KR102042600B1 - A novel peptide from Marphysa sanguinea having anti-inflammatory and anti-oxidant properties and use thereof - Google Patents
A novel peptide from Marphysa sanguinea having anti-inflammatory and anti-oxidant properties and use thereof Download PDFInfo
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- KR102042600B1 KR102042600B1 KR1020180017777A KR20180017777A KR102042600B1 KR 102042600 B1 KR102042600 B1 KR 102042600B1 KR 1020180017777 A KR1020180017777 A KR 1020180017777A KR 20180017777 A KR20180017777 A KR 20180017777A KR 102042600 B1 KR102042600 B1 KR 102042600B1
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
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- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
Abstract
본 발명은 항산화 및 항염증 활성을 갖는 홍갯지렁이 유래 신규 펩타이드 및 이의 이용에 관한 것으로, 구체적으로 서열번호 1의 아미노산 서열로 이루어지는 홍갯지렁이 유래 신규 펩타이드, 이를 유효성분으로 함유하는 피부 노화 방지용 또는 피부 자극 완화용 조성물 및 염증성 질환의 예방, 치료 또는 개선용 조성물에 관한 것이다.
본 발명의 펩타이드는 우수한 항산화 및 항염증 활성을 나타내며 높은 농도에서도 세포독성이 없어 안전하므로, 피부 노화 방지용 또는 피부 자극 완화용 화장료 조성물 및 염증성 질환의 예방, 치료 또는 개선용 약학 조성물 또는 식품 조성물의 유효성분으로 유용하다.The present invention relates to a novel peptide derived from worms having antioxidative and anti-inflammatory activity and its use, specifically, a novel peptide derived from worms consisting of the amino acid sequence of SEQ ID NO: 1, for preventing skin aging or skin stimulation containing the same as an active ingredient It relates to a palliative composition and a composition for preventing, treating or ameliorating an inflammatory disease.
Since the peptide of the present invention exhibits excellent antioxidant and anti-inflammatory activity and is safe since it does not have cytotoxicity at high concentrations, it is effective for preventing or treating skin aging or reducing skin irritation, and for preventing, treating or improving inflammatory diseases. Useful as an ingredient.
Description
본 발명은 항산화 및 항염증 활성을 갖는 홍갯지렁이 유래 신규 펩타이드 및 이의 이용에 관한 것으로, 구체적으로 서열번호 1의 아미노산 서열로 이루어지는 홍갯지렁이 유래 신규 펩타이드, 이를 유효성분으로 함유하는 피부 노화 방지용 또는 피부 자극 완화용 조성물 및 염증성 질환의 예방, 치료 또는 개선용 조성물에 관한 것이다.The present invention relates to a novel peptide derived from worms having antioxidative and anti-inflammatory activity and its use, specifically, a novel peptide derived from worms consisting of the amino acid sequence of SEQ ID NO: 1, for preventing skin aging or skin stimulation containing the same as an active ingredient It relates to a palliative composition and a composition for preventing, treating or ameliorating an inflammatory disease.
염증은 자극, 손상 또는 감염에 대한 조직의 국부적인 보호 반응으로 인식되어 왔으며, 통증, 발적 및 부종의 특징을 나타낸다. 일반적으로 염증은 손상 부위에 백혈구를 모집하는 특이적인 사이토카인 또는 케모카인의 생산을 통해 시작된다.Inflammation has been recognized as a local protective response of tissues to irritation, damage or infection and is characterized by pain, redness and edema. Inflammation generally begins through the production of specific cytokines or chemokines that recruit white blood cells to the site of injury.
염증 과정에서 병원균에 대한 방어 작용을 하기 위해 높은 수준의 활성산소종(ROS)이 생성되며(Kang et al., 2008), 특히 유도성 질소산화물 합성 효소(iNOS)에 의해 생성된 비정상적인 과산화질소(NO)가 단백질, DNA 및 지질과 같은 세포 거대 분자를 손상시켜 몇 가지 불리한 세포 반응을 유발할 수 있는 독성라디칼로 작용한다고 여겨진다(Cui et al. , 2006).High levels of reactive oxygen species (ROS) are produced to protect against pathogens during the inflammatory process (Kang et al., 2008), and abnormal nitrogen peroxide produced by inducible nitric oxide synthase (iNOS) NO) is believed to act as a toxic radical, which can damage cellular giant molecules such as proteins, DNA and lipids and cause some adverse cellular responses (Cui et al., 2006).
최근 보고된 바에 따르면, 과도한 자유라디칼은 말론디알데히드(malondialdehyde, MDA)의 수준을 증가시킴에 따라 지질 과산화를 증가시키며, 이 외에도 글루타치온 과산화효소(glutathione peroxidase, GSH-Px), 글루타치온 환원효소(glutathione reductase, GSH-R) 및 슈퍼옥사이드 디스뮤타아제(superoxide dismutase, SOD)의 수준을 낮추어 신체의 항산화 상태를 감소시킨다(Sontakke et al., 2002).Recently, excessive free radicals increase lipid peroxidation by increasing the level of malondialdehyde (MDA), in addition to glutathione peroxidase (GSH-Px), glutathione reductase (glutathione). Lowering levels of reductase (GSH-R) and superoxide dismutase (SOD) reduces the body's antioxidant status (Sontakke et al., 2002).
염증반응과 활성산소의 발생은 신체를 보호하기 위한 자연스러운 반응이기는 하지만, 과도할 경우 질병을 유발하는 등 신체에 큰 악영향을 미치기 때문에 이를 조절할 수 있는 방법이 필요하며, 이에 따라 여러 가지 물질이 항염증제 또는 항산화제로 개발되어 사용되고 있다.Although inflammatory reactions and the generation of free radicals are natural reactions to protect the body, there is a need for ways to control them, since excessive adverse effects on the body, such as causing diseases, are required. It is developed and used as an antioxidant.
현재는 덱사메타손(dexamethasone)과 같은 코르티코 스테로이드가 항염증제로 주로 사용되고 있는데, 이러한 항염증제는 매우 효과적이기는 하나 부작용이 크기 때문에 보다 안전한 항염증제의 개발이 필요한 실정이다.Currently, corticosteroids such as dexamethasone (dexamethasone) are mainly used as anti-inflammatory drugs. These anti-inflammatory drugs are highly effective but have side effects, and thus, development of safer anti-inflammatory drugs is required.
한편, 갯지렁이는 동물계(Kingdom Animalia) 환형동물문(Phylum Annelida) 다모강(Class Polychaeta)에 속하는 동물군으로, 참갯지렁이, 홍갯지렁이, 청갯지렁이 등 다양한 종이 있고, 주로 바다낚시의 미끼로 잘 알려져 있다.On the other hand, the lugworm is a group belonging to the Kingdom Animalia (Phylum Annelida) Class Polychaeta, and there are various species such as sparrowworm, redworm earthworm, and blueworm earthworm, and are mainly known as bait for sea fishing.
