KR20220097747A - Composition containing small molecular weight enzyme hydrolysates of black goat extracts and effective peptide sequence - Google Patents
Composition containing small molecular weight enzyme hydrolysates of black goat extracts and effective peptide sequence Download PDFInfo
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- KR20220097747A KR20220097747A KR1020200188933A KR20200188933A KR20220097747A KR 20220097747 A KR20220097747 A KR 20220097747A KR 1020200188933 A KR1020200188933 A KR 1020200188933A KR 20200188933 A KR20200188933 A KR 20200188933A KR 20220097747 A KR20220097747 A KR 20220097747A
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- peptide
- seq
- amino acid
- black goat
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Abstract
Description
본 발명은 저분자 흑염소육골즙 효소분해물 및 유효 펩타이드 서열을 함유하는 조성물에 관한 것으로, 더욱 상세하게는 흑염소육골즙으로 효소분해하여 수득한 저분자 흑염소골즙 효소분해물과 이로부터 분리된 유효 펩타이드를 함유하는 조성물에 관한 것이다.The present invention relates to a composition containing a low-molecular-weight black goat bone juice enzyme lysate and an effective peptide sequence. is about
최근 현대인들은 바쁜 일상으로 인해 규칙적인 운동, 균형 잡힌 식사, 충분한 휴식이 부족하고 환경 오염원과 스트레스에 쉽게 노출되어 있어 면역력이 약해지기가 쉽다. 이에 건강기능식품, 화장품, 약품에 대한 관심이 증가하였으며, 이에 따른 세계 시장 규모도 꾸준히 증가하는 추세이다. 특히 건강기능식품 중 기능성 내용별 점유율에서는 '면역기능개선'이 39%로 1위를 차지하여 소비자들은 면역력 증진에 큰 관심을 가지고 있다.Recently, modern people lack regular exercise, balanced meals, and sufficient rest due to their busy daily life, and they are easily exposed to environmental pollutants and stress, so their immunity is likely to weaken. Accordingly, interest in health functional foods, cosmetics, and pharmaceuticals has increased, and the global market size is also steadily increasing. In particular, among health functional foods, 'improving immune function' took the first place with 39% of the market share by functional content, so consumers are very interested in improving immunity.
면역이란 외부에 침입에 방어하는 생체 시스템으로 적응면역과 선천면역 시스템으로 나누어진다. 면역 반응 중 하나인 염증(inflammation)은 상처나 세균 감염과 같은 물리적, 화학적 자극이 있을 때 손상 부위를 회복시키는 중요한 신체 방어 기전 가운데 하나이다. 하지만 과도하거나 지속적인 염증반응이 일어나면 점막 손상을 촉진시켜 결과적으로 부종, 통증, 발열 등을 유발하며, 심혈관 질환 류마티스 관절염, 및 암과 같은 질병을 야기할 수 있다. 염증 질환을 치료하기 위한 항염증제로 스테로이드성과 비스테로이드성으로 구분되며, 이들은 대부분 합성 항염증제로 치료 효과 이외에도 다양한 부작용을 수반하는 경우가 많다. 따라서 효과가 높고 부작용이 적은 항염증제에 대한 개발이 필요한 실정이다.Immunity is a biological system that defends against invasion from the outside and is divided into adaptive immunity and innate immune system. Inflammation, one of the immune responses, is one of the important defense mechanisms of the body to repair the damaged area when there is a physical or chemical stimulus such as a wound or bacterial infection. However, excessive or continuous inflammatory response promotes mucosal damage, resulting in edema, pain, fever, and the like, and may cause diseases such as cardiovascular disease, rheumatoid arthritis, and cancer. Anti-inflammatory agents for the treatment of inflammatory diseases are classified into steroidal and non-steroidal agents, and most of these are synthetic anti-inflammatory agents, which often accompany various side effects in addition to therapeutic effects. Therefore, it is necessary to develop an anti-inflammatory agent with high effectiveness and few side effects.
대식세포는 염증반응에 관여하는 주요 면역세포로 알려져 있으며, 지질다당류(lipopolysaccharide; LPS)나 IFN-γ, TNF-α와 같은 사이토카인 자극에 의해 다양한 MAPKs 인자들(JNK, ERK, p38)이 인산화로 활성화되고, 이는 핵 내의 activator protein-1의 전사를 활성화시켜 염증성 싸이토카인의 발현을 증가시킨다. 또한 세포질의 NF-κB를 핵내로 이동시켜 염증반응을 활성화시키고 염증성 싸이토카인을 생성하고, 질소산화물 합성효소(inducible nitric oxide synthase; iNOS), 사이클로옥시제나제(cycloozygenase; COX-2) 발현을 통한 염증성 단백질 생성 증가시키는 것으로 알려져 있다. Macrophages are known as major immune cells involved in the inflammatory response, and various MAPKs factors (JNK, ERK, p38) are phosphorylated by cytokine stimulation such as lipopolysaccharide (LPS), IFN-γ, and TNF-α. , which activates the transcription of activator protein-1 in the nucleus to increase the expression of inflammatory cytokines. In addition, cytoplasmic NF-κB is moved into the nucleus to activate the inflammatory response and generate inflammatory cytokines. It is known to increase protein production.
산화질소(nitric oxide; NO)는 반응성이 큰 물질로 LPS, IFN-γ, TNF-α와 같은 염증성 자극을 받게되면 다양한 세포에서 iNOS에 의해 생성된다. 소량의 NO은 염증반응에 있어서 혈관확장, 신경전달, 병원체에 대한 세포파괴 등과 같은 긍정적인 생리기능을 담당하지만, 과도하게 생성된 NO은 염증과 암을 포함한 다양한 질병 생성 과정에 관여하며 강력한 산화제로 작용하여 세포에 손상을 일으켜 만성질환으로의 진행에도 관여하는 것으로 알려져 있다.Nitric oxide (NO) is a highly reactive substance and is produced by iNOS in various cells when subjected to inflammatory stimuli such as LPS, IFN-γ, and TNF-α. A small amount of NO is responsible for positive physiological functions such as vasodilation, neurotransmission, and cell destruction of pathogens in the inflammatory response, but excessively produced NO is involved in various disease-generating processes, including inflammation and cancer, and is a powerful oxidizing agent. It is known to be involved in the progression to chronic diseases by causing damage to cells by acting.
프로스타글란딘(prostaglandin; PG)은 여러 다양한 조직에서 물질대사를 조절하는 물질로 혈관의 수축과 확장, 혈압, 발열, 염증 조절과 같은 다양한 기능에 관여한다. 특히 염증 반응에 관여하는 PGE2는 arachidonic acid로부터 COX-2에 의하여 산화되어 생성된다. 이는 조직의 염증 및 통증을 유발한다. 따라서, 상기 산화질소(NO)와 프로스타글란딘 E2의 생성을 억제하는 물질을 탐색함으로써 효과적인 항염증성 물질을 판단할 수 있다.Prostaglandin (PG) is a substance that regulates metabolism in various tissues and is involved in various functions such as vasoconstriction and expansion, blood pressure, fever, and inflammation control. In particular, PGE 2 involved in the inflammatory response is oxidized from arachidonic acid by COX-2 and produced. This causes tissue inflammation and pain. Therefore, effective anti-inflammatory substances can be determined by searching for substances that inhibit the production of nitric oxide (NO) and prostaglandin E 2 .
