KR20200075598A - Composition for Improving Pulmonary Fibrosis Using an Extract of Astilbe rubra - Google Patents

Composition for Improving Pulmonary Fibrosis Using an Extract of Astilbe rubra Download PDF

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KR20200075598A
KR20200075598A KR1020180164452A KR20180164452A KR20200075598A KR 20200075598 A KR20200075598 A KR 20200075598A KR 1020180164452 A KR1020180164452 A KR 1020180164452A KR 20180164452 A KR20180164452 A KR 20180164452A KR 20200075598 A KR20200075598 A KR 20200075598A
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안은경
홍성수
고혜진
정규혁
곽종환
방인재
김하룡
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Abstract

The present invention discloses a composition for alleviating pulmonary fibrosis using an extract of Astilbe rubra, which has no cytotoxicity to a lung cancer cell line A549, inhibits EMT in a concentration-dependent manner, inhibits the expression of E-cadherin and vimentin involved in EMT activation, inhibits the expression of EMT-related transcription factors, Snail etc., inhibits the production of collagen, which is a pulmonary fibrosis substance, and inhibits oxidative stress applied to A549 cells.

Description

노루오줌 추출물을 이용한 폐섬화증 개선용 조성물{Composition for Improving Pulmonary Fibrosis Using an Extract of Astilbe rubra}Composition for Improving Pulmonary Fibrosis Using an Extract of Astilbe rubra

본 발명은 노루오줌(Astilbe rubra) 추출물을 이용한 폐섬화증 개선용 조성물에 관한 것이다.The present invention relates to a composition for improving pulmonary ischemia using Astilbe rubra extract.

폐섬유화 (pulmonary fibrosis) 또는 특발성 폐섬유화 (idiopathic pulmonary fibrosis, IPF)는 폐포벽에 결합조직, 특히 콜라겐이 과도하게 참착되는 폐 간질의 섬유화를 특징으로 하는 원인 불명의 질환이다. 폐섬유화증은 서서히 진행하는 폐 기능 저하로 인해 호흡부전으로 진행하여 진단 이후 평균 생존기간이 2-3년 이내인 치명적인 질환이다. 폐섬유화의 유병률은 보고마다 차이가 있으나, 인구 10만 명 중 2-29명으로 알려져 있으며, 국내에서는 희귀 난치성 질환으로 지정되어 있다.Pulmonary fibrosis or idiopathic pulmonary fibrosis (IPF) is an unexplained disease characterized by fibrosis of connective tissue, especially lung interstitium, in which collagen is excessively attached to the alveolar wall. Pulmonary fibrosis is a fatal disease that progresses to respiratory failure due to slow progression of lung function, and the average survival time after diagnosis is within 2-3 years. Although the prevalence of pulmonary fibrosis varies from report to report, it is known to be 2-29 out of 100,000 population, and it is designated as a rare incurable disease in Korea.

또한, 방사선 치료는 유방암 및 폐암 등 다양한 흉부암에 사용되어오고 있는데, 폐 장기에 방사선 치료를 받는 전체 환자들의 약 70 - 80% 정도가 경증 혹은 중증도의 폐 장기 부작용이 발생한다. 그 대표적인 부작용의 하나가 폐섬유화인데, 폐섬유화는 폐의 기능 소실을 가져와 환자의 폐활량을 감소시켜 삶의 질에 부정적 영향을 미치게며 심한 경우 사망에 이르게 한다.In addition, radiation therapy has been used for various chest cancers, such as breast cancer and lung cancer, and about 70-80% of all patients receiving radiation therapy in the lung organs have mild or severe lung organ side effects. One of the typical side effects is pulmonary fibrosis. Pulmonary fibrosis causes loss of function of the lungs, reducing the patient's lung capacity, negatively affecting the quality of life, and in severe cases, death.

폐섬유화의 원인으로는 폐 손상, 독성 물질 또는 미세먼지나 초미세먼지 등의 독성 환경에의 노출, 항암제, 자가면역질환, 또는 특발성 간질성 폐렴(idiopathic interstitial pneumonia) 등 다양하다(Proc Am Thorac Soc 2006, 3:285-92; Am J Respir Crit Care Med 2002, 165:277-304). 폐섬유화증의 기본적인 조직학적 변화는 폐포 중격에 세포의 침윤, 부종, 삼출 등의 염증성변화와 콜라겐, 프로테오글라이칸(proteoglycan), 피브로넥틴(fibronectin),당단백과 같은 세포외 기질(extracellular matrix)의 침윤, 섬유화 등에 의한 폐실질의 파괴이다(Am J Respir Crit Care Med 2002, 165:277-304; N Engl J Med 2001, 345:517-25).The causes of pulmonary fibrosis are various such as lung damage, exposure to toxic substances or toxic environments such as fine dust or ultrafine dust, anticancer drugs, autoimmune diseases, or idiopathic interstitial pneumonia (Proc Am Thorac Soc) 2006, 3:285-92; Am J Respir Crit Care Med 2002, 165:277-304). The basic histological changes in pulmonary fibrosis include inflammatory changes such as cell infiltration, edema, and exudation in the alveolar septa, and extracellular matrix such as collagen, proteoglycan, fibronectin, and glycoproteins. Destruction of the lung parenchyma by infiltration, fibrosis, etc. (Am J Respir Crit Care Med 2002, 165:277-304; N Engl J Med 2001, 345:517-25).

과거에는 폐섬유화의 병인을 염증으로 보고 항염증치료를 시도하였으나, 실제 그 효과는 미미하였고(Annals of Internal Medicine 2001, 134:136-51; Chest 1996, 110:1058-67), 그래서 최근에는 염증이 아닌 상피-중간엽 이행(epithelial-mesenchymal transition, EMT)이 새로운 병인 및 치료 목표로 다루어 지고 있다(Annals of Internal Medicine 2001, 134:136-51; Chest 2007, 132:1311-21; The Journal of Clinical Investigation 2009, 119:213-24). In the past, the pathogenesis of pulmonary fibrosis was regarded as inflammation, and anti-inflammatory treatment was attempted, but the effect was minimal (Annals of Internal Medicine 2001, 134:136-51; Chest 1996, 110:1058-67), so recently Non-epithelial-mesenchymal transition (EMT) is being addressed as a new pathogenesis and treatment target (Annals of Internal Medicine 2001, 134:136-51; Chest 2007, 132:1311-21; The Journal of Clinical Investigation 2009, 119:213-24).

