KR100575253B1 - Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same - Google Patents
Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same Download PDFInfo
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Abstract
본 발명은 비자나무 추출물로부터 분리된 신규 아비에탄 디터페노이드계 화합물 및 이를 유효성분으로 포함하는 심장순환계 질환의 예방 및 치료용 약학적조성물에 관한 것으로, 상세하게는 본 발명의 신규 화합물은 저밀도 지질 단백질에 대한 우수한 항산화 활성을 나타내므로, 이를 포함하는 조성물은 저밀도 지질 단백질의 산화에 의해 유발되는 고지혈증 또는 동맥경화증과 같은 심장순환계 질환의 예방 및 치료를 위한 용도로 광범위하게 사용될 수 있다.The present invention relates to a novel aviethane diterpenoid compound isolated from a non-tree extract and a pharmaceutical composition for the prevention and treatment of cardiac circulatory diseases comprising the same as an active ingredient. Since the composition exhibits excellent antioxidant activity against proteins, the composition comprising the same can be widely used for the prevention and treatment of cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low density lipid protein.
아비에탄 디터페노이드계 화합물, 고지혈증, 동맥경화, 약학적 조성물.Abiethane diterpenoid compound, hyperlipidemia, arteriosclerosis, pharmaceutical composition.
Description
본 발명은 비자나무 추출물로부터 분리된 신규 아비에탄 디터페노이드계 화합물 및 이를 유효성분으로 포함하는 심장순환계 질환의 예방 및 치료용 약학적조성물에 관한 것이다.The present invention relates to a novel aviethane diterpenoid compound isolated from a non-tree extract and a pharmaceutical composition for the prevention and treatment of cardiovascular diseases comprising the same as an active ingredient.
최근 성인병 증가와 아울러 동맥경화증 등 혈관장애질환이 크게 증가되고 있다. 동맥경화는 뇌동맥 또는 관상동맥에서 일어나기 쉬운데, 뇌동맥경화증의 경우에는 두통, 현기증, 정신장애를 나타내고 뇌연화증의 원인이 되며, 관상동맥경화증의 경우에는 심장부에 동통과 부정맥을 일으켜 협심증, 심근경색 등의 원인이 되는 것으로 알려져 있다. 또한 이로 인해 고혈압, 심장병, 뇌일혈 등이 유발되어, 동맥경화증으로 인한 질병이 현대 사회에 있어, 특히 50∼60대의 남성들에게 가장 큰 사망요인으로 부각되고 있다.Recently, as well as an increase in adult disease, vascular disorders such as arteriosclerosis have been greatly increased. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral arteriosclerosis, headache, dizziness, and mental disorders are indicated and cause encephalopathy, and in the case of coronary arteriosclerosis, pain and arrhythmia in the heart cause angina and myocardial infarction. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading cause of death in modern society, especially among men in their 50s and 60s.
혈중 콜레스테롤 농도가 높으면 관상동맥성 심혈관 질환이 유발되기 쉬우므로, 혈중 콜레스테롤 농도를 줄이기 위해서는 콜레스테롤 및 지방의 섭취를 줄이는 식이요법을 시행하거나 지질대사와 관련된 효소를 저해함으로써 콜레스테롤의 흡수를 억제해야 한다.High blood cholesterol levels are likely to cause coronary cardiovascular disease. Therefore, to reduce blood cholesterol levels, it is necessary to reduce the absorption of cholesterol by administering a diet that reduces cholesterol and fat intake or by inhibiting enzymes related to lipid metabolism.
따라서, 이러한 질병을 예방하려는 목적으로 종전부터 콜레스테롤 흡수의 억제와 생합성의 저해를 통한 혈장 저밀도 지질 단백질(low-density lipoprotein; LDL)량을 감소시키려는 시도가 진행되어 왔다 (Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, N. Engl. J. Med., 320:915-924, 1989).Therefore, attempts have been made to reduce plasma low-density lipoprotein (LDL) levels through inhibition of cholesterol absorption and inhibition of biosynthesis for the purpose of preventing such diseases (Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, N. Engl. J. Med. , 320: 915-924, 1989).
최근에는 죽상경화(atherosclerosis)의 요인으로 혈액내 LDL 산화물의 생성이 주요관심의 대상이 되고 있으며(Circulation, 91:2488-2496, 1995; Arterioscler. Thromb. Vasc. Biol., 17:3338-3346, 1997), 특히 LDL의 과도산화와 구조변형을 통해 생성된 HM-LDL(highly modified LDL)의 대식세포(macrophage)로의 유입에 따른 거품세포(foam cell) 생성이 밝혀짐에 따라 LDL 퍼옥사이드의 생성요인과 제거에 관한 연구가 활발히 진행되고 있다(Curr. Atheroscler. Res., 2:363-372, 2000).Recently, the production of LDL oxide in the blood as a factor of atherosclerosis has been the main concern ( Circulation , 91: 2488-2496, 1995; Arterioscler. Thromb. Vasc. Biol., 17: 3338-3346, 1997), in particular, the production of LDL peroxide, as the formation of foam cells following the influx of HM-LDL (highly modified LDL) produced by macrotransformation into the macrophage, resulting from overoxidation and structural modification of LDL Research into factors and elimination is being actively conducted ( Curr. Atheroscler. Res., 2: 363-372, 2000).
혈관벽내에 플라그(plaque) 형성과 파열은 심근경색 발병에 주요한 요인이며, 동맥경화는 혈관벽의 손상에 대한 만성 염증과정으로, 손상기작보다는 오히려 방어기작으로 제시되고 있다(Circ. Res., 89:298-304, 2001).Plaque formation and rupture in the vessel wall are major factors in the development of myocardial infarction, and atherosclerosis is a chronic inflammatory process for damage of the vessel wall and is suggested as a defense mechanism rather than an injury mechanism ( Circ. Res., 89: 298). -304, 2001).
현재 고지혈증 치료제로 사용되고 있는 프로부콜(Probucol), N,N'-디페닐렌 디아민(N,N'-diphenylenediamine), 페놀계 합성 항산화제인 BHA (butylatedhydroxyanisol)와 BHT(butylated hydroxy toluene)는 LDL 콜레스테롤을 감소시키고, 산화정도를 약화시키며 병변형성을 감소시켜 항산화력은 우수하나, 부작용이 많아 사용이 제한되고 있다.Probucol, N, N'-diphenylenediamine (N, N'-diphenylenediamine) and phenolic synthetic antioxidants BHA (butylatedhydroxyanisol) and BHT (butylated hydroxy toluene), which are currently being used for the treatment of hyperlipidemia, use LDL cholesterol. It is reduced, weakens the degree of oxidation and reduces the formation of lesions, the antioxidant power is excellent, but many side effects are limited use.
따라서, 고지혈증이나 동맥경화 환자에 있어서 LDL 항산화제와 함께 지질강하제의 병행투여 요법에 대한 관심도가 높아지고 있다.Therefore, there is increasing interest in the combined administration of lipid lowering agents with LDL antioxidant in hyperlipidemia and atherosclerosis patients.
