KR20120049043A - Antiinflammatory composition comprising enzymatic hydrolysates of mytilus coruscus - Google Patents
Antiinflammatory composition comprising enzymatic hydrolysates of mytilus coruscus Download PDFInfo
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- KR20120049043A KR20120049043A KR1020100110607A KR20100110607A KR20120049043A KR 20120049043 A KR20120049043 A KR 20120049043A KR 1020100110607 A KR1020100110607 A KR 1020100110607A KR 20100110607 A KR20100110607 A KR 20100110607A KR 20120049043 A KR20120049043 A KR 20120049043A
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- KR
- South Korea
- Prior art keywords
- inflammatory
- hydrolyzate
- buffer
- inflammatory composition
- mussel
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
Description
본 발명은 홍합 가수분해물을 유효성분으로 함유하는 항염증 조성물에 관한 것으로서, 더욱 상세하게는 홍합 단백질 가수분해물로부터 분리된 펩티드와 약학적으로 허용되는 담체를 포함하는 항염증 조성물 및 홍합 단백질 가수분해물의 제조방법과 상기 가수분해물로부터 펩티드를 분리 정제하는 방법 및 항염증 효능을 갖는 홍합 단백질 유래 펩티드의 1차 구조 서열에 관한 것이다.The present invention relates to an anti-inflammatory composition containing mussel hydrolyzate as an active ingredient, and more particularly, to an anti-inflammatory composition and a mussel protein hydrolyzate comprising a peptide isolated from the mussel protein hydrolyzate and a pharmaceutically acceptable carrier. The present invention relates to a method for preparing and separating and purifying a peptide from the hydrolyzate, and to a primary structural sequence of a mussel protein-derived peptide having anti-inflammatory efficacy.
해양생물은 종의 다양성과 더불어 서식환경의 특이성이라는 요인에 의하여 기존의 육상생물이 보유?생산할 수 없는 화학물질 및 생체 분자들을 함유하고 있으며, 이들의 생합성?분해에 관여하는 특이한 신종 효소와 보조인자들도 존재하고있어서 신약물질과 새로운 생리 기능성 물질의 소재로서 관심이 집중되고 있다. 더욱이 육상생물을 대상으로 활발히 진행되어 왔던 선도물질의 탐색 및 개발이 한계에 이르기 시작함에 따라 자연히 해양생물로 관심대상이 전환되었다. 미국, 일본, 유럽 등과 같은 해양 선진국들에 의해서 현재까지 밝혀진 해양생물 유래의 항암성, 항산화성, 항곰팡이성, 항균성, 항바이러스성 및 항염증성을 나타내는 약리활성 물질들이 이미 다수 보고되어 있다. 또한 해양자원으로부터 식량자원의 생산에 관한 연구의 중요성도 점점 증가하고 있다.Marine organisms contain chemicals and biomolecules that cannot be possessed or produced by existing terrestrial organisms due to species diversity and specificity of the habitat environment, and are unique new enzymes and cofactors involved in their biosynthesis and degradation. They also exist, attracting attention as a material for new drugs and new physiological functional substances. Moreover, as the exploration and development of leading materials, which have been actively conducted on land, began to reach their limits, the interests naturally turned to marine life. A large number of pharmacologically active substances have been reported that show anti-cancer, antioxidant, anti-fungal, antimicrobial, antiviral and anti-inflammatory properties from marine organisms, which have been discovered to date by marine advanced countries such as the US, Japan and Europe. The importance of research on the production of food resources from marine resources is also increasing.
일반적으로, 패류(貝類)는 삼면이 바다로 둘러싸인 우리나라의 지역특성에 의해 연안지대에 다양한 종류가 서식하여 예로부터 중요한 식품자원으로 이용되어 온 것으로서, 1990년대 이후부터 패류의 인공양식이 보편화되면서 양식 생산의 비중이 90%를 상회하게 되고 그 생산량도 매년 증가함에 따라 어가의 소득증대 및 어촌지역의 고용창출효과 측면 외에도 보다 위생적이고 풍미가 좋은 원료를 안정적으로 공급함과 동시에 패류 특유의 풍미와 영양특성을 살린 고부가가치 가공기술이 수산업 발전을 위한 주요 관심대상이 되고 있다.In general, shellfish have been used as important food resources since ancient times, due to the regional characteristics of Korea surrounded by the sea on three sides, which have been used as important food resources since the 1990s. As the share of production exceeds 90% and its output increases every year, in addition to the income increase of fish prices and the job creation effect in the fishing villages, it also provides a more hygienic and savory raw material and provides the unique flavor and nutritional characteristics of shellfish. High value-added processing technology that utilizes this technology has become a major concern for fishery development.
특히 최근에는 동식물 단백질을 각종 효소를 이용하여 가수분해 시킨 가수분해물을 분리 정제하여 이들의 생리활성 검토에 관한 연구가 진행되고 있다. 이 가수분해물들은 항산화 활성 및 혈압강하 작용이 있다는 것으로 밝혀지면서 피부노화방지 및 고혈압 등의 성인병 예방치료제로서도 이용 가능하다는 결과가 보고되고 있다. 또한, 단백질 가수분해물은 각종 가공식품이나 조미료, 샴푸, 화장품 등 기타 여러 분야에서 필수적으로 이용되고 있다.In particular, recently, studies have been conducted to examine their physiological activities by separating and purifying hydrolysates obtained by hydrolyzing animal and plant proteins using various enzymes. These hydrolysates have been found to have antioxidant and blood pressure lowering effects, and have been reported to be used as anti-aging agents for skin aging and hypertension. In addition, protein hydrolysates are used in a variety of other fields such as processed foods, seasonings, shampoos, cosmetics.
그러나 국내에서는 거의 단백질의 산(acid) 가수분해물을 이용하고 있는 실정이며, 단백질을 산이나 알칼리로 가수분해 할 경우 트립토판(tryptophan), 시스테인(cysteine)과 같은 필수 아미노산이 손실될 뿐만 아니라 리지노알라닌(lysinoalanine)과 같은 독성이 있는 부산물이 생성되어 일본을 비롯한 선진국에서는 단백질의 산가수분해물의 안전성 문제가 대두되고 있다.
