KR101469961B1 - Method for separating porpyrin-related compounds from laminaria japonica for use in the prevention or treatment of diabetic complication - Google Patents
Method for separating porpyrin-related compounds from laminaria japonica for use in the prevention or treatment of diabetic complication Download PDFInfo
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- KR101469961B1 KR101469961B1 KR1020140024156A KR20140024156A KR101469961B1 KR 101469961 B1 KR101469961 B1 KR 101469961B1 KR 1020140024156 A KR1020140024156 A KR 1020140024156A KR 20140024156 A KR20140024156 A KR 20140024156A KR 101469961 B1 KR101469961 B1 KR 101469961B1
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- South Korea
- Prior art keywords
- extract
- diabetic
- soluble fraction
- hexane
- ethyl acetate
- Prior art date
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- Medicines Containing Plant Substances (AREA)
Abstract
본 발명은 다시마 추출물 또는 그로부터 분리한 폴피린계 화합물(porpyrin-related compounds)을 함유하는 당뇨성 합병증 억제에 활성을 갖는 약학 조성물 또는 건강 식품에 관한 것으로, 상기 추출물은 알도즈 환원 효소 및 최종당화산물 생성 억제 활성을 나타내고, 천연 약재로서 안전성이 확보되어 있으므로 당뇨성 합병증 예방 또는 치료용 약학조성물과 식품 조성물로로 유용하게 이용될 수 있다.The present invention relates to a pharmaceutical composition or a health food having an activity to inhibit diabetic complications, which comprises a sea tangle extract or porpyrin-related compounds isolated therefrom, wherein the extract comprises an aldose reductase and a final glycation product Production inhibitory activity, and safety as a natural medicinal ingredient is ensured. Therefore, it can be usefully used as a pharmaceutical composition and a food composition for the prevention or treatment of diabetic complications.
Description
본 발명은 다시마(Laminaria japonica) 추출물 또는 이로부터 분리한 폴피린계 화합물을 유효 성분으로 함유하는 당뇨성 합병증의 예방 및 치료용 약학 조성물에 관한 것이다. 본 발명의 조성물은 당뇨병 합병증의 원인인 소르비톨 축적을 일으키는 알도즈 환원효소 및 최종당화산물 생성을 억제 활성을 갖고 있어 당뇨병 합병증의 예방 및 치료용 조성물로 다양하게 이용될 수 있다.
The present invention relates to a pharmaceutical composition for preventing and treating diabetic complications comprising Laminaria japonica extract or a polypyrin compound isolated therefrom as an active ingredient. The composition of the present invention has various activities for inhibiting the production of aldose reductase and final glycation end product which cause sorbitol accumulation which is a cause of diabetic complication and thus can be used variously as a composition for prevention and treatment of diabetic complications.
당뇨병은 인슐린의 분비량이 부족하거나 정상적인 기능이 이루어지지 않아 혈중 포도당 농도가 높아져 소변에 포도당을 배출하는 질환으로 당뇨병이 인간사회에서 문제가 되는 것은, 그 특유한 합병증 때문이다. 인슐린이 개발되기 전 시대의 당뇨병은 혼수, 감염증 등이 인류의 생명을 위협했으나 현재의 화학 및 생화학의 발전에 힘입어 식사요법, 혈당강하제 요법, 인슐린 요법 등으로 갑작스런 생명의 위협을 막을 수 있게 되었다.
Diabetes is a disease that releases glucose into the urine due to insufficient secretion of insulin or a lack of normal function, resulting in elevated blood glucose concentration. Diabetes is a problem in human society because of its peculiar complications. Before the development of insulin, diabetes mellitus, coma and infectious diseases threatened the life of mankind. However, thanks to the current development of chemistry and biochemistry, diabetes, hypoglycemic therapy, and insulin therapy can prevent sudden life threats .
당뇨병은 그 자체보다는 합병증이 더 위험하기 때문에, 오늘날 당뇨병 치료에 있어서 가장 큰 목표는 당뇨성 합병증의 유발이나 진행을 억제하는데 있다. 합병증은 당뇨병이 오래 지속되어 나타나는 현상으로 보통 10~15년을 경과한 후에 생기는 만성 합병증이 주이며, 그 대표적인 만성 합병증으로 당뇨성 망막증, 당뇨성 신증, 당뇨성 신경증 등이 있다. 당뇨성 신경증은 당뇨병으로 인해 신경계에 장애가 오는 것으로 말초신경의 장애, 건반사의 소실, 운동신경의 마비, 자율신경 장애 등으로 발바닥이 저릿저릿하고, 화끈거리고, 통증이 심하며, 성기능의 장애가 오고, 뇨나 대변을 가리지 못하는 증상을 가져오기도 한다. 당뇨성 신증은 미세혈관 합병증의 하나로 신 사구체 모세혈관의 경화성 병변에 의해 일어나는 것으로 특별한 증상이 없어도 소변검사를 통해 단백질이 나타나면 신증이 있음을 예측할 수 있다. 혈압의 상승은 당뇨병 신증을 악화시키는 요인으로도 작용하는데 보통 10~15년 이상 당뇨병을 앓은 사람들의 약 5% 정도가 당뇨성 신증이 온다. 당뇨성 망막증은 미세 혈관의 합병증 중의 하나로 당뇨병 환자에게 실명을 가져오는 심각한 합병증이다. 최근에 당뇨병 조절과 합병증과의 관계연구에 의하면 인슐린 치료를 강화시켜 혈당이 정상화되면 당뇨병 합병증 발생을 크게 감소시킬 수 있는 것으로 나타났다 (Seaquist E. R. et al., New Eng. J. Haematol., 320, pp1161-1165, 1989).
Because diabetes is more dangerous than complications in itself, the greatest goal in the treatment of diabetes today is to inhibit the development or progression of diabetic complications. Complications are long-standing diabetes mellitus. Chronic complications usually develop after 10 to 15 years. Typical chronic complications include diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy. Diabetic neuropathy is a disorder of the nervous system due to diabetes. It causes discomfort of the nervous system, loss of key nerves, loss of the key nerves, motor nerve paralysis, autonomic nervous disorders, etc., and the digestive system is hot, hot, painful, It can also cause symptoms that do not cover the stomach. Diabetic nephropathy is a microvascular complication that is caused by a sclerotic lesion of the renal glomerular capillary. It is possible to predict the presence of nephrotic syndrome if the protein appears in the urine without any special symptoms. Elevated blood pressure also acts as a factor in worsening diabetes mellitus. About 5% of people who have diabetes mellitus for more than 10 to 15 years usually come with diabetic nephropathy. Diabetic retinopathy is one of the complications of microvascular complications that cause blindness in diabetic patients. Recently, studies on the relationship between diabetes control and complications have shown that diabetes mellitus can be significantly reduced by normalizing blood glucose levels by enhancing insulin therapy (Seaquist ER et al., New Eng. J. Haematol., 320, pp1161 -1165, 1989).
당뇨병에 있어서 고혈당이 합병증을 유발시키는 기전으로 polyol pathway의 이상 (Sato Y. and Rifkin D. B., J. Cell. Biol., 109, pp309-315, 1989), 체내에서 고혈당과 단백질의 결합, 산화적 스트레스(Williamson J. R. et al., Diabetes, 42, pp801-813, 1993), myoinositol의 감소와 Na+, K+-ATPase활성의 감소 (Greene D. A. et al., New Eng. J. Med., 316, pp599-606, 1987) 등이 발표되었다. 그 중 polyol pathway 는 포도당 대사경로의 하나로, 이는 포도당이 솔비톨을 거쳐 과당으로 변환되는 경로로 두 개의 반응계로 구성되며, 2종의 효소가 관여한다 (Gabbay K. H. and O'Sullivan J. B., Diabetes, 17, pp239, 1968; Dvornik D.et al., Science, 182, pp1146, 1973). 포도당은 에너지원으로 이용되는 중요한 물질로서 정상인의 경우 세포내에 흡수된 후에 대부분은 헥소키나아제에 의하여 글루코오스-6-포스페이트로 되어 해당계에서 대사되고, 불과 몇 % 만이 polyol pathway를 거쳐 대사된다 (Collins J. G. and Corder C. N., Invest. Ophthalmol. Visual Sci., 16, pp242, 1977). 그러나 당뇨병에 의해 고혈당 상태가 되면 세포내의 포도당 흡수가 인슐린에 의존하지 않는 조직에서 포도당의 세포막 투과는 세포 밖의 포도당 농도에 의존하므로 수정체, 망막, 각막, 말초신경, 혈관, 신사구체, 적혈구 등과 같은 인슐린 비의존성 조직의 세포내 포도당 농도는 상승하게 되고, 이들 세포내에 다량 존재하는 polyol pathway의 필수효소인 알도즈 환원효소에 의하여 포도당의 대사가 항진되어 솔비톨이 과잉생성 된다 (Collins J. G. and Corder C. N., Invest. Ophthalmol. Visual Sci., 16, pp242, 1977; Dvornik D. et al., Science, 182, pp1146, 1973). 그 결과 솔비톨이 축적되는 부위에 따라 발병 증상이 나타나는데, 당뇨성 백내장, 당뇨성 망막증, 당뇨성 각막증, 당뇨성 신경증, 당뇨성 신증 등의 당뇨병 합병증을 일으키게 된다 (Heath H. and Hamlett Y. C., Diabetologia, 12, pp43, 1976). 알도즈 환원효소를 촉매하는 포도당이 솔비톨로 되는 환원반응은 그 평형에서 역반응이 일어나지 않으며, sorbitol dehydrogenase의 활성이 알도즈 환원 효소의 활성보다 낮기 때문에 솔비톨에서 과당으로의 변환량이 아주 적으므로 결과적으로 polyol pathway의 항진은 솔비톨을 축적시키게 된다 (Collins J. G. and Corder C. N., Invest. Ophthalmol. Visual Sci., 16, pp 242, 1977). 당알콜인 솔비톨은 극성이 높아 세포막 외로 확산이 어렵기 때문에 솔비톨이 세포내에 축적되며, 이로 인해 세포내 삼투압을 상승시켜, 수정체내로 수분유입을 촉진하여 세포의 팽화를 일으킨다 (Gabbay K. H. and O'Sullivan J. B., Diabetes, 17, pp239, 1968; Dvornik D. et al., Science, 182, pp1146, 1973). 이 팽화는 수정체 섬유세포의 투과성을 항진시키고, Na+ , Cl의 유입과 K+, 아미노산, 펩티드, ATP, 미오-이노시톨(myo-inositol) 등의 유출을 일으킨다. Na+ 이나 K+ 등의 전해질의 평형파괴에 의해 삼투압이 더 증가하게 되고, 세포의 팽화를 촉진하여 세포막은 정상을 유지할 수 없게 된다. 그와 동시에 세포내 단백질 변성이 증가되어 수정체가 혼탁하게 되는데, 이와 같이 수정체가 혼탁해져 나타나는 백내장 증상을 당뇨성 합병증 진행과정의 한 지표로 사용한다. 따라서 솔비톨을 생성하는 효소인 알도즈 환원효소를 억제하게 되면, 솔비톨의 생성을 억제하는 물질이 당뇨성 백내장의 발병을 억제하는 것이 가능하다고 알려져 있다.
