KR20190006058A - Composition for Treating Osteoporosis by Chronological Aging - Google Patents

Composition for Treating Osteoporosis by Chronological Aging Download PDF

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KR20190006058A
KR20190006058A KR1020190002830A KR20190002830A KR20190006058A KR 20190006058 A KR20190006058 A KR 20190006058A KR 1020190002830 A KR1020190002830 A KR 1020190002830A KR 20190002830 A KR20190002830 A KR 20190002830A KR 20190006058 A KR20190006058 A KR 20190006058A
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김두운
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전남대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/734—Alginic acid
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/28—Oligosaccharides

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Abstract

The present invention provides a composition for improving, preventing or treating osteoporosis comprising alginate oligosaccharide as an active ingredient. According to the present invention, the composition is a composition effective for osteoporosis due to natural aging, and facilitates the production of alginate oligosaccharide as an effective substance to be economically advantageous.

Description

자연노화 골다공증 치료용 조성물{Composition for Treating Osteoporosis by Chronological Aging}Composition for Treating Osteoporosis by Chronological Aging}

본 발명은 알긴산 올리고당(Alginate Oligosaccharide)을 유효성분으로 포함하는 자연노화에 따른 골다공증 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating osteoporosis according to natural aging, comprising alginate oligosaccharide as an active ingredient.

알긴산은 해조류인 미역, 감태, 태황, 패, 톳 등의 갈조류의 세포막 사이를 채우고 있는 해조 다당류로서 β-D-만뉴론산(β-D-mannuronic acid; M) 및 α-L-글루쿠론산(α-L-guluronic acid; G)의 2종류의 우론산(uronic acid)이 각종 비율로 1, 4 글리코시드 결합을 한 헤테로형으로 양질의 식이섬유 기능을 나타내는 다당류 이다. 알긴산은 분자 속에 우론산의 카르복시기(COOH-)가 있으므로 산의 성질을 나타내는 특징을 갖는다. 해조류별 함유량은 종류, 장소, 계절, 부위에 따라 다르지만 여름철에 수확한 갈조류의 경우 전체 건조중량의 16-34%의 알긴산이 함유되어 있고 전체 알긴산 소비량의의 약 30%가 식품첨가물로 사용되고 있다. 제약업계에서는 정제의 선형제 등으로 사용되며 아직까지 약물로써 부작용이 알려진바 없으며, 현재까지 식용 가공품으로만 이용되고 있어 부가가치가 낮은 실정임에 따라 알긴산 저분자화를 통해 고부가 의약품 원료로 개발 할 경우 그 가치를 배가 시킬 수 있다.Alginic acid is a seaweed polysaccharide that fills the cell membranes of brown algae such as seaweed seaweed, Ecklonia cava, Taehwang, scallion, and tot. β-D-mannuronic acid (M) and α-L-glucuronic acid ( α-L-guluronic acid; G), two types of uronic acid, are hetero-type with 1, 4 glycosidic bonds in various ratios, and are polysaccharides that exhibit high-quality dietary fiber functions. Alginic acid has the characteristics of an acid because it has a carboxy group (COOH-) of uronic acid in its molecule. The content of each seaweed varies depending on the type, place, season, and part, but brown algae harvested in summer contains 16-34% of the total dry weight of alginic acid, and about 30% of the total amount of alginic acid consumption is used as food additives. In the pharmaceutical industry, it is used as a linear agent for tablets, and there is no known side effect as a drug. Until now, it is used only as an edible processed product, so its value-added is low. You can double the value.

산업적으로 이용 가능한 알긴산 올리고당 생산을 위해서는 첫째, 고온고압 혹은 방사선으로 연화시킨 후 분리하는 방법과 둘째, 산과 알칼리 처리를 이용하는 방법, 셋째, 알긴산 분해효소를 이용한 효소적 제조방법이 있다. 알긴산 분해효소인 알지네이트 라이아제(alginate lyase)는 알긴산의 β-1,4-글리코시드 결합을 분해하여 4,5-불포화 비환원성 말단을 갖는 산물을 생산하는데, 최근에는 효소분해를 통해 생산한 알긴산 올리고당의 기능성이 우수함이 보고됨에 따라 효소적 제조에 대한 연구가 진행되고 있다.In order to produce alginate oligosaccharides that can be used industrially, there are first, a method of separating after softening with high temperature and high pressure or radiation, second, a method using acid and alkali treatment, and third, an enzymatic method using an alginate degrading enzyme. Alginate lyase, an alginate degrading enzyme, breaks down the β-1,4-glycosidic bond of alginic acid to produce a product with a 4,5-unsaturated non-reducing end. As the functionality of oligosaccharides is reported to be excellent, studies on enzymatic preparation are in progress.

골다공증 발생의 중요한 원인 요소 중 하나가 노령이다. 골의 재형성은 조골세포의 골 생성과 파골세포의 골 흡수 균형에 의해 이루어진다. 연령 증가에 따라 골 생성에 비해 골감소가 증가하고, 이에 따른 지속적인 골 감소는 골다공증을 유발하게 된다. 그러나 골다공증에 대한 약물의 효능을 검증하고자 하는 대부분의 연구에서는 건강하고 젊은 실험동물을 사용하고 있으며, 원인요소를 감안하는 노령의 실험동물을 사용한 연구는 매우 찾아보기 어렵다. 이점은 실험적 연구결과와 실제 임상에서 나타날 수 있는 효능 사이의 장벽으로 존재하고 있다. 그러므로 본 연구는 실제 골다공증의 발병 연령이 노령인 점을 감안하여 실험동물을 출생 후 17개월된 노령 쥐를 사용함으로써 실험적 개선을 시도하였다.One of the important causative factors of osteoporosis development is old age. Bone remodeling is achieved by the balance of bone production by osteoblasts and bone resorption by osteoclasts. As age increases, bone loss increases compared to bone formation, and continuous bone loss accordingly causes osteoporosis. However, most studies that attempt to verify the efficacy of drugs on osteoporosis use healthy and young experimental animals, and studies using aged experimental animals considering the causal factors are very difficult to find. This exists as a barrier between experimental results and potential clinical efficacy. Therefore, this study tried to improve the experimental animals by using 17 months old rats in consideration of the fact that the actual age of onset of osteoporosis is old.

특히 폐경 후 여성의 에스트로겐 레벨은 떨어져 결과적으로 파골세포의 활성이 상승되며 조골세포의 활성은 상승되지 않는다. 최근 항골다공증(anti-osteoporotic)의 주요 타켓 중 하나로 ERRa(estrogen-related receptor)은 조골세포의 분화를 상승시켜 골다공증 치료제로 사용된다. ERR은 에스트로겐/엔드로겐 연관 질환에서 주요 역할을 한다. Laflamme 연구진이 2005년 성인여성을 대상으로한 다형성과 BMD의 연관성에 관한 실험에서 ERRa의 발현이 높은 BMD의 발현으로 연관된다고 주장하였으며 이는 Liu 연구진의 2003년 ERRa의 발현이 자궁의 에스트로겐을 상향-조절시키는 기능을 확인하였으며, 골에서의 ERRa의 기능은 성숙된 조골세포로의 분화와 증식을 상승시키는 리셉터로 작용한다고 보고되어 있다(Stein, R.A. et al., 2008. Bianco, S. et al., 2009). 이 때 ERRa의 조절자로 PGC-1a이 작용하게 된다. PGC-1a은 전사 인자인 ERRa의 활성화 시켜 핵 내 DNA 코딩된 미토콘드리아 단백질의 발현을 증가시킨다. 이들 단백질들은 mtDNA의 복제와 발현을 증가시켜 미토콘드리아성 바이오제네시스를 상승시키는 역할을 한다(Lloye M. Dillon. et al., 2012).In particular, postmenopausal women's estrogen levels fall, and as a result, the activity of osteoclasts rises, and the activity of osteoblasts does not. Recently, as one of the major targets of anti-osteoporotic, ERRa (estrogen-related receptor) is used as a treatment for osteoporosis by increasing the differentiation of osteoblasts. ERR plays a major role in estrogen/endrogen-related diseases. In a 2005 experiment on the association between polymorphism and BMD in adult women, Laflamme and colleagues argued that the expression of ERRa was associated with the expression of high BMD. Liu's 2003 expression of ERRa up-regulated uterine estrogen. It has been confirmed that the function of ERRa in bone acts as a receptor to increase differentiation and proliferation into mature osteoblasts (Stein, RA et al., 2008. Bianco, S. et al., 2009). At this time, PGC-1a acts as a regulator of ERRa. PGC-1a increases the expression of DNA-encoded mitochondrial proteins in the nucleus by activating the transcription factor ERRa. These proteins play a role in increasing mitochondrial biogenesis by increasing the replication and expression of mtDNA (Lloye M. Dillon. et al., 2012).

장내 생태계는 출생과 동시에 미생물이 서식하기 시작하여 유익균과 유해균이 균형을 유지하며 장내 균총을 형성하고 인간과 공생하며 상호작용을 통해 인간의 건강에 직·간접으로 영향을 미친다. Rhee SH 연구진에 따르면 내분비 세포는 장내 미생물 균 총의 균형에 따라 영향을 받아 호르몬 방출을 통하여 숙주의 면역세포와 신경세포에 영향을 미친다고 보고하였다. 특히, 노화가 진행됨에 따라 장내 균총의 불균형이 야기되고 이때 장내 미생물은 골 관련 치주염, 관절염, 골다공증을 야기한다(Schogor ALB. et al., 2014). Klara sjogren 연구진에 따르면 장내 미생물이 마우스의 골량을 조절하며, 이는 파골세포가 조절하는 골 흡수에 영향을 줄 수 있다고 보고하였다. 특히 후벽균(Firmicutes)은 노년기에 접어들면서 급격히 증가하게 되나 낮은 비율에서 항생제와 높은 상호작용을 보인다(Zapata et al., 2015). 락토바실러스 루테리(Lactobacillus reuteri)의 경우 영유아들에게 발생하는 설사와 습진, 알레기를 예방하지만, 노년기에는 염증을 유발하거나 심혈관 질환을 증가시킬 수 있다고 보고되어졌다(Forsythe, P. et al., 2007, Van Niel. et al., 2002, Mikelsaar et al., 2010). 건강하게 장수하려면 노년기에 프리보텔라(Prevotella) 종 등의 유익균이 많아야 하는 반면, 후벽균 및 락토바실러스 루테리 등의 균이 적은 상태로 장내 균총을 유지시켜야 한다.In the intestinal ecosystem, microorganisms begin to inhabit upon birth, maintaining a balance between beneficial and harmful bacteria, forming intestinal flora, coexisting with humans, and directly and indirectly affecting human health through interaction. According to Rhee SH researchers, endocrine cells are affected by the balance of the intestinal microbial flora, and have an effect on the host's immune cells and neurons through hormone release. In particular, as aging progresses, imbalance of the intestinal flora is caused, and at this time, the intestinal microbes cause bone-related periodontitis, arthritis, and osteoporosis (Schogor ALB. et al., 2014). According to Klara Sjogren's team, intestinal microbes control bone mass in mice, which may affect bone resorption controlled by osteoclasts. In particular, Firmicutes increases rapidly as they enter old age, but shows high interaction with antibiotics at a low rate (Zapata et al., 2015). Lactobacillus reuteri prevents diarrhea, eczema, and allergies in infants and toddlers, but it has been reported that it can cause inflammation or increase cardiovascular disease in old age (Forsythe, P. et al., 2007, Van Niel. et al., 2002, Mikelsaar et al., 2010). In order to live a healthy longevity, the number of beneficial bacteria such as Prevotella species should be increased in old age, while the intestinal flora should be maintained with fewer bacteria such as posterior bacillus and Lactobacillus luteri.

