KR102648047B1 - Method for preparing ferment extract of Eremochloa ophiuroides, ferment extract of Eremochloa ophiuroides and complex functional cosmetic composition comprising the same - Google Patents
Method for preparing ferment extract of Eremochloa ophiuroides, ferment extract of Eremochloa ophiuroides and complex functional cosmetic composition comprising the same Download PDFInfo
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- KR102648047B1 KR102648047B1 KR1020220022527A KR20220022527A KR102648047B1 KR 102648047 B1 KR102648047 B1 KR 102648047B1 KR 1020220022527 A KR1020220022527 A KR 1020220022527A KR 20220022527 A KR20220022527 A KR 20220022527A KR 102648047 B1 KR102648047 B1 KR 102648047B1
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- extract
- centipede grass
- fermented
- centipede
- skin
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Classifications
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- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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Abstract
본 발명은 센티페드그라스 발효 추출물의 제조방법, 센티페드그라스 발효 추출물 및 이를 함유하는 복합 기능성 화장료 조성물에 관한 것으로, 보다 상세하게는 센티페드그라스 잎 추출물을 마련하는 단계 및 유산균을 이용하여 상기 센티페드그라스 잎 추출물을 발효하는 단계를 포함하는, 센티페드그라스 발효 추출물의 제조방법; 유산균을 이용하여 센티페드그라스 잎 추출물을 발효하여 획득되는, 센티페드그라스 발효 추출물; 및 유산균을 이용하여 센티페드그라스 잎 추출물을 발효한 발효 결과물인 센티페드그라스 발효 추출물을 유효성분으로 함유하는, 복합 기능성 화장료 조성물에 관한 것이다.The present invention relates to a method for producing a centipede grass fermented extract, a centipede grass fermented extract, and a complex functional cosmetic composition containing the same. More specifically, it relates to the step of preparing centipede grass leaf extract and the centipede grass fermentation extract using lactic acid bacteria. A method for producing a centipede grass fermented extract, comprising fermenting the grass leaf extract; Centipede grass fermented extract, obtained by fermenting centipede grass leaf extract using lactic acid bacteria; and a complex functional cosmetic composition containing, as an active ingredient, centipede grass fermented extract, which is a result of fermentation of centipede grass leaf extract using lactic acid bacteria.
Description
본 발명은 센티페드그라스 발효 추출물의 제조방법, 센티페드그라스 발효 추출물 및 이를 함유하는 복합 기능성 화장료 조성물에 관한 것으로, 센티페드그라스(Centipede grass, Eremochloa ophiuroides) 의 추출물을 이용하여 보다 향상된 미백, 항산화 및 주름개선 효능을 발현할 수 있는 센티페드그라스 발효 추출물의 제조방법, 센티페드그라스 발효 추출물 및 이를 함유하는 복합 기능성 화장료 조성물을 제공하는 것이다.The present invention relates to a method for producing a centipede grass fermented extract , a centipede grass fermented extract, and a complex functional cosmetic composition containing the same, and provides improved whitening, antioxidant and The present invention provides a method for producing a centipede grass fermented extract capable of exhibiting anti-wrinkle efficacy, a centipede grass fermented extract, and a complex functional cosmetic composition containing the same.
최근 피부 미용에 대한 관심이 높아지면서, 항산화 및 미백에 대한 수요도 꾸준히 증가하고 있다. 멜라닌은 자외선으로부터 피부를 보호하기 위해서 필수적이지만, 멜라닌의 과다한 생성과 축적은 기미, 주근깨, 검버섯, 심해지면 피부암 등의 색소침착 질환을 유발한다. 비타민 C, 하이드로퀴논, 코직산과 같은 미백제가 많이 개발되었지만, 햇빛에 의해 쉽게 분해되거나, 장기간 사용시 알레르기 등을 유발하는 부작용으로 인해 천연물 유래의 부작용이 최소화 된 기능성 화장품 등의 자연친화적인 천연화장품에 대한 관심이 지속적으로 높아지고 있다.Recently, as interest in skin care has increased, demand for antioxidants and whitening has been steadily increasing. Melanin is essential to protect the skin from ultraviolet rays, but excessive production and accumulation of melanin causes pigmentation diseases such as blemishes, freckles, age spots, and in severe cases, skin cancer. Although many whitening agents such as vitamin C, hydroquinone, and kojic acid have been developed, they are easily decomposed by sunlight or cause allergies when used for a long period of time. Therefore, there is a need for eco-friendly natural cosmetics such as functional cosmetics with minimal side effects derived from natural products. Interest continues to grow.
최근까지 멜라닌 생성을 억제하는 합성 화학물인 하이드로퀴논이 미백 화장품의 주 원료로 사용되었으나, 하이드로퀴논이 발암 물질로 지정돼 이를 대체할 수 있는 천연소재의 화장품 개발이 시급한 상황이다. 특히 동남아시아를 중심으로 미백 및 기능성 화장품에 대한 관심이 집중되고 있기 때문에 이를 위한 신규 화장품 개발이 필요한 상황이다. 그 외 종래 제품들도 에칠아스코르빌에텔, 아스코빌글루코사이드, 나이아신아마이드 등과 같은 합성 화학 성분이 대부분이다. Until recently, hydroquinone, a synthetic chemical that inhibits melanin production, was used as the main ingredient in whitening cosmetics, but hydroquinone has been designated as a carcinogen, so there is an urgent need to develop cosmetics made of natural materials that can replace it. In particular, as interest in whitening and functional cosmetics is focused mainly in Southeast Asia, there is a need to develop new cosmetics for this purpose. Most other conventional products also contain synthetic chemical ingredients such as ethyl ascorbyl ether, ascorbyl glucoside, and niacinamide.
한편, 피부는 각종 오염 물질과 스트레스 등으로 인해 손상을 입게 되는데, 이와 같은 손상으로 피부의 세포 증식이 제대로 이루어지지 않게 되면 피부는 더욱 더 큰 손상을 입게 되어 빠르게 피부 탄력이 저하되고 수분이 상실되며 주름이 생성되게 된다. 한편, 피부가 노화되면 피부 탄력에 중요한 역할을 하고 있는 매트릭스 내의 콜라겐, 뮤코다당류, 히알루론산 등의 양이 줄어들고, 불용성 콜라겐의 양이 증가한다. 이렇게 되면 진피는 피부의 수분을 유지하기 어렵게 되고 피부는 거칠어지게 된다. 나아가 피지샘과 땀샘의 기능도 감소하여 피지와 땀의 양이 점차로 줄어들게 된다. 이로 인해 피지막이 적당히 생성되지 못하고 피부 수화에 대한 보호력을 잃게 되어 결국 피부는 건조하게 되며 투명성을 잃게 되어 피부 단백질의 탄력이 감소되고 주름이 생성된다. 한편 피부의 보습 작용도 기능을 다하지 못하면 피부 탄력 저하의 원인이 된다. 즉 피부의 보습력 상실은 피부 노화의 중요한 요인이 되고 있다. Meanwhile, the skin is damaged by various pollutants and stress. When skin cells do not proliferate properly due to such damage, the skin suffers even greater damage, quickly losing skin elasticity and losing moisture. Wrinkles are formed. Meanwhile, as the skin ages, the amount of collagen, mucopolysaccharide, and hyaluronic acid in the matrix, which play an important role in skin elasticity, decreases, and the amount of insoluble collagen increases. When this happens, it becomes difficult for the dermis to maintain skin moisture and the skin becomes rough. Furthermore, the function of the sebaceous and sweat glands decreases, and the amount of sebum and sweat gradually decreases. As a result, the sebum film is not properly created and the skin loses its ability to protect against hydration, which ultimately causes the skin to dry out and lose transparency, which reduces the elasticity of skin proteins and creates wrinkles. On the other hand, if the skin's moisturizing effect does not perform its function, it can cause a decrease in skin elasticity. In other words, loss of skin's moisturizing ability is an important factor in skin aging.
