KR20170121061A - Anti-thrombotic composition comprising serine protease extracted from Diopatra Sugokai - Google Patents
Anti-thrombotic composition comprising serine protease extracted from Diopatra Sugokai Download PDFInfo
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- KR20170121061A KR20170121061A KR1020170047965A KR20170047965A KR20170121061A KR 20170121061 A KR20170121061 A KR 20170121061A KR 1020170047965 A KR1020170047965 A KR 1020170047965A KR 20170047965 A KR20170047965 A KR 20170047965A KR 20170121061 A KR20170121061 A KR 20170121061A
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- serine protease
- composition
- thrombotic
- house
- hairy
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Abstract
Description
본 발명은 털보집갯지렁이로부터 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 hair follicle worm.
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, 털보집갯지렁이(Diopatra Sugokai)는 우리나라 남해와 서해 연안의 모래진흙 조간대 중·하부지역에서 비교적 흔히 발견되는 육식성의 서관 형성 갯지렁이류이다. 갯지렁이는 지렁이와 비슷한 종류의 환영동물(annelid)이나, 지렁이(Eisenia andrei) 추출물에 대한 연구조차 많이 진행되지 않았으며 갯지렁이에 대한 항혈전 연구는 거의 보고된 바가 없다.On the other hand, Diopatra Sugokai ) is a carnivorous westerlings that are relatively common in sandy mud tidal zones in the southern and southern coastal waters of Korea. There have been few studies on annelids or earthworms (Eisenia andrei) extracts of earthworms similar to earthworms, and there have been few reports of antithrombotic studies on the worms.
이에 본 발명자들은 털보집갯지렁이를 활용한 연구를 수행하던 중 털보집갯지렁이로부터 분리된 세린프로테아제에서 항혈전능을 발견해 본 발명을 완성하였다. Accordingly, the present inventors have completed the present invention by discovering antithrombotic activity in serine proteases isolated from hairy house worms while performing research using a hairy house wormworm.
본 발명의 목적은 털보집갯지렁이로부터 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 a hair follicle worm.
상기 목적을 달성하기 위하여, 본 발명은 털보집갯지렁이로부터 분리된 세린프로테아제를 포함하는 항혈전용 조성물을 제공한다. In order to attain the above object, the present invention provides a composition for exclusive use in hemostasis comprising serine protease separated from a hair follicle worm.
또한 본 발명은 털보집갯지렁이로부터 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 the hair follicle worms.
또한 본 발명은 털보집갯지렁이로부터 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 a hair follicle worm.
본 발명에 따른 털보집갯지렁이로부터 분리된 세린프로테아제는 직, 간접적인 혈전 용해능이 넓은 pH와 온도에서 나타나며 또한, 항응고효과 및 세포안정성을 나타낸다. 더불어 세포 독성도 없는 바, 현재 혈전용해제로 유일하게 사용되고 있는 t-PA(tissue-Plasminogen Activator)의 재막힘(reocclusion) 현상과 같은 부작용을 극복할 수 있다. 특히, 기존 치료제가 간접적인 혈전용해능을 위주로 갖고 있는 것에 비해 본 발명에서의 털보집갯지렁이로부터 분리된 세린프로테아제는 간접적인 혈전용해능 뿐만 아니라 직접적인 혈전용해능까지 갖고 있어, 뛰어난 혈전용해능으로 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 hair follicle worms according to the present invention exhibits direct or indirect fibrinolytic ability at a wide pH and temperature and exhibits anticoagulant effect and cell stability. In addition, there is no cytotoxicity, and it is possible to overcome the side effects such as the reocclusion phenomenon of t-PA (tissue-plasminogen activator) which is currently used as the thrombolytic agent. In particular, although the conventional therapeutic agent mainly has an indirect thrombolytic ability, the serine protease isolated from the hair follicle worms of the present invention has not only an indirect thrombolytic ability but also a direct thrombolytic ability, And anti-thrombotic pharmaceutical composition and food composition effective for diseases such as hypertension, heart disease, stroke, myocardial infarction and the like due to platelet aggregation.
도 1은 털보집갯지렁이로부터 분리된 세린프로테아제의 피브린 용해활성을 확인하기 위한 피브린 평판 분석(Fibrin plate assay) 결과를 나타낸 도이다.
