KR101879780B1 - Anti-thrombotic composition comprising serine protease extracted from Marphysa Sanguinea - Google Patents
Anti-thrombotic composition comprising serine protease extracted from Marphysa Sanguinea Download PDFInfo
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- KR101879780B1 KR101879780B1 KR1020170047982A KR20170047982A KR101879780B1 KR 101879780 B1 KR101879780 B1 KR 101879780B1 KR 1020170047982 A KR1020170047982 A KR 1020170047982A KR 20170047982 A KR20170047982 A KR 20170047982A KR 101879780 B1 KR101879780 B1 KR 101879780B1
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- serine protease
- rockwool
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
본 발명은 바위털갯지렁이로부터 분리된 세린 프로테아제를 포함하는 항혈전용 조성물, 약학적 조성물 및 식품 조성물에 관한 것이다. 본 발명에 따른 바위털갯지렁이로부터 분리된 세린프로테아제는 간접적인 혈전 용해능이 넓은 pH와 온도에서 나타나며 세포독성이 없고 또한, 항응고효과 및 세포안정성을 나타낸다. 혈전용해제의 용해작용에 있어 직접적인 혈전용해능은 그 활성은 매우 좋지만 독성과 무분별하게 단백질은 분해한다는 점에서 그 사용에 제한되는바, 본 발명의 세린프로테아제는 직접적인 혈전용해능이 크지 않고 간접적인 혈전용해능을 주로 나타내므로 출혈과 같은 기존혈전용해제의 부작용을 극복할 수 있다. 따라서, 혈전 및 혈소판 응집으로 인한 고혈압, 심장병, 뇌졸중, 심근경색 등의 질병에 효과적인 항혈전 약학 조성물 및 식품 조성물로서 유용하게 사용될 수 있다. The present invention relates to an antioxidant composition, a pharmaceutical composition and a food composition comprising a serine protease separated from a rockwool crab. The serine protease isolated from the rockwool lug according to the present invention exhibits indirect fibrinolytic activity at a wide pH and temperature, has no cytotoxicity, exhibits anticoagulant effect and cell stability. The direct thrombolytic ability in the dissolution action of the thrombolytic agent is very good, but the activity thereof is very good. However, the use of the serine protease of the present invention is not sufficient in that the thrombin dissolution ability of the present invention is not so large and the indirect thrombolysis And the side effects of conventional thrombolytic agents such as bleeding can be overcome. Therefore, it can be effectively used as an antithrombotic pharmaceutical composition and food composition effective for diseases such as hypertension, heart disease, stroke, myocardial infarction and the like due to thrombus and platelet aggregation.
Description
본 발명은 바위털갯지렁이로부터 분리된 세린 프로테아제를 포함하는 항혈전용 조성물, 약학적 조성물 및 식품 조성물에 관한 것이다.The present invention relates to an antioxidant composition, a pharmaceutical composition and a food composition comprising a serine protease separated from a rockwool crab.
혈전은 통상적으로 손상된 혈관의 과도한 출혈을 막기 위해서 형성되는데, 혈소판이 내피하층의 표면과 접촉하게 되면 다수의 양성 피드백 제어 기구가 혈전을 생성시켜 상처 입은 혈관계를 차단시키는 반응이 개시된다. 이러한 혈소판 응집, 피브린 생성 및 중합화의 과정을 지혈이라 일컬으며 상처 부위로부터 과도한 출혈을 막는데 있어 매우 중요하다.The thrombus is usually formed to prevent excessive bleeding of damaged blood vessels. When the platelets come into contact with the surface of the subcutaneous layer, a large number of positive feedback control mechanisms generate thrombus and initiate a reaction to block the injured vascular system. This process of platelet aggregation, fibrin formation and polymerization is called hemostasis and is very important in preventing excessive bleeding from the wound site.
혈전은 출혈하는 혈관 내에서 형성되는 것이 바람직하며, 손상되지 않은 혈관에서 혈전이 생성될 경우 병인으로 작용한다. 손상되지 않은 혈관에서 내피 세포 표면에 작은 변화가 생기거나 내피 내층의 파괴를 초래하는 부상이 생기는 경우 혈전이 생성될 수 있다. 이와 같이 혈관 내피세포 표면 또는 내층에 비교적 작은 변형이 생겨나더라도 혈소판이 콜라겐과 접촉하여 지혈의 과정이 개시되는데, 이들 작은 변형은 여러 가지의 원인으로부터 일어난다. 이들 원인의 하나로 울체(즉, 심방 또는 혈관에 있어서 혈액의 감소된 운동)를 예시할 수 있으며 이것은 산소의 결핍으로 인한 손상을 유발하여 전단력을 감소시킴으로써, 초기에 응집하기 시작할 때 방출되는 응집 전구 물질 중 하나인 세로토닌의 길항제를 투여함으로써 혈소판 응집을 억제할 수 있음이 보고되었다 (Bush et al, Drug Development Research, 7:319-340, 1986).The thrombus is preferably formed in a bleeding vessel, and acts as a pathogen when thrombus is generated in an uninjured vessel. Thrombosis may be produced if there is a small change in the endothelial cell surface in the intact blood vessel or an injury that causes the endothelial layer to break down. Thus, even if relatively small deformation occurs on the surface or inner layer of vascular endothelial cells, platelets come into contact with the collagen to initiate a process of hemostasis. These small deformations arise from a variety of causes. One of these causes can be exemplified by the scoliosis (i.e., reduced motion of the blood in the atrium or blood vessels), which can lead to damage due to oxygen deficiency and thereby reduce the shear force so that the aggregate precursor (Bush et al., Drug Development Research, 7: 319-340, 1986). It has been reported that administration of antagonists of serotonin, one of the drugs, can inhibit platelet aggregation.
혈전을 제거하기 위한 방법으로 크게 2가지 방법이 제시되고 있는데, 그 한가지는 피브린을 용해시키거나, 압축, 제거하는 방법으로서 혈전의 주 구성물질이 피브린이기 때문에 상당히 호응을 받고 있다. 현재 혈관에 관련된 질병을 치료하기 위해서 개발된 혈전 용해제로는 플라스미노겐을 활성화시켜 불용성의 피브린을 용해성 피브린 분해산물로 만드는 여러 플라스미노겐 활성 인자들이 있다. 또 다른 방법은 트롬빈 유사 효소를 이용하여 피브린의 전구물질인 피브리노겐의 양을 줄이는 방법이다. 트롬빈 유사 효소와 트롬빈에 의해서 형성된 피브린은 피브리노겐의 Aa 사슬로부터 피브린펩타이드 A를 내는 것은 동일하나 트롬빈 유사효소로부터 생성된 피브린은 불안전한 형태의 des-A-피브린을 형성하게 된다. 이것은 플라스민에 의해 트롬빈으로부터 생성된 피브린보다 분해가 잘 이루어지며, 상기 des-A-피브린은 플라스미노겐을 활성화시킨다고 보고된 바 있다 (Stocker, K. F., Medical Use of Snake Venom Proteins, 161-173, CRC press, 1990).Two methods are proposed as a method for removing thrombus. One of them is a method of dissolving, compressing and removing fibrin, and it is very popular because the main constituent of thrombus is fibrin. Currently, thrombolytic agents developed to treat vascular-related diseases include several plasminogen activators that activate plasminogen to turn insoluble fibrin into a soluble fibrin degradation product. Another method is to reduce the amount of fibrinogen, a precursor of fibrin, by using thrombin-like enzymes. Fibrin formed by thrombin-like enzymes and thrombin produces fibrin peptide A from the Aa chain of fibrinogen, but fibrin produced from thrombin-like enzymes forms an unsafe form of des-A-fibrin. It has been reported that des-A-fibrin activates plasminogen, which is more readily degraded than fibrin produced from thrombin by plasmin (Stocker, KF, Medical Use of Snake Venom Proteins, 161-173, CRC press, 1990).