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.Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

대한민국 등록특허 10-1277706Korean Patent Registration 10-1277706

Bonnelye, E. et al. The orphan nuclear estrogen receptor-related receptor a (ERRa) is expressed throughout osteoblast differentiation and regulates bone formation in vitro. J. Cell Biol. (2001) 153, 971983. Bonnelye, E. et al. The orphan nuclear estrogen receptor-related receptor a (ERRa) is expressed throughout osteoblast differentiation and regulates bone formation in vitro. J. Cell Biol. (2001) 153, 971983. Laflamme, N. et al. A frequent regulatory variant of the Estrogen-Related Receptor a gene associated with BMD in FrenchCanadian premenopausal women. J. Bone Miner. Res. (2005) 20, 938944. Laflamme, N. et al. A frequent regulatory variant of the Estrogen-Related Receptor a gene associated with BMD in FrenchCanadian premenopausal women. J. Bone Miner. Res. (2005) 20, 938944. Liu, D. et al. Estrogen stimulates estrogen-related receptor (alpha) gene expression through conserved hormone response elements. Endocrinology. (2003) 144, 48944904. Liu, D. et al. Estrogen stimulates estrogen-related receptor (alpha) gene expression through conserved hormone response elements. Endocrinology. (2003) 144, 48944904. Stein, R.A. et al. Estrogen-Related Receptor a is critical for the growth of estrogen receptor negative breast cancer. Cancer Res. (2008) 68, 88058812. Stein, R.A. et al. Estrogen-Related Receptor a is critical for the growth of estrogen receptor negative breast cancer. Cancer Res. (2008) 68, 880588 12. Bianco, S. et al. Modulating ERRa activity inhibits cell proliferation J. Biol. Chem. (2009) 284, 2328623292. Bianco, S. et al. Modulating ERRa activity inhibits cell proliferation J. Biol. Chem. (2009) 284, 2328623292. Lloye M. Dillon. et al. The Role of PGC-1 Coactivators in Aging Skeletal Muscle and Heart. IUBMB Life. (2012) 64(3), 231241. Lloye M. Dillon. et al. The Role of PGC-1 Coactivators in Aging Skeletal Muscle and Heart. IUBMB Life. (2012) 64(3), 231241. Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat Rev Gastroenterol Hepatol. 2009;6(5):30614. Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat Rev Gastroenterol Hepatol. 2009;6(5):30614. Schogor ALB, Huws SA, Santos GTD, Scollan ND, Hauck BD, et al. (2014) Ruminal Prevotella spp. May Play an Important Role in the Conversion of Plant Lignans into Human Health Beneficial Antioxidants. PLoS ONE 9(4). Schogor ALB, Huws SA, Santos GTD, Scollan ND, Hauck BD, et al. (2014) Ruminal Prevotella spp. May Play an Important Role in the Conversion of Plant Lignans into Human Health Beneficial Antioxidants. PLoS ONE 9(4). SjoKlara, et al. The gut microbiota regulates bone mass in mice. Journal of bone and mineral research 27.6 (2012): 1357-1367. SjoKlara, et al. The gut microbiota regulates bone mass in mice. Journal of bone and mineral research 27.6 (2012): 1357-1367. Zapata, Heidi J., and Vincent J. Quagliarello, The Microbiota and Microbiome in Aging: Potential Implications in Health and Age-Related Diseases. journal of the american geriatrics society 63.4 (2015): 776-781. Zapata, Heidi J., and Vincent J. Quagliarello, The Microbiota and Microbiome in Aging: Potential Implications in Health and Age-Related Diseases. journal of the american geriatrics society 63.4 (2015): 776-781. Van Niel, C. W., Feudtner, C., Garrison, M. M., & Christakis, D. A. (2002). Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis. Pediatrics, 109(4), 678-684. Van Niel, C. W., Feudtner, C., Garrison, M. M., & Christakis, D. A. (2002). Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis. Pediatrics, 109(4), 678-684. Forsythe, P., Inman, M. D., & Bienenstock, J. (2007). Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. American journal of respiratory and critical care medicine, 175(6), 561-569. Forsythe, P., Inman, M. D., & Bienenstock, J. (2007). Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. American journal of respiratory and critical care medicine, 175(6), 561-569. Mikelsaar, Marika, et al. (2010). Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people. Anaerobe, 16(3), 240-246. Mikelsaar, Marika, et al. (2010). Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people. Anaerobe, 16(3), 240-246.

본 발명자들은 알긴산 분해효소의 폴리만뉴론산 분해에 의해 생성된 알긴산 올리고당의(alginate oligosaccharide; AOS)의 효용성을 조사하고자 노력하였다. 그 결과, 상기 알긴산 올리고당이 골밀도를 증가시키고 장내 균총을 개선시킴을 확인하여 자연노화에 따른 골다공증을 개선시키는 효과를 규명함으로써 본 발명을 완성하였다.The present inventors have tried to investigate the effectiveness of alginate oligosaccharide (AOS) produced by the decomposition of polymanneuronic acid by alginate degrading enzyme. As a result, it was confirmed that the alginic acid oligosaccharide increases bone density and improves the intestinal flora, thereby completing the present invention by finding out the effect of improving osteoporosis caused by natural aging.

따라서, 본 발명의 목적은 골다공증 개선, 예방 또는 치료용 약제학적 조성물을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for improving, preventing or treating osteoporosis.

본 발명의 다른 목적은 골다공증 개선, 예방 또는 치료용 식품 조성물을 제공하는 데 있다.Another object of the present invention is to provide a food composition for improving, preventing or treating osteoporosis.

본 발명의 또 다른 목적은 골다공증 개선, 예방 또는 치료용 건강기능식품 조성물을 제공하는 데 있다.Another object of the present invention is to provide a health functional food composition for improving, preventing or treating osteoporosis.

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 by the following detailed description, claims and drawings.

본 발명의 일 양태에 따르면, 본 발명은 알긴산 올리고당(alginate oligosaccharide)을 유효성분으로 포함하는 골다공증 개선, 예방 또는 치료용 약제학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition for improving, preventing or treating osteoporosis, comprising alginate oligosaccharide as an active ingredient.

본 발명자들은 알긴산 분해효소의 폴리만뉴론산 분해에 의해 생성된 알긴산 올리고당의(alginate oligosaccharide; AOS)의 효용성을 조사하고자 노력하였다. 그 결과, 상기 알긴산 올리고당이 골밀도 및 뼈 잔기둥 간 간격을 증가시킴을 확인하여 자연노화에 따른 골다공증을 개선시키는 효과를 규명하였다.The present inventors have tried to investigate the effectiveness of alginate oligosaccharide (AOS) produced by the decomposition of polymanneuronic acid by alginate degrading enzyme. As a result, it was confirmed that the alginic acid oligosaccharide increases the bone mineral density and the spacing between the bone ridges to find out the effect of improving osteoporosis due to natural aging.

상기 알긴산 올리고당은 알긴산 분해효소의 분해에 의해 생성된 폴리만뉴론산, 폴리굴루로닉산, 또는 폴리만뉴론산 및 폴리글루로닉산이 혼합된 형태의 알긴산 올리고당이다.The alginic acid oligosaccharide is an alginic acid oligosaccharide in the form of a mixture of polymanneuronic acid, polyguluronic acid, or polymanneuronic acid and polygluronic acid produced by decomposition of alginic acid degrading enzyme.

본 발명의 일 구현예에 따르면, 상기 알긴산 올리고당은 100-5000 Da 크기이다.According to an embodiment of the present invention, the alginic acid oligosaccharide has a size of 100-5000 Da.

본 발명의 다른 구현예에 따르면, 상기 알긴산 올리고당은 100-4500 Da, 100-4000 Da, 100-3500 Da 또는 100-3000 Da이다.According to another embodiment of the present invention, the alginic acid oligosaccharide is 100-4500 Da, 100-4000 Da, 100-3500 Da or 100-3000 Da.

상기 알긴산 올리고당은 다음의 일 예(3개의 당이 결합된 알긴산 올리고당)의 구조식 1을 갖는다:The alginic acid oligosaccharide has Structural Formula 1 of the following example (alginic acid oligosaccharide to which three sugars are bonded):

[구조식 1][Structural Formula 1]

Figure pat00001
Figure pat00001

본 발명의 일 구현예에 따르면, 상기 알긴산 올리고당은 1 내지 10개의 당으로 구성된다.According to one embodiment of the present invention, the alginic acid oligosaccharide is composed of 1 to 10 sugars.

본 발명의 다른 구현예에 따르면, 상기 알긴산 올리고당은 1 내지 9개, 1 내지 8개 또는 1 내지 7개의 단당으로 구성된다.According to another embodiment of the present invention, the alginic acid oligosaccharide is composed of 1 to 9, 1 to 8, or 1 to 7 monosaccharides.

상기 알긴산 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 3).The alginic acid oligosaccharide is 175 for 1 sugar, 351 for 2 sugars, 527 for 3 sugars, 704 for 4 sugars, 880 for 5 sugars, 1056 for 6 sugars, and 1232 for 7 sugars. (m/z) (Fig. 3).

본 발명의 일 구현예에 따르면, 상기 알긴산 올리고당은 폴리만뉴론산(poly-mannuronate) 기질로 하여 알긴산 라이아제(alginate lyase)에 의해 분해되어 제조된다.According to one embodiment of the present invention, the alginic acid oligosaccharide is prepared by being decomposed by alginate lyase using 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 alginate composed of polyguluronate and polymannuronate to reduce molecular weight.

본 발명의 특정 구현예에 따르면, 상기 알긴산 라이아제는 해양생물 장내균주 유래 신규 알긴산 분해 효소(대한민국 특허 10-2015-0143496)이다.According to a specific embodiment of the present invention, the alginic acid lyase is a novel alginic acid degrading enzyme derived from a marine organism enterobacteriaceae (Korean Patent 10-2015-0143496).

상기 알긴산 라이아제는 서열목록 제2서열 또는 제4서열의 아미노산 서열로 이루어진다.The alginic acid lyase consists of the amino acid sequence of the second or fourth sequence in the sequence listing.

상기 알긴산 라이아제는 서열목록 제1서열 또는 제3서열의 뉴클레오타이드 서열로 이루어진다.The alginic acid lyase consists of the nucleotide sequence of the first sequence or the third sequence of the sequence listing.

본 발명의 상기 알긴산 올리고당은 최소 2종의 당으로 구성된다.The alginic acid oligosaccharide of the present invention is composed of at least two kinds of sugars.

본 발명의 일 구현예에 따르면, 상기 알긴산 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 alginic acid oligosaccharide includes 1 to 10 manneuronic or glutonic acid. According to another embodiment of the present invention, it contains 1 to 9 manneuronic acid or glutonic acid. According to another embodiment of the present invention, it comprises 1 to 8 manneuronic or glutonic acids. According to a specific embodiment of the present invention, it comprises 1 to 7 manneuronic or glutonic acids.

본 발명의 알긴산 올리고당은 비환원성 말단의 포화형 만뉴론산 올리고당과 비교하여 물 분자가 제거되어 물 분자의 질량값인 18 정도의 작은 질량값을 갖는다(도 3).The alginic acid oligosaccharide of the present invention has a small mass value of about 18, which is the mass value of the water molecule, since water molecules are removed compared to the saturated manneuronic acid oligosaccharide at the non-reducing terminal (FIG. 3).

본 발명의 상기 알긴산 올리고당은 만뉴론산 및 글루론산으로 이루어져 있다.The alginic acid oligosaccharide of the present invention consists of manneuronic acid and glutonic acid.

본 발명의 일 구현예에 따르면, 상기 알긴산 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 manneuronic acid:gluronic acid in the alginic acid oligosaccharide is 1.2-5.0:1. According to another embodiment of the present 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 specific embodiment of the invention, it is 2.0-3.0:1.

즉, 본 발명의 알긴산 올리고당은 글루론산보다 만뉴론산을 1.2-5.0, 1.2-4.5, 1.8-4.0 또는 2.0-3.0배 우세하게 포함한다(도 4).That is, the alginic acid oligosaccharide of the present invention contains manneuronic acid 1.2-5.0, 1.2-4.5, 1.8-4.0 or 2.0-3.0 times predominantly over glutonic acid (FIG. 4).

하기 실시예에서 입증된 바와 같이, 본 발명의 알긴산 올리고당은 비환원성 말단의 공액이중결합형 만뉴론산 올리고당이다.As demonstrated in the examples below, the alginic acid oligosaccharide of the present invention is a conjugated double bonded manneuronic acid oligosaccharide at a non-reducing end.

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.In the present specification, the term "non-reducing" refers to an anomer [a hemiacetal of a monosaccharide (formation of a ring between C-1 and C-5) and a hemicetal (ring formation between C-2 and C-5) in the structure of an oligosaccharide. A phenomenon in which a diastereomer is formed in which the positions of a hydrogen atom attached to a carbon and a hydroxyl group are changed in a cyclization reaction that generates )) It refers to a property that does not have carbon.