흑염소는 '3저 4고 식품'으로 불리는데, 3저는 저지방, 저콜레스테롤, 저오염(친환경 식품)을 뜻하고, 4고는 고단백, 고칼슘, 고철분, 고비타민을 말한다. 옛날부터 아이들 성장, 골다공증 에방, 빈혈, 정력증진, 피부질환 증상 완화, 성인병(동맥경화, 고혈압, 심장병) 완화 등 다양한 효능이 있다고 알려져 보양식, 건강식으로 이용되고 있다. 특히, 비타민 E의 함량이 높고, 적색육 중에서 티아민(thiamin)이 0.1 mg/100 g, 리보플라빈(riboflavin)이 0.56 mg/100 g으로 가장 높은 함량을 가지고 있다고 알려져 있다. 또한, 흑염소는 다른 식육(소, 돼지, 닭)에 비해 칼슘과 철의 함량이 높고 포화지방산과 콜레스테롤 함유량이 적다. 우리나라는 흑염소를 주로 육골즙의 형태로 섭취하고 있으며 흑염소육골즙 또한 환자 노약자, 임산부의 기력회복 및 원기회복에 좋다고 알려져 있다. 하지만 흑염소육골즙의 면역증진 및 항염증 효과에 대한 연구는 아직 미흡한 실정이다.Black goat is called '3 low, 4 high food', 3 low means low fat, low cholesterol and low pollution (eco-friendly food), and 4 high means high protein, high calcium, high iron and high vitamin. Since ancient times, it has been known to have various effects such as children's growth, prevention of osteoporosis, anemia, stamina, alleviation of skin disease symptoms, and alleviation of adult diseases (arteriosclerosis, high blood pressure, heart disease). In particular, it is known that the content of vitamin E is high, and among red meat, thiamin is 0.1 mg/100 g and riboflavin has the highest content of 0.56 mg/100 g. In addition, compared to other meats (cow, pig, chicken), black goat has a higher content of calcium and iron and lower content of saturated fatty acids and cholesterol. In Korea, black goat is mainly consumed in the form of meat and bone juice, and black goat meat and bone juice is also known to be good for the recovery of energy and vitality of the elderly and pregnant women. However, studies on the immune-enhancing and anti-inflammatory effects of black goat meat bone juice are still insufficient.
따라서 본 발명자들은 부작용이 없는 천연 소재인 흑염소육골즙으로 효소 분해하여 수득한 효소 분해물로부터 항염증 효능이 있는 유효 펩타이드를 개발하고자 하였다.Therefore, the present inventors tried to develop an effective peptide with anti-inflammatory effect from the enzymatic decomposition product obtained by enzymatic decomposition with black goat meat bone juice, a natural material without side effects.
본 발명은 흑염소육골즙을 효소분해하여 저분자 흑염소육골즙 효소분해물을 수득하고, 이로부터 분리한 유효 펩타이드를 개발함으로써 이를 함유하여 뛰어난 항염 효과를 발휘하는 식품 조성물, 화장료 조성물 및 약학 조성물을 제공하고자한다.The present invention aims to provide a food composition, a cosmetic composition and a pharmaceutical composition that exhibits excellent anti-inflammatory effect by enzymatic decomposition of black goat meat and bone juice to obtain a low-molecular black goat meat and bone juice enzymatic decomposition product, and developing an effective peptide isolated therefrom. .
본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 항염용 식품 조성물을 제공한다.The present invention provides an anti-inflammatory food composition comprising, as an active ingredient, a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 to provide.
또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 항염용 화장료 조성물을 제공한다.In addition, the present invention is an anti-inflammatory cosmetic comprising the peptide having the amino acid sequence shown in SEQ ID NO: 1, the peptide having the amino acid sequence shown in SEQ ID NO: 2, and the peptide having the amino acid sequence shown in SEQ ID NO: 3 as an active ingredient. A composition is provided.
또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a peptide having the amino acid sequence set forth in SEQ ID NO: 1, a peptide having the amino acid sequence set forth in SEQ ID NO: 2, and a peptide having the amino acid sequence set forth in SEQ ID NO: 3 as an active ingredient. A pharmaceutical composition for prevention or treatment is provided.
또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 항염용 식품 조성물을 제공한다.In addition, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides an anti-inflammatory food composition, characterized in that.
한편, 본 발명의 식품 조성물에 있어서, 상기 흑염소육골즙 효소분해물은, 바람직하게 알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것일 수 있다.On the other hand, in the food composition of the present invention, the enzyme lysate of black goat meat bone juice may be prepared by preferably treating alcalase, proteAX and trypsin.
또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 항염용 화장료 조성물을 제공한다.In addition, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides an anti-inflammatory cosmetic composition, characterized in that.
한편, 본 발명의 화장료 조성물에 있어서, 상기 흑염소육골즙 효소분해물은, 바람직하게 알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것일 수 있다.On the other hand, in the cosmetic composition of the present invention, the enzyme decomposition product of black goat meat bone juice may be prepared by preferably treating alcalase, proteAX, and trypsin.
또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that.
한편, 본 발명의 약학 조성물에 있어서, 상기 흑염소육골즙 효소분해물은, 바람직하게 알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것일 수 있다.On the other hand, in the pharmaceutical composition of the present invention, the enzyme decomposition product of black goat meat bone juice may be prepared by treating with alcalase, proteAX, and trypsin.
본 발명은 흑염소육골을 열수 추출하여 수득한 흑염소육골즙을 최적의 효소 구성으로 효소분해하여 효소분해물을 수득하고, 이로부터 서열번호 1, 2, 3의 아미노산 서열을 갖는 유효 펩타이드를 분리하는 방법을 제공한다. 상기와 같이 수득한 본 발명의 흑염소육골즙 가수분해물 및 이로부터 분리된 유효 펩타이드는 산화질소 및 프로스타글란딘 E2 생성 억제를 통한 높은 항염 효과를 발휘하여 항염용 기능성 식품, 기능성 화장품이나, 염증성 질환의 예방 또는 치료를 위한 기능성 약품으로 유용하게 사용될 수 있다.The present invention enzymatically decomposes black goat meat bone juice obtained by hot water extraction of black goat meat bone with an optimal enzyme composition to obtain an enzymatic lysate, and a method for isolating an effective peptide having the amino acid sequence of SEQ ID NOs: 1, 2 and 3 therefrom. to provide. The hydrolyzate of black goat meat bone juice of the present invention obtained as described above and the effective peptide isolated therefrom exert a high anti-inflammatory effect by inhibiting the production of nitric oxide and prostaglandin E 2 Or it may be usefully used as a functional drug for treatment.
도 1은 본 발명의 저분자 흑염소육골즙 효소분해물을 제조하는 과정을 나타낸 모식도이다.
도 2는 본 발명의 저분자 흑염소육골즙 효소분해물 처리에 따른 마우스 대식세포의 생존율을 측정한 결과 그래프이다.
도 3은 본 발명의 저분자 흑염소육골즙 효소분해물 처리에 따른 마우스 대식세포의 산화질소(NO) 생성량을 측정한 결과 그래프이다.
도 4는 본 발명의 저분자 흑염소육골즙 효소분해물 처리에 따른 마우스 대식세포의 프로스타글란딘 E2(PGE2) 생성량을 측정한 결과 그래프이다.
