KR20220046902A - Composition Comprising Korean Mistletoe Extract For Preventing Or Treating Cancer - Google Patents
Composition Comprising Korean Mistletoe Extract For Preventing Or Treating Cancer Download PDFInfo
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- KR20220046902A KR20220046902A KR1020200130155A KR20200130155A KR20220046902A KR 20220046902 A KR20220046902 A KR 20220046902A KR 1020200130155 A KR1020200130155 A KR 1020200130155A KR 20200130155 A KR20200130155 A KR 20200130155A KR 20220046902 A KR20220046902 A KR 20220046902A
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- colorectal cancer
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- mistletoe extract
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Abstract
Description
본 발명은 한국산 겨우살이 추출물을 유효성분으로 포함하는 암 예방 및 치료용 조성물에 관한 것으로 상기 조성물은 COX-2의 발현을 억제시키고 15-PGDH의 발현을 증가시키며 및 STAT3 인산화를 억제하므로 만성염증으로 인한 대장암의 발병을 예방하고 치료하는 약학조성물 또는 건강기능식품으로 제공되는 특징이 있다. The present invention relates to a cancer prevention and treatment composition comprising a Korean mistletoe extract as an active ingredient, and the composition suppresses the expression of COX-2, increases the expression of 15-PGDH, and inhibits STAT3 phosphorylation, thereby causing chronic inflammation. It is characterized in that it is provided as a pharmaceutical composition or health functional food for preventing and treating the onset of colorectal cancer.
대장암은 유전체 불안정성(genomic instability)에 기인하여 이형성 전구병변을 통해 발생되는 산발성 대장암(sporadic colorectal carcinoma, SCC)과 장상피세포에 발생한 염증이 반복 또는 장기화되는 과정을 통해 발생하는 염증성 대장암(colitis-associated colorectal cancer, CAC)로 구분된다. Colorectal cancer is sporadic colorectal carcinoma (SCC), which occurs through dysplastic precursor lesions due to genomic instability, and inflammatory colorectal cancer (SCC) that occurs through repeated or prolonged inflammation in intestinal epithelial cells ( colitis-associated colorectal cancer (CAC).
상기 SCC는 전체 대장암의 65 내지 85%를 차지하며 세포 분열시 염색체가 불안정하게 분리되거나 비정상적인 염색체의 배수체를 보이는 염색체 불안정성(chromosomal instability) 및 DNA 보수효소 유전자(DNA misnatch repair gene)의 기능손실로 인한 현미부수체 불안정성(microstatellite instability)에 의해 발생한 용종을 거쳐 암화되는 선종암종연쇄(adenoma-carcinoma sequence)경로를 거치는 것으로 알려져 있다. The SCC accounts for 65 to 85% of all colorectal cancers and is caused by chromosomal instability and loss of function of the DNA misnatch repair gene, in which chromosomes are unstable during cell division or show an abnormal chromosomal ploidy. It is known that it goes through the adenoma-carcinoma sequence, which is cancerous through polyps caused by microstatellite instability.
이에 반하여 상기 CAC는 전체 대장암의 15 내지 35%를 차지하며 장상피세포에서 발생한 염증이 반복 및 장기화되어 저이형성(low-grade dysplasia) 단계로부터 고이형성(high-grad dysplasia) 단계를 거쳐 암화되는 염증이형성암종연쇄(inflammation-dysplasia-carcinoma sequence)경로를 거지는 것으로 알려져 있다.On the other hand, the CAC accounts for 15 to 35% of all colorectal cancers, and the inflammation generated in the intestinal epithelial cells is repeated and prolonged, leading to cancer from the low-grade dysplasia stage to the high-grade dysplasia stage. Inflammation-dysplasia-carcinoma sequence (inflammation-dysplasia-carcinoma sequence) is known to pass through.
상기 SCC와 CAC는 중요한 분자기전에 관여하는 유전자를 대부분 공유하는 공통점이 있으나 상기 유전자의 시간적 단계와 변이 빈도에 따라 매우 다른 양상을 보이는 특징이 있다.The SCC and CAC share most of the genes involved in important molecular mechanisms in common, but they show very different aspects depending on the temporal stage and mutation frequency of the genes.
특히, CAC의 경우 만성염증으로부터 발병되는 특징이 있어 염증성 장질환을 야기하는 유전자가 CAC를 유발한다는 것이 설득력 있는 기전으로 받아들여지고 있다. In particular, since CAC is characterized by chronic inflammation, it is accepted as a convincing mechanism that a gene causing inflammatory bowel disease induces CAC.
염증과정 중에는 산화스트레스가 증가하게 되고 상기 산화스트레스는 DNA의 변이를 일으켜 암화의 원인이 된다. 상기 산화스트레스는 iNOS 및 COX-2와 같은 유전자들이 과발현에 의해 유발된다. 또한 염증을 겪는 장상피세포는 정상적인 장상피세포에 비하여 성장속도와 사멸속도가 증가하게 되는데 이 과정에서 성장과 사멸의 불균형을 초래하게 되면 암화가 진행되는 것이다. During the inflammatory process, oxidative stress increases, and the oxidative stress causes DNA mutations, which causes cancer. The oxidative stress is induced by overexpression of genes such as iNOS and COX-2. In addition, the rate of growth and death of intestinal epithelial cells suffering from inflammation is increased compared to that of normal intestinal epithelial cells.
따라서 상기 만성염증을 완화하는 방법으로 염증이형성암종연쇄(inflammation-dysplasia-carcinoma sequence)경로를 통한 CAC의 발병을 억제하기 위하여 대장염 초기 치료제인 COX-2 억제제에 대한 연구가 진행되고 있으나 염증완화를 통한 암화 예방에 관한 것일 뿐 직접적으로 암세포의 성장을 억제하는 효과는 입증된 바 없어 대장암을 치료제 사용할 수 없는 한계가 있었다.Therefore, in order to suppress the onset of CAC through the inflammation-dysplasia-carcinoma sequence pathway as a method of alleviating the chronic inflammation, research on a COX-2 inhibitor, an initial treatment for colitis, is being conducted, but the It is only related to cancer prevention through cancer treatment, but there is a limitation in that it cannot be used as a treatment for colorectal cancer because the effect of directly inhibiting the growth of cancer cells has not been proven.
겨우살이(mistletoe)는 나무에 붙어 생장하며 스스로 광합성도 하면서 나무에 기생하여 살아가는 반기생 식물이다. 겨우살이 추출물은 고혈압, 발작, 체력 소모, 불안증, 관절염, 어지럼증, 퇴행성관절염 등과 같은 다양한 질병에 대한 치료제로서 민간에서 사용되어 왔으며 췌장암 및 대장암 환자의 생존기간을 연장시키는 효과가 확인되어 항화학용법 및 방사선 치료의 부작용을 완화시키는 주사제로 사용된다. Mistletoe is a semi-parasitic plant that grows attached to trees and lives as a parasitic tree while photosynthesizing itself. Mistletoe extract has been used in the private sector as a treatment for various diseases such as high blood pressure, seizures, physical exhaustion, anxiety, arthritis, dizziness, and degenerative arthritis. It is used as an injection to relieve the side effects of radiation therapy.
KR10-1951403에는 겨우살이 추출물이 대장염 생쥐모델에서 염증유발인자(IL-6) 및 골수세포형과산화효소(myeloperoxidase)의 발현을 감소시키므로 염증성 장질환 예방 및 장질환 치료용도로서 사용 가능하다는 것을 기재하고 있으며; KR10-1971986에는 겨우살이 추출물이 대장암 세포주(HCT116)의 성장을 억제하므로 항암용 조성물로서 사용 가능하다는 것으로 기재하고 있으며; KR10-2011-0090409에는 겨우살이 추출물이 대식세포에서 COX-2의 발현을 억제하여 염증을 완화하므로 항염활성을 가지는 천연화장료로 사용 가능하다는 결과를 기재하고 있다.KR10-1951403 describes that mistletoe extract can be used for the prevention and treatment of inflammatory bowel disease because it reduces the expression of proinflammatory factor (IL-6) and myeloperoxidase in a mouse model of colitis. ; KR10-1971986 describes that mistletoe extract can be used as an anticancer composition because it inhibits the growth of colon cancer cell line (HCT116); KR10-2011-0090409 describes the results that mistletoe extract can be used as a natural cosmetic with anti-inflammatory activity because it relieves inflammation by suppressing the expression of COX-2 in macrophages.
상기 결과들은 겨우살이 추출물이 대장염 또는 대장암을 억제한다는 결과를 보여주나 장상피세포에서 COX-2의 억제를 통해 염증을 완화시키거나 상세한 항암기작에 대한 언급이 없다. 다만 대식세포에서 겨우살이 추출물에 의한 COX-2 발현억제가 확인되나 이는 대식세포에 한정된 것으로 장상피세포에서 동일한 효과가 있는 지에 대하여 유추할 수 없는 한계가 있다.The above results show that the mistletoe extract suppresses colitis or colorectal cancer, but there is no mention of a detailed anticancer mechanism or alleviating inflammation through inhibition of COX-2 in intestinal epithelial cells. However, inhibition of COX-2 expression by mistletoe extract was confirmed in macrophages, but it is limited to macrophages, and there is a limit that cannot be inferred whether the same effect is obtained in intestinal epithelial cells.
따라서 겨우살이 추출물이 장상피세포에서 COX-2 억제하여 염증을 완화시키는 동시에 대장암을 억제하는 효과가 있다는 것이 실험적으로 증명된다면 만성염증으로 인해 발병하는 대장암을 예방하는 COX-2 억제제인 동시에 대장암 세포를 사멸시키는 대장암 치료제로서 사용 가능할 것으로 기대된다. Therefore, if it is experimentally proven that mistletoe extract has the effect of inhibiting COX-2 in intestinal epithelial cells to relieve inflammation and at the same time suppress colorectal cancer, it is a COX-2 inhibitor that prevents colorectal cancer caused by chronic inflammation and colorectal cancer. It is expected to be usable as a treatment for colorectal cancer that kills cells.
