KR101333669B1 - Composition comprising erythro-(7s,8r)-7-acetoxy-3,4,3',5'-tetramethoxy-8-o-4'-neolignan for treating or preventing vascular diseases - Google Patents

Composition comprising erythro-(7s,8r)-7-acetoxy-3,4,3',5'-tetramethoxy-8-o-4'-neolignan for treating or preventing vascular diseases Download PDF

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KR101333669B1
KR101333669B1 KR1020120122775A KR20120122775A KR101333669B1 KR 101333669 B1 KR101333669 B1 KR 101333669B1 KR 1020120122775 A KR1020120122775 A KR 1020120122775A KR 20120122775 A KR20120122775 A KR 20120122775A KR 101333669 B1 KR101333669 B1 KR 101333669B1
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acetoxy
neolignan
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erythro
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이정형
민병선
강정원
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강원대학교산학협력단
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Abstract

The present invention relates to a composition containing erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan for preventing or treating vascular diseases. The composition containing erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan effectively suppresses platelet aggregation, thereby being used as a composition for effectively preventing and suppressing vascular diseases including thrombosis, arteriosclerosis, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after percutaneous transluminal coronary angioplasty, cerebral infarction, cerebral hemorrhage, and stroke. [Reference numerals] (AA,BB,CC) Blood platelet cohesion activation (%)

Description

에리스로-(7에스,8알)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 치료 또는 예방용 조성물 {Composition comprising erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan for treating or preventing vascular diseases}Composition for the treatment or prophylaxis of vascular disease containing erythro- (7S, 8 tablets) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan { Composition comprising erythro- (7S, 8R) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan for treating or preventing vascular diseases}

본 발명은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 치료 또는 예방용 조성물에 관한 것이다.The invention erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', the treatment or prevention of disease comprising the 5'-tetra-methoxy -8-O-4'- neo lignans It relates to a composition for.

현대 사회에서 혈관질환은 심각한 사회적 문제로 대두되고 있다. 2010년 통계청에서 발표한 사망원인 통계를 보면, 고혈압성 질환, 허혈성 심장 질환, 뇌혈관질환을 포함한 혈관질환은 우리나라 사망원인의 2위로서, 악성 종양 다음으로 높은 순위를 차지하고 있으며, 남성은 55세 이상, 여성은 65세 이상에서 혈관질환의 사망률이 크게 증가한다. 상기 혈관질환으로는 혈전증, 동맥경화증, 고혈압, 협심증, 심근경색, 허혈성 심장질환, 심부전, 경혈관 동맥 성형술 후 발생하는 합병증, 뇌경색, 뇌출혈, 및, 뇌졸중 등이 있다. 혈관질환 환자들은 이를 치료하기 위해 지속적인 약물을 투여받지만 현대 의학에서 완치가 되기는 쉽지 않다. 또 약물을 오래 투여하다가 보면 그에 대한 부작용도 크다. In modern society, vascular disease is a serious social problem. According to the statistics of the causes of death published by the National Statistical Office in 2010, vascular diseases including hypertension, ischemic heart disease, and cerebrovascular disease are the second leading causes of death in Korea, ranking second only to malignant tumors. In women above 65, the mortality rate of vascular disease increases significantly. The vascular diseases include thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after carotid angioplasty, cerebral infarction, cerebral hemorrhage, and stroke. Patients with vascular diseases are given constant medications to treat them, but they are not easy to cure in modern medicine. If you take the medicine for a long time, the side effect is big.

혈소판은 혈관에 상처가 발생했을 때, 손상된 조직을 봉합하고 체내 항상성(hemostasis)을 유지시키는 역할을 하며, 병리학적인 상태(pathophysiological conditions)에서의 적절하지 않는 혈소판의 활성은 심각한 혈전증, 동맥경화, 고혈압이나 혈관염증 등의 혈관질환을 유도할 수 있다(Furie and Furie, 2008; Mackman, 2008; Huo and Ley, 2004; Perros et al., 2008; Gawaz et al., 2005). 이 때의 혈관 상처는 프로트롬빈 세포외 매트릭스 단백질(prothrombotic extracellular matrix proteins)의 형성 및 혈소판의 활성을 유도하는데, 트롬빈과 PAF(platelete activating factor), 아라키돈산(arachidonic acid, AA)이 특정 수용체를 통해 혈소판의 활성을 유도하는 역할을 하며, 활성화된 혈소판은 물리화학적인 변화, 외형변화, 부착, 분비, 응고 등의 과정을 거쳐 외부 상황에 반응한다(Jackson, 2007). 이중에서도 특히, 트롬빈은 PAR-1(protease-activated receptor 1), PAR-4(protease-activated receptor 4)의 절단 및 활성화로 인해 혈소판을 활성화하는데(Sambrano et al., 2001), 이 때, 상기 PAR-1 및 PAR-4를 통해 G 단백질(Gq, G12/13, and Gi)이 활성화되고, PLCβ(phospholipase Cβ) 및 PI3K(phosphatidyl inositol-3 kinas)를 이용한 신호전달을 통해 세포질 내의 Ca2 +의 농도가 높아지고, 세포내부의 cAMP 함량이 낮아진다. Platelets serve to seal damaged tissues and maintain hemostasis when blood vessels injure, and inadequate platelet activity in pathophysiological conditions can lead to severe thrombosis, arteriosclerosis, and high blood pressure. Or vascular diseases such as vascular inflammation (Furie and Furie, 2008; Mackman, 2008; Huo and Ley, 2004; Perros et al., 2008; Gawaz et al., 2005). Vascular wounds then induce the formation of prothrombotic extracellular matrix proteins and the activity of platelets, where thrombin and platelet activating factor (PAF) and arachidonic acid (AA) are released through specific receptors. The activated platelets respond to external conditions through physicochemical changes, appearance changes, adhesion, secretion, and coagulation (Jackson, 2007). In particular, thrombin activates platelets due to cleavage and activation of protease-activated receptor 1 (PAR-1) and protease-activated receptor 4 (PAR-4) (Sambrano et al., 2001). via PAR-1, and PAR-4 G protein (Gq, G12 / 13, and Gi) Ca 2 in the cytoplasm through the signal transmission using is activated, PLCβ (phospholipase Cβ) and PI3K (phosphatidyl inositol-3 kinas) + The concentration of is increased and the cAMP content in the cell is low.

혈소판이 상기와 같이 혈관질환과 밀접한 관련이 있기에, 혈전증(thrombosis)의 치료를 위해 제조된 약제인 항혈소판 제제는 고혈압이나 동맥경화 등의 혈관질환의 치료제로 이용될 수 있다. 천연 항혈소판 제제로서, 플라보노이드(flavonoids), 알카로이드(alkaloids), 스틸베노이드(stilbenoids), 페놀 화합물(phenolic compounds) 등이 사용될 수 있다고 보고된 바 있는데(Xiang et al., 2008), 근래에는 이와 같은 천연물 유래의 화합물이 항혈전 제제로서 각광을 받고 있는 추세이다. Since platelets are closely related to vascular diseases as described above, antiplatelet preparations, which are drugs prepared for the treatment of thrombosis, can be used as therapeutic agents for vascular diseases such as hypertension or arteriosclerosis. Flavonoids, alkaloids, stilbenoids and phenolic compounds have been reported to be used as natural antiplatelet agents (Xiang et al., 2008). Compounds derived from the same natural products are in the spotlight as antithrombogenic agents.

