KR20190022085A - Composition comprising tamarixetin for preventing or treating sepsis or septic shock - Google Patents
Composition comprising tamarixetin for preventing or treating sepsis or septic shock Download PDFInfo
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- KR20190022085A KR20190022085A KR1020170107891A KR20170107891A KR20190022085A KR 20190022085 A KR20190022085 A KR 20190022085A KR 1020170107891 A KR1020170107891 A KR 1020170107891A KR 20170107891 A KR20170107891 A KR 20170107891A KR 20190022085 A KR20190022085 A KR 20190022085A
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- sepsis
- lps
- treatment
- septic shock
- inflammatory
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Description
본 발명은 타마리세틴을 유효성분으로 포함하는 패혈증 또는 패혈증성 쇼크의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating sepsis or septic shock comprising tamaricine as an active ingredient.
패혈증(sepsis)은 병원성 그람 음성 세균이 생체에 감염된 경우 세포벽 성분인 리포폴리사카라이드(lipopolysaccharide, LPS)가 독소로 작용하여 생체의 면역체계가 과도하게 활성화됨으로써 유발되는 염증 반응으로서, 전신에 감염증을 일으키거나 증상이 심할 경우 쇼크를 동반하기도 한다. 구체적으로 패혈증은 악성종상, 백혈병, 악성림프종, 후천성면역부전증후군(AIDS), 교원병, 신부전, 간질환, 뇌혈관장해, 당뇨병 등의 기초질환을 가진 환자나 고령자, 미숙아 같은 체액성면역부전 또는 세포성면역부전을 가진 저항력이 약한 숙주가 부신스테로이드나 항종양제의 화학요법, 코발트 조사 등의 방사선치료 또는 유치카테테르, 혈액투석, 장기이식, 심장수술 등의 처치, 수술을 받은 경우에 주로 발병한다. 패혈증은 병원 중환자 병동에 입원한 환자가 사망하는 주요한 원인이 되며, 이에 의한 치사율이 보통 30% 이상인 매우 심각한 질병이다. 의학기술의 발전에도 불구하고 아직도 전 세계적으로 수술 후유증으로 감염이 되어 패혈증이 발생하는 경우가 많으며, 신생아나 노인들과 같이 체내의 면역력이 약한 사람이 감염되었을 경우 패혈증으로 진행되는 경우도 많다. 대표적으로, 신생아 패혈증의 경우 만삭아 1,000명 중 3명 정도가 발생하는 것으로 알려져 있고, 미숙아는 발병률이 3 내지 4배 증가하는 것으로 알려져 있다. 패혈증에 걸렸을 경우 대게 항생제 치료를 받게 되지만, 적절한 처치가 늦어져 균들이 많이 증식했을 경우나 항생제에 내성이 강한 균주에 의해 감염이 되었을 경우에는 항생제만으로는 효과적인 치료를 할 수 없으며, 다양한 항생제에 대한 내성을 갖는 병원균이 점차 증가하고 있어 새로운 패혈증 치료제의 개발이 시급하다. Sepsis is an inflammatory reaction caused by excessive activation of the body's immune system by acting as a toxin, a lipopolysaccharide (LPS) that is a cell wall component when a pathogenic gram-negative bacterium infects a living body. Shock may be accompanied if it causes or symptoms are severe. Specifically, the sepsis is caused by humoral immune deficiency such as malignant neoplasia, leukemia, malignant lymphoma, acquired immunodeficiency syndrome (AIDS), collagen, renal failure, liver disease, cerebrovascular disorder, A weakly resistant host with a sexually immune deficiency is predominantly caused by chemotherapy of adrenal steroids or antitumor agents, radiation therapy such as cobalt irradiation or treatment such as fetal catheterization, hemodialysis, organ transplantation, cardiac surgery, do. Sepsis is a very serious disease with a mortality rate of more than 30%, which is the main cause of death in hospitalized patients in the hospital ICU. Despite advances in medical technology, there are still cases of post-operative sepsis due to post-operative infection worldwide, and in many cases, sepsis occurs when a person with weak immunity, such as neonates or elderly people, is infected. Typically, it is known that about 3 out of 1,000 newborns develop in the case of neonatal sepsis, and premature infants are known to increase the incidence by 3 to 4 times. In the case of septicemia, antibiotics are usually used for treatment. However, in cases where the antibiotics are resistant to antibiotics, the antibiotics alone can not provide effective treatment. And the development of a new treatment for sepsis is urgent.
타마리세틴은 자연에서 가장 일반적인 플라보노이드 형태인 퀘르세틴과 유사한 모핵구조로 가지는 화합물이다. 한국등록특허 0544711호, 한국공대특허 10-2005-0076452호에는 퀘르세틴이 포함된 조성물의 항암효과가 개시되어 있으며, 한국등록특허 1154616호에는 캄페롤 및 퀘르세틴을 함유하는 조성물의 히알루론산 생성 촉진 효과를 개시하고 있고, 한국등록특허 1217181호에는 탄닌산 및 퀘르세틴을 유효성분으로 포함하는 조성물의 아토피성 피부염에 대한 치료효능이 개시되어 있으나, 항패혈 효과는 떨어지는 것으로 나타났다. Tamaricetin is a compound that has a nucleotide structure similar to quercetin, the most common form of flavonoid in nature. Korean Patent No. 0544711 and Korean Patent Publication No. 10-2005-0076452 disclose an anticancer effect of a composition containing quercetin and Korean Patent No. 1154616 discloses a composition containing camphorol and quercetin to promote the hyaluronic acid production- Korean Patent No. 1217181 discloses a therapeutic effect of a composition comprising tannic acid and quercetin as an active ingredient for atopic dermatitis, but the antiepileptic effect is reduced.
본 발명자들은 상기와 같은 요구를 충족시키기 위하여 연구를 거듭한 결과 피토캐미칼인 타마리세틴이 내독소혈증 및 박테리아 감염에 의한 패혈증을 치료 또는 예방할 수 있음을 확인함으로써 본 발명을 완성하였다. The present inventors have completed the present invention by confirming that tamoxifen, tamaricetin, can treat or prevent sepsis caused by endotoxemia and bacterial infection as a result of repeated studies to satisfy the above-mentioned demand.
본 발명이 해결하고자 하는 과제는 타마리세틴 유도체를 유효성분으로 포함하는 패혈증 패혈증 또는 패혈증성 쇼크의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.The object of the present invention is to provide a pharmaceutical composition for preventing or treating sepsis sepsis or septic shock comprising Tamaricetin derivatives as an active ingredient.
상기한 과제를 해결하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물을 유효성분으로 포함하는 패혈증 또는 패혈증성 쇼크의 예방 또는 치료용 약학적 조성물을 제공한다:In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating sepsis or septic shock comprising a compound represented by the following formula (1) as an active ingredient:
[화학식 1][Chemical Formula 1]
상기 화학식에서, In the above formulas,
R1은 C1 - 6알킬이다.R 1 is C 1 - 6 is alkyl.
본 발명에 의하면, 상기 패혈증 또는 패혈증성 쇼크는 내독소혈증(endotoxemia) 또는 박테리아 감염으로 유발된 것일 수 있다. According to the present invention, the sepsis or septic shock may be caused by an endotoxemia or bacterial infection.
본 발명에 의하면, 상기 박테리아는 황색포도상구균(Staphylococcus aureus), 바실러스 세레우스(Bacillus cereus), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 대장균(Escherichia coli), 살모넬라 엔테라이티디스(Salmonella enteritidis) 및 캄필로박터 제주니(Campylobacter jejuni) 중에서 선택되는 어느 하나 이상의 균일 수 있다. According to the present invention, the bacteria are selected from the group consisting of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Escherichia coli, Salmonella enteritidis and Salmonella enteritidis. Campylobacter jejuni, and the like.
