KR102506493B1 - Composition for preventing or treating edema comprising Prasiola japonica extract or fraction thereof as active ingredient - Google Patents
Composition for preventing or treating edema comprising Prasiola japonica extract or fraction thereof as active ingredient Download PDFInfo
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- KR102506493B1 KR102506493B1 KR1020200154950A KR20200154950A KR102506493B1 KR 102506493 B1 KR102506493 B1 KR 102506493B1 KR 1020200154950 A KR1020200154950 A KR 1020200154950A KR 20200154950 A KR20200154950 A KR 20200154950A KR 102506493 B1 KR102506493 B1 KR 102506493B1
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- edema
- extract
- freshwater
- preventing
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Abstract
본 발명은 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 조성물에 관한 것으로, 부종 또는 염증관련 단백질 및 염증 신호전달 관련 단백질의 발현 등을 효과적으로 억제하여 항부종용 조성물로 용이하게 사용 가능하다.The present invention relates to a composition for preventing or treating edema containing freshwater seaweed extract as an active ingredient, and can be easily used as an anti-edema composition by effectively inhibiting the expression of edema or inflammation-related proteins and inflammatory signaling-related proteins. .
Description
본 발명은 민물김의 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 조성물에 관한 것으로, 보다 구체적으로는, 민물김의 에탄올-클로로포름 분획물을 유효성분으로 부종 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating edema comprising an extract of freshwater laver as an active ingredient, and more particularly, to a composition for preventing or treating edema using an ethanol-chloroform fraction of freshwater laver as an active ingredient.
부종(edema)은 조직 내에 림프액이나 조직 삼출물 등의 액체가 고여 과잉 존재하는 상태를 의미하는 것으로, 국소 부종과 전신 부종으로 분류하여 원인을 평가해볼 수 있다. 국소 부종은 정맥 부전, 림프관의 폐색, 염증이나 국소적인 과민반응에 의하여 발생하나, 전신 부종은 대개 심부전증이나 간경변증, 폐성심, 영양결핍, 갑상선 기능 저하증이나 신증후군과 같은 질병의 증상으로 나타난다. 일부 환자에서는 일반적인 부종의 원인이나 기전을 찾을 수 없는 경우도 있는데 이를 특발성 부종이라고 한다.Edema refers to a condition in which fluid such as lymph or tissue exudates accumulates in tissues and is excessively present. Local edema is caused by venous insufficiency, occlusion of lymphatic vessels, inflammation, or local hypersensitivity reactions, but systemic edema usually appears as a symptom of diseases such as heart failure, liver cirrhosis, cor pulmonale, malnutrition, hypothyroidism, or nephrotic syndrome. In some patients, no common cause or mechanism of edema can be found, which is called idiopathic edema.
특발성 부종은 일반적으로 여성에게서 나타나며, 이러한 여성 부종은 일반적인 부종의 증상 외에 동반되는 증상으로 생리전후, 배란기, 출산 전후로 더욱 심해진다. 특히, 산전산후 부종은 여성이 출산 전과 출산 후에 생기는 부종으로 몸이 붓는 현상을 말한다. 산전산후 부종의 원인은 출산 전후 과정의 피로, 스트레스, 호르몬의 작용으로 인해 우리 몸에 나트륨이 축적되면서 발생하거나, 또는 운동량이 부족하여 원활한 혈액 순환이 이루어지지 않아 더욱 심해지며, 자궁이나 회음부위 또는 제왕절개 부위에 염증반응으로 나타나기도 한다.Idiopathic edema generally occurs in women, and such female edema is accompanied by symptoms in addition to general symptoms of edema, and becomes more severe before and after menstruation, during ovulation, and before and after childbirth. In particular, prenatal and postpartum edema refers to a phenomenon in which a woman's body swells due to edema that occurs before and after childbirth. The causes of edema before and after childbirth are caused by accumulation of sodium in our body due to fatigue, stress, and hormones during the process before and after childbirth, or due to lack of exercise and poor blood circulation. It may also appear as an inflammatory reaction at the caesarean section site.
산후 부종은 흔히들 누구에게나 있는 증상이라고 생각하거나 곧 없어질 것으로 생각하는 것이 가장 큰 문제인데, 이는 평소 심장이 허약하거나 관절염 등이 있는 사람에게 잘 나타나며, 다른 증상을 치료하는데 시간을 빼앗기거나 산후 허약을 치료하다가 치료시기를 놓치는 경우도 많다.The biggest problem is that postpartum edema is commonly thought to be a symptom that everyone has or that it will soon disappear. There are many cases where the treatment period is missed during treatment.
또한, 대부분의 산모들은 산후 부종을 개선하기 위해 이뇨작용을 촉진시키는 식품을 섭취하는데 이 경우 신진대사가 저하되고 영양분이 손실되어 산후풍, 관절통으로 고생하는 경우가 있으며, 평생을 심각한 출산후유증으로 고생하는 경우도 있어, 적절한 치료 또는 개선을 위한 방법이 요구된다.In addition, most mothers consume food that promotes diuretic action to improve postpartum edema, but in this case, metabolism is lowered and nutrients are lost, resulting in postpartum fever and joint pain. In some cases, a method for appropriate treatment or improvement is required.
민물김(Prasiola japonica)은 물김이라는 이름으로 구전되어 왔으며, 현대에 이르러 민물김으로 알려지고 있다. 민물김은 홍조류인 바다김과 달리 녹조류(Green algae, Phylum Chlorophyta)로서 바다가 아닌 민물에서 생육하며, 민물파래과(Prasiolacease)에 포함된다(Park, M. K. et al., J. Plant Bio., 13, 1-10, 1970). 일본에서는 민물김을 카와노리(하천김)라는 이름으로 부르고 있으며, 하천김과 같은 민물 조류를 인공배양하여 높은 상업적 이윤을 창출하고 있다. 우리나라 민물김은 1938년 일본 학자인 Okada가 함경남도 문천군 문천면에서 최초로 채취하여 Prasiola formosana var. coreana Okada로 명명하여 발표한 것이 효시이며 (Okada, Y., J. Jpn. Bot., 15, 449-452, 1939), 삼척군의 집단에 대한 형태 및 생태학적 연구가 수행된 바가 있다(Park, M. K. et al., J. Plant Bio., 13, 1-10, 1970). 우리나라에서는 산업화에 따른 인위적 영향으로 40여년 전에 이미 사라진 종으로 알려졌으나 아직 강원도 삼척시의 소한천에 민물김이 분포하고 있음이 밝혀졌다.Freshwater seaweed (Prasiola japonica) has been passed down orally under the name of water seaweed, and is now known as freshwater seaweed. Unlike sea laver, which is a red algae, freshwater laver is a green algae (Phylum Chlorophyta) that grows in freshwater, not in the sea, and is included in Prasiolacase (Park, M. K. et al., J. Plant Bio., 13, 1-10, 1970). In Japan, freshwater laver is called kawanori (river laver), and freshwater algae such as river laver are artificially cultivated to create high commercial profits. Korea's freshwater laver was first collected in 1938 by Okada, a Japanese scholar, in Muncheon-myeon, Muncheon-gun, Hamgyeongnam-do, and Prasiola formosana var. It was first published under the name coreana Okada (Okada, Y., J. Jpn. Bot., 15, 449-452, 1939), and morphological and ecological studies on the population of Samcheok-gun were conducted (Park, M. K. et al., J. Plant Bio., 13, 1-10, 1970). In Korea, it was known as a species that had already disappeared 40 years ago due to the artificial influence of industrialization, but it was found that freshwater laver is still distributed in small agar in Samcheok City, Gangwon-do.
