimental Example 1> Functional analysis of fermented extracts using Japanese chives
1. 티로시네이즈 억제 효과1. Tyrosinase inhibitory effect
본 발명의 톳 발효 추출물이 티로시네이즈에 미치는 효과를 분석하기 위하여, 톳 발효 추출물 10 μl, 티로시네이즈(110 units/ml) 20 μl, 그리고 1.5 mM L-DOPA 용액, 50 mM 인산나트륨 버퍼(sodium phosphate buffer, pH 6.5) 및 증류수가 10:10:1의 비율로 함유된 혼합물 170 μl을 각각 첨가하여 혼합하였다. 이후, 혼합물을 37℃에서 10분간 반응시킨 다음 Tecan F-200 멀티 웰 플레이트 판독기(Mannedorf, Zurich, Swit-Zerland)를 사용하여 450 nm에서 흡광도를 측정하였다.In order to analyze the effect of the fermented shiitake extract of the present invention on tyrosinase, 10 μl of fermented hibiscus extract, 20 μl of tyrosinase (110 units/ml), and 1.5 mM L-DOPA solution, 50 mM sodium phosphate buffer ( 170 μl of a mixture containing sodium phosphate buffer, pH 6.5) and distilled water in a ratio of 10:10:1 was added and mixed. Thereafter, the mixture was reacted at 37° C. for 10 minutes, and then absorbance was measured at 450 nm using a Tecan F-200 multi-well plate reader (Mannedorf, Zurich, Swit-Zerland).
그 결과, 도 2와 같이, 톳을 이용한 발효 추출물이 처리 후 48시간까지 농도 의존적으로 티로시네이즈 억제 효과를 나타내는 것을 확인하였다. As a result, as shown in FIG. 2 , it was confirmed that the fermented extract using Japanese chives exhibited a concentration-dependently tyrosinase inhibitory effect up to 48 hours after treatment.
2. DPPH 라디칼 소거 효과2. DPPH radical scavenging effect
톳 발효 추출물이 DPPH 라디칼 소거 활성에 미치는 효과를 분석하기 위하여, DPPH 9 mg을 메탄올 30 ml에 녹인 후, 톳 발효 추출물 40 μl, DPPH 용액 40 μl, MeOH 120 μl를 혼합하고 실온에서 30분간 반응시켰다. 이후, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 517 nm에서 흡광도를 측정하였다.To analyze the effect of fermented shiitake extract on DPPH radical scavenging activity, 9 mg of DPPH was dissolved in 30 ml of methanol, 40 μl of fermented hibiscus extract, 40 μl of DPPH solution, and 120 μl of MeOH were mixed and reacted at room temperature for 30 minutes. . Then, the absorbance was measured at 517 nm using a multi-well plate reader (Tecan F-200).
그 결과, 도 3과 같이, 톳을 이용한 발효 추출물이 처리 후 48시간까지 농도 의존적으로 DPPH 라디칼 소거 효과를 나타내는 것을 확인하였다. As a result, as shown in FIG. 3 , it was confirmed that the fermented extract using Japanese chives exhibited a DPPH radical scavenging effect in a concentration-dependent manner up to 48 hours after treatment.
3. 콜라게네이즈 억제 효과3. Collagenase inhibitory effect
톳 발효 추출물이 콜라게네이즈에 미치는 효과를 분석하기 위하여, collagenase activity colorimetric assay kit(K792, Biovision, USA)를 사용하였으며, 제조사에서 제공된 프로토콜을 참조하여 실험을 수행하였다. 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 345 nm에서 흡광도를 측정하였다.To analyze the effect of the fermented shiitake extract on collagenase, a collagenase activity colorimetric assay kit (K792, Biovision, USA) was used, and the experiment was performed with reference to the protocol provided by the manufacturer. Absorbance was measured at 345 nm using a multi-well plate reader (Tecan F-200).
그 결고, 도 4와 같이, 톳을 이용한 발효 추출물이 처리 후 48시간까지 농도 의존적으로 콜라게네이즈 억제 효과를 나타내는 것을 확인하였다.As a result, as shown in FIG. 4 , it was confirmed that the fermented extract using Japanese chives exhibited a collagenase inhibitory effect in a concentration-dependent manner up to 48 hours after treatment.