한편, 바위털갯지렁이(Marphysa Sanguinea)는 낚시용 미끼로서 수출되는 주요 종 가운데 고급종에 속한다. 전 연안에 서식하나, 특히 경기도, 충청남도, 전라남도에 위치한 황해한 갯벌 하부에 널리 분포하며, 연안 저질의 바윗돌 사이와 자갈 사이에 많이 서식한다. 갯지렁이는 지렁이와 비슷한 종류의 환영동물(annelid)이나, 지렁이(Eisenia andrei) 추출물에 대한 연구조차 많이 진행되지 않았으며 갯지렁이의 항혈전 효과는 거의 보고된 바가 없다.Meanwhile, rock wool lug (Marphysa Sanguinea ) belongs to the advanced species among the major species exported as bait for fishing. It is widely distributed in the lower part of Yellow Sea tidal flats located in Gyeonggi-do, Chungcheongnam-do and Jeollanam-do, and it is found in the coastal rocks and between the gravels. There have been few studies on the annelid or earthworm (Eisenia andrei) extracts of earthworms similar to earthworms, and the antithrombotic effects of the worms have been rarely reported.
이에 본 발명자들은 바위털갯지렁이를 활용한 연구를 수행하던 중 바위털갯지렁이로부터 분리된 세린프로테아제에서 항혈전능을 발견해 본 발명을 완성하였다. Therefore, the present inventors have completed the present invention by discovering antithrombogenic activity in serine proteases isolated from rockwool lugwort during research using rockwool lugwort.
본 발명의 목적은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 항혈전용 조성물, 약학적 조성물 및 식품 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for exclusive use in hemodilution, a pharmaceutical composition and a food composition comprising serine protease separated from rockwool crab.
상기 목적을 달성하기 위하여, 본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 항혈전용 조성물을 제공한다. In order to accomplish the above object, the present invention provides a composition for exclusive use in hemostasis comprising serine protease separated from rockwool crab.
또한 본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 혈전질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention also provides a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising serine protease separated from rockwool crabs.
또한 본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 혈전질환의 예방 또는 개선용 식품 조성물을 제공한다.The present invention also provides a food composition for preventing or ameliorating a thrombotic disease comprising serine protease separated from rockwool crabs.
본 발명에 따른 바위털갯지렁이로부터 분리된 세린프로테아제는 간접적인 혈전 용해능이 넓은 pH와 온도에서 나타나며 세포독성이 없고 또한, 항응고효과 및 세포안정성을 나타낸다. 혈전용해제의 용해작용에 있어 직접적인 혈전용해능은 그 활성은 매우 좋지만 독성과 무분별하게 단백질은 분해한다는 점에서 그 사용에 제한되는바, 본 발명의 세린프로테아제는 직접적인 혈전용해능이 크지 않고 간접적인 혈전용해능을 주로 나타내므로 출혈과 같은 기존혈전용해제의 부작용을 극복할 수 있다. 따라서, 혈전 및 혈소판 응집으로 인한 고혈압, 심장병, 뇌졸중, 심근경색 등의 질병에 효과적인 항혈전 약학 조성물 및 식품 조성물로서 유용하게 사용될 수 있다. The serine protease isolated from the rockwool lug according to the present invention exhibits indirect fibrinolytic activity at a wide pH and temperature, has no cytotoxicity, exhibits anticoagulant effect and cell stability. The direct thrombolytic ability in the dissolution action of the thrombolytic agent is very good, but the activity thereof is very good. However, the use of the serine protease of the present invention is not sufficient in that the thrombin dissolution ability of the present invention is not so large and the indirect thrombolysis And the side effects of conventional thrombolytic agents such as bleeding can be overcome. Therefore, it can be effectively used as an antithrombotic pharmaceutical composition and food composition effective for diseases such as hypertension, heart disease, stroke, myocardial infarction and the like due to thrombus and platelet aggregation.
도 1은 본 발명의 바위털갯지렁이로부터 분리된 세린프로테아제의 혈전 용해 효과를 확인하기 위한 피브린 평판 분석(Fibrin plate assay) 결과를 나타낸 도이다.
도 2은 바위털갯지렁이로부터 분리된 세린프로테아제의 간접적 피브린 용해 활성을 확인하기 위한 실험과 그 결과를 나타낸 도이다. 도 2의 (A)는 피브린 평판 실험을 나타낸 것으로, U1은 플라스미노겐과 대조군인 uPA을 처리한 것이고, U2는 플라스미노겐이 없이 대조군 uPA를 처리한 것이며, M1은 플라스미노겐과 바위털갯지렁이로부터 분리된 세린프로테아제를 처리한 영역이며, M2는 플라스미노겐이 없이 바위털갯지렁이로부터 분리된 세린프로테아제만을 처리한 영역을 나타낸다. 도 2의 (B)는 상기 피브린 평판 실험의 시간 경과에 따른 용해된 영역의 측정결과를 그래프화한 것을 나타낸 것이다. 도2의 (C)는 플라스미노겐 용해활성을 확인하기 위한 SDS-PAGE의 결과를 나타낸 것으로, M은 사이즈 마커를 로딩한 레인, P는 플라스미노겐을 로딩한 레인이며, S는 플라스미노겐과 바위털갯지렁이로부터 분리된 세린프로테아제를 처리해 로딩한 레인이다.
도3은 바위털갯지렁이로부터 분리한 세린프로테아제의 분자량을 확인하기 위해 SDS-PAGE를 수행하여 그 결과를 나타낸 도이다(M : 사이즈 마커, Lane 1 : 바위털갯지렁이로부터 분리된 세린프로테아제).
도 4는 바위털갯지렁이에서 분리된 세린프로테아제의 피브린 용해 활성에 대한 pH(A)와 온도(B)의 효과를 알아보기 위해 아조카세인 분석을 수행하여 그 결과를 나타낸 도이다.
도 5는 바위털갯지렁이로부터 분리된 세린프로테아제의 농도에 따른 피브리노겐 분해능을 확인하기 위하여 SDS-PAGE를 수행한 결과를 나타낸 도이다.
도 6은 바위털갯지렁이로부터 분리된 세린프로테아제의 독성을 MTT 분석을 통해 확인한 결과를 나타낸 도이다. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of fibrin plate assay for confirming the thrombolytic effect of serine protease separated from rockwool lugwort of the present invention. FIG.
FIG. 2 is a graph showing an experiment for confirming the indirect fibrinolytic activity of serine protease separated from rockworms, and the results thereof. Fig. 2 (A) shows a fibrin plate experiment, in which U1 was treated with plasminogen and uPA as a control group, U2 was treated with control uPA without plasminogen, M1 was treated with plasminogen, Treated with serine protease separated from the worms, and M2 represents the area treated with only serine protease separated from the rock hairy worm without plasminogen. FIG. 2 (B) is a graph showing the measurement result of the dissolved region with time in the fibrin plate test. 2 (C) shows the result of SDS-PAGE for confirming the plasminogen-lytic activity, wherein M is a lane loaded with a size marker, P is a lane loaded with plasminogen, S is plasminogen And lane treated with serine protease separated from rockwurst.
FIG. 3 is a graph showing the results of SDS-PAGE (M: size marker, Lane 1: serine protease separated from rockwool crab) to confirm the molecular weight of serine protease separated from rockwool gut.
FIG. 4 is a graph showing the results of azo casein analysis to examine the effect of pH (A) and temperature (B) on the fibrinolytic activity of serine proteases separated from rockwool crabs.
FIG. 5 is a graph showing the result of performing SDS-PAGE in order to confirm the fibrinogen-degrading ability according to the concentration of serine protease separated from rockwool crab.
FIG. 6 is a graph showing the results of MTT analysis of the toxicity of serine proteases isolated from rockwool crabs.
본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 항혈전용 조성물을 제공한다.The present invention provides a composition exclusively for hematopoiesis comprising serine protease separated from rockwool crabs.
이하, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
상기 본 발명의 세린프로테아제는 바위털갯지렁이로부터 유래한 가공하지 않은 단백질로부터 분리된 세린프로테아제로서, 혈전 용해능, 항응고효과 및 세포안정성을 가져 혈전의 주 구성물질인 피브린을 효과적으로 제거함으로써, 항혈전용 조성물로 유용하게 사용될 수 있다.The serine protease of the present invention is a serine protease isolated from an unprocessed protein derived from a rockwool lugwort, and has a thrombolytic ability, an anticoagulant effect and a cell stability, thereby effectively removing fibrin, which is a main component of the thrombus, May be useful as a composition.