민물김과 같은 녹조류 유래 추출물이 항고혈압 및 미코스포린-유사 아미노산(mycosporine-like amino acids, MAAs)에 의한 자외선 차단 등의 활성을 가지는 것으로 보고되었다(Cha, S. H. et al., J. Korean Soc. Food Sci. Nutr., 35, 307-314, 2006; Groniger, A. et al., J. Photochem. Photobiol. B., 66, 54-59, 2002). 한편 일본에서 민물유래 식용김으로 사용하고 있는 스이젠지노리(Suizenzinori, Aphanothece sacrum)에는 고 함량의 철분 및 칼슘 등 미네랄 성분이 풍부한 것으로 조사되었으며, 알러지성 피부염을 억제하는 성분인 사크란 (Sacran, sulfated polysaccharide)이 함유되어 있어 약리학적 활용도가 높게 평가되고 있다(Ngatu, N. R. et al., Ann. Allergy Asthma Immunol., 108, 117-122, 2012). 그러나, 국내 자생 민물김에 대한 생리활성물질 성분분석 및 약리학적 분석에 대한 연구는 아직까지 많이 이루어지지 않고 있는 실정이다.It has been reported that green algae-derived extracts such as freshwater seaweed have antihypertensive and UV-blocking activities by mycosporine-like amino acids (MAAs) (Cha, S. H. et al., J. Korean Soc. Food Sci. Nutr., 35, 307-314, 2006; Groniger, A. et al., J. Photochem. Photobiol. B., 66, 54-59, 2002). On the other hand, Suizenzinori (Aphanothece sacrum), which is used as edible seaweed derived from freshwater in Japan, was found to be rich in minerals such as high iron and calcium, and it was found to contain sacran (sulfated polysaccharide), an ingredient that suppresses allergic dermatitis. ), its pharmacological utilization is highly evaluated (Ngatu, N. R. et al., Ann. Allergy Asthma Immunol., 108, 117-122, 2012). However, studies on the analysis of components of physiologically active substances and pharmacological analysis of freshwater laver native to Korea have not yet been conducted.
한편, 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 조성물과 관련된 종래선행문헌으로서, 선행문헌 [Seo, D. W. et al., J. Biomed. Res., 14(2), 83-90, 2013] 및 [Sa, J. H. et al., Rep. Inst. Health & Environ., 25, 52-62, 2014]에는 민물김 추출물의 생리활성(항암 효과, 항염증 효과, 항산화 효과, 항당뇨 효과, 자외선 차단효과)이 개시되었으며, 한국등록특허 제10-1853687호에는 방사무늬김 추출물의 스테롤 분획물을 포함하는 항염증 조성물 및 방사무늬김 추출물의 스테롤 분획물의 제조방법이 개시되었고, 한국등록특허 제10-1015702호에는 해조추출물을 포함하는 염증 및 피부자극 개선 및 완화용 조성물이 개시되었으며, 한국등록특허 제10-1479096호에는 생약혼합물의 추출물을 함유하는 산전산후부종 예방 또는 개선용 건강기능식품이 개시된 바 있다. 그러나, 본 발명과 같이 민물김 추출물의 부종 예방 또는 치료 효과에 대해 직접적인 확인 실험을 개시한 이전 보고는 아직 없다.On the other hand, as a prior art document related to a composition for preventing or treating edema containing freshwater seaweed extract as an active ingredient, prior literature [Seo, D. W. et al., J. Biomed. Res., 14(2), 83-90, 2013] and [Sa, J. H. et al., Rep. Inst. Health & Environ., 25, 52-62, 2014] discloses the physiological activity (anti-cancer effect, anti-inflammatory effect, antioxidant effect, anti-diabetic effect, sunscreen effect) of freshwater seaweed extract, and Korean Patent No. 10-1853687 discloses an anti-inflammatory composition containing a sterol fraction of an extract of radiation laver and a method for preparing the sterol fraction of an extract of radiation laver, and Korean Patent No. A composition for relief has been disclosed, and Korean Patent Registration No. 10-1479096 discloses a health functional food for preventing or improving prenatal and postpartum edema containing an extract of a herbal mixture. However, as in the present invention, there is no previous report disclosing a direct confirmation experiment on the preventive or therapeutic effect of freshwater seaweed extract on edema.
이에 따라 본 발명자들은 민물김 추출물을 연구하는 과정에서, 상기 민물김 추출물 또는 분획물을 부종이 유발된 동물 모델에 처리하는 경우 부종의 정도가 현저하게 감소하였으며, 특히 산후 부종에 효과적임을 확인함으로써 본 발명을 완성할 수 있었다.Accordingly, the inventors of the present invention, in the process of researching freshwater laver extract, confirmed that the degree of edema was significantly reduced when the freshwater laver extract or fraction was treated in an animal model in which edema was induced, and was particularly effective for postpartum edema. was able to complete
본 발명의 목적은 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 약학 조성물 또는 부종 예방 또는 개선용 건강기능식품을 제공하는 데 있다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating edema, or a health functional food for preventing or improving edema, containing freshwater seaweed extract as an active ingredient.
본 발명은 본 발명은 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating edema, comprising an extract of freshwater seaweed as an active ingredient.
상기 민물김은 녹조류로 홍조류인 바다김과는 달리 바다가 아닌 민물에서 생육하며, 민물파래과(Prasiolacease)에 속한다.The freshwater laver is green algae, and unlike sea laver, which is a red algae, grows in freshwater rather than the sea, and belongs to the freshwater paraeaceae (Prasiolacase).
상기 민물김 추출물은 민물김을 물, C1 내지 C4의 저급 알코올, 에틸아세테이트, n-부탄올, 아세톤, 헥산, 디클로로메탄으로 이루어진 군에서 선택되는 1종 이상의 용매로 추출하여 얻을 수 있으며, 상기 C1 내지 C4의 저급 알코올로는 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 및 이소부탄올로 이루어진 군에서 선택될 수 있다. 상기 민물김 추출물은 바람직하게는 민물김의 에탄올 추출물이다.The freshwater seaweed extract can be obtained by extracting freshwater seaweed with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohol, ethyl acetate, n-butanol, acetone, hexane, and dichloromethane. The C4 lower alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. The freshwater laver extract is preferably an ethanol extract of freshwater laver.
상기 민물김 추출물은 민물김의 에탄올 추출물을 헥산, 클로로포름, n-부탄올 및 물로 이루어진 군에서 선택되는 1종 이상의 용매로 더 추출한 분획물일 수 있다. 바람직하게는 상기 민물김 추출물은 민물김의 에탄올 추출물의 클로로포름 분획물일 수 있다. 상기 민물김의 에탄올 추출물의 클로로포름 분획물은 민물김의 에탄올 추출물을 농축 및 물에 현탁하고 클로로포름으로 더 추출하여 클로로포름 층에 포함된 추출물이다.The freshwater laver extract may be a fraction obtained by further extracting an ethanol extract of freshwater laver with at least one solvent selected from the group consisting of hexane, chloroform, n-butanol, and water. Preferably, the freshwater seaweed extract may be a chloroform fraction of the freshwater seaweed ethanol extract. The chloroform fraction of the ethanol extract of freshwater laver is an extract contained in the chloroform layer by concentrating and suspending the ethanol extract of freshwater laver in water and further extracting with chloroform.
상기 민물김 추출물은 바람직하게는 민물김을 알코올 또는 알코올 수용액으로 추출한 다음 농축 및 물에 현탁하고, 이를 헥산, 클로로포름, n-부탄올 및 물로 순차적으로 추출한 헥산 분획물, 클로로포름 분획물, n-부탄올 분획물 및 물 분획물로 이루어진 군으로부터 선택된 민물김 추출물 분획물일 수 있다. 더욱 바람직하게는 민물김을 에탄올 또는 에탄올 수용액으로 추출한 다음 농축 및 물에 현탁하고, 이를 헥산, 클로로포름, n-부탄올 및 물로 순차적으로 추출한 분획물 중 클로로포름 분획물일 수 있다.The freshwater seaweed extract is preferably a hexane fraction, a chloroform fraction, an n-butanol fraction and water obtained by extracting freshwater seaweed with alcohol or an aqueous alcohol solution, then concentrating and suspending it in water, and sequentially extracting it with hexane, chloroform, n-butanol and water. It may be a freshwater seaweed extract fraction selected from the group consisting of fractions. More preferably, it may be a chloroform fraction among fractions obtained by extracting freshwater seaweed with ethanol or aqueous ethanol solution, concentrating and suspending in water, and sequentially extracting it with hexane, chloroform, n-butanol and water.
