KR20030046810A - Pharmaceutical composition for prevention or treatment of arthritis containing degraded chondroitin sulfate - Google Patents
Pharmaceutical composition for prevention or treatment of arthritis containing degraded chondroitin sulfate Download PDFInfo
- Publication number
- KR20030046810A KR20030046810A KR1020010077071A KR20010077071A KR20030046810A KR 20030046810 A KR20030046810 A KR 20030046810A KR 1020010077071 A KR1020010077071 A KR 1020010077071A KR 20010077071 A KR20010077071 A KR 20010077071A KR 20030046810 A KR20030046810 A KR 20030046810A
- Authority
- KR
- South Korea
- Prior art keywords
- chondroitin sulfate
- pharmaceutical composition
- arthritis
- chondroitin
- preventing
- Prior art date
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Classifications
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- 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
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
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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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- 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
- A23V2250/50—Polysaccharides, gums
- A23V2250/51—Polysaccharide
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Rheumatology (AREA)
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- Dermatology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
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Abstract
Description
본 발명은 콘드로이친 황산의 분해산물을 포함하는 관절염의 예방 또는 치료용 약학적 조성물에 관한 것으로써, 보다 상세하게는 콘드로이친 황산을 효소적 또는 화학적으로 분해하여 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 제조하는 방법, 상기 콘드로이친 황산의 분해산물을 유효성분으로 포함하는 관절염의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of arthritis comprising a degradation product of chondroitin sulfate, and more particularly, to a method for preparing chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides by enzymatic or chemical degradation of chondroitin sulfate. It relates to a pharmaceutical composition for the prevention or treatment of arthritis, comprising the decomposition product of the chondroitin sulfate as an active ingredient.
글리코사미노글리칸(Glycosaminoglycan, 이하 "GAG" 라고 약칭함)은 생합성 과정에서 부분적인 변화를 통해 구조적으로 다양하고 복잡한 형태를 지니게 된다. GAG류의 기본적인 골격은 우론산(uronic acid)과 글루코사민(glucosamine)의 반복이면서 부분적으로 설페이트화가 이루어져 있는데, 이러한 복잡한 화학 구조에 의해 여러 종류의 생리 활성을 나타낼 수 있게 된다.Glycosaminoglycans (abbreviated as "GAG") are structurally diverse and complex through partial changes in the biosynthesis process. The basic backbone of GAGs is the repetition and partial sulphation of uronic acid and glucosamine, and these complex chemical structures enable various types of physiological activity.
동물의 GAG류로는 히알우론산(hyaluronic acid), 콘드로이친 황산(chondroitin sulfate), 케라틴 황산(keratin sulfate), 헤파린 황산 (heparan sulfate), 헤파린(heparin) 등이 알려져 있다. 이들 GAG류는 주로 연결조직에 분포하며 헤파린은 예외적으로 간, 허파, 피부 점막내의 동맥에 있는 마스트(mast) 세포 내의 과립에 존재한다.Animal GAGs include hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, heparin, and the like. These GAGs are mainly distributed in connective tissues and heparin is exceptionally present in granules in mast cells in arteries in the liver, lungs, and skin mucosa.
의약품으로 널리 사용되는 글리코사미노글리칸으로는 헤파린이 있으며, 헤파린은 항응고제로서 혈전 치료제, 수술후, 신장투석, 인공혈관 등에 혈전 생성의 예방 및 치료 목적으로 광범위하게 사용되고 있고 최근에 들어서는 부작용을 낮춘 저분자량의 헤파린이 동일한 목적으로 사용되고 있다. 또한, 콘드로이친 황산B인 더마탄 황산(dermatan sulfate)은 항혈전제로 임상 실험중에 있고, 히알우론산은 화장품의 첨가물로 많이 쓰이고 있다.Heparin is a widely used glycosaminoglycan used as a medicine. Heparin is an anticoagulant. It is widely used for the prevention and treatment of thrombosis in thrombosis, postoperative, renal dialysis, and artificial blood vessels. Heparin of molecular weight is used for the same purpose. In addition, dermatan sulfate, a chondroitin sulfate B, is in clinical trials as an antithrombotic agent, and hyaluronic acid is widely used as an additive in cosmetics.
콘드로이친은 크게 세 종류로서, 황산기의 위치와 우론산의 종류에 따라서 콘드로이친 황산 A, 콘드로이친 황산 B, 콘드로이친 황산 C로 분류하고 있다. 콘드로이친 황산 A와 C는N-아세틸-D-갈락토사민과 D-글루쿠론산이 1,3결합과 1,4결합이 번갈아가며 연결된 2당(糖)류의 중합체이며, 2당당(糖當) 한 개의 황산 에스테르기를 갖는다. 콘드로이친 황산 B는 D-글루쿠론산 대신에 L-이두론산을 함유하고 있으며, 이들을 β-헤파린 또는 더마탄 황산이라고도 한다.Chondroitin is largely classified into three types, depending on the position of the sulfate group and the type of uronic acid, which are classified into chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C. Chondroitin sulfates A and C are disaccharides polymers in which N -acetyl-D-galactosamine and D-glucuronic acid are alternately linked with 1,3 and 1,4 bonds. ) Has one sulfuric acid ester group. Chondroitin sulfate B contains L-iduronic acid instead of D-glucuronic acid, which is also referred to as β-heparin or dematane sulfuric acid.
콘드로이친 황산은 연골, 피부, 혈관 및 뼈 등 많은 결합 조직에 존재한다. 콘드로이친 황산은 특히 관절의 연골 조직의 주요 구성 성분이며, 관절 조직의 기능과 탄성에서 중요한 역할을 한다. 정상 관절 연골에서 콘드로이친 황산은 글루코사민으로부터 연골세포나 활막세포에 의해 생산되고, 일단 이러한 물질이 결합 조직 기질로 유리되면 프로테오글라이칸 중합체로 결합된다. 이러한 콘드로이친 프로테오글라이칸으로서 대표적인 종류가 아그리칸(aggrecan)이다. 아그리칸은 관절 연골에서 고농도로 존재하며, 연골 조직의 특정적인 물리·화학적 성질을 부여하게 된다. 이 아그리칸을 이루는 콘드로이친 황산의 정도와 그 구성에서의 변화가 골관절염이나 류마티스성 관절염 같은 관절 질환의 개시와 진행에 중요한 역할을 하는 것으로 알려져 있다(Poole A.R. et al.,J. Clin. Invest., 1994, 94; 25-33). 또한, 중합체의 정전기적 특징과 이것들의 제 2형 콜라겐과의 연관성은 탄성과 점도와 같은 결합 조직의 특징을 결정짓는 중요한 요소이다.Chondroitin sulfate is present in many connective tissues such as cartilage, skin, blood vessels and bones. Chondroitin sulfate is a major component of joint cartilage tissue in particular and plays an important role in the function and elasticity of joint tissue. Chondroitin sulfate in normal articular cartilage is produced from glucosamine by chondrocytes or synovial cells, and once these substances are released into the connective tissue matrix, they are bound to proteoglycan polymers. A typical type of such chondroitin proteoglycans is aggrecan. Agricans are present at high levels in articular cartilage and confer specific physical and chemical properties of cartilage tissue. Changes in the degree and composition of chondroitin sulfate that make up this agrican are known to play an important role in the initiation and progression of joint diseases such as osteoarthritis and rheumatoid arthritis (Poole AR et al., J. Clin. Invest . , 1994, 94; 25-33). In addition, the electrostatic properties of polymers and their association with type 2 collagen are important factors that determine the characteristics of connective tissues such as elasticity and viscosity.
만성 염증 질환인 류마티스성 관절염은 윤활 조직과 윤활액으로 면역계 경쟁성(immuno-competent) 세포의 침윤, 윤활액의 섬유모세포의 증식, 관절 연골과 연골 하 조직에 침윤하여 파괴하는 판누스(pannus) 조직의 형성 등을 특징으로 하고 있다. 그 병인은 아직 확실하지 않지만, 염증 반응이 기본이 되는 여러 세포나 분자 레벨의 기전과, 관절 연골과 연골하 조직의 세포 외 매트릭스의 분해와 관련이 있다는 것이 점점 더 명확해지고 있다. 류마티스성 관절염에 대한 많은 보고가 염증 반응과 관절 손상의 매개체가 될 지도 모르는 분자의 발견에 초점이 맞추어지고 있다. 이러한 분자 중 하나가 종양괴사인자(tumor necrosis factor α, TNF-α)이다. 이 염증성 사이토카인은 연골 분해와 뼈의 재흡수를 촉진하고, 콜라게나제(collagenase)나 아그리카나제(aggrecanases)를 포함한 많은 단백 분해 효소의 유리와 활성화를 유도하게 된다. 따라서 궁극적으로 이러한 TNF-α와 같은 분자들의 생성과 활성화는 관절의 통합성과 기능의 만성적 파괴로 이어진다 (Kumar S. et al.,Currrent Opinion in Pharmacology, 2001, 1; 307-313). 이러한 보고들은 류마티스성 관절염에서 TNF-α의 중요성을 입증하고 있으며, 이러한 사이토카인을 중성화할 수 있는 생물학적 제제의 개발에도 노력을 기울이고 있다.Rheumatoid arthritis, a chronic inflammatory disease, involves the infiltration of immune-competent cells with lubricating tissue and lubricating fluid, the proliferation of fibroblasts in the lubricating fluid, the infiltration and destruction of pannus tissue infiltrating articular cartilage and subchondral tissue. Formation and the like. The etiology is not yet clear, but it is becoming increasingly clear that the inflammatory response is associated with the mechanisms at the cellular or molecular level and the breakdown of the extracellular matrix of articular cartilage and subchondral tissue. Many reports of rheumatoid arthritis have focused on the discovery of molecules that may be mediators of inflammatory responses and joint damage. One such molecule is tumor necrosis factor α (TNF-α). This inflammatory cytokine promotes cartilage breakdown and bone resorption and induces the release and activation of many proteolytic enzymes, including collagenase and aggrecanases. Ultimately, the production and activation of these TNF-α-like molecules leads to chronic destruction of joint integrity and function (Kumar S. et al., Currrent Opinion in Pharmacology , 2001, 1; 307-313). These reports demonstrate the importance of TNF-α in rheumatoid arthritis and are working on the development of biological agents that can neutralize these cytokines.