본 발명의 바위털갯지렁이(Marphysa Sanguinea)로부터 분리된 세린프로테아제는 주로 조간대에서 수심 50m에 이르는 바닷가 바위 틈과 개펄에서 서식하며, 4∼6월에 3만개 안팎의 알을 낳는 한국, 일본, 타이완, 오스트레일리아 등지의 온대 해역에 분포하는 바위털갯지렁이류로부터 분리한 세린프로테아제를 의미한다. The inventive rocky lagoon ( Marphysa Sanguinea ) is mainly distributed in the temperate waters of Korea, Japan, Taiwan, Australia, etc., producing eggs of about 30,000 eggs in April ~ June, Means a serine protease separated from rockworms.
상기 '분리'는 추출, 정제와 상호 교환적으로 사용될 수 있다. The 'separation' can be used interchangeably with extraction and purification.
바위털갯지렁이로부터 세린프로테아제의 분리는 황산암모늄(ammonium sulfate) 침전법과 이온교환 컬럼(ion-exchange column) 두 가지 과정을 거치는 것이 바람직하나, 이에 한정되지 않는다. 상기 황산암모늄 침전법은 황산암모늄의 이온성으로 인해 단백질 주위의 이온을 뺏고 상호간의 뭉침을 이용한 단백질 침전 방법을 말한다. The separation of the serine protease from the rockworms is preferably performed through two steps, but not limited to ammonium sulfate precipitation and ion exchange column. The ammonium sulfate precipitation method refers to a protein precipitation method that uses ionization of ammonium sulfate to lose ions around the protein and mutual aggregation.
바위털갯지렁이를 20 mM 인산 완충액(Na2HPO4, KH2PO4 및 pH 7.0)과 함께 넣고 분쇄하는 단계를 거친 후, 4℃에서 1 내지 3시간 동안 자가침전시킬 수 있으며, 바람직하게는 2시간 동안 자가침전 되도록 둘 수 있다. 그 후에 초고속 원심분리기를 이용해 30분 동안 7000g 내지 9000g에서 원심분리하여 조직을 제거하는 단계를 거칠 수 있으며, 바람직하게는 8000g에서 원심분리하여 조직을 제거할 수 있다. 또한, 조직을 제거한 상등액에서 황산암모늄 침전법을 이용하여 세린프로테아제를 회수할 수 있는 20%에서 55wt% 사이의 황산암모늄 포화농도를 이용해 조추출물을 얻어내는 단계를 거치는 것이 바람직하나, 이에 한정되지 않는다. 이렇게 얻은 조추출물을 가지고 추가적인 분리 정제를 위해 이온교환 컬럼(Hitrap DEAE Sepharose)을 이용하였으며, 시작 완충액은 20 mM Tris-HCL 완충액, 용출 완충액은 NaCl 완충액을 사용할 수 있다. 시작 완충액와 함께 1ml/min의 유량(flow rate)으로 조추출물을 로딩한 후에 NaCl 완충액을 0에서 1M까지 단계적 분획을 나누어 분리 정제를 수행하는 단계를 거칠 수 있으며, 이렇게 얻어진 추출물들을 -20℃에서 보관함이 바람직하나, 이에 한정되지 않는다.After the step of crushing the rockworms with 20 mM phosphate buffer (Na 2 HPO 4 , KH 2 PO 4, and pH 7.0), they can be self-precipitated at 4 ° C for 1 to 3 hours, preferably 2 For a period of time. Thereafter, centrifugation can be performed at 7000 g to 9000 g for 30 minutes using a ultra-high-speed centrifuge to remove the tissue, and preferably tissue can be removed by centrifugation at 8000 g. It is also preferred, but not limited, to obtain the crude extract using a saturated ammonium sulfate concentration of between 20% and 55 wt%, which can recover the serine protease using the ammonium sulfate precipitation method in the supernatant from which the tissue has been removed . An ion exchange column (Hitrap DEAE Sepharose) was used for further separation and purification with the crude extract thus obtained, and 20 mM Tris-HCL buffer for starting buffer and NaCl buffer for elution buffer. After loading the crude extract at a flow rate of 1 ml / min with the starting buffer, a step of fractionating the NaCl buffer from 0 to 1 M may be carried out to separate and purify the extracts. The extracts thus obtained are stored at -20 ° C But is not limited thereto.
본 발명의 프로테아제는 동식물의 조직이나 세포, 미생물에 널리 존재하는 단백질과 펩티드결합을 가수분해하는 효소이다. 분해양식에 의하여 엑소펩티다아제와 엔도펩티다아제로 크게 둘로 나누며, 단백질분해효소라고도 한다. 본 발명의 세린프로테아제는 촉매작용하는 필수아미노산 잔기(殘基)나 조건에 의하여 세린 효소를 가진 프로테아제를 포함하는 것을 의미한다.The protease of the present invention is an enzyme that hydrolyzes peptide bonds between proteins and microorganisms that are widely present in tissues, cells, and microorganisms. It is largely divided into exopeptidase and endopeptidase according to the degradation mode and is also called protease. The serine proteases of the present invention are meant to include proteases with serine enzymes depending on the catalytic essential amino acid residues or conditions.
본 발명의 바위털갯지렁이로부터 분리된 세린프로테아제는 간접적인 혈전용해능을 가지고 있어, 플라스미노겐을 플라스민으로 활성화시키는 간접적인 혈전용해능을 가진 항혈전용 조성물일 수 있다.The serine protease isolated from the rocky tail worms of the present invention has an indirect thrombolytic ability and can be an anti-blood-only composition having an indirect thrombolytic ability to activate plasminogen by plasmin.
또한, 직접적인 혈전용해능은 그 활성이 매우 좋지만 독성과 무분별하게 단백질을 분해한다는 점에서 그 사용에 제한되는바, 본 발명의 유효성분인 바위털갯지렁이로부터 분리된 세린프로테아제는 직접적인 혈전용해능 없이 간접적인 혈전용해능만을 나타내므로 출혈과 같은 기존 혈전용해제의 부작용을 극복할 수 있다.In addition, the direct thrombolytic ability is very good, but the use thereof is limited in that it decomposes the protein in a toxic and indiscriminate manner. As a result, the serine protease isolated from the rock wool lug, which is an effective ingredient of the present invention, And thus can overcome the side effects of conventional thrombolytic agents such as bleeding.
용어 "직접적인 혈전용해능"은 혈전의 주 구성물질인 불용성인 피브린 자체를 용해시키는 능력을 말한다. "간접적인 혈전용해능"은 피브린 자체의 용해가 아닌 불용성의 피브린을 용해시켜 피브린 분해산물로 만드는 플라스민의 전구물질인 플라스미노겐을 플라스민으로 활성화시켜 피브린을 용해시키는 능력을 말한다.The term "direct thrombolytic ability" refers to the ability to dissolve the insoluble fibrin itself, which is the main constituent of the thrombus. "Indirect thrombolytic ability" refers to the ability to dissolve fibrin by activating plasmin, a precursor of plasmin, which dissolves insoluble fibrin, which is not a fibrin itself, but produces fibrin degradation products, with plasmin.
