KR102608023B1 - Pharmaceutical composition for preventing or treating allergic airway inflammatory comprising fucoidan Isolated from the Sporophylls of Undaria pinnatifida - Google Patents
Pharmaceutical composition for preventing or treating allergic airway inflammatory comprising fucoidan Isolated from the Sporophylls of Undaria pinnatifida Download PDFInfo
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- KR102608023B1 KR102608023B1 KR1020210074251A KR20210074251A KR102608023B1 KR 102608023 B1 KR102608023 B1 KR 102608023B1 KR 1020210074251 A KR1020210074251 A KR 1020210074251A KR 20210074251 A KR20210074251 A KR 20210074251A KR 102608023 B1 KR102608023 B1 KR 102608023B1
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- fucoidan
- ova
- group
- sporophylls
- cells
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Abstract
본 발명은 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증의 예방 또는 치료용 약학 조성물 및 알러지성 기도 염증 개선용 식품 조성물을 제공한다. 본 발명의 미역 포자엽 유래 푸코이단 포함 조성물은 MDA 수치, 염증성 세포 활성, 점액 과다 분비를 감소시키는 효능을 나타내므로, 제약, 의학, 식품 분야에서 천식, 폐렴 및 만성 폐색성 폐질환과 같은 알러지성 기도 염증 질환을 예방 및 치료하는데 유용하게 사용될 수 있다.The present invention provides a pharmaceutical composition for preventing or treating allergic airway inflammation and a food composition for improving allergic airway inflammation, including fucoidan derived from seaweed sporophylls. The composition containing fucoidan derived from the seaweed sporophyll of the present invention has the effect of reducing MDA levels, inflammatory cell activity, and mucus hypersecretion, and is therefore used in the pharmaceutical, medical, and food fields to prevent allergic airway inflammation such as asthma, pneumonia, and chronic obstructive pulmonary disease. It can be useful in preventing and treating diseases.
Description
본 발명은 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 또는 치료용 조성물에 관한 것으로서, 더욱 상세하게는, 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증의 예방 또는 치료용 약학적 조성물 및 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 개선용 식품 조성물에 관한 것이다.The present invention relates to a composition for the prevention or treatment of allergic airway inflammation containing fucoidan derived from seaweed sporophylls, and more specifically, to a pharmaceutical composition for preventing or treating allergic airway inflammation containing fucoidan derived from seaweed sporophylls and from seaweed sporophylls. It relates to a food composition for improving allergic airway inflammation containing fucoidan.
미세먼지(Particulate matter, PM)는 주요 대기오염물질로 중금속, 다환 방향족 탄화수소 및 유기탄소 등으로 이루어져 있다. 직경이 2.5μm (PM2.5) 미만인 미세먼지는 공기 중에 남아있으면서 호흡 시 기도 깊숙히 침투할 수 있다. 미세먼지 내 다환 방향족 탄화수소 및 중금속은 기도에서 활성 산소를 생성하고 염증을 유발시켜 천식을 발병시키거나 기존 천식환자의 경우는 증상을 더욱 악화시킨다. Particulate matter (PM) is a major air pollutant and consists of heavy metals, polycyclic aromatic hydrocarbons, and organic carbon. Fine dust with a diameter of less than 2.5μm (PM2.5) remains in the air and can penetrate deep into the airway when breathed. Polycyclic aromatic hydrocarbons and heavy metals in fine dust generate oxygen radicals in the respiratory tract and cause inflammation, causing asthma or worsening symptoms in existing asthma patients.
천식은 염증 세포의 유입으로 인한 기도 폐쇄, 기도 과민 반응 및 점액 과다 분비를 일으키는 기도의 염증성 질환이다. 천식의 염증은 비만세포, 호중구, T helper cell type 2 (Th2), 림프구, 상피세포 및 여러가지 사이토카인들(Interleukin [IL]-4, 5, 6, 9, 13, 25, 33)이 중요한 역할을 하는 것으로 알려져 있다. 조직과 사이토카인, 면역세포들과 상피세포 그리고 외부의 요인들은 복잡하게 상호작용을 하여 기도의 염증과 손상을 일으킨다.Asthma is an inflammatory disease of the airways that causes airway obstruction, airway hyperresponsiveness, and excessive mucus secretion due to the influx of inflammatory cells. Mast cells, neutrophils, T helper cell type 2 (Th2), lymphocytes, epithelial cells, and various cytokines (Interleukin [IL]-4, 5, 6, 9, 13, 25, 33) play an important role in the inflammation of asthma. It is known to do. Tissues, cytokines, immune cells, epithelial cells, and external factors interact complexly to cause inflammation and damage to the airway.
현재 치료법으로는 염증 반응을 억제하는 코르티코스테로이드(Corticosteroids)가 효과적인 것으로 알려져 있다. 그러나 일부 환자에게 장시간 사용시 코르티코스테로이드(Corticosteroids)가 폐에 돌이킬 수 없는 변화를 일으키는 부작용을 나타내는 것으로 보고되었다. 따라서 천식을 부작용이 없는 천연물을 이용해 치료하는 천연물 치료제가 필요한 상황이다. Corticosteroids , which suppress the inflammatory response, are currently known to be effective treatments. However, it has been reported that corticosteroids cause side effects that cause irreversible changes in the lungs when used for a long time in some patients. Therefore, there is a need for a natural product treatment that treats asthma using natural products without side effects.
푸코이단(Fucoidans)은 다시마, 미역 등 여러종의 갈조류에서 얻어지는 물질로 프로테오글라이칸(Proteoglycan)의 일종으로 주요구성은 후코스(L-Fucose)에 황산기, 그리고 약간의 단백질로 되어있다. 후코스(L-Fucose)를 기본으로 미량의 갈락토스(D-Galactose), 만노스(D-Mannose), 자일로스(Xylose), 우론산(Uronic acid) 등의 당이 복합구조를 이루는 다당체이다. 상기 푸코이단은 Th2 면역 반응을 약화시켜 항종양 효과를 나타낸다고 알려져 있으며(Maruyama, H. et al, Int. Arch. Allergy Immunol. 2005, 137, 289-294.), 항바이러스, 항암 및 항혈전 효과가 있는 것으로 알려져 있다. Fucoidans are substances obtained from various types of brown algae, such as kelp and seaweed, and are a type of proteoglycan . The main composition is fucose ( L-Fucose ), a sulfate group, and some proteins. It is a polysaccharide that forms a complex structure based on L-Fucose and small amounts of sugars such as D-Galactose , D-Mannose , Xylose , and Uronic acid . The fucoidan is known to have an anti-tumor effect by weakening the Th2 immune response (Maruyama, H. et al, Int. Arch. Allergy Immunol. 2005, 137, 289-294.), and has anti-viral, anti-cancer and anti-thrombotic effects. It is known to exist.
염증은 병원균 침입이나 조직 손상에 의해 일어나는 선천적 면역 반응으로, 다양한 면역 세포에 의해 발생되는 복합적인 과정이다. 염증 과정 중에는 대식세포와 같은 면역 세포가 병원균 침입에 반응하여 식작용 (phagocytosis)을 하거나 사이토카인과 같은 염증성 인자들을 분비하여 병원균을 제거하는 과정이 포함된다. 만약 이러한 염증 반응이 조절되지 못하면 체내 손상이 유발된다. 실제로 박테리아 침입시 일어나는 과도한 염증에 일어나는 패혈증과 같은 전신 염증의 경우는 30~60%로 높은 사망률을 보이고, 이러한 높은 사망률의 원인은 염증으로 인한 장기의 기능 손실로 인한 장기 부전이다. 따라서 다량 분비되는 염증성 인자들로 인해 다양한 질병 및 사망에 이를 수 있기 때문에 과도한 염증에 대책이 필요하다 (배기춘 외, Kor J Herbology 2012；27 (6): 55-61).Inflammation is an innate immune response caused by pathogen invasion or tissue damage, and is a complex process caused by various immune cells. During the inflammatory process, immune cells such as macrophages respond to invading pathogens by performing phagocytosis or secreting inflammatory factors such as cytokines to eliminate pathogens. If this inflammatory response is not controlled, damage to the body is caused. In fact, systemic inflammation such as sepsis, which occurs due to excessive inflammation caused by bacterial invasion, has a high mortality rate of 30 to 60%, and the cause of this high mortality rate is organ failure due to loss of organ function due to inflammation. Therefore, since inflammatory factors secreted in large quantities can lead to various diseases and death, measures against excessive inflammation are necessary (Bae Ki-chun et al., Kor J Herbology 2012；27 (6): 55-61).
산화 스트레스는 신체에서 에너지원으로 이용된 산소가 불완전하게 환원되는 경우 발생하는 활성산소 등에 의해서 발생된다. 정상적인 상태에서도 발생을 하지만, 과도하게 일어나는 경우 여러 질병 발생의 주요 요인이 된다. 당뇨에서는 산화 스트레스가 근육과 지방에서 당의 섭취를 방해하고, 췌장의 β세포에서 인슐린 분비를 감소시킨다. 혈관에서는 면역세포와 상호 작용에 의한 염증을 유발시키고, 혈관기능의 장애를 초래하다. 또한, 대사성 질환과도 관계가 있는데, 비만 지방조직에 다량의 탐식세포가 있다는 사실이 밝혀졌으며, 활성화된 탐식세포는 산화 스트레스를 활발히 생성하기 때문에 비만 지방조직의 산화 스트레스 생성 증가에 역할을 하는 것으로 생각된다 (최영은 등, J Korean Acad Fam Med 2006; 27: 773~781).Oxidative stress is caused by free radicals that occur when oxygen used as an energy source in the body is incompletely reduced. It occurs even under normal conditions, but when it occurs excessively, it becomes a major factor in the occurrence of various diseases. In diabetes, oxidative stress interferes with sugar uptake from muscle and fat and reduces insulin secretion from pancreatic β cells. In blood vessels, it causes inflammation due to interaction with immune cells and causes impairment of vascular function. In addition, it is also related to metabolic disease. It has been revealed that there are a large number of phagocytic cells in obese adipose tissue, and since activated phagocytic cells actively generate oxidative stress, they are believed to play a role in increasing the production of oxidative stress in obese adipose tissue. (Choi Young-eun et al., J Korean Acad Fam Med 2006; 27: 773-781).
천연물은 동서양을 막론하고 다양한 용도로 활용되었으며, 안전성 측면에서 우수하고 개발기간이 짧으며 성공 가능성이 높아 그 중요성이 높아 그 중요성이 부각되고 있어, 항알러지, 항염 또는 항산화 효과가 있는 우수한 천연물을 개발하는 것이 요구되고 있다.Natural products have been used for a variety of purposes both in the East and the West, and their importance is highlighted due to their excellent safety, short development period, and high probability of success, leading to the development of excellent natural products with anti-allergy, anti-inflammatory, or antioxidant effects. It is being asked to do so.
본 발명이 이루고자 하는 기술적 과제는 미역 포자엽 유래 푸코이단을 포함하는 조성물이 활성산소 형성과 염증을 일으키는 사이토카인 분비를 억제시킬 뿐만 아니라, 알러지성 기도염증의 발병에 주된 역할을 하는 호산구 및 호중구와 같은 과립구의 침윤을 현저하게 감소시키고, 염증 반응의 진행에 관여하는 대식세포, CD4+ T세포의 침윤을 약화시켜 미세먼지로 인한 알러지성 염증 치료에 효과를 나타내어 천식, 폐렴과 같은 알러지성 기도 염증 질환을 예방 및 치료할 수 있는 조성물을 제공하는 것이다.The technical problem to be achieved by the present invention is that a composition containing fucoidan derived from seaweed sporophylls not only suppresses the formation of reactive oxygen species and the secretion of cytokines that cause inflammation, but also suppresses granulocytes such as eosinophils and neutrophils, which play a major role in the development of allergic airway inflammation. Significantly reduces infiltration and weakens the infiltration of macrophages and CD4+ T cells involved in the progression of inflammatory reactions, thereby being effective in treating allergic inflammation caused by fine dust and preventing allergic airway inflammatory diseases such as asthma and pneumonia. and providing a composition capable of treatment.
본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
상기 기술적 과제를 달성하기 위하여, 본 발명의 일측면에 따라, 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 예방 또는 치료용 약학 조성물이 제공된다.In order to achieve the above technical problem, according to one aspect of the present invention, a pharmaceutical composition for preventing or treating allergic airway inflammation containing fucoidan derived from seaweed sporophylls is provided.
상기 기술적 과제를 달성하기 위하여, 본 발명의 다른 측면에 따라, 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 개선용 식품 조성물이 제공된다.In order to achieve the above technical problem, according to another aspect of the present invention, a food composition for improving allergic airway inflammation containing fucoidan derived from seaweed sporophylls is provided.
본 발명의 실시예에 따르면, 미역 포자엽 유래 푸코이단은 미세먼지로 인한 기도 염증과 과다 점액분비를 억제하고, 미세먼지 유입시의 산화적 스트레스를 감소시키고, 염증성 세포를 활성화시키며, 점액 과다 분비를 감소시키는데 효과가 있는 것으로 밝혀졌다. 따라서, 본 발명의 미역 포자엽 유래 푸코이단을 포함하는 조성물은 제약, 의학, 식품 분야에서 미세먼지로 인한 천식, 폐렴 및 만성 폐색성 폐질환과 같은 알러지성 기도 염증 질환을 예방 및 치료하는데 유용하게 사용될 수 있다.According to an embodiment of the present invention, fucoidan derived from seaweed sporophylls suppresses airway inflammation and excessive mucus secretion caused by fine dust, reduces oxidative stress when fine dust enters, activates inflammatory cells, and reduces excessive mucus secretion. It has been found to be effective in doing so. Therefore, the composition containing fucoidan derived from seaweed sporophylls of the present invention can be usefully used in the pharmaceutical, medical, and food fields to prevent and treat allergic airway inflammatory diseases such as asthma, pneumonia, and chronic obstructive pulmonary disease caused by fine dust. there is.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.
