KR101957632B1 - Composition for preventing, improving or treating of fibrosis - Google Patents
Composition for preventing, improving or treating of fibrosis Download PDFInfo
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- KR101957632B1 KR101957632B1 KR1020180031003A KR20180031003A KR101957632B1 KR 101957632 B1 KR101957632 B1 KR 101957632B1 KR 1020180031003 A KR1020180031003 A KR 1020180031003A KR 20180031003 A KR20180031003 A KR 20180031003A KR 101957632 B1 KR101957632 B1 KR 101957632B1
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- fibrosis
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Abstract
본 발명은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 조성물에 관한 것으로, 상기 조성물은 상피-간엽 이행을 억제하고, 폐섬유화에 의하여 감소된 폐용적을 증가시키며, 간섬유화 및 산화적 스트레스를 개선하는 효능이 있으므로 섬유화증의 예방, 개선 또는 치료 용도로 유용하게 이용될 수 있다.The present invention relates to a composition comprising, as an active ingredient, a red head extract, fractions thereof or oleanolin acid acetate isolated therefrom, which inhibits epithelial-mesenchymal transition, increases lung volume reduced by pulmonary fibrosis, Hepatic fibrosis, and oxidative stress, it can be usefully used for prevention, amelioration or treatment of fibrosis.
Description
본 발명은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트(oleanolic acid acetate)를 유효성분으로 포함하는 섬유화증의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, ameliorating or treating fibrosis comprising aspirin toxin extract, fractions thereof or oleanolic acid acetate isolated therefrom as an active ingredient.
섬유화증(fibrosis)은 정상적인 조직이 파괴되면서 섬유성 결합조직으로 대체되는 섬유화로 인하여 조직의 기능을 상실하는 증상을 말하며, 섬유화 과정이 만성적으로 진행되기 때문에 완벽한 치료가 어려운 실정이다.Fibrosis refers to the loss of function of the tissue due to fibrosis, which is replaced with fibrous connective tissue as normal tissue is destroyed, and complete treatment is difficult because the fibrosis progresses chronically.
섬유화증의 하나인 특발성 폐섬유화증(idiopathic pulmonary fibrosis, IPF)은 만성적으로 진행하는 폐의 섬유화를 특징으로 하는 원인불명의 질환으로 주로 폐에 국한되어 나타나며 조직학적으로 특징적인 통상형 간질성 폐렴(usual interstitial pneumonia, UIP) 소견을 보인다. 이 질환의 유병률은 인구 10만 명당 2 내지 29명으로 알려져 있으며 국내에서는 희귀 난치성 질환으로 지정되어 있다. 상기 질환에 대한 임상 경과는 다양하며, 일반적으로 서서히 진행되는 폐의 기능 저하로 인하여 호흡부전으로 진행하고, 진단 이후 평균 생존기간이 2 내지 3년 이내인 치명적인 질환으로 알려져 있다.Idiopathic pulmonary fibrosis (IPF), one of the fibrosis, is an unidentified disease characterized by the chronic fibrosis of the lung. It is mainly localized in the lungs and is characterized by histologically distinctive interstitial pneumonia normal interstitial pneumonia, UIP). The prevalence of this disease is known to be 2 to 29 per 100 000 population and is designated as a rare disease of incurable disease in Korea. The clinical course of the disease is variable, and is generally known to be a fatal disease that progresses to respiratory failure due to a gradual onset of pulmonary dysfunction and an average survival time within 2 to 3 years after diagnosis.
최근 국내에서는 가습기 살균제에 함유된 폴리헥사메틸렌 구아디닌(Polyhexamethylene guanidine, PHMG) 및 염화에톡시에틸구아디닌 (Oligo(2-)ethoxy ethoxyethyl guanidine chloride; PGH)에 노출된 소비자에서 기도 및 폐 손상, 호흡 곤란, 기침 등과 같은 증상을 동반하는 폐섬유화증 환자가 발생하여 사회, 경제적으로 큰 파장을 일으키고 있다. 이러한 파장은 유사물질인 클로로메틸이소티아졸리논(5-chloro-2-methyl-4-isothiazolin-3-one)과 메틸이소티아졸리논(2-methyl-4-isothiazolin-3-one)을 사용한 제품의 판매 중단에까지 이르렀으며, 폐섬유화증의 발병 원인이 명확하지 않음으로 인하여 국민적 불안감이 더욱 증폭되고 있다.Recently, in Korea, exposure to polyhexamethylene guanidine (PHMG) and oligo (2-) ethoxy ethoxyethyl guanidine chloride (PGH) in humidifier disinfectants has been associated with airway and lung injury , Respiratory distress, cough, and other symptoms are accompanied by pulmonary fibrosis patients, causing a great wave of social and economic. These wavelengths were measured using a similar material, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one The product has been discontinued, and the cause of the onset of pulmonary fibrosis is unclear, making the national anxiety more intense.
상기 물질들 이외에도 폐섬유화증의 발병 원인으로는 화학물질, 흡연, 미세먼지, 바이러스 감염 및 유전적 요인 등이 위험 인자로 거론되고 있으나, 확실한 발병 인자는 아직까지 밝혀지지 않는 상태이며, 또한 병인기전에 대한 기초 연구가 부족하여 뚜렷한 치료 표적도 아직까지 발굴되지 않은 상태이다.In addition to these substances, the causes of pulmonary fibrosis include chemicals, smoking, micro dust, virus infection, and genetic factors. However, the definite etiologic factor has not yet been clarified, The lack of basic research on the past has not yet uncovered a clear therapeutic target.
폐섬유화증의 치료에는 스테로이드(glucocorticoids), 면역 억제제 (immunosuppressive agents) 및 사이토카인 제제(anti-viral cytokines)가 대표적으로 사용되고 있으나, 2011년 특발성 폐섬유화증에 대한 ATS/ERS 가이드라인에 의하면 스테로이드와 면역 억제제인 아자티오프린(azathioprine)의 병용요법이 오히려 사망률을 증가시키는 결과를 보였다.Although glucocorticoids, immunosuppressive agents and anti-viral cytokines have been used for treatment of pulmonary fibrosis, according to the ATS / ERS guidelines for idiopathic pulmonary fibrosis in 2011, The combination therapy of the immunosuppressant azathioprine resulted in an increase in mortality.
상기 가이드라인과 같이 면역 또는 염증 억제 방식을 이용하는 기존의 폐섬유화증 치료법은 한계를 보이고 있기 때문에 염증을 억제할 뿐만 아니라 섬유화 진행을 직접 저해하는 약물의 개발이 시급한 실정이다. 최근 섬유화 진행에 중요한 TGF-β 신호전달 체계를 직접적으로 저해하는 물질이 임상시험 중에 있으나, TGF-β는 섬유화를 촉진시키는 역할뿐만 아니라 염증 자체를 억제시키는 작용이 있기 때문에 폐섬유화를 치료하기 위하여 TGF-β를 전반적으로 억제하는 방법은 많은 부작용을 초래할 수 있다.Since the conventional method of treating pulmonary fibrosis using the immunity or inflammation-suppressing method as in the above guideline is limited, it is urgently required to develop drugs that directly inhibit the progress of fibrosis as well as suppress inflammation. Recently, a substance that directly inhibits the TGF-β signal transduction system, which is important for fibrosis progression, is in clinical trials. However, since TGF-β acts not only to promote fibrosis but also to suppress inflammation itself, The overall inhibition of -β can lead to many side effects.
이러한 배경 하에서 본 발명자들은 독성 및 부작용이 거의 없는 천연물 유래의 섬유화증 예방 및 치료용 물질을 찾기 위해 연구한 결과, 적소두(팥 및 이팥) 추출물 및 추출물에서 정제된 화합물인 올레아놀린산 아세테이트가 폐섬유화증 및 간섬유화증을 억제하는 효능이 있음을 확인하여 본 발명을 완성하였다.Under these circumstances, the inventors of the present invention have studied to find substances for the prevention and treatment of fibrosis caused by natural products, which have little toxicity and side effects. As a result, it has been found that oleanolic acid acetate, a compound purified from red bean (red bean and red bean) And liver fibrosis. The present invention has been completed based on this finding.
본 발명의 일 목적은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 섬유화증(fibrosis) 예방 또는 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for preventing or treating fibrosis comprising aspirin extract, fractions thereof or oleanolin acid acetate isolated therefrom as an active ingredient.
본 발명의 다른 목적은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 폐질환 예방 또는 개선용 식품 조성물과 개인 위생용품을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or alleviating pulmonary disease and a personal hygiene article, which comprises asparagus extract, fractions thereof or oleanolin acid acetate isolated therefrom as an active ingredient.
본 발명의 또 다른 목적은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공하는 것이다.It is still another object of the present invention to provide a food composition for preventing or improving liver disease, which comprises asparagus extract, fractions thereof or oleanolin acid acetate isolated therefrom as an active ingredient.
본 발명의 일 양상은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 섬유화증(fibrosis) 예방 또는 치료용 약학적 조성물을 제공한다.An aspect of the present invention provides a pharmaceutical composition for preventing or treating fibrosis comprising aspirin extract, fractions thereof, or oleanolin acid acetate isolated therefrom as an active ingredient.
