KR101671498B1 - Non-reducing end of Unsaturated Mannuronic Acid Oligosaccharides and Composition comprising the same as Active Ingredient - Google Patents
Non-reducing end of Unsaturated Mannuronic Acid Oligosaccharides and Composition comprising the same as Active Ingredient Download PDFInfo
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- KR101671498B1 KR101671498B1 KR1020160014484A KR20160014484A KR101671498B1 KR 101671498 B1 KR101671498 B1 KR 101671498B1 KR 1020160014484 A KR1020160014484 A KR 1020160014484A KR 20160014484 A KR20160014484 A KR 20160014484A KR 101671498 B1 KR101671498 B1 KR 101671498B1
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- acid oligosaccharide
- present
- unsaturated
- oligosaccharide
- composition
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Abstract
본 발명은 폴리만뉴론산(poly-mannuronate) 기질로 하여 알긴산 라이아제(alginate lyase)에 의해 분해된 분자량 100-3000 Da 이하의 비환원성 말단의 불포화형 만뉴론산 올리고당에 관한 것으로, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당 및 이를 유효성분으로 포함하는 비만, 당뇨 및 갱년기 증후군의 개선, 예방 또는 치료용 약제학적 조성물 및 장내 유익균 증진용 프로바이오틱스를 제공한다. 본 발명에 의하면, 본 발명의 항비만, 항당뇨, 에스트로겐 활성 및 장내 미생물 균총 조절 효과는 비환원성 말단의 포화형 만뉴론산 올리고당의 만뉴론산 올리고당과 비교하여 현저히 우수하다.The present invention relates to a non-reducing terminal unsaturated mannuronic acid oligosaccharide having a molecular weight of 100-3000 Da or less, decomposed by an alginate lyase, as a poly-mannuronate substrate, The present invention provides a pharmaceutical composition for improving, preventing or treating obesity, diabetes and menopausal syndrome comprising the unsaturated maleuronic acid oligosaccharide as an active ingredient and a probiotic for promoting beneficial bacteria in the intestines. According to the present invention, the anti-obesity, anti-diabetic, estrogenic activity and intestinal microflora controlling effect of the present invention are remarkably superior to the mannuronic acid oligosaccharides of the non-reducing terminal saturated mannuronic acid oligosaccharide.
Description
본 발명은 비환원성 말단의 불포화형 만뉴론산 올리고당 및 이를 유효성분으로 포함하는 조성물에 관한 것이다.The present invention relates to a non-reducing terminal unsaturated maleuronic acid oligosaccharide and a composition comprising the same as an effective ingredient.
현대사회에는 비만(2014년 기준, OECD 성인 18%가 비만) 및 당뇨(2011년 기준, 6.9 %가 당뇨병)에 의해 나타나는 복합적 대사증후군(metabolic syndrome)이 증가됨에 따라 기능성 식품 시장이 확대되고 있다.In the modern world, the functional food market is expanding as the metabolic syndrome of obesity (based on 2014, 18% of adults in OECD obesity) and diabetes (6.9% of diabetes in 2011) is increased.
대사증후군은 비만과 인슐린 저항성에 의해 발생되는 제 2형 당뇨병과 다양한 여러 대사이상이 복합적으로 발생하는 증후군을 지칭하는 말이다. 대사증후군은 인구의 고령화와 고열량의 식이습관 등에 의하여 급증함에 따라 사회적인 비용이 많이 요구됨으로서 근본적인 원인 예방 및 의약품 또는 식품 소재 개발이 시급한 실정이다.Metabolic syndrome refers to a syndrome in which
인간의 장내 생태계는 출생과 동시에 미생물이 서식하기 시작하여 유익균과 유해균이 균형을 유지하며 장내 미생물 균총을 형성하고 인간과 공생하며 상호작용을 통해 인간의 건강에 직·간접으로 영향을 미친다. 최근 미국 국립보건원(NIH)은 ‘인간 미생물 군집 프로젝트’를 통해 장내 미생물 균총과 질병과의 관계를 연구하여 미생물 균총의 불균형이 염증성 장질환 등의 발병에 중요한 원인을 제공함에 따라 장내 미생물 균총의 정상화의 중요성을 대두시켰다.1,2 The intestinal ecosystem of humans begins to live with microorganisms at the same time as birth, balances beneficial bacteria and harmful microorganisms, forms intestinal microflora, affects human health directly or indirectly through interaction with humans. Recently, the National Institutes of Health (NIH) has been studying the relationship between intestinal microflora and disease through the 'Human Microbial Community Project'. As the imbalance of microflora provides important causes for inflammatory bowel disease, . 1,2
2006년 과학저널 ‘네이처’에 발표된 비만과 장내 세균의 연관성 논문에 따르면 마른 사람과 비만인 사람 사이 장내 세균 분포가 다르며, 쥐를 이용한 실험을 통해 비만 쥐에서는 퍼미테쿠스(Firmicutes)에 속하는 세균이 박테로이데트(Bacteroidetes)에 속하는 세균보다 상대적으로 높은 구성비율을 보임을 확인하였다,3 이 후 장내 미생물과 인체에 대한 연구가 다양한 영역에서 진행되고 있으며, 장내미생물총 개선을 통한 비만 억제 및 질환 치료 등을 위하여 장내 미생물 균총 개선제 개발이 필요하다.According to a 2006 article published in the journal Nature, Obesity and the relationship between intestinal bacteria According to the article, the distribution of intestinal bacteria is different between dry and obese people. Through the experiment using mice, the bacteria belonging to Firmicutes foil teroyi was confirmed that the relative show a high composition ratio and more bacteria belonging to Bernadette (Bacteroidetes), 3 a, and the study of the intestinal flora and human health is going on in various fields after, obesity inhibition and treatment of diseases through the intestinal microflora improvement total It is necessary to develop an intestinal microflora improving agent.
해양생물은 지구상 생물종의 약 80%에 해당하는 약 50만 종의 생물이 서식하고 있는 것으로 추정되나, 이 가운데 유용생물자원으로 개발되고 있는 것이 1% 미만으로 개발 잠재력이 매우 높다. 해조류 유래 기능성 소재로서 대표적인 해조 다당류 알긴산(alginic acid)은 다시마, 미역등과 같은 갈조류에 15-35% 함유되어 있으며, β-D-만뉴론산(β-D-mannuronic acid; M)과 α-L-글루론산(α-L-guluronic acid; G)의 2종류의 우론산(uronic acid)이 각종 비율로 1, 4 글리코시드(glycoside) 결합을 한 폴리우로나이드(polyuronide)의 특징을 갖는다. 알긴산 유래 올리고당은 구성당에 따라 만뉴론산 올리고당(MOS, mannuro-oligosaccharide)과 글루론산 올리고당(GOS, guluro-oligosaccharide) 및 만뉴론산 글루론산 혼합 올리고당(AOS, alginate oligosaccharide)으로 나눌 수 있으며 또한 당 말단의 이중결합에 따라 구분할 수 있다.Although marine life is estimated to be about 500,000 species, about 80% of the world's species, less than 1% of them are being developed as useful biological resources and their development potential is very high. Alginic acid, a representative seaweed polysaccharide as a functional material derived from seaweed, is contained in 15-35% of brown algae such as sea tangle and seaweed, and β-D-mannuronic acid (M) and α-L (L-guluronic acid; G) is characterized by a polyuronide with 1, 4 glycoside bonds at various ratios of uronic acid. The alginate-derived oligosaccharide can be divided into mannuro-oligosaccharide (MOS), guluro-oligosaccharide (GOS), and alginate oligosaccharide (mannuronic acid oligosaccharide) It can be classified according to the double bond.
해양 유래 다당류는 오래전부터 인간생활에 이용되어 왔으며, 세계적으로 해양생물 유래의 항암, 항산화, 항고혈압 및 항균물질 등의 생리활성에 대한 연구가 활발히 진행되고 있다. 전세계적으로 생산되는 알긴산 원조 생산량은 약 10만 톤으로 이중 30% 가량이 식품첨가물로 이용되고 있으나 이를 고부가 의약품 원료로 개발 할 경우 그 가치를 배가 시킬 수 있다. 알긴산 유래 올리고당의 경우 구조적인 특정에 따라 고등식물의 뿌리 성장촉진, 비피도박테리움 에스피(Bifidobacterium sp.) 성장촉진, 항염증, 항산화, 항균 작용 등의 생물학적 활성이 보고되는 등 가시적인 연구 성과가 이루어짐에 따라 응용분야가 폭넓게 확산되고 있다.Background Art [0002] Marine-derived polysaccharides have been used for a long time in human life, and studies on bioactivity such as anticancer, antioxidant, antihypertensive and antimicrobial substances derived from marine life have been actively conducted worldwide. The production of alginic acid aids worldwide is about 100,000 tons, of which about 30% is used as a food additive, but this value can be doubled if it is developed as a high value-added drug raw material. In the case of oligosaccharide derived from alginate, biological activities such as promotion of root growth of higher plants, promotion of growth of Bifidobacterium sp., Anti-inflammation, antioxidation and antibacterial activity have been reported according to structural specificity. As a result, applications are spreading widely.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 알긴산 올리고당을 소재로 하는 의약품 및 식품으로의 이용을 증진시키고자 노력하였다. 그 결과, 항비만, 항당뇨, 장내 미생물 균총 개선 및 에스트로겐 효능과 같은 다양한 생리활성을 갖는 알긴산 유래 비환원성 말단의 불포화형 만뉴론산 올리고당을 규명함으로써 본 발명을 완성하였다.The present inventors have sought to promote the use of alginic acid oligosaccharide as a medicine and food. As a result, the present invention has been completed by identifying unsaturated maleuronic acid oligosaccharides derived from alginic acid-derived non-reducing end having various physiological activities such as anti-obesity, anti-diabetic, intestinal microflora improvement and estrogenic activity.
따라서, 본 발명의 목적은 비환원성 말단의 불포화형 만뉴론산 올리고당을 제공하는 데 있다.Accordingly, an object of the present invention is to provide an unsaturated maleuronic acid oligosaccharide of non-reducing end.
본 발명의 다른 목적은 비만 개선, 예방 또는 치료용 조성물을 제공하는 데 있다.It is another object of the present invention to provide a composition for improving, preventing or treating obesity.
본 발명의 또다른 목적은 당뇨 개선, 예방 또는 치료용 조성물을 제공하는 데 있다.It is still another object of the present invention to provide a composition for improving, preventing or treating diabetes.
본 발명의 다른 목적은 장내 유익균 증진용 프로바이오틱스를 제공하는 데 있다.Another object of the present invention is to provide a probiotic for promoting beneficial bacteria in the intestines.
본 발명의 또다른 목적은 갱년기 증후군 개선, 예방 또는 치료용 조성물을 제공하는 데 있다.It is still another object of the present invention to provide a composition for improving, preventing or treating menopausal symptoms.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 폴리만뉴론산(poly-mannuronate) 기질로 하여 알긴산 라이아제(alinate lyase)에 의해 분해된 분자량 100-3000 Da 이하의 비환원성 말단의 불포화형 만뉴론산 올리고당을 제공한다.According to one aspect of the present invention, the present invention provides a non-reducing terminal unsaturated-type mannuronic acid oligosaccharide having a molecular weight of 100-3000 Da or less, decomposed by an alginate lyase, as a poly- mannuronate substrate, .