도 5는 본 발명의 저분자 흑염소육골즙 효소분해물로부터 분배크로마토그래피를 이용한 유효 펩타이드 분획(F1~F5)의 크로마토그램 결과 그래프이다.
도 6은 본 발명의 저분자 흑염소육골즙 효소분해물로부터의 유효 펩타이드 분획의 항산화 활성(ORAC)을 분석한 결과 그래프이다.
도 7은 본 발명의 저분자 흑염소육골즙 효소분해물로부터의 유효 펩타이드 분획의 산화질소, 프로스타글란딘 E2 생성량을 측정한 결과 그래프이다.1 is a schematic diagram showing a process for preparing a low-molecular-weight black goat meat and bone juice enzyme lysate of the present invention.
Figure 2 is a graph showing the results of measuring the survival rate of mouse macrophages according to the low-molecular weight black goat meat bone juice enzyme lysate treatment of the present invention.
Figure 3 is a graph showing the result of measuring the amount of nitric oxide (NO) production in mouse macrophages according to the low molecular weight black goat meat bone juice enzyme lysate treatment of the present invention.
4 is a graph showing the result of measuring the amount of prostaglandin E 2 (PGE 2 ) produced in mouse macrophages according to the treatment with the enzyme lysate of low molecular weight black goat meat bone juice of the present invention.
5 is a graph showing the results of chromatograms of the effective peptide fractions (F1 to F5) using partition chromatography from the enzyme lysate of low molecular weight black goat meat bone juice of the present invention.
6 is a graph showing the results of analyzing the antioxidant activity (ORAC) of the effective peptide fraction from the enzyme lysate of low molecular weight black goat meat bone juice of the present invention.
7 is a graph showing the results of measuring the production of nitric oxide and prostaglandin E 2 in the effective peptide fraction from the enzyme lysate of low molecular weight black goat meat bone juice of the present invention.
옛날부터 흑염소는 아이들 성장, 골다공증 에방, 빈혈, 정력증진, 피부질환 증상 완화, 성인병(동맥경화, 고혈압, 심장병) 완화 등 다양한 효능이 있다고 알려져 보양식, 건강식으로 이용되고 있다. 또한, 흑염소는 다른 식육(소, 돼지, 닭)에 비해 칼슘과 철의 함량이 높고 포화지방산과 콜레스테롤 함유량이 적다. 우리나라는 흑염소를 주로 육골즙의 형태로 섭취하고 있으며 흑염소육골즙 또한 환자 노약자, 임산부의 기력회복 및 원기회복에 좋다고 알려져 있다. 하지만 흑염소육골즙의 면역증진 및 항염증 효과에 대한 연구는 아직 미흡한 실정이다. 따라서 본 발명은 면역증진 및 항염증 효과를 발휘하기 위한 소재로 흑염소를 이용하여 최적의 방법을 찾고자 예의 노력하였다. Since ancient times, black goat has been known to have various effects such as children's growth, prevention of osteoporosis, anemia, stamina, alleviation of skin disease symptoms, and alleviation of adult diseases (arteriosclerosis, high blood pressure, heart disease), and has been used as a health food and health food. In addition, compared to other meats (cow, pig, chicken), black goat has a higher content of calcium and iron and lower content of saturated fatty acids and cholesterol. In Korea, black goat is mainly consumed in the form of meat and bone juice, and black goat meat and bone juice is also known to be good for the recovery of energy and vitality of the elderly and pregnant women. However, studies on the immune-enhancing and anti-inflammatory effects of black goat meat bone juice are still insufficient. Therefore, in the present invention, intensive efforts were made to find an optimal method using black goat as a material for enhancing immunity and exhibiting anti-inflammatory effects.
이에 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 항염용 식품 조성물을 제공한다. 또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 항염용 화장료 조성물을 제공한다. 또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 유효성분으로 함유하는 것을 특징으로 하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다. Accordingly, the present invention provides an anti-inflammatory food composition comprising, as an active ingredient, a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 provides In addition, the present invention is an anti-inflammatory cosmetic comprising the peptide having the amino acid sequence shown in SEQ ID NO: 1, the peptide having the amino acid sequence shown in SEQ ID NO: 2, and the peptide having the amino acid sequence shown in SEQ ID NO: 3 as an active ingredient. A composition is provided. In addition, the present invention provides a peptide having the amino acid sequence set forth in SEQ ID NO: 1, a peptide having the amino acid sequence set forth in SEQ ID NO: 2, and a peptide having the amino acid sequence set forth in SEQ ID NO: 3 as an active ingredient. A pharmaceutical composition for prevention or treatment is provided.
한편, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 항염용 식품 조성물을 제공한다. 또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 항염용 화장료 조성물을 제공한다. 또한, 본 발명은 서열번호 1에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 2에 기재된 아미노산 서열을 갖는 펩타이드, 서열번호 3에 기재된 아미노산 서열을 갖는 펩타이드를 포함하는 흑염소육골즙 효소분해물을 유효성분으로 함유하는 것을 특징으로 하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides an anti-inflammatory food composition, characterized in that. In addition, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides an anti-inflammatory cosmetic composition, characterized in that. In addition, the present invention contains, as an active ingredient, an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 It provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that.
한편, 본 발명에 있어서, 상기 흑염소육골즙은, 당 업계에서 추출에 사용하는 방법이라면 어느 것이든 제한되지 않으나, 바람직하게 흑염수육골 무게 대비 2~4배의 물을 첨가하여 열수 추출하는 것이 좋다. 더욱 바람직하게는 110~130℃에서 8~12시간 열수 추출하는 것이 좋다. On the other hand, in the present invention, the black goat meat bone juice is not limited as long as it is a method used for extraction in the art, but it is preferable to extract it with hot water by adding 2 to 4 times the weight of the black goat meat bone. . More preferably, hot water extraction is performed at 110-130° C. for 8-12 hours.
한편, 본 발명에 있어서, 상기 흑염소육골즙 효소분해물은, 효소분해를 수행하기 전 지방을 제거하는 것이 좋으며, 바람직하게 서로 다른 종류의 가수분해효소를 사용하여 여러 번 수행하는 것이 좋다. 더욱 바람직하게는 알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)의 3가지 종류의 효소를 사용하되 이들을 알칼라제, 프로테 에이엑스, 트립신 순서대로 각각 1~3시간씩 처리하는 것이 좋다. 이때, 프로테 에이엑스(proteAX)는 프로테아제의 일종이다.On the other hand, in the present invention, it is preferable to remove fat from the enzymatic decomposition product of black goat meat bone juice before enzymatic decomposition, and it is preferable to perform it several times using different types of hydrolytic enzymes. More preferably, three types of enzymes, alcalase, proteAX, and trypsin, are used, but these are used in the order of alcalase, prote AX and trypsin for 1 to 3 hours, respectively. It's better to deal with them one by one. At this time, prote AX (proteAX) is a kind of protease.
한편, 본 발명에 있어서, 상기 흑염소육골즙 효소분해물은, 효소처리한 후 저분자(3kDa 이하)로 분리하는 것이 좋다.On the other hand, in the present invention, the enzymatic lysate of black goat meat bone juice is preferably separated into low molecular weight (3 kDa or less) after enzyme treatment.
