KR100324254B1 - Mushroom extract with thrombolytic activity - Google Patents
Mushroom extract with thrombolytic activity Download PDFInfo
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- KR100324254B1 KR100324254B1 KR1020010037964A KR20010037964A KR100324254B1 KR 100324254 B1 KR100324254 B1 KR 100324254B1 KR 1020010037964 A KR1020010037964 A KR 1020010037964A KR 20010037964 A KR20010037964 A KR 20010037964A KR 100324254 B1 KR100324254 B1 KR 100324254B1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
- A23L31/10—Yeasts or derivatives thereof
- A23L31/15—Extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
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- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
본 발명은 혈전용해능을 갖는 버섯 추출물에 관한 것으로, 보다 상세하게는 혈전용해효소를 함유하고 있어 혈전 관련 질환의 치료제 및 건강 보조 식품으로 유용하게 사용될 수 있는, 혈전용해능을 가진 3종의 균류(버섯류), 즉 구름버섯(Coriolus versicolor), 모래밭버섯(Pisolithus tinctorius) 및 장식솔버섯(Tricholomopsis decora)의 추출물에 관한 것이다.The present invention relates to a mushroom extract having a thrombolytic ability, more specifically, three kinds of fungi with thrombolytic activity, which contain thrombolytic enzymes, which can be usefully used as a therapeutic agent and health supplement for thrombosis related diseases. (Mushrooms), or cloud mushrooms (Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decora)of Relates to an extract.
Description
본 발명은 혈전용해효소를 함유하고 있어 혈전 관련 질환의 치료제 및 건강보조 식품으로 유용하게 사용될 수 있는, 혈전용해능을 가진 3종의 균류(버섯류), 즉 구름버섯(Coriolus versicolor), 모래밭버섯(Pisolithus tinctorius) 및 장식솔버섯(Tricholomopsis decora)의 추출물에 관한 것이다.The present invention contains three types of fungi (mushrooms) having thrombolytic activity, that is, cloud mushrooms, which can be usefully used as a therapeutic agent and health supplement food for thrombosis-related diseases.Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decora)of Relates to an extract.
혈관이 손상을 받아 출혈이 일어나면 혈액이 응고되는 체내 방어시스템이 작동하게 되는데 이 과정은 활성화된 트롬빈(thrombin)에 의해 피브리노겐 (fibrinogen)이 피브린(fibrin)으로 전환됨으로써 유도된다. 이렇게 생성된 혈전 (fibrin clots)은 상처 회복 후 플라스민과 같은 혈전용해효소 (fibrinolytic enzyme)에 의해 용해되는데, 만약 생성된 혈전이 체내에 과도하게 축적되거나 혈전 용해 기작이 원활하게 작동하지 못할 경우에는 혈전증(thrombosis)이 유발되어 인체에 치명적인 손상을 줄 수도 있다. 예를 들어, 뇌혈관에 혈전이 생성되는 뇌혈전증이 일어나면 반신불수가 될 수 있고, 뇌의 미세 혈관이 파열되어 뇌출혈이 일어나면 생명에 치명적인 손상을 일으킬 수 있으며, 혈전에 의해 심장혈관이 막히면 심부전증이나 심장마비가 유발될 수 있다.When the blood vessels are damaged and the bleeding occurs, the body's defense system that coagulates blood is activated. This process is induced by the conversion of fibrinogen to fibrin by activated thrombin. Fibrin clots are solubilized by fibrinolytic enzymes, such as plasmin, after wound healing. If the resulting clots accumulate excessively in the body or the thrombolytic mechanism does not work well, Thrombosis can be triggered and cause fatal damage to the body. For example, cerebellar thrombosis, a blood clot that forms in the cerebrovascular vessels, can lead to an incapacitated stroke, and rupture of the blood vessels in the brain can lead to life-threatening damage. Paralysis may be caused.