However, in Korea, acid hydrolyzate of protein is almost used, and when hydrolyzing protein with acid or alkali, essential amino acids such as tryptophan and cysteine are lost, as well as lignolanine Toxic by-products such as lysinoalanine have been produced, which has raised the issue of the safety of acid hydrolysates of proteins in developed countries, including Japan.
염증반응(inflammation)은 생체 또는 조직에 물리적 작용, 화학적 물질, 세균감염 등의 기질적 변화를 가져오는 침습이 가해질 때 그 손상부위를 수복 재생하려는 기전이다. 일단 자극이 가해지면 국소적으로 히스타민(histamine), 세로토닌(serotonine), 브라디키닌(bradykinin), 프로스타글란딘(prostagladnins), 히드록실-에이코사테라에노이 산(hydorxyl-eicosatetraenoic acid, HETE) 및 류코트리엔(leukotriene)과 같은 혈관 활성물질이 유리되어 혈관 투과성이 증대되면서 단핵포식세포(mononuclear phagocytes), 대식세포(macrophage), 호중구(neutrophil), 자연살해세포(NK cell)가 손상부위로 침윤되어 염증을 유발하게 된다.Inflammation is a mechanism for repairing and repairing an injured area when an invasion that causes a physical change in a living body or tissue, such as a physical action, a chemical substance, or a bacterial infection, is applied. Once stimulated, histamine, serotonin, bradykinin, prostagladnins, hydroxyl-eicosatetraenoic acid (HETE) and leukotriene (HETE) Vascularly active substances such as leukotriene are released to increase vascular permeability, causing monuclear phagocytes, macrophage, neutrophils, and natural killer cells to invade the damaged area and cause inflammation. Done.
감염이나 조직손상에 의해 발생하는 적당한 염증은 손상부위가 빠르게 회복 되기 위한 정상적인 반응이다. 그러나 장기간에 걸친 지속적인 염증반응은 손상부위에 대한 회복을 지연시키고 주위 세포에 대한 상해를 동반하므로, 주변조직에 대한 방어력을 떨어뜨려 증세를 더욱 악화시킬 수 있어, 정상적인 염증반응을 유지하기 위한 항염증 제제가 필요한 것이다. 현재 일반적으로 사용되는 대부분의 항염증제는 스테로이드계의 약물이나, 스테로이드계의 약물은 인체의 저항성을 약화시키거나 염증 부위의 조직재생을 지연시키는 부작용을 가지고 있어 대체약물의 개발이 필요하다.Appropriate inflammation, caused by infection or tissue damage, is a normal reaction for rapid recovery of the damaged area. However, long-term sustained inflammatory response delays recovery to the damaged area and injuries to surrounding cells, which can worsen the symptoms by lowering the defenses of surrounding tissues, and thus anti-inflammatory to maintain a normal inflammatory response. Formulation is required. Currently, most anti-inflammatory drugs commonly used are steroid-based drugs, but steroid-based drugs have a side effect of weakening the body's resistance or delaying tissue regeneration at an inflammation site, and thus, alternative drugs need to be developed.
이에 본 발명자들은 독성과 부작용이 없는 천연물 유래의 항염증제를 개발하기 위하여 예의 노력한 결과, 본 발명에 따른 홍합 단백질 가수분해물의 우수한 항염증 효과를 확인하고 본 발명을 완성하였다.Thus, the present inventors have made diligent efforts to develop anti-inflammatory agents derived from natural products without toxicity and side effects, confirming the excellent anti-inflammatory effect of the mussel protein hydrolyzate according to the present invention and completed the present invention.
결국 본 발명의 주된 목적은 홍합 단백질 가수분해물로부터 분리된 펩티드를 유효성분으로 함유하고, 독성 및 부작용 없이 효과적인 천연 항염증 조성물을 제공하는 것이다.After all, the main object of the present invention is to provide a natural anti-inflammatory composition containing a peptide isolated from the mussel protein hydrolyzate as an active ingredient, effective without toxicity and side effects.
본 발명의 또 다른 목적은 홍합 단백질 가수분해물의 제조방법 및 상기 가수분해물로부터 강력한 항염증 효과를 갖는 기능성 펩티드를 분리 정제하는 방법을 제공하는 것이다.It is another object of the present invention to provide a method for preparing a mussel protein hydrolyzate and a method for separating and purifying a functional peptide having a strong anti-inflammatory effect from the hydrolyzate.
상기 목적을 달성하기 위하여, 본 발명은 홍합 단백질 가수분해물로부터 분리된 펩티드와 약학적으로 허용되는 담체를 포함하는 항염증 조성물 및 홍합 단백질 가수분해물의 제조방법과 상기 가수분해물로부터 펩티드를 분리 정제하는 방법에 관한 것이다.
In order to achieve the above object, the present invention provides a method for producing an anti-inflammatory composition and a mussel protein hydrolyzate comprising a peptide isolated from the mussel protein hydrolyzate and a pharmaceutically acceptable carrier and a method for separating and purifying the peptide from the hydrolyzate It is about.
본 발명은 홍합 단백질 가수분해물로부터 분리된 펩티드를 유효성분으로 함유하는 항염증 조성물을 제공한다.The present invention provides an anti-inflammatory composition containing a peptide isolated from mussel protein hydrolyzate as an active ingredient.
상기 홍합 단백질 가수분해물은 잘게 분쇄한 동결건조 된 홍합을 완충액에 용해시킨 후, 상기 용액에 단백질 가수분해 효소를 첨가한 뒤 교반시켜 가수분해 반응을 진행시킨 다음, 열수 중에서 가열하고 이를 필터, 농축 및 건조하여 제조된 것을 특징으로 한다.The mussel protein hydrolyzate is a finely pulverized lyophilized mussels dissolved in a buffer, the protein hydrolase is added to the solution and stirred to proceed the hydrolysis reaction, and then heated in hot water and filtered, concentrated and It is characterized by being manufactured by drying.
