KR101928883B1 - Cosmetic or Food Composition comprising Hydrolysates of Hippocampus abdominalis - Google Patents
Cosmetic or Food Composition comprising Hydrolysates of Hippocampus abdominalis Download PDFInfo
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- KR101928883B1 KR101928883B1 KR1020160144576A KR20160144576A KR101928883B1 KR 101928883 B1 KR101928883 B1 KR 101928883B1 KR 1020160144576 A KR1020160144576 A KR 1020160144576A KR 20160144576 A KR20160144576 A KR 20160144576A KR 101928883 B1 KR101928883 B1 KR 101928883B1
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- South Korea
- Prior art keywords
- hippocampus
- hydrolyzate
- kda
- fraction
- abdominalis
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Abstract
본 발명은 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 식품 조성물 및 화장료 조성물에 관한 것이다.
본 발명의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물은 알킬 라디칼 소거활성을 나타내고, 산화적 스트레스에 의한 세포 보호효과를 나타내고, ROS 생성을 억제하는 바, 항산화용 식품 조성물, 화장료 조성물 등에 유용하게 사용될 수 있다.The invention Big Valley hippocampus (Hippocampus abdominalis ) or a fraction thereof as an active ingredient, and to a cosmetic composition for antioxidation.
The Hippocampus < RTI ID = 0.0 > abdominalis or fractions thereof exhibit an alkyl radical scavenging activity, exhibit a cytoprotective effect by oxidative stress, inhibit ROS production, and can be usefully used in food compositions for antioxidation, cosmetic compositions and the like.
Description
본 발명은 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 식품 조성물 또는 화장료 조성물에 관한 것이다.The invention Big Valley hippocampus (Hippocampus abdominalis or fractions thereof as an active ingredient. The present invention also relates to a food composition or a cosmetic composition for antioxidation.
생체 내에서 안정한 상태로 존재하던 산소가 화학약품, 공해물질, 광화학 반응과 같은 환경적 및 생화학적 요인 등에 의해 수퍼옥사이드 음이온 라디칼 (superoxide anion radical, O2-), 히드록실 라디칼 (hydroxyl radical, OH-), 과산화수소 (hydrogen peroxide, H2O2), 일중항산소 (singlet oxygen, 1O2) 등과 같이 반응성이 큰 활성산소 (reactive oxygen species: ROS)로 전환되면 인체의 세포구성 성분인 단백질, 지질 및 DNA 등에 대하여 비선택적, 비가역적인 파괴를 유도하여 노화를 유발하는 것으로 알려져 있다 (Cross 등, Ann. Intern. Med ., 1987, 107, 526; Alderson 등, Proc. Natl. Acad. Sci. USA, 1988, 85, 2706). 이와 같이, 산화적 스트레스가 노화를 비롯하여 각종 질환을 일으키는 중요한 원인임이 밝혀지면서 생체 내 활성산소를 효과적으로 제거하는 활성산소 소거제를 개발하려는 연구가 활발히 진행되고 있다. 특히, 활성산소 소거활성이 탁월하면서 보다 안전한 새로운 천연활성산소 소거제의 개발이 절실히 요구되고 있다.Oxygen, which is present in a stable state in the living body, is decomposed by superoxide anion radical (O2 - ), hydroxyl radical (OH - ), and hydroxyl radical (OH) by the environmental and biochemical factors such as chemicals, pollutants, ), Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2) DNA and the like are induced to induce non-selective and irreversible disruption to induce senescence (Cross et al., Ann. Intern. Med., 1987, 107, 526, Alderson et al., Proc. Natl. Acad Sci USA 1988 , 85, 2706). As such, it has been found that oxidative stress is an important cause of various diseases such as aging, and studies are actively conducted to develop active oxygen scavenging agents that effectively remove active oxygen in vivo. In particular, there is a desperate need for the development of a safer new natural active oxygen scavenger which is superior in active oxygen scavenging activity.
한편, 해마는 실고기과에 속하는 경골어류로서 실고기목(Syngnathiformes), 실고기과(Syngnathidae)에 속하는 52속 215종 중에서 해마속(Hippocampus) 어류 32종이 분류되며 열대에서 아한대의 광범위한 해역에 분포하고 있는 것으로 보고되어 있고, 이 중 70% 정도가 인도-태평양해역에 분포한다(Myers, 1979; Vari, 1982). 우리나라에는 해마(Hippocampus coronatus), 가시해마(Hippocampus histrix), 복해마(Hippocampus kuda), 산호해마(Hippocampus mohnikei), 그리고 점해마(Hippocampus trimaculatus) 등의 5종이 서식하고 있다(Kim and Lee, 1995). 해마는 중국에서 전통적인 약제로서 천식, 간 기능 장애, 성기능 향상 그리고 일반적인 통증에 효능이 있는 것으로 알려져 있어 중국을 비롯한 동남아시아에 서는 약제로서의 수요가 많고 유럽과 북미에서는 관상용과 건조시켜 기념품이나 골동품 등으로 상품화하고 있다(Vincent, 1996; Choi et al., 2006).The hippocampus is a tibial fish belonging to the genus Mytilidae, and among the 52 genera of 215 species belonging to Syngnathiformes and Syngnathidae, 32 species of hippocampus fish are classified and distributed in a wide range of tropics to biceps (Myers, 1979; Vari, 1982), with about 70% of these being found in the Indian-Pacific waters. In Korea, seahorses (Hippocampus coronatus ), thorn seahorse ( Hippocampus histrix , Hippocampus kuda , Hippocampus mohnikei), and is five paper such that the hippocampus (Hippocampus trimaculatus) format (Kim and Lee, 1995). Hippocampus is a traditional medicines in China and has been known to have efficacy for asthma, liver dysfunction, sexual function improvement and general pain. Therefore, there is a great demand for medicines in Southeast Asia including China. In Europe and North America, it is customized and dried for souvenirs and antiques (Vincent, 1996; Choi et al., 2006).
빅벨리해마(Hippocampus abdominalis)는 큰가시고기목(Syngnathifromes), 실고기과(Syngnathidae)에 속하며 온대와 열대해역에 서식하고 해마들 중에서도 크기가 가장 크고 아름다운 체색과 체형을 지니고 있어 관상가치가 높아 국제 해수 관상생물 시장에서도 인기가 많다. 이미 뉴질랜드, 호주, 중국에서 해마양식이 시도되었고(Woods, 2007), 최근 제주에서는 식용으로 지정된 빅벨리해마의 완전 양식이 성공하였다. 하지만 이전에 많은 생리활성 연구가 진행된 북해마(Hippocampus kuda Bleeker), 갈귀해마(Hippocampus guttulatus), Short-snouted seahorse (Hippocampus hippocampus)종 등에 비해 빅벨리해마는 생리활성 연구가 많이 부족한 상황이다. Hippocampus abdominalis ) belongs to Syngnathifromes and Syngnathidae. It lives in temperate and tropical waters. It has the biggest size among hippocampus and has beautiful body color and body shape. There are many. Hippocampal farming has already been attempted in New Zealand, Australia and China (Woods, 2007), and the complete breeding of the Big Veli hippocampus, recently designated as edible in Jeju, has been successful. However, it has been reported that Hippocampus kuda Bleeker), galgwi seahorse (Hippocampus guttulatus ), short-snouted seahorse ( Hippocampus hippocampus ) species, and so on.
본 발명의 목적은 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 식품 조성물을 제공하는 것이다.An object of the present invention is big Valley hippocampus (Hippocampus abdominalis protein hydrolyzate or a fraction thereof as an active ingredient.
본 발명의 다른 목적은, 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 화장료 조성물 을 제공하는 것이다.It is another object of the present invention, the big Valley hippocampus (Hippocampus abdominalis ) or a fraction thereof as an active ingredient.
본 발명의 다른 목적은, 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 피부노화 방지용 화장료 조성물을 제공한다. It is another object of the present invention, the big Valley hippocampus (Hippocampus abdominalis ) or a fraction thereof as an active ingredient. The present invention also provides a cosmetic composition for preventing skin aging.
본 발명의 다른 목적은, 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 처리하는 단계를 포함하는, 인간을 제외한 개체에서 산화적 스트레스를 억제하는 방법을 제공하는 것이다. It is another object of the present invention, the big Valley hippocampus (Hippocampus abdominalis , or a fraction thereof, in a subject other than humans. The present invention also provides a method for inhibiting oxidative stress in a subject other than human.
본 발명자는 소화효소 2종(trypsin, pepsin)과 식품용 가수분해 효소 5종(neutrase, protamex, alcalase, kojizyme, flavourzyme)을 이용한 빅벨리해마 가수분해물들의 제조와 가수분해물 유래의 각 분자량별 분획물을 이용해 자유 라디칼에 대한 소거능을 평가하여, 빅벨리해마가 높은 항산화능이 있는 것을 확인하여, 항산화용 식품 조성물과 화장료 조성물에 활용하고자 하였다. The present inventors have found that the production of hydrolysates of Big Belly hippocampus using two kinds of digestive enzymes (trypsin, pepsin) and five kinds of food hydrolyzing enzymes (neutrase, protamex, alcalase, kojizyme and flavorzyme) and hydrolyzate- , The scavenging ability against free radicals was evaluated and it was confirmed that the Bigbellie hippocampus had a high antioxidant ability and it was intended to be utilized in a food composition for antioxidation and a cosmetic composition.