In the present study, we investigated the effects of diabetes on hyperglycemia and hyperglycemia in patients with diabetes mellitus. Reduction of myoinositol and reduction of Na +, K + -ATPase activity (Greene DA et al., New Eng. J. Med., 316, pp 599-606 , 1987). Among them, the polyol pathway is one of the glucose metabolism pathways, which is composed of two reaction systems, that is, glucose is transformed into fructose via sorbitol, and two enzymes are involved (Gabbay KH and O'Sullivan JB, pp. 239, 1968; Dvornik D. et al., Science, 182, pp. 1146, 1973). Glucose is an important substance used as an energy source. In normal people, after being absorbed into cells, glucose is converted to glucose-6-phosphate by hexokinase, metabolized in the system, and only a few percent are metabolized through the polyol pathway (Collins JG and Corder CN, Invest. Ophthalmol. Visual Sci., 16, pp242, 1977). However, in hyperglycemic state due to diabetes, glucose uptake by glucose is dependent on the concentration of glucose outside the cell in a tissue in which glucose uptake in the cell does not depend on insulin, and thus insulin such as lens, retina, cornea, peripheral nerve, blood vessel, The intracellular glucose concentration in the non-dependent tissues is increased, and the metabolism of glucose is enhanced by the aldose reductase, which is an essential enzyme of the polyol pathway existing in a large amount in these cells, resulting in an excess of sorbitol (Collins JG and Corder CN, Dvornik D. et al., Science, 182, pp. 1146, 1973). ≪ / RTI > As a result, symptoms of onset of sorbitol accumulation occur, leading to diabetic complications such as diabetic cataract, diabetic retinopathy, diabetic keratosis, diabetic neuropathy, and diabetic nephropathy (Heath H. and Hamlett YC, Diabetologia , 12, pp43, 1976). The conversion of glucose to sorbitol, which catalyzes Aldose reductase, does not reverse the equilibrium reaction, and since sorbitol dehydrogenase activity is lower than the activity of aldose reductase, the amount of conversion from sorbitol to fructose is very small, The pathway uptake accumulates sorbitol (Collins JG and Corder CN, Invest. Ophthalmol. Visual Sci., 16, pp. 242, 1977). Sorbitols, which are sugar alcohols, have a high polarity and are difficult to diffuse out of the cell membrane. Therefore, sorbitol accumulates in the cells, thereby raising the intracellular osmotic pressure, promoting water infiltration into the fertilized body and causing cell expansion (Gabbay KH and O'Sullivan JB, Diabetes, 17, pp239, 1968; Dvornik D. et al., Science, 182, pp1146, 1973). This enlargement enhances the permeability of the crystalline fiber cells and causes the inflow of Na +, Cl and the leakage of K +, amino acids, peptides, ATP, myo-inositol and the like. The osmotic pressure is further increased by the equilibrium destruction of the electrolyte such as Na + or K +, and the expansion of the cell is promoted, and the cell membrane can not maintain the normal state. At the same time, intracellular protein denaturation increases and the lens becomes turbid. The cataract symptom, which is caused by clouding of the lens, is used as an index of progress of diabetic complication. Therefore, it is known that inhibiting the aldose reductase, an enzyme that generates sorbitol, inhibits the onset of diabetic cataracts.
당뇨병에서 합병증을 유발시키는 기전으로 polyol pathway의 이상 이외에 혈중의 과량의 당과 단백질과의 결합에 의한 단백질 당화 (glycation)가 있다. 단백질의 당화는 단백질의 아미노기와 당의 카르보닐기 사이의 반응에 의해 초기화되고, 가역적인 쉬프베이스 (Schiff base)를 생성한다. 이 반응은 수 시간에 걸쳐서 이루어지며, 불안정한 구조로 형성된 쉬프베이스는 더 안정한 구조인 케토아민 (ketoamine) 혹은 아마도리 생성물(Amadori Products)로 재배열하게 된다. 아마도리 생성물의 형성은 수일간에 걸쳐서 생성되며, 생성물은 비가역적이다. 당이 결합된 단백질은 더 많은 결합을 하여 3-deoxyglucosones (3-DG)와 같은 다이카보닐(dicarbonyl) 중간 생성물을 형성하고, 이를 최종당화산물 (Advanced Glycation Endproducts, AGEs)이라 부른다.In addition to the abnormality of the polyol pathway, there is protein glycation due to excessive sugar and protein binding in the blood. The glycation of a protein is initiated by the reaction between the amino group of the protein and the carbonyl group of the sugar, creating a reversible Schiff base. This reaction takes place over several hours, and the unstable structure of the Schiff base rearranges into a more stable structure, ketoamine or amadori products. Perhaps the formation of the Li-product is produced over several days, and the product is irreversible. The sugar-bound protein binds more and forms a dicarbonyl intermediate such as 3-deoxyglucosones (3-DG), which is called Advanced Glycation Endproducts (AGEs).
생성된 최종당화산물은 복합체로서 단백질과 교차결합을 하고, 갈변화와 형광을 생성한다. 수많은 최종당화산물은 조직에서 검출되는데 크게 세 가지로 분류될 수 있다.The resulting final glycation products cross-link with the protein as a complex, producing galena change and fluorescence. Numerous end saccharides are detected in tissues and can be roughly classified into three types.
첫째, 형광성을 띠며, 교차결합된 최종 당화산물로 펜토시딘 (pentosidine)과 크로스린 (crossline)이 있다. 펜토시딘은 라이신 (lysine)과 아르기닌 (arginine) 잔기 사이에 교차결합을 형성하고, 당뇨가 유발되면 그 농도는 증가한다 (McCance D. R. et al., J. Clin. Invest., 91, pp2470-2478, 1993). 크로스린 (crossline)은 당뇨 쥐의 신장에서 처음으로 발견되었으며, in vitro와 in vivo에서 형성 될 수 있다.First, there is a fluorescent, cross-linked end glycation product, pentosidine and crossline. Pentocidin forms a cross-link between lysine and arginine residues, and the concentration increases when diabetes is induced (McCance DR et al., J. Clin. Invest., 91, pp2470-2478 , 1993). Crosslines were first found in the kidneys of diabetic rats and can be formed in vitro and in vivo.
둘째, 비형광성이며, 교차결합된 최종당화산물로 imidazolium dilysine과 교차결합된 것과 AFGP (alkyl formyl glycosyl pyrrole) 교차결합된 것, 그리고 ALI (arginine-lysine imidazole)이 교차결합된 것이 있다. Imidazolium dilysine이 교차결합된 것은 GOLD (glyoxal lysine dimer) 또는 MOLD (methylglyoxal lysine dimer)로 알려져 있으며, 글리옥살 (glyoxal) 유도체와 라이신 잔기 사이의 반응에 의해 형성된다 (Frye E. B. et al., J. Biol. Chem., 273, pp18714-18716, 1998). ALI는 내부세포와 교차결합한다 (Al-Abed Y. and Bucala R., Bioconjugate Chem., 11, pp39-45, 2000).Second, there is a non-fluorescent, cross-linked final glycation end product that is cross-linked with imidazolium dilysine, cross-linked with AFGP (alkyl formyl glycosyl pyrrole), and cross-linked with arginine-lysine imidazole (ALI). Imidazolium dilysine cross-linked is known as GOLD (glyoxal lysine dimer) or MOLD (methylglyoxal lysine dimer) and is formed by the reaction between a glyoxal derivative and a lysine residue (Frye EB et al., J. Biol Chem., 273, pp18714-18716, 1998). ALI cross-links internal cells (Al-Abed Y. and Bucala R., Bioconjugate Chem., 11, pp39-45, 2000).
세 번째, 교차결합이 없는 최종당화산물로 피랄린 (pyrraline)과 CML (N-carboxy methyllysine)이 있다. 피랄린은 교차결합이 없는 최종당화산물이며, 인간의 피부, 혈장, 뇌의 플라크(plaque)에서 발견되며 (Smith M. A. et al., Proc. Natl. Acad. Sci., 91, pp5710-5714, 1994), CML은 단백질 존재하에 다중불포화 지방산의 금속촉매에 의한 산화과정에서 아마도리 생성물의 산화적 분해에 의해 생성된다 (Reddy S. et al., Biochemistry, 34, pp10872-10878, 1995). 그것은 최종당화산물의 주성분 중 하나이며, 그것의 농도는 당뇨병 환자 피부의 콜라겐에서 정상인 경우보다 두 배 증가하였다 (Dyer D. G. et al., J. Clin. Invest., 91, pp2463-2469, 1993). 당뇨 합병증 발병에서 최종당화산물의 다른 작용은 아직 명확하게 밝혀지지 않았다. 예를 들어 혈청에서 CML의 농도는 망막증이 있는 당뇨병 환자의 경우 증가하나 신증을 가진 환자에서는 증가하지 않았다. 그러나 펜토시딘의 농도는 두 개의 군에서 모두 증가하는 것으로 나타났다.Third, there is pyrroline and CML (N-carboxy methyllysine) as final glycation products without cross-linking. Pyralin is the ultimate glycation product without cross-linking and is found in plaques of human skin, plasma, and brain (Smith MA et al., Proc. Natl. Acad Sci., 91, pp 5710-5714, 1994 ), CML is produced by the oxidative degradation of perhaps the lipid product in the oxidation of polyunsaturated fatty acids by metal catalysts in the presence of protein (Reddy S. et al., Biochemistry, 34, pp10872-10878, 1995). It is one of the major components of the end glycated product, and its concentration has doubled compared to normal in collagen of diabetic skin (Dyer D. G. et al., J. Clin. Invest., 91, pp2463-2469, 1993). Other effects of the final glycated product in the onset of diabetic complications have not yet been elucidated. For example, the concentration of CML in serum was increased in diabetic patients with retinopathy but not in patients with nephropathy. However, the concentration of pentosidine was increased in both groups.