현재까지 혈전의 제거를 위한 연구가 계속 진행되어 왔는데, 특히 스트렙토키나제(streptokinase), 유로키나제(urokinase), t-PA(tissue-type plasminogen activator)와 같은 플라스미노겐 활성인자(plasminogen activator)를 정맥주사하여 혈전용해계(fibrinolytic system)를 활성화하는 치료법이 가장 보편적인 방법으로 사용되고 있다. 그러나, 상기 물질들은 전신출혈(systemic haemorrhage)이 유발되는 등 부작용이 크며 가격이 매우 비쌀 뿐만 아니라, 유로키나제를 제외하고는 경구투여가 불가능한 실정이다. 따라서 보다 경제적이고 경구투여가 가능한 혈전증 치료제에 대한 연구가 다양하게 시도되고 있으며, 직접 경구 투여하거나 경구 투여와 혈관 주사를 병용함으로써 혈액 내의 혈전용해능을 증가시킬 수 있는 제제들이 개발되고 있다. 현재, 경구투여가 가능한 혈전제로는 지렁이(Lumbricus lubellus)로부터 분리된 6가지의 혈전용해효소가 알려져 있다.To date, studies on the elimination of blood clots have been conducted. In particular, plasminogen activators such as streptokinase, urokinase, and t-PA (tissue-type plasminogen activator) are injected intravenously. Therapies that activate the fibrinolytic system are the most common methods. However, these substances have a large side effect such as causing systemic haemorrhage and are very expensive, and are not orally administered except for urokinase. Therefore, various economic and orally administrable thrombosis therapeutics have been tried in various ways, and agents that can increase the thrombolytic ability in the blood by direct oral administration or in combination with oral administration and vascular injection have been developed. Currently, six thrombolytic enzymes isolated from the worm ( Lumbricus lubellus ) are known as orally administrable thrombicides .
한편, 광범위한 탐색 기술을 활용하여 자연계의 여러 생물에 존재하는 혈전 용해성 물질을 새로운 혈전 용해제로 개발려는 많은 시도가 이루어졌다.On the other hand, many attempts have been made to develop thrombolytic substances present in various organisms as natural thrombolytic agents using a wide range of screening techniques.
식물의 경우, 은행잎에서 추출한 성분이 혈류 순환을 원활히 해주는 것으로 알려져 이를 사용한 제품들이 개발되어 상품화되었고, 동물에서도 흡혈박쥐의 침샘조직으로부터 분리한 신규 플라스미노겐 활성인자(plasminogen activator) (Gardell, S.J.,et al., Circulation84, 244-253, 1991)를 비롯하여, 뱀독에서 분리된 살모나제(salmonase)(Chung, K.H. and Kim, D.S.,Thromb. Haemost.65, 953, 1991〕, 거머리에서 분리된 헤멘틴(hementin), 및 지네에서 분리된 피브린 분해효소 (fibrinolytic enzyme)〔Kim, K.Y., Chung, K.H., and Moon, H.M.,Thromb. Haemost.69, 839, 1993) 등이 혈전용해 작용이 있는 것으로 알려 있다.In the case of plants, products extracted from ginkgo biloba are known to facilitate blood circulation, and products using them have been developed and commercialized, and new plasminogen activator (Gardell, SJ,) isolated from salivary glands of vampire bats in animals. et al., Circulation 84, 244-253, 1991), as well as salmonase isolated from snake venom (Chung, KH and Kim, DS, Thromb. Haemost. 65, 953, 1991), isolated from leeches. Hementin and fibrinolytic enzyme isolated from centipedes (Kim, KY, Chung, KH, and Moon, HM, Thromb. Haemost. 69, 839, 1993) It is known.
또한, 미생물에서도 여러가지 혈전 용해 물질이 확인되었는데, 스트렙토키나제가 대료적인 예이며, 그밖에도 1948년에 보고된 스태필로키나제, 아스퍼질러스 오리제에서 발견된 CA-7, 브리노라제, 피브리노리신, 바실러스 속 균주에서 발견된 피브린 분해효소 등이 혈전 용해제로 보고되어 있다.In addition, various thrombolytic substances have been identified in microorganisms, such as streptokinase, as well as CA-7, brinorase and fibrinori, which were found in staphylokinase and aspergillus aurise, reported in 1948. God, Bacillus Fibrin degrading enzymes found in strains have been reported as thrombolytic agents.
