KR20220120498A - Pharmaceutical composition for the prevention or treatment of cancer containing nanovesicles derived from the strains of Leuconostoc mesenteroides as an active ingredient - Google Patents
Pharmaceutical composition for the prevention or treatment of cancer containing nanovesicles derived from the strains of Leuconostoc mesenteroides as an active ingredient Download PDFInfo
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- KR20220120498A KR20220120498A KR1020220022360A KR20220022360A KR20220120498A KR 20220120498 A KR20220120498 A KR 20220120498A KR 1020220022360 A KR1020220022360 A KR 1020220022360A KR 20220022360 A KR20220022360 A KR 20220022360A KR 20220120498 A KR20220120498 A KR 20220120498A
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Abstract
Description
본 발명은 류코노스톡 메센테로이데스 (leuconostoc mesenteroides) 균주, 구체적으로 류코노스톡 메센테로이데스 LB-LM1(leuconostoc mesenteroides LB-LM1, 기탁번호 KCCM12701P) 균주 유래 나노소포체를 유효성분으로 포함하는 암의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention is leuconostoc mesenteroides (leuconostoc mesenteroides) strain, specifically, leuconostoc mesenteroides LB-LM1 (leuconostoc mesenteroides LB-LM1, accession number KCCM12701P) prevention of cancer comprising a strain-derived nanovesicles as an active ingredient , to a composition for improvement or treatment.
암은 세계적으로 높은 사망률을 보이며, 서구 사회에서는 심혈관 질환 다음으로 가장 일반적인 사망 원인이다. 특히, 식생활이 서구화되어 고지방식의 섭취가 일반화되고, 환경 오염 물질의 급격한 증가, 음주량의 증가 등으로 대장암, 유방암, 전립선암 등이 지속적으로 증가하는 추세에 있으며, 인구의 고령화와 더불어 흡연 인구의 증가 및 대기 오염으로 인해 폐암이 증가하고 있는 실정이다. 이러한 실정에서 암의 조기 예방 및 치료를 가능하게 하여 인간 건강의 증진, 건강한 삶의 질 향상 및 인류 보건 증진에 기여할 수 있는 항암 물질의 창출이 절실히 요구되고 있다.Cancer has the highest mortality rate worldwide and is the second most common cause of death in Western societies after cardiovascular disease. In particular, the consumption of high-fat diets has become common due to the westernization of diets, and the incidence of colorectal cancer, breast cancer, and prostate cancer continues to increase due to the rapid increase in environmental pollutants and the increase in alcohol consumption. Lung cancer is increasing due to an increase in the number of cancer cells and air pollution. In this situation, there is an urgent need for the creation of anticancer substances that can contribute to the promotion of human health, the improvement of healthy life quality, and the promotion of human health by enabling the early prevention and treatment of cancer.
한편, 유산균은 인간과 동물의 구강, 장, 질, 분변, 그리고 김치와 같은 발효식품 등에 널리 분포하면서 사람과 동물의 건강과 밀접한 관련을 맺고 있다. 유산균은 정장작용, 유해균 억제, 면역조절, 혈중 콜레스테롤 저하, 항암 등 다양한 건강증진 효과를 나타내고 있다.On the other hand, lactic acid bacteria are widely distributed in the oral cavity, intestine, vagina, feces, and fermented foods such as kimchi of humans and animals, and are closely related to the health of humans and animals. Lactobacillus has various health-promoting effects such as bowel action, suppression of harmful bacteria, immune regulation, lowering blood cholesterol, and anti-cancer.
또한, 세균과 같은 원핵세포와 사람 등의 숙주세포인 진핵세포는 세포밖으로 소포체(vesicles)를 분비하고, 분비된 소포체가 여러 기능을 수행함이 보고되었다. 세균에서 분비된 세포밖 소포체는 내독소(lipopolysaccharide; LPS)와 세균 유래 단백질과 유전자를 함유하고 있고, 크기가 20-1000 나노미터(nm) 크기라서 통상적으로 나노소포체(nanovesicle)라고 칭한다. 사람 또는 동물의 여러 분비물, 배설물 또는 조직 세척액 등에서 세포밖 소포체가 발견되었음이 보고되었으며, 조직에 존재하는 세포밖 소포체는 소포체를 분비하는 조직의 상태를 반영하는 것으로 알려져 있고, 이를 이용해 질병의 진단 및 치료에 이용될 수 있음이 보고되었다.In addition, it has been reported that prokaryotic cells such as bacteria and eukaryotic cells, which are host cells such as humans, secrete vesicles out of the cell, and the secreted endoplasmic reticulum performs various functions. The extracellular vesicles secreted from bacteria contain endotoxin (lipopolysaccharide; LPS) and bacteria-derived proteins and genes. It has been reported that extracellular vesicles have been found in various secretions, feces, or tissue washing fluids of humans or animals, and the extracellular vesicles present in tissues are known to reflect the state of the tissue secreting the ER, and it is used to diagnose and diagnose diseases and It has been reported that it can be used for treatment.
현재, 한국공개특허 제10-2005-0041808호에서는 류코노스톡 시트리움 균주의 항암 활성을 개시하고 있고, 한국공개특허 제10-2019-0105522호에서는 락토바실러스 속 세균 유래 나노소포 및 이의 용도에 대해서 기재하고 있으나, 류코노스톡 메센테로이데스 균주 유래 나노소포체를 포함한 조성물이 암의 예방 또는 치료에 효과가 있음을 확인한 구체적인 실험예나 실시예가 기재된 바 없다.Currently, Korean Patent Application Laid-Open No. 10-2005-0041808 discloses the anticancer activity of Leukonostok citrium strain, and Korean Patent Publication No. 10-2019-0105522 discloses Lactobacillus genus bacterium-derived nanovesicles and their use. However, specific experimental examples or examples confirming that the composition containing the leukonostok mecenteroides strain-derived nanovesicles are effective in the prevention or treatment of cancer has not been described.