증가된 당화와 축적된 조직 최종당화산물은 효소의 활성을 변화시키고, 리간드 결합을 감소시키며, 면역성을 변화시킬 수 있기 때문에 당뇨 합병증과 관련이 있다 (Vlassara H. and Dalace M. R., J. Intern. Med., 251, pp87-101, 2002). 최근 연구에서 당뇨병 환자에서 ACE-면역 복합체를 형성하는 혈청 최종당화산물의 작용에 대한 자동 항체가 존재함을 보고하였고, 그것은 아테롬성동백경화증에 중요한 역할을 한다. 당화로 유도된 자유라디칼은 단백질 분해와 핵산과 지질에 산화를 일으킬 수 있다 (Baynes J. W., Diabates, 40, pp405-412, 1991; Turk Z. et al., Clin. Chim. Acta., 303, pp105-115, 2001). 또한 최종당화산물은 인지질에서도 형성될 수 있는데, 포스파티딜에탄올아민 (phosphatidylethanolamine)과 포스파티딜세린(phosphatidylserine) 잔기와 같은 인지질에서 포도당과 아미노산 사이에 직접적인 반응에 의해서 지질 과산화를 유도할 수 있다 (Vlassara H., Annal. Med., 28, pp419-426, 1996).Increased glycation and accumulated tissue glycosylation products are associated with diabetic complications because they alter the activity of enzymes, reduce ligand binding, and alter immune responses (Vlassara H. and Dalace MR, J. Intern. Med , 251, pp87-101, 2002). Recent studies have reported the presence of autoantibodies to the action of serum final glycation end products that form ACE-immune complexes in diabetic patients, which play an important role in atherosclerosis. Free radicals derived from glycation can cause proteolysis and oxidation of nucleic acids and lipids (Baynes JW, Diabates, 40, pp 405-412, 1991; Turk Z. et al., Clin. Chim. Acta., 303, -115, 2001). The final glycation products can also be formed in phospholipids, which can induce lipid peroxidation by direct reaction between glucose and amino acids in phospholipids such as phosphatidylethanolamine and phosphatidylserine residues (Vlassara H., Annal Med., 28, pp 419-426, 1996).
체내에는 3-DG를 불활성화 시키고, 최종당화산물의 생성을 억제하는 α-ketogluteraldehyde dehydrogenase와 같은 효소가 간에 존재하여 당화와 최종당화산물의 생성에 대한 체내를 보호할 수 있는 기전을 가지고 있으며 (Hata F. et al., Diabates Res. Clin., 5, pp5413, 1988), 항산화제는 당화로 유도된 자유라디칼에 대해 생체를 방어할 수 있는 기능을 가지고 있다. 그러나 in vivo에서 당화와 최종당화산물에 대한 자연적 방어의 효능은 아직 잘 알려져 있지 않다.In the body, 3-DG is inactivated and enzymes such as α-ketogluteraldehyde dehydrogenase, which inhibits the production of final glycation products, are present in the liver to protect the body against glycation and the production of final glycation products (Hata F. et al., Diabates Res. Clin., 5, pp5413, 1988), antioxidants have the ability to defend the body against free radicals induced by glycation. However, the efficacy of natural defense against glycosylation and end glycation products in vivo is not yet known.
당뇨병 환자에서 치료의 목적은 식이 조절에 의해 고혈당증을 감소시키는 것이다. 그러나, 식이 조절은 어렵기 때문에 약리학적인 효과를 가진 화합물을 사용하여 고혈당을 저하시키고, 고혈당으로 인한 최종당화산물 생성을 감소시킬 수 있다. 일단 당뇨병이 발생하여 혈당이 금방 떨어지지 않으면 당뇨합병증 예방제를 복용하여 당뇨합병증을 예방하여야 한다.The goal of treatment in diabetic patients is to reduce hyperglycemia by dietary control. However, since dietary control is difficult, compounds with pharmacological effects can be used to lower hyperglycemia and reduce the production of final glycation end products due to hyperglycemia. Once the diabetes has occurred and blood sugar does not drop quickly, diabetic complications should be prevented to prevent diabetic complications.
현재, 단백질 당화 억제제로 유일한 합성제제인 아미노구아니딘 HCl(aminoguanidine HCl)은 친핵성 히드라진(hydrazine)으로 축합반응의 산물과 결합하여 단백질과의 교차결합을 방지함으로써 최종당화산물의 생성을 억제하여 합병증으로 진전되는 것을 지연 또는 방지한다(Brownlee M et al., Sciences, 232, 1629-1632, 1986; Edelstein D et al., Diabetes, 41, 26-29, 1992). 아미노구아니딘 HCl은 당뇨합병증의 예방 및 치료에 가장 유망한 합성 의약품으로 제 3상 임상실험까지 진행되었으나, 장기간 투여 시 독성이 유발되는 문제점이 있어 보다 안전한 약제의 개발이 요망되고 있다.
At present, aminoguanidine HCl, which is the only synthetic agent for inhibiting protein glycation, is a nucleophilic hydrazine which binds to the product of the condensation reaction to prevent cross-linking with the protein, thereby inhibiting the production of the final glycation end product. (Brownlee M et al., Sciences, 232, 1629-1632, 1986; Edelstein D et al., Diabetes, 41, 26-29, 1992). Aminoguanidine HCl is the most promising synthetic drug for the prevention and treatment of diabetic complications. It has been progressed to Phase III clinical trials. However, it has been desired to develop more safe drugs due to toxicity during long-term administration.
한편, 다시마(Laminaria)는 갈조식물 다시마목 다시마과의 한 속으로, 전통적인 해조식품이다. 한국, 일본, 캄차카반도, 사할린섬 등의 태평양 연안에 분포하며, 크기는 1.5 내지 3.5m 이고, 나비는 25 내지 40cm 이다. 다시마속 식물은 태평양 연안에 20여 종이 자라고 있으며, 10 m 이상의 큰 종류도 있는데, 주요 종으로는 참다시마(L. japonica), 오호츠크다시마(L.ochotensis), 애기다시마(L. religiosa)등이 있다. 다시마에는 카로틴류, 크산토필류, 엽록소 등의 여러 가지 색소 외에 탄소동화작용으로 만들어지는 마니트, 라미나린 등의 탄수화물과 세포벽의 성분인 알긴산이 많이 들어 있고, 요오드, 비타민 B2, 글루탐산 등의 아미노산이 들어 있다고 알려져 있다.
On the other hand, sea tangle (Laminaria) is a traditional seaweed food, which is a genus of the sea tangle of kelp. It is distributed in the Pacific coasts of Korea, Japan, Kamchatka Peninsula and Sakhalin Island, and is 1.5 to 3.5 m in size and 25 to 40 cm in butterfly. There are more than 20 species in the Pacific coast, and there are also large species of more than 10m in diameter. The main species are L. japonica, L.ochotensis and L. religiosa. have. In kelp, there are many pigments such as carotene, xanthophyll and chlorophyll, carnitals such as mannit and laminarin, which are made by carbon assimilation, and alginic acid, which is a component of cell wall, and amino acids such as iodine, vitamin B2 and glutamic acid Is known to contain.
그러나, 알도스 환원 효소 및 최종당화산물을 억제함으로써 당뇨성 합병증 질환을 치료 및 예방할 수 있는 다시마 추출물을 함유하는 조성물에 관한 보고는 없는 실정이다. 따라서, 본 발명자는 알도즈 환원 효소 및 회종당화산물 생성을 억제하여 당뇨성 합병증을 예방 및 치료하는 새로운 추출물 및 폴피린계 화합물을 다시마에서 분리 동정한 후, 그 효과를 확인하여 본 발명을 완성하였다.
However, there is no report on a composition containing kelp extract which can treat and prevent diabetic complication diseases by inhibiting aldose reductase and final glycation products. Accordingly, the present inventors have isolated and identified novel extracts and polyphosphoric acid compounds that inhibit the production of aldose reductase and glycosylated products and prevent and treat diabetic complications from kelp, and confirmed the effects thereof, thereby completing the present invention .
본 발명은 상기와 같은 과제를 위하여 알도즈 환원 효소 및 최종당화산물 생성 억제 활성을 갖는 새로운 추출물 및 화합물로서 다시마 추출물 또는 이로부터 분리한 폴피린계 화합물을 유효 성분으로 함유하는 약학 조성물을 제공하는 것을 목적으로 한다.
DISCLOSURE OF THE INVENTION The present invention provides a novel extract and a compound having an aldose reductase and a final glycoprotein production inhibiting activity as described above and a pharmaceutical composition comprising an extract of sea tangle or a polypyrin compound isolated therefrom as an active ingredient The purpose.
본 발명은 상기와 같은 목적을 위하여 다시마(Laminaria japonica) 추출물 또는 이로부터 분리한 폴피린계 화합물을 유효 성분으로 함유하는 당뇨성 합병증의 예방 및 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of diabetic complications comprising Laminaria japonica extract or a polypyrin compound isolated therefrom as an active ingredient.
