KR20210033609A - Especially extraction and use it for application of antithrombotic functional low molecular collagen from Fig - Google Patents
Especially extraction and use it for application of antithrombotic functional low molecular collagen from Fig Download PDFInfo
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- KR20210033609A KR20210033609A KR1020190115089A KR20190115089A KR20210033609A KR 20210033609 A KR20210033609 A KR 20210033609A KR 1020190115089 A KR1020190115089 A KR 1020190115089A KR 20190115089 A KR20190115089 A KR 20190115089A KR 20210033609 A KR20210033609 A KR 20210033609A
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Abstract
Description
본 발명은 무화과로부터 항혈전 기능의 식품 성분을 추출하는 방법 및 그 항혈전성 추출물에 관한 것으로, 더욱 상세하게는 무화과 원료인 미숙과 및 반건조 무화과(완숙과 및 미숙과) 그리고 폐기되는 무화과나무 줄기 무화과 나무 뿌리, 그리고 무화과 잎을 이용하여 분쇄한 다음 유기산 수용액을 첨가하여 실온에서도 손쉽게 추출할 방법으로 고수율로 무화과 겔을 얻을 수 있다. 즉, 기존 물이나 알코올로 추출하는 방법보다 3배 이상의 수율이 향상되고 미숙과 및 반건조 무화과를 이용함으로써 쉽게 잘 여과가 잘되어 공정이 빠르고 또한 무화과를 열수로 추출하여 침전하는데 많은 양의 유기 용매가 들 수 있으나 본 공정에서는 소량의 유기산 및 유기 용매와 산 알칼리 처리로도 최적의 추출방법이 추진되어 고순도와 고수율을 확인할 수 있어서 산업용으로 타당성이 매우 크다 할 수 있다. 그래서 무화과로부터 콜라겐과 같은 응집유도체의 자극에 의해 활성화되어 응집반응을 일으키는 혈소판에 대하여 항응고작용을 나타내는 항혈전 기능을 갖는 무화과를 추출하는 방법 및 그 방법에 따라 추출된 항혈전성 추출물에 관한 것으로 화장품 소재 또는 혈전증 예방 또는 치료용 약학적 조성물 및 피부 질환 치료 소제, 구강 청결제 이용 용도The present invention relates to a method for extracting a food component with antithrombotic function from a fig and an antithrombotic extract thereof, and more particularly, to an unripe fruit and semi-dried fig (ripe fruit and unripe fruit) as a raw material of a fig, and a fig tree to be discarded. Fig gel can be obtained in high yield by pulverizing the stem fig tree root and fig leaf, and then adding an organic acid solution to extract it easily at room temperature. In other words, the yield is improved by more than 3 times compared to the existing method of extraction with water or alcohol, and the process is fast because it is easily filtered well by using unripe fruits and semi-dried figs, and a large amount of organic solvent is used to extract and precipitate figs with hot water. However, in this process, the optimum extraction method is promoted even with a small amount of organic acid, organic solvent and acid alkali treatment, so that high purity and high yield can be ascertained, so it can be said to be highly feasible for industrial use. Therefore, it relates to a method for extracting figs having antithrombotic function that exhibits anticoagulant activity against platelets that are activated by stimulation of aggregation inducers such as collagen and causes agglutination reaction from figs, and an antithrombotic extract extracted according to the method. Use of cosmetic materials or pharmaceutical compositions for preventing or treating thrombosis, as well as cleaning agents for skin disease treatment and mouthwash
정상적인 혈액 순환은 체내에서의 혈액 응고 반응계와 혈전 용해 반응계가 상호 보완적으로 조절되면서 혈액 순환을 쉽게 하며, 이들 중 혈액 응고 반응계의 기작은 혈관 벽에 혈소판이 점착, 응집하여 혈소판 혈전을 형성한 후, 혈액 응고 계가 활성화되어 혈소판 응집기를 중심으로 피브린 혈전이 형성되는 것으로 보고되어 있다.Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolysis reaction system in the body are complementarily regulated. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate to the blood vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombi are formed around the platelet agglutinator.
한편, 피브린 혈전의 생성은 수많은 혈액응고인자들의 여러 단계 반응을 거쳐 피브린 응고에 관여하는 트롬빈이 활성화되어, 최종적으로 피브리노젠으로부터 피브린 단량체를 생성하게 하며, 피브린 단량체들은 칼슘에 의해 중합되어, 혈소판과 내피세포에 결합하게 되며 XIII 인자에 의해 교차 결합한 피브린 폴리머를 형성하면서 영구적인 혈전을 생성하게 된다. 또한, 트롬빈은 혈소판, V 인자, VII 인자들을 활성화시켜 혈액 응고반응을 촉진시키는 등 혈전 생성에 중추적 역할을 하게 된다. 따라서, 트롬빈의 활성 저해물질은 과다한 혈액응고 이상으로 발생하는 다양한 혈전성 질환에 매우 유용한 예방 및 치료제로 사용될 수 있다. 한편 내인성 혈전 생성경로에는 XII 인자, XI 인자, IX 인자, X 인자의 순차적 활성화에 이은 프로트롬빈의 활성화가 최종적으로 트롬빈을 활성화하는 것으로 알려져 혈액응고 인자의 특이적 저해 역시 중요한 혈전성 질환 치료제의 개발 타겟이 되고 있다.On the other hand, the formation of fibrin thrombi causes thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, and fibrin monomers are polymerized by calcium and platelets. It binds to endothelial cells and forms a fibrin polymer cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as very useful prophylactic and therapeutic agents for various thrombotic diseases caused by excessive blood coagulation. Meanwhile, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by the activation of prothrombin, is known to ultimately activate thrombin. Has become.
현재까지 혈전성 질환의 예방과 치료에 헤파린, 쿠마린, 아스피린, 유로키네이즈 등의 다양한 항응고제, 항혈소판제, 혈전용해제 등이 사용되고 있으나, 이들은 가격이 매우 높을 뿐 아니라 출혈성 부작용과 위장장애 및 과민 반응 등으로 그 사용이 한정되고 있는 실정이다.To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but these are not only very expensive, but also bleeding side effects, gastrointestinal disorders and irritability, etc. As a result, its use is limited.
그러나, 천연물인 무화과의 유용 생리활성 연구는 최근 들어 매우 활발하게 진행되고 있으며, 무화과와 관련된 특허 문헌으로는,However, studies on the useful physiological activity of figs, a natural product, have been very actively conducted in recent years, and as a patent document related to figs,
공개특허 등록번호 10-1080994 고지베리, 무화과와 배초향의 열수 추출물을 포함하는 조성물에 관한 것으로, 항산화 활성, 면역증강, 미백, 주름 개선, 콜라젠 분해효소 억제 효과는 있을 수 있으나 고지베리 무화과 및 배초향의 열수 추출에 따른 활성도 저하와 복합물질로 부작용이 수반되는 문제점으로 활용도가 낮다고 볼 수 있으며, 출원번호 10-2017-0029586 : 전자기장 발생 및 제어를 이용한 혈전제거 방법에 따르면, 자성 나노입자를 혈전에 부착시켜 자성이 있는 혈전을 전자기장 발생을 통해 견인하고, 혈관 경로를 통한 전자기장의 전파를 통해 이동시킴으로써, 혈류를 회복시킬 수 있다. 이는 전자 장치로 외적인 적용문제점이 있으며 혈관 내부에 직접 주사제나 개선재로 이용할 수 없는 문제점이 있다 할 수 있다. Patent Registration No. 10-1080994 relates to a composition comprising a hot water extract of Goji berry, fig and pear chorion, and may have antioxidant activity, immunity enhancement, whitening, wrinkle improvement, and collagen decomposing enzyme inhibitory effects, but It can be seen that the utilization is low due to the problem that the activity decreases due to hot water extraction and side effects are accompanied by a complex substance. Application No. 10-2017-0029586: According to the method of removing blood clots using electromagnetic field generation and control, magnetic nanoparticles are attached to blood clots. Thus, the magnetic blood clots are pulled through the generation of an electromagnetic field, and the blood flow can be restored by moving through the propagation of the electromagnetic field through the vascular path. This is an electronic device, and there is an external application problem, and there may be a problem that it cannot be used as an injection agent or an improvement material directly inside the blood vessel.
등록번호 10-0512285: 천연무화과 분리성분을 함유한 약용 비누조성물에 관한 특허는 본 발명자가 직접 심혈을 기울여 무화과나무 잎 열매 전반적인 분리 실험을 하여 성분 스펙트럼까지 확보한 상태로 이를 바탕으로 물성에 대한 신속한 추출방법을 개량하여 본 출원에 응용하고자 하였다. 한국/10-2017-0133212: 회리바람꽃 추출물을 유효성분으로 포함하는 항알레르기용 및 아토피 피부염 개선용 화장료 조성물. 10-2016-0085387 : 생물전환 추출물을 포함하는 아토피 가려움증 개선용 화장료 조성물 및 그 제조방법. 10-2013-0152314:아토피 또는 피부 트러블 예방 및 개선용 조성물. 공개특허 10-2012-0019968 무화과로부터 항혈전 기능의 식품 성분을 함유하는 방법 및 항혈전서 추출물에 관한 연구는 수용액 저온추출방법으로 실시하고 있으나 실제 같은 방법을 적용하여 시도한 결과 열을 가하지 않고는 추출이 안 되며 또한 정제과정에서 에틸아세테이트로 분획 추출하는 것으로 되어있으나 수용액과의 층이 생겨서 추출이 안 되며 특히 아조트로픽 믹스춰(공비 혼합물 현상)으로 불가능하였으며 물 없이 에틸아세테이트로는 추출 불가로 생각함 그래서 본 제조과정에서 언급한 유기산 수용액으로 전처리를 하여 높은 수율로 발명에 따른 최적 조건으로 완성하였다 할 수 있다.Registration No. 10-0512285: Patent for a medicinal soap composition containing a natural fig separating component is a state in which the present inventors directly conduct an experiment on the overall separation of fig tree leaves, and obtain a component spectrum. It was intended to be applied to the present application by improving the extraction method. Korea/10-2017-0133212: A cosmetic composition for anti-allergic and atopic dermatitis improvement comprising a Hoeriwind flower extract as an active ingredient. 10-2016-0085387: A cosmetic composition for improving atopic itching comprising a bioconversion extract and a method for producing the same. 10-2013-0152314: Atopic or skin trouble prevention and improvement composition. A study published in the patent how to contain the food component of the anti-thrombotic functions from fig 10-2012-0019968 and antithrombotic standing extract, but subjected to the aqueous solution of the low-temperature extraction is extraction without application of results attempted by applying physical methods, such as column In addition, it is supposed to extract fractions with ethyl acetate during the purification process, but it cannot be extracted due to the formation of a layer with an aqueous solution. In particular, it was not possible with an azotropic mixture (azeotropic mixture phenomenon), and it was considered impossible to extract with ethyl acetate without water. So, it can be said that the pretreatment was performed with the organic acid aqueous solution mentioned in the present manufacturing process, and it was completed in the optimum conditions according to the invention in high yield.