또한, 상기 완충액은 인산염 완충액(phosphate buffer) 또는 글리신-염산 완충액(glycine-HCl buffer)이며, 상기 단백질 가수분해 효소는 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 파파인(Papain), 펩신(pepsin), 트립신(Trypsin), 알파-키모트립신(α-Chymotrypsin), 알칼라제(Alcalase) 및 프로타맥스(Protamex)로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 한다.In addition, the buffer is a phosphate buffer (glycine-HCl buffer) or glycine-HCl buffer (protease), the proteolytic enzyme Flavorzyme (Neutrase), Papain (Papain), pepsin ( pepsin), Trypsin, Alpha-chymotrypsin, α-Chymotrypsin, Alcalase, and Protamex.
또한, 본 발명에 있어서, 상기 가수분해 단계는 pH 1.0 ~ 10.0의 완충액 중에서 반응온도 25 ~ 70℃, 반응시간 6~10시간 동안 가수분해하는 것을 특징으로 하며 바람직하게는 상기 완충액의 pH가 2.0~8.0, 반응온도는 30~60℃ 반응시간 8시간 가열하고 감압증발기로 농축 후 동결건조시키는 것이 좋다.In addition, in the present invention, the hydrolysis step is characterized in that the hydrolysis of the reaction temperature of 25 ~ 70 ℃,
또한, 상기 홍합 단백질 가수분해물로부터 분리된 펩티드는 (1) 홍합 단백질 가수분해물을 TFF(Tangetial Flow Filtration) 시스템에 의해 분자량별로 분리한 후 각 획분의 생리 활성을 측정하는 단계; (2) 상기 분리된 획분 중 생리활성이 가장 뛰어난 획분을 크로마토그래피법으로 분리하는 단계; (3) 상기 분리된 각 획분의 생리활성을 측정하는 단계; 및 (4) 상기 분리된 획분 중 생리활성이 가장 뛰어난 획분을 정제하는 단계;를 포함하는 방법으로 제조하며, 상기 (2) 내지 (4) 단계를 반복 실시하여 단일물질로 분리 정제하는 것을 특징으로 한다.In addition, the peptide isolated from the mussel protein hydrolyzate comprises the steps of: (1) separating the mussel protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system to measure the physiological activity of each fraction; (2) separating the fraction having the most physiological activity among the separated fractions by chromatography; (3) measuring the biological activity of each of the separated fractions; And (4) purifying the fraction having the highest physiological activity among the separated fractions. The method is prepared by the method comprising the steps of: (2) to (4) by repeating steps to separate and purify into a single substance. do.
또한, 상기 (2) 단계에 있어서, 펩티드는 이온교환 컬럼(DEAEFF 16/10)이 장착된 단백질 액체 크로마토그래피를 사용하여 분리한 후, 분취용 C18 컬럼(GROM-SIL 120 C18, 20 * 250 mm)이 장착된 고성능크로마토그래피, 두 번의 분석용 C18 컬럼(GROM-SIL 120 C18, 4.0 * 250 mm)이 장착된 고성능크로마토그래피를 이용한 정제과정을 거쳐 강한 항염증 능력을 갖는 것을 특징으로 한다.In the step (2), the peptide was separated using protein liquid chromatography equipped with an ion exchange column (DEAEFF 16/10), and then a preparative C 18 column (GROM-
또한, 본 발명은 분리 정제한 펩티드를 Edman degradation 방법에 의해 PTH 분석컬럼을 이용하여 아미노산 서열을 분석하는 것을 특징으로 한다. In addition, the present invention is characterized by analyzing the amino acid sequence using the PTH analysis column by the Edman degradation method of the separated and purified peptide.
또한, 본 발명은 상기 조성물을 유효성분으로 함유하는 항염증제 및 건강기능식품을 제공한다.The present invention also provides an anti-inflammatory agent and health functional food containing the composition as an active ingredient.
또한, 본 발명의 홍합 단백질 가수분해물 및 이로부터 분리된 기능성 펩티드는 항염증제와 더불어 노화 및 각종 질환의 억제 또는 치료를 위한 의약품, 건강보조제, 화장료, 식품, 식품 첨가제 등에 유용하게 이용될 수 있으며, 이에 의해 제한되지 않는다.In addition, the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom may be usefully used in anti-inflammatory drugs, health supplements, cosmetics, food, food additives, etc. for the inhibition or treatment of aging and various diseases, It is not limited by.
또한, 본 발명의 홍합 단백질 가수분해물 및 이로부터 분리된 기능성 펩티드가 의약품으로 이용될 경우에는 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있으며, 통상적인 방법에 따라 약학적으로 허용되는 제형으로 제조될 수 있는데, 이렇게 이루어진 약학조성물을 권리범위로 포함한다. 또한 상기 약학 조성물을 항염증제로 사용하는 의약적 용도 역시 본 발명의 권리범위에 포함된다.In addition, when the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as a medicine, it may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions, It can be prepared in a pharmaceutically acceptable formulation, including the pharmaceutical composition made in this scope. In addition, the pharmaceutical use of the pharmaceutical composition as an anti-inflammatory agent is also included in the scope of the present invention.
상기 담체 또는 부형제로는 물, 덱스트린, 칼슘카보네이드, 락토스, 프로필렌글리콜, 리퀴드, 파라핀, 생리식염수, 덱스트로스, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물류가 있으며, 이들은 1종 이상 사용될 수 있으나, 이에 한정되는 것은 아니며 통상의 담체 및 부형제는 모두 사용 가능하다. 또한 항염증 조성물을 약제화 하는 경우 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등을 더욱 포함할 수 있다.The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid, paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more, but are not limited thereto. Both carriers and excipients can be used. In addition, when the anti-inflammatory composition is formulated, conventional fillers, extenders, binders, disintegrating agents, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.