예를 들어, 대한민국 등록특허 제10-0644764호의 경우 주름 생성 억제 및 주름 개선 효과를 가지고, 항염활성을 가져 탁월한 상처 치유 효과를 나타내는 조성물이 개시되어 있으나, 주름개선, 화이트닝, 안티에이징 등 복합 기능성을 가지는 화장품 조성물을 제조하는 데는 한계를 가지고 있다.For example, in the case of Republic of Korea Patent No. 10-0644764, a composition is disclosed that has the effect of suppressing wrinkle formation and improving wrinkles, and has anti-inflammatory activity, showing an excellent wound healing effect, but it also has complex functions such as wrinkle improvement, whitening, and anti-aging. Eggplant has limitations in manufacturing cosmetic compositions.
따라서, 천연 소재를 사용함으로써 인체 부작용에 대한 위험성이 없으며, 피부에 효과적인 복합 기능성을 획득할 수 있어, 안정성과 유효성을 동시에 갖는 기능성 자연유래 성분을 획득할 수 있다면 관련 분야에서 널리 적용될 수 있을 것으로 기대된다.Therefore, by using natural materials, there is no risk of side effects to the human body, and complex functionality effective for the skin can be obtained. If functional natural ingredients that are both safe and effective can be obtained, it is expected to be widely applied in related fields. do.
이에 본 발명의 한 측면은 향상된 복합 기능성 효과를 갖는 센티페드그라스 발효 추출물의 제조방법을 제공하는 것이다. Accordingly, one aspect of the present invention is to provide a method for producing a centipede grass fermented extract with improved complex functional effects.
본 발명의 다른 측면은 향상된 복합 기능성 효과를 갖는 센티페드그라스 발효 추출물을 제공하는 것이다. Another aspect of the present invention is to provide a Centipedegrass fermented extract with improved complex functional effects.
본 발명의 또 다른 측면은 상기 본 발명의 센티페드그라스 발효 추출물을 포함하는 복합 기능성 화장료 조성물을 제공하는 것이다.Another aspect of the present invention is to provide a complex functional cosmetic composition containing the fermented Centipedegrass extract of the present invention.
본 발명의 일 견지에 의하면, 센티페드그라스 잎 추출물을 마련하는 단계; 및 유산균을 이용하여 상기 센티페드그라스 잎 추출물을 발효하는 단계를 포함하는, 센티페드그라스 발효 추출물의 제조방법이 제공된다.According to one aspect of the present invention, preparing a centipede grass leaf extract; And a method for producing a fermented centipede grass extract is provided, comprising fermenting the centipede grass leaf extract using lactic acid bacteria.
본 발명의 다른 견지에 의하면, 유산균을 이용하여 센티페드그라스 잎 추출물을 발효하여 획득되는, 또는 상기 본 발명의 제조 방법에 의해 제조되는, 발효 센티페드그라스 추출물이 제공된다. According to another aspect of the present invention, a fermented centipede grass extract is provided, which is obtained by fermenting a centipede grass leaf extract using lactic acid bacteria or produced by the production method of the present invention.
본 발명의 또 다른 견지에 의하면, 발효 센티페드그라스 추출물을 포함하는 복합 기능성 화장료 조성물이 제공된다.According to another aspect of the present invention, a complex functional cosmetic composition containing fermented centipede grass extract is provided.
본 발명에 의한 센티페드그라스 발효 추출물의 제조 기술에 의해 센티페드그라스 추출물의 효능을 현저하게 향상시킬 수 있으며, 따라서 피부에 안전한 천연 유래 성분을 이용하여 기능성이 증대된 새로운 발효 추출물을 제공하여 주름 개선, 미백, 항산화 등에 효과적인 고기능 복합 기능성 화장료 조성물로 활용이 가능할 것으로 기대된다.The efficacy of the centipede grass extract can be significantly improved by the manufacturing technology of the centipede grass fermented extract according to the present invention, and therefore, a new fermented extract with increased functionality is provided using natural ingredients that are safe for the skin, thereby improving wrinkles. It is expected that it can be used as a highly functional complex cosmetic composition that is effective in whitening, antioxidant, etc.
도 1은 센티페드그라스 발효 추출물의 피부미백 효과를 나타낸 것이다. 이때 컨트롤(control)은 멸균 증류수를 의미하는 것이다.
도 2는 센티페드그라스 발효 추출물의 주름개선 효과를 나타낸 것이다. 이때 컨트롤(control)은 멸균 증류수를 의미하는 것이다.
도 3은 센티페드그라스 발효 추출물의 항산화 효과를 나타낸 것이다. 이때 컨트롤(control)은 멸균 증류수를 의미하는 것이다.
도 4는 센티페드그라스 발효 추출물의 제조공정을 도식적으로 나타낸 것이다.Figure 1 shows the skin whitening effect of centipede grass fermented extract. In this case, control refers to sterilized distilled water.
Figure 2 shows the wrinkle-improving effect of centipede grass fermented extract. In this case, control refers to sterilized distilled water.
Figure 3 shows the antioxidant effect of centipede grass fermented extract. In this case, control refers to sterilized distilled water.
Figure 4 schematically shows the manufacturing process of centipede grass fermentation extract.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명에 의하면, 센티페드그라스 추출물의 효능을 증대시키기 위한 방법이 제공되며, 보다 상세하게 센티페드그라스 추출물을 발효하는 공정에 의해 종래 센티페드그라스 추출물의 효능의 한계를 극복할 수 있는 것이다. According to the present invention, a method for increasing the efficacy of centipede grass extract is provided, and in more detail, the limitations of the efficacy of conventional centipede grass extract can be overcome by fermenting the centipede grass extract.
센티페드그라스는 생육이 늦은 난지형 잔디로서 뗏장을 형성하며 포복 생장을 하는 다년생 잔디이다. 벼(Poaceae)과에 속하며, 학명은 에레모클로아 오피우로이데스(Eremochloa ophiuroides) (Munro) Hack이며, 주로 중국과 동아시아, 인도차이나, 미국 북동부, 메소아메리카와 캐러비안 지역에 분포하고 있으며, 각종 토양 조건에 폭 넓게 잘 적응되지만, 특히 습하며 산성토양 및 비옥도가 낮은 사질 토양에서 잘 견딘다. 본 발명은 상기 센티페트그라스의 지상부, 즉 바람직하게는 잎을 사용한다. Centipede grass is a slow-growing, warm-season grass that forms turf and is a perennial grass that grows creeping. It belongs to the Poaceae family, and its scientific name is Eremochloa ophiuroides (Munro) Hack. It is mainly distributed in China, East Asia, Indochina, the northeastern United States, Mesoamerica, and the Caribbean, and is used in various soils. It is well adapted to a wide range of conditions, but is especially tolerant of moist, acidic soils and sandy soils of low fertility. The present invention uses the above-mentioned centipede grass, that is, preferably the leaves.
본 발명의 센티페드그라스 발효 추출물의 제조방법은 센티페드그라스 잎 추출물을 마련하는 단계; 및 유산균을 이용하여 상기 센티페드그라스 잎 추출물을 발효하는 단계를 포함하는 것이다. The method for producing a centipede grass fermented extract of the present invention includes preparing a centipede grass leaf extract; and fermenting the centipede grass leaf extract using lactic acid bacteria.