구체적으로, 빅벨리해마의 펩신(pepsin) 가수분해물, 트립신(trysin) 가수분해물, 뉴트라제(neutrase) 가수분해물, 프로타멕스(protamex) 가수분해물, 알칼레이즈(alcalase) 가수분해물, 코지자임(kojizyme) 가수분해물, 플라보자임(flavourzyme) 가수분해물, 빅벨리해마의 물 추출물의 펩신 가수분해물, 및 빅벨리해마의 물 추출물의 트립신 가수분해물을 제조하였으며, 가수분해물의 수율이 높을수록 단백질 함량도 높은 것을 확인하였다. Specifically, the pepsin hydrolyzate, the trysin hydrolyzate, the neutrase hydrolyzate, the protamex hydrolyzate, the alcalase hydrolyzate, the cozymidine kojizyme hydrolysates, flavozyme hydrolysates, pepsin hydrolysates of water extracts of Big belly hippocampus and trypsin hydrolysates of water extracts of Big belly hippocampus were prepared. The higher the yield of hydrolyzate, the higher the protein content Respectively.
또한, 상기 빅벨리해마의 물(DW) 추출물에 비해 상기 효소들의 가수분해물의 수율과 단백질 함량이 높음을 확인하였으며, 구체적으로 빅벨리해마의 물 추출물의 펩신 가수분해물(DP, Distilled water-pepsin hydrolysate)과 빅벨리해마의 물 추출물의 트립신 가수분해물(DT, Distilled water-trypsin hydrolysate), 식품용 가수분해 효소 5종(Neutrase, Protamex, Alcalase, Kojizyme, Flavourzyme)에서 가수분해가 많이 진행되는 것을 확인하였다. In addition, it was confirmed that the yield and protein content of hydrolysates of the enzymes were higher than that of the water extract (DW) of the Bigbellie hippocampus. Specifically, it was confirmed that the extracts of pepsin (DP, distilled water-pepsin hydrolyzate (DT, Distilled water-trypsin hydrolyzate) and five food hydrolytic enzymes (Neutrase, Protamex, Alcalase, Kojizyme, Flavourzyme) of water extract of Bigbellie hippocampus .
또한, 빅벨리해마의 물 추출물의 펩신 가수분해물(DP, Distilled water-pepsin hydrolysate)이 alkyl radical 소거능이 우수함을 확인하였으며, 이에 따라, 상기 빅벨리해마의 가수분해물을 항산화용 식품 조성물 또는 화장료 조성물에 사용할 수 있음을 확인하고 본 발명을 완성하였다. In addition, it was confirmed that the digoxin hydrolyzate (DP, Distilled water-pepsin hydrolyzate) of the water extract of the Bigbell hippophae was excellent in the alkyl radical scavenging ability. Thus, the hydrolyzate of the Bigbell hippocampus was applied to the antioxidant food composition or cosmetic composition And the present invention has been completed.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 식품 조성물을 제공한다.In one aspect of the present invention, there is provided an antioxidant food composition comprising a protein hydrolyzate of Hippocampus abdominalis or a fraction thereof as an active ingredient.
구체적으로, 본 발명의 항산화용 식품 조성물은 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 포함할 수 있다. Specifically, the antioxidative food composition of the present invention may comprise a protein hydrolyzate of Hippocampus abdominalis or a fraction thereof.
본 발명에서, 용어 "빅벨리해마(Hippocampus abdominalis)"는 큰가시고기목(Syngnathifromes), 실고기과(Syngnathidae)에 속하며 온대와 열대해역에 서식하고 해마들 중에서도 크기가 가장 크고 아름다운 체색과 체형을 지니고 있어 관상가치가 높아 국제 해수 관상생물 시장에서도 인기가 많다. 이미 뉴질랜드, 호주, 중국에서 해마양식이 시도되었고, 최근 제주에서는 식용으로 지정된 빅벨리해마의 완전 양식이 성공하였다. 하지만 이전에 많은 생리활성 연구가 진행된 북해마(Hippocampus kuda Bleeker), 갈귀해마(Hippocampus guttulatus), Short-snouted seahorse (Hippocampus hippocampus)종 등에 비해 빅벨리해마는 생리활성 연구가 많이 부족한 상황이다. 본 발명에서는 상기 빅벨리해마가 알킬 라디칼(Alkyl radical)을 소거하고, 산화적 스트레스를 억제하여 항산화 활성이 있음을 규명하였다. In the present invention, the term " Hippocampus & abdominalis "belongs to Syngnathifromes and Syngnathidae. It lives in temperate and tropical waters. It has the largest size among hippocampus and has beautiful body color and body shape. Hippocampus kuda has already been attempted in New Zealand, Australia and China, and recently the complete breeding of the Big Veli hippocampus designated as edible in Jeju has succeeded. However, the hippocampus kuda Bleeker , Hippocampus guttulatus ), Short-snouted seahorse ( Hippocampus hippocampus ) than the Big Bell hippocampus . In the present invention, it was confirmed that the Bigbell hippopotamus scavenges an alkyl radical and inhibits oxidative stress and has an antioxidative activity.
본 발명에서, 상기 빅벨리해마의 일반 성분은 해마 건조 시료의 일반성분은 AOAC (Helrich K. 1990. Official Methods of Analysis of AOAC Intl. 15th ed.; Association of Official Analysis Chemists, Washington, DC, USA. Chapter 11. 1-31.)방법에 준하여 측정하였으며, 수분, 회분, 조단백질, 조지방을 건조무게를 기준으로 성분을 분석하였으며, 해마의 조단백질의 함량으로 58.47%을 확인하여 해마의 대부분이 조단백질로 이루어져 있음을 확인하였다(표 1).In the present invention, the general component of the Bigbell hippocampus is a general component of the hippocampal dry sample, which is AOAC (Helich K. 1990. Official Methods of Analysis of AOAC Int. 15th ed .; Association of Official Analysis Chemists, Washington, DC, USA.
본 발명에서 상기 가수분해물은 상기 빅벨리해마(Hippocampus abdominalis)를 소화효소 2종(trypsin, pepsin)과 식품용 가수분해 효소 5종(neutrase, protamex, alcalase, kojizyme, flavourzyme)을 이용하여, 빅벨리해마의 펩신(pepsin) 가수분해물, 트립신(trysin) 가수분해물, 뉴트라제(neutrase) 가수분해물, 프로타멕스(protamex) 가수분해물, 알칼레이즈(alcalase) 가수분해물, 코지자임(kojizyme) 가수분해물, 플라보자임(flavourzyme) 가수분해물, 빅벨리해마의 물 추출물의 펩신 가수분해물, 및 빅벨리해마의 물 추출물의 트립신 가수분해물로 이루어진 군에서 선택되는 1종 이상일 수 있다. The hydrolyzate from the invention is the big belly hippocampus (Hippocampus abdominalis) digestive enzymes two kinds (trypsin, pepsin) and food hydrolases for the five kinds (neutrase, protamex, alcalase, kojizyme, using flavourzyme), the big Valley hippocampus pepsin (pepsin) hydrolyzate, trypsin (trysin) Hydrolyzate, hydrolyzate, neutrase hydrolyzate, protamex hydrolyzate, alcalase hydrolyzate, kojizyme hydrolyzate, flavorzyme hydrolyzate, big belly hippocampus, A pepsin hydrolyzate of a water extract, and a trypsin hydrolyzate of a water extract of a big belly hippocampus.
본 발명에서, 용어, "항산화"는 산화를 억제하는 작용을 의미하는 것으로, 인체는 산화촉진물질(prooxidant)과 산화억제물질(antioxidant)이 균형을 이루고 있으나 여러 가지 요인들에 의하여 이런 균형상태가 불균형을 이루게 되고 산화촉진 쪽으로 기울게 되면, 산화적 스트레스(oxidative stress)가 유발되어 잠재적인 세포손상 및 병리적 질환을 일으키게 된다. 이러한 산화적 스트레스의 직접적 원인이 되는 활성 산소종(reactive oxygen species, ROS)은 불안정하고 반응성이 높아 여러 생체물질과 쉽게 반응하고, 체내 고분자들을 공격하여 세포와 조직에 비가역적인 손상을 일으키거나 돌연변이, 세포독성 및 발암 등을 초래하게 된다. NO, HNO2, ONOO-와 같은 활성 질소종(reactive nitrogen species, RNS), ROS와 같은 활성산소는 체내에서 세포를 산화시켜 파괴시키며, 그에 따라 각종 질환에 노출되게 된다. 또한, 상기 항산화는 세포 내 대사 또는 자외선의 영향으로 인한 산화적 스트레스에 따라 반응성이 높은 자유 라디칼(free radical) 또는 활성 산소종 (Reactive oxygen species, ROS)에 의한 세포의 산화를 억제하는 기능을 의미하며, 자유 라디칼 또는 활성 산소종을 제거하여 이로 인한 세포의 손상이 감소되는 것을 포함한다.In the present invention, the term "antioxidant" means an antioxidant action. The human body has a balance between a prooxidant and an antioxidant, but due to various factors, When imbalanced and leaning toward oxidation promotion, oxidative stress is induced, leading to potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. Active oxygen species such as reactive nitrogen species (RNS), NO, HNO 2 , and ONOO - oxidize and destroy cells in the body, thereby exposing them to various diseases. In addition, the antioxidant has a function of inhibiting oxidation of cells due to highly reactive free radicals or reactive oxygen species (ROS) according to oxidative stress caused by intracellular metabolism or ultraviolet rays And eliminating free radicals or reactive oxygen species, thereby reducing damage to the cells.
본 발명에서, 상기 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물은 산화적 스트레스를 억제할 수 있다. In the present invention, the big Valley hippocampus (Hippocampus Abdominalis protein hydrolysates or fractions thereof can inhibit oxidative stress.