또한, 본 발명의 홍합 단백질 가수분해물 및 이로부터 분리된 기능성 펩티드가 화장료로 이용될 경우에는 주름개선 및 미백 기능성 화장품, 유연화장수, 영양화장수, 아이크림, 영양크림, 맛사지크림, 클렌징크림, 에센스 등의 제형으로 제조될 수 있으며, 각 제형의 화장료 조성물, 각 제형의단백질 가수분해물 및 기능성 펩티드를 포함하여 주름개선 및 미백 원료 또는 통상의 화장료 원료로서 이외의 성분들을 화장료의 제형 또는 사용 목적 등에 따라 당업자가 어려움 없이 적의 선정하여 배합 할 수 있다.In addition, when the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as cosmetics, wrinkle improvement and whitening functional cosmetics, softening lotion, nourishing cosmetics, eye cream, nourishing cream, massage cream, cleansing cream, essence, etc. It can be prepared in the formulation of the formulation, including the cosmetic composition of each formulation, the protein hydrolyzate and functional peptide of each formulation, other ingredients as wrinkles and whitening raw materials or conventional cosmetic raw materials according to the formulation or purpose of use of cosmetics, etc. Can be selected and compounded without difficulty.
상기와 같은 본 발명에 따르면 홍합 단백질 가수분해물, 상기 가수분해물로부터 분리, 정제된 항염증 펩티드 및 상기 가수분해물을 포함하는 조성물은 항염증 활성을 가지므로 기능성 의약품 및 건강기능식품으로 이용될 수 있다.According to the present invention as described above, the mussel protein hydrolyzate, the anti-inflammatory peptide isolated from the hydrolyzate, and the composition comprising the hydrolyzate have anti-inflammatory activity and thus can be used as functional medicines and health functional foods.
또한 본 발명의 홍합 단백질 가수분해물은 천연물 유래의 물질로써 독성 및 부작용 없이 안전하게 사용될 수 있다.In addition, the mussel protein hydrolyzate of the present invention can be used safely without toxicity and side effects as a substance derived from natural products.
도 1은 홍합 단백질 가수분해물의 제조 공정을 나타낸 모식도이다.
도 2는 홍합 단백질 가수분해물의 항염증 효과를 나타낸 것이다(1, Flavour- zyme; 2, Neutrase; 3, Protamex; 4, Alcalase; 5, Papain; 6, α-chymotrypsin; 7, Trypsin; 8, Pepsin).
도 3은 홍합 단백질 가수분해물(플라보자임)의 분자량별 NO 생성 억제율을 나타낸 것이다(>30,000 Da; 10,000 ~ 30,000 Da; 5.000 ~ 10,000 Da; 5,000 Da<).
도 4는 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리한 것을 나타낸 것이다.
도 5는 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리한 것을 동결건조하여 항염증 활성을 측정한 결과를 나타낸 것이다.
도 6은 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하여 효능이 입증된 분획물을 분취용 컬럼이 장착된 고성능 크로마토그래피를 이용하여 분리한 것을 나타낸 것이다.
도 7은 홍합 단백질 가수분해물(틀라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하여 효능이 입증된 분획물을 분취용 컬럼이 장착된 고성능 크로마토그래피를 이용하여 분리한 것을 동결건조하여 항염증 활성을 측정한 결과를 나타낸 것이다.
도 8은 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하여 분취용 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리한 후 효능이 입증된 분획물을 분석용 컬럼이 장착된 고성능크로마토그래피로 분리한 것을 나타낸 것이다.
도 9는 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000 Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하여 분취용 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리한 후 효능이 입증된 분획물을 분석용 컬럼이 장착된 고성능크로마토그래피로 분리한 것을 동결건조하여 항염증 활성을 측정한 결과를 나타낸 것이다.
도 10은 본 발명의 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하고 분취용 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리한 후 효능이 입증된 분획물을 분석용 컬럼이 장착된 고성능크로마토그래피로 분리한 후 효능이 입증된 분획물을 다시 한 번 분석용 컬럼이 장착된 고성능크로마토그래피로 분리한 것을 나타낸 것이다.
도 11은 본 발명의 홍합 단백질 가수분해물(플라보자임)의 분자량 10,000 ~ 30,000Da에 해당하는 단백질을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피로 분리하고 분취용 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리한 후 효능이 입증된 분획물을 분석용 컬럼이 장착된 고성능크로마토그래피로 분리한 후 효능이 입증된 분획물을 다시 한 번 분석용 컬럼이 장착된 고성능크로마토그래피로 분리하여 동결건조한 것의 산화질소생성 억제율을 나타낸 것이다.1 is a schematic diagram showing a process for producing mussel protein hydrolyzate.
Figure 2 shows the anti-inflammatory effect of mussel protein hydrolysates (1, Flavor-zyme; 2, Neutrase; 3, Protamex; 4, Alcalase; 5, Papain; 6, α-chymotrypsin; 7, Trypsin; 8, Pepsin ).
Figure 3 shows the NO production inhibition rate by molecular weight of the mussel protein hydrolyzate (flavozyme) (> 30,000 Da; 10,000 ~ 30,000 Da; 5.000 ~ 10,000 Da; 5,000 Da <).
Figure 4 shows the separation of the protein corresponding to the molecular weight of 10,000 ~ 30,000 Da of the mussel protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column.
Figure 5 shows the result of measuring the anti-inflammatory activity by lyophilizing the separation of the protein corresponding to the molecular weight of 10,000 ~ 30,000 Da of the mussel protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column .
6 is a high performance chromatograph equipped with a preparative column that separates the protein corresponding to a molecular weight of 10,000 to 30,000 Da of the mussel protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column. It shows the separation using the graph.
7 is a high performance chromatograph equipped with a preparative column that separates the protein corresponding to a molecular weight of 10,000 to 30,000 Da of the mussel protein hydrolyzate (Tlabozyme) by protein liquid chromatography equipped with an anion exchange column. The results obtained by measuring the anti-inflammatory activity by lyophilization of the isolates using chromatography.