국내 특허출원(출원번호 10-2010-0093901) : 생약 추출물 또는 이의 유산균 발효물을 포함하는 아토피 피부염의 예방 또는 치료용 조성물로 본 발명과는 제조방법이 다르며, 등록번호 10-1181402: 콜라겐 분말 3 내지 7 중량부; 및 오가피, 노근, 모과, 앵두, 포도씨, 및 저두강으로 이루어진 식용식물혼합물을 용매로 추출 후 감압 농축된 식용식물추출물을 특징으로 하는 어린 콜라겐과 식용식물추출물을 포함하는 떡 조성물 및 면류용 조성물에 관한 것으로 본 발명과는 상이하며 공개특허 10-2015-0014410: 페놀화합물을 유효성분으로 포함하는 항혈전용 조성물에 관한 것은 분자량이 커서 용해도 문제점이 있는 것으로 알려짐. 공개특허 10-2004-0027283: 은 콜라겐을 제조할 수 있는 원료의 다양화와 종래의 기술보다 단순화된 공정으로 식물성 또는 마린 콜라겐 펩타이드를 제조할 수 있는 방법으로 펩타이드 분자량이 200만 넘어도 분자간의 수소결합과 반데르발스힘 쌍극자 능력에 따라 엉킴 현상이 두드러지며 마치 공기 접촉으로 산화가 일어나 떡이 되어 쉽게 사용할 수 없는 단점이 있다 할 수 있다. 그래서 본 발명자도 무화과 효소가 약 35개 아미노산 결합으로 이룬 펩타이드 형태로써 예의 연구한 결과 유기산 수용액처리로 부분 가수분해되는 잇점을 가지므로써 보다 효율적으로 추출할 수 있었다.Domestic patent application (application number 10-2010-0093901): A composition for the prevention or treatment of atopic dermatitis containing a herbal extract or a fermented product thereof, and its manufacturing method is different from the present invention, and registration number 10-1181402: Collagen powder 3 To 7 parts by weight; And the edible plant mixture consisting of Ogapi, Nogeun, Chinese quince, cherry, grape seed, and edible plant extract concentrated under reduced pressure after extracting the edible plant mixture with a solvent. It is different from the present invention and relates to an antithrombotic composition containing a phenolic compound as an active ingredient is known to have a solubility problem due to its high molecular weight. Laid-Open Patent 10-2004-0027283: Diversification of raw materials for producing silver collagen and a method for producing vegetable or marine collagen peptides through a more simplified process than conventional techniques. It can be said that the entanglement phenomenon is remarkable depending on the ability of the bonding and the Van der Waals force dipole, and there is a disadvantage that it cannot be easily used because it is oxidized by air contact and becomes rice cake. Therefore, the inventors also studied fig enzymes in the form of peptides formed by about 35 amino acid bonds, and as a result of intensive research, they were able to extract more efficiently because they had the advantage of being partially hydrolyzed by an aqueous organic acid solution treatment.
이와 관련하여 연구자들은 다양한 천연물로부터의 항혈전 활성을 보고하고 있는데 무화과에서부터 분리한 에탄올 추출물로부터 이러한 활성이 보고되기도 하였다.(Anwarul HG et.al., Journal of Ethnopharmacology119 (2008) 1-5) 그러나, 천연물로부터의 얻게되는 유효성분들은 용매 선택에 따른 극성도(polarity)에 따라서 추출되는 성분들의 종류가 차이가 나게되며 기능성 효과도 달라질 수 있다.In this regard, researchers have reported antithrombotic activity from various natural products, and this activity has also been reported from ethanol extracts isolated from figs (Anwarul HG et.al., Journal of Ethnopharmacology119 (2008) 1-5). In the active ingredients obtained from natural products, the types of the extracted ingredients differ depending on the polarity according to the solvent selection, and the functional effects may also vary.
무화과를 잘라내서 추출하려고 해도 바로 물러져서 형태를 알 수 없을 정도로 죽이되고 목적 화합물을 추출하려고 해도 규조토나 베드 필터를 사용해도 걸러지지 않은 문제점이 있다 할 수 있다. 원심분리를 한다 하여도 많은 양에서 소량 얻어지는 단위로 무화과 수용액 죽을 원심분리를 해도 물과 겔의 수소결합으로 인해서 분리가 깨끗하게 되지 않고 겔 화합물이 떠서 줄을 이루어 경사 법으로 따라서 재차 여과를 해야 하는 문제점이 야기되고 있다. 이런 과정은 이론상 방법을 탈피해서 실제 분리과정에서 확인할 수 있는 문제로 어려움이 있으며 설사 얻어진다 해도 수율이 현저하게 낮아 산업적으로 이용할 가치가 낮다 할 수 있다.Even if the fig is cut and extracted, it is immediately softened and killed to the extent that the shape is unknown. Even if the target compound is attempted to be extracted, there is a problem that it is not filtered even if a diatomaceous earth or a bed filter is used. Even if centrifugation is carried out, the fig aqueous solution is a unit that is obtained from a large amount to a small amount. Even if the porridge is centrifuged, the separation is not clean due to hydrogen bonding between the water and the gel. Is being caused. This process is difficult as a problem that can be confirmed in the actual separation process by breaking away from the theoretical method, and even if it is obtained, the yield is remarkably low, and it can be said that the value of industrial use is low.
그래서 본 발명자는 예의 연구한 결과 무화과 원료인 미숙과 및 반건조 무화과(완숙과 및 미숙과) 그리고 폐기되는 무화과나무 줄기 무화과나무 뿌리, 그리고 무화과 잎을 분쇄하여 유기산 수용액으로 실온에서 추출하여도 회수할 방법을 발견하고 충분히 교반하여 추출한 다음 불용성 무화과를 거르고 회수된 여액을 감압 농축한 다음 소량의 염기성 수용액을 첨가하여 중화시킨 다음 알코올을 첨가하여 생성된 겔 화합물을 거르고 알코올로 수회 세 착하여 얻어진 결과 기존 열수 추출방법보다 70% 이상의 고수율로 순수한 무화과 겔을 얻었다. 따라서, 본 발명자들은 항혈전 및 혈류개선 활성을 나타내는 기능성 식품 소재 개발을 목표로, 무화과 원료 추출물을 조제한 후 인간 혈장과 인간 트롬빈을 이용하여 트롬빈 직접저해에 따른 혈전생성 저해활성을 평가하고, 인간 농축 혈소판을 이용한 무화과의 혈소판 응집저해 활성을 측정하여, 무화과 원료 추출물 및 이의 순차적 분획물에서 매우 우수한 항혈전 활성을 확인하여 본 발명을 완성하였다.Therefore, as a result of intensive research, the present inventors crushed fig raw materials, unripe and semi-dried figs (finished and unripe), and discarded fig tree trunks, fig tree roots, and fig leaves, and recovered even after extraction at room temperature with an aqueous organic acid solution. After discovering a method, extracting it with sufficient stirring, filtering insoluble figs, concentrating the recovered filtrate under reduced pressure, neutralizing it by adding a small amount of basic aqueous solution, filtering the gel compound produced by adding alcohol, and washing it several times with alcohol. A pure fig gel was obtained with a higher yield of 70% or more than the hot water extraction method. Therefore, the present inventors aim to develop a functional food material that exhibits antithrombosis and blood flow improvement activity, after preparing a fig raw material extract, using human plasma and human thrombin to evaluate the thrombiogenesis inhibitory activity according to direct thrombin inhibition, and human concentration. The present invention was completed by measuring the platelet aggregation inhibitory activity of figs using platelets, and confirming very excellent antithrombotic activity in the fig raw material extract and sequential fractions thereof.
그리고 다른 한편으로 랍도바이러스 (Rhabdoviridae) 과의 일원인 소포성 구내염 바이러스 (VSV) 은 비분절화된, 네거티브-센스, 단일 가닥 RNA 게놈을 갖는다. 그의 11 kb 게놈은 바이러스의 5 종의 구조 단백질을 코딩하는 5 개의 유전자를 갖는다; 뉴클레오캡 시드 단백질 (N), 이는 복제된 RNA 의 캡슐화를 위해 화학량론적 양으로 필요함; 인단백질 (P), 이는 RNA-의존성 RNA 중합효소 (L) 의 보조인자임; 매트릭스 단백질 (M) 및 결합 당단백질 (G) (예를 들어, 참고문헌으로 Gallione 등, 1981, Rose 및 Gallione, 1981; Rose 및 Schubert,. 1987 및 Schubert 등, 1985; U.S. 특허 6,033,886; U.S. 특허 6,168,943).And on the other hand, vesicular stomatitis virus (VSV), a member of the family Rhabdoviridae, has an unsegmented, negative-sense, single-stranded RNA genome. Its 11 kb genome has 5 genes encoding 5 structural proteins of the virus; Nucleocap seed protein (N), which is required in stoichiometric amounts for encapsulation of the replicated RNA; Phosphoprotein (P), which is a cofactor of RNA-dependent RNA polymerase (L); Matrix protein (M) and binding glycoprotein (G) (see, for example, Gallione et al., 1981, Rose and Gallione, 1981; Rose and Schubert,.1987 and Schubert et al., 1985; US Patent 6,033,886; US Patent 6,168,943 ).
VSV 는, 가장 일반적으로는 소, 말, 돼지 및 설치류인 각종 포유류 숙주에 옮겨질 수 있는 절지동물 유래 바이러스이다. 인간의 VSV 감염은 일반적인 것이 아니며, 일반적으로 자각증상이 없거나 또는 합병증 없이 3 내지 8 일 동안 동반하는 약한 감기 비슷한 증상을 특징으로 한다. VSV 는 인간 병소로 간주되지 않으므로, VSV 에 대한 기존의 면역성은 인간 집단에서는 드물고, VSV 유래성 벡터의 개발은 면역원성 조성물 및 유전자 요법과 같은 영역에 촛점을 두고 있다. 그리고 구내염은 구강점막에 생기는 염증으로서 구강염이라고도 하며 세균, 바이러스 및 진균 등의 감염에 의한 것이 많으며, 그 외에 영양불량, 호흡기질환, 위장질환, 임신 등으로 저항력이 약해졌을 때도 발생한다. 증상으로는 타액 분비의 증가, 동 통, 구취 및 물집 등이 나타나며, 심한 증상으로는 구내 및 혀족의 궤양 증세가 나타난다. 경미한 증상에서는 피오구타닌 액, 머큐러크롬 혹은 붕사(硼砂)·글리세린 및 2%의 질산은수(窒酸銀水)를 치료부위에 바르는 것으로 치료한다. 그러나 심한 증상에서는 트리암시놀론 아세토니드를 적함으로써 치료가 가능하다.VSV is an arthropod-derived virus that can be transferred to various mammalian hosts, most commonly cattle, horses, pigs and rodents. VSV infection in humans is not common and is usually characterized by mild cold-like symptoms that accompany no asymptomatic or complications for 3 to 8 days. Since VSV is not considered a human lesion, existing immunity to VSV is rare in the human population, and the development of VSV-derived vectors focuses on areas such as immunogenic compositions and gene therapy. And stomatitis is an inflammation that occurs in the oral mucosa, which is also called stomatitis, and is often caused by infections such as bacteria, viruses and fungi, and also occurs when resistance is weakened due to malnutrition, respiratory diseases, gastrointestinal diseases, and pregnancy. Symptoms include increased salivation, pain, bad breath and blisters, and severe symptoms include ulcers in the mouth and tongue. For mild symptoms, it is treated by applying fiogutanine solution, mercurochrome or borax, glycerin and 2% silver nitrate to the treatment area. However, in severe symptoms, treatment is possible by dropping triamcinolone acetonide.
