KR102153338B1 - Pharmaceutical composition for preventing or treating an inflammatory disease, comprising an extract of Canarium subulatum as an active ingredient - Google Patents
Pharmaceutical composition for preventing or treating an inflammatory disease, comprising an extract of Canarium subulatum as an active ingredient Download PDFInfo
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- KR102153338B1 KR102153338B1 KR1020180145586A KR20180145586A KR102153338B1 KR 102153338 B1 KR102153338 B1 KR 102153338B1 KR 1020180145586 A KR1020180145586 A KR 1020180145586A KR 20180145586 A KR20180145586 A KR 20180145586A KR 102153338 B1 KR102153338 B1 KR 102153338B1
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- South Korea
- Prior art keywords
- extract
- canarium
- pharmaceutical composition
- inflammatory diseases
- western
- Prior art date
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Abstract
본 발명은 항염증 활성을 갖는 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방, 개선 또는 치료용 조성물에 관한 것으로, 카나리움 서부라텀의 메탄올 추출물은 염증반응으로 생성되는 사이토카인과 그에 따른 반응물의 생성을 효과적으로 감소시킬 수 있으며 또한, 면역 활성에 관여하는 신호전달 단백질의 발현을 억제 시킬 수 있으며, 나아가 상기 추출물의 염증억제 효과는 위염을 유발한 동물실험에서도 확인되었는바 의약품, 의약부외품 소재 개발 및 관련 산업에 유용하게 이용될 수 있을 것으로 기대된다.The present invention relates to a composition for preventing, improving or treating inflammatory diseases, comprising an extract of Canarium subulatum having anti-inflammatory activity as an active ingredient, wherein the methanol extract of Canarium subulatum is produced by an inflammatory reaction. It can effectively reduce the generation of cytokines and their reactants, and can inhibit the expression of signaling proteins involved in immune activity. Furthermore, the anti-inflammatory effect of the extract was also confirmed in animal experiments that caused gastritis. It is expected to be useful in the development of pharmaceuticals and quasi-drug materials and related industries.
Description
본 발명은 항염증 활성을 갖는 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating inflammatory diseases, comprising an extract of Canarium subulatum having anti-inflammatory activity as an active ingredient.
염증 반응(inflammation)은 조직(세포)의 손상이나 외부감염원(박테리아, 곰팡이, 바이러스, 다양한 종류의 알레르기 유발물질)에 감염되었을 때, 각종 염증 매개인자 및 면역세포가 관련되어 효소의 활성화, 염증매개물질 분비, 체액 침윤, 세포 이동, 조직 파괴 등의 복합적인 생리적 반응과 홍반, 부종, 발열, 통증 등의 외적 증상을 나타낸다. 정상인 경우의 염증반응은 외부감염원을 제거하고 손상된 조직을 재생하여 생명체 기능 회복 작용을 하지만, 항원이 제거되지 않거나 내부물질이 원인이 되어 염증반응이 과도하거나 지속적으로 일어나는 경우 에는 오히려 점막손상을 촉진하고, 일부에서는 류마티스 관절염, 골다공증, 패혈증, 혈관 질환, 암 등을 유도한다. 즉, 염증 반응은 상처, 미생물 감염 등에 대항하는 숙주의 방어기제에 따른 병리학적인 기작 중 가장 중요한 반응이지만, 지속적이고 과도한 염증반응은 조직을 손상시킨다.Inflammation is when tissue (cells) is damaged or infected by external infectious agents (bacteria, fungi, viruses, various types of allergens), various inflammatory mediators and immune cells are involved, resulting in the activation of enzymes and mediating inflammation. It exhibits complex physiological reactions such as substance secretion, body fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, swelling, fever, and pain. In normal cases, the inflammatory reaction removes external infectious agents and regenerates damaged tissues to restore the function of living organisms. However, if the inflammatory reaction is excessive or persistent due to the absence of antigens or internal substances, it promotes mucosal damage. , In some cases, it induces rheumatoid arthritis, osteoporosis, sepsis, vascular disease, and cancer. In other words, the inflammatory reaction is the most important reaction among pathological mechanisms according to the host's defense mechanism against wounds and microbial infections, but persistent and excessive inflammatory reactions damage tissues.
대식세포(macrophage)는 이러한 염증 반응을 조절하는 가장 대표적인 면역세포로서, 유해한 환경에서 숙주의 방어기작에 중요한 역할을 하며, 자가면역질환과 같은 다양한 질병의 진행과정에 관여한다. 지질다당류와 같은 염증 자극인자들은 대식세포를 활성화시킬 수 있으며, 활성화된 대식세포는 종양괴사인자-알파 (tumor necrosis factor-α, TNF-α), 인터루킨-6 (interleukin-6, IL-6) 및 IL-1β와 같은 다양한 염증성 매개체를 생성하고, 유도형 NO생성효소 (inducible nitric oxide synthase, iNOS)와 고리형 산소화효소2 (cyclooxygenase-2, COX-2)를 합성하여 니트릭 옥사이드(nitric oxide, NO) 및 PGE 2 (prostaglandin E 2 )를 생성한다.Macrophages are the most representative immune cells that regulate this inflammatory response, play an important role in the host's defense mechanism in a harmful environment, and are involved in the process of various diseases such as autoimmune diseases. Inflammation stimulating factors such as lipopolysaccharides can activate macrophages, and activated macrophages are tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) And IL-1β, and by synthesizing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nitric oxide , NO) and PGE 2 (prostaglandin E 2 ).
한편, Lipopolysaccharide(LPS)는 그람 음성균의 외막 구성성분이며 염증성 인자로 알려져 있으며, 골수에서 생산되는 대식세포(macrophage)는 미량의 LPS에 의해 활성화되고 다양한 사이토카인(cytokine), 과산화 수소(hydrogen peroxide, H2O2), 및 니트릭 옥사이드 등의 각종 세포독성물질을 분비하여 면역기구로서 기능한다. On the other hand, Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and is known as an inflammatory factor, and macrophages produced in the bone marrow are activated by a trace amount of LPS, and various cytokines, hydrogen peroxide, and H 2 O 2 ) secretes various cytotoxic substances such as nitric oxide and functions as an immune system.
최근 질병을 예방 또는 치료할 수 있는 기능은 식품이나 식물체도 가지고 있다는 것이 보고되고 있고, 보다 건강하고 오래 살고자 하는 인류의 필요에 따라, 세계적으로 다양한 자원으로부터 다양한 생리기능을 가진 물질을 탐색하는 연구가 활발히 진행되고 있으며, 그 중에서도 특히 식물자원에 포함된 화합물에 많은 관심이 집중되고 있다.Recently, it has been reported that foods and plants have functions that can prevent or treat diseases, and research to search for substances with various physiological functions from various resources around the world has been conducted according to the needs of humans to live healthier and longer. It is actively progressing, and among them, a lot of attention is being focused on compounds contained in plant resources.
