KR20150123205A - Composition Comprising Colored-Bean Extracts - Google Patents
Composition Comprising Colored-Bean Extracts Download PDFInfo
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- KR20150123205A KR20150123205A KR1020150142337A KR20150142337A KR20150123205A KR 20150123205 A KR20150123205 A KR 20150123205A KR 1020150142337 A KR1020150142337 A KR 1020150142337A KR 20150142337 A KR20150142337 A KR 20150142337A KR 20150123205 A KR20150123205 A KR 20150123205A
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- Prior art keywords
- ethanol
- fraction
- extract
- thrombotic
- resin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
Description
본 발명은 물, C1-C5 알코올 또는 그 혼합물; 및 유기산을 이용하여 추출한 유색콩 추출물 또는 그 분획물을 유효성분으로 함유하는 항혈전 조성물에 관한 것이다.The present invention relates to water, a C 1 -C 5 alcohol or mixture thereof; And an anti-thrombocyte composition containing, as an active ingredient, a colored soybean extract or a fraction thereof extracted with an organic acid.
현대사회는 식생활 양식의 변화와 내외부적인 환경 스트레스로 인해 중장년층에서 소위 성인병이라 일컬어지는 순환기계 질환이 큰 문제가 되고 있다. 2008년도 통계청 자료에 따르면, 동맥경화증, 뇌출혈, 뇌졸중, 뇌경색 등의 뇌혈관 질환과 심장 질환은 암발생 사망률을 제외하면 사망원인 1, 2위를 차지하고 있는 것으로 보고되었다. 이들 질환의 주요 원인은 혈전(thrombus) 으로서, 혈전증이란 과잉의 혈소판 응집반응에 의해 일어나는 병리현상이다. 혈관이 손상되었을 때 혈소판은 콜라겐(collagen), 트롬빈(thrombin), ADP(adenosine diphosphate) 등과 같은 각종 작용물질(agonists)의 자극에 응답해서 활성화되어 점착반응 (adhesion), 방출반응(secretion) 및 응집반응 (aggregation)을 일으킨다. 이 과정은 지혈(hemostasis) 뿐만 아니라 혈전증 (thrombosis) 등을 포함하는 순환계 질환에 중요한 역할을 담당한다.In the modern society, due to changes in dietary patterns and internal and external environmental stresses, circulatory diseases called so - called adult diseases are becoming a big problem in middle - aged and elderly people. According to 2008 National Statistical Office data, cerebrovascular diseases such as arteriosclerosis, cerebral hemorrhage, stroke, cerebral infarction, and heart disease were reported to occupy the first and second places of deaths except for cancer incidence. The main cause of these diseases is thrombus. Thrombosis is a pathological phenomenon caused by excessive platelet aggregation reaction. When blood vessels are damaged, the platelets are activated in response to the stimulation of various agonists such as collagen, thrombin, and adenosine diphosphate (ADP), to promote adhesion, secretion and aggregation Causing aggregation. This process plays an important role in circulatory diseases including hemostasis as well as thrombosis.
한 예로 관상동맥질환을 들 수 있으며, 이는 전세계적으로 가장 흔한 사망원인으로 알려져 있다. 특히 급성관동맥 증후군(acute coronary syndrome) 은 직접적인 사망을 일으키는 관상동맥질환이다. 급성관동맥증후군은 불안정협심증 (Unstable angina), ST분절 비상승 심근경색(Non-ST-segment elevation myocardial infarction; NSTE; Non Q-wave MI), ST 분절 상승 심근경색(ST-elevation myocardial infarction; STE MI; Q-wave MI) 등으로 분류될 수 있으며, 전자의 2가지는 치료, 예후가 유사하기에 묶어서 NSTE ACS(Non-ST-elevation Acute Coronary Syndrome)로 칭하기도 한다. 만성안정성협심증(Chronic Stable Angina)에서는 죽상경화에 의해 관상동맥 유효직경이 감소되어 허혈을 유발하나 급성관상동맥증후군은 그 기전이 다르다. 급성관상동맥증후군은 죽상경화반의 파열 혹은 미란으로 유발된 관상동맥내 급성혈전(Intracoronary Acute Thrombosis)에 의해 혈류가 급속히 감소 혹은 차단되어 일어난다.One example is coronary artery disease, which is known to be the most common cause of death worldwide. In particular, acute coronary syndrome is a coronary artery disease that causes direct death. The acute myocardial infarction (ST-segment elevation myocardial infarction (NSTE), ST-elevation myocardial infarction (STE MI) ; Q-wave MI), and two of the former are also referred to as NSTE ACS (Non-ST-elevation Acute Coronary Syndrome) as they are similar in treatment and prognosis. In chronic stable angina, the effective diameter of the coronary artery is decreased by atherosclerosis, causing ischemia, but the mechanism of acute coronary syndrome is different. Acute coronary syndrome is caused by the rapid decrease or blockage of blood flow by intracoronary acute thrombosis caused by rupture or erosion of atherosclerotic plaque.
보다 구체적으로 설명하면, 급성관상동맥증후군에서 혈관 내 혈전형성과정은 외상으로 혈관손상이 온 후 지혈(hemostasis)이 일어나는 과정과 같다. 즉 취약성 동맥경화반(Vulnerable atherosclerotic plaque)에 파열 혹은 미란이 발생하면 내피세포 밑의 결체조직이 혈액에 노출된다. 혈액 속의 혈소판은 이에 부착되고(Platelet adhesion), 기계적 생화학적 자극으로 혈소판이 활성화되어 TxA2(thromboxane A2), ADP(adenosine diphosphate), 에피네프린(epinephrine) 등이 분비된다(Platelet activation). 이들 물질은 혈소판 표면의 당단백질 수용체(GP IIb/IIIa receptor)를 활성화시키고, 활성화된 GP IIb/IIIa 수용체는 피브리노겐(fibrinogen)을 매개로 혈소판들이 서로 엉켜 붙게 한다(Platelet aggregation). 혈소판은 수많은 경로를 통해 활성화되지만 최종적으로 GP IIb/IIIa 수용기를 통해 피브리노겐을 매개로 응집되므로, 이 과정이 혈소판 응집의 최종 공통경로(final common pathway)가 된다. 일차적으로 생성된 혈전은 혈소판이 풍부한 소위 “백색 혈전(white thrombus)”이며 지혈과정의 1차 지혈(primary hemostasis)에 해당된다. More specifically, in the acute coronary syndrome, the process of intravascular thrombosis is the same as the process in which hemostasis occurs after vascular injury to the trauma. That is, if rupture or erosion occurs in the vulnerable atherosclerotic plaque, the connective tissue beneath the endothelial cells is exposed to blood. Platelet adhesion in the blood (platelet adhesion), activation of platelets by mechanical biochemical stimulation, and release of TxA2 (thromboxane A2), adenosine diphosphate (ADP), and epinephrine (Platelet activation). These substances activate the glycoprotein receptor (GP IIb / IIIa receptor) on the platelet surface, and the activated GP IIb / IIIa receptor causes platelet aggregation through fibrinogen mediated platelet aggregation. Thrombocytes are activated through a number of pathways, but eventually become aggregated via fibrinogen via GP IIb / IIIa receptors, thus rendering the final common pathway of platelet aggregation. The primary thrombus is the so-called " white thrombus " rich in platelets and corresponds to the primary hemostasis of the hemostatic process.
이 때 생긴 PRT(platelet rich thrombus)는 죽상경화반 손상부위의 혈관벽에 생성되며 관상동맥을 완전히 폐색시키지 않는 것이 보통이다(Mural thrombus). 이는 임상적으로 NTSE ACS에 해당된다. PRT(Platelet rich thrombus) 상에 2차적으로 혈액 응고과정(coagulation)이 활성화되며 연쇄반응에 의해 트롬빈(factor IIa)이 생성된다. 트롬빈은 피브리노겐(fibrinogen)을 피브린(fibrin)으로 활성화시키고, 피브린은 그물 모양으로 얽히게 되면서(fibrin meshwork) 적혈구 등 혈액세포들이 포함된 혈전을 형성한다. 형성된 혈전은 소위 “적색 혈전(red thrombus)”이며 지혈과정의 2차 지혈(secondary hemostasis) 에 해당된다. 국소적인 혈전/혈전용해 균형(Thrombosis/thrombolysis balance)이 혈전생성 방향으로 지속되면 혈관이 완전 폐색되어(Occlusive Thrombus) 임상적으로 STE MI로 발현된다. The resulting platelet rich thrombus (PRT) is usually formed in the vessel wall of the atherosclerotic lesion and does not completely occlude the coronary artery (Mural thrombus). This clinically corresponds to the NTSE ACS. Secondary blood coagulation is activated on the PRT (Platelet rich thrombus), and thrombin (factor IIa) is produced by the chain reaction. Thrombin activates fibrinogen as a fibrin, and fibrin forms a thrombus containing blood cells such as red blood cells (fibrin meshwork). The formed thrombus is the so-called "red thrombus" and corresponds to the secondary hemostasis of the hemostatic process. If the local thrombosis / thrombolysis balance persists in the direction of thrombus formation, the blood vessel is completely occluded (Occlusive Thrombus) and clinically expressed as STE MI.