본 발명의 목적은 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공하기 위한 것이다.It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a Leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
또한, 본 발명의 다른 목적은 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 암의 예방 또는 개선용 식품 조성물 또는 식품 첨가제 조성물을 제공하기 위한 것이다.In addition, another object of the present invention is to provide a food composition or a food additive composition for the prevention or improvement of cancer comprising a Leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
또한, 본 발명의 또 다른 목적은 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 가축의 암 예방 또는 개선용 사료 조성물 또는 사료 첨가제 조성물을 제공하기 위한 것이다.In addition, another object of the present invention is to provide a feed composition or a feed additive composition for preventing or improving cancer of livestock comprising a Leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
이에, 본 발명자들은 한국미생물보존센터에 기탁되어 있는 류코노스톡 메센테로이데스 LB-LM1 균주(Leuconostoc mesenteroides LB-LM1)를 동정 및 분리하고, 이로부터 나노소포체를 분리하여 이를 유효성분으로 포함하는 조성물을 이용하여 대장암 등 여러 암종에 대한 예방 및 치료 효과를 확인하고자 노력한 결과, 우수한 예방 및 치료 효과를 확인하여 본 발명을 완성하였다.Accordingly, the present inventors identify and isolate the Leuconostoc mesenteroides LB-LM1 strain deposited at the Korea Microbial Conservation Center (Leuconostoc mesenteroides LB-LM1), isolate the nano-ER, and a composition comprising it as an active ingredient As a result of trying to confirm the preventive and therapeutic effects on various carcinomas such as colorectal cancer using
상기 목적을 달성하기 위하여, 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above object, there is provided a pharmaceutical composition for preventing or treating cancer comprising a leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
또한, 본 발명은 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 암의 예방 또는 개선용 식품 조성물 또는 식품 첨가제 조성물을 제공한다.In addition, the present invention provides a food composition or food additive composition for the prevention or improvement of cancer comprising a Leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
또한, 본 발명은 류코노스톡 메센테로이데스 균주 유래 나노소포체를 유효성분으로 포함하는 가축의 암 예방 또는 개선용 사료 조성물 또는 사료 첨가제 조성물을 제공한다.In addition, the present invention provides a feed composition or feed additive composition for preventing or improving cancer of livestock comprising the Leukonostok mecenteroides strain-derived nano-ER as an active ingredient.
본 발명에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체는 다양한 암종에서 암세포의 사멸과 대식세포의 염증성 물질 (TNF-alpha)을 증가시킴으로써, 사람 또는 동물의 암의 치료 또는 개선 등의 용도를 위한 조성물로서 유용하게 이용될 수 있다.Leukonostok mecenteroides strain-derived nanovesicles according to the present invention by increasing apoptosis of cancer cells and inflammatory substances (TNF-alpha) of macrophages in various carcinomas, for the treatment or improvement of human or animal cancer. It can be usefully used as a composition.
도 1은 본 발명의 일 실시예에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체를 폐암 A549 세포주에 처리한 후 세포 성장률(cell viability assay)을 측정한 그래프를 나타낸 도이다.
도 2는 본 발명의 일 실시예에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체를 췌장암 AsPC-1 세포주에 처리한 후 세포 성장률(cell viability assay)을 측정한 그래프를 나타낸 도이다.
도 3은 본 발명의 일 실시예에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체를 대장암 HCT116 세포주에 처리한 후 세포 성장률(cell viability assay)을 측정한 그래프를 나타낸 도이다.
도 4는 본 발명의 일 실시예에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체를 방광암 T24 세포주에 처리한 후 세포 성장률(cell viability assay)을 측정한 그래프를 나타낸 도이다.
도 5는 본 발명의 일 실시예에 따른 류코노스톡 메센테로이데스 균주 유래 나노소포체를 처리하여 단핵구세포 (THP-1)에서의 TNF-α 발현 정도를 측정한 그래프를 나타낸 도이다.1 is a view showing a graph measuring the cell growth rate (cell viability assay) after treating the Leukonostok mecenteroides strain-derived nano-ER in the lung cancer A549 cell line according to an embodiment of the present invention.
Figure 2 is a diagram showing a graph measuring the cell growth rate (cell viability assay) after treating the Leukonostok mecenteroides strain-derived nano-ER to the pancreatic cancer AsPC-1 cell line according to an embodiment of the present invention.
3 is a view showing a graph measuring the cell growth rate (cell viability assay) after treating the leukonostok mecenteroides strain-derived nano-ER in the colon cancer HCT116 cell line according to an embodiment of the present invention.
Figure 4 is a diagram showing a graph measuring the cell growth rate (cell viability assay) after treating the leukonostok mecenteroides strain-derived nano-ER to the bladder cancer T24 cell line according to an embodiment of the present invention.
5 is a diagram showing a graph measuring the level of TNF-α expression in monocytes (THP-1) by treating the leukonostok mecenteroides strain-derived nanovesicles according to an embodiment of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
본 발명의 류코노스톡 메센테로이데스 균주는 류코노스톡 메센테로이데스 LB-LM1(Leuconostoc mesenteroides, LB-LM1, 기탁번호 KCCM12701P) 균주일 수 있으며, 이에 한정되지 않는다. The Leuconostoc mesenteroides strain of the present invention may be a Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides, LB-LM1, accession number KCCM12701P) strain, but is not limited thereto.
본 발명의 류코노스톡 메센테로이데스 균주는 LB-LM1(Leuconostoc mesenteroides, LB-LM1, 기탁번호 KCCM12701P) 균주 유래 나노소포체를 유효성분으로 포함하는 조성물은 암의 예방 또는 치료 효과를 가지며, 약제학적 조성물로 이용될 수 있다.Leuconostoc mesenteroides strain of the present invention is LB-LM1 (Leuconostoc mesenteroides, LB-LM1, Accession No. KCCM12701P) The composition comprising the strain-derived nano-ER as an active ingredient has a preventive or therapeutic effect on cancer, and a pharmaceutical composition can be used as
상기 류코노스톡 메센테로이데스 LB-LM1(Leuconostoc mesenteroides, LB-LM1)는 유산균 균주이다. 상기 류코노스톡 메센테로이데스 LB-LM1는 프로바이오틱스로서, 유산균의 일반적인 정장 효과 및 면역 증강 효과를 갖는다.The Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides, LB-LM1) is a lactic acid bacteria strain. The leukonostok mecenteroides LB-LM1 is a probiotic, and has a general intestinal effect and immune enhancing effect of lactic acid bacteria.