본 발명의 상기 센티페드그라스 잎 추출물을 마련하는 단계는 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합 용매로 추출하여 수행되는 것일 수 있으며, 저급 알코올은 예를 들어 에탄올, 메탄올 등일 수 있으나, 이에 제한되는 것은 아니며, 1,3-부틸렌글리콜과 같은 다가알코올을 포함하는 것이다. 바람직하게 본 발명의 상기 센티페드그라스 잎 추출물을 마련하는 단계는 열수 추출에 의해 수행되는 것이다.The step of preparing the centipede grass leaf extract of the present invention may be performed by extraction with water, a C 1 to C 4 lower alcohol, or a mixed solvent thereof, and the lower alcohol may be, for example, ethanol, methanol, etc. , but is not limited thereto, and includes polyhydric alcohols such as 1,3-butylene glycol. Preferably, the step of preparing the centipede grass leaf extract of the present invention is performed by hot water extraction.
보다 상세하게, 본 발명의 추출물 획득을 위해 사용되는 추출 방법으로는 침지 추출, 진탕 추출, 열수 추출 또는 환류 추출등을 이용할 수 있으나, 이에 한정하지 않는다. 예를 들어 상기 추출 용매를 센티페드그라스 잎 1 kg 기준으로 1 내지 50 L의 양을 첨가하여 추출할 수 있으며, 4 내지 30 L의 양을 첨가하여 추출하는 것이 바람직하나 이에 한정하지 않는다. 추출 온도는 4 내지 150℃, 예를 들어 10 내지 120℃, 25 내지 100℃, 또는 50 내지 120 ℃인 것이 바람직하나 이에 한정하지 않으며, 추출 시간은 2 내지 5일인 것이 바람직하나 이에 한정하지 않는다. 아울러, 추출 회수는 1 내지 3회인 것이 바람직하나 이에 한정하지 않는다. 예를 들어 열수 추출물은 50 내지 120 ℃, 바람직하게는 50 이상 내지 100 ℃ 미만의 온도에서 1 내지 24시간 동안 침지함으로써 추출을 수행하여 획득될 수 있다.More specifically, the extraction method used to obtain the extract of the present invention may include, but is not limited to, immersion extraction, shaking extraction, hot water extraction, or reflux extraction. For example, the extraction solvent can be extracted by adding 1 to 50 L of the extraction solvent based on 1 kg of centipede grass leaves, and extraction is preferably performed by adding 4 to 30 L of the extract, but is not limited to this. The extraction temperature is preferably, but not limited to, 4 to 150°C, for example, 10 to 120°C, 25 to 100°C, or 50 to 120°C, and the extraction time is preferably, but not limited to, 2 to 5 days. In addition, the number of extractions is preferably 1 to 3 times, but is not limited thereto. For example, a hot water extract can be obtained by performing extraction by soaking at a temperature of 50 to 120°C, preferably 50 to 100°C for 1 to 24 hours.
한편, 본 발명에 있어서 센티페드그라스 잎 추출물은 액상으로 추출되어 그 자체로 사용될 수 있으나, 이에 제한되는 것은 아니며, 상기 추출물은 1회 이상의 여과를 거친 후 사용될 수 있고, 또한 상기 추출물은 농축 혹은 건조하여 사용할 수 있다. 예를 들어 상기 여과는 와트만 필터 종이 0.2 내지 8 μm, 예를 들어 0.2 내지 0.5 μm 기공 사이즈(pore size)의 시린지 필터 또는 이들의 조합을 이용할 수 있다. 또한, 예를 들어 상기 농축은 감압농축일 수 있으나 이에 한정하지 않으며, 또한 상기 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조 등에 의해 수행될 수 있으나 이에 한정하지 않는다. Meanwhile, in the present invention, the centipede grass leaf extract can be extracted as a liquid and used as such, but is not limited to this, and the extract can be used after filtering one or more times, and the extract can be concentrated or dried. You can use it. For example, the filtration may use Whatman filter paper, a syringe filter with a pore size of 0.2 to 8 μm, for example, 0.2 to 0.5 μm, or a combination thereof. In addition, for example, the concentration may be reduced pressure concentration, but is not limited thereto, and the drying may be performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.
따라서, 본 발명에 있어서 "유효성분으로 함유하는"은 추출물 자체, 이의 농축물, 이의 건조물 등을 모두 포함하는 것이다.Therefore, in the present invention, “containing as an active ingredient” includes the extract itself, its concentrate, its dried product, etc.
한편, 본 발명의 상기 유산균을 이용하여 상기 센티페드그라스 잎 추출물을 발효하는 단계는 락토바실러스(lactobacillus sp.) 속 및/또는 리모실락토바실러스 속(Limosilactobacillus sp.) 균주로부터 선택된 적어도 하나의 유산균을 사용할 수 있으며, 예를 들어 상기 유산균은 리모실락토바실러스 퍼멘텀(Limosilactobacillus fermentum), 락티플란티바실러스 플란타룸(Lactiplantibacillus plantarum), 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락티카세이바실러스 카세이(Lacticaseibacillus casei), 및 락토바실러스 람노서스(Lactobacillus rhamnosus)로 이루어진 그룹으로부터 선택된 적어도 하나를 포함하는 것으로 바람직하게는 락토바실러스 아시도필루스(Lactobacillus acidophilus)를 포함하는 유산균을 이용하는 것이다. Meanwhile, the step of fermenting the centipede grass leaf extract using the lactic acid bacteria of the present invention involves at least one lactic acid bacteria selected from the genus Lactobacillus and/or Limosilactobacillus sp . Can be used, for example, the lactic acid bacteria include Limosilactobacillus fermentum , Lactiplantibacillus plantarum , Lactobacillus acidophilus , Lactica ceibacilus casei ( Lacticaseibacillus casei ), and Lactobacillus rhamnosus, preferably containing at least one selected from the group consisting of Lactobacillus acidophilus .
상기 유산균은 센티페드그라스 잎 추출물에 1 × 107 내지 1 × 108 CFU/mL 의 양으로 접종될 수 있으며, 예를 들어 3 × 107 내지 9 × 107 CFU/mL 의 양으로 접종될 수 있다. The lactic acid bacteria can be inoculated into Centipedegrass leaf extract in an amount of 1 × 10 7 to 1 × 10 8 CFU/mL, for example, in an amount of 3 × 10 7 to 9 × 10 7 CFU/mL. there is.
유산균을 접종한 후 35 내지 40℃의 온도에서 24 시간 내지 144 시간, 예를 들어 47 시간 내지 120 시간 동안 배양할 수 있다. 한편, 유산균을 접종한 후 배양액의 교반 속도는 150 내지 200 rpm, 예를 들어 160 내지 180 rpm에서 배양할 수 있다. After inoculation with lactic acid bacteria, the culture can be performed at a temperature of 35 to 40°C for 24 to 144 hours, for example, 47 to 120 hours. Meanwhile, after inoculation with lactic acid bacteria, the culture medium can be cultured at a stirring speed of 150 to 200 rpm, for example, 160 to 180 rpm.
발효된 결과물은 원심분리를 통해 펠렛을 제거하고 상층액을 취하여 사용할 수 있으며, 예를 들어 원심분리는 2000 내지 4000 rpm에서 수행될 수 있다. The fermented product can be used by removing the pellet through centrifugation and taking the supernatant. For example, centrifugation can be performed at 2000 to 4000 rpm.