본 발명의 일실시예에서는 상기 빅벨리해마의 물 추출물의 펩신 가수분해물(DP, Distilled water-pepsin hydrolysate)과 빅벨리해마의 물 추출물의 트립신 가수분해물(DT, Distilled water-trypsin hydrolysate)이 우수한 alkyl radical 소거활성을 나타내는 것을 확인하였다(표 2). 또한, AAPH로 산화적 스트레스를 유도한 후 세포생존률을 확인하였으며, 그 결과, DP는 농도의존적으로 AAPH 산화에 의한 세포 보호효과를 나타내는 것을 확인하였다(도 2B). 또한, AAPH로 세포에 자극을 주고 세포 내 AAPH 산화에 의한 ROS 생성 정도를 측정한 결과, 농도의존적으로 AAPH에 의한 세포의 ROS 생성을 줄여 주는 것을 확인하여, 상기 빅벨리해마의 단백질 가수분해물 또는 이의 분획물이 알킬 라디칼을 소거하고, 산화적 스트레스를 억제하는 것을 확인하였다. In one embodiment of the present invention, the distilled water-pepsin hydrolyzate (DP) of the water extract of the Bigbell horse hippocampus and the distilled water-trypsin hydrolyzate (DT) of the water extract of the Big- radical scavenging activity (Table 2). In addition, the cell survival rate was confirmed after inducing oxidative stress by AAPH. As a result, it was confirmed that DP showed a cytoprotective effect by AAPH oxidation in a concentration-dependent manner (Fig. 2B). In addition, the cells were stimulated with AAPH and the degree of ROS production by the oxidation of AAPH in the cells was measured. As a result, it was confirmed that the ROS production by the AAPH was decreased by the concentration-dependent manner, and the protein hydrolyzate of the Bigbell hippocampus It was confirmed that the fractions cleared the alkyl radicals and suppressed the oxidative stress.
본 발명에서, 상기 분획물은 상기 단백질 가수분해물을 서로 다른 크기의 한외여과막을 이용하여 분자량 별로 분리하여 얻은 분획물이다. 상기 한외여과는 막 여과법에 있어서 한외여과막을 사용하여 분할 원리에 기초해 분자크기로 분리하는 여과방법이다. 구체적으로, 상기 분획물은 5 kDa과 10 kDa의 크기의 한외여과막을 이용하여 한외여과를 수행하여 수득될 수 있다. In the present invention, the fraction is a fraction obtained by separating the protein hydrolysates by molecular weight using an ultrafiltration membrane of different sizes. The ultrafiltration is a filtration method for separating into a molecular size based on the principle of division using an ultrafiltration membrane in membrane filtration. Specifically, the fractions can be obtained by performing ultrafiltration using an ultrafiltration membrane having a size of 5 kDa and 10 kDa.
본 발명의 일실시예에서는 상기 빅벨리해마의 물 추출물의 펩신 가수분해물을 5 kDa과 10 kDa의 크기의 한외여과막을 이용하여 분자량량 별로 분획물을 나누었으며, 구체적으로, 가수분해물을 ≥10 kDa(10 kDa 이상), 5-10 내지 kDa, ≤5 kDa(5 kDa 이하)의 분획물로 분배하였다(도 3A). 도 3A의 SDS-PAGE 전기영동 결과를 통해 해마 펩신 가수분해물과 가수분해물의 각 분자량별 분획물의 분자량 크기에 따라 다양한 밴드 패턴이 나타나 분자량별로 분획물이 잘 분리된 것을 확인하였다. In one embodiment of the present invention, the pepsin hydrolyzate of the water extract of the Bigbell hippocampus was fractionated by molecular weight by using an ultrafiltration membrane having a size of 5 kDa and 10 kDa. Specifically, the hydrolyzate was hydrolyzed with a hydrolyzate of? 10 kDa 10 kDa or more), 5-10 to kDa, and 5 kDa (5 kDa or less) fractions (Fig. 3A). As a result of SDS-PAGE electrophoresis of FIG. 3A, various band patterns were observed depending on the molecular weight of the hydrolyzate of hippopotate and hydrolyzate of each molecular weight, and thus it was confirmed that the fractions were well separated by molecular weight.
이에 따라, 본 발명에서, 상기 분획물은 10 kDa 이상, 5-10 kDa, 5 kDa 이하의 단백질을 포함할 수 있으나, 구체적으로 5 kDa 이하의 단백질을 포함할 수 있다. Accordingly, in the present invention, the fractions may include proteins of 10 kDa or more, 5-10 kDa, 5 kDa or less, and specifically, proteins of 5 kDa or less.
본 발명의 일실시예에서는 상기 분획물들 중 5 kDa 이하의 분획물이 가장 강한 alkyl radical 소거능을 확인할 수 있었고 IC50 값은 0.15±0.01 mg/mL으로 DP의 각 분자량별 분획물 중 저분자 분획물에 뛰어난 alkyl radical 소거능이 확인되어 빅벨리해마의 물 추출물의 펩신 가수분해물(DP, Distilled water-pepsin hydrolysate)의 저분자 펩타이드가 활성을 나타내는 것을 확인하였다. In one embodiment of the present invention, the fractions below 5 kDa were able to confirm the strongest alkyl radical scavenging activity and the IC 50 value was 0.15 ± 0.01 mg / mL, and excellent alkyl radical (DP, Distilled water-pepsin hydrolyzate) of the water extract of Bigbellie hippocampus was active.
이에 따라, 본 발명의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 항산화 목적으로 식품 조성물에 첨가할 수 있다.Accordingly, the Hippocampus of the present invention abdominalis ) or fractions thereof may be added to the food composition for antioxidant purposes.
본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐 또는 액제 등의 형태를 포함할 수 있으며, 본 발명의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 첨가할 수 있는 식품의 종류에는 별다른 제한이 없으며, 예를 들어 각종 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있다.Food compositions of the present invention may include a form of pills, powders, granules, infiltrant, tablets, capsules or liquid preparation, such as Big Valley hippocampus of the present invention (Hippocampus abdominalis , or fractions thereof can be added. There are no particular restrictions on the kinds of foods to which the protein hydrolyzate or fractions thereof can be added. For example, various beverages, gums, tea, vitamin complexes, and health supplement foods.
상기 식품 조성물에는 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물 이외에도 다른 성분을 추가할 수 있으며, 그 종류는 특별히 제한되지 않는다. 예를 들어, 통상의 식품과 같이 여러 가지 생약 추출물, 식품학적으로 허용가능한 식품보조첨가제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있으며, 이에 제한되지 않는다.The food composition has a big belly hippocampus (Hippocampus abdominalis , or fractions thereof, and the kind of the protein hydrolyzate is not particularly limited. For example, it may contain various herbal medicine extracts, food-acceptable food-aid additives or natural carbohydrates such as ordinary food, but is not limited thereto.
본 발명에서 용어, "식품보조첨가제"란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.The term "food supplementary additive " in the present invention means a component which can be added to foods in a supplementary manner, and is appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food-aid additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. However, the types of the food auxiliary additives of the present invention are not limited by the above examples.
상기 천연 탄수화물의 예는 포도당, 과당 등의 단당류; 말토스, 수크로스 등의 이당류; 및 덱스트린, 시클로덱스트린 등의 다당류와, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이 있으며, 상기한 것 이외의 향미제로서 천연 향미제(타우마틴 등), 스테비아 추출물(레바우디오시드 Ａ, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavorings (such as tau mart), stevia extracts (rebaudioside A, Etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
본 발명의 식품 조성물에는 건강기능성 식품이 포함될 수 있다. 본 발명에서 사용된 용어 "건강기능성 식품"이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 기능성이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능성 식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The food composition of the present invention may include a health functional food. The term " health functional food " as used in the present invention refers to foods prepared and processed in the form of tablets, capsules, powders, granules, liquids, and rings using raw materials and ingredients having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be manufactured by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. Also, unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is used for a long time by using food as a raw material, and it is excellent in portability.
유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물은 원료 조성물 중 1 내지 50 중량%, 바람직하게는 5 내지 10 중량%의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로도 사용될 수 있다.The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the protein hydrolyzate or fraction thereof of the Hippocampus abdominalis of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight, in the raw composition, , But is not limited thereto. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능성 식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.
본 발명은 다른 하나의 양태로, 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 항산화용 화장료 조성물을 제공한다.The invention in another aspect, the big Valley hippocampus (Hippocampus abdominalis ) or a fraction thereof as an active ingredient.
본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있다.Ingredients included in the cosmetic composition of the present invention may include ingredients conventionally used in cosmetic compositions in addition to the protein hydrolyzate of Hippocampus abdominalis or its fraction as an active ingredient.
본 발명에서 상기 가수분해물은 빅벨리해마의 펩신(pepsin) 가수분해물, 트립신(trysin) 가수분해물, 뉴트라제(neutrase) 가수분해물, 프로타멕스(protamex) 가수분해물, 알칼레이즈(alcalase) 가수분해물, 코지자임(kojizyme) 가수분해물, 플라보자임(flavourzyme) 가수분해물, 빅벨리해마의 물 추출물의 펩 가수분해물, 및 빅벨리해마의 물 추출물의 트립신 가수분해물로 이루어진 군에서 선택되는 1종일 수 있다. In the present invention, the hydrolyzate is preferably selected from the group consisting of pepsin hydrolyzate, trysin hydrolyzate, neutrase hydrolyzate, protamex hydrolyzate, alcalase hydrolyzate, A kojizyme hydrolyzate, a flavorzyme hydrolyzate, a peptidolytic hydrolyzate of a water extract of a Bigbell hippocampus, and a trypsin hydrolyzate of a water extract of a Bigbell hippocampus .
상기 분획물은 5 kDa과 10 kDa의 크기의 한외여과막을 이용하여 한외여과를 수행하여 수득할 수 있으며, 5 kDa 이하의 단백질을 포함할 수 있다. The fractions may be obtained by ultrafiltration using ultrafiltration membranes of 5 kDa and 10 kDa, and may contain proteins of 5 kDa or less.