상기 류코노스톡 메센테로이데스 LB-LM1는 서열번호 1(SEQ ID NO: 1)의 핵산서열을 갖는 것을 특징으로 한다.The leukonostok mecenteroides LB-LM1 is characterized in that it has a nucleic acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1).
본 발명에 있어서, '프로바이오틱스(probiotics)'는 사람을 포함한 동물의 위장관 내에서 숙주의 장내 미생물 환경을 개선하여 숙주의 건강에 유익한 영향을 주는 살아있는 미생물'이라는 의미로 이해된다. 프로바이오틱스는 프로바이오틱 활성을 갖는 살아있는 미생물로 단일 또는 복합균주 형태로 사람이나 동물에 건조된 세포 형태나 발효산물 형태로 급여될 경우, 숙주의 장내 균총에 유익한 영향을 미칠 수 있다.In the present invention, 'probiotics' is understood to mean 'living microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of animals including humans'. Probiotics are living microorganisms with probiotic activity, and when fed to humans or animals in the form of single or complex strains, in the form of dried cells or fermentation products, it can have a beneficial effect on the intestinal flora of the host.
상기 암은 대장암, 방광암, 유방암, 흑색종양, 갑상선암, 부갑상선암, 직장암, 인후암, 후두암, 식도암, 췌장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두담낭암, 구강암, 결장암, 항문 부근암, 간암, 폐암 및 혈액암으로 이루어지는 군으로부터 선택되는 어느 하나의 암일 수 있으나, 이에 한정되지 않는다.The cancer is colorectal cancer, bladder cancer, breast cancer, black tumor, thyroid cancer, parathyroid cancer, rectal cancer, throat cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, double gallbladder cancer, oral cancer, colon cancer, perianal cancer, liver cancer , may be any one cancer selected from the group consisting of lung cancer and blood cancer, but is not limited thereto.
상기 나노소포는 다양한 세균에서 분비되는 나노 크기의 막으로 된 구조물을 의미한다. 상기 나노소포는 류코노스톡 메센테로이데스 균주에서 유래된 것으로 평균 직경은 1 내지 1000nm 일 수 있으며, 바람직하게는 10 내지 500nm일 수 있으나, 이에 한정되지 않는다.The nanovesicles refer to structures made of nano-sized membranes secreted by various bacteria. The nanovesicles are derived from the Leukonostok mecenteroides strain and may have an average diameter of 1 to 1000 nm, preferably 10 to 500 nm, but is not limited thereto.
상기 나노소포는 류코노스톡 메센테로이데스 균주로부터 자연적 또는 인공적인 방법으로 분리될 수 있으며, 인공적인 방법에는 당업계에서 통상적으로 사용되는 모든 방법이 제한없이 사용될 수 있다. 예컨대, 상용화된 엑소좀 분리키트(예컨대, EXO-BB, ExoQuick®-ULTRA, ExoQuick®-TC, CapturemTM Exosome Isolation Kit, Total Exosome Isolation Kit, ExoTrapTM Exosome Isolation Spin Column Kit, Exo2DTM 등)를 활용하거나, 용액 내성분들 간 비중 차이에 따른 분리(예컨대, 원심분리법), 크기에 따른 분리(예컨대, 한외여과 또는 진공 필터), 특정 기질에 대한 친화도에 기반한 분리(예컨대, 친화성 크로마토그래피)를 포함하나, 이에 제한되지 않으며 비균질 시료 내에서 목적 물질의 고유의 물성에 기반한 분리 방법으로서 당업계에서 통상적으로 사용되는 모든 방법이 제한 없이 사용될 수 있다.The nanovesicles may be isolated from the Leukonostok mecenteroides strain by a natural or artificial method, and any method commonly used in the art may be used without limitation in the artificial method. For example, a commercially available exosome isolation kit (eg, EXO-BB, ExoQuick®-ULTRA, ExoQuick®-TC, CapturemTM Exosome Isolation Kit, Total Exosome Isolation Kit, ExoTrapTM Exosome Isolation Spin Column Kit, Exo2DTM, etc.) is used, or a solution Separation according to the difference in specific gravity between resistant components (eg, centrifugation), separation according to size (eg, ultrafiltration or vacuum filter), separation based on affinity for a specific substrate (eg, affinity chromatography), It is not limited thereto, and any method commonly used in the art may be used without limitation as a separation method based on the intrinsic properties of the target material in the heterogeneous sample.
본 발명에 따른 류코노스톡 메센테로이데스 LB-LM1 균주 유래 소포체를 포함하는 조성물은 경구 또는 비경구로 투여할 수 있다. 비경구 투여인 경우에는 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐 내 투여 및 직장 내 투여 등으로 투여할 수 있으며, 바람직하게는 정맥 내 주입방법으로 투여할 수 있으나, 이에 한정되지 않는다.The composition comprising the endoplasmic reticulum derived from the Leukonostok mecenteroides LB-LM1 strain according to the present invention may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc. may be administered, preferably by intravenous injection. can be administered, but is not limited thereto.
상기 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여시간, 투여경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다.A suitable dosage of the composition may be variously prescribed according to factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient.
본 발명의 조성물이 약제학적 조성물로 활용될 경우, 본 발명의 약제학적 조성물은 상기 유효성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다.When the composition of the present invention is utilized as a pharmaceutical composition, the pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, bog Release, sweetener, binder, coating agent, swelling agent, lubricant, lubricant or flavoring agent and the like can be used.
상기 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.
예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연당, 옥수수감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐아세테이트, 소듐클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸셀룰로스, 아가, 벤토니트, 잔탄검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충식염수, 알부민 주사 용액, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. ate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers, which are sterile and biocompatible, include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
나아가 해당 분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.Furthermore, it can be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the field.