도 2는 본 발명의 털보집갯지렁이로부터 분리된 세린프로테아제의 간접적 혈전용해 활성을 확인하기 위한 피브린 평판 분석 결과를 나타낸 도이다.
도 3은 털보집갯지렁이로부터 분리된 세린프로테아제의 피브린노겐 용해 활성을 확인하기 위한 SDS-PAGE의 결과를 나타낸 도이다.
도 4는 혈전용해를 위해 털보집갯지렁이로부터 분리된 세린프로테아제를 혈관 내에 투여하였을 때 혈관의 pH와 온도 환경에서 효과를 나타내는지 확인하기 위한 아조카세인 분석을 실시하여 그 결과를 나타낸 도이다.
도 5는 털보집갯지렁이로부터 분리된 세린프로테아제의 분자량을 확인하기 위한 SDS-PAGE 실험 후 결과를 나타낸 도이다.
도 6은 털보집갯지렁이로부터 분리된 세린프로테아제의 정제방법에 따른 활성을 분석하기 위한 피브린 자이모그래피를 수행한 후 결과를 나타낸 도이다.
도 7은 혈전용해를 위해 털보집갯지렁이로부터 분리된 세린프로테아제를 혈관 내에 투여하였을 때 내피세포에 독성을 나타내는지 확인하기 위해 MTT 분석을 하여 그 결과를 나타낸 도이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of fibrin plate assay for confirming fibrinolytic activity of serine protease separated from a hair follicle.
FIG. 2 is a graph showing the results of fibrin plate analysis for confirming the indirect thrombolytic activity of serine proteases isolated from the hair follicle worms of the present invention. FIG.
3 is a graph showing the results of SDS-PAGE for confirming the fibrinogen-dissolving activity of serine protease separated from the hair follicle worms.
FIG. 4 is a graph showing the results of an azo casein analysis to determine whether the serine protease isolated from the hair follicle lugwort for thrombus dissolution is effective in the pH and temperature environment of the blood vessel when administered into the blood vessel.
FIG. 5 is a graph showing the results after SDS-PAGE experiments for confirming the molecular weight of serine protease separated from the hair follicle worms.
FIG. 6 is a graph showing the results of fibrinogenography for analyzing the activity of the serine protease separated from the hair follicle evacuation worms.
FIG. 7 is a graph showing the results of MTT analysis to determine whether endothelial cells are toxic when serine protease separated from hair follicle worms for blood clotting is administered into blood vessels.
본 발명은 털보집갯지렁이로부터 분리된 세린프로테아제를 포함하는 항혈전용 조성물을 제공한다.The present invention provides a composition for anti-venom exclusive use comprising a serine protease separated from a hair follicle worm.
이하, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
상기 본 발명의 세린프로테아제는 털보집갯지렁이로부터 유래한 가공하지 않은 단백질로부터 분리된 세린프로테아제로서, 혈전 용해능, 항응고효과 및 세포안정성을 가져 혈전의 주 구성물질인 피브린을 효과적으로 제거함으로써, 항혈전용 조성물로서 유용하게 사용될 수 있다.The serine protease of the present invention is a serine protease isolated from an unprocessed protein derived from a hair follicle worm, and has a thrombolytic effect, an anticoagulant effect and a cell stability, thereby effectively removing fibrin, Can be usefully used as a composition.
본 발명의 털보집갯지렁이로(Diopatra Sugokai)부터 분리된 세린프로테아제는 우리나라 남해와 서해 연안의 모래진흙 조간대 중·하부지역에서 비교적 흔히 발견되는 육식성의 서관 형성 갯지렁이류인 털보집갯지렁이로부터 분리한 세린프로테아제를 의미한다. A home teolbo lug of the present invention (Diopatra The serine protease isolated from Sugokai means serine protease separated from the hairy house worm, which is a relatively common carnivorous wormhole , which is found in the middle and lower parts of the sandy cliffs in the south and west coasts of Korea.
상기 '분리'는 추출, 정제와 상호 교환적으로 사용될 수 있다. The 'separation' can be used interchangeably with extraction and purification.