본 명세서에 사용된 용어, '적소두(赤小豆)'는 콩과 식물인 팥(Phaseolus angularis Wight)의 종자를 의미하는데, 원주 모양으로 조금 납작하고, 종피는 적갈색으로 매끈하여 광택이 있다. 약리활성의 측면에서는 이뇨, 소염, 배농, 해열, 전신부종, 간경화, 황달, 종기, 화농성 질환, 수종, 각기병, 소갈, 이질설사 등의 치료를 위한 민간요법에 사용되어 왔다.As used herein, the term 'red bean' refers to the seed of the bean ( Phaseolus angularis Wight), which is round in the shape of a cylinder, and the seed coat is glossy in reddish brown. In terms of pharmacological activity, it has been used in folk remedies for the treatment of diuresis, inflammation, drainage, fever, systemic edema, cirrhosis, jaundice, swelling, purulent diseases, species, organs, gonorrhea and diarrhea.
본 발명의 일 구체예에 따르면 상기 적소두는 팥(Phaseoli angularis Wight) 또는 이팥(또는 덩굴팥, Phaseolus calcaratus Roxburgh)의 종자를 의미하는 것으로 사용될 수 있으나 이에 제한되지는 않는다.According to one embodiment of the invention is the jeoksodu beans (Phaseoli angularis Wight) or red beans (or red beans, Phaseolus It is used to mean seeds calcaratus Roxburgh), but is not limited to this.
상기 적소두 추출물은 적소두에 용매를 첨가하여 수득한 추출물을 의미하며 당업계에 공지된 통상의 방법에 따라 제조될 수 있다. 예를 들어 적소두를 분쇄한 후 추출에 통상적으로 사용되는 용매를 첨가하고, 적절한 온도와 압력을 가하여 적소두 추출물을 제조할 수 있다. 상기 적소두 추출물은 추출물 원액, 추출물의 희석액 또는 농축액, 추출물을 건조하여 제조되는 건조물, 또는 이들의 정제물 등을 모두 포함한다.The red head extract means an extract obtained by adding a solvent to red head, and can be prepared according to a conventional method known in the art. For example, a red toenite extract can be prepared by pulverizing a red head, adding a solvent conventionally used for extraction, and applying appropriate temperature and pressure. The red head doe extract includes all of the extract solution, the diluted solution or concentrate of the extract, the dried product produced by drying the extract, or the purified product thereof.
본 발명의 일 구체예에 따르면 상기 용매는 정제수, 헥산, 1,3-부틸렌글리콜, 탄소수 1 내지 4의 알코올, 에틸아세테이트, 디에틸에테르, 디클로로메탄, 아세톤 및 이들의 혼합물로 이루어진 군에서 선택될 수 있으며, 정제수 또는 탄소수 1 내지 4의 알코올을 이용하는 것이 바람직하다.According to one embodiment of the present invention, the solvent is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, an alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, And it is preferable to use purified water or an alcohol having 1 to 4 carbon atoms.
본 명세서에 사용된 용어, '분획물'은 다양한 구성성분을 포함하는 혼합물로부터 특정 성분 또는 특정 그룹을 분리하는 분획방법에 의하여 얻어진 결과물을 의미한다. 본 발명의 일 실시예에 따르면 적소두의 추출물을 n-헥산, 에틸아세테이트 등의 용매를 이용한 용매 분획방법으로 분획한 결과물일 수 있으며, 극성 용매 분획물과 비극성 용매 분획물을 모두 포함하고, 구체적으로는 헥산 분획물, 에틸아세테이트 분획물 및 물 분획물 등이 모두 사용될 수 있다.As used herein, the term " fraction " refers to a product obtained by a fractionation method that separates a particular component or a particular group from a mixture comprising various components. According to an embodiment of the present invention, the extract of Red Fox may be a fraction obtained by fractionating the solvent by a solvent fractionation method such as n-hexane or ethyl acetate, and it includes both a polar solvent fraction and a non-polar solvent fraction, Fractions, ethyl acetate fractions and water fractions can all be used.
본 명세서에 사용된 용어, '섬유화증(fibrosis)'은 정상적인 조직이 파괴되면서 섬유성 결합조직으로 대체되는 섬유화로 인하여 조직의 기능을 상실하는 증상을 의미하며, 예를 들어 피부에 상처가 생긴 후 섬유화가 발생하는 경우를 '흉터'라고 한다.As used herein, the term " fibrosis " refers to a symptom of loss of tissue function due to fibrosis, which is replaced with fibrous connective tissue when normal tissue is destroyed. For example, When fibrosis occurs, it is called "scar".
본 발명의 일 구체예에 따르면 상기 섬유화증은 폐섬유화증, 간섬유화증 및 신장섬유화증으로 이루어진 군에서 선택될 수 있다. 간섬유화증(liver fibrosis)은 간의 반복적인 손상 및 회복으로 인하여 간 조직이 딱딱하게 굳는 증상을 말하며, 신장섬유화증(kidney fibrosis)은 신장조직에서 발생하는 과다한 염증반응, 상피세포의 섬유세포화와 같은 원인으로 인하여 신장조직이 섬유화되어 신장 기능을 상실하게 되는 증상을 의미한다.According to one embodiment of the present invention, the fibrosis may be selected from the group consisting of pulmonary fibrosis, hepatic fibrosis and renal fibrosis. Liver fibrosis is a condition in which hepatic tissue is hardened due to repetitive damage and recovery of the liver. Kidney fibrosis is caused by excessive inflammatory reaction in the kidney tissue, fibrosis of epithelial cells, It means a symptom that kidney tissue becomes fibrosis due to the same cause and loss of kidney function.
폐섬유화증(pulmonary fibrosis)은 만성적 간질성 폐질환의 일종으로서, 폐조직세포가 섬유세포로 변화되어, 호흡곤란, 기침, 청색증(cyanosis), 곤봉지(clubbing) 등의 증상을 유발하는 질환을 의미한다. 지금까지는 스테로이드계 치료제, 인터페론 감마, 아세틸시스테인, 피르페니돈, 보세탄 등을 치료제로서 사용하고 있으나, 특이적인 치료효과를 나타내는 제제는 아직 보고되어 있지 않다.Pulmonary fibrosis is a type of chronic interstitial lung disease in which pulmonary tissue cells are transformed into fibrous cells and cause diseases such as dyspnea, cough, cyanosis, and clubbing it means. Up to now, steroid therapy, interferon gamma, acetylcysteine, pyrennidone, and bosetan have been used as therapeutic agents, but no agents showing a specific therapeutic effect have yet been reported.
본 발명의 일 구체예에 따르면 상기 폐섬유화증은 급성 및 만성 기관지염, 천식, ssRNA 및 dsRNA 바이러스 감염증, 세균 및 진균 감염증, 폐렴, 폐혈증, 만성폐쇄성폐질환, 폐결핵, 폐(석회화) 결절, 폐기종, 특발성 폐섬유화증 및 간질성 폐질환으로 이루어진 군으로부터 선택될 수 있다.According to one embodiment of the present invention, the pulmonary fibrosis is selected from the group consisting of acute and chronic bronchitis, asthma, ssRNA and dsRNA viral infection, bacterium and fungal infection, pneumonia, hematemesis, chronic obstructive pulmonary disease, pulmonary tuberculosis, pulmonary nodule, emphysema, Idiopathic pulmonary fibrosis and interstitial lung disease.
본 발명의 일 구체예에 따르면 상기 조성물은 섬유화 과정의 주요 증상의 하나인 상피-간엽 이행을 억제할 수 있으며, 상피-간엽 이행에 관여하는 E-cadherin, N-cadherin, Vimentin, Snail, α-SMA 및 Collagen I의 발현을 억제할 수 있다. 또한, 상기 조성물은 폐섬유화증 동물모델에서 섬유화의 진행을 억제하고, 감소된 폐용적을 증가시킬 수 있으므로 섬유화증 개선 및 치료 용도로 유용하게 이용될 수 있다.According to one embodiment of the present invention, the composition is capable of inhibiting epithelial-mesenchymal transition, which is one of the main symptoms of fibrosis process. E-cadherin, N-cadherin, Vimentin, Snail, SMA, and Collagen I. < / RTI > In addition, the composition can inhibit the progression of fibrosis in an animal model of pulmonary fibrosis, and can be used for improving and treating fibrosis because it can increase the reduced lung volume.
본 발명의 약학적 조성물은 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 상기에서 '약학적으로 허용되는'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 약학적으로 허용되는 담체로는 예를 들면, 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등과 같은 경구 투여용 담체 및 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).
상기 본 발명에 따른 약학적 조성물은 상술한 바와 같은 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법에 따라 적합한 형태로 제형화 될 수 있다. 즉, 본 발명의 약학적 조성물은 공지의 방법에 따라 다양한 비경구 또는 경구 투여용 형태로 제조될 수 있다. 비경구 투여용 제형의 대표적인 것으로는 주사용 제형으로 등장성 수용액 또는 현탁액이 바람직하다. 주사용 제형은 적합한 분산제 또는 습윤제 및 현탁화제를 사용하여 당업계에 공지된 기술에 따라 제조할 수 있다. 예를 들면, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화될 수 있다. 또한, 경구 투여용 제형으로는, 이에 한정되지는 않으나, 분말, 과립, 정제, 환약 및 캡슐 등이 있다.The pharmaceutical composition according to the present invention may be formulated into a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods. Representative examples of formulations for parenteral administration include isotonic aqueous solutions or suspensions in the form of injection. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.
상기와 같은 방법으로 제형화된 약학적 조성물은 유효량으로 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 상기에서 '유효량'이란 환자에게 투여하였을 때, 예방 또는 치료 효과를 나타내는 양을 말한다. 본 발명에 따른 약학적 조성물의 투여량은 투여 경로, 투여 대상, 연령, 성별 체중, 개인차 및 질병 상태에 따라 적절히 선택할 수 있다.The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the route of administration, subject to be administered, age, gender, individual difference, and disease state.