본 발명에서, 상기 빅벨리해마(Hippocampus abdominalis), 가수분해물, 분획물, 항산화에 대한 설명은 전술한 바와 같다. In the present invention, the big Valley hippocampus (Hippocampus abdominalis , hydrolysates, fractions, and antioxidants are described above.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으며, 이에 제한되지 않는다. 구체적으로는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powdered foundation, emulsion foundation, wax foundation and spray, and the like. Specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.
본 발명은 다른 하나의 양태로, 상기 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 유효성분으로 포함하는, 피부노화 방지용 화장료 조성물을 제공한다.In another aspect of the present invention, the above-mentioned Hippocampus abdominalis ) or a fraction thereof as an active ingredient. The present invention also provides a cosmetic composition for preventing skin aging.
본 발명에서, 상기 빅벨리해마(Hippocampus abdominalis), 가수분해물, 분획물, 화장료 조성물에 대한 설명은 전술한 바와 같다. In the present invention, the big Valley hippocampus (Hippocampus abdominalis , hydrolysates, fractions, and cosmetic compositions are as described above.
생체 내에서 안정한 상태로 존재하던 산소가 화학약품, 공해물질, 광화학 반응과 같은 환경적 및 생화학적 요인 등에 의해 수퍼옥사이드 음이온 라디칼 (superoxide anion radical, O2-), 히드록실 라디칼 (hydroxyl radical, OH-), 과산화수소 (hydrogen peroxide, H2O2), 일중항산소 (singlet oxygen, 1O2) 등과 같이 반응성이 큰 활성산소 (reactive oxygen species: ROS)로 전환되면 인체의 세포구성 성분인 단백질, 지질 및 DNA 등에 대하여 비선택적, 비가역적인 파괴를 유도하여 노화를 유발하게 된다. Oxygen, which is present in a stable state in vivo, is converted to superoxide anion radical (O2 - ), hydroxyl radical (OH - ), and hydroxyl radical by the environmental and biochemical factors such as chemicals, pollutants, ), Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2) Non-selective and irreversible destruction of DNA and the like, leading to aging.
본 발명에서, 상기 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물은 산화적 스트레스에 의해 유발된 세포생존력을 증가시키고, 활성산소(ROS)의 생성을 저해하는 바, 피부 노화를 억제하거나 방지할 수 있다. In the present invention, the big Valley hippocampus (Hippocampus Abdominalis protein hydrolysates or fractions thereof increase cell viability induced by oxidative stress and inhibit the production of reactive oxygen species (ROS), thereby inhibiting or preventing skin aging.
본 발명은 다른 하나의 양태로서, 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물을 처리하는 단계를 포함하는, 인간을 제외한 개체에서 산화적 스트레스를 억제하는 방법을 제공한다.The present invention relates to a another aspect, the big Valley hippocampus (Hippocampus abdominalis , or fractions thereof, in a subject other than human. The present invention also provides a method for inhibiting oxidative stress in a subject other than human.
본 발명에서, 상기 빅벨리해마(Hippocampus abdominalis), 가수분해물, 분획물에 대한 설명은 전술한 바와 같다.In the present invention, the big Valley hippocampus (Hippocampus abdominalis ), hydrolyzate, and fractions are as described above.
본 발명에서 용어 "개체"란 산화적 스트레스가 발생하였거나 산화적 스트레스에 의해 유발되는 노화 등을 비롯한 다양한 질환이 발생한 인간을 포함한 모든 동물을 의미한다. 소, 돼지, 양, 닭, 개, 인간 등을 포함하는 포유동물, 조류 등을 포함한다.The term "individual" in the present invention refers to all animals including humans suffering from various diseases including oxidative stress or aging caused by oxidative stress. Cattle, sheep, pigs, sheep, chickens, dogs, humans, and the like.
본 발명의 빅벨리해마(Hippocampus abdominalis)의 단백질 가수분해물 또는 이의 분획물은 알킬 라디칼 소거활성을 나타내고, 산화적 스트레스에 의한 세포 보호효과를 나타내고, ROS 생성을 억제하는 바, 항산화용 식품 조성물, 화장료 조성물 등에 유용하게 사용될 수 있다.The Hippocampus < RTI ID = 0.0 > abdominalis or fractions thereof exhibit an alkyl radical scavenging activity, exhibit a cytoprotective effect by oxidative stress, inhibit ROS production, and can be usefully used in food compositions for antioxidation, cosmetic compositions and the like.
도 1은, 빅벨리해마의 건조에 따른 사이즈 변화를 나타낸다.
도 2(A)는 vero cells의 DP 처리에 따른 세포 독성을 나타낸 것이다. 세포는 DP 50, 100 및 200 μg/mL을 처리하였다. 도 2(B)는 AAPH-유도된 산화적 손상에 대한 빅벨리해마의 물 추출물의 펩신 가수분해물(DP)의 보호효과를 나타낸다. AAPH 처리를 통해, 산화적 스트레스를 유발하고, MTT 분석을 실시하였다. 도 2(C)는 AAPH-유도된 산화된 세포에서 DP의 세포내 ROS 생성 억제 효과를 나타낸 것이다. 실험은 세번 수행되었으며, 데이터는 평균 ± SD로 나타내었다. a-cP<0.05는 각 그룹별 유의적인 차이가 있음을 의미한다.
도 3(A)는 DP의 분자량 별 분획물의 SDS-PAGE 패턴을 나타낸다. 도 3(B)는 빅벨리해마의 물 추출물의 펩신 가수분해물(DP)을 이용하여 제조된 분자량 별 분획물의 알킬 라디칼 소거활성을 나타낸다. 샘플들 (0.125 mg/mL, 및 0.25 mg/mL)의 알킬 라디칼 소거활성은 ESR spectrometry를 이용하여 측정하였다. a-cP<0.05는 각 그룹별 유의적인 차이가 있음을 의미한다.
도 4(A)는 vero cells의 생존력에 대한 DP의 분자량 별 분획물의 세포독성 효과를 나타낸다. 세포는 빅벨리해마의 물 추출물의 펩신 가수분해물(DP)의 분자량 별 분획물 (50, 100 μg/mL)로 처리하였다. 24시간 배양 후에, 세포 생존력을 MTT 분석을 이용하여 측정하였다.
도 4(B)는 AAPH-유도된 산화적 손상(산화적 스트레스)에 대해 정상 세포에서 DP의 분자량 별 분획물의 보호 효과를 나타낸 것이다. 세포 생존력은 MTT 분석을 이용하여 측정하였다.
도 4(C)는 DCFH-DA assay에 의한 세포내 ROS를 측정한 것이다. 그래프는 세포내 ROS 소거 활성을 나타낸다. 각 실험은 세번 수행되었으며, 데이터는 평균 ± SD를 나타낸다. a-dP<0.05는 각 그룹별 유의적인 차이가 있음을 의미한다.
도 5는 DP 5 kDa 이하 분획물의 AAPH에 의해 산화된 Zebrafish embryo의 보호효과를 나타낸다. Fig. 1 shows a change in size due to drying of the Big Belly hippocampus.
Fig. 2 (A) shows cytotoxicity due to DP treatment of vero cells. Cells were treated with
Figure 3 (A) shows the SDS-PAGE pattern of the molecular weight fractions of DP. FIG. 3 (B) shows the alkyl radical scavenging activity of the molecular weight fractions prepared using the pepsin hydrolyzate (DP) of the water extract of Bigbellie hippocampus. The alkyl radical scavenging activity of the samples (0.125 mg / mL, and 0.25 mg / mL) was measured using ESR spectrometry. ac P <0.05 means that there is a significant difference for each group.
Figure 4 (A) shows the cytotoxic effect of the molecular weight fraction of DP on the viability of vero cells. Cells were treated with molecular weight fractions (50, 100 μg / mL) of pepsin hydrolyzate (DP) from the water extract of the Big Belly hippocampus. After 24 hours of culture, cell viability was measured using MTT assay.
Figure 4 (B) shows the protective effect of molecular weight fractions of DP in normal cells against AAPH-induced oxidative damage (oxidative stress). Cell viability was measured using MTT assay.
Fig. 4 (C) shows the measurement of intracellular ROS by the DCFH-DA assay. The graph shows the intracellular ROS scavenging activity. Each experiment was performed in triplicate, and the data represent the mean ± SD. ad P <0.05 means that there is a significant difference for each group.
Figure 5 shows the protective effect of the Zebrafish embryo oxidized by AAPH in the
이하, 본 발명을 실시예에 의해 보다 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
<A: 실험 재료 및 방법><A: Materials and Methods>
실시예Example 1: One: 빅벨리해마의Big belly hippocampus 준비 Ready
본 실험에서 사용한 해마는 제주에 위치한 한국해수관상어종묘센터(CCORA)에서 약 20 cm 크기의 빅벨리해마(Hippocampus abdominalis)를 제공 받았고, 동결건조 된 해마는 분쇄 후 사용되었다. 상기와 같은 빅벨리해마를 도 1에 나타내었다. The hippocampus used in this experiment is a 20 cm sized Bigbell hippocampus ( Hippocampus ) in the Korean Sea Water Seedling Center (CCORA) abdominalis ), and lyophilized hippocampus was used after grinding. The above-mentioned Big Belly hippocampus is shown in Fig.