항혈전 약제는 1 차 지혈에 관여하는 혈소판을 억제하는 항혈소판제제(Antiplatelet agent)와 2 차 지혈의 혈액응고(Coagulation)를 억제하는 항응고제(Anticoagulant)로 분류할 수 있다. 항혈소판제제로는 TxA2의 생성을 억제하는 아스프린(aspirin), ADP 수용체 차단제(ADP receptor blocker)인 클로피도그렐(clopidogrel)과 티클로피딘(ticlopidine), GP IIb/IIIa 수용체 차단제(GP IIb/IIIa receptor blocker)인 압식시맙(abciximab) 등이 있다. 또한 데오필린(theopylline), 몰시도민(molsidomine), 베라파밀(verapamil), 니페디핀(nifedipine), 니트로글리세린(nitroglycerine) 등은 cAMP와 cGMP의 생성을 촉진하여 Ca2 +의 동원을 억제하는 것으로 알려져 있다. 항응고제로서는 항트롬빈 III(antithrombin III)를 활성화시켜 트롬빈을 분해하는 헤파린(heparin), DTI(direct thrombin inhibitor)인 히루딘(hirudin), 비타민K 길항제인 와파린(warfarin) 등이 있다. Anti-thrombotic agents can be classified as antiplatelet agents that inhibit platelets that are involved in primary hemostasis and anticoagulants that inhibit blood coagulation of secondary hemostasis. Antiplatelet agents include aspirin, ADP receptor blockers, clopidogrel and ticlopidine, GP IIb / IIIa receptor blockers (GP IIb / IIIa receptor blockers), which inhibit the production of TxA2 Abciximab, and the like. Also Deo pilrin (theopylline), mole sidomin (molsidomine), verapamil (verapamil), nifedipine (nifedipine), nitroglycerine (nitroglycerine), etc. are known for promoting the formation of cAMP and cGMP inhibit the mobilization of Ca 2 +. Anticoagulants include heparin which activates antithrombin III to decompose thrombin, hirudin as direct thrombin inhibitor (DTI), and warfarin as a vitamin K antagonist.
그러나, 위에서 언급된 약물들은 의약품이긴 하지만 인체에 지혈과다억제, 불임, 소화기 장애 등의 여러 부작용을 야기하는 문제점이 있다. 따라서, 항혈전 약물의 부작용을 획기적으로 줄이면서도 안전한 약물로 개발하는 것이 필요하다.However, although the above-mentioned drugs are medicines, there are problems that cause various side effects such as hemostatic overcontrol, infertility, digestive disorders, etc. in the human body. Therefore, it is necessary to develop a safe drug while dramatically reducing side effects of antithrombotic drugs.
본 발명의 일실시예의 목적은 항혈전 효능이 인정되는 조성물을 제공하는 것이다.It is an object of one embodiment of the present invention to provide a composition that is antithrombotic efficacy.
본 발명의 또 다른 일실시예의 목적은 항혈전 효능이 인정되는 약학 조성물 또는 건강식품 조성물을 제공하는 것이다.It is an object of another embodiment of the present invention to provide a pharmaceutical composition or a health food composition in which the antithrombotic effect is recognized.
물, C1-C5 알코올 또는 그 혼합물; 및 유기산을 이용하여 추출한 유색콩 추출물 또는 그 분획물을 유효성분으로 함유하는 조성물이 개시된다.Water, C 1 -C 5 alcohol or mixtures thereof; And a composition containing, as an active ingredient, a colored soybean extract or a fraction thereof extracted with an organic acid.
본 발명의 일실시예에 따른 조성물은, 우수한 항혈전 효능이 인정되고, 부작용이 없는 천연물질을 사용하였으며, 약학 및 건강식품 분야 등에서 다양하게 활용 가능하다.The composition according to an embodiment of the present invention has excellent antithrombogenic effect, uses natural materials without side effects, and can be used in various fields such as medicine and health food.
도 1은 울타리콩의 20% 에탄올 유기산 추출물을 HP-20 레진으로 분획한 50% 에탄올 분획물의 농도에 따른 혈소판 응집 억제 효능을 나타낸 그래프이다;
도 2는 각 콩의 20% 에탄올 유기산 추출물을 HP-20 레진으로 분획화한 50% 에탄올 분획물의 경구 투여시 혈전 생성 억제 효능을 나타낸 그래프이다;
도 3은 울타리콩의 20% 에탄올 유기산 추출물과 그 추출물의 HP-20 레진 분획물 및 유기산을 넣지 않고 추출한 추출물의 분획물에 따른 경구 투여시 혈전 생성 억제 효능을 나타낸 그래프이다;
도 4는 울타리콩의 20% 에탄올 유기산 추출물을 HP-20 레진으로 분획한 50% 에탄올 분획물의 용량변화에 따른 경구 투여시 혈전 생성 억제 효능을 나타낸 그래프이다.FIG. 1 is a graph showing platelet aggregation inhibitory effect according to the concentration of 50% ethanol fraction obtained by fractionating 20% ethanolic organic acid extract of fence beans with HP-20 resin;
FIG. 2 is a graph showing the effect of 50% ethanol fraction obtained by fractionating 20% ethanolic organic acid extract of each soybean with HP-20 resin in the thrombogenic effect upon oral administration;
FIG. 3 is a graph showing the effect of orally administered 20% ethanolic organic acid extract of fence bean, HP-20 resin fraction of the extract, and fraction of the extract extracted without addition of organic acid to inhibit thrombus formation;
FIG. 4 is a graph showing the effect of oral administration of 50% ethanol fraction obtained by fractionation of 20% ethanolic organic acid extract of fence bean with HP-20 resin to inhibit thrombus formation.
종래의 콩에 관한 연구는, 콩에서 약리활성을 나타내는 유효성분을 분리, 정제하는 방향으로 진행되었으며, 콩 자체의 의약적 용도에 대한 연구는 부족하였다. 또한 그 추출방법에 있어서도, 천연물에서 통상적으로 이용하는 추출법인, 고농도의 유기 용매를 이용한 추출방법을 사용하였다. Conventional studies on soybeans proceeded in the direction of separating and purifying the active ingredient showing pharmacological activity in soybeans, and research on the medicinal use of soybeans itself was lacking. In addition, an extraction method using an organic solvent at a high concentration, which is an extraction method commonly used for natural products, was used for the extraction method.
콩 추출물에는 밝혀진 성분 외에 아직도 여러 가지 미지의 성분들이 있으며, 이들 중 일부 성분은 인체에 유용한 약리효과를 나타내기도 한다. 본 발명의 발명자들은 천연물 및 생약 추출에서 일반적으로 사용되는 추출법에 대한 인식을 탈피하여, 물 또는 저농도의 저급 알코올; 및 유기산을 추출 용매로 사용하여 콩으로부터 추출물을 획득하였고, 이 추출물이 고농도 유기 용매를 추출 용매로 사용하는 경우보다 더 강력한 항혈전 효능이 있음을 밝혀냈다.In addition to the ingredients found in soybean extracts, there are still a number of unknown ingredients, some of which may have beneficial pharmacological effects on the human body. The inventors of the present invention have found that by avoiding the recognition of extraction methods commonly used in natural products and herbal medicine extracts, they can be applied to water or a low concentration of lower alcohol; And organic acid were used as extraction solvents to obtain extracts from soybeans. These extracts showed stronger anti - thrombotic effects than those using high concentration organic solvents as extraction solvents.
본 발명의 일실시예에 따른 조성물은, 물 또는 저농도의 유기용매; 및 유기산을 이용하여 추출한 콩 추출물을 함유하는 것을 특징으로 한다. 일실시예에서, 본 발명에 따른 조성물은, 물, C1-C5 알코올 또는 그 혼합물; 및 유기산을 이용하여 추출한 유색콩 추출물을 함유하는 항혈전 조성물일 수 있다. 또 다른 일실시예에서, 본 발명에 따른 조성물은, 물, C1-C5 알코올 또는 그 혼합물; 및 유기산을 이용하여 추출한 유색콩 추출물의 분획물을 함유하는 항혈전 조성물일 수 있다.The composition according to one embodiment of the present invention comprises water or a low concentration organic solvent; And soybean extract extracted with an organic acid. In one embodiment, the composition according to the present invention comprises water, a C 1 -C 5 alcohol or mixture thereof; And an anti-thrombotic composition containing a colored soybean extract extracted with an organic acid. In another embodiment, the composition according to the invention comprises water, a C 1 -C 5 alcohol or mixture thereof; And an anti-thrombotic composition containing a fraction of a colored soybean extract extracted with an organic acid.
일실시예에서, 상기 유기산은 시트르산(citric aicd), 아세트산(acetic acid), 뷰티르산(butyric acid), 팔미트산(palmitic acid), 옥살산(oxalic acid), 타타르산(tartaric acid), 카르복실산(carboxylic acid) 및 설폰산(sulfonic acid)으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으며, 보다 구체적으로는 시트르산(citric acid)일 수 있다. 유색콩으로부터 유효성분을 추출할 때, 저농도의 유기용매와 함께 유기산을 사용함으로써, 콩으로부터의 추출효율을 증가시키고 유효성분에 대한 선택성을 높일 수 있다.In one embodiment, the organic acid is selected from the group consisting of citric acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, May be any one selected from the group consisting of carboxylic acid and sulfonic acid, and more specifically citric acid. By using an organic acid together with a low concentration organic solvent when extracting the active ingredient from the colored beans, it is possible to increase the extraction efficiency from the soybean and increase the selectivity to the active ingredient.
