KR102606382B1 - Novel Pseudoalteromonas tetraodonis A2 strain - Google Patents
Novel Pseudoalteromonas tetraodonis A2 strain Download PDFInfo
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- KR102606382B1 KR102606382B1 KR1020210146681A KR20210146681A KR102606382B1 KR 102606382 B1 KR102606382 B1 KR 102606382B1 KR 1020210146681 A KR1020210146681 A KR 1020210146681A KR 20210146681 A KR20210146681 A KR 20210146681A KR 102606382 B1 KR102606382 B1 KR 102606382B1
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Abstract
본 발명은 수탁번호 KACC 92372P로 기탁된 슈도알테로모나스 테트라오도니스(Pseudoalteromonas tetraodonis) A2 균주, 상기 균주의 배양액의 에틸아세테이트 분획물, 상기 분획물을 유효성분으로 함유하는 항염증용 조성물 및 상기 균주를 배양하는 단계; 및 균주 배양액을 에틸아세테이트로 분획하여 에틸아세테이트 분획물을 수득하는 단계;를 포함하는 항염증성 물질의 제조 방법에 관한 것이다.The present invention relates to Pseudoalteromonas tetraodonis A2 strain deposited under accession number KACC 92372P, an ethyl acetate fraction of the culture medium of the strain, an anti-inflammatory composition containing the fraction as an active ingredient, and culturing the strain. steps; and fractionating the strain culture medium with ethyl acetate to obtain an ethyl acetate fraction.
Description
본 발명은 신규 슈도알테로모나스 테트라오도니스 A2 균주에 관한 것으로, 구체적으로 세포독성이 낮으면서, 대식세포의 일산화질소 생성 저해 활성, 프로스타글란딘 E2 생성 저해 활성, 유도형 산화질소 합성효소(iNOS) 및 사이클로옥시게나제-2(COX-2)의 발현 억제 활성, 인터루킨 6(IL-6) 및 인터루킨 1 베타(IL-1β)의 생성 억제와 같은 우수한 항염증 활성을 갖는 물질을 제조하는데 활용할 수 있는 신규 슈도알테로모나스 테트라오도니스 A2 균주에 관한 것이다.The present invention relates to a novel Pseudoalteromonas tetraodonis A2 strain, specifically, low cytotoxicity, inhibitory activity on nitric oxide production in macrophages, inhibitory activity on prostaglandin E2 production, inducible nitric oxide synthase (iNOS), and It can be used to manufacture substances with excellent anti-inflammatory activities, such as inhibiting the expression of cyclooxygenase-2 (COX-2) and inhibiting the production of interleukin 6 (IL-6) and interleukin 1 beta (IL-1β). It relates to the new Pseudoalteromonas tetraodonis A2 strain.
자연계의 수많은 미생물들 중 상업적 및/또는 산업적으로 유용한 미생물을 발굴하여 이를 활용하고자 하는 많은 연구들이 이루어지고 있다. 지금까지 많은 유용한 미생물들이 발굴 및 연구되었지만, 여전히 다른 유용한 미생물을 새롭게 발굴하여 이용가능한 유용 미생물의 풀(pool)을 늘리는 것이 필요하며, 특히 보다 우수한 특성을 나타낼 수 있는 미생물의 발굴이 필요하다.Among the numerous microorganisms in the natural world, many studies are being conducted to discover and utilize commercially and/or industrially useful microorganisms. Although many useful microorganisms have been discovered and studied so far, it is still necessary to discover other useful microorganisms to increase the pool of available useful microorganisms, and in particular, it is necessary to discover microorganisms that can exhibit better characteristics.
한편, 항염증은 의학계 또는 기능성 화장품이나 식품 분야에서 많은 관심을 나타내고 있는 활성이다. 많은 질환들이 염증 반응과 관련되어 있음이 명확해짐에 따라 염증성 질환의 예방 및 치료를 목적으로 한 여러 염증 매개 물질의 생성 및/또는 활성 억제에 대한 관심이 높아지고 있다.Meanwhile, anti-inflammation is an activity that is receiving a lot of attention in the medical field, functional cosmetics, and food fields. As it becomes clear that many diseases are related to inflammatory responses, interest in inhibiting the production and/or activity of various inflammatory mediators for the purpose of preventing and treating inflammatory diseases is increasing.
염증은 물리적 화학적 자극에 의한 외상이나 조직 손상으로부터 신체를 방어하기 위한 생체 조직의 면역 반응으로, 다양한 면역세포에 의해 진행되는 일련의 생물학적 과정이다. 일반적으로 염증 반응은 손상된 조직에서 여러 면역 관련 세포들이 분비하는 일산화질소(NO), 프로스타글란딘(prostaglandin, PG) 및 TNF-α(tumor necrosis factor-α), IL-6(interleukin-6), IL-1β(interleukin-1β)와 같은 전염증성 사이토카인(Pro-inflammatory cytokines)을 포함한 다양한 염증 촉진성 매개 물질에 의해 유도되는데, 이들은 통증, 부종, 열 등의 염증성 증상을 발현하여 다양한 질병의 매개체 역할을 한다.Inflammation is an immune response of living tissue to defend the body from trauma or tissue damage caused by physical or chemical stimuli, and is a series of biological processes carried out by various immune cells. In general, the inflammatory response is caused by nitric oxide (NO), prostaglandin (PG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-2 secreted by various immune-related cells in damaged tissues. It is induced by various pro-inflammatory mediators, including pro-inflammatory cytokines such as 1β (interleukin-1β), which act as mediators of various diseases by causing inflammatory symptoms such as pain, swelling, and fever. do.
체내의 주요 염증 세포로 알려진 대식세포(macrophage)는 세포 표면에 발현하는 TLR(toll-like receptor)을 통해 그람 음성 세균의 외부 세포막 독소 물질인 LPS(lipopoly-saccharide)를 인식하여 세포 내 전사 인자 NF-κB(nuclear factor-κB)의 신호 전달 경로를 활성화하는데, 대식세포의 핵 안으로 이동한 NF-κB는 염증 관련 유전자 iNOS(inducible nitric oxide synthase) 및 COX-2(cyclooxygenase-2)의 발현을 유도함으로써 NO 및 PGE2와 같은 염증 매개 물질의 생성을 증가시킨다. 그 중 NO는 고 반응성 라디칼(radical)의 일종으로, 낮은 농도에서는 신호 전달 및 박테리아의 사멸을 통한 면역 작용 등의 인체의 중요한 생리적역할을 수행하지만, 과 발현될 경우 체내에 적절하지 않은 염증을 유발하여 유전자의 변이, 조직과 신경의 손상을 야기하는 것으로 알려져 있다. 또한 COX-2의 촉매적 활성을 증가시키고, 신호전달 캐스케이드를 촉발하는 등 COX-2의 급성적인 발현을 유도하여 PGE2의 생성을 증가시킴으로써 여러 난치성 질환을 유발하는 것으로도 보고되었다. 이러한 염증성 질환의 원인이 되는 과도한 NO 및 PGE2의 생성이 유도형 iNOS 및 COX-2에 의한 것임을 고려할 때, iNOS와 COX-2 유전자 발현을 억제하는 물질이 염증반응조절 소재로서의 활용 가능성이 높다고 볼 수 있다.Macrophages, known as the main inflammatory cells in the body, recognize LPS (lipopoly-saccharide), an external cell membrane toxin of Gram-negative bacteria, through TLR (toll-like receptor) expressed on the cell surface and produce the intracellular transcription factor NF. It activates the signaling pathway of -κB (nuclear factor-κB), and NF-κB that moves into the nucleus of macrophages induces the expression of inflammation-related genes iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2). By doing so, it increases the production of inflammatory mediators such as NO and PGE2. Among them, NO is a type of highly reactive radical. At low concentrations, it performs important physiological roles in the human body, such as signaling and immune function through bacterial killing, but when overexpressed, it causes inappropriate inflammation in the body. It is known to cause genetic mutation and damage to tissues and nerves. It has also been reported to cause several incurable diseases by increasing the catalytic activity of COX-2, triggering a signal transduction cascade, and increasing the production of PGE2 by inducing acute expression of COX-2. Considering that the excessive production of NO and PGE2, which causes these inflammatory diseases, is caused by inducible iNOS and COX-2, it can be seen that substances that inhibit iNOS and COX-2 gene expression have a high possibility of being used as materials for controlling inflammatory responses. there is.