본 발명자는 이러한 갯지렁이로부터 생산되는 생리활성 물질에 관해 연구해 오던 중, 갯지렁이가 해양 환경에 잘 적응한 동물이며 해양 환경에서는 다양한 박테리아, 바이러스, 곰팡이, 기생충 등에 매우 쉽게 노출될 수 있기 때문에 염증반응이나 활성산소를 조절하기 위한 메커니즘이 존재할 가능성이 높다고 생각하였고, 이를 바탕으로 항염증 또는 항산화 활성을 갖는 물질을 개발할 수 있을 것으로 기대하였다.The present inventors have been studying the bioactive substances produced from such worms, and since worms are well adapted to the marine environment, they can be easily exposed to various bacteria, viruses, fungi, and parasites in marine environments. It is thought that there is a high possibility that there exists a mechanism for regulating reactive oxygen, and based on this, it was expected that a substance having anti-inflammatory or antioxidant activity could be developed.
이에 홍갯지렁이를 대상으로 관련 연구를 수행한 결과, 서열번호 1의 아미노산 서열로 이루어지는 홍갯지렁이 유래 신규 펩타이드가 우수한 항산화 및 항염증 활성을 가지며, 높은 농도에서도 세포독성을 나타내지 않아 안전하게 이용할 수 있음을 확인하고 본 발명을 완성하게 되었다.As a result of conducting a related research on the worms, it was confirmed that the novel peptides derived from the worms consisting of the amino acid sequence of SEQ ID NO: 1 have excellent antioxidant and anti-inflammatory activity, and do not show cytotoxicity even at high concentrations and thus can be used safely. This invention was completed.
따라서 본 발명의 주된 목적은 우수한 항산화 및 항염증 활성을 가지며 안전하게 이용할 수 있는 갯지렁이 유래 신규 펩타이드를 제공하는데 있다.Accordingly, the main object of the present invention is to provide a novel worm-derived peptide that has excellent antioxidant and anti-inflammatory activity and can be used safely.
본 발명의 다른 목적은 상기 펩타이드를 이용한 피부 노화 방지 또는 피부 자극 완화 용도의 조성물을 제공하는데 있다.Another object of the present invention to provide a composition for preventing skin aging or skin irritation using the peptide.
본 발명의 또 다른 목적은 상기 펩타이드를 이용한 염증성 질환의 예방, 치료 또는 개선 용도의 조성물을 제공하는데 있다.Another object of the present invention to provide a composition for the prevention, treatment or amelioration of inflammatory diseases using the peptide.
본 발명의 한 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어지는 펩타이드를 제공한다.According to one aspect of the invention, the invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
본 발명의 다른 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어지는 펩타이드를 유효성분으로 함유하는 피부 노화 방지용 화장료 조성물을 제공한다.According to another aspect of the invention, the present invention provides a cosmetic composition for preventing skin aging containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 또 다른 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어지는 펩타이드를 유효성분으로 함유하는 피부 자극 완화용 화장료 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a cosmetic composition for alleviating skin irritation containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 또 다른 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어지는 펩타이드를 유효성분으로 함유하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.According to another aspect of the invention, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 약학 조성물에 있어서, 상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위염, 크론병, 대장염, 통풍, 척추염, 류마티스염, 루푸스, 관절염, 건막염, 근육염, 간염, 췌장염, 방광염, 신장염, 복막염 및 패혈증으로 이루어진 군으로부터 선택된 질환일 수 있다.In the pharmaceutical composition of the present invention, the inflammatory disease is dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastritis, Crohn's disease, colitis, gout, spondylitis, rheumatism, lupus, Arthritis, tendonitis, myositis, hepatitis, pancreatitis, cystitis, nephritis, peritonitis and sepsis.
본 발명의 또 다른 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열로 이루어지는 펩타이드를 유효성분으로 함유하는 염증성 질환의 예방 또는 개선용 식품 조성물을 제공한다.According to another aspect of the invention, the present invention provides a food composition for the prevention or improvement of inflammatory diseases containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
본 발명의 식품 조성물에 있어서, 상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위염, 크론병, 대장염, 통풍, 척추염, 류마티스염, 루푸스, 관절염, 건막염, 근육염, 간염, 췌장염, 방광염, 신장염, 복막염 및 패혈증으로 이루어진 군으로부터 선택된 질환일 수 있다.In the food composition of the present invention, the inflammatory disease is dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastritis, Crohn's disease, colitis, gout, spondylitis, rheumatism, lupus, Arthritis, tendonitis, myositis, hepatitis, pancreatitis, cystitis, nephritis, peritonitis and sepsis.
본 발명의 펩타이드는 우수한 항산화 및 항염증 활성을 나타내며 높은 농도에서도 세포독성이 없어 안전하므로, 피부 노화 방지용 또는 피부 자극 완화용 화장료 조성물 및 염증성 질환의 예방, 치료 또는 개선용 약학 조성물 또는 식품 조성물의 유효성분으로 유용하다.Since the peptide of the present invention exhibits excellent antioxidant and anti-inflammatory activity and is safe since it does not have cytotoxicity at high concentrations, it is effective for preventing or treating skin aging or reducing skin irritation, and for preventing, treating or improving inflammatory diseases. Useful as an ingredient.
도 1은 본 발명의 펩타이드(NCW)를 포함하여 홍갯지렁이로부터 유래한 펩타이드들의 DPPH 라디칼 소거 활성 실험 결과를 나타낸 그래프이다. NCW, 본 발명의 펩타이드; VC, 비타민 C; EAV ~ MGL, 홍갯지렁이 유래의 다른 펩타이드.
도 2는 본 발명 펩타이드(NCW)의 DPPH 라디칼 소거 활성에 대한 IC50을 대조군인 비타민 C(VC)와 비교하여 나타낸 그래프이다.
도 3은 본 발명 펩타이드(NCW)의 세포독성 실험 결과를 나타낸 그래프이다.
도 4는 본 발명 펩타이드(NCW)의 지질 산화 억제 활성 실험 결과를 나타낸 그래프이다.
도 5는 본 발명 펩타이드(NCW)의 항산화 효소 활성에 대한 효과를 실험한 결과 그래프이다.
도 6은 본 발명 펩타이드(NCW)의 일산화질소 생성에 대한 효과를 실험한 결과 그래프이다.
도 7은 본 발명 펩타이드(NCW)의 전염증성 사이토카인 생성에 대한 효과를 실험한 결과 그래프이다.
도 8은 본 발명 펩타이드(NCW)의 iNOS 및 COX-2 단백질 발현에 대한 효과를 실험한 결과이다.1 is a graph showing the results of DPPH radical scavenging activity of the peptides derived from the worm earthworm including the peptide of the present invention (NCW). NCW, peptides of the present invention; VC, vitamin C; EAV-MGL, another peptide derived from the wormworm.
Figure 2 is a graph showing the IC50 for DPPH radical scavenging activity of the peptide (NCW) of the present invention compared to vitamin C (VC) as a control.
Figure 3 is a graph showing the cytotoxicity test results of the peptide (NCW) of the present invention.
Figure 4 is a graph showing the results of the lipid oxidation inhibitory activity of the peptide (NCW) of the present invention.
5 is a graph showing the results of experiments on the antioxidant enzyme activity of the peptide (NCW) of the present invention.
6 is a graph showing the results of experiments on the effect of the invention on the production of nitrogen monoxide (NCW).
7 is a graph showing the results of experiments on the proinflammatory cytokine production of the peptide (NCW) of the present invention.
8 shows the results of experiments on the expression of iNOS and COX-2 proteins of the present invention peptide (NCW).