이 외에도 한국과 일본을 중심으로 한 발효식품에서의 혈전용해효소에 대한 연구는 직접 섭취가 가능한 식품을 대상으로 한다는 점에서 주목할 만하다. 일본의 전통대두 발효식품인 낫토(natto)와 절임식품인 시오카라(Shiokara)에서 혈전용해효소가 존재함이 알려져 생산 균주를 분리하고 효소를 정제하였는데, 이 중 낫토로부터 분리된 낫토키나제(nattokinase)라는 효소는 경구 투여하는 경우에도 생체내의 혈전용해능을 높일 수 있다는 보고가 있어 현재 일본에서는 혈전 용해능을 지닌 건강식품으로서 낫토의 판매량이 급증하고 있다(Sumi, H.,et al.,Experimentia43, 1110, 1997 ; 須見洋行,Bioindustry7, 725, 1990). 우리나라에서도 일본의 낫토와 유사한 콩을 원료로 한 전통발효식품인 청국장에서 혈전용해능을 가진 바실러스(Bacillus)균주를 분리 및 정제하였다. 또한, 본 발명자들은 청국장과 마찬가지로 우리나라의 전통대두 발효식품인 된장으로부터 낫토와 청국장에서 분리한 균주에 비해 2~3배 이상의 높은 혈전용해효소를 생산하는 균주를 분리하고 이들 균주가 생산하는 혈전용해 단백질을 발명하여 특허 출원한 바 있다 (대한민국 특허출원 제 96-75523호).In addition, the research on thrombolytic enzymes in fermented foods, especially in Korea and Japan, is noteworthy in that they focus on foods that can be consumed directly. Natto (natto), a traditional Japanese soybean fermented food, and shiokara (pickled food) were known to have thrombolytic enzymes, and production strains were isolated and enzymes were purified. Among them, nattokinase isolated from natto. Has been reported to increase thrombolytic capacity in vivo even when administered orally. Currently, the sales of natto are rapidly increasing in Japan as a health food with thrombolytic ability (Sumi, H., et al. , Experimentia 43 , 1110, 1997; Bioindustry 7, 725, 1990). In Korea, Bacillus strains with thrombolytic activity were isolated and purified from Chungkookjang, a traditional fermented food made from soybeans similar to Japanese natto. In addition, the present inventors isolate strains that produce thrombolytic enzymes two to three times higher than strains isolated from natto and cheonggukjang from doenjang, a traditional soybean fermented food in Korea, as well as cheonggukjang and the thrombolytic proteins produced by these strains. Has been invented and applied for a patent (Korean Patent Application No. 96-75523).
한편, 버섯은 간단한 요리로 직접 섭취가 가능하며 또한 버섯을 이용한 제품은 부작용이 적고 경구투여가 가능하므로 버섯에 대한 많은 연구가 진행되어 왔다. 그러나, 이는 주로 메탈로엔도펩티다제(metalloendopeptidase)와 항진균 활성에 관한 것으로, 혈전용해에 관한 연구는 보고된 바가 없다.On the other hand, mushrooms can be directly ingested by simple cooking, and since the product using mushrooms has fewer side effects and oral administration, much research has been conducted on mushrooms. However, it is mainly related to metalloendopeptidase and antifungal activity, and no studies on thrombolysis have been reported.
버섯은 예로부터 식용 및 약용으로 널리 이용되어 왔으며 그 종류 또한 다양하다. 현재 한국산 버섯은 990여종이 분류되어 있으며, 이 중 100여종이 식용 가능한 버섯으로 확인되어 있다. 또한 버섯의 경우 균(fungi) 자체의 직접 섭취가 가능하므로 혈전용해효과 면에서 매우 유리하다고 할 수 있다.Mushrooms have been widely used for food and medicinal purposes since ancient times. Currently, 990 kinds of Korean mushrooms are classified, and about 100 of them are identified as edible mushrooms. In addition, mushrooms can be said to be very advantageous in terms of the thrombolytic effect because the fungi themselves can be directly ingested.
이에 본 발명자들은 한국산 버섯 중에서 혈전용해능이 우수한 균주 및 혈전분해효소를 발견하고자 노력한 결과, 구름버섯(Coriolus versicolor), 모래밭버섯(Pisolithus tinctorius) 및 장식솔버섯(Tricholomopsis decora)의 추출물로부터 혈전용해능이 탁월한 혈전용해성 효소 단백질을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors tried to find strains and thrombolytic enzymes excellent in thrombolytic activity in Korean mushrooms, cloud mushrooms (Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decoraThe present invention was completed by identifying the thrombolytic enzyme protein having excellent thrombolytic activity from the extract of N.
본 발명의 목적은 혈전용해능을 갖는 버섯 추출물을 제공하는 것이다.An object of the present invention is to provide a mushroom extract having a thrombolytic ability.
도 1은 피브린 플레이트 측정방법(fibrin plate assay)에 따라 플라스민과 모래밭버섯(Pisolithus tinctorius)의 혈전용해능을 측정한 결과를 나타낸 것이고, Figure 1 shows the results of measuring the thrombolytic activity of the plasmin and sand mushroom ( Pisolithus tinctorius ) according to the fibrin plate assay (fibrin plate assay),
A : 모래밭버섯 추출용액 B : 플라스민 (1.0 NHI 유닛/㎖)A: Sand mushroom extract solution B: Plasmin (1.0 NHI unit / ml)
도 2는 피브린을 기질로 사용한 12% SDS-피브린 자이모그래피(zymography) 활성측정법을 통해 버섯 추출액 내의 혈전용해 효소 단백질을 확인한 결과를 나타낸 것이다. Figure 2 shows the results of identifying the thrombolytic enzyme protein in the mushroom extract through the 12% SDS-fibrin zymography activity assay using fibrin as a substrate.