한편, 비자(榧子, Torreya nucifera)는 주목과(Taxaceae)에 속하는 상록 침엽교목으로 전 세계적으로 우리나라와 일본에만 제한되어 분포한다. 비자나무는 식용, 관상용, 공업용, 약용으로 쓰이고, 종자는 먹거나 기름을 짜내서 이용한다. 또한, 한방과 민간에서는 과실을 구충, 발모, 건위, 조경, 장출혈 등에 약재로 이용하고, 목재는 건축재, 기구재, 선박용재 등에 사용한다(김태정, 한국의 자원식물 Ⅰ, p40, 서울대학교 출판부, 1996; 육창수, 아세아 생약도감, p23, 도서출판 경원, 1997). 비자나무의 잎과 종자에서 분리·보고된 성분으로는 세스퀴터페노이드 (Sakai T., et al., Bull. Chem. Soc. Japan, 38:381, 1965), 라브단(labdane) 계열과 아비에탄(abietane) 계열 디터페노이드(Sayama Y., et al, Agric. Bio. Chem., 35:1068, 1971; Harrison L. and Asakawa Y., Phytochemistry, 26:1211, 1987), 그리고 플라보노이드(Kariyone T. and Sawaka T., Ykugaku Zasshi, 78:1010, 1958) 등이 있다.On the other hand, the visa ( Torreya nucifera ) is an evergreen conifer tree belonging to the family Taxaxaceae and is distributed only in Korea and Japan worldwide. Viburnum is used for edible, ornamental, industrial and medicinal purposes. Seed is eaten or squeezed oil. In oriental medicine and private medicine, fruit is used as a medicine for insect repellent, hair growth, health, landscaping, intestinal bleeding, and wood is used for building materials, equipment materials, and marine materials. Ⅰ, p40, Seoul National University Press, 1996; Yuk Chang-soo, Asian Herbal Medicine Book, p23, Book Publishing Kyungwon, 1997). Sesquiterpenoids (Sakai T., et al., Bull. Chem. Soc. Japan , 38: 381, 1965), labdane family Ethane family diterpenoids (Sayama Y., et al, Agric. Bio. Chem ., 35: 1068, 1971; Harrison L. and Asakawa Y., Phytochemistry , 26: 1211, 1987), and flavonoids (Kariyone) T. and Sawaka T., Ykugaku Zasshi, 78: 1010, 1958).
이에, 본 발명자들은 부작용이 적은 새로운 고지혈증, 동맥경화증 치료제를 천연물에서 탐색하던 중, 비자나무 추출물로부터 분리된 아비에탄 디터페노이드계 화합물에서 저밀도 지질 단백질에 대한 우수한 항산화 활성을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by finding excellent antioxidative activity against low-density lipid protein in abiethane diterpenoid-based compound isolated from non-tree extract while searching for a new hyperlipidemia and atherosclerosis treatment agent with fewer side effects in natural products. It was.
본 발명은 비자나무 추출물로부터 분리된 신규 아비에탄 디터페노이드계 화합물 및 이를 유효성분으로 포함하는 심장순환계 질환의 예방 및 치료용 약학적조성물을 제공하고자 한다.
The present invention is to provide a novel aviethane diterpenoid compound isolated from the extract of the non-tree and a pharmaceutical composition for the prevention and treatment of cardiovascular diseases comprising the same as an active ingredient.
상기 목적에 따라, 본 발명은 하기 일반식 Ⅰ로 표기되는 아비에탄 디터페노이드계 화합물을 제공한다.In accordance with the above object, the present invention provides an aviethane diterpenoid compound represented by the following general formula (I).
상기 식에서,Where
R은 메틸에스테르 또는 디메톡시메틸이다.R is methyl ester or dimethoxymethyl.
상기 일반식 Ⅰ의 아비에탄 디터페노이드계 화합물은 12-히드록시아비에틱-8,11,13-트리엔-18-오익에시드메틸에스테르 및 12-히드록시아비에틱-8,11,13-트리엔-18-디메틸아세탈이고, 약학적으로 허용되는 염의 형태로 사용될 수 있으며, 통상의 방법에 의해 제조되는 모든 염, 수화물 및 용매화물이 포함된다.The aviethane diterpenoid compounds of the general formula (I) include 12-hydroxyabietic-8,11,13-triene-18-oxyacidmethylester and 12-hydroxyabietic-8,11,13 -Triene-18-dimethylacetal, can be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates and solvates prepared by conventional methods.
본 발명은 상기 일반식 Ⅰ의 아비에탄 디터페노이드계 화합물을 유효성분으로 포함하고, 약학적으로 허용 가능한 담체, 희석제 또는 부형제를 포함하는 심장순환계 질환의 예방 및 치료용 약학적조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of cardiac circulatory diseases, including the aviethane diterpenoid compound of Formula I as an active ingredient and a pharmaceutically acceptable carrier, diluent or excipient.
또한, 본 발명은 비자나무 잎(Torreya nucifera leaf)의 추출물로부터 아비에탄 디터페노이드계 화합물을 제조하는 제조방법을 제공한다.The present invention also provides a preparation method for preparing an abiethane diterpenoid compound from an extract of Torreya nucifera leaf.
상기의 아비에탄 디터페노이드계 화합물은 비자나무 잎의 추출물로부터 통상의 추출 및 분리공정을 통하여 수득가능한데,The above-mentioned abiethane diterpenoid compound can be obtained from the extract of the non-tree leaves through a conventional extraction and separation process,
구체적으로 본 발명의 화합물은Specifically, the compound of the present invention
비자나무 잎으로부터 에탄올 가용성 조추출물을 제조하는 제 1단계;A first step of preparing an ethanol soluble crude extract from the non-tree leaves;
상기 1단계의 조추출물로부터 비극성 용매 가용추출물을 제조하는 제 2단계; 및A second step of preparing a non-polar solvent soluble extract from the crude extract of step 1; And
상기 2단계의 비극성 용매 가용추출물, 바람직하게는 에틸아세테이트 가용추 출물을 크로마토그래피로 분획화하여 화합물을 분리 및 정제하는 제 3단계의 제조공정을 통하여 제조 가능하다.The non-polar solvent soluble extract of the second step, preferably ethyl acetate soluble extract can be prepared by a chromatographic fractionation through a third step of the production process to separate and purify the compound.
예를 들어, 비자나무 잎의 추출물로부터 아비에탄 디터페노이드계 화합물을 수득하기 위한 분리공정을 보다 구체적으로 설명하면,For example, the separation process for obtaining the abiethane diterpenoid compound from the extract of the non-tree leaves will be described in more detail.