한편, 하기 실험에 의하면, 본 발명에서 개시하는 저분자 흑염소육골즙 효소분해물은 세포 독성을 나타내지 않아 안전한 조성물로 이용될 수 있었고, 산화질소 생성 억제 및 프로스타글란딘 E2 생성 억제를 통해 항염 효과가 뛰어난 것을 확인하였다. 또한, 상기 저분자 흑염소육골즙 효소분해물로부터 분리된 유효 펩타이드 분획은 우수한 항산화 활성을 나타내면서, 상기 저분자 흑염소육골즙 효소분해물에 비해서도 훨씬 개선된 항염 활성(산화질소 생성 억제, 프로스타글란딘 E2 생성 억제)을 나타내는 것을 확인하였다. 이를 통해 분리된 유효 펩타이드 분획으로부터 가장 뛰어난 항염 활성을 나타냈던 펩타이드에 대한 서열을 규명한 결과, 서열번호 1(Gly-Ala-Xaa-Gly-Phe-Xaa-Gly-Ala-Arg)의 아미노산 서열을 가지고 서열번호 2(Val-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Xaa-Gly-Pro-Xaa-Gly-Pro-Ala)의 아미노산 서열을 가지며, 서열번호 3(Gly-Val-Xaa-Gly-Pro-Xaa-Gly-Ala-Val-Gly-Pro-Ala)의 아미노산 서열을 가지는 것을 확인하였다. 이와 같이 본 발명의 저분자 흑염소육골즙 효소분해물 및 이로부터 분리한 유효 펩타이드는 뛰어난 항염 효과를 발휘하여 기능성 식품, 화장품 및 약품의 소재로 이용될 수 있음을 확인할 수 있었다.On the other hand, according to the following experiment, the enzyme lysate of low molecular weight black goat meat bone juice disclosed in the present invention did not show cytotoxicity, so it could be used as a safe composition, and it was confirmed that the anti-inflammatory effect was excellent through inhibition of nitric oxide production and prostaglandin E 2 production did In addition, the effective peptide fraction isolated from the low-molecular black goat meat bone juice enzyme lysate exhibits excellent antioxidant activity and much improved anti-inflammatory activity (inhibition of nitric oxide production, inhibition of prostaglandin E 2 production) compared to the low-molecular black goat meat bone juice enzyme lysate. confirmed that. As a result of identifying the sequence of the peptide that showed the best anti-inflammatory activity from the isolated effective peptide fraction, the amino acid sequence of SEQ ID NO: 1 (Gly-Ala-Xaa-Gly-Phe-Xaa-Gly-Ala-Arg) was determined. has the amino acid sequence of SEQ ID NO: 2 (Val-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Xaa-Gly-Pro-Xaa-Gly-Pro-Ala), and SEQ ID NO: 3 (Gly-Val -Xaa-Gly-Pro-Xaa-Gly-Ala-Val-Gly-Pro-Ala) was confirmed. As described above, it was confirmed that the low-molecular-weight black goat meat bone juice enzyme lysate of the present invention and the effective peptide isolated therefrom exhibit excellent anti-inflammatory effects and can be used as a material for functional foods, cosmetics, and pharmaceuticals.
한편, 본 발명에 있어서, 서열목록을 작성할 시, 서열번호 1 내지 3에서 'Xaa'라고 기재한 부분은 '4Hyp(4-Hydroxyproline)'이며 이와 같은 사항은 각 서열번호의 특징 부분에 기재되어 있다.On the other hand, in the present invention, when preparing the sequence list, the part described as 'Xaa' in SEQ ID NOs: 1 to 3 is '4Hyp(4-Hydroxyproline)', and such matters are described in the characteristic part of each SEQ ID NO. .
한편, 본 발명의 식품 조성물은 바람직하게 육류, 곡류, 카페인 음료, 일반음료, 초콜렛, 빵류, 스넥류, 과자류, 피자, 젤리, 면류, 껌류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나인 것인 것이 좋으나, 이에 한정되는 것은 아니다.On the other hand, the food composition of the present invention is preferably meat, grains, caffeinated beverages, general drinks, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, vitamin complexes and other health It is preferable that any one of the supplements is selected, but the present invention is not limited thereto.
한편, 본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 일 예로, 용액, 현탁액, 유탁액, 페이스트, 화장수, 젤, 수용성 리퀴드, 크림, 에센스, 계면활성제-함유 클렌징, 오일, 수중유(O/W)형 및 유중수(W/O)형 중 선택되는 어느 하나의 기초 화장료 제형; 스킨; 로션; 아이크림; 수딩젤; 연고; 마스크팩용 제형; 바디워시용 제형; 필링젤; 수중유형 및 유중수형 메이크업베이스; 파운데이션; 스킨커버; 립스틱, 립그로스, 페이스파우더, 투웨이케익, 아이새도, 치크칼라 및 아이브로우 펜슬류 중 선택되는 어느 하나의 색조화장료 제형; 두피용 제형; 중에서 선택되는 어느 하나인 것일 수 있다.On the other hand, the cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, lotion, gel, water-soluble liquid, cream, essence, surfactant- Any one basic cosmetic formulation selected from containing cleansing, oil, oil-in-water (O/W) type and water-in-oil (W/O) type; skin; Lotion; eye cream; soothing gel; Ointment; Formulations for mask packs; formulation for body wash; peeling gel; Oil-in-water and water-in-oil makeup base; foundation; skin cover; Any one color cosmetic formulation selected from lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencil; formulations for the scalp; It may be any one selected from among.
또한, 본 발명의 화장료 조성물은 화장 분야에서 통상적으로 사용되는 보조제 예컨대 친수성 또는 친유성 활성제, 보존제, 항산화제, 용매, 방향제, 충전제, 차단제, 안료, 흡취제, 염료 등을 함유할 수 있다. 이들 다양한 보조제의 양은 당해 분야에서 통상적으로 사용되는 양이며, 예컨대 조성물 총 중량에 대해 0.001 내지 30 중량% 이다. 다만, 어떠한 경우라도 보조제 및 그 비율은 본 발명에 따른 화장료 조성물의 바람직한 성질에 악영향을 미치지 않도록 선택될 것이다.In addition, the cosmetic composition of the present invention may contain adjuvants commonly used in the cosmetic field, such as hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, deodorants, dyes, and the like. The amount of these various adjuvants is an amount conventionally used in the art, for example, 0.001 to 30% by weight relative to the total weight of the composition. However, in any case, the auxiliary agent and its ratio will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.
또한, 본 발명의 화장료 조성물에 있어서, 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, Talc or zinc oxide may be used.
또한, 본 발명의 화장료 조성물에 있어서, 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 일 예로 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤조에이트, 프로필렌글리콜, 1,3-부틸렌글리콜, 글리세롤 지방족 에스테르, 폴리에틸렌글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzoate, propylene glycol, 1,3-butylene glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used.
또한, 본 발명의 화장료 조성물에 있어서, 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라가칸트 등이 이용될 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester Suspending agents such as, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.
또한, 본 발명의 화장료 조성물에 있어서, 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, 프로탄/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.In addition, in the cosmetic composition of the present invention, when the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.
또한, 본 발명의 화장료 조성물에 있어서, 제형이 계면활성제 함유 클렌저인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 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.In addition, in the cosmetic composition of the present invention, when the formulation is a surfactant-containing cleanser, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.