실시예Example 2: 단백질 가수분해 효소의 준비 2: Preparation of protein hydrolyzing enzyme
단백질 효소가수분해에 사용된 소화효소 2종(trypsin, pepsin)은 Sigma Chemical Co. (USA)에서 구입하였으며 식품용 가수분해 효소 5종(neutrase, protamex, alcalase, kojizyme, flavourzyme)은 Novozyme Co. (Denmark)에서 구입하였다. 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH), α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorofluorescein diacetate (DCF-DA), sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA)는 Sigma-Aldrich (St. Louis, MO, USA)에서 구입하여 사용하였다. Pierce® BCA protein assay kit는 Thermo Scientific™ (Rockford, IL, USA)에서 구입하여 사용하였고 RPMI 1640 배지와 fetal bovine serum (FBS)는 Gibco/BRL (Canada)에서 구입하여 사용하였다. 이 밖에 모든 기타 시약은 분석용 특급시약을 사용하였다.Two digestive enzymes (trypsin, pepsin) used for protein hydrolysis were purchased from Sigma Chemical Co. (Neutra, protamex, alcalase, kojizyme, flavourzyme) were purchased from Novozyme Co., USA. (Denmark). 2-azobis- (2-amidinopropane) dihydrochloride (AAPH), α- (4-Pyridyl-1-oxide) -N- (SDS), dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Pierce® BCA protein assay kit was purchased from Thermo Scientific ™ (Rockford, IL, USA) and RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco / BRL (Canada). In addition, all other reagents used the special reagents for analysis.
실시예Example 3: 해마의 일반성분 분석 3: General composition analysis of hippocampus
해마 건조 시료의 일반성분은 AOAC (Helrich K. 1990. Official Methods of Analysis of AOAC Intl. 15th ed.; Association of Official Analysis Chemists, Washington, DC, USA. Chapter 11. 1-31.) 방법에 준하여 측정하였다. 수분함량은 105℃ 건조기에 24시간 유지하여 측정하였으며, 회분은 직접회화법으로 회분기를 이용하여 550℃에서 3시간 동안 회화한 뒤 측정하였다. 조단백은 Kjeldahl 법으로 측정 하였으며, 조지방은 Soxhlet 추출법을 이용하여 측정하였다.The general composition of hippocampal dry samples was determined according to the method of AOAC (Helich K. 1990. Official Methods of Analysis of AOAC Intl. 15th ed .; Association of Official Analysis Chemists, Washington, DC, USA. Respectively. Moisture content was measured by maintaining the moisture content at 105 ° C for 24 hours. The ash was measured by direct filtration method at 550 ° C for 3 hours using a shaking flask. Crude protein was measured by Kjeldahl method and crude fat was measured by Soxhlet extraction method.
실시예Example 4: 해마의 단백질 4: Protein of hippocampus 효소가수분해물의Enzyme hydrolyzate 제조 Produce
해마의 단백질 효소 가수분해물은 Ko et al. (2013)의 방법에 따라 제조되었고, 가수분해 효소 7종을 이용하여 해마의 효소적 가수분해물을 제조하여 물(distilled water, DW) 추출물과 비교하였다. 해마 동결건조물을 이용하여 효소 대 기질비를 1:100으로 하였으며, 각 효소별 최적온도 및 pH 조건에 따라 24시간 가수분해를 진행하였다. 한편 가수분해 수율을 높일 목적으로 DW 24시간 추출을 먼저 진행 한 후 pepsin과 trypsin을 각각 12시간 가수분해를 진행하여 DW-pepsin 가수분해물(빅벨리해마의 물 추출물의 펩신 가수분해물, DP)와 DW-trypsin 가수분해물(빅벨리해마의 물 추출물의 트립신 가수분해물, DT)를 추가적으로 제조하였다. 가수분해를 정지시키기 위해 100℃에서 10분간 효소의 반응을 불활성화시킨 후 pH를 7.0으로 조정해 주었다. 이 후 20분간 원심분리한 뒤 잔사를 제외한 상층액을 pore size 7 μm의 여과지를 사용하여 추출물을 여과하고 동결건조 후 분말화하여 최종적으로 7종의 단백질 효소가수분해물을 얻었다.Protein enzyme hydrolysates of hippocampus are described in Ko et al. (2013). Enzymatic hydrolysates of hippocampus were prepared using 7 kinds of hydrolytic enzymes and compared with distilled water (DW) extracts. The enzyme - to - substrate ratio was adjusted to 1: 100 by using hippocampal lyophilisate, and hydrolysis was carried out for 24 hours according to optimal temperature and pH conditions for each enzyme. In order to increase the yield of hydrolysis, DW was first extracted for 24 hours, and hydrolysis of pepsin and trypsin were performed for 12 hours, respectively. The DW-pepsin hydrolyzate (pepsin hydrolyzate of water extract of big belly hippocampus, DP) -trypsin hydrolyzate (trypsin hydrolyzate of water extract of Bigvalley hippocampus, DT) was additionally prepared. In order to stop the hydrolysis, the enzyme reaction was inactivated at 100 ° C for 10 minutes, and the pH was adjusted to 7.0. After centrifugation for 20 minutes, the supernatant, except for the residues, was filtered with a filter paper of pore size 7 μm, and lyophilized and pulverized to obtain seven kinds of protein hydrolysates.
실시예Example 5: 단백질 5: Protein 효소가수분해물의Enzyme hydrolyzate 수율 및 단백질 함량 측정 Yield and protein content measurement
각 추출조건에 따른 효소가수분해물의 수율은 단백질 효소가수분해 후 얻어진 가수분해물 일부의 무게를 100℃ 드라이오븐에서 수분을 제거한 나머지 고형분 함량을 기준으로 수율을 측정하였다. 7종의 단백질 효소 가수분해물의 총 단백질의 함량은 Pierce® BCA protein assay kit를 사용하여 측정하였고, 단백질의 정량의 표준물질은 BSA를 사용하였다.The yield of the enzyme hydrolyzate according to each extraction condition was measured by the weight of the hydrolyzate obtained after the hydrolysis of the protein enzyme, and the yield was determined based on the remaining solid content after removal of water in a 100 ° C dry oven. The total protein content of the seven protein hydrolysates was determined using the Pierce BCA protein assay kit. BSA was used as a standard for protein quantification.
실시예Example 6: Alkyl radical 6: Alkyl radical 소거능Scatters 측정 Measure
상기 가수분해물들의 alkyl radical 소거능은 Kang et al. (2013)의 방법에 따라 측정하였다. 즉, 시료 20 μL, DW 20 μL, 40 mM AAPH 20 μL, 40 mM 4-POBN 20 μL를 혼합 후 37℃에서 30분간 반응시킨 다음 capillary tube에 옮겨 전자스핀공명; Electron spin resonance (JES-FA ESR, JEOL, Tokyo, Japan)을 사용하여 signal 값을 구하였다. 대조구는 시료 대신 용매인 DW를 사용하였으며 분석조건은 다음과 같이 하였다.The alkyl radical scavenging ability of the hydrolysates was determined by Kang et al. (2013). That is, 20 μL of sample, 20 μL of DW, 20 μL of 40 mM AAPH and 20 μL of 40 mM 4-POBN were mixed and reacted at 37 ° C. for 30 minutes, and transferred to a capillary tube for electron spin resonance; Signal values were obtained using electron spin resonance (JES-FA ESR, JEOL, Tokyo, Japan). The control DW was used as the solvent instead of the sample. The analysis conditions were as follows.
Power: 1 mW, Amplitude: 1000, Modulation width: 0.2 mT,Power: 1 mW, Amplitude: 1000, Modulation width: 0.2 mT,
Sweep width: 10 mT, Sweep time: 30 s, Time constant : 0.03 sSweep width: 10 mT, Sweep time: 30 s, Time constant: 0.03 s
실시예Example 7: 세포 배양 7: Cell culture
Vero cell은 10% FBS, 1% antibiotic을 포함하는 RPMI 1640 배지를 사용하여 배양 하였다. Cell culture flask에 적정농도로 37℃, 5% CO2 조건에서 세포상태에 따라 2-3일간 계대 배양하여 실험에 사용하였다.Vero cells were cultured in RPMI 1640 medium containing 10% FBS and 1% antibiotic. Cell culture flasks were subcultured for 2-3 days at 37 ° C and 5% CO 2 , depending on cell conditions.
실시예Example 8: 8: VeroVero cells에서의 독성 평가 Evaluation of toxicity in cells
시료에 대한 세포독성을 확인하기 위해 Ko et al. (2012)의 방법을 따라 MTT assay 방법으로 측정하였다. 먼저 Vero cells(1×105 cells/mL)으로 96 wellplate에 190 μL를 분주하고, 37℃, 5% CO2 조건에서 24시간 배양하여 시료들을 농도 별로 처리하였다. 이 후 37℃ 인큐베이터에서 24시간 배양하였으며 2 mg/mL 농도의 MTT를 50 μL 처리하였다. 그리고 37℃에서 3시간 배양하고 형성된 불용성 결정을 dimethylsufoxide(DMSO)로 완전히 녹인 후에 빛을 차단시켜 12시간 보관 후에 ELISA reader를 사용하여 540 nm 흡광도에서 측정한 뒤 값을 구하였다.To confirm the cytotoxicity of the samples, Ko et al. (2012). First, 190 μL of each well was seeded in 96-well plates with Vero cells (1 × 10 5 cells / mL) and cultured at 37 ° C and 5% CO 2 for 24 hours. After incubation for 24 hours in a 37 ° C incubator, 50 μL of MTT at 2 mg / mL was treated. After incubation at 37 ° C for 3 hours, the insoluble crystals formed were completely dissolved in dimethylsufoxide (DMSO), and after light was blocked for 12 hours, the absorbance at 540 nm was measured using an ELISA reader.