육두구(Myristica fragrans)는 인도네시아, 태국, 일본, 중국 등에서 자생하며, 향신료로 주로 사용되고 있다(Chung et al., 2006). 육두구는 항산화성(Filleur et al., 2001; Sadhu et al., 2003; Jin et al., 2005), 항발암성(Chung et al., 2006), 항간독성(Sohn et al., 2007), 항당뇨(Han et al., 2008), 항비만(Nguyen et al., 2010), 항염(Cui et al., 2008; Kang et al., 2012), 뇌신경 보호 작용(Jin et al., 2005) 등의 다양한 생물학적 활성을 갖는다. 리그난(lignan)은 육두구로부터 분리되는 주요 활성 화합물로서, 네오리그난(neolignan)은 육두구로부터 분리된 새로운 타입의 리그난에 속한다.Nutmeg ( Myristica fragrans ) is native to Indonesia, Thailand, Japan, and China, and is mainly used as a spice (Chung et al., 2006). Nutmeg is antioxidative (Filleur et al., 2001; Sadhu et al., 2003; Jin et al., 2005), anticarcinogenic (Chung et al., 2006), antihepatic toxicity (Sohn et al., 2007), Antidiabetic (Han et al., 2008), anti-obesity (Nguyen et al., 2010), anti-inflammatory (Cui et al., 2008; Kang et al., 2012), neuroprotective activity of the brain (Jin et al., 2005) And various biological activities. Lignan is the main active compound isolated from nutmeg, and neolignan belongs to a new type of lignan isolated from nutmeg.

본 발명자들은 혈소판의 응집을 억제하는 물질들에 관한 연구를 하던 중, 육두구 유래의 화합물인 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난이 트롬빈이나 PAF 유도로 인한 혈소판의 응집 억제 효과가 우수함을 확인함으로써 본 발명을 완성할 수 있었다. The inventors of the present invention, while studying the substances that inhibit platelet aggregation, erythro- (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'-tetrame, a compound derived from nutmeg The present invention was completed by confirming that oxy-8-O-4'-neolignan was excellent in inhibiting the aggregation of platelets due to thrombin or PAF induction.

한편, 한국등록특허 제399529호에 육두구를 포함하는 생약 추출물을 함유하는 콜레스테롤 에스터레이즈 저해제 조성물이 동맥경화 또는 고지혈증 예방 및 치료 효과가 있다는 것이 개시되어 있기는 하지만, 상기 선행기술은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난이 포함되어 있는 본 발명과는 기술적 구성이 전혀 다른 발명이라고 할 수 있다.Meanwhile, Korea, registered in cholesterol ester raised inhibitor composition containing the crude drug extract containing nutmeg Patent No. 399 529 No. discloses that the arteriosclerosis or hyperlipidemia preventive and therapeutic effect because, although the prior art erythro - (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan is an invention that is completely different from the technical configuration of the present invention. have.

한국등록특허 제399529호 (생약 추출물을 포함하는 콜레스테롤 에스터레이즈 저해제 조성물, 2003.09.16. 등록)Registered Korean Patent No. 399529 (Cholesterol esterase inhibitor composition comprising herbal extracts, registered on September 16, 2003)

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본 발명의 목적은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 치료 또는 예방용 조성물을 제공하는 데에 있다.An object of the present invention are erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo treatment of vascular disease comprising the lignan Or to provide a prophylactic composition.

본 발명은 하기 화학식 1의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to erythromycin of the formula 1 - (7 S, 8 R ) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo vascular diseases containing lignan It relates to a composition for the prophylaxis or treatment of.

[화학식 1][Formula 1]

Figure 112012089581211-pat00001
Figure 112012089581211-pat00001

상기 혈관질환은 혈전증, 동맥경화증, 고혈압, 협심증, 심근경색, 허혈성 심장질환, 심부전, 경혈관 동맥 성형술 후 발생하는 합병증, 뇌경색, 뇌출혈, 및, 뇌졸중으로 이루어진 군으로부터 선택되는 질환일 수 있다.The vascular disease may be a disease selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after carotid angioplasty, cerebral infarction, cerebral hemorrhage, and stroke.

또한 본 발명은 상기 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 예방 또는 개선용 건강기능식품을 제공한다.In another aspect, the present invention is to prevent the vascular disease containing the erythro- (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'- neolignan Or provide a dietary supplement for improvement.

이하 본 발명을 자세하게 설명한다.Hereinafter, the present invention will be described in detail.

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은, 육두구 추출물을 제조하는 단계; 및, 크로마토그래피를 이용하여 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 순수하게 분리 정제하는 단계;를 통해 얻을 수 있다. 바람직하게는, 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 가죽나무로부터 유기용매(알코올, 에테르, 아세톤 등)에 의한 추출, 헥산과 물의 분배, 칼럼크로마토그래피에 의한 방법 등, 식물체 성분의 분리 추출에 이용되는 공지의 방법을 단독 또는 적합하게 조합하여 용이하게 얻을 수가 있다. Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans is, to prepare a nutmeg extract; And, by chromatography erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans purely isolated and purified the It can be obtained through; Preferably, the erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans are organic leather from wood Known methods used for the separate extraction of plant components, such as extraction with a solvent (alcohol, ether, acetone, etc.), distribution of hexane and water, methods by column chromatography, etc. can be easily obtained alone or as a suitable combination.

본 발명에서 사용되는 상기 크로마토그래피로는 실리카겔 칼럼 크로마토그래피(silica gel column chromatography), 엘에이취-20 칼럼 크로마토그래피(LH-20 column chromatography), 이온교환수지 크로마토그래피(ion exchange resin chromatography), 박층크로마토그래피(TLC; thin layer chromatography) 및 고성능 액체 크로마토그래피(high performance liquid chromatography) 등이 이용될 수 있다.The chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, ion exchange resin chromatography, thin layer chromatography. Thin layer chromatography (TLC), high performance liquid chromatography, and the like can be used.

또한, 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 육두구로부터 쉽게 분리할 수 있을 뿐만 아니라 안정도도 높으므로 식품, 의약품의 첨가제로 이용할 수 있다.Also, erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo Lignans can be readily separated from nutmeg As well as high stability, it can be used as an additive in food and medicine.

본 발명은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 포함하는 혈관질환의 예방 또는 치료용 약학 조성물을 제공한다. 상기 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 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. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. The present invention provides for the prevention or treatment of vascular diseases comprising erythro- (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan It provides a pharmaceutical composition. The pharmaceutical compositions may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid formulations of the present invention, erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ' It is prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like with, 5'-tetramethoxy-8-O-4'-neolignan. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 약학 조성물의 투여량은 치료받을 대상의 연령, 성별, 체중, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 1㎎/㎏/일 내지 500㎎/㎏/일이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo dosage of the pharmaceutical compositions containing the lignans It will depend on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 500 mg / kg / day. 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.

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 약학 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다. Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', pharmaceutical compositions containing 5'-tetra-methoxy -8-O-4'- neo lignans are mice, cattle And mammals, such as humans. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans are so few toxic side effects and prevention of This is a medicine that can be used with confidence even for long-term use.

또 다른 측면에 있어서, 본 발명은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 예방 또는 개선용 건강기능식품을 제공한다. 상기 건강기능식품은 각종 식품, 음료, 식품 첨가물 등일 수 있다.In a further aspect, the invention erythro - (S 7, 8 R) -7- acetoxy -3,4,3 ', containing the 5'-tetra-methoxy -8-O-4'- neo lignans Provides a dietary supplement for the prevention or improvement of vascular diseases. The health functional food may be various foods, beverages, food additives and the like.