특히 E-cadherin은 EMT 초기에 N-cadherin으로 전환되면서 TGF-β 경로와 연관되어 EMT를 촉진시키고 폐섬유화증을 일으키는 것으로 알려지는 등, Vimentin과 함께 EMT의 중요한 분자로 보고되어 있으며, Snail1, Snail2(Slug) 및 Snail3가 핵 내에서 EMT 관련 전사인자로는 알려져 있다(The Journal of Clinical Investigation 2009, 119:213-24; Nat Rev Mol Cell Biol 2006, 7:131-42; Cell Biol Int 2002, 26:463-76).In particular, E-cadherin is converted to N-cadherin in the early stage of EMT, and is associated with the TGF-β pathway, promoting EMT and causing pulmonary fibrosis.It has been reported to be an important molecule of EMT along with Vimentin, Snail1, Snail2 (Slug) and Snail3 are known as EMT-related transcription factors in the nucleus (The Journal of Clinical Investigation 2009, 119:213-24; Nat Rev Mol Cell Biol 2006, 7:131-42; Cell Biol Int 2002, 26 :463-76).

최근에는 산화적 스트레스가 폐섬유화증의 원인이 된다는 연구결과들이 보고됨에 따라(Am J Respir Crit Care Med. 2005, 172(4):417-22; Biochim Biophys Acta. 1832(7):1028-40), 폐섬유증의 치료에 항산화제를 사용하여 적용하려는 연구가 활발하게 진행되고 있다. 예컨대 폐섬유증 치료에 항산화 활성을 갖는 커큐민 유도체를 할 수 있을 개시한 한국특허공개 제2006-0030786호나 트레프로스티닐을 개시하고 있는 WO 2008/098196가 그러한 예이다.Recently, as studies have reported that oxidative stress causes pulmonary fibrosis (Am J Respir Crit Care Med. 2005, 172(4):417-22; Biochim Biophys Acta. 1832(7):1028-40 ), Studies to use antioxidants in the treatment of pulmonary fibrosis are being actively conducted. For example, Korean Patent Publication No. 2006-0030786, which discloses a curcumin derivative having antioxidant activity in the treatment of pulmonary fibrosis, or WO 2008/098196, which discloses treprostinil, is an example.

본 발명은 EMT 억제 활성, 항산화 활성 등을 가지는 노루오줌 추출물의 폐섬유화증 개선 활성을 개시한다.The present invention discloses the activity of improving pulmonary fibrosis of roe urine extract having EMT inhibitory activity, antioxidant activity, and the like.

본 발명의 목적은 EMT 억제 활성, 항산화 활성 등을 가지는 노루오줌 추출물을 이용한 폐섬유화증 개선용 조성물을 제공하는 데 있다.An object of the present invention is to provide a composition for improving pulmonary fibrosis using a roe urine extract having EMT inhibitory activity, antioxidant activity, and the like.

본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other or specific objects of the present invention will be presented below.

본 발명자들은 아래의 실시예 및 실험예에서 확인되는 바와 같이, 노루오줌 추출물이 폐암세포주인 A549에 대해 세포독성은 없으면서, 농도 의존적으로 EMT를 억제하고 EMT 활성화에 관여하는 E-cadherin과 Vimentin의 발현을 억제하며 EMT 관련 전사인자 Snail 등의 발현을 억제하는 것을 확인하였을 뿐만 아니라 폐섬유화 물질인 콜라겐의 생성을 억제하고 A549 세포에 가해진 산화적 스트레스를 억제함을 확인하였다. The present inventors confirmed that E-cadherin and Vimentin involved in concentration-dependent inhibition of EMT and involved in EMT activation, while no urination extract was cytotoxic to A549, a lung cancer cell line, as confirmed in the Examples and Experimental Examples below. And inhibited the expression of EMT-related transcription factor Snail, etc., as well as inhibiting the production of collagen, a fibrosis material, and inhibiting oxidative stress applied to A549 cells.

본 발명은 이러한 실험 결과에 기초하여 제공되는 것으로, 본 발명은 일 측면에 있어서 노루오줌 추출물을 유효성분으로 포함하는 폐섬유화증 개선용 조성물로 파악할 수 있고, 다른 측면에 있어서는 폐암세포의 전이 억제용 조성물로 파악할 수 있다.The present invention is provided on the basis of the results of these experiments, and the present invention can be identified as a composition for improving pulmonary fibrosis, which includes, in one aspect, a roe-pee extract as an active ingredient, and in another aspect, for inhibiting metastasis of lung cancer cells. It can be grasped by the composition.

본 명세서에서, "노루오줌 추출물"은 추출 대상인 노루오줌 줄기, 가지, 잎, 뿌리, 전초 또는 이들의 혼합물 등을 물, 탄소수 1 내지 4의 저급 알콜(메탄올, 에탄올, 부탄올 등), 메틸렌클로라이드, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, N,N-디메틸포름아미드(DMF), 메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합 용매를 사용하여 침출하여 얻어진 추출물(즉 상기 추출 용매에 가용성인 추출물), 이산화탄소, 펜탄 등 초임계 추출 용매를 사용하여 얻어진 추출물 또는 그 추출물을 분획하여 얻어진 분획물을 의미하며, 추출 방법은 활성물질의 극성, 추출 정도, 보존 정도를 고려하여 냉침, 환류, 가온, 초음파 방사, 초임계 추출 등 임의의 방법을 적용할 수 있다. 분획된 추출물의 경우 추출물을 특정 용매에 현탁시킨 후 극성이 다른 용매와 혼합·정치시켜 얻은 분획물, 상기 조추출물을 실리카겔 등 극성 또는 비극성 고성상을 충진된 칼럼에 흡착시킨 후 소수성 용매, 친수성 용매 또는 이들의 혼합 용매를 이동상으로 하여 얻은 분획물을 포함하는 의미이다. 또한 상기 추출물의 의미에는 동결건조, 진공건조, 열풍건조, 분무건조 등의 방식으로 추출 용매가 제거된 농축된 액상의 추출물 또는 고형상의 추출물이 포함된다. 바람직하게는 추출용매로서 물, 에탄올 또는 이들의 혼합 용매를 사용하여 얻어진 추출물, 더 바람직하게는 추출용매로서 물과 에탄올의 혼합 용매를 사용하여 얻어진 추출물을 의미한다. In the present specification, "noru-pee extract" is water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, etc. Ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), methyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or mixed solvents thereof. Extract obtained by leaching using (ie, extract soluble in the extraction solvent), carbon dioxide, extract obtained using a supercritical extraction solvent such as pentane or a fraction obtained by fractionating the extract, the extraction method is the polarity of the active substance, Any method such as cold acupuncture, reflux, warming, ultrasonic radiation, and supercritical extraction may be applied in consideration of the degree of extraction and storage. In the case of fractionated extracts, the fractions obtained by suspending the extracts in a specific solvent and mixing and policing with a solvent having a different polarity, adsorb the polar extracts such as silica gel on a column filled with a polar or non-polar high-phase phase, such as a hydrophobic solvent, a hydrophilic solvent, or It means that the fraction obtained by using these mixed solvents as a mobile phase is included. Also, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably, it is an extract obtained using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained using a mixed solvent of water and ethanol as an extraction solvent.