추출 또는 분획시 사용되는 용매는 사용되는 민물김 중량 대비 1~100배 부피를 사용할 수 있으며 상기 과정을 1~5회 반복하는 것도 가능하다.The solvent used during extraction or fractionation may be used in an amount of 1 to 100 times the volume of freshwater seaweed used, and the above process may be repeated 1 to 5 times.
또한, 상기 추출물 또는 분획물의 추출 또는 분획 시간은 특별히 제한되는 것은 아니나, 10분 내지 1일 이내에 추출 또는 분획하는 것이 바람직하며, 통상의 추출기기, 초음파분쇄추출기 또는 분획기 등을 이용하여 통상적으로 사용되는 모든 추출 또는 분획방법, 예컨대, 가압 추출, 침지(냉침, 온침), 초음파 추출, 환류 추출법 등을 사용할 수 있다.In addition, the extraction or fractionation time of the extract or fraction is not particularly limited, but it is preferable to extract or fractionate within 10 minutes to 1 day, and is usually used using a conventional extraction device, ultrasonic crusher, or fractionator. All possible extraction or fractionation methods, such as pressure extraction, immersion (cold or warm needle), ultrasonic extraction, reflux extraction, and the like can be used.
또한, 상기 추출물 또는 분획물은 상기 과정으로부터 얻은 추출 또는 분획액, 추출 또는 분획액의 희석액이나 농축액, 추출 또는 분획액의 건조물, 추출 또는 분획액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출 또는 분획액 자체 및 이를 이용하여 형성 가능한 모든 제형의 추출물 또는 분획물을 포함한다.In addition, the extract or fraction may be an extraction or fraction obtained from the above process, a dilution or concentrate of the extraction or fraction, a dried product of the extraction or fraction, a crude product or purification of the extraction or fraction, or a mixture thereof. Or the fraction itself and extracts or fractions of all formulations that can be formed using the same.
상기 부종은 간질환, 신장질환, 심장질환, 내분비질환, 영양결핍, 부신피질호르몬제 사용, 비스테로이드성 진통제 사용 또는 항고혈압약제 사용에 의한 전신부종을 포함한다. 또한 상기 부종은 산후 부종을 포함한다.The edema includes systemic edema caused by liver disease, kidney disease, heart disease, endocrine disease, nutritional deficiency, use of cortical hormones, use of non-steroidal analgesics, or use of antihypertensive drugs. The edema also includes postpartum edema.
상기 부종 예방 또는 치료는 INOS, COX-2, TNF-α 또는 MMP-9 유전자의 발현 유전자의 발현을 억제하는 것일 수 있다.The prevention or treatment of edema may be suppressing the expression of INOS, COX-2, TNF-α or MMP-9 gene expression.
본 발명에 따른 약학 조성물은 일반적으로 사용되는 약학적으로 허용 가능한 담체와 함께 적합한 형태로 제형화될 수 있다. “약학적으로 허용 가능”이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증 등과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 또한, 상기 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, etc., or similar reactions when administered to humans. In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods.
상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로즈, 수크로스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아라비아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미결정셀룰로오스, 폴리비닐 피롤리돈, 물, 파라옥시벤조산메틸, 파라옥시벤조산프로필, 탈크, 스테아르산마그네슘 및 광물유를 포함할 수 있으나, 이에 한정되는 것은 아니다. 제제화할 경우에는 보통 사용하는 충진제, 안정화제, 결합제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 추출물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 미결정셀룰로오스, 수크로스 또는 락토오스, 저치환도히드록시프로필셀룰로오스, 히프로멜로오스 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아르산마그네슘, 탈크 같은 활택제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 유동파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다. 비경구 투여용 제형으로 제제화하기 위하여 상기 추출물 또는 이의 약학적으로 허용되는 염을 멸균되고/되거나 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질과 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다.Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. When formulated, it is prepared using diluents or excipients such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient for the extract of the present invention, for example, starch, microcrystalline cellulose, sucrose or lactose, It is prepared by mixing low-substituted hydroxypropyl cellulose, hypromellose, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used. In order to formulate a formulation for parenteral administration, the extract or its pharmaceutically acceptable salt is sterilized and/or preservatives, stabilizers, hydrating agents or emulsifying accelerators, salts for adjusting osmotic pressure and/or adjuvants such as buffers, and other treatments It can be mixed in water with useful substances to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form.
본 발명에 개시된 추출물을 유효성분으로 포함하는 약학 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. 투여량은 치료받을 대상의 연령, 성별, 체중, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여시간, 투여경로, 약물의 흡수, 분포 및 배설률, 사용되는 다른 약물의 종류 및 처방자의 판단 등에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 1㎎/㎏/일 내지 500㎎/㎏/일이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.A pharmaceutical composition comprising the extract disclosed in the present invention as an active ingredient may be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection. The dose depends on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the time of administration, the route of administration, the absorption, distribution and excretion rate of the drug, the type of other drug used, and the prescription It will depend on judgment, etc. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.
또 다른 일면에 있어서, 본 발명은 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 개선용 건강기능식품에 관한 것이며, 보다 바람직하게는 민물김 추출물을 유효성분으로 포함하는 특발성 부종인 산후 부종의 예방 또는 개선용 건강기능식품에 관한 것이다.In another aspect, the present invention relates to a health functional food for preventing or improving edema comprising freshwater laver extract as an active ingredient, and more preferably preventing postpartum edema, idiopathic edema, containing freshwater laver extract as an active ingredient. Or health functional food for improvement.
상기 건강기능식품은 유용한 기능성을 가진 원료나 성분을 사용하여 제조 또는 가공한 식품을 지칭하는 것으로, 예를 들어 건강보조식품, 기능성 식품, 영양제, 보조제 등을 모두 포함한다.The health functional food refers to food manufactured or processed using raw materials or ingredients having useful functionality, and includes, for example, health supplement food, functional food, nutritional supplements, supplements, and the like.
상기 민물김 추출물은 전체 건강기능식품 총 중량에 대하여 바람직하게는 0.001중량% 내지 50중량%, 더 바람직하게는 0.001중량% 내지 40중량%, 가장 바람직하게는 0.001중량% 내지 30중량%로 하여 첨가될 수 있다.The freshwater laver extract is added in an amount of preferably 0.001% to 50% by weight, more preferably 0.001% to 40% by weight, and most preferably 0.001% to 30% by weight based on the total weight of the total health functional food It can be.
본 발명의 건강기능식품은 정제, 캡슐제, 환제 및 액제 등의 형태를 포함하며, 본 발명 민물김 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제 등이 있다.The health functional food of the present invention includes forms such as tablets, capsules, pills and liquids, and foods to which the freshwater seaweed extract of the present invention can be added include, for example, various foods, beverages, chewing gum, tea, and vitamins. complexes, etc.
본 발명은 민물김 추출물을 유효성분으로 포함하는 부종 예방 또는 치료용 조성물에 관한 것으로, 상기 민물김 추출물을 부종이 유발된 동물 모델에 처리하면 부종의 두께, 부피가 농도 의존적으로 감소하며, 특히 부종 또는 염증관련 단백질 및 염증 신호전달 관련 단백질의 발현 등을 효과적으로 억제하여 항부종용 조성물로 용이하게 사용 가능하다.The present invention relates to a composition for preventing or treating edema comprising an extract of freshwater laver as an active ingredient, and when the extract of freshwater laver is treated in an animal model in which edema is induced, the thickness and volume of edema are reduced in a concentration-dependent manner, in particular, edema Alternatively, it can be easily used as an anti-edema composition by effectively inhibiting the expression of inflammation-related proteins and inflammatory signaling-related proteins.