본 발명의 바위털갯지렁이로부터 분리된 세린프로테아제는 pH 3 내지 10에서 모두 활성을 나타낼 수 있으며, 바람직하게는 pH 4 내지 6 또는 pH 8 내지 10의 조건에서 활성을 나타낼 수 있다. 또한 본 발명의 세린프로테아제는 4 내지 70℃에서 활성을 나타낼 수 있고, 바람직하게는 24 내지 70℃에서 활성을 나타낼 수 있고, 더욱 바람직하게는 37 내지 70℃의 온도조건에서 활성을 나타내는 특징을 가진 세린프로테아제일 수 있다.The serine protease isolated from the rocky tail worms of the present invention may exhibit activity at
본 발명의 바위털갯지렁이로부터 분리된 세린프로테아제는 서열번호 1로 표시되는 N-말단을 갖는 것을 특징으로 할 수 있다. 상기 서열번호 1의 폴리펩티드는 바위털갯지렁이로부터 분리된 세린프로테아제의 N 말단 서열 15잔기를 말한다. 또한 이는 바람직하게는 서열번호 1로 표시되는 아미노산 서열을 갖는 폴리펩티드 및 상기 폴리펩티드의 기능적 동등물을 포함한다. 상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 폴리펩티드를 말한다.The serine protease separated from the rocky tail worms of the present invention may be characterized by having the N-terminal end represented by SEQ ID NO: 1. The polypeptide of SEQ ID NO: 1 refers to the
본 발명의 서열번호 1의 폴리펩티드는 이의 천연형 아미노산 서열을 갖는 폴리펩티드뿐만 아니라 이의 아미노산 서열 변이체가 또한 본 발명의 범위에 포함된다. 서열번호 1의 폴리펩티드의 변이체란 서열번호 1의 폴리펩티드 천연 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 폴리펩티드을 의미한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 펩티드에서의 아미노산 교환은 당해 분야에 공지되어 있다 (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 경우에 따라서는 인산화(phosphorylation), 황화(sulfation), 아세틸화(acetylation), 당화(glycosylation), 메틸화(methylation), 파렌실화(farnesylation) 등으로 수식(modification)될 수도 있다.The polypeptide of SEQ ID NO: 1 of the present invention is also included within the scope of the present invention as well as polypeptides having a native amino acid sequence thereof, as well as amino acid sequence variants thereof. A variant of the polypeptide of SEQ ID NO: 1 means a polypeptide having a sequence that differs from the native amino acid sequence of the polypeptide of SEQ ID NO: 1 by one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution or a combination thereof. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. And may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, and the like in some cases.
바위털갯지렁이로부터 분리된 세린프로테아제는 조성물 100 중량부 대비 0.01내지 80중량부, 바람직하게는 1 내지 70 중량부, 더욱 바람직하게는 15 내지 70중량부로 포함될 수 있다.The serine protease separated from the rockworms can be contained in an amount of 0.01 to 80 parts by weight, preferably 1 to 70 parts by weight, more preferably 15 to 70 parts by weight based on 100 parts by weight of the composition.
또한 본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 혈전질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising serine protease separated from rockwool crabs.
본 발명의 바위털갯지렁이로부터 분리된 세린프로테아제는 간접적인 혈전용해능뿐만 아니라 직접적인 혈전용해능을 가져, 상기 세린프로테아제를 포함하는 약학적 조성물은 혈전질환 특히 허혈성 질환에 대한 예방 및 치료 효과를 가질 수 있다.The serine protease isolated from the rock-winged lugwort of the present invention has indirect thrombolytic ability as well as direct thrombolytic ability, and the pharmaceutical composition containing the serine protease has a preventive and therapeutic effect on the thrombotic disease, particularly the ischemic disease have.
상기 혈전질환은 허혈성 질환일 수 있고, 바람직하게는 상기 허혈성 질환은 고혈압, 심장병, 뇌졸중 및 심근경색일 수 있으며, 더욱 바람직하게는 뇌졸중일 수 있다.The thrombotic disease may be an ischemic disease, and preferably the ischemic disease may be hypertension, heart disease, stroke and myocardial infarction, more preferably stroke.
상기 혈전질환 예방 또는 치료용 조성물은 상기 바위털갯지렁이로부터 분리된 세린프로테아제를 유효성분으로 단독 포함할 수 있고, 이외 제형, 사용방법 및 사용목적에 따라 추가성분 즉, 약제학적으로 허용되거나 영양학적으로 허용되는 담체, 부형제, 희석제 또는 부성분을 추가로 포함할 수 있다.The composition for the prevention or treatment of thrombotic diseases may contain serine protease isolated from the rocky waggon alone as an active ingredient and may further contain additional components such as pharmaceutically acceptable or nutritionally acceptable substances depending on the formulation, Acceptable carriers, excipients, diluents or subcomponents.
보다 상세하게는 상기 혈전질환 예방 또는 치료용 조성물은 상기 유효성분 외에 추가로 영양제, 비타민, 전해질, 풍미제, 착색제, 중진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 추가로 함유할 수 있다.More specifically, the composition for preventing or treating thrombotic diseases may further comprise, in addition to the active ingredient, a nutrient, a vitamin, an electrolyte, a flavoring agent, a colorant, a thickening agent, a pectic acid or a salt thereof, an alginic acid or a salt thereof, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink.
또한, 상기 담체, 부형제 또는 희석제는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아키시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀루로오스, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이드, 프로필렌글리콜, 리퀴드 파라핀, 생리식염수로 이루어진 군에서 선택된 1 이상일 수 있으나, 이에 한정되는 것은 아니며 통상의 담체, 부형제 또는 희석제 모두 사용가능하다. 상기 성분들은 상기 유효성분인 바위털갯지렁이로부터 분리된 세린프로테아제에 독립적으로 또는 조합하여 추가될 수 있다.The carrier, excipient or diluent may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acicia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline In the group consisting of celluloses, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, But it is not limited thereto, and any conventional carrier, excipient or diluent can be used. The components may be added independently or in combination with a serine protease separated from the active ingredient rockwool crab.
또한, 상기 혈전질환 예방 또는 치료용 조성물은 약제화하는 경우, 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등을 더욱 포함할 수 있으며, 일 예로 경구 또는 비경구로 사용할 수 있다.The composition for preventing or treating thrombotic diseases may further comprise conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, flavors, emulsifiers or preservatives, As an example, it can be used either orally or parenterally.
구체적으로 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Particularly, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have.
또한, 본 발명의 혈전질환 예방 또는 치료용 조성물의 제형은 사용방법에 따라 바람직한 형태일 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 할 수 있다. 구체적인 제형의 예로는 경고제, 과립제, 로션제, 리니멘트제, 리모나데제, 산제, 시럽제, 안연고제, 액제, 에어로솔제, 엑스제(EXTRACTS), 엘릭실제, 연고제, 유동엑스제, 유제, 현탁제, 전제, 침제, 점안제, 정제, 좌제, 주사제, 주정제, 캅셀제, 크림제, 환제, 연질 또는 경질 젤라틴 캅셀 등이 있다.In addition, the formulation of the composition for the prevention or treatment of thrombotic diseases of the present invention may be in a desired form depending on the method of use, and may be formulated so as to provide rapid, sustained or delayed release of the active ingredient, The method can be adopted. Exemplary formulations include, but are not limited to, excipients, granules, lotions, liniments, rimonadense, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, Suspensions, premixes, infusions, eye drops, tablets, suppositories, injections, main tablets, capsules, creams, pills, soft or hard gelatin capsules.
더 나아가 본 발명의 혈전질환 예방 또는 치료용 조성물은 당해 기술 분야의 공지된 적절한 방법을 사용하여 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 바람직하게 제형화 될 수 있다.Further, the composition for the prevention or treatment of thrombotic diseases of the present invention may be prepared by using a suitable method known in the art or by a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA And the like.
본 발명에 따른 혈전질환 예방 또는 치료용 조성물의 투여량은, 투여방법, 복용자의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 당업자에 의해 적절하게 선택될 수 있다. 일예로, 본 발명의 혈전질환 예방 또는 치료용 조성물은 상기 조성물을 기준으로 할 때, 0.0001 mg/kg 내지 1000 mg/kg으로, 보다 효과적이기 위해서는 0.01mg/kg 내지 100 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dose of the composition for the prevention or treatment of thrombotic diseases according to the present invention can be suitably selected by those skilled in the art in consideration of the administration method, the age, sex, and weight of the recipient, severity of disease, and the like. For example, the composition for preventing or treating thrombotic diseases of the present invention may be administered at a dose of 0.0001 mg / kg to 1000 mg / kg based on the above composition, and 0.01 mg / kg to 100 mg / kg for more effective have. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
또한, 본 발명의 혈전질환 예방 또는 치료용 조성물은, 공지의 혈전질환 억제활성을 갖는 화합물 또는 식물 추출물을 더욱 포함할 수 있다.In addition, the composition for preventing or treating thrombosis of the present invention may further comprise a known compound or plant extract having thrombosis-inhibiting activity.
또한 본 발명은 바위털갯지렁이로부터 분리된 세린프로테아제를 포함하는 혈전질환의 예방 또는 개선용 식품 조성물을 제공한다.The present invention also provides a food composition for preventing or ameliorating a thrombotic disease comprising serine protease separated from rockwool crabs.