본 명세서에서 용어 “공액이중결합형”은 단일겹합과 이중결합이 반복되어 형성된 구조를 의미한다. 비환원성 만뉴론산 올리고당의 경우 효소분해로 인하여 생성된 비환원성 말단이 이중결합 하나만 있는 형태이다. In the present specification, the term "conjugated double bond type" refers to a structure formed by repeating a single compound and a double bond. In the case of non-reducing manneuronic acid oligosaccharides, the non-reducing end produced by enzymatic decomposition has only one double bond.

상기 폴리만뉴론 알긴산의 분해는 산-알칼리 가수분해, 방사선 조사, 고온고압, 효소에 의해 실시할 수 있으나, 이에 한정되지 않는다.The decomposition of polymanneuron alginic acid may be performed by acid-alkali hydrolysis, irradiation, high temperature and high pressure, and enzymes, but is not limited thereto.

본 명세서의 용어 “비환원성 말단의 공액이중결합형 만뉴론산 올리고당”은 폴리만뉴론산 수용액에 알긴산 분해효소하여 제조된 것으로, “알긴산 올리고당”과 동일한 의미를 갖는다.The term "non-reducing end conjugated double bonded manneuronic acid oligosaccharide" is prepared by alginic acid degrading enzyme in an aqueous polymanneuronic acid solution, and has the same meaning as "alginic acid oligosaccharide".

본 발명의 조성물의 유효성분인 알긴산 올리고당은 골다공증 개선, 예방 또는 치료 효과를 갖는다.Alginic acid oligosaccharide, which is an active ingredient of the composition of the present invention, has an effect of improving, preventing or treating osteoporosis.

상기 골다공증은 뼈의 성분인 칼슘이 급격히 빠져나와 정상적인 뼈에 비하여 골밀도가 낮아져 “구멍이 많이 난 뼈”를 말하며, 폐경, 노화, 뼈에 해로운 약물의 사용 등의 여러 가지 원인에 의하여 뼈가 많이 손실되고 약해져 경미한 충격에도 쉽게 골절이 일어나는 질환이다.Osteoporosis refers to “a bone with a lot of holes” because calcium, a component of the bone, rapidly escapes and bone density is lower than that of normal bone. A lot of bone is lost due to various causes such as menopause, aging, and the use of drugs that are harmful to bone. It is a disease that is weakened and fractures easily even with a slight impact.

본 발명의 일 구현예에 따르면, 상기 골다공증은 노인성 골다공증이다.According to one embodiment of the present invention, the osteoporosis is senile osteoporosis.

본 명세서에서 용어 ‘노인성 골다공증’은 노화(aging)에 의한 골다공증을 의미한다.In the present specification, the term "senile osteoporosis" refers to osteoporosis caused by aging.

하기 실시예에서 입증된 바와 같이, 상기 조성물은 골밀도를 증가시키고 장내 균총을 개선시키는 효과를 갖는다.As demonstrated in the examples below, the composition has the effect of increasing bone mineral density and improving intestinal flora.

본 발명의 조성물은 골다공증의 개선, 예방 또는 치료용 조성물은 약제학적 조성물로 제조될 수 있다.The composition of the present invention may be prepared as a pharmaceutical composition for improving, preventing or treating osteoporosis.

본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 (a) 상술한 본 발명의 알긴산 올리고당의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물이다. 본 명세서에서 용어 “약제학적 유효량”은 상술한 알긴산 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-described alginic acid oligosaccharide of the present invention; And (b) a pharmaceutically acceptable carrier. In the present specification, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the above-described alginic 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 prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like 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 applied by way of oral administration.

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.A suitable dosage of the pharmaceutical composition of the present invention is formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. A typical dosage of the pharmaceutical composition of the present invention is in the range of 0.001 μg/kg-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 is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

본 명세서에서 용어 “유효성분으로 포함하는”이란 상술한 알긴산 올리고당의 효능 또는 활성을 달성하는 데 충분한 양을 포함하는 것을 의미한다. 상술한 알긴산 올리고당이 본 발명의 조성물에 포함된 양적 상한은 당업자가 적절한 범위 내에서 선택하여 실시할 수 있다.In the present specification, the term "included as an active ingredient" means including an amount sufficient to achieve the above-described efficacy or activity of the alginic acid oligosaccharide. The upper limit of the quantity of alginic acid oligosaccharides contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

본 발명의 다른 양태에 따르면, 본 발명은 알긴산 올리고당을 유효성분으로 포함하는 골다공증 개선 또는 예방용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition for improving or preventing osteoporosis comprising alginate oligosaccharide as an active ingredient.

본 발명의 알긴산 올리고당을 유효성분 포함하는 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 알긴산 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 알긴산 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 alginic acid oligosaccharide of the present invention is prepared as a food composition, it includes not only alginic acid oligosaccharide as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, and fats. , Nutrients, seasonings and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. can be additionally included in addition to the alginic acid oligosaccharide of the present invention. have.

본 발명의 또른 양태에 따르면, 본 발명은 알긴산 올리고당을 유효성분으로 포함하는 골다공증 개선 또는 예방용 식품 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition for improving or preventing osteoporosis comprising alginate oligosaccharide as an active ingredient.

본 발명의 알긴산 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 알긴산 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.It can be prepared as a functional food composition containing the active ingredient alginic acid oligosaccharide of the present invention. When the composition of the present invention is prepared as a functional food composition, it includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, a flavoring agent or natural carbohydrate may be included as an additional component in addition to alginic acid oligosaccharide as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg, maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrin, cyclodextrin, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumarin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) can be used.

본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:

(a) 본 발명은 알긴산 올리고당을 유효성분으로 포함하는 골다공증 개선, 예방 또는 치료용 조성물을 제공한다.(a) The present invention provides a composition for improving, preventing or treating osteoporosis comprising alginate oligosaccharide as an active ingredient.

(b) 본 발명은 자연노화에 따른 골다공증에 효과적인 조성물로, 유효물질인 알긴산 올리고당의 제조가 용이하여 경제적인 이점을 갖는다.(b) The present invention is an effective composition for osteoporosis caused by natural aging, and has an economic advantage because it is easy to prepare an active substance, alginic acid oligosaccharide.

도 1은 알긴산분해효소를 이용하여 만뉴론산(poly mannuronate, poly M)에 대한 디폴리머리제이션 결과를 상업용 알긴산 올리고당(marker)을 표준물질로 사용하여 TLC(Thin Layer Chromatography)로 보여준다.
도 2는 비환원성 말단의 공액이중결합형 만뉴론산 올리고당(CMOS)의 질량분석 결과를 나타낸다.
도 3은 비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 이중결합 비율을 나타낸다.
도 4는 비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 당 구성을 원평광 이색성 분광 측정법(CD, circular dichorism) 방법으로 확인한 결과를 나타낸다.
도 5는 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 처리에 따른 PGC-1α의 mRNA 발현양를 나타낸다.
도 6은 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당 급이가 대퇴골의 골밀도 향상에 미치는 영향을 나타낸다. 멸균음용수를 급여한 그룹(SDW), 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당을 음용수로 급여한 그룹(AOS), 멸균음용수 급이와 운동을 병행한 그룹(SDW_Exer) 및 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당 음용수 급이와 운동을 병행한 그룹(AOS_Exer)별 골밀도를 나타낸다.
도 7은 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 급이가 정강이 뼈의 골밀도 향상에 미치는 영향을 나타낸다. 멸균음용수를 급여한 그룹(SDW), 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당을 음용수로 급여한 그룹(AOS), 멸균음용수 급이와 운동을 병행한 그룹(SDW_Exer) 및 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당 음용수 급이와 운동을 병행한 그룹(AOS_Exer)별 골밀도를 나타낸다.
도 8은 일반 사료 급이에 따른 노령쥐의 대퇴골의 단면 이미지를 나타낸다.
도 9는 일반 사료 급이 및 운동에 따른 노령쥐의 대퇴골 단면 이미지를 나타낸다.
도 10은 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 음용 섭취와 운동에 따른 노령쥐의 대퇴골의 단면 이미지를 나타낸다.
도 11은 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 음용 섭취에 따른 노령 쥐의 장내 균총을 나타낸다.
도 12는 멸균수 음용 섭취 1개월령(C1-M)과 17개월령(C17-M)쥐와 실험군인 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당 음용 섭취에 따른 1개월령(A1-M) 및 17개월령(A17-M)의 장내균총을 나타낸다.1 shows the results of depolymerization of mannuronate (poly M) using alginate degrading enzyme by TLC (Thin Layer Chromatography) using a commercial alginic acid oligosaccharide (marker) as a standard material.
Figure 2 shows the results of mass spectrometry of the conjugated double bonded manneuronic acid oligosaccharide (CMOS) at the non-reducing end.
3 shows the double bond ratio of the conjugated double bond type manneuronic acid oligosaccharide at the non-reducing end.
4 shows the results of confirming the sugar composition of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end by circular dichorism (CD) method.
5 shows the amount of mRNA expression of PGC-1α according to the treatment of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end.
Figure 6 shows the effect of feeding the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end on the improvement of bone mineral density in the femur. The group fed sterile drinking water (SDW), the group fed the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end as drinking water (AOS), the group fed sterile drinking water and exercised together (SDW_Exer), and the conjugate of the non-reducing end Bone mineral density for each group (AOS_Exer) that was fed with drinking water and exercised with double-bonded manneuronic acid oligosaccharide is shown.
Figure 7 shows the effect of feeding the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end on the improvement of bone mineral density in the shin bone. The group fed sterile drinking water (SDW), the group fed the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end as drinking water (AOS), the group fed sterile drinking water and exercised together (SDW_Exer), and the conjugate of the non-reducing end Bone mineral density for each group (AOS_Exer) that was fed with drinking water and exercised with double-bonded manneuronic acid oligosaccharide is shown.
8 shows a cross-sectional image of the femur of an old rat according to normal feed feeding.
9 shows a cross-sectional image of the femur of an old rat according to general feed feeding and exercise.
10 shows a cross-sectional image of the femur of an old rat according to drinking intake and exercise of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end.
Figure 11 shows the intestinal flora of old rats according to drinking intake of conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end.
Figure 12 is 1 month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17-month-old (A1-M) and 17 It shows the intestinal flora of month old (A17-M).

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예 1: 비환원성 말단의 공액이중결합형 만뉴론 올리고당 생산Example 1: Production of conjugated double bonded manneuron oligosaccharide at non-reducing end

본 실시예에서는 0.3% 폴리만뉴론산 수용액에 알긴산 분해효소[대한민국 특허 10-2015-0143496의 알긴산 라이아제] 첨가 후 45℃에서 24시간 분해하고 TLC(Thin Layer Chromatography)를 이용하여 디폴리머리제이션(depolymerization)을 확인하였고, 알긴산 분해 올리고당 혼합물인 비환원성 말단의 공액이중결합형 만뉴론산 올리고당을 확보하였다. 상기 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 혼합물을 한외여과막 시스템(VivaFlow 50, Sartorius)을 이용하여 분자량 3000 Da 이하의 분획물을 확보하였으며, 확보한 분자량 3000 Da 이하의 분획물은 동결건조를 통해 분말형태로 시험 전까지 20℃에 보관하였다.In this example, after addition of alginic acid degrading enzyme [alginic acid lyase of Korean Patent 10-2015-0143496] to 0.3% polymanneuronic acid aqueous solution, depolymerization was performed at 45° C. for 24 hours and then depolymerized using TLC (Thin Layer Chromatography). (depolymerization) was confirmed, and a conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal, which is a mixture of alginic acid-decomposed oligosaccharides, was obtained. A fraction having a molecular weight of 3000 Da or less was obtained using an ultrafiltration membrane system (VivaFlow 50, Sartorius) of the conjugated double bonded manneuronic acid oligosaccharide mixture at the non-reducing terminal, and the obtained fraction having a molecular weight of 3000 Da or less was powdered through lyophilization. It was stored at 20°C until the test in form.

도 1에 제시한 바와 같이, 알긴산 분해효소를 이용하여 생산한 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 TLC를 통해 1-7당(DP 1-7)으로 구성되어 있음을 확인하였으며, TLC의 스팟(spot)의 세기(intensity)를 비교하였을 때, 상대적으로 2-5당이 높은 비율로 존재함을 확인하였다.As shown in Figure 1, it was confirmed that the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal produced using alginic acid degrading enzyme was composed of 1-7 sugars (DP 1-7) through TLC, and TLC When comparing the intensity of the spot (spot), it was confirmed that there is a relatively high ratio of 2-5 sugars.