본 발명자들은 다양한 자연계 시료로부터 유용한 미생물, 특히 상기와 같은 염증과 관련된 활성을 효과적으로 조절함으로써 우수한 항염증 활성을 나타낼 수 있는 물질을 생산하거나 이에 활용할 수 있는 새로운 미생물을 발굴하고자 하였다.The present inventors sought to discover useful microorganisms from various natural samples, especially new microorganisms that can produce or utilize substances that can exhibit excellent anti-inflammatory activity by effectively controlling the above-mentioned inflammation-related activities.
따라서 본 발명의 주된 목적은 우수한 항염증 물질을 생산하거나 이러한 항염증 물질의 생산에 활용할 수 있는 새로운 미생물을 제공하는데 있다.Therefore, the main purpose of the present invention is to produce excellent anti-inflammatory substances or to provide new microorganisms that can be used to produce such anti-inflammatory substances.
본 발명의 한 양태에 따르면, 본 발명은 수탁번호 KACC 92372P로 기탁된 슈도알테로모나스 테트라오도니스(Pseudoalteromonas tetraodonis) A2 균주를 제공한다.According to one aspect of the present invention, the present invention provides Pseudoalteromonas tetraodonis A2 strain deposited under accession number KACC 92372P.
본 발명의 다른 양태에 따르면, 본 발명은 상기 균주의 배양액의 에틸아세테이트 분획물을 제공하다.According to another aspect of the present invention, the present invention provides an ethyl acetate fraction of the culture medium of the above strain.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 분획물을 유효성분으로 함유하는 항염증용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides an anti-inflammatory composition containing the above fraction as an active ingredient.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 균주를 배양하는 단계; 및 균주 배양액을 에틸아세테이트로 분획하여 에틸아세테이트 분획물을 수득하는 단계;를 포함하는 항염증성 물질의 제조 방법을 제공한다.According to another aspect of the present invention, the present invention includes culturing the strain; and fractionating the strain culture medium with ethyl acetate to obtain an ethyl acetate fraction.
본 발명의 균주는 세포독성이 낮으면서, 대식세포의 일산화질소 생성 저해 활성, 프로스타글란딘 E2 생성 저해 활성, 유도형 산화질소 합성효소(iNOS) 및 사이클로옥시게나제-2(COX-2)의 발현 억제 활성, 인터루킨 6(IL-6) 및 인터루킨 1 베타(IL-1β)의 생성 억제와 같은 우수한 항염증 활성을 갖는 물질을 제조하는데 활용할 수 있으며, 본 발명의 방법을 사용하면 상기와 같은 우수한 항염증 활성을 갖는 물질을 쉽게 제조할 수 있다.The strain of the present invention has low cytotoxicity, inhibits nitric oxide production in macrophages, inhibits prostaglandin E 2 production, and expresses inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). It can be used to manufacture a substance with excellent anti-inflammatory activity, such as suppressing activity, inhibiting the production of interleukin 6 (IL-6) and interleukin 1 beta (IL-1β), and using the method of the present invention, excellent anti-inflammatory properties such as the above can be obtained. Substances with inflammatory activity can be easily manufactured.
도 1은 본 발명 슈도알테로모나스 테트라오도니스(Pseudoalteromonas tetraodonis) A2 균주의 미생물 수탁증을 나타낸다.
도 2는 본 발명 A2 균주의 16S rRNA 유전자 서열을 나타낸다.
도 3은 본 발명 A2 균주의 16S rRNA 유전자 염기서열을 토대로 제작된 NJ 계통수(Neighbor-Joining phylogenetic tree)를 나타낸다.
도 4는 본 발명 A2 균주의 균체를 투과 전자 현미경으로 촬영한 사진이다.
도 5는 본 발명 A2 균주를 고체 배양한 사진이다.
도 6은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 세포 독성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄.
도 7은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 일산화질소(NO) 생성 저해 활성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄.
도 8은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 프로스타글란딘 E2(PGE2) 생성 저해 활성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄. 결과값을 3회 반복 실험의 평균±표준편차로 나타냄.
도 9는 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 유도형 산화질소 합성효소(iNOS) 발현 저해 활성을 실험한 결과이다. β-엑틴을 대조군으로 사용함.
도 10은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 사이클로옥시게나제-2(COX-2) 발현 저해 활성을 실험한 결과이다. β-엑틴을 대조군으로 사용함.
도 11은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 종양괴사인자-알파(TNF-α) 생성 저해 활성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄.
도 12는 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 인터루킨 6(IL-6) 생성 저해 활성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄.
도 13은 RAW 264.7 세포를 대상으로 본 발명 A2 균주 배양액의 에틸아세테이트 분획물의 인터루킨 1 베타(IL-1β) 생성 저해 활성을 실험한 결과이다. 결과를 대조군에서 얻은 값에 비교한 백분율로 나타냄.Figure 1 shows the microbial accession certificate of the Pseudoalteromonas tetraodonis A2 strain of the present invention.
Figure 2 shows the 16S rRNA gene sequence of strain A2 of the present invention.
Figure 3 shows a NJ phylogenetic tree (Neighbor-Joining phylogenetic tree) constructed based on the 16S rRNA gene base sequence of the A2 strain of the present invention.
Figure 4 is a photograph taken with a transmission electron microscope of the bacterial cells of strain A2 of the present invention.
Figure 5 is a photograph of solid culture of the A2 strain of the present invention.
Figure 6 shows the results of testing the cytotoxicity of the ethyl acetate fraction of the culture medium of the A2 strain of the present invention on RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group.
Figure 7 shows the results of testing the nitric oxide (NO) production inhibitory activity of the ethyl acetate fraction of the culture medium of the A2 strain of the present invention in RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group.
Figure 8 shows the results of testing the prostaglandin E 2 (PGE2) production inhibitory activity of the ethyl acetate fraction of the culture medium of strain A2 of the present invention in RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group. Results are expressed as the average ± standard deviation of three repeated experiments.