본 발명의 다른 양상은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 폐질환 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for preventing or ameliorating pulmonary disease, comprising asparagus extract, fractions thereof, or oleanolin acid acetate isolated therefrom as an active ingredient.
상기 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트는 천연물에서 유래된 것으로 안전성이 입증되었으므로 식품 조성물 용도로 사용될 수 있다. 상기 식품 조성물은 폐질환을 예방 또는 개선하기 위하여 식품 및 음료 등에 첨가되거나, 또는 식품 및 음료의 원료로 이용될 수 있다. 상기 식품 조성물을 포함하는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다. 상기 식품 조성물을 포함하는 식품은 필수 성분으로서 상기 식품 조성물을 함유하는 것 외에 식품학적으로 허용 가능한 식품보조 첨가제, 예컨대, 천연 탄수화물 및 다양한 향미제 등을 추가 성분으로서 함유할 수 있다.The red head extract, fractions thereof, or oleanolin acid acetate isolated therefrom are derived from natural products and can be used for food composition because they have proved safe. The food composition may be added to foods and beverages to prevent or ameliorate pulmonary diseases, or may be used as a raw material for foods and beverages. Examples of foods containing the food composition include various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and they can be used as powder, granule, tablet, capsule or beverage. In addition to containing the food composition as an essential ingredient, the food comprising the food composition may further comprise a food-acceptable food-aid additive, such as natural carbohydrates and various flavors, as an additional ingredient.
상기 식품 조성물은 그 사용 목적 및 용도에 따라 적절하게 선택된 추가적인 여타의 첨가물을 추가로 더 포함할 수 있으며, 통상적으로 사용되는 첨가물 등 다른 유효성분이나 색소, 계면활성제, 방부제 등의 첨가물과 혼합하여 사용할 수 있다. 상기 첨가제는 그 사용 목적 및 용도에 따라 분말화, 과립화, 정제화 또는 액상화하여 제조될 수 있으며, 제품화를 위하여 포장을 위해 통상의 방법을 사용할 수 있다.The food composition may further include other additives appropriately selected according to the purpose and use of the food, and it may be mixed with other active ingredients such as additives used in common use, additives such as coloring matters, surfactants, preservatives, etc. . The additive may be prepared by pulverizing, granulating, tableting or liquefying according to the purpose and use of the additive. For commercialization, a conventional method may be used for packaging.
본 발명의 또 다른 양상은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 폐질환 예방 또는 개선용 개인 위생용품을 제공한다.Another aspect of the present invention provides personal hygiene products for preventing or improving pulmonary disease, comprising aspirin extract, fractions thereof, or oleanolin acid acetate isolated therefrom as an active ingredient.
상기 개인 위생용품은 비누, 화장품, 물티슈, 에어프레쉬너, 세정겔 등의 형태로 제조될 수 있으나 이에 제한되지 아니한다.The personal care product may be manufactured in the form of a soap, a cosmetic, a wet tissue, an air freshner, a cleaning gel, but is not limited thereto.
본 발명의 또 다른 양상은 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공한다. Another aspect of the present invention provides a food composition for prevention or improvement of liver disease comprising aspirin extract, fractions thereof or oleanolin acid acetate isolated therefrom as an active ingredient.
상기 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트는 간독성 지표인 AST(aspartate aminotransferase)와 ALT(alanine aminotransferase)를 수치를 개선하고, 간섬유화증 진행에 의한 세포괴사를 억제하며, 간세포의 산화적 스트레스를 감소시킬 수 있으므로 간질환 예방 및 개선 용도로 유용하게 사용될 수 있다.The red head extract, its fractions or oleanolic acid acetates isolated therefrom can be used to improve the levels of hepatotoxicity aspartate aminotransferase (AST) and alanine aminotransferase (ALT), inhibit cellular necrosis by progression of hepatic fibrosis, It can be used for prevention and improvement of liver disease.
본 발명의 일 실시예에 따른 적소두 추출물, 이의 분획물 또는 이로부터 분리한 올레아놀린산 아세테이트를 유효성분으로 포함하는 조성물은 상피-간엽 이행을 억제하여 섬유화의 진행을 지연시키고, 폐섬유화에 의하여 감소된 폐용적을 증가시키며, 간섬유화 및 산화적 스트레스를 개선시킬 수 있다.The composition comprising the red dragonfly extract, the fraction thereof or the oleanolin acid acetate isolated therefrom as an active ingredient according to an embodiment of the present invention inhibits epithelial-mesenchymal transition to delay the progress of fibrosis, Increase the volume, and improve liver fibrosis and oxidative stress.
도 1은 비소세포폐암(NSCLC) 세포주에 올레아놀린산 아세테이트(oleanolic acid acetate, OAA) 또는 적소두 추출물을 처리한 후 세포 생존율을 측정한 결과를 보여준다.
도 2는 A549 세포에서 EMT(epithelial-mesenchymal transition)를 유도한 후 OAA 투여에 의한 세포 증식 변화를 확인한 결과를 보여준다.
도 3은 A549 세포에서 EMT를 유도한 후 적소두 추출물 또는 OAA를 처리하여 세포 이동 정도를 확인한 결과를 보여준다.
도 4는 A549 세포에서 EMT를 유도한 후 매트리겔을 이용하여 적소두 추출물 및 OAA의 세포 침투 억제 효과를 확인한 결과를 보여준다.
도 5는 A549 세포에 TGF-β1을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 단백질 발현 변화를 확인한 결과를 보여준다.
도 6은 A549 세포에 IL-6을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 단백질 발현 변화를 확인한 결과를 보여준다.
도 7은 A549 세포에 TGF-
도 8은 블레오마이신을 투여하여 폐섬유화증을 유도한 동물모델에서 OAA 투여에 의한 폐섬유화 억제 효과 및 폐용적 변화를 측정한 결과를 보여준다.
도 9는 사염화탄소를 투여하여 급성 간섬유화증을 유도한 동물모델에서 체중, 간 무게, AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)를 측정한 결과를 보여준다.
도 10은 사염화탄소를 투여하여 간섬유화증을 유도한 동물모델에서 체중, 간 무게, AST(aspartate aminotransferase) 및 ALT(alanine aminotransferase)를 측정한 결과를 보여준다.
도 11은 간섬유화증 동물모델의 간조직을 헤마토자일린&에오신으로 염색한 결과를 보여준다.
도 12는 간섬유화증 동물모델의 간조직에서 마손 삼색 염색(Masson trichrome stain)을 수행한 결과를 보여준다.
도 13은 간섬유화증 동물모델의 간조직에서 유도성 산화질소 합성효소(inducible NO synthase) 및 글루타티온 과산화효소(glutathione peroxidase)의 mRNA 발현량을 측정한 결과를 보여준다.FIG. 1 shows the cell survival rate after treatment of non-small cell lung cancer (NSCLC) cell line with oleanolic acid acetate (OAA) or red head extract.
FIG. 2 shows the results of confirming the cell proliferation change induced by OAA after inducing an epithelial-mesenchymal transition (EMT) in A549 cells.
FIG. 3 shows the results of confirming the degree of cell migration by inducing EMT in A549 cells and then treating the cells with red toenne extract or OAA.
FIG. 4 shows the results of confirming the inhibitory effect on the infiltration of red toenite and OAA by inducing EMT in A549 cells and using Matrigel.
FIG. 5 shows the results of confirming the changes in protein expression by treatment with TGF-β1 and A549 cells after induction of EMT and treatment with red cells or OAA.
FIG. 6 shows the results of confirming the expression of protein by A549 cells treated with IL-6 after induction of EMT and treatment with Red Soybean extract or OAA.
Figure 7 shows that A549 cells express TGF-
FIG. 8 shows the results of measurement of lung fibrosis inhibitory effect and lung volume change by OAA administration in an animal model in which pulomycin was administered to induce pulmonary fibrosis.
FIG. 9 shows the results of measurement of body weight, liver weight, aspartate aminotransferase (ALT) and alanine aminotransferase (ALT) in an animal model in which acute liver fibrosis was induced by administration of carbon tetrachloride.
FIG. 10 shows the results of measurement of body weight, liver weight, aspartate aminotransferase (ALT) and alanine aminotransferase (ALT) in an animal model in which hepatic fibrosis was induced by administration of carbon tetrachloride.
Fig. 11 shows the result of staining liver tissue of hepatic fibrosis animal model with hematoxylin and eosin.
Figure 12 shows the results of performing masson trichrome staining in liver tissue of an animal model of hepatic fibrosis.
FIG. 13 shows the results of measurement of mRNA expression levels of inducible NO synthase and glutathione peroxidase in liver tissue of an animal model of hepatic fibrosis.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.
실시예Example 1: 적소두 추출물, 1: Red head extract, 분획물Fraction 및 활성 성분의 분리 And separation of active ingredients
1-1: 적소두 추출물 제조1-1: Manufacture of Red Pepper Extract
적소두(팥 또는 이팥)를 물로 깨끗이 세척하여 그늘에서 건조시킨 후, 와링 블랜더(waring blender)로 분쇄하여 분말로 제조하였다. 적소두 분말 20 ㎏에 메탄올 100 ℓ를 첨가하고, 실온에서 3일 동안 냉침 추출하였다. 3일 후 추출액을 여과지(Whatman, 미국)로 감압 여과하고, 진공회전농축기로 메탄올을 제거하여 적소두 추출물 450 g을 수득하였다.Red bean (red bean or red bean) was thoroughly washed with water, dried in the shade, and pulverized with a waring blender to prepare powder. 100 L of methanol was added to 20 kg of the red powder, and the mixture was cold-extracted at room temperature for 3 days. After 3 days, the extract was filtered under reduced pressure through a filter paper (Whatman, USA), and methanol was removed by vacuum rotary evaporator to obtain 450 g of red head extract.