실시예Example 2: 2: 비환원성Non-reducing 말단의 Terminal 공액이중결합형Conjugated double bond type 만뉴론산Manneuronic acid 올리고당 구성 및 구조적 특징 분석 Analysis of oligosaccharide composition and structural characteristics

상기 비환원성 말단의 공액이중결합형 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 cm의 큐벳을 이용하여 190-250 ㎚ 영역에서 측정하였으며, 일괄성 있는 CD 시그널을 얻기 위하여 상기 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 1 mg/mL 농도로 사용하였다. 만뉴론산:글루론산 구성비를 알아보기 위하여 피크(peak, 200 ㎚ 흡광도 값) 및 트로프(trough, 215 ㎚ 흡광도 값)를 측정하여 만뉴론산과 글루론산의 비율을 계산하였으며, 계산식은 다음과 같다:Circular dichroism (circular dichroism, CD, J-715 spectropolarimeter, JASCO) was used to measure the circular dichroism spectral signal to measure the ratio of manneuronic acid in the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end. . The CD signal was measured in the region of 190-250 nm using a 1 cm cuvette at room temperature, and the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end was used at a concentration of 1 mg/mL to obtain a uniform CD signal. I did. In order to determine the composition ratio of manneuronic acid: gluconic acid, the ratio of manneuronic acid and gluconic acid was calculated by measuring the peak (peak, absorbance value of 200 ㎚) and trough (absorbance value of 215 ㎚), and the calculation formula is as follows:

(1) 피크/트로프 < 1, 만뉴론산/글루론산 = 2.0(피크/트로프) (1) Peak/Trop <1, Manneuronic/Gluronic Acid = 2.0 (Peak/Trop)

(2) 피크/트로프 > 1, 만뉴론산/글루론산 = 27(피크/트로프) + 40 (2) Peak/Trop> 1, Manneuronic/Gluronic Acid = 27 (Peak/Trop) + 40

도 2에서 알 수 있듯이, 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 만뉴론산/글루론산 비율이 2.59(피크=3.82, 트로프=3.20)이며, 비환원성 말단의 공액이중결합형 형성으로 인하여 215 ㎚의 흡광도값 측정이 방해됨을 확인할 수 있었다.As can be seen from Figure 2, the conjugated double bond type manneuronic oligosaccharide at the non-reducing end has a manneuronic acid/gluronic acid ratio of 2.59 (peak = 3.82, trough = 3.20), and 215 due to the formation of a conjugated double bond type at the non-reducing end. It was confirmed that the measurement of the absorbance value of nm was hindered.

비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 구성당을 분석하기 위하여, 준비된 시료는 이온 교환 수지 컬럼(Hitrap DEAE Sepharose FF, GE Healthcare)을 이용하여 정제한 후 동결건조를 실시하였다. 정제된 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 물에 용해 후 UPLC/MS 시스템에 주입함으로써 구성당 분석을 수행하였다.In order to analyze the constituent sugar of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end, the prepared sample was purified using an ion exchange resin column (Hitrap DEAE Sepharose FF, GE Healthcare) and then lyophilized. Conjugated double bonded manneuronic acid oligosaccharide at the purified non-reducing end was dissolved in water and then injected into a UPLC/MS system to perform constituent sugar analysis.

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 Amylamin, 25 mM HFIP)와 용매 B(15 mM Amylamin, 25 mM HFIP in Acetonitrile)의 선형 구배를 조절하였다. C18-UPLC로 분리되어 나온 용리액은 질량분석기 (Quadrupole-Time of Flight, Q-TOF, Waters)로 분석하였다. Q-TOF는 ESI 네거티브 모드에서 분석하였으며, 캐필러리와 샘플링콘(sampling cone)의 전압은 각각 3 kV와 40 V, 탈용매(desolvation)는 유속 600 L/h, 온도 300℃, 소스 온도는 120℃ 조건에서 실시하였다. TOF MS 데이타는 스캔시간은 0.5 초 m/z 100-1300 범위에서 분석하였다. 분석의 정확도를 위해 모든 분석에 2 ng/㎕의 로이신 엔케팔린(leucine enkephalin, ESI 네거티브 모드에서 554.2619 Da)을 락스프레이(lack spray)로 사용하였다.Ultra Performance Liquid Chromatography (UPLC, Waters) was set up using an ACQUITY UPLC BEH C18 (1.7 μm 1.0 × 100 mm, Waters) column, and Solvent A (15 mM Amylamin, Waters) was used for 12 minutes at 0.4 ml/min. 25 mM HFIP) and solvent B (15 mM Amylamin, 25 mM HFIP in Acetonitrile) were adjusted to have a linear gradient. The eluent separated by C18-UPLC was analyzed by mass spectrometry (Quadrupole-Time of Flight, Q-TOF, Waters). Q-TOF was analyzed in ESI negative mode, the voltage of the capillary and the sampling cone was 3 kV and 40 V, respectively, the desolvation was 600 L/h, the temperature was 300°C, and the source temperature was It was carried out under the conditions of 120 ℃. TOF MS data was analyzed in the range of 0.5 sec m/z 100-1300 scan time. For the accuracy of the analysis, 2 ng/µl of leucine enkephalin (554.2619 Da in ESI negative mode) was used as a lock spray for all analyzes.

도 3에서 알 수 있듯이, 비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 질량분석 결과 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 1-7당으로 구성되어 있으며, 특히 기보고된 만뉴론산 올리고당 질량값 보다 18이 적은 피크들이 관찰됨에 따라 물 분자가 제거되어 비환원성 말단의 공액이중결합형이 형성된 올리고당이 형성되며 비공액이중결합형 만뉴론산 올리고당(nonconjugated carbonyl mannuronic acid oligosaccharide, NCMOS)보다 우세하게 존재함을 알 수 있었다. 비환원성 말단의 공액이중결합형 만뉴론산 올리고당(CMOS)과 비공액이중결합형 만뉴론산 올리고당(NCMOS)의 비율을 나타낸 결과는 도 4에 제시되었다.As can be seen in Figure 3, the result of mass spectrometry of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end, the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end is composed of 1-7 sugars, and in particular, the previously reported manneuronic acid oligosaccharides As peaks 18 less than the mass value are observed, water molecules are removed to form an oligosaccharide with a conjugated double bond at the non-reducing end, and dominate over nonconjugated carbonyl mannuronic acid oligosaccharide (NCMOS). I could see that it exists. The results showing the ratio of the conjugated double bonded manneuronic acid oligosaccharide (CMOS) and the non-conjugated double bonded manneuronic acid oligosaccharide (NCMOS) at the non-reducing end are shown in FIG. 4.

도 4와 같이 비환원성 말단의 공액이중결합형 만뉴론산 올리고당은 비공액이중결합형 만뉴론산 올리고당 보다 평균 2배 이상의 비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 단당들이 존재함을 확인하였다. 비공액이중결합형 만뉴론산 올리고당 분자량은 다음과 같다: 1당 (m/z 175), 2당 (m/z 369), 3당 (m/z 545), 4당 (m/z 722), 5당 (m/z 898) 6당 (m/z 1074), 7당 (m/z 1250).As shown in Figure 4, the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal was confirmed that there were monosaccharides of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal at an average of two times or more than the non-conjugated double bonded manneuronic acid oligosaccharide. The molecular weight of the non-conjugated double bonded manneuronic acid oligosaccharide is as follows: sugar 1 (m/z 175), sugar 2 (m/z 369), sugar 3 (m/z 545), sugar 4 (m/z 722), Per 5 (m/z 898) Per 6 (m/z 1074), Per 7 (m/z 1250).

실시예 3: PGC-1α의 mRNA 발현 평가Example 3: Evaluation of mRNA expression of PGC-1α

비환원성 말단의 공액이중결합형 만뉴론산 올리고당이 PGC-1α의 상위조절자인 AMPK의 인산화를 촉진함이 확인됨에 따라 PGC-1α의 발현을 mRNA 수준에서 확인해 보고자 하였다. As it was confirmed that the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end promotes the phosphorylation of AMPK, the upper regulator of PGC-1α, we tried to confirm the expression of PGC-1α at the mRNA level.

MCF-7 세포는 10%의 우태아혈청, 페니실린-스트렙토마이신 (100 U/㎖) 및 1% 소 인슐린이 포함된 DMEM/F12 배지에서 37 ℃ 온도에 배양하여 PGC-1α 발현양을 측정하기 위한 세포모델로 사용하였다. MCF-7세포에 비환원성 말단의 불포화형 만뉴론산 올리고당 (0.1 ㎎/㎖, MOS)과 에스트라디올 (10 nM, E2)를 48시간 처리 후 GeneJET RNA 정제 키트 방법에 따라 RNA 추출 후 cDNA를 합성하여 PGC-1α의 발현양을 실-시간 PCR으로 확인하였다. 도 5에 제시한 바와 같이, 비환원성 말단의 불포화형 만뉴론산 올리고당을 처리 하여 PGC-1α의 mRNA 발현을 확인한 결과 무처리구 대비 64배 발현을 유도하였으며 비환원성 말단의 불포화형 만뉴론산 올리고당과 에스트라디올을 동시 처리한 경우 (E2+MOS) 128.33배 증가하여 에스트라디올 단독 처리 실험구 (E2) 대비 5.8배 발현이 증가하였다. 항골다공증(anti-osteoporotic)의 주요 타켓 중 하나로 ERRa(estrogen-related receptor)은 조골세포의 분화를 상승시켜 골다공증 치료제로 사용된다. PGC-1a은 전사 인자인 ERRa의 활성화 시켜 핵 내 DNA 코딩된 미토콘드리아 단백질의 발현을 증가시킨다. ERRa의 조절자로 작용하는 PGC-1a의 발현이 비환원성 말단의 불포화형 만뉴론산 올리고당에 의해 증가되는것을 확인하였다.MCF-7 cells were cultured at 37°C in DMEM/F12 medium containing 10% fetal bovine serum, penicillin-streptomycin (100 U/ml) and 1% bovine insulin to measure the amount of PGC-1α expression. It was used as a cell model. MCF-7 cells were treated with non-reducing terminal unsaturated manneuronic acid oligosaccharide (0.1 mg/ml, MOS) and estradiol (10 nM, E2) for 48 hours, followed by RNA extraction according to the GeneJET RNA purification kit method, and then cDNA was synthesized. The amount of expression of PGC-1α was confirmed by real-time PCR. As shown in Figure 5, as a result of confirming the mRNA expression of PGC-1α by treating the unsaturated manneuronic acid oligosaccharide at the non-reducing end, it induced 64 times expression compared to the untreated group. In the case of simultaneous treatment (E2+MOS), the expression increased by 128.33 times compared to the experimental group treated with estradiol alone (E2) by 5.8 times. As one of the major targets of anti-osteoporotic, ERRa (estrogen-related receptor) is used as a treatment for osteoporosis by increasing the differentiation of osteoblasts. PGC-1a increases the expression of DNA-encoded mitochondrial proteins in the nucleus by activating the transcription factor ERRa. It was confirmed that the expression of PGC-1a, which acts as a regulator of ERRa, is increased by the unsaturated manneuronic acid oligosaccharide at the non-reducing terminal.