필요에 따라 추가적으로 1회 이상의 여과 단계를 수행할 수 있으며, 예를 들어 와트만 여과지, 실린지 여과, 또는 이들의 조합 등에 의해 수행될 수 있으나, 이에 제한되는 것은 아니다. If necessary, one or more additional filtration steps may be performed, for example, using Whatman filter paper, syringe filtration, or a combination thereof, but are not limited thereto.
이러한 과정을 통해 획득되는 본 발명의 센티페드그라스 발효 추출물은 바람직하게는 저온에서 보관되는 것으로, 예를 들어 1 내지 15℃, 바람직하게는 3 내지 10℃에서 보관될 수 있다. The centipede grass fermentation extract of the present invention obtained through this process is preferably stored at low temperature, for example, 1 to 15°C, preferably 3 to 10°C.
본 발명에 의한 상기 방법으로부터 획득되는 센티페드그라스 발효 추출물, 즉 환언하면 유산균을 이용하여 센티페드그라스 잎 추출물을 발효하여 획득되는 센티페드그라스 발효 추출물은 우수한 항산화, 피부 보호 또는 이들의 조합의 효과를 발현할 수 있으며, 특히 항산화 효과, 자외선 차단 효과, 주름 개선 효과, 피부 탄력 개선 효과, 피부 미백 효과 등을 발현할 수 있다. The centipede grass fermentation extract obtained from the method according to the present invention, that is, the centipede grass fermentation extract obtained by fermenting centipede grass leaf extract using lactic acid bacteria, has excellent antioxidant, skin protection, or combination thereof effects. In particular, it can exhibit antioxidant effects, UV protection effects, wrinkle improvement effects, skin elasticity improvement effects, and skin whitening effects.
이에 본 발명에 의하면 유산균을 이용하여 센티페드그라스 잎 추출물을 발효한 발효 결과물인 센티페드그라스 발효 추출물을 유효성분으로 함유하는, 복합 기능성 화장료 조성물이 제공된다. Accordingly, according to the present invention, a complex functional cosmetic composition is provided, containing as an active ingredient a centipede grass fermented extract, which is the result of fermentation of centipede grass leaf extract using lactic acid bacteria.
상기 본 발명의 화장료 조성물은 지외선 차단용, 항산화용, 미백용, 피부보호용 또는 이들의 조합 용도인 것일 수 있다. 상기 피부보호 용도는 피부 손상 방지 및/또는 개선 용도을 포함하는 것이다. 상기 용어 "피부 손상"은 외부의 물리적 손상, 화학 물질, 세균, 곰팡이, 바이러스 등의 침투, 자외선에 대한 노출, 피부의 수분 손실, 노화 등에 의한 주름, 자유라디칼(활성산소) 등에 의한 산화, 피부 색소 침착, 피부 염증, 지루성 피부염, 붉어짐(발적, 홍반), 부종, 태선화, 습진, 소양감(가려움), 및 아토피 피부염 등을 포함하는 피부 조직 또는 세포의 임상적 및 미용적 관점에서의 손상을 포함한다. The cosmetic composition of the present invention may be used for ultraviolet ray blocking, antioxidant, whitening, skin protection, or a combination thereof. The skin protection use includes the use of preventing and/or improving skin damage. The term "skin damage" refers to external physical damage, penetration of chemicals, bacteria, mold, viruses, etc., exposure to ultraviolet rays, moisture loss from the skin, wrinkles due to aging, etc., oxidation by free radicals (active oxygen), etc., skin damage, etc. Includes clinical and cosmetic damage to skin tissue or cells, including pigmentation, skin inflammation, seborrheic dermatitis, redness (erythema), edema, lichenification, eczema, pruritus, and atopic dermatitis. do.
상기 피부는 얼굴, 손, 팔, 다리, 발, 가슴, 배, 등, 엉덩이, 및 두피를 포함하는 신체의 모든 피부 부위를 포함한다.The skin includes all skin areas of the body, including the face, hands, arms, legs, feet, chest, stomach, back, buttocks, and scalp.
이때, 상기 화장료 조성물은 상기 센티페드그라스 발효 추출물을 전체 화장료 조성물의 중량을 기준으로 0.1 내지 2 중량%, 예를 들어 0.5 내지 1.5 중량%, 바람직하게는 1 중량%를 함유하는 것일 수 있으며, 다만 본 발명의 조성물에 포함된 센티페드그라스 추출물의 농도, 및 액상 또는 건조 분말 등의 형태에 따라 화장료 조성물에 포함되는 센티페드그라스 추출물의 함유량은 다양할 수 있는 것으로, 상기 범위에 제한되는 것은 아니다. At this time, the cosmetic composition may contain 0.1 to 2% by weight, for example, 0.5 to 1.5% by weight, preferably 1% by weight, based on the weight of the total cosmetic composition, of the centipedegrass fermented extract. Depending on the concentration of the centipede grass extract contained in the composition of the present invention and the form of liquid or dry powder, the content of the centipede grass extract contained in the cosmetic composition may vary, and is not limited to the above range.
상기 화장료 조성물은, 예를 들면 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀), 비이온형의 소낭 분산제의 형태, 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태로 제공될 수 있다. 또한, 포말(foam)의 형태 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 제조될 수 있다.The cosmetic compositions include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules, or ionic (liposome) and non-ionic vesicular dispersants. It may be provided in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. Additionally, it can be prepared in the form of foam or in the form of an aerosol composition further containing compressed propellant.
또한, 상기 화장료 조성물은 본 발명의 센티페드그라스 발효 추출물에 추가로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속이온봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다.In addition, the cosmetic composition contains fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, in addition to the centipedegrass fermented extract of the present invention. Surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, lipid vesicles or in cosmetics. It may contain auxiliaries commonly used in the cosmetic field, such as any other commonly used ingredients.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.
실시예Example
1. 센티페드그라스 발효 추출물의 제조1. Preparation of centipede grass fermentation extract
(1) 센티페드그라스 열수추출물의 제조(1) Preparation of centipede grass hot water extract
센티페드그라스 종자를 정읍에 위치한 한국원자력연구원 첨단방사선연구소의 포장에서 증식하여 센티페드그라스 잎 시료를 채취하였다. 채취한 잎 시료는 20~24 ℃에서 일주일간 자연건조 후, 105 ℃에서 24시간 동안 건조시켜 시료 내 수분함량을 측정하였다. 채취한 잎 시료의 수분함량(Water Content, WC) 측정 공식은 하기 [식 1]과 같다. Centipede grass seeds were propagated in fields at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute located in Jeongeup, and centipede grass leaf samples were collected. The collected leaf samples were naturally dried at 20-24 ℃ for a week and then dried at 105 ℃ for 24 hours to measure the moisture content in the sample. The formula for measuring the water content (WC) of the collected leaf samples is as follows [Equation 1].