본 발명자들은 카나리움 서부라텀(Canarium subulatum) 추출물의 항염증 효능을 밝히기 위해, 대식세포와 위염 마우스 모델을 이용하여 염증반응에 관여하는 사이토카인과 신호전달 단백질의 활성의 변화를 분석한 결과 표적 단백질을 실험적으로 확인하였는바, 이에 기초하여 본 발명을 완성하였다.The present inventors analyzed changes in the activity of cytokines and signaling proteins involved in the inflammatory response using a macrophage and gastritis mouse model to reveal the anti-inflammatory efficacy of the extract of Canarium subulatum. Was confirmed experimentally, the present invention was completed based on this.
이에, 본 발명의 목적은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방, 개선 또는 치료용 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases, comprising the extract of Canarium subulatum as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.
또한, 본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.
본 발명의 일 구현예로, 상기 염증성 질환은 위염일 수 있다.In one embodiment of the present invention, the inflammatory disease may be gastritis.
본 발명의 다른 구현예로, 상기 추출물은 메탄올을 용매로 추출한 것일 수 있다.In another embodiment of the present invention, the extract may be obtained by extracting methanol with a solvent.
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 퀘르세틴(Quercetin), 루테올린(luteolin), 및 캠페롤 (Kaempferol)로 구성된 군에서 선택되는 1개 이상의 성분을 포함할 수 있다.In another embodiment of the present invention, the canarium western latum extract may include one or more components selected from the group consisting of quercetin, luteolin, and campferol.
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 일산화 질소 (nitric oxide, NO) 생성을 억제할 수 있다.In another embodiment of the present invention, the canarium western ratom extract may inhibit the production of nitric oxide (NO).
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 유도형 NO 생성 효소 (Inducible nitric oxide synthase, iNOS) 발현을 억제할 수 있다.In another embodiment of the present invention, the canarium western ratum extract may inhibit the expression of an inducible nitric oxide synthase (iNOS).
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제할 수 있다.In another embodiment of the present invention, the canarium western ratum extract may inhibit phosphorylation of IκBα, IKKα/β, Syk, p85 or Src.
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제함으로써, 뉴클리어 팩터 κB(nuclear factor κB, NF-κB)의 활성을 억제할 수 있다.In another embodiment of the present invention, the canarium western ratum extract inhibits the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, thereby inhibiting the activity of Nuclear Factor κB (NF-κB) can do.
본 발명의 또 다른 구현예로, 상기 카나리움 서부라텀 추출물은 p50 및 p65의 핵 전이(nuclear translocation)를 감소시킬 수 있다.In another embodiment of the present invention, the canarium western ratum extract may reduce nuclear translocation of p50 and p65.
또한, 본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 염증성 질환의 예방 또는 치료방법을 제공한다.In addition, the present invention provides a method for preventing or treating inflammatory diseases, comprising administering to an individual a pharmaceutical composition comprising an extract of Canarium subulatum as an active ingredient.
또한, 본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는 약학적 조성물의, 염증성 질환의 예방 또는 치료용도를 제공한다.In addition, the present invention provides a pharmaceutical composition comprising an extract of Canarium subulatum as an active ingredient, for preventing or treating inflammatory diseases.
카나리움 서부라텀의 추출물은 염증반응으로 생성되는 사이토카인과 그에 따른 반응물의 생성을 효과적으로 감소시킬 수 있으며 면역 활성에 관여하는 신호전달 단백질의 발현을 억제 시킬 수 있고, 나아가 카나리움 서부라텀의 메탄올 추출물의 염증억제 효과는 위염을 유발한 동물실험에서도 확인되었는바 의약품, 의약부외품 소재 개발 및 관련 산업에 유용하게 이용될 수 있을 것으로 기대된다.The extract of canarium western ratum can effectively reduce the generation of cytokines and reactants produced by the inflammatory reaction and can inhibit the expression of signaling proteins involved in immune activity. The anti-inflammatory effect of is expected to be useful in the development of pharmaceuticals and quasi-drug materials and related industries as it was confirmed in animal experiments that caused gastritis.
도 1은 카나리움 서부라텀의 메탄올 추출물(이하 Cs-ME)이 세포에서 염증 유발 물질인 리포폴리사카라이드(lipopolysaccharide, 이하 LPS)에 의해 유발된 니트릭 옥사이드 생성을 억제하는 효과를 나타낸 것이다.
도 2는 Cs-ME의 복막의 마크로파지(Peritoneal macrophages)에서 LPS에 의한 니트릭 옥사이드 생성 억제 효과를 나타낸 것이다.
도 3은 Cs-ME가 RAW 264.7 세포 및 복막의 마크로파지에 대하여 세포 독성이 없음을 나타낸 것이다.
도 4는 액체 크로마토그래피를 통한 Cs-ME의 주요 성분 분석결과를 나타낸 것이다.
도 5는 Cs-ME가 LPS에 의한 유도형 NO 생성 효소(Inducible nitric oxide synthase, iNOS) mRNA 발현 감소 효과가 있음을 나타낸 것이다.
도 6은 Cs-ME를 RAW 264.7 세포에에 처리한 경우 LPS에 의한 단백질 p50와 p65의 핵 내 이동 양상의 감소효과를 나타낸 것이다.
도 7은 Cs-ME에 의한 세포 내 NF-κB 신호전달 단백질인 IKKα/β 및 IκBα 의 인산화 감소효과를 나타낸 것이다.
도 8은 초기 시간에서 Cs-ME에 의한 세포 내 NF-κB 신호전달 단백질인 Src, 및 Syk의 인산화 감소 효과를 나타낸 것이다.
도 9는 Src 과발현을 통한 염증 발생시 Cs-ME 처리에 의한 신호전달 단백질인 Src 및 p85의 인산화 감소 효능을 나타낸 것이다.
도 10은 Syk 과발현을 통한 염증 발생시 Cs-ME 처리에 의한 신호전달 단백질인 Syk의 인산화 감소 효능을 나타낸 것이다.
도 11은 급성 위염 동물 모델에서 Cs-ME의 위 염증 감소 효능을 평가한 결과이다.
도 12는 Cs-ME, 퀘르세틴(Quercetin), 및 캠페롤 (Kaempferol)의 RAW264.7 세포에서 니트릭 옥사이드(NO) 생성능 억제 효과를 비교한 결과이다.
도 13은 염증 신호 전달 체계에서 Cs-ME의 표적 단백질인 Src/Syk을 통한 염증 억제 효능을 설명한 것이다.1 shows the effect of suppressing the production of nitric oxide caused by lipopolysaccharide (LPS), which is an inflammation-inducing substance in cells, of a methanol extract (hereinafter Cs-ME) of canarium western ratum.
Figure 2 shows the effect of inhibiting the production of nitric oxide by LPS in the peritoneal macrophages of Cs-ME.
Figure 3 shows that Cs-ME has no cytotoxicity to RAW 264.7 cells and macrophages of the peritoneum.
4 shows the results of analysis of major components of Cs-ME through liquid chromatography.
Figure 5 shows that Cs-ME has the effect of reducing the expression of inducible nitric oxide synthase (iNOS) mRNA by LPS.
6 shows the effect of reducing the intranuclear migration of proteins p50 and p65 by LPS when Cs-ME is treated on RAW 264.7 cells.
7 shows the effect of reducing phosphorylation of IKKα/β and IκBα, which are intracellular NF-κB signaling proteins by Cs-ME.