실시예Example 9: 9: VeroVero cells에서의 of cells ROSROS 생성 측정 Production measurement
산화적 스트레스를 유도하였을 때 해마 가수분해물에 의한 ROS 소거능을 확인하기 위해 Ko et al. (2012)의 방법을 따라 AAPH 자극을 주어 ROS의 변화를 측정하였다. 먼저 1×105 세포농도의 vero cells을 만들어 96 wellplate에 180 μL를 분주하였고, 37℃ 인큐베이터에서 24시간 배양하여 시료들을 농도별로 처리하였다. 이 후 37℃ 인큐베이터에서 30분 배양하여 4 mM AAPH 10 μL를 처리하였고, 37℃ 인큐베이터에서 30분간 반응시켜 0.05% DCF-DA 에탄올용액을 10 μL 처리하고 빛을 차단시킨 후 37℃ 인큐베이터에서 10분간 반응시킨 후 ELISA reader를 사용하여 측정하였다.In order to confirm ROS scavenging ability by hydrolysates of hippocampus when inducing oxidative stress, Ko et al. (2012), AAPH stimulation was applied to measure changes in ROS. First, vero cells were prepared at a concentration of 1 × 10 5 cells, and 180 μL of the cells were placed in a 96-well plate. The cells were cultured in a 37 ° C. incubator for 24 hours. After incubation for 30 min in a 37 ° C incubator, the cells were treated with 10 μL of 4 mM AAPH for 30 min in a 37 ° C incubator, treated with 10 μL of 0.05% DCF-DA ethanol solution and incubated for 10 min in a 37 ° C incubator The reaction was measured using an ELISA reader.
실시예Example 10: 10: VeroVero cells에서 from cells AAPH에On AAPH 의한 by 산화적Oxidative 손상에 대한 보호효과 측정 Measuring protection against damage
AAPH로 세포에 산화적 스트레스를 유도하였을 때의 해마 가수분해물의 보호효과에 대한 활성을 확인하기 위해 Ko et al.(2012)의 방법을 따라 AAPH 자극을 주어 시료처리에 대한 세포 생존력을 측정하였다. 먼저 1×105 세포농도의 vero cells을 만들어 96 well plate에 180 μL를 seeding하였고, 37℃ 인큐베이터에서 24시간 배양하였으며 시료를 농도 별로 처리하였다. 이 후 37℃ 인큐베이터에서 1시간 배양하였으며 4 mM AAPH 10 μL를 처리하였다. 그 다음 37℃ 인큐베이터에서 24시간 배양하였으며 2 mg/mL 농도의 MTT를 50 μL 처리하였다. 이후 37℃ 인큐베이터에서 3시간 반응시키고, 37℃에서 3시간 배양한 후 형성된 불용성 결정을 DMSO로 완전히 녹여 빛을 차단시키고 12시간 보관 후에 ELISA reader를 사용하여 540 nm 흡광도에서 측정한 뒤 값을 구하였다.To determine the protective effect of hippocampal hydrolysates upon induction of oxidative stress in cells with AAPH, AAPH stimulation was performed according to the method of Ko et al. (2012) to measure the cell viability for the sample treatment. First, vero cells were prepared at a concentration of 1 × 10 5 cells and seeded in a 96-well plate at 180 μL. The cells were cultured in a 37 ° C incubator for 24 hours. After incubation for 1 hour in a 37 ° C incubator, 10 μL of 4 mM AAPH was added. Then, the cells were cultured in a 37 ° C incubator for 24 hours and treated with 50 μL of MTT at a concentration of 2 mg / mL. After incubation at 37 ° C for 3 hours, the cells were incubated at 37 ° C for 3 hours. The insoluble crystals formed were completely dissolved in DMSO, blocked for 12 hours, and then measured for absorbance at 540 nm using an ELISA reader .
대조구는 시료처리 없이 산화적 스트레스만 유도시킨 실험군과 산화적 스트레스를 유도시키지 않은 실험군으로 구분하였다.The control group was divided into two groups, one group inducing oxidative stress without sample treatment and the other group not inducing oxidative stress.
실시예Example 11: 11: ZebrafishZebrafish embryo에서의 in embryo AAPHAAPH 산화 보호효과 측정 Measurement of oxidation protection effect
Zebrafish (Danio rerio)는 인간과 유전적 상동성이 유사한 척추동물로서 초기발생단계에서 산화적 스트레스를 유도하였을 때 시료처리에 의한 항산화 활성으로부터의 보호효과를 확인하고자 시간경과에 따른 생존율 변화를 측정하였다(Kim et al., 2014). 7-9 hpf (hours post-fertilization)에 zebrafish embryo를 12 well plates에 각각 15마리씩 넣어준 뒤 embryo medium 1.8 mL를 맞추고 농도별(25, 50, 100, 200 μg/mL) 시료를 100 μL씩 처리하였다. 1시간 노출시킨 후 15 mM 농도의 AAPH를 100 μL 처리하였고 이후 생존율 변화를 관찰하였다. 대조구는 시료처리 없이 산화적 스트레스만 유발시킨 실험군과 산화적 스트레스를 유도시키지 않은 실험군으로 하였다.Zebrafish (Danio rerio) is a vertebrate animal with similar genetic homology to humans. To determine the protective effect of antioxidant activity on the oxidative stress induced at the early development stage, we measured the change in survival rate over time (Kim et al., 2014). Add zebrafish embryos to each well of 7-9 hpf (hours post-fertilization) in 15 wells of 12 well plates, and then add 1.8 mL of embryo medium and treat with 100 μL of each concentration (25, 50, 100 or 200 μg / mL) Respectively. After 1 hour of exposure, 100 μL of 15 mM AAPH was treated and then the survival rate was observed. The control group was an experimental group that did not induce oxidative stress but a sample group that did not induce oxidative stress without sample treatment.
실시예Example 12: 데이터 분석 12: Data Analysis
실험결과의 통계처리는 각각의 시료에 대한 평균±표준편차로 나타내었다. SPSS 프로그램(SPSS Inc. Ver. 12.0)을 사용하여 Oneway ANOVA-test를 실시하여 조사 항목들 간의 유의성 검증은 Turkey’s multiple range test로 P<0.05 수준에서 실시하였다.Statistical analysis of the experimental results is shown as the mean ± standard deviation for each sample. Oneway ANOVA-test was performed using SPSS program (SPSS Inc. Ver. 12.0) and the significance test was conducted at the level of P <0.05 with Turkey's multiple range test.
<B: <B: 실험예Experimental Example > >
실험예Experimental Example 1: 해마의 일반성분 분석 1: General compositional analysis of hippocampus
해마의 일반성분 분석 결과는 표 1에 나타냈으며, 수분, 회분, 조단백질, 조지방을 건조무게를 기준으로 확인하였다. 해마의 조단백질의 함량으로 58.47%을 확인하여 해마의 대부분이 조단백질로 이루어짐을 확인하였다.The results of the analysis of general components of hippocampus are shown in Table 1, and moisture, ash, crude protein and crude fat were determined on the basis of dry weight. The crude protein content of hippocampus was 58.47%, confirming that most of the hippocampus was composed of crude protein.
실험예Experimental Example 2: 해마의 단백질 효소 2: protein enzyme of hippocampus 가수분해물들의Hydrolyzate 수율 및 단백질 함량 Yield and protein content
단백질 가수분해 효소는 아미노산 또는 펩타이드 혼합물을 만드는 효소이며, 일반적으로 단백질 가수분해 효소를 이용해 시료를 가수분해 하였을 때 효소 양과 가수분해 시간에 비례하여 DW 추출물에 비해 높은 수율과 단백질 함량을 나타냄을 확인할 수 있다(Tsai et al., 2006). 물(DW)로 24시간 추출한 추출물의 수율은 1 g의 건조해마로부터 374.5±3.54 mg였으나, 7종의 단백질 효소 가수분해를 이용하여 효소 가수분해물을 제조한 경우, 증가된 수율을 확인할 수 있었다. 빅벨리해마의 물 추출물의 펩신 가수분해물(DP, Distilled water-pepsin hydrolysate)의 경우 다른 단백질 효소 가수분해물에 비해 가장 높은 786.67±15.28 mg의 수율을 확인하였고, 빅벨리해마의 물 추출물의 트립신 가수분해물(DT, Distilled water-trypsin hydrolysate)의 경우 이보다 낮은 520.00±25.17mg의 수율을 확인할 수 있었다(표 2). 식품용 가수분해 효소 5종을 이용한 효소 가수분해물에서는 전반적으로 높은 수율을 확인할 수 있었다. 이 중 DP와 대등하게 높은 수율을 보인 효소 가수분해물은 neutrase로 가수분해한 효소 가수분해물과 alcalase로 가수분해한 효소 가수분해물로 확인 되었으며, 각각 783.33±20.0 mg, 780.00±10.0 mg의 수율을 확인할 수 있었다(표 2). Proteolytic enzymes are enzymes that make amino acid or peptide mixtures. In general, hydrolysis of proteins using proteolytic enzymes shows higher yields and protein contents than DW extracts in proportion to the amount of enzymes and hydrolysis time (Tsai et al., 2006). The yield of extracts extracted from water (DW) for 24 hours was 374.5 ± 3.54 mg from 1 g dried hippocampus. However, the yields of hydrolysates of enzyme hydrolysates were increased by 7 kinds of protein hydrolysis. The yield of 786.67 ± 15.28 mg of Pepsin hydrolyzate (DP, distilled water-pepsin hydrolyzate) of the Bigbell hippocampus was higher than that of other protein hydrolysates, and the trypsin hydrolyzate (DT, Distilled water-trypsin hydrolysate), the yield of 520.00 ± 25.17 mg was lower (Table 2). The enzyme hydrolysates using five kinds of hydrolytic enzymes for food were found to have a high overall yield. Among them, enzyme hydrolysates showing high yields comparable to those of DP were identified as enzymes hydrolyzed by neutrase and hydrolyzed by alcalase, and the yields of 783.33 ± 20.0 mg and 780.00 ± 10.0 mg were confirmed, respectively (Table 2).