털보집갯지렁이로부터 분리된 세린프로테아제의 분리방법은 믹서기에 털보집갯지렁이 30g과 인산 완충액 60ml을 가하고 분쇄하는 단계를 거친 후, 액체상태인 상기 분쇄물을 세린프로테아제를 포함한 단백질이 인산 완충액에 충분히 녹을 수 있도록 1 내지 3시간, 바람직하게는 2시간 동안 자가분해(auto-lysis) 시키는 단계를 포함할 수 있다. 또한 분리방법은 자가분해된 시료를 초고속 원심분리기를 이용하여 단백질들이 많이 포함된 상등액만 얻는 단계를 거쳐, 단백질을 침전시키기 위해 상등액에 황산암모늄을 10 내지 30wt%, 바람직하게는 20wt%를 첨가하고 원심분리기를 이용하여 단백질 침전물을 얻는 단계를 거칠 수 있으나, 이에 한정되지 않는다. 이후 얻어진 상등액에 황산암모늄을 20 내지 60 wt%를 넣을 수 있으며, 바람직하게는 50wt%로 황산암모늄 포화침전을 진행할 수 있다. 7000g내지 9000g에서 원심분리기를 사용하며, 바람직하게는 8000g로 원심분리기를 사용해 단백질을 침전시키고 상등액을 버린 후, 남은 잔여물에 인산 완충액을 소량 넣어 털보집갯지렁이로부터 분리한 세린프로테아제를 얻는 것이 바람직하나, 이에 한정되지 않는다.The separation method of serine protease separated from the hair follicle house worms is performed by adding 30 g of the hair follicle worms and 60 ml of phosphate buffer to a blender and then pulverizing the mixture. The protein in the liquid phase, which contains serine protease, Lysis for 1 to 3 hours, preferably for 2 hours. In the separation method, the autolysed sample is subjected to a step of obtaining a supernatant containing a large amount of proteins by using an ultra-high-speed centrifugal separator. To precipitate the protein, ammonium sulfate is added to the supernatant in an amount of 10 to 30 wt%, preferably 20 wt% But the present invention is not limited thereto. Ammonium sulfate may be added to the resulting supernatant in an amount of 20 to 60 wt%, preferably 50 wt%, and saturated ammonium sulfate precipitation may proceed. It is preferable to precipitate the proteins using centrifuges at 7000 g to 9000 g, preferably 8000 g, to remove the supernatant, and then add a small amount of phosphate buffer to the remaining residue to obtain serine protease separated from the hair follicle worms , But is not limited thereto.
상기 분리방법에서 황산암모늄를 이용한 포화 침전방법을 이용할 수 있는데, 이런 포화 침전방법은 황산암모늄의 이온성으로 인해 단백질 주위의 이온을 뺏고 상호간의 뭉침을 이용한 단백질 침전 방법을 말한다. 상기 황산암모늄를 이용한 포화 침전방법은 농도구배법을 이용할 수 있다. 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.In this separation method, a saturated precipitation method using ammonium sulfate can be used. Such a saturated precipitation method refers to a protein precipitation method that uses ions to separate ions around the protein due to the ionic property of ammonium sulfate and uses mutual aggregation. The saturated precipitation method using ammonium sulfate can be performed by a concentration gradient method. 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 털보집갯지렁이로부터 분리된 세린프로테아제는 플라스미노겐을 플라스민으로 활성화시키는 간접적인 혈전용해능 뿐만 아니라 직접적으로 혈전인 피브린을 용해시키는 직접적인 혈전용해능을 가진 항혈전용 조성물일 수 있다. The serine protease isolated from the hair follicle worms of the present invention may be an anti-blood-only composition having direct thrombolytic ability to directly dissolve thrombus fibrin as well as indirect thrombolytic ability to activate plasminogen by plasmin.
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 털보집갯지렁이로부터 분리된 세린프로테아제는 pH 3 내지 10에서 모두 활성을 나타낼 수 있으며, 바람직하게는 pH 4 내지 5 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 hair follicle worms of the present invention may exhibit activity at a pH of 3 to 10, preferably at a pH of 4 to 5 or a pH of 8 to 10. In addition, the serine protease of the present invention may exhibit activity at 4 to 70 ° C, preferably at 24 to 70 ° C, more preferably at 37 to 70 ° C, Serine protease.
털보집갯지렁이로부터 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 hairy house worms may 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 털보집갯지렁이로부터 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 the hair follicle worms.