본 발명의 류코노스톡 메센테로이데스 LB-LM1(Leuconostoc mesenteroides, LB-LM1, 기탁번호 KCCM12701P) 균주 유래 나노소포체를 유효성분으로 포함하는 조성물은 암의 예방 또는 개선용 식품 조성물로 이용될 수 있다.Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides, LB-LM1, Accession No. KCCM12701P) strain-derived composition comprising the nano-ER as an active ingredient of the present invention can be used as a food composition for preventing or improving cancer.
상기 식품 조성물은 건강기능식품의 형태일 수 있다.The food composition may be in the form of a health functional food.
상기 "건강기능식품"은 건강기능식품에 관한 법률에 따라 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미(제3조 제1호)하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻는 것(동조 제2호)을 의미한다.The above "health functional food" means food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act (Article 3, No. 1), and "functionality" means It refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body (Article 2).
상기 식품 조성물은 식품 첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합여부는 다른 규정이 없는 한 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The food composition may additionally contain food additives, and the suitability as a "food additive" is determined according to the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. and criteria.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀롤로오스, 구아검 등의 천연첨가물, L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합 제제류 들을 들 수 있다.As items listed in the "Food Additives Codex", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as depigmentation, licorice extract, crystalline cellulose, and guar gum, L- Mixed preparations such as sodium glutamate preparation, noodle-added alkali preparation, preservative preparation, and tar color preparation can be mentioned.
본 발명의 유효성분이 포함된 식품으로는 빵, 떡류, 건과류, 캔디류, 초콜릿류, 츄잉껌, 쨈류와 같은 과자류 아이스크림류, 빙과류, 아이스크림 분말류와 같은 아이스크림 제품류 우유류, 저지방 우유류, 유당분해 우유, 가공유류, 산양유, 발효유류, 버터유류, 농축유류, 유크림류, 버터유, 자연치즈, 가공치즈, 분유류, 유청류와 같은 유가공품류 식육가공품, 알가공품, 햄버거와 같은 식육제품류 어묵, 햄, 소세지, 베이컨 등의 어육가공품과 같은 어육제품류 라면류, 건면류, 생면류, 유탕면류, 호화건먼류, 개량숙면류, 냉동면류, 파스타류와 같은 면류 과실음료, 채소류음료, 탄산음료, 두유류, 요구르트 등의 유산균음료, 혼합음료와 같은 음료 간장, 된장, 고추장, 춘장, 청국장, 혼합장, 식초, 소스류, 토마토케첩, 카레, 드레싱과 같은 조미식품 마가린, 쇼트닝 및 피자를 들 수 있으나, 이에 한정되는 것은 아니다.Foods containing the active ingredient of the present invention include bread, rice cakes, dried fruits, candies, chocolates, chewing gum, confectionery products such as jams, ice cream products, ice cream products, ice cream products such as ice cream powder milk, low-fat milk, lactose-decomposed milk, Processed milk, goat milk, fermented milk, buttermilk, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, milk powder, milk products such as whey Processed meat products, processed eggs, meat products such as hamburgers Fish cakes, ham, Fish and meat products such as sausage and bacon processed fish and meat products Ramen, dried noodles, raw noodles, fried noodles, luxurious dried noodles, improved soft noodles, frozen noodles, pasta, etc. Fruit drinks, vegetable drinks, carbonated drinks, soy milk, Lactic acid bacteria drinks such as yogurt, beverages such as mixed drinks Soy sauce, soybean paste, gochujang, chunjang, cheonggukjang, mixed soy sauce, vinegar, sauces, tomato ketchup, curry, seasoning foods such as dressings, margarine, shortening and pizza, but limited to this it's not going to be
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, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있다. 그밖에 본 발명의 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may include a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may include fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination.
본 발명의 유효성분을 포함한 음료 조성물은 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가성분으로 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, (예를 들어, 포도당, 과당 등); 디사카라이드, (예를 들어 말토스, 슈크로스 등); 및 폴리사카라이드, (예를 들어 덱스트린, 시클로덱스트린 등)과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.The beverage composition including the active ingredient of the present invention is not particularly limited in other ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); disaccharides, (eg maltose, sucrose, etc.); and conventional sugars such as polysaccharides (eg, dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have.
또한, 본 발명의 류코노스톡 메센테로이데스 LB-LM1(Leuconostoc mesenteroides, LB-LM1, 기탁번호 KCCM12701P) 균주 유래 나노소포체를 유효성분으로 포함하는 조성물은 가축의 암 예방 또는 개선용 사료 조성물로 이용될 수 있다.In addition, the composition comprising the leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides, LB-LM1, accession number KCCM12701P) strain-derived nano-ER as an active ingredient of the present invention can be used as a feed composition for preventing or improving livestock cancer. can
상기 조성물이 사료 첨가제로서 제조될 경우, 상기 조성물은 20 내지 90% 고농축액이거나 분말 또는 과립 형태로 제조될 수 있다. 상기 사료 첨가제는 구연산, 후말산, 아디픽산, 젖산, 사과산등의 유기산이나 인산 나트륨, 인산 칼륨, 산성 피로인산염, 폴리인산염(중합인산염) 등의 인산염이나, 폴리페놀, 카테킨, 알파-토코페롤, 로즈마리 추출물, 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제 중 어느 하나 또는 하나 이상을 추가로 포함할 수 있다. 사료로서 제조될 경우, 상기 조성물은 통상의 사료 형태로 제제화될 수 있으며, 통상의 사료 성분을 함께 포함할 수 있다.When the composition is prepared as a feed additive, the composition may be 20 to 90% highly concentrated or may be prepared in powder or granular form. The feed additives include organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, rosemary Extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, any one or more of natural antioxidants such as phytic acid may be further included. When prepared as a feed, the composition may be formulated in the form of a conventional feed, and may include common feed ingredients together.