또한 동결건조된 해마 조시료 1 g으로부터 얻을 수 있는 총 단백질은 DW로 24시간을 추출한 추출물이 93.38±3.04 mg으로 확인되었으며, 그 외 모든 효소 가수분해물들에서는 DW로 24시간을 추출한 추출물 대비 약 2배 가량 높은 총 단백질을 얻을 수 있는 것으로 확인되었다(표 2). 소화효소를 이용하여 가수분해한 효소 가수분해물에서는 DP(Distilled water-pepsin hydrolysate) 효소 가수분해물이 221.21±7.02 mg의 총 단백질을 얻을 수 있는 것으로 확인되었고, 식품용 가수분해 효소 5종을 이용한 효소 가수분해물에서는 전반적으로 높은 총 단백질을 얻을 수 있는 것으로 확인되었다(표 2). 이와 같은 결과는 가수분해물의 수율이 높을수록 단백질 함량도 높은 것이 확인되어 DW 추출물에 비해 수율과 단백질 함량이 높았던 DP와 DT, 식품용 가수분해 효소 5종(Neutrase, Protamex, Alcalase, Kojizyme, Flavourzyme)에서 가수분해가 많이 진행되었고 그에 따라 많은 양의 저분자 펩타이드가 함유되어 있을 것으로 판단되었다.The total protein obtained from 1 g of the lyophilized hippocampus sample was found to be 93.38 ± 3.04 mg in the extract of DW for 24 hours. In the other enzymatic hydrolysates, about 2 (Table 2). ≪ tb > < TABLE > The digested enzyme hydrolyzate hydrolyzed DP (distilled water-pepsin hydrolyzate) showed 221.21 ± 7.02 mg of total protein, while the enzyme hydrolyzate of 5 It was confirmed that the degradation product obtained a high total protein (Table 2). The results showed that the higher the yield of hydrolyzate, the higher the protein content. DP and DT, which were higher in yield and protein content than the DW extract, and the five hydrolytic enzymes (Neutrase, Protamex, Alcalase, Kojizyme, Flavourzyme) It was judged that a large amount of low molecular weight peptide was contained.
실험예Experimental Example 3: 해마 단백질 효소 3: hippocampal protein enzyme 가수분해물들의Hydrolyzate Alkyl radical Alkyl radical 소거능Scatters
Alkyl radical 소거활성에 대한 해마 단백질 효소 가수분해물들의 효과를 평가하였다. 이 중 우수한 활성을 가진 추출물의 IC50값(mg/mL)을 산출한 결과 DP가 0.15±0.00 mg/mL, DT가 0.15±0.00 mg/mL, Neutrase로 가수분해한 효소 가수분해물이 0.18±0.16 mg/mL으로 확인되었으며, DP와 DT가 전체 가수분해물 중 가장 우수한 alkyl radical 소거활성을 나타내는 것을 확인하였다(표 2). 이 결과는 앞에 수율과 단백질 함량의 결과와도 관련이 있다. 수율과 단백질 함량이 높았던 DP가 활성 저분자 펩타이드의 양이 많이 함유한다는 것을 암시하며, 따라서 수율과 단백질 함량이 높고, alkyl radical 소거능이 우수한 DP를 이용하여 이후 실험에 사용하였다.The effects of hippocampal enzyme hydrolysates on the alkaline radical scavenging activity were evaluated. The IC50 value (mg / mL) of the extract with excellent activity was calculated to be 0.15 ± 0.00 mg / mL of DP, 0.15 ± 0.00 mg / mL of DT, 0.18 ± 0.16 mg of enzyme hydrolyzate hydrolyzed with Neutrase / mL, and DP and DT showed the best alkyl radical scavenging activity among all hydrolysates (Table 2). These results are also related to the yield and protein content results. It was suggested that DP, which had high yield and protein content, contained a large amount of active low molecular peptide, and was used in the subsequent experiments using DP having high yield and protein content and excellent alkyl radical scavenging ability.
실험예Experimental Example 4: DP의 세포독성 및 4: cytotoxicity of DP and AAPHAAPH 산화 보호효과 Oxidation protection effect
MTT assay 법은 MTT 시약이 세포 내로 흡수된 후 미토콘드리아의 숙신산탄수소효소(succinate dehydrogenase)에 의해 보라색의 불용성 포르마잔(formazan)을 형성하는데 이 물질의 세포 내 축적은 미토콘드리아의 활성, 넓게는 세포의 활성을 의미하므로 세포의 생장율 및 살아있는 세포수의 측정에 사용되는 대표적인 방법이다(Yang and Boo, 2013). DP의 세포 독성은 도 2A에 나타내었다. 시료를 50 μg/mL, 100 μg/mL, 200 μg/mL의 농도로 각각 처리하여 독성이 없음을 확인하였고, 독성이 없는 농도를 이용해 4 mM AAPH로 산화적 스트레스를 유도하여 세포생존률을 확인하였다. DP는 농도의존적으로 AAPH 산화에 의한 세포 보호효과를 확인할 수 있었으며 (도 2B), AAPH로 세포에 자극을 주고 세포 내 AAPH 산화에 의한 ROS 생성 정도를 측정한 결과, 농도의존적으로 AAPH에 의한 세포의 ROS 생성을 줄여 주는 것을 확인하였다(도 2C).In MTT assay, the MTT reagent is absorbed into cells and then formed into a purple insoluble formazan by the succinate dehydrogenase of mitochondria. The intracellular accumulation of this substance is mitochondrial activity, (Yang and Boo, 2013). This is a representative method used to measure the growth rate of cells and the number of living cells. The cytotoxicity of DP is shown in Figure 2A. The samples were treated with 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. No toxicity was observed, and the cell viability was determined by inducing oxidative stress with 4 mM AAPH . DP was able to confirm the cell protection effect by AAPH oxidation in a concentration dependent manner (FIG. 2B). As a result of stimulating the cells with AAPH and measuring the degree of ROS production by intracellular AAPH oxidation, It was confirmed that ROS generation was reduced (FIG. 2C).
이와 같은 결과는 DP에 의해 다른 효소의 작용이나 생리적인 변화없이 단분자적으로 변성되는 AAPH 산화로 인한 세포 보호효과와 ROS 생성 감소로 항산화 활성이 있음을 시사한다. 단분자적으로 변성된 AAPH는 두 carbon radical과 질소로 분해되는데, 이 carbon radical은 다른 carbon radical과 다시 결합하여 안정적인 구조를 취하기도 하지만 대부분은 산소와 반응하여 peroxy radical을 생성하게 되며, 이 peroxy radical은 세포의 손상을 유발하고, 생체 내 단백질, 지질 등에 산화적 스트레스를 주는 요인으로 작용하게 된다(Niki, 1990).These results suggest that DP has antioxidant activity due to decrease of ROS production and cytoprotective effect due to AAPH oxidation which is monomolecularly denatured without any other enzyme action or physiological change. The monomolecularly modified AAPH is decomposed into two carbon radicals and nitrogen, which reacts with other carbon radicals to form a stable structure. However, most of them react with oxygen to form peroxy radicals, and this peroxy radical (Niki, 1990), which induces cell damage and causes oxidative stress in vivo proteins and lipids.
실험예Experimental Example 5: DP 및 5: DP and 분자량 별By molecular weight 분획물의Fraction SDS PAGE를 이용한 분석 및 각 분자량 분획물의 Alkyl radical 소거 활성 Analysis by SDS PAGE and Alkyl radical scavenging activity of each molecular weight fraction
DP의 각 분자량 별로 분획물을 나누는 것은 각각 다른 크기의 한외여과막을 사용하여 나누었다. DP를 5 kDa과 10 kDa의 크기 막으로 가수분해물을 ≥10 kDa(10 kDa 이상), 5-10 내지 kDa, ≤5 kDa(5 kDa 이하)의 각 분획물로 분배하였고, 도 3A의 SDS-PAGE 전기영동 결과를 통해 해마 펩신 가수분해물과 가수분해물의 각 분자량별 분획물의 분자량 크기에 따라 다양한 밴드 패턴이 나타나 분자량별로 분획물이 잘 분리된 것을 확인하였다. The fractions of each molecular weight of DP were divided using different sizes of ultrafiltration membranes. DP was divided into fractions of ≥10 kDa (10 kDa or more), 5-10 to kDa, and ≤5 kDa (5 kDa or less) with hydrolysates of 5 kDa and 10 kDa, respectively, and SDS-PAGE From the results of electrophoresis, various band patterns were observed according to the molecular weight of each molecular weight fraction of hippopotamus hydrolyzate and hydrolyzate, and it was confirmed that the fractions were well separated by molecular weight.
각 분획물들 중, ≤5 kDa(5 kDa 이하) 분획물이 가장 강한 alkyl radical 소거능을 확인할 수 있었고 IC50 값은 0.15±0.01 mg/mL으로 확인되었다(도 3B). 이와 같은 결과는 DP의 각 분자량별 분획물 중 저분자 분획물에 뛰어난 alkyl radical 소거능이 확인되어 DP의 저분자 펩타이드가 활성을 나타냄을 제시한다. Among the fractions, fractions of ≤ 5 kDa (5 kDa or less) were able to confirm the strongest alkyl radical scavenging activity and the IC50 value was 0.15 ± 0.01 mg / mL (FIG. 3B). These results suggest that low molecular weight peptides of DP are effective for the detection of excellent alkyl radical scavenging ability in low molecular weight fractions of each molecular weight fraction of DP.