현재 구내염 치료제로서 트리암시놀론 아세토니드를 함유하는 제제로는 연고제(오라메디 연고TM, 동국약품) 및 구강 내 부착정제(아프타치 정TM, 동화약품) 등이 시판되고 있다. 트리암시놀론 아세토니드 연고제는 유효성분을 세틸알코올이나 연고기제 등으로 제형화 한 제품인데 이것은 사 시 면봉이나 가제로 연고를 치료부위에 바르는 제제로서 사하기가 불편하고 또한 치료 부위에 잘 달라붙지 않는 문제점이 있다. 또한 구강 내 부착정제는 에틸셀룰로오스 및 하이드록시프로필 셀룰로스 등의 고분자로 정제화하였으며 정제 표면을 물이나 침으로 약간 팽윤시킨 다음 투여 부위에 부착시켜 약효를 발현하도록 하는 제품이다. 그러나 이 제품은 부착은 잘되어 있으나 약효가 오래 발현하도록 하기에는 구강 내에서 이물감 및 거부감이 느껴지는 등 문제점을 가지고 있다. 따라서, 기존 연고의 불편성이나 구강 내 부착정제의 이물감 및 거부감을 해결하기 위해 새로운 구내염 치료제의 개발이 절실히 요구되며, 용매로는 에탄올을 사용한다. 에탄올은 분사 후 상처 부위를 소독하는 효과를 가지고 있으며 빨리 휘발되어 약제가 빨리 침착되는데 도움을 준다.Currently, as a formulation containing triamcinolone acetonide as a treatment for stomatitis, ointments (Olamedi OintmentTM, Dongguk Pharm) and oral tablets (Aptachi TabletTM, Donghwa Pharm) are commercially available. Triamcinolone acetonide ointment is a product formulated with active ingredients such as cetyl alcohol or soft meat. This is a formulation in which the ointment is applied to the treatment area with a strabismus swab or gauze. It is inconvenient to use and does not stick well to the treatment area. have. In addition, oral tablets are tableted with polymers such as ethyl cellulose and hydroxypropyl cellulose, and the surface of the tablet is slightly swollen with water or saliva, and then attached to the administration site to express the medicinal effect. However, although this product is well attached, it has problems such as feeling foreign bodies and feelings of rejection in the oral cavity in order for the drug to develop for a long time. Therefore, in order to solve the discomfort of the existing ointment or the feeling of foreign body and rejection of the oral tablet, it is urgently required to develop a new treatment for stomatitis, and ethanol is used as a solvent. Ethanol has the effect of disinfecting the wound after spraying, and it volatilizes quickly, helping to deposit the drug quickly.
본 발명의 조성물은 대롱이 달린 분사제기에 넣어서 제조하여 분사가 가능하다. 따라서 이 조성물을 치료부위에 분사하면 대롱에 의해 전체 구강이 아니라 구내염 발생 부위에만 분사되어 기존 트리암시놀론 아세토니드 제제에 비해 편리성 및 생체부착성이 우수하며 또한 천연물이므로 그대로 먹을 수 있으며 다른 화합물에 비해서 독성이 없는 장점이 있다 할 수 있다. 그리고 구내염 발생부위에서 에탄올 용매가 순간적으로 살균작용을 할 뿐만 아니라 이어서 즉시 휘발함으로써 분자성 피막을 형성하여 무화과 알콜성 에틸아세테이트 분획 추출물이 구내염 발생부위에서 기존 약제에 비해서 천연물임에도 불구하고 탁월하게 약효 작용을 나타낸다.The composition of the present invention can be sprayed by being prepared by putting it in a propellant equipped with a large bowl. Therefore, if this composition is sprayed on the treatment area, it is sprayed only on the site of stomatitis, not the entire oral cavity by the daerong, and has superior convenience and bioadhesiveness compared to the existing triamcinolone acetonide formulation. It is also a natural product, so it can be eaten as it is. It can be said that there are advantages to this. In addition, the ethanol solvent not only instantly sterilizes at the site where stomatitis occurs, but also immediately volatilizes to form a molecular film, so that the fig alcoholic ethyl acetate fraction extract is superior to the existing drugs at the site where the stomatitis occurs, although it has an excellent medicinal effect. Represents.
본 발명의 제제화 방법은 해당 업계 종사자들에게 잘 알려진 방법에 의하며, 방향제 등 통상의 부형제 및 희석제 등을 포함하는 약제 학적 형태로 제조할 수 있다.The formulation method of the present invention is a method well known to those in the relevant industry, and can be prepared in a pharmaceutical form including conventional excipients and diluents such as fragrances.
이하, 본 발명의 조성물을 하기 실시예에 의해 더욱 상세히 설명하는바, 본 발명의 조성물은 하기의 실시예에 국한되는 것은 아니다.Hereinafter, the composition of the present invention will be described in more detail by the following examples, but the composition of the present invention is not limited to the following examples.
본 발명은 상기와 같은 종래 기술의 문제점을 해결하기 위하여 도출된 것으로서, 본 발명은 무화과 원료인 미숙과 및 반건조 무화과(완숙과 및 미숙과) 그리고 폐기되는 무화과나무 줄기 무화과나무 뿌리, 그리고 무화과 잎을 이용하여( 이하 무화과 원료로 칭한다)올 선택하여 유효성분으로 함유하는 혈전증의 예방 또는 치료용 약학적 조성물을 제공한다. 무화과 원료를 선택적으로 선정하여 잘게 절단하고 추출 용매로 유기산 수용액을 사용하여 실온에서 유용한 수용성 성분들을 추출하는 단계, 추출용액을 거르고 불용성 성분을 제거하는 단계, 회수된 용액을 감압농축을 하여 포화용액으로 한 다음 소량의 염기성 묽은 용액을 첨가하여 중화시킨 다음 에탄올 넣고 세척한 다음 거르고 알코올 용액으로 여러 차례 씻어 무화과로부터 항혈전성 성분을 추출하는 방법 및 항혈전 효과가 우수한 무화과의 항혈전성 추출물에 관한 것이다. 상기 무화과의 항혈전성 추출물은 in vitro 실험에서 임피던스법(Impedencemethod)을 이용한 항혈소판 응집능을 측정한 결과 매우 우수한 항혈전 효과를 나타내었다.The present invention was derived to solve the problems of the prior art as described above, the present invention is the fig raw material immature and semi-dried figs (ripe and unripe), and discarded fig tree stem fig tree root, and fig leaves By using (hereinafter referred to as a fig raw material) to provide a pharmaceutical composition for preventing or treating thrombosis containing as an active ingredient. Selecting the fig raw material selectively, cutting it finely, extracting useful water-soluble components at room temperature using an organic acid aqueous solution as an extraction solvent, filtering the extraction solution to remove insoluble components, and concentrating the recovered solution under reduced pressure to obtain a saturated solution. Then, a small amount of basic dilute solution is added to neutralize it, then put in ethanol, washed, filtered and washed several times with an alcohol solution to extract antithrombotic components from figs, and to antithrombotic extracts of figs with excellent antithrombotic effect. . The antithrombotic extract of the fig showed very excellent antithrombotic effect as a result of measuring the antiplatelet aggregation ability using the impedance method in an in vitro experiment.
본 발명에서 해결하고자 하는 과제는 식용 및 약용으로 이용되고 있는 무화과 원료 추출물을 유효성분으로 함유하는 인간 트롬빈 직접저해, 프로트롬빈 저해 및 내인성 혈액응고인자 저해에 따른 혈전성 질환의 예방 또는 치료용 약학적 조성물 및 상기 추출물을 포함하는 것으로 항혈전성 효과가 우수하면서도 부작용을 일으키지 않는 유효성분을 추출하는 방법 및 그 추출법에 따라 추출된 항혈전성 추출물을 제공하는 것을 목적으로 하나 또한 구내염에 적용하여 효능을 검진한 결과 효과가 화학합성제 제보다 더 우수함을 확인하여 활용방법 등을 나타내고자 하였다.The problem to be solved in the present invention is a pharmaceutical composition for the prevention or treatment of thrombotic diseases caused by direct inhibition of human thrombin, inhibition of prothrombin, and inhibition of endogenous coagulation factors containing fig raw material extracts used for edible and medicinal purposes And a method for extracting an active ingredient that has excellent antithrombotic effect and does not cause side effects by containing the above extract, and to provide an antithrombotic extract extracted according to the extraction method, but also applied to stomatitis to examine the efficacy As a result, it was confirmed that the effect was better than that of the chemical synthesis agent, and it was intended to show the method of application.
상술한 목적을 달성하기 위한 본 발명의 특징에 의하면,According to the features of the present invention for achieving the above object,
본 발명은 무화과 원료로부터 항혈전 기능의 콜라겐 저분자 효율적 추출방법 및 그 용도에 관한 것으로서,The present invention relates to a method for efficient extraction of low-molecular collagen with antithrombotic function from a fig raw material and its use,
i. 무화과(미숙과 열매, 반건조 무화과) 선택적 분쇄단계( 100), ii.유기산 열수추출 및 여과 단계 (200),III.추출용액 회수, 중화 및 감압농축 후 포화용액으로제조 단계 단계(300), iv.알코올 세척 후 겔화 단계(400), v.여과 및 알코올 수회 세척단계 단계(500), vi.무화과 Gel-1 회수 단계(600)로 구성된 제조방법을 통해 건조 무화과를 제조한다.i. Figs (unripe fruits, semi-dried figs) selective pulverization step (100), ii. organic acid hot water extraction and filtration step (200), III. extraction solution recovery, neutralization and concentration under reduced pressure and then production step step (300) as a saturated solution, iv. After alcohol washing, to prepare a dried fig through a manufacturing method consisting of a
본 발명의 혈전증의 예방 또는 치료용 약학적 조성물 및 건강 기능 식품의 유효성분으로서의 무화과 원료 추출물은 콜라겐(collagen)과 같은 응집유도체의 자극에 의해 응집반응을 일으키는 혈소판의 활성화를 방지함으로 이에 따른 혈액응고를 막아 혈관내 혈전의 생성을 억제할 수 있는 것이다. 또한 무화과 수확 후 폐기되는 잎을 이용하므로 불용 자원의 재이용 및 환경 보존에 기여할 수 있는 장점이 있다.The fig raw material extract as an active ingredient in the pharmaceutical composition for preventing or treating thrombosis and the health functional food of the present invention prevents the activation of platelets that cause an agglutination reaction by stimulation of an aggregation inducer such as collagen, thereby causing blood coagulation. It can prevent the formation of blood clots in blood vessels. In addition, since it uses leaves that are discarded after harvesting figs, it has the advantage of contributing to the reuse of unused resources and preservation of the environment.