본 발명에 의하면, 상기 R1이 C1 - 3알킬일 수 있다.According to the present invention, the R 1 C 1 - 3 may be alkyl.
본 발명 의하면, 상기 조성물은 항염증 사이토카인 IL-10의 발현을 증가시키는 것일 수 있다. According to the present invention, the composition may increase the expression of the anti-inflammatory cytokine IL-10.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물을 유효성분으로 포함하는 패혈증 또는 패혈증성 쇼크의 예방 또는 개선용 식품첨가물을 제공한다. The present invention also provides a food additive for preventing or ameliorating septicemia or septic shock comprising the compound represented by Formula 1 as an active ingredient.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물을 유효성분으로 포함하는 항염증성 사이토카인 IL-10의 발현 증강용 조성물을 제공한다.The present invention also provides a composition for enhancing the expression of an anti-inflammatory cytokine IL-10, which comprises a compound represented by the following formula (1) as an active ingredient.
본 발명에 따른 타마리세틴 또는 이의 유도체는 내독소혈증과 박테리아 감염에 의한 패혈증에 대해 예방 또는 치료하는 효과를 나타내므로 항패혈증 후보물질로 유용하게 적용시킬 수 있으며, 염증발생을 효과적으로 억제함으로써 면역증진의 효과를 나타낼 수 있으므로 패혈증 또는 패혈증성 쇼크의 예방, 치료 또는 개선용조성물의 유효성분으로 유용하게 사용될 수 있다.The tamaritetin or its derivative according to the present invention exhibits an effect of preventing or treating sepsis due to endotoxinemia and bacterial infection, and thus can be effectively applied as a candidate antiepileptic agent. In addition, And thus can be effectively used as an effective ingredient of a composition for preventing, treating or ameliorating sepsis or septic shock.
도 1은 본 발명의 일 실시예에 따른 타마리세틴의 세포독성을 평가한 결과이다.
도 2는 LPS 처리시, 타마리세틴의 처리 시점에 따른 사이토카인 발현량을 평가한 결과이다.
도 3은 LPS 처리 후, 타마리세틴 투여에 따른 결과이다.
도 4는 타마리세틴 처리에 따른 사이토카인 발현양을 웨스턴블롯으로 확인한 결과이다.
도 5는 타마리세틴 처리에 따른 사이토카인 발현양을 웨스턴블롯으로 확인한 결과이다.
도 6은 타마리세틴 처리에 의한 분광형광 광도계를 이용한 형광 퀸칭(Quenching) 실험결과이다.
도 7은 타마리세틴 처리에 의한 내독소혈증 마우스 모델의 생존율 변화를 확인한 결과이다.
도 8a는 내독소혈증 마우스모델 혈액에서의 염증성 사이토카인 발현양이며, 도 8b는 내독소혈증 마우스모델 폐에서 측정된 염증성 사이토카인 발현양이다.
도 9는 내독소혈증 마우스모델에서 간 및 신장의 손상도 지표를 확인을 위해 혈액 중의 AST, ALT, BUN 농도를 측정한 결과이다.
도 10은 폐 조직을 헤마톡실린&에오신 법을 이용하여 세포 침윤도을 검사한 결과이다.
도 11a는 E.Coli DH5α에 의한 염증 유도에서, 타마리세틴의 처리 시점에 따른 사이토카인 발현량을 평가한 결과이며, 도 11b는 E.Coli K1에 의한 염증 유도에서, 타마리세틴의 처리 시점에 따른 사이토카인 발현량을 평가한 결과이다.
도 12은 타마리세틴 처리에 의한 패혈증 쇼크 마우스 모델의 생존율 변화를 확인한 결과이다.
도 13a는 패혈증 쇼크 마우스모델 혈액에서의 염증성 사이토카인 발현양이며, 도 13b는 패혈증 쇼크 마우스모델 폐에서 측정된 염증성 사이토카인 발현양이다.
도 14는 패혈증 쇼크 마우스 모델에서 간 및 신장의 손상도 지표를 확인을 위해 혈액 중의 AST, ALT, BUN 농도를 측정한 결과이다.
도 15은 폐 조직을 헤마톡실린&에오신 법을 이용하여 세포 침윤도을 검사한 결과이다.
도 16은 패혈증 쇼크 마우스 모델에서 타마리세틴의 투여에 따른 총균수 변화를 측정한 것이다.
도 17은 패혈증 쇼크 마우스 모델에서 타마리세틴의 투여에 따른 혈청 내 독소 농도 변화를 Limulus Amebocyte Lysate(LAL) 발색 종점 분석으로 분석한 결과이다.
도 18은 타마리세틴의 투여에 따른 IL-10 항염증 인자의 증가를 확인하기 위하여 타마리세틴에 의해 지라에서 면역세포의 증식을 확인한 결과이다.
도 19는 타마리세틴 및 퀘르세틴의 처리에 의한 내독소혈증 마우스 모델에서 IL-10의 발현양을 분석한 결과이다.
도 20은 타마리세틴 및 퀘르세틴의 처리에 의한 패혈증 쇼크 마우스 모델의 생존율 변화를 확인한 결과이다.
도 21은 퀘르세틴 처리에 의한 항박테리아 활성을 평가한 결과이다.FIG. 1 shows the results of evaluating cytotoxicity of tamaricetin according to an embodiment of the present invention.
Fig. 2 shows the results of evaluating the amount of cytokine expression according to the treatment time of tamarisk in LPS treatment.
FIG. 3 shows the results of administration of tamariceptin after LPS treatment.
FIG. 4 shows the result of Western blotting the amount of cytokine expressed by treatment with tamaritetin.
FIG. 5 shows the result of Western blotting the amount of cytokine expressed by treatment with tamaritetin.
FIG. 6 is a fluorescence quenching experiment result using a spectrophotometric spectrophotometer by tamariceptin treatment.
FIG. 7 shows the results of confirming the survival rate change of a mouse model of endotoxemia by treatment with tamarisket.
Figure 8a is the amount of inflammatory cytokine expression in the endotoxemia mouse model blood and Figure 8b is the amount of inflammatory cytokine expression measured in the endotoxemia mouse model lung.
FIG. 9 shows the results of measurement of AST, ALT, and BUN concentrations in the blood to confirm liver and kidney damage index in the endotoxemia mouse model.
FIG. 10 shows the result of examining the degree of cell invasion using lung hematoxylin and eosin.
FIG. 11A shows the results of evaluating the amount of cytokine expression according to the treatment time of tamariceptin at the induction of inflammation by E. coli DH5α, FIG. 11B shows the results at the time of treatment of tamaricetin at the induction of inflammation by E. coli K1 And the amount of cytokine expression was evaluated.
FIG. 12 shows the results of confirming the survival rate change of a sepsis shock mouse model by tamariceptin treatment.
FIG. 13A is the amount of inflammatory cytokine expression in the blood of the sepsis shock mouse model, and FIG. 13B is the amount of inflammatory cytokine expression measured in the septic shock mouse model lung.
FIG. 14 shows the results of measurement of AST, ALT, and BUN concentrations in the blood to confirm liver and kidney damage index in a sepsis shock mouse model.
Fig. 15 shows the result of examining the degree of cell invasion using lung hematoxylin and eosin.
FIG. 16 is a graph showing changes in the total number of bacteria according to the administration of tamarisket in a sepsis shock mouse model.
FIG. 17 shows the result of analyzing the concentration of serum endotoxin according to the administration of tamarisket in a sepsis shock mouse model by the end point of Limulus Amebocyte Lysate (LAL) color.
FIG. 18 shows the results of confirming the proliferation of immune cells in the spleen by tamarisetin in order to confirm the increase of IL-10 anti-inflammatory factor upon administration of tamaricetin.
FIG. 19 shows the results of analysis of the expression level of IL-10 in a mouse model of endotoxemia by treatment with tamaritetin and quercetin.