1-2: 1-2: 분획물Fraction 제조 Produce
상기 수득한 적소두 추출물에서 활성 분획물을 분리하기 위하여 적소두 추출물을 물 1ℓ에 현탁시키고, 동량의 n-헥산을 첨가하여 혼합한 후 분획하였다. 이 과정을 4회 반복하여 수가용성 분획물 1ℓ와 n-헥산 가용성 분획물 4ℓ를 수득하였다. 이후, 상기 n-헥산 가용성 분획물을 감압 농축하여 n-헥산 가용 추출물 50 g을 수득하였다.To separate the active fractions from the obtained red yeast extract, the red head extract was suspended in 1 liter of water, and the same amount of n-hexane was added thereto, followed by mixing and fractionation. This process was repeated four times to obtain 1 liter of water soluble fraction and 4 liter of n-hexane soluble fraction. Thereafter, the n-hexane soluble fraction was concentrated under reduced pressure to obtain 50 g of the n-hexane soluble extract.
또한, 상기 수가용성 분획물 1ℓ에 동량의 에틸아세테이트(C4H8O2)를 첨가하여 혼합한 후 분획하고, 이 과정을 3회 반복하여 수가용성 분획물 1ℓ와 에틸아세테이트 가용성 분획물 3ℓ를 다시 수득하였다. 상기 수득한 에틸아세테이트 가용성 분획물을 감압농축하여 에틸아세테이트 가용 추출물 35 g을 수득하고, 남은 수가용성 분획물을 감압농축하여 35 g을 수득하였으며, 이를 물 분획물로 사용하였다.Further, the same amount of ethyl acetate (C 4 H 8 O 2 ) was added to 1 liter of the water-soluble fraction, followed by mixing and fractionation. This procedure was repeated three times to obtain 1 liter of the water-soluble fraction and 3 liter of the ethyl acetate-soluble fraction . The obtained ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 35 g of ethyl acetate soluble extract. The remaining water-soluble fraction was concentrated under reduced pressure to obtain 35 g of a water fraction.
1-3: 적소두 추출물 및 1-3: Redhead extract and 분획물의Fraction HPLCHPLC 분석 analysis
상기 실시예 1-1 및 1-2에서 수득한 각각의 적소두 추출물 및 분획물을 대상으로 하여 HPLC 분석을 수행하였다. HPLC analysis was performed on each of the red head extracts and fractions obtained in Examples 1-1 and 1-2.
HPLC는 Agilent Technologies 1200 series를 사용하였고, 분석용 컬럼은 YMC(일본)의 J'sphere ODSH80(YMC, 4 ㎛, 4.6 ㎜ I.D.x150 ㎜) 컬럼을 사용하였다. 분석용매는 5% 내지 90% 아세토나이트릴(CH3CN)을 1 ㎖/min 속도로 흘려주면서 210 ㎚에서 분석하였고, 시료는 10 ㎕를 주입하였다. HLPC 분석조건은 하기 표 1에 기재하였다.Agilent Technologies 1200 series was used for HPLC, and J'sphere ODSH80 (YMC, 4 μm, 4.6 mm ID x 150 mm) column of YMC (Japan) was used for the analysis column. The analytical solvent was analyzed at 210 nm while flowing 5% to 90% acetonitrile (CH 3 CN) at a flow rate of 1 ml / min, and 10 μl of the sample was injected. The HLPC analysis conditions are shown in Table 1 below.
HPLC 분석 결과 카테킨-7-글루코피라노시드(catechin-7-glucopyranoside, catechin-7-glu), 루틴(rutin), 올레아놀린산 아세테이트(oleanolic acid acetate, OAA) 및 스티그마스테롤(stigmasterol)의 피크가 각각 5.5, 24.5, 35.5, 35.5분대에 나타나는 것을 관찰할 수 있었으며, 팥 및 이팥의 HPLC 크로마토그램은 상호 유사한 패턴을 나타내는 것을 확인할 수 있었다.HPLC analysis showed that the peaks of catechin-7-glucopyranoside, catechin-7-glu, rutin, oleanolic acid acetate (OAA) and stigmasterol 5.5, 24.5, 35.5, and 35.5, respectively. HPLC chromatograms of red bean and red bean showed similar patterns.
1-4: 유효성분의 정제1-4: Purification of active ingredients
상기 실시예 1-2에서 수득한 n-헥산 분획물 80 g을 헥산:에틸아세테이트(100:1→1:1)로 구성된 단계농도 구배(step gradient) 용매 시스템을 이용하여 실리카겔 컬럼 크로마토그래피(silica gel column chromatography)에 적용하여 5개의 활성분획(Fr.1 내지 5)을 수득하였다. 상기 활성분획 중에서 3번 및 4번 분획에 메탄올을 첨가하여 재결정과정을 수행함으로써, 흰색 분말상을 나타내는 2종의 화합물을 정제하였다.80 g of the n-hexane fraction obtained in Example 1-2 was purified by silica gel column chromatography using a step gradient solvent system consisting of hexane: ethyl acetate (100: 1 to 1: 1) column chromatography to obtain five active fractions (Fr.1 to 5). Among the above active fractions, methanol was added to
상기 정제한 2종의 화합물을 기기분석(1H-, 13C-NMR, MS) 및 문헌 값(Voutquenne L. et al. Phytochemistry 2003, 64, 781-789; Kongduang D. et al. Tetrahedron letters 2008, 49, 4067-4072)에 적용한 결과 각각 올레아놀린산 아세테이트(oleanolic acid acetate; 화학식 1)임을 확인할 수 있었다.The two purified compounds were analyzed by instrumental analysis ( 1 H-, 13 C-NMR, MS) and literature values (Voutquenne L. et al. Phytochemistry 2003, 64, 781-789; Kongduang D. et al. Tetrahedron letters 2008 , 49, 4067-4072). As a result, it was confirmed that oleanolic acid acetate (Formula 1) was used.
[화학식 1] 올레아놀린산 아세테이트[Formula 1] < EMI ID =
4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid acetate10a, 12aR, 14bS-10-hydroxy-2,2,6a, 6b, 9,9,12a-heptamethyl-1,3,4,5,6,6a, 7a, 6a, 6aR, 6aS, 6bR, 8aR, 8,8a, 10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid acetate
실시예Example 2: 2: 올레아놀린산Oleanolic acid 아세테이트 처리에 의한 세포 생존율 변화 확인 Determination of cell viability by acetate treatment
상기 실시예 1에서 수득한 적소두 추출물 및 올레아놀린산 아세테이트가 세포 생존율에 영향을 미치는지 확인하기 위하여 MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]로 세포 생존율을 확인하였다.The cell survival rate was determined by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] in order to examine the effect of the red cells extract and the oleanolin acetate obtained in Example 1 on cell viability Respectively.
비소세포폐암(non-small cell lung cancer, NSCLC) 세포주를 1x104/㎕ 농도로 배지에 부유시킨 후 96-웰 플레이트(96-well plate)에 200 ㎕씩 분주하여 24시간 동안 배양하였다. 24시간 후 올레아놀린산 아세테이트(이하, OAA로 기재함; 0, 1, 3, 6, 10 및 30 uM) 또는 적소두 추출물(0, 5, 10, 30 및 60 ng/㎖)을 포함하는 배지를 웰에 200 ㎕씩 첨가하여 각각 24시간 또는 48시간 동안 배양하였다.Non-small cell lung cancer (NSCLC) cell lines were suspended in the medium at a concentration of 1 x 10 < 4 > / uL and 200 [mu] l was added to 96-well plates for 24 hours. After 24 hours, medium containing oleanolin acetate (hereinafter referred to as OAA; 0, 1, 3, 6, 10 and 30 uM) or red head extract (0, 5, 10, 30 and 60 ng / Were added to each well and cultured for 24 hours or 48 hours, respectively.
배양이 끝난 후 PBS(phosphate buffered saline)에 MTT를 희석(0.5 ㎎/㎖)하여 각 웰에 20 ㎕씩 분주하고, CO2 배양기에서 4시간 동안 배양하였다. 4시간 후 MTT 용액을 제거하고, 각 웰에 DMSO(dimethyl sulfoxide)를 200 ㎕씩 분주하여 포르마잔(formazan) 침전물을 5분 동안 용해시킨 다음 595 ㎚에서 흡광도를 측정하였다.After the incubation, MTT was diluted (0.5 ㎎ / ㎖) in PBS (phosphate buffered saline), and 20 ㎕ was added to each well and cultured in a CO 2 incubator for 4 hours. After 4 hours, the MTT solution was removed and 200 μl of DMSO (dimethyl sulfoxide) was added to each well. The formazan precipitate was dissolved for 5 minutes and the absorbance was measured at 595 nm.
측정 결과, 적소두 추출물 처리군에서는 세포 생존율에 영향이 없지만, OAA 처리군에서는 OAA의 농도가 증가할수록 세포 생존율이 감소하는 것을 확인할 수 있었다.As a result, the cell survival rate was not affected in the group treated with Red Pepper Extract, but the cell survival rate was decreased with increasing OAA concentration in the OAA treated group.
도 1은 비소세포폐암 세포에 OAA 또는 적소두 추출물을 처리하여 24시간 또는 48시간 동안 배양한 후 세포 생존율을 측정한 결과를 보여주는 그래프이다.FIG. 1 is a graph showing cell survival rate after culturing non-small cell lung cancer cells for 24 hours or 48 hours after treatment with OAA or Red Pepper Extract. FIG.