본 발명의 털보집갯지렁이로부터 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 hair follicle 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 thrombotic diseases, particularly ischemic diseases 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 털보집갯지렁이로부터 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 preventing or treating thrombotic diseases may contain serine protease isolated from the hairy house wormworm alone as an active ingredient and may further contain additional components such as pharmaceutically acceptable or nutritionally acceptable agents 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 털보집갯지렁이로부터 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 can be added to the serine protease separated from the hairborne house worm, which is the active ingredient, independently or in combination.
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 제형화될 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 100mg/kg으로 투여할 수 있다. 투여는 하루에 한 번 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 . The administration may be carried out once a day or divided into several doses. 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 털보집갯지렁이로부터 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 a hair follicle worm.
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.
털보집갯지렁이로부터 분리된 Fleece 세린프로테아제의Serine protease 분리방법 How to separate
털보집갯지렁이로부터 세린프로테아제를 분리하기 위해 믹서기에 털보집갯지렁이 30g과 인산 완충액 60ml을 넣고 갈아준다. 액체상태인 세린프로테아제를 포함한 단백질이 인산 완충액에 충분히 녹을 수 있도록 2시간 동안 자가분해(auto-lysis) 시켜준다. 샘플을 초고속 원심분리기를 이용하여 단백질들이 많이 포함된 상등액만 얻는다. 단백질을 침전시키기 위해 상등액에 황산암모늄을 20wt%넣고 원심분리기를 이용하여 다시 침전시킨다. 이때, 황산암모늄를 이용한 포화 침전은 농도구배법을 이용한 것으로, 황산암모늄의 이온성으로 인해 단백질 주위의 이온을 뺏고 상호간의 뭉침을 이용한 단백질 침전 방법이다. 이후 얻어진 상등액에 위와 같이 50wt% 황산암모늄 포화침전을 진행한다. 마지막으로 8000g로 원심분리기를 사용해 단백질을 침전시켜 상등액을 버린 후, 남은 잔여물에 인산 완충액을 소량 넣어 털보집갯지렁이로부터 분리한 세린프로테아제를 얻는다.To remove the serine protease from the fleece house lobster, add 30 grams of fleece house lobsters and 60 ml of phosphate buffer to the mixer. The protein, including liquid serine protease, is auto-lysed for 2 hours to allow it to fully dissolve in phosphate buffer. Using a high-speed centrifuge, obtain only the supernatant containing a large amount of proteins. To precipitate the protein, 20 wt% ammonium sulfate is added to the supernatant and the precipitate is centrifuged again. At this time, the saturated precipitation using ammonium sulfate is performed by a concentration gradient method, which is a protein precipitation method using ion clusters of ions around the protein due to the ionic property of ammonium sulfate. The resulting supernatant is then subjected to 50 wt% ammonium sulfate saturation precipitation as above. Finally, the protein is precipitated with a centrifuge at 8000 g, the supernatant is discarded, and a small amount of phosphate buffer is added to the remaining residue to obtain a serine protease separated from the hair follicle.
실시예Example 1. 혈전 용해 실험 1. Thrombolysis experiment
털보집갯지렁이에서 분리한 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 털보집갯지렁이에서 추출한 세린 프로테아제를 첨가하고 그 효과를 PBS대조군과 비교하여 확인하였다. 또한 간접적 혈전 용해 활성을 확인하기 위해서 플라스미노겐이 풍부한 배지와 플라스미노겐이 없는 배지에 세린프로테아제를 처리하고 4, 24시간 간격으로 피브린 평판분석을 수행하여, 이를 도 1 및 도 2에 나타내었다.A fibrin plate assay was performed to investigate the thrombolytic effect of serine protease isolated from the hairy house rug. 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 was supplemented with serine protease extracted from the hairy house lugworm and its effect was confirmed by comparing with the PBS control. In order to confirm indirect thrombolytic activity, serine protease was treated with plasminogen-rich medium and plasminogen-free medium, and fibrin plate analysis was performed at intervals of 4 hours and 24 hours, as shown in FIGS. 1 and 2 .