본 발명은 또한, 본 발명에 따른 다시마(Laminaria japonica) 추출물 또는 이로부터 분리한 폴피린계 화합물을 유효 성분으로 함유하는 당뇨성 합병증 예방 및 치료용 식품 조성물을 제공한다.
The present invention also provides a food composition for the prevention and treatment of diabetic complications, comprising the extract of Laminaria japonica according to the present invention or a polypyrin compound isolated therefrom as an active ingredient.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명에 있어서, 상기 폴리핀계 화합물은 하기 화학식 1 로 나타내어지는 페오폴바이드 에이 또는 화학식 2로 나타내어지는 페오피틴 에이인 것을 특징으로 한다. In the present invention, the polyphenolic compound is characterized by being a perofibrone aide represented by the following formula (1) or a pheophytin a represented by the following formula (2).
<화학식 1>≪
<화학식 2>(2)
본 발명에 있어서, 상기 다시마 추출물은 다시마를 C1 내지 C4의 저급 알콜로 추출한 다시마 조추출물인 것을 특징으로 한다.In the present invention, the kelp extract is characterized by being seaweed extract extracted from lower alcohols C1 to C4.
본 발명에 있어서, 상기 다시마 조추출물은 다시마 에탄올 추출물인 것을 특징으로 한다.In the present invention, the kelp crude extract is characterized by being seaweed ethanol extract.
본 발명에 있어서, 상기 다시마 추출물은 다시마 조추출물을 ｎ-헥산, 에틸 아세테이트 또는 n-부탄올로 구성된 군으로부터 선택된 비극성 용매로 추출한 다시마 비극성 용매 가용성 추출물인 것을 특징으로 한다.
In the present invention, the sea tangle extract is characterized by being a sea tangle non-polar solvent-soluble extract obtained by extracting a sea tangle extract with a non-polar solvent selected from the group consisting of n-hexane, ethyl acetate and n-butanol.
본 발명의 다시마 조추출물, 다시마 비극성 용매 가용성 추출물은 다음과 같이 수득될 수 있다.The kelp crude extract, kelp non-polar solvent soluble extract of the present invention can be obtained as follows.
우선 다시마를 동결건조한 후 마쇄하여 분말화한다. 이어서, 다시마 분말을 용매로 추출한다. 이 때 추출 용매로는 물, 메탄올, 에탄올 및 부탄올과 같은 저급 알콜 또는 이들의 약 1:0.1 내지 1:10의 혼합비를 갖는 혼합 용매를 다시마 건조 중량의 약 3 내지 20배, 바람직하게는 약 3 내지 5배의 양으로 사용할 수 있고, 이중 에탄올이 바람직하다. First, the sea tangle is freeze-dried and then ground and pulverized. Then, the tangle powder is extracted with a solvent. The extraction solvent may be water, a lower alcohol such as methanol, ethanol, and butanol, or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 thereof, to about 3 to 20 times, preferably about 3 To 5 times, and ethanol is preferred.
다시마의 추출은 20 내지 100 ℃, 바람직하게는 20 내지 70 ℃의 추출 온도에서 약 0.5시간 내지 2일, 바람직하게는 1 시간 내지 1일 동안 수행할 수 있고, 추출 방법으로는 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등을 사용하여 1회 내지 5회, 바람직하게는 3회 연속 추출한 후, 감압여과하고 여액을 진공회전농축기로 20 내지 100 ℃, 바람직하게는 40 내지 70 ℃에서 감압농축하여 물, 저급알콜 또는 이들의 혼합용매에 가용한 다시마 조추출물을 수득할 수 있다.The extraction of kelp can be carried out at an extraction temperature of 20 to 100 DEG C, preferably 20 to 70 DEG C for about 0.5 to 2 days, preferably 1 to 1 day, and extraction methods include hot water extraction, , Followed by filtration under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 占 폚, preferably 40 to 70 占 폚 by a vacuum rotary condenser To obtain a sea tangle extract which is soluble in water, lower alcohols or a mixed solvent thereof.
이어서, 다시마 조추출물로부터 비극성용매 가용추출물을 추출한다. Then, the non-polar solvent-soluble extract is extracted from the sea tangle extract.
상기 다시마 조추출물을 물에 현탁한 후, n-헥산, 에틸아세테이트, n-부탄올 순으로 용매를 이용하여 비극성용매 가용추출물을 얻을 수 있다. After the kelp crude extract is suspended in water, a non-polar solvent-soluble extract can be obtained by using a solvent in the order of n-hexane, ethyl acetate and n-butanol.
보다 구체적으로는 다시마 조추출물에 n-헥산, 바람직하게는 n-헥산: 물: 에탄올 혼합물을 일정 비율, 바람직하게는 10: 9: 1로 혼합하여 n-헥산 가용성 분획물 및 수가용성 분획물을 수득할 수 있다. 이어서, 상기 수가용성 분획물에 에틸아세테이트를 가하여 에틸 아세테이트 가용성 분획물, 즉 본 발명의 에틸 아세테이트 가용성추출물 및 수가용성 분획물을 수득할 수 있고, 마지막으로 상기 수가용성 분획물을 부탄올로 추출하여 부탄올 가용성 분획물을 수득할 수 있다. More specifically, a mixture of n-hexane, preferably n-hexane: water: ethanol, is added to the sea tangle extract to a certain ratio, preferably 10: 9: 1 to obtain a n-hexane soluble fraction and a water soluble fraction . Ethyl acetate is then added to the water soluble fraction to obtain an ethyl acetate soluble fraction, that is, an ethyl acetate soluble extract and a water soluble fraction of the present invention. Finally, the water soluble fraction is extracted with butanol to obtain a butanol soluble fraction can do.
이어서, 상기 비극성용매 가용성추출물로부터 폴피린계 화합물을 분리해 낼 수 있다. 우선, 상기 비극성용매 가용성추출물을 유기용매로 컬럼크로마토그래피하여 분획물을 수득하고, 이 분획물을 실리카 겔 칼럼 크로마토그래피로 분리한 후, 정제하여 본 발명에 따른 폴피린계 화합물을 분리 및 동정할 수 있다. 본 발명에 따른 바람직한 비극성용매 가용성추출물인 n-헥산 분획물에 대하여 설명하면, 우선 n-헥산 : 에틸 아세테이트의 혼합용매, 바람직하게는 n-헥산 : 에틸 아세테이트의 20:1 내지 0:100 의 혼합 용매로 시간당 일정용량, 바람직하게는 1000 ㎖의 속도로 칼럼 크로마토그래피를 수행하여 HF1 부터 HF18까지 18개의 분획물을 수득할 수 있다.The polypyrinic compound can then be isolated from the non-polar solvent soluble extract. First, the nonpolar solvent-soluble extract is subjected to column chromatography using an organic solvent to obtain fractions. The fractions are separated by silica gel column chromatography and then purified to isolate and identify the polypyrin compound according to the present invention . The n-hexane fraction, which is a preferred non-polar solvent-soluble extract according to the present invention, is firstly mixed with a mixed solvent of n-hexane: ethyl acetate, preferably n-hexane: ethyl acetate in a mixed solvent of 20: Column chromatography at a rate of a predetermined volume per hour, preferably 1000 ml, to obtain 18 fractions from HF1 to HF18.
상기 HF9 분획물은 다시 다이아이온 HF-20 칼럼 크로마토그래피(Diaion HP-20 column chomatography)로 에탄올, 메탄올, 그리고 아세톤을 이용하여 HF9-1 부터 HF 9-4까지 4개의 분획물을 수득할 수 있다. 상기 HF 9-4 분획물을 다시 실리카겔 칼럼 크로마토그래피로 n-헥산 : 아세톤의 10:1 -> 0 : 1 의 혼합 용매로 분리한 후, RP-18로 정제하여 상기 화학식 1, 화학식 2로 표시되는 화합물을 분리 및 동정할 수 있다.
The HF9 fraction can be further fractionated into four fractions from HF9-1 to HF9-4 using ethanol, methanol, and acetone with Diaion HP-20 column chromatography (Diaion HP-20 column chromatography). The HF 9-4 fraction was further purified by silica gel column chromatography using a mixed solvent of n-hexane: acetone 10: 1 -> 0: 1 and then purified by RP-18 to obtain the HF 9-4 fraction Compounds can be isolated and identified.
상기에서 수득된 본 발명에 따른 다시마 조추출물 또는 비극성용매 가용성추출물, 또는 그로부터 분리된 폴피린계 화합물의 알도즈 환원효소 및 최종당화산물 생성 억제 효능을 조사하기 위하여, 알도즈 환원효소 및 최종당화산물 생성반응을 이용하여 이들의 생성 억제효과를 측정한 결과, 본 발명에 따른 다시마 조추출물 또는 비극성 용매 가용성추출물, 또는 그로부터 분리된 폴피린계 화합물이 알도즈 환원효소 및 최종당화산물 생성을 억제함을 확인하였다.
In order to investigate the aldose reductase activity and the inhibitory effect on the final glycation endogenesis of the seaweed extract or the non-polar solvent-soluble extract or the polyphosphoric acid compound isolated therefrom according to the present invention, the aldose reductase and the final glycation product As a result of measuring the inhibitory effect of these compounds by the production reaction, it was found that the kelp crude extract or the non-polar solvent soluble extract according to the present invention, or the polypyrin compound isolated therefrom suppressed the production of aldose reductase and final glycation products Respectively.
또한, 본 발명은 알도즈 환원효소 및 최종당화산물 생성을 억제 활성을 갖는 다시마 조추출물, 다시마 비극성용매 가용추출물 또는 다시마 폴피린계 화합물과 같은 다시마 추출물을 유효성분으로 포함하는, 당뇨성 합병증 예방 또는 치료용 약학조성물을 제공한다.The present invention also relates to a method for preventing or treating diabetic complications comprising an aldose reductase and a kelp extract having an activity of inhibiting the production of a final glycation end product, a kelp non-polar solvent extract or a sea tangle extract, A pharmaceutical composition for therapeutic use is provided.