본 명세서에서 식품이란 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미이고, 상기 식품 조성물은 식품, 식품 첨가제, 건강 기능성 식품 및 음료를 모두 포함하는 의도이다.The term " food " as used herein means a natural product or a processed product containing one or more nutrients. Preferably, the term " food " The food composition is intended to include food, food additives, health functional foods and beverages.
본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 캔디, 차, 비타민 복합제, 기능성 식품 등이 있다. 추가로, 본 발명에서 식품에는 특수영양식품(예, 조제유류, 영, 유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예, 라면류, 국수류 등), 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임 식품(각종 김치류, 장아찌 등), 음료(예, 과실, 채소류 음료, 두유류, 발효음료류, 아이스크림류 등), 천연조미료(예, 라면 스프 등), 비타민 복합제, 알코올 음료, 주류 및 그 밖의 건강보조식품류를 포함하나 이에 한정되지 않는다. 상기 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gums, candies, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, natural flavors (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The food, beverage or food additive may be prepared by a conventional production method.
본 발명에서 기능성 식품이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미하며, 바람직하게는 본 발명의 기능성 식품은 혈전질환의 예방 또는 개선에 관한 체조절기능을 생체에 대하여 충분히 발현할 수 있는 식품을 의미한다. 상기 기능성 식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, The functional food of the present invention is preferably a food which is processed so that the body control function for prevention or improvement of thrombotic diseases can be sufficiently expressed in a living body It means food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.
이하 본 발명의 이해를 돕기 위하여 바람직한 실시예 및 제제예를 제시한다. 그러나 하기 실시예 및 제제예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 이에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments and examples of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and thus the content of the present invention is not limited thereto.
바위털Rock hair 갯지렁이로부터 분리된 Detached 세린프로테아제의Serine protease 분리 방법 How to separate
바위털갯지렁이로부터 세린프로테아제의 분리는 황산암모늄(ammonium sulfate) 침전법과 이온교환 컬럼(ion-exchange column) 두 가지 과정을 거쳤다. 바위털갯지렁이를 20 mM 인산 완충액(Na2HPO4, KH2PO4 및 pH 7.0)과 함께 넣고 분쇄한 후 4℃에서 2시간 동안 자가침전 되도록 두었다. 2시간 후 초고속 원심분리기를 이용해 30분 동안 8000g에서 원심분리하여 조직을 제거해준다. 조직을 제거한 상등액에서 황산암모늄 침전법을 이용하여 세린프로테아제를 회수할 수 있는 20%에서 55wt% 사이의 황산암모늄 포화농도를 이용해 조추출물을 얻어내었다. 이렇게 얻은 조추출물을 가지고 추가적인 분리 정제를 위해 이온교환 컬럼(Hitrap DEAE Sepharose)을 이용하였다. 시작 완충액은 20 mM Tris-HCL 완충액, 용출 완충액은 NaCl 완충액을 사용하였다. 시작 완충액와 함께 1ml/min의 유량(flow rate)으로 조추출물을 로딩한 후에 NaCl 완충액을 0에서 1M까지 단계적 분획을 나누어 분리 및 정제를 진행하였다. 이렇게 얻어진 추출물들을 -20℃에서 보관하였다.Separation of serine protease from rockworms was carried out in two steps: ammonium sulfate precipitation and ion exchange column. Rockworms were pulverized with 20 mM phosphate buffer (Na 2 HPO 4 , KH 2 PO 4 and pH 7.0) and allowed to autogenously precipitate at 4 ° C for 2 hours. After 2 hours, centrifuge at 8000g for 30 minutes using a high-speed centrifuge to remove tissue. The crude extract was obtained from ammonium sulfate precipitation in supernatant from which the tissue was removed using a saturated ammonium sulfate concentration of between 20% and 55 wt% which was capable of recovering serine protease. An ion exchange column (Hitrap DEAE Sepharose) was used for further separation and purification with the crude extract thus obtained. The starting buffer was 20 mM Tris-HCL buffer, and the elution buffer was NaCl buffer. After the crude extract was loaded with the starting buffer at a flow rate of 1 ml / min, the NaCl buffer was separated and purified stepwise from 0 to 1M. The extracts thus obtained were stored at -20 ° C.
실시예Example 1. 바위털갯지렁이에서 분리된 1. Separated from Rockwurst 세린프로테아제의Serine protease 서열분석 Sequencing
바위털갯지렁이에서 분리된 세린프로테아제가 기존의 세린프로테아제와의 얼마나 상동성이 있는지 여부를 확인하기 위해 SDS-PAGE로서 확인된 단일 밴드를 PVDF membrane으로 옮긴 후 E-mass에 분석 의뢰하여 N-말단 서열분석을 수행하였다. 구체적으로 NCBI blast에 등재된 다른 알려진 세린프로테아제인 Ixodes scapularis, Pseudoalteromonas rubra , Culex quinquefasciatus , Helobdella robusta 로부터 분리된 세린프로테아제의 N-말단 서열 15잔기와 바위털갯지렁이에서 분리된 세린프로테아제의 N-말단 잔기를 비교하였으며, 그 결과를 표 1에 나타내었다. A single band identified as SDS-PAGE was transferred to a PVDF membrane and analyzed by E-mass to confirm whether the serine protease separated from the rockworms had been homologous to the existing serine protease. The N-terminal sequence Analysis was performed. Specifically, other known serine proteases listed in NCBI blast, Ixodes scapularis, Pseudoalteromonas rubra , Culex The N-terminal sequence of the serine protease isolated from quinquefasciatus , Helobdella robusta was compared with the N-terminal residue of serine protease isolated from the rockwool lug, and the results are shown in Table 1.
[표 1][Table 1]
표 1에서 확인한 바와 같이, 바위털갯지렁이에서 분리된 세린프로테아제의 N-말단은 기존에 알려진 총 4종의 세린프로테아제와의 상동성이 모두 낮았다. 익소디즈 스카풀라리스(Ixodes scapularis ) 와의 동일성이 73.3%로 기존의 세린프로테아제 중에서 가장 유사한 것으로 확인되어, 바위털갯지렁이에서 분리된 세린프로테아제는 다른 세린프로테아제와는 상동성이 매우 낮은 신규한 세린프로테아제임을 확인하였다.As shown in Table 1, the N-terminus of serine protease separated from the rockworms had low homology with all four known serine proteases. Ixodes scapularis ) was found to be the most similar among the existing serine proteases, indicating that the serine protease isolated from the rock hair crab was a novel serine protease having a very low homology with other serine proteases.
실시예Example 2. 2. 세린프로테아제Serine protease 저해제의 저해 효과를 통한 Through the inhibitory effect of inhibitors 세린프로테아제Serine protease 검증 Verification
바위털갯지렁이로부터 분리된 물질이 세린프로테아제가 맞는지 여부를 확인하기 위하여 프로테아제 저해제를 처리하고 이에 따른 상대적 활성의 변화를 확인하였다. PMSF, 트립신 저해제(trypsin inhibitor), EDTA, 아프로티닌(aprotinin)은 잘 알려진 세린프로테아제 저해제이며, 해당 저해제를 통해 활성이 억제되는 경우, 분리된 물질이 세린프로테아제인 것으로 확인할 수 있다. 또한, 체내의 혈액 속에 존재하는 금속이온이 세린프로테아제의 효소 활성에 영향을 미칠 수 있기 때문에 금속이온에 의해 효소활성이 저해되는지 여부를 확인하기 위한 실험을 수행하였다. 상기 인자들을 처리하여 아조카세인 분석(Azocasein assay)을 수행하고 그 결과를 표 2에 나타내었다.The protease inhibitor was treated to confirm whether the material separated from the rockworms matched the serine protease, and the change in the relative activity thereof was confirmed. PMSF, trypsin inhibitor, EDTA, and aprotinin are well-known serine protease inhibitors. If the activity is inhibited by the inhibitor, it can be confirmed that the isolated substance is a serine protease. In addition, since metal ions present in the blood in the body may affect enzyme activity of serine proteases, experiments were conducted to confirm whether enzyme activity was inhibited by metal ions. The above factors were processed to perform an azocasein assay, and the results are shown in Table 2.