류마티스성 관절염은 골관절염과는 매우 다른 질병이다. 골관절염은 연골조직의 마모로서 생기는 질환이며, 류마티스 관절염은 자가면역 질환의 일종이다. 즉, 백혈구가 과잉으로 생성되어 관절에 대하여 상해를 주는 것이다. 일반적으로 류마티스성 관절염은 골관절염에 비해서 급격히 일어나며 관절이 변형되어 기능이 장해를 받게되며, 통증이 심하며 경우에 따라서는 관절을 바꾸어야 할 필요도 있다.Rheumatoid arthritis is a very different disease from osteoarthritis. Osteoarthritis is a disease caused by abrasion of cartilage tissue, and rheumatoid arthritis is a kind of autoimmune disease. That is, white blood cells are excessively generated to injure the joints. In general, rheumatoid arthritis occurs more rapidly than osteoarthritis, and the joint is deformed and the function is impaired, the pain is severe, and in some cases it may be necessary to change the joint.
콘드로이친 황산/글루코사민이 골관절염에만 주로 효능이 있는 것처럼 알려져 있지만 최근의 연구 결과에 따르면 콘드로이친 황산/글루코사민을 미리 10일전에 투여한 랫트에 가용성 콜라겐 II를 주사하여 관절염을 유발 시켰을 경우 염증성 관절염의 빈도가 54%만이 관절염이 발생하였다. 이에 반해 비처리군 중 96.5%가 유발되었다. 이러한 결과는 콘드로이친 황산과 글루코사민의 혼합물이 류마티스성 관절염의 예방 및 치료에도 가능성이 있다는 것을 제시한다(Beren J. et al.,Proceedings of the Society for Experimental Biology and Medicine, 2001, 226:144-151).Although chondroitin sulfate / glucosamine is known to be primarily effective in osteoarthritis, recent studies have shown that the frequency of inflammatory arthritis is increased when soluble collagen II is injected into rats treated with chondroitin sulfate / glucosamine 10 days beforehand. Only% developed arthritis. In contrast, 96.5% of the untreated groups were induced. These results suggest that a mixture of chondroitin sulfate and glucosamine may also be useful in the prevention and treatment of rheumatoid arthritis (Beren J. et al., Proceedings of the Society for Experimental Biology and Medicine , 2001, 226: 144-151). .
관절은 연골 조직으로 구성되어 있으며 혈관이 없기 때문에 새로이 생성이 되지 않는다. 따라서 연골 조직의 주성분인 GAG류를 보충해 주어야만 한다. 그러나, 콘드로이친 황산의 평균 분자량이 15,000이상이므로 투여한 콘드로이친 황산의약 70%가 효소에 의해서 대사를 받아 N-아세틸갈락토사민, 글루쿠론산, 황산 이온으로 분해되어 흡수되는데, 이러한 과정을 거쳐 약 8%가 연골조직에 도달하는 것으로 알려져 있다(Palmieri L. et al.,Arzneim.-Forsh./Drug Res, 1990, 40:319-323).Joints are composed of cartilage tissue and do not have new blood vessels. Therefore, GAGs, the main component of cartilage tissue, must be supplemented. However, since the average molecular weight of chondroitin sulfate is more than 15,000, about 70% of chondroitin sulfate administered is metabolized by an enzyme and decomposed into N-acetylgalactosamine, glucuronic acid, and sulfate ions. % Is known to reach cartilage tissue (Palmieri L. et al., Arzneim.-Forsh./Drug Res , 1990, 40: 319-323).
그러나, 본 발명자들은 큰 분자량과 극성을 지닌 콘드로이친 황산이 경구로 투여될 경우에 흡수가 어렵다는 것을 소장세포(Caco-2 세포주)를 통해서 확인하였으며, 이에 반하여 효소로 처리하여 분자량을 감소시킨 콘드로이친 이당류와 화학적으로 분해한 콘드로이친 올리고당은 소장세포를 통하여 흡수가 증가하는 것을 확인할 수 있었다.However, the present inventors have confirmed through the small intestine cells (Caco-2 cell line) that the absorption of chondroitin sulfate having a large molecular weight and polarity orally when administered orally, on the contrary, with the chondroitin disaccharides reduced molecular weight by treatment with enzyme Chemically degraded chondroitin oligosaccharides were found to increase absorption through small intestine cells.
이에, 본 발명자들은 콘드로이친 황산을 올리고당 또는 저분자량으로 제조한 후 구강으로 투여하는 경우 소장세포를 통해 흡수율이 좋아지는 것을 알고, 콘드로이친 황산을 분해하고 그 분해산물인 콘드로이친 황산 이당류와 올리고당이 관절염 유발시 생기는 부종이나 발적을 감소시키고, 혈청 중 항 콜라겐 항체의 수치를 낮추거나 류마티스성 관절염에 결정적인 역할을 하는 사이토카인인 TNF-α의 수치를 매우 효과적으로 낮춤으로써 류마티스성 관절염의 예방 및 치료에 유용하게 사용될 수 있음을 밝힘으로써 본 발명을 완성하였다.Thus, the present inventors know that when the chondroitin sulfate is produced in oligosaccharide or low molecular weight and then administered orally, the absorption rate is improved through the small intestine cells, and the chondroitin sulfate disaccharides and oligosaccharides, which are the degradation products of chondroitin sulfate, are generated when arthritis is induced. It can be useful for the prevention and treatment of rheumatoid arthritis by reducing edema and redness, lowering the level of anti-collagen antibodies in serum or lowering the level of TNF-α, a cytokine that plays a decisive role in rheumatoid arthritis. The present invention has been completed by revealing that there is.
본 발명의 목적은 관절염 예방 또는 치료에 유용한 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition useful for preventing or treating arthritis.
도 1은 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당이 HLE의 활성에 미치는 영향을 확인한 그래프이고, 1 is a graph confirming the effects of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides of the active ingredient of the present invention on the activity of HLE,
대조군 : 반응 용액(0.05M 트리스 완충용액,pH 8.0)만 처리,Control group: treated only with reaction solution (0.05M Tris buffer, pH 8.0),
CS 이당류 : 콘드로이친 황산 이당류(50 ㎎/㎏)로 처리,CS disaccharide: treated with chondroitin sulfate disaccharide (50 mg / kg),
CS 올리고당 : 콘드로이친 황산 올리고당 (300 ㎎/㎏)으로 처리,CS oligosaccharide: treated with chondroitin sulfate oligosaccharide (300 mg / kg),
CS : 콘드로이친 황산 (1,200 ㎎/㎏)으로 처리,CS: treated with chondroitin sulfate (1,200 mg / kg),
도 2는 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당이 부종과 발적에 미치는 영향을 확인하기 위하여 뒷발 두께를 측정한 그래프이고, Figure 2 is a graph measuring the thickness of the hind paws to determine the effect of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides active edema and redness of the pharmaceutical composition of the present invention,
● : 관절염 대조군, ▼ : 음성 대조군,●: arthritis control, ▼: negative control,
■ : 콘드로이친 황산 이당류(50 ㎎/㎏),■: Chondroitin sulfate disaccharide (50 mg / kg),
◆ : 콘드로이친 황산올리고당 (300 ㎎/㎏),◆: Chondroitin sulfate oligosaccharide (300 ㎎ / ㎏),
▲ : 콘드로이친 황산 (1,200 ㎎/㎏),▲: chondroitin sulfate (1,200 mg / kg),
도 3은 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당이 항 콜라겐 항체의 생산에 미치는 영향을 측정한 그래프이고, Figure 3 is a graph measuring the effect of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides, the active ingredient of the pharmaceutical composition of the present invention on the production of anti-collagen antibodies,
대조군 : 생리 식염수로 처리,Control group: treated with saline solution,
CS 이당류 : 콘드로이친 황산 이당류(50 ㎎/㎏)로 처리,CS disaccharide: treated with chondroitin sulfate disaccharide (50 mg / kg),
CS 올리고당 : 콘드로이친 황산 올리고당 (300 ㎎/㎏)으로 처리,CS oligosaccharide: treated with chondroitin sulfate oligosaccharide (300 mg / kg),
CS : 콘드로이친 황산 (1,200 ㎎/㎏)으로 처리,CS: treated with chondroitin sulfate (1,200 mg / kg),
도 4는 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당이 사이토카인인 TNF-α에 미치는 영향을 측정한 그래프이고, Figure 4 is a graph measuring the effect of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides of the active ingredient of the present invention on the cytokine TNF-α,
대조군 : 생리 식염수로 처리,Control group: treated with saline solution,
CS 이당류 : 콘드로이친 황산 이당류(50 ㎎/㎏)로 처리,CS disaccharide: treated with chondroitin sulfate disaccharide (50 mg / kg),
CS 올리고당 : 콘드로이친 황산 올리고당 (300 ㎎/㎏)으로 처리,CS oligosaccharide: treated with chondroitin sulfate oligosaccharide (300 mg / kg),
CS : 콘드로이친 황산 (1,200 ㎎/㎏)으로 처리,CS: treated with chondroitin sulfate (1,200 mg / kg),
도 5는 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당의 관절염 개선 효과를 조직학적 소견으로 확인한 사진이고, 5 is a photograph confirming the arthritis-improving effect of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides as an active ingredient of the pharmaceutical composition of the present invention,
도 6은 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 및 콘드로이친 황산 올리고당이 Caco-2 세포를 통해서 흡수되어지는 양을 측정한 그래프이다. Figure 6 is a graph measuring the amount of chondroitin sulfate disaccharides and chondroitin sulfate oligosaccharides of the active ingredient of the present invention is absorbed through Caco-2 cells.