실시예Example 3: 3: TGFTGF -- β1β1 유도성 상피- Inducible epithelium- 간엽Liver 이행(epithelial- The epithelial- mesenchymalmesenchymal transition, transition, EMTEMT )에 대하여 )about OAAOAA 투여에 의한 Administration EMTEMT 억제 효과 확인 Confirmation of inhibition
상피-간엽 이행(epithelial-mesenchymal transition, 이하 EMT로 기재함)은 상피세포(epithelial cell)가 간엽세포(mesenchymal cell)로 전환되어 가는 과정으로 폐섬유화증의 발병 과정에서 중요한 역할을 하는 것으로 알려져 있다. EMT가 진행되면 상피세포의 증식 및 이동이 증가하기 때문에 적소두 추출물 및 OAA의 EMT 억제 효능을 하기와 같이 확인하였다.The epithelial-mesenchymal transition (hereinafter referred to as EMT) is known to play an important role in the pathogenesis of pulmonary fibrosis, as epithelial cells are transformed into mesenchymal cells . As the proliferation and migration of epithelial cells increase with the progress of EMT, the efficacy of EMR suppression of the red head extract and OAA was confirmed as follows.
폐암 유래 세포인 A549 세포 및 섬유아세포인 NIH-3T3 세포를 1x104/㎖로 배지에 부유시킨 후 96-웰 플레이트에 200 ㎕씩 분주하여 24시간 동안 배양하였다. 24시간 후 각 웰에 TGF-β1을 처리하고, OAA(30 uM) 또는 피르페니돈(Pirfenidone; 0.3 ㎎/㎖)을 포함하는 배지를 200 ㎕씩 첨가하여 48시간 동안 배양하였다. 배양이 끝나면 Cell Counting Kit-8(CCK-8 kit; Dojindo, 일본) 용액을 각 웰에 20 ㎕씩 분주하고, CO2 배양기에서 1시간 동안 배양하였다. 이후 450 ㎚에서 흡광도를 측정하였다.A549 cells and NIH-3T3 fibroblasts, which are lung cancer-derived cells, were suspended in 1 × 10 4 / ml of the culture medium, and then 200 μl of each was added to a 96-well plate and cultured for 24 hours. After 24 hours, each well was treated with TGF-? 1 and cultured for 48 hours with 200 占 퐇 of medium containing OAA (30 uM) or Pirfenidone (0.3 mg / ml). After the incubation, 20 μl of the Cell Counting Kit-8 (CCK-8 kit; Dojindo, Japan) solution was dispensed into each well and cultured in a CO 2 incubator for 1 hour. The absorbance was then measured at 450 nm.
측정 결과, TGF-1 투여에 의하여 증가된 EMT 관련 세포 증식이 OAA 투여에 의하여 A549 세포 및 NIH-3T3 세포 모두에서 유의적으로 감소된 것을 확인할 수 있었다.As a result, 1-induced EMT-related cell proliferation was significantly reduced in both A549 cells and NIH-3T3 cells by OAA administration.
도 2는 A549 세포에 TGF-β1을 투여하여 EMT를 유도한 후 OAA 투여에 의한 세포 증식 변화를 확인한 결과를 보여주는 그래프이다. 도 2에서 Pir은 피르페니돈을 의미하고, 도 2의 A는 A549 세포, 도 2의 B는 NIH-3T3 세포에서 확인한 결과이다.FIG. 2 is a graph showing the results of confirming cell proliferation changes by OAA after induction of EMT by administering TGF-β1 to A549 cells. In Fig. 2, Pir means pypenidone, A in Fig. 2 shows A549 cells, and Fig. 2 shows results in NIH-3T3 cells.
실시예Example 4: 4: TGFTGF -- β1β1 유도성 Inductive EMT에EMT 대한 적소두 추출물 및 Red Toxin extract and OAA의Of OAA 효과 확인 Check the effect
4-1. 4-1. 스크래치scratch 운드Wound 힐링healing (Scratch wound healing) 방법을 이용한 적소두 추출물 및 (Scratch wound healing) OAA의Of OAA 세포 이동 억제 효과 확인 Confirm cell migration inhibition effect
A549 세포를 6-웰 플레이트에 1x105/㎖로 분주하여 24시간 동안 배양하였다. 24시간 후 200 ㎕ 팁(tip)으로 단일 세포층을 스크래치(scratch)하고, OAA(10 및 30 uM) 또는 적소두 추출물(30 및 60 ㎍/㎖)을 포함하는 배지를 각 웰에 1 ㎖씩 첨가하였다. 48시간 동안 배양한 후 세포의 이동 정도를 현미경으로 확인하고, 이미지를 촬영하였다.A549 cells were plated on a 6-well plate at 1 x 10 < 5 > / ml and cultured for 24 hours. After 24 hours, the single cell layer was scratched with 200 [mu] l tip and the medium containing OAA (10 and 30 uM) or red head extract (30 and 60 [mu] g / ml) was added to each well in an amount of 1 ml . After incubation for 48 hours, the degree of migration of the cells was confirmed by a microscope and images were taken.
확인 결과 대조군(Control)과 비교하여 TGF-β1을 처리한 세포에서 세포 이동이 증가한 것을 알 수 있었으며, 적소두 추출물 또는 OAA를 처리한 경우 세포 이동이 감소한 것을 확인할 수 있었다. 특히 고농도의 OAA를 처리한 경우 세포 이동이 현저하게 감소하였다.As a result, it was found that the cell migration was increased in the TGF-β1-treated cells compared with the control (Control), and the cell migration was reduced when the red cells or OAA was treated. Especially, treatment with high concentration of OAA significantly decreased cell migration.
도 3의 A는 A549 세포에 TGF-β1을 투여하여 EMT를 유도한 후 적소두 추출물 또는 OAA를 처리하여 세포 이동 정도를 촬영한 사진이다.FIG. 3 A is a photograph showing the degree of cell migration by administering TGF-β1 to A549 cells and inducing EMT, followed by treatment with reddish-purine extract or OAA.
4-2. 4-2. ECISECIS (Electrical Cell-Substrate Impedance Sensing) 방법을 이용한 적소두 추출물 및 (Reduced toddler extracts using Electrical Cell-Substrate Impedance Sensing) OAA의Of OAA 세포 이동 억제 효과 확인 Confirm cell migration inhibition effect
ECIS는 세포의 형태학적 변화 및 여러 가지 약물에 대한 반응을 실시간으로 모니터링하는 장치이며, TGF-β1 처리에 의하여 세포 이동이 증가하면 저항값이 증가하기 때문에 OAA 또는 적소두 추출물 처리에 의한 세포 이동 변화를 간접적으로 측정할 수 있다.ECIS is a device that monitors the morphological changes of cells and responses to various drugs in real time. As the cell migration increases by TGF-β1 treatment, the resistance value increases. Therefore, the cell migration by OAA or Reduced Toxin It can be measured indirectly.
전극이 장착되어 있는 웰 플레이트에서 A549 세포를 배양하고, 상기 실시예 3-1과 동일한 방법으로 OAA(10 및 30 uM) 또는 적소두 추출물(30 및 60 uM)을 처리하였다. 이 과정에서 발생되는 전류에 대한 저항값(Impedance)의 변화를 측정하였다.A549 cells were cultured in a well plate equipped with an electrode and treated with OAA (10 and 30 uM) or red head extract (30 and 60 uM) in the same manner as in Example 3-1. The change of the resistance value (Impedance) against the current generated in this process was measured.
측정 결과 대조군(Con A549)과 비교하여 TGF-β1을 처리한 경우 저항값이 증가하여 세포 이동이 증가하는 것을 알 수 있었으며, OAA를 30 uM 처리한 경우 저항값이 현저하게 감소하여 세포 이동이 감소하는 것을 확인할 수 있었다.As a result of the measurement, the resistance value was increased and the cell migration was increased in the case of treatment with TGF-β1 compared to the control (Con A549). When 30 μM of OAA was treated, the resistance value was remarkably decreased, .
도 3의 B는 A549 세포에 TGF-β1을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA를 처리하여 세포 이동 정도를 ECIS로 확인한 결과를 보여주는 그래프이다.FIG. 3B is a graph showing the results of ECIS analysis of cell migration by treating TGF-β1 with A549 cells to induce EMT, and then treating the cells with ODS or O7A.
실시예Example 5: 5: TGFTGF -- β1β1 유도성 Inductive EMT에EMT 대하여 적소두 추출물 및 Against red toddler extract and OAA의Of OAA 세포 침투 억제 효과 확인 Confirm cell infiltration inhibition effect
트랜스-웰 인서트(Trans-well insert; Corning, 미국)에 매트리겔(matrigel; Corning)을 2 ㎎/㎖로 코팅하였다. 무혈청 배지(serum free media) 100 ㎕에 A549 세포를 약 2x104개가 되도록 희석하고, 여기에 OAA(10 및 30 uM) 또는 적소두 추출물(30 및 60 ㎍/㎎)을 첨가하여 트랜스-웰 인서트에 분주하였다. 리시브 챔버(Receive chambers)에는 10% 혈청을 포함하는 배지를 분주하여 세포를 48시간 동안 배양하였다. 배양이 끝난 후 인서트에 남아 있는 세포는 제거하고, 리시브 챔버로 침투한 세포를 크리스탈 바이올렛(crystal violet)으로 염색하여 현미경으로 확인하였다. A trans-well insert (Corning, USA) was coated with 2 mg / ml of matrigel (Corning). A549 cells were diluted to about 2 x 10 4 in 100 μl of serum-free media and OAA (10 and 30 μM) or red head extract (30 and 60 μg / mg) was added thereto and added to the trans-well insert Respectively. Receive chambers were seeded with medium containing 10% serum and the cells were cultured for 48 hours. After the incubation, the cells remaining in the insert were removed, and the cells infiltrated into the receive chamber were stained with crystal violet and confirmed by a microscope.