본 발명자들은 알긴산 올리고당을 소재로 하는 의약품 및 식품으로의 이용을 증진시키고자 노력하였다. 그 결과, 항비만, 항당뇨, 장내 미생물 균총 개선 및 에스트로겐 효능과 같은 다양한 생리활성을 갖는 알긴산 유래 비환원성 말단의 불포화형 만뉴론산 올리고당을 규명하였다.The present inventors have sought to promote the use of alginic acid oligosaccharide as a medicine and food. As a result, an unsaturated maleuronic acid oligosaccharide of alginic acid-derived non-reducing end having a variety of physiological activities such as anti-obesity, anti-diabetic, intestinal microflora improvement and estrogen potency was identified.
본 명세서에서 용어 “비환원성”은 올리고당의 구조에 있어서, 아노머[단당류의 헤미아세탈(C-1과 C-5 사이의 고리 형성), 헤미케탈(C-2와 C-5 사이의 고리 형성)을 생성하는 고리화 반응에서 한 탄소에 붙은 수소원자와 수산화기의 위치가 바뀐 부분입체 이성질체가 생기는 현상) 탄소를 갖지 못한 성질을 의미한다.As used herein, the term " non-reducing " refers to the structure of oligosaccharides, including the anomeric monosaccharide hemiacetal (ring formation between C-1 and C-5), hemiketal (ring formation between C-2 and C-5 ) In the cyclization reaction to produce a diastereomer that changes the position of hydrogen atoms and hydroxyl groups attached to one carbon.
본 명세서에서 용어 “불포화형”은 수소원자의 탄소사슬(carbon chain)이 불포화된 형태를 의미하고, “포화형”은 수소원자의 탄소사슬이 포화된 형태를 의미한다.As used herein, the term " unsaturated " means a form in which a carbon chain of a hydrogen atom is unsaturated, and " saturated type " means a form in which a carbon chain of a hydrogen atom is saturated.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 아노머 탄소를 갖지 못하고 수소원자의 탄소사슬이 불포화된 형태의 모든 만뉴론산 올리고당을 의미한다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention means all mannuronic acid oligosaccharides having no anomeric carbon and unsaturated carbon chain of hydrogen atom.
상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 다음의 일 예(3개의 당이 결합된 만뉴론산 올리고당)의 구조식 1을 갖는다:The non-reducing terminal unsaturated maleuronic acid oligosaccharide has the
[구조식 1][Structural formula 1]
상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 1 당인 경우에 175, 2 당인 경우에 351, 3 당인 경우에 527, 4 당인 경우에 704, 5 당인 경우에 880, 6 당인 경우에 1056, 7 당인 경우에 1232의 Z-평균 분자량(m/z)을 갖는다(도 2).The unsaturated monounsaturated mannitol oligosaccharide at the non-reducing end is 175, 2, 351, 3, 52, 4, 70, 5, 8, 6, Average molecular weight (m / z) of 1232 (Fig. 2).
또한, 본 명세성서 용어 “비환원성 말단의 포화형 만뉴론산 올리고당”은 폴리만뉴론산을 산 가수분해하여 얻은 포화형 만뉴론산 올리고당을 의미한다.In addition, this specification means the saturated mannuronic acid oligosaccharide obtained by acid hydrolysis of polymannuronic acid, the term " non-reducing terminal saturated mannuronic acid oligosaccharide ".
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 폴리만뉴론산(poly-mannuronate) 기질로 하여 알긴산 라이아제(alginate lyase)에 의해 분해되어 제조된다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention is prepared by digesting with an alginate lyase as a poly-mannuronate substrate.
본 발명의 일 구현예에 따르면, 상기 알긴산 라이아제는 폴리굴루론산(polyguluronate) 및 폴리만뉴론산(polymannuronate)로 구성된 알긴산(alginate)를 분해하여 저분자화 하는 효소를 의미한다.According to one embodiment of the present invention, the alginic acid lyase refers to an enzyme that decomposes and low-molecularizes alginate composed of polyguluronate and polymannuronate.
본 발명의 특정 구현예에 따르면, 상기 알긴산 라이아제는 전복 장내 균주 유래의 AlyDW11(대한민국 등록특허 10-1277706)이다.According to a specific embodiment of the present invention, the alginate lyase is AlyDW11 (Korea Patent No. 10-1277706) derived from an abalone intestinal strain.
본 발명의 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 하나 이상의 당으로 구성된다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention is composed of one or more saccharides.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 1개 내지 10개의 만뉴론산 또는 글루론산을 포함한다. 본 발명의 다른 구현예에 따르면, 1개 내지 9개의 만뉴론산 또는 글루론산을 포함한다. 본 발명의 또다른 구현예에 따르면, 1개 내지 8개의 만뉴론산 또는 글루론산을 포함한다. 본 발명의 특정 구현예에 따르면, 1개 내지 7개의 만뉴론산 또는 글루론산을 포함한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharide comprises 1 to 10 mannuronic acid or glutaric acid. According to another embodiment of the invention, it comprises from 1 to 9 mannuronic acid or glutaric acid. According to another embodiment of the present invention, it comprises 1 to 8 mannuronic acid or glutaric acid. According to a particular embodiment of the invention, it comprises from 1 to 7 mannuronic acid or glutaric acid.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 비환원성 말단의 포화형 만뉴론산 올리고당과 비교하여 물 분자가 제거되어 물 분자의 질량값인 18 정도의 작은 질량값을 갖는다(도 2).The non-reducing terminal monounsaturated mannuronic acid oligosaccharide of the present invention has a smaller mass value of about 18, which is the mass value of water molecules, as compared with the non-reducing terminal monounsaturated mannuronic acid oligosaccharide.
본 발명의 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 만뉴론산 및 글루론산으로 이루어져 있다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention is composed of mannuronic acid and glutaric acid.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당의 만뉴론산:글루론산의 비율은 1.2-5.0:1이다. 본 발명의 다른 구현예에 따르면, 1.2-4.5:1이다. 본 발명의 또다른 구현예에 따르면, 1.8-4.0:1이다. 본 발명의 특정 구현예에 따르면, 2.0-3.0:1이다.According to one embodiment of the present invention, the ratio of the mannuronic acid: glucuronic acid in the non-reducing terminal unsaturated maleic acid oligosaccharide is 1.2-5.0: 1. According to another embodiment of the invention, it is 1.2-4.5: 1. According to another embodiment of the present invention, it is 1.8-4.0: 1. According to a particular embodiment of the invention, it is 2.0-3.0: 1.
즉, 본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 만뉴론산을 글루론산보다 1.2-5.0, 1.2-4.5, 1.8-4.0 또는 2.0-3.0배 우세하게 포함한다(도 4).Namely, the non-reducing terminal unsaturated maleic acid oligosaccharide of the present invention predominantly contains mannuronic acid at 1.2-5.0, 1.2-4.5, 1.8-4.0, or 2.0-3.0 times higher than that of glutaric acid (FIG. 4).
본 발명의 다른 양태에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분으로 포함하는 비만 개선, 예방 또는 치료용 조성물을 포함한다.According to another aspect of the present invention, there is provided a composition for improving, preventing or treating obesity comprising the non-reducing terminal unsaturated maleuronic acid oligosaccharide as an active ingredient.
본 명세서에서 사용되는 용어 “비만”은 건강에 이상을 초래할 정도로 지방 조직이 체내에 과잉으로 축적된 상태를 의미한다.As used herein, the term " obesity " refers to a state in which excess fat tissue accumulates in the body such that it causes an abnormality in health.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 지방 축적을 억제한다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention inhibits fat accumulation.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 중성지방의 축적에 대하여 20-60%, 25-50% 또는 30-40% 저해하고, 비환원성 말단의 포화형 만뉴론산 올리고당은 10-30%, 10-25% 또는 10-30% 저해한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide inhibits 20-60%, 25-50%, or 30-40% of the accumulation of triglycerides and the non-reducing terminal saturated, Lonic acid oligosaccharides inhibit 10-30%, 10-25%, or 10-30%.
본 발명의 다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 비환원성 말단의 포화형 만뉴론산 올리고당과 비교하여 중성지방의 축적을 2배 이상 저해한다.According to another embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharide inhibits the accumulation of triglycerides more than twice as compared with the non-reducing terminal saturated maleic acid oligosaccharides.
상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 비만을 유도하는 장내 미생물 균총을 조절한다.The non-reducing terminal unsaturated mannuronic acid oligosaccharides regulate intestinal microflora to induce obesity.
본 명세서에서, 용어 “장내 미생물 균총(intestinal microflora 또는 gut microflora)”은 구성하는 동물의 소화관에서 생장하는 미생물 군락집단(complex community)을 의미한다. 장내 미생물 균총을 구성하는 개개의 미생물은 물질생산 또는 분해능에 의해 숙주에게 피해 또는 이익을 주기도 하는데 장내 균총 전체적으로 볼 때는 비타민 공급, 감염방지 및 장 기능(연동운동 및 흡수)을 돕는데 관여하고 있고 이로 인해 균총의 조성은 변비, 장 관련 질환 발생 등과 밀접한 관계가 있는 것으로 알려져 있다(Mistuoka T. Bifidobacteria Microflora, 1(1): 3, 1982).As used herein, the term " intestinal microflora or gut microflora " refers to a microorganism community that grows in the digestive tract of the constituent animal. The individual microorganisms that make up intestinal microflora are harmful or beneficial to the host by their ability to produce or degrade the substance. They are involved in helping the vitamins supply, infection prevention and intestinal function (peristaltic movement and absorption) The composition of the microflora is known to be closely related to constipation, intestinal diseases, and the like (Mistuoka T. Bifidobacteria Microflora, 1 (1): 3, 1982).
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 장내 미생물 균주의 생장을 감소시켜 장내 미생물 균총을 조절한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide reduces intestinal microbial strain growth and regulates intestinal microflora.
본 발명의 다른 구현예에 따르면, 상기 장내 미생물 균주는 로즈버리아 종(Roseburia sp .) 및 락토바실러스 종(Lactobacillus sp .)로 구성된 군으로부터 선택되는 균주이다.According to another embodiment of the present invention, the intestinal microbial strain is selected from the group consisting of Roseburia species sp . ) And Lactobacillus sp . , Which is a strain selected from the group consisting of Lactobacillus sp .
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 비만을 유도하는 지방-분화와 관련된 유전자의 발현을 억제한다.The non-reducing terminal unsaturated maleuronic acid oligosaccharides of the present invention inhibit the expression of genes associated with fat-differentiation leading to obesity.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 aP2(adipocyte protein 2), C/EBPα(CAAT enhancer binding protein α) 및 PPARγ(Peroxisome Proliferator-Activated Receptor γ)의 발현을 억제한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide can be used for the expression of aP2 (adipocyte protein 2), C / EBP alpha (CAAT enhancer binding protein a) and PPAR gamma (Peroxisome Proliferator-Activated Receptor y) .
본 발명의 다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 aP2, C/EBPα 및 PPARγ의 발현을 각각 15-35%, 50-70% 및 30-50% 억제한다. 이러한 효과는 비환원성 말단의 포화형 만뉴론산 올리고당과 비교하여 1.5 내지 7.0배 우수하다.According to another embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharides inhibit the expression of aP2, C / EBPa and PPARy by 15-35%, 50-70% and 30-50%, respectively. This effect is 1.5 to 7.0 times better than the non-reducing terminal saturated mannuronic acid oligosaccharide.
본 발명의 조성물은 비만 개선, 예방 또는 치료용 조성물은 비만 예방 또는 치료용 약제학적 조성물, 비만 개선 또는 예방용 식품 조성물 또는 기능성 식품 조성물로 제조될 수 있다.The composition of the present invention can be manufactured from a pharmaceutical composition for preventing or treating obesity, a food composition for improving or preventing obesity, or a functional food composition, for the purpose of improving, preventing or treating obesity.