상기 목적을 달성하기 위하여, 본 발명은 콘드로이친 황산의 분해산물인 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당의 제조방법을 제공한다.In order to achieve the above object, the present invention provides a method for producing chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides that are decomposition products of chondroitin sulfate.
또한, 본 발명은 상기 콘드로이친 황산의 분해산물을 포함하는 관절염의 예방 또는 치료용 약학적 조성물 또는 기능성 식품을 제공한다.In another aspect, the present invention provides a pharmaceutical composition or functional food for the prevention or treatment of arthritis, including the decomposition product of chondroitin sulfate.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 콘드로이친 황산의 분해산물인 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당의 제조방법을 제공한다.The present invention provides a method for preparing chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides, which are decomposition products of chondroitin sulfate.
본 발명에서는 콘드로이친 황산을 효소적 또는 화학적으로 분해하여 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 제조하였다.In the present invention, chondroitin sulfate was enzymatically or chemically decomposed to prepare chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides.
본 발명에서 콘드로이친 황산을 분해하여 콘드로이친 황산 이당류를 제조하기 위하여 바람직한 실시예로 박테로이드 스테르코리스(Bacteroides stercoris) HJ15 균주로부터 정제한 콘드로이티나제 AC(Ahn et al.,Canadian J. Microbiol. 1998:423-429)를 사용하였다.Chondroitin AC (Ahn et al., Canadian J. Microbiol . 1998) purified from Bacteroides stercoris HJ15 strain as a preferred embodiment for the preparation of chondroitin sulfate disaccharides by digesting chondroitin sulfate in the present invention. : 423-429).
본 발명의 콘드로이친 황산 이당류를 제조하는 방법은The method for producing the chondroitin sulfate disaccharide of the present invention
(1) 콘드로이친 황산에 콘드로이티나제 AC를 첨가하는 단계;(1) adding chondroitin AC to chondroitin sulfate;
(2) 상기 단계 1의 반응액을 동결 건조시킨 후 정제하는 단계로 구성된다.(2) freeze-drying the reaction solution of step 1 and then purified.
또한, 본 발명에서 콘드로이친 황산을 화학적으로 분해하여 콘드로이친 황산 올리고당을 제조하였다. 대표적인 화학적인 방법은 산이나, 강염기로 분해시킬 수 있으나 황산기 등의 이탈도 나타나므로 좋은 방법이 아니다. 이에 반하여, 구리 등의 중금속을 촉매로 하여 과산화수소를 이용해 OH-라디칼을 만들어 자유기에 의해서 유래된 해중합 반응은 비교적 반응이 온화한 조건에 수행이 가능하고 반응시간을 조절할 수 있다(Volpi et al.,Analytical Biochemistry1992,200:100-107).In the present invention, chondroitin sulfate was chemically decomposed to prepare chondroitin sulfate oligosaccharides. Representative chemical methods can be decomposed into acids or strong bases, but the release of sulfuric acid groups, etc. is not a good method. On the contrary, depolymerization reactions derived from free radicals by using OH - radicals using hydrogen peroxide as catalysts of copper and the like can be performed under relatively mild conditions and control reaction time (Volpi et al., Analytical) . Biochemistry 1992, 200: 100-107).
본 발명의 콘드로이친 황산 올리고당을 제조하는 방법은The method for producing chondroitin sulfate oligosaccharide of the present invention
(1) 콘드로이친 황산을 녹인 후 구리 아세테이트 모노하이드레이트(Copper acetate monohydrate)를 첨가하여 교반하는 단계,(1) dissolving chondroitin sulfuric acid and then stirring by adding copper acetate monohydrate;
(2) 상기 단계 1의 반응액을 중성 pH로 유지시키며 과산화수소를 첨가하는 단계,(2) maintaining the reaction solution of step 1 at neutral pH and adding hydrogen peroxide,
(3) 상기 단계 2의 반응액에서 구리 이온을 제거하고, 낮은 온도 조건에서 분획을 분리하는 단계; 및(3) removing copper ions from the reaction solution of step 2 and separating fractions at low temperature; And
(4) 상기 단계 3에서 선택한 분획의 시료를 동결건조하고, 이를 정제하는 단계로 구성된다.(4) lyophilizing the sample of the fraction selected in step 3 and purifying it.
상기 콘드로이친 황산을 화학적으로 분해하여 얻어진 콘드로이친 황산 올리고당의 평균 분자량은 5,000 Da이다.The average molecular weight of chondroitin sulfate oligosaccharide obtained by chemically decomposing the chondroitin sulfate is 5,000 Da.
또한, 본 발명은 상기 콘드로이친 황산의 분해산물을 포함하는 관절염의 예방 또는 치료용 약학적 조성물 또는 기능성 식품을 제공한다.In another aspect, the present invention provides a pharmaceutical composition or functional food for the prevention or treatment of arthritis, including the decomposition product of chondroitin sulfate.
본 발명은 콘드로이친 황산이 효소적 또는 화학적 분해산물인 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 유효성분으로 포함하는 관절염의 예방 또는 치료용 약학적 조성물 또는 기능성 식품을 제공한다.The present invention provides a pharmaceutical composition or functional food for the prevention or treatment of arthritis, wherein chondroitin sulfate includes an enzymatic or chemical decomposition product of chondroitin sulfate disaccharide or chondroitin sulfate oligosaccharide as an active ingredient.
본 발명의 약학적 조성물 또는 기능석 식품은 류마티스 관절염 또는 골관절염에 모두 효과적이며, 특히 류마티스성 관절염에 더욱 효과적이다.The pharmaceutical composition or functional stone food of the present invention is effective for both rheumatoid arthritis or osteoarthritis, and more particularly for rheumatoid arthritis.
인간 백혈구 엘라스타제(Human Leukocyte Elastase; 이하 'HLE'라 칭한다)는 세린 프로테아제(serine protease)로 결합조직의 구성성분인 엘라스틴과 콜라젠을 포함한 여러 기질을 분해할 수 있다. HLE의 이러한 분해능으로 인하여 HLE는 류마티스성 관절염, 폐기종(pulmonary emphysema), 낭성섬유증(cystic fibrosis), 만성기관지염(chronic bronchitis), 급성호흡기병(acute respiratory distress syndrome) 및 사구체신염(glomerulonephritis)과 같은 질병과 밀접한 관계가 있다. 따라서 HLE 특이적 저해제는 류마티스성 관절염이나 낭성섬유증과 같은 염증성 질병의 치료제로 효과적이다.Human Leukocyte Elastase (hereinafter referred to as 'HLE') is a serine protease that can break down several substrates, including elastin and collagen, which are components of connective tissue. Due to this resolution of HLE, HLE causes diseases such as rheumatoid arthritis, pulmonary emphysema, cystic fibrosis, chronic bronchitis, acute respiratory distress syndrome, and glomerulonephritis. It is closely related to. Therefore, HLE-specific inhibitors are effective for the treatment of inflammatory diseases such as rheumatoid arthritis and cystic fibrosis.
본 발명자들은 상기 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당이 HLE의 활성에 미치는 영향을 살펴본 결과, 분해하지 않은 콘드로이친 황산의 경우에는 생리 식염수를 처리한 대조군과 차이를 보이지 않고 HLE 억제 활성을 거의 보이지 않으나, 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당은 HLE 활성이 감소하는 효과를 나타낸다(도 1참조).The present inventors have examined the effects of the chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides on the activity of HLE, and in the case of undecomposed chondroitin sulfate, there was little difference from the control treated with physiological saline and showed little HLE inhibitory activity. Sulfuric acid disaccharides or chondroitin sulfate oligosaccharides show an effect of decreasing HLE activity (see FIG. 1 ).