상기 사료 및 사료 첨가제는 곡물, 예를 들면 분쇄 또는 파쇄된 밀, 귀리, 보리, 옥수수 및 쌀; 식물성 단백질 사료, 예를 들면 평지, 콩, 및 해바라기를 주성분으로 하는 사료; 동물성 단백질 사료, 예를 들면 혈분, 육분, 골분 및 생선분; 당분 및 유제품, 예를 들면 각종 분유 및 유장 분말로 이루어지는 건조성분 등을 더 포함할 수 있으며, 이외에도 영양보충제, 소화 및 흡수향상제, 성장촉진제 등을 더 포함할 수 있다.The feed and feed additives include grains such as milled or crushed wheat, oats, barley, corn and rice; plant protein feeds, such as feeds based on rape, soybean, and sunflower; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, may further include dry ingredients made of various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.
상기 사료 첨가제는 동물에게 단독으로 투여하거나 식용 담체 중에서 다른 사료 첨가제와 조합하여 투여할 수도 있다. 또한, 상기 사료 첨가제는 탑드레싱으로서 또는 이들을 동물사료에 직접 혼합하거나 또는 사료와 별도의 경구제형으로 용이하게 동물에게 투여할 수 있다. 상기 사료 첨가제를 동물사료와 별도로 투여할 경우, 당해 기술분야에 잘 알려진 바와 같이 약제학적으로 허용 가능한 식용 담체와 조합하여, 즉시 방출 또는 서방성 제형으로 제조할 수 있다. 이러한 식용 담체는 고체 또는 액체, 예를 들어 옥수수전분, 락토오스, 수크로오스, 콩플레이크, 땅콩유, 올리브유, 참깨유 및 프로필렌글리콜일 수 있다. 고체 담체가 사용될 경우, 사료 첨가제는 정제, 캡슐제, 산제, 트로키제 또는 함당정제 또는 미분산성 형태의 탑 드레싱일 수 있다. 액체 담체가 사용될 경우, 사료 첨가제는 젤라틴 연질 캡슐제, 또는 시럽제나 현탁액, 에멀젼제, 또는 용액제의 제형일 수 있다.The feed additive may be administered to the animal alone or in combination with other feed additives in an edible carrier. In addition, the feed additive can be easily administered to the animal as a top dressing, directly mixing them with animal feed, or in an oral formulation separate from the feed. When the feed additive is administered separately from animal feed, it can be combined with a pharmaceutically acceptable edible carrier as well known in the art to prepare an immediate release or sustained release formulation. Such edible carriers may be solid or liquid, for example cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in microdispersed form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup or suspension, emulsion, or solution.
또한, 상기 사료 및 사료 첨가제는 보조제, 예를 들어 보존제, 안정화제, 습윤제 또는 유화제, 용액 촉진제 등을 함유할 수 있다. 상기 사료 첨가제는 침주, 분무 또는 혼합하여 동물의 사료에 첨가하여 이용될 수 있다.In addition, the feed and feed additives may contain adjuvants, for example, preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, and the like. The feed additive may be used by being added to animal feed by immersion, spraying, or mixing.
본 발명의 사료 또는 사료 첨가제는 포유류, 가금 및 어류를 포함하는 다수의 동물 식이에 적용할 수 있다.The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.
상기 포유류로서 돼지, 소, 말, 양, 토끼, 염소, 설치동물 및 실험용 설치동물인 쥐, 햄스터, 기니피그뿐만 아니라, 애완동물(예: 개, 고양이) 등에게 사용할 수 있으며, 상기 가금류로서 닭, 칠면조, 오리, 거위, 꿩, 및 메추라기 등에도 사용할 수 있고, 상기 어류로서 잉어, 붕어 및 송어 등에 이용될 수 있으나, 이에 한정되는 것은 아니다.Pigs, cows, horses, sheep, rabbits, goats, rodents and experimental rodents as the mammals, as well as rats, hamsters, and guinea pigs, can be used for pets (eg, dogs, cats), etc., and chickens as the poultry, It can also be used for turkey, duck, geese, pheasant, and quail, and the like, and may be used for carp, crucian carp and trout as the fish, but is not limited thereto.
실시예Example 1. 균주의 분리 및 배양 1. Isolation and culture of strains
1-1. 균주의 분리 및 동정1-1. Isolation and identification of strains
본 발명자는 김치로부터 유산균을 분리하였다. 먼저, 김치 시료를 파쇄한 후 김치 추출물의 원액을 agar 배지에 도말하여 30℃에서 24시간 이상 배양하였다. 배양 후 형성된 단일 콜로니를 계대배양하여 균단일집락을 선별하였고, 선별된 균단일집락은 루프로 수거하여 broth에 배양하여 16S RNA 염기서열 분석을 통하여 최종적으로 동정하였다. 세균의 동정은 16S rRNA 서열을 National Center for Biotechnology Information(NCBI, www.ncbi.nlm.nih.gov)의 Basic Local Alignment Search Tool (BLAST) 검색 엔진의 유사도 분석을 통해 수행하였다. 미생물의 동정 및 분류를 위한 16S rRNA 염기서열 분석 결과, 서열번호 1의(SEQ ID NO: 1)의 핵산서열을 갖는 것으로 나타났다. 상기 서열번호 1의 핵산서열을 갖는 본 발명의 미생물을 류코노스톡 메센테로이데스 LB-LM1으로 명명하였으며, 한국미생물보존센터에 2020년 04월 24일자로 기탁하였다(기탁번호 KCCM12701P).The present inventors isolated lactic acid bacteria from kimchi. First, after crushing the kimchi sample, the stock solution of the kimchi extract was smeared on an agar medium and cultured at 30° C. for more than 24 hours. A single colony formed after culture was subcultured to select a single colony, and the selected single colony was collected in a loop, cultured in broth, and finally identified through 16S RNA sequencing. Identification of bacteria was performed through similarity analysis of the 16S rRNA sequence with the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov). As a result of 16S rRNA sequencing analysis for identification and classification of microorganisms, it was found to have a nucleic acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1). The microorganism of the present invention having the nucleic acid sequence of SEQ ID NO: 1 was named Leukonostok mecenteroides LB-LM1, and was deposited at the Korea Microorganism Conservation Center on April 24, 2020 (Accession No. KCCM12701P).