본 명세서에서 언급된 특허문헌 및 참고문헌은 각각의 문헌이 참조에 의해 개별적이고 명확하게 특정된 것과 동일한 정도로 본 명세서에 참조로 삽입된다. The patents and references mentioned in this specification are hereby incorporated by reference to the same extent as if each publication were individually and expressly specified by reference.
본 발명은 상기 문제점을 해결하기 위하여 장상피세포 및 대장암세포주에서 한국산 겨우살이 추출물을 이용한 염증완화효과 및 항암효과를 확인한 결과 한국산 겨우살이 추출물이 COX-2의 발현을 억제하여 염증을 완화시키는 동시에 15-PGDH의 발현을 향상시키고 STAT3의 인산화를 억제시키므로 대장암세포의 증식을 억제한다는 것을 실험적으로 확인하였다. 따라서 본 발명의 목적은 한국산 겨우살이 추출물을 유효성분으로 포함하는 염증성 대장암 예방 및 치료용 약학조성물 또는 염증성 대장암 예방 또는 개선용 건강기능식품을 제공하는데 있다.In order to solve the above problems, the present invention confirmed the anti-inflammatory and anticancer effects using Korean mistletoe extract in intestinal epithelial cells and colon cancer cell lines. It was confirmed experimentally that it suppressed the proliferation of colorectal cancer cells by enhancing the expression of PGDH and inhibiting the phosphorylation of STAT3. Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating inflammatory colorectal cancer, or a health functional food for preventing or improving inflammatory colorectal cancer, comprising a Korean mistletoe extract as an active ingredient.
본 발명의 다른 목적 및 기술적 특징은 이하의 발명의 상세한 설명, 청구의 범위 및 도면에 의해 보다 구체적으로 제시된다. Other objects and technical features of the present invention are more specifically set forth by the following detailed description of the invention, claims and drawings.
본 발명은 한국산 겨우살이 추출물을 유효성분으로 포함하는 염증성 대장암 예방 및 치료용 약학조성물 또는 염증성 대장암 예방 또는 개선용 건강기능식품을 제공한다.The present invention provides a pharmaceutical composition for preventing and treating inflammatory colorectal cancer, or a health functional food for preventing or improving inflammatory colorectal cancer, comprising a Korean mistletoe extract as an active ingredient.
상기 약학 조성물 및 건강기능식품은 COX-2 발현을 억제하여 대장의 염증을 완화시키며 동시에 15-PGDH의 발현을 증가시키고 STAT3 인산화를 억제하여 염증에 의한 암화를 억제하는 것을 특징으로 한다.The pharmaceutical composition and health functional food suppress the expression of COX-2 to relieve inflammation of the colon, and at the same time increase the expression of 15-PGDH and inhibit STAT3 phosphorylation to suppress cancerous cancer caused by inflammation.
상기 한국산 겨우살이 추출물은 참나무를 숙주로 하는 한국산 겨우살이(Viscum album coloratum)로부터 수추출한 것이며 랙틴(lectin) 함량이 100㎍/㎖인 것을 특징으로 한다.The Korean mistletoe extract is water-extracted from Korean mistletoe (Viscum album coloratum) using oak as a host, and the lectin content is 100 μg/ml.
본 발명의 한국산 겨우살이 추출물을 유효성분으로 포함하는 염증성 대장암 예방 및 치료용 약학조성물 및 염증성 대장암 예방 또는 개선용 건강기능식품은 COX-2의 발현을 감소시키는 동시에 15-PGDH의 발현을 증가시키고 STAT3 인산화를 억제시키므로 COX-2 억제제로서 염증성 대장암을 예방하는 효과 뿐 아니라 대장암을 치료 및 개선 할 수 있는 장점이 있다.The pharmaceutical composition for the prevention and treatment of inflammatory colorectal cancer and the health functional food for the prevention or improvement of inflammatory colorectal cancer comprising the Korean mistletoe extract as an active ingredient of the present invention decrease the expression of COX-2 and increase the expression of 15-PGDH, As it inhibits STAT3 phosphorylation, it has the advantage of preventing inflammatory colorectal cancer as a COX-2 inhibitor as well as treating and improving colorectal cancer.
도 1은 본 발명의 한국산 겨우살이 추출물에 대한 대장상피세포(CCD841)의 세포생존력(cell viability) 측정 결과를 보여준다. 패널 A는 한국산 겨우살이 추출물(KME)를 투여한 후 24시간 동안 배양한 대장상피세포(CCD841)의 생존력 측정 결과를 보여주며 패널 B는 KME를 투여한 후 48시간 동안 배양한 대장상피세포(CCD841)의 생존력 측정 결과를 보여준다.
도 2는 본 발명의 한국산 겨우살이 추출물에 대한 대장암 세포주(HCT116)의 세포성장 변화결과를 보여준다. 패널 A는 KME를 투여한 후 24시간 동안 배양한 대장암 세포주(HCT116)의 세포성장 변화결과를 보여주며, 패널 B는 KME를 투여한 후 48시간 동안 배양한 대장암 세포주(HCT116)의 세포성장 변화결과를 보여주며, 패널 C는 KME를 투여한 후 72시간 동안 배양한 대장암 세포주(HCT116)의 세포성장 변화결과를 보여준다.
도 3은 본 발명의 한국산 겨우살이 추출물에 대한 간암 세포주(SK-Hep1)의 세포성장 변화결과를 보여준다. 패널 A는 KME를 투여한 후 24시간 동안 배양한 간암 세포주(SK-Hep1)의 세포성장 변화결과를 보여주며, 패널 B는 KME를 투여한 후 48시간 동안 배양한 간암 세포주(SK-Hep1)의 세포성장 변화결과를 보여주며, 패널 C는 KME를 투여한 후 72시간 동안 배양한 간암 세포주(SK-Hep1)의 세포성장 변화결과를 보여준다.
도 4는 본 발명의 한국산 겨우살이 추출물에 대한 대장암 세포주(HCT116)의 세포이동 정도의 변화를 보여준다.
도 5는 본 발명의 IL-6에 의한 대장상피세포(CCD841)의 염증관련 분자지표의 발현 변화를 보여준다. 패널 A는 IL-6의 투여량에 따라 염증관련 분자지표의 발현량이 조절되는 것을 보여주며, 패널 B는 IL-6의 투여량에 따른 STAT3의 인산화(p-STAT3) 정도를 보여주며, 패널 C는 IL-6의 투여량에 따른 15-PGDH의 발현 정도를 보여준다.
도 6은 본 발명의 한국산 겨우살이 추출물에 대한 대장상피세포(CCD841)의 염증 관련 분자지표의 발현 변화를 보여준다.
도 7은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델에 대한 실험을 보여준다.
도 8은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 질병활동지수 분석결과를 보여준다.
도 9는 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 대장길이 분석결과를 보여준다. 패널 A는 DSS를 투여하여 형성한 대장염 동물모델에 KME를 투여한 후 이를 희생하여 대장의 길이를 측정한 결과를 보여주며 패널 B는 상기 대장 길이를 정량적으로 측정한 결과를 그래프로 보여준다.
도 10은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 대장조직 분석결과를 보여준다.
도 11은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 염증지표(COX-2)의 발현 분석결과를 보여준다. 패널 A는 한국산 겨우살이 추출물에 따른 COX-2의 발현 변화를 보여주며, 패널 B는 한국산 겨우살이 추출물에 따른 COX-2의 발현 변화를 정량적으로 측정한 결과를 그래프로 보여준다.
도 12은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 종양억제유전자(15-PGDH)의 발현 분석결과를 보여준다.
도 13은 본 발명의 한국산 겨우살이 추출물이 투여된 대장염 동물모델의 종양억제유전자(15-PGDH)의 발현 분석결과를 보여준다. 1 shows the measurement results of cell viability of colonic epithelial cells (CCD841) to the Korean mistletoe extract of the present invention. Panel A shows the viability measurement results of colonic epithelial cells (CCD841) cultured for 24 hours after administration of Korean mistletoe extract (KME), and panel B shows colorectal epithelial cells cultured for 48 hours after KME administration (CCD841). shows the viability measurement results.
Figure 2 shows the cell growth change of the colon cancer cell line (HCT116) to the Korean mistletoe extract of the present invention. Panel A shows the cell growth results of the colorectal cancer cell line (HCT116) cultured for 24 hours after administration of KME, and panel B shows the cell growth of the colon cancer cell line (HCT116) cultured for 48 hours after administration of KME. Shows the change results, and panel C shows the cell growth changes of the colon cancer cell line (HCT116) cultured for 72 hours after KME administration.
3 shows the cell growth change results of the liver cancer cell line (SK-Hep1) to the Korean mistletoe extract of the present invention. Panel A shows the results of cell growth changes in the liver cancer cell line (SK-Hep1) cultured for 24 hours after KME administration, and Panel B shows the results of cell growth changes in the liver cancer cell line (SK-Hep1) cultured for 48 hours after KME administration. Shows the cell growth change results, and panel C shows the cell growth changes of the liver cancer cell line (SK-Hep1) cultured for 72 hours after KME administration.
Figure 4 shows the change in the degree of cell migration of the colon cancer cell line (HCT116) to the Korean mistletoe extract of the present invention.
5 shows changes in the expression of inflammation-related molecular markers in colonic epithelial cells (CCD841) by IL-6 of the present invention. Panel A shows that the expression level of inflammation-related molecular indicators is regulated according to the dose of IL-6, panel B shows the degree of phosphorylation (p-STAT3) of STAT3 according to the dose of IL-6, and panel C shows the expression level of 15-PGDH according to the dose of IL-6.