[식 1][Equation 1]
· WC(Water Contents, %) = 수분함량 · WC(Water Contents, %) = moisture content
· SW1(Sample Weight, g) = 자연건조 전 측정한 센티페드그라스 잎 시료의 무게· SW 1 (Sample Weight, g) = Weight of centipede grass leaf sample measured before natural drying
· SW2(Dried Sample Weight, g) = 20~24 ℃에서 일주일간 자연건조 및 105 ℃에서 24시간 동안 건조 후 측정한 센티페드그라스 잎 시료의 무게SW 2 (Dried Sample Weight, g) = Weight of centipede grass leaf sample measured after natural drying at 20~24 ℃ for a week and drying at 105 ℃ for 24 hours
상기 [식 1]을 통해 측정된 센티페드그라스 잎 시료 내 수분함량은 8.93 ± 0.48%로 측정되었다. 건조된 센티페드그라스 잎 250 g에 물 5 L를 첨가하여 90 ℃에서 6시간 이상 침지시켜 얻은 추출물을 와트만 거름종이(Whatman filter papers, No. 2, 110 mm)를 이용하여 2회 여과하였다. 이후에 0.45 μm 시린지 필터(syringe filter)를 이용하여 1회 여과하여 센티페드그라스 열수 추출물을 수득하였다. The moisture content in the centipede grass leaf sample measured through [Equation 1] was measured to be 8.93 ± 0.48%. The extract obtained by adding 5 L of water to 250 g of dried centipede grass leaves and soaking them at 90°C for more than 6 hours was filtered twice using Whatman filter papers (No. 2, 110 mm). Afterwards, centipedegrass hot water extract was obtained by filtering once using a 0.45 μm syringe filter.
(2) 센티페드그라스 열수 추출물의 밀도 측정(2) Density measurement of Centipedegrass thermal water extract
본 발명의 센티드그라스 열수 추출물의 밀도(Density, ρ)를 측정하기 위하여 다음과 같이 실험을 실시하였다. To measure the density (ρ) of the scented grass hydrothermal extract of the present invention, the following experiment was performed.
① 동일한 부피의 삼각플라스크(초자류; 300 mL) 5개를 초음파 세척 후, 105 ℃에서 24시간 동안 완전 건조한다.① Wash five Erlenmeyer flasks (glassware; 300 mL) of the same volume ultrasonically and completely dry them at 105°C for 24 hours.
② 상기 ①의 삼각플라스크(초자류; 300 mL) 5개를 충분히 식힌 후, 각각의 무게를 측정한다.② After sufficiently cooling the 5 Erlenmeyer flasks (glassware; 300 mL) in ① above, measure the weight of each.
③ 상기 ②의 삼각플라스크에 상기 1.(1)에서 수득한 센티페드그라스 열수 추출물을 100 mL씩 넣은 후, 105 ℃에서 24시간 동안 완전 건조한다.③ Add 100 mL of Centipedegrass hydrothermal extract obtained in 1.(1) to the Erlenmeyer flask of ② above, then completely dry at 105°C for 24 hours.
④ 상기 ③에서 완전 건조된 삼각플라스크의 무게를 재측정한다. ④ Re-measure the weight of the completely dried Erlenmeyer flask in ③ above.
⑤ 상기 과정을 통해 측정된 무게를 하기 [식 2]에 대입하여 밀도를 계산한다. ⑤ Calculate the density by substituting the weight measured through the above process into [Equation 2] below.
[식 2][Equation 2]
· ρ(Density, g/L) = 센티페드그라스 열수추출물의 밀도· ρ(Density, g/L) = Density of Centipedegrass hydrothermal extract
· FW1(Flask Weight, g) = 완전 건조된 삼각플라스크의 무게· FW 1 (Flask Weight, g) = Weight of completely dried Erlenmeyer flask
· FW2(Flask & Sample Weight, g) = 105 ℃에서 24시간 동안 건조된 센티페드그라스 열수 추출물이 들어있는 완전 건조된 삼각플라스크의 무게· FW 2 (Flask & Sample Weight, g) = Weight of completely dried Erlenmeyer flask containing centipede grass hydrothermal extract dried at 105 ℃ for 24 hours
· SV(Sample Volume, L) = 센티페드그라스 열수추출의 부피(100 mL)· SV(Sample Volume, L) = Volume of centipede grass hot water extraction (100 mL)
그 결과, 상기 [식 2]를 통해 계산된 센티페드그라스 열수 추출물의 밀도는 6.4 ± 0.1 mg/mL로 측정되었다. As a result, the density of Centipedegrass hydrothermal extract calculated through [Equation 2] was measured to be 6.4 ± 0.1 mg/mL.
(3) 센티페드그라스 열수 추출물의 추출 수율 측정(3) Measurement of extraction yield of Centipedegrass thermal water extract
본 발명의 센티페드그라스 열수 추출물의 추출 수율(Extraction Yield)은 하기 [식 3]을 이용하여 계산하였다. The extraction yield (Extraction Yield) of the centipede grass hot water extract of the present invention was calculated using the following [Equation 3].
[식 3] [Equation 3]
· EY(Extraction Yield, %) = 추출수율· EY(Extraction Yield, %) = Extraction yield
· ESW1(Non-extracted Sample Weight, g) = 열수 추출에 사용된 건조된 센티페드그라스 잎 시료의 무게· ESW 1 (Non-extracted Sample Weight, g) = weight of dried centipede grass leaf sample used for hot water extraction
· ESW2(Extracted Sample Weight, g) = 열수 추출 후에 105 ℃에서 24시간 동안 완전 건조 후 측정한 센티페드그라스 열수 추출물의 무게· ESW 2 (Extracted Sample Weight, g) = Weight of Centipedegrass hot water extract measured after hot water extraction and complete drying at 105 ℃ for 24 hours.
그 결과, 상기 [식 3]을 통해 계산된 센티페드그라스 열수 추출물의 추출 수율은 9.04%로 측정되었다. As a result, the extraction yield of centipede grass thermal water extract calculated through [Equation 3] was measured to be 9.04%.
(4)(4) 센티페드그라스 발효 추출물의 제조Preparation of centipede grass fermented extract
센티페드그라스 발효 추출물의 제조를 위하여 다음과 같이 발효 단계를 수행하였다. 한편, 도 4는 센티페드그라스 발효추출물의 제조공정을 도식적으로 나타낸 것이다.To prepare centipede grass fermented extract, the fermentation steps were performed as follows. Meanwhile, Figure 4 schematically shows the manufacturing process of centipede grass fermented extract.
① 고압멸균기로 멸균한 동일한 부피의 삼각플라스크(초자류; 250 mL)에 MRS 배양액 50 mL과 상기 1.(1)에서 수득한 센티페드그라스 열수 추출물을 각각 0 mL, 10 mL, 20 mL을 추가한 후 전체 부피가 100 mL이 되도록 멸균 증류수를 삼각플라스크에 추가하였다. 즉, 하기 표 1과 같이 센티페드그라스 열수 추출물이 전체 부피의 0%, 10%, 20%가 되도록 하였다. ① Add 50 mL of MRS culture medium and 0 mL, 10 mL, and 20 mL of Centipedegrass hydrothermal extract obtained in 1.(1) above to an Erlenmeyer flask (glassware; 250 mL) of equal volume sterilized with a high-pressure sterilizer. Then, sterilized distilled water was added to the Erlenmeyer flask so that the total volume was 100 mL. That is, as shown in Table 1 below, the centipede grass hydrothermal extract was set to 0%, 10%, and 20% of the total volume.