건조된 비자나무 잎에 에탄올을 가하여 상온에서 3주 동안 방치한 다음, 여과지로 여과하고 농축하여 조추출물인 유성물질을 얻는 제 1단계;Ethanol was added to the dried non-leaved leaves and left for 3 weeks at room temperature, followed by filtration with filter paper and concentration to obtain an oily substance as a crude extract;
상기 1단계의 조추출물에 물을 가하여 현탁시키고, 비극성 용매, 바람직하게는 n-헥산, 클로로포름 및 에틸아세테이트 순으로 분획화하여, n-헥산 가용추출물, 클로로포름 가용추출물 및 에틸아세테이트 가용추출물을 얻는 단계; 및Suspending by adding water to the crude extract of step 1, fractionated in the order of a non-polar solvent, preferably n-hexane, chloroform and ethyl acetate, to obtain n-hexane soluble extract, chloroform soluble extract and ethyl acetate soluble extract ; And
상기 2단계의 에틸아세테이트 가용추출물을 에틸아세테이트와 n-헥산의 혼합용매를 이동상 용매로, 바람직하게는 n-헥산:에틸아세테이트=90:10 내지 50:50(v/v)의 용매조건으로, 더욱 바람직하게는, 두 번에 걸쳐 실리카겔 컬럼 크로마토그래피를 수행하여, 황산화 활성이 높은 분획물을 선정하고, 분취용 박층 크로마토그래피(prep-TLC)를 수행하는데, 실리카겔을 고정상으로 하고, 클로로포름:메탄올 80:1(v/v)비의 전개용매조건으로 전개시켜 분획한 후, 수득한 분획물을 모아 세파덱스 LH-20(sephadex LH-20) 컬럼을 고정상으로 하고 클로로포름:메탄올 1:1(v/v)비를 이동상으로 하는 오픈 컬럼 크로마토그래피를 수행하여 정제된 분획물을 얻고, 이 분획물로부터 본 발명의 아비에탄 디터페노이드계 화합물들을 분리하는 제 3단계의 제조공정을 통하여 수득이 가능하다.In the ethyl acetate soluble extract of step 2, a mixed solvent of ethyl acetate and n-hexane is used as a mobile phase solvent, preferably n-hexane: ethyl acetate = 90: 10 to 50:50 (v / v) in a solvent condition. More preferably, silica gel column chromatography is performed twice to select a fraction having high sulfate activity, and preparative thin layer chromatography (prep-TLC) is performed, wherein the silica gel is fixed, and chloroform: methanol is used. Fractions were carried out under a developing solvent condition of 80: 1 (v / v) ratio, and the obtained fractions were collected, and a Sephadex LH-20 column was used as a fixed phase, and chloroform: methanol 1: 1 (v / v). v) The purified fractions are obtained by performing open column chromatography with the ratio of the mobile phase, which can be obtained through the third step of the production process of separating the aviethane diterpenoid compounds of the present invention from the fractions. .
또한, 본 발명의 화합물들은 당업계에서 잘 알려진 합성방법(Herbert O. House, Modern Synthetic Reactions, The Benjamin Cummings Publishing Company, 1972)이나 제조공정을 통하여 당업자가 용이하게 제조가능하다.In addition, the compounds of the present invention can be easily prepared by those skilled in the art through synthetic methods (Herbert O. House, Modern Synthetic Reactions, The Benjamin Cummings Publishing Company, 1972) or a manufacturing process well known in the art.
상기의 제조공정을 통하여 분리된 화합물들은 분광학적 데이타(spectroscopic data)를 이용하여 구조분석이 가능하다.Compounds separated by the above manufacturing process can be structurally analyzed using spectroscopic data.
또한, 상기의 분리된 화합물들의 저밀도 지질단백질에 대한 항산화 활성을 티비에이알에스(TBARS) 방법으로 측정한 결과, 저밀도 지질단백질의 산화에 대해 저해효과를 나타내어, 본 발명 화합물들의 우수한 항산화 활성을 확인하였다(표 1 참조).In addition, the antioxidant activity of the isolated compounds of the low density lipoprotein was measured by TBARS method, and showed the inhibitory effect on the oxidation of the low density lipoprotein, confirming the excellent antioxidant activity of the compounds of the present invention. (See Table 1).
본 발명의 화합물들은 저밀도 지질단백질의 산화에 대해 저해효과를 나타내는 항산화 활성이 있으므로, 저밀도 지단백질의 산화에 의해 유발되는 고지혈증 또는 동맥경화증과 같은 심장순환계 질환의 질병 및 치료에 사용 가능하다.Since the compounds of the present invention have an antioxidant activity that exhibits an inhibitory effect on the oxidation of low density lipoproteins, the compounds of the present invention can be used for diseases and treatment of cardiovascular diseases such as hyperlipidemia or arteriosclerosis caused by the oxidation of low density lipoproteins.
본 발명의 조성물은, 조성물 총 중량에 대하여 상기 화합물을 0.0001 내지 50 중량%로 포함한다.The composition of the present invention comprises 0.0001 to 50% by weight of the compound based on the total weight of the composition.
본 발명의 조성물은 상기 아비에탄 디터페노이드계 화합물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above-mentioned aviethane diterpenoid compound.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가 할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당해 기술분야의 적정한 방법으로 또는 레ald턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers Other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it is preferably formulated according to the respective disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). can do.
본 발명의 조성물은 목적하는 방법에 따라 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일일 투여량은 화합물의 경우 약 0.1~100㎎/㎏ 이고, 바람직하게는 0.5~10㎎/㎏ 이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다.The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on diet, administration time, administration method, excretion rate and severity of disease. The daily dosage is about 0.1 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.
본 발명의 화합물을 마우스에 경구 투여하여 독성 실험을 수행한 결과, 경구 투여 독성시험에 의한 50% 치사량(LD50)은 적어도 1,000㎎/㎏ 이상인 안전한 물질로 판단된다.As a result of oral administration of a compound of the present invention to mice, 50% lethal dose (LD 50 ) by oral administration toxicity test is judged to be a safe substance of at least 1,000 mg / kg or more.
본 발명의 조성물은 심장순환계 질환의 예방 및 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of cardiovascular diseases.
본 발명의 조성물은 심장순환계 질환의 개선을 목적으로 건강식품에 첨가될 수 있다. 본 발명의 화합물을 식품 첨가물로 사용할 경우, 상기 아비에탄 디터페노이드계 화합물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에는 본 발명의 화합물은 원료에 대하여 1~20 중량%, 바람직하게는 5~10 중량%의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The composition of the present invention can be added to health foods for the purpose of improving cardiovascular disease. When the compound of the present invention is used as a food additive, the aviethane diterpenoid-based compound may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of foods or beverages, the compounds of the present invention are added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물 의 비율은 본 발명의 조성물 100㎖당 일반적으로 약 0.01 내지 0.04g, 바람직하게는 약 0.02 내지 0.03g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 실시예, 실험예 및 제제예에 의해 상세히 설명한다. 단, 하기 실시예, 실험예 및 제제예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 실험예 및 제제예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples. However, the following Examples, Experimental Examples, and Formulation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Formulation Examples.
실시예 1. 본 발명의 화합물 제조Example 1 Preparation of Compounds of the Invention
단계 1 : 비자나무 잎의 조추출물의 제조Step 1: Preparation of Crude Extracts of Non-leaf Leaves
건조된 비자나무(제주도, 대한민국)잎 2.16 kg에 100% 에탄올(EtOH) 18 ℓ를 가하여 실온에서 3주 동안 방치시킨 후, 여과지로 여과하고 농축하여 비자나무 잎의 조추출물인 유성물질 42 g을 수득하였다.18 liters of 100% ethanol (EtOH) was added to 2.16 kg of dried non-leaf (Jeju-do, Korea) leaves, and left for 3 weeks at room temperature. Obtained.