실험예Experimental Example 6: DP 6: DP 분자량 별By molecular weight 분획물의Fraction 세포독성 및 Cytotoxicity and AAPH에On AAPH 의해 산화된 Oxidized by VeroVero cells의 보호효과 Protection effect of cells
DP 분자량별 분획물들의 세포 독성은 도 4A에 나타내었다. MTT assay를 통하여 세포생존율을 측정한 결과 ≥10 kDa(10 kDa 이상), 5-10 내지 kDa, ≤5 kDa(5 kDa 이하)의 각 분획물을 50 μg/mL, 100 μg/mL의 농도로 처리하였을 때, Vero cells에 대한 세포독성은 나타나지 않았다(도 4A). The cytotoxicity of DP molecular weight fractions is shown in Figure 4A. Cell viability was measured by MTT assay and the fractions of ≥10 kDa (10 kDa), 5-10 kDa, and ≤5 kDa (5 kDa and below) were treated at concentrations of 50 μg / mL and 100 μg / mL , There was no cytotoxicity against Vero cells (Fig. 4A).
4 mM AAPH의 산화적 스트레스를 유도하였을 때 각 분획물을 50 μg/mL, 100 μg/mL의 농도로 처리하여 Vero cells에 세포생존률을 측정한 결과, Vero cells의 생존률이 농도 의존적으로 증가하는 경향을 확인하였다. 펩신 가수분해물이 저분자화 됨에 따라 세포생존률이 증가하는 경향을 확인하였고, ≤5 kDa의 분획물(Fration)의 100 μg/mL 농도 처리에 의해 세포생존률이 77.17±2.12%로 증가하였다(도 4B). When oxidative stress of 4 mM AAPH was induced, the survival rate of Vero cells was increased in a concentration-dependent manner by measuring the cell viability of Vero cells treated with 50 μg / mL and 100 μg / mL of each fraction Respectively. The cell viability tended to increase with the low molecular weight of pepsin hydrolyzate, and the cell viability increased to 77.17 ± 2.12% by treatment with a fraction of ≤5 kDa at a concentration of 100 μg / mL (FIG. 4B).
4 mM AAPH로 산화적 스트레스를 유도하여 분자량별 분획물을 50 μg/mL, 100 μg/mL 농도로 처리하였을 때, Vero cells에서 Total ROS 생성이 농도의존적으로 감소하는 경향이 확인되었고, ≤5 kDa(5 kDa 이하)의 분획물의 100 μg/mL 농도 처리에 의해 Total ROS 생성이 61.01±0.49%로 감소하였다. 또한 펩신 가수분해물이 저분자화 됨에 따라 ≤5 kDa(5 kDa 이하)의 분획물이 Total ROS 생성을 가장 많이 감소시키는 것을 확인하였다(도 4C). 세포에서의 결과도 앞에 ESR(Electron spin resonance)을 이용한 alkyl radical 소거능 결과와 마찬가지로 저분자 분획물이 항산화 활성을 갖음을 확인했고, DP의 우수한 항산화 활성을 in vivo에서도 확인하기 위해 연구를 진행함에 있어 ≤5 kDa(5 kDa 이하)의 분획물을 사용하여 zebrafish embryo 실험에 사용하였다.The concentration of total ROS in Vero cells was found to decrease in a concentration-dependent manner when treated with 50 μg / mL and 100 μg / mL of molecular weight fractions by inducing oxidative stress with 4 mM AAPH and ≤5
실험예Experimental Example
7: DP 5 7:
Zebrafish 모델은 다른 동물모델들에 비해 많은 이점들을 지닌다. 예를 들면, 작은 크기이고 투명한 embryo, 낮은 비용, 쉽게 다룰 수 있는 실험 모델로써 zebrafish는 약학 관련 연구에서 사전 독성평가나 유전기능의 연구형태 등으로 사용되어 아주 인기 있는 in vivo 모델로 사용되고 있다(Kang et al., 2015; Ko et al., 2014).Zebrafish models have many advantages over other animal models. For example, zebrafish as a small size, transparent embryo, low cost, easy-to-handle experimental model has been used as a very popular in vivo model for pharmacology-related studies, such as prior toxicity assessment or study of genetic function (Kang et al., 2015; Ko et al., 2014).
70% epiboly 단계의 zebrafish embryo에 15 mM AAPH의 산화적 스트레스를 유도하여 경과시간에 따른 zebrafish embryo의 생존율을 측정한 결과, 산화적 스트레스 유도 후 3시간이 경과하여 DP를 50, 200 μg/mL 농도 처리했을 때 생존율이 농도의존적으로 증가하는 경향이 확인 되었으며, 24시간, 48시간, 72시간으로 경과함에도 이러한 경향은 유지되었다. 산화적 스트레스를 입지 않은 대조구와 산화적 스트레스를 입은 대조구를 비교하였을 때 200 μg/mL의 농도처리는 약 50%에 가까운 생존율을 유지시켜 주는 것으로 확인되었으며 이로부터 펩신 가수분해물의 ≤5 kDa (5 kDa 이하) 분획물이 AAPH로 유도된 산화적 스트레스로 인한 보호효과가 확인되었다(도 5). 이와 같은 결과는 in vivo 모델 zebrafish embryo에서 DP 저분자 분획물의 AAPH 산화 보호효과를 확인함으로써 해마 가수분해물이 동물 모델에서도 항산화 활성이 있음을 제시한다.In the zebrafish embryo at 70% epiboly stage, the oxidative stress of 15 mM AAPH was induced and the survival rate of zebrafish embryo was measured at 3, 4, The survival rate tended to increase in a concentration - dependent manner, and this tendency was maintained even after 24 hours, 48 hours, and 72 hours. When compared to the control without oxidative stress and the control with oxidative stress, it was found that the treatment of 200 μg / mL maintained the survival rate close to 50%, from which it was found that ≤5 kDa (5 kDa) fractions were protected by oxidative stress induced by AAPH (FIG. 5). These results suggest that hippocampal hydrolysates have antioxidant activity in animal models by confirming the AAPH oxidation protection effect of DP low molecular fraction in zebrafish embryo in vivo model.
요컨데, 본 발명에서는 해수관상어센터로부터 기증받은 빅벨리해마를 다양한 효소를 사용해 가수분해하여 각 가수분해물의 수율, 단백질 함량과 alkyl 라디칼 소거능 측정을 통해 우수한 효소 가수분해물을 선정하여 기능성소재 응용가능성을 검토하였다. 효소 가수분해물 중 우수 효소 가수분해물인 DP는 Vero Cells에 대한 세포독성이 없이 농도의존적으로 ROS 생성을 감소시켰고, AAPH에 대한 산화반응을 억제함으로서 항산화 효능을 가진다는 것을 확인하였다. DP를 한외여과막을 통해 각 분자량 별로 분획물을 나누고, alkyl 라디칼 소거능을 확인하였을 때, 저분자량인 ≤ 5 kDa의 분획물이 가장 우수한 라디칼 소거능을 보였고, vero cells에 대한 세포독성을 보이지 않는 농도들에서 농도의존적으로 ROS 생성과 AAPH에 대한 산화억제 효과를 보임으로 저분자량의 DP 분획물이 항산화 효능을 가진다는 것을 확인하였다. 또한, ≤ 5 kDa의 분획물은 AAPH가 감소시킨 zebrafish embryo의 생존율을 향상시켰다는 것을 확인하였다. 이 모든 결과로부터, 빅벨리해마 유래 DP 가수분해In summary, in the present invention, hydrolysis of a Bigvalley hippocampus donated from a seawater ornamental fish center was performed using various enzymes, and the hydrolyzate of each hydrolyzate, protein content and alkyl radical scavenging ability were selected, Respectively. Among the enzyme hydrolysates, DP, which is an excellent enzyme hydrolyzate, was found to have antioxidative effect by inhibiting the oxidation reaction to AAPH, and it was found that ROS production was decreased in a concentration dependent manner without cytotoxicity to Vero Cells. DP fraction was fractionated by ultrafiltration membrane and the alkyl radical scavenging ability was confirmed. The fraction with low molecular weight of ≤ 5 kDa showed the most excellent radical scavenging ability and the concentration at the concentration that does not show cytotoxicity to vero cells Dependent ROS production and oxidation inhibition to AAPH, confirming that the low molecular weight DP fraction has antioxidant activity. It was also confirmed that fractions of ≤ 5 kDa enhanced the survival rate of zebrafish embryos with reduced AAPH. From all these results, it was found that DP hydrolysis
물은 항산화 효과를 가지며, 향후 기능성 화장품 소재나 식품으로 활용하기 위한 매우 효과적인 재료가 될 수 있다고 판단된다. Water has an antioxidant effect and can be a very effective material for functional cosmetics and foods in the future.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
Claims (11)
A food composition for antioxidation comprising pepsin hydrolyzate of water extract of Hippocampus abdominalis or fraction thereof as an active ingredient.
The food composition of claim 1, wherein the composition inhibits oxidative stress.
The food composition according to claim 1, wherein the fraction is obtained by performing an ultrafiltration using an ultrafiltration membrane having a size of 5 kDa and 10 kDa.
The food composition of claim 1, wherein said fraction comprises no more than 5 kDa protein.
A cosmetic composition for antioxidation comprising pepsin hydrolyzate of water extract of Hippocampus abdominalis or fraction thereof as an active ingredient.
The cosmetic composition according to claim 6, wherein the fraction is obtained by performing an ultrafiltration using an ultrafiltration membrane having a size of 5 kDa and 10 kDa.
7. The cosmetic composition according to claim 6, wherein the fraction comprises no more than 5 kDa protein.
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