KR100515563B1 - Intestinal glucose absorption inhibitor isolated from Punica granatum and its purification method - Google Patents
Intestinal glucose absorption inhibitor isolated from Punica granatum and its purification method Download PDFInfo
- Publication number
- KR100515563B1 KR100515563B1 KR10-2003-0028664A KR20030028664A KR100515563B1 KR 100515563 B1 KR100515563 B1 KR 100515563B1 KR 20030028664 A KR20030028664 A KR 20030028664A KR 100515563 B1 KR100515563 B1 KR 100515563B1
- Authority
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- South Korea
- Prior art keywords
- pomegranate
- glucose
- glucose absorption
- methanol
- purification method
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 48
- 239000008103 glucose Substances 0.000 title claims abstract description 48
- 238000010521 absorption reaction Methods 0.000 title claims abstract description 39
- 235000014360 Punica granatum Nutrition 0.000 title claims abstract description 35
- 239000003112 inhibitor Substances 0.000 title abstract description 20
- 238000000034 method Methods 0.000 title abstract description 12
- 238000000746 purification Methods 0.000 title abstract description 11
- 230000000968 intestinal effect Effects 0.000 title description 2
- 244000294611 Punica granatum Species 0.000 title 1
- 241000219991 Lythraceae Species 0.000 claims abstract description 34
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 28
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 14
- 239000000284 extract Substances 0.000 claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
- 230000002401 inhibitory effect Effects 0.000 claims description 30
- 210000000813 small intestine Anatomy 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 3
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- 239000000401 methanolic extract Substances 0.000 abstract description 11
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- QUTFFEUUGHUPQC-ILWYWAAHSA-N (2r,3r,4s,5r)-3,4,5,6-tetrahydroxy-2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]hexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)NC1=CC=C([N+]([O-])=O)C2=NON=C12 QUTFFEUUGHUPQC-ILWYWAAHSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 235000009569 green tea Nutrition 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
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- 150000003385 sodium Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010033546 Pallor Diseases 0.000 description 1
- 229940126902 Phlorizin Drugs 0.000 description 1
- 241001083505 Punica Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000002269 analeptic agent Substances 0.000 description 1
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- 244000309464 bull Species 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
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- 230000004069 differentiation Effects 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
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- 230000037406 food intake Effects 0.000 description 1
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- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229940094952 green tea extract Drugs 0.000 description 1
- 235000020688 green tea extract Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000004093 hydrolase inhibitor Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
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- 235000011090 malic acid Nutrition 0.000 description 1
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- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 238000003808 methanol extraction Methods 0.000 description 1
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- 239000002420 orchard Substances 0.000 description 1
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- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- IOUVKUPGCMBWBT-UHFFFAOYSA-N phloridzosid Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-UHFFFAOYSA-N 0.000 description 1
- IOUVKUPGCMBWBT-GHRYLNIYSA-N phlorizin Chemical compound O[C@@H]1[C@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 IOUVKUPGCMBWBT-GHRYLNIYSA-N 0.000 description 1
- 235000019139 phlorizin Nutrition 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
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- 235000019698 starch Nutrition 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
-
- 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
- A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
Abstract
본 발명은 석류로부터 분리되어 당뇨의 유발과 밀접한 관련이 있는 포도당흡수저해제 및 그 정제방법에 관한 것으로 이러한 포도당흡수저해제는 석류의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획한 후 소수성 실리카겔 컬럼 크로마토그래피, 박층 크로마토그래피 및 HPLC 등 일련의 과정을 거쳐 정제되었으며 추출물, 정제단계의 물질, 정제물 등은 포도당 흡수를 저해하게 되고 이로 인해 혈중 당의 농도가 저하됨으로써 비만 및 당뇨 예방용 기능성식품이나 약물에 유용하게 사용될 수 있게 되는 것이다.The present invention relates to a glucose uptake inhibitor and a purification method thereof, which are isolated from pomegranate and closely related to the induction of diabetes mellitus. The glucose uptake inhibitor is obtained by fractionation of methanol extract of pomegranate in the order of nucleic acid, chloroform, ethyl acetate, It is purified through a series of processes such as chromatography, thin layer chromatography and HPLC. Extracts, substances in purification stages, and purified products inhibit glucose absorption, which lowers the concentration of glucose in the blood, which is useful for functional foods and drugs for preventing obesity and diabetes. It will be useful.