본 발명의 혈전증의 예방 또는 치료용 약학적 조성물 및 건강 기능 식품의 유효성분으로서의 무화과 원료 추출물은, 우수한 혈액응고인자 저해 효과에 의한 항혈전 활성을 나타내는 에틸아세테이트 분획은 혈전 생성을 효율적으로 억제할 수 있는 효과가 있으며, 혈행개선을 통해 허혈성 뇌졸중 및 출혈성 뇌졸중과 같은 혈전증의 예방 및 치료용으로 사용할 수 있는 뛰어난 효과가 있다. 특히, 본 발명의 무화과 원료 추출물은 급성경구독성이 나타나지 않으며, 열 안정성이 우수하고, pH 2의 산성조건 및 혈장 내에서도 인간 트롬빈 직접저해, 프로트롬빈 저해, 혈액응고인자 저해 효과의 손실이 나타나지 않아, 추출액, 분말, 환, 정 등의 다양한 형태로 가공되어 상시 복용이 가능한 형태로 조제할 수 있는 뛰어난 효과가 있으므로 제약 산업 및 식품 산업상 매우 유용한 발명으로 더 나아가서 무화과의 소비를 안정적으로 촉진할 수 있어서 무화과 재배 농가의 소득을 증대시킬 수 있는 이점이 있을 것으로 예상합니다.The fig raw material extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis and the health functional food of the present invention, the ethyl acetate fraction that exhibits antithrombotic activity due to an excellent blood coagulation factor inhibitory effect can effectively inhibit thrombus formation. It has an excellent effect that can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In particular, the fig raw material extract of the present invention does not exhibit acute oral toxicity, has excellent thermal stability, and does not directly inhibit human thrombin, inhibit prothrombin, and inhibit blood coagulation factor even in acidic conditions of pH 2 and plasma. Since it has an excellent effect that it can be prepared in a form that can be taken at all times by being processed into various forms such as powder, pill, and tablet, it is a very useful invention in the pharmaceutical industry and food industry, and it is possible to stably promote the consumption of figs. It is expected that there will be benefits that can increase the income of growers.
도 1 은 추출 실험 공정 process: 무화과잎을 추출하는 추출공정을 나타내고( 기타 무화과 원료를 선택에 따라 각각 병행하여 같은 공정을 이용하여 수득함),
도 2는 미숙과 무화과, 반건조무화과 추출공정을 나타내고,
도 3 은 무화과 줄기 및 무화과 뿌리를 추출하는 추출공정을 나타내고,
도 4 는 무화과 잎 분쇄 추출공정을 나타냄1 shows the extraction experiment process process: the extraction process of extracting fig leaves (other fig raw materials are obtained using the same process in parallel, respectively, depending on the selection),
2 shows the extraction process of unripe figs and semi-dried figs,
3 shows an extraction process for extracting fig stems and fig roots,
4 shows a fig leaf crushing extraction process
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 무화과 원료 추출물을 함유하는 인간 트롬빈 직접저해, 프로트롬빈 저해 및 내인성 혈액응고인자 저해 활성을 갖는 혈전증 예방 또는 치료용 약학적 조성물과 건강 기능 식품에 관한 것이다. 더욱 상세하게는, 본 발명의 발명자들은 일정 방법으로 수득한 무화과 원료 추출물로부터 항혈전 활성 성분을 회수하였고, 이러한 성분은 경구 급성독성이 나타나지 않으면서, 열 안정성과 산 안정성이 우수한 특징을 가짐을 확인함으로써 상기 추출물을 혈전증의 예방 또는 치료용 약학적 조성물 및 건강 기능 식품으로 활용하고자 하였다.The present invention relates to a pharmaceutical composition for preventing or treating thrombosis having direct inhibition of human thrombin, prothrombin inhibition, and endogenous coagulation factor inhibitory activity, and a health functional food containing a fig raw material extract. More specifically, the inventors of the present invention recovered the antithrombotic active ingredient from the fig raw material extract obtained by a certain method, and these ingredients did not show oral acute toxicity, and confirmed that it has excellent characteristics of thermal stability and acid stability. By doing so, it was intended to use the extract as a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis.
구체적으로, 본 발명자들은 무화과를 이용하여 혈전증의 예방 또는 치료용 약학적 조성물 및 건강 기능 식품을 개발하기 위하여 무화과의 에탄올 추출물을 조제하고, 이를 순차적 유기용매 분획하여 핵산 분획물, 에틸아세테이트 분획물, 부탄올 분획물 및 물 잔류물 등을 수득하고, 이를 각각 인간 혈장과 인간 트롬빈에 대한 트롬빈 직접저해(Thrombin Time), 프로트롬빈 저해(Prothrombin Time) 및 활성부분 트롬보플라스틴 타임(activated Partial Thromboplastin Time: aPTT), 혈소판 응집저해능을 평가하였다. 그 결과, 무화과 에탄올 추출물에서 트롬빈 타임,프로트롬빈 타임 및 활성부분 트롬보플라스틴 타임 연장 활성은 거의 인정되지 않았으나, 이의 유기용매 분획물 중 특히, 에틸아세테이트 분획에서는 아스피린(시판 항혈전제)보다 강력한 트롬빈 타임 연장효과 및 아스피린과 유사한 프로트롬빈 타임, 활성부분 트롬보플라스틴 타임 연장효과, 혈소판 응집저해 효과를 확인함으로써 본 발명을 완성하였다.Specifically, the present inventors prepared ethanol extracts of figs to develop pharmaceutical compositions and health functional foods for the prevention or treatment of thrombosis using figs, and sequentially fractionated with organic solvents to obtain nucleic acid fractions, ethyl acetate fractions, butanol fractions. And water residues, and the like, respectively, to human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time) and active partial thromboplastin time (activated Partial Thromboplastin Time: aPTT), platelets The ability to inhibit aggregation was evaluated. As a result, the thrombin time, prothrombin time, and active portion of the thrombin time prolonging activity in the fig ethanol extract were hardly recognized, but in the organic solvent fraction, especially the ethyl acetate fraction, the thrombin time more powerful than aspirin (commercial antithrombotic agent). The present invention was completed by confirming the prolonging effect and prothrombin time similar to that of aspirin, the active part thromboplastin time prolonging effect, and platelet aggregation inhibitory effect.
이와 같은 무화과의 효과를 확인하기 위하여, 본 발명의 발명자들은 무화과 원료 추출물 및 유기용매 분획물을 제조하고, 추출물 및 분획물의 인간트롬빈 직접저해 효과,프로트롬빈 저해효과, 혈액응고인자(VIII 인자, IX 인자, XI 인자 및 XII 인자) 저해에 의한 활성부분 트롬보플라스틴 타임 연장 효과를 평가하여, 본 발명의 상기 추출물 등이 상업적으로 이용되고 있는 항혈전제 아스피린(상품명: 프로텍트)보다 우수한 항혈전 활성을 가짐을 확인하고, 인간 적혈구 용혈활성이 나타나지 않으며, 상기 물질의 열 및 산 안정성을 조사한 다음, 상기 물질을 흰쥐에 경구투여하여 급성독성검사를 실시함으로써 상기 추출물 또는 분획물이 인체 독성이 거의 없음을 확인하였다. 따라서, 본 발명은 무화과 원료 추출물을 유효성분으로 함유하는 혈전증 예방 또는 치료용 약학적 조성물 및 건강 기능식품을 제공하는 것을 특징으로 한다.In order to confirm the effect of such figs, the inventors of the present invention prepared a fig raw material extract and an organic solvent fraction, and the direct inhibitory effect of human thrombin, prothrombin inhibitory effect, blood coagulation factor (factor VIII, factor IX, By evaluating the effect of prolonging the active part thromboplastin time by inhibition of factor XI and factor XII), the extract of the present invention has superior antithrombotic activity than a commercially used antithrombotic agent aspirin (brand name: Protect). Was confirmed, human red blood cell hemolytic activity did not appear, and the heat and acid stability of the substance were investigated, and then the substance was orally administered to rats to conduct an acute toxicity test, thereby confirming that the extract or fraction had little toxicity to the human body. . Accordingly, the present invention is characterized by providing a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing the fig raw material extract as an active ingredient.
본 발명의 무화과 원료 추출물을 포함하는 약학적 조성물은 각각의 사용 목적에 맞게 통상의 방법에 따라 산제,과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사용액의 주사제 등 다양한 형태로 제형화 하여 사용할 수 있으며, 경구 투여하거나 정맥 내, 복강 내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다. 또한, 본 발명의 약학적 조성물은 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 더 포함할 수도 있다.The pharmaceutical composition containing the fig raw material extract of the present invention is formulated according to a conventional method according to the purpose of each use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms such as injections, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.
바람직한 구체 예로서, 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 바람직한 구체 예로서, 본 발명의 약학적 조성물에서 무화과 원료 추출물의 유효량은 환자의 나이, 성별, 체중에 따라달라질 수 있으며, 일반적으로는 체중 kg 당 1 내지 5,000 mg, 바람직하게는 100 내지 3,000 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다. 본 발명의 약학적 조성물은 다양한 경로를 통하여 대상에 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내(intracerebroventricular) 주사에 의해 투여될 수 있다. 본 발명의 구체적인 방법을 실시 예를 통하여 더욱 상세하게 설명한다. 하기 실시 예는 본 발명의 바람직한 하나의 구체예일 뿐이며, 본 발명의 권리범위가 하기 실시예의 범위로 한정되는 것은 아니다.As a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and as a preferred embodiment, the effective amount of the fig raw material extract in the pharmaceutical composition of the present invention is the age and sex of the patient. , It may vary depending on the body weight, and in general, 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method. The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection. The specific method of the present invention will be described in more detail through examples. The following examples are only one preferred specific example of the present invention, and the scope of the present invention is not limited to the scope of the following examples.
이하 본 발명을 실시 예를 통해 보다 상세하게 설명하고자 한다.Hereinafter, the present invention will be described in more detail through examples.
[실시예 ][Example]
실시예 1: 무화과 원료 중에서 미숙과 및 반건조 무화과(완숙과 및 미숙과)를 분쇄하고 추출 용매로 유기산 용액으로 물과 구연산과의 부피비율이(0.01~0.5 : 1)인 용액을 사용하여 상온에서 수용성 성분들을 추출하는 단계, 추출용액을 거르고 감압 농축하여 포화용액으로 한 다음 냉각시킨 후 소량의 염기성 화합물을 이용하는 것으로 약염기인 묽은 2N-탄산수소나트륨 용액을 첨가하여 침전물을 거르고 알코올 용액 또는 에틸아세테이트로 신속하게 세척한 다음 분리하여 각각 냉동건조 시키거나 이를 별도의 알코올 용액에 침적시켜 보관하면서 혈전증 예방 또는 치료용 약학적 조성물을 제조하였다.Example 1: Unripe fruit and semi-dried figs (ripe fruit and unripe fruit) are pulverized in fig raw materials, and using a solution having a volume ratio of water and citric acid as an organic acid solution as an extraction solvent (0.01 to 0.5: 1) at room temperature Extracting water-soluble components from the extract, filtering the extraction solution, concentrating under reduced pressure to make a saturated solution, cooling, and using a small amount of basic compound. Add dilute 2N-sodium hydrogen carbonate solution, which is a weak base, to filter the precipitate, and then an alcohol solution or ethyl acetate. It was washed quickly with and then separated and freeze-dried, or immersed in a separate alcohol solution and stored to prepare a pharmaceutical composition for preventing or treating thrombosis.