도 1은 푸코이단이 폐 및 혈청에서 MDA 및 8-OHdG 수준에 미치는 영향을 나타내는 그래프이다(폐에서 (A-G) MDA 양성 세포, (H) MDA 양성 세포의 백분율 및 (I) MDA 수준, 혈청에서 (J) MDA 및 (K) 8-OHdG 수준, * (p <0.05), ** (p <0.01), *** (p <0.005), †(p <0.05) †††(p <0.0005)).
도 2는 푸코이단이 기관지 폐포 세척액(BALF)에서 PM으로 악화된 기도 과민성 및 염증세포 침윤에 미치는 영향을 나타내는 그래프이다((A) 기도 과민반응은 메타콜린농도(0-50 mg/mL)의 증가에 대한 반응으로 Penh값으로 표시됨, (B) BALF의 염증세포 수임. * (p <0.05), ** (p <0.005), †(p < 0.05), ††(p < 0.005)).
도 3은 PM으로 인한 생체 내 조직학적 변화와 푸코이단의 영향을 나타낸 이미지로 H&E염색 결과 (A-G) 기관 및 (H-N) 폐부분의 대표적 이미지, (O) 기관 부분(A-G)에 대한 염증 점수의 중앙값과 사 분위수 범위를 보여주는 산점도, (폐) 폐부분의 산점도를 나타낸 도면이다((A, H) 대조군, (B, I) PM군, (C, J) OVA군, (D, K) OVA + PM군, (E, L) OVA + PM + 푸코이단 (100mg / Kg) 군, (F, M) OVA + PM + 푸코이단 (400 mg / Kg) 군, (G, N) OVA + PM + 프레드니손(Prednisone) (5 mg / Kg)군, (O-P) 0, 정상; 1, 침윤 된 염증 세포가 거의 없음; 2, 세포층 깊이의 염증세포 침윤; 3, 2-4개의 세포층 깊이의 염증세포 침윤; 및 4, 4개 이상의 세포층 깊이의 염증세포의 고리, (A-N)의 스케일바는 25μm, *** (p < 0.0005), †(p < 0.05), ††† (p < 0.0005)).
도 4는 마우스의 기관(A-G)과 폐(H-N)의 콩고 레드 염색 결과를 나타낸 그래프이다((A, H) 대조군, (B, I) PM군, (C, J) OVA군, (D, K) OVA + PM군, (E, L) OVA + PM + 푸코이단(100mg / Kg)군, (F, M) OVA + PM + 푸코이단(400 mg / Kg)군, (G, N) OVA + PM + 프레드니손(Prednisone)(5 mg / Kg)군, (O) 기관 내 부위 당 콩고 적색 염색 세포 수, (P) 폐 부위 당 콩고 적색 염색 세포 수, (A-N)의 스케일바는 25μm, ** (p < 0.005), *** (p < 0.0005), †††(p < 0.0005), ## (p < 0.005), ### (p < 0.0005)).
도 5는 마우스의 기관(A-G) 및 폐(H-N)에 있는 Gr-1+ 세포의 면역조직 화학적 분석을 나타낸 그래프이다((A,H) 대조군, (B, I) PM군, (C, J) OVA군, (D, K) OVA + PM군, (E, L) OVA + PM + 푸코이단(100mg / Kg)군, (F, M) OVA + PM + 푸코이단(400mg / Kg)군, (G, N) OVA + PM + 프레드니손(Prednisone) (5mg / Kg)군, (A-N)의 스케일바는 25μm, * (p < 0.05), *** (p < 0.0005), ††(p < 0.005), †††(p < 0.0005), # (p < 0.05), ### (p < 0.0005)).
도 6은 마우스의 기관(A-G) 및 폐 (H-N)에 있는 F4/80+ 대식세포 세포의 면역조직 화학적 분석을 나타낸 그래프이다((A, H) 대조군, (B, I) PM 군, (C, J) OVA 군, (D, K) OVA + PM군 (E, L) OVA + PM + 푸코이단(100mg / Kg)군, (F, M) OVA + PM + 푸코이단(400mg / Kg)군, (G, N) OVA + PM + 프레드니손(Prednisone) (5 mg / Kg)군, (O) 기관 내 부위 당 F4/80+ 세포의 수, (P) 폐의 부위 당 F4/80+ 세포의 수, (A-N)의 스케일바는 25μm, * (p < 0.05), *** (p < 0.0005), † (p < 0.05), †† (p < 0.005), ††† (p < 0.0005)).
도 7은 마우스의 기관(A-G) 및 폐 (H-N)에 있는 CD4+ T 세포의 면역조직 화학적 분석을 나타낸 그래프이다((A, H) 대조군, (B, I) PM 군, (C, J) OVA 군, (D, K) OVA + PM군 (E, L) OVA + PM + 푸코이단(100mg / Kg)군, (F, M) OVA + PM + 푸코이단(400mg / Kg)군, (G, N) OVA + PM + 프레드니손(Prednisone) (5 mg / Kg)군, (O) 기관 내 부위 당 CD4+ T 세포의 수, (P) 폐의 부위 당 CD4+ T 세포의 수, (A-N)의 스케일바는 25μm, ** (p < 0.005), *** (p < 0.0005), † (p < 0.05), ††† (p < 0.0005)).
도 8은 푸코이단이 혈청 내 면역글로불린 수준에 미치는 영향 결과를 나타낸 그래프이다((A) 총 IgE, (B) OVA-특이적 IgE, (C) OVA-특이적 IgG1 및 (D) 혈청 내 OVA 특이적 IgG2a, * (p < 0.05), *** (p < 0.0005), † (p < 0.05), †† (p < 0.005), # (p < 0.05)).
도 9는 마우스 기관의 톨루이딘 블루 염색을 나타낸 도면이다((A) 대조군, (B) PM군, (C) OVA군, (D) OVA + PM군, (E) OVA + PM + 푸코이단(100mg/Kg), (F) OVA + PM + 푸코이단 (400mg/Kg), (G) OVA + PM + 프레드니손(Prednisone) (5 mg / Kg). (A-G) 비만 세포 이동, (H) 부위 당 비만 세포 수, (I) 비만 세포 탈과립 비율, (A-N)의 스케일바는 25μm, *** (p < 0.0005), †† (p < 0.005), # (p < 0.05)).
도 10은 PM에 노출된 마우스의 기관(a-g, A-G) 및 폐 (h-n, H-N)에서 PAS -양성 배상세포에 대한 푸코이단의 효과의 결과 도면이다((O)기관 및 (P) 폐의 PAS 양성 세포 비율이 표시되며, 화살촉은 점액 분비 배상세포를 나타냄. (a-g), (h-n), (A-N)의 스케일바는 25μm, (H-N)은 오일 침지렌즈로 (배율 X1000)으로 캡쳐됨. * (p < 0.05), *** (p < 0.0005), †† (p < 0.005), ††† (p < 0.0005), # (p < 0.05), ## (p < 0.005)).
도 11은 푸코이단이 PM에 노출된 마우스 기관의 점막하 선에서(A-N) 점액 분비에 미치는 영향을 나타낸 도면이다((A, H) 대조군, (B, I) PM군, (C, J) OVA군, (D, K) OVA + PM군, (E, L) OVA + PM + 푸코이단(100mg / Kg)군, (F, M) OVA + PM + 푸코이단(400 mg /Kg)군, (G, N) OVA + PM + 프레드니손(Prednisone) (5 mg/Kg)군, (A-G)의 스케일바는 25μm, (H-N)은 오일 침지렌즈로 (배율 X1000)으로 캡쳐됨).
도 12는 혈청, BALF 및 폐 내 단백질 수준을 ELISA를 사용하여 측정한 도면이다(혈청 : (A) IL-4, (B) IL-17a, (C) IL-33, BALF : (D) IL-4, (E) IL-17a, (F) IL-33 및 폐 : (G) IL-4, (H) IL-17a, (I) IL-33), * (p < 0.05), ** (p < 0.005), † (p < 0.05), # (p < 0.05)).Figure 1 is a graph showing the effect of fucoidan on MDA and 8-OHdG levels in lung and serum ((AG) MDA positive cells in lung, (H) percentage of MDA positive cells and (I) MDA level in serum ( J) MDA and (K) 8-OHdG levels, * (p < 0.05), ** (p < 0.01), *** (p < 0.005), † (p < 0.05) ††† (p < 0.0005) ).
Figure 2 is a graph showing the effect of fucoidan on airway hyperresponsiveness and inflammatory cell infiltration worsened from bronchoalveolar lavage fluid (BALF) to PM ((A) Airway hyperresponsiveness increases with methacholine concentration (0-50 mg/mL) Expressed as Penh value in response to, (B) Number of inflammatory cells in BALF. * (p < 0.05), ** (p < 0.005), † (p < 0.05), †† (p < 0.005)).
Figure 3 is an image showing in vivo histological changes due to PM and the effect of fucoidan. Representative images of the (AG) trachea and (HN) lung sections as a result of H&E staining, and (O) median inflammation score for the tracheal section (AG). Scatterplot showing the interquartile range, (Lung) This is a diagram showing the scatterplot of the lung section ((A, H) control group, (B, I) PM group, (C, J) OVA group, (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100 mg / Kg) group, (F, M) OVA + PM + fucoidan (400 mg / Kg) group, (G, N) OVA + PM + Prednisone ) (5 mg/Kg) group, (OP) 0, normal; 1, few infiltrated inflammatory cells; 2, inflammatory cell infiltration at a depth of 2 to 4 cell layers; and 4. , a ring of inflammatory cells more than four cell layers deep, scale bar in (AN) is 25 μm, *** (p < 0.0005), † (p < 0.05), ††† (p < 0.0005)).
Figure 4 is a graph showing the results of Congo red staining of the trachea (AG) and lung (HN) of mice ((A, H) control group, (B, I) PM group, (C, J) OVA group, (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100 mg / Kg) group, (F, M) OVA + PM + fucoidan (400 mg / Kg) group, (G, N) OVA + PM + Prednisone (5 mg / Kg) group, (O) number of Congo red-stained cells per intratracheal area, (P) number of Congo red-stained cells per lung area, (AN) scale bar is 25μm, ** ( p < 0.005), *** (p < 0.0005), ††† (p < 0.0005), ## (p < 0.005), ### (p < 0.0005)).
Figure 5 is a graph showing immunohistochemical analysis of Gr-1 + cells in the trachea (AG) and lung (HN) of mice ((A, H) control group, (B, I) PM group, (C, J) ) OVA group, (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100mg / Kg) group, (F, M) OVA + PM + fucoidan (400mg / Kg) group, (G , N) OVA + PM + Prednisone (5mg / Kg) group, (AN) scale bar is 25μm, * (p < 0.05), *** (p < 0.0005), †† (p < 0.005) , ††† (p < 0.0005), # (p < 0.05), ### (p < 0.0005)).
Figure 6 is a graph showing immunohistochemical analysis of F4/80 + macrophage cells in the trachea (AG) and lung (HN) of mice ((A, H) control group, (B, I) PM group, (C , J) OVA group, (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100mg / Kg) group, (F, M) OVA + PM + fucoidan (400mg / Kg) group, ( G, N) OVA + PM + Prednisone (5 mg / Kg) group, (O) number of F4/80 + cells per region in the trachea, (P) number of F4/80 + cells per region in the lung, Scale bar in (AN) is 25 μm, * (p < 0.05), *** (p < 0.0005), † (p < 0.05), †† (p < 0.005), ††† (p < 0.0005)).
Figure 7 is a graph showing immunohistochemical analysis of CD4 + T cells in the trachea (AG) and lung (HN) of mice ((A, H) control group, (B, I) PM group, (C, J) OVA group, (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100mg / Kg) group, (F, M) OVA + PM + fucoidan (400mg / Kg) group, (G, N) ) OVA + PM + Prednisone (5 mg / Kg) group, (O) number of CD4 + T cells per area in the trachea, (P) number of CD4 + T cells per area in the lung, (AN) scale Bars are 25 μm, ** (p < 0.005), *** (p < 0.0005), † (p < 0.05), ††† (p < 0.0005)).
Figure 8 is a graph showing the results of the effect of fucoidan on the level of immunoglobulins in serum ((A) total IgE, (B) OVA-specific IgE, (C) OVA-specific IgG1, and (D) OVA specific in serum. enemy IgG2a, * (p < 0.05), *** (p < 0.0005), † (p < 0.05), †† (p < 0.005), # (p < 0.05)).
Figure 9 is a diagram showing toluidine blue staining of mouse organs ((A) control group, (B) PM group, (C) OVA group, (D) OVA + PM group, (E) OVA + PM + fucoidan (100 mg/ Kg), (F) OVA + PM + Fucoidan (400mg/Kg), (G) OVA + PM + Prednisone (5 mg / Kg). (AG) Mast cell migration, (H) Mast cell number per site , (I) Mast cell degranulation rate, (AN) scale bar is 25 μm, *** (p < 0.0005), †† (p < 0.005), # (p < 0.05)).
Figure 10 is a result diagram of the effect of fucoidan on PAS-positive goblet cells in the trachea (ag, AG) and lung (hn, HN) of mice exposed to PM (PAS positivity in (O) trachea and (P) lung Cell ratios are indicated, and arrowheads indicate mucus-secreting goblet cells. Scale bars for (ag), (hn), and (AN) are 25 μm, and (HN) was captured with an oil immersion lens at (magnification X1000). * ( p < 0.05), *** (p < 0.0005), †† (p < 0.005), ††† (p < 0.0005), # (p < 0.05), ## (p < 0.005)).