Figure 9 shows the results of testing the inhibitory activity of the inducible nitric oxide synthase (iNOS) expression of the ethyl acetate fraction of the culture medium of strain A2 of the present invention in RAW 264.7 cells. β-actin was used as a control.
Figure 10 shows the results of testing the cyclooxygenase-2 (COX-2) expression inhibitory activity of the ethyl acetate fraction of the A2 strain culture medium of the present invention in RAW 264.7 cells. β-actin was used as a control.
Figure 11 shows the results of testing the tumor necrosis factor-alpha (TNF-α) production inhibitory activity of the ethyl acetate fraction of the culture medium of the A2 strain of the present invention in RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group.
Figure 12 shows the results of testing the interleukin 6 (IL-6) production inhibitory activity of the ethyl acetate fraction of the culture medium of the A2 strain of the present invention in RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group.
Figure 13 shows the results of testing the interleukin 1 beta (IL-1β) production inhibitory activity of the ethyl acetate fraction of the culture medium of strain A2 of the present invention in RAW 264.7 cells. Results are expressed as a percentage compared to the values obtained in the control group.
본 발명의 신규 슈도알테로모나스 테트라오도니스(Pseudoalteromonas tetraodonis) A2 균주는 제주도 구좌읍의 구좌농공단지 양식장 인근 침출지 시료에서 분리되어 국립농업과학원 미생물은행(Korean Agricultural Culture Collection, KACC)에 수탁번호 KACC 92372P로 기탁되어 있다(도 1).The new Pseudoalteromonas tetraodonis A2 strain of the present invention was isolated from a leach field sample near the Gujwa Agricultural and Industrial Complex fish farm in Gujwa-eup, Jeju Island, and was deposited in the National Institute of Agricultural Science Microbial Bank (Korean Agricultural Culture Collection, KACC), accession number KACC 92372P. It is deposited as (Figure 1).
본 발명의 균주는 서열번호 1의 16S rRNA 유전자 서열을 가진다(도 2).The strain of the present invention has the 16S rRNA gene sequence of SEQ ID NO: 1 (FIG. 2).
본 발명의 균주는 슈도알테로모나스 테트라오도니스 GFC와 비교하여 D-만니톨(D-mannitol), 시트르산염(citrate), 발레르산염(valerate), L-프롤린(L-proline), D-말토스(D-maltose), 젖산염(lactate) 및 L-알라닌(L-alanine)의 이용가능여부에서 차이가 있다. 구체적으로, 본 발명의 균주는 D-만니톨을 기질로 이용할 수 있지만 GFC 균주는 D-만니톨을 기질로 이용할 수 없고, 본 발명의 균주는 시트르산염을 기질로 이용할 수 없지만 GFC 균주는 시트르산염을 기질로 이용할 수 있고, 본 발명의 균주는 발레르산염을 기질로 이용할 수 없지만 GFC 균주는 발레르산염을 기질로 이용할 수 있고, 본 발명의 균주는 L-프롤린을 기질로 이용할 수 있지만 GFC 균주는 L-프롤린을 기질로 이용할 수 없고, 본 발명의 균주는 D-말토스를 기질로 이용할 수 없지만 GFC 균주는 D-말토스를 기질로 이용할 수 있고, 본 발명의 균주는 젖산염을 기질로 이용할 수 없지만 GFC 균주는 젖산염을 기질로 이용할 수 있고, 본 발명의 균주는 L-알라닌을 기질로 이용할 수 있지만 GFC 균주는 L-알라닌을 기질로 이용할 수 없다.Compared to Pseudoalteromonas tetraodonis GFC, the strain of the present invention contains D-mannitol, citrate, valerate, L-proline, and D-maltose. There are differences in the availability of (D-maltose), lactate, and L-alanine. Specifically, the strain of the present invention can use D-mannitol as a substrate, but the GFC strain cannot use D-mannitol as a substrate, and the strain of the present invention cannot use citrate as a substrate, but the GFC strain cannot use citrate as a substrate. The strain of the present invention cannot use valerate as a substrate, but the GFC strain can use valerate as a substrate, and the strain of the present invention can use L-proline as a substrate, but the GFC strain cannot use L-proline. cannot be used as a substrate, and the strain of the present invention cannot use D-maltose as a substrate, but the GFC strain can use D-maltose as a substrate, and the strain of the present invention cannot use lactate as a substrate, but the GFC strain can use lactate as a substrate, and the strain of the present invention can use L-alanine as a substrate, but the GFC strain cannot use L-alanine as a substrate.
본 발명의 균주는 마린(marine) 배지, 예를 들어 마린 고체 배지(marine agar) 또는 마린 액체 배지(marine broth)에서, 배양 온도를 약 30℃로 하여 배양할 수 있다.The strain of the present invention can be cultured in a marine medium, for example, marine solid medium (marine agar) or marine liquid medium (marine broth), at a culture temperature of about 30°C.
본 발명 균주의 배양액의 에틸아세테이트(ethyl acetate) 분획물은 높은 농도에서도 대식세포에 대해 세포 독성을 거의 나타내지 않을 수 있다. 예를 들어, 대식세포인 RAW 264.7 세포에 200㎍/㎖의 농도로 처리한 경우에도 대조군 RAW 264.7 세포와 비교하여 80% 이상의 세포 생존율을 나타낼 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention may show little cytotoxicity to macrophages even at high concentrations. For example, even when RAW 264.7 cells, which are macrophages, are treated at a concentration of 200 μg/ml, a cell survival rate of more than 80% can be achieved compared to control RAW 264.7 cells.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 일산화질소(NO)의 생성을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the production of nitric oxide (NO) in macrophages in a concentration-dependent manner.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 프로스타글란딘 E2(PGE2)의 생성을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the production of prostaglandin E 2 (PGE2) in macrophages in a concentration-dependent manner.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 유도형 산화질소 합성효소(iNOS)의 발현을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the expression of inducible nitric oxide synthase (iNOS) in macrophages in a concentration-dependent manner.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 사이클로옥시게나제-2(COX-2)의 발현을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the expression of cyclooxygenase-2 (COX-2) in macrophages in a concentration-dependent manner.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 인터루킨 6(IL-6)의 생성을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the production of interleukin 6 (IL-6) in macrophages in a concentration-dependent manner.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 대식세포에서 농도 의존적으로 인터루킨 1 베타(IL-1β)의 생성을 저해할 수 있다.The ethyl acetate fraction of the culture medium of the strain of the present invention can inhibit the production of interleukin 1 beta (IL-1β) in macrophages in a concentration-dependent manner.
상기와 같은 인자들, 즉 일산화질소, 프로스타글란딘 E2, 유도형 산화질소 합성효소(iNOS), 사이클로옥시게나제-2(COX-2), 인터루킨 6(IL-6) 및 인터루킨 1 베타(IL-1β)은 염증 반응에 중요한 역할을 하는 인자들로, 본 발명 균주의 배양액의 에틸아세테이트 분획물은 이러한 인자들을 효과적으로 조절함으로써 우수한 항염증 활성을 나타낼 수 있다.The above factors, namely nitric oxide, prostaglandin E 2 , inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6) and interleukin 1 beta (IL- 1β) are factors that play an important role in the inflammatory response, and the ethyl acetate fraction of the culture medium of the strain of the present invention can exhibit excellent anti-inflammatory activity by effectively controlling these factors.