본 발명자들은 콘드로이친 황산 이당류 및 올리고당이 관절염에 미치는 효과를 알아보기 위해 콜라겐으로 유발된 류마티스성 관절염 동물 모델을 사용하여, 콘드로이친 황산과 비교하여 그 효과를 보면, 정상 대조군은 콘드로이친 황산을 투여한 그룹과 그 부종과 발적을 비교했을 때 차이가 나타나지 않고, 육안적 소견 역시 거의 비슷한 양상을 나타낸다. 반면에 콘드로이친 황산 이당류나 올리고당을 각각 투여한 그룹에서는 부종과 발적이 감소하고, 뒷발 두께도 감소한다(도 2참조).The present inventors, using collagen-induced rheumatoid arthritis animal model to determine the effect of chondroitin sulfate disaccharides and oligosaccharides on arthritis, compared to chondroitin sulfate, the normal control group and the group administered chondroitin sulfate There is no difference when comparing the edema and redness, and the gross findings are almost identical. On the other hand, in the group administered with chondroitin sulfate disaccharide or oligosaccharide, respectively, edema and redness decreased, and hind foot thickness also decreased (see FIG. 2 ).
또한, 항 콜라겐 항체의 생성에 미치는 영향을 보면 정상 관절염 대조군과 콘드로이친 황산이나 콘드로이친 황산 이당류를 투여한 그룹에서는 그 차이가 없으나, 콘드로이친 황산 올리고당을 투여한 그룹에서는 항 콜라겐 항체의 생성이 감소한다(도 3참조).In addition, the effects on the production of anti-collagen antibodies were not different between the normal arthritis control group and the group administered with chondroitin sulfate or chondroitin sulfate disaccharide, but the production of anti-collagen antibody decreased in the group administered with chondroitin sulfate oligosaccharide ( FIG. 3 ).
또한, 혈청 중 사이토카인의 농도는 콘드로이친 황산의 경우에 정상 대조군과 차이가 나타나지 않으나, 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 투여한 두 그룹에서는 각각 혈중 TNF-α의 농도가 감소한다(도 4참조).In addition, the concentration of cytokines in serum did not differ from the normal control in the case of chondroitin sulfate, but the concentration of TNF-α in the blood decreased in the two groups administered chondroitin sulfate disaccharide or chondroitin sulfate oligosaccharide, respectively (see FIG. 4 ). .
정상 대조군과 비교하여 관절염 유발 그룹은 관절 조직의 구조에 큰 변화가 관찰된다. 콘드로이친 황산을 투여한 그룹은 종전의 보고에서와는 달리, 판누스(pannus) 생성이나 그 중증도 면에서 관절염 유발 그룹과 별 차이를 보이지 않고, 유발된 관절염의 개선 효과를 찾아볼 수 없다. 그러나 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 투여한 그룹에서는 관절염의 개선 효과를 육안으로 확인할 수 있고, 특히 콘드로이친 황산 올리고당을 투약한 그룹은 정상 대조군과 거의 비슷한 양상을 나타낸다(도 5참조).Compared to the normal control group, arthritis-induced group showed a big change in the structure of the joint tissue. Unlike the previous report, the group receiving chondroitin sulfate did not show any difference in the arthritis-inducing group in terms of pannus production or its severity, and there was no improvement in the induced arthritis. However, in the group administered with chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides, the effect of improving arthritis can be visually observed, and in particular, the group administered with chondroitin sulfate oligosaccharides shows a similar pattern to the normal control group (see FIG. 5 ).
본 발명의 약학적 조성물은 경구 및 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다.The pharmaceutical compositions of the present invention can be administered orally and parenterally and can be used in the form of general pharmaceutical preparations.
즉, 본 발명의 약학적 조성물은 실제로 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.In other words, the pharmaceutical composition of the present invention can be administered in various parenteral formulations, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. do. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
또한, 본 발명의 약학적 조성물은 생리식염수 또는 유기용매와 같이 약제로 허용된 여러 전달체(carrier)와 혼합하여 사용될 수 있고, 안정성이나 흡수성을 증가시키기 위하여 글루코스, 수크로스 또는 덱스트란과 같은 탄수화물, 아스코르브 산(ascorbic acid) 또는 글루타치온과 같은 항산화제(antioxidants), 킬레이팅 물질(chelating agents), 저분자 단백질 또는 다른 안정화제(stabilizers)들이 약제로 사용될 수 있다.In addition, the pharmaceutical composition of the present invention can be used in admixture with a variety of carriers (pharmaceutically acceptable carrier) such as physiological saline or organic solvents, carbohydrates such as glucose, sucrose or dextran, in order to increase the stability or absorption Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.
본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 또는 올리고당의 유효용량은 주사제의 경우는 10 - 50 ㎎/㎏, 경구 투여제의 경우는 100 - 500 ㎎/㎏이고 하루 1∼2회 투여될 수 있다.The effective dose of chondroitin sulfate disaccharide or oligosaccharide as an active ingredient of the pharmaceutical composition of the present invention is 10-50 mg / kg for injection, 100-500 mg / kg for oral administration and is administered 1-2 times a day. Can be.
본 발명의 약학적 조성물에서 콘드로이친 황산 이당류 및 올리고당의 총 유효량은 거환(bolus) 형태 혹은 상대적으로 짧은 기간 동안 확산(infusion) 등에 의해 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)이 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 상기 약학적 조성물의 농도는 약의 투여 경로 및 치료 횟수 뿐 만 아니라 환자의 나이 및 건강상태 등 다양한 요인들을 고려하여 환자의 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 이 분야의 통상적인 지식을 가진 자라면 상기 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 유효성분으로 포함하는 약학적 조성물로서의 특정한 용도에 따른 적절한 유효 투여량을 결정할 수 있을 것이다.The total effective amount of chondroitin sulfate disaccharide and oligosaccharide in the pharmaceutical composition of the present invention may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration of the pharmaceutical composition is determined by taking into consideration not only the route of administration and the frequency of treatment of the drug, but also various factors such as the age and health of the patient, the general knowledge of the art is considered in this regard. Those who have will be able to determine the appropriate effective dosage for a particular use as a pharmaceutical composition comprising the chondroitin sulfate disaccharide or chondroitin sulfate oligosaccharide as an active ingredient.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당 제조Example 1 Preparation of Chondroitin Sulfate Disaccharide or Chondroitin Sulfate Oligosaccharide
<1-1> 콘드로이친 황산 이당류 제조<1-1> Chondroitin Sulfate Disaccharide Preparation
본 발명자들은 콘드로이친 황산으로부터 이당류를 제조하기 위하여 하기와 같이 콘드로이티나제 AC를 이용하여 콘드로이친 황산을 분해하여 본 발명의 콘드로이친 황산 이당류를 제조하였다.The present inventors prepared the chondroitin sulfate disaccharide of the present invention by decomposing chondroitin sulfate using chondroitin AC to produce disaccharides from chondroitin sulfate.
박테로이드 스테르코리스(Bacteroides stercoris) HJ15 균주로부터 콘드로이티나제 AC를 QAE-셀룰로스(QAE-celluose), DEAE-셀룰로스, CM-세파덱스(CM-Sephadex), 하이드록시아파티트(Hydroxyapatite), 히트랩(Hitrap)-헤파린, 히트랩 SP 컬럼 크로마토그래피를 이용하여 정제하여 콘드로이티나제 AC를 얻었다. 상기 정제시에 수율은 3.96%이고 비활성도는 48.91 μmole/min/㎎이며, 초기의 효소 정제도에 비교하여 520배 증가하였다.Chondroitinase AC from the Bacteroides stercoris HJ15 strain was subjected to QAE-celluose, DEAE-cellulose, CM-Sephadex, hydroxyapatite, Purification was performed using Hittrap-heparin, Heatlab SP column chromatography to obtain chondroitinase AC. Yield was 3.96% and specific activity was 48.91 μmole / min / mg at the time of purification, an increase of 520 times compared to the initial enzyme purification.
콘드로이친 황산 2 g을 13 ㎖의 50 mM 인산완충액(pH 7.0)에서 녹여서 상기에서 콘드로이티나제 AC 2 유니트를 첨가하여 48시간 반응시켰다. 반응액을 동결 건조시킨 후 젤 여과 크로마토그래피로 분리 정제하여 본 발명의 콘드로이친 황산 이당류를 제조하였다.2 g of chondroitin sulfate was dissolved in 13 ml of 50 mM phosphate buffer (pH 7.0) and reacted for 48 hours by adding chondroitin AC 2 units. The reaction solution was freeze-dried and separated and purified by gel filtration chromatography to prepare the chondroitin sulfate disaccharide of the present invention.
<1-2> 콘드로이친 황산 올리고당 제조<1-2> chondroitin sulfate oligosaccharide preparation
본 발명자들은 콘드로이친 황산 올리고당을 제조하기 위하여, 콘드로이친 황산을 하기와 같이 화학적으로 분해하였다.To prepare chondroitin sulfate oligosaccharides, the present inventors chemically decomposed chondroitin sulfate as follows.