단계 2 : 비자나무 잎의 비극성 용매 가용추출물의 제조Step 2: Preparation of Non-Polar Solvent Soluble Extract of Non-tree Leaves
상기 단계 1에서 얻은 비자나무 잎 조추출물에 물 1000 ㎖을 가하여 현탁시키고, n-헥산(n-hexane), 클로로포름(CHCl3) 및 에틸아세테이트(EtOAc) 순으로 분획하여, n-헥산 가용추출물(n-hexane-soluble) 75 g, 클로로포름 가용추출물(CHCl3-soluble) 37 g 및 에틸아세테이트 가용추출물(EtOAc-soluble) 18 g을 각각 수득하였다.Suspended by adding 1000 ml of water to the crude extract of the non-tree leaves obtained in step 1, n-hexane ( n -hexane), chloroform (CHCl 3 ) and ethyl acetate (EtOAc) fractions, n-hexane soluble extract ( 75 g of n- hexane-soluble, 37 g of chloroform soluble extract (CHCl 3 -soluble) and 18 g of ethyl acetate soluble extract (EtOAc-soluble) were obtained, respectively.
에틸아세테이트층으로부터 얻은 노란색 유성물질을 하기 실험예 3과 같은 방법으로 항산화을 관찰한 결과, 40 ㎍/㎖ 농도에서 저밀도 지질단백질에 대한 85%의 항산화 효과를 나타내어, 우수한 항산화 활성이 있음을 확인하였다.As a result of observing the antioxidant of the yellow oily substance obtained from the ethyl acetate layer in the same manner as in Experimental Example 3, it showed an antioxidant effect of 85% against the low density lipoprotein at a concentration of 40 ㎍ / ㎖, it was confirmed that there is excellent antioxidant activity.
단계 3 : 비자나무 잎의 에틸아세테이트 가용추출물의 분획화 및 본 발명의 화합물의 분리Step 3: Fractionation of Ethyl Acetate Soluble Extract from Non-Tree Leaves and Isolation of Compounds of the Invention
상기 단계 2에서 수득한 에틸아세테이트 가용추출물 18 g을 실리카겔 컬럼 크로마토그래피와 이동상 용매로 n-헥산과 에틸아세테이트의 혼합용매를 사용하여 두 단계로 분리하였다.18 g of the ethyl acetate soluble extract obtained in step 2 were separated in two steps using silica gel column chromatography and a mixed solvent of n-hexane and ethyl acetate as the mobile phase solvent.
우선, 첫 번째로 실리카겔 컬럼 크로마토그래피(실리카겔 : Merk, Art 9385, 컬럼크기 : φ7 × 40 cm)에 n-헥산:EtOAc = 98:2, 97:3, 95:5, 10:1, 5:1, 3:1, 1:1 및 EtOAc 100%(v/v)의 이동상 용매조건으로 각각 1.5 ℓ씩을 전개시켜, 17개 분획물(Fr.1∼17)을 수득하였으며, 각 분획물의 항산화 활성을 하기 실험예 3과 같은 방법으로 관찰한 결과, 가장 우수한 항산화 활성을 나타내는 8번 분획물(Fr.8, 헥산:EtOAc=5:1 및 3:1 용매조건, 수득량 593 ㎎)대하여, 두 번째 실리카겔 컬럼 크로마토그래피법을 수행하였다. 이때, n-헥산:EtOAc=98:2, 95:5, 10:1, 5:1, 3:1, 1:1 및 EtOAc 100%(v/v)의 이동상 용매조건으로 각각 100 ㎖씩을 전개시켜, 11개 분획물(Fr.1∼11)을 수득하였으며, 각 분획물의 항산화 활성을 관찰하였다. 관찰 결과, 항산화 활성이 가장 뛰어난 8번부터 10번 분획의 혼합물(Fr.8∼10, n-헥산:EtOAc = 5:1 내지 1:1 용매조건, 149 ㎎)은 프렙 TLC(prep TLC, Silica gel 60F254, Merck, Art 5744, CHCl3/MeOH = 80:1의 이동상 용매조건) 및 세파덱스 LH-20 컬럼(sephadex LH-20 column, Sigma-Aldrich Co., USA, CHCl3/MeOH = 1:1의 용매조건)을 이용하여 정제된 두 가지의 화합물을 수득하였는데, 각 수득량은 1 kg당 하기 실시예 2의 구조분석 결과 확인된 12-히드록시아비에틱-8,11,13-트리엔-18-오익에시드메틸에스테르 화합물이 17.5mg 및 12-히드록시아비에틱-8,11,13-트리엔-18-디메틸아세탈 화합물이 18 mg이었다.First, silica gel column chromatography (silica gel: Merk, Art 9385, column size: 7 × 40 cm) n-hexane: EtOAc = 98: 2, 97: 3, 95: 5, 10: 1, 5: 1.5 L each of 1, 3: 1, 1: 1 and EtOAc 100% (v / v) mobile phase solvents were developed to obtain 17 fractions (Fr.1 to 17), and the antioxidant activity of each fraction was determined. As a result of observation in the same manner as in Experimental Example 3, a second silica gel was used for fraction 8 (Fr. 8, hexane: EtOAc = 5: 1 and 3: 1 solvent conditions, yield 593 mg) showing the best antioxidant activity. Column chromatography was performed. In this case, n-hexane: EtOAc = 98: 2, 95: 5, 10: 1, 5: 1, 3: 1, 1: 1 and 100 ml each of 100 ml (v / v) of mobile phase solvent were developed. To obtain 11 fractions (Fr. 1 to 11), the antioxidant activity of each fraction was observed. As a result, a mixture of fractions 8 to 10 having the highest antioxidant activity (Fr. 8 to 10, n-hexane: EtOAc = 5: 1 to 1: 1 solvent condition, 149 mg) was prepared with prep TLC (prep TLC, Silica). gel 60F 254 , Merck, Art 5744, mobile phase solvent condition of CHCl 3 / MeOH = 80: 1) and Sephadex LH-20 column (sephadex LH-20 column, Sigma-Aldrich Co., USA, CHCl 3 / MeOH = 1) Two compounds purified using (solvent condition of 1) were obtained, and each yield was 12-hydroxyabietic-8,11,13- as confirmed by the structural analysis of Example 2 below per kg. 17.5 mg of the triene-18-oxyacidmethylester compound and 18 mg of the 12-hydroxyabietic-8,11,13-triene-18-dimethylacetal compound.
실시예 2. 본 발명의 화합물의 구조 분석Example 2. Structural Analysis of Compounds of the Invention
상기 실시예 1을 통하여 얻은 물질은 VG 고분해능 GC/MS 분광기(VG high resolution GC/MS spectrometer, Election Ionization MS, Autospec-Ultima, Micromass, UK)를 사용하여 분자량 및 분자식을 결정하였으며, 선광도는 편광기 (DIP-181 digital polarimeter, Jasco, Japan)를 사용하여 측정하였다. 또한 핵자기 공명(NMR) 분석(AMX 500, Bruker, Germany)을 통하여 1H NMR, 13C NMR, 호모-코지(HOMO-COSY), HMQC(1H-Detected heteronuclear Multiple-Quantum Coherence), HMBC(Heteronuclear Multiple-Bond Coherence), DEPT(Distortionless Enhancement by Polarization)스펙트럼을 얻고, 분자구조를 결정하였다.The material obtained in Example 1 was determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima, Micromass, UK to determine the molecular weight and molecular formula. It was measured using (DIP-181 digital polarimeter, Jasco, Japan). Nuclear magnetic resonance (NMR) analysis (AMX 500, Bruker, Germany) also showed 1 H NMR, 13 C NMR, Homo-Cozy, HMQC ( 1 H-Detected heteronuclear Multiple-Quantum Coherence), HMBC ( Heteronuclear Multiple-Bond Coherence (DEP) and Distortionless Enhancement by Polarization (DEPT) spectra were obtained and the molecular structure was determined.