20 shows the results of confirming the survival rate changes of a sepsis-shock mouse model by treatment with tamaritetin and quercetin.
Fig. 21 shows the result of evaluation of antibacterial activity by quercetin treatment.
본 발명은 하기 화학식 1로 표시되는 화합물을 유효성분으로 포함하는 패혈증 또는 패혈증성 쇼크의 예방 또는 치료용 약학적 조성물을 제공한다:The present invention provides a pharmaceutical composition for preventing or treating sepsis or septic shock comprising a compound represented by the following formula (1) as an active ingredient:
[화학식 1][Chemical Formula 1]
상기 화학식에서, In the above formulas,
R1은 C1 - 6알킬이다.R 1 is C 1 - 6 is alkyl.
상기 화학식 1로 표시되는 화합물은 타마리세틴 또는 이의 유도체이다. 본 발명에 있어서, 상기 화학식 1의 화합물은 염증성 사이토카인을 감소시키고, 항염증성 사이토카인을 증가시키며, 바이러스의 감염에 의해 유발되는 패혈증을 완화시켜 패혈증 또는 패혈증 쇼크로 인한 사망률을 현저히 낮춤으로써 패혈증의 예방 또는 치료 효과를 나타낸다.The compound represented by Formula 1 is tamaricin or a derivative thereof. In the present invention, the compound of the formula (1) reduces inflammatory cytokines, increases anti-inflammatory cytokines, alleviates sepsis caused by virus infection, significantly reduces the mortality due to sepsis or sepsis shock, Prevention or treatment effect.
본 발명에 있어서, 상기 패혈증 또는 패혈증성 쇼크는 내독소혈증(endotoxemia) 또는 박테리아 감염으로 유발된 것일 수 있다. In the present invention, the sepsis or septic shock may be caused by an endotoxemia or bacterial infection.
본 발명에 있어서, "패혈증"이란 미생물에 감염되어 전신에 심각한 염증 반응이 나타나는 상태를 말한다. 체온이 38도 이상으로 올라가는 발열 증상 혹은 36도 이하로 내려가는 저체온증, 호흡수가 분당 24회 이상으로 증가(빈호흡), 분당 90회 이상의 심박수(빈맥), 혈액 검사상 백혈구 수의 증가 혹은 현저한 감소 중 두 가지 이상의 증상을 보이는 경우, 이를 전신성 염증 반응 증후군(systemic inflammatory response syndrome; SIRS)이라고 부른다. 이러한 전신성 염증 반응 증후군이 미생물의 감염에 의한 것일 때 패혈증이라고 한다. 신체의 감염병소에서 병원균이 지속적 또는 단속적으로 혈류에 들어와 여러 장기조직에 정착하여 병소를 만들고 심한 전신 증상을 보이는 것이다. 원인균으로는 포도상구균, 연쇄상구균, 대장균, 녹농균, 결핵균, 폐렴간균, 진균, 혐기성균 등이 있다. In the present invention, " sepsis " refers to a state in which a serious inflammatory reaction occurs in the whole body by infection with microorganisms. The number of breaths increased more than 24 times per minute (ventilation), heart rate more than 90 times per minute (tachycardia), increase in leukocyte count in blood test, or significant decrease in respiratory rate If you have symptoms that are more than a branch, it is called systemic inflammatory response syndrome (SIRS). This systemic inflammatory response syndrome is called sepsis when it is caused by microbial infection. Pathogens from the body's infectious lesions persist or intermittently enter the bloodstream, settle in various organs, create lesions, and show severe systemic symptoms. The causative bacteria include Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Pneumococcus, Fungi, and anaerobic bacteria.
본 발명에 의하면 상기 원인균은 구체적으로, 황색포도상구균(Staphylococcus aureus), 바실러스 세레우스(Bacillus cereus), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 대장균(Escherichia coli), 살모넬라 엔테라이티디스(Salmonella enteritidis) 및 캄필로박터 제주니(Campylobacter jejuni) 중에서 선택되는 어느 하나 이상의 균일 수 있다. According to the present invention, the causative microorganism is specifically selected from the group consisting of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Escherichia coli, Salmonella enteritidis ) And Campylobacter jejuni. ≪ / RTI >
본 발명에 의하면, 상기 R1이 C1 - 3알킬인 것이 바람직하며, 보다 바람직하게는 R1이 메틸(타마리세틴, tamarixrtin)인 것일 수 있다. 상기 R1이 메틸인 경우, LPS에 의해 유도된 내독소혈증(endotoxemia)과 E.coli 박테리아 감염으로 유발된 패혈증 또는 패혈증성 쇼크가 현저하게 완화되는 것을 확인하였다. According to the present invention, wherein R 1 is C 1 - 3 alkyl, and preferably is, there is more preferably R 1 may be methyl (L-Marie paroxetine, tamarixrtin). When R 1 is methyl, it was confirmed that endotoxemia induced by LPS and sepsis or septic shock induced by E. coli bacterial infection were remarkably alleviated.
또한, 상기 화학식 1로 표시되는 화합물은 우수한 패혈증 효과를 나타낼 뿐 아니라 면역증진 효과 역시 탁월하여 패혈증 또는 패혈증성 쇼크의 예방 또는 개선용 식품첨가물로 유용하게 사용될 수 있다. In addition, the compound represented by the above formula (1) not only exhibits an excellent sepsis effect but also excellently enhances immunity, and thus can be useful as a food additive for preventing or ameliorating sepsis or septic shock.
또한, 본 발명은 하기 화학식 1로 표시되는 화합물을 유효성분으로 포함하는 항염증성 사이토카인 IL-10의 발현 증강용 조성물을 제공한다.The present invention also provides a composition for enhancing the expression of an anti-inflammatory cytokine IL-10, which comprises a compound represented by the following formula (1) as an active ingredient.
본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 본 발명에 따른 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 당해 기술 분야에 알려진 적합한 제제는 문헌(Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA)에 개시되어 있는 것을 사용하는 것이 바람직하다. The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or other oral formulations, external preparation, suppository and sterilized injection solution, . Suitable formulations known in the art are preferably those disclosed in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton PA.
본 발명의 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학적 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 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, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글라이콜(propylene glycol), 폴리에틸렌 글라이콜, 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. When the pharmaceutical composition of the present invention is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.The term " administering " as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.
본 발명의 약학적 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.001 내지 1000 mg/kg으로 투여할 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 the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention can be administered at a daily dose of 0.001 to 1000 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 바람직하게는 주사제 형태로 투여될 수 있다. The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The pharmaceutical composition of the present invention can preferably be administered in the form of an injection.
본 발명에 따른 약학적 조성물은 상기 유효성분 외에 패혈증 또는 패혈증성 쇼크의 예방 또는 치료 또는 면역 증강 효과를 갖는 공지된 물질을 하나 이상 추가로 포함할 수 있다. The pharmaceutical composition according to the present invention may further include one or more known substances having an effect of preventing or treating septicemia or septic shock or having an immunostimulating effect in addition to the above-mentioned active ingredients.
본 발명에 따른 약학적 조성물은 상기 유효성분 외에 기관지 확장제, 항히스타민제 또는 소염진통제를 추가로 포함할 수 있다.The pharmaceutical composition according to the present invention may further comprise a bronchodilator, an antihistamine or an anti-inflammatory agent in addition to the above-mentioned active ingredients.