1-2. 균주의 배양1-2. culture of strains
류코노스톡 메센테로이데스 LB-LM1 균주를 식물성 액체배지 30ml에 0.1% 접종하여 37℃에서 18시간 동안 정치배양하였다. 배양 후, 3,500rpm에서 10분 동안 원심분리하여 배양액은 나노소포체 분리를 위해 따로 보관하였고, 균체는 PBS(phosphate buffered saline)용액으로 3회 세척하여 남아 있는 배지 성분을 제거하여 실험에 사용하였다.0.1% of the Leukonostok mecenteroides LB-LM1 strain was inoculated into 30 ml of a vegetable broth and cultured at 37° C. for 18 hours stationary. After incubation, centrifuged at 3,500 rpm for 10 minutes, the culture solution was stored separately for nano-vesicle separation, and the cells were washed 3 times with PBS (phosphate buffered saline) solution to remove the remaining medium components and used for the experiment.
실시예Example 2. 2. 나노소포체의of nano-endoplasmic reticulum 분리 separation
상기 균주 배양 후 얻어진 배양액과 16% PEG 6000이 포함되어 있는 1M NaCl 용액을 동량으로 섞어 4℃에서 15시간 반응시켰다. 이후 반응시킨 용액을 10,000g, 4℃에서 20분간 원심분리하여 나노소포체 pellet을 얻었다. 상기 수득한 나노소포체 pellet은 5% PEG6000이 포함되어 있는 0.5M NaCl 용액에 재부유(resuspension) 하여 세척한 후 11,000 rpm으로 4℃에서 20분간 원심분리하였다. 이후 상층액은 버리고 pellet을 1X PBS에 재부유(resuspension) 하여 최종적으로 류코노스톡 메센테로이데스 유래 나노소포체를 분리하였다.The culture solution obtained after culturing the strain and 1M NaCl solution containing 16% PEG 6000 were mixed in the same amount and reacted at 4° C. for 15 hours. Thereafter, the reacted solution was centrifuged at 10,000 g, 4° C. for 20 minutes to obtain a nano-endoplasmic reticulum pellet. The obtained nano-endoplasmic reticulum pellet was resuspended in 0.5M NaCl solution containing 5% PEG6000 and washed, followed by centrifugation at 11,000 rpm at 4°C for 20 minutes. After that, the supernatant was discarded and the pellet was resuspended in 1X PBS to finally separate the leukonostok mecenteroides-derived nanovesicles.
실시예Example 3. In vitro 항암 활성 효능 분석 3. In vitro anticancer activity analysis
3-1. 세포 성장률(cell viability assay) 측정 결과3-1. Cell viability assay measurement result
본 발명은 폐암 세포주 (A549), 췌장암 (AsPC-1), 방광암 (T24), 대장암 (HCT116) 세포를 배양한 후 류코노스톡 메센테로이데스 LB-LM1 균주 유래 나노소포체를 처리한 세포의 세포 성장률을 crystal violet 염색을 통해 측정하였으며, 공배지 유래 particle을 처리한 암세포의 흡광도를 100% 성장률 기준으로 항암 활성 효능을 확인하였다. 세포 성장률 측정을 위해 각 암세포를 96well plate에 1x104 cell을 plating 한 후 5% CO2, 37℃ 세포배양기에서 24시간 배양하였다. 이후 공배지 유래 particle과 상기 분리된 나노소포체, 또는 균주를 24시간 동안 처리하였다. 이후 배양액을 제거한 후 200 ul의 PBS로 3회 세척하였다. 남은 세포를 50 ul의 crystal violet을 넣어 10분간 세포를 염색한 후 PBS로 3회 세척하였다. 이후 plate를 30분간 상온에서 air-dry한 후 세포에 염색된 crystal violet을 100% ethanol로 녹여서 spectrophotometer에서 590 nm에서 흡광도를 측정한 결과를 하기 도 1 내지 도 4에 나타내었다.The present invention is lung cancer cell line (A549), pancreatic cancer (AsPC-1), bladder cancer (T24), colon cancer (HCT116) cells after culturing cells treated with Leukonostok mecenteroides LB-LM1 strain-derived nanovesicles The growth rate was measured through crystal violet staining, and the anticancer activity was confirmed based on the absorbance of the cancer cells treated with the particles derived from the empty medium based on the 100% growth rate. To measure the cell growth rate, 1x10 4 cells were plated on each cancer cell in a 96-well plate, and then cultured for 24 hours in a cell incubator at 5% CO 2 , 37°C. Thereafter, the particles from the empty medium and the isolated nano-vesicles, or strains were treated for 24 hours. After removing the culture medium, it was washed 3 times with 200 ul of PBS. The remaining cells were stained with 50 ul of crystal violet for 10 minutes, and then washed 3 times with PBS. After the plate was air-dried at room temperature for 30 minutes, crystal violet stained with 100% ethanol was dissolved in the cells, and the absorbance was measured in a spectrophotometer at 590 nm. The results are shown in FIGS. 1 to 4 below.
도 1 내지 도 4에 나타난 바와 같이, 류코노스톡 메센테로이데스 LB-LM1 균주 유래 나노소포체를 처리한 그룹에서 공배지 유래 particle을 처리한 그룹과 균주를 처리한 그룹에 비해 암세포 사멸이 유의적으로 증가함이 확인되었다. 특히, 폐암 세포주인 A549 세포주에서 50%, , 췌장암 세포주인 AsPC-1 세포주에서 63%, 대장암 세포주인 HCT116 세포주에서 43%로 매우 높은 세포성장 억제율을 보였다. 또한, 방광암 세포주인 T24 세포주에서도 60%로 본 발명에 의한 조성물 처리를 한 경우 매우 높은 수준으로 암세포 사멸이 증가됨이 확인되었다. 상기 결과로 비추어 볼 때 류코노스톡 메센테로이데스 균주 유래 나노소포체가 암세포의 세포성장을 효과적으로 억제함으로써 항암활성을 나타낼 수 있음이 확인되었다. As shown in FIGS. 1 to 4, in the group treated with the leukonostok mecenteroides LB-LM1 strain-derived nanovesicles, cancer cell death was significantly reduced compared to the group treated with particles derived from the common medium and the group treated with the strain. increase was confirmed. In particular, it showed a very high cell growth inhibition rate at 50% in the lung cancer cell line A549 cell line, 63% in the pancreatic cancer cell line AsPC-1 cell line, and 43% in the colon cancer cell line HCT116 cell line. In addition, it was confirmed that cancer cell death was increased to a very high level when the composition according to the present invention was treated at 60% in the T24 cell line, which is a bladder cancer cell line. In light of the above results, it was confirmed that the leukonostok mecenteroides strain-derived nanovesicles could exhibit anticancer activity by effectively inhibiting the cell growth of cancer cells.