8 is a protein corresponding to a molecular weight of 10,000 ~ 30,000 Da of the mussel protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column was separated by high performance chromatography equipped with a preparative column After the fraction proved efficacy was shown by high performance chromatography equipped with an analytical column.
9 is a protein corresponding to a molecular weight of 10,000 ~ 30,000 Da of mussel protein hydrolyzate (flavozyme) by protein liquid chromatography equipped with an anion exchange column was separated by high performance chromatography equipped with a preparative column After separating the fractions proved efficacy by high performance chromatography equipped with an analytical column shows the results of measuring the anti-inflammatory activity by lyophilization.
Figure 10 is a protein of the mussel protein hydrolyzate (flavozyme) of the present invention is separated by a protein liquid chromatography equipped with an anion exchange column molecular weight of 10,000 ~ 30,000 Da and using high performance chromatography equipped with a preparative column After separation by fractions proved efficacy was separated by high performance chromatography equipped with analytical column, the fractions proved once again separated by high performance chromatography equipped with analytical column.
11 is a protein liquid chromatography of the mussel protein hydrolyzate (flavozyme) of the present invention with a molecular weight of 10,000 ~ 30,000 Da separated by an anion exchange column equipped with protein chromatography using preparative column equipped with high performance chromatography Nitrogen oxides produced by lyophilization by separating fractions with high efficiency chromatography equipped with an analytical column, and then separating fractions with high efficiency chromatography equipped with an analytical column. Inhibition rate is shown.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예Example 1. 홍합의 단백질 1. Protein of mussels 가수분해물의Hydrolyzate 제조 Produce
본 실험에 사용된 홍합 시료는 여수 수산시장에서 구입하였고, 가수분해물을 제조하기 위해 -20℃에 저장하여 사용하였다. 잘게 분쇄한 동결건조 된 홍합은 0.1M 완충액 (buffer, pH 2.0~8.0)을 홍합 : 완충액 = 1 : 50(w/w)의 비율로 용해한 후, 30분간 150rpm에서 교반하면서 30~60℃로 예열한 다음, 단백질 가수분해 효소 : 홍합 = 1 : 50 (w/w)의 비율로 단백질 가수분해 효소, 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 파파인(Papain), 펩신(pepsin), 트립신(Trypsin), 알파-키모트립신(α-Chymotrypsin), 알칼라제(Alcalase) 및 프로타맥스(Protamex)을 첨가하여 8시간 동안 180rpm에서 교반하여 30~60℃에서 가수분해한 후, 100℃에서 10분간 가열하여 가수분해 반응을 종료시켰다.Mussel samples used in this experiment were purchased from Yeosu fish market, and stored at -20 ° C to prepare hydrolyzate. Finely pulverized lyophilized mussel was dissolved in 0.1M buffer (buffer, pH 2.0 ~ 8.0) at the ratio of mussel: buffer = 1: 50 (w / w), then preheated to 30 ~ 60 ℃ with stirring at 150rpm for 30 minutes. Proteinase, mussel = 1: 50 (w / w), followed by proteinase, flavozyme, neutrase, papain, pepsin and trypsin. (Trypsin), alpha-chymotrypsin, α-Chymotrypsin, Alcalase and Protamex were added and stirred at 180 rpm for 8 hours to hydrolyze at 30-60 ° C., and then at 100 ° C. The hydrolysis reaction was terminated by heating for 10 minutes.
반응이 종료된 액은 와트만 필터 41번(Whatman filter No. 41)을 사용하여 필터한 후 감압증발기로 농축시킨 후, -80℃로 동결건조하여 홍합 단백질 가수분해물을 제조하였다. 이때 사용된 효소의 조성 및 반응 조건은 하기 표 1에 나타내었다.After the reaction was completed, the solution was filtered using Whatman filter No. 41 (Whatman filter No. 41), concentrated under a reduced pressure evaporator, and lyophilized at −80 ° C. to prepare a mussel protein hydrolyzate. The composition and reaction conditions of the enzyme used at this time are shown in Table 1 below.
단, 상기에서 PB는 인산염 완충액(Phosphate Buffer), GHB는 글리신-염산 완충액(Glycine-HCl Buffer)을 의미한다.
However, PB in the above means phosphate buffer (Phosphate Buffer), GHB means glycine-HCl buffer (Glycine-HCl Buffer).
실험예Experimental Example 1. 홍합 단백질 1. Mussel Protein 가수분해물의Hydrolyzate 항염증 효과 측정 Anti-inflammatory effect measurement
산화질소(Nitric oxide)는 체내에서 L-아르기닌이 시트룰린을 생성할 때 생성되는 것으로, 과잉 생성 시 우리 몸의 산화를 촉진시키는 자유 라디칼(free radial)로 알려져 있다. 홍합 유래 단백질 가수분해물의 항염증 효과는 마크로파지 세포 Raw 264.7에 LPS와 홍합의 단백질 가수분해물을 처리하여, 마크로파지 내 산화질소 생성에 미치는 영향을 측정하였다.Nitric oxide is produced when L-arginine produces citrulline in the body, and it is known as a free radical that promotes oxidation of the body when excess production occurs. Anti-inflammatory effect of mussel-derived protein hydrolyzate was measured by treating the macrophage cell Raw 264.7 with LPS and mussel protein hydrolysates to produce nitric oxide in macrophages.