도 1은 카라기난(Carrageenan)으로 부종이 유도된 마우스 오른발에서의 민물김 추출물(Pj-EE) 과 이의 각 용매 분획물 및 비교대상 추출물 처리에 따른 부종 정도를 확인한 사진이다.
도 2는 카라기난(Carrageenan)으로 부종이 유도된 마우스 오른발에서의 민물김 추출물(Pj-EE) 과 이의 각 용매 분획물 및 비교대상 추출물 처리에 따른 부종 부피(A) 및 오른발 두께(B)의 변화를 나타낸 그래프이다.
도 3은 카라기난(Carrageenan)으로 부종이 유도된 마우스 오른발에서의 민물김 추출물(Pj-EE) 과 이의 각 용매 분획물 및 비교대상 추출물 처리에 따른 염증 관련 유전자 INOS(A), COX-2(B), TNF-α(C), MMP-9(D) 유전자의 발현 변화를 나타낸 그래프이다.
도 4는 민물김 에탄올 추출물의 클로로포름 분획물 (Pj-EE-CF)의 농도별 처리에 따른 RAW 264.7 세포의 세포생존률을 나타낸 그래프이다.
도 5는 LPS로 유도한 RAW 264.7 세포주에서 민물김 에탄올 추출물의 클로로포름 분획물 (Pj-EE-CF)의 농도별 처리에 따른 NO 생성 억제 효과를 나타낸 그래프이다.
도 6은 HEK 293 세포에서 MyD88 및 TRIF에 의한 NF-κB 및 AP-1 활성화에 미치는 민물김 에탄올 추출물의 클로로포름 분획물 (Pj-EE-CF)의 효과를 나타낸 그래프이다.
도 7은 Carageenan 유도 마우스 Paw 부종 모델에서 민물김 에탄올 추출물의 클로로포름 분획물 (Pj-EE-CF)에 의한 염증 신호전달 관련 단백질의 발현 변화를 나타낸 그래프이다.Figure 1 is a photograph confirming the degree of edema according to the treatment of freshwater seaweed extract (Pj-EE) in the right foot of a mouse induced by carrageenan (Carrageenan), each solvent fraction thereof, and a comparative extract.
Figure 2 shows changes in edema volume (A) and thickness (B) of the right foot according to the treatment of freshwater seaweed extract (Pj-EE), each solvent fraction thereof, and the comparative extract in the right foot of a mouse edema induced by carrageenan. is the graph shown.
Figure 3 is a freshwater seaweed extract (Pj-EE) from the right foot of a mouse edema induced by carrageenan, each solvent fraction thereof, and inflammation-related genes INOS (A) and COX-2 (B) according to the treatment of the comparative extract , TNF-α (C), and MMP-9 (D) gene expression changes are graphs.
Figure 4 is a graph showing the cell viability of RAW 264.7 cells according to the concentration of the chloroform fraction (Pj-EE-CF) of freshwater laver ethanol extract.
Figure 5 is a graph showing the NO production inhibitory effect according to the concentration treatment of the chloroform fraction (Pj-EE-CF) of freshwater seaweed ethanol extract in the RAW 264.7 cell line induced by LPS.
Figure 6 is a graph showing the effect of the chloroform fraction (Pj-EE-CF) of freshwater laver ethanol extract on the activation of NF-κB and AP-1 by MyD88 and TRIF in HEK 293 cells.
7 is a graph showing changes in the expression of proteins related to inflammatory signaling by the chloroform fraction (Pj-EE-CF) of freshwater seaweed ethanol extract in the Carageenan-induced mouse Paw edema model.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.
<< 실시예Example 1. One. 민물김freshwater seaweed 추출물 제조> Extract Preparation>
본 발명의 민물김(Prasiola japonica)은 삼척시에서 채취한 민물김을 사용하였다.Freshwater laver (Prasiola japonica) of the present invention was used freshwater laver collected in Samcheok City.
삼척시에서 채취한 민물김은 불순물들을 제거하기 위해 수돗물로 세척하였고, 70%[v/v] 에탄올을 이용하여 살균 처리 후, 증류수로 세척하여 동결 건조하였다. 동결 건조한 민물김을 곱게 파쇄하고, 파쇄된 민물김 100g에 80%[v/v] 에탄올 1ℓ을 넣고 실온에서 24시간 동안 침지시켰다. 이러한 과정을 3회 반복하여 얻은 추출액을 와트만 여과지(filter paper)로 여과시킨 후, 증발기(evaporator)를 이용하여 용매를 증발시켜 민물김 에탄올 추출물(Pj-EE)을 확보하였다.Freshwater laver collected from Samcheok was washed with tap water to remove impurities, sterilized using 70% [v/v] ethanol, washed with distilled water and freeze-dried. The freeze-dried freshwater laver was finely crushed, and 1 liter of 80% [v/v] ethanol was added to 100 g of the crushed freshwater laver and immersed at room temperature for 24 hours. The extract obtained by repeating this process three times was filtered with Whatman filter paper, and then the solvent was evaporated using an evaporator to obtain freshwater seaweed ethanol extract (Pj-EE).
상기 수득한 민물김 에탄올 추출물(Pj-EE)은 물에 용해시킨뒤 헥산을 넣어 녹아나오는 상층액을 따로 분리한 뒤 진공농축기를 이용하여 상층액에 포함된 용매를 제거시켰다. 용매가 제거되어 남아 있는 용매 분획물을 3번 반복하여 수득하여 헥산 분획물(Pj-EE-HF) 을 제조하였다. 그 뒤 녹아 나오지 않는 하층부 용액에 클로로포름을 넣고 상기 과정과 같은 과정을 통해 클로로포름 분획물(Pj-EE-CF)을 수득하였고, 이후 남아있는 침전용용액에 n-부탄올(Pj-EE-BF), 물(Pj-EE-WF)을 각각 순차적으로 넣어 분획물을 제작하였다. 한편, 민물김의 비교군으로, 중국김(Prasiola yunnanica), 파래(Enteromorpha linza), 구포미역(Undaria pinnatifida), 매생이(Capsosiphon fulvescens)의 에탄올 추출물을 상기 민물김 에탄올 추출물(Pj-EE) 수득과정과 동일한 방법으로 수득하여 준비하였다.The obtained freshwater seaweed ethanol extract (Pj-EE) was dissolved in water, and then hexane was added to separate the dissolved supernatant, and then the solvent contained in the supernatant was removed using a vacuum concentrator. The solvent fraction remaining after the solvent was removed was obtained repeatedly three times to prepare a hexane fraction (Pj-EE-HF). After that, chloroform was added to the lower layer solution that did not dissolve, and a chloroform fraction (Pj-EE-CF) was obtained through the same process as above, and then n-butanol (Pj-EE-BF) and water were added to the remaining precipitation solution. (Pj-EE-WF) were sequentially added to prepare fractions. On the other hand, as a comparison group of freshwater laver, Chinese laver ( Prasiola yunnanica ), green algae ( Enteromorpha linza ), Gupo seaweed ( Undaria pinnatifida ), Maesaengi ( Capsosiphon ) The ethanol extract of fulvescens ) was obtained and prepared in the same manner as in the process of obtaining the ethanol extract of freshwater seaweed (Pj-EE).