본 발명의 항염증용 조성물은 상기와 같은 본 발명 균주의 배양액의 에틸아세테이트 분획물을 유효성분으로 함유하는 것을 특징으로 한다.The anti-inflammatory composition of the present invention is characterized by containing the ethyl acetate fraction of the culture medium of the strain of the present invention as described above as an active ingredient.
이때 분획물은 용매를 제거한 건조물의 형태인 것이 바람직하다.At this time, it is preferable that the fraction is in the form of a dried product from which the solvent has been removed.
본 발명의 항염증용 조성물은 조성물의 형태, 용도 등의 다양한 목적에 따라 상기 분획물을 다양한 정도로 포함할 수 있다. 예를 들어, 조성물 중 분획물을 0.01 내지 100중량%로 함유할 수 있으나, 이로 제한되지 않는다.The anti-inflammatory composition of the present invention may contain the above fractions to various degrees depending on various purposes such as the form and purpose of the composition. For example, the composition may contain 0.01 to 100% by weight of the fraction, but is not limited thereto.
일 실시형태에서, 본 발명의 항염증용 조성물에는, 실질적으로, 본 발명의 분획물 만이 유효성분으로서 포함된다. 다시 말해, 본 발명의 항염증용 조성물 중 본 발명의 분획물 만이 유일한 항염증 물질로서 포함되고, 다른 항염증 물질은 포함되지 않는다. 다른 실시형태에서, 본 발명의 항염증용 조성물에는 본 발명의 분획물 이외에도 다른 항염증 물질이 함께 포함된다. 이때 다른 항염증 물질에는 다른 속의 미생물 및 다른 종의 미생물에서 유래된 물질이 포함되며, 기존에 항염증 활성이 알려진 화합물 또한 포함된다.In one embodiment, the anti-inflammatory composition of the present invention contains substantially only the fraction of the present invention as an active ingredient. In other words, among the anti-inflammatory compositions of the present invention, only the fraction of the present invention is included as the only anti-inflammatory substance and no other anti-inflammatory substances are included. In another embodiment, the anti-inflammatory composition of the present invention includes other anti-inflammatory substances in addition to the fraction of the present invention. At this time, other anti-inflammatory substances include substances derived from other genera and species of microorganisms, and also include compounds known to have anti-inflammatory activity.
본 발명의 항염증용 조성물은 항염증용 약학 조성물, 항염증용 식품 조성물 또는 항염증용 화장료 조성물일 수 있다.The anti-inflammatory composition of the present invention may be an anti-inflammatory pharmaceutical composition, an anti-inflammatory food composition, or an anti-inflammatory cosmetic composition.
또한, 상기와 같은 항염증 효과를 바탕으로, 본 발명의 조성물은 염증성 질환의 예방, 치료 또는 개선용 조성물(예를 들어, 염증성 질환의 예방 또는 치료용 약학적 조성물, 염증성 질환의 예방 또는 개선용 식품 조성물 또는 염증성 질환(염증성 피부 질환)의 예방 또는 개선용 화장료 조성물)일 수 있다.In addition, based on the anti-inflammatory effect as described above, the composition of the present invention is a composition for preventing, treating or improving inflammatory diseases (e.g., a pharmaceutical composition for preventing or treating inflammatory diseases, a composition for preventing or improving inflammatory diseases) It may be a food composition or a cosmetic composition for preventing or improving inflammatory diseases (inflammatory skin diseases).
이때 염증성 질환은 염증을 주병변으로 하는 질환을 의미하는 것으로, 예를 들어 위염, 장염, 간염, 폐렴, 신장염, 방광염, 관절염, 피부염, 알레르기, 아토피, 결막염, 치주염, 비염, 중이염 및 인후염으로 이루어진 군으로부터 선택된 질환일 수 있으나, 이로 제한되지 않는다.At this time, inflammatory disease refers to a disease whose main lesion is inflammation, for example, gastritis, enteritis, hepatitis, pneumonia, nephritis, cystitis, arthritis, dermatitis, allergy, atopy, conjunctivitis, periodontitis, rhinitis, otitis media, and pharyngitis. It may be a disease selected from the group, but is not limited thereto.
본 발명의 항염증용 조성물은 본 발명의 분획물 그 자체, 또는 약학적, 식품학적 또는 화장품학적으로 허용된 담체와 혼합한 조성물일 수 있다.The anti-inflammatory composition of the present invention may be a fraction of the present invention itself, or a composition mixed with a pharmaceutically, foodologically or cosmetically acceptable carrier.
본 발명의 항염증용 조성물은 임상 투여 시에 경구 또는 비경구로 투여가 가능하며, 비경구 투여시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고, 일반적인 의약품 제제의 형태로 사용될 수 있을 것이다.The anti-inflammatory composition of the present invention can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine intrathecal injection, and cerebrovascular injection. It may be administered by intra- or intrathoracic injection and may be used in the form of a general pharmaceutical preparation.
본 발명의 항염증용 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있을 것이다.The anti-inflammatory composition of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response regulators.
본 발명 항염증용 조성물의 일일 투여량은 예를 들어 조성물에 함유된 본 발명의 분획물을 기준으로 체중 1㎏ 당 약 0.0001 내지 200㎎일 수 있으며, 하루 1회 내지 수회 나누어 투여될 수 있으나 투여대상의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양할 것이다.The daily dosage of the anti-inflammatory composition of the present invention may be, for example, about 0.0001 to 200 mg per 1 kg of body weight based on the fraction of the present invention contained in the composition, and may be administered once to several times a day in divided doses, but the subject of administration The range will vary depending on the body weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명 균주의 배양액의 에틸아세테이트 분획물은 상기와 같은 우수한 항염증 활성을 나타내는 항염증성 물질로, 본 발명은 이러한 항염증성 물질의 제조 방법을 제공한다.The ethyl acetate fraction of the culture medium of the strain of the present invention is an anti-inflammatory substance that exhibits excellent anti-inflammatory activity as described above, and the present invention provides a method for producing such an anti-inflammatory substance.
본 발명의 항염증성 물질의 제조 방법은 본 발명의 균주를 배양하는 단계; 및 균주 배양액을 에틸아세테이트로 분획하여 에틸아세테이트 분획물을 수득하는 단계;를 포함하는 것을 특징으로 한다.The method for producing an anti-inflammatory substance of the present invention includes culturing the strain of the present invention; And fractionating the strain culture with ethyl acetate to obtain an ethyl acetate fraction.
상기 배양하는 단계는 바람직하게는 액체 배지, 바람직하게는 마린 액체 배지에서 배양하는 단계로, 바람직하게는 약 30℃에서 2 내지 5일 동안, 바람직하게는 2 내지 4일 동안, 보다 바람직하게는 약 3일 동안 배양하는 단계이다.The culturing step is preferably a step of culturing in a liquid medium, preferably a marine liquid medium, preferably at about 30° C. for 2 to 5 days, preferably for 2 to 4 days, more preferably for about 2 to 5 days. This is the incubation stage for 3 days.