상기 본 발명에 따른 다시마 조추출물 또는 비극성용매 가용성추출물, 또는 그로부터 분리된 폴피린계 화합물은 독성 및 부작용은 거의 없으므로 예방 목적으로 장기간 사용 시에도 안심하고 사용할 수 있다.The kelp crude extract or the non-polar solvent soluble extract according to the present invention or the polypyrin compound separated therefrom has little toxicity and side effects, so that it can be safely used for prolonged use for preventive purposes.
본 발명에 따른 약학조성물은 다시마 조추출물 또는 비극성용매 가용성추출물, 또는 그로부터 분리된 폴피린계 화합물을 총 조성물 중량에 대하여 0.5 내지 50 중량%로 함유한다.The pharmaceutical composition according to the present invention contains a sea tangle extract or a non-polar solvent soluble extract or a polypyrin compound separated therefrom in an amount of 0.5 to 50% by weight based on the total weight of the composition.
또한, 본 발명에 따른 약학조성물은 통상의 약학조성물의 제조에 사용되는 적절한 담체, 부형제 및 희석제를 추가로 포함할 수 있다. 사용가능한 이들 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오즈, 메틸 셀룰로오즈, 미정질 셀룰로오즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 타르크, 스테아린산 마그네슘 및 광유 등을 포함할 수 있다.In addition, the pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents for use in the preparation of conventional pharmaceutical compositions. These carriers, excipients and diluents which can be used include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
또한, 본 발명에 따른 추출물 또는 화합물의 약학적 투여 형태는, 이들의 약학적으로 허용가능한 염의 형태로 사용할 수 있고, 단독으로 또는 다른 약학적 활성 화합물과 결합한 형태 뿐만 아니라 적절한 집합으로 사용하는 것도 가능하다.In addition, the pharmaceutical form of the extract or the compound according to the present invention can be used in the form of a pharmaceutically acceptable salt thereof, and can be used not only as a form which is used alone or in combination with other pharmaceutically active compounds, Do.
본 발명에 따른 약학조성물의 제형화는, 통상의 제형화에 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 수행될 수 있다.The formulation of the pharmaceutical composition according to the present invention can be carried out using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like, which are commonly used for formulation.
경구투여용 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되고, 이들 고형제제는 상기 본 발명에 따른 추출물 또는 화합물을 적어도 하나 이상의 부형제, 바람직하게는 전분, 칼슘 카보네이트, 수크로스 또는 락토오스, 젤라틴 등과 혼합하여 제조된다. 또한, 이들 외에도 마스네슘 스테아레이트, 타르크와 같은 윤활제를 사용할 수도 있다.The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing the above-mentioned extract or compound according to the present invention with at least one excipient, preferably starch, calcium carbonate, sucrose Or lactose, gelatin and the like. In addition to these, a lubricant such as magnesium stearate or talc may be used.
경구투여용 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 포함되고, 통상적으로 사용되는 물, 액상 파라핀 이외에 여러 가지 부형제, 예를 들어 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are usually used.
비경구투여를 위한 제제로는 멸균 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성 용제 또는 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레에이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제로는 당업계에서 통상적으로 사용되는 위텝솔, 마크로골, 트윈61, 카카오지, 라우린지, 글리세로젤라틴 등을 사용할 수 있다.Agents for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the left agent, there can be used, for example, witepsol, macrogol, tween 61, cacao paper, laurin, and glycerogelatin which are commonly used in the art.
또한, 본 발명에 따른 추출물 또는 화합물의 투여량은 환자의 상태, 체중, 질병의 상태, 약물 형태, 투여경로 및 기간에 따라 다양할 수 있으나, 당업자들에 의해 적절하게 선택되어질 수 있다. 그러나, 바람직한 약리효과를 위하여, 본 발명에 따른 추출물 또는 화합물은 1 일 0.0001 내지 100 mg/kg으로 투여하는 것이 바람직하다.In addition, the dose of the extract or the compound according to the present invention may vary depending on the condition of the patient, the body weight, the disease state, the drug form, the administration route and the period, but may be appropriately selected by those skilled in the art. However, for the desired pharmacological effect, the extract or the compound according to the present invention is preferably administered at 0.0001 to 100 mg / kg per day.
투여 횟수는 1일 1회 투여할 수도 있고, 수회로 나누어 투여할 수도 있다. 하지만 이러한 투여량이 본 발명의 범위를 한정하거나 제한하는 것은 아니다.
The number of doses may be administered once a day or divided into several doses. However, such dosages do not limit or limit the scope of the present invention.
또한, 본 발명은 알도즈 환원효소 및 최종당화산물 생성을 억제활성을 갖는 다시마 조추출물, 다시마 비극성용매 가용추출물 또는 다시마 폴피린계 화합물과 같은 다시마 추출물을 유효성분으로 포함하는, 당뇨성 합병증 예방 또는 치료용 식품 조성물을 제공한다.The present invention also relates to a method for preventing or treating diabetic complications comprising an aldose reductase and a kelp extract having an activity of inhibiting the production of a final glycation end product, a kelp non-polar solvent extract or a sea tangle extract, A therapeutic food composition is provided.
상기의 본 발명에 따른 식품 조성물은 식품에 침지, 분무 또는 혼합하여 첨가할 수 있으며, 이러한 첨가 비율은 식품 전체 중량에 대하여 0.01 내지 10 중량%의 범위에서 선택되는 것이 일반적이다.The food composition according to the present invention can be added to the food by immersion, spraying or mixing, and the addition ratio is generally selected in the range of 0.01 to 10% by weight based on the total weight of the food.
상기 식품은 과일, 야채, 과일이나 야채의 건조제품이나 절단제품, 과일쥬스, 야채쥬스, 이들의 혼합 쥬스이거나 칩류, 면류, 축산가공식품, 수산가공식품, 유가공식품, 발효유식품, 곡류식품, 미생물발효식품, 제과제빵, 양념류, 육가공류, 산성음료수, 감초류, 허브류 중 어느 하나 이상을 포함한다.
The food may be a dry product or a cut product of a fruit, a vegetable, a fruit or a vegetable, a fruit juice, a vegetable juice, a mixed juice thereof, or a chip, a noodle, a livestock processed food, a marine processed food, a milk processed food, a fermented milk food, Fermented food, confectionery bakery, condiments, meat products, acid drinks, licorice, and herb.
본 발명에 따른 다시마 추출물 또는 그로부터 분리된 폴피린의 화합물은 알도즈 환원효소 및 최종당화산물 생성을 억제함으로써, 강력한 당뇨성 합병증 억제활성을 나타내고, 천연재료로서 독성 및 부작용은 거의 없어 안전성이 확보되어 있으므로 예방 목적으로 장기간 사용 시에도 안심하고 사용할 수 있는 효과가 있어, 당뇨성 합병증 예방 또는 치료용 약학조성물 또는 식품조성물로 유용하게 이용될 수 있다.
The sea tangle extract according to the present invention or the compound of papyrin isolated therefrom suppresses the production of aldose reductase and the final glycation products, thereby exhibiting a potent diabetic complication-inhibiting activity. As a natural material, there is little toxicity and side effects, Therefore, it can be used safely even for prolonged use for preventive purpose, and can be usefully used as a pharmaceutical composition or food composition for preventing or treating diabetic complications.
도 1A는 본 발명의 일 실시예에 따른 에탄올 조추출물을 HPLC 실시한 결과를 나타낸다.
도 1B는 본 발명의 일 실시예에 따른 n-헥산 추출물을 HPLC 실시한 결과를 나타낸다. FIG. 1A shows the result of HPLC analysis of an ethanol crude extract according to an embodiment of the present invention.
1B shows the results of HPLC analysis of n-hexane extract according to one embodiment of the present invention.
이하, 본 발명을 실시예에 의해 상세히 설명한다. Hereinafter, the present invention will be described in detail with reference to examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.
However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.
<< 실시예 1. 다시마 에탄올 조추출물의 제조>Example 1: Preparation of sea tangle ethanol crude extract >
본 발명에 사용한 다시마는 부산시 기장군 연근해에서 채집하여 사용하였다. 다시마는 동결건조 후, 마쇄하여 얻은 분말 5.5 ㎏을 에탄올 4 L를 넣고 70 ℃에서 일정시간 간격 (12h, 6h, 3h)으로 5회 반복하여 환류 냉각 추출한 후 여지(와트만사, 미국)로 감압 여과한 다음, 여과 추출물은 진공회전농축기로 40 ℃에서 에탄올을 제거한 후 추출된 잔사로서 다시마 조추출물 990.85 g을 수득하였으며, 이 중 2 g을 취하여 알도즈 환원효소 및 최종 당화산물 생성억제 활성 검색을 위한 시료로 사용하였다.
The kelp used in the present invention was collected and used in the coastal waters of Kusang district, Busan city. The kelp was dried by freeze-drying, and 5.5 kg of the powder obtained by grinding was added to 4 L of ethanol and heated at 70 ° C for 5 hours with repeated refluxing and cooling at room temperature (12h, 6h, 3h) Then, the filtrate was extracted with 990.85 g of a sea tangle extract as an extracted residue after removing ethanol at 40 ° C with a vacuum rotary condenser. 2 g of the extract was used to obtain aldose reductase and final glycation endproduct inhibitory activity And used as a sample.
<실시예 2. 다시마 n-헥산 가용 추출물의 제조>≪ Example 2: Preparation of tincture of n-hexane soluble extract &
상기 실시예 1에서 얻은 다시마 조추출물 2ℓ에 n-헥산 2ℓ를 가하여 혼합한 후 3～4차례 분획하여 수가용성 분획물 2 L 및 n-헥산 가용성 분획물 2 L를 얻은 후 n-헥산 가용성 분획물을 건조하여 n-헥산 가용 추출물 100.05 g을 수득하여 시료로 사용하였으며, 이 중 2 g을 취하여 알도즈 환원 효소 억제 활성검색을 위한 시료로 사용하였다.
To 2 liters of the sea tangle extract obtained in Example 1, 2 L of n-hexane was added and mixed, followed by 3 to 4 fractions to obtain 2 L of the water-soluble fraction and 2 L of the n-hexane soluble fraction, and then the n-hexane soluble fraction was dried 100.05 g of the n-hexane soluble extract was obtained and used as a sample. 2 g of the extract was used as a sample for searching the aldose reductase inhibitory activity.