예를 들어, 상기 기관지 확장제로는 β-효능제, 항콜린제, 메틸잔틴 등을 사용할 수 있고, 상기 항히스타민제로는 아크리바스틴(acrivastine), 세티리진(cetirizine), 데슬로라타딘(desloratadine), 펙소페나딘(fexofenadine), 레보세티리진(levocertirizine), 로라타딘(loratadine), 미졸라스틴(mizolastine), 아일메마진(ailmemazine), 클로세티리진(chlocertirizine), 클레마스틴(clemastine), 시프로펩타딘(cypropheptadine), 하이드록시진(hydroxyzine), 케토티펜(ketotifen), 프로멘타진(promenthazine) 등을 사용할 수 있고, 상기 소염진통제로는 아스피린(aspirin), 디클로페낙(diclofenac), 페노프로펜(fenoprofen), 플루비프로펜(flurbiprofen), 이부프로펜(ibuprofen), 인도메타신(indomethacin), 케토프로펜(ketoprofen), 나프록센(naproxen), 피록시캄(piroxicam), 술린닥(sulindac), 셀레콕시브(celecoxib), 발데콕시브(valdecoxib), 로페콕시브(rofecoxib) 등을 사용할 수 있다.For example, the bronchodilator may be a β-agonist, an anticholinergic agent, a methylantanine, etc. The antihistamines include acrivastine, cetirizine, desloratadine, The compounds of the invention may be selected from the group consisting of fexofenadine, levocertirizine, loratadine, mizolastine, ailmemazine, chlocertirizine, clemastine, hydroxypropionate, cyproheptadine, hydroxyzine, ketotifen, promenthazine and the like can be used. Examples of the anti-inflammatory analgesics include aspirin, diclofenac, fenoprofen ( fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, naproxen, piroxicam, sulindac, celecox, Celecoxib, valdecoxib, rofeco (rofeco < / RTI > xib) can be used.
본 발명에 있어서 "식품첨가물"이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 첨가물을 의미한다. The term " food additive " in the present invention refers to a food group imparted with added value to function or express the function of the food by physical, biochemical, biotechnological techniques or the like, Refers to an additive that is processed and designed so that the body's control function regarding prevention, recovery, and the like is sufficiently expressed in a living body.
상기 식품참가물은 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 식품첨가물의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.The foodstuffs may include foodstuff acceptable food-aid additives and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of food additives.
본 발명의 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15중량 % 이하, 바람직하게는 10 중량 % 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the composition of the present invention is used as a food additive, the composition can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, it may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 료에 사용되는 탄산화제 등을 포함할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonic material, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 보다 상세하게 설명한다. 단, 하기 실시예는 본 발명을 더욱 쉽게 이해할 수 있도록 예시하는 것으로 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.
시험예 1. 세포독성 검증Test Example 1. Cytotoxicity Assay
발광 세포 생존 키트(Luminescent Cell viability kit)를 이용하여 수지상 세포에서의 타마리세틴의 세포독성을 분석하였다. 구체적으로 96-웰 마이크로 플레이트에 마우스 골수 유래 수지상 세포(mBM-DCs)를 2×106 세포/웰로 접종하고, 배양액, DMSO 0.1%, 과산화수소(음성대조군) 8.8 mM 및 여러 농도(6.25, 25, 50, 100, 200μM)의 타마리세틴으로 24 시간 동안 배양하여 세포독성을 평가하였다. The cytotoxicity of tamarisetin in dendritic cells was analyzed using a luminescent cell viability kit. Specifically, dendritic cells derived from mouse bone marrow (mBM-DCs) were inoculated at a density of 2 × 10 6 cells / well to a 96-well microplate and cultured in a culture medium containing 0.1% DMSO, 8.8 mM hydrogen peroxide (negative control) 50, 100, 200 [mu] M) tamariceptin for 24 hours to evaluate cytotoxicity.
세포 생존율 분석(Cell Viability Assay)은 발광 세포 생존 키트(Promega, Madison, WI, USA)를 사용하여 대사 활성 세포의 지표인 ATP 존재를 평가하였고, Luminometer를 사용하여 세포 배양 상등액에 존재하는 ATP를 정량화하였다.The cell viability assay (Cell Viability Assay) was used to evaluate the presence of ATP, an indicator of metabolic activation cells, using a luminescent cell survival kit (Promega, Madison, Wis., USA) and quantitate ATP present in the cell culture supernatant using a luminometer Respectively.
하기 도 1에 나타낸 바와 같이, 타마리세틴은 100 μM까지 세포독성을 나타내지 않았으며, 200 μM 이상에서 유의적인 세포독성을 보였다. As shown in FIG. 1, tamarisetin showed no cytotoxicity up to 100 μM, and significant cytotoxicity at 200 μM or more.
시험예 2. LPS 유도된 내독소혈증 개선효과 검증 Test Example 2. Verification of LPS-induced endotoxemia improvement
타마리세틴 전처리 및 LPS 처리에 따른 사이토카인 발현량을 측정하여 내도속혈증에 대한 타마리세틴의 효과를 확인하였다. 수지상 세포를 여러 농도의 타마리세틴으로 30분 동안 전처리한 뒤, LPS(50ng/mL)로 6시간 동안 자극하였다. 배양 배지를 수집하고, 배양 상등액 중의 TNF-α, IL-6, IL-10 및 IL-12p70 발현양을 ELISA를 사용하여 제조사의 지시에 따라 결정하였으며, 이를 하기 도 2에 나타내었다. The amount of cytokine expressed by tamariceptin pretreatment and LPS treatment was measured to confirm the effect of tamaricetin on endotoxin. The dendritic cells were pretreated with tamaritetin at various concentrations for 30 minutes and then stimulated with LPS (50 ng / mL) for 6 hours. The culture medium was collected and the amounts of TNF-α, IL-6, IL-10 and IL-12p70 expressed in the culture supernatant were determined according to the manufacturer's instructions using an ELISA, as shown in FIG.
LPS의 처리는 수지상 세포에 염증성 사이토카인을 발생시켰으며, 타마리세틴의 처리는 염증성 사이토카인을 감소시키고, 항염증성 사이토카인인 IL-10을 증가시키는 것이 확인되었다. LPS treatment induced inflammatory cytokines in dendritic cells, and tamariceptin treatment reduced inflammatory cytokines and increased IL-10, an anti-inflammatory cytokine.
타마리세틴의 처리시점에 따른 LPS 유도된 내독소혈증 개선효과 검증하기 위하여 수지상 세포에 대한 타마리세틴의 처리시점을 각각 LPS 처리 30분 전, 동시, 30분 후, 60분 후, 90분 후 및 120분 후에 처리한 결과를 도 3에 나타내었다. In order to examine the effect of treatment with timaricetin on LPS induced endotoxemia, the treatment time of tamarisetin against dendritic cells was measured 30 minutes before, 30 minutes after, 60 minutes after, 90 minutes after LPS treatment And the treatment after 120 minutes are shown in Fig.
LPS 처리 전에 타마리세틴을 전처리하는 경우 염증성 사이토카인의 발생 저해효과가 가장 우수하였으며, LPS 처리 30 분 이내에 타마리세틴을 처리하는 것은 항염증성 사이토카인의 형성을 증가시키는데 효과적으로 작용하는 것으로 나타났다. 한편, LPS 처리 30 분 이후에 타마리세틴을 처리하는 경우, 염증성 사이토카인을 저해하는데 효과가 떨어지는 것이 확인되었다.Pretreatment of tamarisetin before LPS treatment was most effective in inhibiting the production of inflammatory cytokines. Treatment of tamarisetin within 30 minutes of LPS treatment was effective in increasing the formation of anti-inflammatory cytokines. On the other hand, it was confirmed that when tamarisket was treated after 30 minutes of LPS treatment, the effect of inhibiting inflammatory cytokines was poor.