Figure 11 is a diagram showing the effect of fucoidan on mucus secretion (AN) in the submucosal glands of mouse organs exposed to PM ((A, H) control group, (B, I) PM group, (C, J) OVA group , (D, K) OVA + PM group, (E, L) OVA + PM + fucoidan (100 mg / Kg) group, (F, M) OVA + PM + fucoidan (400 mg / Kg) group, (G, N) ) OVA + PM + Prednisone (5 mg/Kg) group, (AG) scale bar is 25μm, (HN) was captured with oil immersion lens (magnification X1000)).
Figure 12 is a diagram measuring protein levels in serum, BALF, and lung using ELISA (serum: (A) IL-4, (B) IL-17a, (C) IL-33, BALF: (D) IL -4, (E) IL-17a, (F) IL-33 and lung: (G) IL-4, (H) IL-17a, (I) IL-33), * (p < 0.05), ** (p < 0.005), † (p < 0.05), # (p < 0.05)).
본 발명은 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating allergic airway inflammation containing fucoidan derived from seaweed sporophylls.
본 발명의 일 구현예에서, 상기 푸코이단은 미역 포자엽(Undaria pinnatifida Sporophylls)으로부터 추출한 것일 수 있다. In one embodiment of the present invention, the fucoidan may be extracted from Undaria pinnatifida Sporophylls .
본 발명에 있어서, 상기 푸코이단은 모즈쿠, 미역, 다시마, 톳과 같은 다양한 종류의 갈조류에 0.1% 정도 함유되어 있는 다당류(多糖類, Polysaccharide)이다. 특히 푸코이단은 여러가지의 단당류 중에서 후코스(Fucose)와 갈락토스(galactose)를 중심으로 기타의 필수당 성분과 황산기 등이 결합하여 구성되어 있다. 1913년 스웨덴 웁살라대학(Uppsala University) D.Kylin 교수에 의해 최초로 발견되었고 그 후 1991년 러시아에서 본격적인 연구를 통해 푸코이단이 각종 질병의 치료용 혹은 예방용 식품 으로 알려지게 되었고 1996년 제 55회 일본 암학회에 푸코이단의 아포토시스 유도작용을 발표함으로써 세계적인 주목을 받기 시작했다. 푸코이단은 독성 및 부작용이 전혀 없으며 다양한 활성이 있어 식품산업에 새로운 가치를 창출할 수 있는 식품 소재이다. 푸코이단은 음료, 분말, 캡슐, 타정제품 등으로 식품에 이용되며 화장품, 소재 의약품 등의 여러분야에 활용될 수 있는 무궁무진한 원료이다. 푸코이단은 아직도 100% 그 효과가 밝혀지지 않았다. In the present invention, the fucoidan is a polysaccharide contained in approximately 0.1% of various types of brown algae such as mozuku, seaweed, kelp, and hijiki. In particular, fucoidan is composed of various monosaccharides, mainly fucose and galactose , combined with other essential sugar components and sulfate groups. It was first discovered in 1913 by Professor D. Kylin at Uppsala University in Sweden, and through full-scale research in Russia in 1991, fucoidan became known as a food for the treatment or prevention of various diseases, and was presented at the 55th Japan Cancer Conference in 1996. It began to receive global attention by announcing the apoptosis-inducing effect of fucoidan at a conference. Fucoidan is a food material that has no toxicity or side effects and has various activities that can create new value in the food industry. Fucoidan is used in foods such as beverages, powders, capsules, and tablet products, and is an infinite raw material that can be used in various fields such as cosmetics and pharmaceutical ingredients. The 100% effectiveness of fucoidan is still not known.
본 발명의 일 구현예에서, 상기 푸코이단은 분쇄 단계 후 열수 추출하는 단계; 원심분리하여 침전물을 제거하는 단계; 한외여과기로 여과한 후 농축하는 단계; 주정을 사용하여 다당체를 침전시키는 단계; 및 주정을 분리한 후 건조시키는 단계를 포함하는 제조방법으로 제조될 수 있다.In one embodiment of the present invention, the fucoidan is extracted with hot water after the grinding step; Centrifuging to remove sediment; Concentrating after filtering with an ultrafilter; Precipitating polysaccharides using alcohol; and separating the alcohol and then drying it.
본 발명의 다른 일 구현예서, 상기 푸코이단은 2,000 내지 500,000Da의 중량평균분자량(Mw)의 중저분자 푸코이단일 수 있다. 바람직하게는 3,000 내지 100,000Da, 더욱 바람직하게는 3,300 내지 10,000Da 중량평균분자량(Mw)일 수 있다.In another embodiment of the present invention, the fucoidan may be a low-to-medium molecule fucoidan with a weight average molecular weight (Mw) of 2,000 to 500,000 Da. The weight average molecular weight (Mw) may be preferably 3,000 to 100,000 Da, more preferably 3,300 to 10,000 Da.
상기와 같이 얻어진 푸코이단은 활성산소 형성과 염증을 일으키는 사이토카인 분비를 억제시킬 뿐 아니라 미세먼지로 인한 알러지성 염증의 치료에 효과를 나타내어 천식, 폐렴과 같은 알러지성 기도 염증 질환에 사용할 수 있다.Fucoidan obtained as described above not only suppresses the formation of free radicals and the secretion of cytokines that cause inflammation, but is also effective in treating allergic inflammation caused by fine dust, so it can be used for allergic airway inflammatory diseases such as asthma and pneumonia.
미세먼지(PM)은 산화스트레스를 자극해 지질의 산화를 촉진한다. 말론디알데하이드(Malondialdehyde; MDA)는 불포화지방산의 과산화 대사물질로 세포의 산화된 정도를 측정할 수 있는 지표가 된다. Fine dust (PM) stimulates oxidative stress and promotes lipid oxidation. Malondialdehyde (MDA ) is a peroxidized metabolite of unsaturated fatty acids and serves as an indicator to measure the degree of oxidation of cells.
하기 실시예에 나타난 것처럼 푸코이단을 처리한 마우스군은 농도에 비례하여 폐 조직내 MDA 농도를 대조군, PM군 및 OVA군 보다 유의적으로 감소시키며, 이로 인해 산화 스트레스가 감소하여 염증을 약화시킬 수 있다. As shown in the examples below, the fucoidan-treated mouse group significantly reduces the MDA concentration in lung tissue compared to the control group, PM group, and OVA group in proportion to the concentration, which reduces oxidative stress and can attenuate inflammation. .
또한, 고용량의 푸코이단을 처리한 마우스군은 대조군 및 PM에 악화된 군에 비해 기도의 염증반응을 현저하게 약화시킬 수 있으며, 염증세포, 이노시노필, 중성화 및 림프구의 침윤을 크게 감소시킬 수 있다. In addition, the mouse group treated with a high dose of fucoidan was able to significantly weaken the inflammatory response in the airway compared to the control group and the group with worsening PM, and significantly reduce the infiltration of inflammatory cells, inosinophils, neutralizing cells, and lymphocytes. .
상기 알러지성 기도 염증에서 기도염증의 주된 역할을 하는 것은 호산구 및 호중구와 같은 과립구의 침윤이다. 상기 호산구 및 호중구의 침윤을 면역조직 화학염색을 통해 확인해보면, 푸코이단을 처리한 마우스 군에서 과립구 침윤이 현저하게 감소하는 것을 확인할 수 있다. In the allergic airway inflammation, the main role of airway inflammation is infiltration of granulocytes such as eosinophils and neutrophils. When the infiltration of eosinophils and neutrophils was confirmed through immunohistochemical staining, it was confirmed that granulocyte infiltration was significantly reduced in the fucoidan-treated mouse group.
또한, 알러지성 기도 염증의 발병 기전 동안 염증 반응의 진행에 관여하는 풍부한 면역세포는 대식세포이며, 알레르겐을 식별하여 알러지성 기도염증을 일으키는 최초의 물질은 CD4+ T세포이다. 상기 푸코이단은 이러한 대식세포와 CD4+ T세포의 침윤을 약화시키는데 효과가 있다. Additionally, during the pathogenesis of allergic airway inflammation, the abundant immune cells involved in the progression of the inflammatory response are macrophages, and the first agents to identify allergens and cause allergic airway inflammation are CD4 + T cells. The fucoidan is effective in weakening the infiltration of macrophages and CD4 + T cells.
상기 천식의 한가지 특징은 염증성 자극에 대한 점액의 과다 생산으로, 푸코이단은 점액 분비를 감소시킴으로써 PM으로 인한 알러지성 기도 염증을 약화시킬 수 있다. One characteristic of asthma is excessive production of mucus in response to inflammatory stimuli, and fucoidan can attenuate allergic airway inflammation caused by PM by reducing mucus secretion.
또한, 본 발명의 일 구현예에 따르면, 상기 알러지성 기도 염증은 천식, 폐렴 및 만성 폐색성 폐질환으로 이루어진 군에서 선택되는 1종 이상의 질환인 것일 수 있으며, 보다 바람직하게는 천식 및 폐렴이다. Additionally, according to one embodiment of the present invention, the allergic airway inflammation may be one or more diseases selected from the group consisting of asthma, pneumonia, and chronic obstructive pulmonary disease, and more preferably asthma and pneumonia.
본 발명에 있어서, 미역 포자엽 유래 푸코이단은 본 발명의 약학 조성물에 유효성분으로서 포함될 수 있다. 또한, 본 발명의 약학 조성물은 약제학적으로 허용가능한 담체 또는 희석제를 포함할 수 있으며, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화될 수 있다. 상기 약제학적으로 허용가능한 담체는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한다. 또한, 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 포함한다. 경구용 고형 제제는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 포함할 수 있으며, 마그네슘 스테아레이트, 탈크 같은 윤활제 등을 포함할 수 있다. 경구용 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등을 포함하며, 물, 리퀴드 파라핀 등의 희석제, 습윤제, 감미제, 방향제, 보존제 등을 포함할 수 있다. 비경구용 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 크림제, 동결건조 제제, 좌제를 포함하며, 비수성 용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르류 등을 포함한다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In the present invention, fucoidan derived from seaweed sporophylls may be included as an active ingredient in the pharmaceutical composition of the present invention. In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or diluent, and may be prepared in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods. It can be formulated in the form of external preparations, suppositories, and sterile injectable solutions. The pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, and microcrystalline. Contains cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It also includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Oral solid preparations include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. ), gelatin, etc., and may include lubricants such as magnesium stearate and talc. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may include diluents such as water and liquid paraffin, humectants, sweeteners, fragrances, and preservatives. Parenteral preparations include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, creams, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, and vegetable oils such as olive oil. Includes injectable esters such as oil and ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
본 발명의 약학 조성물에 함유되는 유효성분의 투여량은 환자의 상태 및 체중, 질병의 정도, 유효성분 형태, 투여 경로 및 기간에 따라 다르며, 환자에 따라 적절하게 조절될 수 있다. 예를 들면, 상기 유효성분은 1일 0.0001 내지 1000 mg/kg으로, 바람직하게는 0.01 내지 100 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한번 또는 수회 나누어 투여할 수도 있다. 또한, 본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 유효성분을 0.001 내지 90 % 중량 백분율로 포함할 수 있다.The dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, form of the active ingredient, route and period of administration, and can be appropriately adjusted depending on the patient. For example, the active ingredient can be administered at a dose of 0.0001 to 1000 mg/kg per day, preferably 0.01 to 100 mg/kg, and the administration may be administered once a day or in divided doses. Additionally, the pharmaceutical composition of the present invention may contain the active ingredient in a weight percentage of 0.001 to 90% based on the total weight of the composition.
본 발명의 약학 조성물은 랫트, 마우스, 가축, 인간 등의 포유동물에 다양한 경로로, 예를 들면, 경구, 피부, 복강, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내(intracerebroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical composition of the present invention is administered to mammals such as rats, mice, livestock, and humans by various routes, for example, orally, through the skin, abdominal cavity, rectum, or intravenously, into muscle, subcutaneously, intrauterine dura, or intracerebroventricularly. It can be administered by injection.
본 발명은 미역 포자엽 유래 푸코이단을 포함하는 알러지성 기도 염증 개선용 식품 조성물을 제공한다. 상기 푸코이단은 미역 포자엽으로부터 추출한 것일 수 있으며, 구체적인 내용은 전술한 바와 같다. The present invention provides a food composition for improving allergic airway inflammation containing fucoidan derived from seaweed sporophylls. The fucoidan may be extracted from seaweed sporophylls, and specific details are as described above.
본 발명의 일 구현예에서, 상기 푸코이단은 분쇄 단계 후 열수 추출하는 단계; 원심분리하여 침전물을 제거하는 단계; 한외여과기로 여과한 후 농축하는 단계; 주정을 사용하여 다당체를 침전시키는 단계; 및 주정을 분리한 후 건조시키는 단계으로 제조되는 것일 수 있으며, 상기 구체적인 내용은 전술한 바와 같다.In one embodiment of the present invention, the fucoidan is extracted with hot water after the grinding step; Centrifuging to remove sediment; Concentrating after filtering with an ultrafilter; Precipitating polysaccharides using alcohol; and may be manufactured by separating the alcohol and then drying it, the specific details of which are the same as those described above.