본 발명의 또다른 양태에 따르면, 본 발명은 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분으로 포함하는 당뇨 개선, 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, there is provided a composition for improving, preventing or treating diabetes comprising an unsaturated maleuronic acid oligosaccharide having a non-reducing terminal as an active ingredient.
본 명세서에서, 용어 “당뇨”는 포도당-비관용(intolerance)을 초래하는 인슐린의 상대적 또는 절대적 부족으로 특징되는 만성질환을 의미한다. 용어 당뇨는 모든 종류의 당뇨병을 포함하며, 예를 들어, 제1형 당뇨, 제2형 당뇨 및 유전성 당뇨를 포함한다. 제1형 당뇨는 인슐린 의존성 당뇨병으로서, β-세포의 파괴에 의해 주로 초래된다. 제2형 당뇨는 인슐린 비의존성 당뇨병으로서, 식사 후 불충분한 인슐린 분비에 의해 초래되거나 또는 인슐린 내성에 의해 초래된다.As used herein, the term " diabetes " refers to a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. The term diabetes includes all types of diabetes, including, for example,
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 글루코오스(glucose)의 흡수(uptake)를 촉진한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharide promotes uptake of glucose.
본 발명의 이러한 효과는 비환원성 말단의 포화형 만뉴론산 올리고당에서는 나타나지 않는 비환원성 말단의 불포화형 만뉴론산 올리고당만의 효과이다.This effect of the present invention is only the effect of the non-reducing terminal unsaturated maleic acid oligosaccharide which is not present in the non-reducing terminal, saturated maleic acid oligosaccharide.
본 발명의 다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 AMPK(AMP-activated Proein Kinase) 경로를 매개하여 당 흡수를 촉진한다.According to another embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharide mediates AMP-activated proin kinase (AMPK) pathway to promote glucose uptake.
본 발명의 조성물은 당뇨 개선, 예방 또는 치료용 조성물은 비만 예방 또는 치료용 약제학적 조성물, 당뇨 개선 또는 예방용 식품 조성물 또는 기능성 식품 조성물로 제조될 수 있다.The composition of the present invention can be manufactured by a pharmaceutical composition for the prevention or treatment of obesity, a food composition for improving or preventing diabetes or a functional food composition.
본 발명의 또다른 양태에 따르면, 본 발명은 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분으로 포함하는 장내 유익균 증진용 프로바이오틱스를 제공한다.According to still another aspect of the present invention, there is provided a probiotic for enhancing intestinal beneficial bacteria, comprising an unsaturated maleuronic acid oligosaccharide of non-reducing end as an active ingredient.
본 발명의 장내 유익균 증진용 프로바이오틱스는 장내 유익균의 생장을 증진시킨다.The probiotics for promoting beneficial bacteria in the intestines of the present invention promote the growth of beneficial bacteria in the intestines.
본 발명의 일 구현예에서, 상기 장내 유익균은 로즈버리아 종(Roseburia sp .) 및 락토바실러스 종(Lactobacillus sp .)로 구성된 군으로부터 선택되는 장내 유익균이다.In one embodiment of the present invention, the enterobacteriaceae is a Roseburia species sp . ) And Lactobacillus sp . ) . ≪ / RTI >
본 명세서의 용어 “프로바이오틱스”는 숙주 유기체에 의 건강에 도움을 주는 생균의 식품 보조물을 의미한다. 본 발명의 프로바이오틱스 조성물은 발효유 제품으로 제조될 수도 있으나 과립, 분말 등의 형태로 제조될 수도 있다.As used herein, the term " probiotics " refers to a food aid of a live organism that contributes to the health of the host organism. The probiotic composition of the present invention may be manufactured as a fermented milk product, or may be prepared in the form of granules, powders, and the like.
본 발명의 프로바이오틱스 조성물의 투여방법은 특별히 한정되지 않지만 환제, 정제 등으로 제조하여 경구 투여할 수도 있고, 분말형 또는 과립형으로 제조하여 음식물에 첨가하여 투여할 수도 있다. 본 발명의 조성물은 예를 들면, 의약품으로서 사용하는 경우의 제형은 각 성분의 분말을 제제화하지 않고 그 자체로 사용할 수 있지만, 산제, 과립제, 세립제, 정제, 당의정제, 캡슐제, 정제, 장용 피복제 등의 형태로 제제화할 수 있다. 희석제로서는 일반의 의약품제제에 사용되는 부형제, 결합제, 붕괴제 등이 사용되고, 이외에 착색제, 안정화제, 보존제, 윤활제 등을 첨가할 수 있다. 본 발명의 음식용 조성물을 식품으로서 사용하는 경우는 이대로 각 성분의 분말을 제제화하지 않고 사용할 수 있지만, 다른 식물섬유, 올리고당, 곡물류, 비타민류 등을 첨가하거나, 또는 향미료, 착색제, 교미제 등을 첨가하여, 섭취하기에 적합한 형태로 형성 가공하는 것도 가능하다. 또한, 식품 첨가물로서, 다른 식품에 첨가 혼합하여 사용할 수도 있다.The method of administering the probiotic composition of the present invention is not particularly limited, but it may be prepared by parenteral, tablet, or the like, or it may be prepared into a powder or granular form and added to foods. The composition of the present invention can be used as a formulation itself, for example, in the form of pharmaceuticals without using any formulating powder of each component, but it can be used as a powder or granules, a fine granule, a tablet, a tablet, a capsule, And the like. As diluents, excipients, binders, disintegrators, and the like used in general pharmaceutical preparations are used. In addition, coloring agents, stabilizers, preservatives, lubricants and the like may be added. When the food composition of the present invention is used as a food, the powder of each component can be used as it is without being formulated. However, it is also possible to add other plant fibers, oligosaccharides, cereals, vitamins or the like or use a spice, a colorant, It is also possible to form and process it in a form suitable for ingestion. As a food additive, it may be added to other foods and mixed.
본 발명의 장내 유익균 증진용 프로바이오틱스는 약제학적 조성물, 식품 조성물 또는 기능성 식품 조성물로 제조될 수 있다.The probiotics for enhancing the beneficial bacteria in the intestines of the present invention can be prepared from pharmaceutical compositions, food compositions or functional food compositions.
본 발명의 다른 양태에 따르면, 본 발명은 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분으로 포함하는 갱년기 증후군 개선, 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, there is provided a composition for improving, preventing or treating menopausal syndrome comprising an unsaturated maleuronic acid oligosaccharide having a non-reducing terminal as an active ingredient.
본 명세서에서 용어“갱년기 증후군”은 여성 내분비 증후군의 일종으로 자연적 손실이든, 외과적 수술에 의한 손실이든 또는 화학적으로 유도된 손실이든 관계없이 난소 기능의 전반적이고 점진적인 감소가 일어나 생리적 기능 및 성기능이 감소 내지 소실되는 과도기를 말하며, 갱년기의 한 과정으로 난소 기능의 정지 후에 일어나는 생리의 영구적인 정지인 폐경이 오게 된다. 폐경기에는 에스트로겐 생성의 감소, 난포자극호르 및 황체화호르몬의 상승 등 호르몬의 변화에 따라 급만성 증상이 나타날 수 있다. 즉, 초기증상으로 열성홍조, 야간 발한 등의 혈관 운동성 증상과 불안감, 집중력 감퇴, 우울증 등의 심리적 증상이 나타날 수 있으며, 폐경 수년 내에 나타나는 비뇨생식계 및 표피증상이, 폐경 수년 후에는 골다공증, 심장혈관 및 뇌혈관 등의 질환 등이 있을 수 있다.As used herein, the term " menopausal syndrome " is a type of female endocrine syndrome that is characterized by a general and gradual decrease in ovarian function, regardless of whether it is a natural loss, a surgical operation loss or a chemically induced loss, resulting in a decrease in physiological function and sexual function Which is a permanent period of menstruation that occurs after the cessation of ovarian function. Postmenopausal women may develop acute chronic symptoms due to changes in hormones, such as decreased estrogen production, elevated follicle stimulating hormone and luteinizing hormone. In other words, psychological symptoms such as vasomotor symptoms such as hot flushing, night sweating, anxiety, loss of concentration and depression may appear as initial symptoms, and urinary and epidermal symptoms appearing within several years of menopause may be followed by osteoporosis, And cerebrovascular diseases.
상기 갱년기 증후군은 안면홍조, 발한, 심장 불편감, 수면 문제, 우울증, 과민성, 불안감, 신체적 피로, 정신적 피로, 성적 문제, 배뇨 문제, 질 건조감, 관절 불편감 및 근육 불편감으로 구성된 군으로부터 선택되는 증후군을 포함한다.The menopausal syndrome is selected from the group consisting of facial flushing, sweating, heart discomfort, sleeping problems, depression, irritability, anxiety, physical fatigue, mental fatigue, sexual problems, urination problems, vaginal dryness, joint discomfort and muscle discomfort Syndrome. ≪ / RTI >
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분으로 포함하는 조성물은 에스트로겐의 활성을 증가시킨다.The composition comprising the non-reducing terminal unsaturated maleic acid oligosaccharide of the present invention as an active ingredient increases the activity of estrogen.
상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 에스트로겐을 리간드로 작용하는 에스트로겐 수용체 경로 및 에스트로겐 비의존적으로 작용하는 에스트로겐 관련 수용체 알파 경로를 경유하여 에스트로겐 반응 요소(estrogen response element; ERE)-매개 하부 유전자의 발현을 통해 에스트로겐을 활성화시킨다.The non-reducing terminal unsaturated mannuronic acid oligosaccharide is an estrogen receptor element (ERE) -mediated subunit of the estrogen receptor element via an estrogen receptor pathway that acts as a ligand and an estrogen-related receptor alpha pathway that acts estrogen- Expression activates estrogen.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 에스트로겐 수용체 α를 경유하여 ERE의 발현을 유도한다.According to one embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide induces the expression of ERE via the estrogen receptor a.
본 발명의 다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 에스트로겐 관련 수용체 알파를 발현시켜 ERE의 발현을 유도한다.According to another embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide expresses the estrogen-related receptor alpha and induces the expression of ERE.
상기 ERE의 발현으로 인해 하부 에스트로겐 유전자의 발현을 통해 에스트로겐의 활성이 증가된다.Expression of the ERE increases estrogen activity through expression of the lower estrogen gene.
본 발명의 또다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 pS2(Presenilin 2), PR(Progesteron Receptor) 및 CTSD(E2-mediated Cathepsin D) mRNA의 발현을 증가시킨다.According to another embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide increases the expression of pS2 (Presenilin 2), PR (Progesterone Receptor) and CTSD (E2-mediated Cathepsin D) mRNA.
본 발명의 다른 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 PGC-1α(Peroxisome proliferator-activated receptor gamma coactivator 1-α), ERRα(Estrogen-related receptor α), GATA3(Trans-acting T-cell-specific Transcription Factor) 및 FOXA1(Forkhead box protein A1) mRNA의 발현을 증가시킨다.According to another embodiment of the present invention, the non-reducing terminal unsaturated maleuronic acid oligosaccharide is selected from the group consisting of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha), estrogen- T-cell-specific transcription factor) and FOXA1 (Forkhead box protein A1) mRNA expression.
상기 GATA3 및 FOXA1는 유방 세포 분화에 중요한 인자이며, 암세포나 악성 종양으로의 분화를 억제하는 역할을 한다.GATA3 and FOXA1 are important factors for differentiation of breast cells and play a role in inhibiting differentiation into cancer cells or malignant tumors.
본 발명의 조성물은 17β-에스트라디올(17β-estradiol)을 추가적으로 포함한다.The composition of the present invention additionally comprises 17 [beta] -estradiol.