또한, 본 발명의 화장료 조성물에 있어서, 계면활성제 함유 클렌저 제형 또는 계면활성제 비함유 클렌저 제형 또는 비누일 경우에는, 피부에 도포한 후 닦아내거나 떼거나 물로 씻어낼 수도 있다. 일례로, 상기 비누는 액상비누, 가루비누, 고형비누 및 오일비누이며, 상기 계면활성제 함유 클렌징 제형은 클렌징폼, 클렌징 워터, 클렌징 수건 및 클렌징 팩이며, 상기 계면활성제 비함유 클렌징 제형은 클렌징크림, 클렌징 로션, 클렌징 워터 및 클렌징 젤이며, 이에 한정되는 것은 아니다.In addition, in the cosmetic composition of the present invention, in the case of a surfactant-containing cleanser formulation or a surfactant-free cleanser formulation or soap, it may be applied to the skin and then wiped off, removed, or washed off with water. For example, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream, cleansing lotions, cleansing waters, and cleansing gels, but not limited thereto.
또한, 본 발명의 화장료 조성물은 본 발명 이외의 다른 화장료 조성물과 중복하여 사용할 수 있다. 또한 본 발명에 따른 화장료 조성물은 통상적인 사용방법에 따라 사용될 수 있으며, 사용자의 피부 상태 또는 취향에 따라 그 사용횟수를 달리할 수 있다.In addition, the cosmetic composition of the present invention can be used overlapping with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention can be used according to a conventional method of use, and the number of times of use can be varied according to the skin condition or taste of the user.
한편, 본 발명의 약학 조성물의 제형은 사용방법에 따라 바람직한 형태로 제조될 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LPTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPESIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다. On the other hand, the dosage form of the pharmaceutical composition of the present invention may be prepared in a desired form according to the method of use, and in particular, methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal It is better to adopt and formulate it. Examples of specific formulations include PLASTERS, GRANULES, LPTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, syrups ( SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), suspensions (SUSPESIONS), preparations (DECOCTIONS), infusions (INFUSIONS), eye drops (OPHTHALMIC SOLUTIONS), tablets (TABLETS), suppositories (SUPPOSITIORIES), injections (INJECTIONS), alcohol tablets (SPIRITS), CATAPLS ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pastas (PASTES), pills (PILLS), it may be any one selected from soft or hard gelatin capsules.
또한, 본 발명의 약학 조성물의 투여량은 투여방법, 복용자의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분을 기준으로 하였을 때 1일 0.1 내지 100 ㎎/㎏(체중)으로 1회 이상 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.In addition, the dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, based on the active ingredient, 0.1 to 100 mg/kg (body weight) per day may be administered at least once. However, the above dosage is only an example for illustration, and may be changed by the condition of the user and the prescription of the doctor.
또한, 본 발명의 약학 조성물은 유효성분 이외에 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용 가능한 담체, 부형제 또는 희석제로는 일 예로, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 1종 이상 사용될 수 있다. 또한, 예방 및 치료제가 약제인 경우 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등이 추가적으로 포함될 수 있다.In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Usable carriers, excipients or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl There are cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and at least one of them may be used. In addition, when the prophylactic and therapeutic agents are pharmaceuticals, fillers, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives may be additionally included.
이하, 본 발명에 대해 하기 실시예 및 실험예에서 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고, 이와 등가의 기술적 사상의 변형까지를 모두 포함한다.Hereinafter, the present invention will be described in more detail in the following Examples and Experimental Examples. However, the scope of the present invention is not limited only to the following examples and experimental examples, and includes all modifications of the technical idea equivalent thereto.
[실시예 1 : 저분자 흑염소육골즙 효소분해물 제조][Example 1: Preparation of low-molecular-weight black goat meat and bone juice enzyme lysate]
흑염소육골 무게에 3배의 물 첨가후 121℃에서 10시간 추출하였으며, 추출된 육골즙을 동결건조하였다. 흑염소육골즙 추출물 내 지방은 Floch 방법을 이용해 제거 후 0.67% Alcalase(pH 8.0, 50℃), 0.67% ProteAX(pH 7.0, 50℃)와 0.67% Trypsin(pH 8.0, 37℃)를 순서대로 첨가하여 각각 2시간씩 총 6시간 분해하여 효소분해물을 제조하였다. 이후 centrifugal filter device(Millipore, USA)를 이용하여 저분자(3kDa 이하)로 분리하였다. 도 1은 본 발명의 저분자 흑염소육골즙 효소분해물을 제조하는 과정을 나타낸 모식도이다.After adding water three times to the weight of black goat meat, extraction was performed at 121° C. for 10 hours, and the extracted meat bone juice was freeze-dried. After removing the fat in the black goat meat bone juice extract using the Floch method, 0.67% Alcalase (pH 8.0, 50℃), 0.67% ProteAX (pH 7.0, 50℃) and 0.67% Trypsin (pH 8.0, 37℃) were added in order. Enzyme lysate was prepared by digestion for a total of 6 hours for 2 hours each. Thereafter, it was separated into small molecules (3 kDa or less) using a centrifugal filter device (Millipore, USA). 1 is a schematic diagram showing a process for preparing a low molecular weight black goat meat and bone juice enzyme decomposition product of the present invention.
[실험예 1 : 저분자 흑염소육골즙 효소분해물의 아미노산 조성 분석][Experimental Example 1: Analysis of amino acid composition of low-molecular black goat meat and bone juice enzyme lysate]
본 실험예에서는 상기 실시예 1의 저분자 흑염소육골즙 효소분해물의 아미노산 조성을 분석하였다.In this experimental example, the amino acid composition of the low-molecular-weight black goat meat and bone juice enzymatic lysate of Example 1 was analyzed.
아미노산 조성은 시료(저분자 흑염소육골즙 효소분해물) 0.5 g과 6N HCl 10 mL를 삼각플라스크에 넣고 혼합한 다음 110℃에서 24시간 가수분해하였다. 가수분해물을 0.2N sodium citrate buffer(pH 2.2)에 희석시키고 0.45 ㎛ 멤브레인 필터로 여과하였다. 시스테인(cysteine)과 메티오닌(methionine)은 과산화포름산(perfomic acid)으로 가수분해한 후 6N HCl로 가수분해하였다. 여과한 시료는 아미노산 분석기(SYKAM S433, Sykam GmbH, Eresing, Germany)를 이용하여 분석하였다.For amino acid composition, 0.5 g of a sample (enzymatic digest of low molecular weight black goat meat bone juice) and 10 mL of 6N HCl were placed in an Erlenmeyer flask, mixed, and then hydrolyzed at 110° C. for 24 hours. The hydrolyzate was diluted in 0.2N sodium citrate buffer (pH 2.2) and filtered through a 0.45 μm membrane filter. Cysteine and methionine were hydrolyzed with perfomic acid and then hydrolyzed with 6N HCl. The filtered sample was analyzed using an amino acid analyzer (SYKAM S433, Sykam GmbH, Eresing, Germany).
실험결과, 하기 표 1과 같이 저분자 흑염소육골즙 효소분해물의 아미노산 조성을 확인할 수 있었다. 주요 아미노산은 글루탐산(glutamic acid)과 글리신(glycine)으로 각각 13.23%, 9.94%를 나타냈다. 또한, 필수아미노산의 비율은 35%, 항산화 및 소수성 아미노산의 비율은 각각 41.24%, 49.55%를 나타냈다.As a result of the experiment, as shown in Table 1 below, the amino acid composition of the low-molecular black goat meat and bone juice enzyme lysate could be confirmed. The major amino acids were glutamic acid and glycine, which were 13.23% and 9.94%, respectively. In addition, the ratio of essential amino acids was 35%, and the ratios of antioxidant and hydrophobic amino acids were 41.24% and 49.55%, respectively.