또 본 명세서에서, "폐암세포의 전이 억제"는 폐암세포가 타 위, 간 등 타 기관으로 전이되어 침윤되는 것을 억제하는 것을 의미한다.In addition, in this specification, "inhibition of metastasis of lung cancer cells" means that lung cancer cells are metastasized to other organs, such as the stomach and liver, and thus inhibited from being infiltrated.

또 본 명세서에서, "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다.In addition, in the present specification, "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.

본 발명의 조성물에서 그 유효성분은 폐섬유화증 개선 효과, 폐암세포의 전이 억제 효과 등을 나타낼 수 있는 한, 용도, 제형 등에 따라 임의의 양(유효량)으로 포함될 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 15 중량 % 범위 내에서 결정될 것이다. 여기서 "유효량"이란 그 적용 대상인 포유동물 바람직하게는 사람에게 의료 전문가 등의 제언에 의한 투여 기간 동안 본 발명의 조성물이 투여될 때, 폐섬유화증 개선 효과 등 의도한 의료적·약리학적 효과를 나타낼 수 있는, 본 발명의 조성물에 포함되는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.The active ingredient in the composition of the present invention can be included in any amount (effective amount) according to the use, formulation, etc., as long as it can exhibit the effect of improving lung fibrosis, metastatic inhibition of lung cancer cells, etc. It will be determined within the range of 0.001% to 15% by weight based on weight. Here, the "effective amount" refers to the intended medical and pharmacological effect, such as the effect of improving pulmonary fibrosis, when the composition of the present invention is administered to a mammal, preferably a person, to which the object is applied, by a recommendation of a medical expert or the like. Refers to the amount of the active ingredient contained in the composition of the present invention. Such an effective amount can be determined empirically within the ordinary skill in the art.

본 발명의 조성물은 구체적인 양태에 있어서 식품 조성물로서 파악할 수 있다.The composition of the present invention can be grasped as a food composition in a specific aspect.

본 발명의 식품 조성물은 어떠한 형태로도 제조될 수 있으며, 예컨대 차, 쥬스, 탄산음료, 이온음료 등의 음료류, 우유, 요구루트 등의 가공 유류(乳類), 껌류, 떡, 한과, 빵, 과자, 면 등의 식품류, 정제, 캡슐, 환, 과립, 액상, 분말, 편상, 페이스트상, 시럽, 겔, 젤리, 바 등의 건강기능식품 제제류 등으로 제조될 수 있다. 또 본 발명의 식품 조성물은 법률상·기능상의 구분에 있어서 제조·유통 시점의 시행 법규에 부합하는 한 임의의 제품 구분을 띨 수 있다. 예컨대 한국 "건강기능식품에관한법률"에 따른 건강기능식품이거나, 한국 "식품위생법"의 식품공전(식약처 고시 "식품의 기준 및 규격"임)상 각 식품유형에 따른 과자류, 두류, 다류, 음료류, 특수용도식품 등일 수 있다.The food composition of the present invention can be produced in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, Chinese medicine, bread, It can be made of foods such as confectionery, noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, bars, etc. In addition, the food composition of the present invention can be classified into any product as long as it complies with the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food in accordance with the Korea "Act on Health Functional Food", or a confectionary, soybean, or tea product according to each food type in the Korea Food and Drug Administration's Food Fair (which is the "Standard and Specification of Food" by the Ministry of Food and Drug Safety). Beverages, special-purpose foods, and the like.

본 발명의 식품 조성물에는 그 유효성분 이외에 식품첨가물이 포함될 수 있다. 식품첨가물은 일반적으로 식품을 제조, 가공 또는 보존함에 있어 식품에 첨가되어 혼합되거나 침윤되는 물질로서 이해될 수 있는데, 식품과 함께 매일 그리고 장기간 섭취되므로 그 안전성이 보장되어야 한다. 식품의 제조?유통을 규율하는 각국 법률(한국에서는 "식품위생법"임)에 따른 식품첨가물공전에는 안전성이 보장된 식품첨가물이 성분 면에서 또는 기능 면에서 한정적으로 규정되어 있다. 한국 식품첨가물공전(식약처 고시 "식품첨가물 기준 및 규격)에서는 식품첨가물이 성분 면에서 화학적 합성품, 천연 첨가물 및 혼합 제제류로 구분되어 규정되어 있는데, 이러한 식품첨가물은 기능 면에 있어서는 감미제, 풍미제, 보존제, 유화제, 산미료, 점증제 등으로 구분된다. Food composition of the present invention may include a food additive in addition to the active ingredient. Food additives can be understood as substances that are added to foods and mixed or infiltrated in general in the manufacture, processing, or preservation of foods, and their safety must be ensured because they are consumed daily and for a long time with foods. Food additives in accordance with national laws governing the manufacture and distribution of food ("Food Sanitation Act" in Korea) are limited in terms of ingredients or functions in terms of food additives with guaranteed safety. In the Korean Food Additives Code (Korea Food and Drug Administration's "Food Additive Standards and Standards"), food additives are divided into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients. , Preservatives, emulsifiers, acidulants, thickeners, etc.