3-2. 3-2. THPTHP -1 대식세포에서 염증성 사이토카인 (-1 inflammatory cytokines in macrophages ( TNFαTNFα ) 유전자 발현 확인) check gene expression
대식세포의 염증성 사이토카인(cytokine)의 증가는 항암효능을 유도하는 것으로 알려져 있음으로 류코노스톡 메센테로이데스 균주 유래 나노소포체 처리에 따른 염증성 사이토카인 유전자의 발현 수준을 알아보기 위해 실시간 중합 효소 연쇄반응(qPCR)을 실시하였다. The increase in inflammatory cytokines in macrophages is known to induce anticancer efficacy. Real-time polymerase chain reaction to determine the expression level of inflammatory cytokine genes according to the treatment of the leukonostok mecenteroides strain-derived nanovesicles. (qPCR) was performed.
단핵구세포 (THP-1)는 페니실린/스트렙토마이신과 10% FBS, 2-mercaptoethanol을 첨가한 RPMI-1640 배지를 사용하여 37°C, 5% CO2 조건에서 배양후 준비하였다. 단핵구세포 (THP-1)는 6 well plate에 2x106 cell의 세포를 plating하고 phorbol 12-myristate 13-acetate (PMA)를 넣어주어 48시간 동안 대식세포로 분화시켰다. 48시간 후 페니실린/스트렙토마이신이 첨가되지 않은 미디아로 교체 후 세포에 LPS, 류코노스톡 메센테로이데스 LB-LM1 균주 유래 나노소포체, 류코노스톡 메센테로이데스 LB-LM1 균주 (2x106 CFU), 류코노스톡 메센테로이데스 균주 배양 상등액, 배지 유래 particle 등을 각각 처리하여 염증성 사이토카인 유전자인 TNFα 유전자의 발현을 확인한 결과를 5에 나타내었다. Monocytes (THP-1) were prepared after incubation at 37°C and 5% CO2 using RPMI-1640 medium supplemented with penicillin/streptomycin, 10% FBS, and 2-mercaptoethanol. Monocytes (THP-1) were differentiated into macrophages for 48 hours by plating 2x10 6 cells in a 6 well plate and adding phorbol 12-myristate 13-acetate (PMA). After 48 hours, the penicillin/streptomycin-free media was replaced with LPS, Leukonostok mecenteroides LB-LM1 strain-derived nano-endoplasmic reticulum, Leukonostok mecenteroides LB-LM1 strain (2x10 6 CFU), Ryu The results of confirming the expression of TNFα gene, an inflammatory cytokine gene, are shown in Fig. 5 by treating each of the culture supernatant of the Konostok mecenteroides strain, the medium-derived particles, and the like.
도 5에 나타난 바와 같이, 류코노스톡 메센테로이데스 LB-LM1 균주 유래 나노소포체를 처리한 군에서 TNF-α의 발현이 유의적으로 증가되는 것을 확인함으로써, 이와 관련된 암종에 항암 활성을 유도할 수 있음을 알 수 있다.As shown in Figure 5, by confirming that the expression of TNF-α is significantly increased in the group treated with the leukonostok mecenteroides LB-LM1 strain-derived nanovesicles, it is possible to induce anticancer activity in this related carcinoma it can be seen that there is
<110> LISCure Biosciences Inc. <120> LB-LM1 <130> KP21-0019-1 <150> KR 10-2021-0023436 <151> 2021-02-22 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1487 <212> DNA <213> Leuconostoc mesenteroides LB-LM1 <400> 1 tctgtcagga tgaacgctgg cggcgtgcct aatacatgca agtcgaacgc acagcgaaag 60 gtgcttgcac ctttcaagtg agtggcgaac gggtgagtaa cacgtggaca acctgcctca 120 aggctgggga taacatttgg aaacagatgc taataccgaa taaaacttag tgtcgcatga 180 cacaaagtta aaaggcgctt cggcgtcacc tagagatgga tccgcggtgc attagttagt 240 tggtggggta aaggcctacc aagacaatga tgcatagccg agttgagaga ctgatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc tgcagtaggg aatcttccac 360 aatgggcgaa agcctgatgg agcaacgccg cgtgtgtgat gaaggctttc gggtcgtaaa 420 gcactgttgt atgggaagaa cagctagaat aggaaatgat tttagtttga cggtaccata 480 ccagaaaggg acggctaaat acgtgccagc agccgcggta atacgtatgt cccgagcgtt 540 atccggattt attgggcgta aagcgagcgc agacggttta ttaagtctga tgtgaaagcc 600 cggagctcaa ctccggaatg gcattggaaa ctggttaact tgagtgcagt agaggtaagt 660 ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggcttactgg actgcaactg acgttgaggc tcgaaagtgt gggtagcaaa caggattaga 780 taccctggta gtccacaccg taaacgatga acactaggtg ttaggaggtt tccgcctctt 840 agtgccgaag ctaacgcatt aagtgttccg cctggggagt acgaccgcaa ggttgaaact 900 caaaggaatt gacggggacc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatcctt tgaagctttt agagatagaa gtgttctctt 1020 cggagacaaa gtgacaggtg gtgcatggtc gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttattg ttagttgcca gcattcagat gggcactcta 1140 gcgagactgc cggtgacaaa ccggaggaag gcggggacga cgtcagatca tcatgcccct 1200 tatgacctgg gctacacacg tgctacaatg gcgtatacaa cgagttgcca gcccgcgagg 1260 gtgagctaat ctcttaaagt acgtctcagt tcggattgta gtctgcaact cgactacatg 1320 aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggtctt 1380 gtacacaccg cccgtcacac catgggagtt tgtaatgccc aaagccggtg gcctaacctt 1440 ttaggaagga gccgtctaag gcaggacaga tgactggggt gaagtct 1487 <110> LISCure Biosciences Inc. <120> LB-LM1 <130> KP21-0019-1 <150> KR 10-2021-0023436 <151> 2021-02-22 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1487 <212> DNA <213> Leuconostoc mesenteroides LB-LM1 <400> 1 tctgtcagga tgaacgctgg cggcgtgcct aatacatgca agtcgaacgc acagcgaaag 60 gtgcttgcac ctttcaagtg agtggcgaac gggtgagtaa