Description
본 발명은 석류로부터 분리한 소장에서의 포도당흡수저해제 및 그 정제방법에 관한 것이다. 더욱 상세하게는 본 발명은 석류 추출물을 유효성분으로 함유하는 소장내 포도당흡수저해활성용 석류 추출물에 관한 것이다. 또한 본 발명은 석류의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트, 메탄올로 분획하는 단계; 상기 획득한 메탄올 분획물을 실리카겔 60G 컬럼에 주입한 다음 클로로포롬 : 메탄올 = 80 : 20으로 용출분획하여 수득하는 단계; 상기 활성분획물을 ODS 겔 플레이트와 메탄올 : 물 = 4 : 1의 전개제로 분획하는 단계; 및 상기 활성 분획을 C18 시메트리 컬럼(Symmetry column)이 부착된 HPLC를 사용하여 90% MeOH로 용출분획함으로써 포도당흡수저해제를 분리하는 소장내 포도당흡수저해활성용 석류 추출물의 정제방법에 관한 것이다.The present invention relates to a glucose absorption inhibitor in the small intestine isolated from pomegranate and a method for purifying the same. More specifically, the present invention relates to a pomegranate extract for inhibiting glucose absorption in the intestine, which contains a pomegranate extract as an active ingredient. In another aspect, the present invention is to fractionate the methanol extract of pomegranate with nucleic acid, chloroform, ethyl acetate, methanol; Injecting the obtained methanol fraction into a silica gel 60G column and then eluting with chloroform: methanol = 80: 20; Fractionating the active fraction with an ODS gel plate and a developing agent of methanol: water = 4: 1; And it relates to a method for purifying the glucose-inhibiting inhibitory pomegranate extract in the small intestine to separate the glucose absorption inhibitor by eluting the active fraction with 90% MeOH using HPLC with a C 18 Symmetry column attached.
세계적으로 당뇨병은 지역에 관계없이 발병하고 있으며 발병율 또한 증가 추세에 있을 뿐만 아니라 합병증에 의해 사망에 이르는 대표적인 성인병 중의 하나이다. 1998년 WHO(세계보건기구)의 보고서에 의하면 향후 25년 이내에 현 1억 4천만명의 당뇨병환자가 3억명으로 2배이상 증가할 것으로 예측됨으로써 당뇨병의 예방, 제어 및 치료법의 개발이 절실히 요구되고 있다(참조 : Oku, A. et al., Diabete, 48 : 1794 (1994)).Diabetes is a worldwide disease, and the incidence rate is not only increasing, but is also one of the most common adult diseases leading to death due to complications. According to a 1998 report by the World Health Organization (WHO), more than double the current 140 million diabetics to 300 million people within the next 25 years, urgently requiring the development of prevention, control and treatment of diabetes. (Oku, A. et al., Diabete, 48: 1794 (1994)).
당뇨병은 인슐린의존성당뇨(IDD, insulin-dependent diabetes)와 인슐린비의존성당뇨(NIDDM, non-insulin-dependent diabetes mellitus)로 구분되며 선진국에서는 NIDDM이 크게 증가하고 있다. 이 NIDDM은 에너지의 과잉섭취와 운동부족 등의 외부환경인자에 영향을 받고 있으며 실제로 이들 외부인자는 비만과도 관련되어 NIDDM 환자 중 여성의 70%와 남성의 50%가 비만으로 나타나 있다. 비만은 인슐린 저항성을 유도하기 때문에 비만치료에 사용되고 있는 식사제한, 과혈당개선, 적절한 에너지 유지 및 흡수저해제, 발열촉진제 등이 NIDDM의 처치에 효과적인 것으로 보고되고 있으나 실제 당뇨환자들에 있어 칼로리섭취와 식사량을 제한하는 일은 쉬운 일이 아니다.Diabetes is divided into insulin-dependent diabetes (IDD) and non-insulin-dependent diabetes mellitus (NIDDM). In developed countries, NIDDM is increasing. The NIDDM is affected by external environmental factors such as excessive intake of energy and lack of exercise. Actually, these external factors are also associated with obesity. Because obesity induces insulin resistance, dietary restriction, hyperglycemia improvement, proper energy maintenance and absorption inhibitory, and fever-stimulating agent, which are used to treat obesity, are reported to be effective in the treatment of NIDDM. Limiting is not easy.