<< 실시예Example 2. 2. 민물김freshwater seaweed 추출물의 of the extract CarrageenanCarrageenan 유도 마우스 Paw 부종 모델에서 In the induced mouse Paw edema model 항부종anti-edema 효과> effect>
ICR (암컷, 8주령) 마우스에 상기 실시예 1에서 준비한 각 시료를 100 ㎎/㎏이 되도록 1 회/일, 1주일간 구강 투여하였다. 7일째 되는 날 각 실험군의 마우스 뒷발(양쪽)에 부종 유도 물질인 카라기난(carrageenan, 1%)을 주사하였다. 카라기난은 주사기를 이용하여 검지와 중지 발가락 사이를 통과하여 발바닥에 주사하여 부종을 유도하고, 3시간 뒤 발을 절제하였다. 상기 과정에서 절제한 발의 부종 정도를 확인하여 도 1에 나타내었으며, 이때 발의 두께, 부종의 부피를 측정하여 도 2A 및 도 2B에 나타내었다. 발의 두께는 두께측정기, 부피는 부종부피측정기 를 이용하여 측정하였다.Each sample prepared in Example 1 was orally administered to ICR (female, 8-week-old) mice once/day for 1 week to a concentration of 100 mg/kg. On the 7th day, carrageenan (1%), an edema inducing substance, was injected into the hind feet (both sides) of mice in each experimental group. Carrageenan was injected into the sole of the foot by passing between the index finger and the middle toe using a syringe to induce edema, and after 3 hours, the foot was excised. The degree of edema of the foot excised in the above process was confirmed and shown in FIG. 1, and at this time, the thickness of the foot and the volume of edema were measured and shown in FIGS. 2A and 2B. Foot thickness was measured using a thickness meter and volume using an edema volume meter.
도 1 및 도 2에서 보는 바와 같이, 카라기난(carrageenan, 1%)을 주사한 마우스 발은 카라기난(carrageenan, 1%)을 주사하지 않은 대조군에 비하여 발의 두께 및 부종의 부피가 모두 2배 이상 증가하여 부종이 유도된 것을 확인하였다. 이때, 민물김 에탄올 추출물(Pj-EE)을 7일간 경구 투여한 마우스의 경우, 부종 유도된 마우스의 발의 두께 및 부종의 부피가 모두 감소하여 민물김 에탄올 추출물(Pj-EE)의 부종 억제를 확인하였다. 또한, 민물김 에탄올 추출물(Pj-EE)의 용매 분획물 중, 클로로포름 분획물(Pj-EE-CF)이 민물김 에탄올 추출물(Pj-EE)보다 발의 두께 및 부종의 부피가 모두 감소하여 민물김 에탄올 추출물(Pj-EE)보다 뛰어난 부종 억제 효과를 나타내는 것을 확인하였다. 이에 반하여 부탄올 및 물 분획물(Pj-EE-BF, Pj-EE-WF)은 민물김 에탄올 추출물(Pj-EE)보다 오히려 부종 억제 효과가 감소되었으며, 헥산 분획물(Pj-EE-HF)의 경우, 발의 두께는 감소되었으나, 부종 자체의 부피는 큰 차이가 없었다.As shown in FIGS. 1 and 2, the foot of the mouse injected with carrageenan (1%) increased more than twice the thickness of the foot and the volume of edema compared to the control group that was not injected with carrageenan (1%). It was confirmed that edema was induced. At this time, in the case of mice orally administered with freshwater laver ethanol extract (Pj-EE) for 7 days, both the thickness of the feet and the volume of edema induced by edema were reduced, confirming the inhibition of edema by freshwater laver ethanol extract (Pj-EE). did In addition, among the solvent fractions of the freshwater laver ethanol extract (Pj-EE), the chloroform fraction (Pj-EE-CF) decreased both the thickness of the feet and the volume of edema than the freshwater laver ethanol extract (Pj-EE), resulting in a freshwater laver ethanol extract (Pj-EE) was confirmed to exhibit an excellent anti-edema effect. In contrast, the butanol and water fractions (Pj-EE-BF, Pj-EE-WF) had a reduced edema inhibitory effect rather than the freshwater seaweed ethanol extract (Pj-EE), and in the case of the hexane fraction (Pj-EE-HF), Although the thickness of the foot decreased, there was no significant difference in the volume of the edema itself.
상기와 같이 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF)은 민물김 에탄올 추출물(Pj-EE)보다 부종 억제 효과가 뛰어났으며, 민물김 에탄올 추출물(Pj-EE)과 유사한 부종억제효과를 보인 중국김, 구포미역 또는 매생이의 에탄올 추출물보다 더욱 뛰어난 부종억제효과를 나타내었다. 이러한 결과는 민물김 에탄올 추출물이 비극성 용매인 클로로포름으로 분획과정 중 부종억제효과를 나타내는 화합물이 농축되면서 뛰어난 부종억제효과를 나타내는 것으로 사료된다.As described above, the chloroform fraction of the freshwater laver ethanol extract (Pj-EE-CF) was superior in the edema inhibitory effect to the freshwater laver ethanol extract (Pj-EE), and the edema inhibitory effect similar to that of the freshwater laver ethanol extract (Pj-EE) It showed a more excellent anti-edema effect than the ethanol extract of Chinese seaweed, Gupo seaweed or Maesaengi, which showed . These results suggest that the freshwater seaweed ethanol extract exhibits an excellent anti-edema effect as the compound exhibiting the anti-edema effect is concentrated during the fractionation process with chloroform, a non-polar solvent.
<< 실시예Example 3. 3. 민물김freshwater seaweed 추출물의 of the extract CarrageenanCarrageenan 유도 마우스 Paw 부종 모델에서 염증 관련 유전자 발현에 미치는 효과> Effects on inflammation-related gene expression in an induced mouse Paw edema model>
상기 실시예 2에서 얻어진 부종이 유도된 발 조직을 TRI-Zol을 이용하여 상기 세포를 분해하고, BCP(1-bromo-3-chloropropane)를 이용하여 중화시켰다. 중화시킨 액체에 isopropanol을 이용하여 mRNA를 침강시켰다. 이어서 원심분리기를 이용하여 침강된 mRNA만을 모아 실험에 이용하였다. 세포내 mRNA는 cDNA kit를 이용하여 cDNA로 합성하여, PCR을 통해 염증 관련 유전자인 INOS, COX-2, TNF-α 및 MMP-9 유전자의 발현양을 확인하여 도 3A 내지 3D에 나타내었다. Matrix metalloproteinase(기질단백분해효소, MMP로 약함)는 아연과 칼슘-의존 엔도펩티다아제(endopeptidase)의 일종으로 MMP-1, MMP-2(gelatinase A), MMP-3, 및 MMP-9(gelatinase B) 등으로 구분되며 결체 조직들의 여러 구성성분을 분해한다. 특히 MMP-9은 기저막의 주요성분인 젤리틴(gelatin)과 교원질을 분해하며 염증반응시 MMP의 작용에 의해 염증세포들의 이동과 침윤이 촉진된다 (Korean J Otolaryngol 2000;43:604-9).The edema-induced foot tissue obtained in Example 2 was lysed with TRI-Zol and neutralized with 1-bromo-3-chloropropane (BCP). mRNA was precipitated in the neutralized liquid using isopropanol. Subsequently, only the precipitated mRNA was collected using a centrifuge and used for the experiment. Intracellular mRNA was synthesized into cDNA using a cDNA kit, and the expression levels of INOS, COX-2, TNF-α and MMP-9 genes, which are inflammation-related genes, were confirmed through PCR, and are shown in FIGS. 3A to 3D. Matrix metalloproteinase (abbreviated as matrix proteolytic enzyme, MMP) is a type of zinc and calcium-dependent endopeptidase, which includes MMP-1, MMP-2 (gelatinase A), MMP-3, and MMP-9 (gelatinase B). It is divided into the back and breaks down various components of connective tissue. In particular, MMP-9 decomposes gelatin and collagen, which are major components of the basement membrane, and promotes migration and infiltration of inflammatory cells by the action of MMP during an inflammatory reaction (Korean J Otolaryngol 2000;43:604-9).