cacgtggaca acctgcctca 120 aggctgggga taacatttgg aaacagatgc taataccgaa taaaacttag tgtcgcatga 180 cacaaagtta aaaggcgctt cggcgtcacc tagagatgga tccgcggtgc attagttagt 240 tggtggggta aaggcctacc aagacaatga tgcatagccg agttgagaga ctgatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc tgcagtaggg aatcttccac 360 aatgggcgaa agcctgatgg agcaacgccg cgtgtgtgat gaaggctttc gggtcgtaaa 420 gcactgttgt atgggaagaa cagctagaat aggaaatgat tttagtttga cggtaccata 480 ccagaaaggg acggctaaat acgtgccagc agccgcggta atacgtatgt cccgagcgtt 540 atccggattt attgggcgta aagcgagcgc agacggttta ttaagtctga tgtgaaagcc 600 cggagctcaa ctccggaatg gcattggaaa ctggttaact tgagtgcagt agaggtaagt 660 ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggcttactgg actgcaactg acgttgaggc tcgaaagtgt gggtagcaaa caggattaga 780 taccctggta gtccacaccg taaacgatga acactaggtg ttaggaggtt tccgcctctt 840 agtgccgaag ctaacgcatt aagtgttccg cctggggagt acgaccgcaa ggttgaaact 900 caaaggaatt gacggggacc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatcctt tgaagctttt agagatagaa gtgttctctt 1020 cggagacaaa gtgacaggtg gtgcatggtc gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttattg ttagttgcca gcattcagat gggcactcta 1140 gcgagactgc cggtgacaaa ccggaggaag gcggggacga cgtcagatca tcatgcccct 1200 tatgacctgg gctacacacg tgctacaatg gcgtatacaa cgagttgcca gcccgcgagg 1260 gtgagctaat ctcttaaagt acgtctcagt tcggattgta gtctgcaact cgactacatg 1320 aagtcggaat cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggtctt 1380 gtacacaccg cccgtcacac catgggagtt tgtaatgccc aaagccggtg gcctaacctt 1440 ttaggaagga gccgtctaag gcaggacaga tgactggggt gaagtct 1487
Claims (12)
상기 류코노스톡 메센테로이데스 균주는 류코노스톡 메센테로이데스 LB-LM1(Leuconostoc mesenteroides, LB-LM1, 기탁번호 KCCM12701P) 균주인 것을 특징으로 하는, 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The Leuconostoc mesenteroides strain is Leuconostoc mesenteroides LB-LM1 (Leuconostoc mesenteroides, LB-LM1, accession number KCCM12701P), characterized in that the strain, the prevention or treatment of cancer pharmaceutical composition.
상기 나노소포체는 직경이 10 내지 500nm인 것을 특징으로 하는, 암의 예방 또는 치료용 조성물.According to claim 1,
The nano-vesicles have a diameter of 10 to 500 nm, characterized in that the composition for the prevention or treatment of cancer.
상기 나노소포체는 류코노스톡 메센테로이데스 균주에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 암의 예방 또는 치료용 조성물.According to claim 1,
The nano-endoplasmic reticulum is characterized in that naturally or artificially secreted from the Leukonostok mecenteroides strain, the composition for the prevention or treatment of cancer.
상기 암은 대장암, 방광암, 유방암, 흑색종양, 갑상선암, 부갑상선암, 직장암, 인후암, 후두암, 식도암, 췌장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두담낭암, 구강암, 결장암, 항문 부근암, 간암, 폐암 또는 혈액암인 것을 특징으로 하는, 암의 예방 또는 치료용 약학 조성물.According to claim 1,
The cancer is colorectal cancer, bladder cancer, breast cancer, black tumor, thyroid cancer, parathyroid cancer, rectal cancer, throat cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, double gallbladder cancer, oral cancer, colon cancer, perianal cancer, liver cancer , A pharmaceutical composition for the prevention or treatment of cancer, characterized in that it is lung cancer or blood cancer.
상기 조성물은 경구 투여 또는 비경구 투여의 방법으로 투여 가능한 것을 특징으로 하는 조성물.According to claim 1,
The composition is a composition, characterized in that it can be administered by the method of oral administration or parenteral administration.
상기 비경구 투여의 방법은 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐 내 투여 또는 직장 내 투여인 것을 특징으로 하는, 암의 예방 또는 치료용 약학 조성물.7. The method of claim 6,
The method of parenteral administration is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration or rectal administration, cancer prevention or treatment pharmaceutical composition .
상기 식품은 건강기능식품인, 암의 예방 또는 개선용 식품 조성물.9. The method of claim 8,
The food is a health functional food, a food composition for the prevention or improvement of cancer.
A feed additive composition for preventing or improving cancer of livestock, comprising, as an active ingredient, a leukonostok mecenteroides strain-derived nano-vesicles.
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| KR20190105522A (en) | 2018-03-05 | 2019-09-17 | 주식회사 엠디헬스케어 | Nanovesicles derived from Lactobacillus bacteria and Use thereof |
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| KR20190105522A (en) | 2018-03-05 | 2019-09-17 | 주식회사 엠디헬스케어 | Nanovesicles derived from Lactobacillus bacteria and Use thereof |
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