실시예 4: 골다공증 치료 실험 설계 및 방법Example 4: Osteoporosis treatment experiment design and method

비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 골다공증 치료 효능에 대한 평가를 실시하였다. 본 실험에서는 1개월령 마우스 12마리[자성, 대조군(1M SDW): 6마리, 실험군(1M AOS): 6마리), 17개월령 마우스 24마리(자성, 대조군(18M SDW): 6마리, 실험군(18M AOS): 6마리, 운동대조군(18M SDW_Exer): 6마리, 운동실험군(18M AOS Exer): 6마리]를 대상으로 하였다. 실험기간은 총 12주로 비환원성 말단의 공액이중결합형 만뉴론산 올라고당 섭취가 노령 쥐의 골다공증 개선에 미치는 영향을 CT 촬영을 통하여 조사하였다. 실험 실시에 앞서 환경 적응을 위하여 모든 쥐를 1주일 동안 실험 케이지에 순응시켰다. 대조집단인 증류수 음용군(n=18, SDW)과 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 음용군(n=18, AOS)으로 구분하여 사육용 케이지에 3마리씩 넣어 사육하였다. 운동그룹의 경우 실험 시작 4주 이후부터 8주간 자유 트레드밀 운동을 실시하였다. 모든 마우스는 멸균사료 및 멸균 음용수를 자유롭게 섭취하였다. 비환원성 말단의 공액이중결합형 만뉴론산 올리고당의 섭취는 매주 200 ppm 농도의 음용수를 자유 섭취하도록 하였다.The efficacy of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal in the treatment of osteoporosis was evaluated. In this experiment, 12 mice of 1 month old (magnetic, control (1M SDW): 6, experimental group (1M AOS): 6), 24 17-month-old mice (magnetic, control (18M SDW): 6, experimental group (18M) AOS): 6 animals, exercise control group (18M SDW_Exer): 6 animals, exercise test group (18M AOS Exer): 6 animals] The experiment period was 12 weeks, conjugated double bonded mannuronate mannuronate at the non-reducing terminal. The effect of ingestion on the improvement of osteoporosis in aged rats was investigated through CT scans All rats were acclimated to the experimental cage for 1 week prior to the experiment for environmental adaptation. ) And the non-reducing terminal conjugated double bonded manneuronic acid oligosaccharide drinking group (n=18, AOS) and reared 3 animals each in cages for breeding In the case of the exercise group, free treadmill exercise for 8 weeks from 4 weeks after the start of the experiment All mice were freely ingested with sterile feed and sterile drinking water The intake of conjugated double bonded manneuronic oligosaccharide at the non-reducing terminal was to freely ingest 200 ppm drinking water every week.

실시예 5: 조직형태계측학적 분석 방법Example 5: Histomorphometric analysis method

골밀도(Bone Mineral density)측정Bone Mineral density measurement

비환원성 말단의 공액이중결합형 만뉴론산 올리고당 자유섭취 이후, 각 개체별 흡입 마취 하여 마이크로 CT의 X선 카메라 장치를 이용하여 용적 당 골량을 측정하였다. 도 6에 나타난 바와 같이 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 식이한 1M AOS, 18M AOS 및 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 식이와 운동을 병행한 18M AOS Exer에서 각각 대퇴골의 골밀도가 증가하였다. 도 7에 나타난 바와 같이, 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 식이한 1M AOS, 18M AOS 및 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 식이와 운동을 병행한 18M AOS Exer에서 각각 정강이의 골밀도가 증가하였다.After free intake of conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end, each individual was inhaled anesthesia and measured bone mass per volume using an X-ray camera device of micro CT. Bone density of the femur in 18M AOS Exer, which was fed with a conjugated double-bonded manneuronic acid oligosaccharide diet at the non-reducing end and 1M AOS, 18M AOS, and a conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end, and exercised in parallel as shown in FIG. Increased. As shown in Figure 7, in the non-reducing end conjugated double bonded manneuronic acid oligosaccharide diet 1M AOS, 18M AOS, and the non-reducing end conjugated double bonded manneuronic acid oligosaccharide diet and exercise combined 18M AOS Exer, each of the shins Bone mineral density increased.

도 8, 9 및 10에 나타난 바와 같이, 각 개체별 흡입 마취하여 마이크로 CT의 X선 카메라 장치를 이용하여 촬영한 대퇴골의 단면 모습이다. 도 8은 일반 사료 급이에 따른 17개월 노령쥐의 대퇴골 단면 이미지를 나타내며, 도 9는 일반 사료 급이 및 운동에 따른 노령쥐의 대퇴골 단면 이미지이다. 도 10은 비환원성 말단의 공액이중결합형 만뉴론산올리고당 음용 섭취와 운동에 따른 노령쥐의 대퇴골의 단면 이미지를 나타낸다. 일반 사료 급이에 따른 대퇴골의 단면 이미지 대비 비환원성 말단의 공액 이중결합형 만뉴론산 올리고당 음용 섭취와 운동에 따른 대퇴골의 단면이미지가 섬유주(trabecular)의 증가를 나타낸다.As shown in FIGS. 8, 9, and 10, a cross-sectional view of a femur photographed using an X-ray camera device of a micro CT by inhaling anesthesia for each individual. FIG. 8 is a cross-sectional image of the femur of a 17-month-old rat according to normal feed feeding, and FIG. 9 is a cross-sectional image of the femur of an old rat according to normal feed feeding and exercise. 10 shows a cross-sectional image of the femur of an aged rat according to drinking intake and exercise of conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end. Compared to the cross-sectional image of the femur according to general feed feeding, the cross-sectional image of the femur according to drinking intake and exercise of the conjugated double-bonded manneuronic acid oligosaccharide at the non-reducing end shows an increase in trabecular.

실시예Example 6: 노령 마우스에서 6: in old mice 비환원성Non-reducing 말단의 Terminal 공액이중결합형형Conjugated double bond type 만뉴론산Manneuronic acid 올리고당 투여 실험실시 Oligosaccharide administration laboratory

노령 쥐는 생후 1개월령 및 17개월령 수컷 C57BL/6J을 한국기초과학지원연구원에서 구입하여 사육환경은 온도 20℃, 상대습도는 50%이며 명암주기는 1일 12시간으로 조절하여 1주 동안 적응 기간 후 무작위로 3마리 씩 대조구와 실험구로 나누어 그룹을 1개월령과 17개월 별 그룹을 선정하였다. 실험구는 공급하는 물에 비환원성 말단의 공액 이중결합형형 만뉴론산 올리고당을 0.2 mg/mL 농도로 공급하였으며, 대조구는 물만 공급하여 10 주동안 실험을 진행 하였다. 실험 종료 후 마우스는 해부하여 장내 내용물을 분리하여 200 mg을 취하여 Fast DNA™SPIN 키트(for Soil kit)를 이용하여 DNA를 키트에 제시된 방법을 토대로 순수한 DNA를 확보하고, 추출한 DNA의 농도와 순도를 확인하기 위해서 나노드롭을 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 초가변영역(hypervariable region)을 포함하는 27F 정방향 프라이머(5'-GAGTTTGATCMTGGCTCAG-3')와 518R 역방향 프라이머(5'-WTTACCGCGGCTGCTGG-3')를 이용하여 94℃에서 5분 동안 초기 변성시키고, 94℃에서 30초, 55℃에서 45초, 72℃에서 1분 30초로 30회 반복하여 증폭 PCR을 수행 후 QIAquick 겔 추출 키트(Qiagen, Germany)를 통해 정제하였다. PCR 산물은 GS 주니어 티타늄 시스템(Roche, Germany) 염기서열분석기를 이용하여 파이로시퀀싱(pyrosequencing)을 진행하였으며 파이로시퀀싱에 필요한 방법과 반응들은 제조회사(Roche)의 매뉴얼에 따라 ㈜천연구소(Chunlab, Korea)에서 수행 하였다.For old mice, 1-month-old and 17-month-old male C57BL/6J were purchased from the Korea Basic Science Institute and the breeding environment was adjusted to a temperature of 20℃ and a relative humidity of 50%, and the contrast period was adjusted to 12 hours per day. Randomly divided into 3 animals, control group and experimental group, and groups of 1 month old and 17 months of age were selected. The experimental group was supplied with a non-reducing terminal conjugated double bonded manneuronic acid oligosaccharide at a concentration of 0.2 mg/mL to the supplied water, and the control was conducted for 10 weeks by supplying only water. After the end of the experiment, the mouse is dissected, the intestinal contents are separated, 200 mg is taken, and the DNA is obtained using the Fast DNA™ SPIN kit (for Soil kit) to obtain pure DNA based on the method presented in the kit, and the concentration and purity of the extracted DNA are determined. To confirm, the DNA concentration and purity were confirmed based on the result of the DNA band extracted through agarose gel electrophoresis after measurement using nanodrops. 27F forward primer (5'-GAGTTTGATCMTGGCTCAG-3') and 518R reverse primer (5') containing a V1-V3 hypervariable region that specifically binds to bacteria for amplification of the 16S rRNA gene of bacteria in the isolated DNA. '-WTTACCGCGGCTGCTGG-3') was initially denatured at 94°C for 5 minutes, repeated 30 times at 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 1 minute and 30 seconds. After performing amplification PCR, QIAquick gel Purified through an extraction kit (Qiagen, Germany). The PCR product was pyrosequencing using a sequencing analyzer of GS Junior Titanium System (Roche, Germany), and the methods and reactions required for pyrosequencing were carried out by Chunlab Co., Ltd. according to the manual of the manufacturer (Roche). , Korea).

실시예Example 7: 노령 마우스에서 7: in old mice 비환원성Non-reducing 말단의 Terminal 공액이중결합형형Conjugated double bond type 만뉴론산Manneuronic acid 올리고당 투여 실험실시 Oligosaccharide administration laboratory

실험 종료 후 실험동물의 내장 내용물을 수집 후 200 mg을 취하여 실시예 6에 기재된 방법대로 DNA를 분리하여 파이로시퀀싱을 실시하였다.After the experiment was completed, 200 mg of the internal contents of the experimental animals were collected, and DNA was separated according to the method described in Example 6 to perform pyro-sequencing.

도 11 및 12에 제시한 바와 같이, 비환원성 말단의 공액이중결합형형 만뉴론산 올리고당을 음용 섭취한 노령쥐의 경우 1개월령의 쥐의 장내 균총과 유사하며, 비환원성 말단의 공액이중결합형형 만뉴론산 올리고당을 음용 섭취하지 않은 17개월령의 쥐의 장내 균총과는 차이가 있음을 확인하였다. 비환원성 말단의 공액이중결합형 만뉴론산 올리고당을 투여한 쥐의 장내 균총의 경우, 노년기에 증가하는 대표 균총인 그람양성세균(Firmicutes)인 클로스트리디움(Clostridium) 속 균총과 락토바실러스 뉴테리(Lactobacillus reuteri)가 비환원성 말단의 공액이중결합형 만뉴론산 올리고당 비음용구(대조군) 대비 낮은 비율로 존재함을 보였고, 프레보텔라(Prevotella) 종, 락토바실러스 인테스티날리스(Lactobacillus intestinalis) 등의 유익균의 증가를 확인하였다.As shown in Figs. It was confirmed that there was a difference from the intestinal flora of 17-month-old rats that did not consume oligosaccharide. In the case of the intestinal flora of mice administered with the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing end, the colony of the genus Clostridium , which is a representative colony of Gram-positive bacteria, which increases in old age, and Lactobacillus reuteri ) was shown to exist in a lower ratio compared to non-drinking devices (control) of the conjugated double bonded manneuronic acid oligosaccharide at the non-reducing terminal, and beneficial bacteria such as Prevotella species, Lactobacillus intestinalis, etc. Confirmed the increase.

공지된 연구에 따르면, 노화가 진행됨에 따라 장내 균총의 불균형이 야기되고 이때 장내 미생물은 골 관련 치주염, 관절염, 골다공증을 야기하며, 장내 미생물이 숙주의 골량을 조절하고, 이는 파골세포가 조절하는 골 흡수에 영향을 줄 수 있다고 알려져 있다.According to known studies, as aging progresses, imbalance of the intestinal flora is caused, and at this time, the intestinal microbes cause bone-related periodontitis, arthritis, and osteoporosis, and the intestinal microbes regulate the bone mass of the host, which is the bone controlled by osteoclasts. It is known that it can affect absorption.

따라서 상기 결과를 토대로 본 발명자들은 본 발명의 골다공증 치료용 조성물이 골을 생성하는 조골세포의 활성을 촉진시켜주며 파골세포의 활성을 억제시키는 자연노화에 따른 골다공증 치료제로 활용 될 수 있는 조성물을 신규 발명하였다.Therefore, based on the above results, the present inventors newly invented a composition that can be used as a therapeutic agent for osteoporosis due to natural aging that promotes the activity of osteoblasts that produce bone and inhibits the activity of osteoclasts. I did.