6 shows changes in the expression of inflammation-related molecular markers in colonic epithelial cells (CCD841) in response to the Korean mistletoe extract of the present invention.
7 shows an experiment on an animal model of colitis administered with a Korean mistletoe extract of the present invention.
8 shows the disease activity index analysis result of the colitis animal model administered with the Korean mistletoe extract of the present invention.
9 shows the results of analysis of colon length in an animal model of colitis to which the Korean mistletoe extract of the present invention was administered. Panel A shows the results of measuring the length of the large intestine by sacrificing KME after administration of KME to an animal model of colitis formed by administering DSS, and panel B shows the results of quantitatively measuring the length of the large intestine as a graph.
10 shows the results of analysis of colonic tissue in an animal model of colitis to which the Korean mistletoe extract of the present invention was administered.
11 shows the results of analysis of the expression of the inflammatory marker (COX-2) in the colitis animal model to which the Korean mistletoe extract of the present invention was administered. Panel A shows the expression change of COX-2 according to the Korean mistletoe extract, and panel B shows the result of quantitatively measuring the expression change of COX-2 according to the Korean mistletoe extract.
12 shows the results of analysis of the expression of the tumor suppressor gene (15-PGDH) in the colitis animal model administered with the Korean mistletoe extract of the present invention.
13 shows the expression analysis results of the tumor suppressor gene (15-PGDH) in the colitis animal model to which the Korean mistletoe extract of the present invention was administered.
본 발명은 한국산 겨우살이 추출물을 유효성분으로 포함하는 염증성 대장암 예방 및 치료용 약학조성물 또는 염증성 대장암 예방 또는 개선용 건강기능식품을 제공한다.The present invention provides a pharmaceutical composition for preventing and treating inflammatory colorectal cancer, or a health functional food for preventing or improving inflammatory colorectal cancer, comprising a Korean mistletoe extract as an active ingredient.
상기 한국산 겨우살이 추출물은 랙틴(lectin) 함량이 100㎍/㎖인 것을 특징으로 하며 한국에서 자생하는 겨우살이(mistletoe)로서 참나무겨우살이(Taxillus yadoriki (Siebold) Danser)의 추출물이며 녹나무(Cinnamomum camphora), 상수리나무(Quercusacutissima Carruth), 삼나무(Cryptomeria japonica), 참식나무(Neolitsea sericea) 및 벚나무(Prunus serrulata)에 기생하는 겨우살이를 채집하여 열수추출 또는 냉수추출로 추출한 것을 의미한다. The Korean mistletoe extract is characterized in that the lectin content is 100 μg/ml, and it is an extract of the oak mistletoe (Taxillus yadoriki (Siebold) Danser) as a mistletoe that grows wild in Korea, and it is a camphor tree (Cinnamomum camphora), oak tree (Quercusacutissima Carruth), cedar (Cryptomeria japonica), cedar (Neolitsea sericea), and cherry tree (Prunus serrulata) parasitic mistletoe collected and extracted by hot water extraction or cold water extraction.
KR10-1971986은 본 발명의 한국산 참나무겨우살이 추출물을 포함하는 항암용 조성물에 관한 것이 기재되어 있다. KR10-1971986에는 대장암 세포주 HCT116에 대하여 참나무겨우살이 추출물을 처리한 결과, 대장암 세포주의 생존률이 감소하므로 대장암 치료제로서 사용 가능하다는 것을 보여준다. KR10-1971986 discloses a composition for anti-cancer comprising the extract of Korean oak mushroom of the present invention. In KR10-1971986, as a result of treatment with the oak mistletoe extract for the colon cancer cell line HCT116, the survival rate of the colorectal cancer cell line decreases, indicating that it can be used as a colorectal cancer treatment.
이에 반하여 본 발명은 실시예를 통하여, 본 발명의 참나무겨우살이 추출물이 염증 유도된 정상 장상피세포(CCD841 CoN)에서 염증유발 유전자인 COX-2(cyclooxygenase-2)는 감소시키고, 종양억제유전자인 15-PGDH(15-hydroxyprostaglandin dehydrogenase)는 증가시키고, 암유발인자인 STAT3(Signal transducer and activator of transcription 3)의 인산화(pSTAT3)는 억제시키므로 정상 장상피세포가 염증성 대장암으로 암화되는 것을 억제하는 것을 확인하였을 뿐 아니라 대장암 세포주의 HCT116의 생존력을 저하시키는 것이 확인되었다. 따라서 본 발명의 참나무겨우살이 추출물은 염증이 발생한 대장세포의 염증을 완화시켜 대장염을 치료할 수 있을 뿐 아니라 만성염증으로 인한 정상 장상피세포의 암화를 예방하고 이미 암화가 진행된 염증성 대장암 환자에게 제공되어 염증성 대장암을 치료할 수 있을 것으로 판단된다. In contrast, the present invention, through the examples, the oak mistletoe extract of the present invention reduces the inflammation-inducing gene COX-2 (cyclooxygenase-2) in inflammation-induced normal intestinal epithelial cells (CCD841 CoN), and the tumor suppressor gene 15 -It was confirmed that it inhibits the transformation of normal intestinal epithelial cells into inflammatory colorectal cancer by increasing PGDH (15-hydroxyprostaglandin dehydrogenase) and inhibiting phosphorylation (pSTAT3) of STAT3 (Signal transducer and activator of transcription 3), a cancer-causing factor. It was confirmed that not only did it reduce the viability of HCT116 of colorectal cancer cell line. Therefore, the oak mistletoe extract of the present invention can treat colitis by alleviating the inflammation of the inflamed colon cells, as well as prevent the cancer of normal intestinal epithelial cells due to chronic inflammation, and is provided to patients with inflammatory colorectal cancer that has already progressed to cancer. It is believed to be able to treat colorectal cancer.
본 발명은 참나무겨우살이 추출물이 정상 대장상피세포의 염증관련 유전자(COX-2) 및 종양관련 유전자(15-PGDH, pSTAT3)를 조절하므로 정상 장상피세포를 염증반응으로부터 보호하고 이를 통하여 염증성 대장암으로의 암화를 예방하며 이미 진행된 염증성 대장암세포에 대하여는 그 성장을 억제하므로 염증성 대장암을 예방하고 염증성 대장암을 치료할 수 있는 약학조성물에 대한 것이다. 따라서 이미 암화가 진행된 상태의 대장암 세포주만을 사용하여 대장암 치료효과를 확인하는 방법으로 염증성 대장암과 산발성 대장암을 모두 포함하는 일반적인 대장암에 대한 항암효과만을 기재한 선행문헌의 결과와 차별성이 있다.According to the present invention, oak mistletoe extract regulates inflammation-related genes (COX-2) and tumor-related genes (15-PGDH, pSTAT3) of normal colonic epithelial cells, thereby protecting normal intestinal epithelial cells from inflammatory responses, thereby preventing inflammatory colorectal cancer. It relates to a pharmaceutical composition capable of preventing cancer and inhibiting the growth of advanced inflammatory colorectal cancer cells, thereby preventing and treating inflammatory colorectal cancer. Therefore, as a method of confirming the therapeutic effect of colorectal cancer using only colorectal cancer cell lines that have already undergone cancerization, it is different from the results of the previous literature that only described the anticancer effect on general colorectal cancer, including both inflammatory colorectal cancer and sporadic colorectal cancer. there is.
상기 COX-2는 세포막 인지질의 아라키돈산으로부터 프로스타글란딘을 형성시켜 대장 점막 조직의 유지 및 항상성을 유지시키는 단백질로서 염증성 장질환과 대장암에서 과발현되어 염증을 악화시키며 암성장을 촉진하는 특징이 있다. 특히 염증성 대장암에서 염증으로 인한 유전자 변이, 음와세포(crypt cell) 증식, 음와세포 대사변이, 담즙성 장-간 순환의 변화, 장관세균총의 변화를 통하여 염증성 대장암의 발병이 중요한 역할을 하는 것으로 알려져 있어 COX-2의 활성을 억제하므로 대장의 염증을 완화하고 염증성 대장암을 예방하는 COX-2 억제제에 대한 연구가 활발히 진행되고 있는 실정이다.The COX-2 is a protein that forms prostaglandins from arachidonic acid in cell membrane phospholipids to maintain colonic mucosal tissue and homeostasis, and is overexpressed in inflammatory bowel disease and colorectal cancer to exacerbate inflammation and promote cancer growth. In particular, in inflammatory colorectal cancer, the onset of inflammatory colorectal cancer plays an important role through gene mutations, crypt cell proliferation, crypt cell metabolism mutations, biliary intestinal-liver circulation changes, and intestinal microbiota changes. It is known that by inhibiting the activity of COX-2, studies on COX-2 inhibitors that relieve inflammation of the colon and prevent inflammatory colorectal cancer are being actively conducted.
상기 15-PGDH는 COX-2 등에 의해 합성된 프로스타글란딘 E2(prostaglandin E2, PGE2)을 분해하는 효소로서 상기 PGE2는 대장암 세포의 분화, 혈관신생, 세포이동을 포함한 다양한 종양발생단계에 작용하므로 임을 진단하고 예후를 판단하는 중요한 암표지자로서 사용된다. 상기 15-PGDH의 발현이 증가하게 되면 대장암세포의 성장이 억제되는 반면, 상기 15-PGDH의 발현이 감소하게 되면 대장암세포의 성장이 증가하는 것이 확인된 바 있다.The 15-PGDH is an enzyme that decomposes prostaglandin E 2 (prostaglandin E 2 , PGE 2 ) synthesized by COX-2, etc., and the PGE 2 is used in various tumorigenesis stages including colorectal cancer cell differentiation, angiogenesis, and cell migration. Therefore, it is used as an important cancer marker for diagnosing cancer and judging the prognosis. When the expression of 15-PGDH is increased, the growth of colorectal cancer cells is inhibited, whereas when the expression of 15-PGDH is decreased, it has been confirmed that the growth of colorectal cancer cells is increased.