콘드로이친 황산 1 g과 초산 나트륨(sodium acetate) 0.5 g을 37℃의 온도에서 증류수로 10 ㎖이 되도록 녹였다. 완전히 녹으면 구리 아세테이트 모노하이드레이트(Copper acetate monohydrate)를 0.46 g 넣어준 후, 50℃로 온도를 계속 유지시키면서 교반하였다. 1 M의 수산화 나트륨을 적당량 첨가하여, 반응물의 pH를 7.5로 일정하게 유지시켜주면서, 9%의 과산화수소를 15분 간격으로 250 ㎕씩 첨가해 주었다. 상기 조건 하에서 6시간 반응을 시킨 후, 키렉스(Chelex) 100 양이온 교환 컬럼을 통과시켜 구리 이온을 제거하였고, 낮은 pH를 가진 분해 산물의 안정성을 위해 낮은 온도 조건에서 분획을 분리하였다. 분획을 받는 동안에는 1 M의 수산화 나트륨을 분해 산물에 첨가하여 pH를 5.9로 일정하게 유지하게 하였다. 이렇게 해서 얻은 시료의 일부는 TSK-G3000SW GPC-HPLC에 의해서 분자량을 동정하는데 사용하였고, 나머지 시료는 동결 건조하였다.1 g of chondroitin sulfate and 0.5 g of sodium acetate were dissolved to 10 ml with distilled water at a temperature of 37 ° C. When completely dissolved, 0.46 g of copper acetate monohydrate was added thereto, followed by stirring while maintaining the temperature at 50 ° C. An appropriate amount of 1 M sodium hydroxide was added to keep the pH of the reaction constant at 7.5, while 250 μl of 9% hydrogen peroxide was added at 15 minute intervals. After reacting for 6 hours under the above conditions, copper ions were removed by passing through a Chelex 100 cation exchange column, and fractions were separated at low temperature conditions for stability of decomposition products having low pH. During the fractionation, 1 M sodium hydroxide was added to the degradation product to keep the pH constant at 5.9. Some of the samples thus obtained were used to identify molecular weight by TSK-G3000SW GPC-HPLC, and the rest of the samples were lyophilized.
동결 건조된 시료는 소량의 증류수에 다시 녹인 후 세파덱스 G-10 혹은 바이오-젤(Bio-Gel) P-2 컬럼을 이용한 겔 크로마토그래피에 의해 염을 제거하여 정제하였다. 상기 과정을 통해 콘드로이친 황산의 올리고당을 얻을 수 있었다. 이러한 실험 방법으로 통하여 최종적인 콘드로이친 올리고당의 수율은 70.4%이었다.The freeze-dried sample was re-dissolved in a small amount of distilled water and purified by removing the salt by gel chromatography using a Sephadex G-10 or Bio-Gel P-2 column. Through the above procedure, oligosaccharides of chondroitin sulfate were obtained. Through this experimental method, the final yield of chondroitin oligosaccharides was 70.4%.
<1-3> 콘드로이친 황산 올리고당의 분자량 결정<1-3> Molecular weight determination of chondroitin sulfate oligosaccharides
본 발명자들은 콘드로이친 황산 올리고당의 분자량을 TSK-G3000SW GPC-HPLC 칼럼을 이용하여 결정하였다. 구체적으로, FPLC(Pharmacia,스웨덴)에 0.1 M NaCl 용액을 1 ㎖/min로 흘려주면서 210 ㎚에서 검출을 하였다. 분자량의 결정을 위하여 사용한 표준품은 평균 분자량이 15,000 Da인 콘드로이친 황산, 12,000 Da인 헤파린 및 5,000 Da인 저분자량 헤파린을 사용하였다. 상기와 같은 방법을 이용하여결정된 콘드로이친 황산 올리고당의 평균 분자량은 약 5,000 Da이었다.The inventors determined the molecular weight of chondroitin sulfate oligosaccharides using a TSK-G3000SW GPC-HPLC column. Specifically, detection was performed at 210 nm while flowing 0.1 M NaCl solution at 1 mL / min in FPLC (Pharmacia, Sweden). Standards used for determination of molecular weight used chondroitin sulfate with an average molecular weight of 15,000 Da, heparin with 12,000 Da and low molecular weight heparin with 5,000 Da. The average molecular weight of chondroitin sulfate oligosaccharides determined using the method described above was about 5,000 Da.
<실시예 2> 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당이 HLE의 활성 억제에 미치는 영향Example 2 Effect of Chondroitin Sulfate Disaccharide or Chondroitin Sulfate Oligosaccharide on the Inhibition of HLE Activity
본 발명자들은 콘드로이친 황산 이당류 또는 올리고당이 HLE 활성 억제에 어떠한 영향을 미치는지 확인하여 보았다.The inventors have identified how chondroitin sulfate disaccharides or oligosaccharides affect HLE activity inhibition.
각각의 시료(콘드로이친 황산, 이당류, 올리고당)를 완충 용액에 가하여 미리 37℃에서 반응시킨 후 미리 가온한 HLE를 가하여, 37℃에서 3분간 반응시켰다. 0.5 mM의 발색 기질(Boc-Ala-Ala-Pro-Ala-p-nitroanilide)을 가하여 37℃에서 90분간 반응시킨 후 0.5 M 초산을 가하여 반응을 정지시켰고, 그 가수 분해 정도를 405 nm에서 흡광도를 측정하여 대조군에 대한 HLE의 활성을 %로 나타내었다.Each sample (chondroitin sulfate, disaccharide, oligosaccharide) was added to a buffer solution and reacted at 37 ° C. in advance, and warmed HLE was added thereto to react at 37 ° C. for 3 minutes. After 0.5 mM chromogenic substrate (Boc-Ala-Ala-Pro-Ala-p-nitroanilide) was added and reacted at 37 ° C. for 90 minutes, 0.5 M acetic acid was added to stop the reaction, and the degree of hydrolysis was absorbed at 405 nm. The measurement was expressed in% of HLE activity against the control.
그 결과, 대조군에 비해 콘드로이친 황산의 경우에는 그 차이가 유의성을 나타내지 않았고 HLE 억제 활성을 거의 보이지 않았으나, 콘드로이친 이당류 및 올리고당은 HLE의 활성의 약 20%의 감소 효과를 나타내었다(도 1).As a result, in the case of chondroitin sulfate compared to the control, the difference was not significant and showed little HLE inhibitory activity, but chondroitin disaccharides and oligosaccharides showed a reduction effect of about 20% of HLE activity ( FIG. 1 ).
<실시예 3> 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당이 관절염에 미치는 효과Example 3 Effect of Chondroitin Sulfate Disaccharide or Chondroitin Sulfate Oligosaccharide on Arthritis
본 발명자들은 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당이 관절염에 미치는 효과를 알아보기 위하여 콜라겐으로 유발된 류마티스성 관절염 동물 모델을 사용하여, 콘드로이친 황산과 비교하여 그 효과를 확인하였다.In order to determine the effect of chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides on arthritis, the present inventors confirmed the effects of collagen-induced rheumatoid arthritis animal models in comparison with chondroitin sulfate.
DBA/1J 생쥐[일본의 SLC Inc.(Shizuoka, Japan)회사로부터 구입]는 5-6주령의 수컷을 사용하였으며, 서울대학교 임상의학연구소 전임상 실험실의 SPF(Specific Pathogen Free) 조건하에서 일주일간 적응시킨 후, 실험을 실시하였다. 사육장은 인공 조명에 의하여 조명 시간을 아침 8시부터 저녁 8시까지 12시간으로 조절하였으며 실내 온도는 18℃ 내지 23℃로 유지하였다.DBA / 1J mice (purchased from SLC Inc. (Shizuoka, Japan) Ltd. of Japan) used males 5-6 weeks of age and adapted for 1 week under SPF (Specific Pathogen Free) conditions at the preclinical laboratory at Seoul National University Institute of Clinical Medicine. Then, the experiment was performed. The kennel was adjusted to 12 hours from 8 am to 8 pm by artificial lighting and the room temperature was maintained at 18 ℃ to 23 ℃.
동물군을 4그룹으로 나누어, 대조군, 콘드로이친 황산 이당류(50 ㎎/㎏), 콘드로이친 황산 올리고당(300 ㎎/㎏), 콘드로이친 황산(1,200 ㎎/㎏)을 14일간 하루 한번 경구로 투여하였다. 15일째 되는 날, 1차 항원을 주사하여 관절염을 유발시켰다. 병아리 제 2형 콜라겐(Chicken Sternal Cartilage Type II collagen)을 2 ㎎/㎖ 농도로 0.01 N 초산에 녹여서 같은 양의 프로인트 애주번트(Freund Adjuvant)를 첨가하여 에멀젼화 시켜서 항원으로 사용하였다. 각 생쥐 꼬리의 근위부를 70% 알코올로 소독한 후 항원 100 ㎕를 피내로 주사하였다. 추가 접종은 1차 항원 주사 후 21일째 되는 날, 동일 방법으로 동일량 피내 주사하였다.The animals were divided into four groups, and the control group, chondroitin sulfate disaccharide (50 mg / kg), chondroitin sulfate oligosaccharide (300 mg / kg) and chondroitin sulfate (1,200 mg / kg) were administered orally once a day for 14 days. On day 15, arthritis was induced by injection of primary antigen. Chicken Sternal Cartilage Type II collagen was dissolved in 0.01 N acetic acid at a concentration of 2 mg / ml, and the same amount of Freund Adjuvant was added to emulsify and used as an antigen. The proximal portion of each mouse tail was disinfected with 70% alcohol and then 100 μl of antigen was injected intradermally. The booster dose was injected intradermally by the same method on the 21st day after the first antigen injection.