4. 엘라스테이즈 억제 효과4. Elastase inhibitory effect
톳 발효 추출물이 엘라스테이즈에 미치는 영향을 분석하기 위하여, 96 웰 플레이트에 0.1 M Tris-HCl buffer 80 μl, 톳 발효 추출물 40 μl, 3 mM N-succinyl-(Ala)3-P-nitroanilide 30 μl, 0.2 unit Elastase 20 μl를 첨가한 후, 30℃에서 20분 동안 반응시키고, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 410 nm에서 흡광도를 측정하였다.To analyze the effect of fermented shiitake extract on elastase, 80 µl of 0.1 M Tris-HCl buffer, 40 µl of fermented hibiscus extract, 30 µl of 3 mM N-succinyl-(Ala) 3 -P-nitroanilide in a 96-well plate , 0.2
그 결과, 도 5와 같이, 톳을 이용한 발효 추출물이 처리 후 48시간까지 농도 의존적으로 엘라스테이즈 억제 효과를 나타내는 것을 확인하였다. As a result, as shown in FIG. 5 , it was confirmed that the ferment extract using H. shiitake showed an elastase inhibitory effect in a concentration-dependent manner up to 48 hours after treatment.
5. 총 페놀 함량 분석5. Total Phenol Content Analysis
톳 발효 추출물이 총 폴리페놀 함량에 미치는 영향을 분석하기 위하여, 96 웰 플레이트에 톳 발효 추출물 10 μl 및 0.2 N Folin-Ciocalteu reagent 50 μl를 첨가하고 혼합한 다음 5분 동안 반응시켰다. 이후, 7.5% 탄산나트륨 용액 40 μl를 첨가하고 실온에서 30분 동안 반응시켰다. 이후, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 730 nm에서 흡광도를 측정하였다. 표준곡선은 0 - 90 μg/ml 범위의 갈산(Galic acid) 표준 용액을 사용하여 설정하였다.In order to analyze the effect of the fermented hibiscus extract on the total polyphenol content, 10 µl of the fermented hibiscus extract and 50 µl of the 0.2 N Folin-Ciocalteu reagent were added to a 96-well plate, mixed, and then reacted for 5 minutes. Then, 40 μl of a 7.5% sodium carbonate solution was added and reacted at room temperature for 30 minutes. Then, the absorbance was measured at 730 nm using a multi-well plate reader (Tecan F-200). The standard curve was set using a standard solution of gallic acid in the range of 0 - 90 μg/ml.
그 결과, 도 6과 같이, 톳을 이용한 발효 추출물은 처리 후 48시간까지 농도 의존적으로 총 페놀의 함량이 증가되는 것을 확인하였다.As a result, as shown in FIG. 6 , it was confirmed that the content of total phenol was increased in a concentration-dependent manner in the fermented extract using H. chives until 48 hours after treatment.
6. 총 플라보노이드 함량 분석6. Total flavonoid content analysis
톳 발효 추출물이 총 플라보노이드 함량에 미치는 영향을 분석하기 위하여, 96 웰 플레이트에 톳 발효 추출물 50 μl 및 2% AlCl3 시약 50 μl를 첨가하고 혼합한 다음 암실에서 30분 동안 반응시켰다. 이후, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 450 nm에서 흡광도를 측정하였다. 표준곡선은 0 - 90 μg/ml 범위의 케르세틴(Quercetin) 표준 용액을 사용하여 설정하였다.To analyze the effect of fermented shiitake extract on total flavonoid content, 50 µl of fermented hibiscus extract and 50 µl of 2% AlCl 3 reagent were added to a 96-well plate, mixed, and then reacted in the dark for 30 minutes. Then, the absorbance was measured at 450 nm using a multi-well plate reader (Tecan F-200). A standard curve was established using a standard solution of Quercetin in the range of 0 - 90 μg/ml.