상기 건강기능식품에 함유된 유효성분으로서의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난의 함량은 식품의 형태, 사용하고자 하는 용도에 따라 적절하게 함유될 수 있으며, 예컨대, 전체 식품 중량의 0.01~50 중량%로 가할 수 있으며, 바람직하게는 0.1~10 중량%의 비율로 가할 수 있다. 또한 건강음료 조성물은 100㎖을 기준으로 0.02~10g, 바람직하게는 0.1~5g의 비율로 가할 수 있다. Erythromycin as the active ingredient contained in the functional food - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo content of lignans Silver may be appropriately contained depending on the form of the food and the intended use thereof, for example, may be added at 0.01 to 50% by weight of the total food weight, preferably at a ratio of 0.1 to 10% by weight. In addition, the health beverage composition may be added in a ratio of 0.02 to 10 g, preferably 0.1 to 5 g based on 100 ml.

본 발명의 건강음료 조성물은 지시된 비율로 필수 성분으로서 상기 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 것 외에 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(예를 들어, 타우마틴, 스테비아 추출물,레바우디오시드 A, 글리시르히진 등), 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 건강음료 조성물 100㎖당 일반적으로 1~20g, 바람직하게는 5~12g이다. The composition for health beverages of the present invention is the erythromycin as an essential component in the indicated ratio - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4' In addition to containing neolignans, there are no particular limitations on the liquid components and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (e.g., taumartin, stevia extract, rebaudioside A, glycyrrhizin, etc.), synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The ratio of the natural carbohydrate is generally 1-20 g, preferably 5-12 g per 100 ml of the health beverage composition.

상기 외에 본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 건강기능식품은 천연 과일 주스 및 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 건강기능식품 100 중량부당 0.001~20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not so important, but is generally selected from 0.001 to 20 parts by weight per 100 parts by weight of the health functional food of the present invention.

상술한 바와 같이 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 조성물은 혈소판 응집 억제 효과가 우수하여 혈전증, 동맥경화증, 고혈압, 협심증, 심근경색, 허혈성 심장질환, 심부전, 경혈관 동맥 성형술 후 발생하는 합병증, 뇌경색, 뇌출혈, 뇌졸중 등을 비롯한 혈관질환을 효과적으로 예방 또는 억제할 수 있는 조성물로 유용하게 사용될 수 있다.Erythromycin of the present invention as described above - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo compositions containing lignan is Excellent platelet aggregation inhibitory effect effectively prevents or inhibits vascular diseases including thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after angioplasty, cerebral infarction, cerebral hemorrhage, stroke It can be usefully used as a composition that can be.

도 1A는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제하는 것을 나타내는 그래프이다.
도 1B는 PAF(platelete activating factor)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제하는 것을 나타내는 그래프이다.
도 1C는 콜라겐(Collagen)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제하는 것을 나타내는 그래프이다.
도 2A는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때의 ATP 방출량을 확인하는 그래프이다.
도 2B는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때의 세로토닌(Serotonin) 방출량을 확인하는 그래프이다.
도 2C는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때의 트롬복산 B2(TXB2) 형성량을 확인하는 그래프이다.
도 3A는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때의 Ca2 + 농도를 확인하는 그래프이다.
도 3B는 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때의 cAMP 방출량을 확인하는 그래프이다.1A is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph showing that neolignan (EATN) is suppressed.
Figure 1B is erythromycin platelet aggregation caused by induction of (platelete activating factor) PAF - ( 7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4 '-A graph showing that neolignan (EATN) inhibits.
1C is erythromycin platelet aggregation due to the induction of collagen (Collagen) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph showing that neolignan (EATN) is suppressed.
Figure 2A is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph confirming the amount of ATP released when neolignan (EATN) is suppressed.
Figure 2B is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph confirming serotonin release amount when neolignan (EATN) is suppressed.
2C is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph confirming the amount of thromboxane B 2 (TXB 2 ) formation when neolignan (EATN) is suppressed.
3A is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- is a graph confirming the Ca 2 + concentration at which the neo lignans (EATN) be suppressed.
Figure 3B is erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- It is a graph confirming the amount of cAMP released when neolignan (EATN) is suppressed.

이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지기 위해, 또한 당업자에게 본 발명의 사상이 충분히 전달될 수 있기 위해 제공되는 것이다.Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the information provided herein is provided to be thorough and complete, and also to fully convey the spirit of the present invention to those skilled in the art.

<< 실시예Example 1.  One. 육두구로부터From nutmeg 본 발명 화합물의 분리>  Isolation of Compounds of the Invention>

에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 육두구(Myristica fragrans)의 씨앗으로부터 분리되었다(Cuong et al., 2011). 이를 위해, 육두구 씨앗 2kg을 잘게 분쇄한 후, 메탄올 3ℓ로 3시간씩, 3회 실온에서 환류 추출한 후 상기 메탄올 추출물을 왓트만 용지(Whatman paper, No.1)로 거르고, 진공상태에서 감압 증류하여 용매를 제거하여 메탄올 잔류물 660g을 획득하였다. 상기 잔류물을 다시 증류수 3.0ℓ에 현탁하고 n-헥산(1000㎖)으로 2회 추출한 후, 다시 에틸아세테이트(1000㎖, 2회)로 추출하여 54g의 에틸아세테이트 분획물을 얻었다. 에틸아세테이트 분획물 54g을 클로로포름과 메탄올의 농도 구배(50:1-0:1 부피비, 각 2ℓ)를 이용한 용출 용매를 이용하여 실리카겔(500g, Merck silica 230-400mesh) 크로마토그래피를 시행하여 9개의 소분획물을 얻었다(Fr.1 - Fr.9). 상기 소분획물 중 다섯 번째 분획물(Fr.5, 3.5g)을 메탄올과 물의 농도 구배(4:1-4:0 부피비, 각 3ℓ) 역상크로마토그래피(100g, 150μm 입자크기)를 실시하여 5개의 소분획물을 얻었다(Fr.5.1-Fr.5.5). Erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans have been isolated from the seeds of nutmeg (Myristica fragrans) ( Cuong et al., 2011). To this end, 2 kg of nutmeg seeds are finely ground, and then extracted by refluxing three times with 3 L of methanol at room temperature three times, and then filtering the methanol extract with Whatman paper, No. 1, and distilling under reduced pressure under vacuum. Solvent was removed to give 660 g of methanol residue. The residue was again suspended in 3.0 L of distilled water and extracted twice with n-hexane (1000 mL), followed by extraction with ethyl acetate (1000 mL, 2 times) to obtain 54 g of ethyl acetate fraction. 54 g of ethyl acetate fractions were subjected to silica gel (500 g, Merck silica 230-400mesh) chromatography using an eluting solvent using a concentration gradient of chloroform and methanol (50: 1-0: 1 volume ratio, 2 L each). (Fr. 1-Fr. 9) was obtained. The fifth fraction (Fr. 5, 3.5 g) of the small fractions was subjected to reverse phase chromatography (100 g, 150 μm particle size) of methanol and water (4: 1-4: 0 volume ratio, 3 L each). Fractions were obtained (Fr.5.1-Fr.5.5).

이 소분획물 중 세번째 소분획물(Fr.5.3, 430㎎)을 semi-preparative Waters HPLC(semi-preparative Waters high-performance liquid chromatography; YMC Pack Pro C18 column, 20×250㎜, 0.1% trifluoroacetic acid을 포함하는 70% 메탄올, flow rate 5㎖/min)를 실시하여 14㎎(tR=48.2min)의 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 분리하였다. 분리된 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 HPLC를 통해 순도 98% 이상임을 확인하였다.The third subfraction (Fr.5.3, 430 mg) of this subfraction was converted to semi-preparative Waters HPLC (semi-preparative Waters high-performance liquid chromatography; YMC Pack Pro C18 column, 20 × 250 mm, 0.1% trifluoroacetic acid). subjected to 70% methanol, flow rate 5㎖ / min) to erythromycin of the present invention of 14㎎ (tR = 48.2min) - ( 7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-Tetramethoxy-8-O-4'-neolignan was isolated. Separate erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans was confirmed that purity of 98% via HPLC It was.