이상의 기기분석결과를 발표된 문헌의 것과 비교 분석한 결과, 하기 물성치 1을 갖는 화학식 1의 화합물을 12-히드록시아비에틱-8,11,13-트리엔-18-오익에시드메틸에스테르(Chem. Nat. Compod (Engl. Transl), 24:447, 1988)로 동정하였다. 또한, 하기 물성치 2를 갖는 노란색 유성물질을 12-히드록시아비에틱-8,11,13-트리엔-18-디메틸아세탈로 동정하였으며, 상기 화합물은 아직 보고되지 않은 신규한 화합물로 확인하였다.Than one device, the analysis result as a comparison result, the following physical data for compounds of the formula I having a 1 to 12-hydroxy-18-triene father tick -8,11,13- ohik Acid methyl ester (Chem literature published Nat. Compod (Engl. Transl ), 24: 447, 1988). In addition, a yellow oily substance having the following physical property 2 was identified as 12-hydroxyabietic-8,11,13-triene-18-dimethylacetal, and the compound was identified as a new compound which has not yet been reported.
1. One. 12-히드록시아비에틱-8,11,13-트리엔-18-오익에시드메틸에스테르12-hydroxyabietic-8,11,13-triene-18-oxyacidmethylester
1) 물성: 노란색 유성1) Physical property: yellow oily
2) 선광도: [α]D 25 +75.7°(c = 0.28, EtOH)2) Radiance: [α] D 25 + 75.7 ° ( c = 0.28, EtOH)
3) 분자량: 3303) Molecular Weight: 330
4) 분자식: C21H30O3 4) Molecular Formula: C 21 H 30 O 3
5) 1H NMR (CDCl3, 500 MHz) δ 1.12 (s, 3H, H-20), 1.15 (d, J = 7.4 Hz, 3H, H-16), 1.16 (d, J = 7.3 Hz, 3H, H-17), 1.19 (s, 3H, H-19), 1.29 (m, 1H, H-6α), 1.41 (m, 1H, H-2α), 1.55-1.77 (m, 5H, H-1α, H-2β, H-3, H-6β), 2.12 (d like, J = 12.7 Hz, 1H, H-1β), 2.14 (dd, J = 1.7, 12.5 Hz, 1H, H-5), 2.74 (m, 2H, H-7), 3.04 (m, 1H, H-15), 3.59 (s, 3H, CO2Me), 4.58 (s, -OH), 6.55 (s, 1H, H-11), 6.74 (s, 1H, H-14).5) 1 H NMR (CDCl 3 , 500 MHz) δ 1.12 (s, 3H, H-20), 1.15 (d, J = 7.4 Hz, 3H, H-16), 1.16 (d, J = 7.3 Hz, 3H , H-17), 1.19 (s, 3H, H-19), 1.29 (m, 1H, H-6α), 1.41 (m, 1H, H-2α), 1.55-1.77 (m, 5H, H-1α , H-2β, H-3, H-6β), 2.12 (d like, J = 12.7 Hz, 1H, H-1β), 2.14 (dd, J = 1.7, 12.5 Hz, 1H, H-5), 2.74 (m, 2H, H-7), 3.04 (m, 1H, H-15), 3.59 (s, 3H, CO 2 Me), 4.58 (s, -OH), 6.55 (s, 1H, H-11) , 6.74 (s, 1 H, H-14).
6) 13C NMR (CDCl3, 125 MHz) δ 16.5 (C-19), 18.5 (C-2), 21.8 (C-6), 22.5 (C-16), 22.7 (C-17), 25.0 (C-20), 26.8 (C-15), 29.2 (C-7), 36.6 (C-1), 36.9 (C-4), 38.0 (C-3), 44.8 (C-5), 47.7 (C-10), 51.9 (-OMe), 110.8 (C-11), 126.7 (C-14), 127.0 (C-8), 131.7 (C-13), 147.9 (C-9), 150.8 (C-12), 179.2 (C-18)6) 13 C NMR (CDCl 3 , 125 MHz) δ 16.5 (C-19), 18.5 (C-2), 21.8 (C-6), 22.5 (C-16), 22.7 (C-17), 25.0 ( C-20), 26.8 (C-15), 29.2 (C-7), 36.6 (C-1), 36.9 (C-4), 38.0 (C-3), 44.8 (C-5), 47.7 (C -10), 51.9 (-OMe), 110.8 (C-11), 126.7 (C-14), 127.0 (C-8), 131.7 (C-13), 147.9 (C-9), 150.8 (C-12 ), 179.2 (C-18)
2. 2. 12-히드록시아비에틱-8,11,13-트리엔-18-디메틸아세탈12-hydroxyabietic-8,11,13-triene-18-dimethylacetal
1) 물성: 노란색 유성1) Physical property: yellow oily
2) 선광도: [α]D 25 -5.8 °(c = 0.3, CHCl3)2) Radiance: [α] D 25 -5.8 ° ( c = 0.3, CHCl 3 )
3) 분자량: 3463) Molecular Weight: 346
4) 분자식: C22H34O3 4) Molecular Formula: C 22 H 34 O 3
5) 1H NMR (CDCl3, 500 MHz) δ 0.9 (s, 3H, H-19), 1.12 (s, 3H, H-20), 1.15 (d, J = 7.2Hz, H-16), 1.16 (d, J = 7.2Hz, H-17), 1.27 (dt, J = 4.2, 12.7Hz, H-1a), 1.36-1.43 (m, 2H, H-3), 1.53-1.62 (m, 2H, H-2), 1.64-1.75 (m, 2H, H-6), 1.81 (dd, J = 1.7, 12.1 Hz, H-5), 2.05 (d like, J = 12.6Hz, H-1b), 6.55 (s, 1H, H-11), 6.75 (s, 1H, H-14).5) 1 H NMR (CDCl 3 , 500 MHz) δ 0.9 (s, 3H, H-19), 1.12 (s, 3H, H-20), 1.15 (d, J = 7.2 Hz, H-16), 1.16 (d, J = 7.2 Hz, H-17), 1.27 (dt, J = 4.2, 12.7 Hz, H-1a), 1.36-1.43 (m, 2H, H-3), 1.53-1.62 (m, 2H, H-2), 1.64-1.75 (m, 2H, H-6), 1.81 (dd, J = 1.7, 12.1 Hz, H-5), 2.05 (d like, J = 12.6 Hz, H-1b), 6.55 (s, 1H, H-11), 6.75 (s, 1H, H-14).
6) 13C NMR (CDCl3, 125 MHz) δ 16.7 (C-19), 18.3 (C-2), 19.4 (C-6), 22.5 (C-16), 22.7 (C-17), 25.2 (C-20), 26.7 (C-15), 29.3 (C-7), 30.4 (C-3), 37.3 (C-10), 38.2 (C-1), 42.6 (C-4), 42.8 (C-5), 58.7 (C-18b), 59.0 (C-18a), 110.9 (C-11), 113.3 (C-18), 126.5 (C-14), 126.9 (C-8), 131.4 (C-13), 148.7 (C-9), 150.7 (C-12).6) 13 C NMR (CDCl 3 , 125 MHz) δ 16.7 (C-19), 18.3 (C-2), 19.4 (C-6), 22.5 (C-16), 22.7 (C-17), 25.2 ( C-20), 26.7 (C-15), 29.3 (C-7), 30.4 (C-3), 37.3 (C-10), 38.2 (C-1), 42.6 (C-4), 42.8 (C -5), 58.7 (C-18b), 59.0 (C-18a), 110.9 (C-11), 113.3 (C-18), 126.5 (C-14), 126.9 (C-8), 131.4 (C- 13), 148.7 (C-9), 150.7 (C-12).