즉, 6-well plate에 5×105개의 마크로파지 세포를 접종하고 24시간 뒤에 8종의 홍합 유래 단백질 가수분해물(1, Flavourzyme; 2, Neutrase; 3, Protamex; 4, Alcalase; 5, Papain; 6, α-chymotrypsin; 7, Trypsin; 8, Pepsin) 0.5mg/ml와 LPS(Lipopolysaccharide) 1/ml을 처리한 후 37℃, CO2 인큐베이터에 24시간 동안 반응시켰다. 24시간 후, 각 배지 50㎕를 첨가하여 실온에서 10분 방치한 다음 여기에 N-(1-나프틸) 에틸렌다이아민 다이하이드로클로라이드 (N-(1-Naphthyl)ethylenediamine dihydrochloride) 50㎕를 첨가하고 실온에서 15분 방치하여 550nm에서 O.D. 값을 측정하였다.That is, 24 hours after inoculating 5 × 10 5 macrophage cells into a 6-well plate, 8 mussel-derived protein hydrolysates (1, Flavorzyme; 2, Neutrase; 3, Protamex; 4, Alcalase; 5, Papain; 6 , α-chymotrypsin; 7, Trypsin; 8, Pepsin) 0.5mg / ml and LPS (Lipopolysaccharide) 1 / ml after treatment was reacted for 24 hours at 37 ℃, CO 2 incubator. After 24 hours, 50 μl of each medium was added and allowed to stand at room temperature for 10 minutes. Then, 50 μl of N- (1-naphthyl) ethylenediamine dihydrochloride was added thereto. After leaving for 15 minutes at room temperature, the OD value was measured at 550 nm.
그 결과, LPS 단독 처리군에 비해 알파키모트립신을 제외한 모든 홍합 단백질 가수분해물 처리군에서 그 생성량이 감소하였다(도 2참조).
As a result, the amount of all mussel protein hydrolyzate treated groups except alpha chymotrypsin was reduced compared to the group treated with LPS alone (see FIG. 2).
실험 예 2. 홍합 단백질 유래 효소 Experimental Example 2. Mussel-derived enzyme 가수분해물로부터From hydrolyzate 항염증 펩티드 소재 분리 및 정제 I Anti-inflammatory Peptide Material Isolation and Purification I
홍합을 플라보자임으로 가수분해시킨 가수분해물의 항염증 펩티드를 분리 및 정제하기 위하여 멤브레인 사이즈에 따라 단백질을 분리, 정재하는 TFF 시스템을 이용하여 5,000 Da 미만, 5,000 ~ 10,000 Da, 10,000 ~ 30,000 Da, 30,000 Da 이상의 분자량별로 4개의 분획물으로 구분하여 NO 생성 억제율을 측정하였다. 그 결과, 4개의 획분 중 10,000 ~ 30,000 Da의 크기에서 가장 높은 활성을 나타냄을 확인 할 수 있었다(도 3참조).
In order to isolate and purify the anti-inflammatory peptide of hydrolyzate hydrolyzed by mussels with flavozyme, the TFF system for separating and purifying proteins according to the membrane size is used for less than 5,000 Da, 5,000 to 10,000 Da, 10,000 to 30,000 Da, 30,000 Inhibition rate of NO production was measured by dividing into four fractions for each molecular weight above Da. As a result, it was confirmed that the highest activity in the size of 10,000 ~ 30,000 Da of the four fractions (see Figure 3).
실험 예 3. 홍합 단백질 유래 효소 Experimental Example 3. Mussel-derived enzyme 가수분해물로부터From hydrolyzate 항염증 펩티드 소재 분리 및 정제 Anti-inflammatory Peptide Material Isolation and Purification IIII
본 실험 예는 홍합 플라보자임 가수분해물로부터 항염증 펩티드를 얻기 위해 분리, 정제하는 두 번째 과정으로, TFF 방법에서 가장 우수한 항염증 활성을 나타낸 분획인 10,000 ~ 30,000 Da의 분획물을 음이온 교환 컬럼이 장착된 단백질 액체 크로마토그래피를 이용하여 분리, 정제하였다.This experiment is a second process of separating and purifying anti-inflammatory peptides from mussel flabozyme hydrolyzate. An anion exchange column is equipped with a fraction of 10,000-30,000 Da, the fraction showing the best anti-inflammatory activity in the TFF method. Protein was purified using liquid chromatography.
단백질 액체 크로마토그래피는 고성능 크로마토그래피 기술을 응용하여 단백질의 정제에 특화시킨 기기로 이온 교환 혹은 크기배재 등에 적합한 충전물을 균일하게 담은 관(분리관)을 사용하는 전자동식 분리장치이다. 본 실험에서는 음이온 교환에 적합한 컬럼을 장착하여 단백질 액체 크로마토그래피를 실시하였으며, 최종적으로 5개의 분획물을 얻었다(도 4참조). 각 분획물을 동결건조하여 항염증 활성을 측정한 결과 도 5에서 보듯이 C 획분에서 가장 높은 활성을 나타내었다.
Protein Liquid Chromatography is a device that specializes in the purification of proteins by applying high-performance chromatography technology. It is a fully automatic separation device using a tube (separation tube) uniformly containing a packing material suitable for ion exchange or size exclusion. In this experiment, protein liquid chromatography was performed by mounting a column suitable for anion exchange, and finally 5 fractions were obtained (see FIG. 4). As a result of measuring the anti-inflammatory activity by lyophilizing each fraction, as shown in Figure 5 it showed the highest activity in the C fraction.
실험 예 4. 홍합 단백질 유래 효소 가수분해물로부터 항염증 펩티드 소재 분리 및 정제 IIIExperimental Example 4. Isolation and Purification of Anti-inflammatory Peptide Materials from Mussel Protein-derived Enzyme Hydrolysates III
본 실험 예는 홍합 플라보자임 가수분해물로부터 항염증 펩티드를 얻기 위해 분리, 정제하는 세 번째 과정으로, 단백질 액체 크로마토그래피를 사용한 위 실험 예 3에서 가장 우수한 항염증 활성을 나타낸 분획인 분획물 C를 분취용 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리, 정제하였다.This experimental example is a third process of separating and purifying anti-inflammatory peptides from mussel flabozyme hydrolyzate. The fraction C, the fraction showing the best anti-inflammatory activity in the above Experimental Example 3 using protein liquid chromatography, was analyzed. It was separated and purified using high performance chromatography equipped with a drinking column.