일실시예에서, 상기 유기 용매는, 특별히 제한되는 것은 아니며, 저급알코올, 보다 구체적으로는 C1-C5 알코올일 수 있다. 상기 C1-C5 알코올은, 예를 들어, 메탄올, 에탄올, 이소프로필알코올, n-프로필알코올, n-부탄올 및 이소부탄올로 구성된 군으로부터 선택되는 어느 하나 또는 둘 이상의 혼합용매일 수 있으며, 구체적으로는 에탄올이다. 예를 들어, 본 발명에 따른 유색콩 추출물은 에탄올 및 유기산을 이용하여 추출한 추출물일 수 있다. In one embodiment, the organic solvent is not particularly limited and may be a lower alcohol, more specifically a C 1 -C 5 alcohol. The C 1 -C 5 alcohol may be any one or a mixture of two or more selected from the group consisting of methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol and isobutanol, It is ethanol. For example, the colored bean extract according to the present invention may be an extract extracted with ethanol and organic acid.
또 다른 일실시예에서, 상기 C1-C5 알코올의 농도는 1 내지 70%(v/v), 또는 1 내지 40%(v/v)이며, 구체적으로는 5 내지 25%(v/v), 보다 구체적으로는 7 내지 20%(v/v)이다. 예를 들어, 상기 C1-C5 알코올은 5 내지 25%(v/v) 농도의 에탄올, 보다 구체적으로는 10% 또는 20%(v/v) 농도의 에탄올이다. In another embodiment, the concentration of the C 1 -C 5 alcohol is 1 to 70% (v / v), or 1 to 40% (v / v), specifically 5 to 25% ), More specifically 7 to 20% (v / v). For example, the C 1 -C 5 alcohol is ethanol at a concentration of 5 to 25% (v / v), more specifically at a concentration of 10% or 20% (v / v).
본 발명에 따른 조성물은, 물 또는 저농도의 저급 알코올; 및 유기산을 이용하여 콩을 추출하게 된다. 본 발명의 발명자들은, 다양한 연구 및 반복적인 실험을 통해, 물을 포함한 여러 종류의 유기용매들 중에서 저급 알코올, 예를 들어 에탄올, 특히 저농도 에탄올을 이용하여, 유기산과 함께 추출한 유색콩 추출물이 혈액 순환을 개선시키고, 비만 및 당뇨에 효과적이며, 고지혈증에 우수한 효과를 갖는다는 점을 확인하고, 본 발명을 완성하였다.The composition according to the present invention comprises water or a low concentration of lower alcohol; And organic acids to extract soybeans. The inventors of the present invention have found that, through various researches and repeated experiments, it has been found that, among the various kinds of organic solvents including water, using a low alcohol such as ethanol, especially low-density ethanol, And is effective for obesity and diabetes, and has an excellent effect on hyperlipemia. Thus, the present invention has been completed.
일실시예에서, 상기 유색콩 추출물은, 물, C1-C5 알코올 또는 그 혼합물; 및 유기산을 이용한 추출물이며, 그 분획물은 유색콩 추출물을 레진으로 분리한 분획물 또는 유색콩 추출물을 용매인 부탄올로 분획화한 분획물이다. 상기 레진 분획물은, 예를 들어, 폴리스티렌(polystylene)과 벤젠(benzene)의 중합체를 포함하는 합성 흡착제 컬럼이며, 구체적으로는 HP-20 레진(resin)이다. 본 발명의 발명자들은, 예를 들어, 저농도 에탄올을 이용하여 추출한 유색콩 추출물에 대한 다양한 분획을 추출하는 실험을 진행하였다. 실험 결과, HP-20 레진 분획물 또는 부탄올 분획물이 물 분획물에 비해 항혈전 효능이 우수한 것으로 확인되었다. In one embodiment, the colored bean extract comprises water, a C 1 -C 5 alcohol or mixture thereof; And an organic acid. The fraction is a fraction obtained by separating a colored soybean extract into a resin or a fraction obtained by fractionating a colored soybean extract with butanol as a solvent. The resin fraction is, for example, a synthetic adsorbent column comprising a polymer of polystylene and benzene, specifically HP-20 resin. The inventors of the present invention conducted an experiment for extracting various fractions of a colored soybean extract, for example, extracted using low-concentration ethanol. As a result, it was confirmed that the HP-20 resin fraction or butanol fraction had better anti-thrombolytic activity than the water fraction.
본 발명에 따른 “유색콩(colored-bean)”이란, 낟알 껍질의 색이 짙은 빛깔을 띠는 콩을 총칭하는 의미이며, 검은색 뿐만 아니라 적색, 황색 또는 청색 등의 빛깔이 나는 경우도 포괄한다. 상기 유색콩의 예로는, 특별히 제한되는 것은 아니며, 예를 들어, 서리태, 서목태, 청태, 황태, 울타리콩, 강낭콩, 얼룩강낭콩, 적두, 거두, 콩나물콩, 대두 및 흑태로 구성된 군으로부터 선택되는 어느 하나 또는 둘 이상일 수 있다. 일실시예에서, 본 발명에 따른 유색콩은 울타리콩일 수 있다. 유색콩의 명칭은 지역이나 분류, 방언의 영향 등으로 다양하게 호칭될 수 있다. 또한, 본 발명에 따른 “유색콩 추출물”이란, 유색콩에 대한 다양한 추출과정을 통해 추출된 물질을 총칭하며, 예를 들어, 유기 용매 등을 이용하여 추출된 물질을 포함하고, 추출된 물질에 대한 다양한 분획물(예, HP-20 레진 분리 분획) 등을 포함할 수 있다.The term " colored bean " according to the present invention is a generic term for soybeans having a dense color of grain shells, and covers not only black but also red, yellow or blue . Examples of the colored beans include, but are not limited to, those selected from the group consisting of, for example, seahorse, seomyeotyeo, chungcheong, kwangtae, fence bean, kidney bean, spotted kidney bean, One or two or more. In one embodiment, the colored beans according to the present invention may be fence beans. The name of the colored bean can be variously referred to as region, classification, influence of dialect, and the like. The term "colored bean extract" according to the present invention refers to a substance extracted through various extraction processes for colored beans and includes, for example, a substance extracted using an organic solvent or the like, Various fractions (e. G., HP-20 resin separated fractions), and the like.
본 발명에 따른 유색콩 추출물 또는 그 분획물을 함유하는 조성물은, 혈소판 응집을 억제하여 혈전생성을 저해하는 효능이 인정되며, 이러한 효능을 통해, 혈액 순환 개선에 효과적이며, 비만, 당뇨 및 고지혈증 등의 치료 또는 예방에 효과적으로 사용될 수 있다.The composition containing the colored bean extract or the fraction thereof according to the present invention is effective for inhibiting platelet aggregation and inhibiting thrombogenesis and is effective for improving blood circulation through such an effect and is effective for preventing obesity, diabetes and hyperlipemia Can be effectively used for treatment or prevention.
본 발명은 또한, 상기 조성물을 유효성분으로 포함하는 약학 조성물을 제공한다. 일실시예에서, 상기 약학 조성물은 혈관 질환의 예방, 경감 또는 치료용 조성물일 수 있다. 본 발명에 따른 조성물을 포함하는 약학 조성물은, 혈전생성 방지, 혈관수축 억제 및/또는 콜레스테롤 억제 효능이 인정된다. 구체적으로는, 상기 약학 조성물은, 항혈전 효능에 기인하는 혈액 순환 개선용일 수 있으며, 비만, 당뇨 및 고지혈증 등을 포함하는 혈관 질환의 경감 또는 치료에 효과적인 약학 조성물일 수 있다. 상기 혈관 질환은, 예들 들어, 비만, 당뇨, 뇌졸증, 뇌출혈, 동맥경화, 협심증, 심근경색, 고혈압, 빈혈, 편두통 또는 고지혈증 등을 포함한다. The present invention also provides a pharmaceutical composition comprising said composition as an active ingredient. In one embodiment, the pharmaceutical composition may be a composition for preventing, alleviating or treating vascular diseases. The pharmaceutical composition comprising the composition according to the present invention is effective in preventing the formation of blood clots, inhibiting vasoconstriction, and / or inhibiting cholesterol. Specifically, the pharmaceutical composition may be for improving blood circulation due to antithrombogenic effect, and may be a pharmaceutical composition effective for relieving or treating vascular diseases including obesity, diabetes and hyperlipemia. Such vascular diseases include, for example, obesity, diabetes, stroke, cerebral hemorrhage, arteriosclerosis, angina pectoris, myocardial infarction, hypertension, anemia, migraine or hyperlipidemia.
본 발명에 따른 조성물을 의약품에 적용할 경우에는, 상기 조성물을 유효성분으로 하여 상용되는 무기 또는 유기의 담체를 가하여 고체, 반고체 또는 액상의 형태로 경구 투여제 혹은 비경구 투여제로 제제화 할 수 있다.When the composition according to the present invention is applied to medicines, it may be formulated into an oral or parenteral dosage form in the form of solid, semi-solid or liquid by adding an inorganic or organic carrier to the composition as an active ingredient.
상기 경구 투여를 위한 제재로서는 정제 (錠劑), 환제 (丸劑), 과립제 (顆粒劑), 연·경 캡슐제, 산제, 세립제, 분제, 유탁제 (乳濁濟), 시럽제, 펠렛제 등을 들 수 있다. 또한, 상기 비경구 투여를 위한 제재로는 주사제, 점적제, 연고, 로션, 스프레이, 현탁제, 유제, 좌제 (坐劑) 등을 들 수 있다. 본 발명의 유효성분을 제제화하기 위해서는 상법에 따라서 실시하면 용이하게 제제화할 수 있으며 계면활성제, 부형제, 착색료, 향신료, 보존료, 안정제, 완충제, 현탁제, 기타 상용하는 보조제를 적당히 사용할 수 있다.Examples of the agent for oral administration include tablets, pills, granules, soft capsules, powders, granules, powders, emulsions, syrups and pellets. . Examples of the parenteral administration agent include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method. Surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffering agents, suspending agents and other adjuvants can be suitably used.