7) EIMS (rel. int.) m/z [M]+ 59 (22%), 75 (100%), 189 (14%), 201 (10%), 346 (33%).7) EIMS (rel. Int.) M / z [M] + 59 (22%), 75 (100%), 189 (14%), 201 (10%), 346 (33%).
실험예 1. TBARS법을 이용한 본 발명 화합물의 항산화 활성 측정Experimental Example 1. Determination of antioxidant activity of the compound of the present invention using the TBARS method
상기 본 발명의 화합물에 대하여 저밀도 지질 단백질에 대한 항산화 활성을 측정하기 위하여, 하기와 같은 실험을 수행하였다.In order to measure the antioxidant activity of the low density lipid protein with respect to the compound of the present invention, the following experiment was performed.
Cu2+은 저밀도 지질 단백질의 산화를 유도(Cu2+-mediated LDL-oxidation)하는 것으로 알려져 있다. 따라서 본 발명에서는 이 때 생성된 불포화 지방산의 산화산물인 디알데하이드(dialdehyde)를 TBA(thiobarbituric acid)법으로 측정하여, 본 발명 화합물들의 항산화 활성을 측정하였다(Packer, L. Ed., Methods in Enzymology. Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego, 1994).Cu 2+ is known to induce oxidation of low density lipid proteins (Cu 2+ -mediated LDL-oxidation). Therefore, in the present invention, dialdehyde (dialdehyde), which is an oxidation product of the unsaturated fatty acid produced at this time, was measured by TBA (thiobarbituric acid) method to determine the antioxidant activity of the compounds of the present invention (Packer, L. Ed., Methods in Enzymology Vol. 234, Oxygen radicals in biological systems Part D. Academic press, San Diego, 1994).
사람으로부터 혈장 300 ㎖를 채취하여, 초원심분리기로 100,000 x g에서 24시간 동안 원심분리하여 상층에 부유된 고밀도 지질단백질(VLDL)/킬로마이크론(chylomicron)층을 제거하고, 나머지 용액의 비중을 1.063 g/㎖로 맞춘 후, 100,000 x g에서 24시간 동안 원심분리하여 다시 상층에 부유된 저밀도 지질 단백질 25 ㎖(1.5～2.5 ㎎ 단백질/㎖)을 분리하였다.300 ml of plasma was collected from humans, and centrifuged at 100,000 xg for 24 hours using an ultracentrifuge to remove the high-density lipoprotein (VLDL) / chylomicron layer suspended in the upper layer, and the remaining solution was 1.063 g. After adjusting to / ml, centrifugation was performed at 100,000 xg for 24 hours to separate 25 ml (1.5-2.5 mg protein / ml) of low density lipid protein suspended in the upper layer.
상기에서 분리한 저밀도 지질 단백질 20 ㎕(단백질 농도, 50～100 ㎍/㎖)을 10 mM 인산완충용액(phosphate-buffered saline, PBS) 210 ㎕와 혼합하고, 상기 실시예 1에서 제조한 본 발명 화합물들의 용액을 각각 10 ㎕씩 첨가하였다. 이때, 본 발명의 화합물들은 DMSO(dimethylsulfoxide)에 녹여 사용하였으며, 실험에 사용하기 전에 여러 농도로 희석하였다. 음성 대조군으로는 용매인 DMSO 만을 첨가한 것을 사용하였으며, 양성 대조군으로는 프로부콜(Probucol)을 첨가한 것을 사용하였다.20 μl (protein concentration, 50-100 μg / ml) of the isolated low density lipid protein was mixed with 210 μl of 10 mM phosphate-buffered saline (PBS), and the compound of the present invention prepared in Example 1 was used. 10 μl of each solution was added. In this case, the compounds of the present invention were dissolved in DMSO (dimethylsulfoxide) and used, and diluted to various concentrations before use in experiments. As a negative control, only the solvent DMSO was added, and as a positive control, probucol was added.
상기 용액에 0.25 mM CuSO4 10 ㎕를 첨가하여 37 ℃에서 4시간 동안 반응시키고, 20% 트리클로로아세트산(trichloroacetic acid, TCA) 용액 1 ㎖를 첨가하여 반응을 중지시켰다. 0.05N NaOH 용액에 녹인 0.67% TBA 용액 1 ㎖를 첨가하고 10초간 교반시킨 후 95 ℃에서 5분 동안 가열하여 발색 반응을 일으킨 다음, 얼음물로 용액을 냉각하였다. 이후, 용액을 3000 rpm에서 5분 동안 원심분리하여 상등액을 분리하였으며, 자외선-가시광선 분광기로 540 ㎚에서의 흡광도를 측정하여 상기 발색 반응으로 생성된 말론디알데하이드(malondialdehyde, MDA)의 양을 측정하였다.10 μl of 0.25 mM CuSO 4 was added to the solution for 4 hours at 37 ° C., and 1 ml of 20% trichloroacetic acid (TCA) solution was added to stop the reaction. 1 ml of a 0.67% TBA solution dissolved in 0.05N NaOH solution was added thereto, stirred for 10 seconds, and heated at 95 ° C. for 5 minutes to give a color reaction. The solution was then cooled with ice water. Thereafter, the supernatant was separated by centrifuging the solution at 3000 rpm for 5 minutes, and the absorbance at 540 nm was measured by an ultraviolet-vis spectrometer to measure the amount of malondialdehyde (MDA) generated by the color reaction. It was.
한편, 말론디알데하이드의 표준곡선을 구하기 위하여, 말론알데하이드 비스(디메틸아세탈)[malonaldehyde bis(dimethylacetal)]의 저장용액을 이용하여 0～10 nmol 말론디알데하이드를 포함하는 PBS 표준용액을 250 ㎕씩 만들어 상기와 같은 방법으로 발색시키고 540 ㎚에서의 흡광도를 측정하여, 말론디알데하이드의 표준곡선을 구하였다. 상기 표준곡선을 이용하여 본 발명의 화합물들로부터 생성된 말론디알데하이드의 양을 정량하였다(표 1 참조).Meanwhile, to obtain a standard curve of malondialdehyde, 250 μl of PBS standard solution containing 0-10 nmol malondialdehyde was prepared using a stock solution of malonaldehyde bis (dimethylacetal). Color development was carried out in the same manner as above and the absorbance at 540 nm was measured to obtain a standard curve of malondialdehyde. The standard curve was used to quantify the amount of malondialdehyde produced from the compounds of the present invention (see Table 1).
상기 표 1에서 보는 바와 같이, 본 발명의 화합물인 12-히드록시아비에틱-8,11,13-트리엔-18-오익에시드메틸에스테르 및 12-히드록시아비에틱-8,11,13-트리 엔-18-디메틸아세탈 화합물들의 낮은 IC50값으로부터 저밀도 지질 단백질에 대한 우수한 항산화 활성을 확인하였다.As shown in Table 1, the compound of the present invention 12-hydroxyabietic-8,11,13-triene-18-oic acid methyl ester and 12-hydroxyabietic-8,11,13 The low IC 50 value of the -triene-18-dimethylacetal compounds confirmed good antioxidant activity against low density lipid proteins.