[실험예 2 : 저분자 흑염소육골즙 효소분해물 처리에 따른 세포 독성 측정][Experimental Example 2: Measurement of cytotoxicity according to low-molecular-weight black goat meat and bone juice enzyme lysate treatment]
본 실험예에서는 상기 실시예 1의 저분자 흑염소육골즙 효소분해물 처리에 따른 마우스 대식세포에 대한 세포독성을 측정하기 위해 MTT 방법(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)을 이용하여 세포 생존율을 측정하였다.In this experimental example, the MTT method (3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide) was used to measure cell viability.
마우스 대식세포(RAW264.7 cell)를 48 well cell culture plate에 5 x 104 cells/well의 농도로 배양한 후 저분자 흑염소육골즙 효소분해물을 10~500 ㎍/mL 농도로 처리하였다. 24시간 배양 후 PBS로 세척한 다음 MTT(5 mg/mL) 용액을 세포에 4시간 동안 처리하여 염색시켰다. 염색된 세포에 DMSO를 넣어 세포를 녹여낸 후 540 nm 파장에서 흡광도를 측정하였다. 이후 하기 수학식 1을 이용하여 세포 생존율을 산출하였다.Mouse macrophages (RAW264.7 cells) were cultured in a 48-well cell culture plate at a concentration of 5 x 10 4 cells/well, and then treated with a low-molecular-weight black goat meat bone juice enzyme lysate at a concentration of 10-500 μg/mL. After incubation for 24 hours, the cells were washed with PBS and stained with MTT (5 mg/mL) solution for 4 hours. After dissolving the cells by adding DMSO to the stained cells, the absorbance was measured at a wavelength of 540 nm. Thereafter, the cell viability was calculated using
실험결과, 도 2와 같이 저분자 흑염소육골즙 효소분해물은 최고 농도 500 ㎍/mL 처리시에도 세포 생존율을 감소시키지 않았으며, 오히려 대식세포 생존율을 증가시켰다. 이와 같은 결과로부터 저분자 흑염소육골즙 효소분해물 세포 독성을 나타내지 않아 안전한 조성물로 이용될 수 있음을 확인하였다.As a result of the experiment, as shown in FIG. 2 , the enzyme lysate of low molecular weight black goat meat bone juice did not decrease the cell viability even at the highest concentration of 500 μg/mL, but rather increased the macrophage viability. From these results, it was confirmed that the low-molecular-weight black goat meat and bone juice enzyme lysate did not show cytotoxicity and could be used as a safe composition.
[실험예 3 : 저분자 흑염소육골즙 효소분해물의 산화질소 및 프로스타글란딘 E[Experimental Example 3: Nitric oxide and prostaglandin E 2 2 생성 억제 효과 분석]Generation inhibitory effect analysis]
본 실험예에서는 상기 실시예 1의 저분자 흑염소육골즙 효소분해물의 항염증 활성을 확인하기 위해 지질다당류(LPS)로 염증을 유도한 마우스 대식세포 내 산화질소(NO) 및 프로스타글란딘 E2(PGE2) 생성 억제 효과를 분석하였다.In this experimental example, nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) in mouse macrophages induced by inflammation with lipopolysaccharide (LPS) to confirm the anti-inflammatory activity of the enzyme lysate of low molecular weight black goat meat bone juice of Example 1 The production inhibitory effect was analyzed.
1) 산화질소(N0) 생성 억제 효과 분석1) Analysis of nitric oxide (N0) production inhibitory effect
마우스 대식세포(RAW264.7 cell)를 48 well cell culture plate에 4 x 105 cells/well의 농도로 배양한 후 저분자 흑염소육골즙 효소분해물을 10~500 ㎍/mL 농도로 처리하였다. 1시간 배양 후 LPS(1 ㎍/mL)로 자극하여 24시간 배양하였다. 배양 종료 후 배양액을 취하여 원심분리(10,000 rpm, 10분)하여 상등액을 분석에 이용하였다. 50 ㎕의 배양을 취하여 동일부피의 그리에스(Griess) 시약과 혼합한 후 550 nm에서 흡광도를 측정하였다. NO의 농도는 아질산나트륨(sodium nitrite) 표준 곡선을 이용하여 정량하였다.Mouse macrophages (RAW264.7 cells) were cultured in a 48-well cell culture plate at a concentration of 4 x 10 5 cells/well, and then treated with a low-molecular-weight black goat meat bone juice enzyme lysate at a concentration of 10-500 μg/mL. After 1 hour of incubation, the cells were stimulated with LPS (1 μg/mL) and incubated for 24 hours. After completion of the culture, the culture solution was taken and centrifuged (10,000 rpm, 10 minutes), and the supernatant was used for analysis. After taking a culture of 50 μl and mixing it with the same volume of Griess reagent, absorbance was measured at 550 nm. The concentration of NO was quantified using a sodium nitrite standard curve.
실험결과, 도 3과 같이 저분자 흑염소육골즙 효소분해물 100, 250, 500 ㎍/mL의 농도에서 NO 생성을 각각 7.82, 6.98, 10.65% 억제시키는 것으로 확인되었다. 이와 같은 결과로부터 염증성 인자 중 산화질소 억제 효과로부터 저분자 흑염소육골즙 효소분해물의 항염증 효과를 확인하였다.As a result of the experiment, as shown in FIG. 3 , it was confirmed that NO production was inhibited by 7.82, 6.98, and 10.65% at concentrations of 100, 250, and 500 μg/mL of low molecular weight black goat meat and bone juice enzyme lysate, respectively. From these results, the anti-inflammatory effect of the enzyme lysate of low molecular weight black goat meat bone juice was confirmed from the inhibitory effect of nitric oxide among inflammatory factors.
2) 프로스타글란딘 E2) Prostaglandin E 22 (PGE(PGE 22 ) 생성 억제 효과 분석) production inhibitory effect analysis
마우스 대식세포(RAW264.7 cell)를 48 well cell culture plate에 4 x 105 cells/well의 농도로 배양한 후 저분자 흑염소육골즙 효소분해물을 10~500 ㎍/mL 농도로 처리하였다. 1시간 배양 후 LPS(1 ㎍/mL)로 자극하여 24시간 배양하였다. 배양 종료 후 배양액을 취하여 원심분리(10,000 rpm, 10분)하여 상등액을 분석에 이용하였다. PGE2의 농도는 ELISA 키트를 이용하여 측정하였다.Mouse macrophages (RAW264.7 cells) were cultured in a 48-well cell culture plate at a concentration of 4 x 10 5 cells/well, and then treated with a low-molecular-weight black goat meat bone juice enzyme lysate at a concentration of 10-500 μg/mL. After 1 hour of incubation, the cells were stimulated with LPS (1 μg/mL) and incubated for 24 hours. After completion of the culture, the culture solution was taken and centrifuged (10,000 rpm, 10 minutes), and the supernatant was used for analysis. The concentration of PGE 2 was measured using an ELISA kit.