감미제는 식품에 적당한 단맛을 부여하기 위하여 사용되는 것으로, 천연의 것이거나 합성된 것 모두 본 발명의 식품 조성물에 사용할 수 있다. 바람직하게는 천연 감미제를 사용하는 경우인데, 천연 감미제로서는 옥수수 시럽 고형물, 꿀, 수크로오스, 프룩토오스, 락토오스, 말토오스 등의 당 감미제를 들 수 있다. Sweeteners are used to impart a moderate sweetness to food, and both natural and synthetic can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

풍미제는 맛이나 향을 좋게 하기 위한 용도로 사용되는 것으로, 천연의 것과 합성된 것 모두 사용될 수 있다. 바람직하게는 천연의 것을 사용하는 경우이다. 천연의 것을 사용할 경우에 풍미 이외에 영양 강화의 목적도 병행할 수 있다. 천연 풍미제로서는 사과, 레몬, 감귤, 포도, 딸기, 복숭아 등에서 얻어진 것이거나 녹차잎, 둥굴레, 대잎, 계피, 국화 잎, 자스민 등에서 얻어진 것일 수 있다. 또 인삼(홍삼), 죽순, 알로에 베라, 은행 등에서 얻어진 것을 사용할 수 있다. 천연 풍미제는 액상의 농축액이나 고형상의 추출물일 수 있다. 경우에 따라서 합성 풍미제가 사용될 수 있는데, 합성 풍미제로서는 에스테르, 알콜, 알데하이드, 테르펜 등이 이용될 수 있다. Flavoring agents are used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, it is the case of using a natural thing. In addition to flavor, when using natural ones, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, or the like, or may be obtained from green tea leaves, perilla, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. Natural flavors may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used. As the synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

보존제로서는 소르브산칼슘, 소르브산나트륨, 소르브산칼륨, 벤조산칼슘, 벤조산나트륨, 벤조산칼륨, EDTA(에틸렌디아민테트라아세트산) 등이 사용될 수 있고, 또 유화제로서는 아카시아검, 카르복시메틸셀룰로스, 잔탄검, 펙틴 등이 사용될 수 있으며, 산미료로서는 연산, 말산, 푸마르산, 아디프산, 인산, 글루콘산, 타르타르산, 아스코르브산, 아세트산, 인산 등이 사용될 수 있다. 산미료는 맛을 증진시키는 목적 이외에 미생물의 증식을 억제할 목적으로 식품 조성물이 적정 산도로 되도록 첨가될 수 있다. 점증제로서는 현탁화 구현제, 침강제, 겔형성제, 팽화제 등이 사용될 수 있다.As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin Etc. can be used, and as acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste. As a thickener, a suspending agent, a sedimentation agent, a gel-forming agent, a swelling agent, etc. can be used.

본 발명의 식품 조성물은 전술한 바의 식품첨가물 이외에, 기능성과 영양성을 보충·보강할 목적으로 당업계에 공지되고 식품첨가물로서 안정성이 보장된 생리활성 물질이나 미네랄류를 포함할 수 있다.The food composition of the present invention, in addition to the food additives as described above, may include bioactive substances or minerals known in the art for the purpose of supplementing and reinforcing functional and nutritional properties and guaranteed stability as food additives.

그러한 생리활성 물질로서는 녹차 등에 포함된 카테킨류, 비타민 B1, 비타민 C, 비타민 E, 비타민 B12 등의 비타민류, 토코페롤, 디벤조일티아민 등을 들 수 있으며, 미네랄류로서는 구연산칼슘 등의 칼슘 제제, 스테아린산마그네슘 등의 마그네슘 제제, 구연산철 등의 철 제제, 염화크롬, 요오드칼륨, 셀레늄, 게르마늄, 바나듐, 아연 등을 들 수 있다. Examples of such bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, and the like, and calcium preparations such as calcium citrate and magnesium stearate as minerals. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

본 발명의 식품 조성물에는 전술한 바의 식품첨가물이 제품 유형에 따라 그 첨가 목적을 달성할 수 있는 적량으로 포함될 수 있다.The food composition of the present invention may include the food additives as described above in an appropriate amount to achieve the purpose of addition according to the product type.

본 발명의 식품 조성물에 포함될 수 있는 기타의 식품첨가물과 관련하여서는 각국 법률에 따른 식품공전이나 식품첨가물공전을 참조할 수 있다.With regard to other food additives that may be included in the food composition of the present invention, reference may be made to food or food additives according to the laws of each country.

본 발명의 조성물은 다른 구체적인 양태에 있어서는 약제학적 조성물로 파악될 수 있다.The composition of the present invention may be identified as a pharmaceutical composition in other specific embodiments.

본 발명의 약제학적 조성물은 유효성분 이외에 약제학적으로 허용되는 담체를 포함하여 당업계에 공지된 통상의 방법으로 투여 경로에 따라 경구용 제형 또는 비경구용 제형으로 제조될 수 있다. 여기서 "약제학적으로 허용되는" 의미는 유효성분의 활성을 억제하지 않으면서 적용(처방) 대상이 적응 가능한 이상의 독성을 지니지 않는다는 의미이다.The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the target of application (prescription) has no toxicity beyond adaptability without inhibiting the activity of the active ingredient.

본 발명의 약제학적 조성물이 경구용 제형으로 제조될 경우, 적합한 담체와 함께 당업계에 공지된 방법에 따라 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 현탁액, 웨이퍼 등의 제형으로 제조될 수 있다. 이때 약제학적으로 허용되는 적합한 담체의 예로서는 락토스, 글루코오스, 슈크로스, 덱스트로스, 솔비톨, 만니톨, 자일리톨 등의 당류, 옥수수 전분, 감자 전분, 밀 전분 등의 전분류, 셀룰로오스, 메틸셀룰로오스, 에틸셀룰로오스, 나트륨 카르복시메틸셀룰로오스, 하이드록시프로필메틸셀룰로오스 등의 셀룰로오스류, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트, 광물유, 맥아, 젤라틴, 탈크, 폴리올, 식물성유 등을 들 수 있다. 제제화활 경우 필요에 따라 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 및/또는 부형제를 포함하여 제제화할 수 있다.When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers And the like. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable And the like. In the case of formulation, if necessary, it can be formulated by including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.