본 발명의 펩타이드는 서열번호 1의 아미노산 서열로 이루어지는 펩타이드로, 홍갯지렁이(Marphysa sanguinea)로부터 유래되었으며 우수한 항산화 및 항염증 활성을 나타낼 수 있다.Peptide of the present invention is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, is derived from Marphysa sanguinea and can exhibit excellent antioxidant and anti-inflammatory activity.
본 발명의 펩타이드는 홍갯지렁이를 대상으로 균질화, 컬럼 크로마토그래피 등을 이용하는 통상의 펩타이드 정제방법을 통해 수득할 수 있으며, 길이가 짧아 통상적인 펩타이드를 합성하는 방법으로도 용이하게 수득할 수 있다.Peptides of the present invention can be obtained through a common peptide purification method using homogenization, column chromatography, etc. for the mungworm earthworm, and can be easily obtained by a method for synthesizing a conventional peptide is short.
본 발명에 따르면, DPPH를 이용한 자유라디칼 소거 실험에서 본 발명의 펩타이드는 대표적인 항산화 물질로 알려진 비타민 C와 비교될 수 있을 정도로 매우 우수한 라디칼 소거능을 갖는 것으로 나타났다. 이는 본 발명의 펩타이드가 매우 우수한 항산화 활성을 갖는다는 것을 의미한다.According to the present invention, free radical scavenging experiments using DPPH have shown that the peptides of the present invention have a very good radical scavenging ability that can be compared with vitamin C, which is known as a representative antioxidant. This means that the peptides of the present invention have very good antioxidant activity.
또한 대식세포인 RAW264.7 세포에 대한 세포독성 실험에서 본 발명의 펩타이드는 50μM의 농도에서도 세포독성이 없는 것으로 나타났다. 이는 본 발명의 펩타이드를 높은 농도로 사용하더라도 안전하며 부작용 발생 가능성이 낮다는 것을 의미한다.In addition, the cytotoxicity test for macrophage RAW264.7 cells showed that the peptide of the present invention is not cytotoxic even at a concentration of 50μM. This means that even when the peptide of the present invention is used at a high concentration, it is safe and the possibility of side effects is low.
또한 지질 산화 억제 활성 실험에서 본 발명의 펩타이드는 LPS로 유도되는 지질 산화를 효과적으로 억제하는 것으로 나타났다. 이는 본 발명의 펩타이드가 염증이 유도되는 상황에서 매우 우수한 항산화 활성을 나타낸다는 것을 의미한다.In addition, the lipid oxidation inhibitory activity experiments showed that the peptide of the present invention effectively inhibited lipid oxidation induced by LPS. This means that the peptides of the present invention exhibit very good antioxidant activity in the context of inducing inflammation.
또한 항산화 효소 활성 실험에서 본 발명의 펩타이드는 항산화 효소인 SOD(superoxide dismutase), CAT(catalase) 및 GSH-Px(glutathione peroxidase)의 활성을 효과적으로 증가시키는 것으로 나타났다. 이는 본 발명의 펩타이드가 세포에 존재하는 항산화 효소를 증가시켜 보다 우수한 항산화 활성을 나타낼 수 있다는 것을 의미한다.In addition, in the antioxidant enzyme activity experiment, the peptide of the present invention was shown to effectively increase the activity of the antioxidant enzymes SOD (superoxide dismutase), CAT (catalase) and GSH-Px (glutathione peroxidase). This means that the peptides of the present invention can exhibit better antioxidant activity by increasing the antioxidant enzymes present in the cells.
또한 일산화질소(NO) 생성 실험에서 본 발명의 펩타이드는 LPS로 유도되는 일산화질소의 생성을 효과적으로 억제하는 것으로 나타났다. 이는 본 발명의 펩타이드가 염증반응을 유발하는 일산화질소의 생성을 억제하여 우수한 항염증 활성을 나타낸다는 것을 의미한다.In addition, in the production of nitrogen monoxide (NO), the peptide of the present invention was shown to effectively inhibit the production of nitrogen monoxide induced by LPS. This means that the peptide of the present invention exhibits excellent anti-inflammatory activity by inhibiting the production of nitric oxide, which causes an inflammatory response.
또한 전염증성 사이토카인 생성 실험에서 본 발명의 펩타이드는 LPS로 유도되는 전염증성 사이토카인의 발현을 효과적으로 억제하는 것으로 나타났다. 이는 본 발명의 펩타이드가 염증반응을 유발하는 전염증성 사이토카인의 발현을 억제하여 우수한 항염증 활성을 나타낸다는 것을 의미한다.In addition, proinflammatory cytokine production experiments showed that the peptide of the present invention effectively inhibited the expression of proinflammatory cytokines induced by LPS. This means that the peptide of the present invention exhibits excellent anti-inflammatory activity by inhibiting the expression of proinflammatory cytokines causing inflammatory reactions.
또한 염증반응과 관련된 대표적인 단백질인 iNOS 및 COX-2 발현 실험에서 본 발명의 펩타이드는 LPS로 유도되는 iNOS 및 COX-2의 발현을 효과적으로 억제하는 것으로 나타났다. 이는 본 발명의 펩타이드가 iNOS의 발현을 억제하여 일산화질소의 생성을 억제함과 동시에 COX-2의 발현을 억제하여 우수한 항염증 활성을 나타낸다는 것을 의미한다.In addition, iNOS and COX-2 expression experiments, which are representative proteins related to the inflammatory response, showed that the peptide of the present invention effectively inhibited the expression of iNOS and COX-2 induced by LPS. This means that the peptide of the present invention inhibits the expression of iNOS, inhibits the production of nitrogen monoxide, and simultaneously inhibits the expression of COX-2, thereby exhibiting excellent anti-inflammatory activity.
이와 같이 본 발명의 펩타이드는 우수한 항산화 및 항염증 활성을 나타낼 수 있고, 또한 높은 농도로도 안전하게 사용할 수 있으므로, 항산화 및 항염증 활성이 요구되는 다양한 분야에 유용하게 이용될 수 있을 것이다.As such, the peptide of the present invention may exhibit excellent antioxidant and anti-inflammatory activity, and may also be safely used at high concentrations, and thus may be usefully used in various fields requiring antioxidant and anti-inflammatory activity.
특히 본 발명의 펩타이드는 상기와 같은 항산화 및 항염증 효과를 바탕으로 피부의 노화를 방지하기 위한 용도 또는 피부의 자극을 완화하기 위한 용도의 화장료 조성물, 그리고 다양한 염증성 질환을 예방, 치료 또는 개선하기 위한 약학 또는 식품 조성물의 유효성분으로 사용될 수 있다.In particular, the peptide of the present invention is based on the antioxidant and anti-inflammatory effects as described above for the purpose of preventing skin aging or to reduce the irritation of the skin cosmetic composition, and to prevent, treat or improve various inflammatory diseases It can be used as an active ingredient in pharmaceutical or food compositions.
상기 염증성 질환은 예를 들어, 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위염, 크론병, 대장염, 통풍, 척추염, 류마티스염, 루푸스, 관절염, 건막염, 근육염, 간염, 췌장염, 방광염, 신장염, 복막염 및 패혈증으로 이루어진 군으로부터 선택된 질환일 수 있으나 이에 제한되는 것은 아니다.The inflammatory diseases include, for example, dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastritis, Crohn's disease, colitis, gout, spondylitis, rheumatitis, lupus, arthritis, fasciitis , Myositis, hepatitis, pancreatitis, cystitis, nephritis, peritonitis and sepsis may be a disease selected from, but is not limited thereto.