<실시예 2. 에리스로-(7Example 2 erythro- (7 SS ,8,8 RR )-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난의 물리화학적 성질 확인>) Physical and Chemical Properties of 7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan>

erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan;erythro- (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan;

Colorless oil; Colorless oil;

[M]+ peak at m/z 429.1923 in the ESI-MS [M] + peak at m / z 429.1923 in the ESI-MS

[α]

Figure 112012089581211-pat00002
27.1 (c 0.14, MeOH); [alpha]
Figure 112012089581211-pat00002
27.1 (c 0.14, MeOH);

IR Vmax (KBr): 3446, 2920, 1624, 1431, 1268, 1164, 1032 cm-1;IR Vmax (KBr): 3446, 2920, 1624, 1431, 1268, 1164, 1032 cm - 1;

UV λmax (MeOH): 277 nm; UV λ max (MeOH): 277 nm;

TOF-MS m/z 371.1 [M OAc]+ (calcd for C24H29O7 , 371.1); TOF-MS m / z 371.1 [M OAc] + (calcd for C 24 H 29 O 7 , 371.1);

1H-NMR (400 MHz, CD3OD): δ 6.93 (1H, br s, H-2), 6.92 (1H, d, J = 8.4 Hz, H-5), 6.84 (1H, br d, J = 8.4 Hz, H-6), 5.86 (1H, d, J = 3.2 Hz, H-7), 4.46 (1H, dq, J = 3.2, 6.4 Hz, H-8), 1.26 (3H, d, J = 6.4 Hz, H-9), 6.52 (2H, br s, H-2′ and H-6′), 3.36 (2H, d, J = 6.8 Hz, H-7′), 6.00 (1H, m, H-8′), 5.08 (1H, dd, J = 1.2, 11.2 Hz, H-9′Z), 5.13 (1H, dd, J = 1.2, 13.6 Hz, H-9′E), 3.82 (6H, s, 3, 4-diOMe), 3.80 (6H, s, 3′,5′-diOMe), 2.18 (3H, s, 7-OCOMe); 1 H-NMR (400 MHz, CD 3 OD): δ 6.93 (1H, br s, H-2), 6.92 (1H, d, J = 8.4 Hz, H-5), 6.84 (1H, br d, J = 8.4 Hz, H-6), 5.86 (1H, d, J = 3.2 Hz, H-7), 4.46 (1H, dq, J = 3.2, 6.4 Hz, H-8), 1.26 (3H, d, J = 6.4 Hz, H-9), 6.52 (2H, br s, H-2 ′ and H-6 ′), 3.36 (2H, d, J = 6.8 Hz, H-7 ′), 6.00 (1H, m, H-8 ′), 5.08 (1H, dd, J = 1.2, 11.2 Hz, H-9′Z), 5.13 (1H, dd, J = 1.2, 13.6 Hz, H-9′E), 3.82 (6H, s, 3, 4-diOMe), 3.80 (6H, s, 3 ′, 5′-diOMe), 2.18 (3H, s, 7-OCOMe);

13C-NMR(100 MHz, CD3OD): δ 131.9 (C-1), 111.8 (C-2), 150.4 (C-3), 150.2 (C-4), 112.8 (C-5), 120.6 (C-6), 78.2 (C-7), 81.6 (C-8), 14.9 (C-9), 137.8 (C-1′), 106.8 (C-2′,6′), 154.7 (C-3′,5′), 134.8 (C-4′), 41.5 (C-7′), 138.9 (C-8′), 116.2 (C-9′), 56.7 (3,4-diOMe), 56.5 (3′,5′-diOMe), 21.3 (7-OCOMe), 172.2 (7-OCOMe). 13 C-NMR (100 MHz, CD 3 OD): δ 131.9 (C-1), 111.8 (C-2), 150.4 (C-3), 150.2 (C-4), 112.8 (C-5), 120.6 (C-6), 78.2 (C-7), 81.6 (C-8), 14.9 (C-9), 137.8 (C-1 ′), 106.8 (C-2 ′, 6 ′), 154.7 (C- 3 ′, 5 ′), 134.8 (C-4 ′), 41.5 (C-7 ′), 138.9 (C-8 ′), 116.2 (C-9 ′), 56.7 (3,4-diOMe), 56.5 ( 3 ′, 5′-diOMe), 21.3 (7-OCOMe), 172.2 (7-OCOMe).

<< 실시예Example 3. 세척 혈소판의 준비> 3. Preparation of Washed Platelets>

혈소판 수집을 위한 혈액은 최소 14일 동안 어떠한 약도 전혀 복용하지 않은 건강한 20~25세의 성인 남성들로부터 얻었다. 수집된 혈액은 3.8% 구연산 나트륨 용액(3.8% trisodium citrate solution, 1:9 citrate/blood, v/v)을 처리하여 응집되지 않게 하였으며, 37℃, 200×g에서 15분 동안 원심분리하여 혈소판 풍부 혈장(platelet-rich plasma, PRP)를 얻었다. 혈소판 풍부 혈장은 세척용 완충액(washing buffer : 85mM sodium citrate, 71mM citric acid, 111mM D-Glusose, 134mM NaCl, 2.9mM KCl, 1mM MgCl2, 10mM HEPES, 12mM NaHCO3, 0.34mM Na2HPO4, 0.35% BSA, pH 7.3)에 현탁시켜 다시 37℃, 2000×g에서 12분 동안 원심분리하였다. 원심분리하여 모은 혈소판 펠렛(pellet)은 현탁용 완충액(suspenstion buffer : 134mM NaCl, 2.9mM KCl, 1mM MgCl2, 10mM HEPES, 12mM NaHCO3, 0.34mM Na2HPO4, 5mM D-Glusose, 0.35% BSA, pH 7.4)에 4×108cells/㎖로 현탁시켰다. Blood for platelet collection was obtained from healthy 20-25 year old adult men who had not taken any medication for at least 14 days. The collected blood was treated with 3.8% sodium citrate solution (3.8% trisodium citrate solution, 1: 9 citrate / blood, v / v) to prevent aggregation and centrifuged at 37 ° C and 200 × g for 15 minutes to enrich platelet. Platelet-rich plasma (PRP) was obtained. Platelet-rich plasma was washed buffer (85 mM sodium citrate, 71 mM citric acid, 111 mM D-Glusose, 134 mM NaCl, 2.9 mM KCl, 1 mM MgCl2, 10 mM HEPES, 12 mM NaHCO 3 , 0.34 mM Na 2 HPO 4 , 0.35% BSA, pH 7.3) and again centrifuged for 12 minutes at 2000 ℃ g, 37 ℃. The platelet pellets collected by centrifugation were suspended buffer (134 mM NaCl, 2.9 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 12 mM NaHCO 3 , 0.34 mM Na 2 HPO 4 , 5 mM D-Glusose, 0.35% BSA). , pH 7.4) was suspended at 4 × 10 8 cells / ml.

<< 실시예Example 4. 혈소판 응집  4. Platelet aggregation 어세이Assay >>

실시예 3에서 얻은 세척 혈소판 0.5㎖(4×108cells/㎖)을 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)에 3분간 전반응시켰다. 이 후, 트롬빈(Thrombin, 0.5U/㎖), PAF(platelete activating factor, 0.4㎍/㎖), 콜라겐(Collagen 5㎍/㎖)을 5분간 반응시켜 혈소판 응집을 유도하였으며, four-channel Chrono-Log aggregometer를 이용하여 혈소판 응집 정도를 측정하였고, 이에 대한 결과는 도 1에 나타내었다. Washed platelets carried 0.5㎖ (4 × 10 8 cells / ㎖) erythromycin of 1 ~ 10μM a obtained in Example 3 - (7 S, 8 R ) -7- acetoxy -3,4,3 ', 5'-tetramethoxy It was pre-reacted with oxy-8-O-4'-neolignan (EATN) for 3 minutes. Subsequently, thrombin (0.5 U / mL), platelet activating factor (PAF) (0.4 μg / mL), and collagen (Collagen 5 μg / mL) were reacted for 5 minutes to induce platelet aggregation. Four-channel Chrono-Log Platelet aggregation was measured using an aggregometer, and the results are shown in FIG. 1.