본 발명에 따른 상기 약학 조성물은 경구, 비경구, 직장, 국소, 경피, 정맥 내, 근육 내, 복강 내, 피하 등으로 투여될 수 있다. The pharmaceutical composition according to the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously and the like.
또한, 상기 활성성분의 투여량은 치료 받을 대상의 연령, 성별, 체중과 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 있다. 일반적인 투여량은 0.001mg/kg/일 내지 2000mg/kg/일, 보다 구체적으로는 0.5mg/kg/일 내지 1500mg/kg/일이다.In addition, the dosage of the active ingredient will vary depending on the age, sex, body weight and the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of those skilled in the art. Typical dosages are from 0.001 mg / kg / day to 2000 mg / kg / day, more specifically from 0.5 mg / kg / day to 1500 mg / kg / day.
또한, 본 발명에 따른 조성물을 포함하는 식품 첨가제, 기능성 식품 또는 건강식품 등을 제공한다. 상기 식품 첨가제, 기능성 식품 또는 건강식품 등은 본 발명에 따른 조성물을 유효성분으로 포함한다. 일실시예에서, 본 발명에 따른 조성물을 포함하는 건강식품 등은 혈액 순환 개선용이며, 보다 구체적으로는 비만, 당뇨 및 고지혈증을 포함하는 혈관 질환의 예방 또는 완화에 효과적인 건강식품 조성물 등일 수 있다. 상기 혈관 질환은, 예를 들어, 비만, 당뇨, 뇌졸증, 뇌출혈, 동맥경화, 협심증, 심근경색, 고혈압, 빈혈, 편두통 또는 고지혈증 등을 포함한다. The present invention also provides a food additive, a functional food or a health food containing the composition according to the present invention. The food additive, the functional food or the health food include the composition according to the present invention as an active ingredient. In one embodiment, the health food or the like containing the composition according to the present invention is for improving blood circulation, and more specifically, it may be a health food composition effective for preventing or alleviating vascular diseases including obesity, diabetes and hyperlipemia. Such vascular diseases include, for example, obesity, diabetes, stroke, cerebral hemorrhage, arteriosclerosis, angina pectoris, myocardial infarction, hypertension, anemia, migraine or hyperlipemia.
또한, 본 발명에 따른 조성물은 포함하는 다양한 형태의 식품 첨가제 또는 기능성 식품을 제공한다. 상기 조성물을 포함하는 발효유, 치즈, 요구르트, 주스, 생균제제 및 건강보조식품 등으로 가공될 수 있으며, 그 외 다양한 식품 첨가제의 형태로 사용될 수 있다. In addition, the composition according to the present invention provides various types of food additives or functional foods containing them. It can be processed into fermented milk, cheese, yogurt, juice, probiotic agent and health supplement food including the above composition, and it can be used in various other food additives.
일실시예에서 상기 조성물은 본 발명이 목적으로 하는 주 효과를 손상시키지 않는 범위 내에서 주 효과에 상승 효과를 줄 수 있는 다른 성분 등을 함유할 수 있다. 예를 들어, 물성 개선을 위하여 향료, 색소, 살균제, 산화방지제, 방부제, 보습제, 점증제, 무기염류, 유화제 및 합성 고분자 물질 등의 첨가제를 더 포함할 수 있다. 그 외에도, 수용성 비타민, 유용성 비타민, 고분자 펩티드, 고분자 다당 및 해초 엑기스 등의 보조 성분을 더 포함할 수도 있다. 상기 성분들은 제형 또는 사용 목적에 따라서 당업자가 어려움 없이 적의 선정하여 배합할 수 있으며, 그 첨가량은 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 선택될 수 있다. 예를 들어, 상기 성분들의 첨가량은, 조성물 전체 중량을 기준으로, 0.01-5 중량%, 보다 구체적으로는 0.01-3 중량% 범위일 수 있다.In one embodiment, the composition may contain other ingredients, etc., which may give rise to synergistic effects on the main effect, to the extent that the main effect of the present invention is not impaired. For example, additives such as perfume, coloring agent, bactericide, antioxidant, preservative, moisturizing agent, thickening agent, inorganic salt, emulsifier and synthetic polymer substance may be further added for improvement of physical properties. In addition, it may further contain auxiliary components such as water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides and seaweed extract. The above components may be mixed and selected without difficulty by those skilled in the art depending on the purpose of formulation or use, and the amount thereof may be selected within a range that does not impair the objects and effects of the present invention. For example, the addition amount of the above components may be in the range of 0.01-5 wt%, more specifically 0.01-3 wt%, based on the total weight of the composition.
본 발명에 따른 조성물의 제형은 용액, 유화물, 점성형 혼합물, 타블렛, 분말 등의 다양한 형태일 수 있으며, 이는 단순 음용, 주사 투여, 스프레이 방식 또는 스퀴즈 방식 등의 다양한 방법으로 투여될 수 있다.The composition according to the present invention may be in various forms such as a solution, an emulsion, a viscous mixture, a tablet, a powder, etc., and may be administered by various methods such as simple drinking, injection administration, spraying or squeezing.
이하, 본 발명의 바람직한 실시예 등을 통해 본 발명을 더욱 상술하지만, 하기 실시예 등은 본 발명의 효과를 예시적으로 확인하기 위한 것이며, 본 발명의 범주가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the preferred embodiments of the present invention. However, the following examples are intended to illustrate the effects of the present invention, and the scope of the present invention is not limited thereto.
[실시예 1] 20% 에탄올 및 유기산을 이용한 서리태 추출물의 제조[Example 1] Preparation of a safflower extract using 20% ethanol and organic acid
건조된 서리태 1 kg을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.1 kg of the dried seaweed was immersed in 5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 째 C, refluxed for 3 hours, and left at room temperature for a certain time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 2] 20% 에탄올 및 유기산을 이용한 서목태 추출물의 제조[Example 2] Preparation of Seomyeongae extract using 20% ethanol and organic acid
건조된 서목태 1 kg을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.1 kg of dried noodles was immersed in 5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 ° C., refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 3] 20% 에탄올 및 유기산을 이용한 강낭콩 추출물의 제조[Example 3] Preparation of kidney bean extract using 20% ethanol and organic acid
건조된 강낭콩 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다. 300 g of dried kidney beans were immersed in 1.5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 4] 20% 에탄올 및 유기산을 이용한 강낭콩(얼룩) 추출물의 제조[Example 4] Preparation of kidney bean (stain) extract using 20% ethanol and organic acid
건조된 강낭콩 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of dried kidney beans were immersed in 1.5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 5] 20% 에탄올 및 유기산을 이용한 콩나물콩 추출물의 제조[Example 5] Preparation of bean sprouts bean extract using 20% ethanol and organic acid
건조된 콩나물콩 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of dried bean sprouts beans were immersed in 1.5 L of a 20% ethanol aqueous solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 6] 20% 에탄올 및 유기산을 이용한 황태 추출물의 제조[Example 6] Preparation of extract of Huangtai using 20% ethanol and organic acid
건조된 황태 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of dried safflower was immersed in 1.5 L of a 20% ethanol aqueous solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 7] 20% 에탄올 및 유기산을 이용한 청태 추출물의 제조[Example 7] Preparation of cheongchae extract using 20% ethanol and organic acid
건조된 청태 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of dried cheongchae was immersed in 1.5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 째 C, refluxed for 3 hours, and left at room temperature for a certain time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 8] 20% 에탄올 및 유기산을 이용한 울타리콩 추출물의 제조[Example 8] Preparation of fence bean extract using 20% ethanol and organic acid
건조된 울타리콩 1 kg을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온 보관하였다.1 kg of dried fence beans was immersed in 5 L of a 20% ethanol aqueous solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was stored at low temperature until use.
[실시예 9] 20% 에탄올 및 유기산을 이용한 대두 추출물의 제조[Example 9] Production of soybean extract using 20% ethanol and organic acid
건조된 대두 1 kg을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 냉장 보관하였다.1 kg of dried soybeans was immersed in 5 L of 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15%, and the prepared powder was stored in the refrigerator until use.
[실시예 10] 20% 에탄올 및 유기산을 이용한 거두 추출물의 제조[Example 10] Preparation of sorghum extract using 20% ethanol and organic acid
건조된 거두 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of the dried reeds were immersed in 1.5 L of a 20% ethanol aqueous solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 11] 20% 에탄올 및 유기산을 이용한 적두 추출물의 제조[Example 11] Preparation of red ginseng extract using 20% ethanol and organic acid
건조된 적두 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of the dried red oak was immersed in 1.5 L of a 20% ethanol aqueous solution containing 1% citric acid at a temperature of 60 DEG C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 12] 20% 에탄올 및 유기산을 이용한 흑태 추출물의 제조[Example 12] Preparation of black tea extract using 20% ethanol and organic acid
건조된 흑태 300 g을 60℃의 온도에서 1% 시트르산(citric acid)을 포함한 20%의 에탄올 수용액 1.5 L에 침지하여 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 분말 시료를 제조하였다. 수율은 3-15% 이었으며, 조제된 분말은 사용시까지 저온에서 보관하였다.300 g of the dried black tea was immersed in 1.5 L of a 20% aqueous ethanol solution containing 1% citric acid at a temperature of 60 ° C, refluxed for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a powder sample. The yield was 3-15% and the prepared powder was kept at low temperature until use.