실시예 2: 무화과 원료 중에서 무화과 뿌리 및 줄기 전체를 분쇄하고 추출 용매로 유기산 용액으로 물과 구연산과의 부피비율이 (0.01~0.5 : 1)인 용액을 사용하여 상온에서 수용성 성분들을 추출하는 단계, 추출용액을 거르고 감압 농축하여 포화용액으로 한 다음 냉각시킨 후 소량의 염기성 화합물을 이용하는 것으로 약염기인 묽은 2N-수산화나트륨용액을 첨가하여 침전물을 거르고 알코올 용액 또는 에틸아세테이트로 신속하게 세척한 다음 분리하여 각각 냉동건조 시키거나 이를 별도의 알코올 용액에 침적시켜 보관하면서 혈전증 예방 또는 치료용 약학적 조성물을 제조하였다.Example 2: A step of pulverizing the whole fig roots and stems from fig raw materials and extracting water-soluble components at room temperature using a solution having a volume ratio of water and citric acid (0.01 to 0.5: 1) as an organic acid solution as an extraction solvent, Filter the extract solution, concentrate under reduced pressure to make a saturated solution, cool, and use a small amount of basic compound. Add dilute 2N-sodium hydroxide solution, which is a weak base, to filter the precipitate, wash quickly with alcohol solution or ethyl acetate, and then separate and separate each A pharmaceutical composition for preventing or treating thrombosis was prepared by lyophilizing or immersing it in a separate alcohol solution and storing it.
실시예 3: 무화과 원료 중에서 무화과 잎을 분쇄하고 추출용매로 유기산용액으로 물과 구연산과의 부피비율이 (0.01~0.5 : 1)인 용액을 사용하여 상온에서 수용성 성분들을 추출하는 단계, 추출용액을 거르고 감압 농축하여 포화용액으로 한 다음 냉각시킨 후 소량의 염기성 화합물을 이용하는 것으로 약염기인 묽은 수산화 칼륨 용액을 첨가하여 침전물을 거르고 알코올 용액 또는 에틸아세테이트로 신속하게 세척한 다음 분리하여 각각 냉동건조시키거나 이를 별도의 알코올 용액에 침적시켜 보관하면서 혈전증 예방 또는 치료용 약학적 조성물을 제조하였다.Example 3: A step of extracting water-soluble components at room temperature by pulverizing fig leaves from fig raw materials and using a solution having a volume ratio of water and citric acid (0.01 to 0.5: 1) as an organic acid solution as an extraction solvent. Filter, concentrate under reduced pressure to make a saturated solution, cool, and use a small amount of basic compound. Add diluted potassium hydroxide solution, which is a weak base, to filter the precipitate, quickly wash it with alcohol solution or ethyl acetate, and separate and freeze-dry them, respectively. A pharmaceutical composition for preventing or treating thrombosis was prepared while immersed in a separate alcohol solution and stored.
실시예 4: 무화과 원료 추출물 및 유기용매 분획물의 조제 2019년 전북 고창에서 재배된 무화과를 구입하여 무화과에 대해 약 10배 부피의 수용성 유기산을 가하고 상온에서 5 시간씩 2 회 추출하였다. 이후, 에탄올 추출물을 증류수에 현탁한 후, 핵산, 에틸아세테이트, 부탄올로 순차적 분획하였으며, 최종적으로 물 잔류물을 얻었다. 무화과 에탄올 추출물의 핵산 분획물, 에틸아세테이트 분획물, 부탄올 분획물 및 물 잔류물의 수득율은 각각 1.62 %, 2.42 %, 18.98 % 및 78.33 %였다. 특히 에틸아세테이트 분획물의 양이 매우 적게 나타났으며, 이는 무화과 100 g에 대해 에틸아세테이트 분획물은 약 46 mg 을 얻게 됨을 의미하며, 에틸아세테이트 분획은 상당히 정제된 것으로 판단된다.Example 4: Preparation of Fig Raw Material Extracts and Organic Solvent Fractions Figs cultivated in Gochang, Jeollabuk-do in 2019 were purchased, about 10 times the volume of water-soluble organic acid was added to the figs, and extracted twice at room temperature for 5 hours. Thereafter, the ethanol extract was suspended in distilled water, followed by sequential fractionation with nucleic acid, ethyl acetate, and butanol, and finally a water residue was obtained. The yields of the nucleic acid fraction, ethyl acetate fraction, butanol fraction and water residue of the fig ethanol extract were 1.62%, 2.42%, 18.98% and 78.33%, respectively. In particular, the amount of the ethyl acetate fraction was very small, which means that about 46 mg of the ethyl acetate fraction was obtained for 100 g of figs, and the ethyl acetate fraction was judged to be considerably purified.
다음으로, 무화과 원료 추출물의 항혈전 활성을 평가하였다. 항혈전 활성은 기존에 보고된 방법에 따라 평가하였으며(Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; 류등 2010. J. Life Science, 20. 922-928), 트롬빈 타임, 프로트롬빈 타임과 에이피티티 타임을 측정하였다. 혈장은 지원자의 전혈로부터 조제하였으며, 채혈 후 즉시 4 ℃에서 5,000 g로 5 분 동안 원심분리하여 혈장을 분리하고 냉동한 상태로 보관하였으며(신선동결혈장), 필요시 상온에서 해동하여 사용하였다. 트롬빈 타임, 프로트롬빈 타임과 에이피티티 측정법은 다음과 같은 과정으로 수행되었다.Next, the antithrombotic activity of the fig raw material extract was evaluated. Antithrombotic activity was evaluated according to previously reported methods (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and APT time were measured. Plasma was prepared from the volunteer's whole blood, and immediately after blood collection, it was centrifuged at 4°C for 5 minutes to separate the plasma and stored in a frozen state (fresh frozen plasma), and if necessary, thawed at room temperature and used. Thrombin time, prothrombin time, and AP measurement were performed as follows.
트롬빈 타임(Thrombin Time)[0059] 37 ℃에서 0.5 U 트롬빈 (Sigma Co., USA) 50 μl와 20 mM CaCl2 50 μl, 다양한 농도의 시료 추출액 10 μl를 Amelung coagulometer KC-1A(Japan)의 튜브에 혼합하여 2 분간 반응시킨 후, 혈장 100 μl를 첨가한 후 혈장이 응고될 때까지의 시간을 측정하였다. 대조로는 아스피린(Sigma Co., USA)을 사용하였으며, 용매 대조구로는 시료 대신 DMSO를 사용하였다. DMSO의 경우 32.1 초의 응고시간을 나타내었다. 열 안정성 측정의 경우에는 다양한농도의 시료용액을 100 ℃에서 30 분간 열처리하고, 실온에서 1 시간 방냉한 후, 잔존활성을 측정하였다. 트롬빈 저해 효과는 3 회 이상 반복한 실험의 평균치로 나타내었으며, 시료 첨가시의 응고시간을 용매 대조구의 응고시간으로 나눈 값에 100을 곱하여 %로 나타내었다. 프로트롬빈 타임( prothrombin time) 표준혈장(MD Pacific Co., China) 70 μl와 다양한 농도의 시료액 10 μl를 Amelung coagulometer KC-1A(Japan)의 튜브에 첨가하여 37 ℃에서 3 분간 가온 후, 130 μl의 PT reagent를 첨가하고 혈장이 응고될 때까지의 시간을 3 회 반복한 실험의 평균치로 나타내었다. 대조로는 아스피린(Sigma Co., USA)을 사용하였으며, 용매 대조구로는 시료 대신 DMSO를 사용하였다. DMSO의 경우 18.1 초의 응고시간을 나타내었다. aPTT (activated Partial Thromboplastin Time) 혈장 100 μl와 다양한 농도의 시료 추출액 10 μl를 Amelung coagulometer KC-1A(Japan)의 튜브에 첨가하여 37 ℃에서 3 분간 가온한 후, 50 μl의 aPTT reagent(Sigma, ALEXINTM)를 첨가하고 다시 37 ℃에서 3 분간 배양하였다.Thrombin Time [0059] 50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl2, and 10 μl of sample extracts of various concentrations at 37°C were placed in a tube of Amelung coagulometer KC-1A (Japan). After mixing and reacting for 2 minutes, 100 μl of plasma was added and the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 32.1 seconds. In the case of thermal stability measurement, sample solutions of various concentrations were heat-treated at 100° C. for 30 minutes, allowed to cool at room temperature for 1 hour, and then the residual activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the coagulation time at the time of sample addition was divided by the coagulation time of the solvent control and multiplied by 100 and expressed as %. Add 70 μl of prothrombin time standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations to the tube of Amelung coagulometer KC-1A(Japan), warm at 37°C for 3 minutes, and then 130 μl The time until the plasma coagulation after addition of the PT reagent of was expressed as the average value of the experiment in which 3 times were repeated. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 18.1 seconds. 100 μl of aPTT (activated Partial Thromboplastin Time) plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A(Japan), heated at 37°C for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXINTM). ) Was added and incubated again at 37° C. for 3 minutes.
이후 50 μl CaCl2(35 mM)을 첨가한 후 혈장이 응고될 때까지의 시간을 측정하였다. 용매 대조구로는 시료 대신 DMSO를 사용하였으며, 이 경우 55.1 초의 응고시간을 나타내었다. aPTT의 결과는 3 회 반복한 실험의 평균치로 나타내었으며, 시료 첨가시의 응고시간을 용매 대조구의 응고시간으로 나눈 값에 100을 곱하여 %로 나타내었다. 상기 설명된 트롬빈 타임, 프로트롬빈 타임과 에이피티 타임 측정법에 따른 항혈전 측정 결과는 다음 표 2에 나타내었다. 대조구로 사용된 아스피린은 5 mg/ml 농도에서 우수한 항혈전 효과를 나타내었으며, 무화과의 뿌리전체부분, 껍질 부분 및 거피후 식용부분의 에탄올 추출물은 5 mg/ml 농도까지 트롬빈 타임, 프로트롬빈 타임, 에이피 타임을 크게 변화시키지는 않았다. 따라서 무화과 원료 추출물을 조제하여 항혈전 활성을 평가하는 경우 항혈전 활성을 검출하기는 어려울 것으로 판단된다.Thereafter, 50 μl CaCl2 (35 mM) was added and the time until plasma coagulated was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 55.1 seconds. The result of aPTT was expressed as the average value of the experiment repeated three times, and the coagulation time at the time of sample addition was divided by the coagulation time of the solvent control and multiplied by 100 to express a %. The antithrombosis measurement results according to the above-described thrombin time, prothrombin time, and AP time measurement methods are shown in Table 2 below. Aspirin used as a control showed excellent antithrombotic effect at a concentration of 5 mg/ml, and ethanol extracts from the whole root, skin, and edible parts of figs were used as thrombin time, prothrombin time, and AP up to a concentration of 5 mg/ml. It didn't change the time significantly. Therefore, it is judged that it is difficult to detect the antithrombotic activity when the fig raw material extract is prepared and the antithrombotic activity is evaluated.