따라서, 본 발명의 화합물들은 저밀도 지질단백질의 산화에 의해 유발되는 것으로 알려진 고지혈증 또는 동맥경화증과 같은 심장순환계 질환의 예방 또는 치료에 유용하게 사용될 수 있다.Accordingly, the compounds of the present invention can be usefully used for the prevention or treatment of cardiovascular diseases such as hyperlipidemia or arteriosclerosis, which are known to be caused by the oxidation of low density lipoproteins.
실험예 2. 급성 독성실험Experimental Example 2. Acute Toxicity Test
본 발명의 화합물들에 대한 급성 독성을 알아보기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the acute toxicity for the compounds of the present invention, the following experiment was performed.
4주령의 특정 병원체 부재(SPF, specific pathogens free) ICR계 마우스를 암수 각각 12 마리씩 4군(암수 각각 3마리/실험군)으로 나누어, 온도 22± 3℃, 습도 55± 10%, 조명 12L/12D의 동물실내에서 사육하였다. 마우스는 실험에 사용되기 전 1주일 정도 순화시켰다. 실험동물용 사료(마우스 및 랫트용, (주)제일제당, 서울, 대한민국) 및 음수는 멸균한 후 공급하였으며 자유 섭취시켰다.SPF (specific pathogens free) ICR mice were divided into 4 groups (3 males and 3 females / experimental group) at 4 weeks of age, temperature 22 ± 3 ℃, humidity 55 ± 10%, illumination 12L / 12D Was bred in the animal room. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (for mice and rats, CheilJedang Co., Seoul, South Korea) and drinking water were sterilized and supplied freely.
상기 실시예 1에서 제조한 본 발명의 화합물들을 0.5% 트윈(tween) 80에 50 mg/㎖ 농도로 조제한 후, 마우스 체중 20 g 당 0.04 ㎖(100 mg/kg), 0.2 ㎖(500 mg/kg) 및 0.4 ㎖(1,000 mg/kg)씩 경구 투여하였다. 시료는 단회 경구 투여하였으며, 투여 후 7일 동안 다음과 같이 부작용 또는 치사 여부를 관찰하였다. 즉, 투여당일은 투여 후 1시간, 4시간, 8시간, 12시간 뒤에, 그리고 투여 익일부터 7일째 까지는 매일 오전, 오후 1회 이상씩 일반증상의 변화 및 사망동물의 유무를 관찰하였다.The compounds of the present invention prepared in Example 1 were prepared at a concentration of 50 mg / ml in 0.5% tween 80, and then 0.04 ml (100 mg / kg) and 0.2 ml (500 mg / kg) per 20 g of mouse body weight. ) And 0.4 ml (1,000 mg / kg) orally. Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of dosing, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours, and the next day after administration.
또한, 투여 7일째에 동물을 치사시켜 해부한 후 육안으로 내부 장기를 검사하였다. 투여당일부터 1일 간격으로 체중의 변화를 측정하여 본 발명의 화합물들에 의한 동물의 체중 감소 현상을 관찰하였다.In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss phenomenon of the animals caused by the compounds of the present invention.
상기와 같은 급성 독성실험 결과, 시료를 투여한 모든 마우스에서 특기할 만한 임상증상이 나타나지 않았고 폐사된 마우스도 없었으며, 또한 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다.As a result of the acute toxicity test as described above, all mice treated with the sample did not show any significant clinical symptoms, and there were no dead mice. Also, no change in toxicity was observed in weight change, blood test, blood biochemical test, autopsy findings, etc. Did.
따라서, 본 발명의 화합물들은 모든 마우스에서 1,000 mg/kg까지 독성변화를 나타내지 않았으며, 경구투여 최소치사량(LD50)이 1,000 mg/kg 이상인 안전한 물질로 판단되었다.Therefore, the compounds of the present invention did not show a change in toxicity in all mice up to 1,000 mg / kg, was determined to be a safe substance oral minimum dose (LD 50 ) of 1,000 mg / kg or more.
이하, 본 발명의 화합물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, the preparation examples of the composition containing the compound of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.
제제예 1. 약학적 제제의 제조Formulation Example 1 Preparation of a Pharmaceutical Formulation
1-1. 산제의 제조1-1. Manufacture of powder
실시예 1의 화합물 2g2 g of compound of Example 1
유당 1g1g lactose
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.The above ingredients were mixed and filled in airtight cloth to prepare a powder.
1-2. 정제의 제조1-2. Manufacture of tablets
실시예 1의 화합물 100㎎100 mg of the compound of Example 1
옥수수전분 100㎎Corn Starch 100mg
유 당 100㎎Lactose 100mg
스테아린산 마그네슘 2㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
1-3. 캡슐제의 제조1-3. Preparation of Capsules
실시예 1의 화합물 100㎎100 mg of the compound of Example 1
옥수수전분 100㎎Corn Starch 100mg
유 당 100㎎Lactose 100mg
스테아린산 마그네슘 2㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
1-4. 주사액제의 제조1-4. Preparation of Injection
실시예 1의 화합물 10 ㎍/㎖10 μg / ml of the compound of Example 1
묽은 염산 BP pH 3.5로 될 때까지Dilute hydrochloric acid BP until pH 3.5
주사용 염화나트륨 BP 최대 1㎖Injectable sodium chloride BP up to 1 ml
적당한 용적의 주사용 염화나트륨 BP 중에 실시예 1의 화합물을 용해시키고, 생성된 용액의 pH를 묽은 염산 BP를 사용하여 pH 3.5로 조절하고, 주사용 염화나트륨 BP를 사용하여 용적을 조절하고 충분히 혼합하였다. 용액을 투명유리로된 5㎖ 타입 I 앰플 중에 충전시키고, 유리를 용해시킴으로써 공기의 상부 격자하에 봉입시키고, 120℃에서 15분 이상 오토클래이브시켜 살균하여 주사액제를 제조하였다.The compound of Example 1 was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule of clear glass, dissolved in glass and enclosed under an upper grid of air, sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.
제제예 2. 식품의 제조Formulation Example 2 Preparation of Food
본 발명의 화합물을 포함하는 식품들을 다음과 같이 제조하였다.Foods containing the compound of the present invention were prepared as follows.
2-1. 조리용 양념의 제조2-1. Preparation of Cooking Seasonings
실시예 1의 화합물 0.2 ~ 10 중량%로 건강 증진용 조리용 양념을 제조하였다.0.2 to 10% by weight of the compound of Example 1 was prepared for health promotion cooking seasoning.
2-2. 토마토 케찹 및 소스의 제조2-2. Preparation of Tomato Ketchup and Sauce
실시예 1의 화합물 0.2 ~ 1.0 중량%를 토마토 케찹 또는 소스에 첨가하여 건강 증진용 토마토 케찹 또는 소스를 제조하였다.0.2 to 1.0% by weight of the compound of Example 1 was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.
2-3. 밀가루 식품의 제조2-3. Manufacture of Flour Food
실시예 1의 화합물 0.1 ~ 5.0 중량%를 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.0.1 to 5.0% by weight of the compound of Example 1 was added to the flour, using this mixture to prepare bread, cakes, cookies, crackers and noodles to prepare a health food.