시험예 3. 웨스턴 블롯Test Example 3. Western blot
LPS에 의한 수지상 세포의 염증작용은 TLR4 경로를 타고 이루어지기 때문에 TLR4 경로의 분자(molecule)에 타마리세틴이 영향을 미치는지 웨스턴블롯을 통해 확인하였다. 구체적으로 단백질(25mg)을 10 % 소듐도데실설페이트폴리아크릴아미드 겔 전기영동으로 분리하고 폴리불화비닐리덴 막으로 옮겼다. 0.05 % Tween-20을 함유하는 트리스 완충액 중 5 % 탈지유로 막을 차단하고 Abs로 배양하였다. 막을 2차 호스래디쉬 퍼옥시다제(horseradish peroxidase)-결합 항체와 함께 배양하고, 향상된 화학 발광 키트(Merck, Germany)를 사용하여 시각화하였다.Since the inflammatory action of dendritic cells by LPS is mediated by the TLR4 pathway, Western blotting confirmed whether tamarisetin affects the TLR4 pathway molecule. Specifically, the protein (25 mg) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% skim milk in Tris buffer containing 0.05% Tween-20 and incubated with Abs. The membranes were incubated with secondary horseradish peroxidase-conjugated antibodies and visualized using an enhanced chemiluminescence kit (Merck, Germany).
면역침강반응(immunoprecipitation, IP)은 상응하는 Ab를 각각의 세포 추출물 1 mldp 추출하고, 4℃ 로테이터(rotator)에서 1 시간 동안 배양하였다. 미리 세척한 단백질 A-아가로스 비드의 50% 슬러리 50 마이크로리터를 각 샘플에 첨가한 다음 4 ℃에서 12시간 동안 추가로 배양하였다. 샘플을 용해 완충액(lysis buffer)에서 4회 세척하고, 상기한 바와 같이, 웨스턴 블롯 분석을 수행하였다. Immunoprecipitation (IP) was performed by 1 mldp extraction of the corresponding Ab from each cell extract and incubation for 1 hour in a 4 ° C rotator. 50 microliters of a 50% slurry of pre-washed protein A-agarose beads was added to each sample and then further incubated at 4 占 폚 for 12 hours. Samples were washed four times in lysis buffer and Western blot analysis performed as described above.
웨스턴 블롯을 통해 Phospho-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Akt, Akt, COX-2, IκBα 및 α-tubulin을 검출하였다. P38, p38, p-JNK, JNK, p-ERK, ERK, p-Akt, Akt, COX-2, IκBα and α-tubulin were detected by Western blotting.
도 4에 나타낸 바와 같이, JNK, p38와 Akt의 phosphorylation이 억제되고 COX-2의 양과 IκBα의 degradation이 감소되는 것으로 확인되었으며, 타마리세틴이 LPS 유도된 NF-κB의 활성화 및 TLR4 및 IRAK4의 결합을 감소시키는 것을 확인하였다.As shown in FIG. 4, it was confirmed that the phosphorylation of JNK, p38 and Akt was inhibited, and the amount of COX-2 and the degradation of IκBα were reduced, and that tamarisetin reduced LPS induced NF-κB activation and binding of TLR4 and IRAK4 .
또한, 도 5에 나타난 바와 같이, LPS가 TLR4와 결합하면 일어나게 되는 TLR4와 세포 내부에 있는 IRAK4와의 결합도 타마리세틴에 의해 약화되는 것을 확인할 수 있다. In addition, as shown in FIG. 5, it can be confirmed that the binding of TLR4, which occurs when LPS binds to TLR4, and IRAK4 in the cell, is attenuated by tamarisetin.
시험예 4. 형광 퀸칭 실험Test Example 4. Fluorescence quenching experiment
JNK1, ERK, p38 MAPK를 발현시키기 위해 인간 단백질의 C-말단 절단된 형태를 pET21b 발현 벡터(Novagen)에 클로닝하여 대장균에서 C- 말단 His-6 표지 형태로 발현시켰다. 단백질을 정제하고 형광 퀸칭 실험을 RF- 5301PC 분광형광 광도계(Shimadzu, Kyoto, Japan)를 이용하여 25℃에서 수행하였다. 타마리세틴은 pH 8.0의 100 mM NaCl을 함유한 50 mM 인산나트륨 완충액에서 10 μM 키나제 용액으로 적정하였으며, 최종 단백질:억제제 비율은 1:10이었다. 샘플을 여기 및 방출 경로 길이가 10 nm 인 2 ml 큐벳에 넣었다. 타마리세틴의 농도 증가에 따른, 트립토판 방출을 측정하여 키나아제의 형광 양자 수율이 결정되었다. In order to express JNK1, ERK, p38 MAPK, the C-terminal truncated form of human protein was cloned into pET21b expression vector (Novagen) and expressed in C-terminal His-6 labeled form in E. coli. The protein was purified and the fluorescence quenching experiment was performed at 25 ° C using an RF-5301PC spectrophotometer (Shimadzu, Kyoto, Japan). Tamaricetin was titrated with 10 μM kinase solution in 50 mM sodium phosphate buffer containing 100 mM NaCl at pH 8.0, with a final protein: inhibitor ratio of 1:10. Samples were placed in a 2 ml cuvette with an excitation and emission path length of 10 nm. The fluorescence quantum yield of the kinase was determined by measuring tryptophan release with increasing concentration of tamariceptin.
도 6에 나타난 바와 같이, 타마리세틴이 JNK 및 p38과의 결합친화도가 높은것이 확인되었다. As shown in Fig. 6, it was confirmed that tamariceptin has a high binding affinity with JNK and p38.
시험예 5. in-vivo 테스트Test Example 5: in-vivo test
마우스(5마리/그룹당)에 식염수에 용해된 10 및 25 mg/kg LPS (E. coli O127:B8, Sigma-Aldrich, St. Louis, MO)를 복강 주사하여 LPS-유발성 내독소혈증 마우스 모델을 얻고, 타마리세틴의 효과를 검증하였다. Mice were injected intraperitoneally with 10 and 25 mg / kg LPS (E. coli O127: B8, Sigma-Aldrich, St. Louis, Mo.) in saline solution to induce LPS-induced endotoxemia , And the effect of tamaricetin was verified.
생존율Survival rate
마우스의 복강에 타마리세틴(1 mg/kg)을 주사하고, 1시간 뒤에 LPS (25 mg/kg)를 주사하였다. 생존율을 72 시간 동안 관찰하였다. Tamarisetin (1 mg / kg) was injected into the abdominal cavity of mice and LPS (25 mg / kg) was injected 1 hour later. The survival rate was observed for 72 hours.
도 7에 나타난 바와 같이, 타마리세틴은 내독소혈증 마우스 모델의 생존율을 향상시켰다. As shown in Figure 7, tamarisetin improved the survival rate of the endotoxemia mouse model.
사이토카인 Cytokine 발현양Expression level 측정 Measure
마우스의 복강에 타마리세틴(1 mg/kg)을 주사하고, 1시간 뒤에 LPS (10 mg/kg)를 주사하였다. LPS 주사 2시간 후 혈청을 수집하였다. 사이토카인(TNF-α, IL-6, IL-10 및 IL-12p70) 수준은 제조사의 지침에 따라 샌드위치 ELISA(enzyme-linked immunosorbent assay)를 사용하여 결정하였으며, LPS 주입 12시간 후 혈정과 폐를 수득하였다. Tamarisetin (1 mg / kg) was injected into the peritoneal cavity of mice and LPS (10 mg / kg) was injected 1 hour later. Serum was collected 2 hours after LPS injection. Levels of cytokines (TNF-alpha, IL-6, IL-10 and IL-12p70) were determined using a sandwich ELISA (enzyme-linked immunosorbent assay) according to the manufacturer's instructions. Twelve hours after LPS injection, .
도 8a는 혈액에서의 염증성 사이토카인 발현양이며, 도 8b는 폐(폐의 균질액)에서 측정된 염증성 사이토카인 발현양이다. 혈액 및 폐에서 염증성 사이토카인인 TNF-α, IL-6의 발현양이 감소되었으며, 항염증성 사이토카인인 IL-10의 발현양은 증가되는 것이 확인되었다.Figure 8A is the amount of inflammatory cytokine expression in the blood, and Figure 8B is the amount of inflammatory cytokine expression measured in the lungs (homogenate of the lungs). The expression levels of TNF-α and IL-6, which are inflammatory cytokines in blood and lung, were decreased and the expression level of IL-10, an anti-inflammatory cytokine, was found to be increased.