본 발명의 화장료 조성물은 본 발명의 펩타이드 그 자체 또는 화장품학적으로 허용된 담체를 혼합하여 다양한 형태로 제형화할 수 있다. 예를 들어, 화장수, 영양로션, 영양크림, 맛사지 크림, 팩 및 영양 에센스로 이루어진 군에서 선택된 형태로 제형화할 수 있다. 이때 조성물 중 본 발명의 펩타이드의 함량은 각 용도에 따라 선택적으로 조절할 수 있으며 크게 제한되지는 않으나, 0.00001 내지 50중량%로 포함하는 것이 바람직할 것으로 판단된다.The cosmetic composition of the present invention may be formulated in various forms by mixing the peptide of the present invention or a cosmetically acceptable carrier. For example, it may be formulated in a form selected from the group consisting of lotion, nutrition lotion, nutrition cream, massage cream, pack and nutrition essence. At this time, the content of the peptide of the present invention in the composition can be selectively adjusted according to each use, but not limited significantly, it is determined that it is preferable to include from 0.00001 to 50% by weight.
본 발명의 약학 조성물은 식품의약품안전처의 통상적인 약학 제제로의 제형화 기준 또는 건강보조식품의 제형 기준에 의거하여 제형화할 수 있다. 통상적인 방법으로, 투여방법, 투여형태 및 치료목적에 따라 본 발명의 펩타이드를 약학적으로 허용 가능한 담체와 함께 혼합하여 희석하거나, 용기 형태의 담체 내에 봉입시켜 제형화할 수 있다. 이때 조성물 중 본 발명의 펩타이드의 함량은 각 용도에 따라 선택적으로 조절할 수 있으며 크게 제한되지는 않으나, 0.00001 내지 50중량%로 포함하는 것이 바람직할 것으로 판단된다.The pharmaceutical composition of the present invention may be formulated based on the formulation criteria of the Ministry of Food and Drug Safety into conventional pharmaceutical formulations or the formulation criteria of dietary supplements. In a conventional manner, the peptides of the present invention may be formulated by diluting the mixture of the present invention with a pharmaceutically acceptable carrier or encapsulating in a container form depending on the administration method, dosage form and therapeutic purpose. At this time, the content of the peptide of the present invention in the composition can be selectively adjusted according to each use, but not limited significantly, it is determined that it is preferable to include from 0.00001 to 50% by weight.
본 발명의 약학 조성물은 임상 투여 시에 경구 또는 비경구로 투여가 가능하며 비경구 투여시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고, 일반적인 의약품 제제의 형태로 사용될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or It can be administered by intrathoracic injection and can be used in the form of a general pharmaceutical formulation.
본 발명의 약학 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
본 발명 약학 조성물의 일일 투여량은 조성물에 함유된 본 발명의 펩타이드를 기준으로 체중 1㎏ 당 약 0.0001 내지 100㎎, 바람직하게는 0.001 내지 50㎎일 수 있으며, 하루 1회 내지 수회 나누어 투여될 수 있으나 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다.The daily dosage of the pharmaceutical composition of the present invention may be about 0.0001 to 100 mg, preferably 0.001 to 50 mg per kg of body weight based on the peptide of the present invention contained in the composition, and may be administered once to several times a day. However, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and the severity of the disease.
본 발명의 약학 조성물은 본 발명의 펩타이드에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the peptide of the present invention.
본 발명의 식품 조성물은 본 발명의 펩타이드 그 자체 또는 식품학적으로 허용된 담체와 혼합한 조성물일 수 있다. 본 발명의 식품 조성물은 식육가공품, 어육제품, 두부, 묵, 죽, 라면이나 국수 등의 면류, 간장, 된장, 고추장, 혼합장 등의 조미식품, 소스, 과자, 발효유나 치즈 등의 유가공품, 김치나 장아찌 등의 절임식품, 과실, 채소, 두유, 발효음료 등의 음료수의 식품 형태가 가능할 것으로 판단된다. 또한 식품학적으로 허용된 담체는 상기 약학적으로 허용된 담체도 사용할 수 있을 것이다.The food composition of the present invention may be a composition mixed with the peptide of the present invention or a food acceptable carrier. The food composition of the present invention is processed meat products, fish products, tofu, jelly, porridge, noodles such as ramen or noodles, soy sauce, miso, seasonings such as miso, red pepper paste, mixed soy sauce, dairy products such as sauces, sweets, fermented milk or cheese, kimchi Foods such as pickles such as pickles, pickles, fruits, vegetables, soy milk, and fermented beverages may be possible. In addition, a pharmaceutically acceptable carrier may also be used as the pharmaceutically acceptable carrier.
본 발명의 화장료 조성물, 약학 조성물 및 식품 조성물에서 본 발명의 펩타이드는 그 자체를 사용하거나 약학적으로 허용 가능한 산부가염 또는 금속 복합체의 형태로도 사용할 수 있을 것으로 판단된다.In the cosmetic compositions, pharmaceutical compositions and food compositions of the invention, it is contemplated that the peptides of the invention may be used by themselves or in the form of pharmaceutically acceptable acid addition salts or metal complexes.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 홍갯지렁이로부터 펩타이드의 분리 및 확인Example 1 Isolation and Identification of Peptides from Snail Earthworm
위장 대사 산물 및 오물을 제거하기 위해 홍갯지렁이(Marphysa sanguinea) 성체(~200g)를 약간 찬 인공해수(2.7~3% marine salt)에 24시간 동안 넣어 둔 다음 물기를 제거한 후 -80℃에서 보관하였다가, Mixer를 이용해 분말화한 후 얼려둔 갯지렁이에 4배 부피의 예열된 1% acetic acid(HAc)(1:4, v/v)를 첨가하고 5분간 끓여 단백질 분해 효소의 활성을 억제시켰다. 이후 조직을 5분간 wet ice 상에서 완전히 균질화(Speed #6, T10 basic ULTRA-TURRAX, IKA, USA)하고, 원심분리(15,000Xg, 30분, 4℃)한 다음 상등액을 수득하여 -80℃에 보관하였다.In order to remove gastrointestinal metabolites and dirt, adult Marphysa sanguinea (~ 200 g) was placed in slightly cold artificial seawater ( 2.7-3 % marine salt) for 24 hours and then drained and stored at -80 ° C. After powdering with Mixer, 4 times of preheated 1% acetic acid (HAc) (1: 4, v / v) was added to frozen wormworms and boiled for 5 minutes to inhibit proteolytic enzyme activity. The tissue is then completely homogenized on wet ice for 5 minutes (Speed # 6, T10 basic ULTRA-TURRAX, IKA, USA), centrifuged (15,000Xg, 30 minutes, 4 ° C), and then the supernatant is obtained and stored at -80 ° C. It was.