8 shows the effect of reducing phosphorylation of Src and Syk, an intracellular NF-κB signaling protein, by Cs-ME at an initial time.
9 shows the effect of reducing phosphorylation of Src and p85 signaling proteins by Cs-ME treatment when inflammation occurs through Src overexpression.
FIG. 10 shows the effect of reducing phosphorylation of Syk, a signaling protein, by Cs-ME treatment when inflammation occurs through Syk overexpression.
11 is a result of evaluating the effect of Cs-ME on reducing gastric inflammation in an acute gastritis animal model.
12 is a result of comparison of the inhibitory effect of Cs-ME, quercetin, and campferol on the ability to produce nitric oxide (NO) in RAW264.7 cells.
13 illustrates the efficacy of suppressing inflammation through Src/Syk, a target protein of Cs-ME, in the inflammatory signaling system.
본 발명자들은 카나리움 서부라텀(Canarium subulatum) 추출물의 항염증 효능을 밝히기 위해, 대식세포와 위염 마우스 모델을 이용하여 염증반응에 관여하는 사이토카인과 신호전달 단백질의 활성의 변화를 분석한 결과 표적 단백질을 실험적으로 확인하였는바, 이에 기초하여 본 발명을 완성하였다.The present inventors analyzed changes in the activity of cytokines and signaling proteins involved in the inflammatory response using a macrophage and gastritis mouse model to reveal the anti-inflammatory efficacy of the extract of Canarium subulatum. Was confirmed experimentally, the present invention was completed based on this.
본 발명의 일 실시예에서는, LPS로 염증 유발된 세포에 Cs-ME를 처리한 경우 용량의존적으로 니트릭 옥사이드 생성이 억제됨을 확인하였다(실시예 2 참조).In one embodiment of the present invention, it was confirmed that when Cs-ME was treated with LPS-induced cells, nitric oxide production was inhibited in a dose-dependent manner (see Example 2).
본 발명의 다른 실시예에서는, RAW 264.7 세포 및 대식세포주에 Cs-ME를 처리한 결과 세포독성이 나타나지 않음을 확인하였다(실시예 3 참조).In another example of the present invention, it was confirmed that the RAW 264.7 cells and macrophages were treated with Cs-ME and no cytotoxicity appeared (see Example 3).
본 발명의 또 다른 실시예에서는, 액체 크로마토그래피를 이용하여 Cs-ME의 주요 성분으로 퀘르세틴(Quercetin), 루테올린(luteolin), 및 캠페롤 (Kaempferol)이 있음을 확인하였다(실시예 4 참조).In another embodiment of the present invention, it was confirmed that quercetin, luteolin, and campferol were present as main components of Cs-ME using liquid chromatography (see Example 4). .
본 발명의 또 다른 실시예에서는, Cs-ME가 LPS에 의한 염증관련 사이토카인인 유도형 NO 생성 효소 (Inducible nitric oxide synthase, iNOS)의 mRNA 발현을 줄이는 효과가 있음을 확인하였다(실시예 5 참조).In another embodiment of the present invention, it was confirmed that Cs-ME has the effect of reducing the mRNA expression of the inducible nitric oxide synthase (iNOS), an inflammation-related cytokine caused by LPS (see Example 5). ).
본 발명의 또 다른 실시예에서는, Cs-ME를 RAW 264.7 세포에 처리하였을 때 LPS에 의한 단백질 p50와 p65의 핵 내 이동 양상의 감소효과가 나타남을 확인하였다(실시예 6 참조).In another example of the present invention, when Cs-ME was treated with RAW 264.7 cells, it was confirmed that the effect of reducing the intranuclear migration of proteins p50 and p65 by LPS was exhibited (see Example 6).
본 발명의 또 다른 실시예에서는, Cs-ME을 처리한 세포 내에서 NF-κB 신호전달 단백질인 IKKα/β, IκBα, Src, Syk 또는 p58의 인산화가 감소함을 확인하였다(실시예 7 내지 10 참조).In another embodiment of the present invention, it was confirmed that phosphorylation of NF-κB signaling proteins IKKα/β, IκBα, Src, Syk or p58 decreased in cells treated with Cs-ME (Examples 7 to 10 Reference).
본 발명의 또 다른 실시예에서는, Cs-ME의 위염 완화효과는 농도의존적으로 증가하고 특히, 100mg/kg Cs-ME 처리군의 경우, 라니티딘(Ranitidine)처리군보다 더 높은 위염 완화효과를 보임을 확인하였다(실시예 11 참조).In another embodiment of the present invention, the gastritis alleviation effect of Cs-ME increases in a concentration-dependent manner, and in particular, the 100mg/kg Cs-ME treatment group shows a higher gastritis alleviation effect than the ranitidine treatment group. Confirmed (see Example 11).
본 발명의 또 다른 실시예에서는, 세포에 Cs-ME를 처리한 경우 캠퍼롤 및 퀘르세틴 각각을 처리한 경우보다 니트릭 옥사이드 생성억제 효과가 더 큰 것을 확인하였다(실시예 12 참조).In another embodiment of the present invention, it was confirmed that when the cells were treated with Cs-ME, the inhibitory effect of nitric oxide production was greater than that of each treatment with camphorol and quercetin (see Example 12).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.
본 발명에서 사용되는 용어, “예방”이란 본 발명에 따른 약학적 조성물의 투여에 의해 염증성 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다. The term "prevention" used in the present invention means any action that suppresses or delays the onset of inflammatory diseases by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 약학적 조성물의 투여에 의해 염증성 질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "treatment" used in the present invention means any action in which symptoms caused by an inflammatory disease are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어 "염증성 질환"이란 염증을 주병변으로 하는 질병으로, 염증성 질환의 비제한적인 예로는 알레르기성 질환, 염증성 장 질환, 죽상동맥경화, 염증성 콜라겐 혈관 질환, 사구체신염, 염증성 피부 질환, 유육종증, 망막염, 위염, 간염, 장염, 복막염, 관절염, 편도선염, 인후염, 기관지염, 폐렴, 췌장염, 패혈증, 및 신장염 등이 있으며, 바람직하게는 위염 일 수 있으나 이에 제한되는 것은 아니다. The term "inflammatory disease" used in the present invention is a disease whose main lesion is inflammation, and non-limiting examples of inflammatory diseases include allergic disease, inflammatory bowel disease, atherosclerosis, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin Disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, peritonitis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, and nephritis, and the like, preferably gastritis, but is not limited thereto.
본 발명에서 상기 카나리움 서부라텀 추출물 획득에 이용되는 추출 용매의 종류는 특별히 제한되지 아니하며, 당해 기술분야에서 공지된 임의의 용매를 사용할 수 있다. 상기 추출 용매의 비제한적인 예로는 물; 메탄올, 에탄올, 프로필알코올, 부틸알코올 등의 C1내지 C4의 저급 알코올; 글리세린, 부틸렌글라이콜, 프로필렌글라이콜 등의 다가 알코올; 및 메틸아세테이트, 에틸아세테이트, 아세톤, 벤젠, 헥산, 디에틸에테르, 디클로로메탄 등의 탄화수소계 용매; 또는 이들의 혼합물을 사용할 수 있으며, 바람직하게 메탄올을 단독으로 사용하거나 2종 이상 혼합하여 사용할 수 있으며, 더욱 바람직하게는 99.9% 메탄올을 사용할 수 있으나 이에 제한되는 것은 아니다.In the present invention, the kind of the extraction solvent used to obtain the canarium western ratum extract is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C 1 to C 4 lower alcohols such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; And hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Alternatively, a mixture thereof may be used, and methanol may be used alone or in combination of two or more, and more preferably 99.9% methanol may be used, but the present invention is not limited thereto.