[실시예 13] 서리태 추출물의 HP-20 레진(resin)을 이용한 분리[Example 13] Separation of edible leaf extract with HP-20 resin
건조된 서리태 1 kg을 1% 시트르산(citric acid)을 포함한 20%의 에탄올 용액 5 L에 침지하여 60℃의 온도에서 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 20% 에탄올 추출물 시료를 제조하였다. 소수성 수지인 DIAION HP-20 (SUPELCO) 을 이용하여 하기와 같이 20% 에탄올 유기산 서리태 추출물에 대한 레진 분리 분획을 얻었다.1 kg of dried seaweed was immersed in 5 L of a 20% ethanol solution containing 1% citric acid, refluxed at 60 ° C. for 3 hours, and left at room temperature for a certain time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a 20% ethanol extract. A hydrophobic resin, DIAION HP-20 (SUPELCO), was used to obtain a resin-separated fraction for the 20% ethanol organic acid surfactant extract as described below.
HP-20 레진을 이용한 분리를 위해, 컬럼에 30 cm 길이가 되도록 HP-20 레진을 충진 한 후, 1 L 에탄올을 이용하여 500 ml씩 2 회, 1 L 50% 에탄올을 이용하여 500 ml씩 2 회, 1 L 증류수를 이용하여 500 ml씩 2 회 각각 세척한 후에 이용하였다. 6 g의 20% 에탄올 유기산 서리태 추출물을 물에 녹인 후 HP-20 레진 충진 칼럼에 가한 후, 750 ml 증류수, 750 ml 50% 에탄올, 500 ml 에탄올을 순차적으로 넣어주면서 흘러나온 용매를 250 ml 삼각 플라스크로 받은 후 각각을 감압 농축하여 동결 건조하여 분획 시료를 제조하였다.For separation using HP-20 resin, fill the column with HP-20 resin to a length of 30 cm, add 2 ml of 500 ml each with 1 L of ethanol, 500 ml of 1 ml of 50 ml of ethanol And washed once with 500 ml of 1 L distilled water each time. 6 g of 20% ethanolic organic acid extract was dissolved in water and added to an HP-20 resin filling column. 750 ml of distilled water, 750 ml of 50% ethanol and 500 ml of ethanol were added successively, , And each fraction was concentrated under reduced pressure and lyophilized to prepare a fraction sample.
[실시예 14] 울타리콩 추출물의 HP-20 레진(resin)을 이용한 분리[Example 14] Isolation of fence bean extract with HP-20 resin
건조된 울타리콩 1 kg을 1% 시트르산(citric acid)을 포함한 20%의 에탄올 용액 5 L에 침지하여 60℃의 온도에서 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 20% 에탄올 유기산 추출물 시료를 제조하였다. 소수성 수지인 DIAION HP-20 (SUPELCO) 을 이용하여 하기와 같이 20% 에탄올 유기산 울타리콩 추출물에 대한 레진 분리 분획을 얻었다. 1 kg of dried fence beans was immersed in 5 L of a 20% ethanol solution containing 1% citric acid, refluxed at 60 ° C. for 3 hours, and then left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a 20% ethanol organic acid extract sample. A hydrophobic resin DIAION HP-20 (SUPELCO) was used to obtain a resin-separated fraction for the 20% ethanol organic acid fence bean extract as described below.
HP-20 레진을 이용한 분리를 위해, 컬럼에 30 cm 길이가 되도록 HP-20 레진을 충진 한 후, 1 L 에탄올을 이용하여 500 ml씩 2 회, 1 L 50% 에탄올을 이용하여 500 ml씩 2 회, 1 L 증류수를 이용하여 500 ml씩 2 회 각각 세척한 후에 이용하였다. 6 g의 20% 에탄올 유기산 울타리콩 추출물을 물에 녹인 후 HP-20 레진 충진 칼럼에 가한 후, 750 ml 증류수, 750 ml 50% 에탄올, 500 ml 에탄올을 순차적으로 넣어주면서 흘러나온 용매를 250 ml 삼각 플라스크로 받은 후 각각을 감압 농축하여 동결 건조하여 분획 시료를 제조하였다.For separation using HP-20 resin, fill the column with HP-20 resin to a length of 30 cm, add 2 ml of 500 ml each with 1 L of ethanol, 500 ml of 1 ml of 50 ml of ethanol And washed once with 500 ml of 1 L distilled water each time. After dissolving 6 g of 20% ethanol organic acid fence bean extract in water, it was added to HP-20 resin filling column, 750 ml of distilled water, 750 ml of 50% ethanol and 500 ml of ethanol were added successively, After receiving them in a flask, they were concentrated under reduced pressure and lyophilized to prepare a fraction sample.
[실시예 15] 강낭콩 추출물의 HP-20 레진(resin)을 이용한 분리[Example 15] Isolate of kidney bean extract with HP-20 resin
건조된 강낭콩 1 kg을 1% 시트르산(citric acid)을 포함한 20%의 에탄올 용액 5 L에 침지하여 60℃의 온도에서 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 20% 에탄올 유기산 추출물 시료를 제조하였다. 소수성 수지인 DIAION HP-20 (SUPELCO) 을 이용하여 하기와 같이 20% 에탄올 유기산 강낭콩 추출물에 대한 레진 분리 분획을 얻었다.1 kg of dried kidney beans was immersed in 5 L of a 20% ethanol solution containing 1% citric acid, refluxed at 60 ° C for 3 hours, and then left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a 20% ethanol organic acid extract sample. A hydrophobic resin DIAION HP-20 (SUPELCO) was used to obtain a resin-separated fraction for a 20% ethanol organic acid kidney bean extract as follows.
HP-20 레진을 이용한 분리를 위해, 컬럼에 30 cm 길이가 되도록 HP-20 레진을 충진 한 후, 1 L 에탄올을 이용하여 500 ml씩 2 회, 1 L 50% 에탄올을 이용하여 500 ml씩 2 회, 1 L 증류수를 이용하여 500 ml씩 2 회 각각 세척한 후에 이용하였다. 6 g의 20% 에탄올 유기산 강낭콩 추출물을 물에 녹인 후 HP-20 레진 충진 칼럼에 가한 후, 750 ml 증류수, 750 ml 50% 에탄올, 500 ml 에탄올을 순차적으로 넣어주면서 흘러나온 용매를 250 ml 삼각 플라스크로 받은 후 각각을 감압 농축하여 동결 건조하여 분획 시료를 제조하였다.For separation using HP-20 resin, fill the column with HP-20 resin to a length of 30 cm, add 2 ml of 500 ml each with 1 L of ethanol, 500 ml of 1 ml of 50 ml of ethanol And washed once with 500 ml of 1 L distilled water each time. 6 g of 20% ethanolic organic acid kidney bean extract was dissolved in water and added to an HP-20 resin filling column. 750 ml of distilled water, 750 ml of 50% ethanol and 500 ml of ethanol were added successively, , And each fraction was concentrated under reduced pressure and lyophilized to prepare a fraction sample.
[실시예 16] 대두 추출물의 HP-20 레진(resin)을 이용한 분리Example 16 Isolation of Soybean Extract Using HP-20 Resin
건조된 대두 1 kg을 1% 시트르산(citric acid)을 포함한 20%의 에탄올 용액 5 L에 침지하여 60℃의 온도에서 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 20% 에탄올 유기산 추출물 시료를 제조하였다. 소수성 수지인 DIAION HP-20 (SUPELCO) 을 이용하여 하기와 같이 20% 에탄올 유기산 대두 추출물에 대한 레진 분리 분획을 얻었다.1 kg of dried soybeans was immersed in 5 liters of a 20% ethanol solution containing 1% citric acid, refluxed at 60 ° C for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a 20% ethanol organic acid extract sample. A hydrophobic resin DIAION HP-20 (SUPELCO) was used to obtain a resin-separated fraction for 20% ethanol organic acid soybean extract as described below.
HP-20 레진을 이용한 분리를 위해, 컬럼에 30 cm 길이가 되도록 HP-20 레진을 충진 한 후, 1 L 에탄올을 이용하여 500 ml씩 2 회, 1 L 50% 에탄올을 이용하여 500 ml씩 2 회, 1 L 증류수를 이용하여 500 ml씩 2 회 각각 세척한 후에 이용하였다. 6 g의 20% 에탄올 유기산 대두 추출물을 물에 녹인 후 HP-20 레진 충진 칼럼에 가한 후, 750 ml 증류수, 750 ml 50% 에탄올, 500 ml 에탄올을 순차적으로 넣어주면서 흘러나온 용매를 250 ml 삼각 플라스크로 받은 후 각각을 감압 농축하여 동결 건조하여 분획 시료를 제조하였다.For separation using HP-20 resin, fill the column with HP-20 resin to a length of 30 cm, add 2 ml of 500 ml each with 1 L of ethanol, 500 ml of 1 ml of 50 ml of ethanol And washed once with 500 ml of 1 L distilled water each time. 6 g of 20% ethanolic organic acid soybean extract was dissolved in water and added to an HP-20 resin filling column. 750 ml of distilled water, 750 ml of 50% ethanol and 500 ml of ethanol were added successively, , And each fraction was concentrated under reduced pressure and lyophilized to prepare a fraction sample.