도 1의 결과를 확인하면, 트롬빈(Thrombin, 도 1A), PAF(도 1B) 및 콜라겐(Collagen, 도 1C)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제하는 것을 확인할 수 있었다.Referring to the results of FIG. 1, platelet aggregation due to the induction of thrombin (FIG. 1A), PAF (FIG. 1B) and collagen (Collagen (FIG. 1C)) was determined by erythro- (7 S , 8 R ) -7-acetoxy. It was confirmed that -3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan (EATN) was inhibited.

<< 실시예Example 5.  5. ATPATP , , 트롬복산Thrombox B B 22 , 세로토닌의 확인>, Confirmation of serotonin

혈소판의 활성 및 응집은 혈관 상처 부위가 콜라겐(collagen), 트롬빈, ADP(adenosine diphosphate) 및 PAF(platelete activating factor) 등과 같은 물질들에 노출되면서 시작된다(Li et al ., 2010). 혈소판은 위의 각각의 물질에 대한 수용체를 가지고 있으며, 수용체에 의해 혈소판이 활성화되고 외분비 작용이 일어나게 된다. 이를 과립분비(grannule secretion) 현상이라고 하며, 이 때 활성화된 혈소판에 의해서 발생되는 과립분비 현상에 의해 분비되는 ATP(adenosine triphosphate), ADP(adenosine diphosphate), 칼슘 이온(Ca2 +), 세로토닌(serotonin) 등은 혈소판 활성화를 촉진한다. 또한 혈소판 활성화 과정 중에 생성되는 트롬복산 A2(thromboxane A2) 또한 혈소판 활성화를 조절하는 데에 중요한 역할을 한다. 따라서 ATP, 트롬복산 A2(실시예에서는 트롬복산 A2의 안정한 대사산물인 트롬복산 B2로 측정), 세로토닌(serotonin) 등의 다양한 물질들이 혈소판 활성 및 응집의 중요한 마커로서 작용한다(Zarbock et al ., 2007 ; Rendu and Brohard-Bohn, 2001). Platelet activity and aggregation begins when the vascular wound is exposed to substances such as collagen, thrombin, adenosine diphosphate (ADP) and platelet activating factor (PAF) (Li et al. al . , 2010). Platelets have receptors for each of the above, and the receptors activate platelets and exocrine. This is called granule secretion, and at this time, ATP (adenosine triphosphate), ADP (adenosine diphosphate), calcium ions (Ca 2 + ), and serotonin (Serotonin) are secreted by granule secretion caused by activated platelets. ) Promotes platelet activation. Further thromboxane A 2 (thromboxane A 2) that are created during the process of platelet activation also plays a key role in regulating platelet activation. Therefore, various substances such as ATP, thromboxane A 2 (measured by thromboxane B 2 , a stable metabolite of thromboxane A 2 ), and serotonin act as important markers of platelet activity and aggregation (Zarbock et. al ., 2007; Rendu and Brohard-Bohn, 2001).

이에 상기 물질들의 함량을 트롬빈과 반응시킨 혈소판 내에서 확인하였다. The content of the substances was confirmed in platelets reacted with thrombin.

실시예Example 5-1.  5-1. ATPATP 방출  Release 어세이Assay

실시예 3에서 얻은 세척 혈소판(4×108cells/㎖)에 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)을 37℃에서 30분간 반응시키고, 트롬빈(0.5U/㎖)을 4분간 반응시켰다. Example 3 washed platelet obtained from (4 × 10 8 cells / ㎖ ) erythromycin of 1 ~ 10μM to - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetramethoxy- 8-O-4'-neolignan (EATN) was reacted at 37 ° C for 30 minutes, and thrombin (0.5 U / ml) was reacted for 4 minutes.

반응을 마친 후에는 샘플을 원심분리(37℃, 12000g, 10min)하여 상층액을 얻었으며, 이 상층액의 ATP의 양은 ATP 어세이 키트(Adenosine 5'-triphosphate bioluminescent somatic cell assay kit, Sigma-Aldrich, cat No 118K9804) 및 루미노미터(luminometer, BioTek Instruments, Winooski, VT, USA)를 이용하여 확인하였으며 이에 대한 결과를 도 2A에 나타내었다. After the reaction, the sample was centrifuged (37 ° C., 12000 g, 10 min) to obtain a supernatant, and the amount of ATP in the supernatant was measured using an ATP assay kit (Adenosine 5'-triphosphate bioluminescent somatic cell assay kit, Sigma-Aldrich). , cat No 118K9804) and a luminometer (luminometer, BioTek Instruments, Winooski, VT, USA) was confirmed using the results are shown in Figure 2A.

도 2A를 참고하면, 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때 ATP 방출량이 감소되는 것을 확인할 수 있다. Referring to Figure 2A, erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O- It can be seen that the amount of ATP released when 4'-neolignan (EATN) inhibits.

실시예Example 5-2. 세로토닌 방출 측정 5-2. Serotonin Release Measurement

에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 세로토닌 방출을 억제하는지를 확인하였다. 이를 위해, 실시예 3에서 얻은 세척 혈소판(4×108cells/㎖)에 이미프라민(Imipramine, 5μM) 및 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)을 동시에 3분간 반응시키고, 트롬빈(0.5U/㎖)을 처리하였다. 혈소판이 여러 가지 물질에 의해서 자극을 받아 활성화되면 세로토닌을 분비하게 되는데, 이 과정에서 분비된 세로토닌이 세포내로 다시 흡수되는 것을 차단하여 좀 더 정확한 실험을 수행하도록 하기 위한 재료로써 이미프라민이 사용되었다.Erythromycin was confirmed whether (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans (EATN) inhibits the release of serotonin. To this end, the washed platelets (4 × 10 8 cells / ml) obtained in Example 3 were prepared with imipramine (Imipramine, 5 μM) and 1-10 μM of erythro- (7 S , 8 R ) -7-acetoxy-3, 4,3 ', 5'-tetramethoxy-8-O-4'-neolignan (EATN) was simultaneously reacted for 3 minutes and treated with thrombin (0.5 U / ml). When platelets are stimulated by various substances and activated, they secrete serotonin. In this process, imipramine was used as a material to prevent the secretion of serotonin from being absorbed back into the cell to perform more accurate experiments.

트롬빈 처리 5분 후, 반응을 중단시키고 37℃, 12,000×g에서 10분간 원심분리하여, 얻은 상층액에서의 세로토닌의 양을 효소 면역 어세이 키트(enzyme immunoassay kit, ADI-900-175, Enzo life science)를 이용하여 확인하였으며, 이에 대한 결과는 도 2B에 나타내었다. After 5 minutes of thrombin treatment, the reaction was stopped and centrifuged for 10 minutes at 12,000 × g at 37 ° C., and the amount of serotonin in the obtained supernatant was measured using an enzyme immunoassay kit (ADI-900-175, Enzo life). science), and the results are shown in FIG. 2B.

도 2B를 참고하면, 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때, 세로토닌(Serotonin) 방출량이 억제되는 것을 확인할 수 있다.Referring to Figure 2B, erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O- When 4'-neolignan (EATN) inhibits, it can be seen that the serotonin release amount is suppressed.