식이 탄수화물의 소장내 흡수는 amlyase와 glucosidase에 의해 소화되어 포도당으로 분해된 후 소장 상피세포에서 흡수, 수송된다. 이 수송에 관여하는 운반체는 GLUT(sodium-independent glucose transporters) 계열과 SGLT (sodium dependent glucose transporters) 계열로 대별되며, 포도당은 SGLT1의 작용에 의해 소장내에서 흡수된다(참조 : Silverman, M. et al., Annu. Rev. Biochem. 60 : 757 (1991), Thorens, B, Annu. Rev. Physiol. 55 : 591 (1993)). SGLT1은 소장내 가장 함량이 높은 포도당운반체로서 상피세포의 BBMV(brush border membrane vesicle)에 존재하기 때문에 포도당 과다 흡수에 의한 비만과 당뇨의 예방용 소재개발에 있어 주목을 받아온 운반체 중의 하나이다(참조 : Bell, G.I. et al., Diabetes Care. 13 : 198 (1990)).Intestinal absorption of dietary carbohydrates is digested by amlyase and glucosidase, broken down into glucose, and then absorbed and transported in small intestinal epithelial cells. Carriers involved in this transport are classified into the class of sodium-independent glucose transporters (GLUT) and the class of sodium dependent glucose transporters (SGLT), and glucose is absorbed in the small intestine by the action of SGLT1 (see Silverman, M. et al. , Annu. Rev. Biochem. 60: 757 (1991), Thorens, B, Annu. Rev. Physiol. 55: 591 (1993)). SGLT1 is one of the carriers with the highest content of glucose in the small intestine and has been attracting attention in developing materials for the prevention of obesity and diabetes caused by excessive absorption of glucose because it is present in BBMV (brush border membrane vesicle) of epithelial cells. , GI et al., Diabetes Care. 13: 198 (1990)).
따라서 SGLT1의 생합성 억제 또는 활성저해에 의한 비만, 당뇨의 예방 및 치료제 개발 전략은 기존의 전분가수분해효소 저해제(참조 : Tsujihara, K. et al., J. Med. Chem. 42 : 5311 (1999), Vedavanam, K. et al., Phytother Res. 13 : 601 (1999), Murakami, N. et al., Chem. Pharm. Bull. 44 : 469 (1996), Welsch, C.A. et al., J . Nutr. 119 : 1698 (1989))에 의한 방법에 비해 음식물의 섭취량과 종류에 관계없이 포도당의 흡수를 직접적으로 제어할 수 있다는 관점에서 유효성이 높다.Therefore, strategies for the prevention and treatment of obesity and diabetes by inhibiting or inhibiting the biosynthesis of SGLT1 are known to be known as starch hydrolase inhibitors (Tsujihara, K. et al., J. Med. Chem. 42: 5311 (1999). , Vedavanam, K. et al., Phytother Res. 13: 601 (1999), Murakami, N. et al., Chem. Pharm. Bull. 44: 469 (1996), Welsch, CA et al., J. Nutr 119: 1698 (1989)) are more effective in terms of direct control of glucose uptake regardless of food intake and type.
기능성식품, 식품의약, 의약의 개발에 있어 요구되는 안전성을 확보하고, 물질의 임상에 대한 위험성을 감소시키기 위해 오랫동안 식품으로 이용해 온 식용생물자원을 대상으로 포도당흡수저해물질을 검색하였다. 검색을 위한 assay계는 분화시 소장의 상피세포와 기능 및 형태가 유사하고 SGLT1을 함유하고 있는 대장암세포주의 하나인 Caco-2와 비방사성물질이면서 취급이 용이한 2-NBDG(2-(N-(7-니트로벤즈-2-옥사-1,3-디아졸-4-일)아미노)-2-디옥시글루코스)를 사용하여 포도당의 소장내 흡수 저해활성을 측정하였다(참조 : Sokoloff. L. J. Neurochem. 28 : 897 (1997)). Glucose absorption inhibitors were searched for edible biological resources that have been used for a long time in order to secure the safety required for the development of functional foods, food medicines, and medicines and to reduce the risks of the substance. The assay system for screening was similar to the epithelial cells of the small intestine during differentiation, and Caco-2, one of colorectal cancer cell lines containing SGLT1, and non-radioactive and easy-to-handle 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2-dioxyglucose) was used to measure glucose intestinal absorption inhibitory activity (see Sokoloff. LJ Neurochem). 28: 897 (1997).