M : 표준 단백질 a : 구름버섯(Coriolus versicolor)M: Standard Protein a: Cloud Mushroom ( Coriolus versicolor )
b : 모래밭버섯 c : 장식솔버섯(Tricholomopsis decora)b: Sand mushroom c: Tricholomopsis decora
본 발명은 혈전용해능을 갖는 버섯 추출물을 제공한다. 이 때 버섯은 구름버섯(Coriolus versicolor), 모래밭버섯(Pisolithus tinctorius)및 장식솔버섯 (Tricholomopsis decora)인 것이 바람직하다.The present invention provides a mushroom extract having a thrombolytic ability. In this case, the mushrooms are preferably Coriolus versicolor , Pisolithus tinctorius, and Tricholomopsis decora .
또한, 본 발명은 상기 버섯 추출물로부터 분리되고, 분자량이 각각 54 kDa, 61 kDa 및 84 kDa인 혈전용해성 효소 단백질을 제공한다.The present invention also provides a thrombolytic enzyme protein isolated from the mushroom extract and having a molecular weight of 54 kDa, 61 kDa and 84 kDa, respectively.
본 발명은 또한 상기 혈전용해성 효소 단백질의 일부 또는 전부를 함유하는버섯 추출물을 유효 성분으로 하는 혈전 관련 질환 치료제용 약학적 조성물 및 건강보조식품을 제공한다. 이 때, 혈전 관련 질환 치료제는 경구 투여가 가능하며, 관상동맥 질환, 맥혈전증 또는 뇌혈관질환을 가진 환자에게 적용할 수 있다.The present invention also provides a pharmaceutical composition for treating thrombosis-related diseases, and a dietary supplement, comprising as an active ingredient a mushroom extract containing some or all of the thrombolytic enzyme protein. At this time, the thrombotic disease treatment agent can be administered orally, and can be applied to patients with coronary artery disease, thrombosis or cerebrovascular disease.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 혈전용해능을 갖는 버섯 추출물 및 이로부터 분리한 54 kDa, 61 kDa, 84 kDa의 효소 단백질을 제공한다.The present invention provides a mushroom extract having a thrombolytic ability and an enzyme protein of 54 kDa, 61 kDa, 84 kDa isolated therefrom.
본 발명에 사용된 50여종의 한국산 버섯은 전국각지에서 수집하여 실험에 사용하였으며, 이 중 혈전용해능을 갖는 3종의 균류(버섯류), 즉 구름버섯, 모래밭버섯 및 장식솔버섯을 선발하여 혈전용해효소 활성을 측정하고, 기질 특이성을 조사하였다.About 50 kinds of Korean mushrooms used in the present invention were collected from all over the country and used in experiments. Among them, three kinds of fungi (mushrooms) having thrombolytic ability, namely cloud mushrooms, sand mushrooms and decorative brush mushrooms, were selected for thrombus Lysase activity was measured and substrate specificity was investigated.
본 발명에서는 버섯 추출물의 혈전용해활성을 측정하기 위해, 추출물의 피브린 플레이트 용해 면적을 측정함으로써 혈전 용해의 우수성을 비교하였다. 이 때, 대조군으로는 혈전 용해제로서 잘 알려진 플라스민 1.0 NIH 유닛(unit)/㎖를 사용하였으며, 모래밭버섯의 추출물이 대조군인 플라스민보다 4.71배 높은 혈전 용해능을 나타내었다(도 1). 이는 한국과 일본의 전통대두 발효식품인 청국장(1.84 플라스민 유닛)과 낫토(1.58 플라스민 유닛)에서 분리한 바실러스(Bacillus)균주의 활성보다 훨씬 높은 활성에 해당한다. 또한 본 발명자들에 의해 한국 전통재래식 된장에서 분리한 혈전 용해 균주들의 활성(2-5 플라스민 유닛)과는 비슷한 활성을 나타내었다(대한민국 특허출원 제 96-75523 참조). 그러나, 바실러스 균주들과는 달리 버섯의 경우 직접 섭취가 가능하다는 점에서 더욱 주목할 만하다.In the present invention, to measure the thrombolytic activity of the mushroom extract, the superiority of thrombolytic dissolution was compared by measuring the fibrin plate dissolution area of the extract. At this time, plasmin 1.0 NIH unit (unit) / ml well-known as a thrombolytic agent was used as a control, and the extract of the sand mushroom showed 4.71 times higher thrombolytic ability than plasmin as a control ( FIG. 1 ). This corresponds to much higher activity than that of Bacillus strains isolated from Chungkookjang (1.84 plasmin unit) and natto (1.58 plasmin unit). In addition, the present inventors showed similar activity to the activity (2-5 plasmin units) of thrombolytic strains isolated from Korean traditional doenjang (see Korean Patent Application No. 96-75523). However, unlike Bacillus strains, it is more noteworthy that the mushroom can be directly ingested.