[표 2][Table 2]
표 2에서 확인한 바와 같이 PMSF, 트립신 저해제(trypsin inhibitor), 아프로티닌(aprotinin)은 잘 알려진 세린프로테아제 저해 인자로 이와 본 발명의 세린프로테아제를 일정농도로 반응시켰을 때 세린프로테아제 활성의 저해가 일어남을 확인하였다. 반면에 금속단백질 분해효소로 알려진 EDTA와 반응 하였을 때는 영향이 없음을 확인하였다. 또한, 바위털갯지렁이로부터 분리된 물질은 Fe3 +, Na+, K+와 같은 금속이온에 의해 영향 받지 않아 효소 활동 저해가 일어나지 않음을 확인하였다. As shown in Table 2, PMSF, trypsin inhibitor and aprotinin are well-known serine protease inhibitors. When serine protease of the present invention is reacted at a certain concentration, inhibition of serine protease activity is confirmed Respectively. On the other hand, the reaction with EDTA, known as metalloproteinases, was not affected. In addition, it was confirmed that the substances isolated from rockworms were not affected by metal ions such as Fe 3 + , Na + , K +, and thus did not inhibit enzyme activity.
따라서 바위털갯지렁이로부터 분리된 본 물질은 세린프로테아제임을 확인하였고, 체내 혈액에 존재하는 금속이온에 의한 효소활성의 저해를 받지 않아 약학적 조성물과 식품 조성물로 활용할 수 있음을 확인하였다.Therefore, it was confirmed that the present material isolated from rockwool mite was serine protease, and it was confirmed that it could be utilized as a pharmaceutical composition and a food composition without being inhibited by the enzyme activity by the metal ion present in the body blood.
실시예Example 3. 바위털갯지렁이에서 분리된 3. Separated from Rockwurst 세린프로테아제의Serine protease 혈전 용해 실험 Thrombolysis experiment
바위털갯지렁이에서 분리된 세린프로테아제의 혈전 용해 효과를 알아보기 위해 피브린 평판 분석(Fibrin plate assay)을 수행하였다. 피브린 평판은 피브리노겐과 트롬빈을 섞어 제조하였다. 피브린은 허혈성 질환에 대한 가장 큰 요인으로 혈전을 이루게 되는 주된 물질이다. 상기 피브린 평판에 바위털갯지렁이에서 분리된 세린프로테아제를 첨가하고 플라스미노겐을 풍부하게 처리한 피브린 평판과 바위털갯지렁이에서 분리된 세린프로테아제에 플라스미노겐을 첨가하지 않은 피브린 평판으로 간접적 피브린 용해 효과를 비교하여 확인하였으며, 이를 도 1에 나타내었다.Fibrin plate assay was performed to investigate the thrombolytic effect of serine protease isolated from rockwool. The fibrin plate was prepared by mixing fibrinogen and thrombin. Fibrin is the major cause of ischemic disease and the main cause of thrombosis. The fibrin plate and the fibrin plate without plasminogen added to the serine protease separated from the fibrin plate and the rock fur lug were treated with the fibrin dissolving effect by adding the serine protease separated from the rock flea to the fibrin plate and the plasminogen- The results are shown in FIG.
도 1에 나타낸 바와 같이, 바위털갯지렁이에서 분리된 세린프로테아제는 플라스미노겐이 없는 평판과 플라스미노겐이 풍부한 평판에서 상이한 용해 활성을 나타내었다. 구체적으로 플라스미노겐이 풍부한 배지에서는 4시간 후부터 용해활성을 나타내어 24시간 후에는 피브린 배지를 전부 용해시키는 우수한 활성을 나타내었다. 반면, 플라스미노겐이 없는 배지에서는 처리 후 24시간동안 분해가 거의 없어 피브린 배지 용해활성이 없는 것을 확인하였다. 이로써, 바위털갯지렁이에서 분리된 세린프로테아제가 피브린 응괴를 분해하는 직접적 용해활성이 아닌 플라스미노겐을 플라스민으로 활성화시키는 과정을 통해 간접적 혈전용해능을 가지는 것을 확인하였다.As shown in Fig. 1, the serine protease separated from the rockworms exhibited different dissolution activity on plasminogen-free plates and plasminogen-rich plates. Specifically, in the plasminogen-rich medium, the lysate was active after 4 hours, and after 24 hours, the fibrin medium was completely dissolved. On the other hand, in the plasminogen - free medium, it was confirmed that there was almost no decomposition for 24 hours after the treatment and no fibrin medium was dissolved. It was confirmed that serine protease separated from rockwool mite had indirect thrombolytic activity through the process of activating plasminogen, which is not a direct lytic activity that decomposes fibrin clot, by plasmin.
상기 결과로부터 본 발명의 세린프로테아제가 간접적 피브린 용해성을 주로 나타내 기존 직접적 용해 활성을 갖는 항혈전제의 부작용으로 판단되었던 출혈을 극복할 수 있을 것임을 확인하였다.From the above results, it was confirmed that the serine protease of the present invention is able to overcome bleeding which is considered to be a side effect of the antithrombotic agent having the direct fibrin solubility mainly due to the indirect fibrin solubility.
실시예Example 4. 바위털갯지렁이에서 분리된 4. Separated from rock-winged lug 세린프로테아제의Serine protease 피브리노겐 간접적 분해능 확인 Identification of fibrinogen indirect resolution
허혈성 뇌졸중의 가장 큰 요인인 혈전(피브린)을 플레이트 상에서 제작하여 그에 대한 혈전 용해능을 확인하기 위한 피브린 평판 분석을 수행하였다. 피브리노겐, 아가로오스, 트롬빈을 섞어 피브린을 제조하였다. 이렇게 완성된 피브린 평판에 세린프로테아제와 대조군(uPA)를 함께 투여하여 시간에 따른 피브린의 용해 범위를 비교하였다. uPA의 농도보다 세린프로테아제의 농도를 10배 높게 첨가하여 실험을 수행하였다. 도 2의 A, B에 나타내었다.Fibrin plate analysis was performed to determine the thrombus solubility of thrombus (fibrin), which is the most important factor of ischemic stroke, on the plate. Fibrinogen, agarose and thrombin were mixed to prepare fibrin. Serum protease and control (uPA) were added to the completed fibrin plate to compare the fibrin dissolution range over time. Experiments were performed by adding 10 times higher concentration of serine protease than the concentration of uPA. Are shown in Figs. 2A and 2B.
도 2의 A에서 확인한 바와 같이, U1은 500ug/ml의 농도로 20ul 첨가한 플라스미노겐과 대조군인 uPA을 처리한 것이며, U2는 플라스미노겐이 없이 대조군uPA를 처리한 것인데 대조군에서는 플라스미노겐의 유무에 따른 피브린 용해 활성의 차이가 없는 것을 확인하였다. 반면에, M1은 각각 500ug/ml의 농도로 20ul 첨가한 플라스미노겐과 바위털갯지렁이로부터 분리된 세린프로테아제를 처리한 영역이며, M2는 플라스미노겐이 없이 바위털갯지렁이로부터 분리된 세린프로테아제만을 처리한 영역인데, 플라스미노겐이 풍부한 영역의 용해된 부분이 더 넓은 것을 확인하였다. As shown in FIG. 2A, U1 was treated with plasminogen added at a concentration of 20 .mu.g at a concentration of 500 .mu.g / ml and uPA as a control group. U2 was treated with a control uPA without plasminogen. In the control group, plasminogen And there was no difference in the fibrinolytic activity depending on the presence or absence of the fibrin. On the other hand, M1 is a region treated with serine protease separated from plasminogen and rock hairy worms added at a concentration of 20 ug / ml at a concentration of 500 ug / ml, M2 is treated with only serine protease separated from rockworms without plasminogen One region, and it was confirmed that the dissolved portion of the region rich in plasminogen was wider.
도 2의 B에서 확인한 바와 같이, 플라스미노겐이 없는 배지(M2)보다 플라스미노겐이 풍부한 배지(M1)가 용해 영역이 더 넓어 간접적 용해 활성이 더 강하게 나타남을 확인하였다. 이는 바위털갯지렁이에서 분리된 세린프로테아제의 간접적 피브린 용해활성이 더 강하다는 점을 입증하는 것으로 허혈성 뇌졸중 후 재관류 과정에서 혈액뇌장벽(blood-brain barrier)의 보호에 더 효과적임을 확인하였다.As shown in Fig. 2B, it was confirmed that the medium (M1) rich in plasminogen was wider in the dissolution zone than the medium (M2) without plasminogen, and the indirect dissolution activity was more strongly exhibited. This demonstrates that the indirect fibrinolytic activity of serine proteases isolated from rockwool was more effective in protecting the blood-brain barrier during reperfusion after ischemic stroke.