본 발명의 다른 구현예에서, 상기 푸코이단은 2,000 내지 500,000Da의 중량평균분자량(Mw)의 중저분자 푸코이단일 수 있으며, 구체적인 내용은 전술한 바와 같다. 또한, 본 발명의 일 구현에 따르면, 상기 알러지성 기도염증은 천식, 폐렴 및 만성 폐색성 폐질환으로 이루어진 군에서 선택되는 1종 이상의 질환일 수 있다. 상기 구체적인 내용은 전술한 바와 같다. In another embodiment of the present invention, the fucoidan may be a low-to-medium molecule fucoidan with a weight average molecular weight (Mw) of 2,000 to 500,000 Da, and specific details are as described above. Additionally, according to one embodiment of the present invention, the allergic airway inflammation may be one or more diseases selected from the group consisting of asthma, pneumonia, and chronic obstructive pulmonary disease. The specific details are the same as described above.
본 발명의 일 구현예에 따른 상기 식품 조성물에 있어서, 식품 종류에 특별한 제한은 없으며, 일례로 육류, 소시지, 빵, 초코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등일 수 있고, 통상적인 의미에서 식품을 모두 포함하는 것일 수 있다.In the food composition according to one embodiment of the present invention, there is no particular limitation on the type of food, and examples include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, dairy products, various soups, It may be beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and may include all foods in the conventional sense.
또한, 본 발명에서 사용되는 푸코이단은 알러지성 기도 염증 예방 또는 치료를 목적으로 하는 건강 기능성 식품 또는 건강 기능성 음료 조성물에 첨가될 수 있다. 이때, 상기 건강 기능성 식품 또는 건강 기능성 음료 조성물 중의 상기 푸코이단의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 기능성 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. In addition, fucoidan used in the present invention can be added to a health functional food or health functional beverage composition for the purpose of preventing or treating allergic airway inflammation. At this time, the amount of fucoidan in the health functional food or health functional beverage composition may be added in the range of 0.01 to 15% by weight of the total weight of the food, and the health functional beverage composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 0.3 g, based on 100 ml. It can be added at a rate of 1 g.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 푸코이단을 함유하는 외에 다른 성분에는 특별한 제한이 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등의 추가 성분을 함유할 수 있다. 상술한 천연탄수화물의 예로는 모노사카라이드, 예를 들어 포도당, 과당 등; 디사카라이드, 예를 들어 말토오스, 수크로오스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 솔비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제인 타우마틴, 스테비아 추출물, 예를 들어 레바우디오시드 A, 글리시르히진 등; 및 합성 향미제, 예를 들어 사카린, 아스파르탐 등을 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1내지 20g, 바람직하게는 약 5 내지 12g이다. The health functional beverage composition of the present invention has no particular limitations on other ingredients other than containing the above-mentioned fucoidan as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like ordinary beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Flavoring agents other than those mentioned above include natural flavoring agent thaumatin, stevia extract, such as rebaudioside A, glycyrrhizin, etc.; and synthetic flavoring agents such as saccharin, aspartame, etc. can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.
상기 외에 본 발명의 푸코이단을 포함하는 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 및 천연 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 푸코이단을 포함하는 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이때, 첨가제의 비율은 그다지 중요하지는 않지만 본 발명의 푸코이단을 포함하는 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition containing fucoidan of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition containing fucoidan of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. At this time, the ratio of the additive is not very important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition containing fucoidan of the present invention.
본 발명의 일 구현예에 따른 식품 조성물은 동물은 물론이고 인간에게도 적용할 수 있다.The food composition according to one embodiment of the present invention can be applied to humans as well as animals.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.
실시예Example
1. 재료 및 방법1. Materials and Methods
1-1. 시료 및 키트1-1. Samples and Kits
오브알브민(Ovalbumin; OVA), 프레드니손(prednisone), 아세틸-β-메틸콜린(acetyl-β-methylcholine), 콩고레드(Congo red), 에오신(eosin), 톨루이딘 블루(toluidine blue), (periodic acid of Schiff; PAS), 류펩틴(leupeptin), 페닐메틸설포닐 플루로라이드(phenylmethylsulfonyl fluroride; PMSF), 소듐 오르토 바나데이트(sodium ortho-vanadate; NOV), 아프로티닌(aprotinin) 및 말론디알데하이드(malondialdehyde; MDA)는 시그마 (세인트루이스, 미주리주, MO, 미국)에서 구입하였다. 헤마톡실렌(hematoxylene)은 다코(Dako)(Carpinteria, CA, 미국)으로부터 구입하고, 메틸 그린(Methyl green)은 플루카TM(브라질 상파울루)에서 구입했으며, 명반보조제 (Alum adjuvant는 써모 피셔 사이언시픽(Thermo Fisher Scientific; 록포드, 일리노이주, 미국)에서 구입했다.Ovalbumin (OVA), prednisone, acetyl-β-methylcholine, Congo red, eosin, toluidine blue, (periodic acid of Schiff; PAS), leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium ortho-vanadate (NOV), aprotinin and malondialdehyde; MDA) was purchased from Sigma (St. Louis, MO, USA). Hematoxylene was purchased from Dako (Carpinteria, CA, USA), methyl green was purchased from Fluka TM (Sao Paulo, Brazil), and alum adjuvant was purchased from Thermo Fisher Scientific. Purchased from Pick (Thermo Fisher Scientific; Rockford, IL, USA).
티오바르비투르산(Thiobarbituric acid)은 알파에이사(워드힐, 메사추세츠주, 미국)에서 구입하고, 트리클로로 아세트산 (Trichloroacetic acid)은 머크(Darmstadt, 독일)에서 구입했으며, 브래드포드 시약은 바이오-레드(리치몬드, CA, 미국)에서 구입했다. Thiobarbituric acid was purchased from Alpha A (Wardhill, MA, USA), trichloroacetic acid was purchased from Merck (Darmstadt, Germany), and Bradford reagent was purchased from Bio-Red. (Richmond, CA, USA).
총 IgE 측정을 위한 ELISA 키트는 베틸연구소(몽고메리, 텍사스주, 미국)에서 구입했다. OVA 특이적 IgE, OVA 특이적 IgG2a, IL-4, IL-6 및 IL-17a 측정을 위한 ELISA 키트는 바이오레전드(샌디에고, 캘리포니아주, 미국)에서 구입했다. OVA- 특이적 IgG1 측정을 위한 ELISA 키트는 케이맨 케미칼(앤아버, 미시건주, 미국)에서 구입했다.ELISA kits for total IgE measurement were purchased from Betil Laboratories (Montgomery, TX, USA). ELISA kits for measuring OVA-specific IgE, OVA-specific IgG2a, IL-4, IL-6, and IL-17a were purchased from Biolegend (San Diego, CA, USA). The ELISA kit for OVA-specific IgG1 measurement was purchased from Cayman Chemical (Ann Arbor, MI, USA).
마우스 IL-33 ELISA 키트는 인비트로젠 (써모피셔 사이언티픽; 월섬, 매사추세츠주, 미국)에서 구입했다. DNA / RNA 산화 손상 ELISA 키트는 케이맨 케미칼(앤아버, 미시건주, 미국)에서 구입했다.Mouse IL-33 ELISA kit was purchased from Invitrogen (Thermo Fisher Scientific; Waltham, MA, USA). DNA/RNA oxidative damage ELISA kit was purchased from Cayman Chemicals (Ann Arbor, MI, USA).
디프 퀵(Diff Quick) 염색 키트는 시스멕스 법인(고베, 일본)에서 구입했다. 자일 렌과 에탄올은 오씨아이 케미칼(OCI Chemicals)(서울, 한국)에서 구입했다.Diff Quick staining kit was purchased from Sysmex Corporation (Kobe, Japan). Xylene and ethanol were purchased from OCI Chemicals (Seoul, Korea).
항-말론디알데히드 항체는 압캠(abcam)(캠브리지, 영국)에서 구입했다.Anti-malondialdehyde antibody was purchased from abcam (Cambridge, UK).
마우스 Gr-1 항체는 R&D 시스템즈(미니애폴리스, 미네소타주, 미국)에서 구입했다. 정제된 항-마우스 F4/80 항체는 바이오레전(샌디에고, 캘리포니아주, 미국)에서 구입했다.Mouse Gr-1 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Purified anti-mouse F4/80 antibody was purchased from Biolegion (San Diego, CA, USA).
CD4 항체는 노부스 바이오로지칼(Novus Biologicals)(LLC, 미국)에서 구입하였으며, 푸코이단은 해원바이오텍 (서울, 한국)에서 공급했다.CD4 antibody was purchased from Novus Biologicals (LLC, USA), and fucoidan was supplied by Haewon Biotech (Seoul, Korea).
1-2. 미세먼지(Particulate matter; PM)1-2. Particulate matter (PM)
본 발명에서는 일본 국립 환경 연구소(National Institute for Environmental Studies; NIES)에서 개발 및 인증된 레퍼런스 물질(CRM) No.28 얼반 에어로졸인 미세먼지(PM)가 사용되었다. 대부분의 PM은 직경이 2.5 μm 미만이었으며 PM 샘플의 대부분은 다환 방향족 탄화수소(PAH)와 납(Pb) 및 바륨(Ba)과 같은 중금속을 포함하고 있다. In the present invention, fine dust (PM), a reference material (CRM) No. 28 urban aerosol developed and certified by the National Institute for Environmental Studies (NIES) of Japan, was used. Most PMs were less than 2.5 μm in diameter, and most of the PM samples contained polycyclic aromatic hydrocarbons (PAHs) and heavy metals such as lead (Pb) and barium (Ba).
1-3. 동물(마우스)1-3. Animal (mouse)
한국 성남 오리엔트바이오(OrientBio)에서 구입한 암컷 Balb/c 마우스 (20 ~ 25g, 7 ~ 8주령)는 시설에 수용되어 물과 함께 NIH-07-승인된 식단을 충분히 공급하였다. 동물 실험은 제주대학교 제도 윤리위원회의 실험 동물 관리 및 사용에 관한 지침에 따라 수행되었다. Female Balb/c mice (20 to 25 g, 7 to 8 weeks old) purchased from OrientBio in Seongnam, Korea, were housed in a facility and provided with sufficient water and an NIH-07-approved diet. Animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals of the Jeju National University Institutional Ethics Committee.
1-4. 실험 디자인1-4. experimental design
미세먼지로 악화된 알러지성 기도 염증 마우스 모델은 이전에 설명된 프로토콜에 약간의 수정을 가하여 이에 따라 활용하였다.A mouse model of allergic airway inflammation exacerbated by fine dust was utilized according to the previously described protocol with some modifications.
마우스는 각 군당 7마리씩 사용했으며, 대조군, PM군, OVA군, OVA+PM군, OVA+PM+푸코이단(100mg/kg)군, OVA+PM+푸코이단(400mg/kg)군 및 OVA+PM+프레드니손(5mg/kg)군으로 구성되었다.Seven mice were used in each group: control group, PM group, OVA group, OVA+PM group, OVA+PM+fucoidan (100mg/kg) group, OVA+PM+fucoidan (400mg/kg) group, and OVA+PM+prednisone (5mg). /kg) group.
OVA 유도 그룹 (OVA군, OVA+PM군, OVA+PM+푸코이단군, OVA+PM+프레드니손군)의 마우스의 복강내로 200uL 식염수에 용해된 OVA와 2mg Al(OH)3로 감작시켰다. 감작 2 주 후, 미세먼지는 5 mg/m3의 농도로 하루에 30분간씩 7일간 분무하였다. Mice in the OVA induction group (OVA group, OVA+PM group, OVA+PM+fucoidan group, OVA+PM+prednisone group) were sensitized intraperitoneally with OVA and 2mg Al(OH) 3 dissolved in 200uL saline solution. Two weeks after sensitization, fine dust was sprayed at a concentration of 5 mg/m 3 for 30 minutes a day for 7 days.
미세먼지에 노출된 마우스는 저용량(100mg/kg) 또는 고용량(400mg/kg)의 푸코이단 또는 식염수에 현탁된 프레드니손 (5mg/kg)을 경구로 섭취했다. 다른 그룹은 식염수를 경구 투여했다 (도 13).Mice exposed to fine dust were orally administered a low dose (100 mg/kg) or high dose (400 mg/kg) of fucoidan or prednisone (5 mg/kg) suspended in saline solution. The other group was administered saline solution orally (Figure 13).
2. 실험방법2. Experimental method
2-1. 혈청 내 MDA 수치 측정2-1. Measurement of MDA levels in serum
지질 분해는 산화 스트레스에 대한 유용한 지표이며, 이는 자유 라디칼 생성을 더욱 증가시킨다. 말론디알데하이드(MDA; Malondialdehyde)는 산화 공격을 받은 다중 불포화 지질의 중요한 최종 산물이다. 제조업체의 지침에 따라 지질 과산화 분석 키트를 사용하여 마우스 혈청의 MDA 수준을 측정했다. MDA 활성은 비색 분석을 사용하여 표준 곡선을 기반으로 마이크로 몰 수준에서 측정되었다.Lipid breakdown is a useful indicator of oxidative stress, which further increases free radical production. Malondialdehyde (MDA) is an important end product of polyunsaturated lipids subjected to oxidative attack. MDA levels in mouse serum were measured using a lipid peroxidation assay kit according to the manufacturer's instructions. MDA activity was measured at the micromolar level based on a standard curve using a colorimetric assay.