또한, 본 발명의 버섯 추출물 내의 혈전분해능을 갖는 것으로 추정되는 효소 단백질을 확인하기 위해, 배양 상등액에 암모늄 설페이트를 처리하여 DEAE-세파실 칼럼 등을 실시함으로써 새로운 효소 단백질을 분리 정제하였으며, 이로부터 약 54 kDa, 61 kDa 및 84 kDa의 분자량을 갖는 효소 단백질을 얻을 수 있었다.In addition, in order to identify enzyme proteins that are presumed to have thrombolytic activity in the mushroom extracts of the present invention, new enzyme proteins were isolated and purified by subjecting the culture supernatant to a DEAE-Sephasil column by treating with ammonium sulfate. Enzyme proteins with molecular weights of 54 kDa, 61 kDa and 84 kDa could be obtained.
상기에서 분리 정제한 효소 단백질의 활성을 확인하기 위해, 버섯의 추출물을 원심 분리하여 균체를 제거하고 그 상등액을 얻은 다음, 이를 SDS-피브린 자이모그래피(SDS-fibrin zymography) 활성확인법으로 분석하였다. 그 결과, 상기 효소 단백질이 혈전 용해 활성을 가짐을 확인할 수 있었다(도 2).In order to confirm the activity of the enzyme protein purified from the above, the extract of the mushroom was centrifuged to remove the cells and obtained the supernatant, which was analyzed by SDS-fibrin zymography activity confirmation method. As a result, it was confirmed that the enzyme protein has thrombolytic activity ( FIG. 2 ).
또한, 본 발명의 효소 단백질을 함유하는 버섯 추출물이 임상적으로 사용하기에 적합한가를 확인하기 위해 그 용혈활성(hemolytic activity)을 검색한 결과, 본 발명의 추출물은 전혀 용혈 활성을 나타내지 않았다.In addition, as a result of searching for hemolytic activity to confirm whether the mushroom extract containing the enzyme protein of the present invention is suitable for clinical use, the extract of the present invention showed no hemolytic activity at all.
기존의 혈전 용해제는 경구투여가 거의 불가능한 반면, 본 발명의 버섯 추출물은 임상투여시에 경구 또는 비경구 투여가 모두 가능하다는 이점을 갖는다.While conventional thrombolytic agents are almost impossible to orally administer, the mushroom extract of the present invention has the advantage that both oral or parenteral administration is possible at the time of clinical administration.
즉, 본 발명의 버섯 추출물은 실제 임상투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제재화할 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 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를들면, 전분, 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose) 또는 락토오스 (Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 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. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜, 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 사용 될 수 있다.That is, the mushroom extract of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used Or using excipients. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which form at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 버섯 추출물의 유효용량은 10∼1000 mg/kg 이고, 바람직하기로는 50∼100 mg/kg 이다.The effective dose of the mushroom extract of the present invention is 10 to 1000 mg / kg, preferably 50 to 100 mg / kg.
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
하기 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
<실시예 1> 버섯 추출물의 제조Example 1 Preparation of Mushroom Extract
본 발명에 사용된 50여종의 한국산 버섯은 전국 각지에서 수집하여 실험에사용하였고, 피브린 플레이트(fibrin plate) 제조에 사용된 피브리노겐 (fibrinogen), 트롬빈(thrombin), 아가로즈(agarose) 및 대조구로 사용된 플라스민과 플라스민에 대한 기질 N-p-Tosyl-Gly-Pro-Lys p-니트로아닐라이드 (nitroanilide) 등은 시그마사 제품을 구입하여 사용하였으며, 그 외 사용된 기타 시약들은 특급시약을 사용하였다.About 50 kinds of Korean mushrooms used in the present invention were collected from all over the country and used in experiments, and used as fibrinogen, thrombin, agarose and control used in the manufacture of fibrin plates. The prepared plasmin and the substrate for plasmin, Np-Tosyl-Gly-Pro-Lys p-nitroanilide, were purchased from Sigma Co., and other reagents were used as a special reagent.
분류된 50여종의 버섯을 100 g의 동일 무게를 80 % 메탄올에 충분히 잠길 정도로 침적하여 한달 간 실온에서 방치한 다음 추출액을 여과하였다. 여액을 감압 농축하여 분말화 한 후 동일량의 메탄올에 현탁시켜 재여과하였다. 여과한 여액을 5ml가 되도록 농축시켜 버섯 추출물을 제조하였다.About 50 kinds of the classified mushrooms were immersed so that 100 g of the same weight was sufficiently submerged in 80% methanol, and left at room temperature for one month, and then the extract was filtered. The filtrate was concentrated under reduced pressure, powdered and then suspended in the same amount of methanol and filtered again. The filtrate was concentrated to 5 ml to prepare a mushroom extract.