확인 결과 대조군(control)과 비교하여 TGF-β1을 처리한 세포의 경우 리시브 챔버로 침투한 세포의 수가 증가한 것을 알 수 있었으며, 이와 반대로 적소두 추출물 또는 OAA를 처리한 세포의 경우 리시브 챔버로 침투한 세포의 수가 감소한 것을 확인할 수 있었다.As a result, it was found that the cells treated with TGF-β1 increased the number of cells infiltrated into the receive chamber as compared with the control (control). On the other hand, in the case of cells treated with red cells or OAA, Of the total number of patients.
도 4는 A549 세포에 TGF-β1을 처리하여 EMT를 유도한 후 매트리겔을 이용하여 적소두 추출물 및 OAA의 세포 침투 억제 효과를 확인한 결과를 보여주는 사진이다.FIG. 4 is a photograph showing the results of confirming the inhibition of cell infiltration of red papilla extract and OAA by using Matrigel after inducing EMT by treating TGF-β1 with A549 cells.
실시예Example 6: 6: TGFTGF -- β1β1 유도성 Inductive EMT에EMT 대하여 적소두 추출물 및 Against red toddler extract and OAAOAA 처리에 의한 단백질 발현 변화 확인 Identification of protein expression changes by treatment
A549 세포를 6-웰 플레이트에 1x105/㎖로 분주하여 24시간 동안 배양하였다. 이후 OAA(10 및 30 uM) 또는 적소두 추출물(30 및 60 ㎍/㎎)을 포함하는 배지를 각 웰에 1 ㎖씩 첨가하여 다시 48시간 동안 배양하였다. 배양이 끝난 후 각 웰에 RIPA 용해 버퍼[50 mM Tris-Cl(pH, 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF(phenylmethylsulfonyl fluoride), 1 ㎍/㎖ aprotinin, 1 ㎍/㎖ leupeptin, 및 1 mM Na3VO4]를 500 ㎕씩 첨가하여 4℃에서 15분 동안 세포를 용해시켰다. 다음으로 4℃에서 원심분리(14,000 rpm 및 10분)하여 상등액만을 수거하고, SDS-PAGE(SDS-polyacrylamide gel electrophoresis)로 단백질을 분리하였다. 분리된 단백질을 PVDF 막(polyvinylidene difluoride membrane)으로 이동시키고, 일차 항체와 4℃에서 12시간 동안 반응시켰다. 12시간 후 각각의 일차항체에 대한 이차항체와 추가로 반응시킨 후 ECL kit(Thermo Fisher Scientific, 미국)를 이용하여 제조사의 프로토콜에 따라 단백질 밴드를 확인하였다.A549 cells were plated on a 6-well plate at 1 x 10 < 5 > / ml and cultured for 24 hours. The medium containing OAA (10 and 30 uM) or red head extract (30 and 60 占 퐂 / mg) was then added to each well in an amount of 1 ml, followed by incubation for another 48 hours. After incubation, the wells were incubated with RIPA lysis buffer [50 mM Tris-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg / ml aprotinin, / ㎖ leupeptin, and 1 mM Na 3 VO 4] was added to each 500 ㎕ dissolve the cells for 15 minutes at 4 ℃. Next, the supernatant was collected by centrifugation (14,000 rpm and 10 minutes) at 4 ° C, and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene difluoride membrane) and reacted with the primary antibody at 4 ° C for 12 hours. After 12 hours, the cells were further reacted with the secondary antibody for each primary antibody, and the protein bands were confirmed using an ECL kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.
확인 결과 TGF-β1 처리에 의하여 증가된 E-cadherin, N-cadherin, Vimentin, Snail, α-SMA 및 Collagen I의 발현이 적소두 추출물 또는 OAA 처리에 의하여 발현이 감소하는 것을 확인할 수 있었다.Expression of E-cadherin, N-cadherin, Vimentin, Snail, α-SMA, and Collagen I was increased by TGF-β1 treatment and decreased by OAA treatment.
또한, IL-6을 처리한 경우 상기 단백질의 발현 이외에도 인산화된 STAT3의 수준이 증가하지만 적소두 추출물 또는 OAA 처리에 의하여 STAT3의 인산화가 감소하는 것을 확인할 수 있었다.In addition, when IL-6 was treated, the level of phosphorylated STAT3 was increased in addition to the expression of the protein, but the phosphorylation of STAT3 was decreased by treatment with Reduced Toxin extract or OAA.
도 5는 A549 세포에 TGF-β1을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 단백질 발현 변화를 확인한 결과를 보여주는 그림이다.FIG. 5 is a graph showing the results of confirming the changes in protein expression by treatment with TGF-β1 and EMR-induced red cells or OAA in A549 cells.
도 6은 A549 세포에 IL-6을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 단백질 발현 변화를 확인한 결과를 보여주는 그림이다.FIG. 6 is a graph showing the results of confirming the expression of protein by A549 cells treated with IL-6 and EMT, followed by treatment with Pfu extract or OAA.
실시예Example 7: 7: TGFTGF -- β1β1 유도성 Inductive EMT에EMT 대하여 적소두 추출물 및 Against red toddler extract and OAAOAA 처리에 의한 세포의 형태 변화 확인 Confirmation of morphological changes of cells by treatment
A549 세포를 4-웰 플레이트에 1x105/㎖로 분주하여 24시간 동안 배양하였다. 이후 OAA(10 및 30 uM) 또는 적소두 추출물(30 및 60 ㎍/㎎)을 포함하는 배지를 각 웰에 1 ㎖씩 첨가하여 다시 48시간 동안 배양하였다. 배양이 끝난 후 세포를 PBS(phosphate buffered saline)로 세척하고, 4% 파라포름알데하이드(paraformaldehyde)로 20분 동안 고정하였다. 상기 고정시킨 세포에 TritonX-100(0.2% in PBS)을 첨가하여 상온에서 반응시키고, 5% 정상 염소 혈청(normal goat serum)으로 상온에서 1시간 동안 블록킹(blocking)시켰다. 블록킹 버퍼(blocking buffer)에 1차 항체를 1:200으로 희석하여 4℃에서 12시간 동안 반응시키고, 각각의 일차항체에 대한 이차항체와 90분 동안 추가로 반응시켰다. 반응이 종료한 후 DAPI가 포함된 마운팅 용액(mounting solution)을 분주하여 현미경으로 관찰하고, 이미지를 촬영하였다.A549 cells were plated on a 4-well plate at 1 × 10 5 / ml and cultured for 24 hours. The medium containing OAA (10 and 30 uM) or red head extract (30 and 60 占 퐂 / mg) was then added to each well in an amount of 1 ml, followed by incubation for another 48 hours. After incubation, the cells were washed with PBS (phosphate buffered saline) and fixed with 4% paraformaldehyde for 20 min. TritonX-100 (0.2% in PBS) was added to the fixed cells, reacted at room temperature, and blocked with 5% normal goat serum at room temperature for 1 hour. The primary antibody was diluted 1: 200 in a blocking buffer, reacted at 4 ° C for 12 hours, and further reacted with the secondary antibody for each primary antibody for 90 minutes. After completion of the reaction, a mounting solution containing DAPI was dispensed and observed under a microscope, and images were taken.
확인 결과, TGF-β1 처리에 의하여 증가된 E-cadherin, N-cadherin, Vimentin 및 α-SMA의 발현이 적소두 추출물 또는 OAA 처리에 의하여 감소하는 것을 확인할 수 있었다.As a result, it was confirmed that the expression of E-cadherin, N-cadherin, Vimentin and α-SMA increased by TGF-β1 treatment was decreased by the treatment with Reduced tofu extract or OAA treatment.
도 7은 A549 세포에 TGF-β1을 처리하여 EMT를 유도한 후 적소두 추출물 또는 OAA 처리에 의한 E-cadherin, N-cadherin, Vimentin 및 α-SMA의 발현을 확인한 결과를 보여주는 사진이다.FIG. 7 is a photograph showing the results of confirming the expression of E-cadherin, N-cadherin, Vimentin and α-SMA by treatment with TGF-β1 and EMT and treatment with OEA extract of A549 cells.
실시예Example 8: 8: OAA의Of OAA 폐섬유화증 억제 효과 확인 Confirmation of inhibition of pulmonary fibrosis
8-1. 폐섬유화증 동물모델 제작8-1. Production of an animal model of pulmonary fibrosis
마우스의 기도를 절개하고, 블레오마이신(bleomycin; 2 ㎎/㎏)을 50 ㎕ 부피로 기도 내에 주입하였다. 주입 후 절개 부위는 5-0 나일론으로 봉입(suture)하였으며, 블레오마이신을 주입한 후 24시간이 지난 시점에 OAA를 투여하였다. 블레오마이신 주입 후 각각 3일, 7일, 10일, 13일, 17일, 20일 및 21일 차에 마우스를 희생시키고, BALF(broncho-alveolar lavage fluid) 및 폐조직을 분리하여 추가 실험에 이용하였다.Mice were intubated and bleomycin (2 mg / kg) was injected intraperitoneally in a volume of 50 ul. After injection, the incision site was sutured with 5-0 nylon, and OAA was administered 24 hours after injection of bleomycin. Mice were sacrificed at 3 days, 7 days, 10 days, 13 days, 17 days, 20 days, and 21 days after bleomycin injection, and BALF (broncho-alveolar lavage fluid) Respectively.