2-2. Thiobarbituric Acid Reactive Substance (TBARS) 분석2-2. Thiobarbituric Acid Reactive Substance (TBARS) Analysis
폐의 지질 과산화를 측정하기 위해, 프로테아제억제제(2mM Na3VO4, 1mM PMSF, 10ug/m Laprotinin, 10ug/mL leupeptin)를 포함하는 RIPA 버퍼(완충액)을 사용하여 tacoTm Prep 비드 비터 (GeneReach Biotechnology Co., 대만)를 사용하여 폐 조직을 균질화했다. 수득된 균질물을 원심분리 (3000X g, 10분, 4 ℃)하고, 상층액의 단백질 농도를 브래드포드(Bradford) 단백질 분석법으로 측정하였다.To measure lipid peroxidation in the lung, RIPA buffer containing protease inhibitors (2mM Na 3 VO 4 , 1mM PMSF, 10ug/m Laprotinin, 10ug/mL leupeptin) was used using a taco Tm Prep bead beater (GeneReach Biotechnology). Co., Taiwan) was used to homogenize the lung tissue. The obtained homogenate was centrifuged (3000X g, 10 minutes, 4°C), and the protein concentration of the supernatant was measured using the Bradford protein assay.
동일한 양의 단백질을 10% (w/v) 트리클로로 아세트산과 혼합하고 4℃에서 10분동안 배양하도록 두었다. 2000X g (15분, 4 ℃)에서 원심분리 한 후, 생성된 상층액을 가열 블록에서 동일한 부피의 0.67%(w/v) 티오바르비투르산(thiobarbituric acid)과 10 분 동안 반응시켰다.Equal amounts of protein were mixed with 10% (w/v) trichloroacetic acid and allowed to incubate at 4°C for 10 minutes. After centrifugation at 2000
상층액은 2000X g (15분, 4℃)에서 원심분리하여 분리하였고, 상층액의 흡광도는 마이크로 플레이트 리더를 사용하여 532nm에서 측정하였다.The supernatant was separated by centrifugation at 2000X g (15 minutes, 4°C), and the absorbance of the supernatant was measured at 532 nm using a microplate reader.
샘플의 말론디알데히드(MDA) 함량은 미리 결정된 MDA 표준 곡선으로 측정되었다. The malondialdehyde (MDA) content of the samples was determined with a predetermined MDA standard curve.
2-3. 기도 과민반응을 위한 메타콜린 검사2-3. Methacholine test for airway hyperresponsiveness
본 실험에서는 PM에 노출된 마우스에서 메타콜린 흡입에 대한 반응으로 후코이단이 기도 저항(Penh 값)에 미치는 영향을 평가했다. 상기 7가지 군의 마우스를 전신 챔버에서 10분동안 다른 농도의 메타콜린(0, 12.5, 25, 50 mg / mL)으로 분무했다. 각 분무 후 즉시 체적 측정실 (Allmedicus, Anyang, Korea)에서 향상된 일시 정지 (Penh)를 측정했다. 판독 값은 3 분 동안 평균화 되었다.In this experiment, we evaluated the effect of fucoidan on airway resistance (Penh value) in response to methacholine inhalation in mice exposed to PM. The seven groups of mice were sprayed with different concentrations of methacholine (0, 12.5, 25, and 50 mg/mL) for 10 minutes in a whole body chamber. Enhanced pauses (Penh) were measured in a plethysmography laboratory (Allmedicus, Anyang, Korea) immediately after each spray. Readings were averaged over 3 minutes.
2-4. 기관지 폐포 세척액(BALF; Bronchoalveolar Lavage Fluid) 수집 및 미분계수(Differential Counting)2-4. Bronchoalveolar Lavage Fluid (BALF) Collection and Differential Counting
푸코이단이 PM으로 악화된 기도의 염증반응을 약화시키는지 여부를 확인하기 위해 BALF의 다양한 염증세포의 수를 평가했다.To determine whether fucoidan attenuates the inflammatory response in the airway exacerbated by PM, the number of various inflammatory cells in BALF was evaluated.
캐뉼라를 사용하여 1ml의 Dulbecco's Phosphate-Buffered Saline(DPBS)를 폐 (기관 내)에 주입하고 부드럽게 BALF를 수집했다. 수집된 BALF를 원심 분리 (300xg, 5 분, 4 ℃에서)하고 상청액을 분리하여 사이토 카인 분석을 수행하였다. 세포를 메탄올에 고정하고 도말을 유리 슬라이드에 준비했다.Using a cannula, 1 ml of Dulbecco's Phosphate-Buffered Saline (DPBS) was injected into the lung (endotracheally) and BALF was gently collected. The collected BALF was centrifuged (300xg, 5 min, 4°C) and the supernatant was separated for cytokine analysis. Cells were fixed in methanol and smears were prepared on glass slides.
도말은 Diff Quick 염색 키트로 염색하고, 첨착체, 호산구, 호중구, 림프구 및 호염기구의 세포 조성은 Olympus DP-72 현미경 카메라 (Olympus, Tokyo, Japan)가 있는 현미경을 사용하여 결정되었다.The smears were stained with the Diff Quick staining kit, and the cellular composition of accretes, eosinophils, neutrophils, lymphocytes, and basophils was determined using a microscope with an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan).
2-5. 푸코이단이 마우스의 기관 및 폐의 병리학적 특징에 미치는 영향2-5. Effects of fucoidan on pathological characteristics of trachea and lungs in mice
PM으로 악화된 염증 반응을 약화시키는 푸코이단의 가능성을 확인하기 위해, 마우스 기관 및 폐 부분을 헤마토실린/에오신(H&E)으로 염색하고 염증 활동의 등급과 함께 조직 병리학적 변화를 관찰했다. PM에 22일동안 노출된 마우스의 기관 및 폐 부분을 10 % 포르말린에 고정한 후, 샘플을 파라핀으로 매립하고 코팅된 유리 위에 단면화(3 μm)했다.To determine the potential of fucoidan to attenuate the inflammatory response exacerbated by PM, mouse trachea and lung sections were stained with hematoxylin/eosin (H&E) and histopathological changes along with the grade of inflammatory activity were observed. After the trachea and lung sections of mice exposed to PM for 22 days were fixed in 10% formalin, the samples were embedded in paraffin and sectioned (3 μm) on coated glass.
상기 조직 부분을 헤마톡실린/에오신(H&E)으로 염색하고 조직 병리학적 검사를 위해 광학현미경(Olympus, Tokyo, Japan)으로 관찰하였다.The tissue sections were stained with hematoxylin/eosin (H&E) and observed under a light microscope (Olympus, Tokyo, Japan) for histopathological examination.
기관 및 폐 섹션의 염증은 다음과 같이 점수를 부여하였다(0, 정상; 1, 침윤된 염증 세포가 거의 없음; 2, 관찰된 염증 세포 한 층 깊이; 3, 관찰된 염증 세포 2-4 세포층 깊이; 및 4, 4 개 이상의 세포층 깊이의 침윤된 염증 세포).Inflammation in tracheal and lung sections was scored as follows (0, normal; 1, few infiltrated inflammatory cells; 2, one layer of inflammatory cells observed; 3, 2-4 cell layers deep of inflammatory cells observed. ; and 4, infiltrated inflammatory cells more than 4 cell layers deep).
콩고 레드 염색을 사용하여 폐 및 기관 조직 부분에서 호산구의 침윤을 확인했다.Congo red staining was used to confirm eosinophil infiltration in lung and tracheal tissue sections.
<방법><Method>
- 탈 파라핀화된 조직을 콩고 레드 작업 용액에 30 분 동안 담근 후 섹션을 흐르는 수돗물로 세척하고 0.5 % 메틸그린으로 역 염색했다.- Deparaffinized tissues were soaked in Congo Red working solution for 30 minutes, then sections were washed with running tap water and counterstained with 0.5% methylgreen.
- 염색된 부분은 현미경으로 관찰되었으며, 대표적인 영역은 Olympus DP-72 현미경 카메라(Olympus, Tokyo, Japan)를 사용하여 캡처되었다.- Stained sections were observed under a microscope, and representative areas were captured using an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan).
- 각 섹션의 3 개 이상의 부위 (각 마우스에서 3 개의 섹션을 취함)에서 부위 당 콩고 레드 염색된 호산구의 수를 Image J(v1.46) 소프트웨어를 사용하여 정량화했다.- The number of Congo red-stained eosinophils per site in at least three areas of each section (three sections were taken from each mouse) was quantified using Image J (v1.46) software.
- 기관 내 비만 세포 침윤은 톨루이딘 블루 염색을 사용하여 평가되다. 탈 파라핀화된 기관 절편을 0.5 % 톨루이딘 블루 용액에 담그고 0.5 % 메틸 그린으로 대조 염색했다.- Mast cell infiltration within the organ was assessed using toluidine blue staining. Deparaffinized tracheal sections were soaked in 0.5% toluidine blue solution and counterstained with 0.5% methyl green.
- 염색된 부분을 현미경으로 관찰하고, 대표적인 영역은 Olympus DP-72 현미경 카메라 (Olympus, Tokyo, Japan)를 사용하여 캡처했다.- Stained areas were observed under a microscope, and representative areas were captured using an Olympus DP-72 microscope camera (Olympus, Tokyo, Japan).
- ImageJ (v1.46) 소프트웨어를 사용하여 각 섹션의 최소 3 개 사이트 (각 마우스에서 3 개 섹션을 가져옴)에서 사이트 당 비만 세포의 수 및 사이트 당 탈과립 된 비만 세포의 수를 정량화했다.-The number of mast cells per site and the number of degranulated mast cells per site were quantified in at least three sites in each section (three sections were taken from each mouse) using ImageJ (v1.46) software.
- 기관 및 폐 절편을 PAS (periodic acid-Schiff)로 염색하고 0.5 % 메틸 그린으로 대조 염색하여 잔 세포의 화생 및 점액 분비를 평가했다.- Tracheal and lung sections were stained with periodic acid-Schiff (PAS) and counterstained with 0.5% methyl green to evaluate goblet cell metaplasia and mucus secretion.
- 각 섹션의 3 개 이상의 부위 (각 마우스에서 3 개 섹션을 취함)에서 PAS 양성 세포 백분율을 ImageJ (v1.46) 소프트웨어를 사용하여 정량화했다.-The percentage of PAS-positive cells in at least three sites in each section (three sections were taken from each mouse) was quantified using ImageJ (v1.46) software.
2-6. 기관 및 폐의 면역조직화학적 분석2-6. Immunohistochemical analysis of trachea and lungs.
유리 슬라이드 위에 놓인 기관 및 폐 부분은 자일렌 시리즈에서 파라핀화 되고, 에탄올 시리즈에서 재수화 되었다.Tracheal and lung sections placed on glass slides were paraffinized in a xylene series and rehydrated in an ethanol series.
비특이적 결합은 토끼나 말 혈청과 함께 0.3% 과산화수소에 30분간 침지시켜 차단하였다. 차단 후, 조직을 마우스 Gr-2항체(1:200) 또는 항 마우스 F4/80 항체(1:200), 항 Cd4 항체(1:400) 또는 MDA 항체(1:400)와 함께 4 ℃에서 밤새 배양했다.Non-specific binding was blocked by immersion in 0.3% hydrogen peroxide with rabbit or horse serum for 30 minutes. After blocking, tissues were incubated with mouse Gr-2 antibody (1:200) or anti-mouse F4/80 antibody (1:200), anti-Cd4 antibody (1:400), or MDA antibody (1:400) overnight at 4°C. cultured.
세척 후, 조직을 45 분 동안 비오틴화 된 항-rat IgG 또는 항-토끼 IgG와 함께 배양 한 다음 Vectastain Elite ABC 키트 (Vector Inc., Burlingame, CA, USA)를 사용하여 아비딘-비오틴 퍼옥시다제 복합체로 처리하였다.After washing, tissues were incubated with biotinylated anti-rat IgG or anti-rabbit IgG for 45 min and then avidin-biotin peroxidase complex using the Vectastain Elite ABC kit (Vector Inc., Burlingame, CA, USA). It was processed.
2-7. Enzyme-Linked Immunosorbent Assay (ELISA) 분석2-7. Enzyme-Linked Immunosorbent Assay (ELISA) analysis
본 실험에서는 알러지성 기도 염증에서 비만 세포 활성화의 핵심 매개체인 혈청 IgE의 레벨을 평가했다.In this experiment, we evaluated the level of serum IgE, a key mediator of mast cell activation in allergic airway inflammation.
헤파린 처리된 주사기로 심장 천자에 삽입하여 22일째 마취된 마우스로부터 혈액을 수집했다. 수집된 혈액을 4 ℃에서 10 분 동안 12,000rpm으로 원심 분리하여 혈청을 혈액 세포에서 분리했다. Blood was collected from anesthetized mice on day 22 by insertion of a heparinized syringe into cardiac puncture. The collected blood was centrifuged at 12,000 rpm for 10 min at 4 °C to separate serum from blood cells.
IL-4, IL-17a, IL-33, 8-OHdG-아세티콜린에스테라제 접합체 (DNA / RNA 산화 손상 추적자), IgE, OVA-specific IgE, OVA-specific IgG1 (1 : 2000 희석된 혈청) 및 OVA-specific IgG2a는 제조업체의 권장사항에 따라 각 ELLSA키트를 사용하여 분석되었다.IL-4, IL-17a, IL-33, 8-OHdG-acetylcholinesterase conjugate (DNA/RNA oxidative damage tracer), IgE, OVA-specific IgE, OVA-specific IgG1 (1:2000 diluted serum) ) and OVA-specific IgG2a were analyzed using each ELLSA kit according to the manufacturer's recommendations.
채혈 후 마우스를 희생시키고 1ml의 DPBS를 캐뉼라를 사용하여 마우스의 폐에 부드럽게 주입하고 BALF를 수집했다. 간단히 말해서, 수집된 BALF(12,000 rpm, 4°C)를 10분 동안 원심분리하고 상층액을 각 ELISA 키트를 사용하여 IL-4, IL-17a, 그리고 IL-33 레벨에 대해서 분석했다.After blood collection, the mouse was sacrificed, and 1 ml of DPBS was gently injected into the lung of the mouse using a cannula, and BALF was collected. Briefly, collected BALF (12,000 rpm, 4°C) was centrifuged for 10 min and the supernatant was analyzed for IL-4, IL-17a, and IL-33 levels using respective ELISA kits.