도 1에서 나타난 바와 같이, 털보집갯지렁이에서 분리한 세린프로테아제는 PBS 대조군과 비교할 때 확연하게 피브린을 용해하여 직접적으로 용해함을 확인하였다. 또한 도 2에 나타난 바와 같이, 플라스미노겐이 없는 피브린평판(plasminogen free)에서보다 플라스미노겐이 풍부한(plasminogen rich)평판에서 용해의 정도가 더 좋은 것이 확인되어 털보집갯지렁이에서 분리한 세린프로테아제가 플라스미노겐을 플라스민으로 활성화시켜 피브린을 용해시키는 용해능이 있음을 확인하였다.As shown in Fig. 1, the serine protease isolated from the hair follicle evacuation worms was confirmed to dissolve fibrin in a markedly dissoluble state when compared with the PBS control. As shown in Fig. 2, it was confirmed that the degree of dissolution was better in plasminogen-rich plate than in plasminogen-free fibrin plate (plasminogen-free), and serine protease isolated from the hairy house lugwort It was confirmed that the plasminogen was activated by the plasmin to have solubility in dissolving fibrin.
이를 통해 털보집갯지렁이에서 분리한 세린프로테아제는 피브린 평판 분석에서 피브린을 직접적으로 녹이는 활성뿐만 아니라 플라스미노겐을 플라스민으로 활성화시켜 간접적으로 피브린을 녹이는 활성을 가지고 있음을 확인하였다.The serine protease isolated from the hairy house gargoyle had the activity of directly dissolving the fibrin in the fibrin plate analysis as well as activating the plasminogen by the plasmin to indirectly dissolve the fibrin.
실시예Example 2. 2. 세린프로테아제Serine protease 저해제의 저해 효과를 통한 Through the inhibitory effect of inhibitors 세린프로테아제Serine protease 검증 Verification
털보집갯지렁이로부터 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. 상기 인자들을 처리하여 아조카세인 분석(Azocasein assay)을 수행하고 그 결과를 표 1에 나타내었다.The protease inhibitor was treated to confirm whether the substance isolated from the hairy house worms was 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. The above factors were processed to perform an azocasein assay, and the results are shown in Table 1.
[표 1] [Table 1]
표 1에 나타낸 바와 같이, 털보집갯지렁이로부터 분리된 물질이 세린프로테아제 저해제로 저해를 받음이 확인되어, 분리된 물질이 신규한 세린프로테아제인 것을 확인하였다. As shown in Table 1, it was confirmed that the substance isolated from the hair follicle worms was inhibited by a serine protease inhibitor, and it was confirmed that the separated substance was a novel serine protease.
실시예3Example 3 . 털보집갯지렁이로부터 분리된 . Fleece 세린프로테아제의Serine protease 피브리노겐 용해 활성 Fibrinogen-lytic activity
털보집갯지렁이로부터 분리된 세린프로테아제가 트롬빈과 피브리노겐이 만나 피브린이 되기 전의 피브리노겐을 용해시키는 활성이 있는지 여부를 확인하기 위하여, 털보집갯지렁이로부터 분리한 세린프로테아제를 10 내지 1000ng으로 피브리노겐에 처리하고 이에 따른 피브린노겐 용해 활성을 확인하였으며 그 결과를 도3에 나타내었다.In order to confirm whether serine protease isolated from the hairy house worms has activity to dissolve fibrinogen before the fusion of thrombin and fibrinogen to fibrin, the fibrinogen is treated with 10 to 1000 ng of serine protease separated from the hairy house wormworm The fibrinogen dissolution activity was confirmed and the results are shown in Fig.
도 3에 나타낸 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. 3, it was confirmed that the alpha, beta and gamma chains of fibrinogen are sequentially cut in a dose-dependent manner, and through this, it is possible to break up to a gamma chain which can not be broken by a normal protease, Which has an activity of effectively cleaving fibrinogen.
실시예4Example 4 . 털보집갯지렁이에서. From a hairy house lug 분리된 Isolated 세린프로테아제의Serine protease pH와 온도에 따른 피브린 용해 활성 Fibrinolytic activity according to pH and temperature
털보집갯지렁이로부터 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 hairy house rug was effective in the pH and temperature environment of blood vessels This is shown in FIG.