간 및 신장의 손상도 지표를 확인하기 위하여 혈액 중의 AST, ALT, BUN 농도를 측정하였으며, 이를 도 9에 나타내었다. 타마리세틴의 처리에 의해, AST, ALT, BUN 발현양이 감소한 것이 확인되었다. The AST, ALT and BUN concentrations in the blood were measured to determine the index of damage to the liver and kidney, which is shown in FIG. It was confirmed that the treatment with tamaritetin decreased the expression levels of AST, ALT and BUN.
H&E stainingH & E staining
폐로부터 조직 블록을 수집하여 12시간동안 4% 파라포름알데하이드에 고정하고 왁스에 포매하였다. 관상절편은 왁스를 제거하고, 헤마톡실린&에오신(H&E)으로 염색한 뒤, 광학현미경으로 세포침윤을 검사하였으며, 디지털 이미지를 얻었다. Tissue blocks were collected from the lungs and fixed in 4% paraformaldehyde for 12 hours and embedded in wax. The coronal sections were removed with wax, stained with hematoxylin and eosin (H & E), examined for cellular infiltration with an optical microscope, and digital images were obtained.
혈청 중 아스파테이트 아미노전이효소(AST), 알라닌 아미노전이효소(ALT) 및 혈중요소질소(BUN) 농도의 수준은 토탈 실험 자동화(Hitachi, Japan) 및 TBA-200FR NEO(Toshiba, Japan) 시스템을 사용하여 분석하였다. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels in serum were measured using total laboratory automation (Hitachi, Japan) and TBA-200FR NEO (Toshiba, Japan) Respectively.
도 10에 나타낸 바와 같이, 폐로 침윤되는 염증 세포가 감소된 것을 H&E staining을 통해 확인할 수 있다.As shown in FIG. 10, it can be confirmed by H & E staining that the inflammatory cells infiltrating into the lung are reduced.
시험예 6. 항 박테리아 활성 평가Test Example 6 Evaluation of antibacterial activity
타마리세틴의 항 박테리아 활성을 평가하기 위하여, LPS와 같은 덴도톡신을 분비해 과도한 염증 반응을 일으키는 균인 E.Coli DH5α와 E.Coli K1을 처리한 수지상세포에서 타마리세틴의 처리(각각, 균처리전, 동시, 균처리 후 타마리세틴을 처리)에 따른 염증성 사이토카인의 발현양을 측정하였다. In order to evaluate the antibacterial activity of tamariceptin, treatment of tamarisetin in dendritic cells treated with E. coli DH5α and E. coli K1, which produce excessive inflammatory responses by secreting tendotoxin such as LPS, And treatment with tamarisket before and after the treatment) was measured.
타마리세틴 (25μM)을 생체 내 자극 30분 전, 동시, 30분 후, 60분 후, 90분 후, 120분 후에 각각 처리하였다. 배양 상등액 중의 사이토카인(TNF-α, IL-6, IL-10 및 IL-12p70) 수준은 제조사의 지침에 따라 샌드위치 ELISA(enzyme-linked immunosorbent assay)를 사용하여 결정하였다. Tamaricetin (25 μM) was treated 30 minutes before, 30 minutes after, 60 minutes after, 90 minutes after, 120 minutes after stimulation in vivo. Levels of cytokines (TNF-alpha, IL-6, IL-10 and IL-12p70) in culture supernatants were determined using a sandwich ELISA (enzyme-linked immunosorbent assay) according to the manufacturer's instructions.
도 11a는 E.Coli DH5α에 의한 염증 유도에서, 타마리세틴의 처리 시점에 따른 사이토카인 발현량을 평가한 결과이며, 도 11b는 E.Coli K1에 의한 염증 유도에서, 타마리세틴의 처리 시점에 따른 사이토카인 발현량을 평가한 결과이다.FIG. 11A shows the results of evaluating the amount of cytokine expression according to the treatment time of tamariceptin at the induction of inflammation by E. coli DH5α, FIG. 11B shows the results at the time of treatment of tamaricetin at the induction of inflammation by E. coli K1 And the amount of cytokine expression was evaluated.
타마리세틴의 처리에 따라 염증성 사이토카인의 발현양은 감소되었으며, 항염증성 사이토카인인 IL-10은 증가되는 것이 확인되어 타마리세틴은 대장균에 접종된 DC 활성화를 약화시키는 것을 입증하였다. The expression of inflammatory cytokines was reduced by treatment with tamarisket and the anti-inflammatory cytokine, IL-10, was found to be increased, demonstrating that tamarisetin attenuated DC activation inoculated on E. coli.
시험예 7. 패혈증 쇼크 마우스 모델에서 타마리세틴의 효과Test Example 7 Effect of Tamaricetin on Sepsis-Shock Mouse Model
생존율Survival rate
마우스(5마리/그룹당)의 복강에 타마리세틴(1 mg/kg)을 주사하고, 1시간 뒤에 루리아-베르타니(Ruria-Bertani) 배지로 희석한 대장균 E.coli K1 (3×107 CFU/mice)을 주사하였다. 45시간 동안 생존율을 관찰하였다. 도 12에 나타난 바와 같이, 타마리세틴은 대장균에 의한 패혈증 쇼크 마우스 모델의 생존율을 향상시켰다. (1 mg / kg) was injected into the peritoneal cavity of mice (5 per group / group) and E. coli K1 (3 x 10 < 7 > CFU / mice). Survival rates were monitored for 45 hours. As shown in Fig. 12, tamarisetin enhanced the survival rate of the E. coli-induced sepsis shock mouse model.
사이토카인 Cytokine 발현양Expression level 측정 Measure
마우스의 복강에 타마리세틴(1 mg/kg)을 주사하고, 1시간 뒤에 E.coli K1 (3×107 CFU/mice)을 주사하였다. 대장균 주입 2시간 후 혈청을 수집하였다. 사이토카인(TNF-α, IL-6, IL-10 및 IL-12p70) 수준은 제조사의 지침에 따라 샌드위치 ELISA(enzyme-linked immunosorbent assay)를 사용하여 결정하였으며, 대장균 주입 12시간 후 혈청과 폐를 수득하였다. Tamarisetin (1 mg / kg) was injected into the abdominal cavity of mice and injected with E. coli K1 (3 × 10 7 CFU / mice) 1 hour later. Serum was collected 2 hours after the injection of E. coli. Levels of cytokines (TNF-alpha, IL-6, IL-10 and IL-12p70) were determined using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Serum and lungs .
도 13a는 혈액에서의 염증성 사이토카인 발현양이며, 도 8b는 폐(폐의 균질액)에서 측정된 염증성 사이토카인 발현양이다. 혈액 및 폐에서 염증성 사이토카인인 TNF-α, IL-6의 발현양이 감소되었으며, 항염증성 사이토카인인 IL-10의 발현양은 증가되는 것이 확인되었다.Fig. 13A is the amount of inflammatory cytokine expression in the blood, and Fig. 8B is the amount of inflammatory cytokine expression measured in the lung (homogenate of the lung). The expression levels of TNF-α and IL-6, which are inflammatory cytokines in blood and lung, were decreased and the expression level of IL-10, an anti-inflammatory cytokine, was found to be increased.
간 및 신장의 손상도 지표를 확인하기 위하여 혈액 중의 AST, ALT, BUN 농도를 측정하였으며, 이를 도 14에 나타내었다. 타마리세틴의 처리에 의해, AST, ALT, BUN 발현양이 감소한 것이 확인되었다. AST, ALT, and BUN concentrations in blood were measured to check for liver and kidney damage index, which is shown in FIG. It was confirmed that the treatment with tamaritetin decreased the expression levels of AST, ALT and BUN.