본 발명의 일 구현예에 따르면, 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 17β-에스트라디올(17β-estradiol)과 동반상승효과(synergic effect)를 나타낸다.According to one embodiment of the present invention, the non-reducing terminal unsaturated mannuronic acid oligosaccharide exhibits a synergic effect with 17? -Estradiol.
본 발명의 조성물은 갱년기 증후군 예방 또는 치료용 약제학적 조성물, 갱년기 증후군 개선 또는 예방용 식품 조성물 또는 기능성 식품 조성물로 제조될 수 있다.The composition of the present invention can be manufactured with a pharmaceutical composition for the prevention or treatment of menopausal syndrome, a food composition for improving or preventing menopausal syndrome or a functional food composition.
본 발명의 (a) 비만 개선, 예방 또는 치료용 조성물; (b) 당뇨 개선, 예방 또는 치료용 조성물; (c) 장내 유익균 증진용 프로바이오틱스; 및 (d) 갱년기 증후군 개선, 예방 또는 치료용 조성물은 약제학적 조성물로 제조될 수 있다.(A) a composition for improving, preventing or treating obesity; (b) a composition for improving, preventing or treating diabetes; (c) probiotics for promoting beneficial bacteria in the intestines; And (d) a composition for improving, preventing or treating menopausal symptoms may be prepared from a pharmaceutical composition.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 (a) 상술한 본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물이다. 본 명세서에서 용어 “약제학적 유효량”은 상술한 비환원성 말단의 불포화형 만뉴론산 올리고당의 효능 또는 활성을 달성하는 데 충분한 양을 의미한다.According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-mentioned non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention; And (b) a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve the efficacy or activity of the above-mentioned non-reducing terminally unsaturated maleic acid oligosaccharide.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.001 ㎍/kg - 100 ㎎/kg 범위 내이다.The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of this invention are in the range of 0.001 [mu] g / kg to 100 mg / kg on an adult basis.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 명세서에서 용어 “유효성분으로 포함하는”이란 상술한 비환원성 말단의 불포화형 만뉴론산 올리고당의 효능 또는 활성을 달성하는 데 충분한 양을 포함하는 것을 의미한다. 상술한 비환원성 말단의 불포화형 만뉴론산 올리고당이 본 발명의 조성물에 포함된 양적 상한은 당업자가 적절한 범위 내에서 선택하여 실시할 수 있다.As used herein, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve the efficacy or activity of the above-mentioned non-reducing terminally unsaturated maleic acid oligosaccharide. The quantitative upper limit of the above-mentioned non-reducing terminal unsaturated maleuronic acid oligosaccharide contained in the composition of the present invention can be selected by a person skilled in the art within a suitable range.
본 발명의 (a) 비만 개선, 예방 또는 치료용 조성물; (b) 당뇨 개선, 예방 또는 치료용 조성물; (c) 장내 유익균 증진용 프로바이오틱스; 및 (d) 갱년기 증후군 개선, 예방 또는 치료용 조성물은 식품 조성물로 제조될 수 있다.(A) a composition for improving, preventing or treating obesity; (b) a composition for improving, preventing or treating diabetes; (c) probiotics for promoting beneficial bacteria in the intestines; And (d) compositions for improving, preventing or treating menopausal symptoms can be prepared with food compositions.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분 포함하는 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 비환원성 말단의 불포화형 만뉴론산 올리고당 뿐 만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다.When the composition containing the non-reducing terminal unsaturated maleic acid oligosaccharide of the present invention as an active ingredient is prepared from a food composition, not only the non-reducing terminal unsaturated maleic acid oligosaccharide as an active ingredient but also the normally added And includes, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, it may contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract And the like.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당을 유효성분 포함하는 기능성 식품 조성물로 제조될 수 있다. 본 발명의 조성물이 기능성 식품 조성물로 제조되는 경우, 식품 제조 시 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분으로서 비환원성 말단의 불포화형 만뉴론산 올리고당 이외에 향미제 또는 천연 탄수화물을 추가 성분으로 포함시킬 수 있다. 예를 들어, 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등); 디사카라이드(예컨대, 말토스, 수크로오스 등); 올리고당; 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등); 및 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)을 포함한다. 향미제로서 천연 향미제(예컨대, 타우마린, 스테비아 추출물 등) 및 합성 향미제(예컨대, 사카린, 아스파르탐 등)을 이용할 수 있다.The functionalized food composition containing the non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention as an active ingredient. When the composition of the present invention is prepared with a functional food composition, it includes components that are conventionally added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of being made with a drink, a flavoring agent or a natural carbohydrate may be added as an additional ingredient in addition to the non-reducing terminally unsaturated maleuronic acid oligosaccharide as an active ingredient. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); oligosaccharide; Polysaccharides (e.g., dextrin, cyclodextrin and the like); And sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). Natural flavoring agents (e.g., tau marin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be used as flavorings.
본 발명의 비환원성 말단의 불포화형 만뉴론산 올리고당은 종래에 알려진 알긴산의 생리활성의 활성성분으로서, 항비만, 항당뇨, 갱년기 증후군 개선 및 장내 미생물 균총의 조절 효과를 나타낸다. 이러한 효과는 비환원성 말단의 포화형 만뉴론산 올리고당과 비교하여 현저히 우수한 효과이며, 이에 상기 효과를 나타내는데 비환원성 말단의 불포화형 만뉴론산 올리고당에서 나타나는 말단당의 이중결합 및 이의 불포화 형태가 중요한 요소임을 의미한다.The non-reducing terminal unsaturated maleuronic acid oligosaccharide of the present invention is an active ingredient of known physiological activity of alginic acid and exhibits an anti-obesity, anti-diabetic effect, improvement of menopausal syndrome and control effect of intestinal microflora. This effect is remarkably excellent as compared with the non-reducing terminal saturated mannuronic acid oligosaccharide, and the above effect is exhibited. This means that the terminal double bond and the unsaturated form thereof in the non-reducing terminal monounsaturated mannuronic acid oligosaccharide are important factors .
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 비환원성 말단의 불포화형 만뉴론산 올리고당, 이를 유효성분으로 포함하는 비만, 당뇨 및 갱년기 증후군의 개선, 예방 또는 치료용 약제학적 조성물 및 장내 유익균 증진용 프로바이오틱스를 제공한다.(a) The present invention provides a pharmaceutical composition for improving, preventing or treating obesity, diabetes and menopausal syndrome comprising an unsaturated maleic acid oligosaccharide of non-reducing end and an effective ingredient thereof, and a probiotic for enhancing intestinal beneficial bacteria.
(b) 본 발명은 알긴산의 활성물질인 비환원성 말단의 불포화형 만뉴론산 올리고당을 제조하여 이의 우수한 항비만, 항당뇨, 에스트로겐 활성 및 장내 미생물 균총 조절 효과를 제공한다.(b) The present invention provides a non-reducing terminal unsaturated maleuronic acid oligosaccharide which is an active substance of alginic acid to provide excellent anti-obesity, anti-diabetic, estrogenic activity and intestinal microflora controlling effect.
(c) 이러한 효과는 종래의 비환원성 말단의 포화형 만뉴론산 올리고당의 형태를 나타내는 만뉴론산 올리고당과 비교하여 현저히 우수하다.(c) This effect is remarkably superior to that of mannuronic acid oligosaccharides which exhibit the form of a conventional non-reducing terminal saturated mannuronic acid oligosaccharide.
도 1은 비환원성 말단의 불포화형 만뉴론산 올리고당의 박막크로마토그래피 결과를 나타낸다.
도 2는 비환원성 말단의 불포화형 만뉴론산 올리고당 및 비환원성 말단의 포화형 만뉴론산 올리고당의 질량분석 결과를 나타낸다.
도 3은 비환원성 말단의 불포화형 만뉴론산 올리고당(Non-reducing end of unsaturated Mannuroic Acid Oligosaccharide; USMOS) 및 비환원성 말단의 포화형 만뉴론산 올리고당(Non-reducing end of saturated Mannuroic Acid Oligosaccharide; SMOS)의 이중결합 비율을 나타낸다.
도 4는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 및 비환원성 말단의 포화형 만뉴론산 올리고당(SMOS)의 당 구성을 원평광 이색성 분광 측정법(CD, circular dichorism) 방법으로 확인한 결과를 나타낸다.
도 5a 및 5b는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 처리에 의한 지방축적 저해 결과 및 실-시간 PCR을 이용한 비만-관련 유전자, aP2, C/EBPα 및 PPARγ의 발현 억제 효과를 나타낸다.
도 6a 내지 6c는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 처리에 따른 당흡수 촉진 결과 및 관련 단백질, p-PAK, p-Akt 및 p-AS160의 발현양을 나타낸다.
도 7a 및 7b는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 복강투여에 따른 비만 유도 랫트의 장내 균총 분석 결과를 나타낸다.
도 8는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS)의 음용섭취에 따른 노령쥐의 장내 미생물 균총을 PCO(principal coordinate) 분석 결과를 나타낸다.
도 9은 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 음용섭취에 따른 노령쥐의 장내 균총 변화를 문(phylum)에서 비교 분석한 결과를 나타낸다.
도 10a 및 10b는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 처리에 따른 pS2, PR 및 CTSD의 mRNA 발현양을 나타낸다.
도 11은 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS) 처리에 따른 PGC-1α, ERRα, GATA3 및 FOXA1의 mRNA 발현양을 나타낸다.
도 12는 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS)의 작용기전을 나타낸다.
도 13은 비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS)의 다양한 생리활성 효과를 나타낸다.1 shows the result of thin film chromatography of an unsaturated maleuronic acid oligosaccharide having a non-reducing end.
Fig. 2 shows the results of mass spectrometry of a non-reducing terminal unsaturated mannuronic acid oligosaccharide and a non-reducing terminal saturated mannuronic acid oligosaccharide.
FIG. 3 is a graph showing the relationship between the number of non-reducing end of unsaturated mannuroic acid oligosaccharide (USMOS) and the number of non-reducing end of saturated mannuroic acid oligosaccharide (SMOS) Lt; / RTI >
FIG. 4 shows the result of confirming the sugar structure of a non-reducing terminal unsaturated maleuronic acid oligosaccharide (USMOS) and a non-reducing terminal saturated type mannuronic acid oligosaccharide (SMOS) by a circular dichroism (CD) method .
FIGS. 5A and 5B show the results of inhibition of lipid accumulation by non-reducing terminal unsaturated mannuronic acid oligosaccharide (USMOS) treatment and the effect of suppressing the expression of obesity-related genes aP2, C / EBPa and PPARγ by real-time PCR.
FIGS. 6A to 6C show the results of stimulating glucose uptake by non-reducing terminal unsaturated mannuronic acid oligosaccharide (USMOS) treatment and the expression amounts of the related proteins, p-PAK, p-Akt and p-AS160.
FIGS. 7A and 7B show intestinal microflora analysis results of obesity-induced rats according to the administration of unsaturated maleic acid oligosaccharide (USMOS) peritoneally administered at the non-reducing end.
Fig. 8 shows PCO (principal coordinate) analysis of intestinal microflora of old rats according to ingestion of non-reducing terminal unsaturated mannuronic acid oligosaccharide (USMOS).
FIG. 9 shows the result of comparative analysis of the intestinal microflora of old rats according to ingestion of non-reducing terminal unsaturated mannuronic acid oligosaccharide (USMOS) in phylum.
Figures 10a and 10b show the amount of mRNA expression of pS2, PR and CTSD following treatment with a non-reducing terminal unsaturated mannitol oligosaccharide (USMOS).
FIG. 11 shows mRNA expression levels of PGC-1 alpha, ERR alpha, GATA3 and FOXA1 according to the treatment with unsaturated maleic acid oligosaccharide (USMOS) at the non-reducing end.