[실시예 17] 서목태 추출물의 HP-20 레진(resin)을 이용한 분리[Example 17] Separation of Seomyeongae extract with HP-20 resin
건조된 서목태 1 kg을 1% 시트르산(citric acid)을 포함한 20%의 에탄올 용액 5 L에 침지하여 60℃의 온도에서 3 시간 동안 환류 추출한 다음, 상온에서 일정 시간 동안 방치하였다. 추출액은 여과하여 감압 농축하고 동결 건조하여 20% 에탄올 유기산 추출물 시료를 제조하였다. 소수성 수지인 DIAION HP-20 (SUPELCO) 을 이용하여 하기와 같이 20% 에탄올 유기산 서목태 추출물에 대한 레진 분리 분획을 얻었다. 1 kg of dried noodle soup was immersed in 5 L of 20% ethanol solution containing 1% citric acid, refluxed at 60 ° C for 3 hours, and left at room temperature for a certain period of time. The extract was filtered, concentrated under reduced pressure and lyophilized to prepare a 20% ethanol organic acid extract sample. DIAION HP-20 (SUPELCO), a hydrophobic resin, was used to obtain a resin-separated fraction for 20% ethanol organic acid Seomyeok extract.
HP-20 레진을 이용한 분리를 위해, 컬럼에 30 cm 길이가 되도록 HP-20 레진을 충진 한 후, 1 L 에탄올을 이용하여 500 ml씩 2 회, 1 L 50% 에탄올을 이용하여 500 ml씩 2 회, 1 L 증류수를 이용하여 500 ml씩 2 회 각각 세척한 후에 이용하였다. 6 g의 20% 에탄올 유기산 서목태 추출물을 물에 녹인 후 HP-20 레진 충진 칼럼에 가한 후, 750 ml 증류수, 750 ml 50% 에탄올, 500 ml 에탄올을 순차적으로 넣어주면서 흘러나온 용매를 250 ml 삼각 플라스크로 받은 후 각각을 감압 농축하여 동결 건조하여 분획 시료를 제조하였다.For separation using HP-20 resin, fill the column with HP-20 resin to a length of 30 cm, add 2 ml of 500 ml each with 1 L of ethanol, 500 ml of 1 ml of 50 ml of ethanol And washed once with 500 ml of 1 L distilled water each time. 6 g of 20% ethanolic organic acid extracts were dissolved in water and added to an HP-20 resin filling column. 750 ml of distilled water, 750 ml of 50% ethanol and 500 ml of ethanol were added successively, , And each fraction was concentrated under reduced pressure and lyophilized to prepare a fraction sample.
[실시예 18] 울타리콩 추출물의 용매별 분획[Example 18] Fractional fraction of fence soybean extract
실시예 8에서 얻어진 20% 에탄올 유기산 울타리콩 추출물 10.18 g을 증류수 100 ml에 녹인 후 분별깔때기를 사용하여 에틸아세테이트(EtOAc) 100 ml로 2 회 추출한 후 남은 물층을 n-부탄올(n-BuOH) 100 ml로 2 회 추출하였다. 얻어진 에틸아세테이트(EtOAc), n-부탄올(n-BuOH) 및 물층을 각각 모아 감압 농축한 후 동결 건조하여 시료를 제조하였다. 수율은 에틸아세테이트(EtOAc) 4.62%, n-부탄올(n-BuOH) 6.58% 및 물층은 8.88%이었으며, 제조된 시료는 사용시까지 냉장 보관하였다.10.18 g of the 20% ethanol organic acid fence soybean extract obtained in Example 8 was dissolved in 100 ml of distilled water and extracted twice with 100 ml of ethyl acetate (EtOAc) using a separatory funnel. The remaining water layer was washed with n-
[실험예 1] 콜라겐에 의해 유도된 사람 혈소판 응집 억제 관찰[Experimental Example 1] Inhibition of human platelet aggregation induced by collagen
에탄올 함량에 따른 추출물의 활성 차이를 비교하기 위하여 에탄올 함량을 달리하여 추출한 울타리콩을 이용하여 하기의 실험을 실시하였다. In order to compare the difference in the activity of the extracts according to the ethanol content, the following experiment was carried out using the fence beans extracted with different ethanol contents.
사람의 혈소판 농축 혈장을 (Platelet Rich Plasma, PRP)를 분리하기 위하여, 3.2% 구연산 나트륨(sodium citrate)을 항응고제로 사용하였으며, 2 주일 이상 약물을 복용하지 않은 건강한 남성의 정맥으로부터 혈액을 채취하였다. 채혈된 혈액 150 g을 15 분간 원심 분리한 후, 상층액(PRP)을 얻고 잔사를 다시 원심분리하여 혈소판 결핍 혈장(platelet poor plasma, PPP)를 분리하였다. 분리된 PRP의 혈소판 수는 세포 카운터를 사용하여 세고, PRP를 PPP로 희석하여 1 ml 당 3x108 개의 혈소판이 포함되도록 희석한 뒤에 실험에 사용하였다.To isolate human platelet rich plasma (PRP), 3.2% sodium citrate was used as an anticoagulant and blood was drawn from a healthy male vein not taking the drug for more than 2 weeks. Platelet poor plasma (PPP) was isolated by centrifuging 150 g of the collected blood for 15 minutes, obtaining supernatant (PRP) and centrifuging the residue again. Platelet counts of isolated PRP were counted using a cell counter, PRP was diluted with PPP and diluted to include 3 × 10 8 platelets per ml.
혈소판의 응집 활성은 혈소판 응집계(lumi-aggregometer, Chrono-Log Co., USA)를 이용하여 흡광도 변화로써 측정하였다. 2 분간 37℃가 되도록 열혼합기(thermomixer)에서 선 배양한 PRP에 울타리콩 추출물을 200 ㎍/ml 농도가 되도록 가하고 7 분간 배양하였다. 배양된 PRP 500 ㎕를 실리콘이 코팅된 혈소판 응집계용 큐벳에 넣고 3 분간 배양하였다. 그런 다음, 혈소판 응집 유발 시료인 콜라겐을 최대 응집을 일으키는 최소 농도인 1-3 ㎍/ml를 가하고, 이 후 6 분간 반응을 관찰하였다. 관찰 결과, 콜라겐만 처리한 대조군의 혈소판 응집 억제율을 0%로 했을 때의 비교값을 표 1(혈소판 응집 억제율(%))에 나타내었다. The platelet aggregation activity was measured by changing the absorbance using a platelet aggregometer (Chromo-Log Co., USA). The fenugreek extract was added to the PRP which had been preincubated in a thermomixer at 37 ° C for 2 minutes to a concentration of 200 μg / ml and cultured for 7 minutes. 500 쨉 l of the cultured PRP was placed in a silicon-coated platelet aggregation cuvette and incubated for 3 minutes. Then, the collagen, which is a platelet aggregation-inducing sample, was added at a concentration of 1-3 μg / ml, which is the maximum concentration causing aggregation, and then the reaction was observed for 6 minutes. As a result of observation, the comparison value when the inhibition rate of platelet aggregation of the control group treated with only collagen was set to 0% is shown in Table 1 (platelet aggregation inhibition rate (%)).
표 1을 참조하면, 물과 10%, 20% 에탄올 추출조건이 다른 조건에 비해 혈소판 응집 억제 효능이 우수한 것으로 나타났으며, 그 중에서도 혈소판 응집 억제 효능이 가장 우수한 것으로 나타난 20% 에탄올 유기산 추출 조건으로 이후 실험을 진행하였다.As shown in Table 1, the extracting conditions of 10% ethanol and 20% ethanol were superior to other conditions in terms of inhibition of platelet aggregation, and 20% ethanol organic acid extracting condition showed the best inhibitory effect on platelet aggregation Then, the experiment was carried out.
[실험예 2] 콩 종류별 사람 혈소판 응집 억제 효능 관찰[Experimental Example 2] Inhibitory effect of human platelet aggregation on soybean
활성이 가장 우수한 것으로 나타난 20% 에탄올 유기산 추출 조건에서, 다양한 콩에 대해 혈소판 응집억제 효능이 나타나는지 살펴보기 위하여, 하기의 실험을 실시하였다. 실험에 사용된 콩은 모두 국산으로 구입하여 사용하였다.The following experiments were conducted to investigate whether platelet aggregation inhibitory effects of various soybean were exhibited under 20% ethanol organic acid extraction conditions in which the activity was most excellent. All of the beans used in the experiment were purchased and used domestically.
거두, 대두, 서리태, 서목태, 울타리콩, 강낭콩, 얼룩 강낭콩, 적두, 청태, 콩나물콩, 황태, 흑태에 대하여 실험을 실시하였다. 대상 콩들은 실시예에 기재된 방법으로 추출하였다. 또한, 항혈소판 응집 억제 효능을 관찰하기 위하여, 실험예 1과 동일한 조건에서 20% 에탄올 유기산 추출물 200 ㎍/ml을 처리하여 실험을 진행하였다. 실험 결과는 하기 표 2(혈소판 응집 억제율(%))에 나타내었다. The experiments were conducted on reed, soybean, seoltei, seomyeutae, fence bean, kidney bean, stain kidney bean, red tongue, cheongtae, bean sprouts bean, Target soybeans were extracted by the method described in the examples. In order to observe anti-platelet aggregation inhibitory effect, the experiment was carried out by treating 200 μg / ml of 20% ethanol organic acid extract under the same condition as Experimental Example 1. The experimental results are shown in Table 2 (platelet aggregation inhibition rate (%)).
표 2를 참조하면, 혈소판 응집 억제 효능은 울타리콩에서 큰 것으로 나타났다.Referring to Table 2, the inhibitory effect on platelet aggregation was found to be large in fence beans.