또한, 본 발명의 카나리움 서부라텀 추출물은 추출 및/또는 분획 과정을 수행한 이후, 감압 여과 과정을 수행하거나 추가로 농축 및/또는 동결건조를 수행하여 농축하거나 용매를 제거할 수 있으며, 상기 수득한 카나리움 서부라텀 추출물은 사용 시까지 급속 냉동 냉장고에 보관할 수 있다.In addition, after performing the extraction and / or fractionation process of the canarium western ratum extract of the present invention, it can be concentrated or removed by performing a vacuum filtration process or further concentration and/or freeze-drying, and the obtained One canarium western ratum extract can be stored in a quick freezer until use.
대식세포(macrophage)는 염증 반응을 조절하는 가장 대표적인 면역세포로서, 유해한 환경에서 숙주의 방어기작에 중요한 역할을 하며, 자가면역질환과 같은 다양한 질병의 진행과정에 관여한다. 지질다당류(LPS)와 같은 염증 자극 인자들은 대식세포를 활성화시킬 수 있으며, 활성화된 대식세포는 종양괴사인자-알파 (tumor necrosis factor-α, TNF-α), 인터루킨-6 (interleukin-6, IL-6) 및 IL-1β와 같은 다양한 염증성 매개체를 생성하고, 유도형 NO생성효소 (inducible nitric oxide synthase, iNOS)와 고리형 산소화효소2 (cyclooxygenase-2, COX-2)를 합성하여 일산화 질소(nitric oxide, NO) 및 PGE 2 (prostaglandin E 2 )를 생성한다.Macrophages are the most representative immune cells that regulate the inflammatory response, play an important role in the host's defense mechanism in a harmful environment, and are involved in the progression of various diseases such as autoimmune diseases. Inflammatory stimulating factors such as lipopolysaccharide (LPS) can activate macrophages, and activated macrophages are tumor necrosis factor-α (TNF-α), interleukin-6 (IL). -6) and IL-1β, and by synthesizing inducible nitric oxide synthase (iNOS) and cyclic oxygenase 2 (cyclooxygenase-2, COX-2) to produce nitrogen monoxide ( nitric oxide, NO) and PGE 2 (prostaglandin E 2) are produced.
한편, nuclear factor κB (NF-κB)는 어디에나 존재하고 복합체 현상의 조절을 담당하는 것에 특화된 단백질이다. 어떤 조건에서 신체의 세포신호를 통제하는 중심적 역할을 하며, 특히 다른 기능 중에서도 NF-κB는 염증의 진행을 통제하는데 중요한 역할을 하는 상기 TNF-α 등의 염증 사이토카인 (pro-inflammatory cytokines), 케모카인 (chemokines), 및 COX-2 등의 유발 효소 (inducible enzymes)를 통제하는 것으로 알려져 있다. 따라서 NFκB는 많은 염증 질환을 치료하는 약에 있어서 중요하면서도 매우 눈에 띄는 치료 타겟을 대표한다고 할 수 있다.On the other hand, nuclear factor κB (NF-κB) is a protein that exists everywhere and is specialized in controlling the complex phenomenon. In certain conditions, NF-κB plays a central role in controlling the cellular signals of the body, and among other functions, NF-κB is pro-inflammatory cytokines such as TNF-α and chemokines, which play an important role in controlling the progression of inflammation. (chemokines), and inducible enzymes such as COX-2 are known to control. Therefore, it can be said that NFκB represents an important and very prominent therapeutic target in drugs for treating many inflammatory diseases.
본 발명의 상기 카나리움 서부라텀 추출물은 퀘르세틴(Quercetin), 루테올린(luteolin), 및 캠페롤 (Kaempferol)로 구성된 군에서 선택되는 1개 이상의 성분을 포함할 수 있다.The canarium western ratum extract of the present invention may contain one or more components selected from the group consisting of quercetin, luteolin, and campferol.
본 발명의 상기 카나리움 서부라텀 추출물은 일산화 질소 (nitric oxide, NO) 생성을 억제할 수 있다.The canarium western ratum extract of the present invention can inhibit the production of nitrogen monoxide (NO).
본 발명의 상기 카나리움 서부라텀 추출물은 유도형 NO 생성 효소 (Inducible nitric oxide synthase, iNOS) 발현을 억제할 수 있다.The canarium western ratum extract of the present invention can inhibit the expression of inducible nitric oxide synthase (iNOS).
본 발명의 상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제할 수 있다.The canarium western ratum extract of the present invention can inhibit the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src.
본 발명의 상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제함으로써, 뉴클리어 팩터 κB(nuclear factor κB, NF-κB)의 활성을 억제할 수 있다.By inhibiting the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, the canarium western ratum extract of the present invention can inhibit the activity of nuclear factor κB (NF-κB).
본 발명의 카나리움 서부라텀 추출물은 p50 및 p65의 핵 전이(nuclear translocation)를 감소시킬 수 있다.Canarium western ratum extract of the present invention can reduce the nuclear translocation of p50 and p65 (nuclear translocation).
본 발명에 따른 카나리움 서부라텀 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 외용제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 바람직하게는 크림, 젤, 패취, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제의 제형을 가질 수 있다. 상기 카나리움 서부라텀 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 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, 현탁제로는 프로필렌글리콜 (propylene glycol), 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, 글리세로제라틴 등이 사용될 수 있다.Pharmaceutical compositions for the prevention or treatment of inflammatory diseases comprising the extract of Canarium western ratum according to the present invention as an active ingredient, respectively, are powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated and used in the form of external preparations and sterile injectable solutions, and preferably has a formulation of cream, gel, patch, spray, ointment, warning agent, lotion, linenment, pasta or cataplasma. have. Carriers, excipients, and diluents that may be included in the composition containing the canarium western ratum extract include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract such as starch, calcium carbonate, sucrose. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
본 발명의 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
본 발명의 다른 양태로서, 본 발명은 카나리움 서부라텀(Canarium subulatum) 추출물을 유효성분으로 포함하는, 염증성 질환 예방 또는 개선용 식품 조성물을 제공한다. As another aspect of the present invention, the present invention provides a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient.
상기 식품 조성물은 건강기능성 식품 조성물을 포함한다.The food composition includes a health functional food composition.
본 발명에서 사용되는 용어, “개선”이란, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.
본 발명의 건강기능성식품 조성물에서 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.In the health functional food composition of the present invention, the active ingredient may be added to food as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, in the manufacture of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.
본 발명의 건강기능성식품 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The health functional food composition of the present invention is not particularly limited to other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients, such as a normal beverage. have. Examples of the natural carbohydrates described above include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the choice of a person skilled in the art.