또한, 세린프로테아제의 간접적 피브린 용해활성을 확인하기 위해 플라스미노겐에 세린프로테아제를 일정 농도로 섞어 플라스미노겐이 분해되는지 확인하기 위한 실험을 수행하였다. 이를 도 2C에 나타내었다. In order to confirm the indirect fibrinolytic activity of serine protease, experiments were conducted to confirm that plasminogen was degraded by mixing serin protease at a certain concentration in plasminogen. This is shown in FIG. 2C.
도 2C에서 확인한 바와 같이, 세린프로테아제에 의해 플라스미노겐이 17 kDa 보다 작은 조각으로 가수분해되었으며, 이를 통해 세린프로테아제가 플라스미노겐을 플라스민으로 가수분해하여 간접적 피브린 용해 활성을 가짐을 확인하였다.As shown in FIG. 2C, plasminogen was hydrolyzed by serine protease into fragments smaller than 17 kDa, which confirmed that serine protease hydrolyzes plasminogen to plasmin and has indirect fibrinolytic activity.
실시예Example 5. 바위털갯지렁이에서 분리된 5. Separated from rock-cut lark 세린프로테아제의Serine protease 정제방법에 따른 활성 및 수율 분석 Activity and yield analysis by purification method
분리 정제 단계에 따른 세린 프로테아제의 각각의 전체 단백질 양, 전체 활성, 특정 활성, 수율을 확인하기 위해서 BCA 분석과 피브린 평판 분석을 수행하였다. 세린프로테아제는 황산 암모늄(ammonium sulfate) 침전법, ion-exchange column 두 단계를 거쳐 바위털갯지렁이로부터 분리 정제하였다. BCA assay를 통한 전체 단백질의 양을 확인하고, 피브린 평판 분석을 통해 크기를 통한 전체 활성을 측정하였다. 특정 활성은 전체 활성/전체 단백질 양으로 했으며, 수율은 전체 활성을 통해 측정하였다. 상기 전체활성은 피브린 평판 분석 후 임의의 단위 unit을 통해 각 정제 단계별로 시료에 들어있는 활성도를 나타낸 척도를 말한다. 또한, 상기 특정활성은 이러한 전체활성을 BCA assay를 이용해 확인한 전체 단백질 양으로 나누어 전체 단백질 양 대비 정제과정 후에 얼마나 더 높은 활성을 나타내는지의 척도를 말하며, 이를 표 3에 나타내었다.BCA analysis and fibrin plate analysis were performed to confirm the total protein content, total activity, specific activity, and yield of each of the serine proteases according to the separation and purification steps. The serine protease was separated and purified from the rockworms by two steps of ammonium sulfate precipitation and ion exchange column. The total amount of protein was determined by BCA assay and total activity through size was measured by fibrin plate analysis. The specific activity was the total active / total protein amount, and the yield was determined by total activity. The total activity is a measure of the activity of the sample in each purification step through a unit of unit after the fibrin plate assay. In addition, the specific activity refers to a measure of how much the total activity is divided by the total protein amount determined by the BCA assay to show the higher activity after the purification process compared to the total protein amount, as shown in Table 3.
[표 3] [Table 3]
세린프로테아제의 전체활성과 특정활성Total activity and specific activity of serine protease
결과적으로, 특정활성이 ammonium sulfate 침전법 후보다 ion-exchange column을 내린 후에 약 1.5배 증가함을 확인하였다. 따라서, 바위털갯지렁이로부터 분리한 세린프로테아제는 황산 암모늄 침전법 후 ion-exchange column을 수행하여 수득하는 것이 우수한 효소활성을 나타낼 수 있음을 확인하였다. As a result, it was confirmed that the specific activity increased about 1.5 times after the ion-exchange column was lowered after the ammonium sulfate precipitation method. Therefore, it has been confirmed that the serine protease separated from the rockworms can exhibit an excellent enzyme activity by performing an ion-exchange column after ammonium sulfate precipitation.
실시예Example 6. 바위털갯지렁이로부터 분리한 6. Separated from Rockwurst 세린프로테아제의Serine protease 분자량 확인 Check molecular weight
세린프로테아제의 분자량을 확인하기 위해 SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)를 수행하였다. 바위털갯지렁이로부터 분리한 세린프로테아제를 ion-exchange column으로 정제 후 SDS-PAGE를 수행하였으며 그 결과를 도 3에 나타내었다. M은 standard size marker이며, Lane 1은 바위털갯지렁이로부터 분리한 세린프로테아제에 관한 것으로 세린프로테아제는 약 27 kDa로 확인되었다.SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed to confirm the molecular weight of serine protease. Serine protease separated from rockworms was purified by ion-exchange column and subjected to SDS-PAGE. The results are shown in FIG. M is a standard size marker,
실시예Example 7. 바위털갯지렁이에서 분리된 7. Separated from rock-winged lobsters 세린프로테아제의Serine protease 피브린 용해 활성에 대한 pH와 온도의 효과Effect of pH and Temperature on Fibrinolytic Activity
바위털갯지렁이로부터 분리된 세린프로테아제를 혈관 내에 투여하였을 때 혈관의 pH와 온도 환경에서 효과를 나타내는지 확인하기 위하여, pH 3~10 범위와 온도 4~70℃일때 아조카세인 분석(Azocasein assay)을 수행하였으며, 이를 도 4에 나타내었다. Azocasein assay was performed at a pH range of 3 to 10 and at a temperature of 4 to 70 ° C in order to confirm whether the serine protease separated from the rockworms was effective in the pH and temperature environment of blood vessels This is shown in FIG.
도 4에서 확인한 바와 같이, 바위갯지렁이로부터 분리한 세린프로테아제를 각 범위의 pH와 온도에 처리한 후의 결과를 확인하였을 때, 산성과 알칼리성에서 모두 활성을 나타내는 것을 볼 수 있으며(A), 온도가 높을수록 활성이 증가하는 것을 확인하였다(B). 특히 허혈성 뇌졸중 이후에는 혈액환경이 산성이 되는데 이런 산성 환경에서 바위갯지렁이로부터 분리한 세린프로테아제가 효소 활성을 나타낼 수 있음을 확인하였다. 따라서, 바위갯지렁이로부터 분리한 세린프로테아제는 넓은 pH 범위와 높은 온도에서 활성을 나타낼 수 있어 인체에 투여 시 좋은 활성을 유지 할 수 있음을 확인하였다.As can be seen in FIG. 4, when the serine protease isolated from the rockworms was treated at various pH and temperature ranges, the activity was found to be both acidic and alkaline (A) (B). ≪ / RTI > Especially after the ischemic stroke, the blood environment becomes acidic. It has been confirmed that the serine protease isolated from the rockworms in this acidic environment can exhibit the enzyme activity. Therefore, it was confirmed that serine protease separated from rockworms can exhibit activity over a wide pH range and high temperature, and can maintain good activity upon administration to human body.
실시예Example 8. 바위털갯지렁이에서 8. Rockwool 분리된 분리된Detached separated 세린프로테아제의Serine protease 피브리노겐 용해 활성 Fibrinogen-lytic activity
바위털갯지렁이로부터 분리된 세린프로테아제가 트롬빈과 피브리노겐이 만나 피브린이 되기 전의 피브리노겐을 용해시키는 활성이 있는지 여부를 확인하기 위하여, 바위털갯지렁이로부터 분리한 세린프로테아제를 10 내지 1000ng 으로 피브리노겐에 처리하고 이에 따른 피브린노겐 용해 활성을 확인하였으며 그 결과를 도 5에 나타내었다.Serine proteases isolated from rockworms were treated with 10-1000 ng of fibrinogen to isolate serine protease from rockwool crabs to determine whether thrombin and fibrinogen met the activity of dissolving fibrinogen before it became fibrin. The fibrinogen dissolution activity was confirmed and the results are shown in Fig.