실시예 5: 무화과 원료 추출물의 순차적 유기용매 분획물의 성분 분석 및 항혈전 활성 평가Example 5: Component analysis and antithrombotic activity evaluation of sequential organic solvent fractions of fig raw material extract
실시예 1의 무화과 에탄올 추출물을 용매 분획한 후, 분획물의After solvent fractionation of the fig ethanol extract of Example 1, the fraction
표 2.무화과 재료 중 줄기 뿌리 부분 전체, 미숙과 및 반건조무화과 부분, 가식부분 껍질, 수용액 잔유물의 항혈전 활성Table 2. Antithrombotic activity of whole stem roots, unripe and semi-dried figs, edible skins, and aqueous solution residues among fig materials
성분 및 항혈전 활성을 조사하였다. 분획물의 총 플라보노이드, 총 폴리페놀, 총당 및 환원당 함량을 측정한 결과는 표 3과 같다.Components and antithrombotic activity were investigated. Table 3 shows the results of measuring the total flavonoid, total polyphenol, total sugar and reducing sugar content of the fraction.
표 3.무화과 재료 유기용매 추출물의 분획 효율 및 분석Table 3: Fractionation efficiency and analysis of fig material organic solvent extract
무화과 에탄올 추출물의 유기용매 분획시 물 잔류물이 대부분을 차지하였으며, 일부가 부탄올 분획으로 이행되었으며, 핵산 분획과 에틸아세테이트 분획은 각각 1.62% 및 0.42%로 매우 낮은 함량을 보였다. 물 분획물과 부탄올 분획물은 대부분이 당류로 구성되어 있었으며, 2.27-3.51 mg/g의 낮은 폴리페놀 함량 및 0.57-8.16 mg/g의 플라보노이드 함량을 보였다. 핵산 분획물의 경우에도 약 16%의 당을 함유하고 있었으며, 2.48 mg/g의 폴리페놀함량 및 3.25 mg/g의 비교적 낮은 플라보노이드 함량을 보였다. 그러나 에틸아세테이트 분획물에서는 당 함량은8.5% 수준이었으나, 4.59 mg/g의 폴리페놀 및 147.36 mg/g의 매우 높은 플라보노이드 함량을 확인하였다. 이러한 결과로 미루어 볼때 무화과의 에틸아세테이트 분획은 무화과가 가진 대부분의 플라보노이드를 가지고 있는 것으로 판단되며, 강력한 항산화력과 유용 생리활성을 나타낼 것으로 예상하였으며, 실제 강력한 항산화력을 나타내었다.In the organic solvent fractionation of the fig ethanol extract, water residues accounted for most, and some of them were transferred to the butanol fraction, and the nucleic acid fraction and ethyl acetate fraction showed very low content of 1.62% and 0.42%, respectively. Most of the water and butanol fractions were composed of sugars, and showed low polyphenol content of 2.27-3.51 mg/g and flavonoid content of 0.57-8.16 mg/g. The nucleic acid fraction also contained about 16% sugar, and showed a polyphenol content of 2.48 mg/g and a relatively low flavonoid content of 3.25 mg/g. However, in the ethyl acetate fraction, the sugar content was at the level of 8.5%, but it was confirmed that the polyphenol content of 4.59 mg/g and the flavonoid content of 147.36 mg/g were very high. From these results, it is judged that the ethyl acetate fraction of figs has most of the flavonoids of figs, and is expected to show strong antioxidant power and useful physiological activity, and actually shows strong antioxidant power.
분획물의 항혈전 활성을 평가한 결과, 다음 표 4에 나타낸 바와 같이 핵산 분획은 5 mg/ml 농도에서 15 배 증가된 트롬빈 타임 및 2.5 배 증가된 프로트롬빈 타임을 나타내어 우수한 트롬빈 저해활성을 나타내었다. 그러나 임상에서 사용되고 있는 아스피린과 동일농도에서 비교하면 상대적으로 약한 저해 활성을 나타내었다. 이는 부탄올 분획물 및 물 잔류물에서도 유사하였으며, 아스피린과 비교하면 미약한 항혈전을 확인하였다. 그러나 에틸아세테이트 분획물의 경우, 5 mg/ml 농도에서 트롬빈 타임, 프로트롬빈 타임 및 에이피티 타임 모두 15 배 이상증가된 연장효과를 나타내어 매우 강력한 항혈전 활성을 확인하였다. 특히 실험에 사용한 아스피린 1.5 mg/ml보다 낮은 1.25 mg/ml 농도에서도 15 배 이상 증가된 트롬빈 타임 연장효과 및 1.47 배, 1.5 배의 연장된 프로트롬빈 타임 및 에이피티 타임을 나타내어, 무화과의 에틸아세테이트 분획물은 아스피린보다 우수한 혈전생성 억제조성물로 개발될 수 있음을 확인하였다. 또한 본 결과는 에틸아세테이트 순차적 용매 분획이 분획 수득율은 매우 낮지만 강력한 활성을 나타내어, 무화과의 항혈전 활성물질을 정제하는 방법으로 매우 효율적임을 제시하고 있다.As a result of evaluating the antithrombotic activity of the fractions, as shown in Table 4 below, the nucleic acid fraction exhibited a 15-fold increase in thrombin time and a 2.5-fold increase in prothrombin time at a concentration of 5 mg/ml, indicating excellent thrombin inhibitory activity. However, compared with aspirin used in clinical practice at the same concentration, it showed relatively weak inhibitory activity. This was similar in the butanol fraction and the water residue, and a weak antithrombosis was confirmed compared to aspirin. However, in the case of the ethyl acetate fraction, thrombin time, prothrombin time, and AP time all showed a 15-fold or more prolonging effect at a concentration of 5 mg/ml, confirming a very strong antithrombotic activity. In particular, even at a concentration of 1.25 mg/ml lower than 1.5 mg/ml of aspirin used in the experiment, the thrombin time prolongation effect increased by more than 15 times and prothrombin time and AP time increased by 1.47 and 1.5 times, and the ethyl acetate fraction of figs It was confirmed that it can be developed as a composition that inhibits thrombogenesis better than aspirin. In addition, this result suggests that the ethyl acetate sequential solvent fraction has a very low fractional yield, but exhibits strong activity, and is therefore very efficient as a method of purifying the antithrombotic active substance of figs.
표 4.무화과 재료 유기용매 추출물 및 물 잔유물의 항혈전 활성도Table 4 Antithrombotic activity of organic solvent extracts of fig materials and water residues
실시예 6: 무화과 원료 추출물, 순차적 유기용매 분획물의 인간 혈소판 응집 저해활성 무화과 원료 추출물 및 분획물의 항혈전 활성 평가의 일환으로 인간 혈소판에 대한 응집저해 활성을 평가하였으며, 그 결과는 도 1 및 표 5에 나타내었다. 먼저 아스피린은 우수한 인간 혈소판 응집저해 활성을 나타내었으며, 농도 의존적인 응집저해 활성을 확인하였다. 무화과 에탄올 추출물 및 유기용매 분획물은 0.25 mg/ml 농도에서 전반적으로 혈소판 응집저해 활성이 우수한바, 아스피린 0.25 mg/ml 농도가 나타내는 응집저해 활성과 비교할 만하였으며, 특히 핵산 분획물과 에틸아세테이트 분획물은 혈소판 응집 연장시간 및 하강 면적에서 아스피린 0.5 mg/ml농도의 효과보다 더욱 강력한 혈소판 응집저해 활성을 나타내었다. 따라서 무화과 원료 추출물 및 분획물은 항혈전제로 아스피린을 대치할 수 있음을 확인하였다. 혈소판 응집저해 활성은 다음의 방법에 따라 평가하였다. 혈소판 응집저해 활성(Platelet aggregation inhibition activity)[0075] 혈소판은 건강한 인간의 전혈로부터 3.2% sodium citrate 용액과 1:9의 비율로 혼합한 후, 1,100 rpm에서 10분간 원심분리하여 상층의 PRP (platelet rich plasma)를 취하고, 이를 washing buffer (138 mM NaCl, 2.7 mMKCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 1 mM EDTA, pH 6.5)로 1회 세척하였다. 이후, suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 0.49 mMMgCl2, 025% gelatin, pH 7.4)에 재 현탁한 후, 3,000 rpm에서 10분간 원심분리한 후 다시 suspending buffer에 재 현탁하였으며, 이때 혈소판 수는 4x109/ml 되도록 조정하였다. 이후 1 ml 현탁액에 2.5 ul collagen을 가해 5분간 반응시키고, whole-blood aggregometer (Chrono-log, USA)를 사용하여 37℃에서 혈소판 응집을 측정하였다.Example 6: Human Platelet Aggregation Inhibitory Activity of Fig Raw Material Extracts and Sequential Organic Solvent Fractions As part of the antithrombotic activity evaluation of fig raw material extracts and fractions, the aggregation inhibitory activity against human platelets was evaluated, and the results are shown in FIGS. 1 and 5 Shown in. First, aspirin showed excellent human platelet aggregation inhibitory activity, and concentration-dependent aggregation inhibitory activity was confirmed. The fig ethanol extract and the organic solvent fraction had excellent overall platelet aggregation inhibitory activity at 0.25 mg/ml concentration, which was comparable to the aggregation inhibitory activity exhibited by aspirin 0.25 mg/ml concentration. In particular, the nucleic acid fraction and ethyl acetate fraction were platelet aggregation. It showed more potent platelet aggregation inhibitory activity than the effect of aspirin 0.5 mg/ml concentration in the extended time and descending area. Therefore, it was confirmed that the fig raw material extract and fraction could replace aspirin as an antithrombotic agent. Platelet aggregation inhibitory activity was evaluated according to the following method. Platelet aggregation inhibition activity [0075] Platelets were mixed with 3.2% sodium citrate solution from healthy human whole blood in a ratio of 1:9, and then centrifuged at 1,100 rpm for 10 minutes to obtain platelet rich (PRP) in the upper layer. plasma), which was washed once with a washing buffer (138 mM NaCl, 2.7 mMKCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, re-suspended in suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM Glucose, 0.49 mMMgCl2, 025% gelatin, pH 7.4), and centrifuged at 3,000 rpm for 10 minutes. It was resuspended in the suspending buffer again, and the platelet count was adjusted to be 4x109/ml. Then, 2.5 ul collagen was added to the 1 ml suspension and reacted for 5 minutes, and platelet aggregation was measured at 37°C using a whole-blood aggregometer (Chrono-log, USA).