그 결과, 도 7과 같이, 톳을 이용한 발효 추출물은 처리 후 48시간까지 농도 의존적으로 총 플라보노이드의 함량이 증가되는 것을 확인하였다. As a result, as shown in FIG. 7 , it was confirmed that the content of total flavonoids was increased in a concentration-dependent manner in the fermented extract using Japanese chives until 48 hours after treatment.
7. 발효 시간별 톳 발효 추출물의 pH 변화 분석7. Analysis of pH change of fermented shiitake extract by fermentation time
톳 발효 추출물의 pH 변화를 분석하기 위하여, filter paper(0.072 mm)를 이용하여 균체 및 톳 분말을 제거하고 pH meter기를 사용하여 pH를 측정하였다.In order to analyze the change in pH of the fermented hibiscus extract, the cells and powder were removed using filter paper (0.072 mm), and the pH was measured using a pH meter.
그 결과, 도 8과 같이, 톳을 이용한 발효 추출물은 처리 후 72시간까지 농도 의존적으로 pH가 감소하는 것을 확인하였다.As a result, as shown in FIG. 8 , it was confirmed that the pH of the fermented extract using Shiitake was reduced in a concentration-dependent manner up to 72 hours after treatment.
8. 멜라닌 함량 분석8. Melanin content analysis
톳 발효 추출물이 멜라닌 함량에 미치는 영향을 분석하기 위하여, 24 웰 플레이트에 B16F10 흑색종 세포(1 X 105 cell/ml)를 접종한 후 100 μM α-MSH의 존재 또는 부재 하에 배양을 수행하였다. 이후, 세포를 다양한 농도의 톳 발효 추출물(10, 50, 100 μg/ml)과 72시간 동안 배양하였다. 세포를 회수하고 PBS로 2회 세척한 다음 100 μl의 1 N NaOH를 이용하여 세포를 용해시켰다. 용해시킨 세포는 80℃에서 1시간 동안 반응시킨 후 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 450 nm에서 흡광도를 측정하였다.To analyze the effect of fermented hibiscus extract on melanin content, B16F10 melanoma cells (1
그 결과, 도 9와 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 멜라닌 함량을 감소시키는 것을 확인하였다. As a result, as shown in FIG. 9, it was confirmed that the fermented hibiscus extract treated group decreased the melanin content in a concentration-dependent manner compared to the control group.
9. 세포성 티로시네이즈 활성 분석9. Cellular Tyrosinase Activity Assay
톳 발효 추출물이 세포성 티로시네이즈 활성에 미치는 영향을 분석하기 위하여, 24 웰 플레이트에 B16F10 흑색종 세포(1 X 105 cell/ml)를 접종한 후 100 μM α-MSH의 존재 또는 부재 하에 배양을 수하였다. 이후, 세포를 다양한 농도의 톳 발효 추출물(10, 50, 100 μg/ml)과 72시간 동안 배양하였다. 이후, 상층액을 제거하고 1% Triton X-100(Sigma-Aldrich, MO, USA) 및 0.1 mM PMSF(phenylmethylsulfonyl fluoride)가 함유된 50 mM 인산나트륨 버퍼(pH 6.5) 100 μl 분주하여 세포를 회수하였다. 이후, -80℃에서 30분간 냉동 후 해동을 3회 반복하였다, 세포 추출물을 4℃, 12,000 rpm에서 30분 동안 원심분리 하여 상등액을 획득하였으며, 단백질 농도는 BCA Protein Assay kit를 사용하여 Bradford 법으로 측정하였다. 96 웰 플레이트의 각 웰에 0.1 μM PBS(pH 6.8)에 용해한 50 μg/100 μl의 단백질과 1.5 mM L-DOPA 100 μl로 구성된 반응 혼합물을 첨가하고 37℃에서 1시간 동안 반응시킨 다음 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 475 nm에서 흡광도를 측정하였다.To analyze the effect of fermented shiitake extract on cellular tyrosinase activity, 24 well plates were inoculated with B16F10 melanoma cells (1
그 결과, 도 10과 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 세포성 티로시네이즈 활성을 감소시키는 것을 확인하였다. As a result, as shown in FIG. 10 , it was confirmed that the fermented shiitake extract treated group decreased the cellular tyrosinase activity in a concentration-dependent manner compared to the control group.