상등액을 Sep-Pak C18 카트리지(Waters Associates, Milford, USA)를 사용한 역상 농축(reverse-phase concentration)에 적용하여 활성 분획을 수집하고, Delta Pak C18 컬럼(Waters Associates, Milford, USA)을 사용한 역상 HPLC[0.1%(v/v) trifluoroacetic acid 함유 0 ~ 60% acetonitrile의 선형농도구배, 60분 이상, 유속 1㎖/min]로 추가 정제하였다. 컬럼 분획물을 225nm UV absorption으로 모니터하고, 분당 분획물을 Biofraction collecter(Bio-rad)로 수집한 후 각 분당 분획물을 -80℃에 보관하였다가 24시간 후 동결 건조기를 이용해 동결건조 하였다.The supernatant was subjected to reverse-phase concentration using a Sep-Pak C18 cartridge (Waters Associates, Milford, USA) to collect the active fractions and reversed phase HPLC using a Delta Pak C18 column (Waters Associates, Milford, USA) [0.1% (v / v) trifluoroacetic acid containing 0 ~ 60% of the acetonitrile linear concentrate, 60 minutes or more, flow rate 1ml / min] was further purified. The column fractions were monitored by 225 nm UV absorption, and the fractions per minute were collected by Biofraction collecter (Bio-rad), and each fraction was stored at -80 ° C and lyophilized after 24 hours using a freeze dryer.
수집한 분획물에 중 활성이 있는 분획물을 mass spectrometer UPLC-Q-TOF (micrOTOF-Q III, BRUKER)로 분석하였다. C18 컬럼(5㎛, 32×150㎜)을 사용하고 용매 A(0.1% FA in water)에 10 ~ 45% 용매 B(100% acetonitrile, 0.1% FA in water)의 선형농도구배(유속 0.1㎖/min)로 용출하여 펩타이드를 분리하였고, m/z 50 ~ 2,000 사이의 양이온 모드를 사용하여 모든 MS/MS 분석을 수행하였다. 펩타이드 서열을 확인하기 위해 MS/MS-ions를 Data Analysis(4.0)로 분석하였고, 확인된 펩타이드 서열을 biotool(3.2)로 확인하여 각 활성 분획의 펩타이드 서열을 결정하였다.The active fractions in the collected fractions were analyzed by mass spectrometer UPLC-Q-TOF (micrOTOF-Q III, BRUKER). Using a C18 column (5 μm, 32 × 150 mm) and adding a 10-45% solvent B (100% acetonitrile, 0.1% FA in water) in a linear thickener (flow rate 0.1 mL /) in solvent A (0.1% FA in water) The peptide was isolated by elution in min) and all MS / MS analyzes were performed using cation mode between m / z 50-2,000. In order to confirm the peptide sequence, MS / MS-ions were analyzed by Data Analysis (4.0), and the identified peptide sequence was confirmed by biotool (3.2) to determine the peptide sequence of each active fraction.
이의 결과, 홍갯지렁이로부터 서열번호 1의 아미노산 서열로 이루어지는 본 발명의 펩타이드를 포함하여 총 9종류의 펩타이드를 확인할 수 있었고, 각각의 펩타이드를 합성하여 이후의 실험에 사용하였다.As a result, a total of nine kinds of peptides including peptides of the present invention consisting of the amino acid sequence of SEQ ID NO: 1 from the blackworm earthworms were identified, and each peptide was synthesized and used in subsequent experiments.
실험예 1. 펩타이드의 라디칼 소거 활성 조사Experimental Example 1. Investigation of the radical scavenging activity of the peptide
상기 실시예 1에서 확인 및 합성한 9종류의 펩타이드를 대상으로 항산화 활성을 조사하기 위해, DPPH(1,1-Diphenyl-2-picrylhydrazyl) 라디칼 소거 활성을 조사하였다.In order to investigate the antioxidant activity of the nine kinds of peptides identified and synthesized in Example 1, DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity was investigated.
각 펩타이드를 125㎍/㎖의 농도로 희석하여 준비한 시료를 사용하여 DPPH 라디칼 소거 활성을 조사한 결과, 도 1과 같이 양성대조군인 비타민 C(vitamin C, VC)와 비슷한 라디칼 소거 활성이 본 발명의 펩타이드(NCW)에서 나타났다.As a result of investigating DPPH radical scavenging activity using a sample prepared by diluting each peptide to 125 μg / ml, as shown in FIG. 1, a radical scavenging activity similar to vitamin C (vitamin C, VC), which is a positive control group, was observed. (NCW) appeared.
이를 바탕으로 본 발명 펩타이드의 IC50을 조사한 결과 17.48㎍/㎖로 나타났는데, 비록 비타민 C의 IC50 2.752㎍/㎖ 보다는 다소 높지만 매우 탁월한 라디칼 소거 활성이 있는 것으로 확인되었다(도 2 참조).On the basis of this, the IC 50 of the peptide of the present invention was found to be 17.48 µg / ml, although it was slightly higher than the IC 50 of 2.752 µg / ml, but it was confirmed to have a very excellent radical scavenging activity (see FIG. 2).
DPPH 분석DPPH Analysis
DPPH 용액(0.1M) 0.2㎖에 펩타이드 시료를 첨가하고 37℃에서 30분 동안 반응시킨 후, 각 용액의 흡광도를 ELISA 마이크로플레이트 판독기(Molecular Devices, Sunnyvale, CA, USA)를 사용하여 540nm에서 측정하고 다음 계산식을 사용하여 DPPH 라디칼 소거능을 결정하였다.Peptide samples were added to 0.2 ml of DPPH solution (0.1M) and reacted at 37 ° C. for 30 minutes, and then the absorbance of each solution was measured at 540 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The following formula was used to determine DPPH radical scavenging ability.
[계산식][formula]
DPPH 라디칼 소거능(%) = [1 - 샘플의 흡광도(540nm) / 대조군의 흡광도(540nm)] × 100DPPH radical scavenging activity (%) = [1-absorbance of the sample (540 nm) / absorbance of the control (540 nm)] × 100
실험예 2. 펩타이드의 세포독성 조사Experimental Example 2. Investigation of Cytotoxicity of Peptides
항산화 활성이 확인된 본 발명 펩타이드의 세포독성을 조사하기 위해, RAW264.7 세포를 다양한 농도(0, 5, 25 및 50μM)의 펩타이드로 24시간 처리한 다음 생존하는 RAW264.7 세포를 MTT 분석을 통해 측정하였다.In order to investigate the cytotoxicity of the peptides of the present invention, the antioxidant activity of which was confirmed, RAW264.7 cells were treated with peptides of various concentrations (0, 5, 25, and 50 μM) for 24 hours, and then surviving RAW264.7 cells were subjected to MTT assay. Measured through.
이의 결과, 도 3과 같이 본 발명의 펩타이드는 50μM의 농도까지 세포독성이 없는 것으로 나타났다.As a result, the peptide of the present invention as shown in Figure 3 was found to be cytotoxic up to a concentration of 50μM.
세포 배양Cell culture
마우스 대식세포인 RAW264.7 세포는 ATCC(American Type Culture Collection)(Manassas, VA, USA)로부터 구입하였고, 10% heat-inactivated FBS, 100U/㎖ penicillin 및 100㎍/㎖ streptomycin을 첨가한 Dulbecco's Modified Eagle's Medium(DMEM)에서 5% CO2 및 37℃의 humidified atmosphere 조건으로 배양하였다.RAW264.7 cells, a mouse macrophage, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and Dulbecco's Modified Eagle's with 10% heat-inactivated FBS, 100U / ml penicillin and 100µg / ml streptomycin Medium (DMEM) was incubated in humidified atmosphere conditions of 5% CO 2 and 37 ℃.