상기 STAT3는 STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, 및 STAT6의 일곱개의 subunit 형태를 가지는 전사인자의 일환으로 상기 STAT3의 C-말단에 위치하는 Tyrosine 705, 및 Serine 727 잔기가 인산화(phosphorylation)되면 활성이 유지된다. 암세포의 경우 활성화된 STAT3가 과발현되는 것이 발견되었으며 Cyclin D1나 c-Myc을 조절하여 암세포의 생장을 증가시키는 것으로 알려져 있다. The STAT3 is a transcription factor having seven subunit forms of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Tyrosine 705 and Serine 727 residues located at the C-terminus of STAT3 are phosphorylated. It will remain active. In the case of cancer cells, it was found that activated STAT3 was overexpressed, and it is known to increase the growth of cancer cells by regulating Cyclin D1 or c-Myc.
본 발명의 실시예에 따르면, 본 발명의 한국산 겨우살이 추출물은 COX-2 발현을 억제하며 15-PGDH의 발현을 증가시키고 STAT3 인산화를 억제하는 것이 실험적으로 확인되었다. 따라서 본 발명의 한국산 겨우살이 추출물은 대장의 염증을 완화시키며 염증성 대장암을 예방 또는 치료할 수 있는 약학조성물로서 사용 가능하다.According to an embodiment of the present invention, it was experimentally confirmed that the Korean mistletoe extract of the present invention suppressed COX-2 expression, increased the expression of 15-PGDH, and inhibited STAT3 phosphorylation. Therefore, the Korean mistletoe extract of the present invention can be used as a pharmaceutical composition to relieve colon inflammation and prevent or treat inflammatory colorectal cancer.
상기 염증성 대장암((colitis-associated colorectal cancer)은 염증이형성암종연쇄(inflammation-dysplasia-carcinoma sequence)경로를 통해 발병하는 암종으로 선종암종연쇄(adenoma-carcinoma sequence)경로를 통해 발병하는 산발성 대장암(sporadic colorectal carcinoma)과 구분된다.The inflammatory colorectal cancer (colitis-associated colorectal cancer) is a carcinoma that occurs through the inflammation-dysplasia-carcinoma sequence pathway, and sporadic colorectal cancer that occurs through the adenoma-carcinoma sequence pathway. (sporadic colorectal carcinoma).
상기 예방은 발병을 억제시키거나 발병을 지연시키는 행위를 의미하며 치료 또는 개선은 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The prevention means an action of suppressing the onset or delaying the onset, and treatment or improvement means any action in which the symptoms of a disease are improved or beneficially changed.
상기 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 추가로 포함 할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 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.The pharmaceutical composition may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The carrier, excipient and diluent include lactose, dextrose, sucrose, 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 oil.
한편, 본 발명의 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화하여 사용될 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 고형제는 상기 혼합물의 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 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 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등On the other hand, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract of the mixture, for example, starch, calcium carbonate, sucrose It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc.
이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.this can be used As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
건강기능식품은 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀,Health functional food uses raw materials or ingredients with useful functions for the human body, such as tablets, capsules,
분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 의미한다. 상기 건강기능식품은 드링크제, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함할 수 있다. 본 발명의 한국산 겨우살이 추출물은 실시예를 통해 안정성이 확인되었으므로 건강기능식품의 사용 목적에 따라 유효성분의 혼합량을 적합하게 결정가능하다. It refers to food manufactured and processed in the form of powder, granules, liquid, and pills. The health functional food includes drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins There are complexes, dairy products, and dairy products, and may include all health functional foods in the ordinary sense. Since the stability of the Korean mistletoe extract of the present invention has been confirmed through Examples, the mixing amount of the active ingredient can be appropriately determined according to the purpose of use of the health functional food.
상기 건강기능식품은 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 또한 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 더 함유할 수 있다.The health functional food may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used. In addition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. may contain more.
하기에서 실시예를 통해 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of Examples.
실시예Example
1. 실험 재료 및 방법1. Experimental Materials and Methods
1.1 세포배양1.1 Cell culture
인체 유래 대장 정상 상피 세포인 CCD841 CoN 및 인체 유래 대장암 세포주인 HCT116 은 ATCC에서 분양받아 사용하였다. Dulbecco’s modified Eagle’s medium (DMEM) 또는 Minimum Essential Medium (MEM) (Hyclone Lab, Inc, Logan, UT, USA), 10% Fetal bovin serum (FBS, HyClone) 100U/ml 페니실린(penicillin) 및 100ug/ml 스트렙토마이신(streptomycin) (Gibco, Grand Island, NY, USA)을 포함하는 배지에서 95%의 습도가 유지되는 37℃, 5% CO2 incubator에서 배양하였다.CCD841 CoN, a human colonic normal epithelial cell, and HCT116, a human colorectal cancer cell line, were purchased from ATCC and used. Dulbecco's modified Eagle's medium (DMEM) or Minimum Essential Medium (MEM) (Hyclone Lab, Inc, Logan, UT, USA), 10% Fetal bovin serum (FBS, HyClone) 100U/ml penicillin and 100ug/ml streptomycin (streptomycin) (Gibco, Grand Island, NY, USA) in a medium containing 95% humidity maintained at 37 ℃, 5% CO 2 Incubated in an incubator.
1.2 세포생존력(cell viability) 측정1.2 Measurement of cell viability
한국산 겨우살이 추출물(korean mistletoe extract, KME)에 대한 세포 생존력(cell viability)의 변화를 측정하기 위하여 대장상피세포(CCD841), 인체 대장암 세포주(HCT116), 인체 간암세포주(SK-Hep1, male)에 KME를 농도에 따라 첨가하고 이를 배양한 후 MTT 분석을 수행하였다. 상기 KME는 참나무를 숙주로 하는 겨우살이 추출물로써 ㈜미슬바이오텍 (Mistle Biotech Co., Ltd., Seoul, Korea)으로부터 제공받았으며 100㎍/ml의 렉틴을 함유하였다. To measure changes in cell viability of Korean mistletoe extract (KME), colonic epithelial cells (CCD841), human colorectal cancer cell line (HCT116), and human liver cancer cell line (SK-Hep1, male) After KME was added according to the concentration and incubated, MTT analysis was performed. The KME was provided by Mistle Biotech Co., Ltd., Seoul, Korea as a mistletoe extract using oak as a host and contained 100 μg/ml of lectin.
상기 MTT 분석은 Thiazolyl Blue Tetrazolium Bromide (Sigma Chemical Co., St Louis, MO, USA ;product No. M2128)를 사용하여 분석하였다. 대장 정상 상피세포 (CCD841 CoN)에 대한 MTT 에세이를 위하여 상기 배양한 CCD841 CoN (1.5 x 105cells/ml)을 48 well 플레이트에 분주하여 24 시간 동안 배양한 후 각 well에 상기 KME가 0㎍/㎖, 0.5㎍/㎖, 1㎍/㎖, 5㎍/㎖ 또는 10㎍/㎖의 농도가 되도록 첨가하여 24시간 또는 48시간 동안 더 배양하였다. 그 후 각 well에 MTT 시약을 넣고 1시간 동안 배양기에서 배양한 후 상기 MTT 시약 제거하고 DMSO를 넣어 흡광도를 측정하였다. The MTT analysis was analyzed using Thiazolyl Blue Tetrazolium Bromide (Sigma Chemical Co., St Louis, MO, USA; product No. M2128). For the MTT assay on colonic normal epithelial cells (CCD841 CoN), the cultured CCD841 CoN (1.5 x 10 5 cells/ml) was dispensed into a 48 well plate and cultured for 24 hours. ㎖, 0.5㎍ / ㎖, 1㎍ / ㎖, 5㎍ / ㎖ or 10㎍ / ㎖ added so as to be further cultured for 24 hours or 48 hours. After that, MTT reagent was put into each well and incubated in an incubator for 1 hour, the MTT reagent was removed, and the absorbance was measured by adding DMSO.
인체 유래 대장암 세포주(HCT116) 및 상기 인체 유래 간암세포주(SK-Hep1, male)의 MTT 분석은 상기와 동일한 방법으로 수행하되, 상기 KME을 0㎍/㎖, 1㎍/㎖, 10㎍/㎖, 100㎍/㎖ 또는 1000㎍/㎖의 농도가 되도록 첨가한 후 24시간 또는 48시간 동안 더 배양하였다.MTT analysis of the human colorectal cancer cell line (HCT116) and the human hepatocellular carcinoma cell line (SK-Hep1, male) was performed in the same manner as above, except that the KME was administered at 0 μg/ml, 1 μg/ml, 10 μg/ml , after addition to a concentration of 100㎍ / ㎖ or 1000㎍ / ㎖ was further cultured for 24 hours or 48 hours.