10. 흑색종 세포의 생존률 분석10. Analysis of viability of melanoma cells
톳 발효 추출물이 흑색종 세포 생존률에 미치는 영향을 분석하기 위하여, 96 웰 플레이트에 B16F10 흑색종 세포를 1 X 105 cell/ml의 농도로 접종하고 24시간 동안 배양하였다. 이후, 세포를 다양한 농도의 톳 발효 추출물(10, 50, 100 μg/ml)과 24시간 동안 배양한 후, 세포를 PBS로 1회 세척하고 100 ㎕의 MTT 용액(5 mg/ml)을 각 웰에 첨가하여 3시간 동안 반응시켰다. 이후, MTT용액을 제거하고 DMSO(dimethyl sulfoxide) 100 ㎕를 첨가한 후, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 540 nm에서 흡광도를 측정하였다.To analyze the effect of fermented shiitake extract on the viability of melanoma cells, B16F10 melanoma cells were inoculated into a 96-well plate at a concentration of 1 X 10 5 cell/ml and cultured for 24 hours. Thereafter, the cells were incubated with various concentrations of fermented hibiscus extract (10, 50, 100 μg/ml) for 24 hours, the cells were washed once with PBS, and 100 μl of MTT solution (5 mg/ml) was added to each well. was added and reacted for 3 hours. Then, after removing the MTT solution and adding 100 μl of dimethyl sulfoxide (DMSO), absorbance was measured at 540 nm using a multi-well plate reader (Tecan F-200).
그 결과, 도 11과 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 흑색종 세포의 생존률을 감소시키는 것을 확인하였다. As a result, as shown in FIG. 11 , it was confirmed that the fermented shiitake extract treated group reduced the survival rate of melanoma cells in a concentration-dependent manner compared to the control group.
11. 섬유아세포의 생존률 분석11. Analysis of viability of fibroblasts
톳 발효 추출물이 섬유아세포 생존률에 미치는 영향을 분석하기 위하여, 96 웰 플레이트에 CCD-986SK 섬유아세포를 1 X 105 cell/ml의 농도로 접종하고 24시간 동안 배양하였다. 이후, 세포를 다양한 농도의 톳 발효 추출물(10, 50, 100 μg/ml)과 24시간 동안 배양한 후, 세포를 PBS로 1회 세척하고 100 ㎕의 MTT 용액(5 mg/ml)을 각 웰에 첨가하여 3시간 동안 반응시켰다. 이후, MTT용액을 제거하고 DMSO(dimethyl sulfoxide) 100 ㎕를 첨가한 후, 멀티 웰 플레이트 판독기(Tecan F-200)를 사용하여 540 nm에서 흡광도를 측정하였다.To analyze the effect of fermented hibiscus extract on fibroblast viability, CCD-986SK fibroblasts were inoculated in a 96-well plate at a concentration of 1 X 10 5 cell/ml and cultured for 24 hours. Thereafter, the cells were incubated with various concentrations of fermented hibiscus extract (10, 50, 100 μg/ml) for 24 hours, the cells were washed once with PBS, and 100 μl of MTT solution (5 mg/ml) was added to each well. was added and reacted for 3 hours. Then, after removing the MTT solution and adding 100 μl of dimethyl sulfoxide (DMSO), absorbance was measured at 540 nm using a multi-well plate reader (Tecan F-200).
그 결과, 도 11과 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 섬유아세포의 생존률을 감소시키는 것을 확인하였다. As a result, as shown in FIG. 11 , it was confirmed that the fermented shiitake extract treated group reduced the survival rate of fibroblasts in a concentration-dependent manner compared to the control group.