<실시예 2> 혈전용해 활성의 갖는 버섯의 선발 및 활성측정Example 2 Selection and Activity Measurement of Mushrooms Having Thrombolytic Activity
50 mM 인산완충용액(pH 7.4, 0.15M NaCl 포함)에 피브리노겐을 최종농도 0.3 % 가 되도록 완전히 용해시킨 다음, 이 용액 5 ml에 동량의 2% 아가로즈(agarose)를 첨가하여 혼합하였다. 혼합한 용액에 트롬빈(100 NIH 유닛/ml) 0.1 ml을 첨가하여 충분히 혼합한 후 즉시 플레이트에 붓고 1시간 동안 실온에서 고화시켜 피브린 플레이트를 제조하였다. 활성 측정을 위해 피브린 플레이트에 파스테르 파이펫(pasteur pippet)으로 지름 5 mm의 구멍을 만들어 상기 실시예 1로부터 제조한 50종의 버섯 추출액 20 μl씩을 주입하고 37 ℃에서 12시간 반응시킨 다음 생성된 투명환의 면적을 계산하였다. 이 때 대조구로서는 정제된 혈전 용해효소인 플라스민(1.0 NIH unit/ml)을 사용하였다.Fibrinogen was completely dissolved in 50 mM phosphate buffer solution (pH 7.4, containing 0.15M NaCl) to a final concentration of 0.3%, and then 5 ml of this solution was mixed with an equal amount of 2% agarose. Fibrin plates were prepared by adding 0.1 ml of thrombin (100 NIH units / ml) to the mixed solution, thoroughly mixing, pouring into plates immediately, and solidifying at room temperature for 1 hour. For measuring activity, a 5 mm diameter hole was made with a pasteur pippet in a fibrin plate, and 20 µl of 50 mushroom extracts prepared in Example 1 were injected and reacted at 37 ° C. for 12 hours. The area of the transparent ring was calculated. At this time, plasmin (1.0 NIH unit / ml), which is a purified thrombolytic enzyme, was used as a control.
그 결과, 구름버섯, 모래밭버섯 및 장식솔버섯 추출액이 혈전용해활성을 나타냄을 알 수 있었다. 이와 같이 선발된 3종의 버섯 추출액의 피브린 플레이트 상에서의 용해면적을 측정하고, 이를 플라스민(1.0 NIH unit/ml)의 용해면적에 대한 상대적인 비율로 환산함으로써 혈전용해능을 산출하였다(표 1).As a result, cloud mushrooms, sand mushrooms And decorative sol mushroom extract showed thrombolytic activity. The thrombolytic ability was calculated by measuring the dissolution area on the fibrin plate of the three mushroom extracts thus selected and converting it in a relative ratio with respect to the dissolution area of plasmin (1.0 NIH unit / ml) (Table 1). .
표 1의 결과로부터 알 수 있듯이, 상기 3종의 버섯 모두가 플라스민 1.0 NIH 유닛 보다 3~4배의 높은 활성을 보였으며, 그 중 모래밭버섯이 가장 높은 활성(4.71 플라스민 unit)을 나타내었다(도 1참조). 이러한 버섯 추출액의 혈전분해활성은 플라스민보다 높은 활성을 보이는 것으로 알려진 바실러스 속 균주의 경우보다도 2-3배 높은 활성이다.As can be seen from the results of Table 1, all three mushrooms showed three to four times higher activity than plasmin 1.0 NIH unit, and among them, sand mushroom showed the highest activity (4.71 plasmin unit). (See FIG. 1 ). The thrombolytic activity of these mushroom extracts is 2-3 times higher than that of the Bacillus strain known to exhibit higher activity than plasmin.
<실시예 3> 혈전용해능 기질특이성 조사<Example 3> Thrombolytic substrate specificity investigation
일반적으로 피브린 플레이트는 플라스민 뿐만 아니라 다른 프로테아제 (protease)에 의해서도 쉽게 분해될 수 있어 플라스민에 대한 특이성이 낮다. 따라서, 플라스민에 대한 특이 기질 (chromogenic substrate, T-6140)인 N-p-Tosyl-Gly-Pro-Lys p-니트로아닐라이드(nitroanilide)를 사용하여 각 버섯 추출액의 혈전분해효소에 대한 기질 특이성을 조사하였다.In general, fibrin plates can be easily degraded not only by plasmin but also by other proteases, thus having low specificity for plasmin. Therefore, Np-Tosyl-Gly-Pro-Lys p-nitroanilide, a chromogenic substrate (T-6140) for plasmin, was used to investigate the substrate specificity for thrombolytic enzymes in each mushroom extract. It was.