상기 외에 본 발명의 건강기능성식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다.In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives can also be appropriately selected by a person skilled in the art.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[[ 실시예Example ]]
실시예Example 1. 실험방법 1. Experiment method
실시예Example 1-1 1-1 카나리움Canarium 서부라텀Western Latum (( CanariumCanarium subulatumsubulatum ) 추출물의 제조) Preparation of extract
카나리움 서부라텀(Canarium subulatum) 메탄올 추출물(Cs-ME)을 제조하기 위해, 카나리움 서부라텀(Canarium subulatum)의 지상부(aerial parts)를 건조시키고, 분쇄하여 미세분말 샘플을 만든 후, 상기 샘플 (125g)을 초음파 분쇄기(sonicator)로 99.9% 메탄올(1,000 ml)을 이용하여 추출물을 추출하였다. 이후, 감압하에서 증발 건조시켜 최종 카나리움 서부라텀(Canarium subulatum) 메탄올 추출물을 얻었다.To prepare a Canarium subulatum methanol extract (Cs-ME), the aerial parts of Canarium subulatum were dried and pulverized to make a fine powder sample, and then the sample ( 125g) was extracted using 99.9% methanol (1,000 ml) with an ultrasonic grinder (sonicator). Thereafter, it was evaporated to dryness under reduced pressure to obtain a final methanol extract of Canarium subulatum.
실시예Example 1-2. 세포배양법 1-2. Cell culture method
뮤린(Murine) 대식세포주인 RAW264.7 세포를 페니실린(penicillin) 100 IU/ml 및 스트렙토마이신(streptomycin) 100 μg/ml과 10%의 소태아혈청(Fetal Bovine Serum, FBS)을 함유하는 RPMI 1640 배지를 이용하여 100 mm 셀컬쳐디쉬(cell culture dish)에 70-80%의 밀도로 배양하였다. 또한, 인간 세포주인 HEK293 세포는 페니실린 100 IU/ml 및 스트렙토마이신 100 μg/ml과 10%의 소태아혈청을 함유하는 DMEM 배지를 이용하여 100 mm 셀컬쳐디쉬에 70-80%의 밀도로 배양하였다.Murine macrophage cell line RAW264.7 cells in RPMI 1640 medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% Fetal Bovine Serum (FBS) Using a 100 mm cell culture dish (cell culture dish) was cultured at a density of 70-80%. In addition, the human cell line HEK293 cells were cultured in a 100 mm cell culture dish at a density of 70-80% using DMEM medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% fetal bovine serum. .
실시예Example 1-3. 1-3. 웨스턴Western 블로팅Blotting (Western blotting)(Western blotting)
뮤린 대식 세포주인 RAW264.7 세포를 페니실린 100 IU/ml 및 스트렙토마이신 100 μg/ml과 10%의 소태아혈청을 함유하는 RPMI 1640 배지를 이용해서 배양한 세포를 7X106 cell/ml 농도로 60mm의 디쉬에서 전 배양시켰다. 그 다음, 각 분획물들을 처리하고 일정시간 후 자극제(stimuli, Lipopolysaccharide)로 자극하고 약물에 따른 일정시간 후 세포 들을 모아서 라이시스 버퍼(lysis buffer)를 사용해 세포를 깨어 웨스턴(western) 표본을 얻어냈다. 그리고 각 표본의 단백질 농도를 소혈청알부민(Bovine Serum Albumin, BSA)을 표준으로 잡고 측정하였다. 60mm of the murine macrophage cell line, RAW264.7 cells the
상기와 같이 얻어진 값을 기준으로 단백질 농도가 되는 각 표본량을 가지고 SDS-PAGE(Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis)를 실행하고, 블로팅(wet blotting) 방법을 사용해 PVDF 멤브레인(membrane)으로 단백질을 블로팅시킨 후 맴브레인을 3% 소혈청알부민을 사용해 블로킹(blocking)시키고, 표적 단백질 항체 및 신호전달 단백질 항체 (p50, p65, Lamin A/C, p-IκBα, IκBα, p-IKKα/β, IKKα/β, p-Src, Src, p-Syk, Syk, p-p85, β-actin) 용액을 사용해 1차 처리하고, 다시 워싱(washing) 단계 후 2차 항체 용액을 처리하고 워싱하였다. 그리고 암실에서 맴브레인에 ECL(enhanced chemiluminescence) 용액을 골고루 분주하여 엑스레이필름(X-ray film)으로 감광하였다.Based on the value obtained as above, perform SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) with each sample amount that becomes the protein concentration, and use a PVDF membrane using a wet blotting method. After blotting the protein, the membrane was blocked with 3% bovine serum albumin, and the target protein antibody and signaling protein antibody (p50, p65, Lamin A/C, p-IκBα, IκBα, p-IKKα/β) , IKKα/β, p-Src, Src, p-Syk, Syk, p-p85, β-actin) solution was used for primary treatment, and the secondary antibody solution was treated and washed again after washing step. In addition, an enhanced chemiluminescence (ECL) solution was evenly dispensed on the membrane in a dark room, and photosensitized with an X-ray film.
실시예Example 2. 대식세포 유래 세포독성 물질인 2. Macrophage-derived cytotoxic substance 니트릭Nitric 옥사이드Oxide (NO) (NO) 생성능Generating ability 측정 Measure
마우스 대식세포주인 RAW264.7를 페니실린 100 IU/ml 및 스트렙토마이신 (100 μg/ml)과 10%의 소태아혈청을 함유하는 RPMI 1640 배지를 이용해서 1×106 cell/ml의 농도로 조절한 후, 96 웰 플레이트에 접종하고, 5% CO2 및 37℃ 조건에서 18시간 동안 전배양하였다. The mouse macrophage cell line RAW264.7 was adjusted to a concentration of 1×10 6 cells/ml using RPMI 1640 medium containing 100 IU/ml of penicillin and streptomycin (100 μg/ml) and 10% fetal bovine serum. Then, it was inoculated into a 96-well plate, and pre-incubated for 18 hours at 5% CO 2 and 37°C.
그 다음, 배지를 제거하고 4배 농도로 조제된 Cs-ME 50 μl와 50 μl의 LPS (최종 농도 1 μg/ml) 함유 배지를 동시에 처리하여 배양하였다. 24시간 후 상층액을 100μl씩 또 다른 96 웰 플레이트에 옮기고 NO 정량은 Griess 용액 (0.5% 나프틸에틸렌디아민 디하이드로클로라이드(naphthylethylenediamine dihydrochloride), 5% 설파닐아마이드(sulfanilamide), 25% H3PO4)을 이용하여 540 nm에서 흡광도를 측정하여 실시하였으며 표준물질로 소듐 니트레이트(sodium nitrite, 0 에서 100 μM)를 사용하여 검량선을 작성하였다.Then, the medium was removed, and 50 μl of Cs-ME prepared at a 4-fold concentration and a medium containing 50 μl of LPS (
그 결과, 도 1, 및 도 2에 나타난 바와 같이 LPS만 투여한 경우 니트릭 옥사이드의 생성이 급격히 증가했으나, Cs-ME를 함께 투여한 경우 용량의존적으로 니트릭 옥사이드의 생성이 억제됨을 확인하였다.As a result, as shown in FIGS. 1 and 2, when only LPS was administered, the production of nitric oxide increased rapidly, but when Cs-ME was administered together, it was confirmed that the production of nitric oxide was suppressed in a dose-dependent manner.