표 5. 무화과 재료 에탄올 추출물 및 분확물의 인간 혈소판 응집저해 활성Table 5. Inhibitory Activity of Human Platelet Aggregation of Ethanol Extracts and Manures of Fig Materials
실시예 7: 무화과 에틸아세테이트 분획물의 활성물질의 화학적 특성 및 안정성 상기 실시예 1에서 얻은 무화과의 에틸아세테이트 분획물을 갑압건조하여 분말 조제한 후, 회수된 활성물질을 대상으로 인간 적혈구 용혈활성, 혈장 안정성, 열 안정성 및 산 안정성을 확인하였다. 조정제된 활성물질은 100℃에서 2 시간 처리, pH 2(0.01 M HCl)에서의 2 시간 처리, 혈장에서 2 시간 처리시에도 에틸아세테이트 분획이 트롬빈 저해 및 혈소판 응집저해 활성의 감소가 나타나지 않아 높은 안정성을 나타내었다. 또한, 5 mg/ml 농도까지 인간 적혈구에 대한 용혈 활성은 나타나지 않았다(표 6).Example 7: Chemical properties and stability of the active substance of the fig ethylacetate fraction After the ethyl acetate fraction of the fig obtained in Example 1 was pressure-dried to prepare a powder, the recovered active substance was subjected to human erythrocyte hemolytic activity, plasma stability, Thermal stability and acid stability were confirmed. Adjusted active substance is treated at 100℃ for 2 hours, pH 2 (0.01 M HCl) for 2 hours, and plasma for 2 hours, the ethylacetate fraction does not inhibit thrombin and platelet aggregation inhibitory activity, resulting in high stability. Shown. In addition, hemolytic activity against human red blood cells was not shown up to the concentration of 5 mg/ml (Table 6).
표 6. 무화과 재료 에탄올 추출물 및 분확물의 인간 적혈구 용혈활성도Table 6. Human Erythrocyte Hemolytic Activity of Ethanol Extracts and Manures of Fig Materials
실시예 8: 무화과 유기산 수용액 추출 후 1차 정제된 혼합물을 이용하여 항혈소판 응집능 측정시험Example 8: Test for measuring antiplatelet aggregation ability using the first purified mixture after extraction of fig organic acid aqueous solution
추출시 용매의 추출온도에 따른 추출물의 항응고 활성을 비교하기 위하여 무화과 재료를 선택하여 분쇄하고 유기산 수용액을 이용하여 이를 각각 10℃, 30℃, 50℃, 70℃, 100℃의 온도로 추출한 후 여과하고 저온 감압 농축하여 포화용액으로 한 다음 에탄올을 첨가하여 결정 화합물을 거르고 여러 차례 세척한 후 임피던스법을 이용한 항혈소판 응집능을 측정하였다. 임피던스법은 혈액내로 전류를 통과시켜 혈소판 응집에 따른 전기적 저항의 변화를 측정하는 방법으로 전혈(Whole blood)에서의 측정이 가능한 방법을 이용하였다. 실험을 위하여 4 주령의 흰쥐(Slc, ICR Mouse)를 (주)중앙실험 동물로부터 공급받아 온도 23±3 ℃, 상대습도 50±10 %, 150~300 Lux의 조도로 12 시간 간격(오전 9 시~오후 9시)으로 조절되는 동물실험실에서 2주 동안 순화시킨 후 4주령의 SD(Sprague-Dewley)rat으로부터 채혈하여 0.38% 구연산나트륨(sodium citrate)을 혈액에 대하여 1:9(v/v)의 비율이 되도록 한 전혈(Whole blood)을 사용하였다. 그리고 상기의 추출방법에 따른 무화과의 온도별 추출물을 5mg/mL의 농도로 희석하여 각각을 전혈에 처리한 후 콜라겐에 의한 응집유도 후 각각의 저항값을 측정하고 이를 아래 항혈소판 활성을 수학공식에 적용하여 각각 계산하였다. 계산한 항혈소판활성은 표7에 나타내었다.In order to compare the anticoagulant activity of the extract according to the extraction temperature of the solvent during extraction, the fig material is selected and pulverized, and extracted with an organic acid aqueous solution at a temperature of 10℃, 30℃, 50℃, 70℃, and 100℃, respectively. After filtration and concentration under reduced pressure to obtain a saturated solution, ethanol was added to filter the crystalline compound, washed several times, and the antiplatelet aggregation ability was measured using an impedance method. The impedance method is a method of measuring the change in electrical resistance due to platelet aggregation by passing an electric current into the blood, and a method capable of measuring in whole blood was used. For the experiment, a 4-week-old rat (Slc, ICR Mouse) was supplied from the Central Experimental Animal Co., Ltd., at a temperature of 23±3 ℃, a relative humidity of 50±10%, and an illuminance of 150~300 Lux for 12 hours (9 am ~ 9 p.m.) after acclimatization for 2 weeks in an animal laboratory controlled by 4 weeks old SD (Sprague-Dewley) rat, and 0.38% sodium citrate is 1:9 (v/v) to the blood. Whole blood was used so that the ratio of And after diluting the extract by temperature of figs according to the above extraction method to a concentration of 5 mg/mL, treating each of the whole blood, inducing aggregation by collagen, measuring each resistance value, and then calculating the antiplatelet activity below in the mathematical formula. Each was calculated by applying. The calculated antiplatelet activity is shown in Table 7.
수학식 항혈소판활성(%) = 1- 시료의 저항값/ 대조군의 저항값 X 100Equation Antiplatelet activity (%) = 1- Resistance value of sample / Resistance value of
표 7. 추출 온도별 항혈소판활성도.Table 7. Antiplatelet activity by extraction temperature.
상기 표1의 결과에서 추출온도가 30-40 ℃ 이상 증가할수록 항 혈소판 활성이 감소함을 알 수 있었다.From the results of Table 1, it was found that the antiplatelet activity decreased as the extraction temperature increased by 30-40 °C or more.
실시예 9: 무화과 유기산 수용액 추출 후 1차 정제된 혼합물을 이용하여 에틸아세테이트 분획물의 일 회 섭취에 의한 독성 시험Example 9: Toxicity test by single intake of ethyl acetate fraction using the first purified mixture after extraction of fig organic acid aqueous solution
실시예 1로부터 얻은 에틸아세테이트 분획물의 일 회 경구독성을 평가하였다. 먼저, 시료를 DMSO 및 물에 녹인후, 400, 800, 1500, 2000 mg/kg 농도로 각각 3마리씩 경구투여 후, 일주일 간 생존 여부와 병적 이상 증후를 관찰하였다. 대조 구로는 DMSO만을 경구투여하였다. 그 결과 생존율은 100%였으며, 병적 이상 징후는 나타나지 않았다. 따라서, 약용으로 사용되어 온 무화과의 에틸아세테이트 분획물은 저독성 또는 무독성으로 구분되며, 이의 낮은 농도의 사용은 부가적인 문제점을 일으키지 않으리라 판단되었다.One-time oral toxicity of the ethyl acetate fraction obtained from Example 1 was evaluated. First, the samples were dissolved in DMSO and water, and then orally administered at a concentration of 400, 800, 1500, and 2000 mg/kg, respectively, 3 animals, and then survival and symptoms of pathological abnormalities were observed for a week. As a control group, only DMSO was administered orally. As a result, the survival rate was 100%, and there were no signs of pathological abnormalities. Therefore, the ethyl acetate fraction of figs, which have been used for medicinal purposes, is classified as either low toxicity or non-toxic, and it was judged that the use of a low concentration thereof would not cause additional problems.
실시예 10: 본 발명의 추출방법 (도-1) 공정방법에 따라 무화과 원료인 미숙과 및 반건조 무화과(완숙과 및 미숙과 건조물) 그리고 폐기되는 무화과 나무 줄기 무화과나무 뿌리, 그리고 무화과 잎을 이용하여 1차적으로 부분 용매 잔유함유로 100g (놘전 건조 안함) 농축액 기준 무화과 잎은 추출 분리 프로세스 방법에 따라서 부탄올층 5.6 g 유기층에서 12g 을 얻었으며 미숙과 및 반건조 무화과(완숙과 및 미숙과 건조물)은 동일 방법으로 부탄올층 15.6 g 유기층에서 62.1 g 이상을 얻었다. 그리고 무화과나무 줄기 무화과나무 뿌리혼합은 동일 방법으로 부탄올층 34.1 g 유기층에서 41.8 g을 얻었다.Example 10: According to the extraction method of the present invention (Fig.-1), using the fig raw material immature fruits and semi-dried figs (dried ripe fruits and unripe fruits) and discarded fig tree trunk fig tree roots, and fig leaves according to the process method As a result, fig leaves based on the concentrated solution of 100g (not dried before thinning) were obtained primarily from 5.6 g of the butanol layer and 12 g from the organic layer according to the extraction and separation process with partial solvent residual content, respectively, and unripe and semi-dried figs (ripe fruit and dried unripe fruit). In the same method, 62.1 g or more was obtained from 15.6 g of a butanol layer and an organic layer. And fig tree trunk and fig tree roots were mixed in the same way to obtain 41.8 g of butanol layer in 34.1 g organic layer.
실시예 11: 본 발명의 추출방법에 따라 제조된 무화과 원료 추출물에 대한 항 혈소판 능을 기존에 보고된바 있는 무화과 에탄올 추출물과 활성을 비교함으로 본 발명에 의한 수용성 유기산 무화과 원료 추출물의 우수성을 확인하였다. 무화과 에탄올 추출물의 제조는 기존 보고자료에 기술한 방법을 충실히 따라 제조하였다. 항 혈소판 능의 활성은 실시예 1에서 기술한 실험법을 따라 측정하였다.Example 11: The superiority of the water-soluble organic acid fig raw material extract according to the present invention was confirmed by comparing the anti-platelet activity of the fig raw material extract prepared according to the present invention with the previously reported fig ethanol extract. . The preparation of fig ethanol extract was faithfully prepared according to the method described in the previous report. The antiplatelet activity was measured according to the experimental method described in Example 1.