MTT 분석MTT analysis
RAW264.7 세포를 96-웰 플레이트에 5×103 cells/well의 농도로 접종하고, 10% FBS 첨가 배지에서 밤새 배양한 다음, 다양한 농도로 펩타이드(PBS에 녹여 사용)를 처리하였다.RAW264.7 cells were seeded in 96-well plates at a concentration of 5 × 10 3 cells / well, incubated overnight in 10% FBS addition medium, and then treated with peptides (dissolved in PBS) at various concentrations.
24시간 배양한 후 세포를 PBS(phosphate-buffered saline)로 세척하고, 100㎍/㎖ MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 함유 배지로 처리하여 37℃에서 2시간 동안 반응시켰다.After 24 hours of incubation, the cells were washed with PBS (phosphate-buffered saline) and treated with 100 µg / ml MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] containing medium. The reaction was carried out at 37 ° C. for 2 hours.
이후 세포를 PBS로 세척하고, DMSO 200㎕에 녹인 다음 540nm에서 흡광도를 측정하여 용해된 보라색 포마잔(formazan)의 양을 결정하였다.Cells were then washed with PBS, dissolved in 200 μl of DMSO, and absorbance was measured at 540 nm to determine the amount of soluble purple formazan.
실험예 3. 펩타이드의 지질 산화 억제 활성 조사Experimental Example 3. Investigation of Lipid Oxidation Inhibitory Activity of Peptides
본 발명 펩타이드의 지질 산화 억제 활성을 조사하기 위해, MDA 측정 방법을 이용하였다.In order to investigate the lipid oxidation inhibitory activity of the peptide of the present invention, MDA measurement method was used.
이의 결과, 도 4와 같이 본 발명의 펩타이드는 5 ~ 50μM의 농도에서 모두 지질 산화 억제 활성을 나타냈으며(p<0.05), 이러한 활성은 농도 의존적인 것으로 나타났다.As a result, as shown in FIG. 4, the peptides of the present invention showed lipid oxidation inhibitory activity at concentrations of 5 to 50 μM (p <0.05), and this activity was found to be concentration dependent.
MDA 측정MDA Measurement
RAW264.7 세포를 혈청이 결핍된 배지에서 밤새 배양한 다음 LPS(2㎍/㎖) 및 다양한 농도의 펩타이드(PBS에 녹여 사용)가 함유된 배지에서 배양하였다.RAW264.7 cells were cultured overnight in serum deficient media and then in medium containing LPS (2 μg / ml) and various concentrations of peptide (used in PBS).
세포를 수거하고 lysis buffer[50mM Tris-HCl, 150mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1mM EDTA, 1mM Na3VO4, 1mM phenylmethyl-sulfonyl fluoride(PMSF), 1㎍/㎖ leupeptin, 1㎍/㎖ pepstatin, 5㎍/㎖ aprotinin 및 20mM NaF)에서 균질화하였다. 용해 현탁액을 12,000g에서 15분 동안 원심분리하고, 상등액을 전체 세포추출물로 사용하였다.Cells were harvested and lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Na 3 VO 4 , 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 1 μg / ml leupeptin, 1 μg / Ml pepstatin, 5 μg / ml aprotinin and 20 mM NaF). The lysis suspension was centrifuged at 12,000 g for 15 minutes and the supernatant was used as the whole cell extract.
Prasad K 등의 방법[Prasad, K., Mantha, S. V., Muir, A. D. and Westcott, N. D. (2000) Protective effect of secoisolariciresinol diglucoside against streptozotocin-induced diabetes and its mechanism. Mol. Cell Biochem. 206(1-2): 141-149]을 사용하여 thiobarbituric acid로 측정하였다.Prasad K et al. [Prasad, K., Mantha, S. V., Muir, A. D. and Westcott, N. D. (2000) Protective effect of secoisolariciresinol diglucoside against streptozotocin-induced diabetes and its mechanism. Mol. Cell Biochem. 206 (1-2): 141-149] and measured with thiobarbituric acid.
전체 세포추출물을 10%(w/v) trichloroacetic acid를 2배로 섞어 침전시켰다.Total cell extracts were precipitated by mixing 10% (w / v) trichloroacetic acid twice.
표준곡선은 malondialdehyde (MDA)의 재료인 malondialdehyde bisdimethylacetal을 사용하여 준비하였다. 12,000×g에서 10분 동안 원심분리 후 상층액은 1시간 동안 boiling water에서 0.67%(w/v) thiobarbituric acid 동일한 양을 첨가하여 반응시켰다. ELISA microplate reader로 532nm에서 흡광도를 측정하여 MDA의 농도를 결정하였다.Standard curves were prepared using malondialdehyde bisdimethylacetal, a material of malondialdehyde (MDA). After centrifugation at 12,000 × g for 10 minutes, the supernatant was reacted by adding the same amount of 0.67% (w / v) thiobarbituric acid in boiling water for 1 hour. The concentration of MDA was determined by measuring absorbance at 532 nm with an ELISA microplate reader.
실험예 4. 항산화 효소 활성에 대한 펩타이드의 효과 조사Experimental Example 4. Investigation of the effect of peptides on antioxidant enzyme activity
항산화 효소인 SOD(superoxide dismutase), CAT(catalase) 및 GSH-Px(glutathione peroxidase)의 활성에 대한 본 발명 펩타이드의 효과를 조사하기 위해, RAW264.7 세포를 LPS(2㎍/㎖)로 자극하기 전에 다양한 농도(0, 5, 25 및 50μM)의 펩타이드로 30분간 전처리한 다음 배양한 세포로부터 단백질을 추출하여 각 효소에 대한 활성을 측정하였다.Stimulating RAW264.7 cells with LPS (2 μg / ml) to investigate the effect of the peptides of the present invention on the activity of the antioxidant enzymes superoxide dismutase (SOD), CAT (catalase) and GSH-Px (glutathione peroxidase) Before 30 minutes pre-treatment with various concentrations of peptide (0, 5, 25 and 50μM) and then protein was extracted from the cultured cells to determine the activity for each enzyme.
이의 결과, 도 5와 같이 본 발명의 펩타이드는 5 ~ 50μM의 농도에서 SOD의 활성을 유의적으로 증가시켰고, 25 ~ 50μM의 농도에서 CAT의 활성을 유의적으로 증가시켰으며, 5 ~ 50μM의 농도에서 GSH-Px의 활성을 유의적으로 증가시켰다.As a result, as shown in Figure 5, the peptide of the present invention significantly increased the activity of SOD at a concentration of 5 ~ 50μM, significantly increased the activity of CAT at a concentration of 25 ~ 50μM, a concentration of 5 ~ 50μM Significantly increased the activity of GSH-Px.
항산화 효소 활성Antioxidant enzyme activity
RAW264.7 세포를 96-웰 플레이트에 5×103 cells/well의 농도로 접종하고, 10% FBS 첨가 배지에서 18시간 배양하였다. 이후 혈청-결핍 배지에서 다양한 농도로 펩타이드(PBS에 녹여 사용)를 처리하여 30분간 전배양한 다음, LPS(2㎍/㎖)를 첨가하고 20시간 배양하였다.RAW264.7 cells were seeded in 96-well plates at a concentration of 5 × 10 3 cells / well and incubated for 18 hours in medium supplemented with 10% FBS. Thereafter, peptides (dissolved in PBS) were treated in various concentrations in serum-deficient medium, pre-incubated for 30 minutes, and then LPS (2 µg / ml) was added and incubated for 20 hours.