② 상기 센티페드그라스 열수 추출물을 각각 0, 10, 20%을 포함하는 ①의 삼각플라스크에 한국생명공학연구원에서 분양 받은 리모실락토바실러스 퍼멘텀(Limosilactobacillus fermentum, 균주번호: 3112), 락티플란티바실러스 플란타룸(Lactiplantibacillus plantarum, 균주번호: 5045), 락토바실러스 아시도필루스(Lactobacillus acidophilus, 균주번호: 3164), 락티카세이바실러스 카세이(Lacticaseibacillus casei, 균주번호: 3110), 및 락토바실러스 람노서스(Lactobacillus rhamnosus, 균주번호: 3237)를 각각 1 × 107 CFU/mL 양으로 접종하고 37℃에서 200 rpm으로 120 시간 배양하였다. 대조군으로 유산균을 미접종한 삼각플라스크를 동일 조건으로 사용하였다. ② Limosilactobacillus fermentum (strain number: 3112) and Lactiplantybacillus distributed by the Korea Research Institute of Bioscience and Biotechnology in the Erlenmeyer flask of ① containing 0, 10, and 20% of the above centipedegrass hot water extract, respectively. Plantarum ( Lactiplantibacillus plantarum , strain number: 5045), Lactobacillus acidophilus ( Lactobacillus acidophilus , strain number: 3164), Lacticaseibacillus casei ( Lacticaseibacillus casei , strain number: 3110), and Lactobacillus rhamnosus ( Lactobacillus rhamnosus (strain number: 3237) was inoculated at an amount of 1 × 10 7 CFU/mL and cultured at 37°C at 200 rpm for 120 hours. As a control, an Erlenmeyer flask uninoculated with lactic acid bacteria was used under the same conditions.
③ 상기 ②의 유산균(Lactobacillus)에 의해 발효된 센티페드그라스 발효 추출물을 100 mL 코니칼 튜브(conical tube)에 옮긴 후 원심분리(centrifuge)하여 미생물 등을 가라앉힌다. ③ Transfer the centipede grass fermentation extract fermented by Lactobacillus in ② above to a 100 mL conical tube and centrifuge to settle microorganisms.
④ 상기 ③에서 상층액으로 확보한 센티페드그라스 발효 추출물을 와트만 여과지(Whatman filter papers No. 2, 110mm)를 이용하여 2회 여과하였다. 이후에 0.45 μm 실린지 필터(syringe filter)를 이용하여 1회 여과한 후 4℃ 냉장고에 보관하였다. ④ The centipede grass fermentation extract obtained as the supernatant in ③ above was filtered twice using Whatman filter paper (Whatman filter papers No. 2, 110mm). Afterwards, it was filtered once using a 0.45 μm syringe filter and stored in a refrigerator at 4°C.
2. 센티페드그라스 발효 추출물의 물성 및 기능성 분석2. Analysis of physical properties and functionality of centipede grass fermented extract
(1) pH 변화 측정(1) Measurement of pH change
상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물의 pH 변화를 측정하였다. 이때 pH 측정은 (Mettle-Toledo)사의 pH 미터(SevenEasy pH meter)를 이용하여 측정하였고, 그 결과는 하기 [표 2]에 나타내었다. 정확한 측정을 위하여 유리전극을 염기성 완충액에 담근 유리전극을 사용하였고 측정 직전 3차 증류수를 이용하여 유리전극 pH 검출부의 이물질을 완전히 제거하였다. 샘플의 pH를 측정한 후에는 유리전극의 pH 검출부를 3차 증류수를 이용하여 씻어 낸 후 다른 샘플의 pH를 측정하였다. The pH change of the centipede grass fermentation extract prepared through 1(4) above was measured. At this time, pH was measured using a Mettle-Toledo pH meter (SevenEasy pH meter), and the results are shown in [Table 2] below. For accurate measurement, a glass electrode soaked in a basic buffer solution was used, and immediately before measurement, foreign substances were completely removed from the pH detection part of the glass electrode using tertiary distilled water. After measuring the pH of a sample, the pH detection part of the glass electrode was washed with distilled water and the pH of another sample was measured.
발효 추출물 centipede grass
fermented extract
*측정된 값은 평균(Mean) ± 표준편차(SD)로 표시됨 *Measured values are expressed as mean ± standard deviation (SD)
상기 표 2에서 나타난 바와 같이, 본 발명의 센티페드그라스 열수 추출물은 발효 후 pH 값이 감소하였다. 특히, 센티페드그라스 열수 추출물이 락토바실러스 아시도필루스(Lactobacillus acidophilus)에 의해 발효된 후 pH값이 6.3에서 4.7로 가장 크게 감소하는 것으로 나타났다. 반면에, 센티페드그라스 열수 추출물의 농도는 pH변화와 무관한 것을 확인할 수 있었다. As shown in Table 2, the pH value of the centipede grass hydrothermal extract of the present invention decreased after fermentation. In particular, the pH value was found to decrease the most from 6.3 to 4.7 after Centipedegrass hot water extract was fermented by Lactobacillus acidophilus . On the other hand, it was confirmed that the concentration of Centipedegrass hot water extract was unrelated to pH changes.
(2) 피부미백 활성 측정(2) Measurement of skin whitening activity
상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물의 피부 미백 활성을 확인하기 위하여 티로시나제 억제 활성을 측정하였다. 피부는 표피와 진피로 구분되며, 표피는 다시 각질층, 과립층, 유극층, 기저층으로 구분된다. 멜라닌을 만드는 세포인 흑색세포(Melanocyte)는 기저층에 존재하며, 피부가 자극을 받으면 티로시나제(Tyrosinase)라는 효소에 의해 흑색세포에 있는 티로신(Tyrosin)이 산화되어 L-DOPA(3,4-Dihydroxy-L-phenylalanine)가 되며, 다시 티로시나제에 의해 도파크롬(Dopa-quinone)이 생성된다. 도파크롬은 두 가지 형태의 Melanin(Eumelanin, Pheomelanin)을 생성한다. 흑색세포의 유사분열에 의해 활성화된 흑색 세포에서는 트로시나아제 합성이 촉진되고 멜라닌 생성이 증가하여 표피 밖으로 이동하게 되며, 기미/주근깨와 같은 색소침착이 발생하게 된다. 따라서, L-DOPA가 티로시나제에 의해 도파크롬이 되는 과정을 저해할 수 있다면 미백효과를 기대할 수 있다. 본 실험에서는 센티페드그라스 발효추출물이 티로시나아제 효소 활성을 억제하는 효과가 있는지를 측정하였다. The tyrosinase inhibitory activity was measured to confirm the skin whitening activity of the centipede grass fermentation extract prepared through 1(4) above. The skin is divided into the epidermis and the dermis, and the epidermis is further divided into the stratum corneum, stratum granulosum, stratum spinosum, and basal layer. Melanocytes, cells that produce melanin, exist in the basal layer. When the skin is stimulated, tyrosin in the melanocytes is oxidized by an enzyme called tyrosinase, producing L-DOPA (3,4-Dihydroxy- It becomes L-phenylalanine), and dopachrome (Dopa-quinone) is again produced by tyrosinase. Dopachrome produces two types of melanin (Eumelanin and Pheomelanin). In black cells activated by mitosis, trocinase synthesis is promoted and melanin production increases, moving out of the epidermis and causing pigmentation such as spots/freckles. Therefore, if L-DOPA can inhibit the process of becoming dopachrome by tyrosinase, a whitening effect can be expected. In this experiment, the effect of centipede grass fermentation extract on inhibiting tyrosinase enzyme activity was measured.