12. 티로시네이즈 및 TRP-1의 단백질 발현 수준 분석12. Analysis of Protein Expression Levels of Tyrosinase and TRP-1
톳 발효 추출물이 티로시네이즈 및 TRP-1 발현에 미치는 영향을 분석하기 위하여, B16F10 흑색종 세포에 톳 발효 추출물(10, 25, 50, 100 μg/ml) 및 α-MSH(1 μM)를 처리하고 72시간 동안 반응시킨 뒤 세포를 회수하였다. 회수된 세포에 RIPA buffer(20 mM Tris-HCl, pH 7.5, 150 mM Nacl, 1% sodium deoxycholate, 1 mM Na2EDTA, 1mM EGTA, 1 % NP-40, 2.5 mM Sodium pyrophophate, 1mM β-glycerophosphate, 1M Na3VO4, 1ug/ml leupeptin)를 첨가하고 4°C에서 20분간 반응시켰다. 이후, 세포 추출물을 4°C, 14,000 g에서 20분 동안 원심분리 하여 상등액을 획득하였으며, 단백질 농도는 BCA Protein Assay kit를 표준으로 사용하여 Bradford 법으로 측정하였다. 단백질은 10% SDS-polyacylamaid gel 전기영동으로 분리한 후, PVDF transfer membrane(Millipore, Billerica, Ma, USA)으로 이동시켰다. 이후, membrane을 5% Skim milk[TBS-T buffer(20 mM Tris-HCl pH 7.6, 136 mM NaCl, 0.1 % Tween-20) 이용]와 실온에서 90분 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 3회 세척한 다음 1차 항체(tyrosinase, TRP-1: Santa Cruz Biotechnology, CA)(1:1000 희석. 1% Skim milk 이용)와 4℃에서 24시간 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 3회 세척한 후, 2차 항체(1:1000 희석)와 실온에서 90분 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 4회 세척한 후, ECL-plus western blotting substrate(Thermo Scientific Inc., Bremen, Germany)로 전개시켰으며, ChemiDoc MP imager(Bio-Rad, CA, USA)를 사용하여 블롯 밴드를 시각화 하였다.To analyze the effect of fermented shiitake extract on tyrosinase and TRP-1 expression, B16F10 melanoma cells were treated with fermented shiitake extract (10, 25, 50, 100 μg/ml) and α-MSH (1 μM). The cells were recovered after incubation for 72 hours. RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM Nacl, 1% sodium deoxycholate, 1 mM Na 2 EDTA, 1 mM EGTA, 1 % NP-40, 2.5 mM Sodium pyrophophate, 1 mM β-glycerophosphate, 1M Na 3 VO 4 , 1ug/ml leupeptin) was added and reacted at 4°C for 20 minutes. Thereafter, the cell extract was centrifuged at 14,000 g at 4 °C for 20 minutes to obtain a supernatant, and the protein concentration was measured by the Bradford method using the BCA Protein Assay kit as a standard. Proteins were separated by 10% SDS-polyacylamaid gel electrophoresis, and then transferred to a PVDF transfer membrane (Millipore, Billerica, Ma, USA). Thereafter, the membrane was reacted with 5% Skim milk [using TBS-T buffer (20 mM Tris-HCl pH 7.6, 136 mM NaCl, 0.1 % Tween-20)] at room temperature for 90 minutes. Thereafter, the membrane was washed 3 times with TBS-T buffer, and then reacted with the primary antibody (tyrosinase, TRP-1: Santa Cruz Biotechnology, CA) (1:1000 dilution, using 1% Skim milk) at 4°C for 24 hours. did it Thereafter, the membrane was washed three times with TBS-T buffer, and then reacted with the secondary antibody (1:1000 dilution) at room temperature for 90 minutes. Thereafter, the membrane was washed 4 times with TBS-T buffer and then developed on ECL-plus western blotting substrate (Thermo Scientific Inc., Bremen, Germany), and ChemiDoc MP imager (Bio-Rad, CA, USA) was used. to visualize the blot band.
그 결과, 도 13과 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 티로시네이즈 및 TRP-1의 발현 수준을 감소시키는 것을 확인하였다. As a result, as shown in FIG. 13 , it was confirmed that the fermented Shiitake extract treated group decreased the expression levels of tyrosinase and TRP-1 in a concentration-dependent manner compared to the control group.