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.As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for Treating Osteoporosis by Chronological Aging <130> PN160225D <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1569 <212> DNA <213> Unknown <220> <223> Aly3 <400> 1 atgaaacaaa ttactctaaa aactttactc gcttcttcta ttctacttgc ggttggttgt 60 gcgagcacga gcacgcctac tgctgatttt ccaaataaca aagaaactgg tgaagcgctt 120 ctgacgccag ttgctgtttc cgctagtagc catgatggta acggacctga tcgtctcgtt 180 gaccaagacc taactacacg ttggtcatct gcgggtgacg gcgagtgggc aacgctagac 240 tatggttcag tacaagagtt tgacgcggtt caggcatctt tcagtaaagg taatcagcgc 300 caatctaaat ttgatatcca agtgagtgtt gatggcgaaa gctggacaac ggtactagaa 360 aaccaactaa gctcaggtaa agcgatcggc ctagagcgtt tccaatttga gccagcagtg 420 caagcacgct tcgtaagata cgttggtcac ggtaacacca aaaacggttg gaacagtgtg 480 actggattag cggcggttaa ctgtagcatc aacgcatgtc ctgctagcca tatcatcact 540 tcagacgtgg tagcagcaga agccgtgatt attgctgaaa tgaaagcggc agaaaaagca 600 cgtaaagatg cgcgcaaaga tctacgctct ggtaacttcg gtgtagcagc ggtttaccct 660 tgtgagacga ccgttgaatg cgacacacgc agtgcactgc cagttcctac aggcctgcca 720 gcgacaccag ttgctggtaa cgcaccgagc gaaaactttg acatgacgca ttggtaccta 780 tctcaaccat ttgaccatga caaaaatggc aaacctgatg acgtttctga gtggaatctt 840 gcaaacggtt accaacaccc tgaaatcttc tacacagctg atgacggcgg cctagtattc 900 aaagcttacg tgaaaggtgt acgtacatct aaaaacacta agtacgcgcg tacagagctt 960 cgtgaaatga tgcgtcgtgg tgatcagtct attagcacta aaggtgttaa caagaataac 1020 tgggtattct caagcgctcc tgaatctgac ttagagtcgg cagcgggtat tgacggcgtt 1080 ctagaagcga cgttgaaaat cgaccatgca acaacgacgg gtaatgcgaa tgaagtaggt 1140 cgctttatca ttggtcagat tcacgatcaa aacgatgaac caattcgttt gtactaccgt 1200 aaactgccaa accaagaaac gggtgctgtt tacttcgcac atgaaagcca agacgcaact 1260 aaagaggact tctaccctct agtgggcgac atgacggctg aagtgggtga cgatggtatc 1320 gcgcttggcg aagtgttcag ctaccgtatt gacgttaaag gcaacacgat gactgtaacg 1380 ctaatgcgtg aaggcaaaga cgatgttgta caagtggttg atatgagcaa cagcggctac 1440 gacgcaggcg gcaagtacat gtacttcaag gccggtgttt acaaccaaaa catcagcggc 1500 gacctagacg attactcaca agcgactttc taccagctag atgtatcgca cgatcaatac 1560 caaaagtaa 1569 <210> 2 <211> 522 <212> PRT <213> Unknown <220> <223> Aly3 <400> 2 Met Lys Gln Ile Thr Leu Lys Thr Leu Leu Ala Ser Ser Ile Leu Leu 1 5 10 15 Ala Val Gly Cys Ala Ser Thr Ser Thr Pro Thr Ala Asp Phe Pro Asn 20 25 30 Asn Lys Glu Thr Gly Glu Ala Leu Leu Thr Pro Val Ala Val Ser Ala 35 40 45 Ser Ser His Asp Gly Asn Gly Pro Asp Arg Leu Val Asp Gln Asp Leu 50 55 60 Thr Thr Arg Trp Ser Ser Ala Gly Asp Gly Glu Trp Ala Thr Leu Asp 65 70 75 80 Tyr Gly Ser Val Gln Glu Phe Asp Ala Val Gln Ala Ser Phe Ser Lys 85 90 95 Gly Asn Gln Arg Gln Ser Lys Phe Asp Ile Gln Val Ser Val Asp Gly 100 105 110 Glu Ser Trp Thr Thr Val Leu Glu Asn Gln Leu Ser Ser Gly Lys Ala 115 120 125 Ile Gly Leu Glu Arg Phe Gln Phe Glu Pro Ala Val Gln Ala Arg Phe 130 135 140 Val Arg Tyr Val Gly His Gly Asn Thr Lys Asn Gly Trp Asn Ser Val 145 150 155 160 Thr Gly Leu Ala Ala Val Asn Cys Ser Ile Asn Ala Cys Pro Ala Ser 165 170 175 His Ile Ile Thr Ser Asp Val Val Ala Ala Glu Ala Val Ile Ile Ala 180 185 190 Glu Met Lys Ala Ala Glu Lys Ala Arg Lys Asp Ala Arg Lys Asp Leu 195 200 205 Arg Ser Gly Asn Phe Gly Val Ala Ala Val Tyr Pro Cys Glu Thr Thr 210 215 220 Val Glu Cys Asp Thr Arg Ser Ala Leu Pro Val Pro Thr Gly Leu Pro 225 230 235 240 Ala Thr Pro Val Ala Gly Asn Ala Pro Ser Glu Asn Phe Asp Met Thr 245 250 255 His Trp Tyr Leu Ser Gln Pro Phe Asp His Asp Lys Asn Gly Lys Pro 260 265 270 Asp Asp Val Ser Glu Trp Asn Leu Ala Asn Gly Tyr Gln His Pro Glu 275 280 285 Ile Phe Tyr Thr Ala Asp Asp Gly Gly Leu Val Phe Lys Ala Tyr Val 290 295 300 Lys Gly Val Arg Thr Ser Lys Asn Thr Lys Tyr Ala Arg Thr Glu Leu 305 310 315 320 Arg Glu Met Met Arg Arg Gly Asp Gln Ser Ile Ser Thr Lys Gly Val 325 330 335 Asn Lys Asn Asn Trp Val Phe Ser Ser Ala Pro Glu Ser Asp Leu Glu 340 345 350 Ser Ala Ala Gly Ile Asp Gly Val Leu Glu Ala Thr Leu Lys Ile Asp 355 360 365 His Ala Thr Thr Thr Gly Asn Ala Asn Glu Val Gly Arg Phe Ile Ile 370 375 380 Gly Gln Ile His Asp Gln Asn Asp Glu Pro Ile Arg Leu Tyr Tyr Arg 385 390 395 400 Lys Leu Pro Asn Gln Glu Thr Gly Ala Val Tyr Phe Ala His Glu Ser 405 410 415 Gln Asp Ala Thr Lys Glu Asp Phe Tyr Pro Leu Val Gly Asp Met Thr 420 425 430 Ala Glu Val Gly Asp Asp Gly Ile Ala Leu Gly Glu Val Phe Ser Tyr 435 440 445 Arg Ile Asp Val Lys Gly Asn Thr Met Thr Val Thr Leu Met Arg Glu 450 455 460 Gly Lys Asp Asp Val Val Gln Val Val Asp Met Ser Asn Ser Gly Tyr 465 470 475 480 Asp Ala Gly Gly Lys Tyr Met Tyr Phe Lys Ala Gly Val Tyr Asn Gln 485 490 495 Asn Ile Ser Gly Asp Leu Asp Asp Tyr Ser Gln Ala Thr Phe Tyr Gln 500 505 510 Leu Asp Val Ser His Asp Gln Tyr Gln Lys 515 520 <210> 3 <211> 2154 <212> DNA <213> Unknown <220> <223> Aly5 <400> 3 atgagctatc aaccactttt acttaacttt gatgaagcag ctgaacttcg taaagaactt 60 ggcaaggata gccttttagg taacgcactg actcgcgata ttaaacaaac tgatgcttac 120 atggcggaag ttggcattga agtaccaggt cacggtgaag gcggcggtta cgagcacaac 180 cgtcataagc aaaactacat ccatatggat ctagcgggcc gtttgttcct tatcactgag 240 gaaacaaaat accgtgacta tatcgttgat atgctaactg cttacgcgac ggtataccca 300 acacttgaaa gcaacgtaag ccgtgactct aaccctccag gtaagctgtt ccaccaaacg 360 ttgaacgaga acatgtggat gctttacgct tcttgtgcgt acagctgtat ttaccacacc 420 atttctgaag agcaaaaacg tctgatcgaa gacgatcttc ttaagcaaat gatcgaaatg 480 ttcgttgtga cttacgcaca cgacttcgat atcgtgcaca accatggcct ttgggcagtg 540 gctgcagtag gtatctgtgg ttacgcaatc aacgatcaag agtctgtaga caaagcgctt 600 tacggcctga aactagacaa agtaagcggc ggtttcttag cgcaacttga ccaactgttc 660 tcgccagatg gctactacat ggaaggtcct tactaccacc gtttctcgct acgtccaatc 720 tacctgtttg cagaggcgat tgaacgtcgt cagcctgaag ttggtatcta tgaattcaac 780 gattcagtga tcaagacaac gtcttactct gtattcaaaa cggcattccc agacggtaca 840 ttgccagctc tgaacgattc atcgaagaca atttctatca acgatgaagg cgttatcatg 900 gcaacgtctg tgtgttacca ccgttacgag cagactgaaa ctctgcttgg tatggctaac 960 caccaacaaa acgtttgggt tcatgcttca ggtaaaacat tgtctgacgc ggttgattca 1020 gcagacgaca tcaaagcatt caactggggt agcctgtttg taaccgacgg ccctgaaggc 1080 gaaaaaggcg gcgtaagcat ccttcgtcac cgtgacgatc aagatgacga cacgatggcg 1140 ttgatctggt ttggtcaaca cggttctgat caccagtacc actctgctct agaccacggt 1200 cactacgatg gcctgcacct aagcgtattc aaccgtggcc acgaagtgct gcacgattac 1260 ggcttcggtc gctgggtaaa cgttgagcct aagtttggcg gtcgttacat cccagagaac 1320 aagtcttact gtaagcagac ggttgctcac aacacggtaa cggttgatca gaaaacgcag 1380 aacaacttca acacagcatt ggctgagtct aagtttggtc agaagcactt cttcgtagca 1440 gacgaccagt ctctacaagg catgagcggc acaatttctg agtactacac aggcgtagac 1500 atgcaacgca gcgtgattct tgctgaactt cctgagttcg aaaagccact tgttatcgac 1560 gtataccgca tcgaagctga cactgaacac cagtacgacc tacccgttca ccactctggt 1620 cagatcatcc gtactgactt cgattacaac atggaaaaaa cgcttaagcc gctaggtgaa 1680 gacaacggtt accagcactt atggaacgtg gcttcaggca aagtgaacga agaaggttct 1740 ctagtaagct ggctacatga cagcagctac tacagcctag taaccagcgc gaatgcgggc 1800 agcgaagtga tttttgctcg cactggtgct aacgatccag acttcaacct taagagtgag 1860 cctgcgttca tcttacgtca gtctggtcaa aaccacgtgt ttgcttctgt actagaaacg 1920 catggttact ttaacgagtc tatcgaagcc tctgtaggcg ctcgtggtct agttaaatca 1980 gtatctgttg tgggccataa cagtgtcggg actgttgttc gcattcagac tacttctggc 2040 aacacttacc actacggtat ctcaaaccaa gctgaagaca cgcagcaagc aactcacact 2100 gttgagttcg cgggtgagac atactcgtgg gaaggatcat ttgctcaact gtaa 2154 <210> 4 <211> 717 <212> PRT <213> Unknown <220> <223> Aly5 <400> 4 Met Ser Tyr Gln Pro Leu Leu Leu Asn Phe Asp Glu Ala Ala Glu Leu 1 5 10 15 Arg Lys Glu Leu Gly Lys Asp Ser Leu Leu Gly Asn Ala Leu Thr Arg 20 25 30 Asp Ile Lys Gln Thr Asp Ala Tyr Met Ala Glu Val Gly Ile Glu Val 35 40 45 Pro Gly His Gly Glu Gly Gly Gly Tyr Glu His Asn Arg His Lys Gln 50 55 60 Asn Tyr Ile His Met Asp Leu Ala Gly Arg Leu Phe Leu Ile Thr Glu 65 70 75 80 Glu Thr Lys Tyr Arg Asp Tyr Ile Val Asp Met Leu Thr Ala Tyr Ala 85 90 95 Thr Val Tyr Pro Thr Leu Glu Ser Asn Val Ser Arg Asp Ser Asn Pro 100 105 110 Pro Gly Lys Leu Phe His Gln Thr Leu Asn Glu Asn Met Trp Met Leu 115 120 125 Tyr Ala Ser Cys Ala Tyr Ser Cys Ile Tyr His Thr Ile Ser Glu Glu 130 135 140 Gln Lys Arg Leu Ile Glu Asp Asp Leu Leu Lys Gln Met Ile Glu Met 145 150 155 160 Phe Val Val Thr Tyr Ala His Asp Phe Asp Ile Val His Asn His Gly 165 170 175 Leu Trp Ala Val Ala Ala Val Gly Ile Cys Gly Tyr Ala Ile Asn Asp 180 185 190 Gln Glu Ser Val Asp Lys Ala Leu Tyr Gly Leu Lys Leu Asp Lys Val 195 200 205 Ser Gly Gly Phe Leu Ala Gln Leu Asp Gln Leu Phe Ser Pro Asp Gly 210 215 220 Tyr Tyr Met Glu Gly Pro Tyr Tyr His Arg Phe Ser Leu Arg Pro Ile 225 230 235 240 Tyr Leu Phe Ala Glu Ala Ile Glu Arg Arg Gln Pro Glu Val Gly Ile 245 250 255 Tyr Glu Phe Asn Asp Ser Val Ile Lys Thr Thr Ser Tyr Ser Val Phe 260 265 270 Lys Thr Ala Phe Pro Asp Gly Thr Leu Pro Ala Leu Asn Asp Ser Ser 275 280 285 Lys Thr Ile Ser Ile Asn Asp Glu Gly Val Ile Met Ala Thr Ser Val 290 295 300 Cys Tyr His Arg Tyr Glu Gln Thr Glu Thr Leu Leu Gly Met Ala Asn 305 310 315 320 His Gln Gln Asn Val Trp Val His Ala Ser Gly Lys Thr Leu Ser Asp 325 330 335 Ala Val Asp Ser Ala Asp Asp Ile Lys Ala Phe Asn Trp Gly Ser Leu 340 345 350 Phe Val Thr Asp Gly Pro Glu Gly Glu Lys Gly Gly Val Ser Ile Leu 355 360 365 Arg His Arg Asp Asp Gln Asp Asp Asp Thr Met Ala Leu Ile Trp Phe 370 375 380 Gly Gln His Gly Ser Asp His Gln Tyr His Ser Ala Leu Asp His Gly 385 390 395 400 His Tyr Asp Gly Leu His Leu Ser Val Phe Asn Arg Gly His Glu Val 405 410 415 Leu His Asp Tyr Gly Phe Gly Arg Trp Val Asn Val Glu Pro Lys Phe 420 425 430 Gly Gly Arg Tyr Ile Pro Glu Asn Lys Ser Tyr Cys Lys Gln Thr Val 435 440 445 Ala His Asn Thr Val Thr Val Asp Gln Lys Thr Gln Asn Asn Phe Asn 450 455 460 Thr Ala Leu Ala Glu Ser Lys Phe Gly Gln Lys His Phe Phe Val Ala 465 470 475 480 Asp Asp Gln Ser Leu Gln Gly Met Ser Gly Thr Ile Ser Glu Tyr Tyr 485 490 495 Thr Gly Val Asp Met Gln Arg Ser Val Ile Leu Ala Glu Leu Pro Glu 500 505 510 Phe Glu Lys Pro Leu Val Ile Asp Val Tyr Arg Ile Glu Ala Asp Thr 515 520 525 Glu His Gln Tyr Asp Leu Pro Val His His Ser Gly Gln Ile Ile Arg 530 535 540 Thr Asp Phe Asp Tyr Asn Met Glu Lys Thr Leu Lys Pro Leu Gly Glu 545 550 555 560 Asp Asn Gly Tyr Gln His Leu Trp Asn Val Ala Ser Gly Lys Val Asn 565 570 575 Glu Glu Gly Ser Leu Val Ser Trp Leu His Asp Ser Ser Tyr Tyr Ser 580 585 590 Leu Val Thr Ser Ala Asn Ala Gly Ser Glu Val Ile Phe Ala Arg Thr 595 600 605 Gly Ala Asn Asp Pro Asp Phe Asn Leu Lys Ser Glu Pro Ala Phe Ile 610 615 620 Leu Arg Gln Ser Gly Gln Asn His Val Phe Ala Ser Val Leu Glu Thr 625 630 635 640 His Gly Tyr Phe Asn Glu Ser Ile Glu Ala Ser Val Gly Ala Arg Gly 645 650 655 Leu Val Lys Ser Val Ser Val Val Gly His Asn Ser Val Gly Thr Val 660 665 670 Val Arg Ile Gln Thr Thr Ser Gly Asn Thr Tyr His Tyr Gly Ile Ser 675 680 685 Asn Gln Ala Glu Asp Thr Gln Gln Ala Thr His Thr Val Glu Phe Ala 690 695 700 Gly Glu Thr Tyr Ser Trp Glu Gly Ser