8-2. 8-2. OAAOAA 투여에 의한 Administration 폐섬유화Pulmonary fibrosis 억제 효과 확인 Confirmation of inhibition
원추-빔형(cone-beam type)의 평판형 X-선 영상센서(flat-panel detector)를 기반으로 하는 볼륨 CT(computed tomography)를 이용하여 CT 영상을 촬영하였다. 이때 엑스선 발생장치의 관전압 및 관전류는 각각 50 kV와 120 ㎄로 하였다. CT는 한 조사(projection)당 0.5°씩 회전하여 360° 회전에 총 720개의 영상을 촬영하여 재조합하였다. 재조합된 영상은 화상을 구성하는 최소 단위의 화소가 1,024x1,024 픽셀(pixel)이며, 총 512개의 재조합 단층 영상을 얻을 수 있었다. CT images were taken using volume CT (computed tomography) based on a cone-beam type flat-panel detector (flat-panel detector). At this time, the tube voltage and the tube current of the x-ray generator were 50 kV and 120 각각, respectively. CT reconstructed a total of 720 images at 360 ° rotation by 0.5 ° rotation per projection. The reconstructed images consisted of 1024x1,024 pixels (pixels) of the smallest units composing the image, and a total of 512 reconstructed tomographic images were obtained.
3D-렌더링 소프트웨어 프로그램인 Xelis(Infinitt Technology, Seoul, Korea)를 이용하여 재조합된 영상을 삼차원으로 확인하였으며, 영상 분석은 시상면(Sagittal plane), 축상면(axial plane) 및 관상면(coronal plane)의 각 방향에서 관찰하였다.The reconstructed image was three-dimensionally analyzed using a 3D-rendering software program Xelis (Infinitt Technology, Seoul, Korea). The image analysis was performed using a sagittal plane, an axial plane, and a coronal plane. .
관찰 결과, 블레오마이신을 주입한 경우 폐섬유화가 진행되지만 블레오마이신 주입 후 OAA를 투여한 경우 OAA 농도의존적으로 폐섬유화가 억제된 것을 확인할 수 있었으며, 시간이 경과해도 폐섬유화가 진행되지 않는 것을 알 수 있었다.Observation showed that pulmonary fibrosis progressed when the bleomycin was injected, but when OAA was administered after bleomycin injection, it was confirmed that OAA concentration-dependent pulmonary fibrosis was suppressed, and that pulmonary fibrosis did not progress even after the lapse of time there was.
도 8은 블레오마이신을 투여하여 폐섬유화증을 유도한 동물모델에서 OAA 투여에 의한 폐섬유화 억제 효과를 보여주는 것으로, 2번째 및 4번째 패널이 재조합 단층 영상을 보여주는 것이다.FIG. 8 shows the inhibitory effect of OAA on pulmonary fibrosis in an animal model in which pulmomycin was induced to induce pulmonary fibrosis, and the second and fourth panels show reconstructed tomographic images.
8-3. 8-3. OAAOAA 투여에 의한 Administration 폐용적Lung volume (Lung volume) 변화 확인 (Lung volume) change confirmation
폐섬유화가 진행되면 폐용적이 감소하기 때문에 폐섬유화증 동물모델에 OAA를 투여한 후 폐용적을 측정하였다. 그 결과 블레오마이신을 투여하면 폐용적이 감소하지만, OAA를 투여하면 감소된 폐용적이 다시 회복하는 것을 확인할 수 있었다.Since pulmonary volume decreases when pulmonary fibrosis progresses, lung volume was measured after OAA administration in an animal model of pulmonary fibrosis. As a result, bleomycin administration decreased lung volume, but OAA administration showed a decrease in lung volume.
도 8은 블레오마이신을 투여하여 폐섬유화증을 유도한 동물모델에서 OAA 투여에 의한 폐용적 변화를 측정한 결과를 보여주는 것으로, 1번째 및 3번째 패널이 폐용적 측정 결과를 보여주는 것이다.FIG. 8 shows the results of measurement of lung volume change by OAA administration in an animal model in which pulmomycin was induced to induce pulmonary fibrosis. The first and third panels show the results of lung volume measurement.
실시예Example 9: 9: OAA의Of OAA 급성 Acute 간섬유화증Liver fibrosis 억제 효과 확인 Confirmation of inhibition
9-1. 급성 9-1. Acute 간섬유화증Liver fibrosis 동물모델 제작 Animal modeling
체중 200 내지 250 g의 4주령 수컷 SD 랫트(SD rat; 오리엔탈바이오, 서울, 대한민국)를 구입하여 SPF 동물 사육실에서 일정 온도(23±3℃), 습도(55±15%) 및 조사량(7:00시부터 19:00시까지) 조건에서 사육하였다. 랫트는 1주일 동안 안정시킨 후 실험에 사용하였으며, 무작위로 5개 그룹으로 분류하였다: 대조군(생리식염수 투여군; Control, CON), 사염화탄소 단독 투여군(carbon tetrachloride; CCl4), 사염화탄소&올레아놀린산(oleanolic acid, OA) 병용 투여군(CCl4+OA 50 ㎎/㎏), 사염화탄소&올레아놀린산 아세테이트 병용 투여군(CCl4+OAA 10 ㎎/㎏) 및 사염화탄소&올레아놀린산 아세테이트 병용 투여군(CCl4+OAA 50 ㎎/㎏). OA와 OAA는 정해진 용량으로 3일 동안 매일 경구투여하고, 경구투여 3일차에 사염화탄소(100 ㎕/g)를 복강 투여한 후 24시간 뒤에 랫트를 희생시켰다.(23 ± 3 ° C), humidity (55 ± 15%) and irradiation dose (7: 1) in an SPF animal breeding room by purchasing a 4-week-old male SD rat (Oriata Bio, Seoul, Korea) 00 to 19:00). Rats were stabilized for one week and used in the experiment and were randomly divided into five groups: control (CON, Control), carbon tetrachloride (CCl 4 ), carbon tetrachloride & oleanolic acid acid, OA) combined administration group (CCl 4 + OA 50 ㎎ / ㎏), carbon tetrachloride & olreahnol acid acetate combined administration group (CCl 4 + OAA 10 ㎎ / ㎏) and carbon tetrachloride & olreahnol acid acetate combined administration group (CCl 4 +
9-2. 급성 9-2. Acute 간섬유화증Liver fibrosis 동물모델에서 체중, 간 무게 및 In animal models, weight, liver weight and 간기능Liver function 지표 확인 Check indicator
랫트를 희생시킨 후 체중을 측정하고, 간을 적출하여 무게를 측정하였다. 또한, 심장에서 혈액을 채취하여 4℃에서 원심분리(3000rpm, 15분)하고, 혈청만을 분리하여 자동분석장치(automatic analyzer; Fuji Dry-Chem NX500i, 일본)로 간 기능과 관련된 생화학적 지표인 AST(aspartate aminotransferase)와 ALT(alanine aminotransferase)를 측정하였다. AST와 ALT는 간세포 내에 존재하는 효소로 급성 간 손상시 혈청 내로 유출되며, 혈중 AST와 ALT 수치의 상승은 일반적으로 간 손상을 의미한다.The rats were sacrificed, their weights were measured, and the liver was weighed and weighed. Blood was collected from the heart and centrifuged at 3000 rpm for 15 minutes at 4 ° C. The serum was separated and analyzed with an automatic analyzer (Fuji Dry-Chem NX500i, Japan) using a biochemical indicator AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were measured. AST and ALT are enzymes present in hepatocytes, which leak into the serum upon acute liver injury, and elevated levels of AST and ALT in the blood usually signify liver damage.
측정 결과, 도 9에 나타난 바와 같이 CCl4 투여군에서는 대조군과 비교하여 체중과 간의 무게가 감소하여 급성 간섬유화가 유발된 것을 알 수 있었다. 또한, CCl4 투여군에서는 간 독성으로 인하여 AST 및 ALT 수치가 크게 증가한 반면, CCl4+OA 병용 투여군 또는 CCl4+OAA 병용 투여군에서는 증가된 AST 및 ALT 수치가 감소하는 것을 확인할 수 있었다. 특히, CCl4+OA 병용 투여군보다 CCl4+OAA 병용 투여군에서 AST 및 ALT 수치 감소 효과가 더 현저하여 OA보다 OAA의 간섬유화 치료 효과가 더 우수한 것을 알 수 있었다.As a result, as shown in FIG. 9, in the CCl 4 administration group, weight and weight of the liver were decreased compared to the control group, and it was found that acute liver fibrosis was induced. In addition, AST and ALT levels were significantly increased in CCl 4 treated group, whereas AST and ALT levels were decreased in CCl 4 + OA treated group or CCl 4 + OAA treated group. In particular, CCl 4 + and OA in CCl 4 + OAA combination group than the combination group AST and ALT levels decreased effect is more noticeable it was found that fiberization treatment effect between the OAA is better than OA.