상기 에틸아세테이트 분획물을 수득하는 단계는 바람직하게는 균주 배양액을 에틸아세테이트와 혼합한 다음 정치하여 생성되는 에틸아세테이트층을 회수하는 단계이다. 이때 바람직하게는 균주 배양액의 중량에 대해 0.5 내지 1.5배 중량의 에틸아세테이트를 혼합한다.The step of obtaining the ethyl acetate fraction is preferably a step of mixing the strain culture medium with ethyl acetate and then allowing it to stand to recover the resulting ethyl acetate layer. At this time, preferably 0.5 to 1.5 times the weight of ethyl acetate is mixed with respect to the weight of the strain culture medium.
또한 바람직하게는 에틸아세테이트로 분획하기 전에 균주 배양액으로부터 균체를 제거한다. 균체의 제거는 배양액에서 균체를 제거하는 통상의 방법, 예를 들어 원심분리 또는 여과하는 방법을 사용하여 수행할 수 있다.Also, preferably, the cells are removed from the strain culture medium before fractionation with ethyl acetate. Removal of bacterial cells can be performed using a conventional method for removing bacterial cells from the culture medium, for example, centrifugation or filtration.
또한 바람직하게는 회수한 에틸아세테이트층에서 용매인 에틸아세테이트를 제거하여 최종 분획물을 얻는다.Also, preferably, the solvent ethyl acetate is removed from the recovered ethyl acetate layer to obtain the final fraction.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.
[실시예][Example]
실시예 1. 균주 발굴Example 1. Strain discovery
본 발명의 슈도알테로모나스 테트라오도니스(Pseudoalteromonas tetraodonis) A2 균주는 제주도 구좌읍에 있는 구좌농공단지 양식장 인근 침출지 시료를 10-5으로 희석하고 마린 아가(marine agar) 배지에 도말한 후 30℃에서 3일간 배양한 후 순수 분리하여 얻었다. The Pseudoalteromonas tetraodonis A2 strain of the present invention was prepared by diluting a leach land sample near the Gujwa Agricultural and Industrial Complex fish farm in Gujwa-eup, Jeju Island to 10 -5 and spreading it on a marine agar medium at 30°C. After culturing for 3 days, pure water was isolated.
순수 분리된 균주의 게놈 DNA를 상용화된 게놈 DNA 추출 키트를 활용하여 추출하였으며, 종 동정을 위하여 세균에 특이적인 16S 리보솜 RNA 유전자를 PCR로 증폭하였다. PCR에 사용된 프라이머는 bacterial universal primer 27F(5'-AGAGTTTGATCCTGGCTCAG-3')와 1492R(5'-GGTTACCTTGTTACGACTT-3')이다. 증폭된 PCR 산물을 정제하여 ㈜제노텍에 의뢰하여 전체 염기서열을 확보하여 조립하였다. 조립된 16S rRNA유전자 염기서열(1,519bp)은 EzBioCloud(https://www.ezbiocloud.net/)를 사용하여 분리된 미생물(특히, type strain)과 상동성을 비교하였다.Genomic DNA of the pure isolated strain was extracted using a commercially available genomic DNA extraction kit, and the 16S ribosomal RNA gene specific to the bacteria was amplified by PCR for species identification. Primers used in PCR were bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified PCR product was purified and commissioned by Genotech Co., Ltd. to secure and assemble the entire base sequence. The assembled 16S rRNA gene base sequence (1,519bp) was compared for homology with isolated microorganisms (especially type strain) using EzBioCloud (https://www.ezbiocloud.net/).
A2 균주의 16S rRNA유전자 염기서열을 토대로 하는 상동성 검색결과, 슈도알테로모나스 테트라오도니스 GFC와 99.79%로 가장 높은 상동성을 보인 것으로부터 동일종으로 동정하여, 슈도알테로모나스 테트라오도니스 A2로 명명하였다.As a result of a homology search based on the 16S rRNA gene base sequence of the A2 strain, it was identified as the same species as it showed the highest homology at 99.79% with Pseudoalteromonas tetraodonis GFC, and Pseudoalteromonas tetraodonis A2 It was named.
실시예 2. 균주 특성 조사Example 2. Investigation of strain characteristics
API 20NE(2.1), API 32GN 및 API ZYM 키트를 사용하여 A2 균주의 다양한 효소 활성 및 당 이용성을 조사하였다. A2 균주의 특성 조사 결과를 슈도알테로모나스 테트라오도니스 GFC의 특성과 비교하여 아래 표 1 내지 3에 나타내었다.Various enzyme activities and sugar availability of strain A2 were investigated using API 20NE (2.1), API 32GN, and API ZYM kits. The results of the investigation of the characteristics of the A2 strain are compared with the characteristics of Pseudoalteromonas tetraodonis GFC and are shown in Tables 1 to 3 below.
A2 균주는 GFC 균주와 비교하여 D-만니톨(D-mannitol), 시트르산염(citrate), 발레르산염(valerate), L-프롤린(L-proline), D-말토스(D-maltose), 젖산염(lactate) 및 L-알라닌(L-alanine)의 이용가능여부에서 차이가 있는 것으로 나타났다.Compared to the GFC strain, the A2 strain contains higher levels of D-mannitol, citrate, valerate, L-proline, D-maltose, and lactate ( It was found that there was a difference in the availability of lactate) and L-alanine.
* w : weak.*w: weak.
* w : weak.*w: weak.
실시예 3. 균주 배양물의 생리활성 분석Example 3. Analysis of physiological activity of strain culture
3-1. 방법3-1. method
3-1-1. 분석 시료의 제조3-1-1. Preparation of analytical samples
A2 균주를 마린 액체 배지(Marine broth, MB)에 접종하여 30℃에서 3일간 정치 배양한 후 4,000rpm으로 15분 동안 원심분리하고 5.0㎛의 공극 크기를 갖는 필터(TOYO, 일본 소재)로 여과하여 상등액을 수득하였다. 이후 에틸아세테이트(ethyl acetate)를 사용하여 분획하고 에틸아세테이트 분획물을 감압농축기로 농축하여 건조 시료화하고 DMSO(dimethyl sulfoxide, Sigma-Aldrich, 미국 미주리주 세인트루이스 소재)를 용매로 사용하여 100mM 스톡(stock)을 제조하고 DMEM(Dulbecco's Modified Eagle's medium, Welgene, 한국 경산 소재)으로 희석하여 50, 100 및 200 ㎍/㎖ 농도로 실험을 진행하였다.Strain A2 was inoculated into marine broth (MB) and cultured at 30°C for 3 days, centrifuged at 4,000 rpm for 15 minutes, and filtered using a filter with a pore size of 5.0 μm (TOYO, Japan). The supernatant was obtained. Afterwards, fractionation was performed using ethyl acetate, and the ethyl acetate fraction was concentrated using a reduced pressure concentrator to dry the sample. DMSO (dimethyl sulfoxide, Sigma-Aldrich, St. Louis, Missouri, USA) was used as a solvent to produce 100mM stock. was prepared, diluted with DMEM (Dulbecco's Modified Eagle's medium, Welgene, Gyeongsan, Korea), and experiments were conducted at concentrations of 50, 100, and 200 μg/ml.