도 3A 내지 3D에서 보는 바와 같이, Carageenan에 의해 유도된 부종 조직은 염증 및 멜라닌 형성 관련 유전자인 INOS, COX-2, TNF-α 및 MMP-9 유전자의 발현이 대조군과 비교하여 10~20 배 증가하였다. 이러한 INOS, COX-2, TNF-α 및 MMP-9 유전자의 발현은 Pj-EE 처리 군에서 크게 감소하였으며, 특히 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF) 처리에서 현저하게 감소함을 확인하여, 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF)이 염증관련 유전자 발현 수준에서 부종 억제 효과를 나타내는 것을 확인하였다.As shown in Figures 3A to 3D, the expression of INOS, COX-2, TNF-α and MMP-9 genes, which are genes related to inflammation and melanin formation, in the edematous tissue induced by Carageenan increased 10 to 20 times compared to the control group. did The expression of these INOS, COX-2, TNF-α and MMP-9 genes was greatly reduced in the Pj-EE treatment group, especially in the treatment with the chloroform fraction (Pj-EE-CF) of freshwater seaweed ethanol extract. As a result, it was confirmed that the chloroform fraction (Pj-EE-CF) of the ethanol extract of freshwater seaweed exhibited an edema inhibitory effect at the level of inflammation-related gene expression.
<< 실시예Example 4. 4. 민물김freshwater seaweed 추출물의 RAW 264.7 세포에서의 세포 Cells from RAW 264.7 cells in the extract 생존률survival rate 확인> OK>
RAW 264.7 세포에서 민물김 에탄올 추출물의 클로로포름 분획물의 세포 독성 여부를 확인하고자, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 어세이를 실시하였다. RAW 264.7 세포 10% FBS(fetal bovine serum)와 1% 페니실린(penicillin) 및 100 IU/㎖의 스트렙토마이신(streptomycin)을 첨가한 RPMI 1640(Roswell Park Memorial Institute) 배지를 사용하여, 37℃, 5% CO₂조건에서 배양하였다.In order to confirm the cytotoxicity of the chloroform fraction of freshwater seaweed ethanol extract in RAW 264.7 cells, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. Raw 264.7
배양한 RAW 264.7 세포를 96웰 플레이트에 웰 당 1×105개의 세포가 되도록 100㎕씩 분주하고, 37℃, 5% CO₂조건에서 18시간 동안 배양하였다. 이후 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF)을 0, 12.5, 25, 50, 100, 200 ㎍/㎖ 가 되도록 시료 100㎕씩 각 웰에 처리하고, 37℃, 5% CO₂조건에서 24시간 동안 배양하였다. 24시간 후에 상기 배양액의 상등액 100㎕를 제거하고, 5㎎/㎖ 농도의 MTT 용액을 10㎕씩 첨가하여 37℃에서 3시간 동안 반응하였다. 이 후 반응액을 제거하고 각 웰 당 반응정지 용액(10% SDS in 0.1N HCl) 100㎕를 가하여 24시간 동안 반응한 뒤, 570㎚에서 흡광도를 측정하여 그 결과를 도 4에 나타내었다. 이때, 아무것도 처리하지 않은 RAW 264.7 세포의 세포 생존율을 100% 기준으로 하여, 시료를 처리한 세포의 생존율을 계산하였다.100 μl of the cultured RAW 264.7 cells were dispensed into a 96-well plate to be 1×10 5 cells per well, and cultured for 18 hours at 37° C. and 5% CO₂ condition. Thereafter, 100 μl of the sample was treated in each well so that the chloroform fraction (Pj-EE-CF) of the ethanol extract of freshwater laver was 0, 12.5, 25, 50, 100, and 200 μg/ml, and Incubated for 24 hours. After 24 hours, 100 μl of the supernatant of the culture medium was removed, and 10 μl of 5 mg/ml MTT solution was added thereto, followed by reaction at 37° C. for 3 hours. Thereafter, the reaction solution was removed, 100 μl of a reaction stop solution (10% SDS in 0.1N HCl) was added to each well, reacted for 24 hours, and absorbance was measured at 570 nm, and the results are shown in FIG. 4 . At this time, the viability of the cells treated with the sample was calculated based on the cell viability of the RAW 264.7 cells that were not treated with anything as a standard of 100%.
도 4에서 보는 바와 같이, 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF)은 25 ㎍/㎖ 처리 시까지 세포 독성을 나타내지 않았으며, 50 ㎍/㎖에서 90% 이상의 세포 생존률을 나타내었다. 따라서, 이후 실험은 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF) 농도를 최대 50 ㎍/㎖로 하여 실험을 수행하였다.As shown in Figure 4, the chloroform fraction of freshwater seaweed ethanol extract (Pj-EE-CF) did not show cytotoxicity until 25 μg/ml treatment, and showed a cell viability of 90% or more at 50 μg/ml. Therefore, subsequent experiments were performed with the maximum concentration of the chloroform fraction (Pj-EE-CF) of the ethanol extract of freshwater seaweed at 50 μg/ml.
<< 실시예Example 5. 5. 민물김freshwater seaweed 추출물의 of the extract LPSLPS 유도 RAW 264.7 세포주에서 산화질소 생성 억제 효과 확인> Confirmation of inhibitory effect on nitric oxide production in induced RAW 264.7 cell line>
RAW 264.7 세포에서 민물김 추출물의 NO 생성에 미치는 효과를 확인하였다. 상기 실시예 4에서 배양한 RAW 264.7 세포를 96웰 플레이트에 웰 당 1×105개의 세포가 되도록 100㎕씩 분주하고, 37℃, 5% CO₂조건에서 18시간 동안 배양하였다. 이후 농도별로 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF) 50㎕씩 각 웰에 처리하고 30분간 배양한 다음, RAW 264.7 세포의 염증반응을 유도하는 LPS(lipopolysaccharide)를 최종 농도가 1㎎/㎖가 되도록 50㎕씩 첨가하여 37℃, 5% CO₂조건에서 24시간 동안 배양하였다. 24시간 후, 새로운 96웰 플레이트에 상기 세포 배양액의 상등액 100㎕과 NO의 양을 정량할 수 있는 Griess 용액(0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H3PO4) 100㎕를 더한 다음 570㎚에서의 흡광도를 측정하여 NO 생성능을 분석하고 이를 도 5에 나타내었다.The effect of freshwater seaweed extract on NO production in RAW 264.7 cells was confirmed. 100 μl of the RAW 264.7 cells cultured in Example 4 were dispensed into a 96-well plate to form 1×10 5 cells per well, and cultured for 18 hours at 37° C. and 5
도 5에서 보는 바와 같이, 염증이 유발된 대식세포인 RAW 264.7 세포에 본 발명의 Pj-EE-CF 을 처리하는 경우 농도 의존적으로 염증 물질인 NO 생성량이 감소하는 것을 확인하였다.As shown in FIG. 5 , it was confirmed that the production of NO, an inflammatory substance, was reduced in a concentration-dependent manner when RAW 264.7 cells, which are inflammatory macrophages, were treated with the Pj-EE-CF of the present invention.
<< 실시예Example 6. 6. 민물김freshwater seaweed 추출물의 of the extract LPSLPS 유도 Judo HEKHEK 293 세포에서의 항염증 효과 확인> Confirmation of anti-inflammatory effect in 293 cells>
HEK 293 세포는 100㎜ 세포배양접시(cell culture dish)에 5% FBS(fetal bovine serum)와 1% 페니실린(penicillin) 및 100 IU/㎖의 스트렙토마이신(streptomycin)을 첨가한 DMEM(Dulbecco's Modified Medium) 배지를 사용하여, 37℃, 5% CO₂조건에서 배양하였다.HEK 293 cells were cultured in Dulbecco's Modified Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin, and 100 IU/ml of streptomycin in a 100 mm cell culture dish. Using the medium, it was cultured at 37 ℃, 5% CO₂ condition.