본 발명의 약제학적 조성물이 비경구용 제형으로 제조될 경우, 적합한 담체와 함께 당업계에 공지된 방법에 따라 점안제, 주사제, 경피 투여제, 비강 흡입제, 좌제의 형태로 제제화될 수 있다. 점안제로 제제화활 경우 적합한 담체로서는 멸균수, 식염수, 5% 덱스트로스 같은 등장 용액 등을 사용할 수 있으며 필요에 따라 염화벤잘코늄, 메필파라벤, 에틸파라벤 등을 방부 목적으로 첨가할 수 있다. 주사제로 제제화할 경우 적합한 담체로서는 멸균수, 에탄올, 글리세롤이나 프로필렌 글리콜 등의 폴리올 또는 이들의 혼합물을 사용할 수 있으며, 바람직하게는 링거 용액, 트리에탄올 아민이 함유된 PBS(phosphate buffered saline)나 주사용 멸균수, 5% 덱스트로스 같은 등장 용액 등을 사용할 수 있다. 경피 투여제로 제제화할 경우 연고제, 크림제, 로션제, 겔제, 외용액제, 파스타제, 리니멘트제, 에어롤제 등의 형태로 제제화할 수 있다. 비강 흡입제의 경우 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 등의 적합한 추진제를 사용하여 에어로졸 스프레이 형태로 제제화할 수 있으며, 좌제로 제제화할 경우 그 기제로는 위텝솔(witepsol), 트윈(tween) 61, 폴리에틸렌글리콜류, 카카오지, 라우린지, 폴리옥시에틸렌 소르비탄 지방산 에스테르류, 폴리옥시에틸렌 스테아레이트류, 소르비탄 지방산 에스테르류 등을 사용할 수 있다.When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of eye drops, injections, transdermal dosage forms, nasal inhalants and suppositories according to methods known in the art with suitable carriers. In the case of formulation formulation with an eye drop, suitable carriers may be isotonic solutions such as sterile water, saline, 5% dextrose, and if necessary, benzalkonium chloride, mefilparaben, ethylparaben, etc. may be added for preservative purposes. When formulated as an injectable agent, a suitable carrier may be sterile water, ethanol, glycerol, polyols such as propylene glycol, or mixtures thereof. Preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine or sterilized for injection Isotonic solutions such as water and 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta agents, linen agents, aerosols, and the like. For nasal inhalants, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be formulated in the form of an aerosol spray using a suitable propellant, and when formulated as a suppository, Witthesol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like.

약제학적 조성물의 구체적인 제제화와 관련하여서는 당업계에 공지되어 있으며, 예컨대 문헌[Remington's Pharmaceutical Sciences(19th ed., 1995)] 등을 참조할 수 있다. 상기 문헌은 본 명세서의 일부로서 간주 된다.Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, see Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. The above documents are regarded as part of this specification.

본 발명의 약제학적 조성물의 바람직한 투여량은 환자의 상태, 체중, 성별, 연령, 환자의 중증도, 투여 경로에 따라 1일 0.001mg/kg ~ 10g/kg 범위, 바람직하게는 0.001mg/kg ~ 1g/kg 범위일 수 있다. 투여는 1일 1회 또는 수회로 나누어 이루어질 수 있다. 이러한 투여량은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 해석되어서는 아니 된다. Preferred dosages of the pharmaceutical compositions of the invention range from 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g per day, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. /kg range. Administration can be done once a day or divided into several times. Such doses should not be construed as limiting the scope of the invention in any aspect.

전술한 바와 같이, 본 발명에 따르면 노루오줌 추출물을 이용한 폐섬유화증 개선용 조성물을 제공할 수 있다. 본 발명의 조성물은 건강기능식품 등의 식품이나 천연물 의약품 등의 약품으로 제품화될 수 있다.As described above, according to the present invention, it is possible to provide a composition for improving pulmonary fibrosis using roe urine extract. The composition of the present invention may be commercialized as a food such as a health functional food or a medicine such as a natural medicine.

도 1은 노루오줌 전초 추출물의 농도(0, 2, 8, 31, 125, 500 ㎍/㎖)에 따른, A549 세포의 세포 증식율을 나타낸 그래프이다.
도 2는 노루오줌 전초 추출물을 다양한 농도(25, 50, 75, 100, 125 ㎍/ml)로 처리한 후 EMT 억제 영향의 용량-반응 관계를 나타낸 그래프이다.
도 3은 노루오줌 전초 추출물을 다양한 농도(25, 50, 75, 100, 125 ㎍/ml)로 처리한 후 TGF-β1 매개 Snail/Smad2/3 발현에 미치는 억제효과를 나타난 그래프이다.
도 4는 A549 세포에서 EMT 유발물질인 TGF-β1과 노루오줌 전초 추출물을 농도별로 처리하였을 때의 세포의 migration에 미치는 영향을 나타낸 그래프이다.
도 5는 A549 세포에서 EMT 유발물질인 TGF-β1과 노루오줌 전초 추출물을 농도별로 처리하였을 때의 세포의 invasion에 미치는 영향을 나타낸 그래프이다.
도 6은 노루오줌 전초 추출물의 다양한 농도(25, 50, 75, 100, 125, 150 ㎍/ml)에서 DPPH assay의한 항산화 효과를 나타낸 그래프이다.
도 7은 노루오줌 전초 추출물의 다양한 농도(50, 75, 100, 125 ㎍/ml)에서 DCF-DA assay의한 활성산소 억제효과를 나타낸 그래프이다.
도 8은 노루오줌 전초 추출물의 다양한 농도(25, 50, 75, 100, 125 ㎍/ml)에서 콜라겐 생성 억제 효과를 나타낸 그래프이다.1 is a graph showing the cell proliferation rate of A549 cells according to the concentration (0, 2, 8, 31, 125, 500 µg/ml) of Noru urine outpost extract.
Figure 2 is a graph showing the dose-response relationship of the EMT inhibitory effect after treatment with a variety of concentrations (25, 50, 75, 100, 125 µg/ml) of the roe urine outpost extract.
Figure 3 is a graph showing the inhibitory effect on TGF-β1 mediated Snail/Smad2/3 expression after treatment with Noru urine outpost extract at various concentrations (25, 50, 75, 100, 125 μg/ml).
Figure 4 is a graph showing the effect on the migration of cells when treated with EMT-inducing TGF-β1 and roe urine outpost extract by concentration in A549 cells.
5 is a graph showing the effect on the invasion of cells when treated with EMT-inducing TGF-β1 and roe urine outpost extract in A549 cells by concentration.
Figure 6 is a graph showing the antioxidant effect of the DPPH assay at various concentrations (25, 50, 75, 100, 125, 150 ㎍ / ml) of roe urine outpost extract.
7 is a graph showing the free radical inhibitory effect of the DCF-DA assay at various concentrations (50, 75, 100, 125 µg/ml) of roe urine outpost extract.
8 is a graph showing the inhibitory effect of collagen production at various concentrations (25, 50, 75, 100, 125 µg/ml) of Noru urine outpost extract.