3-1-2. 세포배양3-1-2. cell culture
본 연구에서 사용된 LPS는 Sigma-Aldrich(미국 미주리주 세인트루이스 소재)에서 구입하였으며, RAW 264.7 세포는 한국세포주은행에서 분양받아 10% FBS(fetal bovine serum)와 100U/㎖ 페니실린(penicillin), 100㎍/㎖ 스트렙토마이신(streptomycin)을 DMEM에 첨가한 배양액을 사용하여 37℃, 5% CO2 배양기에서 배양하였으며, 2일을 주기로 계대배양 하였다.LPS used in this study was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), and RAW 264.7 cells were purchased from the Korea Cell Line Bank and incubated with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg. /㎖ streptomycin (streptomycin) was added to DMEM and cultured in an incubator at 37°C and 5% CO 2 , and subcultured every 2 days.
3-1-3. 세포 독성 평가3-1-3. Cytotoxicity evaluation
24-웰 플레이트에 RAW 264.7 세포를 7.0×104 세포/웰 만큼 분주하여 37℃, 5%, CO2 조건의 배양기에서 24시간 전 배양한 후 시료와 LPS(1㎍/㎖)를 동시 처리하고, 24시간 동안 배양하였다. 이후 MTT를 첨가하여 37℃에서 4시간 동안 반응시킨 다음 상층액을 제거한 후 DMSO를 첨가하여 침전물을 용해시킨 후 96-웰 플레이트에 옮겨 담아 ELISA 리더를 사용하여 570㎚에서 흡광도를 측정하였다. 각 시료 군에 대한 평균 흡광도를 측정하였으며, 대조군의 흡광도 측정값과 비교하여 세포독성을 평가하였다.RAW 264.7 cells were distributed in a 24- well plate at an amount of 7.0 , cultured for 24 hours. Afterwards, MTT was added and reacted at 37°C for 4 hours, the supernatant was removed, DMSO was added to dissolve the precipitate, and the mixture was transferred to a 96-well plate and the absorbance was measured at 570 nm using an ELISA reader. The average absorbance for each sample group was measured, and cytotoxicity was evaluated by comparing it with the absorbance measurement of the control group.
3-1-4. NO 생성 억제 활성 측정3-1-4. Measurement of NO production inhibition activity
24-웰 플레이트에 RAW 264.7 세포를 8.0×104 세포/웰 만큼 분주하고 37℃, 5% CO2 조건의 배양기에서 24시간 전 배양하였다. 이후 시료와 LPS(1㎍/㎖)를 동시 처리하여 24시간 배양한 후 상층액 100㎕와 그리스(Griess) 시약[1%(w/v) sulfanilamide, 0.1%(w/v) naphylethylenediamine in 2.5%(v/v) phosphoric acid] 100㎕를 96-웰 플레이트에서 동량 혼합하여 10분간 암반응시킨 후 ELISA 리더를 사용하여 540㎚에서 흡광도를 측정하였다.RAW 264.7 cells were distributed in a 24-well plate at an amount of 8.0×10 4 cells/well and cultured in an incubator at 37°C and 5% CO 2 for 24 hours. Afterwards, the sample and LPS (1㎍/㎖) were simultaneously treated and cultured for 24 hours, and then 100㎕ of the supernatant and Griess' reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] 100㎕ was mixed in equal amounts in a 96-well plate and reacted in the dark for 10 minutes, and then the absorbance was measured at 540㎚ using an ELISA reader.
3-1-5. PGE2 생성 억제 활성 측정3-1-5. Measurement of PGE2 production inhibition activity
24-웰 플레이트에 RAW 264.7 세포를 8.0×104 세포/웰 만큼 분주하여 24시간 전 배양한 뒤, 시료와 LPS(1㎍/㎖)를 처리하여 24시간 동안 배양하였다. 이후 배양액을 10,000rpm에서 3분 동안 원심분리하여 침전물을 제거한 뒤 상등액을 회수하였다. 회수한 상등액에서 PGE2(Prostaglandin E2)의 함량을 mouse enzyme-linked immnunosorbent assay(ELISA) 키트(R&D Systems Inc., 미국 미네소타주 미니애폴리스 소재)를 이용하여 측정하였다.RAW 264.7 cells were distributed in a 24 -well plate at 8.0 Afterwards, the culture medium was centrifuged at 10,000 rpm for 3 minutes to remove the precipitate, and the supernatant was recovered. The content of PGE2 (Prostaglandin E 2 ) in the recovered supernatant was measured using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA).
3-1-6. 웨스턴 블롯 분석3-1-6. Western blot analysis
RAW 264.7 세포를 4.0×105 세포/웰 만큼 24시간 전 배양한 후 시료와 LPS(1㎍/㎖)를 동시 처리하여 24시간 배양하였다. 이후 세포를 PBS로 2회 세척하고 용해 완충액(lysis buffer)[1×RIPA(Upstate Cell Signaling Solution, 미국 뉴욕 소재), 1mM phenylmethylsulfonyl fluoride(PMSF), 1mM Na3VO4, 1mM NaF, 1㎍/㎖ aprotinin, 1㎍/㎖ pepstatin 및 1㎍/㎖ leupeptin]을 이용하여 1시간 동안 용해시킨 후 원심분리하여 단백질 상층액을 분리하였다. 단백질 농도는 BCA 키트(Bio-Rad, 미국 소재)를 사용하여 정량하였다. 정량한 단백질을 10%의 폴리아크릴아마이드 겔(polyacrylamide gel)에 전기영동하고 PVDF(poly-vinylidene difluoride) 멤브레인(Milipore, 미국 매사추세츠주 버링턴 소재)에 200mA로 2시간 동안 전이시켰다. 단백질이 전이된 멤브레인을 5% 탈지분유를 포함한 0.05% T/TBS(Tween 20/Tris-buffered saline)에 넣고 상온에서 1시간 블로킹(blocking)시킨 후, 1차 항체와 반응시켰다. 1차 항체 반응은 iNOS 항체(1:5,000, Bio-Rad, 미국 소재), COX-2 항체(1:1,000, Rockland Immunochemicals, Inc., 미국 소재), β-엑틴 항체 클론(β-actin antibody clone) AC-74(1:10,000, Sigma, 미국 소재), 포스포-ERK1/2(phospho-ERK1/2) 항체, 포스포-SAPK/JNK(phospho-SAPK/JNK)(Thr180/Tyr185) 항체, 포스포-p38(phospho-p38) 항체, IκB-α 항체, 포스포-NF-κB(phospho-NF-κB) 항체(1:1,000, Cell signaling Tech, 미국 매사추세츠주 댄버스 소재)를 이용하여 4℃에서 하루 밤 동안 반응시킨 후 0.05% T/TBS 용액으로 3회 세척 후 2차 항체(Jackson ImmunoResearch, 미국 소재)를 1:10,000으로 희석하여 상온에서 1시간 반응한 뒤 0.05% T/TBS 용액으로 3회 세척하였다. 단백질은 ECL 키트(Bio-Rad, 미국 소재)를 사용하여 이미징 덴시토미터(imaging densitometer, model GS-700, Bio-rad, 미국 소재)를 통해 측정하였다. 또한, 이미지J 프로그램(NIH, 미국 메릴랜드주 베데스다 소재)을 이용하여 β-엑틴 대비 iNOS와 COX-2 단백질 발현량의 면적을 수치화한 뒤 그래프로 나타내었다.RAW 264.7 cells were cultured at 4.0 Afterwards , the cells were washed twice with PBS and lysed with lysis buffer [1 After dissolving for 1 hour using aprotinin, 1㎍/㎖ pepstatin and 1㎍/㎖ leupeptin], the protein supernatant was separated by centrifugation. Protein concentration was quantified using the BCA kit (Bio-Rad, USA). The quantified protein was electrophoresed on a 10% polyacrylamide gel and transferred to a PVDF (poly-vinylidene difluoride) membrane (Milipore, Burlington, MA, USA) at 200 mA for 2 hours. The membrane to which the protein had been transferred was placed in 0.05% T/TBS (Tween 20/Tris-buffered saline) containing 5% skim milk powder, blocked for 1 hour at room temperature, and then reacted with the primary antibody. The primary antibody reaction consisted of iNOS antibody (1:5,000, Bio-Rad, USA), COX-2 antibody (1:1,000, Rockland Immunochemicals, Inc., USA), and β-actin antibody clone. ) AC-74 (1:10,000, Sigma, USA), phospho-ERK1/2 (phospho-ERK1/2) antibody, phospho-SAPK/JNK (phospho-SAPK/JNK) (Thr180/Tyr185) antibody; 4 using phospho-p38 antibody, IκB-α antibody, and phospho-NF-κB (phospho-NF-κB) antibody (1:1,000, Cell signaling Tech, Danvers, MA, USA). After reacting at ℃ overnight, washing three times with 0.05% T/TBS solution, secondary antibody (Jackson ImmunoResearch, USA) was diluted 1:10,000, reacted at room temperature for 1 hour, and then washed with 0.05% T/TBS solution. Washed 3 times. Proteins were measured using an imaging densitometer (model GS-700, Bio-rad, USA) using an ECL kit (Bio-Rad, USA). In addition, the area of iNOS and COX-2 protein expression compared to β-actin was quantified using the ImageJ program (NIH, Bethesda, Maryland, USA) and displayed in a graph.