상기 배양한 HEK 293 세포를 10% FBS(fetal bovine serum), 1% 페니실린(penicillin) 및 100 IU/㎖의 스트렙토마이신(streptomycin)을 첨가한 DMEM 배지를 사용하여 1×106세포/㎖ 세포가 되도록 농도를 조절한 다음 37℃, 5% CO₂조건에서 18시간 동안 배양하였다. 배양 후 세포가 70% 밀도가 되었을 때 리포펙타민 2000(Lipofectamine 2000)을 이용하여 NF-κB(nuclear factor kappa-light-chain-enhancer of activated B cells) 또는 AP-1(Activator protein 1) 루시퍼라아제 DNA, β-갈락토시다아제(β-galactosidase), Flag-MyD88 또는 CFP-TRIF DNA를 각각 트랜스펙션(co-transfection)하였다. 1㎍ DNA와 1㎕ 리포펙타민 2000을 배지에 각각 희석해 준 다음 상온에서 20분 동안 배양하고, DNA 희석액과 리포펙타민 2000 희석액을 혼합하여 다시 상온에서 20분 배양하였다. 상기 배양액을 HEK 293 세포가 분주된 24웰 플레이트에 넣어주고 24시간 동안 배양한 다음, 민물김 에탄올 추출물의 클로로포름 분획물(Pj-EE-CF)을 농도별(0, 12.5, 25, 50 ㎍/㎖)로 처리하여 24시간 배양하였다. 배양 후 루시퍼라아제 용해 완충액(luciferase lysis buffer)으로 세포를 용해시킨 다음 기질인 루시페린(luciferin)과 1:1로 반응하였다. 반응물은 루미노미터(Luminometer)로 흡광도를 측정하여 도 6에 나타내었다. β-갈락토시다아제의 경우에는 X-gal과 1:1로 반응시킨 후 405nm에서 흡광도를 측정하였다.The cultured HEK 293 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 100 IU/ml of streptomycin at a concentration of 1×10 6 cells/ml. After adjusting the concentration as much as possible, it was cultured for 18 hours at 37℃ and 5% CO₂ conditions. After culture, when the cells reach 70% density, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) or AP-1 (Activator protein 1) luciferase using Lipofectamine 2000 The enzyme DNA, β-galactosidase (β-galactosidase), Flag-MyD88 or CFP-TRIF DNA was transfected (co-transfection), respectively. 1 μg DNA and 1 μl Lipofectamine 2000 were each diluted in the medium, followed by incubation at room temperature for 20 minutes, mixing the diluted DNA solution and Lipofectamine 2000 solution, and incubation at room temperature for 20 minutes. The culture solution was put into a 24-well plate in which HEK 293 cells were dispensed and cultured for 24 hours, and then the chloroform fraction (Pj-EE-CF) of freshwater seaweed ethanol extract was added at concentrations (0, 12.5, 25, 50 μg/ml). ) and cultured for 24 hours. After culturing, the cells were lysed with luciferase lysis buffer and then reacted 1:1 with luciferin as a substrate. The absorbance of the reactant was measured with a luminometer and is shown in FIG. 6 . In the case of β-galactosidase, absorbance was measured at 405 nm after reacting 1:1 with X-gal.
도 6에서 보는 바와 같이, HEK 293 세포에 Pj-EE-CF을 처리하는 경우 MyD88 또는 TRIF에 의한 NF-κB 또는 AP-1 활성이 농도 의존적으로 감소되었다. 이로부터, Pj-EE-CF는 염증 반응을 억제시키는 효과가 우수한 조성물임을 확인하였다.As shown in FIG. 6 , when HEK 293 cells were treated with Pj-EE-CF, MyD88 or TRIF-induced NF-κB or AP-1 activity was decreased in a concentration-dependent manner. From this, it was confirmed that Pj-EE-CF is a composition having an excellent effect of inhibiting the inflammatory response.
<< 실시예Example 7. 7. 민물김freshwater seaweed 추출물의 of the extract LPSLPS 유도 RAW 264.7 세포주에서 염증 신호전달 경로 단백질의 발현에 미치는 효과 확인> Confirmation of the effect on the expression of inflammatory signaling pathway proteins in the induced RAW 264.7 cell line>
배양한 RAW 264.7 세포를 10% FBS(fetal bovine serum)와 1% 페니실린/스트렙토마이신(100 IU/㎖)을 첨가한 RPMI 1640 배지를 사용하여 플레이트(30㎜)에 웰 당 2×106개의 세포가 되도록 2㎖씩 분주하고, 37℃, 5% CO₂조건에서 18시간 동안 배양하였다. 배양 후 상기 배양액의 상등액 1㎖을 제거하고, Pj-EE-CF을 0, 12.5, 25, 50 ㎍/㎖가 되도록 500㎕씩 처리한 다음 37℃, 5% CO₂조건에서 30분간 배양하였다. 여기에 RAW 264.7 세포의 염증반응을 유도하는 LPS(lipopolysaccharide)를 최종 농도가 1㎎/㎖가 되도록 500㎕ 첨가하고 37℃, 5% CO₂조건에서 배양하였다. 배양된 세포를 PBS로 2회 세척하고 PBS를 완전히 제거한 후 세포 용해 및 염색 시약인 RIPA 버퍼 200㎕를 처리하였다. 그 다음 BCA assay로 단백질을 정량한 후 10% SDS-PAGE를 이용하여 전기영동을 수행하였다. 전기영동한 SDS-PAGE 젤의 단백질을 PVDF 막으로 이동시킨 후, 단백질 이동이 완료된 PVDF 막을 상온에서 TBS-T(in 100mM NaCl, 10mM Tris, 0.1%(v/v) Tween-20, pH 7.4 (PBST)containing 3% BSA)로 블록킹하였다. 그 다음 TBS-T를 사용하여 10분간 3회 세척한 후, 3% BSA를 녹인 TBS-T에 1차 항체를 1시간 처리하고, TBS-T로 충분히 세척하였다. 그리고 2차 항체를 1시간 동안 처리하고 PBS-T로 10분씩 3회 세척하였다. 그 다음 ECL detection system을 사용하여 염증 질환 관련 인자인 JNK, p38, ERK, c-FOS, c-Jun 및 이들의 인산화된 단백질 발현을 확인하고 그 결과를 도 7에 나타내었다. 단백질 양의 보정은 PVDF 막을 재활용(stripping)하여 액틴 단백질의 양을 확인함으로써 동량임을 확인하였으며, 각각의 항체는 Cell signaling 및 Santacruz에서 구입하였다.Cultured RAW 264.7 cells were plated (30 mm) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 IU/ml), and 2×10 6 cells were plated per well. 2 ml was dispensed so as to be, and incubated for 18 hours at 37 ℃, 5% CO₂ condition. After culturing, 1 ml of the supernatant of the culture medium was removed, and 500 μl of Pj-EE-CF was treated at 0, 12.5, 25, and 50 μg/ml, and then cultured at 37° C. and 5% CO₂ for 30 minutes. Here, 500 μl of LPS (lipopolysaccharide), which induces an inflammatory response of RAW 264.7 cells, was added to a final concentration of 1 mg/ml, and cultured at 37° C. and 5% CO₂ conditions. After washing the cultured cells twice with PBS and completely removing the PBS, 200 μl of RIPA buffer, which is a cell lysis and staining reagent, was treated. Then, after quantifying the protein by BCA assay, electrophoresis was performed using 10% SDS-PAGE. After transferring the protein from the electrophoresed SDS-PAGE gel to a PVDF membrane, the PVDF membrane on which the protein transfer was completed was incubated with TBS-T (in 100mM NaCl, 10mM Tris, 0.1% (v/v) Tween-20, pH 7.4 ( PBST) containing 3% BSA). Then, after washing three times for 10 minutes using TBS-T, the primary antibody was treated in TBS-T in which 3% BSA was dissolved for 1 hour, and washed sufficiently with TBS-T. Then, the secondary antibody was treated for 1 hour and washed three times with PBS-T for 10 minutes each. Then, the expression of JNK, p38, ERK, c-FOS, c-Jun and their phosphorylated proteins, which are inflammatory disease-related factors, were confirmed using the ECL detection system, and the results are shown in FIG. 7 . The amount of protein was corrected by stripping the PVDF membrane to confirm the amount of actin protein, and each antibody was purchased from Cell Signaling and Santacruz.