표 8. 항 혈소판 능의 활성 비교도Table 8. Comparison of antiplatelet activity activity
상기표 8의 결과에서 무화과 원료 선택과 용매 선택에 따라서 수율 향상과 항혈소판 활성의 더 우수함을 확인할 수 있었다. 그러나 기존보고 된 무화과 원료 추출물의 경우, 물에 의한 추출이나 알코올에 의한 추출방법은 통상적 이론 개념으로 무화과 효소인 picin의 화학적 구조에 따를 물리적 특성상 유용한 혈전 용해제 물질이 추출되지 않으며 추출된다 하더라도 너무 수율이 낮아 상업적 가치가 없다고 본다. 특히 본 발명자가 얻고자 하는 물질은 끈적끈적한 왁스 형태로 융합되어 산소 결합에 따른 불용성 물질로의 변화로 물리적 특성상 고수율로 얻기가 어려웠다. 그러나 본 발명자는 수년간 무화과를 연구한 결과 유기산 수용액을 적용하여 저온에서도 손쉽게 추출하는 방법을 발견하고 이를 이용하여 항혈소판 활성 실험을 실시한 결과 기존 알코올 추출방법이나 물을 이용하여 추출하는 방법은 수율이 현저하게 낮아 유용한 화합물을 얻을 수 없으며 이를 대조 구로 항혈소판능 실험을 비교동정하였으나 활성이 거의 나타내지 않았다. 이는 본 실시예에서 사용된 화합물 추출 전처리 회수방법의 차이로 판단된다. 특히 종전의 알려진 보고에서는 항혈소판 활성 확인을 위하여 PRP(Platelet Rich Plasma)를 사용하고 혈소판 응집 inducer로써 ADP와 Adrenaline을 사용하였으나 본 실시예에서는 전혈내 혈소판만을 이용하였고 혈소판 응집 inducer는 collagen을 사용하여 비교 동정하였으며 또한 종전의 알려진 방법으로도 확인하였다.From the results of Table 8, it was confirmed that the yield was improved and the antiplatelet activity was more excellent depending on the selection of the fig raw material and the selection of the solvent. However, in the case of the previously reported fig raw material extract, extraction with water or alcohol is a common theoretical concept. Due to the physical properties of the fig enzyme picin, a useful thrombolytic substance is not extracted, and even if it is extracted, the yield is too high. I don't think it has any commercial value. In particular, the material to be obtained by the present inventors was difficult to obtain in a high yield due to physical properties due to the change of the material into an insoluble material due to oxygen bonding due to the fusion in the form of a sticky wax. However, as a result of studying figs for many years, the present inventors have found a method of easily extracting even at low temperatures by applying an aqueous organic acid solution, and as a result of conducting an antiplatelet activity experiment using this, the conventional alcohol extraction method or the method of extracting using water has a remarkable yield. It was too low to obtain a useful compound, and the antiplatelet activity test was compared and identified as a control, but it showed little activity. This is judged by the difference in the pretreatment and recovery method for compound extraction used in this example. In particular, in the previous known report, PRP (Platelet Rich Plasma) was used to confirm antiplatelet activity, and ADP and Adrenaline were used as platelet aggregation inducers, but in this example, only whole blood platelets were used, and platelet aggregation inducers were compared using collagen. It was identified and confirmed by previously known methods.
실시예 12 : 구내염 약효시험Example 12: Stomatitis drug efficacy test
감염 당일 랫트를 마취시키고 전기면도기로 등의 털을 제거한 다음 등에 칼로 2 cm의 상처를 내고 피부 표피층을 피부근 층까지 잡아 늘린다. 상처에 직접 104~106 cfu(inoculum size ; 1.12x106 cfu)의 황색포도상구균(Staphylococcus aureus ATCC 6538P)을 감염시킨 다음 균을 주입한 상처부위를 실을 가지고 1 번 정도 꿰메어서 일시적으로 봉합하였다. 마취가 깨는 것을 확인하고 동 부위가 다른 부위에 닿지 않도록 하면서 감염 후 1 일째 되는 날부터 기존 연고(오라메디 연 고TM) 및 실시예 9의 조성물을 각각 1 일 3 회 투여하였다(0.1g/kg/회). 감염 2 일과 3 일째 상처 부위(2x2 cm)를 포함한 조직을 조심스럽게 뜯어내었다. 피부 조직판을 10 ml의 멸균 식염수에 넣어 오랫동안 균질화한 다음 상등액을 연속 희석 하여 아가 플래이트에 도말하고 18시간 후에 자라난 균을 세어 치료효과를 결정한다. 또한 랫트(7주, 200~250 g)는 자유 로이 물과 먹이를 공급하였으며 무작위 추출에 의해 약물 무처치 대조군, 기존 연고 치료군 및 실시예 1의 조성물 치료군 으로 나누어서 실험에 사하였다. 그 결과, 다음 표 5에 나타낸 바와 같이 약물무처치 랫트에서는 감염 후 2 일째 되는 날 균의 수가 가장 높았으나 감염 후 3 일째 되는 날부터는 균수가 감소되기 시작하여 7 일째 되는 날에는 자연 치유되는 현상 을 나타내었다. 실시예 1의 조성물과 기존 연고는 감염 1 일째 되는 날 균수가 가장 높았으며 감염 2 일째 되는 날부터 균 수가 감소되기 시작하였으며 3 일째 되는 날에는 치유현상을 나타내었다. 또한 표 5에서 나타난 것과 같이 유의적인 차이 는 보이지 않았으나 실시예 9의 무화과 추출 조성물은 기존 연고와 비교할 때 기존 연고제와는 더 효능 효과가 우수한 감염 치료 효과가 있는 것으로 판단됐다. 따라서 구내염 치료제 조성물은 기존 연고와 비교할 경우 더 신속하게 나은 구내염 치료효과가 있는 것으로 판단됐다.On the day of infection, anesthetize the rat, remove the hair on the back with an electric razor, and make a 2 cm wound with a knife on the back and stretch the skin epidermal layer to the skin muscle layer. After infecting the wound with Staphylococcus aureus ATCC 6538P (104~106 cfu (inoculum size; 1.12x106 cfu)) directly, the wound area injected with the bacteria was stitched once with a thread and temporarily closed. From the first day after infection, the original ointment (Oramedi OintmentTM) and the composition of Example 9 were each administered three times a day (0.1 g/kg) from the day 1 after infection, confirming that the anesthesia had broken and the same area did not touch other areas (0.1 g/kg /time). On days 2 and 3 of infection, the tissue including the wound site (2x2 cm) was carefully removed. After homogenizing the skin tissue plate in 10 ml of sterile saline for a long time, the supernatant is serially diluted and smeared on an agar plate, and the bacteria grown after 18 hours are counted to determine the therapeutic effect. In addition, rats (7 weeks, 200-250 g) were freely supplied with water and food, and were subjected to the experiment by dividing into a drug-free control group, an existing ointment treatment group, and the composition treatment group of Example 1 by random extraction. As a result, as shown in Table 5 below, the number of bacteria on the 2nd day after infection was the highest in the drug-free rats, but the number of bacteria started to decrease on the 3rd day after infection, and spontaneously healed on the 7th day after infection. Indicated. The composition of Example 1 and the existing ointment had the highest number of bacteria on the first day of infection, and the number of bacteria started to decrease from the second day of infection, and showed a healing phenomenon on the third day. In addition, as shown in Table 5, there was no significant difference, but the fig extract composition of Example 9 was judged to have an excellent infection treatment effect compared to the conventional ointment when compared with the existing ointment. Therefore, the composition for treating stomatitis was judged to have a better treatment effect for stomatitis more quickly when compared to conventional ointments.
표 9. 구내염 치료 효능 비교도Table 9. Comparison of stomatitis treatment efficacy
무화과 원료인 미숙과 및 반건조 무화과(완숙과 및 미숙과) 그리고 폐기되는 무화과나무 줄기 무화과나무 뿌리, 그리고 무화과 잎을 분쇄하여 유기산 수용액으로 실온에서 추출하여도 회수할 방법을 발견하고 충분히 교반하여 추출한 다음 불용성 무화과를 거르고 회수된 여액을 감압 농축한 다음 소량의 염기성 수용액을 첨가하여 중화시킨 다음 알코올을 첨가하여 생성된 겔화합물을 거르고 알코올로 수회 세 착하여 얻어진 결과 기존 열수 추출방법보다 70% 이상의 고수율로 순수한 무화과 겔을 얻었다. 따라서, 본 발명자들은 항혈전 및 혈류개선 활성을 나타내는 기능성 식품 소재 개발을 목표로, 무화과 원료 추출물을 조제한 후 인간 혈장과 인간 트롬빈을 이용하여 트롬빈 직접저해에 따른 혈전생성 저해활성을 평가하고, 인간 농축 혈소판을 이용한 무화과의 혈소판 응집저해 활성을 측정하여, 무화과 원료 추출물 및 이의 순차적 분획물에서 매우 우수한 항혈전 활성을 확인하여 본 발명을 완성하였다. 따라서 무화과 재배 농가의 대량 폐기 처분한 자원을 시제품 원료로 활용할 수 있어서, 국민 건강 증진은 물론 무화과 재배 농가의 소득을 증가해 장차 산업발전에 이바지할 수 있다.Unripe and semi-dried figs, which are raw materials for figs, and fig tree stems, fig tree roots, and fig leaves that are discarded were pulverized and extracted with an organic acid solution at room temperature. Next, the insoluble figs were filtered, the recovered filtrate was concentrated under reduced pressure, neutralized by adding a small amount of basic aqueous solution, and then the gel compound produced by adding alcohol was filtered and washed several times with alcohol. A pure fig gel was obtained in yield. Therefore, the present inventors aim to develop a functional food material that exhibits antithrombosis and blood flow improvement activity, after preparing a fig raw material extract, using human plasma and human thrombin to evaluate the thrombiogenesis inhibitory activity according to direct thrombin inhibition, and human concentration. The present invention was completed by measuring the platelet aggregation inhibitory activity of figs using platelets, and confirming very excellent antithrombotic activity in the fig raw material extract and sequential fractions thereof. Therefore, it is possible to use the resources discarded in large quantities by fig cultivation farmers as raw materials for prototypes, thereby contributing to the future industrial development by increasing the income of fig cultivation farmers as well as promoting national health.
( 100): 무화과(미숙과 열매, 반건조 무화과) 선택적 분쇄단계
(200): 유기산 열수추출 및 여과 단계
(300):추출용액 회수, 중화 및 감압농축 후 포화용액으로 제조 단계
(400): 알코올 세척후 겔화 단계
(500): 여과 및 알코올 구회 세척단계
(600): 무화과 Gel-1 회수 단계
(700) 불용성 고체 제거단계(100): Fig (unripe fruit, semi-dried fig) selective crushing step
(200): Organic acid hot water extraction and filtration step
(300): Extraction solution recovery, neutralization and concentration under reduced pressure, and then preparation step as a saturated solution
(400): gelation step after alcohol washing
(500): filtration and alcohol washing step
(600): Fig Gel-1 recovery step
(700) Insoluble solid removal step
Claims (5)
상기 무화과 원료 추출물은 무화과 원료를 선택하여 유기산 수용액 추출물인 것을 특징으로 하는 혈전증 예방 또는 치료용 약학적 조성물.The method of claim 1,
The fig raw material extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the fig raw material is selected and an organic acid aqueous solution extract.
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KR101080994B1 (en) | 2010-06-04 | 2011-11-09 | 목포대학교산학협력단 | Manufacture apparatus of salt |
JP2013152314A (en) | 2012-01-25 | 2013-08-08 | Konica Minolta Inc | Image forming system |
KR101181402B1 (en) | 2012-02-02 | 2012-09-11 | 윤규형 | The composition for rice cake and noodle comprising fish scale collagen and herb extracts |
KR20170029586A (en) | 2014-07-11 | 2017-03-15 | 유나이티드 오토모티브 일렉트로닉 시스템즈 컴퍼니 리미티드 | Integrated copper bar for secondary-side power circuit of power electronic converter |
KR20150014410A (en) | 2014-11-18 | 2015-02-06 | 경북대학교 산학협력단 | Anti-thrombotic composition comprising phenol compound |
KR20160085387A (en) | 2015-01-07 | 2016-07-18 | 삼성디스플레이 주식회사 | Display device and driving method thereof |
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