프로타제 억제제(2mM Na3VO4, 1mM PMSF, 10ug/mL aprotinin, 10 ug/mL leupeptin)를 포함하는 RIPA 버퍼를 사용하여 tacoTMPrep 비드 비터 (GeneReach Biotechnology Co.)를 사용하여 폐 조직을 균질화했다.Lung tissue was homogenized using a taco TM Prep bead beater (GeneReach Biotechnology Co.) using RIPA buffer containing protease inhibitors (2mM Na3VO4, 1mM PMSF, 10ug/mL aprotinin, 10ug/mL leupeptin).
수득된 균질물을 원심분리(3000X g 4 ℃, 10분)하고, 폐 균질물 상층액의 IL-4, IL-17a, 그리고 IL-33의 레벨을 각각의 ELISA 키트를 사용하여 분석하였다.The obtained homogenate was centrifuged (3000
2-8. 통계 분석2-8. statistical analysis
일원 분산 분석 (One-way analysis of variance ; ANOVA)에 이어 Tukey의 다중 비교를 수행하여 그룹 간의 평균 값을 비교했다. p <0.05는 통계적으로 유의한 것으로 간주되었다.One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons was performed to compare mean values between groups. p <0.05 was considered statistically significant.
3. 실험결과3. Experiment results
3-1. 푸코이단이 마우스의 폐 조직 내 8-OHdG와 Malondialdehyde (MDA) 농도에 미치는 영향3-1. Effect of fucoidan on 8-OHdG and Malondialdehyde (MDA) concentrations in mouse lung tissue
푸코이단이 마우스의 기관과 폐의 병리학적 특성에 미치는 영향을 관찰한 결과, 푸코이단은 OVA+PM군에 비해 폐에서 MDA 양성 세포 비율을 용량 의존적으로 약화시켰다. As a result of observing the effect of fucoidan on the pathological characteristics of the organs and lungs of mice, fucoidan attenuated the proportion of MDA-positive cells in the lung in a dose-dependent manner compared to the OVA+PM group.
PM노출은 대조군과 비교하여 PM군 88.4 %, OVA군 88.6 %, OVA + PM군 89.5 %로 MDA 수준을 크게 향상시켰다. (p <0.05) 반면, OVA + PM군에 비해 저용량 (100mg / kg)의 푸코이단 처리시 51.2% 감소, 고용량 (400mg/kg)의 푸코이단 처리시 70.4% 감소 (p <0.05)되는 것을 관찰했다. 푸코이단의 높은 투여량은 본 연구에서 PM으로 악화된 MDA 수준에 영향을 미치는 양성 대조군인 프레드니손(Prednisone)과 비교했을 때 다소 또는 비슷한 양상을 보였다. PM exposure significantly improved the MDA level by 88.4% in the PM group, 88.6% in the OVA group, and 89.5% in the OVA + PM group compared to the control group. (p <0.05) On the other hand, compared to the OVA + PM group, a 51.2% decrease was observed when treated with a low dose (100 mg / kg) of fucoidan, and a 70.4% decrease (p < 0.05) was observed when treated with a high dose (400 mg / kg) of fucoidan. High doses of fucoidan showed somewhat or similar effects compared to the positive control, prednisone, on MDA levels exacerbated by PM in this study.
추가로, 혈청 내 MDA 수준을 추가로 분석하여, PM에 노출시 대조군과 비교하여 혈청의 MDA 수준을 유의하게 향상시킨 걸 확인하였다(PM군 및 OVA+PM군 모두에서 74.6 % 증가, 둘 다 p <0.005) (도 1J).In addition, we further analyzed the level of MDA in serum and confirmed that exposure to PM significantly improved the level of MDA in serum compared to the control group (74.6% increase in both PM group and OVA+PM group, both p <0.005) (Figure 1J).
푸코이단은 혈청에서 PM으로 악화된 MDA 수준을 용량 의존적으로 감소시켰다. OVA+PM군에 비해 저용량 (100mg/kg) 푸코이단 처리시 44.8 % 감소(p <0.0005), 고용량 (400mg/kg) 푸코이단 처리시 78.0 % 감소 (p <0.0005)되는 것을 확인하였다. Fucoidan dose-dependently reduced PM-exacerbated MDA levels in serum. Compared to the OVA+PM group, it was confirmed that when treated with low dose (100mg/kg) fucoidan, there was a 44.8% decrease (p <0.0005), and when treated with high dose (400mg/kg) fucoidan, there was a 78.0% decrease (p <0.0005).
또한, 뮤린 혈청에서 8-OHdG의 수준을 조사했다. MDA 분석의 경우와 마찬가지로 OVA군(by 81.4%, p < 0.005), PM군 (by 122.2%, p < 0.005), 그리고, OVA + PM군 (by 116.8%, p < 0.005)이 대조군과 비교했을 때, 혈청 8-OHdG 수준이 크게 증가하는 것을 볼 수 있었다. 푸코이단은 OVA+PM군 그룹과 비교하여 혈청에서 8-OHdG 수준을 감소시켰다. (두 푸코이단 용량에서 11.8% 감소). 그동안 프레드니손은 OVA+PM군에 비해 혈청 내 8-OHdG 수준을 25.7% (p < 0.05) 감소시켰다. 이러한 결과는 푸코이단과 프레드니손이 서로 다른 신호 경로를 통해 PM에 의해 유도된 산화 스트레스를 약화시켜 서로 다른 측정방법에 따라 서로 다른 효능을 나타냄을 시사한다.Additionally, the levels of 8-OHdG were examined in murine serum. As in the case of MDA analysis, the OVA group (by 81.4%, p < 0.005), PM group (by 122.2%, p < 0.005), and OVA + PM group (by 116.8%, p < 0.005) compared to the control group. When, serum 8-OHdG levels were seen to increase significantly. Fucoidan reduced 8-OHdG levels in serum compared to the OVA+PM group. (11.8% reduction at both fucoidan doses). Meanwhile, prednisone reduced serum 8-OHdG levels by 25.7% (p < 0.05) compared to the OVA+PM group. These results suggest that fucoidan and prednisone attenuate PM-induced oxidative stress through different signaling pathways, showing different efficacy according to different measurement methods.
푸코이단은 OVA+PM군에 비해 폐에서 MDA 양성 세포 비율을 용량 의존적으로 약화시켰다.Fucoidan attenuated the proportion of MDA-positive cells in the lung in a dose-dependent manner compared to the OVA+PM group.
OVA+PM+Fucoidans군들은 농도에 비례하여 폐 조직내 MDA 농도를 OVA군 혹은 OVA+PM군보다 유의적으로 감소시켰으며 400 mg/kg군은 항염증 치료제인 prednison과 비슷한 효과를 보였다.The OVA+PM+Fucoidans group significantly reduced the MDA concentration in lung tissue in proportion to the concentration compared to the OVA group or OVA+PM group, and the 400 mg/kg group showed a similar effect to prednison, an anti-inflammatory treatment.
3-2. 푸코이단이 마우스의 기관 및 폐에서 PM에 악화된 기도 과민반응 및 염증세포 침윤에 미치는 영향3-2. Effect of fucoidan on PM-exacerbated airway hyperresponsiveness and inflammatory cell infiltration in the trachea and lungs of mice.
본 실험에서는 OVA에 민감한 PM에 노출된 마우스에서 메타콜린 흡입에 대한 반응으로 푸코이단이 기도 저항(Penh 값)에 미치는 영향을 평가했다(도 2A 참고).In this experiment, we evaluated the effect of fucoidan on airway resistance (Penh value) in response to methacholine inhalation in mice exposed to OVA-sensitive PM (see Figure 2A).
PM군, OVA군, OVA+PM군은 고농도의 메타콜린(50 mg/mL)에 노출되었을 때, 대조군에 비해 기도 과민 반응이 현저하게 증가했다(p < 0.0005). When the PM group, OVA group, and OVA+PM group were exposed to high concentrations of methacholine (50 mg/mL), airway hyperresponsiveness significantly increased compared to the control group (p < 0.0005).
PM은 OVA로 인한 기도 과민 반응을 20.7% 악화시켰으며(p < 0.05), 고용량의 푸코이단을 투여받은 그룹이 고농도의 메타콜린(50 mg/mL)에 노출된 OVA+PM 군에 비해 PM에 악화된 기도의 과민 반응을 43.0% 약화시켰다(p <0.0005).PM worsened OVA-induced airway hyperresponsiveness by 20.7% (p < 0.05), and the group administered high-dose fucoidan worsened PM compared to the OVA+PM group exposed to high-dose methacholine (50 mg/mL). Airway hypersensitivity was attenuated by 43.0% (p <0.0005).
그 결과, 푸코이단의 다량 투여는 PM으로 악회된 기도의 과민반응을 약화시키는데 프레드니손과 유사하게 효과적임을 알 수 있다.As a result, it can be seen that large-dose administration of fucoidan is similarly effective as prednisone in attenuating the hyperresponsiveness of the airways worsened by PM.
푸코이단이 PM으로 악화된 기도의 염증반응을 약화시키는지 여부를 확인하기 위해 BALF의 다양한 염증세포의 수를 평가했다(도 2B 참고)To determine whether fucoidan attenuates the inflammatory response in the airway exacerbated by PM, the number of various inflammatory cells in BALF was evaluated (see Figure 2B).
고용량의 푸코이단을 투여받은 그룹은 PM으로 악화되어 BALF에 있는 염증세포, 이노시노필, 중성화, 림프구의 침윤을 크게 감소시켰다(p <0.05).The group receiving high-dose fucoidan worsened PM and significantly reduced the infiltration of inflammatory cells, inosinophils, neutralizing cells, and lymphocytes in BALF (p <0.05).
3-3. 푸코이단이 마우스의 기관 및 폐의 병리학적 특징에 미치는 영향3-3. Effects of fucoidan on pathological characteristics of trachea and lungs in mice
PM으로 악화된 염증 반응을 약화시키는 푸코이단의 가능성을 확인하기 위해, 마우스 기관지와 폐 부분을 헤마토실린/에오신(H&E)으로 염색하고 염증 활동의 등급과 함께 조직 병리학적 변화를 관찰했다(도 3O(기관), 도 3P(폐)).To determine the potential of fucoidan to attenuate the inflammatory response exacerbated by PM, mouse bronchial and lung sections were stained with hematoxylin/eosin (H&E) and histopathological changes along with the grade of inflammatory activity were observed (Figure 3O (trachea), Figure 3P (lung)).
OVA+PM군에서 염증세포의 침윤은 기관과 폐에서 대조군, PM군, OVA군 보다 높았다.The infiltration of inflammatory cells in the OVA+PM group was higher than that in the control group, PM group, and OVA group in the trachea and lung.
고용량의 푸코이단을 투여받은 그룹은 기관(도 3F,O) 및 폐(도 3M,P)에서 PM으로 악화된 염증세포 침투를 약화시켰다. 푸코이단은 PM으로 악화된 염증세포 침윤를 약화시키는데 프레드니손만큼 효과적이었다.The group receiving high doses of fucoidan attenuated the exacerbated inflammatory cell infiltration into the PM in the trachea (Figure 3F,O) and lung (Figure 3M,P). Fucoidan was as effective as prednisone in attenuating inflammatory cell infiltration exacerbated by PM.
3-4. 푸코이단이 마우스의 기관 및 폐에서 호산구(Eosinophils)의 침윤에 미치는 영향3-4. Effect of Fucoidan on Eosinophils Infiltration in Trachea and Lungs of Mice
침윤된 염증 세포 중에서 특히 천식상태에서 호산구의 유입이 관찰되어 폐 염증과 기도 과민 반응을 일으켰다. 호산구 침윤에 대한 푸코이단의 효과를 평가하기 위해 콩고 레드를 사용하여 기관 및 폐 부분을 염색했다(도 4).Among the infiltrated inflammatory cells, an influx of eosinophils was observed, especially in asthmatic conditions, causing lung inflammation and airway hyperresponsiveness. To evaluate the effect of fucoidan on eosinophil infiltration, tracheal and lung sections were stained using Congo red (Figure 4).
PM은 대조군과 비교하여 OVA에 민감한 마우스의 기관에서 호산구 침윤을 500.0 % 증가시켰다. (도 4A, O; p <0.0005). PM 및 OVA군은 OVA+PM군보다 낮은 수준으로 기관내 호산구 침윤을 증가시켰으나, OVA + PM군은 OVA 단독 군에 비해 호산구 침투를 더 악화시켰다. (56.4 %, p <0.005) (도 4C, O).PM increased eosinophil infiltration in the trachea of OVA-sensitive mice by 500.0% compared to controls. (Fig. 4A,O; p < 0.0005). The PM and OVA groups increased intratracheal eosinophil infiltration to a lower level than the OVA + PM group, but the OVA + PM group worsened eosinophil infiltration compared to the OVA alone group. (56.4%, p < 0.005) (Fig. 4C,O).
그러나 푸코이단 처리는 OVA+PM군과 비교하여 용량 반응 방식(선량반응방식)으로 기관 내 PM으로 악화된 호산구 침윤을 약화시켰으며(저용량 및 고용량 푸코이단 처리에 대해 각각 52.0 % 및 72.0 % 약화; 도 4D, O), 프레드니손(도 4G, O)은 예상대로 기관 내 호산구 침윤을 약화시켰다.However, fucoidan treatment attenuated the exacerbated eosinophil infiltration into intratracheal PM in a dose-response manner compared to the OVA+PM group (52.0% and 72.0% attenuation for low-dose and high-dose fucoidan treatment, respectively; Figure 4D). , O), prednisone (Figures 4G, O) attenuated intratracheal eosinophil infiltration, as expected.