<3-1> 콜라겐 유발 관절염의 발생과 중증도 평가에 미치는 영향 측정<3-1> Measurement of the effects on collagen-induced arthritis and its severity assessment
콜라겐으로 면역시킨 후, 3-4일 간격으로 생쥐의 관절염 발생 여부를 관찰하였다. 발적과 부종이 나타나는 분명한 관절염이 있는 경우를 관절염 발생으로 정의하였으며, 뒷발의 두께를 다이얼 두께 게이지(DIAL THICKNESS GAUGE; Mitutoyo Corp., Tokyo, Japan)를 이용하여 측정하였다. 양 뒷발의 두께를 잰 평균값을 이용하였으며, 뒷발 두께(Hind Paw Thickness, mm)로 표시하였다.After immunization with collagen, mice were examined for arthritis at 3-4 day intervals. The incidence of arthritis was defined as the presence of obvious arthritis with redness and edema, and the thickness of the hind paw was measured using a dial thickness gauge (DIAL THICKNESS GAUGE; Mitutoyo Corp., Tokyo, Japan). The average value of both hind paws was used and expressed as Hind Paw Thickness (mm).
그 결과, 생리 식염수를 투여한 대조군은 콘드로이친 황산을 투여한 그룹과 그 부종과 발적을 비교했을시 차이를 나타내지 않았고, 육안적 소견 역시 거의 비슷한 양상을 나타내었다. 반면에 콘드로이친 이당류나 올리고당을 각각 투여한 그룹에서는 부종과 발적이 감소하였고, 뒷발 두께를 측정한 결과 역시 감소함을 확인할 수 있었다(도 2).As a result, the control group treated with physiological saline did not show a difference when comparing edema and redness with the group treated with chondroitin sulfate, and the gross findings showed almost the same pattern. On the other hand, edema and redness decreased in the group administered with chondroitin disaccharide or oligosaccharide, respectively, and the hind paw thickness was also reduced ( FIG. 2 ).
<3-2> 혈청 항 콜라겐 항체의 생성에 미치는 영향 측정<3-2> Determination of the effect on the production of serum anti-collagen antibody
실험 10주째에 심장 천자를 통하여 얻은 혈액을 원심 분리하여 혈청을 구하였으며, 혈청내의 IgG 항콜라겐 항체의 양을 효소면역법(Enzyme-Linked Immunosorbant Assay)으로 측정하였다. 96 마이크로플레이트 웰에 병아리 제 2형 콜라겐(10 ㎍/㎖)을 100 ㎕/웰씩 넣고 4℃에서 하룻밤 동안 미리 부착시켰다. PBS-트윈 20 용액으로 3회 세척한 후, 완전히 물기를 제거하고 PBS-1% BSA를 100 ㎕/웰씩 가하여 25℃에서 1시간 반응시켰다. PBS-트윈 20 용액으로 3회 세척한 후, 혈청을 가하여 37℃에서 2시간 반응시키고, 다시 4℃에서 하룻밤 동안 반응시켰다. 같은 방법으로 세척한 후, 알카라인 포스파타제가 결합된 α 생쥐 IgG(alkaline phosphatase α mouse IgG) 항체를 1/1,000으로 희석한 액을 가하여 다시 37℃에서 1시간 반응 시켰다. 세척 후, 실온에서 발색 기질을 가하여 발색 정도를 관찰하다가 반응 정지액을 가하여 405 nm에서 그 흡광도를 측정하여, 관절염 대조군과 각 그룹간의 항체 생성 차이를 확인하였다.Serum was obtained by centrifugation of blood obtained through cardiac puncture at 10 weeks of experiment, and the amount of IgG anti-collagen antibody in serum was measured by Enzyme-Linked Immunosorbant Assay. 100 μl / well of chick type 2 collagen (10 μg / ml) was added to 96 microplate wells and previously attached at 4 ° C. overnight. After washing three times with PBS-Tween 20 solution, water was completely removed, and 100 μl / well of PBS-1% BSA was added thereto, followed by reaction at 25 ° C. for 1 hour. After washing three times with PBS-Tween 20 solution, serum was added and reacted at 37 ° C. for 2 hours and again at 4 ° C. overnight. After washing in the same manner, a solution of alkaline phosphatase-binding α mouse IgG (alkaline phosphatase α mouse IgG) antibody diluted to 1 / 1,000 was added and reacted again at 37 ° C. for 1 hour. After washing, the color development substrate was added at room temperature to observe the degree of color development, and then the reaction stopper was added to measure the absorbance at 405 nm to confirm the difference in antibody production between the arthritis control group and each group.
그 결과, 생리 식염수를 투여한 관절염 대조군과 콘드로이친 황산이나 콘드로이친 황산 이당류를 투여한 그룹에서는 그 차이를 확인할 수 없었으나, 콘드로이친 황산 올리고당을 투여한 그룹에서는 항 콜라겐 항체가 감소하는 경향을 확인할 수 있었다(도 3).As a result, the difference was not observed in the arthritis control group administered with saline and the group administered chondroitin sulfate or chondroitin sulfate disaccharide, but the anti-collagen antibody tended to decrease in the group administered chondroitin sulfate oligosaccharides. 3 ).
<3-3> 혈청 사이토카인에 미치는 영향 측정<3-3> Measurement of the effect on serum cytokines
관절 부위에서의 염증 반응에 의해 일어나는 사이토카인의 변화는 순환혈에 존재하는 사이토카인의 상대적 균형을 변화시킬 수 있기 때문에 순환혈에 존재하는 특정 사이토카인의 혈중 농도의 변화를 검토하였다.Since changes in cytokines caused by an inflammatory response at the joint site can alter the relative balance of cytokines present in the circulating blood, changes in blood concentrations of specific cytokines present in the circulating blood were examined.
실험 10주째에 얻은 혈청 중 TNF-α의 양을 쿼티킨 M(Quantikine M; R&D Systems Inc., Minneapolis, MN, USA) 키트를 이용하여 효소 면역법으로 정량하였다. 96 마이크로플레이트 웰에 희석액을 50 ㎕씩 넣고 표준 용액과 측정할 혈청 샘플을 50 ㎕씩 첨가한 후 실온에서 2시간 반응시켰다. 세척용액으로 5회 세척 후 항-생쥐 TNF-α 컨쥬게이트(anti-mouse TNF-α conjugate) 용액을 100 ㎕/웰씩 가하고 실온에서 다시 반응시켰다. 2시간 후 세척용액으로 5회 세척하고 완전히 물기를 제거한 후, 발색 용액 100 ㎕/웰을 가하여 30분간 실온에서 반응시킨 후 반응 정지액 100 ㎕/웰을 첨가하여 450 nm에서 흡광도를 측정하여 혈중 TNF-α의 농도를 구하였다.The amount of TNF-α in the serum obtained at the 10th week of the experiment was quantitated by enzyme immunoassay using the Quantikin M (R & D Systems Inc., Minneapolis, MN, USA) kit. 50 μl of diluent was added to 96 microplate wells, and 50 μl of the standard solution and the serum sample to be measured were reacted at room temperature for 2 hours. After washing 5 times with the washing solution, 100 μl / well of the anti-mouse TNF-α conjugate solution was added thereto and reacted again at room temperature. After 2 hours, washed 5 times with washing solution and completely drained. After 100 μl / well of color development solution was added and reacted at room temperature for 30 minutes, 100 μl / well of reaction stop solution was added to measure absorbance at 450 nm. The concentration of -α was obtained.
그 결과, 혈청 중 사이토카인의 농도 역시, 콘드로이친 황산의 경우에는 생리 식염수를 투여한 대조군과 차이를 나타내지 않았으나, 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 투여한 두 그룹에서는 각각 혈중 TNF-α의 농도가 감소하는 것을 확인할 수 있었다(도 4).As a result, the concentration of cytokines in serum did not differ from the control group treated with saline, whereas the concentrations of TNF-α in the blood decreased in the two groups treated with chondroitin sulfate disaccharide or chondroitin sulfate oligosaccharide. It was confirmed that ( Fig. 4 ).