고성능크로마토그래피는 적당한 충전물이 균일하게 담긴 관(분리관) 안에서 기체 시료나 기체화한 액체 또는 고체 시료를 운반기체에 의해 전개시키되 분해하지 않고 기체인 상태로 통과시켜 각 성분을 분리시키는 방법인데, 본 실험에서는 분취용 C18 컬럼을 장착하여 크로마토그래피를 실시하였으며, 최종적으로 3개의 분획물을 얻었다(도 6참조). High performance chromatography is a method in which gas components or gasified liquid or solid samples are developed by a carrier gas in a uniformly packed tube (separation tube), but separated by passing through a gaseous state without decomposition. In this experiment, chromatography was performed using a preparative C 18 column, and finally three fractions were obtained (see FIG. 6).
각 분획물을 동결건조하여 항염증 활성을 측정한 결과 도 7에 보듯이, C-Ⅲ 분획물에서 가장 높은 활성을 나타내었다.
As a result of measuring the anti-inflammatory activity by lyophilization of each fraction, as shown in Figure 7, the C-III fraction showed the highest activity.
실험 예 5: 홍합 단백질 유래 효소 Experimental Example 5: Mussel-derived enzyme 가수분해물로부터From hydrolyzate 항염증 펩티드 소재 분리 및 정제 Anti-inflammatory Peptide Material Isolation and Purification IVIV
본 실험 예는 홍합 플라보자임 가수분해물로부터 항염증 펩티드를 얻기 위해 분리, 정제하는 네 번째 과정으로, 상기 실험 예 4에서 얻은 분획물 C-Ⅲ을 분석용 C18 컬럼이 장착된 고성능크로마토그래피를 이용하여 분리, 정제하였다.This Experimental Example is a fourth process of separating and purifying the anti-inflammatory peptide from the mussel flabozyme hydrolyzate. The fraction C-III obtained in Experimental Example 4 was analyzed using high performance chromatography equipped with an analytical C 18 column. Separated and purified.
최종적으로 4개의 분획물을 얻었으며(도 8), 각 획분을 동결건조하여 항염증 활성을 측정한 결과 도 9에서 보듯이 C-Ⅲ-ⅲ 분획물에서 가장 높은 활성을 나타내었다.
Finally, four fractions were obtained (FIG. 8), and each fraction was lyophilized to measure anti-inflammatory activity. As shown in FIG. 9, C-III-III fraction showed the highest activity.
실험 예 6: 홍합 단백질 유래 효소 가수분해물로부터 항염증 펩티드 소재 분리 및 정제 VExperimental Example 6: Isolation and Purification of Anti-inflammatory Peptide Material from Mussel Protein-Derived Enzyme Hydrolysate V
본 실험 예는 홍합 플라보자임 가수분해물로부터 항염증 펩티드를 얻기 위해 분리, 정제하는 다섯 번째 과정으로, 상기 실험 예 5에서 얻은 분획물 C-Ⅲ-ⅲ 를 다시 한 번 분석용 컬럼이 장착된 고성능크로마토그래피를 이용하여 최종적으로 다시 한 번 더 분리, 정제하여, 최종적으로 1개의 분획물을 얻었으며(도 10), 그 결과 도 11에서 나타난 바와 같이 분획물의 산화질소생성 억제율은 73.0% 소거율을 나타내었다.
This Experimental Example is a fifth process of separating and purifying anti-inflammatory peptides from mussel flabozyme hydrolyzate. The fraction C-III-III obtained in Experimental Example 5 was once again equipped with a high performance chromatograph equipped with an analytical column. Finally, the resultant was separated and purified once more using chromatography, finally obtaining one fraction (FIG. 10). As a result, as shown in FIG. 11, the inhibition rate of nitric oxide production of the fraction was 73.0%. .
실험 예 7: 항염증 활성을 갖는 펩티드 아미노산 배열 확인Experimental Example 7: Confirmation of peptide amino acid sequence having anti-inflammatory activity
본 실험 예는 상기 실험 예 6에서 얻은 항염증 펩티드의 아미노산 배열을 확인하기 위하여, Edman 분해법(Edman, P., Acta Chemica Scandinavica, 283-293, 1950)에 준하여 N-termina로부터 아미노산 배열을 분석하였다.In this experimental example, in order to confirm the amino acid sequence of the anti-inflammatory peptide obtained in Experimental Example 6, Edman digestion method (Edman, P., Acta Chemica The amino acid sequence was analyzed from N-termina according to Scandinavica , 283-293, 1950).
그 결과, 항염증 펩티드의 아미노산 배열은 각각 서열번호 1로 기재되는 Gly-Val-Ser-Leu-Leu-Gln-Gln-Phe-Phe-Leu으로 확인되었다(표 2참조).As a result, the amino acid sequence of the anti-inflammatory peptide was confirmed by Gly-Val-Ser-Leu-Leu-Gln-Gln-Phe-Phe-Leu described in SEQ ID NO: 1, respectively (see Table 2).
이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 의하여 정의된다고 할 것이다.As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> Konkuk University Industrial Cooperation Corp.
<120> Antiinflammatory composition comprising enzymatic hydrolysates of
Mytilus coruscus
<130> P10-E424
<160> 1
<170> KopatentIn 1.71
<210> 1
<211> 10
<212> PRT
<213> Mytilus coruscus
<400> 1
Gly Val Ser Leu Leu Gln Gln Phe Phe Leu
1 5 10
<110> Konkuk University Industrial Cooperation Corp.
<120> Antiinflammatory composition comprising enzymatic hydrolysates of
Mytilus coruscus
<130> P10-E424
<160> 1
<170> KopatentIn 1.71
<210> 1
<211> 10
<212> PRT
<213> Mytilus coruscus
<400> 1
Gly Val Ser Leu Leu Gln Gln
Claims (10)
An anti-inflammatory composition containing a peptide isolated from the mussel protein hydrolyzate as an active ingredient.