Phe Ala Gln Leu 705 710 715 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for Treating Osteoporosis by Chronological Aging <130> PN160225D <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1569 <212> DNA <213> Unknown <220> <223> Aly3 <400> 1 atgaaacaaa ttactctaaa aactttactc gcttcttcta ttctacttgc ggttggttgt 60 gcgagcacga gcacgcctac tgctgatttt ccaaataaca aagaaactgg tgaagcgctt 120 ctgacgccag ttgctgtttc cgctagtagc catgatggta acggacctga tcgtctcgtt 180 gaccaagacc taactacacg ttggtcatct gcgggtgacg gcgagtgggc aacgctagac 240 tatggttcag tacaagagtt tgacgcggtt caggcatctt tcagtaaagg taatcagcgc 300 caatctaaat ttgatatcca agtgagtgtt gatggcgaaa gctggacaac ggtactagaa 360 aaccaactaa gctcaggtaa agcgatcggc ctagagcgtt tccaatttga gccagcagtg 420 caagcacgct tcgtaagata cgttggtcac ggtaacacca aaaacggttg gaacagtgtg 480 actggattag cggcggttaa ctgtagcatc aacgcatgtc ctgctagcca tatcatcact 540 tcagacgtgg tagcagcaga agccgtgatt attgctgaaa tgaaagcggc agaaaaagca 600 cgtaaagatg cgcgcaaaga tctacgctct ggtaacttcg gtgtagcagc ggtttaccct 660 tgtgagacga ccgttgaatg cgacacacgc agtgcactgc cagttcctac aggcctgcca 720 gcgacaccag ttgctggtaa cgcaccgagc gaaaactttg acatgacgca ttggtaccta 780 tctcaaccat ttgaccatga caaaaatggc aaacctgatg acgtttctga gtggaatctt 840 gcaaacggtt accaacaccc tgaaatcttc tacacagctg atgacggcgg cctagtattc 900 aaagcttacg tgaaaggtgt acgtacatct aaaaacacta agtacgcgcg tacagagctt 960 cgtgaaatga tgcgtcgtgg tgatcagtct attagcacta aaggtgttaa caagaataac 1020 tgggtattct caagcgctcc tgaatctgac ttagagtcgg cagcgggtat tgacggcgtt 1080 ctagaagcga cgttgaaaat cgaccatgca acaacgacgg gtaatgcgaa tgaagtaggt 1140 cgctttatca ttggtcagat tcacgatcaa aacgatgaac caattcgttt gtactaccgt 1200 aaactgccaa accaagaaac gggtgctgtt tacttcgcac atgaaagcca agacgcaact 1260 aaagaggact tctaccctct agtgggcgac atgacggctg aagtgggtga cgatggtatc 1320 gcgcttggcg aagtgttcag ctaccgtatt gacgttaaag gcaacacgat gactgtaacg 1380 ctaatgcgtg aaggcaaaga cgatgttgta caagtggttg atatgagcaa cagcggctac 1440 gacgcaggcg gcaagtacat gtacttcaag gccggtgttt acaaccaaaa catcagcggc 1500 gacctagacg attactcaca agcgactttc taccagctag atgtatcgca cgatcaatac 1560 caaaagtaa 1569 <210> 2 <211> 522 <212> PRT <213> Unknown <220> <223> Aly3 <400> 2 Met Lys Gln Ile Thr Leu Lys Thr Leu Leu Ala Ser Ser Ile Leu Leu 1 5 10 15 Ala Val Gly Cys Ala Ser Thr Ser Thr Pro Thr Ala Asp Phe Pro Asn 20 25 30 Asn Lys Glu Thr Gly Glu Ala Leu Leu Thr Pro Val Ala Val Ser Ala 35 40 45 Ser Ser His Asp Gly Asn Gly Pro Asp Arg Leu Val Asp Gln Asp Leu 50 55 60 Thr Thr Arg Trp Ser Ser Ala Gly Asp Gly Glu Trp Ala Thr Leu Asp 65 70 75 80 Tyr Gly Ser Val Gln Glu Phe Asp Ala Val Gln Ala Ser Phe Ser Lys 85 90 95 Gly Asn Gln Arg Gln Ser Lys Phe Asp Ile Gln Val Ser Val Asp Gly 100 105 110 Glu Ser Trp Thr Thr Val Leu Glu Asn Gln Leu Ser Ser Gly Lys Ala 115 120 125 Ile Gly Leu Glu Arg Phe Gln Phe Glu Pro Ala Val Gln Ala Arg Phe 130 135 140 Val Arg Tyr Val Gly His Gly Asn Thr Lys Asn Gly Trp Asn Ser Val 145 150 155 160 Thr Gly Leu Ala Ala Val Asn Cys Ser Ile Asn Ala Cys Pro Ala Ser 165 170 175 His Ile Ile Thr Ser Asp Val Val Ala Ala Glu Ala Val Ile Ile Ala 180 185 190 Glu Met Lys Ala Ala Glu Lys Ala Arg Lys Asp Ala Arg Lys Asp Leu 195 200 205 Arg Ser Gly Asn Phe Gly Val Ala Ala Val Tyr Pro Cys Glu Thr Thr 210 215 220 Val Glu Cys Asp Thr Arg Ser Ala Leu Pro Val Pro Thr Gly Leu Pro 225 230 235 240 Ala Thr Pro Val Ala Gly Asn Ala Pro Ser Glu Asn Phe Asp Met Thr 245 250 255 His Trp Tyr Leu Ser Gln Pro Phe Asp His Asp Lys Asn Gly Lys Pro 260 265 270 Asp Asp Val Ser Glu Trp Asn Leu Ala Asn Gly Tyr Gln His Pro Glu 275 280 285 Ile Phe Tyr Thr Ala Asp Asp Gly Gly Leu Val Phe Lys Ala Tyr Val 290 295 300 Lys Gly Val Arg Thr Ser Lys Asn Thr Lys Tyr Ala Arg Thr Glu Leu 305 310 315 320 Arg Glu Met Met Arg Arg Gly Asp Gln Ser Ile Ser Thr Lys Gly Val 325 330 335 Asn Lys Asn Asn Trp Val Phe Ser Ser Ala Pro Glu Ser Asp Leu Glu 340 345 350 Ser Ala Ala Gly Ile Asp Gly Val Leu Glu Ala Thr Leu Lys Ile Asp 355 360 365 His Ala Thr Thr Thr Gly Asn Ala Asn Glu Val Gly Arg Phe Ile Ile 370 375 380 Gly Gln Ile His Asp Gln Asn Asp Glu Pro Ile Arg Leu Tyr Tyr Arg 385 390 395 400 Lys Leu Pro Asn Gln Glu Thr Gly Ala Val Tyr Phe Ala His Glu Ser 405 410 415 Gln Asp Ala Thr Lys Glu Asp Phe Tyr Pro Leu Val Gly Asp Met Thr 420 425 430 Ala Glu Val Gly Asp Asp Gly Ile Ala Leu Gly Glu Val Phe Ser Tyr 435 440 445 Arg Ile Asp Val Lys Gly Asn Thr Met Thr Val Thr Leu Met Arg Glu 450 455 460 Gly Lys Asp Asp Val Val Gln Val Val Asp Met Ser Asn Ser Gly Tyr 465 470 475 480 Asp Ala Gly Gly Lys Tyr Met Tyr Phe Lys Ala Gly Val Tyr Asn Gln 485 490 495 Asn Ile Ser Gly Asp Leu Asp Asp Tyr Ser Gln Ala Thr Phe Tyr Gln 500 505 510 Leu Asp Val Ser His Asp Gln Tyr Gln Lys 515 520 <210> 3 <211> 2154 <212> DNA <213> Unknown <220> <223> Aly5 <400> 3 atgagctatc aaccactttt acttaacttt gatgaagcag ctgaacttcg taaagaactt 60 ggcaaggata gccttttagg taacgcactg actcgcgata ttaaacaaac tgatgcttac 120 atggcggaag ttggcattga agtaccaggt cacggtgaag gcggcggtta cgagcacaac 180 cgtcataagc aaaactacat ccatatggat ctagcgggcc gtttgttcct tatcactgag 240 gaaacaaaat accgtgacta tatcgttgat atgctaactg cttacgcgac ggtataccca 300 acacttgaaa gcaacgtaag ccgtgactct aaccctccag gtaagctgtt ccaccaaacg 360 ttgaacgaga acatgtggat gctttacgct tcttgtgcgt acagctgtat ttaccacacc 420 atttctgaag agcaaaaacg tctgatcgaa gacgatcttc ttaagcaaat gatcgaaatg 480 ttcgttgtga cttacgcaca cgacttcgat atcgtgcaca accatggcct ttgggcagtg 540 gctgcagtag gtatctgtgg ttacgcaatc aacgatcaag agtctgtaga caaagcgctt 600 tacggcctga aactagacaa agtaagcggc ggtttcttag cgcaacttga ccaactgttc 660 tcgccagatg gctactacat ggaaggtcct tactaccacc gtttctcgct acgtccaatc 720 tacctgtttg cagaggcgat tgaacgtcgt cagcctgaag ttggtatcta tgaattcaac 780 gattcagtga tcaagacaac gtcttactct gtattcaaaa cggcattccc agacggtaca 840 ttgccagctc tgaacgattc atcgaagaca atttctatca acgatgaagg cgttatcatg 900 gcaacgtctg tgtgttacca ccgttacgag cagactgaaa ctctgcttgg tatggctaac 960 caccaacaaa acgtttgggt tcatgcttca ggtaaaacat tgtctgacgc ggttgattca 1020 gcagacgaca tcaaagcatt caactggggt agcctgtttg taaccgacgg ccctgaaggc 1080 gaaaaaggcg gcgtaagcat ccttcgtcac cgtgacgatc aagatgacga cacgatggcg 1140 ttgatctggt ttggtcaaca cggttctgat caccagtacc actctgctct agaccacggt 1200 cactacgatg gcctgcacct aagcgtattc aaccgtggcc acgaagtgct gcacgattac 1260 ggcttcggtc gctgggtaaa cgttgagcct aagtttggcg gtcgttacat cccagagaac 1320 aagtcttact gtaagcagac ggttgctcac aacacggtaa cggttgatca gaaaacgcag 1380 aacaacttca acacagcatt ggctgagtct aagtttggtc agaagcactt cttcgtagca 1440 gacgaccagt ctctacaagg catgagcggc acaatttctg agtactacac aggcgtagac 1500 atgcaacgca gcgtgattct tgctgaactt cctgagttcg aaaagccact tgttatcgac 1560 gtataccgca tcgaagctga cactgaacac cagtacgacc tacccgttca ccactctggt 1620 cagatcatcc gtactgactt cgattacaac atggaaaaaa cgcttaagcc gctaggtgaa 1680 gacaacggtt accagcactt atggaacgtg gcttcaggca aagtgaacga agaaggttct 1740 ctagtaagct ggctacatga cagcagctac tacagcctag taaccagcgc gaatgcgggc 1800 agcgaagtga tttttgctcg cactggtgct aacgatccag acttcaacct taagagtgag 1860 cctgcgttca tcttacgtca gtctggtcaa aaccacgtgt ttgcttctgt actagaaacg 1920 catggttact ttaacgagtc tatcgaagcc tctgtaggcg ctcgtggtct agttaaatca 1980 gtatctgttg tgggccataa cagtgtcggg actgttgttc gcattcagac tacttctggc 2040 aacacttacc actacggtat ctcaaaccaa gctgaagaca cgcagcaagc aactcacact 2100 gttgagttcg cgggtgagac atactcgtgg gaaggatcat ttgctcaact gtaa 2154 <210> 4 <211> 717 <212> PRT <213> Unknown <220> <223> Aly5 <400> 4 Met Ser Tyr Gln Pro Leu Leu Leu Asn Phe Asp Glu Ala Ala Glu Leu 1 5 10 15 Arg Lys Glu Leu Gly Lys Asp Ser Leu Leu Gly Asn Ala Leu Thr Arg 20 25 30 Asp Ile Lys Gln Thr Asp Ala Tyr Met Ala Glu Val Gly Ile Glu Val 35 40 45 Pro Gly His Gly Glu Gly Gly Gly Tyr Glu His Asn Arg His Lys Gln 50 55 60 Asn Tyr Ile His Met Asp Leu Ala Gly Arg Leu Phe Leu Ile Thr Glu 65 70 75 80 Glu Thr Lys Tyr Arg Asp Tyr Ile Val Asp Met Leu Thr Ala Tyr Ala 85 90 95 Thr Val Tyr Pro Thr Leu Glu Ser Asn Val Ser Arg Asp Ser Asn Pro 100 105 110 Pro Gly Lys Leu Phe His Gln Thr Leu Asn Glu Asn Met Trp Met Leu 115 120 125 Tyr Ala Ser Cys Ala Tyr Ser Cys Ile Tyr His Thr Ile Ser Glu Glu 130 135 140 Gln Lys Arg Leu Ile Glu Asp Asp Leu Leu Lys Gln Met Ile Glu Met 145 150 155 160 Phe Val Val Thr Tyr Ala His Asp Phe Asp Ile Val His Asn His Gly 165 170 175 Leu Trp Ala Val Ala Ala Val Gly Ile Cys Gly Tyr Ala Ile Asn Asp 180 185 190 Gln Glu Ser Val Asp Lys Ala Leu Tyr Gly Leu Lys Leu Asp Lys Val 195 200 205 Ser Gly Gly Phe Leu Ala Gln Leu Asp Gln Leu Phe Ser Pro Asp Gly 210 215 220 Tyr Tyr Met Glu Gly Pro Tyr Tyr His Arg Phe Ser Leu Arg Pro Ile 225 230 235 240 Tyr Leu Phe Ala Glu Ala Ile Glu Arg Arg Gln Pro Glu Val Gly Ile 245 250 255 Tyr Glu Phe Asn Asp Ser Val Ile Lys Thr Thr Ser Tyr Ser Val Phe 260 265 270 Lys Thr Ala Phe Pro Asp Gly Thr Leu Pro Ala Leu Asn Asp Ser Ser 275 280 285 Lys Thr Ile Ser Ile Asn Asp Glu Gly Val Ile Met Ala Thr Ser Val 290 295 300 Cys Tyr His Arg Tyr Glu Gln Thr Glu Thr Leu Leu Gly Met Ala Asn 305 310 315 320 His Gln Gln Asn Val Trp Val His Ala Ser Gly Lys Thr Leu Ser Asp 325 330 335 Ala Val Asp Ser Ala Asp Asp Ile Lys Ala Phe Asn Trp Gly Ser Leu 340 345 350 Phe Val Thr Asp Gly Pro Glu Gly Glu Lys Gly Gly Val Ser Ile Leu 355 360 365 Arg His Arg Asp Asp Gln Asp Asp Asp Thr Met Ala Leu Ile Trp Phe 370 375 380 Gly Gln His Gly Ser Asp His Gln Tyr His Ser Ala Leu Asp His Gly 385 390 395 400 His Tyr Asp Gly Leu His Leu Ser Val Phe Asn Arg Gly His Glu Val 405 410 415 Leu His Asp Tyr Gly Phe Gly Arg Trp Val Asn Val Glu Pro Lys Phe 420 425 430 Gly Gly Arg Tyr Ile Pro Glu Asn Lys Ser Tyr Cys Lys Gln Thr Val 435 440 445 Ala His Asn Thr Val Thr Val Asp Gln Lys Thr Gln Asn Asn Phe Asn 450 455 460 Thr Ala Leu Ala Glu Ser Lys Phe Gly Gln Lys His Phe Phe Val Ala 465 470 475 480 Asp Asp Gln Ser Leu Gln Gly Met Ser Gly Thr Ile Ser Glu Tyr Tyr 485 490 495 Thr Gly Val Asp Met Gln Arg Ser Val Ile Leu Ala Glu Leu Pro Glu 500 505 510 Phe Glu Lys Pro Leu Val Ile Asp Val Tyr Arg Ile Glu Ala Asp Thr 515 520 525 Glu His Gln Tyr Asp Leu Pro Val His His Ser Gly Gln Ile Ile Arg 530 535 540 Thr Asp Phe Asp Tyr Asn Met Glu Lys Thr Leu Lys Pro Leu Gly Glu 545 550 555 560 Asp Asn Gly Tyr Gln His Leu Trp Asn Val Ala Ser Gly Lys Val Asn 565 570 575 Glu Glu Gly Ser Leu Val Ser Trp Leu His Asp Ser Ser Tyr Tyr Ser 580 585 590 Leu Val Thr Ser Ala Asn Ala Gly Ser Glu Val Ile Phe Ala Arg Thr 595 600 605 Gly Ala Asn Asp Pro Asp Phe Asn Leu Lys Ser Glu Pro Ala Phe Ile 610 615 620 Leu Arg Gln Ser Gly Gln Asn His Val Phe Ala Ser Val Leu Glu Thr 625 630 635 640 His Gly Tyr Phe Asn Glu Ser Ile Glu Ala Ser Val Gly Ala Arg Gly 645 650 655 Leu Val Lys Ser Val Ser Val Val Gly His Asn Ser Val Gly Thr Val 660 665 670 Val Arg Ile Gln Thr Thr Ser Gly Asn Thr Tyr His Tyr Gly Ile Ser 675 680 685 Asn Gln Ala Glu Asp Thr Gln Gln Ala Thr His Thr Val Glu Phe Ala 690 695 700 Gly Glu Thr Tyr Ser Trp Glu Gly Ser Phe Ala Gln Leu 705 710 715