실시예Example 10: 10: OAA의Of OAA 간섬유화증Liver fibrosis 억제 효과 Inhibitory effect
10-1. 10-1. 간섬유화증Liver fibrosis 동물모델 제작 Animal modeling
상기 실시예 9-1과 동일한 조건에서 랫트를 사육한 후 무작위로 4개 그룹으로 분류하였다: 대조군(생리식염수 투여군; Control, CON), 사염화탄소 단독 투여군(carbon tetrachloride; CCl4), 사염화탄소&올레아놀린산 병용 투여군(CCl4+OA 50 ㎎/㎏) 및 사염화탄소&올레아놀린산 아세테이트 병용 투여군(CCl4+OAA 50 ㎎/㎏). OA와 OAA는 정해진 용량으로 3주 동안 매일 경구투여하고, 사염화탄소(100 ㎕/100 g)는 주 3회씩 3주 동안 복강 투여하여 22일차 되는 날에 랫트를 희생시켰다.The rats were randomly divided into four groups after the rats were raised under the same conditions as in Example 9-1. Control group (Control, CON), carbon tetrachloride (CCl 4 ), carbon tetrachloride & oleanolin combined administration group (CCl 4 + OA 50 ㎎ / ㎏) and carbon tetrachloride & olreahnol acetate acid combination group (CCl 4 + OAA 50 ㎎ / ㎏). OA and OAA were orally administered daily for three weeks at a defined dose, and carbon tetrachloride (100 μl / 100 g) was intraperitoneally administered three times per week for 3 weeks to sacrifice the rat on day 22.
10-2. 10-2. 간섬유화증Liver fibrosis 동물모델에서 체중, 간 무게 및 In animal models, weight, liver weight and 간기능Liver function 지표 확인 Check indicator
랫트를 희생시킨 후 상기 실시예 9-2와 동일한 방법으로 체중, 간 무게, ALT 및 AST 수치를 측정하였다.After sacrificing the rats, body weight, liver weight, ALT and AST levels were measured in the same manner as in Example 9-2.
측정 결과, 도 10에 나타난 바와 같이 CCl4 투여군에서는 대조군과 비교하여 체중과 간의 무게가 감소하여 간섬유화가 유발된 것을 알 수 있었다. 또한, CCl4 투여군에서는 대조군에 비해 AST 및 ALT 수치가 현저하게 증가한 반면, CCl4+OA 병용 투여군 또는 CCl4+OAA 병용 투여군에서는 증가된 AST 및 ALT 수치가 감소하는 것을 확인할 수 있었다. 특히, CCl4+OA 병용 투여군보다 CCl4+OAA 병용 투여군에서 AST 및 ALT 수치 감소 효과가 더 현저하여 OA보다 OAA의 간섬유화 치료 효과가 더 우수한 것을 알 수 있었다.As a result, as shown in FIG. 10, in the CCl 4 administration group, weight and liver weight were decreased compared to the control group, and liver fibrosis was induced. In addition, AST and ALT levels were significantly increased in the CCl 4 treated group compared to the control group, but the AST and ALT levels were decreased in the CCl 4 + OA combination group or the CCl 4 + OAA combination group. In particular, CCl 4 + and OA in CCl 4 + OAA combination group than the combination group AST and ALT levels decreased effect is more noticeable it was found that fiberization treatment effect between the OAA is better than OA.
10-3. 10-3. 간섬유화증Liver fibrosis 동물모델의 Animal model 간조직Liver tissue 병리 분석 Pathology analysis
랫트를 희생시킨 후 간을 분리하고, 일부를 4% 포름알데하이드로 고정시킨 후 파라핀에 포매하여 파라핀 블록을 제작하였다. 파라핀 블록을 박절하여 간조직 절편을 제작하고, 절편에 헤마토자일린&에오신(hematoxylin & eosin; H&E) 염색 또는 마손 삼색 염색(Masson trichrome stain)을 수행한 후 광학현미경으로 관찰하였다.After sacrificing the rats, the liver was separated and fixed with 4% formaldehyde and embedded in paraffin to make a paraffin block. The paraffin blocks were sectioned to produce liver tissue sections. Hematoxylin & eosin (H & E) staining or masson trichrome staining was performed on the sections and observed with an optical microscope.
헤마토자일린&에오신 염색 결과, 도 11에 나타난 바와 같이 CCl4 투여군에서는 간문맥 주위의 세포괴사를 관찰할 수 있었으나, CCl4+OA 병용 투여군 또는 CCl4+OAA 병용 투여군에서는 세포괴사 현상이 완화된 것을 확인할 수 있었다.As a result of hematoxylin & eosin staining, cell necrosis around the portal vein was observed in the CCl 4 administration group as shown in FIG. 11, but in the group treated with CCl 4 + OA or CCl 4 + OAA, .
또한, 마손 삼색 염색으로 간조직 내 콜라겐을 염색한 결과, 도 12에 나타난 바와 같이 CCl4 투여군에서는 간세포의 섬유화가 심한 것을 확인할 수 있었다. 반면, CCl4+OA 병용 투여군 또는 CCl4+OAA 병용 투여군에서는 섬유화 정도가 완화된 것을 알 수 있었다.In addition, as a result of staining of collagen in hepatic tissue with Maeson's tricolor dyeing, it was confirmed that hepatocyte fibrosis was severe in the CCl 4 administration group as shown in Fig. On the other hand, the degree of fibrosis was mitigated in the combination of CCl 4 + OA and CCl 4 + OAA.
10-4. 10-4. 간섬유화증Liver fibrosis 동물모델의 Animal model 간조직에서In liver tissue 항산화 효소의 발현량 분석 Analysis of expression of antioxidant enzyme
랫트를 희생시킨 후 간을 분리하고, Trizol reagent(Invitrogen; 미국)를 첨가하여 균질화하였다. 여기에 클로로폼을 첨가하여 RNA를 분리한 후 이소프로판올을 첨가하여 침전시켰다. 침전된 RNA를 75% 에탄올로 세척한 후 2100 Bioanalyzer system(Agilent Technologies, 미국)으로 RNA의 농도 및 순도를 측정하였다. 분리한 RNA를 주형으로 하여 Taqman reverse transcription reagents kit(Applied Biosystems, Foster City, CA, USA)로 cDNA를 합성하였다. 염증성 인자의 발현 정도는 SYBR Green PCR master mix kit(Applied Biosystem, Foster City, CA, USA)를 사용하여 Real-time PCR로 확인하였다.After sacrificing the rats, the liver was separated and homogenized by addition of Trizol reagent (Invitrogen; USA). The RNA was isolated by adding chloroform thereto, and was precipitated by addition of isopropanol. The precipitated RNA was washed with 75% ethanol and the RNA concentration and purity were measured with a 2100 Bioanalyzer system (Agilent Technologies, USA). CDNA was synthesized with the Taqman reverse transcription reagent kit (Applied Biosystems, Foster City, CA, USA) using the separated RNA as a template. The expression level of inflammatory factor was confirmed by Real-time PCR using SYBR Green PCR master mix kit (Applied Biosystem, Foster City, CA, USA).
간세포내 산화스트레스를 확인하기 위해 유도성 산화질소 합성효소(inducible NO synthase, iNOS)의 mRNA 발현량을 측정한 결과, 도 13에 나타난 바와 같이 CCl4 투여군에서 증가한 iNOS 발현량이 CCl4+OA 병용 투여군 및 CCl4+OAA 병용 투여군에서는 현저하게 감소하는 것을 확인할 수 있었다.As a result of measuring the expression level of inducible NO synthase (iNOS) in the hepatocyte in order to examine the oxidative stress, the amount of iNOS expression increased in the CCl 4 administration group as shown in FIG. 13 was higher in the CCl 4 + OA combination administration group And CCl 4 + OAA, respectively.
산화스트레스에 의해 과잉으로 생성된 활성산소는 글루타티온 과산화효소(glutathione peroxidase, GPx)에 의하여 제거된다. GPx의 mRNA 발현량을 측정한 결과, 도 13에 나타난 바와 같이 CCl4 투여군에서는 산화적 스트레스 및 간세포 손상에 의해 GPx mRNA의 발현량이 대조군보다 감소한 것을 확인할 수 있었다. 그러나 CCl4+OA 병용 투여군 및 CCl4+OAA 병용 투여군에서는 CCl4에 의해 감소된 GPx mRNA의 발현량이 다시 증가하는 것을 확인할 수 있었다.The excess oxygen produced by oxidative stress is removed by glutathione peroxidase (GPx). As a result of measurement of GPx mRNA expression level, it was confirmed that the expression amount of GPx mRNA was decreased by oxidative stress and hepatocyte injury in the CCl 4 administration group as compared with the control group as shown in FIG. However, in the group treated with CCl 4 + OA and the group treated with CCl 4 + OAA, the amount of GPx mRNA decreased by CCl 4 was increased again.
상기 결과들을 통하여 올레아놀린산 아세테이트는 사염화탄소에 의해 유발된 간섬유화를 개선하고, 산화적 스트레스 관련 유전자의 발현을 억제하며, 활성산소를 효과적으로 제거할 수 있으므로 간섬유화증 치료 용도로 유용하게 사용될 수 있음을 알 수 있다.From the above results, oleanolin acetate can be effectively used for the treatment of hepatic fibrosis because it can improve hepatic fibrosis induced by carbon tetrachloride, suppress the expression of oxidative stress-related genes, and effectively remove active oxygen Able to know.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (10)
상기 조성물은 올레아놀린산 아세테이트를 유효성분으로 포함하고,
상기 섬유화증은 폐섬유화증 또는 간섬유화증인 것인 섬유화증 예방 또는 치료용 약학적 조성물.
In a pharmaceutical composition for the prevention or treatment of fibrosis,
Wherein said composition comprises oleanolic acid acetate as an active ingredient,
Wherein the fibrosis is lung fibrosis or liver fibrosis.
The method of claim 1, wherein the pulmonary fibrosis is selected from the group consisting of acute and chronic bronchitis, asthma, ssRNA and dsRNA viral infections, bacterial and fungal infections, pneumonia, hemorrhage, chronic obstructive pulmonary disease, pulmonary tuberculosis, pulmonary nodules, emphysema, Fibrosis, and interstitial lung disease.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
2. The composition of claim 1, wherein the composition inhibits epithelial-mesenchymal transition (EMT).
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