H&E stainingH & E staining
폐로부터 조직 블록을 수집하여 12시간 동안 4% 파라포름알데하이드에 고정하고 왁스에 포매하였다. 관상절편은 왁스를 제거하고, 헤마톡실린&에오신(H&E)으로 염색한 뒤, 광학현미경으로 세포침윤을 검사하였으며, 디지털 이미지를 얻었다. Tissue blocks were collected from the lungs and fixed in 4% paraformaldehyde for 12 hours and embedded in wax. The coronal sections were removed with wax, stained with hematoxylin and eosin (H & E), examined for cellular infiltration with an optical microscope, and digital images were obtained.
혈청 중 아스파테이트 아미노전이효소(AST), 알라닌 아미노전이효소(ALT) 및 혈중요소질소(BUN) 농도의 수준은 토탈 실험 자동화(Hitachi, Japan) 및 TBA-200FR NEO(Toshiba, Japan) 시스템을 사용하여 분석하였다. (×200)Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels in serum were measured using total laboratory automation (Hitachi, Japan) and TBA-200FR NEO (Toshiba, Japan) Respectively. (× 200)
도 15에 나타낸 바와 같이, 폐로 침윤되는 염증 세포가 감소된 것을 H&E staining을 통해 확인할 수 있다.As shown in FIG. 15, it can be confirmed by H & E staining that the inflammatory cells infiltrating into the lung are reduced.
총 gun 균수Number of bacteria 계수 Coefficient
다음으로, 희생시점에서 폐, 간, 신장을 멸균시키고, 1 ml 살균 PBS에 넣었다. 그 다음 통기 후드 하의 얼음 상에서 조직을 균질기로 균질화하였다. 폐, 간, 신장 균질물은 1:1000으로 희석하였고, 각 희석액 10 ㎕를 LB 한천 플레이트에 도말하였다. 플레이트를 37 ℃에서 24 시간 동안 배양 한 후, 콜로니를 계수하였다.Next, at the sacrifice time point, the lungs, liver, and kidneys were sterilized and placed in 1 ml sterile PBS. The tissue was then homogenized with a homogenizer on ice under aeration hood. The lung, liver, and kidney homogenates were diluted 1: 1000 and 10 μl of each dilution was plated on an LB agar plate. Plates were incubated at 37 [deg.] C for 24 hours and colonies counted.
도 16에 나타낸 바와 같이, 타마리세틴의 투여에 따라 총 균수가 감소하였다. As shown in Fig. 16, the total number of bacteria was decreased by administration of tamarisket.
혈액 내 In the blood LPSLPS 검출 detection
LPS의 수준은 제조사의 권고에 따라 Limulus Amebocyte Lysate(LAL) 발색 종점 분석(Lonza Group Ltd, Allendale, New Jersey, USA)을 사용하여 혈액에서 측정 하였다. 분석 전에 혈장을 내독소가 없는 PBS(endotoxin-free PBS)에서 10 배 희석시켰다. 백그라운드 수준의 감산 결과는 분석과 함께 제공되는 Escherichia coli endotoxin 표준과 비교하여 계산되었으며, LPS의 결과는 EU/mL로 표시하였다.LPS levels were measured in the blood using Limulus Amebocyte Lysate (LAL) color endpoint assay (Lonza Group Ltd, Allendale, New Jersey, USA) according to the manufacturer's recommendations. Plasma was diluted 10-fold in endotoxin-free PBS without endotoxin before analysis. The background subtraction results were calculated relative to the Escherichia coli endotoxin standard provided with the assay and the results of LPS were expressed as EU / mL.
도 17에 나타낸 바와 같이, 혈청 내 독소 농도가 타마리세틴의 투여로 감소되었다. As shown in Fig. 17, the concentration of serum endotoxin was reduced by the administration of Tamaricetin.
타마리세틴에 의한 면역 세포 집단의 IL-10 분비 증가를 확인하기 위하여 마우스의 복강에 타마리세틴(1 mg/kg)을 주사하고, 1시간 뒤에 LPS (10 mg/kg)를 주사하였다. LPS 주사 12시간 후 비장을 수집하였다. RBC를 용해시킨 후 비장 세포를 5시간 동안 5 시간 동안 PMA (50 ng/ml), Ionomycin (0.5 μg/ml), Monensin 및 LPS (10 ㎍/ml)로 자극하였다. 비장 세포를 F40 / 80, CD19, CD11c, CD11b, CD4 및 IL-10에 대하여 염색하였으며, 유동 세포 계측기로 평가하였다. Tamarisetin (1 mg / kg) was injected into the peritoneal cavity of mice and LPS (10 mg / kg) was injected 1 hour later to confirm the increase of IL-10 secretion by the immature cell population by tamariceptin. The spleen was collected after 12 hours of LPS injection. After dissolving RBC, spleen cells were stimulated with PMA (50 ng / ml), Ionomycin (0.5 μg / ml), Monensin and LPS (10 μg / ml) for 5 hours for 5 hours. Splenocytes were stained for F40 / 80, CD19, CD11c, CD11b, CD4 and IL-10 and evaluated by flow cytometry.
유세포 분석(Flow cytometry)은 구체적으로 마우스 비장 세포를 분리한 뒤, WBC를 풍부하게 하기 위해, RBC를 실온에서 15분동안 RBC 용해버퍼를 사용하여 용해시켰다. WBC를 차가운 PBS 완충액으로 세척하였다. WBC를 계수기를 사용하여 계수하고, 24-웰 세포 배양 판에 3×106 세포/웰로 접종하였다. 비장 세포는 37 ℃, 5 % CO2를 함유한 환경에서 5 시간 동안 PMA (50 ng/ml), Ionomycin (0.5 μg/ml), Monensin 및 LPS (10 ㎍/ml)로 자극하였다. 표면 항체(ms anti-F40/80-FITC, ms anti-CD19 APC, ms anti-CD11c APC, ms anti-CD11b PE-cy7, ms anti-CD4 PE-cy7) 및 세포 내 항체(ms anti-IL-10 PE)는 PBS에서 준비하였다. 비장세포 WBC를 V-바닥 플레이트에서 회전시키고, 차가운 PBS로 세척하고 30분 동안 표면항체를 염색하였다. 얼음 위에서 30분 후, 세포를 회전시키고, 차가운 PBS로 세척하였다. 그 다음 비장 세포 백혈구를 4 % 파라포름알데히드에 고정시키고 perm buffer로 염색된 세포 내 항체를 사용하여 20 분간 투과시켰다. 그리고 유세포 분석기(BD Biosciences; San Jose, CA)로 비장 세포를 평가했다.Flow cytometry specifically dissociates mouse spleen cells and then dissolves the RBCs using RBC lysis buffer for 15 minutes at room temperature to enrich the WBCs. The WBCs were washed with cold PBS buffer. The WBC counted using a counter and a 24-well cell culture plate was inoculated in 3 × 10 6 cells / well. Splenocytes were stimulated with PMA (50 ng / ml), Ionomycin (0.5 μg / ml), Monensin and LPS (10 μg / ml) for 5 hours in an environment containing 37 ° C and 5% CO 2 . (Anti-IL-8) antibody and anti-IL-8 antibody (anti-CD40 mAb, anti-CD40 mAb, anti- 10 PE) were prepared in PBS. Spleen cell WBCs were spun on V-bottom plates, washed with cold PBS and stained with surface antibodies for 30 minutes. After 30 minutes on ice, cells were spun and washed with cold PBS. The spleen cell white blood cells were then fixed in 4% paraformaldehyde and permeabilized for 20 minutes using an intracellular antibody stained with perm buffer. And splenocytes were assessed by flow cytometry (BD Biosciences; San Jose, Calif.).