Claims (9)

알긴산 올리고당(alginate oligosaccharide)을 유효성분으로 포함하는 골다공증 개선, 예방 또는 치료용 약제학적 조성물.
A pharmaceutical composition for improving, preventing or treating osteoporosis comprising alginate oligosaccharide as an active ingredient.
제 1 항에 있어서, 상기 알긴산 올리고당은 분자량(molecular weight)은 100 내지 5000 Da 인 것을 특징으로 하는 조성물.
The composition of claim 1, wherein the alginate oligosaccharide has a molecular weight of 100 to 5000 Da.
제 1 항에 있어서, 상기 알긴산 올리고당은 만뉴론산:글루론산 비율이 1.2-5.0:1 몰비인 것을 특징으로 하는 조성물.
The composition of claim 1, wherein the alginate oligosaccharide has a mannuronic acid: glucuronic acid ratio of 1.2-5.0: 1.
제 1 항에 있어서, 상기 알긴산 올리고당은 1 내지 10 개의 단당(monosaccharide)으로 구성된 것을 특징으로 하는 조성물.
The composition of claim 1, wherein the alginate oligosaccharide is comprised of from 1 to 10 monosaccharides.
제 1 항에 있어서, 상기 골다공증은 노인성 골다공증인 것을 특징으로 하는 조성물.
The composition according to claim 1, wherein the osteoporosis is senile osteoporosis.
제 1 항에 있어서, 상기 조성물은 골밀도를 증가시키는 것을 특징으로 하는 조성물.
2. The composition of claim 1, wherein the composition increases bone mineral density.
제 1 항에 있어서, 상기 조성물은 장내 균총을 개선시키는 것을 특징으로 하는 조성물.
2. The composition of claim 1, wherein the composition improves intestinal flora.
알긴산 올리고당(alginate oligosaccharide)을 유효성분으로 포함하는 골다공증 개선 또는 예방용 식품 조성물.
A food composition for improving or preventing osteoporosis comprising alginate oligosaccharide as an active ingredient.
알긴산 올리고당(alginate oligosaccharide)을 유효성분으로 포함하는 골다공증 개선 또는 예방용 건강기능식품 조성물.A composition for improving or preventing osteoporosis comprising alginate oligosaccharide as an active ingredient.
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