Caco-2 세포주는 주로 금속과 지질의 흡수 연구에 이용되어 왔고 최근에 이르러 SGLT1이 밝혀짐에 따라 BBMV(brush border membrane vesicles)와 함께 포도당 흡수기작의 규명에 사용되었다(참조 : Yoshida, A. et al., Histochem. J. 27 : 42 (1995), Davidson, N.O. et al., Am. J. Physiol. 262 : c795 (1992), Dyer, J. et al., Biochem. Biophys. Res. Commun. 230 : 624 (1997), Dyer, J. et al., Gut. 41 : 56 (1997), Ferraris, R.P. Biochem. J. 360 : 265 (2001)). 그러나 Caco-2를 이용한 소장내 포도당 흡수저해물질에 관한 연구는 녹차 추출물인 T-1095 물질의 streptozotocin 유도 당뇨쥐의 과혈당 개선효과(참조 : Adachi, T. et al., Metabolism, 49 : 990 (2000)), T-1095 물질의 C57BL/KSJ-db/db mice의 당뇨증상의 개선효과(참조 : Arokawa, K. et al., Br. J. Pharmacol. 132 : 578 (2001)), 녹차 카테킨에 의한 소장 포도당의 수송조절(참조 : Shimizu, M. et al., Biofactors, 13 : 61 (2000)), 녹차 polyphenol의 경쟁적 Na 의존성 포도당 수송저해(참조 : Kobayashi, Y. et al., J. Agric. Food Chem. 48 : 5618 (2000)) 등이 보고되어 있을 뿐 소장에서 포도당 흡수저해제의 개발에 관한 연구는 미미하다.Caco-2 cell lines have been used mainly for the study of metal and lipid uptake, and recently SGLT1 has been used to identify glucose uptake mechanisms with brush border membrane vesicles (BBMV) (Yoshida, A. et. al., Histochem. J. 27: 42 (1995), Davidson, NO et al., Am. J. Physiol. 262: c795 (1992), Dyer, J. et al., Biochem. Biophys. Res. Commun. 230: 624 (1997), Dyer, J. et al., Gut. 41: 56 (1997), Ferraris, RP Biochem. J. 360: 265 (2001)). However, studies on glucose-inhibiting substances in the small intestine using Caco-2 have been shown to improve the hyperglycemia of streptozotocin-induced diabetic rats with T-1095, a green tea extract. )), Improvement effect of diabetes mellitus in C57BL / KSJ-db / db mice of T-1095 substance (Arokawa, K. et al., Br. J. Pharmacol. 132: 578 (2001)), green tea catechin Control of small intestine glucose (see Shimizu, M. et al., Biofactors, 13: 61 (2000)), competitive Na-dependent glucose inhibition of green tea polyphenols (Kobayashi, Y. et al., J. Agric) Food Chem. 48: 5618 (2000)) has been reported, but little research has been made on the development of glucose-absorbing inhibitors in the small intestine.
석류(Punica pranatum)는 지중해 연안과 서남아시아 지방에서 일찍이 재배하기 시작한 과수로 우리나라에는 중국을 거쳐 3세기말 이후에 들어왔으며 현재는 주로 중국(서안), 미국(캘리포니아), 인도 등에서 대량으로 50종 이상이 재배되고 있다. 석류열매의 성분은 주로 tannin(10.4∼21.3%), gallic acid(4.0%), mannitol(8.0%), sucrose(2.7%), pectin(0.6%)과 소량의 malic acid 등으로 구성되어 있다. 대한약전에 수재된 석류피는 조충구제약으로 석류열매는 지사작용과 세균성이질을 고치는 효능이 있는 것으로 알려진 바 있으나 석류열매가 소장에서 포도당 흡수를 저해하는데 탁월한 효과가 있음이 보고되어진 바는 없다.Pomegranate ( Punica pranatum ) is an orchard that has been cultivated early on the Mediterranean coast and southwestern Asia. It came to Korea after the end of the 3rd century through China. Currently, more than 50 kinds of pomegranate are mainly in China (Xian), USA (California), India, etc. It is cultivated. Pomegranate fruit consists mainly of tannin (10.4 ~ 21.3%), gallic acid (4.0%), mannitol (8.0%), sucrose (2.7%), pectin (0.6%) and small amount of malic acid. Pomegranate bark, which has been harvested from the Korean Pharmacopoeia, is a medicinal insecticide, and pomegranate fruit has been known to be effective in correcting branching and bacterial dysfunction, but no pomegranate fruit has been reported to have an excellent effect on inhibiting glucose absorption in the small intestine.