실시예Example 3. Cs-ME 처리한 3. Cs-ME treated 대식세포주에서In macrophage line 세포 cell 생존률Survival rate (cell viability)(cell viability)
MTT(3-[4,5-디메틸티아졸-2-일]-2,5-디피닐테트라졸리움 브로마이드(3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide)) 어세이(assay)법을 이용하여 분석하였다. 96-웰 플레이트에 1 × 106 cell/ml의 세포를 플레이팅하고 37℃에서 각 면역실험 조건에 상응하는 배양시간 동안 CO2 인큐베이터(incubator)에서 배양하였다. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-difinyltetrazolium bromide (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide)) Analysis was performed using an assay method. Cells of 1 × 10 6 cells/ml were plated on a 96-well plate and cultured in a CO 2 incubator at 37° C. for an incubation time corresponding to each immunization test condition.
그 다음, 10 ㎕ MTT 용액 (스톡(stock)농도 : 5 mg/ml)을 첨가하고 3시간 동안 추가반응을 유도하였다. 반응 종료 및 포르마잔 크리스탈(formazan crystal) 용해를 위해 각 웰에 100 ㎕ MTT 스토핑 솔루션(stopping solution, 10% 소듐 도데실 설페이트(Sodium dodecyl sulfate) in 0.01M HCl)을 추가적으로 첨가하였다. 세포 생존율은 MTT가 포르마잔으로 환원된 양을 570 nm에서 흡광도를 측정하여 얻어진 OD 값을 통해 산출하였다.Then, 10 µl MTT solution (stock concentration: 5 mg/ml) was added and further reaction was induced for 3 hours. To complete the reaction and dissolve the formazan crystal, 100 μl MTT stopping solution (10% sodium dodecyl sulfate in 0.01M HCl) was additionally added to each well. Cell viability was calculated through the OD value obtained by measuring the absorbance at 570 nm of the amount of MTT reduced to formazan.
그 결과, 도 3에 나타난 바와 같이 RAW 264.7 세포 및 복막의 마크로파지에 Cs-ME를 처리하여도 모두 세포 생존력이 높게 나타나고 있어 세포 독성효과가 유의하게 나타나지 않음을 확인 하였다.As a result, as shown in FIG. 3, even if Cs-ME was treated on RAW 264.7 cells and peritoneal macrophages, it was confirmed that the cell viability was high, so that the cytotoxic effect was not significant.
실시예Example 4. 액체 4. Liquid 크로마토 그래피를Chromatography 통한 Cs-ME의 주요 성분 분석 Analysis of major components of Cs-ME through
Cs-ME의 유효성분을 확인하기 위해 AZURA Analytical HPLC system(KNAUER Corp.)을 이용하여 HPLC(high performance liquid chromatography)를 수행하였다. To confirm the active ingredient of Cs-ME, high performance liquid chromatography (HPLC) was performed using an AZURA Analytical HPLC system (KNAUER Corp.).
분석 조건(하기 기재)Analysis conditions (described below)
HPLC System: AZURA Analytical HPLC (KNAUER Corp.)HPLC System: AZURA Analytical HPLC (KNAUER Corp.)
컬럼(Column) : Phenomenex, Gemini 5μ C18 110A, 250×4.60mm 5μmColumn: Phenomenex, Gemini 5μ C18 110A, 250×4.60mm 5μm
용매menstruum
용매 A : 0.1% TFA in H2OSolvent A: 0.1% TFA in H 2 O
용매 B : 0.08% TFA in 95% MeCN + 5% H2OSolvent B: 0.08% TFA in 95% MeCN + 5% H 2 O
유속 : 1.0㎖/minFlow rate: 1.0ml/min
파장 : 370nm (Quercetin, Luteolin, Kaempferol)Wavelength: 370nm (Quercetin, Luteolin, Kaempferol)
그 결과, 도 4에 나타난 바와 같이 Cs-ME의 주요 성분으로 퀘르세틴(Quercetin), 루테올린(luteolin), 및 캠페롤 (Kaempferol)이 있음을 확인하였다.As a result, as shown in FIG. 4, it was confirmed that quercetin, luteolin, and campferol were present as major components of Cs-ME.
실시예Example 5. 대식세포로부터 유도형 NO 생성 효소 (Inducible nitric oxide synthase, 5. Inducible nitric oxide synthase from macrophages iNOSiNOS )의 )of 분비능Secretory ability 측정 Measure
사이토카인의 발현 정도를 전사수준에서 조사하기 위해 RAW264.7 세포를 6웰 플레이트에 2 x 106으로 24시간 배양하였으며 Cs-ME(50, 100νg/ml)을 30분간 전처리 한 후 LPS(1νg/ml)을 6시간 처리하였다. 그 다음, 트리졸(Trizol) 시약을 사용하여 전체 RNA를 추출하였다. 추출한 전체 RNA를 First strand cDNA synthesis kit (Thermo scientific)를 사용하여 cDNA를 제조한 다음, 동량의 cDNA를 PCR(polymerase chain reaction)로 증폭하였다. 이때 사용한 표적단백질의 센스(sense) 및 안티센스(antisense) 프라이머(primer) 염기서열은 기존문헌을 참조하여 제조하고, 대조군 유전자로는 GAPDH을 사용하였다. PCR 엠플리케이션(amplication)은 Hipi PCR kit (Elpis biotech)을 사용하여 각 실험군 cDNA와 표적단백질들의 센스 및 안티센스 프라이머, 대조군 GAPDH 프라이머를 dNTP 250 uM, Tris-HCL(pH 8.3) 10 mM, KCl 50 mM, NgCl2 1.5 mM를 포함한 Hipi PCR kit 20ul에서 시행하였다. PCR은 95 ℃에서 45초 간 디네츄어링(denaturing), 55 ℃에서 45초 간 어넬링(annealing) 그리고 72 ℃에서 1분간 익스텐션(extension) 하는 조건으로 시행하며, 총 30 사이클을 수행하였다. PCR로 증폭된 DNA는 1.5 % 아가로스 겔에서 전기영동 하였고 분획된 DNA 밴드의 인텐시티(intensity)를 측정하였다.To investigate the level of cytokine expression at the transcription level, RAW264.7 cells were cultured in a 6-well plate at 2 x 10 6 for 24 hours, and Cs-ME (50, 100 νg/ml) was pretreated for 30 minutes and then LPS (1 νg/ml). ml) was treated for 6 hours. Then, total RNA was extracted using Trizol reagent. The extracted total RNA was prepared using a First strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR (polymerase chain reaction). The sense and antisense primer nucleotide sequences of the target protein used at this time were prepared by referring to existing literature, and GAPDH was used as a control gene. PCR amplification was performed using the Hipi PCR kit (Elpis biotech), and the sense and antisense primers of each experimental group cDNA and target proteins, and the control GAPDH primer were dNTP 250 uM, Tris-HCL (pH 8.3) 10 mM,
그 결과, 도 5에 나타난 바와 같이 Cs-ME가 LPS에 의해 발생한 염증관련 사이토카인인 유도형 NO 생성 효소 (Inducible nitric oxide synthase, iNOS)의 mRNA 발현을 줄이는 효과가 있었으며 특히 Cs-ME를 100(μg/ml) 처리한 경우 그 효과가 명확함을 확인하였다.As a result, as shown in FIG. 5, Cs-ME has the effect of reducing the mRNA expression of the inducible nitric oxide synthase (iNOS), an inflammation-related cytokine caused by LPS, and in particular, Cs-ME is 100 μg/ml), it was confirmed that the effect was clear.