도 4에 나타낸 바와 같이, 털보집갯지렁이로부터 분리한 세린프로테아제를 각 범위의 pH와 온도에 처리한 후의 결과를 확인하였을 때, 산성과 알칼리성에서 모두 활성을 나타내는 것을 볼 수 있으며, 온도가 높을수록 활성이 증가하는 것을 확인할 수 있다. 따라서, 털보집갯지렁이에서 분리한 세린프로테아제는 넓은 pH 범위와 높은 온도에서 활성을 나타낼 수 있어 인체에 투여 시 좋은 활성을 유지할 수 있음을 확인하였다. As shown in FIG. 4, when serine protease isolated from the hair follicle worms was treated at pH and temperature in various ranges, it was found that both acidic and alkaline activities were exhibited. As the temperature increased, In the case of the present invention. Therefore, it was confirmed that serine protease isolated from the hairy house gargoyle can exhibit activity at a wide pH range and at a high temperature, thereby maintaining good activity upon administration to human body.
실시예Example 5. 털보집갯지렁이로부터 분리된 5. Separated from the cliff house 세린프로테아제의Serine protease 분자량 확인 Check molecular weight
털보집갯지렁이로부터 분리된 세린프로테아제의 분자량을 확인하기 위해서 혼합물로부터 단일 타입의 단백질을 분리하는 실험인 SDS-PAGE를 수행했다. SDS-PAGE 실험은 털보집갯지렁이로부터 분리한 crude 단백질과 분리한 표본에서 친화도에 따라 분리된 세린프로테아제를 SDS 젤에 로딩 후, 전기 영동을 수행하였고, 이를 도 5에 나타내었다. SDS-PAGE, an experiment to separate a single type of protein from a mixture, was performed to confirm the molecular weight of the serine protease separated from the hairy house worm. In the SDS-PAGE experiment, serine protease separated according to the affinity was loaded on the SDS gel after separation of the crude protein isolated from the hair follicle evacuation worm, and then electrophoresis was carried out, which is shown in FIG.
도 5에 나타낸 바와 같이, 털보집갯지렁이로부터 분리된 세린프로테아제를 전기 영동 후 염색과 탈염색화 한 후의 결과를 확인하였다. M은 standard size marker이며, Lane 1은 털보집갯지렁이로부터 분리한 crude상태의 세린프로테아제이며, Lane 2는 affinity column과 ion-exchange column을 사용하여 정제한 세린프로테아제로 약 38 kDa임을 확인하였다. As shown in FIG. 5, serine protease separated from the hair follicle evacuation worms was stained and desalted after electrophoresis, and the results were confirmed. M is a standard size marker, and
실시예Example 6. 털보집갯지렁이에서 분리된 6. Separated from the hairy house lug 세린프로테아제의Serine protease 정제방법에 따른 활성 분석 Activity analysis by purification method
털보집갯지렁이로부터 분리된 세린프로테아제의 상기 SDS-PAGE로부터 확인한 분자량의 재확인과 세린프로테아제의 정제방법에 따른 활성을 알아보기 위해 피브린 자이모그래피(zymography)를 수행하였다. 상기 자이모그래피는 (A)는 direct, (B) indirect로 피브린 젤에 세린프로테아제를 첨가하여 전기영동하였고, M은 standard size marker, Lane 1은 대조군인 우로키나제(urokinase), Lane 2는 털보집갯지렁이로부터 분리한 crude상태의 세린프로테아제이며, Lane 3은 affinity column을 사용하여 정제한 세린프로테아제, Lane 4는 affinity column과 ion-exchange column을 사용하여 정제한 세린프로테아제이며, 전기영동의 결과를 도 6에 나타내었다. Fibrin zymography was performed to confirm the molecular weights determined from the SDS-PAGE of the serine protease separated from the hairy house worms and the activity according to the purification method of serine protease.
도 6에서 확인한 바와 같이, (A)와 (B) 모두 털보집갯지렁이로부터 분리된 세린프로테아제는 38kDa에서 피브린을 분해하여 밴드가 나타나 세린프로테아제의 분자량이 재확인되었다. 또한, crude 상태의 세린프로테아제보다 affinity column을 사용하여 정제한 세린프로테아제, affinity column을 사용하여 정제한 세린프로테아제를 ion-exchange column을 사용해 정제한 세린프로테아제로 갈수록 피브린의 분해 활성이 좋아져 밴드가 뚜렷하게 나타남을 확인하였다. 따라서, 털보집갯지렁이로부터 분리한 세린프로테아제는 affinity column과 ion-exchange column을 수행하여 분리하는 것이 우수한 효소활성을 나타낼 수 있음을 확인하였다.As shown in FIG. 6, the serine proteases isolated from the hair follicle worms (A) and (B) decomposed fibrin at 38 kDa and band appeared to confirm the molecular weight of the serine protease. Serine proteases purified using affinity columns rather than crude serine proteases, and serine proteases purified using affinity columns were further purified by serine proteases purified using an ion-exchange column, resulting in improved fibrin cleavage activity and marked banding Respectively. Therefore, it was confirmed that the serine protease isolated from the hairy house worms can exhibit an excellent enzyme activity by performing separation by affinity column and ion exchange column.