2-4. 스프 및 육즙(gravies)의 제조2-4. Preparation of soups and gravy
실시예 1의 화합물 0.1 ~ 1.0 중량%를 스프 및 육즙에 첨가하여 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.0.1 to 1.0% by weight of the compound of Example 1 was added to the soup and gravy to prepare a health promoting meat product, a soup of noodles and a gravy.
2-5. 그라운드 비프(ground beef)의 제조2-5. Preparation of Ground Beef
실시예 1의 화합물 10 중량%를 그라운드 비프에 첨가하여 건강 증진용 그라운드 비프를 제조하였다.10% by weight of the compound of Example 1 was added to the ground beef to prepare a ground beef for health promotion.
2-6. 유제품(dairy products)의 제조2-6. Manufacture of dairy products
실시예 1의 화합물 0.1 ~ 1.0 중량%를 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.0.1 to 1.0% by weight of the compound of Example 1 was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.
2-7. 선식의 제조2-7. Manufacture of wire
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.
검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60메쉬의 분말로 제조하였다.Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.
실시예 1의 화합물을 진공 농축기에서 감압·농축하고, 분무, 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60메쉬로 분쇄하여 건조분말을 얻었다.The compound of Example 1 was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.
상기에서 제조한 곡물류, 종실류 및 실시예 1의 화합물의 건조분말을 다음의 비율로 배합하여 제조하였다.The grains, seeds and the dry powder of the compound of Example 1 prepared above were formulated in the following ratio.
곡물류(현미 30중량%, 율무 15중량%, 보리 20중량%),Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),
종실류(들깨 7중량%, 검정콩 8중량%, 검정깨 7중량%),Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),
실시예 1의 화합물의 건조분말(1 중량%),Dry powder (1% by weight) of the compound of Example 1,
영지(0.5중량%),Ganoderma lucidum (0.5% by weight),
지황(0.5중량%)Foxglove (0.5 wt%)
제제예 3. 음료의 제조Formulation Example 3 Preparation of Beverage
3-1. 탄산음료의 제조3-1. Preparation of Carbonated Drinks
설탕 5~10%, 구연산 0.05~0.3%, 카라멜 0.005~0.02%, 비타민 C 0.1~1%의 첨가물을 혼합하고, 여기에 79~94%의 정제수를 섞어서 시럽을 만들고, 상기 시럽을 85~98℃에서 20~180초간 살균하여 냉각수와 1:4의 비율로 혼합한 다음 탄산가스를 0.5~0.82% 주입하여 실시예 1의 화합물을 함유하는 탄산음료를 제조하였다.5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 After sterilizing at 20 ° C. for 180 seconds, the mixture was mixed with cooling water at a ratio of 1: 4, and 0.5 to 0.82% of carbon dioxide was injected to prepare a carbonated beverage containing the compound of Example 1.
3-2. 건강음료의 제조3-2. Manufacture of health drinks
액상과당(0.5%), 올리고당(2%), 설탕(2%), 식염(0.5%), 물(75%)과 같은 부재료와 실시예 1의 화합물을 균질하게 배합하여 순간 살균을 한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 건강음료를 제조하였다.Instant sterilization by homogeneously mixing the compound of Example 1 with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) Healthy drinks were prepared by packaging in small packaging containers such as glass bottles and plastic bottles.
3-3. 야채쥬스의 제조3-3. Preparation of Vegetable Juice
실시예 1의 화합물 0.5g을 토마토 또는 당근 쥬스 1,000㎖에 가하여 건강 증진용 야채쥬스를 제조하였다.0.5 g of the compound of Example 1 was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.
3-4. 과일쥬스의 제조3-4. Preparation of Fruit Juice
실시예 1의 화합물 0.1g을 사과 또는 포도 쥬스 1,000㎖에 가하여 건강 증진용 과일쥬스를 제조하였다.0.1 g of the compound of Example 1 was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.
본 발명의 아비에탄 디터페노이드계 화합물은 저밀도 지질 단백질에 대한 우수한 항산화 활성을 나타내므로, 이를 포함하는 조성물은 저밀도 지질 단백질의 산화에 의해 유발되는 고지혈증 또는 동맥경화증과 같은 심장순환계 질환의 예방 및 치료를 위한 용도로 광범위하게 사용될 수 있다.Since the aviethane diterpenoid compound of the present invention exhibits excellent antioxidant activity against low density lipid protein, the composition comprising the same prevents and treats cardiac circulatory diseases such as hyperlipidemia or arteriosclerosis caused by oxidation of low density lipid protein. It can be used extensively for the purpose.
Claims (11)
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020040089372A KR100575253B1 (en) | 2004-11-04 | 2004-11-04 | Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same |
| PCT/KR2005/000472 WO2005084141A2 (en) | 2004-03-03 | 2005-02-22 | Novel abietane diterpenoid compound, and composition comprising extract of torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease |
| JP2007501703A JP4777970B2 (en) | 2004-03-03 | 2005-02-22 | Novel abietane diterpenoid compound and sputum extract, or composition for prevention and treatment of cardiovascular disease comprising as an active ingredient an abietane diterpenoid compound or terpenoid compound isolated therefrom |
| US10/591,282 US7517542B2 (en) | 2004-03-03 | 2005-02-22 | Abietane diterpenoid compound, and composition comprising extract of torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease |
| US12/181,583 US7820212B2 (en) | 2004-03-03 | 2008-07-29 | Abietane diterpenoid compound, and composition comprising extract of Torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease |
| US12/265,088 US7825162B2 (en) | 2004-03-03 | 2008-11-05 | Abietane diterpenoid compound, and composition comprising extract of torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease |
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| KR1020040089372A KR100575253B1 (en) | 2004-11-04 | 2004-11-04 | Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same |
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| KR100575253B1 true KR100575253B1 (en) | 2006-05-02 |
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| KR1020040089372A KR100575253B1 (en) | 2004-03-03 | 2004-11-04 | Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101811544B1 (en) | 2016-12-09 | 2017-12-21 | 남종현 | Pain Relief Composition |
| KR101811545B1 (en) | 2016-12-09 | 2017-12-21 | 남종현 | Pharmceutical composition for treating or preventing burn injury |
| CN108129295A (en) * | 2018-01-12 | 2018-06-08 | 云南大学 | A kind of Diterpene derivative and its pharmaceutical composition and purposes |
-
2004
- 2004-11-04 KR KR1020040089372A patent/KR100575253B1/en active IP Right Grant
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101811544B1 (en) | 2016-12-09 | 2017-12-21 | 남종현 | Pain Relief Composition |
| KR101811545B1 (en) | 2016-12-09 | 2017-12-21 | 남종현 | Pharmceutical composition for treating or preventing burn injury |
| WO2018105816A1 (en) * | 2016-12-09 | 2018-06-14 | 남종현 | Pain relief composition |
| WO2018105817A1 (en) * | 2016-12-09 | 2018-06-14 | 남종현 | Pharmaceutical composition for treating or preventing burn injuries |
| CN108129295A (en) * | 2018-01-12 | 2018-06-08 | 云南大学 | A kind of Diterpene derivative and its pharmaceutical composition and purposes |
| CN108129295B (en) * | 2018-01-12 | 2020-09-08 | 云南大学 | Abietane diterpene derivative and pharmaceutical composition and application thereof |
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