3-1-7. 전염증성 사이토카인 생성 억제 활성 측정3-1-7. Measurement of activity to suppress pro-inflammatory cytokine production
24-웰 플레이트에 RAW 264.7 세포를 7.0×104 세포/웰의 농도로 분주하여 37℃, 5% CO2 조건에서 24시간 동안 전 배양한 후, 농도별 시료와 LPS(1㎍/㎖)를 동시에 처리하여 24시간 배양하였다. 이후 배양액을 원심분리(10,000rpm, 3분)하여 침전물을 제거하였고, 상등액을 회수하여 전염증성 사이토카인(TNF-α, IL-6, IL-1β)의 생성량을 측정하였다. 상등액에서의 전염증성 사이토카인 생성량은 Mouse TNF-α ELISA 키트(Invitrogen, 미국 캘리포니아 소재), Mouse IL-6 ELISA 키트(BD Biosciences, 미국 캘리포니아 소재), Mouse IL-1β ELISA 키트(R&D Systems Inc., 미국 미네소타주 미니애폴리스 소재)를 이용하여 측정하였다.RAW 264.7 cells were dispensed into a 24- well plate at a concentration of 7.0 They were treated simultaneously and cultured for 24 hours. Afterwards, the culture was centrifuged (10,000 rpm, 3 minutes) to remove sediment, and the supernatant was recovered to measure the production of proinflammatory cytokines (TNF-α, IL-6, and IL-1β). The amount of pro-inflammatory cytokines produced in the supernatant was measured using the Mouse TNF-α ELISA kit (Invitrogen, California, USA), Mouse IL-6 ELISA kit (BD Biosciences, California, USA), and Mouse IL-1β ELISA kit (R&D Systems Inc., USA). It was measured using (Minneapolis, Minnesota, USA).
3-2. 결과3-2. result
3-2-1. 세포독성 및 NO 저해 활성 측정3-2-1. Measurement of cytotoxicity and NO inhibitory activity
A2 균주 유래 시료(배양액의 에틸아세테이트 분획물)를 50, 100, 200㎍/㎖의 농도로 처리한 결과, 모든 농도에서 80% 이상의 세포 생존율이 확인되었다(도 6).As a result of treating the A2 strain-derived sample (ethyl acetate fraction of the culture medium) at concentrations of 50, 100, and 200 μg/ml, a cell survival rate of more than 80% was confirmed at all concentrations (FIG. 6).
시료가 RAW 264.7 세포에서 NO 생성량에 미치는 영향을 알아보기 위해 LPS(1㎍/㎖)와 시료를 50, 100, 200㎍/㎖의 농도로 처리한 결과, RAW 264.7 세포에서 농도 의존적으로 NO의 생성이 감소하는 것으로 나타났다(도 7).To determine the effect of samples on NO production in RAW 264.7 cells, samples were treated with LPS (1㎍/㎖) at concentrations of 50, 100, and 200㎍/㎖. As a result, NO was produced in a concentration-dependent manner in RAW 264.7 cells. appeared to decrease (Figure 7).
3-2-2. PGE2 생성 억제 활성 측정3-2-2. Measurement of PGE2 production inhibition activity
A2 균주 유래 시료를 50, 100, 200㎍/㎖의 농도로 처리하여 PGE2의 생성량을 조사한 결과, 50㎍/㎖의 농도에서는 감소 경향이 확인되지 않았으나, 100㎍/㎖에서 큰 폭으로 감소하였으며, 가장 고농도인 200㎍/㎖에서는 LPS 무처리 군과 유사한 수준의 감소 활성이 확인되었다(도 8).As a result of examining the amount of PGE2 produced by treating samples derived from the A2 strain at concentrations of 50, 100, and 200㎍/㎖, no decreasing trend was confirmed at the concentration of 50㎍/㎖, but it decreased significantly at 100㎍/㎖. At the highest concentration, 200㎍/㎖, a similar level of reduction activity as that of the LPS untreated group was confirmed (Figure 8).
3-2-3. iNOS 및 COX-2 발현 억제3-2-3. Inhibition of iNOS and COX-2 expression
웨스턴 블롯 수행 결과 50, 100, 200㎍/㎖의 농도로 A2 균주 유래 시료를 처리한 RAW 264.7 세포에서 iNOS의 발현이 감소하는 것으로 나타났으며, NO와 같은 경향으로 감소하는 것으로 나타났다(도 9). 따라서 시료 처리에 의한 NO의 감소는 iNOS의 발현 감소와 관련이 있음을 확인하였다. 뿐만 아니라 COX-2의 발현 또한 PGE2와 유사한 경향을 보이며 감소하는 것으로 볼 때(도 10), PGE2의 감소 활성이 COX-2의 발현 감소와 밀접한 연관이 있는 것으로 판단된다.Western blot results showed that the expression of iNOS decreased in RAW 264.7 cells treated with samples derived from the A2 strain at concentrations of 50, 100, and 200 ㎍/㎖, and decreased in the same trend as NO (Figure 9) . Therefore, it was confirmed that the decrease in NO due to sample treatment was related to the decrease in the expression of iNOS. In addition, given that the expression of COX-2 also decreased with a similar trend to that of PGE2 (Figure 10), it is believed that the decreasing activity of PGE2 is closely related to the decreased expression of COX-2.