Figure 12 shows the mechanism of action of the non-reducing terminal unsaturated maleuronic acid oligosaccharide (USMOS).
Fig. 13 shows various physiological activity effects of the unsaturated maleuronic acid oligosaccharide (USMOS) at the non-reducing end.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다.Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
실시예 1: 알긴산 올리고당의 제조Example 1: Preparation of alginate oligosaccharide
폴리만뉴론산(poly-mannuronate; poly M)을 제조하기 위하여 알긴산 나트륨 1 g(Wako, Osaka Japan)과 함께 0.3 M HCl 100 ㎖을 넣고 100℃에서 2시간 동안 가열하였다. 가열된 알긴산 나트륨-HCl용액을 500 g로 5 분 동안 원심 분리하여 분리된 침전물을 증류수에 용해시켰다. 증류수에 용해시킨 침전물이 0.1 M이 되도록 NaCl을 첨가한 후, pH를 2.8-3.0으로 조절하여 500 g, 5분 동안 원심 분리로 상징액과 침전물을 분리하였다. 분리된 침전물과 상징액은 알코올 침전 후 건조하여 상징액 부분을 poly M으로, 침전물 부분을 poly G로 제조하여 알긴산 분해 효소를 활용한 만뉴론산 올리고당 제조를 위한 기질로 사용하였다(Haug, A et al. A study of the constitution of alginic acid by partial acid hydrolysis. ActaChemicaScandinavica, 1966, 20(1): 183-190. 및 Joo, D. S. et al. Preparation of oligosaccharides from alginic acid by enzymic hydrolysis. Korean Society of Food Science and Technology, 1996, 28(1): 146-151).To prepare poly-mannuronate (poly M), 100 ml of 0.3 M HCl together with 1 g of sodium alginate (Wako, Osaka Japan) was added and the mixture was heated at 100 ° C for 2 hours. The heated sodium alginate-HCl solution was centrifuged at 500 g for 5 minutes to dissolve the separated precipitate in distilled water. NaCl was added so that the precipitate dissolved in the distilled water was adjusted to a pH of 2.8-3.0, and the supernatant and the precipitate were separated by centrifugation at 500 g for 5 minutes. The separated precipitate and supernatant were dried after precipitation of alcohol, and the supernatant was used as a poly M and the precipitate was used as a substrate for the production of mannuronic acid oligosaccharide using alginate degrading enzyme (Haug, A et al., A . study of the constitution of alginic acid by partial acid hydrolysis ActaChemicaScandinavica, 1966, 20 (1):... 183-190 and Joo, DS et al Preparation of oligosaccharides from alginic acid by enzymic hydrolysis Korean Society of Food Science and Technology, 1996, 28 (1): 146-151).
비환원성 말단의 불포화형 만뉴론산 올리고당을 제조하기 위하여 2009년 2월 대한민국 여수 근해에서 서식하는 전복의 장내 DNA 메타지놈 라이브러리에서 선별된 만뉴론산 분해능이 우수한 ORF11에 해당되는 유전자를 pMAL-c2x 발현 벡터에 재조합한 후 BL21(DE3) 균주에 클로닝을 실시하여 제작된 유전자 형질 전환 균주를 이용하였다(대한민국 등록특허 10-1277706).In order to prepare the unsaturated maleic acid oligosaccharide of non-reducing end, the gene corresponding to ORF11 having excellent mannuronic acid resolution selected from the intrathecal DNA metagenomic library of abalone inhabited in Yeosu offshore of Korea in February 2009 was transformed into pMAL-c2x expression vector After the recombination, the BL21 (DE3) strain was cloned into the prepared transformant (Korean Patent No. 10-1277706).
40 L의 앰피실린(ampicillin, 100 ㎍/㎖)이 첨가된 Luria-Bertani(LB)에 500 ㎖의 상기 유전자 형질 전환 균주를 접종하여 37℃에서 배양하고, 600 ㎚에서 흡광도가 0.4-0.5가 되었을 때, 배양액의 IPTG(Isopropyl-β-D-thiogalactopyranoside) 최종 농도가 0.3 mM이 되도록 첨가한 후 12시간 동안 배양하였다. 배양이 끝난 후 배양액을 15분 동안 9,000 g에서 원심 분리하여 세포를 침전시켰다. 침전된 세포를 pH 7.0의 10 mM 인산 완충액(phosphate buffer)으로 부유 시킨 후세포막을 분해하기 위하여 초음파 분해(sonication)를 실시하고 조효소를 분리해내기 위해 15분 동안 9,000 g에서 원심분리를 실시하였다. 원심분리 후 분리된 상층액을 조효소로 사용하였으며, 기질로서 폴리만뉴론산(poly-mannuronate(poly M))을 0.3% 농도로 pH 7.0의 10 mM 인산 완충액 1L에 녹인 후 최종 농도가 1 mM이 되도록 AgNO3을 첨가하였다. 2.5 L 발효기(KBT KB-250, Japan)를 이용하여 45℃에서 48시간 동안 기질 분해 반응을 진행하였다. 반응 후 분자량 3,000 Da이하의 올리고당 혼합물을 얻기 위해 한외 여과막 시스템(VivaFlow 50, Sartorius, Ag, Germany)으로 여과하여 이를 동결 건조 한 후 비환원성 말단의 불포화형 만뉴론산 올리고당을 제조하였다. Luria-Bertani (LB) supplemented with 40 L of ampicillin (100 μg / ml) was inoculated with 500 ml of the above transgenic strain and cultured at 37 ° C., and the absorbance was 0.4-0.5 at 600 nm , IPTG (Isopropyl-β-D-thiogalactopyranoside) was added to the culture to a final concentration of 0.3 mM, and then cultured for 12 hours. After the culture was completed, the culture was centrifuged at 9,000 g for 15 minutes to precipitate the cells. The precipitated cells were suspended in 10 mM phosphate buffer at pH 7.0, sonicated for sonication to break down the cell membrane, and centrifuged at 9,000 g for 15 min to isolate the coenzyme. After centrifugation, the separated supernatant was used as the coenzyme. Poly-mannuronate (poly M) was dissolved in 1 L of 10 mM phosphate buffer (pH 7.0) at a concentration of 0.3% AgNO 3 was added as much as possible. The substrate degradation reaction was carried out at 45 ° C for 48 hours using a 2.5 L fermenter (KBT KB-250, Japan). After the reaction, the mixture was filtered with an ultrafiltration membrane system (
올리고당의 생산 여부를 확인하기 위하여 박막크로마토그래피(thin-layer chromatography)을 실시하였으며, 방법은 다음과 같다. 비환원성 말단의 불포화형 만뉴론산 올리고당을 0.1 ㎎/㎕ 농도로 물에 용해 후, 실리카겔 플레이트(Merck KGaA, Germany)에 3 ㎕ 스파팅을 실시하였다. 전개용매（1-부탄올 : 포름산 : 물 = 4 : 6 : 1)을 이용하여 비환원성 말단의 불포화형 만뉴론산 올리고당 크기별로 분리 후 황산을 가한 발색시약(아니스알데하이드 0.5 ㎖, 아세트산 10 ㎖, MeOH 85 ㎖, H2SO4 5 ㎖)을 사용하여 올리고당의 존재를 확인하였다.Thin-layer chromatography was performed to confirm the production of oligosaccharide. The method was as follows. Unsaturated mannuronate oligosaccharide at the non-reducing end was dissolved in water at a concentration of 0.1 mg / mu l, and then 3 [mu] l of a spotting was performed on a silica gel plate (Merck KGaA, Germany). Separated by the size of the unsaturated maleic acid oligosaccharide at the non-reducing end, the coloring reagent (0.5 ml of anisaldehyde, 10 ml of acetic acid, MeOH 85 (1: 1) ㎖, H 2 SO 4 5 ml) was used to confirm the presence of oligosaccharide.
실시예 2: 비환원성 말단의 불포화형 만뉴론산 올리고당 구성 및 구조적 특징 분석Example 2: Composition and characterization of unsaturated maleuronic acid oligosaccharide having non-reducing end
본 실험예에서는 AlyDW11 알긴산 분해효소를 이용하여 폴리만뉴론산을 분해 후 확보된 비환원성 말단의 불포화형 만뉴론산 올리고당 혼합물을 한외여과막 시스템(VivaFlow 50, Sartorius)을 이용하여 분자량 3000 Da 이하의 분획물을 확보하였다. 비환원성 말단의 불포화형 만뉴론산 올리고당의 구성당을 분석하기 위하여, 준비된 시료를 이온 교환 수지 컬럼(Hitrap DEAE Sepharose FF, GE Healthcare)을 이용하여 정제한 후 동결건조 시켰다. 정제된 비환원성 말단의 불포화형 만뉴론산 올리고당은 물에 용해 후 UPLC/MS 시스템에 주입함으로써 구성당 분석을 수행하였다.In this experiment, a non-reducing terminal unsaturated maleuronic acid oligosaccharide mixture obtained by digesting polymannuronic acid using AlyDW11 alginate degrading enzyme was fractionated with an ultrafiltration membrane system (
초고성능 액체 크로마토그래피(Ultra Performance Liquid Chromatography; UPLC, Waters) 설정은 ACQUITY UPLC BEH C18(1.7 ㎛ 1.0×100 ㎜, Waters) 컬럼을 이용하였으며 0.4 ㎖/분에서 12분 동안 용매 A[15 mM Amylamine 및 25 mM HFIP(Hexafluoroisopropanol)]와 용매 B(15 mM Amylamin 및 25 mM HFIP in Acetonitrile)의 선형 구배를 조절하였다. C18-UPLC로 분리되어 나온 용리액을 질량분석기(Quadrupole-Time of Flight, Q-TOF, Waters)로 분석하였다. Q-TOF는 ESI 네거티브 모드에서 분석하였으며, 캐필러리 및 샘플링콘(sampling cone)의 전압은 각각 3 kV 및 40 V, 탈용매(desolvation)는 유속 600 L/h, 온도 300℃, 소스 온도는 120℃ 조건에서 실시하였다. TOF MS 데이터는 스캔시간은 0.5 초 m/z 100-1300 범위에서 분석하였다. 분석의 정확도를 위해 모든 분석에 2 ng/㎕의 류신 엔케팔린(leucine enkephalin, ESI 네거티브 모드에서 554.2619 Da)을 락스프레이(lock spray)로 사용하였다.Ultra Performance Liquid Chromatography (UPLC, Waters) setup was performed using ACQUITY UPLC BEH C18 (1.7 ㎛ 1.0 × 100 mm, Waters) column and eluting with solvent A [15 mM Amylamine and 25 mM HFIP (Hexafluoroisopropanol)] and solvent B (15 mM Amylamin and 25 mM HFIP in Acetonitrile). The eluent separated by C 18 -UPLC was analyzed with a mass spectrometer (Quadrupole-Time of Flight, Q-TOF, Waters). The Q-TOF was analyzed in the ESI negative mode. The voltages of the capillary and the sampling cone were 3 kV and 40 V respectively, the desolvation was at a flow rate of 600 L / h, the temperature was 300 ° C, Lt; 0 > C. TOF MS data were analyzed at scan time in the range of 0.5 sec m / z 100-1300. For accuracy of analysis, 2 ng / μl leucine enkephalin (554.2619 Da in ESI negative mode) was used as lock spray for all assays.