13. MMP-1 및 MMP-12의 단백질 발현 수준 분석13. Analysis of Protein Expression Levels of MMP-1 and MMP-12
톳 발효 추출물이 MMP-1 및 MMP-12 발현에 미치는 영향을 분석하기 위하여, CCD-986SK 섬유아세포에 톳 발효 추출물(10, 50, 100 μg/ml) 및 자외선을 처리하고 72시간 동안 반응시킨 뒤 세포를 회수하였다. 회수된 세포에 RIPA buffer(20 mM Tris-HCl, pH 7.5, 150 mM Nacl, 1% sodium deoxycholate, 1 mM Na2EDTA, 1mM EGTA, 1 % NP-40, 2.5 mM Sodium pyrophophate, 1mM β-glycerophosphate, 1M Na3VO4, 1ug/ml leupeptin)를 첨가하고 4°C에서 20분간 반응시켰다. 이후, 세포 추출물을 4°C, 14,000 g에서 20분 동안 원심분리 하여 상등액을 획득하였으며, 단백질 농도는 BCA Protein Assay kit를 표준으로 사용하여 Bradford 법으로 측정하였다. 단백질은 10% SDS-polyacylamaid gel 전기영동으로 분리한 후, PVDF transfer membrane(Millipore, Billerica, Ma, USA)으로 이동시켰다. 이후, membrane을 5% Skim milk[TBS-T buffer(20 mM Tris-HCl pH 7.6, 136 mM NaCl, 0.1 % Tween-20) 이용]와 실온에서 90분 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 3회 세척한 다음 1차 항체(MMP-1, MMP-12: Santa Cruz Biotechnology, CA)(1:1000 희석. 1% Skim milk 이용)와 4℃에서 24시간 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 3회 세척한 후, 2차 항체(1:1000 희석)와 실온에서 90분 동안 반응시켰다. 이후, membrane을 TBS-T buffer로 4회 세척한 후, ECL-plus western blotting substrate(Thermo Scientific Inc., Bremen, Germany)로 전개시켰으며, ChemiDoc MP imager(Bio-Rad, CA, USA)를 사용하여 블롯 밴드를 시각화 하였다.In order to analyze the effect of fermented shiitake extract on the expression of MMP-1 and MMP-12, CCD-986SK fibroblasts were treated with fermented hibiscus extract (10, 50, 100 μg/ml) and UV light and reacted for 72 hours. Cells were harvested. RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM Nacl, 1% sodium deoxycholate, 1 mM Na 2 EDTA, 1 mM EGTA, 1 % NP-40, 2.5 mM Sodium pyrophophate, 1 mM β-glycerophosphate, 1M Na 3 VO 4 , 1ug/ml leupeptin) was added and reacted at 4°C for 20 minutes. Thereafter, the cell extract was centrifuged at 14,000 g at 4 °C for 20 minutes to obtain a supernatant, and the protein concentration was measured by the Bradford method using the BCA Protein Assay kit as a standard. Proteins were separated by 10% SDS-polyacylamaid gel electrophoresis, and then transferred to a PVDF transfer membrane (Millipore, Billerica, Ma, USA). Thereafter, the membrane was reacted with 5% Skim milk [using TBS-T buffer (20 mM Tris-HCl pH 7.6, 136 mM NaCl, 0.1 % Tween-20)] at room temperature for 90 minutes. After that, the membrane was washed 3 times with TBS-T buffer, and then treated with primary antibodies (MMP-1, MMP-12: Santa Cruz Biotechnology, CA) (1:1000 dilution, using 1% Skim milk) at 4°C for 24 hours. reacted while Then, the membrane was washed 3 times with TBS-T buffer, and then reacted with the secondary antibody (1:1000 dilution) at room temperature for 90 minutes. Thereafter, the membrane was washed 4 times with TBS-T buffer, and then developed on ECL-plus western blotting substrate (Thermo Scientific Inc., Bremen, Germany), and ChemiDoc MP imager (Bio-Rad, CA, USA) was used. to visualize the blot band.