1.3. 세포 이동 분석(cell migration assay)1.3. cell migration assay
한국산 겨우살이 추출물(korean mistletoe extract, KME)에 대한 세포 이동성(cell motility)을 측정하기 위하여 인체 대장암 세포주 (HCT116)에 대한 세포 이동 분석을 진행하였다. Culture-insert (Ibidi; Martinsried, Germany)를 24 well plate에 부착하여 500μm의 간격을 만든 뒤 상기 배양한 HCT116 (1.5 x 105cells/ml)을 분주하여 24시간 동안 배양하였다. 플레이트에서 insert를 떼어낸 후 각 well에 상기 KME가 0㎍/㎖, 1㎍/㎖, 10㎍/㎖의 농도가 되도록 첨가하여 24시간, 48시간, 72시간 동안 세포를 배양한 후 현미경으로 사진을 찍었다.To measure the cell motility of Korean mistletoe extract (KME), cell migration analysis was performed on a human colorectal cancer cell line (HCT116). Culture-insert (Ibidi; Martinsried, Germany) was attached to a 24-well plate to make an interval of 500 μm, and then the cultured HCT116 (1.5 x 10 5 cells/ml) was dispensed and cultured for 24 hours. After removing the insert from the plate, the KME was added to each well at a concentration of 0 μg/ml, 1 μg/ml, and 10 μg/ml, and the cells were cultured for 24 hours, 48 hours, and 72 hours, and then photographed under a microscope. took the
1.3. 염증 및 암관련 유전자의 조절 확인1.3. Confirmation of regulation of inflammation and cancer-related genes
한국산 겨우살이 추출물에 의한 대장염증 및 대장암 관련 유전자 조절을 확인하기 위하여 대장 정상 상피세포 (CCD841 CoN) 3.5X105cells/㎖을 60㎜ 배양 용기에 분주하여 세포가 60~70% confluence가 될 때까지 배양한 후 Recombinant Human interleukin(IL)-6 (R&D Systems, Inc., Minneapolis, MN) 또는 IL-6와 KME를 농도별로 처리하여 12시간 동안 더 배양하였다. 상기 배양한 세포는 phosphate buffered saline (PBS)로 세척하고 스크래퍼(scrapper)로 긁어 용해 완충용액(lysis buffer)를 첨가하여 1시간 동안 용해시켰다. 상기 세포용해물은 4℃에서 13,000rpm으로 15분간 원심분리하여 상층액만을 추출하여 사용 전까지 -80℃에서 보관했다. 상기 상층액의 단백질 농도는 protein assay Dye Reagent concentrate (Bio-rad, Hercules, CA, USA)를 사용하여 결정되었다. 20μg의 단백질 시료를 SDS-폴리아크릴아마이드 겔(SDS-polyacrylamide gel)에 전기영동하고 Polyvinylidene difluoride (PVDF) membrane(Burlington, MS, USA)에 0.2A에서 1시간 20분 동안 전이시켰다. 상기 PVDF membrane을 Blocking buffer(PBS, 0.1% Tween-20, 5% skim milk)에 넣고 1시간 동안 반응시킨 후 PBST buffer로 10분간 3번씩 씻었다. STAT3 (#9132), pSTAT3(#9145), 15-PGDH(cayman, No.160615), 및 COX-2(#12282)의 항체 용액과 함께 4℃에서 24시간 배양하였다. membrane을 세척한 뒤 2차 항체 용액에 1시간 반응 시킨 뒤 ELPIS-BIOTECH (Daejeon, Korea)의 ECL(enhanced chemiluminascence)를 사용하여 immunoblot을 검출하였다. Blot은 Chemidoc imaging system (CAN ICES-3, Bio-rad)에 의해 검출되었다.In order to confirm the regulation of colon inflammation and colorectal cancer-related genes by Korean mistletoe extract, 3.5X10 5 cells/ml of colonic normal epithelial cells (CCD841 CoN) were dispensed into a 60 mm culture vessel until the cells reached 60-70% confluence. After culturing, Recombinant Human interleukin (IL)-6 (R&D Systems, Inc., Minneapolis, MN) or IL-6 and KME were treated at different concentrations and further cultured for 12 hours. The cultured cells were washed with phosphate buffered saline (PBS), scraped with a scraper, and lysed for 1 hour by adding a lysis buffer. The cell lysate was centrifuged at 13,000 rpm at 4° C. for 15 minutes to extract only the supernatant and stored at -80° C. until use. The protein concentration of the supernatant was determined using protein assay Dye Reagent concentrate (Bio-rad, Hercules, CA, USA). A 20 μg protein sample was electrophoresed on SDS-polyacrylamide gel and transferred to a Polyvinylidene difluoride (PVDF) membrane (Burlington, MS, USA) at 0.2 A for 1 hour and 20 minutes. The PVDF membrane was placed in a blocking buffer (PBS, 0.1% Tween-20, 5% skim milk) and reacted for 1 hour, followed by washing 3 times for 10 minutes with PBST buffer. Incubated with antibody solutions of STAT3 (#9132), pSTAT3 (#9145), 15-PGDH (cayman, No. 160615), and COX-2 (#12282) at 4°C for 24 hours. After washing the membrane, it was reacted with the secondary antibody solution for 1 hour, and immunoblot was detected using ECL (enhanced chemiluminascence) of ELPIS-BIOTECH (Daejeon, Korea). Blots were detected by Chemidoc imaging system (CAN ICES-3, Bio-rad).
1.4. 대장염 동물모델 실험1.4. Colitis animal model experiment
1.4.1. 대장염 동물 모델 1.4.1. Colitis Animal Model
3% 덱스트란황산나트륨(dextran sodium sulfate, DSS)이 포함된 음용수를 제공하여 대장염 마우스모델을 제작하고 KME의 농도에 따른 질병활동지수(disease activity index, DAI) 및 대장 길이(colon length)의 변화, 대장조직의 병리상태 분석, 유전자 발현 분석을 수행하였다.A mouse model of colitis was prepared by providing drinking water containing 3% dextran sodium sulfate (DSS), and the disease activity index (DAI) and colon length change according to the concentration of KME; Pathological analysis of colon tissues and gene expression analysis were performed.
DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 단독 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 복합 투여 대장염 마우스모델 그룹(n=8)을 총 21일 동안 사육한 후 희생하여 상기 분석을 실시하였다. DSS 단독 투여 대장염 마우스모델 그룹(n=8)은 7일 동안의 적응 기간 후 7일 동안 더 사육한 후 DSS 3% 음용수를 제공하며 7일 동안 더 사육하였다. KME 단독 투여 대장염 마우스모델 그룹(n=8)은 7일 동안의 적응 기간 후 KME 200㎎/kg 또는 KME 500㎎/kg을 경구투여하며 14일 동안 더 사육하였다. DSS-KME 복합 투여 대장염 마우스모델 그룹(n=8)은 7일 동안의 적응 기간 후 KME 200㎎/kg 또는 KME 500㎎/kg을 경구투여하며 7일 동안 더 사육하였고 그 후 DSS 3% 음용수를 제공하며 7일 동안 더 사육하였다. DSS-only-administered colitis mouse model group (n=8), KME-only-administered colitis mouse model group (n=8), and DSS-KME combined-administered colitis mouse model group (n=8) were bred for a total of 21 days and then sacrificed. The above analysis was performed. The DSS-only administration colitis mouse model group (n=8) was bred for 7 more days after the adaptation period for 7 days, and then provided with
1.4.2. 질병활동지수분석1.4.2. Disease activity index analysis
질병활동지수(DAI; Disease Activity Index)는 DSS 제공 시작일부터 매일 측정하였다. 혈액의 양과 설사의 중증도에 따라 대장 출혈 및 대변의 일관성을 관찰하고 0에서 4까지 등급을 매겼다. 대변의 일관성은 정상적인 대변일 경우 0점, 묽은 페이스트 형태의 변일 경우 2점, 항문에 묻는 액체형태의 변을 4점으로 하였으며, 대장출혈은 혈흔이 없는 경우 0점, 항문에서 혈흔이 관찰되는 경우 2점, 심한 출혈이 관찰되는 경우에는 4점으로 등급이 매겨졌다. 질병활동지수는 직장 출혈 및 대변 일관성 점수의 합으로 최소 0점에서 최대 8점으로 등급이 매겨졌다.Disease Activity Index (DAI) was measured daily from the start date of DSS provision. Colonic bleeding and stool consistency were observed and graded from 0 to 4 according to the amount of blood and the severity of diarrhea. The consistency of stool was scored as 0 for normal stool, 2 points for thin paste stool, and 4 points for liquid stool in the anus. It was graded as 2 points and 4 points if severe bleeding was observed. The disease activity index was graded from a minimum of 0 to a maximum of 8 as the sum of rectal bleeding and stool consistency scores.
1.4.3. 대장 길이의 변화 분석1.4.3. Analysis of changes in colon length
대장 길이 단축의 완화를 통해 KME의 항염증효과를 확인하기 위하여 상기 사육한 DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 단독 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 복합 투여 대장염 마우스모델 그룹(n=8)의 마우스를 희생시킨 후 대장을 적출하여 길이를 측정하였다.In order to confirm the anti-inflammatory effect of KME through alleviation of colon length shortening, DSS alone administered colitis mouse model group (n = 8), KME alone administered colitis mouse model group (n = 8) and DSS-KME combined administration After sacrificing the mice of the colitis mouse model group (n=8), the colon was removed and the length was measured.