<3-4> 조직학적 관찰<3-4> Histological observation
본 발명자들은 상기 생쥐에서 채혈 후, 양쪽 다리의 관절 부위를 잘라내어 10% 포르말린 용액에 담그어 고정시켜 4℃에서 24시간 방치하였다. 10% 포름산-포르말린(formic acid-formalin) 용액에서 석회질을 제거하고, 70% 에탄올(1시간), 80% 에탄올(1시간), 95% 에탄올(1시간), 95% 에탄올(1시간), 95% 에탄올(2시간), 100% 에탄올(2시간), 자일렌(1시간), 자일렌(2시간) 및 파라핀(2시간)의 순서를 거쳤다. 이후 일정 모양의 파리핀 모형을 만들기 위해 임베딩 센터(embedding center, Lipshaw)를 사용하였다. 파라핀 침투 과정이 끝난 조직을 파라핀 가온 배양조에 넣고 기본주형에 파라핀을 채운 후 조직을 안착시키고 냉동판에 올려놓고 굳은 후 기본 주형을 떼어 놓았다. 파라핀 모형을 박절기에서 4 ㎛ 두께로 자른 후 슬라이드 글라스 위에 올려 60℃ 에서 건조 부착 시켰다. 절편이 부착된 슬라이드 글라스를 60℃의 오븐에 넣어 조직 이외의 파라핀을 녹이고 자일렌에서 10분씩 3회 처리한 후 100%, 95%, 80% 및 70% 에탄올 순으로 2회씩 2분간 처리하여 함수 시켰다. 염색은 함수과정 후 탈이온수에서 10분간 처리한 후 해리스 헤마토실린 염색 용액으로 염색하였다. 흐르는 상수로 세척하고 95% 에탄올로 8분 동안 처리한 후, 0.5% 에오진 용액으로 2분 동안 염색하였다. 95%와 100% 에탄올로 탈수한 후 말리고 자일렌으로 처리한 후 밀봉하여 모든 검경을 100배율로 하였다After collecting blood from the mice, the inventors cut the joints of both legs, immersed in 10% formalin solution, and left for 24 hours at 4 ° C. Decalcify in a 10% formic acid-formalin solution, 70% ethanol (1 hour), 80% ethanol (1 hour), 95% ethanol (1 hour), 95% ethanol (1 hour), 95% ethanol (2 hours), 100% ethanol (2 hours), xylene (1 hour), xylene (2 hours) and paraffin (2 hours) were followed. After that, an embedding center (Lipshaw) was used to create a shape of a flypin model. After the paraffin penetrating process, the tissue was placed in a paraffin warming culture tank and paraffin was filled in the basic mold, and then the tissue was settled, placed on a frozen plate, and solidified. The paraffin model was cut to a thickness of 4 μm in a slicer and placed on a slide glass to dry at 60 ° C. The slide glass with the slices was placed in an oven at 60 ° C. to dissolve paraffin other than the tissue, and treated three times in xylene for 10 minutes, and then treated twice for 2 minutes in the order of 100%, 95%, 80% and 70% ethanol. I was. The dyeing was treated with deionized water for 10 minutes after the hydrolysis process and then stained with Harris hematoclinin dye solution. Washed with running constant, treated with 95% ethanol for 8 minutes, and then stained with 0.5% eosin solution for 2 minutes. After dehydration with 95% and 100% ethanol, dried, treated with xylene and sealed, all speculum was at 100 times magnification.
그 결과, 정상 대조군과 비교하여, 관절염 유발 그룹은 관절 조직의 구조에큰 변화가 관찰되었다. 콘드로이친 황산을 투여한 그룹은 종전의 보고에서와는 달리, 판누스(pannus) 생성이나 그 중증도 면에서 관절염 유발 그룹과 별 차이를 보이지 않았고, 유발된 관절염의 개선 효과를 찾아볼 수 없었다. 그러나 콘드로이친 이당류나 올리고당을 투여한 그룹에서는 관절염의 개선 효과를 육안으로 확인할 수 있었으며, 특히 콘드로이친 올리고당을 투약한 그룹은 거의 정상 대조군과 비슷한 양상을 나타낸 것을 확인할 수 있었다(도 5).As a result, compared with the normal control group, the arthritis-induced group showed a large change in the structure of the joint tissue. Unlike the previous reports, the group receiving chondroitin sulfate did not show any difference in the arthritis-inducing group in terms of pannus production or its severity, and there was no improvement in the induced arthritis. However, in the group administered with chondroitin disaccharides or oligosaccharides, the effect of improving arthritis was visually confirmed, and in particular, the group administered with chondroitin oligosaccharides showed a similar pattern to that of the normal control group ( FIG. 5 ).
<실시예 4> Caco-2 세포주를 이용한 콘드로이친 황산, 콘드로이친 황산 이당류, 콘드로이친 황산 올리고당의 소장세포로의 투과 조사Example 4 Permeation Investigation of Chondroitin Sulfate, Chondroitin Sulfate Disaccharide, and Chondroitin Sulfate Oligosaccharides into Small Intestine Cells Using Caco-2 Cell Line
본 발명자들은 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을이 소장세포에서 흡수가 잘 되는지 확인해 보기 위하여 Caco-2 세포주를 이용하여 관찰하여 보았다. 구체적으로, Caco-2 단층세포를 10 ㎖의 완전배지가 들어있는 75 cm3의 T-플라스크에 접종한 후 세포가 T-플라스크의 벽면에 약 90% 정도 차지할 때 까지 배양하였다. Caco-2 세포가 T-플라스크의 벽면에서 약 90% 정도의 포화에 도달시 트랜스웰(12 웰) 상의 점막층에 Caco-2 세포의 성장을 위한 지지체 역할을 하는 제 1형 콜라겐을 8∼10 ㎍/㎠ 의 농도가 되도록 첨가한 후 UV 램프를 켜고 4∼5 시간 정도 건조시켜 코팅을 한 후 각각 0.5 ㎖과 1.5 ㎖의 완전배지를 트랜스웰의 점막층과 장액막층에 넣어준 후 37℃, 5% CO2배양기에 넣어두었다. 그리고 90% 정도의 포화에 도달한 Caco-2 세포가 들어있는 T-플라스크는 남아있는 완전배지를 완전히 제거한 후 인산완충액를 이용하여 2분 정도 깨끗하게 세척을 해 주고, 트립신-EDTA를 1 ㎖ 첨가한 후 3분 동안 인큐베이터에서 배양하여 플라스크의 기벽에 붙은 Caco-2 세포를 접종하였다. 다음 새로운 완전배지를 첨가하고, 피펫팅을 하여 Caco-2 세포들이 뭉쳐있지 않고 따로따로 떨어져서 존재하도록 만들어주었다. 이렇게 처리한 세포들은 헤모사이토미터를 이용하여 세포 수를 측정하고, 2.5 - 3×105개의 세포수/㎖가 되도록 희석하였다. 이렇게 희석한 Caco-2 세포 용액의 500 ㎕씩을 트랜스웰의 각각의 위에 부과한 후 인큐베이터에서 배양을 하였다. 3주 동안 매일 트랜스웰의 위와 바닥 상의 배지를 갈아주면서 배양을 하여 트랜스웰상의 위의 부분에 접종한 Caco-2 세포가 단층세포를 형성하기 위한 조건을 유지시켜 주었다. 3주 동안 배양한 후 투과실험에 들어가기 앞서 배양한 Caco-2 세포의 전기저항 값을 측정하였다. 트랜스웰의 점막층과 장액층 상의 남아 있는 배지를 제거한 후 투과실험용 배지로 2번 세척을 해 준 콘드로이친 황산과 화학반응에 의해서 분해를 시킨 콘드로이친 황산 올리고당을 100 ㎎/㎖의 농도에서 10 ㎕를 취해서 490 ㎕의 투과실험용 배지와 혼합하여 트랜스웰의 점막층에 넣어준다. 그런 다음 진탕수욕조를 이용하여 37℃, 85 rpm으로 교반을 하면서 배양을 하고, 1시간 간격으로 Caco-2 세포의 전기저항을 측정하고, 콘드로이친 황산과 콘드로이친 황산 올리고당이 들어있는 트란스웰의 점막층을 신선한 투과실험용 배지가 담긴 새로운 웰로 옮겨주었다. 그리고 이전에 남은 트란스웰의 장액층에 존재하는 각각의 투과된 콘드로이친 황산이 포함된 투과실험용 배지는 따로 보관하였다. 이러한 방법을 이용하여 1시간 간격으로 전기저항 값의 측정과 투과실험용 배지를 바꿔주는 작업을 반복하고, 각각의 수치를 기입하였다. 투과실험이 끝나면 따로 보관했던 콘드로이친 황산과 화학반응에 의한 콘드로 이친 황산 올리고당이 투과된 각각의 시간대의 투과실험용 배지를 1,9-디메틸메틸렌 블루를 이용하여 정량을 하였다. 시료가 들어있는 투과실험용 배지들과 검량선 작성을 위한 각기 다른 농도의 콘드로이친 황산과 화학반응에 의한 콘드로이친 황산 올리고당 100 ㎕씩을 96-웰 마이크로플레이트에 넣어준 후 염색용액을 150 ㎕씩 첨가하여 540 nm에서 흡광도를 측정함으로써 투과된 시료의 양을 측정하였다. 한편 콘드로이친 황산 이당류의 경우에는 낮은 전하 밀도때문에 1,9-디메틸메틸렌 블루를 이용한 정량법에 의해서 정량할 수 없으므로, SAX-HPLC(Strong Anion-Exchange High Performance Liquid Chromatography)를 이용하여 각기 다른 농도에서의 콘드로이친 황산 이당류의 검량선을 작성하고, 이를 기준으로 해서 각기 다른 시간대의 투과된 시료들의 양을 결정하였다. 정량을 한 결과 콘드로이친 황산의 경우에는 반응 3시간까지 1 mg 중 8.70 ㎍ 정도로 약 0.87% 정도의 투과가 되었음을 확인할 수 있었고, 화학적으로 분해한 콘드로이친 황산 올리고당의 경우에는 반응 3시간까지 1 mg 중 110.36 ㎍ 정도로 약 11.04% 정도의 투과율을 보였다. 그리고 콘드로이친 황산 분해효소에 의해서 얻은 콘드로이친 황산 이당류의 경우에는 반응 3시간까지 100 ㎍ 중에서 35.08 ㎍ 정도로 약 35.08%의 투과율을 보였다.The present inventors observed the chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides using Caco-2 cell line to check whether the small intestine cells are absorbed well. Specifically, Caco-2 monolayer cells were inoculated into a 75 cm 3 T-flask containing 10 ml of complete medium and cultured until the cells occupy about 90% of the wall of the T-flask. When Caco-2 cells reach about 90% saturation at the wall of the T-flask, 8-10 μg of type 1 collagen acts as a support for the growth of Caco-2 cells in the mucosal layer on the transwell (12 wells). / Cm 2, add UV lamp, dry for 4 ~ 5 hours, and coat 0.5 ml and 1.5 ml complete medium into transwell mucosa and serous membrane, respectively. 37 ℃, 5% It was placed in a CO 2 incubator. The T-flask containing Caco-2 cells reaching 90% saturation was completely removed after thorough removal of the remaining complete medium, followed by rinsing with phosphate buffer for 2 minutes, and 1 ml of trypsin-EDTA. The cells were incubated in the incubator for 3 minutes to inoculate Caco-2 cells on the base wall of the flask. New fresh medium was then added and pipetted to allow Caco-2 cells to be present separately without clumping. The cells thus treated were measured using a hemocytometer and the cells were diluted to 2.5-3 × 10 5 cells / ml. 500 μl of the diluted Caco-2 cell solution was imposed on each of the transwells, and then cultured in an incubator. Daily culture for three weeks was carried out by changing the medium on the top and bottom of the transwell to maintain the conditions for Caco-2 cells inoculated into the upper part of the transwell to form monolayer cells. After incubation for 3 weeks, the electrical resistance value of the cultured Caco-2 cells was measured prior to entering the permeation experiment. After removing the remaining medium