이하 본 발명을 실시예 및 실험예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예 및 실험예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

실시예 1. 노루오줌 전초 에탄올 추출물의 제조 Example 1. Preparation of ethanol extract from roe urine outpost

노루오줌 전초를 음건 세절한 후 70% 에탄올로 60℃ 수욕 상에서 3회 온침하고, 농축하여 노루오줌 전초 70% 에탄올 추출물을 얻었다.After slicing the roe urine outpost, it was incubated three times on a 60°C water bath with 70% ethanol, and concentrated to obtain a 70% ethanol extract from the roe urine outpost.

실험예 1. 노루오줌 추출물의 안정성 평가Experimental Example 1. Stability evaluation of roe urine extract

노루오줌 추출물의 독성 유무를 분석하였다.Toxic urine extract was analyzed for toxicity.

세포 증식능력이나 생존능력을 정량하기 위한 실험으로, 세포내의 미토콘드리아 탈수소효소에 의해 tetrazolium salts (WST-1)에서 formazan이라는 발색물질이 생성되는 것을 microplate reader기를 이용하여 측정하였다. Human lung epithelial cell (A549)을 5×103 densitiy로 100 ㎕/well에 seeding 한 후 5% CO2, 37℃에서 24시간 보관하였다. 세포와 함께 있던 배지는 제거한 후, 2 - 500 ㎍/mL의 다양한 농도로 만들어진 노루오줌 추출물을 well 당 100 ㎕ 씩 처리하였고, 24시간 이후, WST-1을 배지에 1/10 씩 넣어 1시간 incubation한 후 microplate reader기에서 측정하였다. As an experiment to quantify cell proliferation capacity or viability, it was measured by using a microplate reader that a chromogenic material called formazan is produced from tetrazolium salts (WST-1) by mitochondrial dehydrogenase in the cell. Human lung epithelial cells (A549) were seeded at 100 µl/well with 5×10 3 densitiy, and then stored at 5% CO 2 and 37° C. for 24 hours. After removing the medium with the cells, 100 μl per well was treated with the extract of roe urine made at various concentrations of 2-500 μg/mL, and after 24 hours, 1/10 of WST-1 was added to the medium for 1 hour incubation. Then, it was measured on a microplate reader.

모든 데이터는 평균 ± 표준오차로 나타내었고, 대조군과 처리군과의 통계학적인 차이는 Student’s t-test 분석법을 이용하여 유의성을 결정하였다. P 값이 5% 미만일 때, 통계적으로 유의성이 있다고 판정하였다.All data were expressed as mean ± standard error, and statistical differences between control and treatment groups were determined using Student's t-test analysis. When the P value was less than 5%, it was judged to be statistically significant.

도 1에 나타낸 바와 같이 노루오줌 추출물은 125 μg/ml 농도에서 80% 이상의 cell viability를 보였다. 즉 노루오줌 전초 추출물은 비교적 높은 농도에서 거의 세포독성을 나타내지 않는 것으로 확인할 수 있었다.As shown in FIG. 1, the roe-urine extract showed a cell viability of 80% or more at a concentration of 125 μg/ml. That is, it could be confirmed that the extract of roe urine outpost showed little cytotoxicity at a relatively high concentration.

실험예 2. 노루오줌 추출물의 EMT 억제 활성 평가 및 그 기전 연구Experimental Example 2. Evaluation of EMT inhibitory activity of roe urine extract and its mechanism

노루오줌 추출물의 농도(25, 50, 75, 100, 125 ㎍/ml)에 따른 EMT 억제 활성과 그 기전을 탐색하기 위해 웨스턴 블롯 분석을 수행하였다. Western blot analysis was performed to investigate EMT inhibitory activity and its mechanism according to the concentration of Noru urine extract (25, 50, 75, 100, 125 μg/ml).

웨스턴 블롯 분석은 특정 단백질의 유무 또는 발현 여부를 확인하기 위한 실험법으로, 항원-항체반응을 이용하였다. 먼저 단백질 추출방법으로는 세포를 24시간 6.25×104 density로 1 ml/well에 seeding 한 후 5% CO2, 37℃에서 24시간 배양하였다. 세포와 함께 있던 배지는 제거한 후, 다양한 농도의 노루오줌 추출물을 well 당 1mL 씩 처리한다. 48시간 이후, 세포의 농도에 알맞게 lipa buffer를 넣어 ice에서 5분 보관한 후, scrapper를 이용하여 cell lysate를 1.5 mL tube에 옮겨주었다. 10분마다 vortex하여 30분이 지나면, 14,000g, 4℃ 15min 동안 centrifuge하고, 상층액만 수거하여 새로운 1.5mL tube에 옮겨준 후, BCA kit를 이용하여 단백질을 정량하였다. 10 ㎍ 단백질을 계산하여 증류수와 sample buffer로 mixture를 만들어 denaturation하고, gel에 80V 20min, 120V 60min loading하였다. 전기영동이 끝나면, gel을 membrane에 transfer하여 skim milk에서 1h 동안 blocking 하고, 이후, 1차, 2차 항체를 차례로 붙인 후, ECL를 뿌려 X-ray film에 감광하여 확인하였다. Western blot analysis was an experimental method for confirming the presence or absence of a specific protein, and used an antigen-antibody reaction. First, as a protein extraction method, cells were seeded in 1 ml/well at a density of 6.25×10 4 for 24 hours, and then cultured at 5% CO 2 at 37° C. for 24 hours. After removing the medium with the cells, various concentrations of roe-urine extract are treated with 1 mL per well. After 48 hours, the lipa buffer was added to the concentration of the cells and stored for 5 minutes on ice, and then the cell lysate was transferred to a 1.5 mL tube using a scrapper. After vortexing every 10 minutes, after 30 minutes, centrifuge at 14,000 g, 4° C. for 15 minutes, collect only the supernatant, transfer to a new 1.5 mL tube, and quantify the protein using a BCA kit. 10 μg protein was calculated, the mixture was made with distilled water and sample buffer, denaturation was performed, and 80V 20min and 120V 60min were loaded onto the gel. After electrophoresis, the gel was transferred to the membrane, blocked for 1 h in skim milk, and then, after attaching the primary and secondary antibodies in sequence, sprayed with ECL to confirm the light by exposing the X-ray film.

노루오줌 추출물의 EMT 억제 활성에 대한 결과를 도 2에 나타내었고, 그 억제 기전에 대한 결과를 도 3에 나타내었다. The results of EMT inhibitory activity of Noru urine extract are shown in FIG. 2, and the results of the inhibitory mechanism are shown in FIG. 3.