따라서 본 발명자들은 상기와 같이 소장에서 포도당흡수저해제로 사용할 수 있도록 식품신소재를 개발하기 위해 식용생물자원을 대상으로 검색을 수행하던 중 국내에서도 재배되고, 먹어오기도 한 석류의 메탄올 추출물에 높은 포도당흡수저해활성을 확인하고 메탄올 추출물로부터 상기 활성을 갖는 저분자 물질을 정제함으로써 본 발명을 이루게 되었다. Therefore, the present inventors are cultivated in Korea during the search for edible biological resources to develop a new food material to be used as a glucose absorption inhibitor in the small intestine as described above, high glucose absorption inhibition in methanol extract of pomegranate which is also eaten The present invention has been achieved by confirming the activity and purifying the low molecular material having the above activity from the methanol extract.
본 발명의 목적은 상기와 같은 종래의 문제점을 해소하기 위한 것으로, 특히 소장내에서 포도당 흡수를 저해하는 포도당흡수저해제를 제공함을 목적으로 한다. 또한 본 발명의 또 다른 목적은 석류로부터 포도당흡수저해물질을 분리하는 방법을 제공함에 있다. 상기와 같은 목적을 달성하기 위하여 본 발명은 석류로부터 분리한 포도당흡수저해제를 석류로부터 분리함을 특징으로 한다. 또한, 상기와 같은 목적을 달성하기 위하여 본 발명 석류로부터 분리한 소장에서의 포도당흡수저해제 및 그 정제방법은 석류를 분쇄한 후, 실온에서 메탄올로 추출하여 그 농축물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획하여 실리카겔 컬럼 크로마토그래피, HPLC 등 일련의 정제공정을 수행함으로써 석류로부터 분리함을 특징으로 한다. An object of the present invention is to solve the conventional problems as described above, in particular to provide a glucose absorption inhibitor that inhibits glucose absorption in the small intestine. It is another object of the present invention to provide a method for separating glucose absorption inhibitors from pomegranate. In order to achieve the above object, the present invention is characterized in that the glucose absorption inhibitor separated from the pomegranate is separated from the pomegranate. In addition, in order to achieve the above object, the glucose absorption inhibitor in the small intestine separated from the pomegranate of the present invention and the purification method thereof are pulverized pomegranate, extracted with methanol at room temperature, the concentrate is extracted with nucleic acid, chloroform, ethyl acetate It is characterized in that the separation from the pomegranate by performing a series of purification steps such as silica gel column chromatography, HPLC.
소장내 포도당 흡수 저해활성을 갖는 석류 유기용매 추출물을 유효성분으로 함유하는 당뇨병 예방 또는 치료용 조성물을 제공한다. 상기 유기용매는 물, 메탄올, 헥산, 클로로포름, 에틸아세테이트 또는 이들의 혼합용매임을 의미한다. 또한 본 발명은 석류의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트, 메탄올로 분획하는 단계; 상기 획득한 메탄올 분획물을 실리카겔 60G 컬럼에 주입한 다음 클로로포롬 : 메탄올 = 80 : 20으로 용출분획하여 수득하는 단계; 상기 활성분획물을 ODS 겔 플레이트와 메탄올 : 물 = 4 : 1의 전개제로 분획하는 단계; 및 상기 활성 분획을 C18 시메트리 컬럼(Symmetry column)이 부착된 HPLC를 사용하여 90% MeOH로 용출분획함으로써 포도당흡수저해제를 분리하는 소장내 포도당흡수저해활성용 석류 추출물의 정제방법이다.Provided is a composition for preventing or treating diabetes, comprising a pomegranate organic solvent extract having a glucose absorption inhibitory activity in the small intestine as an active ingredient. The organic solvent means water, methanol, hexane, chloroform, ethyl acetate or a mixed solvent thereof. In another aspect, the present invention is to fractionate the methanol extract of pomegranate with nucleic acid, chloroform, ethyl acetate, methanol; Injecting the obtained methanol fraction into a silica gel 60G column and then eluting with chloroform: methanol = 80: 20; Fractionating the active fraction with an ODS gel plate and a developing agent of methanol: water = 4: 1; And distilling the active fraction with 90% MeOH using HPLC with a C 18 symmetry column to purify the glucose inhibitor active pomegranate extract.