on the transwell mucosal layer and serous layer, 10 μl of chondroitin sulfate oligosaccharide decomposed by chemical reaction with chondroitin sulfate, which was washed twice with a permeation experiment medium, at a concentration of 100 mg / mL, was taken 490 Mix with μl permeation medium and put into transmucosal layer. Then, incubate with shaking water bath at 37 ° C. and 85 rpm, measure the electrical resistance of Caco-2 cells at 1 hour intervals, and transmucosal layer of transwell containing chondroitin sulfate and chondroitin sulfate oligosaccharide. Transferred to new wells containing fresh permeation experiment medium. And the permeation experiment medium containing each permeated chondroitin sulfate present in the remaining transwell of the transwell was stored separately. Using this method, the measurement of the electrical resistance value and the change of the medium for permeation experiment were repeated at 1 hour intervals, and each value was entered. After the permeation experiment, the permeation experiment medium at each time period in which chondroitin sulfate and chemically reacted chondroitin sulfate oligosaccharides were permeated was determined using 1,9-dimethylmethylene blue. 100 μl of Chondroitin Sulfate by chemical reaction with different concentrations of Chondroitin Sulfate for the calibration curve preparation and calibration curve containing the sample were put into a 96-well microplate, and 150 μl of the dyeing solution was added at 540 nm. The amount of sample transmitted was measured by measuring the absorbance. On the other hand, chondroitin sulfate disaccharides cannot be quantified by the quantitative method using 1,9-dimethylmethylene blue due to the low charge density. Therefore, chondroitin at different concentrations can be determined using Strong Anion-Exchange High Performance Liquid Chromatography (SAX-HPLC). A calibration curve of sulfuric acid disaccharide was prepared and the amount of permeated samples at different time points was determined based on this. As a result of the quantification, it was confirmed that about 0.87% of chondroitin sulfate was permeated about 8.70 ㎍ of 1 mg until the reaction time of 3 hours, and 110.36 ㎍ of 1 mg until the reaction time of the chemically decomposed chondroitin sulfate oligosaccharides. The transmittance was about 11.04%. In the case of chondroitin sulfate disaccharide obtained by chondroitin sulfate degrading enzyme, the permeability was about 35.08% in 35 μg of 100 μg up to 3 hours of reaction.
<실시예 5> 랫트에 대한 비경구투여 급성 독성실험Example 5 Parenteral Administration Acute Toxicity in Rats
6주령의 특정병원체부재(SPF) SD계 랫트를 사용하여 급성독성실험을 실시하였다. 군당 2 마리씩의 동물에 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 또는 올리고당을 0.5% 메틸셀룰로즈 용액에 현탁하여 500 mg/㎏/㎖의 용량으로 단회 비경구 투여하였다. 시험물질 투여후 동물의 폐사여부, 임상증상, 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적검사를 실시하였으며, 부검하여 육안으로 복강장기와 흉강장기의 이상여부를 관찰하였다. 시험결과, 시험물질을 투여한 모든 동물에서 특기할 만한 임상증상이나 폐사된 동물은 없었으며, 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다. 이상의 결과 실험된 본 발명의 약학적 조성물의 유효성분인 콘드로이친 황산 이당류 또는 올리고당은 랫트에서 1,000 ㎎/㎏까지 독성변화를 나타내지 않으며 비경구투여 최소치사량(LD50)은 500 ㎎/㎏ 이상인 안전한 물질로 판단되었다.Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were administered parenterally at a dose of 500 mg / kg / ml, suspended in 0.5% methylcellulose solution, with chondroitin sulfate disaccharides or oligosaccharides, the active ingredients of the pharmaceutical composition of the present invention. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, chondroitin sulfate disaccharides or oligosaccharides, which are the active ingredients of the pharmaceutical composition of the present invention, do not show toxicological changes up to 1,000 mg / kg in rats and have a minimum parenteral dose (LD 50 ) of 500 mg / kg or more. Judging.
본 발명의 콘드로이친 황산의 분해산물인 콘드로이친 황산 이당류 또는 콘드로이친 황산 올리고당을 유효성분으로 포함하는 류마티스성 관절염 예방 및 치료용 약학적 조성물은 관절염 유발시 생기는 부종이나 발적을 감소시킬 뿐 아니라, 혈청 중 항 콜라겐 항체나 사이토카인인 TNF-α의 수치를 낮춤으로써 류마티스성 관절염의 예방 및 치료에 유용하게 사용될 수 있다.Rheumatoid arthritis prevention and treatment pharmaceutical composition comprising chondroitin sulfate disaccharides or chondroitin sulfate oligosaccharides, which are decomposition products of chondroitin sulfate of the present invention as an active ingredient, not only reduces swelling or redness during arthritis, but also anti-collagen in serum By lowering the level of the antibody or cytokine TNF-α it can be usefully used for the prevention and treatment of rheumatoid arthritis.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100906559B1 (en) * | 2007-08-27 | 2009-07-07 | 한국농축산영농조합 | Preparation of chondroitin sulfate oligosaccharide |
| EP2106798A1 (en) * | 2008-04-02 | 2009-10-07 | Nestec S.A. | Sulfated unsaturated disaccharidic chondroitin sulfate in connective tissue protection and repair |
| KR20220091992A (en) | 2020-12-24 | 2022-07-01 | 충북대학교 산학협력단 | Chicken bone extract with enhanced functionality and chicken stock composition comprising the same as an active ingredient |
| CN117646054A (en) * | 2024-01-29 | 2024-03-05 | 湖南天劲制药有限责任公司 | Process for producing, separating and purifying bone polypeptide for treating arthritis |
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| JP2000053569A (en) * | 1998-03-19 | 2000-02-22 | Sigma-Tau Healthscience Spa | Combined composition in form of nutrient auxiliary or medicine composition for preventive and therapeutic treatment of degenerative or inflammatory arthropathy |
| KR20010046562A (en) * | 1999-11-12 | 2001-06-15 | 박명환 | Composition for treatment of osteoarthritis |
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| KR940005281A (en) * | 1992-07-30 | 1994-03-21 | 야마타니 와타루 | Antirheumatic composition |
| KR19990008198A (en) * | 1995-04-28 | 1999-01-25 | 로빈스탠톤 | Preparation and Use of Sulfated Oligosaccharides |
| JPH11222425A (en) * | 1997-10-27 | 1999-08-17 | Ss Pharmaceut Co Ltd | Intra-articular injection preparation for treating articular disease |
| JP2000053569A (en) * | 1998-03-19 | 2000-02-22 | Sigma-Tau Healthscience Spa | Combined composition in form of nutrient auxiliary or medicine composition for preventive and therapeutic treatment of degenerative or inflammatory arthropathy |
| KR20010046562A (en) * | 1999-11-12 | 2001-06-15 | 박명환 | Composition for treatment of osteoarthritis |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100906559B1 (en) * | 2007-08-27 | 2009-07-07 | 한국농축산영농조합 | Preparation of chondroitin sulfate oligosaccharide |
| EP2106798A1 (en) * | 2008-04-02 | 2009-10-07 | Nestec S.A. | Sulfated unsaturated disaccharidic chondroitin sulfate in connective tissue protection and repair |
| WO2009121519A1 (en) * | 2008-04-02 | 2009-10-08 | Nestec S.A. | Sulfated unsaturated disaccharidic chondroitin sulfate in connective tissue protection and repair |
| US20110028400A1 (en) * | 2008-04-02 | 2011-02-03 | Nestec S.A. | Sulfated unsaturated disaccharidic chondroitin sulfate in connective tissue protection and repair |
| KR20220091992A (en) | 2020-12-24 | 2022-07-01 | 충북대학교 산학협력단 | Chicken bone extract with enhanced functionality and chicken stock composition comprising the same as an active ingredient |
| CN117646054A (en) * | 2024-01-29 | 2024-03-05 | 湖南天劲制药有限责任公司 | Process for producing, separating and purifying bone polypeptide for treating arthritis |
| CN117646054B (en) * | 2024-01-29 | 2024-04-30 | 湖南天劲制药有限责任公司 | Process for producing, separating and purifying bone polypeptide for treating arthritis |
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