LPS 내독소혈증 모델의 지라에서 IL-10을 분비하는 면역세포의 집단을 확인한 결과 도 18에 나타낸 바와 같이, 타마리세틴에 의해 면역 세포들이 증가된 것을 확인되었다.As shown in Fig. 18, it was confirmed that the immune cells were increased by tamarisetin by confirming the group of IL-10-secreting immune cells in the LPS endotoxemia model model.
시험예 8. 비교실험Test Example 8. Comparative Experiment
IL-10 발현IL-10 expression
퀘르세틴을 이용하여 타마리세틴과의 효과를 비교하였다. Quercetin was used to compare the effects of tamarisetin.
수지상 세포를 타마리세틴 (25μM) 및 퀘르세틴 (25μM)으로 30분 동안 전처리한 뒤, LPS(50ng/mL)로 6시간 동안 자극하였다. 배양 배지를 수집하고, 배양 상등액 중의 IL-10 발현양을 ELISA를 사용하여 제조사의 지시에 따라 결정하였으며, 이를 하기 도 19에 나타내었다. The dendritic cells were pretreated with tamarisetin (25 μM) and quercetin (25 μM) for 30 min and stimulated with LPS (50 ng / mL) for 6 h. The culture medium was collected and the amount of IL-10 expressed in the culture supernatant was determined according to the manufacturer's instructions using ELISA, as shown in Fig.
퀘르세틴은 항염증성 사이토카인 IL-10을 전혀 발현시키지 못하였으나, 타마리세틴은 IL-10의 발현양이 현저하게 향상되었다. Quercetin did not express the anti-inflammatory cytokine IL-10 at all, but tamariceptin significantly increased IL-10 expression.
세포생존율Cell survival rate
시험예 7에서와 동일한 방법으로 세포 생존율을 측정하였다. 퀘르세틴의 처리는 17분 후 세포생존율이 급속도로 저하되었으나, 타마리세틴은 45분 이후까지 80%이상이 생존하였다. Cell viability was measured in the same manner as in Test Example 7. Quercetin treatment rapidly decreased the cell viability after 17 min, but remained more than 80% until 45 min after tamarisk.
항박테리아Antibacterial 활성 평가 Activity evaluation
마우스의 복강에 퀘르세틴(1 mg/kg)을 주사하고, 1시간 뒤에 E.coli K1 (3×107 CFU/mice)을 주사하였다. 대장균 주입 2시간 후 혈청을 수집하였다. 희생시점에서 폐, 간, 신장을 멸균시키고, 1 ml 살균 PBS에 넣었다. 그 다음 통기 후드 하의 얼음 상에서 조직을 균질기로 균질화하였다. 폐, 간, 신장 균질물은 1:1000으로 희석하였고, 각 희석액 10 ㎕를 LB 한천 플레이트에 도말하였다. 플레이트를 37 ℃에서 24 시간 동안 배양 한 후, 콜로니를 계수하였다. Quercetin (1 mg / kg) was injected into the abdominal cavity of mice and injected with E. coli K1 (3 x 10 7 CFU / mice) 1 hour later. Serum was collected 2 hours after the injection of E. coli. At the time of sacrifice, the lungs, liver, and kidneys were sterilized and placed in 1 ml sterile PBS. The tissue was then homogenized with a homogenizer on ice under aeration hood. The lung, liver, and kidney homogenates were diluted 1: 1000 and 10 μl of each dilution was plated on an LB agar plate. Plates were incubated at 37 [deg.] C for 24 hours and colonies counted.
LPS의 수준은 제조사의 권고에 따라 Limulus Amebocyte Lysate(LAL) 발색 종점 분석(Lonza Group Ltd, Allendale, New Jersey, USA)을 사용하여 혈액에서 측정하였다. 분석 전에 혈장을 내독소가 없는 PBS(endotoxin-free PBS)에서 10 배 희석시켰다. 백그라운드 수준의 감산 결과는 분석과 함께 제공되는 Escherichia coli endotoxin 표준과 비교하여 계산되었으며, LPS의 결과는 EU/mL로 표시하였다.LPS levels were measured in the blood using Limulus Amebocyte Lysate (LAL) color endpoint assay (Lonza Group Ltd, Allendale, New Jersey, USA) according to the manufacturer's recommendations. Plasma was diluted 10-fold in endotoxin-free PBS without endotoxin before analysis. The background subtraction results were calculated relative to the Escherichia coli endotoxin standard provided with the assay and the results of LPS were expressed as EU / mL.
도 21에 나타낸 바와 같이, 대장균의 감염에 의한 염증 반응으로 조직이 파괴되었으며, 보호막이 망가져 박테리아가 다른 조직으로 퍼지게 된다. 본 발명에 따른 타마리세틴은 염증 반응을 억제한 반면, 퀘르세틴은 폐와 신장에서 대장균의 cfi 양에 전혀 영향을 주지 않았고, 혈액 내 대장균 내독소인 LPS 역시 감소시키지 못하였다.As shown in Fig. 21, the tissue is destroyed by the inflammatory reaction caused by the infection of E. coli, and the protective membrane is destroyed, and the bacteria spread to other tissues. Tamaricetin according to the present invention inhibited the inflammatory response, whereas quercetin did not affect the cfi amount of E. coli in the lungs and kidneys, nor did it reduce the E. coli endotoxin, LPS, in the blood.
Claims (7)
[화학식 1]
상기 화학식에서,
R1은 C1 - 6알킬이다.A pharmaceutical composition for preventing or treating sepsis or septic shock comprising a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]
In the above formulas,
R 1 is C 1 - 6 is alkyl.
상기 패혈증 또는 패혈증성 쇼크는 내독소혈증(endotoxemia) 또는 박테리아 감염으로 유발된 것임을 특징으로 하는, 약학적 조성물.The method according to claim 1,
Wherein said sepsis or septic shock is caused by an endotoxemia or bacterial infection.
상기 박테리아는 황색포도상구균(Staphylococcus aureus), 바실러스 세레우스(Bacillus cereus), 클로스트리디움 퍼프린젠스(Clostridium perfringens), 대장균(Escherichia coli), 살모넬라 엔테라이티디스(Salmonella enteritidis) 및 캄필로박터 제주니(Campylobacter jejuni) 중에서 선택되는 어느 하나 이상의 균인 것을 특징으로 하는, 약학적 조성물.3. The method of claim 2,
The bacteria may be selected from the group consisting of Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Escherichia coli, Salmonella enteritidis and Campylobacter jejuni. (Campylobacter jejuni). ≪ / RTI >
R1이 C1 - 3알킬인 것을 특징으로 하는, 약학적 조성물.The method according to claim 1,
R 1 is C 1 - 3 alkyl, characterized in that the pharmaceutical composition.
항염증 사이토카인 IL-10의 발현을 증가시키는 것을 특징으로 하는, 약학적 조성물.The method according to claim 1,
Lt; RTI ID = 0.0 > anti-inflammatory < / RTI > cytokine IL-10.
[화학식 1]
상기 화학식에서,
R1은 C1 - 6알킬이다.A composition for enhancing the expression of an anti-inflammatory cytokine IL-10 comprising a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]
In the above formulas,
R 1 is C 1 - 6 is alkyl.
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| PCT/KR2018/009652 WO2019039857A1 (en) | 2017-08-25 | 2018-08-22 | Pharmaceutical composition for preventing or treating sepsis or septic shock, comprising tamarixetin as active ingredient |
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| KR20210129817A (en) | 2020-04-21 | 2021-10-29 | 한국식품연구원 | A composition for weight control comprising benzopyran |
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| WO2011021833A2 (en) * | 2009-08-17 | 2011-02-24 | 경희대학교 산학협력단 | Composition for preventing or treating inflammation |
| KR101286743B1 (en) * | 2011-02-18 | 2013-07-15 | 충남대학교산학협력단 | Pharmaceutical composition containing fenofibrate for prevention of sepsis |
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