도 7에서 보는 바와 같이, LPS에 의하여 염증이 유발된 대식세포는 인산화된 c-Jun, c-FOS, ERK, p38의 단백질 발현이 증가하였으며, 이는 본 발명의 Pj-EE-CF을 처리하는 경우, 그 발현이 감소하였다. As shown in FIG. 7, the macrophages inflamed by LPS showed increased protein expression of phosphorylated c-Jun, c-FOS, ERK, and p38, which was observed when the Pj-EE-CF of the present invention was treated. , its expression decreased.
상기 결과를 통하여 본 발명 Pj-EE-CF은 인산화된 p38, ERK, c-FOS, c-Jun의 단백질 발현을 감소시킴으로써 염증 신호전달 경로 단백질 발현을 억제하고 이를 통해 염증 반응이 감소됨을 확인하였으며, 이로부터 항염증용 조성물로 유용하게 사용할 수 있음을 알 수 있었다.Through the above results, it was confirmed that the Pj-EE-CF of the present invention inhibits the expression of inflammatory signaling pathway proteins by reducing the protein expression of phosphorylated p38, ERK, c-FOS, and c-Jun, thereby reducing the inflammatory response, From this, it was found that it can be usefully used as an anti-inflammatory composition.
<< 제제예formulation example 1. 정제의 제조> 1. Preparation of tablets>
본 발명 민물김 추출물 또는 분획물 20g을 각각 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다.20 g of the freshwater seaweed extract or fraction of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, respectively. After adding 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.
<< 제제예formulation example 2. 캡슐제의 제조> 2. Preparation of capsules>
본 발명 민물김 추출물 또는 분획물 100㎎, 옥수수전분 100㎎, 유당 100㎎ 및 스테아린산 마그네슘 2㎎을 혼합한 후 통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing 100 mg of the freshwater seaweed extract or fraction of the present invention, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, the above ingredients were mixed according to a conventional capsule preparation method and filled into gelatin capsules to prepare capsules. did
<< 제제예formulation example 3. 주사제의 제조> 3. Preparation of Injection>
본 발명 민물김 추출물 또는 분획물 1g, 염화나트륨 0.6g 및 아스코르브산 0.1g을 증류수에 용해시켜서 100㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.1 g of freshwater seaweed extract or fraction of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was bottled and sterilized by heating at 20° C. for 30 minutes.
<< 제제예formulation example 4. 건강기능식품의 제조> 4. Manufacture of health functional food>
제제예formulation example 4-1. 건강기능식품의 제조 4-1. Manufacture of health functional food
본 발명의 민물김 추출물 또는 분획물 2g, 비타민 혼합물 적량, 비타민 A 아세테이트 70㎍, 비타민 E 1.0㎎, 비타민 B1 0.13㎎, 비타민 B2 0.15㎎, 비타민 B6 0.5㎎, 비타민 B12 0.2㎍, 비타민 C 10㎎, 비오틴 10㎍, 니코틴산아미드 1.7㎎, 엽산 50㎍, 판토텐산 칼슘 0.5㎎, 무기질 혼합물 적량, 황산제1철 1.75㎎, 산화아연 0.82㎎, 탄산 마그네슘 25.3㎎, 제1인산칼륨 15㎎, 제2인산칼슘 55㎎, 구연산칼륨 90㎎, 탄산칼슘 100㎎, 염화마그네슘 24.8㎎을 섞어 과립으로 제조하였으나, 용도에 따라 다양한 제형으로 변형시켜 제조할 수 있다. 또한, 상기의 비타민 및 미네랄 혼합물의 조성비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합하여 제조할 수 있다.2 g of freshwater laver extract or fraction of the present invention, appropriate amount of vitamin mixture, vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg,
제제예formulation example 4-2. 건강기능성 음료의 제조 4-2. Manufacture of health functional beverages
본 발명의 민물김 추출물 또는 분획물 1g, 구연산 0.1g, 프락토올리고당 100g, 정제수 900g을 섞어 통상의 음료 제조방법에 따라 교반, 가열, 여과, 살균, 냉장하여 음료를 제조하였다.1 g of the freshwater seaweed extract or fraction of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized, and refrigerated according to a conventional beverage manufacturing method to prepare a beverage.
Claims (14)
상기 민물김 추출물은 민물김의 에탄올 추출물을 농축하여 물에 현탁한 후, 헥산, 클로로포름, n-부탄올 및 물로 순차적으로 추출한 분획물 중 클로로포름 분획물인 것을 특징으로 하는 부종 예방 또는 치료용 조성물.In the composition for preventing or treating edema containing freshwater laver (Prasiola japonica) extract as an active ingredient,
The freshwater seaweed extract is a composition for preventing or treating edema, characterized in that the chloroform fraction among the fractions obtained by concentrating the freshwater seaweed ethanol extract, suspending it in water, and sequentially extracting it with hexane, chloroform, n-butanol and water.
상기 부종은 간질환, 신장질환, 심장질환, 내분비질환, 영양결핍, 부신피질호르몬제 사용, 비스테로이드성 진통제 사용 또는 항고혈압약제 사용에 의한 전신부종 또는 국소부종인 것을 특징으로 부종 예방 또는 치료용 조성물.According to claim 1
The edema is liver disease, kidney disease, heart disease, endocrine disease, nutritional deficiency, use of adrenal cortical hormones, use of non-steroidal analgesics, or use of antihypertensive drugs, characterized in that systemic edema or local edema caused by edema prevention or treatment composition.
상기 부종은 산후 부종인 것을 특징으로 부종 예방 또는 치료용 조성물.According to claim 1,
The edema is a composition for preventing or treating edema, characterized in that postpartum edema.
상기 부종 예방 또는 치료는 INOS, COX-2, TNF-α 또는 MMP-9 유전자의 발현을 억제하는 것을 특징으로 부종 예방 또는 치료용 조성물.According to claim 1,
The prevention or treatment of edema is a composition for preventing or treating edema, characterized in that inhibiting the expression of INOS, COX-2, TNF-α or MMP-9 gene.
상기 부종 예방 또는 치료는 MyD88 또는 TRIF에 의한 NF-κB 또는 AP-1 활성이 감소하는 것을 특징으로 부종 예방 또는 치료용 조성물.According to claim 1,
The prevention or treatment of edema is a composition for preventing or treating edema, characterized in that the activity of NF-κB or AP-1 by MyD88 or TRIF is reduced.
상기 부종 예방 또는 치료는 인산화된 p38, ERK, c-FOS 및 c-Jun로 구성된 군으로부터 선택된 1 이상의 염증 신호전달 경로 단백질 발현을 억제하는 것을 특징으로 부종 예방 또는 치료용 조성물.According to claim 1,
The prevention or treatment of edema is a composition for preventing or treating edema, characterized in that inhibiting the expression of one or more inflammatory signaling pathway proteins selected from the group consisting of phosphorylated p38, ERK, c-FOS and c-Jun.
상기 민물김 추출물은 민물김의 에탄올 추출물을 농축하여 물에 현탁한 후, 헥산, 클로로포름, n-부탄올 및 물로 순차적으로 추출한 분획물 중 클로로포름 분획물인 것을 특징으로 하는 부종 예방 또는 개선용 건강기능식품.In the health functional food for preventing or improving edema containing freshwater seaweed (Prasiola japonica) extract as an active ingredient,
The freshwater seaweed extract is a health functional food for preventing or improving edema, characterized in that the chloroform fraction among the fractions obtained by concentrating the freshwater seaweed ethanol extract, suspending it in water, and sequentially extracting it with hexane, chloroform, n-butanol and water.
상기 부종은 산후 부종인 것을 특징으로 하는 부종 예방 또는 개선용 건강기능식품.According to claim 11,
The edema is a health functional food for preventing or improving edema, characterized in that postpartum edema.
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