실시예Example 7. 털보집갯지렁이로부터 분리된 7. Separated from the cliff house 세린프로테아제의Serine protease 세포독성여부Cytotoxicity
털보집갯지렁이로부터 분리된 세린프로테아제를 혈관 내에 투여하였을 때 내피세포에 독성을 나타내는지 확인하기 위하여, 혈관뇌장벽에 분포한 내피세포 (Endothelial cell line (hCMEC/D3)) 에 털보집갯지렁이로부터 분리된 세린프로테아제를 처리하고, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 분석을 통한 비색분석법을 수행하였다. 유의도 표시(*)는 실험적 정확성을 나타내기 위한 통계적 분석이다. 효소활성은 Turkey의 사후검정방법에 따른 분산의 일원분산분석방법(one-way ANOVA)을 따른다. 모든 실험은 3회 반복하였으며, p-value(* P< 0.05, ** P< 0.01, or *** P< 0.001)를 확인하여, 그 결과를 도 7에 나타내었다. To determine whether endothelial cells were toxic when serine protease isolated from the hairy house rug was administered into the blood vessels, the endothelial cell line (hCMEC / D3) distributed in the blood brain barrier was separated from the hairy house rug Serine protease was treated and colorimetric assay was performed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis. The significance mark (*) is a statistical analysis to show experimental accuracy. Enzyme activity follows the one-way ANOVA of variance according to the post test method of Turkey. All experiments were repeated 3 times and p-values (* P <0.05, ** P <0.01, or *** P <0.001) were confirmed, and the results are shown in FIG.
도 7에 나타낸 바와 같이, 털보집갯지렁이로부터 분리된 세린프로테아제를 세포배양접시에 배양된 hCMEC/D3에 일정농도로 처리한 후의 결과를 확인하였을 때, 통계적으로 유의성 있는 결과가 확인되었다. 따라서, 털보집갯지렁이에서 분리된 세린프로테아제는 세포 독성이 없음을 확인하였다. As shown in FIG. 7, when the results obtained after treating the hCMEC / D3 cultured in the cell culture dish with a certain concentration of serine protease separated from the hair follicle evacuation worm, statistically significant results were confirmed. Therefore, it was confirmed that serine protease isolated from the hair follicle worms was not cytotoxic.
제제예Formulation example 1. 약학적 제제의 제조 1. Preparation of pharmaceutical preparations
1. 산제의 제조 1. Manufacturing of powder
털보집갯지렁이로부터 분리된 세린프로테아제 20 ml20 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 10 ml10 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 20 ml 20 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 100 ml100 ml of serine protease isolated from the hairy house rug
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
털보집갯지렁이로부터 분리된 세린프로테아제 100 ml100 ml of serine protease isolated from the hairy house rug
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.
Claims (9)
1) 털보집갯지렁이에 인산완충액을 가하고 분쇄하는 단계;
2) 상기 분쇄물을 1 내지 3시간동안 자가 분해(auto-lysis) 시키는 단계;
3) 자가분해된 시료에 10 내지 30wt% 황산암모늄을 첨가하고 원심분리하여 단백질 포함 상등액을 얻는 단계; 및
4) 상기 상등액에 20 내지 60wt% 황산암모늄을 첨가하고 원심분리하여 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 the hair follicle worms and pulverizing them;
2) auto-lysing the pulverized material for 1 to 3 hours;
3) adding 10 to 30 wt% ammonium sulfate to the autolysed sample and centrifuging to obtain protein-containing supernatant; And
4) adding 20 to 60 wt% ammonium sulfate to the supernatant and centrifuging to obtain serine protease; ≪ / RTI > wherein the composition is prepared by the method of claim 1.
A food composition for preventing or ameliorating a thrombotic disease comprising serine protease separated from a hairy house rug.
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