1.4.4 대장조직의 병리상태 분석1.4.4 Pathological analysis of colon tissue
상기 사육한 DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 단독 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 복합 투여 대장염 마우스모델 그룹(n=8)의 마우스를 희생시켜 대장을 적출한 후 형태학적 분석을 위해 조직을 전처리하였다. 10% 포르말린에 고정된 조직을 농도별로 에탄올에서 탈수시키고 파라핀에 포매한 후 4μm두께로 절단하여 슬라이드글라스에 고정하였다. Xylene으로 3번 파라핀을 제거하고 95% 및 70% 알코올에서 재수화시킨 후 Mayer’s Hematoxylin을 사용하여 H&E 염색을 수행하였다.The colon was removed by sacrificing the mice of the above-bred DSS single-administered colitis mouse model group (n=8), KME single-administered colitis mouse model group (n=8), and DSS-KME combined-administered colitis mouse model group (n=8). After excision, the tissue was pretreated for morphological analysis. Tissues fixed in 10% formalin were dehydrated in ethanol by concentration, embedded in paraffin, cut to a thickness of 4 μm, and fixed on a slide glass. After removing
1.4.5. 형광염색1.4.5. Fluorescent dyeing
상기 제작된 4μm로 제작된 슬라이드글라스를 Xylene으로 3번 파라핀을 제거하고 95%, 80%, 75%의 알코올에서 순차적으로 재수화시킨 후 항원이 노출될 수 있도록 10mmol/L citrate buffer (pH 6.0) for antigen retrieval에서 6분 동안 두 번 끓였다. 비특이적 염색을 줄이기 위하여 0.3% hydrogen peroxide에 30분 동안 반응시켰다. 단백질 발현의 검출을 위해 슬라이드를 15-PGDH(sc-271418) 항체와 함께 4℃에서 24시간 동안 배양하였다. Fluorophore-conjugated 2차 항체 (Alexa fluor 488)와 함께 배양 후 ZEN imaging softwere에 의해 통합된 LSM 700 공 초점 현미경 (ZEISS; Oberkochen, Germany)에서 분석되었다.Remove
1.4.6. 유전자 발현 분석1.4.6. gene expression analysis
상기 사육한 DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 단독 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 복합 투여 대장염 마우스모델 그룹(n=8)의 마우스를 희생시켜 대장을 적출한 후 조직의 유전자 발현을 분석하였다. 이를 위하여 대장조직을 용해 완충용액을 첨가한 후 균질화한 후 13,000rpm에서 10분 동안 원심분리하여 사용하기 전까지 -80℃에서 보관했다. 45μg의 단백질 시료를 SDS-폴리아크릴아마이드 겔(SDS-polyacrylamide gel)에 전기영동하고 Polyvinylidene difluoride (PVDF) membrane(Burlington, MS, USA)에 0.2A에서 1시간 20분 동안 전이시켰다. 상기 PVDF membrane을 Blocking buffer(PBS, 0.1% Tween-20, 5% skim milk)에 넣고 1시간 동안 반응시킨 후 PBST buffer로 10분간 3번씩 씻었다. COX2(#12282)의 항체 용액과 함께 4℃에서 24시간 배양하였다. Membrane을 세척한 뒤 2차 항체 용액에 1시간 반응 시킨 뒤 ELPIS-BIOTECH (Daejeon, Korea)의 ECL(enhanced chemiluminascence)를 사용하여 immunoblot을 검출하였다. Blot은 Chemidoc imaging system (CAN ICES-3, Bio-rad)에 의해 검출되었다.The colon was removed by sacrificing the mice of the above-bred DSS single-administered colitis mouse model group (n=8), KME single-administered colitis mouse model group (n=8), and DSS-KME combined-administered colitis mouse model group (n=8). After extraction, the tissue gene expression was analyzed. To this end, colon tissues were homogenized after addition of a lysis buffer, and centrifuged at 13,000 rpm for 10 minutes and stored at -80°C until use. 45 μg of protein sample was electrophoresed on SDS-polyacrylamide gel and transferred to Polyvinylidene difluoride (PVDF) membrane (Burlington, MS, USA) at 0.2 A for 1 hour and 20 minutes. The PVDF membrane was placed in a blocking buffer (PBS, 0.1% Tween-20, 5% skim milk) and reacted for 1 hour, followed by washing 3 times for 10 minutes with PBST buffer. It was incubated with an antibody solution of COX2 (#12282) at 4°C for 24 hours. After the membrane was washed and reacted with the secondary antibody solution for 1 hour, immunoblot was detected using ECL (enhanced chemiluminascence) of ELPIS-BIOTECH (Daejeon, Korea). Blots were detected by Chemidoc imaging system (CAN ICES-3, Bio-rad).
2. 실험결과2. Experimental results
2.1. 한국산 겨우살이 추출물에 의한 암세포 활성 억제효과2.1. Inhibition of cancer cell activity by Korean mistletoe extract
도 1은 한국산 겨우살이 추출물(KME)이 대장상피세포(CCD841)의 세포 생존력에 미치는 영향을 보여준다. 대장상피세포에 KME를 농도별로 처리하고 24시간 동안 배양한 결과 0.5 내지 10 ㎍/㎖의 범위에서 대장상피세포의 세포 생존력이 저하되지 않는 것으로 확인되었다. 또한 대장상피세포에 KME를 농도별로 처리하고 48시간 동안 배양한 결과 0.5 내지 1㎍/㎖의 범위에서 대장상피세포의 세포 생존력은 저하되지 않았으나 5㎍/㎖ 이상으로 투여시 KME의 농도가 증가함에 따라 생존력이 점차 감소하는 것이 확인되었다.1 shows the effect of Korean mistletoe extract (KME) on the cell viability of colonic epithelial cells (CCD841). As a result of treating colonic epithelial cells with KME at different concentrations and culturing for 24 hours, it was confirmed that the cell viability of colonic epithelial cells did not decrease in the range of 0.5 to 10 μg/ml. In addition, as a result of treating colonic epithelial cells with KME at different concentrations and culturing for 48 hours, the cell viability of colonic epithelial cells did not decrease in the range of 0.5 to 1㎍/ml, but when administered at 5㎍/ml or more, the concentration of KME increased. As a result, it was confirmed that the viability gradually decreased.
도 2는 한국산 겨우살이 추출물(KME)이 대장암 세포주(HCT116)의 세포 생존력에 미치는 영향을 보여준다. 대장암 세포주(HCT116)에 KME를 농도별로 처리하고 24시간 동안 배양한 결과 상기 정상 대장상피세포(CCD841)의 결과와 달리 KME 10 ㎍/㎖이상에서 세포 생존력이 저하되는 것이 확인 되었으며 농도가 증가함에 따라 세포 생존력 역시 점차 감소하는 것이 확인되었다. 상기 결과를 더 확인하기 위하여 KME를 동일한 농도 조건으로 투여하되, 투여 후 배양시간을 48시간 또는 72시간으로 증가시킨 결과 동일한 패턴으로 KME가 대장암 세포주(HCT116)의 세포 생존력을 감소시키는 것이 확인되었다.Figure 2 shows the effect of Korean mistletoe extract (KME) on the cell viability of colorectal cancer cell line (HCT116). Colorectal cancer cell line (HCT116) was treated with KME at different concentrations and cultured for 24 hours. As a result, unlike the results of normal colonic epithelial cells (CCD841), it was confirmed that cell viability was reduced at 10 μg/ml or more of KME, and as the concentration increased, Accordingly, it was confirmed that the cell viability also gradually decreased. In order to further confirm the above results, KME was administered at the same concentration condition, but as a result of increasing the incubation time to 48 hours or 72 hours after administration, it was confirmed that KME reduced the cell viability of the colon cancer cell line (HCT116) in the same pattern. .
도 3은 한국산 겨우살이 추출물(KME)이 간암 세포주(SK-Hep1)의 세포 생존력에 미치는 영향을 보여준다. 간암 세포주(SK-Hep1)에 KME를 농도별로 처리하고 24시간 동안 배양한 결과 상기 대장암 세포주(HCT116)의 결과와 유사하게 KME 10 ㎍/㎖이상에서 세포 생존력이 저하되는 것이 확인 되었으며 농도가 증가함에 따라 세포 생존력 역시 점차 감소하는 것이 확인되었다. 상기 결과를 더 확인하기 위하여 KME를 동일한 농도 조건으로 투여하되, 투여 후 배양시간을 48시간 또는 72시간으로 증가시킨 결과 동일한 패턴으로 KME가 간암 세포주(SK-Hep1)의 세포 생존력을 감소시키는 것이 확인되었다.3 shows the effect of a Korean mistletoe extract (KME) on the cell viability of a liver cancer cell line (SK-Hep1). As a result of treating the liver cancer cell line (SK-Hep1) with KME by concentration and culturing for 24 hours, similar to the result of the colorectal cancer cell line (HCT116), it was confirmed that the cell viability was decreased at 10 μg/ml or more of KME, and the concentration was increased. It was confirmed that the cell viability also gradually decreased. In order to further confirm the above results, KME was administered under the same concentration conditions, but as a result of increasing the incubation time to 48 hours or 72 hours after administration, it was confirmed that KME reduced the cell viability of the liver cancer cell line (SK-Hep1) in the same pattern. became
추가적으로 KME에 의한 대장암세포주(HCT116)의 활성 저하를 확인하기 위하여 암세포의 이동성(cell motility)을 측정하였다(도 4 참조). 측정 결과 KME에 의해 암세포의 이동성이 감소하는 것이 확인되었으며 KME의 농도가 증가함에 따라 암세포의 이동성이 더 감소하는 것이 확인되었다.Additionally, in order to confirm the decrease in the activity of the colorectal cancer cell line (HCT116) by KME, the cell motility of the cancer cells was measured (see FIG. 4 ). As a result of the measurement, it was confirmed that the mobility of cancer cells was decreased by KME, and it was confirmed that the mobility of cancer cells was further decreased as the concentration of KME increased.
정리하면 한국산 겨우살이 추출물(KME)은 정상 대장상피세포에는 독성이 없어 세포 활성을 저하시키지 않는 것으로 확인되나 특이적으로 대장암세포 또는 간암세포에서 세포활성을 저하시키는 것으로 확인되었다.In summary, it was confirmed that Korean mistletoe extract (KME) was not toxic to normal colonic epithelial cells and did not decrease cellular activity, but specifically decreased cellular activity in colorectal cancer cells or liver cancer cells.
2.2. 한국산 겨우살이 추출물에 의한 대장염증 억제효과2.2. Inhibitory effect of colonic inflammation by Korean mistletoe extract
2.2.1. 세포실험결과2.2.1. Cell test results
KME에 의한 대장염증의 완화를 확인하기 위하여 대장상피세포(CCD841)를 배양한 후 염증성 싸이토카인 interleukin-6(IL-6) 및 KME를 처리하고 염증 관련 분자지표의 변화를 관찰하였다. In order to confirm the alleviation of KME-induced colon inflammation, colonic epithelial cells (CCD841) were cultured, treated with inflammatory cytokines interleukin-6 (IL-6) and KME, and changes in inflammation-related molecular indicators were observed.