그 결과, 도 14와 같이, 톳 발효 추출물 처리군은 대조군 대비 농도 의존적으로 MMP-1 및 MMP-12의 발현 수준을 감소시키는 것을 확인하였다.As a result, as shown in FIG. 14 , it was confirmed that the fermented shiitake extract treated group decreased the expression levels of MMP-1 and MMP-12 in a concentration-dependent manner compared to the control group.
하기에 본 발명에 따른 톳 발효 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, a formulation example of a composition comprising a fermented hibiscus extract according to the present invention will be described, but the present invention is not intended to limit the present invention, but merely to describe it in detail.
<제제예 1> 로션의 제조<Formulation Example 1> Preparation of lotion
프로필렌글리콜 3.0 중량부, 카르복시폴리머 0.1 중량부, 방부제 미량과 잔량의 정제수를 혼합교반하면서 80 내지 85℃로 가열하여 제조부에 투입한 후 유화기를 작용시키고, 폴리솔베이트60 1.0 중량부, 솔비탄 세스퀴올레이트 0.5 중량부, 유동 파라핀 10.0 중량부, 솔비탄 스테아레이트 1.0 중량부, 친유형 모노스테아린산 글리세린 0.5 중량부, 스테아린산 1.5 중량부, 글리세릴스테아레이트/PEG-400 스테아레이트 1.0 중량부, 트리에탄올아민 0.2 중량부를 80 내지 85℃로 가열하여 투입한 뒤 유화하였다. 유화가 끝나면 교반기를 이용하여 교반하면서 50℃까지 열 냉각한 뒤 향료 미량을 투입하고, 45℃까지 냉각한 뒤 색소 미량을 투입하고, 35℃에서 실시예 1에 따른 톳 발효 추출물을 투입하여 25℃까지 냉각한 뒤 숙성시켰다.3.0 parts by weight of propylene glycol, 0.1 parts by weight of carboxypolymer, a trace amount of preservative and the remaining amount of purified water are heated to 80 to 85° C. while mixing and stirring, and put into the manufacturing part, and then the emulsifier acts,
<제제예 2> 크림의 제조<Formulation Example 2> Preparation of cream
카르복시폴리머 0.3 중량부, 부틸렌글리콜 5.0 중량부, 글리세린 3.0 중량부 및 잔량의 정제수를 혼합교반하면서 80 내지 85℃로 가열하여 제조부에 투입한 후 유화기를 작용시키고, 스테아린산 2.0 중량부, 세틸알콜 2.0 중량부, 글리세릴모노스테아레이트 2.0 중량부, 폴리옥시에틸렌솔비탄모노스테아레이트 0.5 중량부, 솔비탄세스퀴올레이트 0.5 중량부, 글리세릴모노스테아레이트/글리세릴스테아레이트/폴리옥시에틸렌스테아레이트 1.0 중량부, 왁스 1.0 중량부, 유동파라핀 4.0 중량부, 스쿠알란 4.0 중량부, 카프릴릭/카프릭트리글리세라이드 4.0 중량부를 80 내지 85℃로 가열하여 투입한 뒤 트리에탄올아민 0.5 중량부를 투입하여 유화하였다. 유화가 끝나면 교반기를 이용하여 교반하면서 35℃까지 냉각한 뒤 실시예 1에 따른 톳 발효 추출물을 투입하여 25℃까지 냉각한 뒤 숙성시켰다.0.3 parts by weight of carboxypolymer, 5.0 parts by weight of butylene glycol, 3.0 parts by weight of glycerin, and the remaining amount of purified water are heated to 80 to 85° C. while mixing and stirring, and put into the manufacturing unit, and then the emulsifier acts, stearic acid 2.0 parts by weight, cetyl alcohol 2.0 parts by weight, glyceryl monostearate 2.0 parts by weight, polyoxyethylene sorbitan monostearate 0.5 parts by weight, sorbitan sesquioleate 0.5 parts by weight, glyceryl monostearate/glyceryl stearate/polyoxyethylene stearate 1.0 parts by weight of wax, 1.0 parts by weight of wax, 4.0 parts by weight of liquid paraffin, 4.0 parts by weight of squalane, 4.0 parts by weight of caprylic/capric triglyceride was heated to 80 to 85° C. . After the emulsification was completed, the mixture was cooled to 35° C. while stirring using a stirrer, and then the fermented hibiscus extract according to Example 1 was added, cooled to 25° C., and then aged.