실험결과, 도 4와 같이 저분자 흑염소육골즙 효소분해물 250, 500 ㎍/mL의 농도에서 PGE2 생성을 각각 17.13, 29.86% 억제시키는 것으로 확인되었다. 이와 같은 결과로부터 염증성 인자 중 프로스타글란딘 E2 억제 효과로부터 저분자 흑염소육골즙 효소분해물의 항염증 효과를 확인하였다.As a result of the experiment, as shown in FIG. 4 , it was confirmed that PGE 2 production was inhibited by 17.13 and 29.86%, respectively, at concentrations of 250 and 500 μg/mL of the enzyme lysate of low molecular weight black goat meat bone juice. From these results, the anti-inflammatory effect of the enzyme lysate of low molecular weight black goat meat bone juice was confirmed from the inhibitory effect of prostaglandin E 2 among inflammatory factors.
[실시예 2 : 저분자 흑염소육골즙 효소분해물로부터 유효 펩타이드 분리][Example 2: Separation of effective peptides from low-molecular-weight black goat meat and bone juice enzyme lysate]
실시예 1의 저분자 흑염소육골즙 효소분해물을 200 mg/mL로 증류수에 녹인 후 0.22 ㎛ 필터하여 분배크로마토그래피법을 이용하여 정제하였다. 정제에 이용된 컬럼은 Superdex Peptide 30/100GL (10 x 300 mm)을 이용하였으며, 유속은 0.5 mL/min으로 증류수를 60분 동안 일정한 유속으로 흘려주며 215 nm에서 검출하였다. 검출된 크로마토그램을 기준으로 5개의 유효 펩타이드 분획(F1~F5)를 분리하였으며, 그 결과는 도 5와 같았다.The low-molecular-weight black goat meat and bone juice enzyme lysate of Example 1 was dissolved in distilled water at 200 mg/mL, filtered with 0.22 μm, and purified by partition chromatography. The column used for purification was
[실험예 4 : 유효 펩타이드의 항산화 활성 분석][Experimental Example 4: Analysis of Antioxidant Activity of Effective Peptides]
본 실험예에서는 상기 실시예 2의 유효 펩타이드 분획(F1~F5)의 항산화 활성은 ORAC(Oxygen Radical Absorbance Capacity) 활성 측정을 통해 확인하였다. In this experimental example, the antioxidant activity of the effective peptide fractions (F1 to F5) of Example 2 was confirmed through ORAC (Oxygen Radical Absorbance Capacity) activity measurement.
ORAC 활성은 black 96 well plate에 25 ㎕ 표준시약(trolox) 또는 시료(저분자 흑염소육골즙 효소분해물, 유효 펩타이드 분획(F1~F5))를 넣고, 150 ㎕ fluorescein (80 nM)을 넣어 혼합한 뒤 37℃ 인큐베이터에서 15분 동안 인큐베이션하였다. 이후 25 ㎕ AAPH(150 mM)을 넣고 혼합한 뒤 형광분광광도계를 사용하여 37℃에서 285~520 nm 파장으로 60분 동안 1분 간격으로 형광도의 감소율을 측정하였다. 표준시약(trolox)과 시료의 area under the curve(AUC)를 측정하였으며, 표준시약 농도와 AUC 간의 회귀곡선을 이용하여 μmol trolox equivalent(TE)/g으로 나타내었다. For ORAC activity, put 25 μl of standard reagent (trolox) or sample (low molecular weight black goat meat and bone juice enzyme lysate, effective peptide fraction (F1-F5)) in a black 96 well plate, add 150 μl fluorescein (80 nM), and mix 37 Incubated for 15 minutes in an incubator at °C. Then, 25 μl of AAPH (150 mM) was added and mixed, and the reduction in fluorescence was measured at 1 minute intervals for 60 minutes at a wavelength of 285 to 520 nm at 37° C. using a fluorescence spectrophotometer. The area under the curve (AUC) of the standard reagent (trolox) and the sample was measured, and expressed as μmol trolox equivalent (TE)/g using a regression curve between the standard reagent concentration and AUC.
실험결과, 도 6과 같이 F3, F4, F5가 각각 690.18, 824.64, 979.88 μmol TE/g으로 원물(저분자 흑염소육골즙 효소분해물) 247.44 μmol TE/g보다 최소 2.8배 증가시켰으며, 특히 F5가 가장 높은 항산화 활성을 나타내었다.As a result of the experiment, as shown in FIG. 6, F3, F4, and F5 were 690.18, 824.64, and 979.88 μmol TE/g, respectively, which increased at least 2.8 times than the raw material (low-molecular black goat meat enzymatic decomposition product) 247.44 μmol TE/g, and in particular, F5 was the most It showed high antioxidant activity.
[실험예 5: 유효 펩타이드의 산화질소 및 프로스타글란딘 E[Experimental Example 5: Nitric oxide and prostaglandin E of effective peptides 2 2 생성량 분석 및 펩타이드 서열 규명]Production amount analysis and peptide sequence identification]
본 실험예에서는 상기 실시예 2의 유효 펩타이드 분획(F1~F5)의 항염증 활성을 확인하기 위해 지질다당류(LPS)로 염증을 유도한 마우스 대식세포 내 산화질소(NO) 및 프로스타글란딘 E2(PGE2) 생성 억제 효과를 분석하였다.In this experimental example, in order to confirm the anti-inflammatory activity of the effective peptide fractions (F1 to F5) of Example 2, nitric oxide (NO) and prostaglandin E 2 (PGE) in mouse macrophages induced by inflammation with lipopolysaccharide (LPS) 2 ) The production inhibitory effect was analyzed.
마우스 대식세포(RAW264.7 cell)를 48 well cell culture plate에 4 x 105 cells/well의 농도로 배양한 후 유효 펩타이드 분획 F1~F5을 250 ㎍/mL 농도로 처리하였다. 1시간 배양 후 LPS(1 ㎍/mL)로 자극하여 24시간 배양하였다. 배양 종료 후 배양액을 취하여 원심분리(10,000 rpm, 10분)하여 상등액을 분석에 이용하였다. 실험예 3과 같이 NO는 그리에스(Griess) 시약을 이용하여 분석하였으며, PGE2는 ELISA 키트를 이용하여 분석하였다. 실험결과는 LPS 처리구 내 생산량을 100%로 하여 계산하였다.Mouse macrophages (RAW264.7 cells) were cultured in a 48-well cell culture plate at a concentration of 4 x 10 5 cells/well, and then treated with effective peptide fractions F1 to F5 at a concentration of 250 μg/mL. After 1 hour of incubation, the cells were stimulated with LPS (1 μg/mL) and incubated for 24 hours. After completion of the culture, the culture solution was taken and centrifuged (10,000 rpm, 10 minutes), and the supernatant was used for analysis. As in Experimental Example 3, NO was analyzed using a Griess reagent, and PGE 2 was analyzed using an ELISA kit. Experimental results were calculated with 100% of the production in the LPS treatment group.
실험결과, 도 7의 A)와 같이 F2, F4, F5가 각각 86.26, 90.90, 83.94%로 원물(저분자 흑염소육골즙 효소분해물) 93.02%보다 감소시켰으며, 특히 F5가 가장 낮은 NO 생성량을 나타내었다. 또한, 도 7의 B)와 같이 F2, F4, F5가 각각 33.07, 57.99, 27.79%로 원물(저분자 흑염소육골즙 효소분해물) 82.87%보다 감소시켰으며, 특히 F5가 가장 낮은 PGE2 생성량을 나타내었다. As a result of the experiment, as shown in FIG. 7A), F2, F4, and F5 were 86.26, 90.90, and 83.94%, respectively, which were reduced compared to 93.02% of the raw material (low molecular weight black goat meat and bone juice enzymatic lysate), and in particular, F5 showed the lowest NO production. . In addition, as shown in FIG. 7B), F2, F4, and F5 were 33.07, 57.99, and 27.79%, respectively, which were reduced compared to 82.87% of the raw material (low molecular weight black goat meat and bone juice enzyme lysate), and in particular, F5 showed the lowest PGE 2 production amount. .