0.5 mM 기질용액 0.2 ml(20 mM 인산완충용액, pH 7.0)과 상기 실시예 2로부터 동정된 3종 버섯 추출액 (3 ㎍/0.2 ml)을 37 ℃ 수조에 넣고 1분간 반응시킨 후 50 % 초산용액 0.1 ml을 첨가하여 반응을 중단시켰다. 대조구로서는 정제된 플라스민(1.0 NIH unit/0.2 ml)을 사용하였고, 공시험물(blank)은 추출액 0.2 ml와 인산완충용액 0.2 ml를 측정온도에서 1분간 방치한 후 50 % 초산용액 0.1 ml을 혼합하여 제조하였다. 이렇게 제조된 각각의 시료와 공시험물을 405 nm에서 흡광도를 측정하여 플라스민에 대한 상대적 효소활성을 결정하였다(표 2).0.2 ml of 0.5 mM substrate solution (20 mM phosphate buffer solution, pH 7.0) and three mushroom extracts identified in Example 2 (3 ㎍ / 0.2 ml) were added to a 37 ° C. water bath for 1 minute, followed by 50% acetic acid solution. The reaction was stopped by adding 0.1 ml. As a control, purified plasmin (1.0 NIH unit / 0.2 ml) was used, and blank (blank) was mixed with 0.2 ml of extract and 0.2 ml of phosphate buffer solution at a measuring temperature for 1 minute, and then mixed with 0.1 ml of 50% acetic acid solution. It was prepared by. Each of the samples and blanks thus prepared were measured for absorbance at 405 nm to determine relative enzyme activity against plasmin (Table 2).
표 2의 결과로부터 알 수 있듯이, 구름버섯은 0.24 플라스민 유닛으로 아주 낮은 특이성을 보였고, 모래밭버섯의 경우 0.99 플라스민 유닛의 높은 특이성을 나타내었다. 특히 식용되고 있는 장식솔버섯의 경우 1.32 플라스민 유닛의 가장 높은 피브린 다량체에 대한 효소 특이성을 보였다.As can be seen from the results of Table 2, the cloud mushroom showed a very low specificity of 0.24 plasmin units, and the sand mushroom showed a high specificity of 0.99 plasmin units. In particular, decorative edible mushrooms showed enzymatic specificity for the highest fibrin multimer of 1.32 plasmin units.
<실시예 4> 혈전용해능을 갖는 효소 단백질의 분리 및 정제Example 4 Isolation and Purification of Enzyme Protein Having Thrombolytic Activity
*실시예 2에서 동정한 3종 버섯 추출물을 10,000 xg 에서 20분간 원심 분리하여 상등액을 취하고 여기에 암모늄 설페이트를 처리하였다. 이렇게 얻어진 단백질 침전물을 탈염시킨 후 DEAE-세파셀(DEAE-Sephacel) 컬럼(시그마제품, 미국)을 통과시켜 새로운 효소 단백질을 분리 정제하고, 표준 단백질과 함께 SDS-PAGE 전기영동을 실시하여 상기 효소 단백질이 분리 정제되었음을 확인하였다.* The three mushroom extracts identified in Example 2 were centrifuged at 10,000 xg for 20 minutes to take a supernatant and treated with ammonium sulfate. After desalting the protein precipitate thus obtained, it was passed through a DEAE-Sephacel column (Sigma, USA) to isolate and purify the new enzyme protein, and subjected to SDS-PAGE electrophoresis with the standard protein to carry out the enzyme protein. It was confirmed that this was purified by separation.
<실시예 5> 혈전용해능을 갖는 효소 단백질의 확인Example 5 Identification of Enzyme Protein Having Thrombolytic Capacity
피브린을 기질로 사용하는 SDS-피브린 자이모그래피(SDS-fibrin zymography) 활성 확인법에 따라 상기 실시예 4로부터 분리 정제된 효소 단백질의 활성 및 분자량을 확인하였다. 피브리노겐의 농도가 0.12 %(w/v)가 되도록 폴리아크릴아마이드 (polyacrylamide) 용액에 혼합한 후 즉시 트롬빈(1.0 NIH unit)을 첨가하여 제조한 피브린-폴리아크릴아마이드 겔 (fibrin-polyacrylamide gel)에서 실시하였고, 단백질 정량은 소 혈청 알부민 (bovine serum albumin)을 표준으로 로우리(Lowry)의 방법에 따라 수행하였다.The activity and molecular weight of the enzyme protein separated and purified from Example 4 were confirmed according to SDS-fibrin zymography activity confirmation method using fibrin as a substrate. The mixture was mixed with a polyacrylamide solution so that the concentration of fibrinogen was 0.12% (w / v), followed by fibrin-polyacrylamide gel prepared by adding thrombin (1.0 NIH unit) immediately. Protein quantification was performed according to Lowry's method using bovine serum albumin.