실시예Example 5-3.  5-3. 트롬복산Thrombox B B 22 형성 측정 Formation measurement

다음으로는 트롬복산 A2의 인정한 대사산물인 트롬복산 B2를 측정하여 트롬복산 A2의 상대적인 양을 측정하였다. 실시예 3에서 얻은 세척 혈소판(4×108cells/㎖)에 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)을 37℃에서 3분간 반응시키고, 트롬빈(0.5U/㎖)을 넣어 5분간 더 반응시켰다. 37℃, 12,000×g에서 10분간 원심분리하였다. 원심분리하여 얻은 상층액에서 트롬복산 B2의 양을 효소 면역 어세이 키트(enzyme immunoassay kit, Cat no. 519031, Cayman Chemicals)를 이용하여 확인하였으며, 이에 대한 결과를 도 2C에 나타내었다. Next, the relative amount of thromboxane A 2 was determined by measuring the metabolite thromboxane B 2 recognizes the thromboxane A 2. Example 3 washed platelet obtained from (4 × 10 8 cells / ㎖ ) erythromycin of 1 ~ 10μM to - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetramethoxy- 8-O-4'-neolignan (EATN) was reacted at 37 ° C. for 3 minutes, and thrombin (0.5 U / ml) was added for 5 minutes. Centrifugation was performed at 37 DEG C and 12,000 x g for 10 minutes. The amount of thromboxane B 2 in the supernatant obtained by centrifugation was confirmed using an enzyme immunoassay kit (Cat no. 519031, Cayman Chemicals), and the results are shown in FIG. 2C.

도 2C를 참고하면, 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때, 트롬복산 B2(TXB2) 형성량도 감소되는 것을 확인할 수 있다.Referring to Figure 2C, erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O- When 4'-neolignan (EATN) is suppressed, it can be seen that the amount of thromboxane B 2 (TXB 2 ) is also reduced.

<< 실시예Example 6.  6. 세포내Intracellular CaCa 22 ++ 및  And cycliccyclic AMPAMP 측정> Measurement>

실시예Example 6-1.  6-1. 세포내Intracellular CaCa 22 ++ 측정  Measure

트롬빈은 PAR-1(type 1 proteinase-activated receptor) 및 PAR-4(type 4 proteinase-activated receptor)를 통해 혈소판을 활성화한다. 이 때, PAR-1 및 PAR-4는 다양한 G 단백질(G proteins : Gq, G12/13, and Gi)을 활성화하며, 이 G 단백질들은 포스포리파아제 Cβ(phospholipase Cβ)를 포스포이노시티드(phosphoinositide)로 변환시켜 세포질 내의 Ca2 + 농도를 올리는 역할을 한다. 세포내 Ca2 + 농도는 혈소판 응집 시에 가장 필요한 요소라고 할 수 있기에(Jackson et al., 2003), 혈소판 응집이 억제되면, Ca2 + 농도 또한 줄어들게 된다. 이에, 세포내 Ca2 +를 Fura-2(the pentacarboxylate calcium indicator)의 형광성을 이용하여 확인하였다. Fura-2/AM(Fura-2-acetoxymethyl ester)을 세포에 반응시키면, 상기 Fura-2/AM이 세포막을 투과해서 세포 내로 이동되고, 이 때 cellular esterases가 Fura-2/AM의 acetoxymethyl esters를 제거하여 Fura-2/AM이 Fura-2가 되며 이것이 Ca2 +와 특이적으로 반응해서 형광을 띄게 되는데, 380nm에서 여기(excitation) 시킨 후 510nm에서 형광도를 비교하여 측정한다. Thrombin activates platelets through type 1 proteinase-activated receptor (PAR-1) and type 4 proteinase-activated receptor (PAR-4). At this time, PAR-1 and PAR-4 activate various G proteins (G q , G 12/13 , and G i ), and these G proteins are phospholipase Cβ. was converted to lactide (phosphoinositide) serves to raise the Ca 2 + concentration in the cytosol. In Ca 2 + concentration of cells because it can be said that the required elements at the time of platelet aggregation (Jackson et al., 2003), when the platelet aggregation is inhibited, is also reduced Ca 2 + concentrations. Thus, the cells within the Ca 2 + was confirmed by the fluorescence of Fura-2 (the pentacarboxylate calcium indicator ). Reaction of Fura-2 / AM (Fura-2-acetoxymethyl ester) to cells causes the Fura-2 / AM to penetrate the cell membrane and move into the cell, where cellular esterases remove the acetoxymethyl esters of Fura-2 / AM. the Fura-2 / AM is and the Fura-2 this is determined by comparing the fluorescence at 510nm was to react with Ca 2 + and specifically there is a markedly fluorescence, at 380nm here (excitation).

이를 위해, 실시예 3에서 얻은 세척 혈소판(4×108cells/㎖)에 5μM Fura-2/AM(Fura-2-acetoxymethyl ester)을 37℃에서 1시간 동안 현탁용 완충액 상에서 반응시켰다. 상기 반응시킨 세척 혈소판(4×108cells/㎖)에 1mM CaCl2가 있는 상태에서 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)을 37℃에서 3분간 반응시켰다. 다음으로, 트롬빈(0.5U/㎖)을 5분간 더 반응시키고, 0.1% Triton X-100으로 혈소판을 용해하여 얻은 용해액의 Fura-2의 형광도를 구하였다. To this end, 5 μM Fura-2 / AM (Fura-2-acetoxymethyl ester) was reacted in suspension buffer at 37 ° C. for 1 hour on washed platelets (4 × 10 8 cells / ml) obtained in Example 3. The reaction was washed platelets (4 × 10 8 cells / ㎖ ) to erythromycin of 1 ~ 10μM in the presence of 1mM CaCl 2 - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'Tetramethoxy-8-O-4'-neolignan (EATN) was reacted at 37 ° C for 3 minutes. Next, thrombin (0.5 U / ml) was further reacted for 5 minutes, and the fluorescence of Fura-2 of the lysate obtained by dissolving platelets with 0.1% Triton X-100 was determined.

Fura-2의 형광성(fluorescence)은 Synergy MX Multi-Mode Microplate Reader(BioTek, Instruments, Winooski, VT, USA)을 이용하여 380nm에서 여기(excitation)시킨 후, 510nm에서 방출(emission)되는 형광도를 측정하여 구하였다즉, 340nm는 형광물질인 Fura-2를 excitation시키기 위한 파장이고, 510nm는 여기 (excitation)반응에 의해서 형광으로 방출(emission)되는 파장으로서, 340nm에서 흥분시키면 510nm에서 형광을 발광한다. 따라서 세포내 칼슘의 농도의 계산은 Fura-2의 형광성을 380nm에서 빛을 쪼이고, 510nm에서 방출되는 형광도의 세기를 측정하여 아래의 계산식을 이용하여 세포내 Ca2 +을 계산한다. Fluorescence of Fura-2 was measured by excitation at 380 nm using Synergy MX Multi-Mode Microplate Reader (BioTek, Instruments, Winooski, VT, USA), and then measured fluorescence emission at 510 nm. That is, 340 nm is a wavelength for excitation of Fura-2, a fluorescent material, and 510 nm is a wavelength emitted by fluorescence by an excitation reaction. When 340 nm is excited, fluorescence is emitted at 510 nm. Therefore, the calculation of the concentration of intracellular calcium by jjoyigo light the fluorescence of Fura-2 at 380nm, measuring the fluorescence intensity of the emitted at 510nm using the formula below to calculate the intracellular Ca + 2.