실시예Example 6. 전사인자의 핵 내 이동 평가 6. Evaluation of transcription factor movement within the nucleus
뮤린 대식 세포주인 RAW264.7 세포를 페니실린 100 IU/ml 및 스트렙토마이신 100 μg/ml과 10%의 소태아혈청을 함유하는 RPMI 1640 배지를 이용해서 배양한 세포를 90%의 밀도로 100mm의 디쉬에서 전 배양시킨다. 이후 Cs-ME를 30분 동안 전처리하고 자극제(리포폴리사카라이드(Lipopolysaccharide, LPS))로 자극한 후, 약물에 따른 일정시간 후 아이스-콜드(ice-cold) PBS(phosphate buffered saline)로 워싱 하고 균질화 버퍼(Homogenization buffer) A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg/ml 아프로티닌(Aprotinin), 10 μg/ml 류펩틴(Leupeptin)] 300 μl를 이용하여 세포를 모았다. The murine macrophage RAW264.7 cells were cultured using RPMI 1640 medium containing 100 IU/ml of penicillin and 100 μg/ml of streptomycin and 10% fetal bovine serum in a 100 mm dish at a density of 90% Pre-cultivate. After that, Cs-ME was pretreated for 30 minutes, stimulated with a stimulant (Lipopolysaccharide, LPS), and washed with ice-cold phosphate buffered saline (PBS) after a certain period of time depending on the drug. Homogenization buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg/ml Aprotinin, 10 μg/ml leupeptin ( Leupeptin)] 300 μl were used to collect cells.
그 다음, 소니케이터(Sonicator)를 사용하여 아웃풋(Output) 4의 세기로 세포를 깬 후 8000 rpm으로 15분 동안 4 ℃에서 원심분리 하여 상층액 (Cytosol 분획)을 분리하였다. 펠렛 (Nuclear 분획)은 300 μl 균질화 버퍼 B [1% TritonX-100 in 균질화 버퍼 A]로 부유시킨 후 소니케이터를 사용하여 아웃풋 4의 세기로 세포를 깬 다음 얻어진 분획을 SDS-PAGE와 웨시턴 블로팅법을 이용하여 전사인자의 핵 내 이동 수준을 평가하였다.Then, the cells were broken at the intensity of Output 4 using a Sonicator, and then centrifuged at 8000 rpm for 15 minutes at 4° C. to separate the supernatant (Cytosol fraction). The pellet (Nuclear fraction) was suspended in 300 μl homogenization buffer B [1% TritonX-100 in homogenization buffer A], and then the cells were broken with a strength of output 4 using a sonicator. The level of intranuclear migration of transcription factors was evaluated using the blotting method.
그 결과, 도 6에 나타난 바와 같이 Cs-ME를 RAW 264.7 세포에 처리한 경우 밴드가 미처리군에 비하여 희미하게 나타나는바 LPS에 의한 단백질 p50와 p65의 핵 내 이동 양상의 감소효과가 나타남을 확인하였다.As a result, as shown in FIG. 6, when Cs-ME was treated on RAW 264.7 cells, the band appeared faintly compared to the untreated group, and it was confirmed that the effect of reducing the intranuclear migration patterns of proteins p50 and p65 by LPS was shown. .
실시예Example 7. Cs-ME에 의한 세포 내 7. Intracellular by Cs-ME NFNF -- κBκB 신호전달 단백질 Signaling protein IKKαIKKα /β, /β, IκBαIκBα 의 인산화 감소 Reduction of phosphorylation
Cs-ME에 의한 세포 내 NF-κB 신호전달 단백질인 IKKα/β, IκBα 의 인산화 감소 효과를 확인하기 위해 실시예 1-3의 방법에 따라 웨스턴 블롯을 수행하였다.Western blot was performed according to the method of Example 1-3 in order to confirm the effect of reducing phosphorylation of IKKα/β, IκBα, which are intracellular NF-κB signaling proteins by Cs-ME.
그 결과, 도 7에 나타난 바와 같이 Cs-ME을 처리한 세포 내에서 인산화된 IKKα/β, IκBα가 Cs-ME 처리전보다 감소함을 알 수 있는바 NF-κB 신호전달 단백질인 IKKα/β, IκBα 의 인산화 감소 효과를 확인할 수 있었다.As a result, as shown in FIG. 7, it can be seen that phosphorylated IKKα/β and IκBα in the cells treated with Cs-ME are reduced compared to before Cs-ME treatment. The effect of reducing phosphorylation was confirmed.
실시예Example 8. Cs-ME에 의한 세포 내 8. Intracellular by Cs-ME NFNF -- κBκB 신호전달 단백질 Signaling protein SrcSrc , , SykSyk 의 인산화 감소 Reduction of phosphorylation
Cs-ME에 의한 세포 내 NF-κB 신호전달 단백질인 Src, Syk 의 인산화 감소를 확인하기 위해 실시예 1-3의 방법에 따라 웨스턴 블롯을 수행하였다.Western blot was performed according to the method of Example 1-3 to confirm the decrease in phosphorylation of Src and Syk, which are intracellular NF-κB signaling proteins by Cs-ME.
그 결과, 도 8에 나타난 바와 같이 Cs-ME에 처리한 경우 인산화된 Src 및 Syk가 감소하여 p-Src 및 p-Syk 밴드가 연하게 나타나고 있는바 세포 내 NF-κB 신호전달 단백질인 Src 및 Syk 인산화가 감소함을 확인하였다.As a result, as shown in FIG. 8, when Cs-ME was treated, phosphorylated Src and Syk were decreased, resulting in softer p-Src and p-Syk bands. Intracellular NF-κB signaling proteins Src and Syk It was confirmed that phosphorylation was reduced.
실시예Example 9. 9. SrcSrc 과발현을 통한 염증 발생시 When inflammation occurs through overexpression SrcSrc , p85의 인산화 감소, reduced phosphorylation of p85
Src 과발현을 통한 염증 발생시 Cs-ME를 처리하면 Src, p85의 인산화 감소가 나타나는지 확인하기 위해 실시예 1-3의 방법에 따라 웨스턴 블롯을 수행하였다.Western blot was performed according to the method of Example 1-3 in order to confirm whether the phosphorylation of Src and p85 decreased when Cs-ME was treated when inflammation through Src overexpression occurred.
그 결과, 도 9에 나타난 바와 같이 Src 과발현을 통한 염증 발생시 Cs-ME을 처리하면 신호전달 단백질인 Src, p85의 인산화 감소 효과가 나타남을 확인하였으며 특히 Cs-ME를 100(μg/ml) 처리한 경우 p-p85의 감소가 급격히 나타남을 확인하였다.As a result, as shown in FIG. 9, it was confirmed that when Cs-ME was treated during inflammation through Src overexpression, the effect of reducing phosphorylation of signal transduction proteins Src and p85 appeared. In the case, it was confirmed that the decrease in p-p85 appeared rapidly.