EMT 유발 표준물질로서 TGF-β1을 사용하여 이에 의해 유도되는 E-cadherin, N-cadherin, Vimentin를 EMT 억제 작용을 평가하는 marker로 사용하였다. 도 2에서 확인되는 바와 같이 TGF-β1에 의해 N-cadherin과 Vimentin은 증가하고, E-cadherin은 감소하는 것으로 EMT가 유발되었음을 확인하였다. GAPDH는 housekeeping gene으로 사용되었다. 노루오줌 추출물을 다양한 농도(25, 50, 75, 100, 125 ㎍/ml)로 처리한 후 EMT 억제 영향의 용량-반응 관계를 확인하였는데, 그 결과, 농도 의존적으로 N-cadherin, Vimentin을 감소시키고, E-cadherin을 증가시키는 것을 확인하였으며, 따라서 노루오줌 추출물은 TGF-β1 매개의 EMT에 대한 억제효과가 있음을 확인하였다.TGF-β1 was used as an EMT-inducing standard, and E-cadherin, N-cadherin, and Vimentin induced by this were used as markers for evaluating EMT inhibitory action. As can be seen in Figure 2, it was confirmed that N-cadherin and Vimentin increased by TGF-β1, and EMT was induced by decreasing E-cadherin. GAPDH was used as a housekeeping gene. After treatment with the various extracts (25, 50, 75, 100, and 125 μg/ml), the dose-response relationship of the EMT inhibitory effect was confirmed. As a result, N-cadherin and Vimentin were reduced in a concentration-dependent manner. , It was confirmed to increase E-cadherin, and thus, it was confirmed that the roe-urine extract had an inhibitory effect on TGF-β1-mediated EMT.

노루오줌 전초 추출물의 EMT 억제 기전의 탐색을 위해 TGF-β1 매개 Snail/Smad2/3 발현에 미치는 효과를 확인하였는데, 도 3에 나타난 바와 같이, TGF-β1 (2 ng/ml)으로 인해 증가한 Snail및 p-smad2/3 단백질 발현이 노루오줌 추출물에 의해 농도의존적으로 억제되었다.The effect on TGF-β1-mediated Snail/Smad2/3 expression was confirmed to explore the mechanism of EMT inhibition of Noru urine outpost extract, as shown in FIG. 3, increased Snail due to TGF-β1 (2 ng/ml) and p-smad2/3 protein expression was inhibited in a concentration-dependent manner by roe urine extract.

실험예 2. 노루오줌 추출물의 migration과 invasion에 미치는 영향 평가Experimental Example 2. Evaluation of the effects of roe urine extract on migration and invasion

인간상피세포인 A549 세포에서 EMT 유발물질인 TGF-β1 5 ng/ml과 다양한 농도의 노루오줌 추출물을 처리하여 세포의 migration과 invasion에 미치는 영향을 확인하였다. In human epithelial cells A549 cells, EMT inducer TGF-β1 5 ng/ml and various concentrations of Noru urine extract were treated to confirm the effects on cell migration and invasion.

Migration은 96well plate에 3×104 density로 A549 세포를 seeding 하고, 5% CO2, 37℃에서 24시간 배양한 후. wound maker를 이용하여, 세포에 scratch를 내고 media로 2번 wash한 다음, well당 100㎕으로 노루오줌 추출물을 처리하였다. Incucyte zoom기계에서 15분 이상 안정화시킨 후, 48시간까지 2시간마다 촬영하였다. Migration is after seeding A549 cells with a density of 3×10 4 in a 96-well plate, and incubating at 5% CO 2 at 37° C. for 24 hours. Using a wound maker, the cells were scratched, washed twice with media, and then treated with roe urine extract at 100 μl per well. After stabilizing for more than 15 minutes on the Incucyte zoom machine, it was photographed every 2 hours until 48 hours.

Invasion은 96well plate에 matrigel을 깔아주고 overnight한 후, 3×104 density로 A549를 seeding하고, 5% CO2, 37℃에서 24시간 배양한 후, wound maker를 이용하여, 세포에 scratch를 내고 media로 2번 wash하였다. 배지에 matrigel을 적정량 희석하여 노루오줌 추출물을 다양한 농도로 만든 후, well당 50㎕로 처리하였다. Incucyte zoom기계에서 15분 안정화시킨 후 72시간까지 2시마다 촬영하였다.After invasion, matrigel was placed on a 96-well plate overnight, seeded A549 at 3×10 4 density, incubated for 24 hours at 5% CO 2 , 37℃, and wound cells were used to scratch the cells and media Washed twice. After diluting the matrigel in an appropriate amount in the medium, the roe urine extract was made at various concentrations, and then treated with 50 µl per well. After stabilizing 15 minutes on the Incucyte zoom machine, it was photographed every 2 hours until 72 hours.

결과를 도 4 및 도 5에 나타내었는데, 도 4 및 도 5는 A549 세포에 EMT 유발물질인 TGF-β1 과 노루오줌 추출물을 농도별로 처리하였을 때 노루오줌 추출물이 A549 세포의 migration과 invasion을 농도의존적으로 억제함을 보여준다.The results are shown in Figures 4 and 5, Figures 4 and 5 is a concentration of E549 inducing TGF-β1 and Noru urine extracts in A549 cells by concentration, the Noru urine extract concentration-dependent migration and invasion of A549 cells It shows suppression.

실험예 3. 노루오줌 추출물의 항산화 효과 평가Experimental Example 3. Evaluation of antioxidant effect of roe urine extract

항산화 효과를 확인하기 위해 노루오줌 추출물의 다양한 농도(25, 50, 75, 100, 125 ㎍/㎖)에서 DPPH assay를 실시하였다. To confirm the antioxidant effect, DPPH assay was performed at various concentrations (25, 50, 75, 100, 125 µg/ml) of Noru urine extract.

시료를 농도별로 메탄올에 희석한 후, 시료 1 ml과 0.2 mM DPPH 용액 0.25 ml을 섞어 차광 상태에서 30분 동안 room temperature에서 보관하였고, 그 다음 microplate reader기를 사용하여 517nm에서 흡광도를 측정하였다. DPPH 억제효율은 다음과 같은 공식으로 계산하였으며, A0는 control, A1은 시료의 흡광도이다.After diluting the sample in methanol by concentration, 1 ml of the sample and 0.25 ml of 0.2 mM DPPH solution were mixed and stored at room temperature for 30 minutes in a light-shielded state, and then absorbance was measured at 517 nm using a microplate reader. DPPH inhibition efficiency was calculated by the following formula, A0 is the control, and A1 is the absorbance of the sample.