3-2-4. 전염증성 사이토카인 생성 억제 활성3-2-4. Inhibitory activity on pro-inflammatory cytokine production
A2 균주 유래 시료를 처리하여 전염증성 사이토카인인 TNF-α, IL-6, IL-1β의 생성을 측정하였다. 시료는 TNF-α에서는 큰 억제 효과를 보이지 않으나 IL-6, IL-1β는 농도 의존적으로 억제하는 경향을 보였으며, 특히 IL-1β의 경우 가장 고농도인 200㎍/㎖에서 무처리군과 유사한 저해 수준을 보였다(도 11 내지 13).Samples derived from the A2 strain were processed to measure the production of proinflammatory cytokines TNF-α, IL-6, and IL-1β. The sample did not show a significant inhibitory effect on TNF-α, but showed a tendency to inhibit IL-6 and IL-1β in a concentration-dependent manner. In particular, in the case of IL-1β, inhibition was similar to that of the untreated group at the highest concentration of 200㎍/㎖. level (Figures 11 to 13).
<110> National Institute of Biological Resources <120> Novel Pseudoalteromonas tetraodonis A2 strain <130> ALP21023 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1411 <212> DNA <213> Unknown <220> <223> Pseudoalteromonas tetraodonis A2 <400> 1 gactcacccc cagtcatgaa tcacgtccgt ggtaaacgtc ctcccgaggg ttagactatc 60 tacttctgga gcaacccact cccatggtgt gacgggcggt gtgtacaagg cccgggaacg 120 tattcaccgc gtcattctga tacgcgatta ctagcgattc cgacttcatg gagtcgagtt 180 gcagactcca atccggacta cgacgcactt taagtgattc gcttactctc gcgagttcgc 240 agcactctgt atgcgccatt gtagcacgtg tgtagcccta cacgtaaggg ccatgatgac 300 ttgacgtcgt ccccaccttc ctccggttta tcaccggcag tctccttaga gttctcagca 360 ttacctgcta gcaactaagg ataggggttg cgctcgttgc gggacttaac ccaacatctc 420 acaacacgag ctgacgacag ccatgcagca cctgtatcag agttcccgaa ggcaccaaac 480 catctctggt aagttctctg tatgtcaagt gtaggtaagg ttcttcgcgt tgcatcgaat 540 taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcatttgagt tttaaccttg 600 cggccgtact ccccaggcgg tctacttaat gcgttagctt tgaaaaacag aaccgaggtt 660 ccgagcttct agtagacatc gtttacggcg tggactacca gggtatctaa tcctgtttgc 720 tccccacgct ttcgtacatg agcgtcagtg ttgacccagg tggctgcctt cgccatcggt 780 attccttcag atctctacgc atttcaccgc tacacctgaa attctaccac cctctatcac 840 actctagttt gccagttcga aatgcagttc ccaggttgag cccggggctt tcacatctcg 900 cttaacaaac cgcctgcgta cgctttacgc ccagtaattc cgattaacgc tcgcaccctc 960 cgtattaccg cggctgctgg cacggagtta gccggtgctt cttctgtcag taacgtcaca 1020 gctagcaggt attaactact aacctttcct ccttgactga aagtgcttta caacccgaag 1080 gccttcttca caacacgcgg catggctgca tcaggcttgc gcccattgtg caatattccc 1140 cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc tgatcatcct 1200 ctcaaaccag ctagggatcg tcgccttggt gagccattac ctcaccaact agctaatccc 1260 acttgggcca atctaaaggc gagagccgaa gccccctttg gtccgtagac attatgcggt 1320 attagcagtc gtttccaact gttgtccccc acctcaaggc atgttcccaa gcattactca 1380 cccgtccgcc gctcgtcatc ttctagcaag c 1411 <110> National Institute of Biological Resources <120> Novel Pseudoalteromonas tetraodonis A2 strain <130> ALP21023 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1411 <212> DNA <213> Unknown <220> <223> Pseudoalteromonas tetraodonis A2 <400> 1 gactcacccc cagtcatgaa tcacgtccgt ggtaaacgtc ctcccgaggg ttagactatc 60 tacttctgga gcaacccact cccatggtgt gacgggcggt gtgtacaagg cccgggaacg 120 tattcaccgc gtcattctga tacgcgatta ctagcgattc cgacttcatg gagtcgagtt 180 gcagactcca atccggacta cgacgcactt taagtgattc gcttactctc gcgagttcgc 240 agcactctgt atgcgccatt gtagcacgtg tgtagcccta cacgtaaggg ccatgatgac 300 ttgacgtcgt ccccaccttc ctccggttta tcaccggcag tctccttaga gttctcagca 360 ttacctgcta gcaactaagg atagggttg cgctcgttgc gggacttaac ccaacatctc 420 acaacacgag ctgacgacag ccatgcagca cctgtatcag agttcccgaa ggcaccaaac 480 catctctggt aagttctctg tatgtcaagt gtaggtaagg ttcttcgcgt tgcatcgaat 540 taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcatttgagt tttaaccttg 600 cggccgtact ccccaggcgg tctacttaat gcgttagctt tgaaaaaacag aaccgaggtt 660 ccgagcttct agtagacatc gtttacggcg tggactacca gggtatctaa tcctgtttgc 720 tccccacgct ttcgtacatg agcgtcagtg ttgacccagg tggctgcctt cgccatcggt 780 attccttcag atctctacgc atttcaccgc tacacctgaa attctaccac cctctatcac 840 actctagttt gccagttcga aatgcagttc ccaggttgag cccggggctt tcacatctcg 900 cttaacaaac cgcctgcgta cgctttacgc ccagtaattc cgattaacgc tcgcaccctc 960 cgtattaccg cggctgctgg cacggagtta gccggtgctt cttctgtcag taacgtcaca 1020 gctagcaggt attaactact aacctttcct ccttgactga aagtgcttta caacccgaag 1080 gccttcttca caacacgcgg catggctgca tcaggcttgc gcccattgtg caatattccc 1140 cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc tgatcatcct 1200 ctcaaaccag ctagggatcg tcgccttggt gagccattac ctcaccaact agctaatccc 1260 acttgggcca atctaaaggc gagagccgaa gccccctttg gtccgtagac attatgcggt 1320 attagcagtc gtttccaact gttgtccccc acctcaaggc atgttcccaa gcattactca 1380 cccgtccgcc gctcgtcatc ttctagcaag c 1411
Claims (4)
균주 배양액을 에틸아세테이트와 혼합하고 정치하여 생성되는 에틸아세테이트층을 회수하고 용매인 에틸아세테이트를 제거하여 에틸아세테이트 분획물을 수득하는 단계;를 포함하는 항염증용 조성물의 제조 방법.Cultivating the strain of claim 1; and
A method for producing an anti-inflammatory composition comprising the step of mixing the strain culture medium with ethyl acetate and allowing it to stand, recovering the resulting ethyl acetate layer, and removing the solvent, ethyl acetate, to obtain an ethyl acetate fraction.
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| KR20230061928A (en) | 2023-05-09 |
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