상기 홍합 단백질 가수분해물은
(1) 동결건조된 홍합을 완충액 중에 용해시키는 단계;
(2) 상기 용액에 단백질 가수분해 효소를 첨가하여 가수분해하는 단계;
(3) 상기 가수분해물을 열수 중에서 가열하는 단계;
(4) 상기 가열된 가수분해물을 필터, 농축 및 건조하는 단계;를 포함하는 방법으로 제조된 것을 특징으로 하는 항염증제 조성물.
The method of claim 1,
The mussel protein hydrolyzate is
(1) dissolving lyophilized mussels in buffer;
(2) hydrolysis by adding proteolytic enzyme to the solution;
(3) heating the hydrolyzate in hot water;
(4) filtering, concentrating and drying the heated hydrolyzate; anti-inflammatory composition, characterized in that it was prepared by a method comprising a.
상기 (1) 단계의 완충액은 인산염 완충액(Phosphate buffer) 또는 글리신-염산 완충액(Glycine-HCl buffer) 중 어느 하나인 것을 특징으로 하는 항염증제 조성물.
The method of claim 2,
The buffer of step (1) is an anti-inflammatory composition, characterized in that any one of phosphate buffer (Phosphate buffer) or glycine-HCl buffer (Glycine-HCl buffer).
상기 (1) 단계의 완충액의 pH는 1.0~10.0인 것을 특징으로 하는 항염증제 조성물.
The method of claim 2,
The pH of the buffer of step (1) is the anti-inflammatory composition, characterized in that 1.0 ~ 10.0.
상기 단백질 가수분해 효소는 플라보자임(Flavourzyme), 뉴트라제(Neutrase), 파파인(Papain), 펩신(pepsin), 트립신(Trypsin), 알파-키모트립신(α-Chymotrypsin), 알칼라제(Alcalase) 및 프로타맥스(Protamex)으로 이루어진 군으로부터 선택되는 1종 이상인 것을 특징으로 항염증제 조성물.
The method of claim 2,
The proteolytic enzymes are Flavorzyme, Neutrase, Papain, Pepsin, Trypsin, Alpha-Chymotrypsin, Alcalase And Protamex (Protamex) anti-inflammatory composition, characterized in that at least one member selected from the group consisting of.
상기 (2) 단계의 단백질 가수분해는 반응온도 25 ~ 70℃에서 반응시간 6~10시간으로 효소처리하는 것을 특징으로 하는 항염증제 조성물.
The method of claim 2,
Proteolysis of step (2) is an anti-inflammatory composition, characterized in that the enzyme treatment with a reaction time of 6 to 10 hours at a reaction temperature of 25 ~ 70 ℃.
상기 펩티드는
(1) 홍합 단백질 가수분해물을 TFF(Tangetial Flow Filtration) 시스템에 의해 분자량별로 분리한 후 각 획분의 생리 활성을 측정하는 단계;
(2) 상기 분리된 획분 중 생리활성이 가장 뛰어난 획분을 크로마토그래피법으로 분리하는 단계;
(3) 상기 분리된 각 획분의 생리활성을 측정하는 단계; 및
(4) 상기 분리된 획분 중 생리활성이 가장 뛰어난 획분을 정제하는 단계;를 포함하는 방법으로 제조하며, 상기 (2) 내지 (4) 단계를 반복 실시하여 단일물질로 분리 정제하는 것을 특징으로 하는 항염증제 조성물.
The method of claim 1,
The peptide is
(1) separating the mussel protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system and measuring the physiological activity of each fraction;
(2) separating the fraction having the most physiological activity among the separated fractions by chromatography;
(3) measuring the biological activity of each of the separated fractions; And
(4) purifying the fraction having the highest physiological activity among the separated fractions; prepared by the method comprising a, characterized in that the separation and purification to a single material by repeating the steps (2) to (4) Anti-inflammatory composition.
상기 (2) 단계는 이온결합 컬럼이 장착된 단백질 액체 크로마토그래피, 분취용 C18 컬럼이 장착된 고성능크로마토그래피, 두 번의 분석용 C18 컬럼이 장착된 고성능크로마토그래피를 이용하여 정제과정을 실시하는 것을 특징으로 하는 항염증제 조성물.
The method of claim 7, wherein
In step (2), a purification process is performed using protein liquid chromatography equipped with an ion binding column, high performance chromatography equipped with a preparative C 18 column, and high performance chromatography equipped with two analytical C 18 columns. Anti-inflammatory composition, characterized in that.
An anti-inflammatory agent comprising the composition of any one of claims 1 to 8 as an active ingredient.
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020100110607A KR20120049043A (en) | 2010-11-08 | 2010-11-08 | Antiinflammatory composition comprising enzymatic hydrolysates of mytilus coruscus |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20120049043A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018510910A (en) * | 2015-03-24 | 2018-04-19 | ザ・ニュージーランド・インスティチュート・フォー・プラント・アンド・フード・リサーチ・リミテッド | Water-soluble mussel extract |
| WO2018147516A1 (en) * | 2017-02-08 | 2018-08-16 | 부경대학교 산학협력단 | Composition for preventing or treating bone diseases comprising mussel protein-derived peptide as active ingredient |
-
2010
- 2010-11-08 KR KR1020100110607A patent/KR20120049043A/en not_active Application Discontinuation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018510910A (en) * | 2015-03-24 | 2018-04-19 | ザ・ニュージーランド・インスティチュート・フォー・プラント・アンド・フード・リサーチ・リミテッド | Water-soluble mussel extract |
| WO2018147516A1 (en) * | 2017-02-08 | 2018-08-16 | 부경대학교 산학협력단 | Composition for preventing or treating bone diseases comprising mussel protein-derived peptide as active ingredient |
| KR20180092255A (en) * | 2017-02-08 | 2018-08-17 | 부경대학교 산학협력단 | Composition for preventing and treating bone disease comprising mussel protein-derived peptides as active ingredient |
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