본 발명 석류로부터 분리한 소장에서의 포도당흡수저해제 및 그 정제방법의 기술적 사항을 첨부된 도면을 참고로 하여 상세하게 설명하면 다음과 같다. 본 발명자들은 식용생물자원에서 추출한 추출물 중에서 석류의 메탄올 추출물이 비교적 높은 포도당흡수저해 활성을 가지고 있음을 발견하였다. 이에 석류에 포함된 포도당흡수저해활성 물질을 분리하고자 실온에서 100% 메탄올로 추출한 후 이 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획을 실시한 다음, 포도당 흡수 저해활성이 높았던 메탄올 분획물을 실리카겔 컬럼 크로마토그래피하였다. 그 결과 포도당흡수저해활성이 높은 분획을 모아 ODS 겔 플레이트(gel Plate)로 Prep-TLC를 실시한 후 높은 활성의 분획을 HPLC를 실시하여 정제분획을 얻었다. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The technical details of the glucose absorption inhibitory agent in the small intestine separated from the pomegranate of the present invention and its purification method will be described in detail with reference to the accompanying drawings. The present inventors found that methanol extract of pomegranate has relatively high glucose absorption inhibitory activity among extracts extracted from edible biological resources. In order to isolate the glucose-absorbing inhibitory substance contained in pomegranate, the extract was extracted with 100% methanol at room temperature, and then the extract was fractionated in the order of nucleic acid, chloroform, ethyl acetate, and then the methanol fraction having high glucose absorption inhibitory activity was subjected to silica gel column chromatography. It was. As a result, the fractions with high glucose absorption inhibitory activity were collected and prep-TLC was performed on an ODS gel plate to obtain a purified fraction by HPLC.
다음은 상기와 같이 구성된 본 발명에 대해 실시예를 통해 보다 구체적으로 설명하고자 한다. 이들 실시예는 오로지 본 발명을 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서는 자명할 것이다.Next will be described in more detail with reference to the embodiment of the present invention configured as described above. These examples are only for illustrating the present invention in detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
실시예 1 : 석류로부터 소장내 포도당흡수저해제 분리를 위한 추출조건 선정과 저해능 확인Example 1 Selection of Extraction Conditions and Confirmation of Inhibitory Activity for Separation of Glucose Absorption Inhibitors from Small Pomegranate
여러 가지 식용생물자원에서 냉수, 열수, 메탄올로 추출한 추출물을 검색하던 중 석류의 메탄올 추출물이 포도당흡수저해능을 나타내는 것을 확인하였다. 이에 석류에 포함된 포도당 흡수 저해물질을 분리하고자 석류를 100℃에서 5분간 블랜칭(blanching)하여 생체세포내 효소를 불활성화시키고 분쇄한 후, 실온에서 100% 메탄올로 추출하고, 그 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획하고 남은 것을 메탄올에 녹여 각각을 여과포로 여과하고 잔사를 감압 농축한 후 건조하여 추출물을 얻었다(도 1).While searching for extracts extracted with cold water, hot water and methanol from various edible biological sources, it was confirmed that methanol extract of pomegranate exhibited glucose absorption inhibitory ability. In order to isolate the glucose absorption inhibitors contained in the pomegranate, the pomegranate was blanched at 100 ° C. for 5 minutes to inactivate and pulverize enzymes in living cells, and then extracted with 100% methanol at room temperature. , Chloroform and ethyl acetate were partitioned in this order, and the residue was dissolved in methanol, and each was filtered through a filter cloth. The residue was concentrated under reduced pressure and dried to obtain an extract (FIG. 1).