유사하게, 대조군 (566.6 %, p <0.0005) 및 OVA 군 (74.0 %, p <0.005)에 비해 폐 내에서 OVA + PM군(도 4K, P)에서 호산구의 가장 높은 침윤이 관찰되었다. 그러나 푸코이단은 폐에서 호산구 침투를 용량 의존적으로 약화시켰다 (저용량 및 고용량 후코이단 치료에 대해 각각 45.0 % 및 65.0 % 억제; 도 4M, P). Similarly, the highest infiltration of eosinophils was observed in the OVA + PM group (Figure 4K,P) within the lung compared to the control group (566.6%, p < 0.0005) and OVA group (74.0%, p < 0.005). However, fucoidan attenuated eosinophil infiltration in the lung in a dose-dependent manner (45.0% and 65.0% inhibition for low-dose and high-dose fucoidan treatment, respectively; Fig. 4M,P).
또한, 고용량 푸코이단 치료는 마우스에서 PM으로 악화된 호산구수를 약화시키기 위한 프레드니손 치료만큼 효과적이었다.Additionally, high-dose fucoidan treatment was as effective as prednisone treatment for attenuating PM-exacerbated eosinophil counts in mice.
3-5. 푸코이단이 마우스의 기관 및 폐에서 GR-1+ 세포 침윤에 미치는 영향3-5. Effect of fucoidan on GR-1 + cell infiltration in trachea and lungs of mice
면역조직 화학염색을 통해 PM에 악화된 알러지성 기도 염증에서 기도 염증의 주된 역할을 하는 호산구 및 호중구와 같은 과립구의 침투를 확인했다.Immunohistochemical staining confirmed the infiltration of granulocytes such as eosinophils and neutrophils, which play a major role in airway inflammation in allergic airway inflammation exacerbated by PM.
알러지성 기도 염증의 마우스(PM으로 악화된)의 기관 및 폐 내에서 GR-1+ 세포의 면역조직 화학분석의 대표적인 이미지는 도 5에 나타내었다. Representative images of immunohistochemical analysis of GR-1 + cells within the trachea and lungs of mice with allergic airway inflammation (exacerbated by PM) are shown in Figure 5.
OVA+PM군(도 5D, O)은 대조군과 비교하여 기관의 과립구 유입을 200.0% 향상시켰다(p < 0.0005). The OVA+PM group (Figure 5D, O) improved tracheal granulocyte influx by 200.0% compared to the control group (p < 0.0005).
PM은 OVA군과 비교하여 OVA에 민감한 마우스의 기관에서 GR-1+ 세포 침투를 52.5% 악화시켰다(도 5C, O). 그러나 푸코이단 처리는 PM으로 악화된 기관에서 GR-1+ 세포 유입을 약화시켰다. 또한, OVA + PM군에 비해 저용량 푸코이단 처리 (도 5E, O)에서 36.6 % 감소와 고용량 푸코이단 처리 (도 5F, O)에서 53.3 % 감소를 관찰했다. 프레드니손은 또한 OVA + PM에 비해 과립구 유입을 55.5 % 감소시켰다 (도 5G, O). PM worsened GR-1 + cell infiltration by 52.5% in the organs of OVA-sensitive mice compared to the OVA group (Figures 5C,O). However, fucoidan treatment attenuated GR-1 + cell influx in PM-deteriorated organs. Additionally, we observed a 36.6% decrease in low-dose fucoidan treatment (Figure 5E,O) and a 53.3% decrease in high-dose fucoidan treatment (Figure 5F,O) compared to the OVA+PM group. Prednisone also reduced granulocyte influx by 55.5% compared to OVA+PM (Fig. 5G,O).
추가로 천식 마우스의 폐에서 과립구 침윤에 대한 푸코이단의 효과를 평가했다. 대조군과 비교하여 OVA + PM군 (도 5K, P)에서 과립구 침윤이 247.8 % 증가하는 것을 관찰했다(도 5H, P). PM은 OVA에 비해 OVA 유도 과립구 침윤을 113.3% 악화시켰다(p <0.0005). 그러나 고용량의 푸코이단으로 처리했을 때 (도 5M, P), OVA + PM에 비해 폐에서 과립구 침윤이 66.4%로 현저하게 감소했다(p <0.0005).Additionally, we evaluated the effect of fucoidan on granulocyte infiltration in the lungs of asthmatic mice. We observed a 247.8% increase in granulocyte infiltration in the OVA + PM group (Figure 5K, P) compared to the control group (Figure 5H, P). PM worsened OVA-induced granulocyte infiltration by 113.3% compared to OVA (p <0.0005). However, when treated with high doses of fucoidan (Figure 5M,P), granulocyte infiltration was significantly reduced to 66.4% in the lung compared to OVA + PM (p <0.0005).
이러한 결과로 볼 때, 푸코이단이 OVA에 민감해진 마우스의 폐에서 PM으로 악화된 과립구 침윤을 약화시키는데 프레드니손보다 더 효과적인 것을 알 수 있다. These results show that fucoidan is more effective than prednisone in attenuating granulocyte infiltration exacerbated by PM in the lungs of OVA-sensitized mice.
3-6. 푸코이단이 마우스 기관 및 폐에서 F4/80+ 대식세포의 침윤에 미치는 영향3-6. Effect of fucoidan on infiltration of F4/80 + macrophages in mouse trachea and lungs
대식세포는 알러지성 기도 염증의 발병 기전 동안 염증 반응의 진행에 관여하는 풍부한 면역세포이다. Macrophages are abundant immune cells involved in the progression of inflammatory responses during the pathogenesis of allergic airway inflammation.
푸코이단이 대식세포에 미치는 영향을 연구하기 위해 알러지성 기도 염증 마우스(PM에 악화된) 모델의 기관 및 폐에 있는 F4/80+ 세포의 면역 조직 화학적 분석을 수행했다(도 6).To study the effect of fucoidan on macrophages, we performed immunohistochemical analysis of F4/80 + cells in the trachea and lungs of a mouse model of allergic airway inflammation (exacerbated by PM) ( Fig. 6 ).
OVA + PM군은 대조군과 비교하여 기관 내 대식세포 침윤이 명확하게 강화된 것으로 나타났다 (250.0 % 증가, p <0.0005). 그러나 푸코이단은 OVA+PM 그룹에 비해 기관에서 대식세포의 침윤을 현저하게 감소시켰다. (34.7% 감소, p < 0.05; 45.7% 감소, p < 0.0005).The OVA + PM group showed clearly enhanced intratracheal macrophage infiltration compared to the control group (250.0% increase, p < 0.0005). However, fucoidan significantly reduced macrophage infiltration in the trachea compared to the OVA+PM group. (34.7% decrease, p < 0.05; 45.7% decrease, p < 0.0005).
면역 조직 화학적 분석은 또한 건강한 대조군과 비교하여 OVA+PM 군 마우스의 내강에서 F4/80+ 대식세포 침윤에서 515.3 % (p <0.0005)의 증가를 나타냈다. PM은 OVA만을 사용하여 처리한 군과 비교하여 OVA에 민감한 마우스(OVA+PM, 도 6K, P)의 폐에서 대식세포의 침윤을 악화시켰다(도 6K, M, P)Immunohistochemical analysis also revealed a 515.3% (p < 0.0005) increase in F4/80 + macrophage infiltration in the lumen of OVA+PM group mice compared to healthy controls. PM exacerbated macrophage infiltration in the lungs of OVA-sensitive mice (OVA+PM, Fig. 6K, P) compared to the group treated with OVA alone ( Fig. 6K, M, P ).
3-7. 푸코이단이 마우스의 기관 및 폐에서 CD4+ T 세포 침윤에 미치는 영향3-7. Effect of fucoidan on CD4 + T cell infiltration in trachea and lungs of mice
CD4+ T 세포는 알레르겐을 식별하여 알러지성 기도 염증을 일으키는 최초의 물질이다. 대조군에 비해 OVA+PM군은 CD4+ T 세포 수치가 64.1% (p < 0.005)가 증가하는 것으로 나타났다(도 7A, O). 대조적으로, OVA+PM+Fucoidans 100 mg/kg군, OVA+PM+Fucoidans 400 mg/kg군들의 CD4+ T 세포수는 OVA+PM군보다 각각 15.4%, 56.4%(p < 0.05) 감소하였다. CD4 + T cells are the first to identify allergens and initiate allergic airway inflammation. Compared to the control group, the OVA + PM group showed an increase in CD4 + T cell count by 64.1% (p < 0.005) (Figure 7A, O). In contrast, the CD4 + T cell counts of the OVA+PM+Fucoidans 100 mg/kg group and the OVA+PM+Fucoidans 400 mg/kg group decreased by 15.4% and 56.4% (p < 0.05), respectively, compared to the OVA+PM group.
또한 기관과 폐의 CD4+ T 세포 침윤은 OVA+PM 군이 대조군보다 197.8% 증가하였으나 OVA+PM+Fucoidans 400 mg/kg군은 OVA+PM군보다 59.1%(p < 0.0005) 감소하였으며 프레드니손과 비슷한 효과를 보였다. 이러한 결과는 푸코이단이 CD4+ T 세포의 selectin과 결합하여 CD4+ T 세포의 기능을 억제하기 때문인 것으로 사료된다.Additionally, CD4 + T cell infiltration in the trachea and lungs increased by 197.8% in the OVA+PM group compared to the control group, but decreased by 59.1% (p < 0.0005) in the OVA+PM+Fucoidans 400 mg/kg group compared to the OVA+PM group, similar to prednisone. It showed effect. These results are believed to be because fucoidan binds to the selectin of CD4 + T cells and inhibits the function of CD4 + T cells.
3-8. 푸코이단이 PM 노출 및 OVA에 민감한 마우스에서 항원 특이적 항체인 총 혈청 lgE의 레벨에 미치는 영향3-8. Effect of fucoidan on levels of total serum lgE, an antigen-specific antibody, in PM-exposed and OVA-sensitive mice.
본 실험에서는 알러지성 기도 염증에서 비만 세포 활성화의 핵심 매개체인 혈청 IgE의 레벨을 평가했다. 도 8A에서 볼 수 있듯이, 총 IgE 레벨은 대조군과 비교하여 OVA+PM군에서 280.1 % 현저하게 증가했다 (p <0.05). 또한, PM은 OVA군과 비교하여 OVA에 민감해진 마우스에서 혈청 IgE 수준을 68.3% 증가시켰다 (p <0.05). 푸코이단은 프레드니손만큼 효과적이지 않았지만 고용량 푸코이단 치료는 OVA+PM에 비해 PM으로 악화된 혈청 lgE 레벨을 33.2% 감소시켰다. (p <0.005).In this experiment, we evaluated the level of serum IgE, a key mediator of mast cell activation in allergic airway inflammation. As shown in Figure 8A, the total IgE level was significantly increased by 280.1% in the OVA+PM group compared to the control group (p < 0.05). Additionally, PM increased serum IgE levels by 68.3% in mice sensitized to OVA compared to the OVA group (p <0.05). Although fucoidan was not as effective as prednisone, high-dose fucoidan treatment reduced PM-exacerbated serum lgE levels by 33.2% compared to OVA+PM. (p <0.005).
추가로, 혈청에서 OVA-특이적 면역 글로불린 수준을 추가로 평가했다. Additionally, OVA-specific immunoglobulin levels were further assessed in serum.
도 8B에서 볼 수 있듯이, OVA-특이적 lgE 수준은 건강한 대조군(p <0.005)과 OVA 단독군(18.90 %, p <0.05)에 비해 OVA + PM군에서 현저하게 증가했다. 고용량의 후코이단은 OVA+PM에 비해 OVA-특이적 IgE 수준 (56.3 %, p <0.005)을 크게 약화시켰으며 프레드니손보다 더 효과적이었다. OVA+PM군은 OVA군과 비교하여, OVA-특이적 lgE 수준을 적당히 증가시켰으며, 프레드니손 치료와 유사하게, 고용량 푸코이단 치료는 OVA+PM에 비해 OVA-특이적 IgE 수준을 유의하게 약화시켰다. 그러나 푸코이단은 혈청에서 OVA-특이적 IgG2a 수준에 영향을 미치지 않았다(도8D). 이러한 결과는 PM이 OVA 감작을 악화시켰음을 나타낸다.As shown in Figure 8B, OVA-specific lgE levels were significantly increased in the OVA + PM group compared to the healthy control group (p < 0.005) and the OVA alone group (18.90%, p < 0.05). High doses of fucoidan significantly attenuated OVA-specific IgE levels (56.3%, p <0.005) compared to OVA+PM and were more effective than prednisone. Compared to the OVA group, the OVA+PM group moderately increased OVA-specific IgE levels, and similar to prednisone treatment, high-dose fucoidan treatment significantly attenuated OVA-specific IgE levels compared to OVA+PM. However, fucoidan did not affect OVA-specific IgG2a levels in serum (Figure 8D). These results indicate that PM worsened OVA sensitization.
3-9. 푸코이단이 PM노출과 OVA에 민감한 마우스에서 비만세포의 활성화 및 탈과립에 미치는 영향3-9. Effect of fucoidan on mast cell activation and degranulation in mice sensitive to PM exposure and OVA
톨루이딘 불루 염색을 통해 알러지성 천식의 발병 기전에서 기도 염증을 시작하고 촉진하는 비만세포의 활성화를 조사했다. Using toluidine blue staining, we investigated the activation of mast cells, which initiate and promote airway inflammation in the pathogenesis of allergic asthma.