도 5는 염증성 싸이토카인(cytokine) IL-6에 의한 대장상피세포(CCD841)의 염증 관련 분자지표의 변화를 확인한 결과를 보여준다. IL-6을 처리하여 24시간 동안 배양한 후 유전자 발현을 확인한 결과, Signal transducer and activator of transcription 3 (STAT3)의 발현양은 변화되지 않았으나 IL-6의 농도가 증가함에 따라 STAT3의 인산화가 증가된 것이 확인되었다. 또한 염증 관련 분자지표인 cyclooxygenase-2(COX-2)의 내인성 길항제(antagonist)이며 종양억제 효과가 있는 15-hydroxyprostaglandin dehydrogenase(15-PGDH)의 발현량이 농도에 따라 감소하는 것이 확인되었다.5 shows the results of confirming the change in the inflammation-related molecular markers of colonic epithelial cells (CCD841) by the inflammatory cytokine IL-6. As a result of checking the gene expression after culturing for 24 hours after treatment with IL-6, the expression level of signal transducer and activator of transcription 3 (STAT3) did not change, but phosphorylation of STAT3 increased as the concentration of IL-6 increased. Confirmed. In addition, it was confirmed that the expression level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which is an endogenous antagonist of cyclooxygenase-2 (COX-2), an inflammation-related molecular index, and has a tumor-suppressing effect, decreased with concentration.
도 6은 본 발명의 KME에 대한 대장상피세포(CCD841)의 염증 관련 분자지표의 변화를 보여준다. 상기 도 5에서 IL-6가 25ng/㎖의 농도로 처리된 경우 STAT3의 인산화 및 15-PGDH의 발현이 증가된 결과를 바탕으로 IL-6 25ng/㎖과 KME 5㎍/㎖ 및 10㎍/㎖ 처리한 후 염증 관련 분자지표의 변화를 확인하였다. STAT3의 경우 IL-6와 KME의 처리와 무관하게 유사한 발현량을 보이는 반면, IL-6에 의해 STAT3의 인산화가 증가하며 KME에 의해 STAT3의 인산화가 감소하는 것이 확인되었다. 또한 KME의 농도가 증가함에 따라 STAT3의 인산화가 더 감소하는 것으로 확인되었다. 또한 COX-2의 경우 IL-6에 의해 발현량이 증가하나 IL-6 없이 KME만을 단독으로 처리하는 경우 그 발현량이 변화하지 않는 것이 확인된다. 이에 반하여 IL-6와 KME를 동시에 처리한 경우 COX-2의 발현량이 IL-6을 단독으로 처리한 경우에 대비하여 감소하는 것이 확인되었으며 KME의 농도가 증가함에 따라 COX-2의 발현량 또한 감소하는 것이 확인되었다. 상기 KME에 의한 COX-2의 발현량 조절은 15-PGDH의 발현량 변화에 의해 설명된다. 15-PGDH는 IL-6를 처리한 경우 그 발현량이 감소한 것이 확인되었으며 IL-6와 함께 KME를 처리한 결과 KME의 농도가 증가함에 따라 15-PGDH의 발현량 또한 증가하는 것이 확인되었다. 상기 KME에 의한 COX-2 및 15-PGDH의 발현량 확인 결과는 15-PGDH가 COX-2의 길항제로서 작용한다는 점에서 설명이 가능하다.6 shows changes in inflammation-related molecular markers of colonic epithelial cells (CCD841) for KME of the present invention. 5, based on the results of increased phosphorylation of STAT3 and 15-PGDH expression when IL-6 was treated at a concentration of 25 ng/ml, IL-6 25ng/ml,
따라서 본 발명의 KME는 농도 의존적으로 15-PGDH의 발현량을 증가시키며 이는 COX-2의 발현량 감소로 이어지므로 외적 요인에 의해 발현된 대장염 유발 인자(IL-6)로 인한 대장염을 완화시키거나 증상을 치료할 수 있는 것으로 판단된다. 또한 본 발명의 KME에 의해 농도 의존적으로 발현량이 증가하는 15-PGDH는 종양억제 유전자로 알려져 있으며, KME에 농도 의존적으로 인산화가 저해되는 STAT-3는 인산화를 통해 종양발생을 증가시킨다. 따라서 암세포에 KME를 처리하게 되면 상기 15-PGDH의 발현이 증가하게 되고 pSTAT3가 낮아져 암세포의 활성을 저하시키게 되므로 염증성 대장암 치료제로서 사용이 가능할 것으로 판단된다. Therefore, the KME of the present invention increases the expression level of 15-PGDH in a concentration-dependent manner, which leads to a decrease in the expression level of COX-2. It is considered that the symptoms can be treated. In addition, 15-PGDH, whose expression is increased in a concentration-dependent manner by KME of the present invention, is known as a tumor suppressor gene, and STAT-3, whose phosphorylation is inhibited in a concentration-dependent manner in KME, increases tumorigenesis through phosphorylation. Therefore, when the cancer cells are treated with KME, the expression of 15-PGDH is increased and pSTAT3 is lowered, thereby lowering the activity of cancer cells.
2.2.2. 동물실험결과2.2.2. Animal test results
2.2.2.1. 질병활동지수, 대장 길이 및 대장 조직 분석2.2.2.1. Disease activity index, colon length and colon tissue analysis
한국산 겨우살이 추출물(KME)에 의한 염증 관련 분자지표 및 암 관련지표의 발현 조절을 확인하기 위하여 대장염 동물모델을 제작하였다. 상기 동물모델은 마우스에 3% 덱스트란황산나트륨(dextran sodium sulfate, DSS)이 포함된 음용수를 제공하는 방법으로 제작하였다(도 7 참조).An animal model of colitis was prepared to confirm the expression regulation of inflammation-related molecular indicators and cancer-related indicators by Korean mistletoe extract (KME). The animal model was prepared by providing drinking water containing 3% dextran sodium sulfate (DSS) to mice (see FIG. 7 ).
아무것도 투여하지 않은 마우스 그룹(n=8), DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 200㎎/kg 단독 투여 마우스 그룹(n=8), KME 500㎎/kg 단독 투여 마우스 그룹(n=8), DSS-KME 200㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 500㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8)에 대하여 질병활동지수(DAI)를 분석하였다(도 8 참조). 그 결과 DSS로 처리하지 않은 마우스(아무것도 투여하지 않은 마우스 그룹(n=8), KME 200㎎/kg 단독 투여 마우스 그룹(n=8), 및 KME 500㎎/kg 단독 투여 마우스 그룹(n=8)의 경우 DAI 스코어가 0으로 질병이 발생하지 않았으나 DSS만을 처리한 마우스(DSS 단독 투여 대장염 마우스모델 그룹(n=8))의 경우 사육 3일 이후부터 질병이 발생하기 시작하여 사육 7일만에 DAI 스코어가 평균 6에 달하는 것이 확인되었다. A mouse group unadministered (n=8), a mouse model group administered with DSS alone (n=8), a mouse group administered with
이에 반하여 DSS와 KME를 병행 처리한 마우스(DSS-KME 200㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 500㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8))의 경우 사육 4일 이후부터 질병이 발생하여 사육 7일 후에는 DAI 스코어가 평균 3 내지 5로 DSS만을 처리한 마우스에 비하여 매우 낮은 질병활동지수를 보이는 것으로 확인되었다. 특히 처리된 KME의 농도가 높을수록 질병활동지수가 낮게 유지되는 것이 확인되었다. In contrast, in mice treated with DSS and KME in parallel (DSS-
아무것도 투여하지 않은 마우스 그룹(n=8), DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 200㎎/kg 단독 투여 마우스 그룹(n=8), KME 500㎎/kg 단독 투여 마우스 그룹(n=8), DSS-KME 200㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 500㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8)에 대하여 대장 길이 분석을 수행하였다(도 9 참조). 총 3주간 사육한 마우스를 희생시킨 후 대장을 적출하여 대장 길이를 비교하였다. 실험결과 아무것도 투여하지 않은 마우스의 경우 평균 12㎝의 대장 길이를 보였으며 DSS 단독 처리된 마우스의 경우 평균 6.5㎝의 대장 길이를 보여 DSS에 의해 대장염이 발병하여 대장 길이가 짧아진 것으로 판단되었다. 또한 KME를 단독으로 처리한 마우스의 경우 대장 길이가 평균 11㎝(KME 200㎎/kg 단독 투여 마우스 그룹(n=8)) 및 10.5㎝(KME 500㎎/kg 단독 투여 마우스 그룹(n=8))을 보여 KME에 의한 대장염이 발생하지 않은 것으로 확인되었다. DSS와 KME를 복합 투여 대장염 마우스의 경우 대장 길이는 평균 9.5㎝(DSS-KME 200㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8)) 및 평균 9㎝(DSS-KME 500㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8))로 아무것도 처리하지 않은 마우스 및 KME를 단독 처리한 마우스에 비하여 약간 짧은 대장 길이를 보이는 것이 확인되었다. 상기 결과는 KME가 DSS에 의한 대장염 발병을 완화시킨다는 것을 의미하는 결과로서 상기 KME의 대장상피세포 염증 관련 분자지표 조절 결과와 일치하는 결과로 판단된다.A mouse group unadministered (n=8), a mouse model group administered with DSS alone (n=8), a mouse group administered with
상기 마우스 그룹의 대장조직을 수득하고 H&E 염색을 실시하였다. DSS가 투여된 마우스의 대장 융모조직의 경우 조직이 파괴되어 융모조직을 확인할 수 없는 상황이었으나 KME를 복합 투여하는 경우 융모 조직의 형태가 잘 유지되어 있는 것이 확인되었다. 다만 KME의 농도를 200㎎/kg에서 500㎎/kg으로 증가시킨 경우 융모 조직의 손상이 약간 확인되는데 이는 KME를 단독으로 처리한 결과에도 유사하게 확인된다.The colon tissue of the mouse group was obtained and subjected to H&E staining. In the case of the colon villi of the mice administered with DSS, the tissue was destroyed and the villous tissue could not be identified, but it was confirmed that the form of the villous tissue was well maintained when KME was administered in combination. However, when the concentration of KME is increased from 200 mg/kg to 500 mg/kg, some damage to the villous tissue is confirmed, which is similarly confirmed even when KME is treated alone.