각 분획물에 대한 포도당흡수저해 실험을 하기 위해 Caco-2(human colonic cancer cell)을 6x104 cells/well의 농도로 24 well-plate로 37℃, CO2 incubator에서 13-14일 동안 배양한 후 저해활성 측정 전날 원배지를 제거한 후 PBS로 세척하여 배지내 serum 성분을 제거시킨다음 새로운 serum free DMEM에 시료를 0.1 mg/ml의 농도로 첨가하여 37℃, CO2 incubator에서 20시간 배양시켰다. 배양이 끝나면 배지를 제거하고 PBS로 2회 cell을 세척 후 반응의 종료를 위해 반응액을 제거하고 ice-cold PBS로 3회 수세한 다음 2-NBDG의 형광세기를 spectrophotofluorometer (여기 : 485 nm, 발광 : 535 nm)로 측정하였다. 시료에 의한 포도당 흡수 저해활성은 아래 식에 따라 환산하였다.In order to inhibit glucose absorption of each fraction, Caco-2 (human colonic cancer cell) was incubated for 13-14 days in a CO 2 incubator at 37 ° C and 24 well-plate at a concentration of 6x10 4 cells / well. On the day before the activity measurement, the medium was removed and washed with PBS to remove the serum components in the medium. The samples were added to fresh serum free DMEM at a concentration of 0.1 mg / ml and incubated at 37 ° C. in a CO 2 incubator for 20 hours. After incubation, remove the medium, wash the cells twice with PBS, remove the reaction solution to terminate the reaction, wash three times with ice-cold PBS, and measure the fluorescence intensity of 2-NBDG with a spectrophotofluorometer (excitation: 485 nm, luminescence). : 535 nm). Glucose absorption inhibitory activity by the sample was converted according to the following equation.
Inhibitory effect (%) = [Fs - (Fs / Fc)] ×100 Inhibitory effect (%) = [Fs-(Fs / Fc)] × 100
Fs : 시료를 처리한 실험구의 형광세기 Fs: Fluorescence intensity of the experimental group treated with the sample
Fc : 시료를 처리하지 않은 실험구의 형광세기 Fc: Fluorescence intensity of the experimental group without sample treatment
상기와 같이 분획에 따른 포도당흡수저해활성을 측정한 결과, 메탄올 분획에서 높은 활성을 나타내는 것을 확인하였다(도 2). 클로로포름으로 활성화된 실리카겔 60 컬럼에 시료인 메탄올 분획을 loading하고 클로로포름과 메탄올(CHCl3 : MeOH = 100 : 0 ~ 0 : 100) 혼합용액으로 5 bed volume씩 전개한 후, 포도당 흡수 저해활성을 검토하여 클로로포름-메탄올 80 : 20에서 포도당 흡수 저해활성을 갖는 소분획을 분리하고 이 분획물을 동일한 컬럼을 사용하여 클로로포름과 메탄올 (CHCl3 : MeOH = 85 : 15 ~ 70 : 30) 혼합용액으로 5 bed volume씩 전개한 후 포도당 흡수 저해활성이 가장 높은 80 : 20 용출획분을 분리하였다. 고활성의 상기 분획물을 ODS gel plate (20 ×10 cm), 메탄올 : 물 = 4 : 1의 전개용매와 에탄올에 녹인 5% H2SO4를 발색제로 사용하여 Prep-TLC를 실시하여 활성분획을 분리하였다(도3). 분리된 소분획을 HPLC (u-Bondapak C18 (7.8 ×300 mm)에 주입하고 물과 메탄올 gradient (H2O : MeOH = 10 : 90 ~ 0 : 100) 용매조건에서 2 ml/min의 유속으로 용출하여, 활성획분만을 농축한 후 활성획분을 동일한 조건에서 HPLC를 실시한 결과 좌우 대칭성이 높은 단일 peak를 얻음으로써 비교적 정제도 높게 물질을 정제할 수 있었다(도4). 각 정제단계별 수율과 IC50 값을 종합하면 표 1과 같으며 정제물질의 IC50 값이 매우 낮음을 나타냄으로써 실용화 가능성을 보였다.As a result of measuring glucose absorption inhibitory activity according to the fraction, it was confirmed that the methanol fraction exhibits high activity (FIG. 2). After loading the methanol fraction as a sample on the silica gel 60 column activated with chloroform, it was developed by 5 bed volumes with a mixed solution of chloroform and methanol (CHCl 3 : MeOH = 100: 0 ~ 0: 100), and the glucose absorption inhibitory activity was examined. A small fraction having glucose absorption inhibitory activity was isolated from chloroform-methanol 80: 20, and the fractions were separated by 5 bed volumes with a mixture of chloroform and methanol (CHCl 3 : MeOH = 85: 15 ~ 70: 30) using the same column. After development, the 80:20 eluted fraction with the highest glucose absorption inhibitory activity was isolated. The fraction of the highly active ODS gel plate (20 × 10 cm), methanol: water = 4: 1 using a developing solvent and 5% H 2 SO 4 dissolved in ethanol as a colorant to perform a prep-TLC active fraction Isolated (Figure 3). The separated subfraction was injected into HPLC (u-Bondapak C 18 (7.8 × 300 mm), and the flow rate was 2 ml / min under water and methanol gradient (H 2 O: MeOH = 10: 90 ~ 0: 100) solvent conditions. After eluting and concentrating only the active fractions, HPLC was performed on the active fractions under the same conditions. As a result, a single peak with high symmetry was obtained to obtain a relatively high degree of purification (Fig. 4). The total value of 50 is shown in Table 1, and shows that the IC 50 value of the purified material is very low.