(억제율 %) = [(A0-A1)/A0] X 100 (% inhibition) = [(A0-A1)/A0] X 100

또한 살아있는 세포에서 활성산소를 억제하는지 확인하기 위해 A549 세포에 노루오줌 추출물을 농도별(50, 75, 100, 125 μg/㎖)로 48시간 동안 전처리한 후, 양성대조물질인 H2O2를 5mM로 30분 처리하고 DCF-DA assay를 실시하였다. 48well plate에 5×104의 density로 A549세포를 seeding 하고, 5% CO2, 37℃에서 24시간 배양한 후, 노루오줌 추출물을 well당 500㎕으로 농도별로 처리하였다. 48시간 후, H2O2 5mM을 30분을 처리하고, DCF-DA 시약을 1시간 처리, 그리고 PBS로 1번 wash 한 후, 0.1N NaOH로 세포를 용해시키고 형광값을 측정하였다. In addition, in order to confirm that the free radicals are inhibited in living cells, A549 cells were pretreated for 48 hours at a concentration (50, 75, 100, 125 μg/ml) by extract, and H 2 O 2 , a positive control, was added. DCF-DA assay was performed after treatment for 30 minutes with 5 mM. After seeding A549 cells at a density of 5×10 4 in a 48-well plate, and incubating at 5% CO 2 at 37° C. for 24 hours, the roe-pee extract was treated by concentration at 500 μl per well. After 48 hours, H 2 O 2 5mM was treated for 30 minutes, DCF-DA reagent was treated for 1 hour, and washed once with PBS, cells were lysed with 0.1N NaOH and fluorescence values were measured.

DPPH assay 결과를 도 6에 나타내었고, DCF-DA assay 결과를 도 7에 나타내었다.DPPH assay results are shown in Figure 6, DCF-DA assay results are shown in Figure 7.

도 6에서 확인되는 바와 같이, 노루오줌 추출물(GL0456)은 농도 의존적으로 DPPH 소거 활성을 나타내었고, 도 7에서 확인돠는 바와 같이, 살아있는 세포에서 활성산소를 억제하는지 확인하기 위해 A549 세포에서 노루오줌 추출물을 농도별(50, 75, 100, 125 μg/㎖)로 48시간 동안 전처리한 후, 양성대조물질인 H2O2를 5mM로 30분 처리하고 DCF-DA assay를 실시한 결과, 노루오줌 추출물은 H2O2에 의해 발생한 세포내 활성산소를 농도의존적으로 감소시키는 것으로 나타났다.As can be seen in Figure 6, the roe urine extract (GL0456) exhibited DPPH scavenging activity in a concentration-dependent manner, and as shown in Figure 7, as shown in Figure 7, neulea urine in A549 cells to confirm that it inhibits free radicals in living cells. After pre-treatment of the extracts for 48 hours at concentrations (50, 75, 100, 125 μg/ml), the positive control material H 2 O 2 was treated with 5 mM for 30 minutes, and DCF-DA assay was performed, and the result of the roe-urine extract Has been shown to decrease the concentration of intracellular free radicals caused by H 2 O 2 in a concentration-dependent manner.

실험예 4. 노루오줌Experimental Example 4. Presser urine 추출물의 콜라겐 억제 효과 평가Evaluation of collagen inhibitory effect of extract

노루오줌 추출물의 콜라겐 억제 효과를 확인하기 위해 다양한 농도(25, 50, 75, 100, 125 μg/㎖)에서 웨스턴 블럿 분석을 실시하였다. 웨스턴 블럿 분석 방법은 상기와 동일하나 단백질양을 30㎍으로 하였고, 또한 denaturation을 100℃에서 13분 동안 한 후 TGX 10 precast gel에 120V, 60min loading하였다.Western blot analysis was performed at various concentrations (25, 50, 75, 100, and 125 μg/ml) to confirm the collagen inhibitory effect of the Noru urine extract. The Western blot analysis method was the same as above, but the protein amount was set to 30 µg, and denaturation was performed at 100° C. for 13 minutes, followed by loading 120 V, 60 min on TGX 10 precast gel.

결과가 도 8에 나타나 있는데, 노루오줌 추출물은 농도의존적으로 콜라겐의 생성을 억제함을 확인하였다.The results are shown in FIG. 8, and it was confirmed that the extract of roe urine inhibits the production of collagen in a concentration-dependent manner.

Claims (9)

노루오줌 추출물을 유효성분으로 포함하는 폐섬유화증 개선용 조성물.
A composition for improving pulmonary fibrosis, which includes Noru urine extract as an active ingredient.
제1항에 있어서,
상기 추출물은 노루오줌 전초의 물, 메탄올, 에탄올 또는 이들의 혼합용매 추출물인 것을 특징으로 하는 조성물.
According to claim 1,
The extract is a composition characterized in that the water, methanol, ethanol, or a mixed solvent extract of roe urine outpost.
제1항에 있어서,
상기 폐섬유화증은 미세먼지 또는 초미세먼지가 원인이 되어 발병한 것임을 특징으로 하는 조성물.
According to claim 1,
The pulmonary fibrosis is a composition characterized in that the fine dust or ultrafine dust is caused.
제1항 또는 제2항에 있어서,
상기 조성물은 식품 조성물인 것을 특징으로 하는 조성물.
The method according to claim 1 or 2,
The composition is characterized in that the food composition.
제1항 또는 제2항에 있어서,
상기 조성물은 약제학적 조성물인 것을 특징으로 하는 조성물.
The method according to claim 1 or 2,
The composition is characterized in that the pharmaceutical composition.
노루오줌 추출물을 유효성분으로 포함하는 폐암세포 전이 억제용 조성물.
A composition for inhibiting lung cancer cell metastasis, which includes Noru urine extract as an active ingredient.
제6항에 있어서,
상기 추출물은 노루오줌 전초의 물, 메탄올, 에탄올 또는 이들의 혼합용매 추출물인 것을 특징으로 하는 조성물.

The method of claim 6,
The extract is a composition characterized in that the water, methanol, ethanol, or a mixed solvent extract of roe urine outpost.

제6항 또는 제7항에 있어서,
상기 조성물은 식품 조성물인 것을 특징으로 하는 조성물.
The method of claim 6 or 7,
The composition is characterized in that the food composition.
제6항 또는 제7항에 있어서,
상기 조성물은 약제학적 조성물인 것을 특징으로 하는 조성물.

The method of claim 6 or 7,
The composition is characterized in that the pharmaceutical composition.

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