FuraFura -2의 형광성을 이용하여 Using fluorescence of -2 세포내Intracellular 칼슘농도(F)를 구하기 위한 반응식:  Reaction equation for calculating calcium concentration (F):

세포질 내에서의 [In the cytoplasm [ CaCa 22 ++ ]] ii = 224 = 224 nMnM × ( × ( FFFF minmin /(/ ( FF maxmax FF ) )

224nM는 Ca2 +와 Fura-2의 dissociation 값 (이미 밝혀져 있는 절대값).224nM is Ca 2 + and the dissociation value for Fura-2 (an absolute value that is already been identified).

Fmax는 혈소판 내에서 Ca2 +의 생성을 극대화시킨 조건에서 측정한 최대 형광도 값.F max is the maximum fluorescence value measured under the conditions which maximize the generation of Ca 2 + in the platelets.

Fmin은 Ca2 +의 생성을 최소화시킨 조건에서 측정한 최소 형광도 값.F min is the minimum fluorescence value measured under the conditions that minimize the formation of Ca + 2.

상기 반응식에 대한 참고문헌 ; Smith, J.B. et all, Anal. biochem., 187, 173-178, 1990. Reference to the above reaction scheme; Smith, J. B. et all, Anal. biochem., 187, 173-178, 1990.

상기 [Ca2 +]i는 Schaeffer 및 Blaustein의 방법(1989)을 이용하여 얻은 Ca2 +-Fura-2 해리 상수(dissociation constant)를 이용하여 계산하였다. The [Ca 2 +] i was calculated using the Ca 2 + -Fura-2 dissociation constant (dissociation constant) obtained by the method (1989) of Schaeffer and Blaustein.

최소 형광도(Fmin)는 20mM Tris/3mM EDTA 조건의 킬레이터를 처리하여, Ca2 +을 제거한 후에 측정하였고, 최대 형광도(Fmax) 측정을 위해서는 1mM CaCl2를 처리한 후에 킬레이터를 처리하지 않은 채로 측정하였다.FIG minimum fluorescence (F min) is a chelator then processes the chelator of 20mM Tris / 3mM EDTA conditions, was measured after the removal of Ca 2 +, the maximum fluorescence handle 1mM CaCl 2 in order to (F max) measured It was measured without treatment.

상기 결과는 도 3A에 나타내었으며, 도 3A를 참고하면, 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때, Ca2 + 농도가 감소하는 것을 확인할 수 있다.The results are shown in FIG. 3A. Referring to FIG. 3A, platelet aggregation due to induction of thrombin is induced by erythro- (7 S , 8 R ) -7-acetoxy-3,4,3 ', 5'. When -tetramethoxy-8-O-4'-neolignan (EATN) inhibits, it can be seen that the Ca 2 + concentration decreases.

실시예Example 6-2.  6-2. cycliccyclic AMPAMP 의 측정Measurement of

cyclic AMP는 Ca2 +농도의 항상성 유지를 위해 이용되는 가장 강력한 저해제 중의 하나로서, Ca2 + 농도가 줄어들게 되면, 세포내 cyclic AMP의 농도가 높아진다(Schwarz et al., 2001). 또한, 트롬빈은 PKC(protein kinase C) 및 PDE3A( phosphatidylinositol-4,5-bisphosphate 3-kinase-dependent phosphorylation of phosphodiesterase-3A)를 통해 cyclic AMP의 합성을 억제한다(Zhang and Colman, 2007; Hunter et al., 2009). cyclic AMP is one of the most powerful inhibitors to be used for the homeostasis of Ca 2 + concentrations, Ca 2 + When the concentration is less, the higher the cell concentration of the cyclic AMP (Schwarz et al., 2001). Thrombin also inhibits the synthesis of cyclic AMP through protein kinase C (PKC) and phosphatidylinositol-4,5-bisphosphate 3-kinase-dependent phosphorylation of phosphodiesterase-3A (Zhang and Colman, 2007; Hunter et al. , 2009).

따라서, 혈소판의 응집을 억제하는 본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 cyclic AMP의 농도를 높이는지를 확인하였다. Accordingly, erythromycin of the present invention to inhibit the aggregation of blood platelets - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans ( EATN) was confirmed to increase the concentration of the cyclic AMP.

실시예 3에서 얻은 세척 혈소판(4×108cells/㎖)에 1~10μM의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)을 37℃에서 3분간 반응시키고, 트롬빈(0.5U/㎖)을 넣어 4분간 더 반응시켰다. 이 후, EDTA(ethylenediaminetetraacetic acid, 10mM)를 넣어 반응을 중지시키고, 5분간 전체 반응물을 끓였으며, 끓인 샘플은 0~4℃에서 5분 동안 반응시켜 식혔다. 상기 식힌 샘플은 37℃에서 3,600×g에서 12분간 원심분리하였고, 원심 분리된 상층액에서 효소 면역 어세이 키트(enzyme immunoassay kit, cat No 581002, Cayman Chemicals)를 이용하여 cyclic AMP(cyclic adenosine monophosphate)를 확인하였으며, 이에 대한 결과는 도 3B에 나타내었다. Example 3 washed platelet obtained from (4 × 10 8 cells / ㎖ ) erythromycin of 1 ~ 10μM to - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetramethoxy- 8-O-4'-neolignan (EATN) was reacted at 37 ° C. for 3 minutes, and thrombin (0.5 U / ml) was added for 4 minutes. After that, EDTA (ethylenediaminetetraacetic acid, 10mM) was added to stop the reaction, the entire reaction was boiled for 5 minutes, the boiled sample was cooled by reacting for 5 minutes at 0 ~ 4 ℃. The cooled samples were centrifuged at 3,600 × g for 12 minutes at 37 ° C., and cyclic denodeine monophosphate (cyclic AMP) using an enzyme immunoassay kit (cat No 581002, Cayman Chemicals) in the centrifuged supernatant. It was confirmed that the results are shown in Figure 3B.

도 3B를 참고하면, 트롬빈(Thrombin)의 유도로 인한 혈소판 응집을 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(EATN)이 억제할 때, cyclic AMP(cAMP) 방출이 증가하는 것을 확인할 수 있다.Referring to Figure 3B, erythromycin platelet aggregation due to induction of thrombin (Thrombin) - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O- It can be seen that cyclic AMP (cAMP) release increases when 4'-neolignan (EATN) inhibits.

<< 실시예Example 7. 독성실험>  7. Toxicity Test

실시예Example 7-1.  7-1. 급성독성Acute toxicity

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 단기간에 과량을 섭취하였을 때 급성적(24시간 이내)으로 동물체내에 미치는 독성을 조사하고, 치사율을 결정하기 위하여 본 실험을 수행하였다. 일반적인 마우스인 ICR 마우스 계통 20마리를 대조군과 실험군에 각각 10마리씩 배정하였다. 대조군에는 PEG-400/tween-80/에탄올(8/1/1, v/v/v)만을 투여하고, 실험군은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 상기 PEG-400/tween-80/에탄올(8/1/1, v/v/v)에 녹여 2g/㎏/day의 농도로 각각 경구투여하였다. 투여 24시간 후에 각각의 치사율을 조사한 결과, 대조군과 2g/㎏/day 농도의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 투여한 실험군에서 마우스가 모두 생존하는 것으로 확인되었다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignan-class when the ingestion of excessive for a short time This experiment was conducted to investigate the toxicity in the animal body (within 24 hours) and to determine the mortality rate. Twenty ICR mouse strains, which are common mice, were assigned to 10 control and experimental groups, respectively. The control group, PEG-400 / tween-80 / ethanol (8/1/1, v / v / v ) administration only, and the test group erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan was dissolved in PEG-400 / tween-80 / ethanol (8/1/1, v / v / v) at 2 g / kg / day Each dose was administered orally. Control results of testing the respective mortality 24 hours after administration, and 2g / ㎏ / day Concentration of erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetramethoxy -8 It was confirmed that all mice survived in the experimental group administered -O-4'-neolignan.