[표 1]TABLE 1
실시예 2 : 석류로부터 분리한 포도당흡수저해물질의 급성독성 실험Example 2 Acute Toxicity Test of Glucose Absorption Inhibitors Isolated from Pomegranate
석류에 함유된 포도당흡수저해물질의 급성독성을 조사하기 위해 생쥐를 사용하여 경구투여에 의한 경우 표2에 나타난 바와 같이 시료랑 0 ~ 2,000 mg/kg의 모든 실험군에서 경구투여에 의한 독성은 나타나지 않았으며 LD50이 2,000 mg/kg 이상에 존재한 것으로 추정되었다.To investigate the acute toxicity of glucose-absorbing inhibitors in pomegranate, oral administration using mice did not show toxicity by oral administration in all experimental groups from 0 to 2,000 mg / kg. It is estimated that LD 50 is present at more than 2,000 mg / kg.
[표 2] 경구투여를 이용한 포도당흡수저해성 메탄올 추출물의 급성독성실험[Table 2] Acute Toxicity Test of Glucose Absorption Inhibitory Methanol Extracts by Oral Administration
실시예 3 : 석류로부터 분리한 소장내 포도당흡수저해제의 활성비교Example 3 Activity Comparison of Intestinal Glucose Absorption Inhibitors Isolated from Pomegranate
실시예 1의 방법으로 석류의 메탄올 추출물과 석류로부터 분리한 물질을 농도별로 포도당흡수저해활성을 측정하였다. 비교실험예로 포도당흡수저해제로 널리 알려진 phlorizin(0.1 mg/ml)을 사용하여 소장에서의 포도당 흡수저해활성을 비교한 결과 동일농도에서 정제물질이 약 10% 높은 저해활성을 나타내었다(도 5).In the method of Example 1, the methanol extract of pomegranate and the substance isolated from the pomegranate were measured for glucose absorption inhibitory activity by concentration. As a comparative example, the use of phlorizin (0.1 mg / ml), which is widely known as a glucose absorption inhibitor, was used to compare glucose absorption inhibitory activity in the small intestine. .
이상에서 살펴본 바와 같이, 본 발명 석류로부터 분리한 소장내 포도당흡수저해 효과 및 그 정제방법은 블랜칭(blanching)하여 분쇄한 후, 메탄올로 추출하여 그 잔사를 핵산, 클로로포름, 에틸아세테이트 순으로 분획하여 실리카겔 컬럼 크로마토그래피, HPLC 등 일련의 정제공정을 수행함으로써 얻어졌으며, 당뇨 및 비만 예방용 기능성식품이나 약물에 사용될 수 있는 효과가 있는 유용한 발명이다. As described above, the small intestine glucose absorption inhibitory effect and purification method isolated from the pomegranate of the present invention by blanching and pulverized, extracted with methanol and the residue was fractionated in the order of nucleic acid, chloroform, ethyl acetate Obtained by performing a series of purification processes such as silica gel column chromatography, HPLC, and is a useful invention that can be used in functional foods and drugs for the prevention of diabetes and obesity.
도 1은 석류로부터 메탄올추출 및 추출물의 유기용매 분획 단계를 보인 과정도이다.1 is a process diagram showing the organic solvent fractionation step of methanol extraction and extract from pomegranate.
도 2는 도 1의 각 단계 분획물의 포도당흡수저해활성을 보인 도표이다. FIG. 2 is a graph showing glucose absorption inhibitory activity of each step fraction of FIG. 1.
도 3은 분획된 메탄올 추출물로부터 정제단계를 보인 과정도이다.Figure 3 is a process diagram showing the purification step from the fractionated methanol extract.
도 4는 분리물질의 정제도를 보인 도표이다.Figure 4 is a diagram showing the purification of the separation material.
도 5는 정제물질, 메탄올추출물 및 시판 포도당흡수저해제의 저해활성을 보인 도표이다.Figure 5 is a chart showing the inhibitory activity of the purified material, methanol extract and commercial glucose uptake inhibitors.
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