<실시예 3. 다시마 에틸 아세테이트 가용 추출물의 제조>≪ Example 3: Preparation of tallow ethyl acetate-soluble extract >
상기 실시예 2에서 얻은 수가용성 분획물 2 ℓ에 에틸 아세테이트 2 ℓ를 가하여 혼합한 후 3 ~ 4차례 분획하여 수가용성 분획물 2 ℓ및 에틸 아세테이트 가용성 분획물 2 ℓ를 얻은 후, 이 에틸 아세테이트 가용성 분획물을 건조하여 에틸 아세테이트 가용 추출물 1.6 g을 수득하여 시료로 사용하였으며, 이 중 0.2 g을 취하여 알도즈 환원효소 억제 활성검색을 위한 시료로 사용하였다.
To 2 liters of the water-soluble fraction obtained in Example 2, 2 L of ethyl acetate was added and mixed, followed by 3 to 4 fractions to obtain 2 L of the water soluble fraction and 2 L of the ethyl acetate soluble fraction, and the ethyl acetate soluble fraction was dried , And 1.6 g of the ethyl acetate soluble extract was obtained and used as a sample. 0.2 g of the extract was used as a sample for searching the aldose reductase inhibitory activity.
<실시예 4. 다시마 n-부탄올 가용 추출물의 제조>≪ Example 4: Preparation of tallow n-butanol soluble extract >
상기 실시예 3에서 얻은 수가용성 분획물 2 ℓ에 부탄올 2 ℓ를 가하여 혼합한 후 3 ~ 4차례 분획하여 수가용성 분획물 2 ℓ및 부탄올 가용성 분획물 2 ℓ를 각각 얻은 후, 수가용성 분획물 및 n-부탄올 가용성 분획물을 건조하여 수가용 추출물 및 n-부탄올 가용 추출물 36.52 g을 수득하여 시료로 사용하였으며, 이 중 2 g을 취하여 알도즈 환원 효소 억제 활성 검색을 위한 시료로 사용하였다.
2 L of butanol was added to 2 L of the water-soluble fraction obtained in Example 3, followed by 3 to 4 fractions to obtain 2 L of the water-soluble fraction and 2 L of the butanol-soluble fraction, The fractions were dried to obtain 36.52 g of water extract and n-butanol soluble extract, which was used as a sample. 2 g of the extract was used as a sample for screening for aldose reductase inhibitory activity.
실험예Experimental Example 1. 다시마 1. Kelp 조추출물Crude extract 및 각 용매 And each solvent 분획물의Fraction 알도즈Aldoz 환원효소 ( Reducing enzyme ARAR ) 억제활성) Inhibitory activity
다시마 조추출물 및 각 용매 분획물의 알도즈 환원효소 억제활성을 측정하기 위한 효소원의 조제는 Hayman and Kinoshite (Hayman S. and Kinoshite I. H., J. Biol. Chem., 240, pp877-882, 1965)가 사용한 방법을 수정하여 실시하였다.Preparation of an enzyme source for measuring aldose reductase inhibitory activity of kelp crude extract and each solvent fraction was carried out in accordance with Hayman and Kinoshite (Hayman S. and Kinoshite IH, J. Biol. Chem., 240, pp 877-882, 1965) The method used was modified.
먼저, 효소원을 제조하는데, 흰쥐의 수정체를 적출하고 수정체 1개당 0.5 ㎖ 정도의 인산염 완충액(pH 6.2)를 가하여 균질화하였다. 이를 4 ℃에서 10,000 rpm으로 20분간 원심 분리한 후 그 상등액을 취하여 황산 암모늄으로 40 %까지 포화시키고 원심 분리한 상등액을 취하여 다시 70 %가 되도록 황산 암모늄을 가하여 1시간 가량 저어준 다음 원심 분리하여 얻어진 펠렛을 최소량의 인산염 완충액에 현탁하여 1일 정도 투석한 다음, 효소원으로 하였다. 반응 완충액인 인산 칼륨 완충액 (pH 7.0), 효소원, NADPH (1.6 mM ; Sigma, St. Louis, MO, USA), DMSO에 녹인 시료, 기질로 DL-글리세르알데하이드(DL-glyceraldehyde) (10 mM : Sigma, St. Louis, MO, USA) 순으로 셀에 넣어 U/V 분광광도계(ultrospec 2100 pro, Amersham Biosciences, Sweden)를 이용하여 340 nm 에서 NADPH 흡광도의 감소율을 측정하였다. First, to prepare the enzyme source, the lens of the rat was extracted and homogenized by adding 0.5 ml of phosphate buffer (pH 6.2) per lens. The supernatant was centrifuged at 10,000 rpm for 20 minutes at 4 ° C. The supernatant was then saturated with ammonium sulfate to 40%. The supernatant was centrifuged. Ammonium sulfate was added again to 70% The pellet was suspended in a minimum amount of phosphate buffer solution, dialyzed for about one day, and used as an enzyme source. A sample dissolved in DMSO and a solution of DL-glyceraldehyde (10 mM) were added to a reaction buffer solution (pH 7.0), enzyme source, NADPH (1.6 mM; Sigma, St. Louis, Mo., USA) (Ultrospec 2100 pro, Amersham Biosciences, Sweden) was used to measure the rate of decrease of NADPH absorbance at 340 nm.
대조군은 시료를 녹이지 않은 DMSO 만을 사용하여 측정하고, 양성 대조군은 퀘르세틴(Quercetin : Sigma, St. Louis, MO, USA)을 이용하여 농도별로 측정하였다. 알도즈 환원제 억제활성 실험 % 는 하기의 수학식 1로 구하였고, 선회귀 방정식을 사용하여 통계처리 하였다. In the control group, the samples were measured using only non-dissolved DMSO, and the positive control group was determined by quercetin (Quercetin: Sigma, St. Louis, MO, USA). % Of the aldose reductase inhibitory activity was calculated by the following
<수학식 1>&Quot; (1) "
% 억제율 = 1-(시료의 흡광도-대조군의 흡광도)/표준흡광도×100% Inhibition rate = 1- (absorbance of sample-absorbance of control) / standard absorbance x 100
그 결과, 표 1에서 나타낸 바와 같이 에틸 아세테이트 가용성 분획물에서 억제율이 50.83 ± 0.12 % 로 두드러진 활성을 나타내었다. 다음으로 n-부탄올, n-헥산 가용성 분획물이 31.85 ± 1.01, 30.24 ± 0.36 % 로 두드러진 활성을 나타내었다.As a result, as shown in Table 1, the ethyl acetate soluble fraction exhibited remarkable activity with an inhibition rate of 50.83 ± 0.12%. Then, the soluble fraction of n - butanol and n - hexane showed a remarkable activity of 31.85 ± 1.01 and 30.24 ± 0.36%.
<< 실시예Example 5. 5. 폴피린계Paul Pyrin system 화합물의 분리> Separation of compounds >
상기 실시예 2의 다시마 n-헥산 가용 추출물 100.05 g 중 46 g 을 전개용매로서 n-헥산 : 에틸 아세테이트의 20:1 과 0:100 의 혼합 용매를 사용하여 칼럼 크로마토그래피를 수행하였다. Column chromatography was performed using 46 g of 100.05 g of the ternary n-hexane soluble extract of Example 2 as a developing solvent in a mixed solvent of n-hexane: ethyl acetate at 20: 1 and 0: 100.
고정상으로는 키에셀 겔 60 (Kiesel gel 60, 230-400 매쉬)을 사용하여 시간당 1,000 mL 씩 분획을 수행하여 18개의 하부 분획물 (EF01～EF10)로 나누었다. HF09 (2.3 g)을 에탄올, 메탄올, 그리고 아세톤으로 다이아이온 칼럼 크로마토그래피를 수행하여 4개의 하부 분획물 (HF9-1～HF9-4)로 나누었다. HF09-4를 n-헥산 : 아세톤의 10:1 -> 0:1 의 혼합 용매를 키에셀 겔 칼럼 크로마토그래피와 디클로로메탄 : 메탄올 : 물의 8:22:1 혼합 용매로 RP-18로 정제하여 화합물 1과 2 를 분리하였다. HF 10(1.9 g)에서도 동일한 방법으로 화합물 2를 얻었다.
For the stationary phase, Kiesel gel 60 (Kiesel gel 60, 230-400 mesh) was used to fractionate 1,000 mL per hour and divided into 18 lower fractions (EF01 to EF10). HF09 (2.3 g) was separated into four lower fractions (HF9-1 to HF9-4) by performing a diion column chromatography with ethanol, methanol and acetone. HF09-4 was purified by RP-18 with a mixed solvent of n-hexane: acetone 10: 1 -> 0: 1 in a mixed solvent of dichloromethane: methanol: water 8: 22: 1, 1 and 2 were separated.
각각의 화합물을 NMR 및 MS 등과 같은 각종 분석기기로 분석한 결과는 하기와 같다.The results of analysis of each compound by various analyzers such as NMR and MS are as follows.