2.2.2.2. 염증 및 암 관련 분자지표 발현 분석2.2.2.2. Expression analysis of molecular markers related to inflammation and cancer
아무것도 투여하지 않은 마우스 그룹(n=8), DSS 단독 투여 대장염 마우스모델 그룹(n=8), KME 200㎎/kg 단독 투여 마우스 그룹(n=8), DSS-KME 200㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8) 및 DSS-KME 500㎎/kg 복합 투여 대장염 마우스모델 그룹(n=8)에 대하여 염증 관련 분자 지표인 COX-2 및 종양억제 유전자인 15-PGDH의 발현을 분석하였다(도 11 내지 13 참조).No-administration mouse group (n=8), DSS single administration colitis mouse model group (n=8),
상기 마우스 그룹의 대장조직을 각각 적출한 후 분쇄하고 전기영동한 후 항체를 이용하여 COX-2의 발현을 분석하였다. 분석결과 DSS만을 투여한 마우스의 경우 COX-2 발현이 급격히 증가하며 아무것도 투여하지 않은 마우스 및 KME 200㎎/kg 단독 투여 마우스에서 COX-2 발현이 서로 유사하게 유지되는 것이 확인된다. 이에 반하여 DSS와 KME를 동시에 투여한 마우스의 경우 COX-2 발현량이 DSS만을 투여한 마우스의 COX-2 발현량의 30 내지 60% 수준에 불과하며 특히 DSS-KME 500㎎/kg 복합 투여 대장염 마우스의 경우 DSS만을 투여한 마우스 COX-2 발현량의 30% 수준으로 아무것도 투여하지 않은 마우스 및 KME 200㎎/kg 단독 투여 마우스의 COX-2 발현량과 유사한 수준인 것으로 확인된다(도 11 참조). After extracting each colonic tissue from the mouse group, grinding and electrophoresis, the expression of COX-2 was analyzed using an antibody. As a result of the analysis, it is confirmed that, in the case of mice administered only DSS, COX-2 expression is rapidly increased, and COX-2 expression is maintained similarly in mice not administered with nothing and mice administered with
상기 마우스의 대장조직을 각각 적출한 후 조직내에서 COX-2의 길항제인 15-PGDH의 발현을 분석하였다(도 12 및 13 참조). 실험결과 DSS만을 투여한 마우스의 대장 조직의 경우 15-PGDH의 발현이 급격히 감소한 것이 확인되며 아무것도 투여하지 않은 마우스 및 KME 200㎎/kg 단독 투여 마우스에서는 15-PGDH가 발현된 것이 확인된다. 이에 반하여 DSS와 KME를 동시에 투여한 마우스의 경우 15-PGDH의 발현이 증가하여 KME 단독 투여 마우스와 유사한 수준인 것으로 확인되었다. 상기 동물실험 결과는 정상 대장상피세포 및 대장암세포에서 KME가 염증관련 유전자 및 종양관련 유전자를 조절하여 염증완화 및 암치료효과가 있다는 결과와 일치하는 것으로 판단된다.After each colonic tissue of the mouse was excised, the expression of 15-PGDH, an antagonist of COX-2, was analyzed in the tissue (see FIGS. 12 and 13). As a result of the experiment, it was confirmed that the expression of 15-PGDH was rapidly decreased in the colon tissues of mice administered only DSS, and 15-PGDH was expressed in mice not administered with nothing and mice administered with
3. 결론3. Conclusion
본 발명은 KME의 대장염, 대장암, 및 간암에 대한 치료효과에 관한 것이다. The present invention relates to the therapeutic effect of KME on colitis, colorectal cancer, and liver cancer.
먼저 정상대장상피세포(CCD841)에 KME를 투여하여 KME의 세포독성을 확인 한 결과 세포 독성이 없는 것으로 확인되었다. KME의 대장암세포(HCT116) 및 간암세포(SK-Hep1)에 대한 항암효과를 확인한 결과, 대장암세포 및 간암세포 모두에서 KME가 암세포의 세포생존력(cell viability)을 감소시키는 것이 확인되었으며 상기 결과는 KME가 대장암세포 및 간암세포의 종양억제 유전자인 15-PGDH의 발현을 촉진시키고 암화를 유발하는 STAT3의 인산화를 억제하기 때문으로 확인되었다. First, KME was administered to normal colonic epithelial cells (CCD841) to confirm the cytotoxicity of KME, and as a result, it was confirmed that there was no cytotoxicity. As a result of confirming the anticancer effect of KME on colorectal cancer cells (HCT116) and liver cancer cells (SK-Hep1), it was confirmed that KME reduced the cell viability of cancer cells in both colorectal cancer cells and liver cancer cells. was confirmed to be because it promotes the expression of 15-PGDH, a tumor suppressor gene, and inhibits the phosphorylation of STAT3, which induces cancer in colorectal and liver cancer cells.
KME의 대장염증에 대한 치료효과를 확인한 결과, 염증성 싸이토카인 IL-6을 처리하여 염증을 유도한 대장상피세포에 KME를 처리하면 염증관련 분자지표인 COX-2의 발현이 감소하는 것이 확인되었다. 또한 DSS를 제공하여 대장염증을 유발시킨 마우스에 KME를 투여한 결과 질병활동지수가 감소하고 대장 길이가 유지되며 대장상피조직이 염증유발인자(DSS)로부터 보호되는 것이 확인되었는데 상기 결과는 KME가 COX-2의 길항제인 15-PGDH의 발현을 증가시켜 COX-2의 발현을 감소시키므로 대장상피세포의 COX-2에 의한 염증반응이 감소하였기 때문으로 판단된다. As a result of confirming the therapeutic effect of KME on colitis, it was confirmed that the expression of COX-2, an inflammation-related molecular index, decreased when KME was treated on the colonic epithelial cells induced by the inflammatory cytokine IL-6. In addition, as a result of administering KME to mice that induced colon inflammation by providing DSS, it was confirmed that the disease activity index decreased, colon length was maintained, and colonic epithelial tissue was protected from inflammatory factors (DSS). It is thought that this is because the inflammatory response by COX-2 in colonic epithelial cells was decreased because it decreased the expression of COX-2 by increasing the expression of 15-PGDH, an antagonist of -2.
따라서 본 발명의 KME는 약제학적으로 허용 가능한 염과 함께 약학조성물로서 제공되거나 적절한 첨가제와 함께 건강기능식품으로서 제공되면 대장염증을 완화하고 만성염증으로 인해 발병하는 염증성 대장암을 예방 및 치료, 개선 할 수 있을 것으로 판단된다. 추가적으로 본 발명의 KME는 간암세포주의 생존력을 감소시켰으므로 간암치료제로서 사용할 수 있는 가능성이 있는 것으로 판단된다.Therefore, when the KME of the present invention is provided as a pharmaceutical composition together with a pharmaceutically acceptable salt or as a health functional food together with an appropriate additive, it can relieve colon inflammation and prevent, treat, and improve inflammatory colorectal cancer caused by chronic inflammation. It is considered possible Additionally, since the KME of the present invention reduced the viability of liver cancer cell lines, it is judged that there is a possibility that it can be used as a liver cancer treatment agent.
본 명세서에서 설명된 구체적인 실시예는 본 발명의 바람직한 구현예 또는 예시를 대표하는 의미이며, 이에 의해 본 발명의 범위가 한정되지는 않는다. 본 발명의 변형과 다른 용도가 본 명세서 특허청구범위에 기재된 발명의 범위로부터 벗어나지 않는다는 것은 당업자에게 명백하다.The specific examples described herein are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.
Claims (5)
A pharmaceutical composition for preventing or treating inflammatory colorectal cancer comprising a Korean mistletoe extract as an active ingredient.
The pharmaceutical composition for preventing or treating inflammatory colorectal cancer according to claim 1, wherein the pharmaceutical composition suppresses COX2 expression to relieve inflammation of the colon.
According to claim 1, wherein the pharmaceutical composition increases the expression of 15-PGDH and inhibits STAT3 phosphorylation to inhibit cancer due to inflammation, the pharmaceutical composition for preventing or treating inflammatory colorectal cancer.
The method according to claim 1, wherein the Korean mistletoe extract is water-extracted from Korean mistletoe (VTaxillus yadoriki (Siebold) Danser) with oak as a host, and the lectin content is 100 μg/ml. A therapeutic pharmaceutical composition.
A health functional food for preventing or improving inflammatory colorectal cancer containing Korean mistletoe extract as an active ingredient.
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| KR20110090409A (en) | 2010-02-03 | 2011-08-10 | 문득곤 | Ilomys no.4 complex with an anti-inflammation activity |
| KR101951403B1 (en) | 2017-05-10 | 2019-02-22 | 한국한의학연구원 | A composition for preventing or treating inflammatory bowel disease comprising mistletoe extract as an active ingredient |
| KR101971986B1 (en) | 2017-10-20 | 2019-04-24 | 대한민국 | Composition comprising Silverberry like taxillus extract for preventing or treating cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20110090409A (en) | 2010-02-03 | 2011-08-10 | 문득곤 | Ilomys no.4 complex with an anti-inflammation activity |
| KR101951403B1 (en) | 2017-05-10 | 2019-02-22 | 한국한의학연구원 | A composition for preventing or treating inflammatory bowel disease comprising mistletoe extract as an active ingredient |
| KR101971986B1 (en) | 2017-10-20 | 2019-04-24 | 대한민국 | Composition comprising Silverberry like taxillus extract for preventing or treating cancer |
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