대조군과 비교하여 OVA+PM (도 9D, H)에서 활성화된 비만세포의 현저한 증가(123.5%)를 관찰했다(p < 0.0005, 도 9A, H). 대조적으로, 비만 세포 활성화는 OVA+PM와 비교하여 푸코이단 처리에서 용량 의존적으로 저용량 28.9 %, 고용량 50 % 감소했다(도 9E, H))We observed a significant increase (123.5%) in activated mast cells (p < 0.0005, Figure 9A, H) in OVA+PM (Figure 9D, H) compared to the control group. In contrast, mast cell activation was dose-dependently reduced by 28.9% at low dose and 50% at high dose with fucoidan treatment compared to OVA+PM (Figures 9E,H).
활성화된 비만 세포는 탈과립을 거쳐 히스타민 및 기타 혈관 활성 매개체를 방출하는 것으로 알려져 있다. 도 9I에 나타난 바와 같이, OVA + PM은 PM 및 OVA 단독 그룹에 비해 비만 세포의 가장 높은 탈과립화를 나타냈다. PM은 OVA만을 사용한 치료에 비해 OVA에 의한 비만 세포 탈과립을 133.6 % 악화시켰다.Activated mast cells are known to undergo degranulation and release histamine and other vasoactive mediators. As shown in Figure 9I, OVA + PM showed the highest degranulation of mast cells compared to the PM and OVA alone groups. PM worsened OVA-induced mast cell degranulation by 133.6% compared to treatment with OVA alone.
그러나 푸코이단 처리는 기관 내 비만 세포의 PM 유발 탈과립을 저용량에서 51.9 %, 고용량에서 76.7 % 증가시켰다(p <0.005). 이처럼 고용량 푸코이단 치료는 OVA에 민감해진 마우스에서 비만 세포의 PM-악화 탈과립을 약화시키는데 프레드니손만큼 효과적이었다.However, fucoidan treatment increased PM-induced degranulation of intratracheal mast cells by 51.9% at the low dose and 76.7% at the high dose (p <0.005). As such, high-dose fucoidan treatment was as effective as prednisone in attenuating PM-aggravated degranulation of mast cells in OVA-sensitized mice.
3-10. 푸코이단의 배상세포(Goblet Cell) 증식 및 점액분비량에 미치는 영향3-10. Effect of fucoidan on goblet cell proliferation and mucus secretion
천식의 한가지 특징인 염증성 자극에 대한 점액의 과다생산을 확인하기 위해, PAS 염색을 수행하여 배상세포 증식과 점액 분비를 검사하였으며, (도 10) 기관의 PAS 염색은 대조군과 비교하여 OVA+PM 그룹에서 배상세포 증식 및 점액분비에서 유의한 증가를 보여 심각한 기도 염증의 존재를 확인했다.In order to confirm the overproduction of mucus in response to inflammatory stimuli, which is a characteristic of asthma, PAS staining was performed to examine goblet cell proliferation and mucus secretion (Figure 10). PAS staining of organs was performed in the OVA+PM group compared to the control group. showed a significant increase in goblet cell proliferation and mucus secretion, confirming the presence of severe airway inflammation.
PM군 및 OVA 군은 대조군에 비해 PAS 양성 세포가 상승했지만, OVA+PM 군에서 가장 높은 PAS 양성 세포 비율이 관찰되었다. 그러나 푸코이단 처리는 OVA+PM 그룹에 비해 기관에서 PAS 양성 세포 비율을 저용량 9.1 %, 고용량 36.7 % 약화시켰다(p <0.005). The PM group and OVA group had an increase in PAS-positive cells compared to the control group, but the highest PAS-positive cell ratio was observed in the OVA+PM group. However, fucoidan treatment attenuated the proportion of PAS-positive cells in the trachea by 9.1% in the low dose and 36.7% in the high dose compared to the OVA+PM group (p <0.005).
한편, PM-challanged 및 OVA-민감한 마우스(OVA + PM군)는 대조군과 비교하여 폐에서 점액 및 잔 세포 증식의 분비를 378.0 % 증가시켰다(p <0.0005). PM은 OVA 단독과 비교하여 폐에서 PAS 양성 세포를 49.0 % 유도했다 ((p <0.005, 도10J, P). 그러나 푸코이단 치료는 폐에서 OVA + PM 그룹에 비해 PAS 양성 세포의 수가 저용량에서 35.2 %, 고용량에서 66.8 % 감소했다(p <0.005).Meanwhile, PM-challanged and OVA-sensitive mice (OVA + PM group) had 378.0% increased secretion of mucus and goblet cell proliferation in the lung compared to the control group (p <0.0005). PM induced 49.0% PAS-positive cells in the lung compared to OVA alone ((p < 0.005, Figure 10J,P). However, fucoidan treatment reduced the number of PAS-positive cells in the lung by 35.2% at the low dose compared to the OVA+PM group. , decreased by 66.8% at the high dose (p <0.005).
상기 실험을 통해 PM으로 인해 알러지성 기도 염증이 악화된 마우스에서 배상세포 증식과 점액 분비를 약화시키는 푸코이단의 역할을 확인하였다.Through the above experiment, the role of fucoidan in attenuating goblet cell proliferation and mucus secretion in mice with worsening allergic airway inflammation due to PM was confirmed.
3-11. 푸코이단이 마우스의 Th2 유래 싸이토카인 IL-4(interleukin-4)와 상피세포 유래 IL-33에 미치는 영향3-11. Effect of fucoidan on mouse Th2-derived cytokine IL-4 (interleukin-4) and epithelial cell-derived IL-33
OVA+PM군의 혈청(p < 0.005), 기관지 폐포 세척액(p < 0.05)과 폐(p < 0.05)의 IL-4 수치는 대조군에 비해 유의적으로 증가하였다. PM군은 OVA군 보다 IL-4 수치가 악화되었고, OVA+PM+푸코이단(400 mg/kg) 투여군은 OVA+PM군의 혈청(도12A, p < 0.005), 기관지 폐포 세척액(도 12D, p < 0.05)과 폐(도 12G, p < 0.05)보다 IL-4 값이 유의적으로 감소하였다. OVA+PM+푸코이단 (400 mg/kg)군은 프레드니손과 비슷한 효과를 보였다. 푸코이단은 OVA+PM군의 혈청 내 IL-33 값은 낮추지 못했으나 기관지 폐포 세척액과 폐의 IL-33 수치는 유의적으로 감소시켰다. 이러한 결과는 푸코이단이 미세먼지로 인한 기도의 염증 악화를 억제시키는 효과가 있다는 것을 시사한다.IL-4 levels in serum (p < 0.005), bronchoalveolar lavage fluid (p < 0.05), and lung (p < 0.05) in the OVA+PM group were significantly increased compared to the control group. The PM group had worse IL-4 levels than the OVA group, and the OVA+PM+fucoidan (400 mg/kg) administration group had lower serum (Figure 12A, p < 0.005) and bronchoalveolar lavage fluid (Figure 12D, p < 0.05) and lung (Figure 12G, p < 0.05). The OVA+PM+Fucoidan (400 mg/kg) group showed similar effects to prednisone. Fucoidan did not lower serum IL-33 levels in the OVA+PM group, but significantly reduced IL-33 levels in bronchoalveolar lavage fluid and lungs. These results suggest that fucoidan is effective in suppressing the worsening of airway inflammation caused by fine dust.
마지막으로 혈청, 기관지 폐포 세척액 (BALF) 및 폐에서 Th2 유래 사이토 카인 IL-4, Th17 유래 사이토 카인 IL-17a 및 상피 세포 유래 사이토 카인 IL-33을 평가했다. IL-4 수준은 혈청, BALF 및 폐(도 12B, 12E 및 12H)에서 대조군과 비교하여 OVA+PM에서 유의하게 증가했다.Finally, we assessed the Th2-derived cytokine IL-4, Th17-derived cytokine IL-17a, and epithelial cell-derived cytokine IL-33 in serum, bronchoalveolar lavage fluid (BALF), and lung. IL-4 levels were significantly increased in OVA+PM compared to controls in serum, BALF and lung (Figures 12B, 12E and 12H).
PM은 OVA 단독에 비해 IL-4 분비를 악화시켰고, 고용량 푸코이단 처리는 혈청, BALF 및 폐에서 OVA + PM에 비해 IL-4 수준을 약화시켰다 (모두 p <0.05). 고용량의 푸코이단은 PM으로 악화된 IL-4 수준을 긍정적으로 약화시키는 약물인 프레드니손만큼 효과적이었다.PM exacerbated IL-4 secretion compared to OVA alone, and high-dose fucoidan treatment attenuated IL-4 levels compared to OVA+PM in serum, BALF, and lung (all p < 0.05). High doses of fucoidan were as effective as prednisone, a drug that positively attenuates PM-exacerbated IL-4 levels.
IL-17a의 수준은 대조군과 비교하여 OVA+PM 그룹의 혈청, BALF 및 폐에서도 현저하게 증가했다(도 12F, I). 고용량 푸코이단 치료는 IL-17a의 수준을 약화시켰다. 더욱이 푸코이단은 혈청에서 상피 세포 유래 사이토카인 IL-33의 분비를 약화시키지 않았다.The levels of IL-17a were also significantly increased in serum, BALF, and lung of the OVA+PM group compared to the control group (Figure 12F,I). High-dose fucoidan treatment attenuated the levels of IL-17a. Moreover, fucoidan did not attenuate the secretion of the epithelial cell-derived cytokine IL-33 in serum.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.
Claims (10)
A pharmaceutical composition for preventing or treating allergic airway inflammatory diseases selected from the group consisting of asthma, pneumonia, and chronic obstructive pulmonary disease caused by fine dust, containing fucoidan derived from seaweed sporophylls.
상기 푸코이단은 미역 포자엽으로부터 추출한 것을 특징으로 하는 약학 조성물.
According to paragraph 1,
A pharmaceutical composition, characterized in that the fucoidan is extracted from seaweed sporophylls.
상기 푸코이단은
분쇄 단계 후 열수 추출하는 단계;
원심분리하여 침전물을 제거하는 단계;
한외여과기로 여과한 후 농축하는 단계;
주정을 사용하여 다당체를 침전시키는 단계; 및
주정을 분리한 후 건조시키는 단계
를 포함하는 제조방법으로 제조되는 것을 특징으로 하는 약학 조성물.
According to paragraph 1,
The fucoidan is
A step of hot water extraction after the pulverization step;
Centrifuging to remove sediment;
Concentrating after filtering with an ultrafilter;
Precipitating polysaccharides using alcohol; and
Step of separating and drying the alcohol
A pharmaceutical composition, characterized in that it is manufactured by a manufacturing method comprising.
상기 푸코이단은 2,000 내지 500,000Da 중량평균분자량(Mw)의 중저분자 푸코이단인 것을 특징으로 하는 약학 조성물.
According to paragraph 1,
A pharmaceutical composition, characterized in that the fucoidan is a low-to-medium molecule fucoidan with a weight average molecular weight (Mw) of 2,000 to 500,000 Da.
A food composition for improving allergic airway inflammatory diseases selected from the group consisting of asthma, pneumonia, and chronic obstructive pulmonary disease caused by fine dust, containing fucoidan derived from seaweed sporophylls.
상기 푸코이단은 미역 포자엽으로부터 추출한 것을 특징으로 하는 식품 조성물.
According to clause 6,
A food composition characterized in that the fucoidan is extracted from seaweed sporophylls.
상기 푸코이단은
분쇄 단계 후 열수 추출하는 단계;
원심분리하여 침전물을 제거하는 단계;
한외여과기로 여과한 후 농축하는 단계;
주정을 사용하여 다당체를 침전시키는 단계; 및
주정을 분리한 후 건조시키는 단계
를 포함하는 제조방법으로 제조되는 것을 특징으로 하는 식품 조성물.
According to clause 6,
The fucoidan is
A step of hot water extraction after the pulverization step;
Centrifuging to remove sediment;
Concentrating after filtering with an ultrafilter;
Precipitating polysaccharides using alcohol; and
Step of separating and drying the alcohol
A food composition, characterized in that it is manufactured by a manufacturing method comprising.
상기 푸코이단은 2,000 내지 500,000Da 중량평균분자량(Mw)의 푸코이단인 것을 특징으로 하는 식품 조성물.
According to clause 6,
A food composition, characterized in that the fucoidan is a fucoidan with a weight average molecular weight (Mw) of 2,000 to 500,000 Da.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210074251A KR102608023B1 (en) | 2021-06-08 | 2021-06-08 | Pharmaceutical composition for preventing or treating allergic airway inflammatory comprising fucoidan Isolated from the Sporophylls of Undaria pinnatifida |
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| KR1020210074251A KR102608023B1 (en) | 2021-06-08 | 2021-06-08 | Pharmaceutical composition for preventing or treating allergic airway inflammatory comprising fucoidan Isolated from the Sporophylls of Undaria pinnatifida |
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| KR1020210074251A KR102608023B1 (en) | 2021-06-08 | 2021-06-08 | Pharmaceutical composition for preventing or treating allergic airway inflammatory comprising fucoidan Isolated from the Sporophylls of Undaria pinnatifida |
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| KR101018304B1 (en) | 2008-02-26 | 2011-03-04 | (주)바이온 | Composition Containing Bioavailable Fucoidan for Preventing or Treating Inflammatory Diseases |
| KR101635268B1 (en) * | 2014-07-23 | 2016-06-30 | 제주대학교 산학협력단 | Composition for immune boosting having viability and function of spleen cells comprising Fucoidane |
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