(1) 화합물 1 : 페오폴바이드 에이 (pheophorbide a )(1) Compound 1: pheophorbide a:
<화학식 1>≪
물질의 성상 : 녹색의 무정형 고체Appearance of substance: green amorphous solid
물질의 분자식 :C35H36N4O5 Molecular formula of the substance: C 35 H 36 N 4 O 5
1 H-NMR(400 MHz, CDCl 3 ): δ 9.42(s, 10-H), 9.25(s, 5-H), 8.53(s, 20-H), 7.90(dd, J=18, 12Hz, 31-Hx), 6.13(d, J=11Hz, 32-H), 6.23(s, 132-H), 4.19(brd, J=8, 17-H), 4.43(q, J=9, 18-H), 3.63(q, J=9, 81-CH2), 3.55(s, 121-Me), 3.34(s, 21-Me), 3.13(s, 71-Me), 3.85(s, 132-CO2Me), 2.58(m, 171-CH2), 2.24(m, 172-CH2), 1.63(t, J=8, 82-Me), 1.81(d, J=7, 181-Me), 0.00(NH) 1 H-NMR (400 MHz, CDCl 3): δ 9.42 (s, 10-H), 9.25 (s, 5-H), 8.53 (s, 20-H), 7.90 (dd, J = 18, 12Hz, 3 1 -H x), 6.13 ( d, J = 11Hz, 3 2 -H), 6.23 (s, 13 2 -H), 4.19 (brd, J = 8, 17-H), 4.43 (q, J = 9, 18-H), 3.63 (q, J = 9, 8 1 -CH 2), 3.55 (s, 12 1 -Me), 3.34 (s, 2 1 -Me), 3.13 (s, 7 1 -Me ), 3.85 (s, 13 2 -
13 C-NMR(100 MHz, CDCl 3 ) : 11.1 (C-71), 12.0(C-21), 12.0(C-121), 17.3(C-82), 19.3(C-81), 23.0(C-181), 29.5(C-172), 30.7(C-171), 50.0(C-18), 50.9(C-17), 52.8(C-132, CO2Me), 64.6(C-132), 93.0(C-20), 97.4(C-5), 104.3(C-10), 105.0(C-15), 122.7(C-32), 128.8(C-13), 128.9(C-31), 128.9(C-12), 131.8(C-2), 136.0(C-3), 136.2(C-7), 136.4(C-4), 137.8(C-11), 142.0(C-1), 145.1(C-8), 149.5(C-9), 150.8(C-14), 155.5(C-6), 161.0(C-16), 169.6(C-132, CO2), 172.0(C-19), 177.8(C-172, CO2), 189.6(C-131) 13 C-NMR (100 MHz, CDCl 3): 11.1 (C-7 1), 12.0 (C-2 1), 12.0 (C-12 1), 17.3 (C-8 2), 19.3 (C-8 1 ), 23.0 (C-18 1 ), 29.5 (C-17 2), 30.7 (C-17 1), 50.0 (C-18), 50.9 (C-17), 52.8 (C-13 2,
(2)화합물 2 : 페오피틴 에이(pheophytin a)(2) Compound 2: pheophytin a < RTI ID = 0.0 >
물질의 성상 : 녹색의 무정형 고체Appearance of substance: green amorphous solid
물질의 분자식 : C55H74N4O6 Molecular formula of the substance: C 55 H 74 N 4 O 6
1 H-NMR(400 MHz, CDCl 3 ): δ 9.34(s, 10-H), 9.16(s, 5-H), 8.52(s, 20-H), 7.82(dd, J=18, 11.6Hz, 31-H), 6.19(dd, J=11.6, 1.6Hz, 32-CH2), 6.28(s, 132-H), 4.23(br.d, J=9, 17-H), 4.49(q, J=8, 18-H), 3.49(q, J=9, 81-CH2), 3.90(s, 121-Me), 3.33(s, 21-Me), 3.04(s, 71-Me), 3.64(s, 132-OMe), 2.50-2.64(m, 171-C), 2.21-2.36(m, 172-C), 1.49(t, J=8, 82-Me), 1.82(d, J=8, 181-Me) 1 H-NMR (400 MHz, CDCl 3 ) :? 9.34 (s, 10-H), 9.16 , 3 1 -H), 6.19 ( dd, J = 11.6, 1.6Hz, 3 2 -CH 2), 6.28 (s, 13 2 -H), 4.23 (br.d, J = 9, 17-H), 4.49 (q, J = 8, 18-H), 3.49 (q, J = 9, 8 1 -CH 2), 3.90 (s, 12 1 -Me), 3.33 (s, 2 1 -Me), 3.04 ( s, 7 1 -Me), 3.64 (s, 13 2 -OMe), 2.50-2.64 (m, 17 1 -C), 2.21-2.36 (m, 17 2 -C), 1.49 (t, J = 8, 8 2 -Me), 1.82 (d , J = 8, 18 1 -Me)
Phytygroup: 5.16(t, J=6.5, P2-H), 4.44(dd, J=12.9 6.5, P1-H), 1.88(m, P4-C), 1.59(s, P17-Me), 0.9-1.3(m, P5-P14-C), 0.85(m, P18, 19-CH3), 0.79(P16, 20-CH3) Phytygroup: 5.16 (t, J = 6.5, P2-H), 4.44 (dd, J = 12.9 6.5, P1-H), 1.88 (m, P4-C), 1.59 (s, P17-Me), 0.9-1.3 (m, P5-P14-C ), 0.85 (m, P18, 19-CH 3), 0.79 (P16, 20-CH 3)
13 C-NMR(100 MHz, CDCl 3 ): 11.0(C-71), 12.0(C-21), 12.0(C-121), 16.3(C-P17), 17.3(C-82), 19.2(C-81), 22.6(C-P20), 22.7(C-P16), 23.1(C-181), 24.4(C-P15), 24.7(C-P9), 24.9(C-P5), 27.9(C-P13), 29.8(C-172), 31.2(C-171), 32.6(C-P11), 32.7(C-P7), 36.6(C-P14), 37.2(C-P12), 37.3(C-P10), 37.3(C-P8), 39.3(C-P6), 39.8(C-P4), 50.1(C-18), 51.1(C-17), 52.8(C-132, OMe), 61.5(C-P1), 64.7(C-132), 93.0(C-20), 97.3(C-5), 104.2(C-10), 105.2(C-15), 117.7(C-P2), 122.6(C-32), 128.8(C-13), 128.8(C-31), 128.9(C-12), 131.8(C-2), 135.9(C-7), 136.0(C-3), 136.3(C-4), 137.8(C-11), 141.9(C-1), 142.8(C-P3), 145.0(C-8), 150.8(C-9), 149.6(C-14), 155.4(C-6), 161.2(C-16), 172.9(C-132, OH), 172.1(C-19), 172.9(C-172, CO2), 189.6(C-131)
13 C-NMR (100 MHz, CDCl 3): 11.0 (C-7 1), 12.0 (C-2 1), 12.0 (C-12 1), 16.3 (C-P17), 17.3 (C-8 2) , 19.2 (C-8 1) , 22.6 (C-P20), 22.7 (C-P16), 23.1 (C-18 1), 24.4 (C-P15), 24.7 (C-P9), 24.9 (C-P5 ), 27.9 (C-P13) , 29.8 (C-17 2), 31.2 (C-17 1), 32.6 (C-P11), 32.7 (C-P7), 36.6 (C-P14), 37.2 (C- P12), 37.3 (C-P10), 37.3 (C-P8), 39.3 (C-P6), 39.8 2, OMe), 61.5 (C -P1), 64.7 (C-13 2), 93.0 (C-20), 97.3 (C-5), 104.2 (C-10), 105.2 (C-15), 117.7 ( C-P2), 122.6 (C- 32 ), 128.8 (C-13), 128.8 (C- 31 ), 128.9 (C-3), 136.3 (C-4), 137.8 (C-11), 141.9 (C-1), 142.8 C-14), 155.4 (C -6), 161.2 (C-16), 172.9 (C-13 2, OH), 172.1 (C-19), 172.9 (C-17 2, CO 2), 189.6 (C -13 1 )
실험예Experimental Example 2. 2 개의 2. Two 폴피린계Paul Pyrin system 화합물( compound( porphyrinporphyrin relatedrelated compoundscompounds ) 의 ) Of 알도즈Aldoz 환원효소 ( Reducing enzyme ARAR ) 억제활성 ) Inhibitory activity
상기 실시예 5 에서 수득된 2가지 폴피린 화합물의 알도즈 환원효소에 대한 억제 활성을 상기 실험예 1과 같은 방법으로 실시하여, 그 결과를 하기 표 2에 나타내었다. The inhibitory activity of the two polypyrin compounds obtained in Example 5 on aldose reductase was measured in the same manner as in Experimental Example 1, and the results are shown in Table 2 below.
하기 표 2에 나타낸 바와 같이 페오폴바이드 에이는 알도즈 환원효소 억제활성에서 IC50 12.31±1.19 μM로 측정된 폴피린계 화합물 중 가장 높은 억제 활성을 나타낸 반면에, 페오피틴 에이는 알도즈 환원효소 억제활성을 나타내지 않았다. As shown in Table 2 below, peofolide-ai exhibited the highest inhibitory activity among the polypyrin compounds measured with an IC 50 of 12.31 ± 1.19 μM in the aldose reductase inhibitory activity, while pheophytin A- Showed no enzyme inhibitory activity.
Serial number
sample
실험예Experimental Example 3. 다시마 3. Kelp 조추출물Crude extract 및 각 용매 And each solvent 분획물의Fraction 최종당화산물The final glycation product ( ( AGEsAGEs ) 생성 억제활성) Production inhibitory activity
다시마 조추출물 및 각 용매 분획물의 최종당화산물 생성 억제 활성의 측정은 Vinson (Vinson J. A., J. Nutr. Biochem., 7, pp 659-663, 1996)가 사용한 방법을 수정하여 실시하였다.The determination of the activity of inhibiting the formation of the final glycation end product of the sea tangle extract and the respective solvent fractions was carried out by modifying the method used by Vinson (Vinson J. A., J. Nutr. Biochem., 7, pp 659-663, 1996).