<제제예 3> 워시폼의 제조<Formulation Example 3> Preparation of wash foam
TEA-코코일 글루타메이트 30.0 중량부, 디소듐 라우레스 설포숙시네이트글리세린 10.0 중량부, 글리세린 10.0 중량부, 코카마이드 DEA 2.0 중량부, PEG-120 메칠글루코오스 디올리에이트 1.0 중량부, 메칠글루세스-20 0.5 중량부, PEG-150 펜타에리트리틸 테트라 스테아레이트 0.5 중량부, 테트라소듐 EDTA 0.05 중량부 및 방부제 미량을 순차적으로 제조부에 투입하고 60 내지 65℃로 가열한 후 15분 동안 교반하였다. 교반이 끝나면 정제수의 일부를 투입하여 30분 동안 교분한 후, 다시 정제수의 일부를 천천히 투입하고 30분 동안 교반한 후 35℃까지 냉각하고, 실시예 1에 따른 톳 발효 추출물과 향료를 투입하여 25℃까지 냉각한 뒤 숙성시켰다.TEA-cocoyl glutamate 30.0 parts by weight, disodium laureth sulfosuccinate glycerin 10.0 parts by weight, glycerin 10.0 parts by weight, cocamide DEA 2.0 parts by weight, PEG-120 methylglucose dioleate 1.0 parts by weight, methylgluces- 20 0.5 parts by weight, PEG-150 pentaerythrityl tetrastearate 0.5 parts by weight, tetrasodium EDTA 0.05 parts by weight, and a trace amount of preservative were sequentially added to the preparation unit, heated to 60 to 65° C., and stirred for 15 minutes. After stirring, a portion of purified water was added and stirred for 30 minutes, then a portion of purified water was slowly added again, stirred for 30 minutes, cooled to 35° C., and 25 After cooling to °C, it was aged.
<제제예 4> 건강식품 제조<Formulation Example 4> Health food manufacturing
실시예 1에 따른 톳 발효 추출물 1 ㎎, 비타민 혼합물 적량(비타민 A 아세테이트 70 ㎍, 비타민 E 1.0 ㎎, 비타민 B 1 0.13 ㎎, 비타민 B 2 0.15 ㎎, 비타민 B 6 0.5 ㎎, 비타민 B 12 0.2 ㎍, 비타민 C 10 ㎎, 비오틴 10 ㎍, 니코틴산아미드 1.7㎎, 엽산 50 ㎍, 판토텐산 칼슘 0.5 ㎎) 및 무기질 혼합물 적량(황산제1철 1.75 ㎎, 산화아연 0.82 ㎎, 탄산마그네슘 25.3 ㎎, 제1인산칼륨 15 ㎎, 제2인산칼슘 55 ㎎, 구연산칼륨 90 ㎎, 탄산칼슘 100 ㎎, 염화마그네슘 24.8 ㎎)을 혼합한 다음 과립을 제조하고 통상의 방법에 따라 건강식품을 제조하였다.1 mg of fermented hibiscus extract according to Example 1, an appropriate amount of a vitamin mixture (
<제제예 5> 건강음료 제조<Formulation Example 5> Health drink manufacturing
실시예 1에 따른 톳 발효 추출물 1 ㎎, 구연산 1000 ㎎, 올리고당 100 g, 매실농축액 2 g, 타우린 1 g 및 정제수를 가하여 전체 900 ㎖가 되도록 하며, 통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반가열한 후, 만들어진 용액을 여과하여 멸균된 2 L 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관하였다.1 mg of fermented hibiscus extract according to Example 1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine and purified water were added to make a total of 900 ml. After mixing, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered and acquired in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
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