최종적으로 유효 펩타이드 F5를 선정하여 Triple TOF 5600 Q-TOF LC-MS/MS system을 이용하여 서열을 분석하였다. 그 결과, 대식세포에 대해 항염증 효과를 갖는 저분자 흑염소육골즙 효소분해물 조성물 내에 서열번호 1(Gly-Ala-Xaa-Gly-Phe-Xaa-Gly-Ala-Arg), 서열번호 2(Val-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Xaa-Gly-Pro-Xaa-Gly-Pro-Ala) 및 서열번호 3(Gly-Val-Xaa-Gly-Pro-Xaa-Gly-Ala-Val-Gly-Pro-Ala)의 아미노산 서열을 가지는 항염증성 유효 펩타이드를 확인하였다. 이때, 상기 서열번호 1 내지 3의 'Xaa'로 표시된 부분은 각각 '4Hyp(4-Hydroxyproline)'이다.Finally, the effective peptide F5 was selected and the sequence was analyzed using the Triple TOF 5600 Q-TOF LC-MS/MS system. As a result, SEQ ID NO: 1 (Gly-Ala-Xaa-Gly-Phe-Xaa-Gly-Ala-Arg), SEQ ID NO: 2 (Val-Gly) in the composition of a low-molecular black goat meat bone juice enzyme lysate having an anti-inflammatory effect on macrophages -Pro-Pro-Gly-Pro-Ser-Gly-Pro-Xaa-Gly-Pro-Xaa-Gly-Pro-Ala) and SEQ ID NO:3 (Gly-Val-Xaa-Gly-Pro-Xaa-Gly-Ala- Val-Gly-Pro-Ala), an anti-inflammatory effective peptide having an amino acid sequence was identified. In this case, the portions indicated by 'Xaa' of SEQ ID NOs: 1 to 3 are '4Hyp(4-Hydroxyproline)', respectively.
<110> KNU-Industry Cooperation Foundation <120> Composition containing small molecular weight enzyme hydrolysates of black goat extracts and effective peptide sequence <130> YP-20-218 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (3) <223> 4Hyp(4-Hydroxyproline) <220> <221> UNSURE <222> (6) <223> 4Hyp(4-Hydroxyproline) <400> 1 Gly Ala Xaa Gly Phe Xaa Gly Ala Arg 1 5 <210> 2 <211> 16 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (10) <223> 4Hyp(4-Hydroxyproline) <220> <221> UNSURE <222> (13) <223> 4Hyp(4-Hydroxyproline) <400> 2 Val Gly Pro Pro Gly Pro Ser Gly Pro Xaa Gly Pro Xaa Gly Pro Ala 1 5 10 15 <210> 3 <211> 12 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (3) <223> 4Hyp(4-Hydroxyproline) <220> <221> UNSURE <222> (6) <223> 4Hyp(4-Hydroxyproline) <400> 3 Gly Val Xaa Gly Pro Xaa Gly Ala Val Gly Pro Ala 1 5 10 <110> KNU-Industry Cooperation Foundation <120> Composition containing small molecular weight enzyme hydrolysates of black goat extracts and effective peptide sequence <130> YP-20-218 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (3) <223> 4Hyp (4-Hydroxyproline) <220> <221> UNSURE <222> (6) <223> 4Hyp (4-Hydroxyproline) <400> 1 Gly Ala Xaa Gly Phe Xaa Gly Ala Arg 1 5 <210> 2 <211> 16 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (10) <223> 4Hyp (4-Hydroxyproline) <220> <221> UNSURE <222> (13) <223> 4Hyp (4-Hydroxyproline) <400> 2 Val Gly Pro Pro Gly Pro Ser Gly Pro Xaa Gly Pro Xaa Gly Pro Ala 1 5 10 15 <210> 3 <211> 12 <212> PRT <213> Capra hircus <220> <221> UNSURE <222> (3) <223> 4Hyp (4-Hydroxyproline) <220> <221> UNSURE <222> (6) <223> 4Hyp (4-Hydroxyproline) <400> 3 Gly Val Xaa Gly Pro Xaa Gly Ala Val Gly Pro Ala 1 5 10
Claims (9)
Anti-inflammatory food composition comprising the peptide having the amino acid sequence shown in SEQ ID NO: 1, the peptide having the amino acid sequence shown in SEQ ID NO: 2, and the peptide having the amino acid sequence shown in SEQ ID NO: 3 as an active ingredient.
A cosmetic composition for anti-inflammatory comprising the peptide having the amino acid sequence shown in SEQ ID NO: 1, the peptide having the amino acid sequence shown in SEQ ID NO: 2, and the peptide having the amino acid sequence shown in SEQ ID NO: 3 as an active ingredient.
A pharmaceutical for preventing or treating inflammatory diseases, comprising as an active ingredient a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 composition.
It characterized in that it contains an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 Food composition for anti-inflammatory.
상기 흑염소육골즙 효소분해물은,
알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것을 특징으로 하는 항염용 식품 조성물.
5. The method of claim 4,
The enzyme decomposition product of black goat meat and bone juice,
Anti-inflammatory food composition, characterized in that prepared by treating alcalase (alcalase), prote AX (proteAX) and trypsin (trypsin).
It characterized in that it contains an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 A cosmetic composition for anti-inflammatory.
상기 흑염소육골즙 효소분해물은,
알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것을 특징으로 하는 항염용 화장료 조성물.
7. The method of claim 6,
The enzyme decomposition product of black goat meat and bone juice,
An anti-inflammatory cosmetic composition, characterized in that it is prepared by treating alcalase, proteAX, and trypsin.
It characterized in that it contains an enzyme lysate of black goat meat bone juice comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1, a peptide having the amino acid sequence shown in SEQ ID NO: 2, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 A pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 흑염소육골즙 효소분해물은,
알칼라제(alcalase), 프로테 에이엑스(proteAX) 및 트립신(trypsin)을 처리하여 제조된 것을 특징으로 하는 염증성 질환의 예방 또는 치료용 약학 조성물.
9. The method of claim 8,
The enzyme decomposition product of black goat meat and bone juice,
Alcalase (alcalase), prote AX (proteAX), and a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that prepared by processing trypsin (trypsin).
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| KR20200103564A (en) * | 2019-02-25 | 2020-09-02 | 조선대학교산학협력단 | Composition comprising extracts of Hizikia fusiforme for anti-inflammation or anti-allergy |
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| KR960028910A (en) * | 1995-01-25 | 1996-08-17 | 한동근 | Method for preparing hydrolyzate of Zara enzyme mainly containing low molecular weight peptide and health nutrition composition using same |
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| KR101536215B1 (en) | 2013-12-13 | 2015-07-14 | 주식회사 내추럴솔루션 | Cosmetic compositions for anti-oxidation, anti-inflammation, whitening and anti-wrinkle comprising collagen hydrolysate of jellyfish as an active ingredient |
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