[실험예 3] 각 콩의 분획별 혈소판응집 억제 활성 [Experimental Example 3] Platelet aggregation inhibitory activity of each soybean fraction
앞의 결과에서 활성이 가장 좋았던 20% 에탄올 유기산 추출 조건으로 실시예에 나타낸 것과 같이 다양한 콩에 대해 HP-20 레진을 이용하여 분획화를 진행하였다. In the above results, various soybean was fractionated using HP-20 resin as shown in the example under 20% ethanol organic acid extraction condition having the best activity.
각 콩의 20% 에탄올 유기산 추출물을 HP-20 레진을 이용하여 분획한 분획물인 물 분획물(이하 HP-20 레진 물 분획물)과 50% 에탄올 분획물 (이하 HP-20 레진 50% 에탄올 분획물)에 대해 실험예 1과 동일한 조건에서, 200 ㎍/ml를 처리하여 실험을 진행하였다. 실험 결과는 하기 표 3(혈소판 응집 억제율(%))에 나타내었다.(Hereinafter referred to as HP-20 resin water fraction) and 50% ethanol fraction (hereinafter referred to as HP-20 resin 50% ethanol fraction), which are fractions obtained by fractionating 20% ethanol organic acid extract of each soybean with HP-20 resin Under the same conditions as in Example 1, 200 쨉 g / ml was treated to conduct experiments. The experimental results are shown in Table 3 (Platelet Aggregation Inhibition Rate (%)).
20% 에탄올 유기산 추출물HP-20 resin fractionation
20% ethanol organic acid extract
표 3을 참조하면, 모든 콩에서 HP-20 레진 물 분획물에 비해서 HP-20 레진 50% 에탄올 분획물의 혈소판 응집 억제 효능이 현저히 우수한 것으로 나타났으며, 활성성분은 대부분 50% 에탄올 분획으로 집중되는 것으로 확인되었다. 이런 활성은 농도 의존적으로 나타났고, 울타리콩의 예를 도 1에 나타내었다.Referring to Table 3, it was found that the 50% ethanol fraction of HP-20 resin significantly inhibited the platelet aggregation inhibition of all soybean compared to the HP-20 resin fraction, and the active ingredient was mainly concentrated in the 50% ethanol fraction . This activity is shown in a concentration dependent manner, and an example of a fence bean is shown in Fig.
[실험예 4] 혈소판응집 자극원별 활성 억제 특이성 관찰 [Experimental Example 4] Specific inhibition of activity inhibition by platelet aggregation stimulating agent
활성이 가장 우수했던 각 콩의 HP-20 레진 50% 에탄올 분획물에서, 혈소판 응집 자극원에 따라 특이적 활성 억제 효능을 보이는지 살펴보기 위한 실험을 실시하였다. Experiments were conducted to investigate whether the 50% ethanol fraction of HP-20 resin, which had the highest activity, exhibited a specific inhibitory effect according to the platelet aggregation stimulus source.
혈관이 손상되면 그 부위의 내피세포층이 파괴되면서 혈액내로 노출되는 콜라겐 (collagen), 혈소판에서 분비하는 ADP(adenosine diphosphate), 트롬빈(thrombin)은 혈소판 응집을 자극하는 원인이 되는 것으로 알려져 있다. 따라서 HP-20 레진 50% 에탄올 분획물이 ADP, 트롬빈 (thrombin) 에 의한 활성 억제 특이성을 가지는지 평가를 실시하였다. 평가는 상기 실험예 1과 동일한 방법으로 실시하였으며, 단지 실험예 1의 콜라겐 대신 ADP, 트롬빈 자극을 가하였다. 그 결과는 표 4에(혈소판 응집 억제율(%)) 나타내었다.It is known that collagen, platelet-derived adenosine diphosphate (ADP), and thrombin, which are exposed to blood as the endothelial cell layer is destroyed when the blood vessel is damaged, are known to cause stimulation of platelet aggregation. Therefore, it was evaluated whether HP-20 resin 50% ethanol fraction had specificity for inhibiting ADP and thrombin activity. The evaluation was carried out in the same manner as in Experimental Example 1, except that ADP and thrombin stimulation were added instead of the collagen of Experimental Example 1. The results are shown in Table 4 (platelet aggregation inhibition rate (%)).
표 4에서 알 수 있는 것처럼, HP-20 레진 50% 에탄올 분획물은 콜라겐으로 유도된 혈소판 응집뿐만 아니라 ADP, 트롬빈 등의 다른 자극제로 유도된 혈소판 응집에 대해서도 억제 효과가 나타났으며 특히 서목태, 울타리콩의 경우가 더 뛰어난 것으로 나타났다.As can be seen in Table 4, the 50% ethanol fraction of HP-20 resin inhibited not only platelet aggregation induced by collagen but also platelet aggregation induced by other stimulants such as ADP and thrombin. In particular, Of the respondents.
[실험예 5] 울타리콩 분획별 사람 혈소판 응집 억제 효능 관찰[Experimental Example 5] Inhibitory effect of human platelet aggregation on fenugreek fraction
활성이 가장 우수했던 울타리콩의 용매별 분획에 따라 어떤 부분이 가장 우수한 활성을 나타나는지 살펴보기 위한 실험을 실시하였다. Experiments were carried out to investigate which fraction had the best activity depending on the fraction of the solvent of the fence bean that had the highest activity.
먼저, 에틸아세테이트, 부탄올 및 물 분획을 상기 실시예 18의 방법으로 제조하였다. 각 분획에 대한 혈소판 응집 억제 효능은, 상기 실험예 1과 동일한 방법을 사용하여, 각 분획별 200 ㎍/ml의 농도로 사용하여 실시하였다. 결과는 표 5(혈소판 응집 억제율 (%))에 나타내었다. First, ethyl acetate, butanol and water fractions were prepared by the method of Example 18 above. The inhibitory effect on platelet aggregation on each fraction was carried out by using the same method as in Experimental Example 1 at a concentration of 200 占 퐂 / ml for each fraction. The results are shown in Table 5 (platelet aggregation inhibition rate (%)).
표 5에서 알 수 있는 것처럼, 혈소판 응집 억제 효능은 부탄올 분획이 가장 우수하였으며, 에틸아세테이트와 물 분획이 유사하였다. 따라서, 울타리콩의 저농도 에탄올 추출물에서의 혈소판 응집 억제 효능은 부탄올 분획에 포함된 활성 성분에 의한 것으로 추측할 수 있다. As shown in Table 5, the inhibitory effect on platelet aggregation was most excellent in the butanol fraction, and the ethyl acetate and water fraction were similar. Therefore, the inhibitory effect on the platelet aggregation in the low concentration ethanol extract of the fence bean can be presumed to be due to the active ingredient contained in the butanol fraction.
[실험예 6] 부탄올 분획물의 혈소판응집 자극원별 활성 억제 특이성 관찰[Experimental Example 6] Specific inhibition of activity inhibition by platelet aggregation stimulants of butanol fraction
울타리콩 부탄올 분획물이 혈소판 응집 자극원별로 활성 억제의 특이성을 가지는지 살펴보기 위하여 ADP, 트롬빈 자극에 대해 상기 실험예 4와 동일한 방법으로 실험을 수행하였고, 시료의 처리 농도는 200 ㎍/ml로 실시하였다. 그 결과는 표 6(혈소판 응집 억제율 (%))에 나타내었다. In order to examine the specificity of the inhibition of the activity of inhibition of platelet aggregation stimulant by fentanyl butanol fractions, experiments were carried out in the same manner as Experimental Example 4 for ADP and thrombin stimulation. The treatment concentration of the sample was 200 μg / ml Respectively. The results are shown in Table 6 (platelet aggregation inhibition rate (%)).
표 6에서 알 수 있듯이, 울타리콩 부탄올 분획물은 콜라겐뿐만 아니라, ADP, 트롬빈 자극에 의한 응집을 모두 억제하는 것을 알 수 있다. As shown in Table 6, it can be seen that the fentanyl butanol fraction inhibits aggregation not only by collagen but also by ADP and thrombin stimulation.
[실험예 7] 분획물에 대한 혈소판응집 후 활성화 물질 분비 억제 효능 관찰[Experimental Example 7] Inhibitory effect on secretion of active substance after platelet aggregation on fractions
각 콩의 HP-20 레진 50% 에탄올 분획물 및 울타리콩 부탄올 분획물이 혈소판 응집시 생성되는 트롬복산 생성을 억제시키는 효과를 살펴보기 위해서, 실험예 1에서 사용한 PRP에 시료를 400 ㎍/ml이 되도록 가하고 37℃에서 10 분간 배양한 후, 콜라겐 5 ㎍/ml을 가하고 6 분간 반응시켰다. EDTA 2 mM, 인도메타신(Indomethacin) 50 μM을 가하여 반응을 종결시키고 2000xg에서 20 분간 원심분리하여 상층액만을 취하여 생성된 트롬복산을 효소면역법을 사용하여 정량하였다. 그 결과는 표 7(트롬복산 생성 억제율 (%))에 나타내었다. To examine the effect of 50% ethanol fraction and fenced soybean butanol fraction of HP-20 resin in each soybean inhibiting the production of thromboxane produced during platelet aggregation, a sample was added to PRP used in Experimental Example 1 so as to have a concentration of 400 μg / ml After incubation at 37 ° C for 10 minutes, 5 μg / ml of collagen was added and allowed to react for 6 minutes.
표 7에서 볼 수 있는 것처럼, 각 콩의 HP-20 레진 50% 에탄올 분획물 및 울타리콩 부탄올 분획물은 혈소판 응집후 생성되는 트롬복산 생성을 억제하는 것으로 관찰되었다.As can be seen in Table 7, the HP-20 resin 50% ethanol fraction and fenced soybean butanol fraction of each soybean inhibited the formation of thromboxane produced after platelet aggregation.