도 5에서 확인한 바와 같이, 피브리노겐의 알파, 베타, 감마 체인이 용량 의존적으로 순서대로 끊어지는 것을 확인하였고, 이를 통해, 통상의 프로테아제에 의해 끊어지기 힘든 감마체인까지 끊을 수 있어, 상기 세린프로테아제가 피브리노겐을 효과적으로 절단하는 활성이 있는 우수한 피브리노겐 분해능이 있음을 확인하였다.As shown in FIG. 5, it was confirmed that the alpha, beta and gamma chains of fibrinogen were sequentially cut in a dose-dependent manner, and through this, it was possible to break up to a gamma chain which is not easily broken by a normal protease, Which has an activity of effectively cleaving fibrinogen.
실시예Example 9. 바위털갯지렁이에서 분리된 9. Separated from Rockwurst 세린프로테아제의Serine protease 세포독성여부Cytotoxicity
바위털갯지렁이로부터 분리된 세린프로테아제를 혈관 내에 투여하였을 때 내피세포에 독성을 나타내는지 확인하기 위하여, 혈관뇌장벽에 분포한 내피세포 (Endothelial cell line (hCMEC/D3)) 에 바위털갯지렁이로부터 분리된 세린프로테아제를 처리하고, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 분석을 통한 비색분석법을 인산완충생리식염수(PBS)를 처리한 대조군과 1, 3, 5일차를 비교하여 수행하였으며, 그 결과를 도 6에 나타내었다.In order to confirm that endothelial cells were toxic when serine protease isolated from rockworms was administered into the blood vessels, endothelial cell lines (hCMEC / D3) distributed in the blood brain barrier were separated from rockwool lug Serine protease was treated and the colorimetric analysis by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis was performed in phosphate buffered saline (PBS) , And 5 days were compared. The results are shown in FIG.
도 6에서 확인한 바와 같이, 바위털갯지렁이에서 분리된 세린프로테아제는 대조군과 세포 생존률에서 유의한 차이를 나타내지 않으므로, 세포독성이 없음을 확인하였다.As shown in FIG. 6, serine protease isolated from rockwool lugwort did not show any significant difference in the cell survival rate between the control group and the control group, and thus it was confirmed that there was no cytotoxicity.
제제예Formulation example 1. 약학적 제제의 제조 1. Preparation of pharmaceutical preparations
1. 산제의 제조 1. Manufacturing of powder
바위털갯지렁이로부터 분리된 세린프로테아제 20 ml20 ml of serine protease isolated from rockwool
유당 100 mg
탈크 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above components are mixed and filled in airtight bags to prepare powders.
2. 정제의 제조2. Preparation of tablets
바위털갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease separated from rockworms
옥수수전분 100 mg
유당 100 mg
스테아린산 마그네슘 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
3. 캡슐제의 제조3. Preparation of capsules
바위털갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease separated from rockworms
결정성 셀룰로오스 3 mg
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
4. 주사제의 제조4. Preparation of injections
바위털갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease separated from rockworms
만니톨 180 mg180 mg mannitol
주사용 멸균 증류수 2974 mgSterile sterilized water for injection 2974 mg
Na2HPO42H2O 26 mgNa 2 HPO 4 2H 2 O 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당 (2 ml) 상기의 성분 함량으로 제조한다.(2 ml) per 1 ampoule in accordance with the usual injection preparation method.
5. 액제의 제조5. Manufacture of liquids
바위털갯지렁이로부터 분리된 세린프로테아제 20 ml 20 ml of serine protease isolated from rockwool
이성화당 10 g10 g per isomer
만니톨 5 g5 g mannitol
정제수 적량Purified water quantity
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ml로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.
제제예Formulation example 2. 식품 제제의 제조 2. Manufacture of food preparation
1. 건강식품의 제조1. Manufacture of health food
바위털갯지렁이로부터 분리된 세린프로테아제 100 ml100 ml of serine protease isolated from rockworms
비타민 혼합물 적량Vitamin mixture quantity
비타민 A 아세테이트 70 g 70 g of vitamin A acetate
비타민 E 1.0 mgVitamin E 1.0 mg
비타민 B1 0.13 mgVitamin B1 0.13 mg
비타민 B2 0.15 mg0.15 mg of vitamin B2
비타민 B6 0.5 mgVitamin B6 0.5 mg
비타민 B12 0.2 g 0.2 g of vitamin B12
비타민 C 10 mg
비오틴 10 g Biotin 10 g
니코틴산아미드 1.7 mgNicotinic acid amide 1.7 mg
엽산 50 g Folate 50 g
판토텐산 칼슘 0.5 mgCalcium pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture quantity
황산제1철 1.75 mg1.75 mg of ferrous sulfate
산화아연 0.82 mg0.82 mg of zinc oxide
탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg
제1인산칼륨 15 mgPotassium monophosphate 15 mg
제2인산칼슘 55 mgSecondary calcium phosphate 55 mg
구연산칼륨 90 mgPotassium citrate 90 mg
탄산칼슘 100 mg
염화마그네슘 24.8 mgMagnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
2. 건강음료의 제조2. Manufacture of health drinks
바위털갯지렁이로부터 분리된 세린프로테아제 100 ml100 ml of serine protease isolated from rockworms
비타민 C 15 gVitamin C 15 g
비타민 E(분말) 100 gVitamin E (powder) 100 g
젖산철 19.75 g19.75 g of ferrous lactate
산화아연 3.5 g3.5 g of zinc oxide
니코틴산아미드 3.5 gNicotinic acid amide 3.5 g
비타민 A 0.2 gVitamin A 0.2 g
비타민 B1 0.25 gVitamin B1 0.25 g
비타민 B2 0.3gVitamin B2 0.3g
물 정량Water quantification
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2리터 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다.The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
<110> Korea Institute of Coastal Ecology, Inc.
<120> Anti-thrombotic composition comprising serine protease extracted
from Marphysa Sanguinea
<130> 1-2p-1
<150> KR 10-2016-0049159
<151> 2016-04-22
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 15
<212> PRT
<213> Unknown
<220>
<223> serine protease N-terminal sequence extracted from Marphysa
Sanguinea
<400> 1
Ile Val Gly Gly Ser Glu Ala Thr Pro Tyr Gln Phe Pro Phe Gln
1 5 10 15
<110> Korea Institute of Coastal Ecology, Inc.
<120> Anti-thrombotic composition comprising serine protease extracted
from Marphysa Sanguinea
<130> 1-2p-1
<150> KR 10-2016-0049159
<151> 2016-04-22
<160> 1
<170> KoPatentin 3.0
<210> 1
<211> 15
<212> PRT
<213> Unknown
<220>
<223> serine protease N-terminal sequence extracted from Marphysa
Sanguinea
<400> 1
Ile Val Gly Gly Ser Glu Ala Thr Pro Tyr Gln Phe
Claims (9)
1) 바위털갯지렁이에 인산완충액을 가하고 분쇄하는 단계;
2) 상기 분쇄물을 1 내지 3시간동안 자가 침전 시키는 단계;
3) 원심분리기를 이용하여 자가침전된 분쇄물에서 조직을 제거하는 단계;
4) 조직이 제거된 시료에 20 내지 55wt% 황산암모늄을 첨가해 조추출물을 얻는 단계; 및
5) 상기 조추출물을 이온교환 컬럼을 이용해 분리 정제하여 세린프로테아제를 얻는 단계; 를 포함하여 제조되는 것을 특징으로 하는, 항혈전용 조성물.The method of claim 1, wherein the serine protease is
1) adding phosphoric acid buffer solution to rockworms and pulverizing;
2) self-precipitating the pulverized material for 1 to 3 hours;
3) removing the tissue from the self-precipitated pulp using a centrifuge;
4) adding 20 to 55 wt% ammonium sulfate to the sample from which the tissue has been removed to obtain a crude extract; And
5) Separation and purification of the crude extract using an ion exchange column to obtain serine protease; ≪ / RTI > wherein the composition is prepared by the method of claim 1.
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| KR20170121061A (en) * | 2016-04-22 | 2017-11-01 | (주)한국연안환경생태연구소 | Anti-thrombotic composition comprising serine protease extracted from Diopatra Sugokai |
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| KR20170121061A (en) * | 2016-04-22 | 2017-11-01 | (주)한국연안환경생태연구소 | Anti-thrombotic composition comprising serine protease extracted from Diopatra Sugokai |
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