배양한 세포를 파쇄하고 파쇄물을 각 항산화 효소 활성을 측정하기 위한 단백질 시료로 사용하였다.The cultured cells were crushed and the lysate was used as a protein sample for measuring the antioxidant enzyme activity.
SOD 활성은 SOD assay kit(Oxis Research, Portland, OR USA)를 사용하여 측정하였다. Boric acid와 diethylenetriaminepenta acetic acid(DTPA)가 들어있는 2-amino-2-methyl-1,3-propanediol(pH 8.0)액 900㎕, 40㎕의 단백질 시료(0.1~0.5㎎) 및 1-methyl-2-vinylpyridium trifluoromethanesulfonate액 30㎕를 1㎖ cuvette에 넣고 섞은 후 1분 동안 37℃에 두었다가 DPTA와 ethanol이 들어있는 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorine액 30㎕를 첨가한 후 525nm에서 흡광도의 변화를 5분간 측정하였다. 이후 SOD의 활성을 다음 계산식을 사용하여 계산하였다.SOD activity was measured using a SOD assay kit (Oxis Research, Portland, OR USA). 900 µl of 2-amino-2-methyl-1,3-propanediol (pH 8.0) solution containing boric acid and diethylenetriaminepenta acetic acid (DTPA), 40 µl of protein sample (0.1-0.5 mg) and 1-methyl-2 30 μl of -vinylpyridium trifluoromethanesulfonate solution was added to 1 ml cuvette, mixed, and placed at 37 ° C. for 1 minute, followed by 5,6,6a, 11b-tetrahydro-3,9,10-trihydroxybenzo [c]
[계산식][formula]
units/mg of protein/min = 1분 반응에 따른 흡광도의 변화량(1min) × 1000/43.6 × 단백질양(㎎)units / mg of protein / min = change in absorbance according to 1 minute reaction (1min) × 1000 / 43.6 × amount of protein (mg)
CAT 활성은 Aebi 등의 방법[Aebi, H. (1984) Catalase in Vitro. Methods Enzymol. 105: 121-126.]을 사용하여 측정하였다. 반응액의 최종 양이 1㎖이 되도록 단백질 시료 100㎍에 100mM KH2PO4(pH 7.0) 850㎕, 19mM H2O2 150㎕를 첨가하여 반응을 유도하였다. 4분 동안 10초 간격으로 25℃에서 흡광도(240nm)를 측정하였다. 이후 CAT의 활성을 상기 계산식을 사용하여 계산하였다.CAT activity was determined by Aebi et al. [Aebi, H. (1984) Catalase in Vitro. Methods Enzymol. 105: 121-126.]. The reaction was induced by adding 850 µl of 100 mM KH 2 PO 4 (pH 7.0) and 150 µl of 19 mM H 2 O 2 to 100 µg of the protein sample so that the final amount of the reaction solution was 1 ml. Absorbance (240 nm) was measured at 25 ° C. at 10 second intervals for 4 minutes. The activity of CAT was then calculated using the above formula.
GSH-Px 활성은 50mM Tris/HCl(pH 7.0)이 함유된 900㎕ GSH-Px assay buffer와 1.6mM GSH, 0.32mM NADPH를 포함한 50㎕ NADPH assay reagent에 0.5mM EDTA를 cuvette에 첨가하여 4분 동안 5초 간격으로 340nm에서 흡광도를 측정하였다. 이후 GSH-Px의 활성을 상기 계산식을 사용하여 계산하였다.GSH-Px activity was obtained by adding 0.5 mM EDTA to the cuvette in 900 μl GSH-Px assay buffer containing 50 mM Tris / HCl (pH 7.0) and 50 μl NADPH assay reagent containing 1.6 mM GSH and 0.32 mM NADPH. Absorbance at 340 nm was measured at 5 second intervals. The activity of GSH-Px was then calculated using the above formula.
실험예 5. NO 생성에 대한 펩타이드의 효과 조사Experimental Example 5 Investigation of the Effect of Peptides on NO Production
LPS로 유도되는 NO(일산화질소) 생성에 대한 본 발명 펩타이드의 효과를 조사하기 위해, RAW264.7 세포를 LPS(2㎍/㎖)로 자극하기 전에 다양한 농도(0, 5, 25 및 50μM)의 펩타이드로 2시간 전처리한 후 2㎍/㎖의 LPS를 처리하여 20시간 배양하였다. 배양액 150㎕에 50㎕의 Griess 시약(1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution)을 넣어주고 10분간 상온에서 반응시킨 후 ELISA reader로 540nm에서 흡광도를 측정하였다. NaNO2의 농도별 표준곡선을 이용하여 배양액 내의 NO 농도를 계산하여 NO의 생성량을 측정하였다.To investigate the effect of the peptides of the invention on NO (nitrogen monoxide) production induced by LPS, various concentrations (0, 5, 25 and 50 μM) of RAW264.7 cells were stimulated prior to stimulation with LPS (2 μg / ml). After 2 hours of pretreatment with peptides, 2 μg / ml of LPS was incubated for 20 hours. 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution) was added to 150 μl of the culture solution, and reacted at room temperature for 10 minutes. The absorbance was measured at 540 nm using an ELISA reader. The amount of NO produced was measured by calculating the NO concentration in the culture medium using a standard curve for each concentration of NaNO 2 .
이의 결과, 도 6과 같이 LPS의 처리로 NO 생성이 8배 증가한 반면, 본 발명의 펩타이드를 전처리한 경우 이러한 현상이 농도 의존적으로 감소하였다.As a result, as shown in FIG. 6, the NO production was increased 8-fold by the treatment of LPS, whereas when the peptide of the present invention was pretreated, this phenomenon was reduced in a concentration-dependent manner.
실험예 6. 전염증성 사이토카인의 생성에 대한 펩타이드의 효과 조사Experimental Example 6. Investigation of the effect of peptides on the production of proinflammatory cytokines
LPS로 유도되는 전염증성 사이토카인(pro-inflammatory cytokine)의 발현에 대한 본 발명 펩타이드의 효과를 조사하기 위해, RAW264.7 세포를 LPS(2㎍/㎖)로 자극하기 전에 다양한 농도(0, 5, 25 및 50μM)의 펩타이드로 30분간 전처리한 다음 배양상등액의 IL-1β 및 TNF-α의 농도를 측정하였다.To investigate the effect of the peptides of the present invention on the expression of pro-inflammatory cytokine induced by LPS, various concentrations (0, 5) before stimulating RAW264.7 cells with LPS (2 μg / ml) , 25 and 50 μM) peptides were pretreated for 30 minutes and then the concentrations of IL-1β and TNF-α in the culture supernatant were measured.
이의 결과, 도 7과 같이 본 발명의 펩타이드는 사이토카인 IL-1β와 TNF-α의 생성을 농도가 증가함에 따라 유의적으로 차단하였다(p<0.05).As a result, the peptide of the present invention as shown in Figure 7 significantly blocked the production of cytokines IL-1β and TNF-α with increasing concentration (p <0.05).