실시예Example 10. 10. SykSyk 과발현을 통한 염증 발생시 When inflammation occurs through overexpression Syk의Syk's 인산화 감소 Reduced phosphorylation
Syk 과발현을 통한 염증 발생시 Cs-ME를 처리하면 Syk의 인산화 정도가 감소하는지 확인하기 위해 실시예 1-3의 방법에 따라 웨스턴 블롯을 수행하였다.Western blot was performed according to the method of Example 1-3 in order to check whether the degree of phosphorylation of Syk decreased when Cs-ME was treated when inflammation through Syk overexpression occurred.
그 결과, 도 10에 나타난 바와 같이 Syk 과발현을 통한 염증 발생시 Cs-ME를 처리하면 p-Syk 밴드가 대조군에 비해 용량의존적으로 연해짐을 알 수 있는 바 신호전달 단백질인 Syk의 인산화 감소 효과 있음을 확인하였다.As a result, as shown in FIG. 10, when Cs-ME is treated when inflammation occurs through Syk overexpression, it can be seen that the p-Syk band is dose-dependently softened compared to the control. I did.
실시예Example 11. 급성위염(acute gastritis)유발 동물 모델에서 Cs-ME의 효과 11. Effect of Cs-ME in acute gastritis-induced animal model
마우스에게 에탄올(Ethanol)과 염산(HCl)을 사용하여 급성위염을 유발시켰다. 먼저 ICR 마우스를 다섯 군으로 분류한 뒤, 한 군은 염증반응을 유도하지 않았고 나머지 네 군은 에탄올과 HCl 로 염증반응을 유도하였다. 그 중 두 군은 Cs-ME를 50mg/kg, 100mg/kg으로, 한 군은 컨트롤 컴파운드인 라니티딘(Ranitidine)을, 나머지는 Cs-ME의 비히클(vehicle)인 CMC 0.5%를 이틀간 주입한 뒤 해부 후 위벽의 상태를 측정, 위염 유발 정도를 정량화하였다.Acute gastritis was induced in mice with ethanol (Ethanol) and hydrochloric acid (HCl). First, ICR mice were classified into five groups, and one group did not induce an inflammatory response and the other four groups induced an inflammatory response with ethanol and HCl. Among them, two groups were injected with Cs-ME at 50mg/kg and 100mg/kg, one group was injected with ranitidine, a control compound, and 0.5% CMC, a vehicle of Cs-ME, was injected for two days and then dissected. After measuring the state of the gastric wall, the degree of gastritis induction was quantified.
그 결과, 도 12에 나타나 바와 같이 Cs-ME의 위염 완화효과는 50mg/kg처리군과 100mg/kg처리군을 비교했을 때, 농도의존적으로 증가하였다. 특히, 100mg/kg Cs-ME 처리군의 경우, 라니티딘(Ranitidine)처리군보다 더 높은 위염 완화효과를 보임을 확인하였다.As a result, as shown in FIG. 12, the gastritis alleviation effect of Cs-ME increased in a concentration-dependent manner when comparing the 50 mg/kg treatment group and the 100 mg/kg treatment group. In particular, it was confirmed that the 100mg/kg Cs-ME treatment group showed a higher gastritis alleviation effect than the ranitidine treatment group.
실시예Example 12. Cs-ME, 12. Cs-ME, 퀘르세틴Quercetin (( QuercetinQuercetin ), 및 ), and 캠페롤Camperol ( ( KaempferolKaempferol )의 )of 니트릭Nitric 옥사이드(NO) Oxide (NO) 생성능Generating ability 비교 compare
Cs-ME을 처리한 경우 퀘르세틴(Quercetin), 및 캠페롤 (Kaempferol) 단독처리인 경우보다 니트릭 옥사이드 생성능 저해 효과가 더 크다는 것을 확인하기 위해 실시예 2의 실험방법에 따라 실험을 수행하였다.The experiment was performed according to the experimental method of Example 2 in order to confirm that the Cs-ME treatment had a greater effect of inhibiting the nitric oxide production ability than the case of the treatment with Quercetin and Campferol alone.
그 결과, 도 12에 나타난 바와 같이 Cs-ME를 처리한 경우 캠퍼롤 및 퀘르세틴 각각을 처리한 경우보다 니트릭 옥사이드 생성억제 효과가 더 큰 것을 확인하였다.As a result, it was confirmed that, as shown in FIG. 12, when Cs-ME was treated, the nitric oxide production inhibitory effect was greater than that of each treatment with camphorol and quercetin.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가지는 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.The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.
Claims (11)
상기 염증성 질환은 위염인 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient,
The inflammatory disease is characterized in that gastritis, a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 추출물은 메탄올을 용매로 추출한 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The extract is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that methanol is extracted with a solvent.
상기 카나리움 서부라텀 추출물은 퀘르세틴(Quercetin), 루테올린(luteolin), 및 캠페롤 (Kaempferol)로 구성된 군에서 선택되는 1개 이상의 성분을 포함하는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract is characterized in that it comprises one or more components selected from the group consisting of quercetin, luteolin, and camperol (Kaempferol), a pharmaceutical for the prevention or treatment of inflammatory diseases Ever composition.
상기 카나리움 서부라텀 추출물은 일산화 질소 (nitric oxide, NO) 생성을 억제하는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract is characterized in that it inhibits the production of nitrogen monoxide (nitric oxide, NO), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 카나리움 서부라텀 추출물은 유도형 NO 생성 효소 (Inducible nitric oxide synthase, iNOS) 발현을 억제하는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract is characterized in that to inhibit the expression of inducible nitric oxide synthase (iNOS), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제하는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract is characterized in that inhibiting the phosphorylation of IκBα, IKKα / β, Syk, p85 or Src, a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 카나리움 서부라텀 추출물은 IκBα, IKKα/β, Syk, p85 또는 Src의 인산화를 억제함으로써, 뉴클리어 팩터 κB(nuclear factor κB, NF-κB)의 활성을 억제하는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract inhibits the phosphorylation of IκBα, IKKα/β, Syk, p85 or Src, thereby inhibiting the activity of nuclear factor κB (NF-κB). Pharmaceutical composition for prophylaxis or treatment.
상기 카나리움 서부라텀 추출물은 p50 및 p65의 핵 전이(nuclear translocation)를 감소시키는 것을 특징으로 하는, 염증성 질환의 예방 또는 치료용 약학적 조성물.
The method of claim 1,
The canarium western ratum extract is characterized in that reducing the nuclear translocation of p50 and p65 (nuclear translocation), a pharmaceutical composition for the prevention or treatment of inflammatory diseases.
상기 염증성 질환은 위염인 것을 특징으로 하는, 염증성 질환의 예방 또는 개선용 식품 조성물.
As a food composition for preventing or improving inflammatory diseases, comprising an extract of Canarium subulatum as an active ingredient,
The food composition for preventing or improving inflammatory diseases, characterized in that the inflammatory disease is gastritis.
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