티로시나아제 억제 효과는 도파크롬(dopachrome)방법을 이용하여 측정하였다. 100 ㎕의 15.7 U/mL의 머쉬룸티로시나아제(Tyrosinase from Mushroom), 100 ㎕의 2.5 mM L-티로신, 200 ㎕의 50 mM sodium phophate buffer(pH 6.8), 그리고 300 ㎕의 코직산(Kojic acid) 혹은 센티페드그라스 발효 추출물을 섞은 후 25℃에서 25분간 배양하여 형성된 발색단(melanin)의 흡광도를 475 nm(흡광도 범위 = 0.4 ~ 0.5)에서 측정하였다. 티로시나아제의 억제 정도는 하기 [식 4]에 대입하여 계산하였으며, 그 결과는 도 1 및 표 3에 나타내었다. The tyrosinase inhibition effect was measured using the dopachrome method. 100 ㎕ of 15.7 U/mL of Tyrosinase from Mushroom, 100 ㎕ of 2.5 mM L-tyrosine, 200 ㎕ of 50 mM sodium phophate buffer (pH 6.8), and 300 ㎕ of Kojic acid. Alternatively, the fermented centipede grass extract was mixed and incubated at 25°C for 25 minutes, and the absorbance of the formed chromophore (melanin) was measured at 475 nm (absorbance range = 0.4 to 0.5). The degree of tyrosinase inhibition was calculated by substituting it into [Equation 4] below, and the results are shown in Figure 1 and Table 3.
[식 4][Equation 4]
Tyrosinase inhibition assay (%) = [1- (반응 후 실험군의 흡광도 - 초기실험군의 흡광도)/(반응 후 대조군의 흡광도 - 초기 대조군의 흡광도)] × 100Tyrosinase inhibition assay (%) = [1- (Absorbance of experimental group after reaction - Absorbance of initial experimental group)/(Absorbance of control group after reaction - Absorbance of initial control group)] × 100
*측정된 값은 평균(Mean) ± 표준편차(SD)로 표시됨 *Measured values are expressed as mean ± standard deviation (SD)
도 1 및 상기 표 3에서 확인할 수 있는 바와 같이, 본 발명의 센티페드그라스 발효 추출물은 처리 농도에 의존하여 티로시나아제의 억제 효율이 현저하게 증대되는 것으로 나타났다. 특히, 락토바실러스 아시도필루스(Lactobacillus acidophilus) 균주로 발효된 센티페드그라스 20 % 발효 추출믈은 약 61%의 티로시나아제 저해율을 나타내었으며, 5종의 균주 중에서 가장 높은 효과를 보였다. 결론적으로 센티페드그라스 발효 추출물이 티로시나아제 효소를 억제함으로써 멜라닌 합성을 효과적으로 억제할 수 있어 피부 미백에 유용함을 확인할 수 있었다.As can be seen in Figure 1 and Table 3, the centipede grass fermented extract of the present invention was found to significantly increase the inhibition efficiency of tyrosinase depending on the treatment concentration. In particular, the 20% fermented centipede grass extract fermented with Lactobacillus acidophilus strain showed a tyrosinase inhibition rate of about 61%, showing the highest effect among the five strains. In conclusion, it was confirmed that centipede grass fermented extract can effectively inhibit melanin synthesis by inhibiting the tyrosinase enzyme, making it useful for skin whitening.
(3) 엘라스타아제 활성 저해능 측정(3) Measurement of elastase activity inhibition ability
엘라스타아제(elastase)는 염증이나 질병과 관련된 다양한 유형의 조직에서 탄성섬유 대사에 영향을 미치는 엘라스틴(elastin) 분해 효소로서, 엘라스타아제가 과잉 생성되면 엘라스틴의 분해가 급격히 증가하며 피부가 처지고 주름이 생성된다. 따라서, 엘라스타아제의 활성을 억제하면 피부 노화 및 주름 형성을 방지할 수 있다. 본 실험에서는 센티페드그라스 열수추출물의 주름개선 효과를 확인 하기 위하여 엘라스타아제 활성 억제 정도를 측정하였다. 엘라스타아제 활성 저해는 돼지췌장 엘라스타아제(porcine pancreas elastase)와 기질인 N-Succinyl-Ala-Ala-Ala-p-nitroanilide를 사용하여 측정하였다. 상기 1(2).를 통해 제조된 센티페드그라스 발효 추출물을 농도 별 (10% 및 20%)로 준비한 시료를 400 ㎕씩 시험관에 넣고 50 mM Tris-HCl 완충용액 (pH 8.0)에 녹인 돼지췌장 엘라스타아제 (0.15U/ml) 용액 40 ㎕을 가한 후 DMSO에 녹여 준비된 0.3 mg/ml N-Succinyl-Ala-Ala-Ala-p-nitroanilide를 200 ㎕ 첨가하여 잘 섞은 후 실온에서 3분간 반응시켰다. 엘라스타아제 활성은 엘라스타아제에 의해 N-Succinyl-Ala-Ala-Ala-p-nitroanilide 기질로부터 알라닌 사이트(alanine site)가 분해되어 생성되는 p-nitroanilide(p-nitroanilide)의 생성량을 분광광도계를 이용하여 410 nm에서 측정하여 발색의 감소여부에 따라 시료가 가진 엘라스타아제 활성 저해를 측정하였다. 엘라스타아제의 억제 정도는 하기 [식 5]에 대입하여 계산하였으며, 그 결과는 하기 표 4 및 도 2에 나타내었다. Elastase is an elastin-decomposing enzyme that affects elastic fiber metabolism in various types of tissues related to inflammation or disease. When elastase is overproduced, the breakdown of elastin increases rapidly, causing sagging skin. Wrinkles are formed. Therefore, inhibiting the activity of elastase can prevent skin aging and wrinkle formation. In this experiment, the degree of inhibition of elastase activity was measured to confirm the wrinkle improvement effect of centipede grass hot water extract. Inhibition of elastase activity was measured using porcine pancreas elastase and the substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide. Put 400 ㎕ of samples prepared at different concentrations (10% and 20%) of the centipede grass fermentation extract prepared through 1(2) above into a test tube and dissolve it in 50 mM Tris-HCl buffer solution (pH 8.0). After adding 40 ㎕ of elastase (0.15U/ml) solution, 200 ㎕ of 0.3 mg/ml N-Succinyl-Ala-Ala-Ala-p-nitroanilide prepared by dissolving in DMSO was added, mixed well, and reacted at room temperature for 3 minutes. . Elastase activity is measured using a spectrophotometer to measure the amount of p-nitroanilide (p-nitroanilide) produced by decomposition of the alanine site from the N-Succinyl-Ala-Ala-Ala-p-nitroanilide substrate by elastase. It was measured at 410 nm to measure the inhibition of elastase activity of the sample according to the decrease in color development. The degree of elastase inhibition was calculated by substituting it into [Equation 5] below, and the results are shown in Table 4 and Figure 2.
[식 5][Equation 5]
Elastase inhibition activity (%) = [1- (반응 후 실험군의 흡광도 - 초기실험군의 흡광도)/(반응 후 대조군의 흡광도 - 초기 대조군의 흡광도)] × 100Elastase inhibition activity (%) = [1- (Absorbance of the experimental group after reaction - Absorbance of the initial experimental group)/(Absorbance of the control group after the reaction - Absorbance of the initial control group)] × 100
*측정된 값은 평균(Mean) ± 표준편차(SD)로 표시됨 *Measured values are expressed as mean ± standard deviation (SD)
도 2에서 나타난 바와 같이, 5종의 발효 균주 중 락토바실러스 아시도필루스(Lactobacillus acidophilus)를 통해 발효된 센티페드그라스 발효 추출물이 가장 높은 엘라스타아제 저해 활성을 보였다. 상기 1(4).를 통해 제조된 센티페드그라스 10% 발효추출물(Lactobacillus acidophilus)은 약 12.0%의 엘라스타아제 저해 효과를 보였다. 특히 센티페드그라스 20% 발효추출물에서는 약 57.9%의 엘라스타아제 저해 활성이 나타나 그 효과가 매우 높아지는 것을 확인할 수 있었다.As shown in Figure 2, among the five fermentation strains, centipede grass fermentation extract fermented through Lactobacillus acidophilus showed the highest elastase inhibitory activity. The 10% fermented centipede grass extract ( Lactobacillus acidophilus ) prepared through 1(4) above showed an elastase inhibition effect of about 12.0%. In particular, it was confirmed that about 57.9% of elastase inhibitory activity was observed in the 20% fermented centipede grass extract, greatly increasing the effect.