전염증성 사이토카인 농도 측정Proinflammatory cytokine concentration measurement
RAW264.7 세포를 96-웰 플레이트에 5×103 cells/well의 농도로 접종하고, 10% FBS 첨가 배지에서 18시간 배양하였다. 이후 혈청-결핍 배지에서 다양한 농도로 펩타이드(PBS에 녹여 사용)를 처리하여 30분간 전배양한 다음, LPS(2㎍/㎖)를 첨가하고 20시간 배양하였다.RAW264.7 cells were seeded in 96-well plates at a concentration of 5 × 10 3 cells / well and incubated for 18 hours in medium supplemented with 10% FBS. Thereafter, peptides (dissolved in PBS) were treated in various concentrations in serum-deficient medium, pre-incubated for 30 minutes, and then LPS (2 µg / ml) was added and incubated for 20 hours.
이후, IL-1β 및 TNF-α ELISA kit를 이용하여 배양상등액 중 IL-1β 및 TNF-α의 농도를 측정하였다.Thereafter, IL-1β and TNF-α ELISA kits were used to measure the concentrations of IL-1β and TNF-α in the culture supernatant.
실험예 7. iNOS 및 COX-2 발현에 대한 펩타이드의 효과 조사Experimental Example 7 Investigation of the Effect of Peptides on iNOS and COX-2 Expression
본 발명 펩타이드의 NO 생성 억제 효과가 iNOS 단백질 수준의 감소에 의한 것인지를 확인하기 위해, LPS로 유도되는 iNOS 및 COX-2 단백질 발현에 대한 본 발명 펩타이드의 효과를 웨스턴 블롯으로 조사하였다.To determine whether the NO production inhibitory effect of the peptides of the invention was due to a decrease in iNOS protein levels, the effect of the peptides of the invention on LPS-induced iNOS and COX-2 protein expression was examined by Western blot.
이의 결과, 도 8과 같이 LPS의 처리로 iNOS 및 COX-2의 발현이 증가한 반면, 본 발명의 펩타이드를 처리한 경우 5 ~ 50μM의 농도에서 iNOS 및 COX-2의 발현이 유의적으로 감소하였다(p<0.05).As a result, while iNOS and COX-2 expression increased by the treatment of LPS as shown in Figure 8, the expression of iNOS and COX-2 significantly decreased at a concentration of 5 ~ 50μM treated with the peptide of the present invention ( p <0.05).
웨스턴 블롯Western blot
RAW264.7 세포를 96-웰 플레이트에 5×103 cells/well의 농도로 접종하고, 10% FBS 첨가 배지에서 18시간 배양하였다. 이후 혈청-결핍 배지에서 다양한 농도로 펩타이드(PBS에 녹여 사용)를 처리하여 30분간 전배양한 다음, LPS(2㎍/㎖)를 첨가하고 20시간 배양하였다.RAW264.7 cells were seeded in 96-well plates at a concentration of 5 × 10 3 cells / well and incubated for 18 hours in medium supplemented with 10% FBS. Thereafter, peptides (dissolved in PBS) were treated in various concentrations in serum-deficient medium, pre-incubated for 30 minutes, and then LPS (2 µg / ml) was added and incubated for 20 hours.
배양한 세포로부터 단백질을 추출하고 8~10% SDS-PAGE로 분리한 다음 PVDF 멤브레인(polyvinylidene difluoride membrane)으로 전이시켰다. 5% non-fat skim milk[0.5% Tween-20(TTBS) 함유 Tris-buffered saline(TBS) 중]로 16℃에서 1시간 블록시킨 후 rabbit anti-iNOS 및 anti-COX-2 항체[1:500~1:1000 희석, 5% non-fat skim milk 함유 TTBS 중]를 처리하여 4℃에서 밤새 반응시켰다. Horseradish peroxidase-conjugated anti-rabbit 또는 anti-mouse 항체를 2차 항체(1:3000~1:5000 희석, 5% non-fat skim milk 함유 TTBS 중, 상온에서 2시간 반응)로 사용하였다.Proteins were extracted from the cultured cells, separated by 8-10% SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane. Rabbit anti-iNOS and anti-COX-2 antibodies [1: 500] were blocked with 5% non-fat skim milk [in 0.5% Tween-20 (TTBS) containing Tris-buffered saline (TBS)] for 1 hour at 16 ° C. ~ 1: 1000 dilution, in TTBS containing 5% non-fat skim milk] was reacted overnight at 4 ℃. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody was used as a secondary antibody (1: 3000-1: 5000 dilution, 2 hours reaction at room temperature in TTBS containing 5% non-fat skim milk).
이후 ECL western blot detection reagent(Amersham Biosciences, Piscataway, NJ, USA)를 사용하고 X-ray 필름에 노출시켜 가시화하였다.Then, ECL western blot detection reagent (Amersham Biosciences, Piscataway, NJ, USA) was used and visualized by exposure to X-ray film.
상기 실험예들의 통계분석을 위해 Dunnett의 다중 비교 테스트를 SPSS ver. 12.0 소프트웨어를 사용하여 수행하였다. 모든 실험은 3회 수행하였고 데이터는 평균±표준편차로 나타냈다. P<0.05의 값을 유의한 것으로 간주하였다.Dunnett's multiple comparison test was performed by SPSS ver. 12.0 software was used. All experiments were performed three times and the data are presented as mean ± standard deviation. Values of P <0.05 were considered significant.
<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> A novel peptide from Marphysa sanguinea having anti-inflammatory and anti-oxidant properties and use thereof <130> PA-D17035 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Unknown <220> <223> Marphysa sanguinea <400> 1 Asn Cys Trp Pro Phe Gln Gly Val Pro Leu Gly Phe Gln Ala Pro Pro 1 5 10 15 <110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> A novel peptide from Marphysa sanguinea having anti-inflammatory and anti-oxidant properties and use <130> PA-D17035 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Unknown <220> <223> Marphysa sanguinea <400> 1 Asn Cys Trp Pro Phe Gln Gly Val Pro Leu Gly Phe Gln Ala Pro Pro 1 5 10 15
Claims (7)
상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위염, 크론병, 대장염, 통풍, 척추염, 류마티스염, 루푸스, 관절염, 건막염, 근육염, 간염, 췌장염, 방광염, 신장염, 복막염 및 패혈증으로 이루어진 군으로부터 선택된 질환인 것을 특징으로 하는 조성물.The method of claim 4, wherein
The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastritis, Crohn's disease, colitis, gout, spondylitis, rheumatoiditis, lupus, arthritis, tendonitis, myositis, hepatitis , Pancreatitis, cystitis, nephritis, peritonitis and sepsis.
상기 염증성 질환은 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위염, 크론병, 대장염, 통풍, 척추염, 류마티스염, 루푸스, 관절염, 건막염, 근육염, 간염, 췌장염, 방광염, 신장염, 복막염 및 패혈증으로 이루어진 군으로부터 선택된 질환인 것을 특징으로 하는 조성물.
The method of claim 6,
The inflammatory diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastritis, Crohn's disease, colitis, gout, spondylitis, rheumatoiditis, lupus, arthritis, tendonitis, myositis, hepatitis , Pancreatitis, cystitis, nephritis, peritonitis and sepsis.
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