도 2에서 알 수 있듯이, 비환원성 말단의 불포화형 만뉴론산 올리고당의 질량분석 결과 비환원성 말단의 불포화형 만뉴론산 올리고당은 1개 내지 7개의 당으로 구성되어 있으며, 특히 기 보고된 만뉴론산 올리고당 질량값 보다 18이 적은 피크들이 관찰됨에 따라 물 분자가 제거되어 비환원성 말단의 불포화형 만뉴론산 올리고당이 형성되어 비환원성 말단의 포화형 만뉴론산 올리고당(non-reducing end of Saturated Mannuronic Acid Oligosaccharide; SMOS)보다 우세하게 존재함을 알 수 있었다.As can be seen from FIG. 2, the mass analysis of the unsaturated maleuronic acid oligosaccharide at the non-reducing end revealed that the unsaturated maleuronic acid oligosaccharide at the non-reducing end was composed of one to seven sugars, The water molecules are removed to form an unsaturated-type mannuronic acid oligosaccharide at the non-reducing end, which is superior to the non-reducing end of the saturated mannuronic acid oligosaccharide (SMOS). And that the
비환원성 말단의 불포화형 만뉴론산 올리고당(USMOS)과 비환원성 말단의 포화형 만뉴론산 올리고당(SMOS)의 비율을 나타낸 결과는 도 2에 제시되었다. 도 3과 같이 비환원성 말단의 불포화형 만뉴론산 올리고당은 비환원성 말단의 포화형 만뉴론산 올리고당 보다 평균 2배 이상의 비환원성 말단의 불포화형 만뉴론산 올리고당의 단당들이 존재함을 확인하였다. 비환원성 말단의 포화형 만뉴론산 올리고당 분자량은 다음과 같다. 1당 (m/z 193),당 (m/z 369), 3당 (m/z 545), 4당 (m/z 722), 5당 (m/z 898) 6당 (m/z 1074), 7당 (m/z 1250).The results showing the ratio of the non-reducing terminal unsaturated maleic acid oligosaccharide (USMOS) to the non-reducing terminal saturated type mannuronic acid oligosaccharide (SMOS) are shown in FIG. As shown in FIG. 3, it was confirmed that monomers having an unsaturated maleic acid oligosaccharide having an unsaturated maleic acid oligosaccharide at an average of 2 times or more than that of a non-reducing maleic acid mono oligosaccharide were present in a non-reducing terminal. The molecular weight of the non-reducing terminal saturated mannuronic acid oligosaccharide is as follows. (M / z 892), 5 (m / z 892), 4 (m / z 924), 4 (m / z 369), 3 ), 7 (m / z 1250).
상기 비환원성 말단의 불포화형 만뉴론산 올리고당의 만뉴론산 비율을 측정하기 위하여 원평광 이색성 분광법(Circular Dichroism; CD, J-715 spectropolarimeter, JASCO)을 이용하여 원평광 이색성 분광 신호를 측정하였다. 상기 CD 신호는 상온에서 1 ㎝의 큐벳을 이용하여 190-250 ㎚ 영역에서 측정하였으며, 일관성 있는 CD 시그널을 얻기 위하여 상기 비환원성 말단의 불포화형 만뉴론산 올리고당은 1 ㎎/㎖ 농도로 사용하였다. 만뉴론산:글루론산 구성비를 알아보기 위하여 정점(peak, 200 ㎚ 흡광도 값), 저점(trough, 215 nm 흡광도 값)을 측정하여 만뉴론산과 글루론산의 비율을 계산하였으며, 계산식은 다음과 같다:To measure the mannuronic acid ratio of the unsaturated maleic acid oligosaccharide at the non-reducing end, a circular dichroism spectroscopic signal was measured using Circular Dichroism (CD, J-715 spectropolarimeter, JASCO). The CD signal was measured at a temperature of 190-250 nm using a 1 cm cuvette at room temperature. To obtain a consistent CD signal, the non-reducing terminal monounsaturated mannuronic acid oligosaccharide was used at a concentration of 1 mg / ml. The ratio of mannuronic acid to glutaric acid was calculated by measuring the peak (200 ㎚ absorbance value) and the trough (215 nm absorbance value) in order to determine the composition ratio of mannuronic acid. The calculation formula was as follows:
(1) peak/trough < 1, mannuronic acid/guluronic acid = 2.0(peak/trough)(1) peak / trough <1, mannuronic acid / guluronic acid = 2.0 (peak / trough)
(2) peak/trough > 1, mannuronic acid/guluronic acid = 27(peak/trough) + 40(2) peak / trough> 1, mannuronic acid / guluronic acid = 27 (peak / trough) + 40
도 3에서 알 수 있듯이, 비환원성 말단의 불포화형 만뉴론산 올리고당은 만뉴론산/글루론산 비율이 2.12(peak=6.66, trough=6.42)임을 확인하였다.As shown in FIG. 3, it was confirmed that the ratio of mannuronic acid / glucuronic acid to the unsaturated maleuronic acid oligosaccharide having non-reducing end was 2.12 (peak = 6.66, trough = 6.42).
실시예Example 3: 3: 비환원성Non-reducing 말단의 Distal 불포화형Unsaturated 만뉴론산Mannuronic acid 올리고당에 의한 지방세포의 지방축적 억제 Suppression of fat accumulation of adipocytes by oligosaccharides
3T3-L1 지방전구세포를 DMEM 배지에 배양하여 컨플루언시(confluency)에 이르면 0.5 mM IBMX(isobutylmethylxanthine, IBMX), 1 mM 덱사메사손(dexamethasone) 및 1 ㎍/㎖ 인슐린(이상 MDI)으로 2일 동안 처리 후 1 ㎍/㎖ 인슐린이 포함된 DMEM+혈청 배지로 48시간 간격으로 교체하여 7일 동안 지방세포로 분화를 유도하였다. 비환원성 말단의 불포화형 만뉴론산 올리고당은 배지교체 시 0.2 ㎎/㎖ 농도로 함께 처리하였으며, 7일 후 지방세포의 지방 축적 및 분화 억제 정도를 오일 레드 O 염색(Oil red O stain) 및 RNA 추출을 하여 관찰하여 결과를 도 4에 나타내었다.3T3-L1 adipocyte precursor cells were cultured in DMEM medium and confluency was achieved by adding 0.5 mM IBMX (isobutylmethylxanthine, IBMX), 1 mM dexamethasone, and 1 μg / ml insulin (MDI) After the treatment, the cells were changed into DMEM + serum medium containing 1 ㎍ / ml insulin every 48 hours to induce differentiation into adipocytes for 7 days. Unsaturated mannuronate oligosaccharide with non - reducing terminal was treated with 0.2 ㎎ / ㎖ concentration at the time of replacing medium. After 7 days, the degree of fat accumulation and inhibition of adipocyte was measured by oil red stain and RNA extraction And the results are shown in Fig.
도 5에 제시한 바와 같이, 중성지방을 오일 레드 O 색소로 염색한 후 광학현미경으로 세포를 관찰한 결과 비환원성 말단의 불포화형 만뉴론산 올리고당은 0.2 ㎎/㎖ 농도로 처리 시 대조구 대비 지방세포의 오일 레드 O 염색강도가 현저히 약화되었다. 나아가 오일 레드 O로 염색된 중성지방에서 색소를 추출하여 정량한 결과 비환원성 말단에 이중결합을 형성한 USMOS가 대조구 대비 중성지방 축적을 약 40 % 억제하였으며, 비환원성 말단의 포화형 만뉴론산 올리고당의 경우 지방축적 저해 효과가 대조구 대비 약 15% 정도만 저해함에 따라 이중결합 형성이 항비만 효과에 중요한 요소임을 확인하였다.As shown in FIG. 5, when the neutral fat was stained with oil red O dye and the cells were observed under an optical microscope, the concentration of unsaturated mannuronic acid oligosaccharide at the non-reducing end was 0.2 ㎎ / ㎖, The oil red O dyeing strength was remarkably weakened. In addition, USMOS, which formed a double bond at the non-reducing end, inhibited the accumulation of triglyceride by about 40% compared to the control, and the concentration of the non-reducing terminal saturated maleic acid oligosaccharide The inhibitory effect of lipid accumulation was inhibited by only about 15% of that of the control.
3T3-L1 지방전구세포를 동일한 방법으로 지방세포로 분화시켜 비환원성 말단의 불포화형 만뉴론산 올리고당을 0.2 ㎎/㎖ 농도로 처리 후 GeneJET RNA 정제 키트 방법에 따라 RNA 추출 후 지방 분화 지표인 aP2, 지방 분화 관련 유전자 C/EBPα(CAAT enhancer binding protein α) 및 PPARγ (Peroxisome proliferator-activated receptor) γ 발현억제 결과를 도 4에 나타내었다.3T3-L1 adipose precursor cells were differentiated into adipocytes by the same method to treat the non-reducing terminal monounsaturated mannitol oligosaccharide at a concentration of 0.2 mg / ml. After RNA extraction according to the GeneJET RNA purification kit method, The results of inhibition of the expression-related genes C / EBPα (CAAT enhancer binding protein α) and PPARγ (peroxisome proliferator-activated receptor) γ are shown in FIG.
도 5에 나타낸 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당 처리시 대조구 대비 발현양이 aP2, C/EBPα 및 PPARγ는 각각 25%, 60% 및 40% 억제됨을 확인하였다. 또한 비환원성 말단의 포화형 만뉴론산 올리고당 대비 항비만 효과가 우수함을 확인하였다.As shown in FIG. 5, the expression levels of aP2, C / EBPα and PPARγ were 25%, 60% and 40%, respectively, when treated with the unsaturated maleuronic acid oligosaccharide at the non-reducing end. Also, it was confirmed that the anti-obesity effect was superior to the saturated terminal type mannuronic acid oligosaccharide at the non-reducing end.
실시예Example 4: 4: 비환원성Non-reducing 말단의 Distal 불포화형Unsaturated 만뉴론산Mannuronic acid 올리고당 oligosaccharide 당흡수Sugar absorption 조절 확인 Confirm adjustment
L6 근육세포를 10% 혈청이 함유된 DMEM 배지에 배양 후 2% 혈청 배지로 교체하며 L6 세포를 완전히 분화시킨다. 완전히 분화시킨 L6 근육 세포의 배지를 무혈청 DMEM 배지로 교환하여 비환원성 말단의 불포화형 만뉴론산 올리고당을 0.2 ㎎/㎖, 수준으로 1 시간동안 처리 후, 비환원성 말단의 불포화형 만뉴론산 올리고당이 처리된 배지를 제거하고 미리 가온한 37℃ KRH 완충액(Krebs-Ringer Hepes buffer)로 2회 세척하여 배지 내에 존재하는 포도당을 제거하였다. [3H]-2-데옥시글루코오스([3H]-2-deoxyglucose)를 0.04 mM 농도로 15분 처리한 후 [3H]-2-데옥시글루코오스를 포함한 KRH 완충액을 신속히 제거하고 빙냉한 PBS를 가함으로써 반응을 정지시켰다. 세포 용해 완충액(Cell lysis buffer)를 이용하여 세포를 파쇄한 후 섬광계수기(scintillation counter)로 방사능성(radioactivity)를 측정하여 알긴산 올리고당 처리에 따른 포도당 수송능 증가 효과를 확인하였다.L6 muscle cells are cultured in DMEM medium containing 10% serum, and then replaced with 2% serum medium to completely differentiate L6 cells. The medium of completely differentiated L6 muscle cells was exchanged with serum-free DMEM medium to treat the non-reducing terminal monounsaturated mannuronic acid oligosaccharide at a level of 0.2 mg / ml for 1 hour, and then the non-reducing terminal monounsaturated mannuronic acid oligosaccharide was treated The medium was washed twice with pre-warmed 37 ° C KRH buffer (Krebs-Ringer Hepes buffer) to remove glucose present in the medium. [3 H] -2- deoxy-glucose ([3 H] -2-deoxyglucose ) a 0.04 mM concentration after 15 minutes treatment with [3 H] -2- to quickly remove the KRH buffer containing oxy-glucose and ice-cooling The reaction was stopped by adding PBS. Cells were disrupted using cell lysis buffer and radioactivity was measured with a scintillation counter. The effect of increasing the glucose uptake by alginate oligosaccharide treatment was confirmed.