실시예Example 7-2.  7-2. 실험군Experimental group 및 대조군의 장기 및 조직 독성 실험 And control organ organs and tissue toxicity experiments

장기 독성 실험은 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 최고 농도 2g/㎏/day의 농도로 하여 각 농도로 8주 동안 C57BL/6J 마우스(각 군당 10마리)에 투여하여 실험하였다. 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 투여한 실험군과 PEG-400/tween-80/에탄올(8/1/1, v/v/v)만을 투여한 대조군의 동물들로부터 8주 후 혈액을 채취하여 GPT(glutamate-pyruvate transferase) 및 BUN(blood urea nitrogen)의 혈액 내 농도를 Select E(Vital Scientific NV, Netherland) 기기를 이용하여 측정하였다. 그 결과, 간독성과 관계있는 것으로 알려진 GPT와 신장독성과 관계있는 것으로 알려진 BUN의 경우, 대조군과 비교하여 실험군은 별다른 차이를 보이지 않았다. 또한, 각 동물로부터 간과 신장을 절취하여 통상적인 조직절편 제작과정을 거쳐 광학현미경으로 조직학적 관찰을 시행하였으며 모든 조직에서 특이한 이상이 관찰되지 않았다. Long-term toxicity experiments erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- Neo lignans highest concentration 2g / ㎏ / day The experiments were conducted by administering C57BL / 6J mice (10 mice in each group) for 8 weeks at each concentration. Erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', the experimental group treated with 5'-tetra-methoxy -8-O-4'- neo lignans and PEG-400 / tween-80 Blood was collected after 8 weeks from animals in the control group administered only ethanol (8/1/1, v / v / v) to select blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN). Measurement was performed using an E (Vital Scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidneys were excised from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed in all tissues.

<< 제제예Formulation example 1. 약학적 제제> 1. Pharmaceutical preparations>

1-1. 정제의 제조1-1. Manufacture of tablets

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난 200g을 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다. Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', the 5'-tetra-methoxy -8-O-4'- neo lignans 200g 175.9g lactose, potato starch 180g And 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2. 주사액제의 제조1-2. Injection preparation

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난 1g, 염화나트륨 0.6g 및 아스코르브산 0.1g을 증류수에 용해시켜서 100㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans 1g, 0.6g of sodium chloride and ascorbic acid 0.1 g was dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<< 제제예Formulation example 2. 식품 제조> 2. Food Manufacturing>

2-1. 조리용 양념의 제조2-1. Manufacture of cooking seasonings

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 조리용 양념에 1 중량%로 첨가하여 건강 증진용 조리용 양념을 제조하였다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', the 5'-tetra-methoxy -8-O-4'- neo lignans to 1% by weight of the seasoning for cooking Addition was made to the cooking sauce for health promotion.

2-2. 밀가루 식품의 제조2-2. Manufacture of flour food products

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 밀가루에 0.1 중량%로 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- adding the neo lignans to 0.1% by weight of the wheat flour, and Using this mixture, bread, cakes, cookies, crackers and noodles were prepared to prepare health promoting foods.

2-3. 2-3. 스프soup 및 육즙( And juicy ( graviesgravies )의 제조Manufacturing

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 스프 및 육즙에 0.1 중량%로 첨가하여 건강 증진용 수프 및 육즙을 제조하였다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 0.1% by weight of the 5'-tetra-methoxy -8-O-4'- neo lignans in soups and gravy Added to prepare health soup and gravy.

2-4. 유제품(2-4. dairy product( dairydairy productsproducts )의 제조Manufacturing

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 우유에 0.1 중량%로 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.Erythromycin of the present invention - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- adding the neo lignans to 0.1% by weight of the milk, and By using the milk, various dairy products such as butter and ice cream were prepared.

2-5. 2-5. 야채주스Vegetable juice 제조 Produce

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 토마토주스 또는 당근주스 1,000㎖에 가하여 건강 증진용 야채주스를 제조하였다.Erythromycin of the present invention in (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignan the tomato juice, or carrot juice 1,000㎖ Was added to prepare a health promoting vegetable juice.

2-6. 2-6. 과일주스Fruit juice 제조 Produce

본 발명의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 사과주스 또는 포도주스 1,000㎖에 가하여 건강 증진용 과일주스를 제조하였다.Erythromycin of the present invention in (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignan the apple juice or grape juice 1,000㎖ It was added to prepare a fruit juice for health promotion.

Claims (5)

하기 화학식 1의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난(erythro-(7S,8R)-7-acetoxy-3,4,3',5'-tetramethoxy-8-O-4'-neolignan)을 함유하는 혈관질환의 예방 또는 치료용 조성물.
[화학식 1]
Figure 112012089581211-pat00003
To erythromycin of the formula 1 - (7 S, 8 R ) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignans (erythro- (7S, 8R) -7-acetoxy-3,4,3 ', 5'-tetramethoxy-8-O-4'-neolignan) composition for the prevention or treatment of vascular diseases.
[Formula 1]
Figure 112012089581211-pat00003
 제1항에 있어서,
상기 혈관질환은 혈전증, 동맥경화증, 고혈압, 협심증, 심근경색, 허혈성 심장질환, 심부전, 뇌경색, 뇌출혈, 및, 뇌졸중으로 이루어진 군으로부터 선택되는 질환인 것을 특징으로 하는 혈관질환의 예방 또는 치료용 조성물.The method of claim 1,
The vascular disease is a composition for preventing or treating vascular disease, characterized in that the disease is selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, cerebral infarction, cerebral hemorrhage, and stroke. 제1항에 있어서,
상기 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난은 혈소판 응집 억제 효과가 있는 것을 특징으로 하는 혈관질환의 예방 또는 치료용 조성물. The method of claim 1,
The erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo lignan is characterized in that the platelet aggregation inhibiting effect Composition for preventing or treating vascular disease. 하기 화학식 1의 에리스로-(7S,8R)-7-아세톡시-3,4,3',5'-테트라메톡시-8-O-4'-네오리그난을 함유하는 혈관질환의 예방 또는 개선용 건강기능식품.
[화학식 1]
Figure 112012089581211-pat00004
To general formula (I) of erythro - (7 S, 8 R) -7- acetoxy -3,4,3 ', 5'-tetra-methoxy -8-O-4'- neo prevention of disease comprising the lignan or Health functional food for improvement.
[Formula 1]
Figure 112012089581211-pat00004
 제4항에 있어서,
상기 혈관질환은 혈전증, 동맥경화증, 고혈압, 협심증, 심근경색, 허혈성 심장질환, 심부전, 뇌경색, 뇌출혈, 및, 뇌졸중으로 이루어진 군으로부터 선택되는 질환인 것을 특징으로 하는 혈관질환의 예방 또는 개선용 건강기능식품. 5. The method of claim 4,
The vascular disease is a disease selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, cerebral infarction, cerebral hemorrhage, and stroke. food.
KR1020120122775A 2012-11-01 2012-11-01 Composition comprising erythro-(7s,8r)-7-acetoxy-3,4,3',5'-tetramethoxy-8-o-4'-neolignan for treating or preventing vascular diseases KR101333669B1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109674774A (en) * 2019-02-01 2019-04-26 扬州大学 A kind of application of propane -1- alcoholic compound in preparation treatment cerebral hemorrhage drug

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Journal of Chromatographic Science, 2004, 42, 478-483 *
Natural Product Science, 2012, 18(2), 97-101 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109674774A (en) * 2019-02-01 2019-04-26 扬州大学 A kind of application of propane -1- alcoholic compound in preparation treatment cerebral hemorrhage drug
CN109674774B (en) * 2019-02-01 2021-06-25 扬州大学 Application of propane-1-alcohol compound in preparation of medicine for treating cerebral hemorrhage

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