AGE 반응 시약은 세균 증식을 막기 위해서 0.02% 아지드화 나트륨이 첨가된 50 mM 인산 나트륨 완충액 (pH 7.4)에 10 mg/ml의 소 혈청 알부민(bovine serum albumin) (Sigma, St. Louis, MO, USA)과 0.2 M 과당(fructose)과 0.2 M 글루코오스(glucose)를 넣어 만든다. 10% DMSO에 녹인 추출물과 분획물은 1600, 800, 400, 200, 100, 50 μg/ml의 농도로, 화합물은 200, 100, 50, 25 μg/ml 의 농도로 희석 시킨다. AGE 반응 시약 950 μl 에 반응시료 50 μl를 넣은 후, 37 ℃에서 7 일간 배양한다. 배양 후, 반응 생성물의 형광강도를 분광형광검출기 (spectrofluorometric detector)로 여기 파장과 방출 파장을 350 nm과 450 nm에서 측정한다. 최종당화산물 생성 억제활성 실험 % 는 하기의 수학식 2로 구하였고, 선회귀 방정식을 사용하여 통계처리 하였다.AGE reaction reagents were mixed with 10 mg / ml bovine serum albumin (Sigma, St. Louis, MO, USA) in 50 mM sodium phosphate buffer (pH 7.4) supplemented with 0.02% sodium azide to prevent bacterial growth. USA), 0.2 M fructose and 0.2 M glucose. Dilute the extracts and fractions dissolved in 10% DMSO at concentrations of 1600, 800, 400, 200, 100 and 50 μg / ml and the compounds at concentrations of 200, 100, 50 and 25 μg / ml. Add 50 μl of reaction sample to 950 μl of AGE reaction reagent, and incubate at 37 ° C for 7 days. After incubation, the fluorescence intensity of the reaction product is measured with a spectrofluorometric detector at excitation wavelengths and emission wavelengths of 350 nm and 450 nm. The percent inhibition activity of the final glycation end product was determined by the following formula (2) and statistically treated using linear regression equations.
<수학식 2>&Quot; (2) "
% 억제율 = 1-(시료의 흡광도-대조군의 흡광도)/표준흡광도×100% Inhibition rate = 1- (absorbance of sample-absorbance of control) / standard absorbance x 100
그 결과, 표 3에서 나타낸 바와 같이 에틸 아세테이트 가용성 분획물과 n-헥산 분획물에서 IC50 값이 150.32 ± 2.84 ㎍ /㎖ 과 167.65 ± 1.76 ㎍ /㎖ 로 강력한 최종당화산물 형성 억제 활성을 나타내었다. As a result, as shown in Table 3, the IC 50 values of the ethyl acetate-soluble fraction and the n-hexane fraction were 150.32 ± 2.84 μg / ml and 167.65 ± 1.76 μg / ml, respectively.
실험예 4. 2 개의 폴피린계 화합물 (porphyrin related compounds) 의 최종 당화산물 (AGEs) 생성 억제활성 EXPERIMENTAL EXAMPLE 4 Inhibitory activity on formation of the final glycation products (AGEs) of two porphyrin related compounds
상기 실시예 5에서 수득된 2 가지 폴피린계 화합물의 최종당화산물 생성 억제활성을 상기 실험예 3과 같은 방법으로 실시하여, 그 결과를 하기 표 4에 나타내었다. The activity of inhibiting the formation of the final glycation end products of the two polypyrin compounds obtained in Example 5 was measured in the same manner as in Experimental Example 3. The results are shown in Table 4 below.
하기 표 4에 나타낸 바와 같이 2 가지 폴피린계 화합물 중에 페오폴바이드 에이가 IC50 49.43±1.32 μM로 강력한 최종당화산물 생성 억제 활성이 나타났다. 다음으로 페오피틴 에이가 IC50 228.71 ± 0.07 μM 로 대조약물인 아미노구아니딘과 비교하였을 때, 훨씬 높은 억제활성을 가지는 것으로 나타났다.As shown in the following Table 4, among the two polypyrin compounds, perofolvide A had an IC 50 of 49.43 ± 1.32 μM, indicating a strong inhibitory activity against the formation of final glycation end products. Next, pheophytin A was found to have a much higher inhibitory activity when compared to the control drug aminoguanidine, with an IC 50 of 228.71 ± 0.07 μM.
Serial number
sample
실험예 5. 추출 용매에 따른 폴피린계 화합물 (porphyrin related compounds)의 추출 효능 분석 EXPERIMENTAL EXAMPLE 5. Extraction Efficiency of Porphyrin Related Compounds According to Extraction Solvents
상기 실시예 1 의 에탄올 조추출물과 상기 실시예 2의 다시마 n-헥산 추출물에 대해서 활성 성분의 정량 분석을 위하여 HPLC 분석을 실시하였다.The ethanol crude extract of Example 1 and the kelp n-hexane extract of Example 2 were subjected to HPLC analysis for quantitative analysis of active ingredients.
균질화 이후 5,000rpm에서 20분간 원심분리하고 상층액을 취해 질소로 flushing하였다. 위의 과정을 3 회 반복 회수하였고, 이후 2 mL 이동상을 첨가하고 2,000rmp에서 2분간 원심분리 이후 상층액을 0.45μm filter로 필터하여 2010pump(Jasco Europe S.R.L., Como, Italy)가 장착된 HPLC(Jasco Europ S.R.L., Como, Italy) system을 이용하여 분석하였다. After homogenization, the mixture was centrifuged at 5,000 rpm for 20 minutes, and the supernatant was taken and flushed with nitrogen. The above procedure was repeated three times, and then 2 mL of mobile phase was added. After centrifugation at 2,000 rpm for 2 minutes, the supernatant was filtered with a 0.45 μm filter and analyzed by HPLC (Jasco Europe SRL, Como, Italy) Europ SRL, Como, Italy) system.
HPLC의 분석조건은 UV 360 nm, 용매는 0.5M 암모니움 아세테이트와 메탄올의 80:20 혼합 용액 A, 메탄올과 아세톤의 70 : 30 혼합 용액 B로 만들고, A:B 100 : 0 혼합 용매를 35분간 흘려 보내고, ODS Hypersil column (5 um, 4.6 mm I.D. x 250 mm, 25 ℃), 그리고 유속 0.8 mL/min였다.The HPLC analysis conditions were as follows: UV 360 nm; solvent: a mixture of A: 80: 20 mixture of 0.5M ammonia acetate and methanol; A: 70: 30 mixture B of methanol and acetone; A: B 100: Flow rate, ODS Hypersil column (5 [mu] m, 4.6 mm ID x 250 mm, 25 [deg.] C) and a flow rate of 0.8 mL / min.
상기 실시예 1 의 에탄올 조추출물을 HPLC 실시한 결과를 도 1에, 상기 실시예 2의 다시마 n-헥산 추출물을 HPLC 실시한 결과를 도 2에 나타내었다. The results of HPLC analysis of the ethanol crude extract of Example 1 are shown in FIG. 1, and the results of HPLC of the ternary n-hexane extract of Example 2 are shown in FIG.
도 1, 도 2에서 보는 바와 같이 페오폴바이드 에이 피크 (피크 1)와 페오피틴 에이 피크 (피크 2)를 각각 확인할 수 있으며, 각각의 추출물에서 페오폴바이드 에이, 페오피틴 에이가 주요 화합물이며, 다시마 n-헥산 추출물의 경우 추출능이 더 뛰어남을 확인할 수 있다.(Peak 1) and pheophytin Apeak (peak 2), respectively, as shown in Fig. 1 and Fig. 2. As shown in Fig. 1 and Fig. 2, peofitide ae peak , And the extractability of kelp-n-hexane extract was superior.
Claims (3)
(1) 다시마(Laminaria japonica)를 건조하고 마쇄하여 분말화하는 단계;
(2) 상기 (1)단계에 의해 제조된 다시마 분말에 에탄올을 가하고, 70 ℃의 온도에서 환류냉각 추출한 후, 감압여과한 다음, 40 ℃에서 감압농축하여 에탄올 조추출물을 수득하는 단계;
(3) 상기 에탄올 조추출물로부터 가용성 분획물을 수득하는 단계; 및
(4) 상기 가용성 분획물로부터 하기 화학식 1 또는 화학식 2로 표시되는 폴피린계 화합물을 분리하는 단계를 포함하고,
상기 단계 (3)은 (a) 에탄올 조추출물에 n-헥산을 가하고 혼합한 후 분획하여 n-헥산 가용성 분획물을 수득하는 단계;
(b) 상기 (a) 단계에 의해 수득한 n-헥산 가용성 분획물에 에틸아세테이트를 가하고 혼합한 후 분획하여 에틸아세테이트 가용성 분획물을 수득하는 단계; 또는
(c) 상기 (b)단계에 의해 수득한 에틸아세테이트 가용성 분획물에 n-부탄올을 가하고 혼합한 후 분획하여 n-부탄올 가용성 분획물을 수득하는 단계를 포함하고,
상기 당뇨성 합병증은 당뇨성 백내장, 당뇨성 망막증, 당뇨성 각막증, 당뇨성 신경증 및 당뇨성 신증으로 이루어진 군으로부터 선택되는 것인, 폴피린계 화합물의 분리 방법.
<화학식 1>
<화학식 2>
1. A method for separating a polypyrin compound from a sea tangle extract for use in the prevention or treatment of diabetic complications, which has an activity of inhibiting the production of an aldose reductase and a final glycation end product,
(1) drying and crushing and pulverizing tallow (Laminaria japonica);
(2) adding ethanol to the kelp powder prepared in step (1), refluxing and cooling at 70 ° C, filtering under reduced pressure, and concentrating under reduced pressure at 40 ° C to obtain an ethanol crude extract;
(3) obtaining a soluble fraction from the ethanol crude extract; And
(4) separating the polypyrin compound represented by the following formula (1) or (2) from the soluble fraction,
The step (3) comprises the steps of: (a) adding n-hexane to an ethanol crude extract, mixing and fractionating to obtain a n-hexane soluble fraction;
(b) adding ethyl acetate to the n-hexane-soluble fraction obtained in the step (a), mixing and fractionating to obtain an ethyl acetate-soluble fraction; or
(c) adding n-butanol to the ethyl acetate-soluble fraction obtained by the step (b), mixing and fractionating to obtain an n-butanol soluble fraction,
Wherein the diabetic complication is selected from the group consisting of diabetic cataract, diabetic retinopathy, diabetic keratopathy, diabetic neuropathy, and diabetic nephropathy.
≪ Formula 1 >
(2)
상기 (3) 단계의 (a) 내지 (c)는 3회 내지 4회 실시되는 것을 특징으로 하는, 폴피린계 화합물의 분리 방법.
The method according to claim 1,
The method of separating a polyphosphoric acid compound according to any one of claims 1 to 3, wherein the steps (a) to (c) are carried out three to four times.
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