도 5에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당을 처리 시 양성대조구인 인슐린(0.2 μM)과 비교하여 유사한 세포 내 당 흡수가 촉진됨을 확인하였다. 비환원성 말단의 포화형 만뉴론산 올리고당을 0.2 ㎎/㎖ 농도로 처리 시 당흡수가 일어나지 않음에 따라 당흡수 촉진에 비환원성 말단의 이중결합의 형성이 중요함을 확인하였으며, 당 흡수에 중요 단백질인 AMPK(AMP-activated protein kinase)의 억제제인 C.C(Compound C, 1 μM)와 비환원성 말단의 불포화형 만뉴론산 올리고당을 동시 처리 시 당흡수가 억제됨에 따라 비환원성 말단의 불포화형 만뉴론산 올리고당이 AMPK 경로를 통하여 당흡수롤 촉진함을 확인하였다.As shown in Fig. 5, it was confirmed that the similar absorption of intracellular sugar was promoted by comparing the unsaturated maleuronic acid oligosaccharide of non-reducing end with that of insulin (0.2 μM), which is a positive control. It was confirmed that the formation of non - reducing terminal double bond was important for the stimulation of glucose uptake due to the absence of glucose uptake when the non - reducing terminal saturated mannuronic acid oligosaccharide was treated at a concentration of 0.2 mg / ml. The simultaneous treatment of CC (Compound C, 1 μM), which is an inhibitor of AMPK (AMP-activated protein kinase), and the unsaturated maleic acid oligosaccharide of non-reducing end, inhibited glucose uptake and accordingly, the non-reducing terminal monounsaturated mannuronic acid oligosaccharide was AMPK It was confirmed that the absorption roll was promoted through the pathway.
도 6에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당 처리 시 당흡수 촉진과 관련한 수송체 발현에 영향을 주는 PAK, Akt 및 AS160의 인산화 증가를 알아보고자 농도별로 근육세포에 처리하여 단백질을 추출 후 웨스턴 블럿 방법을 통하여 확인한 결과 비환원성 말단의 불포화형 만뉴론산 올리고당 농도 의존적으로 증가함을 확인하였다. 따라서 비환원성 말단의 불포화형 만뉴론산 올리고당은 당흡수 촉진을 위하여 AMPK 경로 및 PAK, Akt, AS160의 인산화를 촉진하여 당수송체(GLUT4, glucose transporter 4)의 발현에도 영향을 줄 것으로 예상하였다.As shown in FIG. 6, in order to investigate the increase of phosphorylation of PAK, Akt and AS160 affecting transporter expression related to glucose uptake upon treatment with unsaturated maleic acid oligosaccharide of non-reducing end, Was determined by Western blotting method and it was confirmed that the concentration of unsaturated mannuronic acid oligosaccharide at the non-reducing end was increased. Therefore, it was expected that the non-reducing terminal unsaturated mannuronic acid oligosaccharide would promote the AMPK pathway and the phosphorylation of PAK, Akt and AS160 to promote glucose uptake, thereby affecting the expression of glucose transporter 4 (GLUT4).
실시예Example 5: 5: 비환원성Non-reducing 말단의 Distal 불포화형Unsaturated 만뉴론산Mannuronic acid 올리고당의 장내 The intestines of oligosaccharides 균총Lichen 조절 효능 확인 Confirmation of control efficacy
비환원성 말단의 불포화형 만뉴론산 올리고당의 장내 균총 개선 효능을 확인하기 위하여 비만 동물 모델인 랫트와 노령 동물 모델인 마우스를 이용하였다. 비만을 유도한 랫트는 생후 3주령의 수컷 SD 랫트를 ㈜중앙동물에서 구입하여 3일 동안 적응시킨 후 사육환경은 온도 20±2℃, 상대습도는 50±10%이며 명암주기는 1일 12시간으로 조절하여 10주 동안 고지방식이를 유도하여 실험군에는 비환원성 말단의 불포화형 만뉴론산 올리고당 0.25 ㎎/㎏ 농도로 48시간 주기로 복강 투여를 실시하며 실험을 진행하였다. 노령 쥐는 생후 1개월령 및 17개월령 수컷 C57BL/6J을 한국기초과학지원연구원에서 구입하여 사육환경은 온도 20±2℃, 상대습도는 50±10%이며 명암주기는 1일 12시간으로 조절하여 10주 동안 실험을 실시하였으며 실험군은 공급하는 물에 비환원성 말단의 불포화형 만뉴론산 올리고당을 0.2 ㎎/㎏ 농도로 공급하였다. 실험 종료 후 실험동물의 내장 내용물을 수집 후 200 ㎎을 취하여 Fast DNA™SPIN Kit for Soil kit를 이용하여 DNA를 kit에 제시된 방법을 토대로 순수한 DNA를 확보하고, 추출한 DNA의 농도와 순도를 확인하기 위해서 나노드롭(nanodrop)을 활용하여 측정한 후, 아가로스겔 전기영동을 통해 추출된 DNA 밴드 결과를 토대로 DNA 농도와 순도를 확인하였다. 분리된 DNA 내 세균의 16S rRNA 유전자 증폭을 위하여 세균에 특이적으로 결합하는 V1-V3 과가변영역(hypervariable region)을 포함하는 27F 정방향 프라이머(GAGTTTGATCMTGGCTCAG)와 518R 역방향 프라이머(WTTACCGCGGCTGCTGG)를 이용하여 94℃에서 5분 동안 초기 변성(denaturation) 시키고, 94 ℃에서 30초, 55℃에서 45초, 72℃에서 1분 30초로 30회 반복하여 증폭 PCR을 수행 후 QIAquick 겔 추출 키트(Qiagen, Germany)를 통해 정제한 PCR 산물은 GS Junior Titanium system(Roche, Germany) 염기서열분석기를 이용하여 파이로시퀀싱(pyrosequencing)을 진행하였으며 파이로시퀀싱에 필요한 방법과 반응들은 제조회사(Roche)의 매뉴얼에 따라 ㈜천연구소(Chunlab, Korea)에서 수행하였다.In order to confirm the effect of the unsaturated maleuronic acid oligosaccharide at the non-reducing end on the intestinal flora, an obese animal model rat and a mouse model of an old animal model were used. Obesity-induced rats were purchased from a 3-week-old male SD rats at 3 weeks of age and were adapted for 3 days. The incubation environment was 20 ± 2 ℃ and the relative humidity was 50 ± 10% . The experimental group was administered with the non - reducing terminal unsaturated mannuronic acid oligosaccharide at a concentration of 0.25 ㎎ / ㎏ for 48 hours. The aged mice were purchased from Korea Basic Science Research Institute at 1 month and 17 months old male C57BL / 6J. The incubation period was 20 ± 2 ℃ and the relative humidity was 50 ± 10% Experimental group was fed with 0.2 ㎎ / ㎏ of unsaturated mannuronic acid oligosaccharide at non - reducing end. After completion of the experiment, 200 mg of the internal contents of the test animal were collected and the pure DNA was obtained based on the method described in the kit using the Fast DNA ™ SPIN Kit for Soil kit and the concentration and purity of the extracted DNA were checked After measuring using nanodrop, DNA concentration and purity were confirmed based on the result of DNA band extracted through agarose gel electrophoresis. For the amplification of the 16S rRNA gene of bacteria in the isolated DNA, 27F forward primer (GAGTTTGATCMTGGCTCAG) and 518R reverse primer (WTTACCGCGGCTGCTGG) containing V1-V3 and a hypervariable region specifically binding to bacteria were used to amplify the 94S Denatured for 5 minutes and amplified by repeating 30 times at 94 ° C for 30 seconds, at 55 ° C for 45 seconds, and at 72 ° C for 1 minute and 30 seconds, followed by amplification with QIAquick gel extraction kit (Qiagen, Germany) The purified PCR products were subjected to pyrosequencing using a GS Junior Titanium system (Roche, Germany) sequencer. Methods and reactions required for pyrosequencing were performed according to the manufacturer's (Roche) (Chunlab, Korea).
도 7에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당을 투여한 비만 랫트 장내 균총의 경우 그람양성세균(Firmicutes)에 속하는 항비만 지표균주인 Roseburia sp. 및 Lactobacillus sp.가 대조군(비만 유도 랫트, 고지방식이, high fat diet; HFD) 대비 각각 약 4% 및 2% 증가 하여 균총의 변화가 일어났으며, 또한 그람음성세균(Bacteroidetes)에 속하는 Clostridium sp. 및 Ruminococcus sp. 균주가 각각 약 1%씩 감소됨을 확인하였다.4,5 As shown in FIG. 7, in the case of the intestinal microflora administered with the unsaturated maleic acid oligosaccharide at the non-reducing end, Roseburia sp., An anti-obesity indicator strain belonging to gram-positive bacteria (Firmicutes) And Lactobacillus sp. Were increased by about 4% and 2%, respectively, compared to the control (obesity induction rat, high fat diet; HFD) and Clostridium sp . And Ruminococcus sp. And the strains were reduced by about 1%, respectively. 4,5
도 8에 제시한 바와 같이, PCO 분석을 통해 비환원성 말단의 불포화형 만뉴론산 올리고당을 음용섭취한 노령쥐의 경우 1개월령의 쥐의 장내 균총과 유사하며 17개월령의 쥐의 장내 균총과는 차이가 있음을 확인하였다. 또한 비환원성 말단의 불포화형 만뉴론산 올리고당 섭취 1개월령과 유사한 장내 균총을 형성함을 확인하였다.As shown in Fig. 8, PCO analysis showed that the age-matched male rats fed with the unsaturated maleic acid oligosaccharide at the non-reducing end were similar to the intestinal microflora of 1 month old rats and were different from the intestinal microflora of 17 months old rats Respectively. Also, it was confirmed that intestinal microflora was formed similar to 1 month of ingestion of unsaturated maleuronic acid oligosaccharide at non - reducing end.
도 9에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당을 음용섭취한 17개월령 노령쥐의 경우 대조구(17개월령 마우스) 대비 그람음성세균(Bacteroidetes)가 약 22% 증가하며, 상대적으로 그람양성세균(Firmicutes) 균이 22% 감소하여 1개월령 쥐의 장내 균총과 유사하게 변화하였음을 확인하였다. As shown in FIG. 9, the 17-month old aged rat ingested with the unsaturated maleic acid oligosaccharide at the non-reducing end increased about 22% of the gram-negative bacteria (Bombyceae) compared to the control (17 months old mouse) It was confirmed that Firmicutes were decreased by 22% and similar to intestinal microflora of 1 month old rats.