(4) 엘라스타아제 DPPH 자유라디컬 생성 저해 활성 측정(4) Measurement of elastase DPPH free radical generation inhibition activity
상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물의 항산화 효과를 평가하기 위해 DPPH 자유라디칼 소거능 실험을 진행하였다. 1mM DPPH(1,1-diphenyl-2-picryl hydrazyl)는 메탄올 내에서 프리라디칼을 생성한다. 메탄올 500 ㎕과 1 mM DPPH 500㎕에 센티페드그라스 발효 추출물을 농도 별로 첨가하였다. 혼합물을 약 20초간 강하게 교반한 뒤, 4 ℃에서 30분 동안 보관한 다음, 517nm에서 흡광도를 측정하였다. L-Ascorbic acid를 양성 대조군으로 사용하여 흡광도를 측정하고, 자유 라디칼 소거능은 하기 [식 6]에 대입하여 계산하였으며, 그 결과는 하기 도 3 및 [표 5]에서 나타내었다. A DPPH free radical scavenging activity test was conducted to evaluate the antioxidant effect of the centipede grass fermented extract prepared through 1(4) above. 1mM DPPH (1,1-diphenyl-2-picryl hydrazyl) generates free radicals in methanol. Centipedegrass fermentation extract was added at different concentrations to 500 μl of methanol and 500 μl of 1 mM DPPH. The mixture was stirred vigorously for about 20 seconds, stored at 4 °C for 30 minutes, and then the absorbance was measured at 517 nm. L-Ascorbic acid was used as a positive control to measure absorbance, and the free radical scavenging ability was calculated by substituting it into [Equation 6] below, and the results are shown in Figure 3 and Table 5 below.
[식 6] [Equation 6]
DPPH radical scavenging activity (%) = [1- (반응 후 실험군의 흡광도 - 초기실험군의 흡광도)/(반응 후 대조군의 흡광도 - 초기 대조군의 흡광도)] × 100DPPH radical scavenging activity (%) = [1- (absorbance of experimental group after reaction - absorbance of initial experimental group)/(absorbance of control group after reaction - absorbance of initial control group)] × 100
*측정된 값은 평균(Mean) ± 표준편차(SD)로 표시됨 *Measured values are expressed as mean ± standard deviation (SD)
도 3에 나타난 바와 같이 상기 1.(1)에서 수득한 센티페드그라스 열수 추출물은 약 90 내지 92%의 자유라디칼 소거능을 나타내었다. 또한, 5종의 발효 균주로 발효된 센티페드그라스 발효 추출물은 약 83~94%의 자유라디칼 소거능을 보였으며, 센티페드그라스 추출물의 농도와는 무관한 것으로 확인되었다. 결과적으로, 센티페드그라스 추출물 대비 센티페드그라스 추출물이 유산균(Lactobacillus)에 의해 발효과정을 거친 후에도 항산화 효과에는 변화가 없음을 확인하였다. As shown in Figure 3, the centipede grass hydrothermal extract obtained in (1) above showed a free radical scavenging ability of about 90 to 92%. In addition, centipede grass fermented extracts fermented with five types of fermentation strains showed a free radical scavenging ability of about 83-94%, and it was confirmed to be unrelated to the concentration of centipede grass extract. As a result, it was confirmed that there was no change in the antioxidant effect of the centipede grass extract compared to the centipede grass extract even after fermentation by lactic acid bacteria (Lactobacillus).
3. 센티페드그라스 발효 추출물의 유기산 분석3. Organic acid analysis of centipede grass fermentation extract
상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물 중 티로시나아제 및 엘라스타아제 활성 저해능이 가장 높았던 락토바실러스 아시도필루스(Lactobacillus acidophilus)를 통해 발효된 센티페드그라스 발효 추출물의 유기산 분석을 위하여 HPLC분석을 수행하였다. Organic acid analysis of centipede grass fermented extract fermented using Lactobacillus acidophilus, which had the highest tyrosinase and elastase activity inhibition ability among centipede grass fermented extracts prepared through 1(4). HPLC analysis was performed for this purpose.
구체적으로, Agilient Technologies 1200 series HPLC에 20 μl의 시료 용액(상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물)을 주입하고, 1 % 포름산(용액 A) 및 100 % 메탄올(용액 B)로 구성된 이동상을 사용하였다. 상기 이동상을 복합 공정(combined step) 및 30분 동안 100:0(용액 A:B)에서 50:50의 직선 구배로, 이어서 60분 동안 50:50에서 0:100의 직선 구배로 분획물 성분을 용출시켰다. 유속은 0.5 μl/min이고 용출된 화합물은 360 nm의 파장에서 탐지하였다. Specifically, 20 μl of sample solution (centipede grass fermentation extract prepared through 1(4) above) was injected into the Agilent Technologies 1200 series HPLC, and 1% formic acid (solution A) and 100% methanol (solution B) were added to the Agilent Technologies 1200 series HPLC. A mobile phase consisting of was used. The mobile phase was used in a combined step and the fraction components were eluted with a linear gradient of 100:0 (solution A:B) to 50:50 for 30 minutes, followed by a linear gradient of 50:50 to 0:100 for 60 minutes. I ordered it. The flow rate was 0.5 μl/min and the eluted compounds were detected at a wavelength of 360 nm.
그 결과, 하기 표 6에서 확인할 수 있는 바와 같이 상기 1(4).를 통해 제조된 센티페드그라스 발효 추출물에서 말산(malic acid), 락트산(lactic acid) 및 아세트산(acetic acid)의 함량이 증가한 것을 확인하였다. As a result, as can be seen in Table 6 below, the content of malic acid, lactic acid, and acetic acid increased in the centipede grass fermentation extract prepared through 1 (4). Confirmed.
*측정된 값은 평균(Mean) ± 표준편차(SD)로 표시됨 *Measured values are expressed as mean ± standard deviation (SD)
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. This will be self-evident to those with ordinary knowledge in the field.
Claims (11)
유산균을 이용하여 상기 센티페드그라스 잎 추출물을 발효하는 단계를 포함하며,
상기 유산균은 락토바실러스 아시도필루스(Lactobacillus acidophilus)를 포함하는, 센티페드그라스 발효 추출물의 제조방법.
Preparing a centipede grass leaf hot water extract by hot water extraction; and
It includes the step of fermenting the centipede grass leaf extract using lactic acid bacteria,
The lactic acid bacteria include Lactobacillus acidophilus . A method of producing a centipede grass fermentation extract.
The method of claim 1, wherein the hot water extraction is performed at a temperature of 50 ℃ to 120 ℃ for 1 hour to 24 hours. Method for producing centipede grass fermented extract, carried out during.
The method of claim 1, wherein the centipede grass leaf hot water extract is obtained using 1 to 50 L of water based on 1 kg of centipede grass leaves.
A centipede grass fermentation extract prepared from any one of claims 1, 4, and 5.
A centipede grass fermented extract obtained by fermenting a hot water extract of centipede grass leaves using Lactobacillus acidophilus .
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