상기 분리 및 정제된 효소 단백질 분획 20 ng을 각 레인(lane)에 로딩(loading)한 후 10 mA의 일정한 전류를 걸어 전기영동을 실시하였다. SDS에 의해 불활성화된 효소를 재활성화하기 위하여 겔을 2.5 % 트리톤(Triton) X-100를 포함한 트리스(Tris) 완충용액(50 mM, pH 7.4)에서 30분간 방치하여 SDS를 제거한 후, 다시 증류수로 트리톤 X-100을 제거하였다. 분획된 단백질의 혈전 용해 활성은 활성 반응 완충용액(200 mM NaCl, 10 mM CaCl2, 0.02 % NaN3가 포함된 30 mM 트리스 완충용액, pH 7.4)에 겔을 침적하여 37 ℃ 배양기에서 12시간 반응을 시켰다. 피브린 분해능을 지닌 단백질 분획은 활성염색법에 의해 겔 상의 피브린을 분해하여 코마시 블루(Coomassie blue)로 염색시에 투명대를 형성하므로, 상기 단백질 분획이 혈전 용해 활성을 가짐을 확인할 수 있었다.20 ng of the separated and purified enzyme protein fractions were loaded in each lane, and electrophoresis was performed by applying a constant current of 10 mA. To reactivate the enzyme inactivated by SDS, the gel was left in Tris buffer solution (50 mM, pH 7.4) containing 2.5% Triton X-100 for 30 minutes to remove SDS, and then distilled water Triton X-100 was removed. Thrombolytic activity of the fractionated protein was reacted for 12 hours in a 37 ℃ incubator by dipping the gel in the activity reaction buffer (200 mM NaCl, 10 mM CaCl 2 , 30 mM Tris buffer containing 0.02% NaN 3 , pH 7.4) Let. The protein fraction having fibrin degradability decomposes fibrin on the gel by active staining to form a zona pellucida when stained with Coomassie blue, and thus it was confirmed that the protein fraction has thrombolytic activity.
또한, 3종의 버섯 모두에서 분자량이 54, 61 kDa의 공통된 활성을 가지는 단백질을 확인하였으며, 장식솔버섯은 84 kDa의 강한 활성을 가지는 혈전용해효소를 가지고 있음을 확인하였다 (도 2).In addition, all three mushrooms identified a protein having a common activity of 54 and 61 kDa in molecular weight. It was confirmed that it has a thrombolytic enzyme having a strong activity (2).
<실시예 6> 용혈 활성 측정Example 6 Measurement of Hemolytic Activity
본 발명의 균주가 생산하는 새로운 효소 단백질이 용혈 활성(hemolytic activity)이 있는지를 확인하는 시험을 실시하였다.A test was conducted to determine whether the new enzyme protein produced by the strain of the present invention had hemolytic activity.
실시예 1로부터 얻은 버섯 추출물을 플레이트에 놓고 여기에 사람의 혈액을 첨가한 후 배양하였다. 24시간 동안 배양한 후 현미경으로 관찰한 결과, 혈액이 분해되지 않고 그대로 유지되는 것을 알 수 있었으며, 이로부터 본 발명의 버섯 추출물은 용혈 활성이 없는 것으로 판단되었다.The mushroom extract obtained from Example 1 was placed on a plate and cultured after adding human blood thereto. After culturing for 24 hours and observed under a microscope, it was found that the blood is maintained without being degraded, from which the mushroom extract of the present invention was determined to have no hemolytic activity.
본 발명의 혈전용해능을 갖는 버섯 추출물은 주로 정맥 주사를 통해 투여되는 기존의 혈전용해제와 비교해 볼 때, 경구 투여가 가능하므로 혈관 주사제와 병용하여 사용할 수 있는 이점이 있다.Mushroom extract having a thrombolytic ability of the present invention has an advantage that can be used in combination with a vascular injection because it can be administered orally as compared to conventional thrombolytics administered mainly via intravenous injection.
또한, 본 발명의 버섯 추출물은 용혈 활성을 나타내지 않으므로, 경구 투여 약제로 개발할 수 있을 뿐만 아니라 혈전 용해 치료제 및 건강 보조 식품 등으로 널리 이용될 수 있다.In addition, the mushroom extract of the present invention does not exhibit hemolytic activity, and thus can be developed as an oral dosage agent, and can be widely used as a thrombolytic therapeutic agent and health supplement.
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