실시예Example 6: 6: 비환원성Non-reducing 말단의 Distal 불포화형Unsaturated 만뉴론산Mannuronic acid 올리고당의 에스트로겐 Estrogen of oligosaccharides 증감제Increase / decrease agent 기능 확인 Verify features
본 연구에서 사용된 17β-estradiol은 Sigma (St. Louis, MO, USA)에서 구입하여 사용하였으며, DMEM/F12(Dulbecco’s modified Eagle’s medium/F12), 우태아혈청(Fetal bovine serum), Opti-MEM 배지 및 페니실린-스트렙토마이신은 Gibco (NY, USA)에서 구입하였다. PBS는 WelGENE(Daegu, Korea), 세포 계수 키트(CCK-8)는 Dojindo Molecular Technologies (Tokyo, Japan), RNeasy 소형 키트는 QIAGEN(Hiden, Germany), 소 인슐린은 Cell Applications(San Diego, USA), FuGENE HD는 Promega(Madison, WI, USA)에서 구입하여 사용하였다.The 17β-estradiol used in this study was purchased from Sigma (St. Louis, MO, USA) and used in DMEM / F12 (Dulbecco's modified Eagle's medium / F12), fetal bovine serum, Opti-MEM medium And penicillin-streptomycin were purchased from Gibco (NY, USA). Cells (San Diego, USA), small intestine, QIAGEN (Hiden, Germany), and RNeasy mini kit were purchased from Dojindo Molecular Technologies (Tokyo, Japan), PBS (WelGENE, Daegu, Korea) FuGENE HD was purchased from Promega (Madison, Wis., USA).
MCF-7 세포는 10%의 우태아혈청, 페니실린-스트렙토마이신(100 U/㎖) 및 1% 소 인슐린이 포함된 DMEM/F12 배지에서 37℃ 온도에 배양하여 에스트로겐 증감제 활성을 측정하고자 ERE(estrogen response element)-루시퍼레이즈 활성 및 pS2, PR, CTSD, PGC-1α, ERR, GATA3 및 FOXO1 발현양을 확인하였다.MCF-7 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, penicillin-streptomycin (100 U / ml) and 1% bovine insulin at 37 ° C to measure estrogen- estrogen response element-luciferase activity and expression levels of pS2, PR, CTSD, PGC-1 ?, ERR, GATA3 and FOXO1.
비환원성 말단의 불포화형 만뉴론산 올리고당을 0.1 ㎎/㎖ 농도로 48시간 처리 후 ERE-루시퍼레이즈 활성을 확인하고자 pEGFP-C1-ERα, 3X ERE TATA luc 및 pRL-SV40을 FuGENE HD 시약을 이용하여 MCF-7 세포에 트랜스펙션(transfection) 후 용해하여 루시퍼레이즈 분석을 실시하였으며, GeneJET RNA 정제 키트 방법에 따라 RNA 추출 후 pS2, PR, CTSD, PGC-1α, ERR, GATA3 및 FOXO1의 발현양을 실-시간 PCR로 확인하였다.After treatment of the non-reducing terminal monounsaturated mannuronic acid oligosaccharide at a concentration of 0.1 mg / ml for 48 hours, pEGFP-C1-ER alpha, 3X ERE TATA luc and pRL-SV40 were incubated with FuGENE HD reagent to determine the ERE- -7 cells were transfected and luciferase was analyzed. The amount of expression of pS2, PR, CTSD, PGC-1α, ERR, GATA3 and FOXO1 was measured after RNA extraction according to GeneJET RNA purification kit method. - time PCR.
도 10에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당을 처리시 비환원성 말단의 포화형 만뉴론산 올리고당과 다르게 에스트로겐 시그널 하부 유전자인 pS2의 발현이 대조구 대비 약 5배 증가하였으며, 또한 에스트로겐(E2, 17β-estradiol)과 비환원성 말단의 불포화형 만뉴론산 올리고당 병행 처리 시 에스트로겐 수용체α(ERα. estrogen receptorα)에 의한 ERE 루시퍼레이즈 활성 및 pS2, PR의 발현이 증가하고, CTSD의 발현을 감소시켜 비환원성 말단의 불포화형 만뉴론산 올리고당이 선택적으로 에스트로겐 수용체α 하부 시그널의 유전자의 발현을 조절함을 확인하였다.As shown in FIG. 10, unlike the non-reducing terminal mono-oligosaccharide of mannuron acid, the expression of pS2, an estrogen signal lower gene, was increased about 5-fold when the unsaturated maleic acid oligosaccharide of non-reducing terminal was treated and the estrogen (E2, 17β-estradiol) and unreduced terminal unsaturated mannitol oligosaccharides increased the expression of ERE luciferase activity and pS2, PR by the estrogen receptor α (ERα. Estrogen receptor α) and decreased the expression of CTSD It was confirmed that the unsaturated maleuronic acid oligosaccharide at the non-reducing end selectively regulates the expression of the gene of the estrogen receptor alpha lower signal.
도 11에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당 처리 시 PGC-1α(peroxisome proliferator-activated receptor c coactivator-1a) 및 전사 파트너인 ERRα(estrogen related receptor α)의 발현을 증가시켜, ERα 경로와 함께 작용하여 GATA3(GATA binding protein 3)와 FOXO1 (forkhead box protein A1)의 mRNA 발현을 증가시킴으로서 에스트로겐 증감제 효능을 갖는 것을 확인하였다. As shown in Fig. 11, the expression of PGC-1 alpha (transposon) and transcription partner ERR [alpha] (estrogen related receptor [alpha]) were increased during the treatment of unsaturated maleuronic acid oligosaccharide at the non- ERα pathway and increased the mRNA expression of GATA3 (GATA binding protein 3) and FOXO1 (forkhead box protein A1), confirming the estrogen sensitizing effect.
실시예Example 7: 7: 비환원성Non-reducing 말단의 Distal 불포화형Unsaturated 만뉴론산Mannuronic acid 올리고당의 Oligosaccharide 항비만Anti-obesity , , 항당뇨Anti-diabetic 및 에스트로겐 감수성 증대 기전 모식도 And Estrogen Sensitivity Enhancement Mechanism
비환원성 말단의 불포화형 만뉴론산 올리고당의 항비만, 항당뇨 및 에스트로겐 감수성 증진기전에 대한 전체적인 모식도를 도 12에 제시하였다.An overall schematic diagram of an anti-obesity, anti-diabetic and estrogen sensitivity enhancing mechanism of the unsaturated maleuronic acid oligosaccharide of non-reducing end is shown in FIG.
도 12에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당 처리에 따른 항비만, 항당뇨 및 에스트로겐 감수성 증대 기전을 종합한 결과, 비환원성 말단의 불포화형 만뉴론산 올리고당 처리시 AMPK 활성화를 통해 PGC-1α의 발현이 증가 되고, PGC-1α의 전사 파트너인 ERRs(estrogen related receptor α, β, γ)를 활성화 시켜 지방산 β 산화를 촉진시킴으로서 항비만 효과를 갖는 것을 확인할 수 있었다. 또한 근육 내 AMPK는 지방산의 합성과 분해를 매개함으로서 지방산 β 산화 대사를 촉진하는 작용 및 PGC-1발현을 통해 미토콘드리아 관련 유전자들의 발현을 증가시키는 것으로 보고됨에 따라, 비환원성 말단의 불포화형 만뉴론산 올리고당은 AMPK 활성화를 통해 또한 PCG-1α 발현을 증가시켜, 미토콘드리아의 유전자의 발현 및 수의 증가를 통해 인슐린 저항성을 개선하는 효능을 갖는 것으로 기대되었다.As shown in FIG. 12, the synthesis of anti-obesity, anti-diabetic and estrogen sensitivity enhancing mechanisms by treatment of unsaturated maleic acid oligosaccharides at the non-reducing end resulted in the activation of AMPK in the treatment of unsaturated maleic acid oligosaccharide at the non- It was confirmed that the expression of PGC-1α is increased and the anti-obesity effect is promoted by activating the transcription partners ERCs (estrogen related receptors α, β, γ) of PGC-1α and promoting fatty acid β oxidation. In addition, it has been reported that intramuscular AMPK increases the expression of mitochondrial-related genes through the action of promoting fatty acid β-oxidation by mediating the synthesis and degradation of fatty acids and PGC-1 expression. Thus, Was expected to have an effect of increasing insulin resistance through the activation of AMPK and also by increasing the expression of PCG-1 alpha and the expression and number of mitochondrial genes.
비환원성 말단의 불포화형 만뉴론산 올리고당은 에스트로겐 증감제로서 에스트로겐과 함께 처리시 ERα(estrogen receptor α) 경로를 통해 GATA3 및 FOXO1의 mRNA 발현을 증가시키고, ERRα 및 PGC-1α의 발현을 증가시킴에 따라, 비환원성 말단의 불포화형 만뉴론산 올리고당은 PGC-1α에 의존하는 ERα 경로에 의해 ERE를 활성화 시켜 에스트로겐 증감제의 기능을 갖는 것으로 확인하였다.Unreduced terminal unsaturated mannuronic acid oligosaccharides increase the mRNA expression of GATA3 and FOXO1 through ERα (estrogen receptor α) pathway and increase the expression of ERRα and PGC-1α when treated with estrogen as an estrogen sensitizer , The non-reducing terminal unsaturated mannuronic acid oligosaccharide was confirmed to have an estrogen sensitizer function by activating ERE by ERα pathway which is dependent on PGC-1α.
또한, 비환원성 말단의 불포화형 만뉴론산 올리고당은 체내에서 장내 균총을 개선하여 항비만 지표 균주(Roseburia sp., Lactobacillus sp.)를 증가시키고 비만 지표균주(Clostridium sp. Ruminococcus sp.)를 감소시켜 복합적인 항비만, 항당뇨, 장내 균총 개선 및 에스트로겐 감수성 증대 효능을 갖는 것으로 확인하였다.Unsaturated mannuronic acid oligosaccharide of non-reducing end improves intestinal microflora in the body to increase anti-obesity indicator strain ( Roseburia sp., Lactobacillus sp.) And reduce obesity indicator strain ( Clostridium sp. Ruminococcus sp.) Anti-obesity, anti-diabetic, intestinal flora improvement and estrogen sensitivity enhancement.
<참고문헌><References>
1. Qin J1 et al. A human gut microbial gene catalogue established by metagenomic sequencing. nature. 2010. 59-651. Qin J1 et al. A human gut microbial gene catalog established by metagenomic sequencing. nature . 2010. 59-65
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4. Nadal I et al. ts in clostridia, bacteroides and immunoglobulin-coating fecal bacteria associated with weight loss in obese adolescents. Int J Obes(Lond). 2009. 758-7674. Nadal I et al. ts in clostridia, bacteroides and immunoglobulin-coating fecal bacteria associated with weight loss in obese adolescents. Int J Obes (Lond). 2009. 758-767
5. Neyrinck AM et al. Prebiotic Effects of Wheat Arabinoxylan Related to the Increase in Bifidobacteria, Roseburia and Bacteroides/Prevotella in Diet-Induced Obese Mice. PLoS One. 2011. e209445. Neyrinck AM et al. Prebiotic Effects of Wheat Arabinoxylan Related to the Increase in Bifidobacteria, Roseburia and Bacteroides / Prevotella in Diet-Induced Obese Mice. PLoS One . 2011. e20944
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
Claims (5)
A composition for promoting intestinal benefit-bacterium comprising, as an active ingredient, an unsaturated-type mannuronic acid oligosaccharide having a molecular weight of 100-3000 Da, which is degraded by alginate lyase, using poly-mannuronate as a substrate .
The composition of claim 1, wherein the non-reducing terminal unsaturated maleic acid oligosaccharide is a non-reducing terminal unsaturated maleic acid oligosaccharide, wherein the unsaturated maleic acid oligosaccharide comprises from 1 to 10 mannuronic acid or glutaric acid.
The composition according to claim 1, wherein the non-reducing terminal unsaturated maleic acid oligosaccharide is a non-reducing terminal unsaturated maleic acid oligosaccharide, wherein the ratio of mannuronic acid: glutaric acid is 1.2-5.0: 1. .
The composition according to claim 1, wherein the intestinal beneficial bacteria are enterobacteriaceae selected from the group consisting of Roseburia sp. And Lactobacillus sp .
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