[실험예 8] SD 래트를 이용한 정맥혈전생성 억제 효과 관찰[Experimental Example 8] Observation of inhibitory effect on venous blood formation using SD rats
서리태, 울타리콩, 서목태, 대두 분획물을 실제 섭취하였을 경우 생체 내에서 혈전생성억제 효능이 있는지 알아보기 위한 실험을 실시하였다. 체중 220-250 g의 숫컷 SD (Sprague Dawley) 래트 (rat)를 대상으로 정맥혈전생성능 시험을 실시하였다. In order to investigate the inhibitory effect of threonine, fentanyl, fenugreek, and soybean fractions on the thrombogenesis in vivo, an experiment was conducted. Sprague Dawley male rats weighing 220-250 g were subjected to a venous blood circulation performance test.
SD 래트에 200 mg의 각 콩 분획물을 식염수 (saline)에 용해시켜 10 ml/kg의 용량으로 경구 투여한 뒤, 1 시간 동안 방치하였다. 1 시간 후, 우레탄 (Urethane) 12.5 g/kg을 복강 투여하여 전신 마취시킨 후, 개복하고 후대정맥 (caudal vena cava) 이 잘 보이도록 지방조직을 걷어내었다. 지방조직을 걷어낼 때 주변의 혈관이 손상되지 않도록 주의하였다. 5% FeCl3 용액을 필터페이퍼 (filter paper, 2 mm x 4 mm) 에 3 ㎕를 적셔 후대정맥 위에 5 분간 적용한 후 제거하였다. 30 분 후, 혈전을 함유하고 있는 후대정맥을 12 mm 길이로 결찰한 후 절제하였다. 식염수에 혈전 덩어리를 떼어낸 후 수분은 제거하고 무게를 측정하여 각 시험군을 비교하였고, 그 결과를 도 2에 나타내었다. To the SD rats, 200 mg of each soy fraction was dissolved in saline and orally administered at a dose of 10 ml / kg and left for 1 hour. After 1 hour, Urethane 12.5 g / kg was administered intraperitoneally to the anesthetized group, and the fat tissues were taken out so that the caudal vena cava was visible. When removing the fat tissue, care was taken not to damage the surrounding blood vessels. 3 μl of 5% FeCl 3 solution in filter paper (2 mm x 4 mm) was applied onto the vena cava for 5 minutes and then removed. After 30 minutes, the vena cava containing thrombus was ligated to a length of 12 mm and then resected. After removing the blood clots from the saline solution, the water was removed and the weight was measured. The test groups were compared and the results are shown in FIG.
도 2를 참조하면, 서리태와 울타리콩의 HP-20 레진 50% 에탄올 분획물 200mg/kg을 경구 투여하였을 때, 유의적으로 혈전 생성이 억제되는 것을 알 수 있다. Referring to FIG. 2, it can be seen that the thrombogenesis is significantly inhibited when oral administration of 200 mg / kg of the 50% ethanol fraction of HP-20 resin of foliar soybean and fried soybeans is administered.
또한, 동일한 방법으로 울타리콩 20% 에탄올 유기산 추출물과 HP-20 레진 50% 에탄올 분획물 및 추출시 유기산을 함유하지 않고 추출하여 HP-20 레진으로 분획한 분획물에 대한 비교시험 결과를 도 3에 나타내었다. 도 3을 참조하면, 울타리콩 20% 에탄올 유기산 추출물과 HP-20 레진 50% 에탄올 분획물의 200 mg/kg 투여 시 유의적으로 혈전 생성이 억제되는 것을 알 수 있었다. In addition, the results of a comparative test for the fractions obtained by extracting 20% ethanolic organic acid extract of fence bean, 50% ethanol fraction of HP-20 resin, and extracting organic acid-free fraction and HP-20 resin by the same method are shown in FIG. . 3, it was found that thrombogenesis was significantly inhibited when 200 mg / kg of 20% ethanol organic acid extract of fence bean and 50% ethanol fraction of HP-20 resin were administered.
마지막으로, 동일한 방법으로 울타리콩 20% 에탄올 유기산 추출물을 HP-20 레진 50% 에탄올 분획물 100, 200 mg/kg과 시판되고 있는 항혈전제인 clopidogrel 2.5 mg/kg에 대하여 시험하였다. 그 결과는 도 4에 나타내었다. 이 결과를 참조하면, 음성대조용매 투여군에 비하여 농도의존적으로 혈전 생성이 억제되는 것을 알 수 있으며, 200 mg/kg 투여군은 Clopidogrel 2.5 mg/kg 투여군과 유사한 효능을 나타내는 것을 알 수 있다. 따라서, 울타리콩의 HP-20 레진 50% 에탄올 분획물은 우수한 항혈전제로 작용함을 알 수 있다.Finally, in the same manner, 20% ethanolic organic acid extract of fenugreek was tested against HP-20 resin 50
[실험예 9] SD 래트(rat)로부터 분리한 혈소판 응집 억제 효능 관찰 [Experimental Example 9] Inhibition of Platelet Aggregation by SD Rat
상기에서 효능을 관찰했던 울타리콩 HP-20 레진 50% 에탄올 분획물과 울타리콩의 부탄올 분획물, 또한 식품으로 많이 섭취하는 대두의 HP-20 레진 50% 에탄올 분획물에 대하여 래트 (rat) 로부터 분리한 혈소판의 응집 억제 효과가 있는지 살펴보기 위하여 상기 실험예 1과 동일한 방법으로 자극원인 콜라겐의 농도를 15 ㎍/ml 농도로 실험을 수행하였다. 각 콩 분획의 처리 농도는 400 ㎍/ml 이었으며 그 결과는 표 8(혈소판 응집 억제율 (%))에 나타내었다.The 50% ethanol fraction of the fence bean HP-20 resin, the butanol fraction of the fence bean, and the 50% ethanol fraction of the soybean HP-20 resin consumed as food were observed in the above, and the platelets isolated from the rat In order to examine whether there is an aggregation inhibiting effect, experiments were carried out in the same manner as in Experimental Example 1, at a concentration of 15 / / ml of stimulus collagen. The treatment concentration of each soybean fraction was 400 / / ml, and the results are shown in Table 8 (Platelet aggregation inhibition rate (%)).
표 8에서 알 수 있는 것처럼, 울타리콩의 HP-20 레진 50% 에탄올 분획물 뿐만 아니라 부탄올 분획물과 대두의 HP-20 레진 50% 에탄올 분획물 모두 래트에서 혈소판의 응집 억제 효능이 매우 우수하였다. As can be seen in Table 8, the inhibition of aggregation of platelets in rat 50% Ethanol fraction, butanol fraction and HP-20 resin 50% Ethanol fraction of soybean HP-20 resin were excellent.
Claims (8)
시트르산을 이용하여 추출한 유색콩 추출물을 함유하고, 상기 유색콩은 울타리콩인 혈소판 응집 억제용 조성물.Aqueous ethanol solution; And
Wherein the colored soybean is a fence bean, wherein the colored bean extract is extracted with citric acid.
시트르산을 이용하여 추출한 유색콩 추출물의 분획물을 함유하고, 상기 유색콩은 울타리콩이며, 상기 분획물은 상기 추출물을 폴리스티렌(polystylene)과 벤젠(benzene)의 중합체를 포함하는 합성 흡착제 컬럼을 사용하여 에탄올로 분획화한 에탄올 분획물인 혈소판 응집 억제용 조성물.Aqueous ethanol solution; And
Wherein the colored soybean is a fence bean and the fraction is extracted with ethanol using a synthetic adsorbent column comprising a polymer of polystyrene and benzene A fractionated platelet aggregation inhibiting composition which is a fractionated ethanol fraction.
에탄올 수용액의 농도는 1 내지 40% (v/v)인 혈소판 응집 억제용 조성물.3. The method according to claim 1 or 2,
Wherein the concentration of the aqueous ethanol solution is 1 to 40% (v / v).
에탄올 수용액은 5 내지 25% (v/v) 농도인 혈소판 응집 억제용 조성물 3. The method according to claim 1 or 2,
The aqueous ethanol solution is a 5 to 25% (v / v) concentration platelet aggregation inhibiting composition
상기 혈관 질환은, 혈전에 의한 뇌졸증, 혈전에 의한 동맥경화, 혈전에 의한 협심증, 혈전에 의한 심근경색, 혈전에 의한 고혈압 또는 혈전에 의한 편두통인 혈전에 의한 혈관 질환의 경감 또는 치료용 약학 조성물.6. The method of claim 5,
The aforementioned vascular disease is a pharmaceutical composition for alleviating or treating vascular diseases caused by thrombotic stroke, thrombotic arteriosclerosis, thrombotic angina, thrombotic myocardial infarction, thrombotic hypertension, or thrombotic migraine caused by thrombus.
상기 혈관 질환은, 혈전에 의한 뇌졸증, 혈전에 의한 동맥경화, 혈전에 의한 협심증, 혈전에 의한 심근경색, 혈전에 의한 고혈압 또는 혈전에 의한 편두통인 혈전에 의한 혈관 질환의 예방 또는 완화용 건강식품 조성물.8. The method of claim 7,
The vascular diseases include health food compositions for preventing or alleviating vascular diseases caused by thrombosis caused by thrombosis, thrombus-induced arteriosclerosis, thrombotic angina, thrombus-induced myocardial infarction, thrombotic hypertension, or thrombus .
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